Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You and Your Team.

Learn More →

p38β MAPK upregulates atrogin1/MAFbx by specific phosphorylation of C/EBPβ

p38β MAPK upregulates atrogin1/MAFbx by specific phosphorylation of C/EBPβ Background: The p38 mitogen-activated protein kinases (MAPK) family plays pivotal roles in skeletal muscle metabolism. Recent evidence revealed that p38α and p38β exert paradoxical effects on muscle protein homeostasis. However, it is unknown why p38β, but not p38α, is capable of mediating muscle catabolism via selective activation of the C/EBPβ that upregulates atrogin1/MAFbx. Methods: Tryptic phosphopeptide mapping was carried out to identify p38α- and p38β-mediated phosphorylation sites in C/EBPβ. Chromosome immunoprecipitation (ChIP) assay was used to evaluate p38α and p38β effect on C/EBPβ binding to the atrogin1/MAFbx promoter. Overexpression or siRNA-mediated gene knockdown of p38α and p38β, and site-directed mutagenesis or knockout of C/EBPβ, were used to analyze the roles of these kinases in muscle catabolism in C2C12 myotubes and mice. Results: Cellular expression of constitutively active p38α or p38β resulted in phosphorylation of C/EBPβ at multiple serine and threonine residues; however, only p38β phosphorylated Thr-188, which had been known to be critical to the DNA-binding activity of C/EBPβ. Only p38β, but not p38α, activated C/EBPβ-binding to the atrogin1/MAFbx promoter. A C/EBPβ mutant in which Thr-188 was replaced by alanine acted as a dominant-negative inhibitor of atrogin1/MAFbx upregulation induced by either p38β or Lewis lung carcinoma (LLC) cell-conditioned medium (LCM). In addition, knockdown of p38β specifically inhibited C/EBPβ activation and atrogin1/MAFbx upregulation induced by LCM. Finally, expression of active p38β in mouse tibialis anterior specifically induced C/EBPβ phosphorylation at Thr-188, atrogin1/MAFbx upregulation and muscle mass loss, which were blocked in C/EBPβ-null mice. Conclusions: The α and β isoforms of p38 MAPK are capable of recognizing distinct phosphorylation sites in a substrate. The unique capacity of p38β in mediating muscle catabolism is due to its capability in phosphorylating Thr-188 of C/EBPβ. Keywords: Cachexia, E3 protein, Gene regulation, DNA-binding, Thr-188 Background exercise) stimuli [6]. On one hand, p38 stimulates The p38 mitogen-activated protein kinases (MAPK) muscle satellite cell proliferation [7] and differentiation family plays a pivotal role in skeletal muscle by mediat- [8], which increases muscle mass; on the other hand, ing diverse cellular activities, and interestingly, some of p38 stimulates muscle protein degradation leading to which result in paradoxical effects. For example, p38 muscle atrophy [3-5]. Intriguingly, p38 has the capacity mediates both insulin stimulation of glucose uptake [1] to activate different protein substrates depending on the and TNF-α stimulation of insulin resistance [2] in cellular environment [7]. It is of great interest to under- muscle. In the context of skeletal muscle protein homeo- stand how the p38 MAPK family is able to mediate the stasis, p38 responds to both catabolic (lipopolysacchar- discrete and sometimes opposing effects in response to ide, cytokines, and ROS) [3-5] and anabolic (insulin and diverse physiological and pathological stimuli. The fam- ily of MAPK is composed of at least four members (α, β, * Correspondence: yi-ping.li@uth.tmc.edu γ and δ), which enable the transduction of a variety Department of Integrative Biology and Pharmacology, University of Texas of extracellular signals into distinct nuclear responses Health Science Center at Houston, 6431 Fannin Street, Houston, TX 77030, USA © 2012 Zhang and Li; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Zhang and Li Skeletal Muscle 2012, 2:20 Page 2 of 9 http://www.skeletalmusclejournal.com/content/2/1/20 [9-11]. The α, β, and γ isoforms are found in muscle common phosphorylation sites with p38α, it specifically cells. Recently, it became clear that p38α MAPK plays phosphorylates the Thr-188 residue of C/EBPβ, which