p38MAPK is a novel DNA damage response‐independent regulator of the senescence‐associated secretory phenotype

Adam Freund; Christopher K Patil; Judith Campisi

doi:10.1038/emboj.2011.69

p38MAPK is a novel DNA damage response‐independent regulator of the senescence‐associated secretory phenotype

Freund, Adam; Patil, Christopher K; Campisi, Judith 2011-04-20 00:00:00 The EMBO Journal (2011) 30, 1536–1548 & 2011 European Molecular Biology Organization All Rights Reserved 0261-4189/11 | | T THE H E www.embojournal.org E E EMB M MB BO O O J JO OURN URN A AL L p38MAPK is a novel DNA damage response-independent regulator of the senescence-associated secretory phenotype 1,2,3 2,3 senescence is a common response to oncogene activation Adam Freund , Christopher K Patil 2,3, in vivo (Prieur and Peeper, 2008) suggest that senescence and Judith Campisi * may be as important as apoptosis for suppressing the Department of Molecular and Cell Biology, University of California, 2 development of cancer. Berkeley, CA, USA, Buck Institute for Age Research, Novato, CA, USA The senescent phenotype is multi-faceted. The chief hall- and Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA mark of senescent cells—the halt of proliferation (hereafter, growth arrest)—is essentially irreversible in that it cannot be Cellular senescence suppresses cancer by forcing poten- reversed by physiological stimuli. This growth arrest is INK4a tially oncogenic cells into a permanent cell cycle arrest. established and maintained by the p53 and p16 /pRB Senescent cells also secrete growth factors, proteases, and pathways. Although many questions remain, we broadly inﬂammatory cytokines, termed the senescence-associated understand the interwoven and complementary mechanisms secretory phenotype (SASP). Much is known about path- by which these pathways regulate the growth arrest (Collins ways that regulate the senescence growth arrest, but far and Sedivy, 2003; Ohtani et al, 2004; Gil and Peters, 2006; less is known about pathways that regulate the SASP. Campisi and d’Adda di Fagagna, 2007; Rodier et al, 2007). By We previously showed that DNA damage response (DDR) contrast, we understand little about how other senescence- signalling is essential, but not sufﬁcient, for the SASP, associated phenotypes are controlled. which is restrained by p53. Here, we delineate another Senescent cells develop an enlarged morphology, upregu- crucial SASP regulatory pathway and its relationship late a senescence-associated b-galactosidase (SA-bgal) acti- to the DDR and p53. We show that diverse senescence- vity (Dimri et al, 1995), and show widespread changes in inducing stimuli activate the stress-inducible kinase chromatin organization and gene expression (Campisi and p38MAPK in normal human ﬁbroblasts. p38MAPK inhibi- d’Adda di Fagagna, 2007). Of particular biological impor- tion markedly reduced the secretion of most SASP factors, tance, senescent cells increase the expression and secretion constitutive p38MAPK activation was sufﬁcient to induce of numerous cytokines, chemokines, matrix metalloprotei- an SASP, and p53 restrained p38MAPK activation. Further, nases (MMPs), and other proteins that can alter local tissue p38MAPK regulated the SASP independently of the environments. We termed this feature the senescence- canonical DDR. Mechanistically, p38MAPK induced the associated secretory phenotype (SASP) (Coppe et al, 2008, SASP largely by increasing NF-jB transcriptional activity. 2010b). These ﬁndings assign p38MAPK a novel role in SASP The SASP can be beneﬁcial or deleterious, depending regulation—one that is necessary, sufﬁcient, and inde- on the biological context. Among the beneﬁts, some SASP pendent of previously described pathways. components reinforce the senescence growth arrest The EMBO Journal (2011) 30, 1536–1548. doi:10.1038/ (Kortlever et al, 2006; Acosta et al, 2008; Kuilman et al, emboj.2011.69; Published online 11 March 2011 2008; Wajapeyee et al, 2008). Others signal the immune Subject Categories: signal transduction; genome stability & system to clear senescent cells (Xue et al, 2007), and SASP dynamics MMPs can suppress ﬁbrotic scar formation (Krizhanovsky Keywords: aging; cancer; inﬂammation; NF-kB; tumour et al, 2008; Jun and Lau, 2010). Among the deleterious suppression effects, SASP MMPs can disrupt mammary morphogenesis in cell culture models (Parrinello et al, 2005). Further, in culture and in vivo, SASP factors can promote malignant phenotypes, including cell proliferation (Bavik et al, 2006; Coppe et al, 2010b), angiogenesis (Coppe et al, 2006), Introduction epithelial-to-mesenchymal transitions and invasiveness (Coppe et al, 2008), and accelerated growth of xenografted Cellular senescence halts the proliferation of cells at risk tumours (Krtolica et al, 2001; Liu and Hornsby, 2007). for malignant transformation. Many potentially oncogenic Moreover, many SASP factors are pro-inﬂammatory. Thus, stimuli, ranging from DNA damage to the activation of certain senescent cells, which increase with age in vivo (Dimri et al, oncogenes, can induce a senescence response (Campisi and 1995; Paradis et al, 2001; Jeyapalan et al, 2007; Zhou et al, d’Adda di Fagagna, 2007). Recent data showing that cellular 2008), may contribute to the low-level chronic inﬂammation that is a hallmark of aged mammalian tissues and many *Corresponding author. The Buck Institute for Age Research, 8001 Redwood Blvd, Novato, CA 94945, USA. age-related diseases (Freund et al, 2010; Coppe et al, 2010a). Tel.: þ 1 415 209 2066; Fax: þ 1 415 493 3640; This important anti-cancer defense, then, might be antago- E-mail: [email protected] nistically pleiotropic and, ironically, promote cancer in aged tissues. Given the known and proposed importance of the Received: 21 October 2010; accepted: 18 February 2011; published online: 11 March 2011 SASP, it is crucial to understand its regulation. 1536 The EMBO Journal VOL 30 NO 8 2011 &2011 European Molecular Biology Organization | | p38 regulates the senescence secretory phenotype A Freund et al Although the senescence growth arrest and SASP are often chemokines, growth factors, MMPs and shed receptors and coordinately induced, the pathways that regulate them do not ligands (Coppe et al, 2008, 2010b). INK4a completely overlap. For example, p16 expression is We show that p38MAPK activity is necessary and sufﬁcient sufﬁcient to induce a senescence growth arrest, but does for development of an SASP in cells induced to senesce by not induce or modify the SASP (Coppe et al, 2010a). Likewise, direct DNA damage or oncogenic RAS. In these contexts, p53 is required for the growth arrest (Campisi and d’Adda di p38MAPK is not activated rapidly, but rather with delayed Fagagna, 2007; Courtois-Cox et al, 2008), but not the SASP; kinetics characteristic of the SASP. Further, p53 restrains the in fact, cells lacking functional p53 secrete markedly higher SASP by restraining p38MAPK activation, and p38MAPK levels of most SASP factors. Thus, p53 restrains the SASP activation occurs independently of the DDR. Further, (Coppe et al, 2008), suggesting it suppresses tumorigenesis in p38MAPK regulates the SASP mainly through NF-kB trans- part by limiting the development of a pro-inﬂammatory tissue criptional activity, which we show is required for the environment. The pathways by which p53 restrains the SASP expression of most SASP factors. These ﬁndings assign the are not known. p38MAPK pathway a novel role in senescence regulation. The SASP is not an acute (rapid, transient) inﬂammatory response. It does not develop immediately after cells experi- Results ence a senescence-inducing stimulus but, once established, it persists for long intervals (Coppe et al, 2008, 2010b; Rodier p38MAPK is activated during the senescence response et al, 2009). One regulator of the SASP is the DNA damage to genotoxic stress p38MAPK is activated by tyrosine and threonine phosphory- response (DDR), the signalling cascade that senses and lation in response to a variety of stresses (Cuenda and ultimately repairs DNA damage. The DDR is initiated by V12 Rousseau, 2007), including oncogenic RAS (Ha-RAS ) phosphatidylinositol 3-kinase-like protein kinases such as ATM and ATR, which phosphorylate chromatin modifying expression (Wang et al, 2002), which indirectly causes DNA proteins such as gH2AX, adaptor proteins such as NBS1, damage (Di Micco et al, 2006). To determine whether and downstream kinases such as CHK1 and CHK2, which p38MAPK activation is a direct genotoxic stress response, we X-irradiated (XRA; 10 Gy) presenescent (PRE) normal ultimately activate p53 (Ciccia and Elledge, 2010). ATM, human ﬁbroblasts (strain HCA2) to synchronously induce CHK2, and NBS1 are essential for establishing and maintain- senescence (SEN(XRA)). After XRA, cells were cultured for ing the expression of several SASP proteins, particularly inﬂammatory cytokines such as IL-6 and IL-8 (Rodier et al, 10 days, during which time they developed classic markers of 2009). However, canonical DDR signalling is not sufﬁcient for senescence: growth arrest (no increase in cell number; low the SASP: a transient DDR, caused by low-level ionizing 5-bromodeoxyuridine (BrdU) labeling), an enlarged ﬂattened radiation, does not induce an SASP (Rodier et al, 2009). morphology, and SA-bgal activity (Supplementary Figures S1A and B; not shown). To assess p38MAPK activation, we Additionally, the SASP, like some other features of the senes- prepared whole cell lysates at intervals after XRA until cells cent phenotype (e.g., cell enlargement, SA-bgal activity), takes several days to develop after the damaging event, developed a complete senescent phenotype 8–10 days later. whereas the canonical DDR is activated immediately after We analysed levels of total and phosphorylated p38MAPK damage. Thus, at least one additional, slower event—which and its downstream target Hsp27 (Beyaert et al, 1996; Davis cooperates with the DDR, but is independent of rapid- et al, 2005) by western blot. Unlike the rapid and robust response to LPS, p38MAPK phosphorylation (p38-P) in- response DDR factors—must be required for the SASP. creased only slightly in the 24 h immediately after XRA Here, we show that this event is activation of p38MAPK, a (Supplementary Figure S1C). p38-P and Hsp27-P levels did member of the mitogen-activated protein kinase (MAPK) family. Like other MAPK members, p38MAPK is activated not rise substantially until 2–4 days after XRA, reaching peak by phosphorylation, and this generally occurs rapidly (within levels at 8–10 days (Figure 1A), which were sustained for minutes) and transiently (subsiding within a few hours) in weeks (not shown). Thus, the p38MAPK response to senes- response to acute cellular stress (Cuenda and Rousseau, cence-inducing genotoxic stress differed markedly in kinetics from the response to an acute stress (e.g., LPS stimulation) 2007). p38MAPK is important for the senescence growth (Cuenda and Rousseau, 2007) that does not induce senes- arrest due to its ability to activate both the p53 and pRb/ p16 growth arrest pathways. p38MAPK inhibition moderately cence. Importantly, the kinetics of p38MAPK activation delays replicative senescence (caused by dysfunctional closely paralleled the kinetics with which the SASP develops telomeres, which resemble DNA double-strand breaks) (Coppe et al, 2008; Rodier et al, 2009). (Iwasa et al, 2003), and the rapid senescence of cells from patients with Werner’s syndrome, a premature aging disorder p38MAPK activity is required for the SASP caused by a defective DNA repair protein (Davis and Kipling, p38MAPK activation during senescence was inhibited by the 2009). Further, p38MAPK activity is required for the senes- small molecule SB203580 (SB). SB displaces ATP from the cence arrest caused by oncogenic RAS, and constitutive p38MAPK ATP-binding pocket (Young et al, 1997), thereby p38MAPK activity can induce a growth arrest in normal preventing p38MAPK from phosphorylating its targets human cells (Wang et al, 2002; Deng et al, 2004). Whether without preventing p38MAPK phosphorylation itself. As p38MAPK regulates the SASP had not been explored. determined by Hsp27-P levels, daily treatment with 10 mM p38MAPK is known to upregulate speciﬁc cytokines such as SB, which has minimal off-target effects (Cuenda et al, 1995; IL-6, IL-8, and TNFa in some biological contexts (Ono and Wilson et al, 1997), prevented p38MAPK activation after XRA Han, 2000; Zhang et al, 2007), but the upregulation is (Figure 1A). generally an acute response, whereas the SASP is chronic To determine the signiﬁcance of the coincident rise in and includes 440 secreted proteins comprising cytokines, p38MAPK activity and the SASP, we added SB to SEN(XRA) &2011 European Molecular Biology Organization The EMBO Journal VOL 30 NO 8 2011 1537 | | p38 regulates the senescence secretory phenotype A Freund et al Figure 1 p38MAPK is activated with slow kinetics during genotoxic stress-induced senescence and is required for the SASP. (A) p38MAPK phosphorylation increases during DNA damage-induced senescence. Cells were irradiated and whole cell lysates collected at the indicated days thereafter. þ : p38MAPK was continuously inhibited by 10 mM SB203580 (SB) beginning 48 h before irradiation. Left: western blot analysis of indicated proteins. Right: western blot quantitation, normalized to PRE levels. p38-P, phosphorylated p38MAPK; Hsp27-P, phosphorylated heat shock protein 27. (B) p38MAPK inhibition decreases intracellular IL-6 in SEN(XRA) cells. PRE and SEN(XRA) cells immunostained for IL-6. þ SB: p38MAPK was inhibited by SB023580 (SB) for 48 h before ﬁxation. (C) p38MAPK inhibition decreases IL-6, IL-8, and GM-CSF secreted by SEN(XRA) cells. Conditioned media (CM) were collected from PRE and SEN(XRA) cells and analysed by ELISA. þ SB: p38MAPK was inhibited by SB023580 (SB) for 48 h before CM collection. (D) p38MAPKa depletion decreases secreted IL-6. Cells were infected with lentivirus expressing either of two shRNAs against p38MAPKa (shp38a) or a control shRNA (shGFP) and selected. Cells were irradiated and allowed to senesce (SEN(XRA)). CM were analysed by ELISA. (E) p38MAPK inhibition suppresses the SEN(XRA) SASP. CM from PRE and SEN(XRA) cells, with (SB) or without p38MAPK inhibition, were analysed by antibody arrays. Shown are factors for which the SEN(XRA) level was signiﬁcantly increased (Po0.05) over PRE. For each protein, signals from all conditions were averaged to generate the baseline. Signals above baseline are yellow; signals below baseline are blue. The heat map key shows log -fold changes from baseline. The hierarchical clustering relationship between sample proﬁles is shown graphically as a dendrogram (left). *: Factors signiﬁcantly decreased by p38MAPK inhibition (Po0.05). (F) p38MAPK depletion decreases the ability of senescent cells to stimulate cancer cell invasiveness. CM from cells described in (D) were analysed for the ability to stimulate invasiveness of MDA-MB-231 breast cancer cells in a Boyden chamber invasion assay. cells for 48 h, then assessed IL-6, an indicator of SASP activity regulates the SASP, we depleted cells of p38MAPKa, the most (Coppe et al, 2008, 2010a; Bhaumik et al, 2009; Orjalo et al, abundant isoform, by RNA interference (RNAi). Using lenti- 2009) by immunostaining (intracellular levels) and enzyme- viruses, we expressed in SEN(XRA) cells either of two linked immunoadsorbent assay (ELISA) of conditioned unrelated short hairpin (sh) RNAs that speciﬁcally target medium (CM) (secreted levels). Both assays showed that p38MAPKa (Supplementary Figure S1F). We then assayed SB reduced IL-6 levels to near-PRE levels (Figures 1B and IL-6 levels in CM from cells expressing control (shGFP) or C); ELISA showed that SB also signiﬁcantly reduced secretion p38MAPKa-speciﬁc (shp38a) shRNAs. Both shp38a shRNAs of the SASP components IL-8 and GM-CSF (Figure 1C) signiﬁcantly decreased secreted IL-6 levels in SEN(XRA) cells (Po0.05). Further, SB signiﬁcantly reduced secreted IL-6 (Figure 1D; Po0.01). A p38MAPKb shRNA did not reduce levels in CM from SEN(XRA) WI-38, an unrelated human secreted IL-6 levels (not shown). These data conﬁrm that ﬁbroblast strain (Supplementary Figure S1D), and replica- p38MAPK is essential for induction of the SASP, and identify tively senescent (SEN(REP)) HCA2 and WI-38 cells (Supple- p38MAPKa as the major functional isoform. mentary Figure S1E). Thus, the ability of p38MAPK inhibition The SASP is a complex network comprising 440 proteins to signiﬁcantly reduce senescence-associated IL-6 secretion (Coppe et al, 2008, 2010b). To determine which SASP factors was not conﬁned to XRA-induced senescence or a single cell are p38MAPK regulated, we analysed CM from PRE and strain. These ﬁndings suggest that p38MAPK activation is SEN(XRA) cells, with or without p38MAPK inhibition, using necessary for secretion of at least some SASP components. arrays containing antibodies against 120 secreted proteins. This Although SB is a well-characterized p38MAPK inhibitor, analysis identiﬁed 37 proteins that were signiﬁcantly upregu- one report showed it can partially inhibit protein kinase B at lated in SEN(XRA) cells relative to PRE (Figure 1E). Most of the concentration used here (10 mM) (Lali et al, 2000), and it these proteins (68%, 25/37) declined signiﬁcantly (Po0.05) inhibits both the a and b p38MAPK isoforms (Enslen et al, following p38MAPK inhibition (SEN(XRA)þ SB) (Figure 1E, 1998). To determine whether the effect of SB on SASP compo- asterisks; Supplementary Figure S1G); the remaining SASP nents was p38MAPK speciﬁc, and to identify the isoform that proteins exhibited non-signiﬁcant decreases (Figure 1E). The 1538 The EMBO Journal VOL 30 NO 8 2011 &2011 European Molecular Biology Organization | | p38 regulates the senescence secretory phenotype A Freund et al p38MAPK-regulated proteins included cytokines, chemokines, SEN(RAS), 78% (19/23) were signiﬁcantly decreased by growth factors, shed ligands and, importantly, 9 of the 10 most p38MAPK inhibition in both cases (Supplementary Figure robustly secreted SASP proteins. Hierarchical clustering (Eisen S2C). Thus, a majority of the SASP factors induced by both et al, 1998) of the array results showed that SEN(XRA) cells genotoxic stress (XRA) and oncogene activation (RAS) de- treated with SB had an SASP proﬁle that more closely resem- pends on p38MAPK activity. bled PRE cells than untreated SEN(XRA) cells (Figure 1E). p38MAPK inhibition slightly increased secreted levels of a few p53 restrains the SASP by restraining p38MAPK activity proteins, but not signiﬁcantly (Supplementary Figure S1H). p53 restrains the SASP via an unknown mechanism (Coppe The SASP also includes several MMPs, most prominently et al, 2008). To determine the relationship between p53 and MMP1 and MMP3 (Coppe et al, 2010b). p38MAPK inhibition p38MAPK during development of the SASP, we inactivated had little effect on secreted MMP1 or MMP3 levels; even p53 using retrovirally-delivered GSE22, a peptide that pre- when we started p38MAPK inhibition before XRA and contin- vents p53 tetramerization and thus p53 transcriptional acti- ued until sample collection, only MMP3 declined; MMP1 was vity (Ossovskaya et al, 1996). Because p53 monomers are not affected (Supplementary Figure S1I). Thus, p38MAPK not rapidly degraded, GSE22 activity can be monitored by is a less potent regulator of SASP MMPs, but a strong the accumulation of p53 protein (Figure 2C). We induced positive regulator of many SASP chemokines, cytokines, and p53-deﬁcient cells to senesce with XRA (SEN(XRA)þ GSE), growth factors. and compared phosphorylated p38MAPK levels with those in SEN(XRA) and SEN(RAS) cells. p38MAPK inhibition mitigates a paracrine effect of Activated p38MAPK levels were highest in SEN(XRA)þ senescent cells GSE cells, followed by SEN(RAS) and SEN(XRA) (Figure 2C). CM from senescent cells stimulate the ability of cancer cells The relative levels of p38MAPK phosphorylation matched to invade a basement membrane (Coppe et al, 2008). To the relative levels of IL-6 secretion (Figures 2C versus D), determine whether p38MAPK inhibition mitigates this effect, suggesting that p53 and RAS regulate the intensity of the we measured the ability of CM from PRE or SEN(XRA) cells SASP by regulating the level of p38MAPK activation. p53 expressing either a control (shGFP) or p38MAPKa (shp38a) also regulated the kinetics of SASP development by regulating shRNA to stimulate the invasiveness of MDA-MB-231 human the timing of p38MAPK activation. When p53 was inacti- breast cancer cells. SEN(XRA) CM stimulated B6-fold more vated by GSE22, p38MAPK phosphorylation occurred more invasion than PRE CM (Figure 1F, Po0.001), as expected. rapidly after XRA compared with cells with wild-type p53 p38MAPK depletion markedly reduced this stimulatory acti- (Figure 2E). To determine whether this increase in p38MAPK vity (Figure 1F, Po0.001), indicating that p38MAPK inhibi- activity was responsible for the ampliﬁed SASP in p53- tion can mitigate an important biological consequence of deﬁcient cells, we inhibited p38MAPK with SB. The ampliﬁed the SASP. levels of IL-6, IL-8, and GM-CSF were almost completely suppressed by p38MAPK inhibition (Figure 2F). We obtained similar results when we depleted cells of p53 by RNAi p38MAPK inhibition mitigates the SASP induced by (Supplementary Figures S2D and E). We conclude that oncogenic RAS expression Senescence can be induced by certain oncogenes, including p53 restrains p38MAPK activity after senescence induction, V12 the oncogenic form of H-RAS (RAS ) (Serrano et al, thereby restraining the SASP. 1997). As reported (Di Micco et al, 2006), oncogenic V12 H-RAS expression caused hyperproliferation for several p38MAPK activity is sufﬁcient to induce an SASP days, resulting in DNA damage and ultimately a senescence To study the effect of constitutive p38MAPK activation, growth arrest 8–10 days later (SEN(RAS)) (not shown). Like we infected PRE cells with a constitutively active mutant SEN(XRA) cells, and as reported (Wang et al, 2002; Deng (MKK6EE) of MAP kinase kinase 6 (MKK6), which directly et al, 2004), SEN(RAS) cells showed increased levels of phosphorylates p38MAPK. As expected, MKK6EE expres- activated (phosphorylated) p38MAPK (Figure 2A). Also as sion caused phosphorylation of endogenous p38MAPK reported (Coppe et al, 2008), SEN(RAS) cells expressed an (Figure 3A). Moreover, cells ceased proliferation several ampliﬁed SASP, secreting several proteins at signiﬁcantly days later (Figure 3B). This growth arrest was accompanied higher levels than those secreted by SEN(XRA) cells and by increased SA-bgal activity (Supplementary Figure S3A), several factors not present in the SEN(XRA) SASP. In decreased BrdU incorporation (Supplementary Figure S3B), HCA2 cells, the SEN(RAS) SASP included 83 proteins and a senescent morphology (Supplementary Figure S3C). (Figure 2B; Supplementary Figure S2A). p38MAPK inhibition These responses were prevented by p38MAPK inhibition. (SEN(RASþ SB)) signiﬁcantly reduced (Po0.05) secreted To determine whether constitutive p38MAPK activity levels of 78% (65/83) of these proteins (Figure 2B; (due to MKK6EE expression) is sufﬁcient to induce an Supplementary Figure S2A, asterisks), including 9 of the 10 SASP, we used antibody arrays and identiﬁed 19 factors most robustly secreted SASP proteins. The remaining SASP that were signiﬁcantly upregulated in MKK6EE-expressing proteins were non-signiﬁcantly reduced (Supplementary cells relative to PRE controls (Figure 3C). p38MAPK inhibi- Figure S2A). p38MAPK inhibition also signiﬁcantly reduced tion (MKK6EEþ SB) signiﬁcantly reduced the secreted levels MMP1 and MMP3 levels in SEN(RAS) cells, although to a of most of these proteins (84%, 16/19) (Figure 3C, asterisks); lesser extent than most cytokines and chemokines (Supple- the remaining proteins were non-signiﬁcantly reduced mentary Figure S2B). The SASP proteins affected by p38MAPK (Figure 3C). Notably, 7 of the 10 most upregulated factors inhibition in SEN(RAS) cells overlapped with many of in SEN(XRA) cells, and 9 of the 10 most upregulated factors those affected by p38MAPK inhibition in SEN(XRA) cells: in SEN(RAS) cells, increased signiﬁcantly upon MKK6EE of the 23 factors upregulated in both SEN(XRA) and expression (Figure 3D). We validated the array results by &2011 European Molecular Biology Organization The EMBO Journal VOL 30 NO 8 2011 1539 | | p38 regulates the senescence secretory phenotype A Freund et al V12 Figure 2 p38MAPK drives the ampliﬁed SASPs induced by RAS or p53 inactivation. (A) p38MAPK is phosphorylated during RAS -induced V12 senescence. PRE cells were infected with lentivirus expressing oncogenic RAS , selected, and allowed to senesce (SEN(RAS)) for 10 days. Whole cell lysates were then collected and analysed by western blot. Presenescent controls (PRE) were infected with insertless vector. (B) p38MAPK inhibition suppresses the SEN(RAS) SASP. Cells were infected as described in (A). CM from PRE and SEN(RAS) cells were analysed by antibody arrays. þ SB: p38MAPK was inhibited by SB203580 for 48 h before CM collection. Shown are the top 40 factors for which the SEN(RAS) level was signiﬁcantly increased over PRE. PRE and SEN(RAS) values were averaged to generate the baseline. Heat map and dendrogram were generated as in Figure 1E. *: Factors signiﬁcantly decreased by p38MAPK inhibition. (C) Ampliﬁed p38MAPK phosphoryla- tion in SEN(RAS) cells and SEN(XRA) cells lacking functional p53 (SEN(XRA)þ GSE). Cells were infected with lentivirus expressing GSE22 V12 (GSE) or an insertless vector, selected, then irradiated (XRA) or infected with lentivirus expressing oncogenic RAS (RAS) and allowed to senesce. Whole cell lysates were analysed by western blot. (D) SEN(RAS) and SEN(XRA) cells lacking functional p53 secrete ampliﬁed IL-6 levels. Cells were treated as in (C), then CM were collected and analysed by ELISA. (E) p53 inactivation accelerates p38MAPK phosphorylation after XRA. Cells were infected with lentivirus lacking insert (Vector) or expressing GSE22 (GSE), selected, and irradiated. Whole cell lysates were collected at speciﬁed time points and analysed by western blot. (F) GSE-ampliﬁed levels of IL-6, IL-8, and GM-CSF are p38MAPK dependent. Cells were infected as in (E) and irradiated. CM were collected 3 days later and analysed by ELISA. SB: p38MAPK was inhibited by SB203580 for 48 h before CM collection. ELISA measurements of secreted IL-6 (Supplementary Figure addition to inducing growth arrest, SA-bgal activity, and a S3D). Constitutive p38MAPK activity was also sufﬁcient to senescent morphology. induce MMP1 and MMP3, although to a lesser extent than it induced the cytokines and chemokines (Supplementary p38MAPK regulates the SASP independent of the DDR Figure S3E). Together, these data show that high-level con- The DDR is required for expression of a subset of SASP stitutive p38MAPK activity is sufﬁcient to induce a robust proteins, including IL-6 and IL-8 (Rodier et al, 2009). SASP that resembles the SEN(XRA) and SEN(RAS) SASPs, in To determine whether p38MAPK inhibition decreases the 1540 The EMBO Journal VOL 30 NO 8 2011 &2011 European Molecular Biology Organization | | p38 regulates the senescence secretory phenotype A Freund et al Figure 3 Constitutive p38MAPK activation is sufﬁcient to induce an SASP. (A) PRE cells were infected with lentivirus expressing a constitutively active MAP kinase kinase 6 mutant (MKK6EE). Whole cell lysates were collected 8 days after infection and analysed by western blot. Presenescent controls (PRE) were infected with insertless vector. (B) MKK6EE induces p38MAPK-dependent growth arrest. Cells were infected as in (A) and counted at the indicated intervals thereafter. þ SB: p38MAPK was continuously inhibited by SB203580 beginning 48 h before infection. (C) MKK6EE induces an SASP. Cells were infected as described in (A); CM were collected 8 days after infection. þ SB: p38MAPK was inhibited by SB203580 for 48 h before CM collection. Secreted proteins were analysed using antibody arrays. Shown are proteins for which the MKK6EE level was signiﬁcantly increased (Po0.05) over PRE. PRE and MKK6EE values were averaged to generate the baseline. Heat map and dendrogram were generated as in Figure 1E. *: Factors signiﬁcantly decreased by p38MAPK inhibition (Po0.05). (D) The MKK6EE SASP resembles SEN(XRA) and SEN(RAS) SASPs. Shown are the 10 most upregulated factors in the SEN(XRA) and SEN(RAS) SASPs. þ : Factors signiﬁcantly increased by MKK6EE expression. SASP by inhibiting the DDR, we induced senescence by XRA Figure S4C). Further, cells made senescent by MKK6EE and measured the activities of several DDR proteins with or expression did not exhibit an increase in global ATM or without p38MAPK inhibition (SB). p38MAPK inhibition had CHK2 phosphorylation (Figure 4C). MKK6EE expression did no effect on the rapid (within 2 h) phosphorylation of ATM, not increase global DNA damage signalling at any time after CHK2, or p53(Ser15), nor on the transient stabilization of infection, as measured by p53(Ser15) phosphorylation, un- p53 and expression of p21 after XRA (Figure 4A). p38MAPK like RAS expression (Supplementary Figure S4D). Finally, inhibition also did not affect the low-level activation of these though depletion of ATM or CHK2 by RNAi reduced IL-6, DDR proteins that persists after XRA (42 days) (Rodier et al, IL-8, and GM-CSF secretion by SEN(XRA) and SEN(RAS) 2009, 2011) (Figure 4A). Thus, p38MAPK inhibition does not cells, as described (Rodier et al, 2009), 490% depletion of suppress the SASP by suppressing the DDR. these DDR factors (Supplementary Figure S4E) had no effect In the presence of existing DNA damage, p38MAPK can on IL-6, IL-8, or GM-CSF secretion induced by MKK6EE replenish short-lived DNA damage foci via an ROS feedback (Figure 4D, P40.05). Thus, p38MAPK does not regulate the loop (Passos et al, 2010). Supporting those ﬁndings, though SASP by modulating the DDR. Conversely, the DDR does not p38MAPK inhibition had no effect on the formation or regulate the SASP by modulating p38MAPK activity. Neither resolution of 53BP1 foci during the ﬁrst 4 days after XRA, it ATM nor CHK2 depletion suppressed p38MAPK phosphory- slightly decreased foci number 6–8 days after XRA (Supple- lation in SEN(XRA) (Figure 4E, top) or SEN(RAS) (Figure 4E, mentary Figure S4A, Po0.01) and in replicatively senescent bottom) cells. Together, these ﬁndings indicate that p38MAPK cells (Supplementary Figure S4B). However, in contrast to activity uniquely regulates the SASP independently of XRA, REP, and RAS, constitutive p38MAPK activity (MKK6EE the DDR. expression) did not induce DNA damage foci in most cells: If p38MAPK is sufﬁcient to induce an SASP and also most SEN(RAS), SEN(XRA), and (SEN(REP) cells harboured independent of the DDR, how can the DDR be required for X3 53BP1 foci per nucleus, but most cells induced to senesce the SEN(XRA) and SEN(RAS) SASPs? MKK6EE induces acti- by MKK6EE harboured o3 53BP1 foci and were not signi- vation of endogenous p38MAPK to a much greater extent ﬁcantly different from PRE cells (Figure 4B, P40.05). There than XRA or RAS. To determine whether a lower level of was a slight increase in the percentage of MKK6EE-induced p38MAPK activation is sufﬁcient to induce the SASP, we senescent cells with 410 53BP1 foci/nucleus, but these blunted p38MAPK activity in MKK6EE cells with varying cells accounted for only B3% of the total (Supplementary doses of SB203580 and measured downstream signalling via &2011 European Molecular Biology Organization The EMBO Journal VOL 30 NO 8 2011 1541 | | p38 regulates the senescence secretory phenotype A Freund et al Figure 4 p38MAPK induces an SASP independent of the DNA damage response. (A) p38MAPK inhibition does not prevent the DDR. Whole cell lysates were collected at speciﬁed intervals after irradiation and analysed by western blot. Where indicated, p38MAPK was continuously inhibited by SB203580(þ ) beginning 48 h before irradiation. ATM-P, Ser 1981 phosphorylated ATM; CHK2-P, Thr 68 phosphorylated CHK2. (B) Constitutive p38MAPK activation does not induce 53BP1 foci. Cells were ﬁxed 8 days after MKK6EE expression (MKK6EE), 10 days after RAS expression (SEN(RAS)), 8 days after irradiation (SEN(XRA)), or after replicative senescence (69 population doublings) (SEN(REP)) and immunostained for 53BP1. Foci were quantiﬁed using CellProﬁler to count cells with X3 53BP1 foci per nucleus. Error bars indicate 95% conﬁdence interval. (C) Constitutive p38MAPK activation does not induce a DDR. Whole cell lysate was collected 8 days after MKK6EE infection and analysed by western blot. Presenescent controls (PRE) were infected with insertless vector. ATM-P, Ser 1981 phosphorylated ATM; CHK2-P, Thr 68 phosphorylated CHK2. (D) Neither ATM nor CHK2 depletion prevents the SASP induced by constitutive p38MAPK activation. PRE cells were irradiated (SEN(XRA)), infected with RAS lentivirus (SEN(RAS)), or infected with MKK6EE lentivirus (MKK6EE). Simultaneously, cells were infected with lentivirus expressing shRNAs against ATM (shATM #12), CHK2 (shChk2 #2, shChk2 #12), or GFP (shGFP; control) and selected. CM from 8 days after infection/irradiation were analysed by ELISA. (E) ATM or CHK2 depletion does not prevent p38MAPK phosphorylation at senescence. Top: cells were irradiated; 6 days later cells were infected with lentivirus expressing shRNAs against ATM (shATM #12), CHK2 (shChk2 #2, shChk2 #12), or GFP (shGFP; control) and selected. Bottom: cells were simultaneously infected with RAS lentivirus, lentivirus expressing an shRNA against ATM (shATM #12), CHK2 (shChk2 #2, shChk2 #12), or GFP (shGFP; control) and selected. Whole cell lysates from 8 days after irradiation/infection were analysed by western blot. p38-P: phosphorylated p38. Hsp27 phosphorylation. p38MAPK was sufﬁcient to induce levels (Supplementary Figure S4G). Thus, though the level of a substantial increase in IL-6 only when active at a higher p38MAPK signalling in MKK6EE cells is sufﬁcient to induce level than that seen in SEN(XRA) cells (Supplementary an SASP without the DDR, the level of p38MAPK signalling Figure S4F). When p38MAPK signalling was inhibited to in other types of senescence is not sufﬁcient, and therefore a level that matched SEN(XRA) cells (as measured by requires cooperation with the DDR in order to induce Hsp27-P), it was not sufﬁcient to induce IL-6 to SEN(XRA) the SASP. 1542 The EMBO Journal VOL 30 NO 8 2011 &2011 European Molecular Biology Organization | | p38 regulates the senescence secretory phenotype A Freund et al p38MAPK inhibition suppresses SASP component decreased reporter activity (Po0.001). We conclude that mRNA levels NF-kB activity is regulated by p38MAPK during senescence. Many SASP factors are upregulated at the level of mRNA Depletion of ATM also decreased NF-kB reporter activity— abundance (Coppe et al, 2008, 2010b). To understand the to roughly the same level as p38MAPK inhibition (Figure 5E, mechanism by which p38MAPK regulates the SASP, we used Po0.001). Although increased concentrations of SB203580 quantitative RT–PCR to determine mRNA levels of six SASP had no additional effect on NF-kB reporter output (data not factors (GM-CSF, IL-6, IL-8, GROa, MCP-2, and IL-1a) that shown), the combined treatment of SB203580 and ATM declined signiﬁcantly upon p38MAPK inhibition. For all depletion had a synergistic effect, lowering NF-kB transcrip- six factors, p38MAPK inhibition (SB) markedly decreased tional activity to PRE control levels (Figure 5E, SBþ shATM). mRNA abundance in SEN(XRA) cells (Figure 5A). We These data reinforce the idea that ATM depletion and SB obtained similar results in another cell strain (Supple- affect NF-kB via different pathways. mentary Figure S5A). For GM-CSF, IL-6, and IL-8, the decrease in mRNA abundance matched the decrease in NF-jB is required for the SASP secreted protein level (Supplementary Figure S5B). In addi- Because p38MAPK regulates both the SASP and NF-kB tion, constitutive p38MAPK activation was sufﬁcient to activity, we asked whether NF-kB is required for the SASP. induce SASP mRNAs, as determined by IL-6 and IL-8 We expressed either of two unrelated shRNAs against RelA, mRNA levels upon MKK6EE expression (Supplementary both of which efﬁciently decreased RelA levels without sub- Figure S5C). While these results do not rule out the possibi- stantially affecting RelB or C-Rel levels (Supplementary lity that p38MAPK stimulates the SASP by inﬂuencing other Figure S5G). RelA depletion signiﬁcantly decreased 73% processes (e.g., translation and secretion), the data suggest (27/37) of the SASP proteins secreted by SEN(XRA) cells that p38MAPK induces the SASP primarily by increasing (Figure 5E, asterisks), including MMP1 and MMP3 (Supple- mRNA abundance. mentary Figure S5H), and non-signiﬁcantly decreased the remaining SASP proteins (Figure 5F). Hierarchical clustering p38MAPK controls NF-jB activity in senescent cells showed that RelA-depleted SEN(XRA) cells had an SASP Because p38MAPK is known to regulate the activity of multi- proﬁle that more closely resembled PRE cells than unmodiﬁed ple transcription factors (TFs) depending on context (Zarubin SEN(XRA) cells (Figure 5F). There was substantial overlap and Han, 2005), we examined the promoters of p38MAPK- between the RelA-dependent and p38MAPK-dependent induced factors (Figure 3D) for overrepresented transcription (Figure 1) SASP factors: 76% (19/25) of p38MAPK-dependent factor-binding sites (TFBS). We interrogated 200 bp upstream factors were also RelA dependent (Figure 5G, Venn diagram). of each transcriptional start site using the 243 TF weight Of the 10 most robustly secreted SASP proteins from Figure 1, 8 matrices in the TRANSFAC database. NF-kB-binding motifs were both p38MAPK- and RelA-dependent (Figure 5G, table). were most statistically overrepresented (Figure 5B). Activated Using IL-6, IL-8, and GM-CSF as SASP markers, we veriﬁed the NF-kB is enriched at the IL-8 and GROg promoters following RelA dependence of the SASP for SEN(RAS) cells (Figure 5H), MEK-induced senescence (Acosta et al, 2008). We therefore and two cell strains (Supplementary Figure S5I). Finally, asked whether NF-kB activity increases during multiple we demonstrated that IL-6, IL-8, and GM-CSF secretion types of senescence, and whether the increase is p38MAPK induced by MKK6EE were RelA dependent (Supplementary dependent. Figure S5J). Thus, p38MAPK acts primarily through NF-kBto Inactive NF-kB dimers are sequestered in the cytoplasm by induce the SASP. the inhibitor IkB. NF-kB activating signals cause IkB degrada- tion, allowing NF-kB nuclear translocation. Three NF-kB Discussion family members (RelA, RelB, and C-Rel) have DNA binding and transactivation domains, but RelA is most strongly The SASP develops when cells experience a stress severe associated with inﬂammatory cytokine gene transcription enough to cause a senescence response. These stresses are (Karin, 2006; Perkins, 2007). RelA was mostly cytoplasmic primarily genotoxic, leading to activation of a DDR. Persistent in PRE cells, but noticeably more nuclear in SEN(XRA) cells DDR signalling is necessary for the expression of several SASP (Figure 5C). Further, NF-kB DNA-binding activity increased factors (Rodier et al, 2009); depletion of DDR proteins such B5 fold in SEN(REP), SEN(RAS), and SEN(XRA) cells (two as ATM, NBS1, or CHK2 suppresses the expression of SASP strains) (Supplementary Figures S5D and E, Po0.001). components, including IL-6 and IL-8. However, the DDR is Constitutive p38MAPK activity (MKK6EE expression) was activated immediately after damage, whereas the SASP takes sufﬁcient to induce this activity (Supplementary Figure days to develop, indicating that canonical DDR signalling is S5D). Importantly, following XRA, NF-kB DNA-binding acti- not sufﬁcient for SASP expression. Thus, there must be other, vity increased slowly with kinetics that followed p38MAPK delayed molecular events that are required for development activation (Supplementary Figure S5F): NF-kB DNA-binding of the SASP and are regulated independently of the DDR. We activity remained near-PRE levels for 8 h after XRA, began to show here that activation of the p38MAPK/NF-kB pathway is rise 24 h after XRA and reached maximal levels 8–10 days such an event. later (Supplementary Figure S5F). Additionally, chromatin Unlike the rapid and transient response to acute stresses immunoprecipitation showed increased RelA binding to the such as LPS stimulation, the kinetics of p38MAPK activation promoters of three SASP genes (Figure 5D, Po0.01). A after DNA damage were slow and chronic, coinciding with lentiviral-delivered luciferase reporter driven by an NF-kB- expression of the SASP. p38MAPK inhibition effectively responsive promoter showed that NF-kB transcriptional acti- collapsed the senescence-associated cytokine network, pre- vity was 430-fold higher in SEN(XRA) compared with PRE venting the pro-invasion paracine effects of senescent cells. cells (Figure 5E), and p38MAPK inhibition (SB) signiﬁcantly Further, robust p38MAPK activation, caused by MKK6EE &2011 European Molecular Biology Organization The EMBO Journal VOL 30 NO 8 2011 1543 | | p38 regulates the senescence secretory phenotype A Freund et al Figure 5 p38MAPK induces SASP mRNAs by increasing NF-kB activity. (A) p38MAPK inhibition decreases SASP mRNA levels. Total RNA was extracted from PRE and SEN(XRA) HCA2 cells; mRNA levels for indicated genes were analysed by qRT–PCR. þ SB: p38MAPK was inhibited with SB203580 for 48 h before sample collection. For each gene, the four signals were averaged to generate the baseline. Signals above baseline are red; signals below baseline are green. The heat map key shows log -fold changes from baseline. p38MAPK inhibition signiﬁcantly decreased (Po0.05) mRNA levels for all genes assayed. (B) Transcription factor (TF) binding sites (BS) in MKK6EE-induced genes. Genes encoding SASP proteins upregulated by MKK6EE expression (Figure 3C) were analysed for statistically overrepresented TFBS in the 200 bp upstream of the transcriptional start site. ‘% of sequences’ indicates percentage of sequences with X1 binding site for each indicated weight matrix. TFBS are sorted by P-value. (C) RelA partially localizes to the nucleus during damage-induced senescence. PRE and SEN(XRA) cells were immunostained for RelA; representative images are shown. (D) Increased RelA binding to the promoters of SASP genes. Cells were irradiated, allowed to senesce (SEN(XRA)), and lysates were analysed by chromatin immunoprecipitation using an antibody against RelA. RelA binding to the promoters of the indicated genes is represented relative to Histone H3 binding and is normalized to SEN(XRA) binding for each gene. (E) p38MAPK inhibition and ATM depletion reduce NF-kB transcriptional activity in senescent cells. Cells were infected with lentivirus expressing an NF-kB luciferase reporter construct, irradiated, and allowed to senesce (SEN(XRA)). Cells were lysed and luciferase activity was measured. SB: p38MAPK was inhibited with SB203580 for 48 h before lysis. shATM: cells were infected with lentivirus expressing shRNA against ATM 5 days before lysis. (F) RelA depletion suppresses the SASP of SEN(XRA) cells. Cells were infected with lentivirus expressing either of two shRNAs against RelA (shRelA) or GFP (shGFP; control), selected, irradiated, and allowed to senesce (SEN(XRA)). Secreted proteins were detected by antibody arrays as in Figure 1. Shown are factors for which the SEN(XRA) level was signiﬁcantly increased (Po0.05) over PRE. For each protein, the six signals were averaged to generate the baseline. Heat map and dendrogram were generated as in Figure 1E. Asterisks indicate factors that are signiﬁcantly decreased by both RelA shRNAs (Po0.05). (G) Most p38MAPK-dependent SASP proteins are NF-kB dependent. Left: proportional Venn diagram displaying the overlap between p38MAPK-dependent factors (red), RelA-dependent factors (blue), and the SEN(XRA) SASP (yellow). In all, 76% of p38MAPK-dependent factors are also RelA-dependent (dashed area). Right: the 10 most upregulated SEN(XRA) SASP factors from Figure 1E. þ : Proteins dependent on RelA or p38MAPK. (H) SEN(RAS)-induced IL-6, IL-8, and GM- CSF are RelA-dependent. Cells were infected with lentivirus expressing either of two shRNAs against RelA (shRelA) or GFP (shGFP; control) V12 and selected. Cells were then infected with lentivirus lacking an insert (PRE) or expressing RAS and allowed to senesce (SEN(RAS)). CM were analysed by ELISA. expression, was sufﬁcient to induce an SASP, suggesting that p53 and then delivered a senescence-inducing genotoxic p38MAPK activity is limiting for SASP development. stress, p38MAPK was activated faster and to a higher level. Of particular importance, we found that p53 restrains the The increased activation correlated with the ampliﬁed SASP SASP by restraining p38MAPK activity. When we inactivated that develops in the absence of p53 (Coppe et al, 2008), and 1544 The EMBO Journal VOL 30 NO 8 2011 &2011 European Molecular Biology Organization | | p38 regulates the senescence secretory phenotype A Freund et al p38MAPK inhibition reduced the ampliﬁed SASP. Given that phosphorylated by ATM or its downstream targets. Those transformed and primary cells can differ in the kinetics with low-afﬁnity sites, then, are phosphorylated by p38MAPK only which the p38MAPK/NF-kB pathway is activated (Janssens if p38MAPK signalling is high (e.g., due to MKK6EE expres- and Tschopp, 2006), our data suggest that p53 represses the sion). In SEN(XRA) and SEN(RAS) cells, p38MAPK signalling p38MAPK pathway immediately after DNA damage in normal is not high enough to result in phosphorylation of the low- cells. This repression may allow time to repair the damage afﬁnity sites, making both p38MAPK and the DDR necessary before cells commit to developing an SASP, which signals to for NF-kB activity. the tissue microenvironment. p38MAPK was crucial for the expression of many SASP The p38MAPK pathway acted in parallel to the DDR. cytokines and chemokines, which are largely pro-inﬂamma- Inhibition of p38MAPK did not affect activation of important tory and pro-carcinogenic (Davalos et al, 2010; Freund et al, DDR factors such as ATM, CHK2, or p53. Additionally, 2010; Coppe et al, 2010a). However, p38MAPK was less constitutive p38MAPK activation, despite inducing an SASP, important for the SASP MMPs, which may be beneﬁcial in neither induced ATM or CHK2 activation nor increased DNA the short term. The MMPs have matrix-degrading and ﬁbro- damage foci in most cells. Furthermore, ATM or CHK2 lytic activity, which may limit ﬁbrosis during wound healing depletion had no effect on the p38MAPK-induced SASP. (Krizhanovsky et al, 2008; Jun and Lau, 2010). The two most Thus, we show for the ﬁrst time that senescence-associated highly secreted MMPs are MMP1 and MMP3 (Coppe et al, pro-inﬂammatory cytokine secretion can occur in the absence 2010b). p38MAPK activation induced these MMPs, but to of a DDR. Additionally, neither ATM nor CHK2 depletion a lesser extent than many cytokines and chemokines. altered p38MAPK phosphorylation at senescence, suggesting Additionally, although prolonged p38MAPK inhibition that p38MAPK is not downstream of the canonical DDR. reduced MMP levels in SEN(XRA) and SEN(RAS) cells, the p38MAPK inhibition had a small effect on the resolution of effect was substantially smaller than the reduction in IL-6, DNA damage foci in a subset of senescent cells, supporting IL-8, GM-CSF, and other SASP factors. Thus, other MMP- reports that p38MAPK can replenish short-lived DNA damage regulating pathways must be active at senescence. More foci (Passos et al, 2010). However, as the p38MAPK-induced importantly, our data suggest it may be possible to reduce SASP did not require ATM or CHK2, the DDR-ROS feedback some SASP factors without affecting others, potentially loop that maintains a subset of DNA damage foci (Passos mitigating the deleterious effects without strongly mitigating et al, 2010) does not seem to be the mechanism by which the beneﬁts. p38MAPK regulates the SASP. p38MAPK establishes the senescence growth arrest by INK4A NF-kB was the crucial effector of p38MAPK signalling mediating RAS-induced expression of p16 and directly during senescence. NF-kB-binding sites were the most and indirectly phosphorylating p53 (Sun et al, 2007; Kwong INK4a statistically overrepresented TFBS in the identiﬁed set of et al, 2009). Neither p16 nor p53 are required for the p38MAPK-induced genes, and NF-kB activity was increased SASP (Coppe et al, 2008, 2010a). Thus, although the SASP can in DNA damage-induced, oncogene-induced, and replicative indirectly reinforce the growth arrest through an IL-6/IL-8 senescence. Moreover, p38MAPK increased SASP factor autocrine feedback loop (Acosta et al, 2008; Kuilman et al, mRNA abundance, and p38MAPK was required for senes- 2008), the above ﬁndings and our data that p38MAPK cence-induced NF-kB activity. ATM was also required for regulates the SASP via NF-kB suggest that p38MAPK may senescence-induced NF-kB activity and acted synergistically deﬁne a divergence point for events that activate the SASP with p38MAPK, supporting the independence of the DDR and versus the growth arrest. p38MAPK pathways. The SASPs induced by DNA damage, Our identiﬁcation of the p38MAPK/NF-kB pathway as a RAS, or constitutive p38MAPK activity all required NF-kB, necessary and sufﬁcient, DNA damage-independent regulator demonstrating its role in mediating p38MAPK signalling. of the SASP provides new insights into how senescent cells NF-kB may not be the only means by which p38MAPK might be a source of the chronic inﬂammation that is a increases SASP gene expression—other TFBS such as hallmark of aging and many age-related diseases. C/EBP sites were also overrepresented in p38MAPK-induced SASP genes and are indirectly regulated by p38MAPK (Cortez Materials and methods et al, 2007), and p38MAPK can also affect mRNA stability (Wang et al, 1999; Radtke et al, 2010). Nevertheless, the SASP Cell culture network was largely dependent on NF-kB, and p38MAPK Primary human ﬁbroblasts and MDA-MB-231 cells were cultured in a 10% CO ,3%O atmosphere (Coppe et al, 2008). Unless noted was, in turn, necessary and sufﬁcient for NF-kB activity. 2 2 otherwise, ‘ﬁbroblast’ or ‘cells’ in the text and legends refer to p38MAPK activity was sufﬁcient for SASP activity, but only HCA2 ﬁbroblasts. Presenescent (PRE) HCA2 cells completed o35 at high levels. At the level of activation caused by XRA or population doublings and had a 24-h BrdU labeling index of 460%. RAS, p38MAPK was not sufﬁcient to activate the SASP—the Cells were made replicatively senescent (SEN(REP)) by repeated subculture (Dimri et al, 1995; Krtolica et al, 2001). For DNA DDR was also necessary. We propose a model in which the damage-induced senescence (SEN(XRA)), cells were grown to DDR and the p38MAPK pathways each post-translationally conﬂuence, exposed to 10 Gy X-ray and, unless noted otherwise, modify speciﬁc NF-kB sites, with varying degrees of analysed 8–10 days later; PRE cells were mock irradiated. For efﬁciency. Both p38MAPK and ATM are required for NF-kB oncogene-induced senescence (SEN(RAS)), cells were infected with V12 lentivirus expressing RAS and analysed 8–10 days after infection. activity at senescence, and NF-kB requires simultaneous Where indicated, cells were given 10 mM SB203580 (Calbiochem, phosphorylation on multiple sites for its activity (Karin, 559395) for the speciﬁed intervals with daily media changes. 2006; Perkins, 2007). We suggest that p38MAPK or its down- stream target(s) have a high afﬁnity for some sites, which Vectors, viruses, and infections become phosphorylated by the amount of p38MAPK signal- MKK6EE (provided by Dr Eisuke Nishida of Kyoto University), ling caused by XRA or RAS, but low afﬁnity for sites normally Genetic suppressor element 22 (GSE) (Ossovskaya et al, 1996), and &2011 European Molecular Biology Organization The EMBO Journal VOL 30 NO 8 2011 1545 | | p38 regulates the senescence secretory phenotype A Freund et al V12 RAS were subcloned into Gateway destination vector 670-1 (Hs 00174131 m1), IL-8 (Hs 00174103 m1), GROa (Hs 00236937 (Campeau et al, 2009). Insertless vector was used as a control for m1), GM-CSF (Hs 00929873 m1), and MCP-2 (Hs 99999026 m1). PRE cells. Lentiviral vectors encoding shRNAs against GFP (RHS4459), p38a (TRCN0000000509, TRCN0000010051), and RelA NF-jB assays (TRCN0000014686, TRCN0000014687) were from Open Biosystems. NF-kB DNA-binding activity in whole cell lysate was assayed using TM Lentiviral vectors encoding ATM shRNAs, CHK2 shRNAs, p53 the TransAM NF-kB p65 Transcription Factor Assay Kit from shRNA (Rodier et al, 2009) and virus production were as described Active Motif (40096). To assay transcriptional activity, cells were (Naldini et al, 1996; Beausejour et al, 2003). Viral titres were infected with Cignal NF-kB Lentiviral Reporter (SABiosciences, CLS- adjusted to infect B90% of cells. Cells were infected overnight with 013L) and selected. After senescence induction, cells were lysed and polybrene, allowed to recover for 48 h, selected for 48 h, and assayed for luciferase using the Luciferase Assay System (Promega, allowed to recover for at least another 48 h before use. E1500). All values for both assays were normalized to cell number. Immunoﬂuorescence Chromatin immunoprecipitation Cells were ﬁxed, permeabilized, blocked, and incubated with Chromatin immunoprecipitation was performed with the MAGnify primary antibodies overnight at 41C in PBSþ 1% BSA. Primary ChIP system (Invitrogen, #49-2024) and signal was detected with antibodies were from R&D Systems (IL6, AF206NA, 1:60), Novus Roche’s UPL system. In all, 3 mg of RelA antibody (Santa Cruz, Biologicals (53BP1, NB 100-305, 1:2000), and BD Biosciences #SC-372X) or Histone H3 (Abcam, #AB-1791) was used. Primers (BrdU, 347580, 1:100). Images were quantitated using CellProﬁler used: IL-6 promoter: F-cacagaagaactcagatgactgg, R-aaaaccaaagatgttc (Carpenter et al, 2006). tgaactga; IL-8 promoter: F-catcagttgcaaatcgtgga, R-gaacttatgcaccctc atcttttc; GM-CSF promoter: F-ccccttactggactgaggttg, R-cccactgacagtt Senescence-associated b-galactosidase assay cacatgg. Cells were ﬁxed and stained for SA-bgal using BioVision’s Sene- scence Detection Kit (#K320-250) for 24 h. Staining was visualized Analysis of TFBS by light microscopy; positive cells were counted manually. TFM-Explorer (Defrance and Touzet, 2006) was used to identify the top 10 statistically overrepresented partial weight matrices (PWMs) BrdU incorporation in the 200 bp upstream of the transcription start sites of genes Subconﬂuent cells were incubated for 24 h with 10 mM BrdU and encoding proteins signiﬁcantly induced by MKK6EE expression. We immunostained for BrdU as described (Rodier et al, 2011). searched all vertebrate PWMs available in the TRANSFAC database using a cluster density ratio of 2.5. P-values were calculated Western blot analysis compared with a background model incorporating all RefSeq genes On-plate lysis was performed with either denaturing (5% SDS, (24 328). 10 mM Tris) or non-denaturing (Cell Lysis Buffer, Cell Signalling, 9803) buffer containing protease and phosphatase inhibitors. Statistical analyses Primary antibodies are listed in Supplementary Table S1. Signals Statistical analyses of main ﬁgures are in Supplementary Table S1; were quantiﬁed with LI-COR Odyssey software. statistical analyses of supplementary ﬁgures are in Supplementary Table S2. Statistical signiﬁcance between distributions of signals ELISAs and conditioned media was evaluated using a two-tailed Student’s t-test and assumption ELISA kits to detect IL-6 (D6050), IL-8 (D8000C), and GM-CSF of equal variance. Statistical signiﬁcance between binary assays (DGM00) were from R&D Systems. MMP1 and MMP3 were detected (i.e., positive and negative scores) was evaluated using a w -test. with AlphaLISA kits from Perkin-Elmer (AL242C, AL284C). CM were prepared by washing with serum-free DMEM and incubating Supplementary data in serum-free DMEM for 24 h. All ELISA data were normalized to Supplementary data are available at The EMBO Journal Online cell number. (http://www.embojournal.org). Antibody arrays Acknowledgements CM samples were diluted to equivalent cell numbers in serum-free DMEM. Antibody arrays were from Raybiotech (AAH-CYT-G1000-8). We thank Drs Eisuke Nishida (Kyoto University) for the pSRalpha- Arrays were scanned using a GenePix 4200A Professional micro- myc-MKK6-EE vector, Pierre Desprez (California Paciﬁc array scanner at 10 mm resolution. Signal intensities were Medical Center) for critically reading the manuscript, and Shruti quantitated using LI-COR Odyssey software, and normalized to Waghray for technical assistance. This work was supported by positive controls for each sample, which were then normalized grants from the National Institutes of Health (AG09909, across all samples. AG017242, and AG25901, JC), a National Science Foundation Graduate Research Fellowship (AF), and a fellowship from the Invasion assay Larry L Hillblom Foundation (CP). CM were diluted to equivalent cell numbers in serum-free DMEM. Author contributions: AF and JC conceived the project; AF Invasion assays were performed as described (Coppe et al, 2006) performed most of the experiments and prepared the ﬁgures; CKP using Matrigel Invasion Chambers (8 mm pore, BD Biosciences participated in the array analyses; AF, CKP, and JC participated in 354480). interpreting the data; AF, CKP, and JC wrote the paper. Quantitative RT–PCR Taqman analyses were performed by the UCSF Genome Core. Conﬂict of interest Samples were normalized to b-glucuronidase (GUS). Primers were from Applied Biosystems: IL-1a (Hs 00174092 m1), IL-6 The authors declare that they have no conﬂict of interest. References Acosta JC, O’Loghlen A, Banito A, Guijarro MV, Augert A, Raguz S, Beausejour CM, Krtolica A, Galimi F, Narita M, Lowe SW, Yaswen P, Fumagalli M, Da Costa M, Brown C, Popov N, Takatsu Y, Campisi J (2003) Reversal of human cellular senescence: roles of Melamed J, d’Adda di Fagagna F, Bernard D, Hernando E, Gil J the p53 and p16 pathways. EMBO J 22: 4212–4222 (2008) Chemokine signalling via the CXCR2 receptor reinforces Beyaert R, Cuenda A, Vanden Berghe W, Plaisance S, Lee JC, senescence. Cell 133: 1006–1018 Haegeman G, Cohen P, Fiers W (1996) The p38/RK mitogen- Bavik C, Coleman I, Dean JP, Knudsen B, Plymate S, Nelson PS activated protein kinase pathway regulates interleukin-6 syn- (2006) The gene expression program of prostate ﬁbroblast sene- thesis response to tumor necrosis factor. EMBO J 15: 1914–1923 scence modulates neoplastic epithelial cell proliferation through Bhaumik D, Scott G, Schokrpur S, Patil CK, Orjalo AV, Rodier F, paracrine mechanisms. Cancer Res 66: 794–802 Lithgow G, Campisi J (2009) MicroRNAs miR-146a/b negatively 1546 The EMBO Journal VOL 30 NO 8 2011 &2011 European Molecular Biology Organization | | p38 regulates the senescence secretory phenotype A Freund et al modulate the senescence-associated inﬂammatory mediators IL-6 Freund A, Orjalo AV, Desprez PY, Campisi J (2010) Inﬂammatory and IL-8. Aging 1: 402–411 networks during cellular senescence: causes and consequences. Campeau E, Ruhl VE, Rodier F, Smith CL, Rahmberg BL, Fuss JO, Trends Mol Med 16: 238–246 Campisi J, Yaswen P, Cooper PK, Kaufman PD (2009) A versatile Gil J, Peters G (2006) Regulation of the INK4b-ARF-INK4a tumour viral system for expression and depletion of proteins in mamma- suppressor locus: all for one or one for all. Nat Rev Mol Cell Biol lian cells. PLoS One 4: e6529 7: 667–677 Campisi J, d’Adda di Fagagna F (2007) Cellular senescence: when Iwasa H, Han J, Ishikawa F (2003) Mitogen-activated protein kinase bad things happen to good cells. Nat Rev Mol Cell Biol 8: 729–740 p38 deﬁnes the common senescence-signalling pathway. Genes Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman Cells 8: 131–144 O, Guertin DA, Chang JH, Lindquist RA, Moffat J, Golland P, Janssens S, Tschopp J (2006) Signals from within: the DNA-damage- Sabatini DM (2006) CellProﬁler: image analysis software for induced NF-kappaB response. Cell Death Differ 13: 773–784 identifying and quantifying cell phenotypes. Genome Biol 7: R100 Jeyapalan JC, Ferreira M, Sedivy JM, Herbig U (2007) Accumulation Ciccia A, Elledge SJ (2010) The DNA damage response: making it of senescent cells in mitotic tissue of aging primates. Mech Ageing safe to play with knives. Mol Cell 40: 179–204 Dev 128: 36–44 Collins CJ, Sedivy JM (2003) Involvement of the INK4a/Arf gene Jun JI, Lau LF (2010) The matricellular protein CCN1 induces locus in senescence. Aging Cell 2: 145–150 ﬁbroblast senescence and restricts ﬁbrosis in cutaneous wound Coppe JP, Desprez PY, Krtolica A, Campisi J (2010a) The sene- healing. Nat Cell Biol 12: 676–685 scence-associated secretory phenotype: the dark side of tumor Karin M (2006) Nuclear factor-[kappa]B in cancer development and suppression. Annu Rev Pathol 5: 99–118 progression. Nature 441: 431–436 Coppe JP, Kauser K, Campisi J, Beausejour CM (2006) Secretion of Kortlever RM, Higgins PJ, Bernards R (2006) Plasminogen activator vascular endothelial growth factor by primary human ﬁbroblasts inhibitor-1 is a critical downstream target of p53 in the induction at senescence. J Biol Chem 281: 29568–29574 of replicative senescence. Nat Cell Biol 8: 877–884 Coppe JP, Patil CK, Rodier F, Krtolica A, Beausejour CM, Parrinello S, Krizhanovsky V, Yon M, Dickins RA, Hearn S, Simon J, Miething C, Hodgson JG, Chin K, Desprez PY, Campisi J (2010b) A human-like Yee H, Zender L, Lowe SW (2008) Senescence of activated stellate senescence-associated secretory phenotype is conserved in mouse cells limits liver ﬁbrosis. Cell 134: 657–667 cells dependent on physiological oxygen. PLoS One 5: e9188 Krtolica A, Parrinello S, Lockett S, Desprez P-Y, Campisi J (2001) Coppe JP, Patil CK, Rodier F, Sun Y, Munoz DP, Goldstein J, Nelson Senescent ﬁbroblasts promote epithelial cell growth and tumori- PS, Desprez PY, Campisi J (2008) Senescence-associated secretory genesis: a link between cancer and aging. Proc Natl Acad Sci USA phenotypes reveal cell-nonautonomous functions of oncogenic 98: 12072–12077 RAS and the p53 tumor suppressor. PLoS Biol 6: 2853–2868 Kuilman T, Michaloglou C, Vredeveld LCW, Douma S, van Doorn R, Cortez DM, Feldman MD, Mummidi S, Valente AJ, Steffensen B, Desmet CJ, Aarden LA, Mooi WJ, Peeper DS (2008) Oncogene- Vincenti M, Barnes JL, Chandrasekar B (2007) IL-17 stimulates induced senescence relayed by an interleukin-dependent inﬂam- MMP-1 expression in primary human cardiac ﬁbroblasts via p38 matory network. Cell 133: 1019–1031 MAPK- and ERK1/2-dependent C/EBP-beta, NF-kappaB, and AP-1 Kwong J, Hong L, Liao R, Deng Q, Han J, Sun P (2009) p38alpha activation. Am J Physiol Heart Circ Physiol 293: H3356–H3365 and p38gamma mediate oncogenic Ras-induced senescence Courtois-Cox S, Jones SL, Cichowski K (2008) Many roads lead to through differential mechanisms. J Biol Chem 284: 11237–11246 oncogene-induced senescence. Oncogene 27: 2801–2809 Lali FV, Hunt A, Turner S, Foxwell B (2000) The pyridinyl imidazole Cuenda A, Rouse J, Doza YN, Meier R, Cohen P, Gallagher TF, inhibitor SB203580 blocks phosphoinositide-dependent protein Young PR, Lee JC (1995) SB 203580 is a speciﬁc inhibitor of a kinase activity, protein kinase B phosphorylation, and retino- MAP kinase homologue which is stimulated by cellular stresses blastoma hyperphosphorylation in interleukin-2-stimulated and interleukin-1. FEBS Lett 364: 229–233 T cells independently of p38 mitogen-activated protein kinase. Cuenda A, Rousseau S (2007) p38 MAP-kinases pathway regulation, J Biol Chem 275: 7395–7402 function and role in human diseases. Biochim Biophys Acta Liu D, Hornsby PJ (2007) Senescent human ﬁbroblasts increase the 1773: 1358–1375 early growth of xenograft tumors via matrix metalloproteinase Davalos AR, Coppe JP, Campisi J, Desprez PY (2010) Senescent cells secretion. Cancer Res 67: 3117–3126 as a source of inﬂammatory factors for tumor progression. Cancer Naldini L, Blomer U, Gage FH, Trono D, Verma IM (1996) Efﬁcient Metastasis Rev 29: 273–283 transfer, integration, and sustained long-term expression of the Davis T, Baird D, Haughton M, Jones CJ, Kipling D (2005) transgene in adult rat brains injected with a lentiviral vector. Prevention of accelerated cell aging in Werner syndrome using Proc Natl Acad Sci USA 93: 11382–11388 a p38 mitogen-activated protein kinase inhibitor. J Gerontol A Biol Ohtani N, Yamakoshi K, Takahashi A, Hara E (2004) The p16INK4a- Sci Med Sci 60: 1386–1393 RB pathway: molecular link between cellular senescence and Davis T, Kipling D (2009) Assessing the role of stress signalling tumor suppression. J Med Invest 51: 146–153 via p38 MAP kinase in the premature senescence of Ataxia Ono K, Han J (2000) The p38 signal transduction pathway: activa- Telangiectasia and Werner syndrome ﬁbroblasts. Biogerontology tion and function. Cell Signal 12: 1–13 10: 253–266 Orjalo AV, Bhaumik D, Gengler BK, Scott GK, Campisi J (2009) Cell Defrance M, Touzet H (2006) Predicting transcription factor binding surface-bound IL-1{alpha} is an upstream regulator of the senes- sites using local over-representation and comparative genomics. cence-associated IL-6/IL-8 cytokine network. Proc Natl Acad Sci BMC Bioinformatics 7: 396 USA 106: 17031–17036 Deng Q, Liao R, Wu B-L, Sun P (2004) High intensity ras signalling Ossovskaya VS, Mazo IA, Chernov MV, Chernova OB, Strezoska Z, induces premature senescence by activating p38 pathway in Kondratov R, Stark GR, Chumakov PM, Gudkov AV (1996) Use of primary human ﬁbroblasts. J Biol Chem 279: 1050–1059 genetic suppressor elements to dissect distinct biological effects Di Micco R, Fumagalli M, Cicalese A, Piccinin S, Gasparini P, of separate p53 domains. Proc Natl Acad Sci USA 93: 10309–10314 Luise C, Schurra C, Garre M, Nuciforo PG, Bensimon A, Paradis V, Youssef N, Dargere D, Ba N, Bonvoust F, Bedossa P (2001) Maestro R, Pelicci PG, d’Adda di Fagagna F (2006) Oncogene- Replicative senescence in normal liver, chronic hepatitis C, and induced senescence is a DNA damage response triggered by DNA hepatocellular carcinomas. Hum Pathol 32: 327–332 hyper-replication. Nature 444: 638–642 Parrinello S, Coppe J-P, Krtolica A, Campisi J (2005) Stromal- Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, epithelial interactions in aging and cancer: senescent ﬁbroblasts Medrano EE, Linskens M, Rubelj I, Pereira-Smith O, Peacocke alter epithelial cell differentiation. J Cell Sci 118: 485–496 M, Campisi J (1995) A biomarker that identiﬁes senescent human Passos JF, Nelson G, Wang C, Richter T, Simillion C, Proctor CJ, cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA Miwa S, Olijslagers S, Hallinan J, Wipat A, Saretzki G, Rudolph 92: 9363–9367 KL, Kirkwood TB, von Zglinicki T (2010) Feedback between p21 Eisen MB, Spellman PT, Brown PO, Botstein D (1998) Cluster and reactive oxygen production is necessary for cell senescence. analysis and display of genome-wide expression patterns. Mol Syst Biol 6: 347 Proc Natl Acad Sci USA 95: 14863–14868 Perkins ND (2007) Integrating cell-signalling pathways with Enslen H, Raingeaud J, Davis RJ (1998) Selective activation of p38 NF-kappaB and IKK function. Nat Rev Mol Cell Biol 8: 49–62 mitogen-activated protein (MAP) kinase isoforms by the MAP Prieur A, Peeper DS (2008) Cellular senescence in vivo: a barrier to kinase kinases MKK3 and MKK6. J Biol Chem 273: 1741–1748 tumorigenesis. Curr Opin Cell Biol 20: 150–155 &2011 European Molecular Biology Organization The EMBO Journal VOL 30 NO 8 2011 1547 | | p38 regulates the senescence secretory phenotype A Freund et al Radtke S, Wuller S, Yang XP, Lippok BE, Mutze B, Mais C, Wang SW, Pawlowski J, Wathen ST, Kinney SD, Lichenstein HS, de Leur HS, Bode JG, Gaestel M, Heinrich PC, Behrmann I, Manthey CL (1999) Cytokine mRNA decay is accelerated by an Schaper F, Hermanns HM (2010) Cross-regulation of cytokine inhibitor of p38-mitogen-activated protein kinase. Inﬂamm Res signalling: pro-inﬂammatory cytokines restrict IL-6 signalling 48: 533–538 through receptor internalisation and degradation. J Cell Sci 123: Wang W, Chen JX, Liao R, Deng Q, Zhou JJ, Huang S, Sun P (2002) 947–959 Sequential activation of the MEK-extracellular signal-regulated Rodier F, Campisi J, Bhaumik D (2007) Two faces of p53: aging and kinase and MKK3/6-p38 mitogen-activated protein kinase path- tumor suppression. Nucleic Acids Res 35: 7475–7484 ways mediates oncogenic ras-induced premature senescence. Rodier F, Coppe JP, Patil CK, Hoeijmakers WA, Munoz DP, Mol Cell Biol 22: 3389–3403 Raza SR, Freund A, Campeau E, Davalos AR, Campisi J Wilson KP, McCaffrey PG, Hsiao K, Pazhanisamy S, Galullo V, (2009) Persistent DNA damage signalling triggers senescence- Bemis GW, Fitzgibbon MJ, Caron PR, Murcko MA, Su MS associated inﬂammatory cytokine secretion. Nat Cell Biol 11: (1997) The structural basis for the speciﬁcity of pyridinyl- 973–979 imidazole inhibitors of p38 MAP kinase. Chem Biol 4: 423–431 Rodier F, Munoz DP, Teachenor R, Chu V, Le O, Bhaumik D, Coppe Xue W, Zender L, Miething C, Dickins RA, Hernando E, J-P, Campeau E, Beausejour C, Kim S-H, Davalos AR, Campisi J Krizhanovsky V, Cordon-Cardo C, Lowe SW (2007) Senescence (2011) DNA-SCARS: distinct nuclear structures that sustain and tumour clearance is triggered by p53 restoration in murine damage-induced senescence growth arrest and inﬂammatory liver carcinomas. Nature 445: 656–660 cytokine secretion. J Cell Sci 124: 68–81 Young PR, McLaughlin MM, Kumar S, Kassis S, Doyle ML, McNulty Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW (1997) D, Gallagher TF, Fisher S, McDonnell PC, Carr SA, Huddleston Oncogenic ras provokes premature cell senescence asso- MJ, Seibel G, Porter TG, Livi GP, Adams JL, Lee JC (1997) ciated with accumulation of p53 and p16INK4a. Cell 88: Pyridinyl imidazole inhibitors of p38 mitogen-activated protein 593–602 kinase bind in the ATP site. J Biol Chem 272: 12116–12121 Sun P, Yoshizuka N, New L, Moser BA, Li Y, Liao R, Xie C, Chen J, Zarubin T, Han J (2005) Activation and signalling of the p38 MAP Deng Q, Yamout M, Dong M-Q, Frangou CG, Yates JR, Wright PE, kinase pathway. Cell Res 15: 11–18 Han J (2007) PRAK is essential for ras-induced senescence and Zhang J, Shen B, Lin A (2007) Novel strategies for inhibition of the tumor suppression. Cell 128: 295–308 p38 MAPK pathway. Trends Pharmacol Sci 28: 286–295 Wajapeyee N, Serra RW, Zhu X, Mahalingam M, Green MR (2008) Zhou S, Greenberger JS, Epperly MW, Goff JP, Adler C, Leboff MS, Oncogenic BRAF induces senescence and apoptosis through Glowacki J (2008) Age-related intrinsic changes in human bone- pathways mediated by the secreted protein IGFBP7. Cell 132: marrow-derived mesenchymal stem cells and their differentiation 363–374 to osteoblasts. Aging Cell 7: 335–343 1548 The EMBO Journal VOL 30 NO 8 2011 &2011 European Molecular Biology Organization | | http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals http://www.deepdyve.com/lp/springer-journals/p38mapk-is-a-novel-dna-damage-response-independent-regulator-of-the-sXkp1y9AzJ

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References (70)

Tyler Zarubin, Jiahuai Han (2005)
Activation and signaling of the p38 MAP kinase pathway
Cell Research, 15
R. Kortlever, P. Higgins, R. Bernards (2006)
Plasminogen activator inhibitor-1 is a critical downstream target of p53 in the induction of replicative senescence
Nature Cell Biology, 8
Anne Carpenter, T. Jones, M. Lamprecht, C. Clarke, In Kang, O. Friman, D. Guertin, Joo Chang, Robert Lindquist, J. Moffat, P. Golland, D. Sabatini (2006)
CellProfiler: image analysis software for identifying and quantifying cell phenotypes
Genome Biology, 7
Jiyan Zhang, B. Shen, A. Lin (2007)
Novel strategies for inhibition of the p38 MAPK pathway.
Trends in pharmacological sciences, 28 6
N. Ohtani, Kimi Yamakoshi, Akiko Takahashi, E. Hara (2004)
The p16INK4a-RB pathway: molecular link between cellular senescence and tumor suppression.
The journal of medical investigation : JMI, 51 3-4
A. Cuenda, J. Rouse, Y. Doza, R. Meier, Philip Cohen, T. Gallagher, P. Young, John Lee (1995)
SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin‐1
FEBS Letters, 364
Matthieu Defrance, H. Touzet (2006)
Predicting transcription factor binding sites using local over-representation and comparative genomics
BMC Bioinformatics, 7
H. Iwasa, Jiahuai Han, F. Ishikawa (2003)
Mitogen‐activated protein kinase p38 defines the common senescence‐signalling pathway
Genes to Cells, 8
S.-W. Wang, J. Pawlowski, S. Wathen, S. Kinney, H. Lichenstein, C. Manthey (1999)
Cytokine mRNA decay is accelerated by an inhibitor of p38-mitogen-activated protein kinase
Inflammation Research, 48
Adam Freund, Arturo Orjalo, P. Desprez, J. Campisi (2010)
Inflammatory networks during cellular senescence: causes and consequences.
Trends in molecular medicine, 16 5
T. Davis, D. Kipling (2009)
Assessing the role of stress signalling via p38 MAP kinase in the premature senescence of Ataxia Telangiectasia and Werner syndrome fibroblasts
Biogerontology, 10
F. Rodier, Jean-Philippe Coppé, C. Patil, W. Hoeijmakers, Denise Muñoz, Saba Raza, Adam Freund, E. Campeau, Albert Davalos, J. Campisi (2009)
Persistent DNA damage signaling triggers senescence-associated inflammatory cytokine secretion
Nature cell biology, 11
Jessie Jeyapalan, Mark Ferreira, J. Sedivy, U. Herbig (2007)
Accumulation of senescent cells in mitotic tissue of aging primates
Mechanisms of Ageing and Development, 128
Jean-Philippe Coppé, P. Desprez, A. Krtolica, J. Campisi (2010)
The senescence-associated secretory phenotype: the dark side of tumor suppression.
Annual review of pathology, 5
R. Beyaert, Ana Cuendal, W. Berghe, S. Plaisance, John Lee, G. Haegeman, '. PhilipCohen, W. Fiers (1996)
The p38/RK mitogen‐activated protein kinase pathway regulates interleukin‐6 synthesis response to tumor necrosis factor.
The EMBO Journal, 15
J Kwong, L Hong, R Liao, Q Deng, J Han, P Sun (2009)
p38alpha and p38gamma mediate oncogenic Ras‐induced senescence through differential mechanisms
J Biol Chem, 284
Jean-Philippe Coppé, Jean-Philippe Coppé, Christopher Patil, Christopher Patil, Francis Rodier, Francis Rodier, A. Krtolica, C. Beauséjour, S. Parrinello, J. Hodgson, K. Chin, P. Desprez, P. Desprez, P. Desprez, Judith Campisi, Judith Campisi (2010)
A Human-Like Senescence-Associated Secretory Phenotype Is Conserved in Mouse Cells Dependent on Physiological Oxygen
PLoS ONE, 5
S. Janssens, J. Tschopp (2006)
Signals from within: the DNA-damage-induced NF-kappaB response.
Cell death and differentiation, 13 5
Jean-Philippe Coppé, C. Patil, F. Rodier, Yu Sun, Denise Muñoz, J. Goldstein, P. Nelson, P. Desprez, J. Campisi (2008)
Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor
PLoS Biology, 6
N. Wajapeyee, Ryan Serra, Xiaochun Zhu, M. Mahalingam, Michael Green (2008)
Oncogenic BRAF Induces Senescence and Apoptosis through Pathways Mediated by the Secreted Protein IGFBP7
Cell, 132
A. Krtolica, S. Parrinello, S. Lockett, P. Desprez, J. Campisi (2001)
Senescent fibroblasts promote epithelial cell growth and tumorigenesis: A link between cancer and aging
Proceedings of the National Academy of Sciences of the United States of America, 98
M. Serrano, Athena Lin, M. McCurrach, D. Beach, S. Lowe (1997)
Oncogenic ras Provokes Premature Cell Senescence Associated with Accumulation of p53 and p16INK4a
Cell, 88
T. Davis, D. Baird, M. Haughton, C. Jones, D. Kipling (2005)
Prevention of accelerated cell aging in Werner syndrome using a p38 mitogen-activated protein kinase inhibitor.
The journals of gerontology. Series A, Biological sciences and medical sciences, 60 11
Albert Davalos, Jean-Philippe Coppé, J. Campisi, P. Desprez (2010)
Senescent cells as a source of inflammatory factors for tumor progression
Cancer Metastasis Reviews, 29
Weiping Wang, Joan Chen, Rong Liao, Qingdong Deng, Jennifer Zhou, Shuang Huang, Peiqing Sun (2002)
Sequential Activation of the MEK-Extracellular Signal-Regulated Kinase and MKK3/6-p38 Mitogen-Activated Protein Kinase Pathways Mediates Oncogenic ras-Induced Premature Senescence
Molecular and Cellular Biology, 22
W. Xue, L. Zender, C. Miething, R. Dickins, E. Hernando, V. Krizhanovsky, C. Cordon-Cardo, S. Lowe (2007)
Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas
Nature, 445
V. Paradis, N. Youssef, D. Dargère, N. Bâ, Franck Bonvoust, J. Deschatrette, P. Bedossa (2001)
Replicative senescence in normal liver, chronic hepatitis C, and hepatocellular carcinomas.
Human pathology, 32 3
A. Prieur, D. Peeper (2008)
Cellular senescence in vivo: a barrier to tumorigenesis.
Current opinion in cell biology, 20 2
N. Perkins (2007)
Integrating cell-signalling pathways with NF-kappaB and IKK function.
Nature reviews. Molecular cell biology, 8 1
M. Karin (2006)
Nuclear factor-κB in cancer development and progression
Nature, 441
D. Cortez, M. Feldman, S. Mummidi, A. Valente, B. Steffensen, M. Vincenti, J. Barnes, B. Chandrasekar (2007)
IL-17 stimulates MMP-1 expression in primary human cardiac fibroblasts via p38 MAPK- and ERK1/2-dependent C/EBP-beta , NF-kappaB, and AP-1 activation.
American journal of physiology. Heart and circulatory physiology, 293 6
Arturo Orjalo, D. Bhaumik, Bridget Gengler, G. Scott, J. Campisi (2009)
Cell surface-bound IL-1α is an upstream regulator of the senescence-associated IL-6/IL-8 cytokine network
Proceedings of the National Academy of Sciences, 106
L. Naldini, U. Blömer, F. Gage, D. Trono, Inder Verma (1996)
Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector.
Proceedings of the National Academy of Sciences of the United States of America, 93 21
Goberdhan Dimri, X. Lee, G. Basile, M. Acosta, G. Scott, C. Roskelley, E. Medrano, M. Linskens, I. Rubelj, O. Pereira-smith (1995)
A biomarker that identifies senescent human cells in culture and in aging skin in vivo.
Proceedings of the National Academy of Sciences of the United States of America, 92 20
Peiqing Sun, N. Yoshizuka, L. New, B. Moser, Yi-lei Li, Rong Liao, Changchuan Xie, Jianming Chen, Qingdong Deng, M. Yamout, M. Dong, Costa Frangou, J. Yates, P. Wright, Jiahuai Han (2007)
PRAK Is Essential for ras-Induced Senescence and Tumor Suppression
Cell, 128
Alberto Ciccia, S. Elledge (2010)
The DNA damage response: making it safe to play with knives.
Molecular cell, 40 2
M Karin (2006)
Nuclear factor‐[kappa]B in cancer development and progression
Nature, 441
F. Rodier, Denise Muñoz, Robert Teachenor, Victoria Chu, O. Le, D. Bhaumik, Jean-Philippe Coppé, E. Campeau, C. Beauséjour, Sahn-Ho Kim, Albert Davalos, J. Campisi (2011)
DNA-SCARS: distinct nuclear structures that sustain damage-induced senescence growth arrest and inflammatory cytokine secretion
Journal of Cell Science, 124
D. Bhaumik, G. Scott, S. Schokrpur, C. Patil, Arturo Orjalo, F. Rodier, G. Lithgow, J. Campisi (2009)
MicroRNAs miR-146a/b negatively modulate the senescence-associated inflammatory mediators IL-6 and IL-8
Aging, 1
V. Krizhanovsky, Monica Yon, R. Dickins, Stephen Hearn, Janelle Simon, C. Miething, H. Yee, L. Zender, S. Lowe (2008)
Senescence of Activated Stellate Cells Limits Liver Fibrosis
Cell, 134
T. Kuilman, C. Michaloglou, Liesbeth Vredeveld, Sirith Douma, R. Doorn, C. Desmet, L. Aarden, W. Mooi, D. Peeper (2008)
Oncogene-Induced Senescence Relayed by an Interleukin-Dependent Inflammatory Network
Cell, 133
Qingdong Deng, Rong Liao, Bai-Lin Wu, Peiqing Sun (2004)
High Intensity ras Signaling Induces Premature Senescence by Activating p38 Pathway in Primary Human Fibroblasts*
Journal of Biological Chemistry, 279
H. Enslen, J. Raingeaud, R. Davis (1998)
Selective Activation of p38 Mitogen-activated Protein (MAP) Kinase Isoforms by the MAP Kinase Kinases MKK3 and MKK6*
The Journal of Biological Chemistry, 273
Dan Liu, P. Hornsby (2007)
Senescent human fibroblasts increase the early growth of xenograft tumors via matrix metalloproteinase secretion.
Cancer research, 67 7
J. Acosta, A. O’Loghlen, A. Banito, M. Guijarro, Arnaud Augert, S. Raguz, M. Fumagalli, M. Costa, Celia Brown, N. Popov, Yoshihiro Takatsu, J. Melamed, F. Fagagna, D. Bernard, E. Hernando, J. Gil (2008)
Chemokine Signaling via the CXCR2 Receptor Reinforces Senescence
Cell, 133
M. Eisen, P. Spellman, P. Brown, D. Botstein (1998)
Cluster analysis and display of genome-wide expression patterns.
Proceedings of the National Academy of Sciences of the United States of America, 95 25
João Passos, G. Nelson, Chunfang Wang, T. Richter, Cedric Simillion, Carole Proctor, S. Miwa, Sharon Olijslagers, Jennifer Hallinan, Anil Wipat, G. Saretzki, Karl Rudolph, Tom Kirkwood, T. Zglinicki (2010)
Feedback between p21 and reactive oxygen production is necessary for cell senescence
Molecular Systems Biology, 6
Shuanhu Zhou, J. Greenberger, M. Epperly, J. Goff, C. Adler, M. LeBoff, J. Glowacki (2008)
Age‐related intrinsic changes in human bone‐marrow‐derived mesenchymal stem cells and their differentiation to osteoblasts
Aging Cell, 7
Valeria Ossovskaya, Valeria Ossovskaya, Ilya Mazo, Chernov Mv, Olga Chernova, Z. Strezoska, Roman Kondratov, George Stark, P. Chumakov, Andrei Gudkov (1996)
Use of genetic suppressor elements to dissect distinct biological effects of separate p53 domains.
Proceedings of the National Academy of Sciences of the United States of America, 93 19
Joon-Il Jun, L. Lau (2010)
The Matricellular Protein CCN1/CYR61 Induces Fibroblast Senescence and Restricts Fibrosis in Cutaneous Wound Healing
Nature cell biology, 12
E. Campeau, Victoria Ruhl, F. Rodier, Corey Smith, Brittany Rahmberg, Jill Fuss, J. Campisi, P. Yaswen, P. Cooper, P. Kaufman (2009)
A Versatile Viral System for Expression and Depletion of Proteins in Mammalian Cells
PLoS ONE, 4
J. Gil, G. Peters (2006)
Regulation of the INK4b–ARF–INK4a tumour suppressor locus: all for one or one for all
Nature Reviews Molecular Cell Biology, 7
K. Wilson, P. Mccaffrey, K. Hsiao, Sam Pazhanisamy, Vincent Galullo, G. Bemis, M. Fitzgibbon, P. Caron, M. Murcko, M. Su (1997)
The structural basis for the specificity of pyridinylimidazole inhibitors of p38 MAP kinase.
Chemistry & biology, 4 6
Jinny Kwong, Lixin Hong, Rong Liao, Qingdong Deng, Jiahuai Han, Peiqing Sun (2009)
p38α and p38γ Mediate Oncogenic ras-induced Senescence through Differential Mechanisms*
Journal of Biological Chemistry, 284
C. Båvik, Ilsa Coleman, J. Dean, B. Knudsen, S. Plymate, P. Nelson (2006)
The gene expression program of prostate fibroblast senescence modulates neoplastic epithelial cell proliferation through paracrine mechanisms.
Cancer research, 66 2
C. Collins, J. Sedivy (2003)
Involvement of the INK4a/Arf gene locus in senescence
Aging Cell, 2
S. Parrinello, Jean-Philippe Coppé, A. Krtolica, J. Campisi (2004)
Stromal-epithelial interactions in aging and cancer: senescent fibroblasts alter epithelial cell differentiation
Journal of Cell Science, 118
S. Courtois-Cox, S. Jones, K. Cichowski (2008)
Many roads lead to oncogene-induced senescence
Oncogene, 27
F. Lali, A. Hunt, Sarah Turner, B. Foxwell (2000)
The Pyridinyl Imidazole Inhibitor SB203580 Blocks Phosphoinositide-dependent Protein Kinase Activity, Protein Kinase B Phosphorylation, and Retinoblastoma Hyperphosphorylation in Interleukin-2-stimulated T Cells Independently of p38 Mitogen-activated Protein Kinase*
The Journal of Biological Chemistry, 275
A. Cuenda, S. Rousseau (2007)
p38 MAP-kinases pathway regulation, function and role in human diseases.
Biochimica et biophysica acta, 1773 8
Koh Ono, Jiahuai Han (2000)
The p38 signal transduction pathway: activation and function.
Cellular signalling, 12 1
C. Beauséjour, A. Krtolica, F. Galimi, M. Narita, S. Lowe, P. Yaswen, J. Campisi (2003)
Reversal of human cellular senescence: roles of the p53 and p16 pathways
The EMBO Journal, 22
Sophie Janssens, Jürg Tschopp (2006)
Signals from within: the DNA-damage-induced NF-κB response
Cell Death and Differentiation, 13
S. Radtke, S. Wüller, Xiang-ping Yang, B. Lippok, Barbara Mütze, C. Mais, Hildegard Leur, J. Bode, M. Gaestel, P. Heinrich, I. Behrmann, F. Schaper, H. Hermanns (2010)
Cross-regulation of cytokine signalling: pro-inflammatory cytokines restrict IL-6 signalling through receptor internalisation and degradation
Journal of Cell Science, 123
J. Campisi, F. Fagagna (2007)
Cellular senescence: when bad things happen to good cells
Nature Reviews Molecular Cell Biology, 8
D. Cortez, M. Feldman, S. Mummidi, A. Valente, B. Steffensen, M. Vincenti, J. Barnes, B. Chandrasekar (2007)
IL-17 stimulates MMP-1 expression in primary human cardiac fibroblasts via p38 MAPK- and ERK1/2-dependent C/EBP-β, NF-κB, and AP-1 activation
American Journal of Physiology-heart and Circulatory Physiology, 293
F. Rodier, J. Campisi, D. Bhaumik (2007)
Two faces of p53: aging and tumor suppression
Nucleic Acids Research, 35
Jean-Philippe Coppé, K. Kauser, J. Campisi, C. Beauséjour (2006)
Secretion of Vascular Endothelial Growth Factor by Primary Human Fibroblasts at Senescence*
Journal of Biological Chemistry, 281
R. Micco, M. Fumagalli, Angelo Cicalese, S. Piccinin, P. Gasparini, C. Luise, C. Schurra, M. Garrè, P. Nuciforo, A. Bensimon, R. Maestro, P. Pelicci, F. Fagagna (2006)
Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication
Nature, 444
P. Young, Megan Mclaughlin, Sanjay Kumar, S. Kassis, M. Doyle, D. Mcnulty, T. Gallagher, S. Fisher, P. Mcdonnell, S. Carr, M. Huddleston, G. Seibel, T. Porter, G. Livi, J. Adams, John Lee (1997)
Pyridinyl Imidazole Inhibitors of p38 Mitogen-activated Protein Kinase Bind in the ATP Site*
The Journal of Biological Chemistry, 272

Publisher: Springer Journals
Copyright: Copyright © European Molecular Biology Organization 2011
ISSN: 0261-4189
eISSN: 1460-2075
DOI: 10.1038/emboj.2011.69
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Abstract

The EMBO Journal (2011) 30, 1536–1548 & 2011 European Molecular Biology Organization All Rights Reserved 0261-4189/11 | | T THE H E www.embojournal.org E E EMB M MB BO O O J JO OURN URN A AL L p38MAPK is a novel DNA damage response-independent regulator of the senescence-associated secretory phenotype 1,2,3 2,3 senescence is a common response to oncogene activation Adam Freund , Christopher K Patil 2,3, in vivo (Prieur and Peeper, 2008) suggest that senescence and Judith Campisi * may be as important as apoptosis for suppressing the Department of Molecular and Cell Biology, University of California, 2 development of cancer. Berkeley, CA, USA, Buck Institute for Age Research, Novato, CA, USA The senescent phenotype is multi-faceted. The chief hall- and Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA mark of senescent cells—the halt of proliferation (hereafter, growth arrest)—is essentially irreversible in that it cannot be Cellular senescence suppresses cancer by forcing poten- reversed by physiological stimuli. This growth arrest is INK4a tially oncogenic cells into a permanent cell cycle arrest. established and maintained by the p53 and p16 /pRB Senescent cells also secrete growth factors, proteases, and pathways. Although many questions remain, we broadly inﬂammatory cytokines, termed the senescence-associated understand the interwoven and complementary mechanisms secretory phenotype (SASP). Much is known about path- by which these pathways regulate the growth arrest (Collins ways that regulate the senescence growth arrest, but far and Sedivy, 2003; Ohtani et al, 2004; Gil and Peters, 2006; less is known about pathways that regulate the SASP. Campisi and d’Adda di Fagagna, 2007; Rodier et al, 2007). By We previously showed that DNA damage response (DDR) contrast, we understand little about how other senescence- signalling is essential, but not sufﬁcient, for the SASP, associated phenotypes are controlled. which is restrained by p53. Here, we delineate another Senescent cells develop an enlarged morphology, upregu- crucial SASP regulatory pathway and its relationship late a senescence-associated b-galactosidase (SA-bgal) acti- to the DDR and p53. We show that diverse senescence- vity (Dimri et al, 1995), and show widespread changes in inducing stimuli activate the stress-inducible kinase chromatin organization and gene expression (Campisi and p38MAPK in normal human ﬁbroblasts. p38MAPK inhibi- d’Adda di Fagagna, 2007). Of particular biological impor- tion markedly reduced the secretion of most SASP factors, tance, senescent cells increase the expression and secretion constitutive p38MAPK activation was sufﬁcient to induce of numerous cytokines, chemokines, matrix metalloprotei- an SASP, and p53 restrained p38MAPK activation. Further, nases (MMPs), and other proteins that can alter local tissue p38MAPK regulated the SASP independently of the environments. We termed this feature the senescence- canonical DDR. Mechanistically, p38MAPK induced the associated secretory phenotype (SASP) (Coppe et al, 2008, SASP largely by increasing NF-jB transcriptional activity. 2010b). These ﬁndings assign p38MAPK a novel role in SASP The SASP can be beneﬁcial or deleterious, depending regulation—one that is necessary, sufﬁcient, and inde- on the biological context. Among the beneﬁts, some SASP pendent of previously described pathways. components reinforce the senescence growth arrest The EMBO Journal (2011) 30, 1536–1548. doi:10.1038/ (Kortlever et al, 2006; Acosta et al, 2008; Kuilman et al, emboj.2011.69; Published online 11 March 2011 2008; Wajapeyee et al, 2008). Others signal the immune Subject Categories: signal transduction; genome stability & system to clear senescent cells (Xue et al, 2007), and SASP dynamics MMPs can suppress ﬁbrotic scar formation (Krizhanovsky Keywords: aging; cancer; inﬂammation; NF-kB; tumour et al, 2008; Jun and Lau, 2010). Among the deleterious suppression effects, SASP MMPs can disrupt mammary morphogenesis in cell culture models (Parrinello et al, 2005). Further, in culture and in vivo, SASP factors can promote malignant phenotypes, including cell proliferation (Bavik et al, 2006; Coppe et al, 2010b), angiogenesis (Coppe et al, 2006), Introduction epithelial-to-mesenchymal transitions and invasiveness (Coppe et al, 2008), and accelerated growth of xenografted Cellular senescence halts the proliferation of cells at risk tumours (Krtolica et al, 2001; Liu and Hornsby, 2007). for malignant transformation. Many potentially oncogenic Moreover, many SASP factors are pro-inﬂammatory. Thus, stimuli, ranging from DNA damage to the activation of certain senescent cells, which increase with age in vivo (Dimri et al, oncogenes, can induce a senescence response (Campisi and 1995; Paradis et al, 2001; Jeyapalan et al, 2007; Zhou et al, d’Adda di Fagagna, 2007). Recent data showing that cellular 2008), may contribute to the low-level chronic inﬂammation that is a hallmark of aged mammalian tissues and many *Corresponding author. The Buck Institute for Age Research, 8001 Redwood Blvd, Novato, CA 94945, USA. age-related diseases (Freund et al, 2010; Coppe et al, 2010a). Tel.: þ 1 415 209 2066; Fax: þ 1 415 493 3640; This important anti-cancer defense, then, might be antago- E-mail: [email protected] nistically pleiotropic and, ironically, promote cancer in aged tissues. Given the known and proposed importance of the Received: 21 October 2010; accepted: 18 February 2011; published online: 11 March 2011 SASP, it is crucial to understand its regulation. 1536 The EMBO Journal VOL 30 NO 8 2011 &2011 European Molecular Biology Organization | | p38 regulates the senescence secretory phenotype A Freund et al Although the senescence growth arrest and SASP are often chemokines, growth factors, MMPs and shed receptors and coordinately induced, the pathways that regulate them do not ligands (Coppe et al, 2008, 2010b). INK4a completely overlap. For example, p16 expression is We show that p38MAPK activity is necessary and sufﬁcient sufﬁcient to induce a senescence growth arrest, but does for development of an SASP in cells induced to senesce by not induce or modify the SASP (Coppe et al, 2010a). Likewise, direct DNA damage or oncogenic RAS. In these contexts, p53 is required for the growth arrest (Campisi and d’Adda di p38MAPK is not activated rapidly, but rather with delayed Fagagna, 2007; Courtois-Cox et al, 2008), but not the SASP; kinetics characteristic of the SASP. Further, p53 restrains the in fact, cells lacking functional p53 secrete markedly higher SASP by restraining p38MAPK activation, and p38MAPK levels of most SASP factors. Thus, p53 restrains the SASP activation occurs independently of the DDR. Further, (Coppe et al, 2008), suggesting it suppresses tumorigenesis in p38MAPK regulates the SASP mainly through NF-kB trans- part by limiting the development of a pro-inﬂammatory tissue criptional activity, which we show is required for the environment. The pathways by which p53 restrains the SASP expression of most SASP factors. These ﬁndings assign the are not known. p38MAPK pathway a novel role in senescence regulation. The SASP is not an acute (rapid, transient) inﬂammatory response. It does not develop immediately after cells experi- Results ence a senescence-inducing stimulus but, once established, it persists for long intervals (Coppe et al, 2008, 2010b; Rodier p38MAPK is activated during the senescence response et al, 2009). One regulator of the SASP is the DNA damage to genotoxic stress p38MAPK is activated by tyrosine and threonine phosphory- response (DDR), the signalling cascade that senses and lation in response to a variety of stresses (Cuenda and ultimately repairs DNA damage. The DDR is initiated by V12 Rousseau, 2007), including oncogenic RAS (Ha-RAS ) phosphatidylinositol 3-kinase-like protein kinases such as ATM and ATR, which phosphorylate chromatin modifying expression (Wang et al, 2002), which indirectly causes DNA proteins such as gH2AX, adaptor proteins such as NBS1, damage (Di Micco et al, 2006). To determine whether and downstream kinases such as CHK1 and CHK2, which p38MAPK activation is a direct genotoxic stress response, we X-irradiated (XRA; 10 Gy) presenescent (PRE) normal ultimately activate p53 (Ciccia and Elledge, 2010). ATM, human ﬁbroblasts (strain HCA2) to synchronously induce CHK2, and NBS1 are essential for establishing and maintain- senescence (SEN(XRA)). After XRA, cells were cultured for ing the expression of several SASP proteins, particularly inﬂammatory cytokines such as IL-6 and IL-8 (Rodier et al, 10 days, during which time they developed classic markers of 2009). However, canonical DDR signalling is not sufﬁcient for senescence: growth arrest (no increase in cell number; low the SASP: a transient DDR, caused by low-level ionizing 5-bromodeoxyuridine (BrdU) labeling), an enlarged ﬂattened radiation, does not induce an SASP (Rodier et al, 2009). morphology, and SA-bgal activity (Supplementary Figures S1A and B; not shown). To assess p38MAPK activation, we Additionally, the SASP, like some other features of the senes- prepared whole cell lysates at intervals after XRA until cells cent phenotype (e.g., cell enlargement, SA-bgal activity), takes several days to develop after the damaging event, developed a complete senescent phenotype 8–10 days later. whereas the canonical DDR is activated immediately after We analysed levels of total and phosphorylated p38MAPK damage. Thus, at least one additional, slower event—which and its downstream target Hsp27 (Beyaert et al, 1996; Davis cooperates with the DDR, but is independent of rapid- et al, 2005) by western blot. Unlike the rapid and robust response to LPS, p38MAPK phosphorylation (p38-P) in- response DDR factors—must be required for the SASP. creased only slightly in the 24 h immediately after XRA Here, we show that this event is activation of p38MAPK, a (Supplementary Figure S1C). p38-P and Hsp27-P levels did member of the mitogen-activated protein kinase (MAPK) family. Like other MAPK members, p38MAPK is activated not rise substantially until 2–4 days after XRA, reaching peak by phosphorylation, and this generally occurs rapidly (within levels at 8–10 days (Figure 1A), which were sustained for minutes) and transiently (subsiding within a few hours) in weeks (not shown). Thus, the p38MAPK response to senes- response to acute cellular stress (Cuenda and Rousseau, cence-inducing genotoxic stress differed markedly in kinetics from the response to an acute stress (e.g., LPS stimulation) 2007). p38MAPK is important for the senescence growth (Cuenda and Rousseau, 2007) that does not induce senes- arrest due to its ability to activate both the p53 and pRb/ p16 growth arrest pathways. p38MAPK inhibition moderately cence. Importantly, the kinetics of p38MAPK activation delays replicative senescence (caused by dysfunctional closely paralleled the kinetics with which the SASP develops telomeres, which resemble DNA double-strand breaks) (Coppe et al, 2008; Rodier et al, 2009). (Iwasa et al, 2003), and the rapid senescence of cells from patients with Werner’s syndrome, a premature aging disorder p38MAPK activity is required for the SASP caused by a defective DNA repair protein (Davis and Kipling, p38MAPK activation during senescence was inhibited by the 2009). Further, p38MAPK activity is required for the senes- small molecule SB203580 (SB). SB displaces ATP from the cence arrest caused by oncogenic RAS, and constitutive p38MAPK ATP-binding pocket (Young et al, 1997), thereby p38MAPK activity can induce a growth arrest in normal preventing p38MAPK from phosphorylating its targets human cells (Wang et al, 2002; Deng et al, 2004). Whether without preventing p38MAPK phosphorylation itself. As p38MAPK regulates the SASP had not been explored. determined by Hsp27-P levels, daily treatment with 10 mM p38MAPK is known to upregulate speciﬁc cytokines such as SB, which has minimal off-target effects (Cuenda et al, 1995; IL-6, IL-8, and TNFa in some biological contexts (Ono and Wilson et al, 1997), prevented p38MAPK activation after XRA Han, 2000; Zhang et al, 2007), but the upregulation is (Figure 1A). generally an acute response, whereas the SASP is chronic To determine the signiﬁcance of the coincident rise in and includes 440 secreted proteins comprising cytokines, p38MAPK activity and the SASP, we added SB to SEN(XRA) &2011 European Molecular Biology Organization The EMBO Journal VOL 30 NO 8 2011 1537 | | p38 regulates the senescence secretory phenotype A Freund et al Figure 1 p38MAPK is activated with slow kinetics during genotoxic stress-induced senescence and is required for the SASP. (A) p38MAPK phosphorylation increases during DNA damage-induced senescence. Cells were irradiated and whole cell lysates collected at the indicated days thereafter. þ : p38MAPK was continuously inhibited by 10 mM SB203580 (SB) beginning 48 h before irradiation. Left: western blot analysis of indicated proteins. Right: western blot quantitation, normalized to PRE levels. p38-P, phosphorylated p38MAPK; Hsp27-P, phosphorylated heat shock protein 27. (B) p38MAPK inhibition decreases intracellular IL-6 in SEN(XRA) cells. PRE and SEN(XRA) cells immunostained for IL-6. þ SB: p38MAPK was inhibited by SB023580 (SB) for 48 h before ﬁxation. (C) p38MAPK inhibition decreases IL-6, IL-8, and GM-CSF secreted by SEN(XRA) cells. Conditioned media (CM) were collected from PRE and SEN(XRA) cells and analysed by ELISA. þ SB: p38MAPK was inhibited by SB023580 (SB) for 48 h before CM collection. (D) p38MAPKa depletion decreases secreted IL-6. Cells were infected with lentivirus expressing either of two shRNAs against p38MAPKa (shp38a) or a control shRNA (shGFP) and selected. Cells were irradiated and allowed to senesce (SEN(XRA)). CM were analysed by ELISA. (E) p38MAPK inhibition suppresses the SEN(XRA) SASP. CM from PRE and SEN(XRA) cells, with (SB) or without p38MAPK inhibition, were analysed by antibody arrays. Shown are factors for which the SEN(XRA) level was signiﬁcantly increased (Po0.05) over PRE. For each protein, signals from all conditions were averaged to generate the baseline. Signals above baseline are yellow; signals below baseline are blue. The heat map key shows log -fold changes from baseline. The hierarchical clustering relationship between sample proﬁles is shown graphically as a dendrogram (left). *: Factors signiﬁcantly decreased by p38MAPK inhibition (Po0.05). (F) p38MAPK depletion decreases the ability of senescent cells to stimulate cancer cell invasiveness. CM from cells described in (D) were analysed for the ability to stimulate invasiveness of MDA-MB-231 breast cancer cells in a Boyden chamber invasion assay. cells for 48 h, then assessed IL-6, an indicator of SASP activity regulates the SASP, we depleted cells of p38MAPKa, the most (Coppe et al, 2008, 2010a; Bhaumik et al, 2009; Orjalo et al, abundant isoform, by RNA interference (RNAi). Using lenti- 2009) by immunostaining (intracellular levels) and enzyme- viruses, we expressed in SEN(XRA) cells either of two linked immunoadsorbent assay (ELISA) of conditioned unrelated short hairpin (sh) RNAs that speciﬁcally target medium (CM) (secreted levels). Both assays showed that p38MAPKa (Supplementary Figure S1F). We then assayed SB reduced IL-6 levels to near-PRE levels (Figures 1B and IL-6 levels in CM from cells expressing control (shGFP) or C); ELISA showed that SB also signiﬁcantly reduced secretion p38MAPKa-speciﬁc (shp38a) shRNAs. Both shp38a shRNAs of the SASP components IL-8 and GM-CSF (Figure 1C) signiﬁcantly decreased secreted IL-6 levels in SEN(XRA) cells (Po0.05). Further, SB signiﬁcantly reduced secreted IL-6 (Figure 1D; Po0.01). A p38MAPKb shRNA did not reduce levels in CM from SEN(XRA) WI-38, an unrelated human secreted IL-6 levels (not shown). These data conﬁrm that ﬁbroblast strain (Supplementary Figure S1D), and replica- p38MAPK is essential for induction of the SASP, and identify tively senescent (SEN(REP)) HCA2 and WI-38 cells (Supple- p38MAPKa as the major functional isoform. mentary Figure S1E). Thus, the ability of p38MAPK inhibition The SASP is a complex network comprising 440 proteins to signiﬁcantly reduce senescence-associated IL-6 secretion (Coppe et al, 2008, 2010b). To determine which SASP factors was not conﬁned to XRA-induced senescence or a single cell are p38MAPK regulated, we analysed CM from PRE and strain. These ﬁndings suggest that p38MAPK activation is SEN(XRA) cells, with or without p38MAPK inhibition, using necessary for secretion of at least some SASP components. arrays containing antibodies against 120 secreted proteins. This Although SB is a well-characterized p38MAPK inhibitor, analysis identiﬁed 37 proteins that were signiﬁcantly upregu- one report showed it can partially inhibit protein kinase B at lated in SEN(XRA) cells relative to PRE (Figure 1E). Most of the concentration used here (10 mM) (Lali et al, 2000), and it these proteins (68%, 25/37) declined signiﬁcantly (Po0.05) inhibits both the a and b p38MAPK isoforms (Enslen et al, following p38MAPK inhibition (SEN(XRA)þ SB) (Figure 1E, 1998). To determine whether the effect of SB on SASP compo- asterisks; Supplementary Figure S1G); the remaining SASP nents was p38MAPK speciﬁc, and to identify the isoform that proteins exhibited non-signiﬁcant decreases (Figure 1E). The 1538 The EMBO Journal VOL 30 NO 8 2011 &2011 European Molecular Biology Organization | | p38 regulates the senescence secretory phenotype A Freund et al p38MAPK-regulated proteins included cytokines, chemokines, SEN(RAS), 78% (19/23) were signiﬁcantly decreased by growth factors, shed ligands and, importantly, 9 of the 10 most p38MAPK inhibition in both cases (Supplementary Figure robustly secreted SASP proteins. Hierarchical clustering (Eisen S2C). Thus, a majority of the SASP factors induced by both et al, 1998) of the array results showed that SEN(XRA) cells genotoxic stress (XRA) and oncogene activation (RAS) de- treated with SB had an SASP proﬁle that more closely resem- pends on p38MAPK activity. bled PRE cells than untreated SEN(XRA) cells (Figure 1E). p38MAPK inhibition slightly increased secreted levels of a few p53 restrains the SASP by restraining p38MAPK activity proteins, but not signiﬁcantly (Supplementary Figure S1H). p53 restrains the SASP via an unknown mechanism (Coppe The SASP also includes several MMPs, most prominently et al, 2008). To determine the relationship between p53 and MMP1 and MMP3 (Coppe et al, 2010b). p38MAPK inhibition p38MAPK during development of the SASP, we inactivated had little effect on secreted MMP1 or MMP3 levels; even p53 using retrovirally-delivered GSE22, a peptide that pre- when we started p38MAPK inhibition before XRA and contin- vents p53 tetramerization and thus p53 transcriptional acti- ued until sample collection, only MMP3 declined; MMP1 was vity (Ossovskaya et al, 1996). Because p53 monomers are not affected (Supplementary Figure S1I). Thus, p38MAPK not rapidly degraded, GSE22 activity can be monitored by is a less potent regulator of SASP MMPs, but a strong the accumulation of p53 protein (Figure 2C). We induced positive regulator of many SASP chemokines, cytokines, and p53-deﬁcient cells to senesce with XRA (SEN(XRA)þ GSE), growth factors. and compared phosphorylated p38MAPK levels with those in SEN(XRA) and SEN(RAS) cells. p38MAPK inhibition mitigates a paracrine effect of Activated p38MAPK levels were highest in SEN(XRA)þ senescent cells GSE cells, followed by SEN(RAS) and SEN(XRA) (Figure 2C). CM from senescent cells stimulate the ability of cancer cells The relative levels of p38MAPK phosphorylation matched to invade a basement membrane (Coppe et al, 2008). To the relative levels of IL-6 secretion (Figures 2C versus D), determine whether p38MAPK inhibition mitigates this effect, suggesting that p53 and RAS regulate the intensity of the we measured the ability of CM from PRE or SEN(XRA) cells SASP by regulating the level of p38MAPK activation. p53 expressing either a control (shGFP) or p38MAPKa (shp38a) also regulated the kinetics of SASP development by regulating shRNA to stimulate the invasiveness of MDA-MB-231 human the timing of p38MAPK activation. When p53 was inacti- breast cancer cells. SEN(XRA) CM stimulated B6-fold more vated by GSE22, p38MAPK phosphorylation occurred more invasion than PRE CM (Figure 1F, Po0.001), as expected. rapidly after XRA compared with cells with wild-type p53 p38MAPK depletion markedly reduced this stimulatory acti- (Figure 2E). To determine whether this increase in p38MAPK vity (Figure 1F, Po0.001), indicating that p38MAPK inhibi- activity was responsible for the ampliﬁed SASP in p53- tion can mitigate an important biological consequence of deﬁcient cells, we inhibited p38MAPK with SB. The ampliﬁed the SASP. levels of IL-6, IL-8, and GM-CSF were almost completely suppressed by p38MAPK inhibition (Figure 2F). We obtained similar results when we depleted cells of p53 by RNAi p38MAPK inhibition mitigates the SASP induced by (Supplementary Figures S2D and E). We conclude that oncogenic RAS expression Senescence can be induced by certain oncogenes, including p53 restrains p38MAPK activity after senescence induction, V12 the oncogenic form of H-RAS (RAS ) (Serrano et al, thereby restraining the SASP. 1997). As reported (Di Micco et al, 2006), oncogenic V12 H-RAS expression caused hyperproliferation for several p38MAPK activity is sufﬁcient to induce an SASP days, resulting in DNA damage and ultimately a senescence To study the effect of constitutive p38MAPK activation, growth arrest 8–10 days later (SEN(RAS)) (not shown). Like we infected PRE cells with a constitutively active mutant SEN(XRA) cells, and as reported (Wang et al, 2002; Deng (MKK6EE) of MAP kinase kinase 6 (MKK6), which directly et al, 2004), SEN(RAS) cells showed increased levels of phosphorylates p38MAPK. As expected, MKK6EE expres- activated (phosphorylated) p38MAPK (Figure 2A). Also as sion caused phosphorylation of endogenous p38MAPK reported (Coppe et al, 2008), SEN(RAS) cells expressed an (Figure 3A). Moreover, cells ceased proliferation several ampliﬁed SASP, secreting several proteins at signiﬁcantly days later (Figure 3B). This growth arrest was accompanied higher levels than those secreted by SEN(XRA) cells and by increased SA-bgal activity (Supplementary Figure S3A), several factors not present in the SEN(XRA) SASP. In decreased BrdU incorporation (Supplementary Figure S3B), HCA2 cells, the SEN(RAS) SASP included 83 proteins and a senescent morphology (Supplementary Figure S3C). (Figure 2B; Supplementary Figure S2A). p38MAPK inhibition These responses were prevented by p38MAPK inhibition. (SEN(RASþ SB)) signiﬁcantly reduced (Po0.05) secreted To determine whether constitutive p38MAPK activity levels of 78% (65/83) of these proteins (Figure 2B; (due to MKK6EE expression) is sufﬁcient to induce an Supplementary Figure S2A, asterisks), including 9 of the 10 SASP, we used antibody arrays and identiﬁed 19 factors most robustly secreted SASP proteins. The remaining SASP that were signiﬁcantly upregulated in MKK6EE-expressing proteins were non-signiﬁcantly reduced (Supplementary cells relative to PRE controls (Figure 3C). p38MAPK inhibi- Figure S2A). p38MAPK inhibition also signiﬁcantly reduced tion (MKK6EEþ SB) signiﬁcantly reduced the secreted levels MMP1 and MMP3 levels in SEN(RAS) cells, although to a of most of these proteins (84%, 16/19) (Figure 3C, asterisks); lesser extent than most cytokines and chemokines (Supple- the remaining proteins were non-signiﬁcantly reduced mentary Figure S2B). The SASP proteins affected by p38MAPK (Figure 3C). Notably, 7 of the 10 most upregulated factors inhibition in SEN(RAS) cells overlapped with many of in SEN(XRA) cells, and 9 of the 10 most upregulated factors those affected by p38MAPK inhibition in SEN(XRA) cells: in SEN(RAS) cells, increased signiﬁcantly upon MKK6EE of the 23 factors upregulated in both SEN(XRA) and expression (Figure 3D). We validated the array results by &2011 European Molecular Biology Organization The EMBO Journal VOL 30 NO 8 2011 1539 | | p38 regulates the senescence secretory phenotype A Freund et al V12 Figure 2 p38MAPK drives the ampliﬁed SASPs induced by RAS or p53 inactivation. (A) p38MAPK is phosphorylated during RAS -induced V12 senescence. PRE cells were infected with lentivirus expressing oncogenic RAS , selected, and allowed to senesce (SEN(RAS)) for 10 days. Whole cell lysates were then collected and analysed by western blot. Presenescent controls (PRE) were infected with insertless vector. (B) p38MAPK inhibition suppresses the SEN(RAS) SASP. Cells were infected as described in (A). CM from PRE and SEN(RAS) cells were analysed by antibody arrays. þ SB: p38MAPK was inhibited by SB203580 for 48 h before CM collection. Shown are the top 40 factors for which the SEN(RAS) level was signiﬁcantly increased over PRE. PRE and SEN(RAS) values were averaged to generate the baseline. Heat map and dendrogram were generated as in Figure 1E. *: Factors signiﬁcantly decreased by p38MAPK inhibition. (C) Ampliﬁed p38MAPK phosphoryla- tion in SEN(RAS) cells and SEN(XRA) cells lacking functional p53 (SEN(XRA)þ GSE). Cells were infected with lentivirus expressing GSE22 V12 (GSE) or an insertless vector, selected, then irradiated (XRA) or infected with lentivirus expressing oncogenic RAS (RAS) and allowed to senesce. Whole cell lysates were analysed by western blot. (D) SEN(RAS) and SEN(XRA) cells lacking functional p53 secrete ampliﬁed IL-6 levels. Cells were treated as in (C), then CM were collected and analysed by ELISA. (E) p53 inactivation accelerates p38MAPK phosphorylation after XRA. Cells were infected with lentivirus lacking insert (Vector) or expressing GSE22 (GSE), selected, and irradiated. Whole cell lysates were collected at speciﬁed time points and analysed by western blot. (F) GSE-ampliﬁed levels of IL-6, IL-8, and GM-CSF are p38MAPK dependent. Cells were infected as in (E) and irradiated. CM were collected 3 days later and analysed by ELISA. SB: p38MAPK was inhibited by SB203580 for 48 h before CM collection. ELISA measurements of secreted IL-6 (Supplementary Figure addition to inducing growth arrest, SA-bgal activity, and a S3D). Constitutive p38MAPK activity was also sufﬁcient to senescent morphology. induce MMP1 and MMP3, although to a lesser extent than it induced the cytokines and chemokines (Supplementary p38MAPK regulates the SASP independent of the DDR Figure S3E). Together, these data show that high-level con- The DDR is required for expression of a subset of SASP stitutive p38MAPK activity is sufﬁcient to induce a robust proteins, including IL-6 and IL-8 (Rodier et al, 2009). SASP that resembles the SEN(XRA) and SEN(RAS) SASPs, in To determine whether p38MAPK inhibition decreases the 1540 The EMBO Journal VOL 30 NO 8 2011 &2011 European Molecular Biology Organization | | p38 regulates the senescence secretory phenotype A Freund et al Figure 3 Constitutive p38MAPK activation is sufﬁcient to induce an SASP. (A) PRE cells were infected with lentivirus expressing a constitutively active MAP kinase kinase 6 mutant (MKK6EE). Whole cell lysates were collected 8 days after infection and analysed by western blot. Presenescent controls (PRE) were infected with insertless vector. (B) MKK6EE induces p38MAPK-dependent growth arrest. Cells were infected as in (A) and counted at the indicated intervals thereafter. þ SB: p38MAPK was continuously inhibited by SB203580 beginning 48 h before infection. (C) MKK6EE induces an SASP. Cells were infected as described in (A); CM were collected 8 days after infection. þ SB: p38MAPK was inhibited by SB203580 for 48 h before CM collection. Secreted proteins were analysed using antibody arrays. Shown are proteins for which the MKK6EE level was signiﬁcantly increased (Po0.05) over PRE. PRE and MKK6EE values were averaged to generate the baseline. Heat map and dendrogram were generated as in Figure 1E. *: Factors signiﬁcantly decreased by p38MAPK inhibition (Po0.05). (D) The MKK6EE SASP resembles SEN(XRA) and SEN(RAS) SASPs. Shown are the 10 most upregulated factors in the SEN(XRA) and SEN(RAS) SASPs. þ : Factors signiﬁcantly increased by MKK6EE expression. SASP by inhibiting the DDR, we induced senescence by XRA Figure S4C). Further, cells made senescent by MKK6EE and measured the activities of several DDR proteins with or expression did not exhibit an increase in global ATM or without p38MAPK inhibition (SB). p38MAPK inhibition had CHK2 phosphorylation (Figure 4C). MKK6EE expression did no effect on the rapid (within 2 h) phosphorylation of ATM, not increase global DNA damage signalling at any time after CHK2, or p53(Ser15), nor on the transient stabilization of infection, as measured by p53(Ser15) phosphorylation, un- p53 and expression of p21 after XRA (Figure 4A). p38MAPK like RAS expression (Supplementary Figure S4D). Finally, inhibition also did not affect the low-level activation of these though depletion of ATM or CHK2 by RNAi reduced IL-6, DDR proteins that persists after XRA (42 days) (Rodier et al, IL-8, and GM-CSF secretion by SEN(XRA) and SEN(RAS) 2009, 2011) (Figure 4A). Thus, p38MAPK inhibition does not cells, as described (Rodier et al, 2009), 490% depletion of suppress the SASP by suppressing the DDR. these DDR factors (Supplementary Figure S4E) had no effect In the presence of existing DNA damage, p38MAPK can on IL-6, IL-8, or GM-CSF secretion induced by MKK6EE replenish short-lived DNA damage foci via an ROS feedback (Figure 4D, P40.05). Thus, p38MAPK does not regulate the loop (Passos et al, 2010). Supporting those ﬁndings, though SASP by modulating the DDR. Conversely, the DDR does not p38MAPK inhibition had no effect on the formation or regulate the SASP by modulating p38MAPK activity. Neither resolution of 53BP1 foci during the ﬁrst 4 days after XRA, it ATM nor CHK2 depletion suppressed p38MAPK phosphory- slightly decreased foci number 6–8 days after XRA (Supple- lation in SEN(XRA) (Figure 4E, top) or SEN(RAS) (Figure 4E, mentary Figure S4A, Po0.01) and in replicatively senescent bottom) cells. Together, these ﬁndings indicate that p38MAPK cells (Supplementary Figure S4B). However, in contrast to activity uniquely regulates the SASP independently of XRA, REP, and RAS, constitutive p38MAPK activity (MKK6EE the DDR. expression) did not induce DNA damage foci in most cells: If p38MAPK is sufﬁcient to induce an SASP and also most SEN(RAS), SEN(XRA), and (SEN(REP) cells harboured independent of the DDR, how can the DDR be required for X3 53BP1 foci per nucleus, but most cells induced to senesce the SEN(XRA) and SEN(RAS) SASPs? MKK6EE induces acti- by MKK6EE harboured o3 53BP1 foci and were not signi- vation of endogenous p38MAPK to a much greater extent ﬁcantly different from PRE cells (Figure 4B, P40.05). There than XRA or RAS. To determine whether a lower level of was a slight increase in the percentage of MKK6EE-induced p38MAPK activation is sufﬁcient to induce the SASP, we senescent cells with 410 53BP1 foci/nucleus, but these blunted p38MAPK activity in MKK6EE cells with varying cells accounted for only B3% of the total (Supplementary doses of SB203580 and measured downstream signalling via &2011 European Molecular Biology Organization The EMBO Journal VOL 30 NO 8 2011 1541 | | p38 regulates the senescence secretory phenotype A Freund et al Figure 4 p38MAPK induces an SASP independent of the DNA damage response. (A) p38MAPK inhibition does not prevent the DDR. Whole cell lysates were collected at speciﬁed intervals after irradiation and analysed by western blot. Where indicated, p38MAPK was continuously inhibited by SB203580(þ ) beginning 48 h before irradiation. ATM-P, Ser 1981 phosphorylated ATM; CHK2-P, Thr 68 phosphorylated CHK2. (B) Constitutive p38MAPK activation does not induce 53BP1 foci. Cells were ﬁxed 8 days after MKK6EE expression (MKK6EE), 10 days after RAS expression (SEN(RAS)), 8 days after irradiation (SEN(XRA)), or after replicative senescence (69 population doublings) (SEN(REP)) and immunostained for 53BP1. Foci were quantiﬁed using CellProﬁler to count cells with X3 53BP1 foci per nucleus. Error bars indicate 95% conﬁdence interval. (C) Constitutive p38MAPK activation does not induce a DDR. Whole cell lysate was collected 8 days after MKK6EE infection and analysed by western blot. Presenescent controls (PRE) were infected with insertless vector. ATM-P, Ser 1981 phosphorylated ATM; CHK2-P, Thr 68 phosphorylated CHK2. (D) Neither ATM nor CHK2 depletion prevents the SASP induced by constitutive p38MAPK activation. PRE cells were irradiated (SEN(XRA)), infected with RAS lentivirus (SEN(RAS)), or infected with MKK6EE lentivirus (MKK6EE). Simultaneously, cells were infected with lentivirus expressing shRNAs against ATM (shATM #12), CHK2 (shChk2 #2, shChk2 #12), or GFP (shGFP; control) and selected. CM from 8 days after infection/irradiation were analysed by ELISA. (E) ATM or CHK2 depletion does not prevent p38MAPK phosphorylation at senescence. Top: cells were irradiated; 6 days later cells were infected with lentivirus expressing shRNAs against ATM (shATM #12), CHK2 (shChk2 #2, shChk2 #12), or GFP (shGFP; control) and selected. Bottom: cells were simultaneously infected with RAS lentivirus, lentivirus expressing an shRNA against ATM (shATM #12), CHK2 (shChk2 #2, shChk2 #12), or GFP (shGFP; control) and selected. Whole cell lysates from 8 days after irradiation/infection were analysed by western blot. p38-P: phosphorylated p38. Hsp27 phosphorylation. p38MAPK was sufﬁcient to induce levels (Supplementary Figure S4G). Thus, though the level of a substantial increase in IL-6 only when active at a higher p38MAPK signalling in MKK6EE cells is sufﬁcient to induce level than that seen in SEN(XRA) cells (Supplementary an SASP without the DDR, the level of p38MAPK signalling Figure S4F). When p38MAPK signalling was inhibited to in other types of senescence is not sufﬁcient, and therefore a level that matched SEN(XRA) cells (as measured by requires cooperation with the DDR in order to induce Hsp27-P), it was not sufﬁcient to induce IL-6 to SEN(XRA) the SASP. 1542 The EMBO Journal VOL 30 NO 8 2011 &2011 European Molecular Biology Organization | | p38 regulates the senescence secretory phenotype A Freund et al p38MAPK inhibition suppresses SASP component decreased reporter activity (Po0.001). We conclude that mRNA levels NF-kB activity is regulated by p38MAPK during senescence. Many SASP factors are upregulated at the level of mRNA Depletion of ATM also decreased NF-kB reporter activity— abundance (Coppe et al, 2008, 2010b). To understand the to roughly the same level as p38MAPK inhibition (Figure 5E, mechanism by which p38MAPK regulates the SASP, we used Po0.001). Although increased concentrations of SB203580 quantitative RT–PCR to determine mRNA levels of six SASP had no additional effect on NF-kB reporter output (data not factors (GM-CSF, IL-6, IL-8, GROa, MCP-2, and IL-1a) that shown), the combined treatment of SB203580 and ATM declined signiﬁcantly upon p38MAPK inhibition. For all depletion had a synergistic effect, lowering NF-kB transcrip- six factors, p38MAPK inhibition (SB) markedly decreased tional activity to PRE control levels (Figure 5E, SBþ shATM). mRNA abundance in SEN(XRA) cells (Figure 5A). We These data reinforce the idea that ATM depletion and SB obtained similar results in another cell strain (Supple- affect NF-kB via different pathways. mentary Figure S5A). For GM-CSF, IL-6, and IL-8, the decrease in mRNA abundance matched the decrease in NF-jB is required for the SASP secreted protein level (Supplementary Figure S5B). In addi- Because p38MAPK regulates both the SASP and NF-kB tion, constitutive p38MAPK activation was sufﬁcient to activity, we asked whether NF-kB is required for the SASP. induce SASP mRNAs, as determined by IL-6 and IL-8 We expressed either of two unrelated shRNAs against RelA, mRNA levels upon MKK6EE expression (Supplementary both of which efﬁciently decreased RelA levels without sub- Figure S5C). While these results do not rule out the possibi- stantially affecting RelB or C-Rel levels (Supplementary lity that p38MAPK stimulates the SASP by inﬂuencing other Figure S5G). RelA depletion signiﬁcantly decreased 73% processes (e.g., translation and secretion), the data suggest (27/37) of the SASP proteins secreted by SEN(XRA) cells that p38MAPK induces the SASP primarily by increasing (Figure 5E, asterisks), including MMP1 and MMP3 (Supple- mRNA abundance. mentary Figure S5H), and non-signiﬁcantly decreased the remaining SASP proteins (Figure 5F). Hierarchical clustering p38MAPK controls NF-jB activity in senescent cells showed that RelA-depleted SEN(XRA) cells had an SASP Because p38MAPK is known to regulate the activity of multi- proﬁle that more closely resembled PRE cells than unmodiﬁed ple transcription factors (TFs) depending on context (Zarubin SEN(XRA) cells (Figure 5F). There was substantial overlap and Han, 2005), we examined the promoters of p38MAPK- between the RelA-dependent and p38MAPK-dependent induced factors (Figure 3D) for overrepresented transcription (Figure 1) SASP factors: 76% (19/25) of p38MAPK-dependent factor-binding sites (TFBS). We interrogated 200 bp upstream factors were also RelA dependent (Figure 5G, Venn diagram). of each transcriptional start site using the 243 TF weight Of the 10 most robustly secreted SASP proteins from Figure 1, 8 matrices in the TRANSFAC database. NF-kB-binding motifs were both p38MAPK- and RelA-dependent (Figure 5G, table). were most statistically overrepresented (Figure 5B). Activated Using IL-6, IL-8, and GM-CSF as SASP markers, we veriﬁed the NF-kB is enriched at the IL-8 and GROg promoters following RelA dependence of the SASP for SEN(RAS) cells (Figure 5H), MEK-induced senescence (Acosta et al, 2008). We therefore and two cell strains (Supplementary Figure S5I). Finally, asked whether NF-kB activity increases during multiple we demonstrated that IL-6, IL-8, and GM-CSF secretion types of senescence, and whether the increase is p38MAPK induced by MKK6EE were RelA dependent (Supplementary dependent. Figure S5J). Thus, p38MAPK acts primarily through NF-kBto Inactive NF-kB dimers are sequestered in the cytoplasm by induce the SASP. the inhibitor IkB. NF-kB activating signals cause IkB degrada- tion, allowing NF-kB nuclear translocation. Three NF-kB Discussion family members (RelA, RelB, and C-Rel) have DNA binding and transactivation domains, but RelA is most strongly The SASP develops when cells experience a stress severe associated with inﬂammatory cytokine gene transcription enough to cause a senescence response. These stresses are (Karin, 2006; Perkins, 2007). RelA was mostly cytoplasmic primarily genotoxic, leading to activation of a DDR. Persistent in PRE cells, but noticeably more nuclear in SEN(XRA) cells DDR signalling is necessary for the expression of several SASP (Figure 5C). Further, NF-kB DNA-binding activity increased factors (Rodier et al, 2009); depletion of DDR proteins such B5 fold in SEN(REP), SEN(RAS), and SEN(XRA) cells (two as ATM, NBS1, or CHK2 suppresses the expression of SASP strains) (Supplementary Figures S5D and E, Po0.001). components, including IL-6 and IL-8. However, the DDR is Constitutive p38MAPK activity (MKK6EE expression) was activated immediately after damage, whereas the SASP takes sufﬁcient to induce this activity (Supplementary Figure days to develop, indicating that canonical DDR signalling is S5D). Importantly, following XRA, NF-kB DNA-binding acti- not sufﬁcient for SASP expression. Thus, there must be other, vity increased slowly with kinetics that followed p38MAPK delayed molecular events that are required for development activation (Supplementary Figure S5F): NF-kB DNA-binding of the SASP and are regulated independently of the DDR. We activity remained near-PRE levels for 8 h after XRA, began to show here that activation of the p38MAPK/NF-kB pathway is rise 24 h after XRA and reached maximal levels 8–10 days such an event. later (Supplementary Figure S5F). Additionally, chromatin Unlike the rapid and transient response to acute stresses immunoprecipitation showed increased RelA binding to the such as LPS stimulation, the kinetics of p38MAPK activation promoters of three SASP genes (Figure 5D, Po0.01). A after DNA damage were slow and chronic, coinciding with lentiviral-delivered luciferase reporter driven by an NF-kB- expression of the SASP. p38MAPK inhibition effectively responsive promoter showed that NF-kB transcriptional acti- collapsed the senescence-associated cytokine network, pre- vity was 430-fold higher in SEN(XRA) compared with PRE venting the pro-invasion paracine effects of senescent cells. cells (Figure 5E), and p38MAPK inhibition (SB) signiﬁcantly Further, robust p38MAPK activation, caused by MKK6EE &2011 European Molecular Biology Organization The EMBO Journal VOL 30 NO 8 2011 1543 | | p38 regulates the senescence secretory phenotype A Freund et al Figure 5 p38MAPK induces SASP mRNAs by increasing NF-kB activity. (A) p38MAPK inhibition decreases SASP mRNA levels. Total RNA was extracted from PRE and SEN(XRA) HCA2 cells; mRNA levels for indicated genes were analysed by qRT–PCR. þ SB: p38MAPK was inhibited with SB203580 for 48 h before sample collection. For each gene, the four signals were averaged to generate the baseline. Signals above baseline are red; signals below baseline are green. The heat map key shows log -fold changes from baseline. p38MAPK inhibition signiﬁcantly decreased (Po0.05) mRNA levels for all genes assayed. (B) Transcription factor (TF) binding sites (BS) in MKK6EE-induced genes. Genes encoding SASP proteins upregulated by MKK6EE expression (Figure 3C) were analysed for statistically overrepresented TFBS in the 200 bp upstream of the transcriptional start site. ‘% of sequences’ indicates percentage of sequences with X1 binding site for each indicated weight matrix. TFBS are sorted by P-value. (C) RelA partially localizes to the nucleus during damage-induced senescence. PRE and SEN(XRA) cells were immunostained for RelA; representative images are shown. (D) Increased RelA binding to the promoters of SASP genes. Cells were irradiated, allowed to senesce (SEN(XRA)), and lysates were analysed by chromatin immunoprecipitation using an antibody against RelA. RelA binding to the promoters of the indicated genes is represented relative to Histone H3 binding and is normalized to SEN(XRA) binding for each gene. (E) p38MAPK inhibition and ATM depletion reduce NF-kB transcriptional activity in senescent cells. Cells were infected with lentivirus expressing an NF-kB luciferase reporter construct, irradiated, and allowed to senesce (SEN(XRA)). Cells were lysed and luciferase activity was measured. SB: p38MAPK was inhibited with SB203580 for 48 h before lysis. shATM: cells were infected with lentivirus expressing shRNA against ATM 5 days before lysis. (F) RelA depletion suppresses the SASP of SEN(XRA) cells. Cells were infected with lentivirus expressing either of two shRNAs against RelA (shRelA) or GFP (shGFP; control), selected, irradiated, and allowed to senesce (SEN(XRA)). Secreted proteins were detected by antibody arrays as in Figure 1. Shown are factors for which the SEN(XRA) level was signiﬁcantly increased (Po0.05) over PRE. For each protein, the six signals were averaged to generate the baseline. Heat map and dendrogram were generated as in Figure 1E. Asterisks indicate factors that are signiﬁcantly decreased by both RelA shRNAs (Po0.05). (G) Most p38MAPK-dependent SASP proteins are NF-kB dependent. Left: proportional Venn diagram displaying the overlap between p38MAPK-dependent factors (red), RelA-dependent factors (blue), and the SEN(XRA) SASP (yellow). In all, 76% of p38MAPK-dependent factors are also RelA-dependent (dashed area). Right: the 10 most upregulated SEN(XRA) SASP factors from Figure 1E. þ : Proteins dependent on RelA or p38MAPK. (H) SEN(RAS)-induced IL-6, IL-8, and GM- CSF are RelA-dependent. Cells were infected with lentivirus expressing either of two shRNAs against RelA (shRelA) or GFP (shGFP; control) V12 and selected. Cells were then infected with lentivirus lacking an insert (PRE) or expressing RAS and allowed to senesce (SEN(RAS)). CM were analysed by ELISA. expression, was sufﬁcient to induce an SASP, suggesting that p53 and then delivered a senescence-inducing genotoxic p38MAPK activity is limiting for SASP development. stress, p38MAPK was activated faster and to a higher level. Of particular importance, we found that p53 restrains the The increased activation correlated with the ampliﬁed SASP SASP by restraining p38MAPK activity. When we inactivated that develops in the absence of p53 (Coppe et al, 2008), and 1544 The EMBO Journal VOL 30 NO 8 2011 &2011 European Molecular Biology Organization | | p38 regulates the senescence secretory phenotype A Freund et al p38MAPK inhibition reduced the ampliﬁed SASP. Given that phosphorylated by ATM or its downstream targets. Those transformed and primary cells can differ in the kinetics with low-afﬁnity sites, then, are phosphorylated by p38MAPK only which the p38MAPK/NF-kB pathway is activated (Janssens if p38MAPK signalling is high (e.g., due to MKK6EE expres- and Tschopp, 2006), our data suggest that p53 represses the sion). In SEN(XRA) and SEN(RAS) cells, p38MAPK signalling p38MAPK pathway immediately after DNA damage in normal is not high enough to result in phosphorylation of the low- cells. This repression may allow time to repair the damage afﬁnity sites, making both p38MAPK and the DDR necessary before cells commit to developing an SASP, which signals to for NF-kB activity. the tissue microenvironment. p38MAPK was crucial for the expression of many SASP The p38MAPK pathway acted in parallel to the DDR. cytokines and chemokines, which are largely pro-inﬂamma- Inhibition of p38MAPK did not affect activation of important tory and pro-carcinogenic (Davalos et al, 2010; Freund et al, DDR factors such as ATM, CHK2, or p53. Additionally, 2010; Coppe et al, 2010a). However, p38MAPK was less constitutive p38MAPK activation, despite inducing an SASP, important for the SASP MMPs, which may be beneﬁcial in neither induced ATM or CHK2 activation nor increased DNA the short term. The MMPs have matrix-degrading and ﬁbro- damage foci in most cells. Furthermore, ATM or CHK2 lytic activity, which may limit ﬁbrosis during wound healing depletion had no effect on the p38MAPK-induced SASP. (Krizhanovsky et al, 2008; Jun and Lau, 2010). The two most Thus, we show for the ﬁrst time that senescence-associated highly secreted MMPs are MMP1 and MMP3 (Coppe et al, pro-inﬂammatory cytokine secretion can occur in the absence 2010b). p38MAPK activation induced these MMPs, but to of a DDR. Additionally, neither ATM nor CHK2 depletion a lesser extent than many cytokines and chemokines. altered p38MAPK phosphorylation at senescence, suggesting Additionally, although prolonged p38MAPK inhibition that p38MAPK is not downstream of the canonical DDR. reduced MMP levels in SEN(XRA) and SEN(RAS) cells, the p38MAPK inhibition had a small effect on the resolution of effect was substantially smaller than the reduction in IL-6, DNA damage foci in a subset of senescent cells, supporting IL-8, GM-CSF, and other SASP factors. Thus, other MMP- reports that p38MAPK can replenish short-lived DNA damage regulating pathways must be active at senescence. More foci (Passos et al, 2010). However, as the p38MAPK-induced importantly, our data suggest it may be possible to reduce SASP did not require ATM or CHK2, the DDR-ROS feedback some SASP factors without affecting others, potentially loop that maintains a subset of DNA damage foci (Passos mitigating the deleterious effects without strongly mitigating et al, 2010) does not seem to be the mechanism by which the beneﬁts. p38MAPK regulates the SASP. p38MAPK establishes the senescence growth arrest by INK4A NF-kB was the crucial effector of p38MAPK signalling mediating RAS-induced expression of p16 and directly during senescence. NF-kB-binding sites were the most and indirectly phosphorylating p53 (Sun et al, 2007; Kwong INK4a statistically overrepresented TFBS in the identiﬁed set of et al, 2009). Neither p16 nor p53 are required for the p38MAPK-induced genes, and NF-kB activity was increased SASP (Coppe et al, 2008, 2010a). Thus, although the SASP can in DNA damage-induced, oncogene-induced, and replicative indirectly reinforce the growth arrest through an IL-6/IL-8 senescence. Moreover, p38MAPK increased SASP factor autocrine feedback loop (Acosta et al, 2008; Kuilman et al, mRNA abundance, and p38MAPK was required for senes- 2008), the above ﬁndings and our data that p38MAPK cence-induced NF-kB activity. ATM was also required for regulates the SASP via NF-kB suggest that p38MAPK may senescence-induced NF-kB activity and acted synergistically deﬁne a divergence point for events that activate the SASP with p38MAPK, supporting the independence of the DDR and versus the growth arrest. p38MAPK pathways. The SASPs induced by DNA damage, Our identiﬁcation of the p38MAPK/NF-kB pathway as a RAS, or constitutive p38MAPK activity all required NF-kB, necessary and sufﬁcient, DNA damage-independent regulator demonstrating its role in mediating p38MAPK signalling. of the SASP provides new insights into how senescent cells NF-kB may not be the only means by which p38MAPK might be a source of the chronic inﬂammation that is a increases SASP gene expression—other TFBS such as hallmark of aging and many age-related diseases. C/EBP sites were also overrepresented in p38MAPK-induced SASP genes and are indirectly regulated by p38MAPK (Cortez Materials and methods et al, 2007), and p38MAPK can also affect mRNA stability (Wang et al, 1999; Radtke et al, 2010). Nevertheless, the SASP Cell culture network was largely dependent on NF-kB, and p38MAPK Primary human ﬁbroblasts and MDA-MB-231 cells were cultured in a 10% CO ,3%O atmosphere (Coppe et al, 2008). Unless noted was, in turn, necessary and sufﬁcient for NF-kB activity. 2 2 otherwise, ‘ﬁbroblast’ or ‘cells’ in the text and legends refer to p38MAPK activity was sufﬁcient for SASP activity, but only HCA2 ﬁbroblasts. Presenescent (PRE) HCA2 cells completed o35 at high levels. At the level of activation caused by XRA or population doublings and had a 24-h BrdU labeling index of 460%. RAS, p38MAPK was not sufﬁcient to activate the SASP—the Cells were made replicatively senescent (SEN(REP)) by repeated subculture (Dimri et al, 1995; Krtolica et al, 2001). For DNA DDR was also necessary. We propose a model in which the damage-induced senescence (SEN(XRA)), cells were grown to DDR and the p38MAPK pathways each post-translationally conﬂuence, exposed to 10 Gy X-ray and, unless noted otherwise, modify speciﬁc NF-kB sites, with varying degrees of analysed 8–10 days later; PRE cells were mock irradiated. For efﬁciency. Both p38MAPK and ATM are required for NF-kB oncogene-induced senescence (SEN(RAS)), cells were infected with V12 lentivirus expressing RAS and analysed 8–10 days after infection. activity at senescence, and NF-kB requires simultaneous Where indicated, cells were given 10 mM SB203580 (Calbiochem, phosphorylation on multiple sites for its activity (Karin, 559395) for the speciﬁed intervals with daily media changes. 2006; Perkins, 2007). We suggest that p38MAPK or its down- stream target(s) have a high afﬁnity for some sites, which Vectors, viruses, and infections become phosphorylated by the amount of p38MAPK signal- MKK6EE (provided by Dr Eisuke Nishida of Kyoto University), ling caused by XRA or RAS, but low afﬁnity for sites normally Genetic suppressor element 22 (GSE) (Ossovskaya et al, 1996), and &2011 European Molecular Biology Organization The EMBO Journal VOL 30 NO 8 2011 1545 | | p38 regulates the senescence secretory phenotype A Freund et al V12 RAS were subcloned into Gateway destination vector 670-1 (Hs 00174131 m1), IL-8 (Hs 00174103 m1), GROa (Hs 00236937 (Campeau et al, 2009). Insertless vector was used as a control for m1), GM-CSF (Hs 00929873 m1), and MCP-2 (Hs 99999026 m1). PRE cells. Lentiviral vectors encoding shRNAs against GFP (RHS4459), p38a (TRCN0000000509, TRCN0000010051), and RelA NF-jB assays (TRCN0000014686, TRCN0000014687) were from Open Biosystems. NF-kB DNA-binding activity in whole cell lysate was assayed using TM Lentiviral vectors encoding ATM shRNAs, CHK2 shRNAs, p53 the TransAM NF-kB p65 Transcription Factor Assay Kit from shRNA (Rodier et al, 2009) and virus production were as described Active Motif (40096). To assay transcriptional activity, cells were (Naldini et al, 1996; Beausejour et al, 2003). Viral titres were infected with Cignal NF-kB Lentiviral Reporter (SABiosciences, CLS- adjusted to infect B90% of cells. Cells were infected overnight with 013L) and selected. After senescence induction, cells were lysed and polybrene, allowed to recover for 48 h, selected for 48 h, and assayed for luciferase using the Luciferase Assay System (Promega, allowed to recover for at least another 48 h before use. E1500). All values for both assays were normalized to cell number. Immunoﬂuorescence Chromatin immunoprecipitation Cells were ﬁxed, permeabilized, blocked, and incubated with Chromatin immunoprecipitation was performed with the MAGnify primary antibodies overnight at 41C in PBSþ 1% BSA. Primary ChIP system (Invitrogen, #49-2024) and signal was detected with antibodies were from R&D Systems (IL6, AF206NA, 1:60), Novus Roche’s UPL system. In all, 3 mg of RelA antibody (Santa Cruz, Biologicals (53BP1, NB 100-305, 1:2000), and BD Biosciences #SC-372X) or Histone H3 (Abcam, #AB-1791) was used. Primers (BrdU, 347580, 1:100). Images were quantitated using CellProﬁler used: IL-6 promoter: F-cacagaagaactcagatgactgg, R-aaaaccaaagatgttc (Carpenter et al, 2006). tgaactga; IL-8 promoter: F-catcagttgcaaatcgtgga, R-gaacttatgcaccctc atcttttc; GM-CSF promoter: F-ccccttactggactgaggttg, R-cccactgacagtt Senescence-associated b-galactosidase assay cacatgg. Cells were ﬁxed and stained for SA-bgal using BioVision’s Sene- scence Detection Kit (#K320-250) for 24 h. Staining was visualized Analysis of TFBS by light microscopy; positive cells were counted manually. TFM-Explorer (Defrance and Touzet, 2006) was used to identify the top 10 statistically overrepresented partial weight matrices (PWMs) BrdU incorporation in the 200 bp upstream of the transcription start sites of genes Subconﬂuent cells were incubated for 24 h with 10 mM BrdU and encoding proteins signiﬁcantly induced by MKK6EE expression. We immunostained for BrdU as described (Rodier et al, 2011). searched all vertebrate PWMs available in the TRANSFAC database using a cluster density ratio of 2.5. P-values were calculated Western blot analysis compared with a background model incorporating all RefSeq genes On-plate lysis was performed with either denaturing (5% SDS, (24 328). 10 mM Tris) or non-denaturing (Cell Lysis Buffer, Cell Signalling, 9803) buffer containing protease and phosphatase inhibitors. Statistical analyses Primary antibodies are listed in Supplementary Table S1. Signals Statistical analyses of main ﬁgures are in Supplementary Table S1; were quantiﬁed with LI-COR Odyssey software. statistical analyses of supplementary ﬁgures are in Supplementary Table S2. Statistical signiﬁcance between distributions of signals ELISAs and conditioned media was evaluated using a two-tailed Student’s t-test and assumption ELISA kits to detect IL-6 (D6050), IL-8 (D8000C), and GM-CSF of equal variance. Statistical signiﬁcance between binary assays (DGM00) were from R&D Systems. MMP1 and MMP3 were detected (i.e., positive and negative scores) was evaluated using a w -test. with AlphaLISA kits from Perkin-Elmer (AL242C, AL284C). CM were prepared by washing with serum-free DMEM and incubating Supplementary data in serum-free DMEM for 24 h. All ELISA data were normalized to Supplementary data are available at The EMBO Journal Online cell number. (http://www.embojournal.org). Antibody arrays Acknowledgements CM samples were diluted to equivalent cell numbers in serum-free DMEM. Antibody arrays were from Raybiotech (AAH-CYT-G1000-8). We thank Drs Eisuke Nishida (Kyoto University) for the pSRalpha- Arrays were scanned using a GenePix 4200A Professional micro- myc-MKK6-EE vector, Pierre Desprez (California Paciﬁc array scanner at 10 mm resolution. Signal intensities were Medical Center) for critically reading the manuscript, and Shruti quantitated using LI-COR Odyssey software, and normalized to Waghray for technical assistance. This work was supported by positive controls for each sample, which were then normalized grants from the National Institutes of Health (AG09909, across all samples. AG017242, and AG25901, JC), a National Science Foundation Graduate Research Fellowship (AF), and a fellowship from the Invasion assay Larry L Hillblom Foundation (CP). CM were diluted to equivalent cell numbers in serum-free DMEM. Author contributions: AF and JC conceived the project; AF Invasion assays were performed as described (Coppe et al, 2006) performed most of the experiments and prepared the ﬁgures; CKP using Matrigel Invasion Chambers (8 mm pore, BD Biosciences participated in the array analyses; AF, CKP, and JC participated in 354480). interpreting the data; AF, CKP, and JC wrote the paper. Quantitative RT–PCR Taqman analyses were performed by the UCSF Genome Core. Conﬂict of interest Samples were normalized to b-glucuronidase (GUS). Primers were from Applied Biosystems: IL-1a (Hs 00174092 m1), IL-6 The authors declare that they have no conﬂict of interest. References Acosta JC, O’Loghlen A, Banito A, Guijarro MV, Augert A, Raguz S, Beausejour CM, Krtolica A, Galimi F, Narita M, Lowe SW, Yaswen P, Fumagalli M, Da Costa M, Brown C, Popov N, Takatsu Y, Campisi J (2003) Reversal of human cellular senescence: roles of Melamed J, d’Adda di Fagagna F, Bernard D, Hernando E, Gil J the p53 and p16 pathways. EMBO J 22: 4212–4222 (2008) Chemokine signalling via the CXCR2 receptor reinforces Beyaert R, Cuenda A, Vanden Berghe W, Plaisance S, Lee JC, senescence. Cell 133: 1006–1018 Haegeman G, Cohen P, Fiers W (1996) The p38/RK mitogen- Bavik C, Coleman I, Dean JP, Knudsen B, Plymate S, Nelson PS activated protein kinase pathway regulates interleukin-6 syn- (2006) The gene expression program of prostate ﬁbroblast sene- thesis response to tumor necrosis factor. EMBO J 15: 1914–1923 scence modulates neoplastic epithelial cell proliferation through Bhaumik D, Scott G, Schokrpur S, Patil CK, Orjalo AV, Rodier F, paracrine mechanisms. Cancer Res 66: 794–802 Lithgow G, Campisi J (2009) MicroRNAs miR-146a/b negatively 1546 The EMBO Journal VOL 30 NO 8 2011 &2011 European Molecular Biology Organization | | p38 regulates the senescence secretory phenotype A Freund et al modulate the senescence-associated inﬂammatory mediators IL-6 Freund A, Orjalo AV, Desprez PY, Campisi J (2010) Inﬂammatory and IL-8. Aging 1: 402–411 networks during cellular senescence: causes and consequences. Campeau E, Ruhl VE, Rodier F, Smith CL, Rahmberg BL, Fuss JO, Trends Mol Med 16: 238–246 Campisi J, Yaswen P, Cooper PK, Kaufman PD (2009) A versatile Gil J, Peters G (2006) Regulation of the INK4b-ARF-INK4a tumour viral system for expression and depletion of proteins in mamma- suppressor locus: all for one or one for all. Nat Rev Mol Cell Biol lian cells. PLoS One 4: e6529 7: 667–677 Campisi J, d’Adda di Fagagna F (2007) Cellular senescence: when Iwasa H, Han J, Ishikawa F (2003) Mitogen-activated protein kinase bad things happen to good cells. Nat Rev Mol Cell Biol 8: 729–740 p38 deﬁnes the common senescence-signalling pathway. Genes Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman Cells 8: 131–144 O, Guertin DA, Chang JH, Lindquist RA, Moffat J, Golland P, Janssens S, Tschopp J (2006) Signals from within: the DNA-damage- Sabatini DM (2006) CellProﬁler: image analysis software for induced NF-kappaB response. Cell Death Differ 13: 773–784 identifying and quantifying cell phenotypes. Genome Biol 7: R100 Jeyapalan JC, Ferreira M, Sedivy JM, Herbig U (2007) Accumulation Ciccia A, Elledge SJ (2010) The DNA damage response: making it of senescent cells in mitotic tissue of aging primates. Mech Ageing safe to play with knives. Mol Cell 40: 179–204 Dev 128: 36–44 Collins CJ, Sedivy JM (2003) Involvement of the INK4a/Arf gene Jun JI, Lau LF (2010) The matricellular protein CCN1 induces locus in senescence. Aging Cell 2: 145–150 ﬁbroblast senescence and restricts ﬁbrosis in cutaneous wound Coppe JP, Desprez PY, Krtolica A, Campisi J (2010a) The sene- healing. Nat Cell Biol 12: 676–685 scence-associated secretory phenotype: the dark side of tumor Karin M (2006) Nuclear factor-[kappa]B in cancer development and suppression. Annu Rev Pathol 5: 99–118 progression. Nature 441: 431–436 Coppe JP, Kauser K, Campisi J, Beausejour CM (2006) Secretion of Kortlever RM, Higgins PJ, Bernards R (2006) Plasminogen activator vascular endothelial growth factor by primary human ﬁbroblasts inhibitor-1 is a critical downstream target of p53 in the induction at senescence. J Biol Chem 281: 29568–29574 of replicative senescence. Nat Cell Biol 8: 877–884 Coppe JP, Patil CK, Rodier F, Krtolica A, Beausejour CM, Parrinello S, Krizhanovsky V, Yon M, Dickins RA, Hearn S, Simon J, Miething C, Hodgson JG, Chin K, Desprez PY, Campisi J (2010b) A human-like Yee H, Zender L, Lowe SW (2008) Senescence of activated stellate senescence-associated secretory phenotype is conserved in mouse cells limits liver ﬁbrosis. Cell 134: 657–667 cells dependent on physiological oxygen. PLoS One 5: e9188 Krtolica A, Parrinello S, Lockett S, Desprez P-Y, Campisi J (2001) Coppe JP, Patil CK, Rodier F, Sun Y, Munoz DP, Goldstein J, Nelson Senescent ﬁbroblasts promote epithelial cell growth and tumori- PS, Desprez PY, Campisi J (2008) Senescence-associated secretory genesis: a link between cancer and aging. Proc Natl Acad Sci USA phenotypes reveal cell-nonautonomous functions of oncogenic 98: 12072–12077 RAS and the p53 tumor suppressor. PLoS Biol 6: 2853–2868 Kuilman T, Michaloglou C, Vredeveld LCW, Douma S, van Doorn R, Cortez DM, Feldman MD, Mummidi S, Valente AJ, Steffensen B, Desmet CJ, Aarden LA, Mooi WJ, Peeper DS (2008) Oncogene- Vincenti M, Barnes JL, Chandrasekar B (2007) IL-17 stimulates induced senescence relayed by an interleukin-dependent inﬂam- MMP-1 expression in primary human cardiac ﬁbroblasts via p38 matory network. Cell 133: 1019–1031 MAPK- and ERK1/2-dependent C/EBP-beta, NF-kappaB, and AP-1 Kwong J, Hong L, Liao R, Deng Q, Han J, Sun P (2009) p38alpha activation. Am J Physiol Heart Circ Physiol 293: H3356–H3365 and p38gamma mediate oncogenic Ras-induced senescence Courtois-Cox S, Jones SL, Cichowski K (2008) Many roads lead to through differential mechanisms. J Biol Chem 284: 11237–11246 oncogene-induced senescence. Oncogene 27: 2801–2809 Lali FV, Hunt A, Turner S, Foxwell B (2000) The pyridinyl imidazole Cuenda A, Rouse J, Doza YN, Meier R, Cohen P, Gallagher TF, inhibitor SB203580 blocks phosphoinositide-dependent protein Young PR, Lee JC (1995) SB 203580 is a speciﬁc inhibitor of a kinase activity, protein kinase B phosphorylation, and retino- MAP kinase homologue which is stimulated by cellular stresses blastoma hyperphosphorylation in interleukin-2-stimulated and interleukin-1. FEBS Lett 364: 229–233 T cells independently of p38 mitogen-activated protein kinase. Cuenda A, Rousseau S (2007) p38 MAP-kinases pathway regulation, J Biol Chem 275: 7395–7402 function and role in human diseases. Biochim Biophys Acta Liu D, Hornsby PJ (2007) Senescent human ﬁbroblasts increase the 1773: 1358–1375 early growth of xenograft tumors via matrix metalloproteinase Davalos AR, Coppe JP, Campisi J, Desprez PY (2010) Senescent cells secretion. Cancer Res 67: 3117–3126 as a source of inﬂammatory factors for tumor progression. Cancer Naldini L, Blomer U, Gage FH, Trono D, Verma IM (1996) Efﬁcient Metastasis Rev 29: 273–283 transfer, integration, and sustained long-term expression of the Davis T, Baird D, Haughton M, Jones CJ, Kipling D (2005) transgene in adult rat brains injected with a lentiviral vector. Prevention of accelerated cell aging in Werner syndrome using Proc Natl Acad Sci USA 93: 11382–11388 a p38 mitogen-activated protein kinase inhibitor. J Gerontol A Biol Ohtani N, Yamakoshi K, Takahashi A, Hara E (2004) The p16INK4a- Sci Med Sci 60: 1386–1393 RB pathway: molecular link between cellular senescence and Davis T, Kipling D (2009) Assessing the role of stress signalling tumor suppression. J Med Invest 51: 146–153 via p38 MAP kinase in the premature senescence of Ataxia Ono K, Han J (2000) The p38 signal transduction pathway: activa- Telangiectasia and Werner syndrome ﬁbroblasts. Biogerontology tion and function. Cell Signal 12: 1–13 10: 253–266 Orjalo AV, Bhaumik D, Gengler BK, Scott GK, Campisi J (2009) Cell Defrance M, Touzet H (2006) Predicting transcription factor binding surface-bound IL-1{alpha} is an upstream regulator of the senes- sites using local over-representation and comparative genomics. cence-associated IL-6/IL-8 cytokine network. Proc Natl Acad Sci BMC Bioinformatics 7: 396 USA 106: 17031–17036 Deng Q, Liao R, Wu B-L, Sun P (2004) High intensity ras signalling Ossovskaya VS, Mazo IA, Chernov MV, Chernova OB, Strezoska Z, induces premature senescence by activating p38 pathway in Kondratov R, Stark GR, Chumakov PM, Gudkov AV (1996) Use of primary human ﬁbroblasts. J Biol Chem 279: 1050–1059 genetic suppressor elements to dissect distinct biological effects Di Micco R, Fumagalli M, Cicalese A, Piccinin S, Gasparini P, of separate p53 domains. Proc Natl Acad Sci USA 93: 10309–10314 Luise C, Schurra C, Garre M, Nuciforo PG, Bensimon A, Paradis V, Youssef N, Dargere D, Ba N, Bonvoust F, Bedossa P (2001) Maestro R, Pelicci PG, d’Adda di Fagagna F (2006) Oncogene- Replicative senescence in normal liver, chronic hepatitis C, and induced senescence is a DNA damage response triggered by DNA hepatocellular carcinomas. Hum Pathol 32: 327–332 hyper-replication. Nature 444: 638–642 Parrinello S, Coppe J-P, Krtolica A, Campisi J (2005) Stromal- Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, epithelial interactions in aging and cancer: senescent ﬁbroblasts Medrano EE, Linskens M, Rubelj I, Pereira-Smith O, Peacocke alter epithelial cell differentiation. J Cell Sci 118: 485–496 M, Campisi J (1995) A biomarker that identiﬁes senescent human Passos JF, Nelson G, Wang C, Richter T, Simillion C, Proctor CJ, cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA Miwa S, Olijslagers S, Hallinan J, Wipat A, Saretzki G, Rudolph 92: 9363–9367 KL, Kirkwood TB, von Zglinicki T (2010) Feedback between p21 Eisen MB, Spellman PT, Brown PO, Botstein D (1998) Cluster and reactive oxygen production is necessary for cell senescence. analysis and display of genome-wide expression patterns. Mol Syst Biol 6: 347 Proc Natl Acad Sci USA 95: 14863–14868 Perkins ND (2007) Integrating cell-signalling pathways with Enslen H, Raingeaud J, Davis RJ (1998) Selective activation of p38 NF-kappaB and IKK function. Nat Rev Mol Cell Biol 8: 49–62 mitogen-activated protein (MAP) kinase isoforms by the MAP Prieur A, Peeper DS (2008) Cellular senescence in vivo: a barrier to kinase kinases MKK3 and MKK6. J Biol Chem 273: 1741–1748 tumorigenesis. Curr Opin Cell Biol 20: 150–155 &2011 European Molecular Biology Organization The EMBO Journal VOL 30 NO 8 2011 1547 | | p38 regulates the senescence secretory phenotype A Freund et al Radtke S, Wuller S, Yang XP, Lippok BE, Mutze B, Mais C, Wang SW, Pawlowski J, Wathen ST, Kinney SD, Lichenstein HS, de Leur HS, Bode JG, Gaestel M, Heinrich PC, Behrmann I, Manthey CL (1999) Cytokine mRNA decay is accelerated by an Schaper F, Hermanns HM (2010) Cross-regulation of cytokine inhibitor of p38-mitogen-activated protein kinase. Inﬂamm Res signalling: pro-inﬂammatory cytokines restrict IL-6 signalling 48: 533–538 through receptor internalisation and degradation. J Cell Sci 123: Wang W, Chen JX, Liao R, Deng Q, Zhou JJ, Huang S, Sun P (2002) 947–959 Sequential activation of the MEK-extracellular signal-regulated Rodier F, Campisi J, Bhaumik D (2007) Two faces of p53: aging and kinase and MKK3/6-p38 mitogen-activated protein kinase path- tumor suppression. Nucleic Acids Res 35: 7475–7484 ways mediates oncogenic ras-induced premature senescence. Rodier F, Coppe JP, Patil CK, Hoeijmakers WA, Munoz DP, Mol Cell Biol 22: 3389–3403 Raza SR, Freund A, Campeau E, Davalos AR, Campisi J Wilson KP, McCaffrey PG, Hsiao K, Pazhanisamy S, Galullo V, (2009) Persistent DNA damage signalling triggers senescence- Bemis GW, Fitzgibbon MJ, Caron PR, Murcko MA, Su MS associated inﬂammatory cytokine secretion. Nat Cell Biol 11: (1997) The structural basis for the speciﬁcity of pyridinyl- 973–979 imidazole inhibitors of p38 MAP kinase. Chem Biol 4: 423–431 Rodier F, Munoz DP, Teachenor R, Chu V, Le O, Bhaumik D, Coppe Xue W, Zender L, Miething C, Dickins RA, Hernando E, J-P, Campeau E, Beausejour C, Kim S-H, Davalos AR, Campisi J Krizhanovsky V, Cordon-Cardo C, Lowe SW (2007) Senescence (2011) DNA-SCARS: distinct nuclear structures that sustain and tumour clearance is triggered by p53 restoration in murine damage-induced senescence growth arrest and inﬂammatory liver carcinomas. Nature 445: 656–660 cytokine secretion. J Cell Sci 124: 68–81 Young PR, McLaughlin MM, Kumar S, Kassis S, Doyle ML, McNulty Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW (1997) D, Gallagher TF, Fisher S, McDonnell PC, Carr SA, Huddleston Oncogenic ras provokes premature cell senescence asso- MJ, Seibel G, Porter TG, Livi GP, Adams JL, Lee JC (1997) ciated with accumulation of p53 and p16INK4a. Cell 88: Pyridinyl imidazole inhibitors of p38 mitogen-activated protein 593–602 kinase bind in the ATP site. J Biol Chem 272: 12116–12121 Sun P, Yoshizuka N, New L, Moser BA, Li Y, Liao R, Xie C, Chen J, Zarubin T, Han J (2005) Activation and signalling of the p38 MAP Deng Q, Yamout M, Dong M-Q, Frangou CG, Yates JR, Wright PE, kinase pathway. Cell Res 15: 11–18 Han J (2007) PRAK is essential for ras-induced senescence and Zhang J, Shen B, Lin A (2007) Novel strategies for inhibition of the tumor suppression. Cell 128: 295–308 p38 MAPK pathway. Trends Pharmacol Sci 28: 286–295 Wajapeyee N, Serra RW, Zhu X, Mahalingam M, Green MR (2008) Zhou S, Greenberger JS, Epperly MW, Goff JP, Adler C, Leboff MS, Oncogenic BRAF induces senescence and apoptosis through Glowacki J (2008) Age-related intrinsic changes in human bone- pathways mediated by the secreted protein IGFBP7. Cell 132: marrow-derived mesenchymal stem cells and their differentiation 363–374 to osteoblasts. Aging Cell 7: 335–343 1548 The EMBO Journal VOL 30 NO 8 2011 &2011 European Molecular Biology Organization | |

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The EMBO Journal – Springer Journals

Published: Apr 20, 2011

Keywords: aging; cancer; inflammation; NF‐κB; tumour suppression

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p38MAPK is a novel DNA damage response‐independent regulator of the senescence‐associated secretory phenotype

p38MAPK is a novel DNA damage response‐independent regulator of the senescence‐associated secretory phenotype

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p38MAPK is a novel DNA damage response‐independent regulator of the senescence‐associated secretory phenotype

p38MAPK is a novel DNA damage response‐independent regulator of the senescence‐associated secretory phenotype

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