an essential role in myogenic differentiation [12,13]. activates C/EBPβ binding to the atrogin1/MAFbx pro- On the other hand, p38γ MAPK appears to regulate moter and upregulates this gene in response to a tumor. the expansion of myogenic precursor cells [14], endur- ance exercise-induced mitochondrial biogenesis and Methods angiogenesis [15], as well as glucose uptake [16]. But, Tryptic phosphopeptide mapping was carried out to little was known about the function of p38β until our identify p38α- and p38β-mediated phosphorylation sites most recent discovery of its role in regulating the in C/EBPβ. ChIP assay was used to evaluate p38α and atrogin1/MAFbx gene [17]. p38β effect on C/EBPβ-binding to the atrogin1/MAFbx Cachexia, a wasting disease characterized by loss of promoter. Overexpression or siRNA-mediated gene muscle mass with or without loss of fat mass, is frequently knockdown of p38α and p38β, and site-directed muta- associated with such diseases as cancer, sepsis, AIDS, con- genesis or knockout of C/EBPβ, were used to analyze gestive heart failure, diabetes, chronic renal failure and the roles of these kinases in muscle catabolism in C2C12 chronic obstructive pulmonary disease (COPD). Cachexia myotubes and mice. is distinct from starvation-, disuse-, aging-, primary de- pression-, malabsorption- and hyperthyroidism-induced Tryptic phosphopeptide mapping muscle mass loss and is associated with increased morbid- HEK293T cells (American Type Culture Collection, ity and mortality [18,19]. The prominent clinical feature of Manassas, VA, USA) cultured in 150 mm culture plates cachexia is weight loss with anorexia, inflammation, in- that were ~50% confluent were co-transfected with a sulin resistance, and increased muscle protein break- plasmid encoding LAP with a FLAG tag (Addgene) and down. Increased muscle protein breakdown in cachexia a plasmid encoding constitutively active p38α or p38β is at least partially due to accelerated muscle proteoly- [26] (10 μg each) using deacylated polyethylenimine sis by the ubiquitin-proteasome pathway, a common (PEI) 22000 [27], a gift from Dr. Guangwei Du (Univer- pathway of muscle mass loss due to pathological as sity of Texas Health Science Center at Houston, Hous- well as physiological causes [20]. However, the signal- ton, TX, USA). The cell culture medium was replaced ing mechanism of the activation of the ubiquitin- with fresh medium at 24 h. Cells were lysed in RIPA buf- proteasome pathway in cachexia appears to be different fer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 2 mM from that of physiological muscle atrophy. It is well EDTA, 1% NP-40, 0.1% SDS, 2 mM phenylmethylsul- established that a depression in AKT activity activates phonylfluoride (PMSF), 0.5% sodium deoxycholate, 1 FoxO1/3 transcription factors, which upregulates two mM NaF, 1/100 protease inhibitor cocktail, and 1/100 key ubiquitin ligases (E3 proteins), atrogin1/MAFbx and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MuRF1, in animal models of physiological muscle atro- MO, USA) after an additional incubation of 24 h. LAP phy caused by fasting, denervation and disuse [21-23]. in cell lysates was precipitated using FLAG-M2 magnetic In animal models of cachexia, however, AKT is often beads (Sigma-Aldrich) and subjected to 10% SDS-PAGE. activated, which leads to the inactivation of FoxO1/3 The gel was then stained with Coommassie Blue R-250. [4,5,24]. In fact, it has been shown in animal models of The LAP band was cut out and subjected to tryptic cachexia that upregulation of MuRF1 is mediated by phosphopeptide mapping conducted by Taplin Mass NF-κB [25], and upregulation of atrogin1/MAFbx is Spectrometry Facility at Harvard Medical School using mediated by p38 MAPK [4,5]. an LTQ-Orbitrap mass spectrometer (Thermo Electron, We showed most recently that among the known p38 West Palm Beach, FL, USA). MAPK isoforms only p38β MAPK is capable of upregu- lating atrogin1/MAFbx via the activation of transcription Myogenic cell culture and transfection factor C/EBPβ in response to tumor cell-conditioned Murine C2C12 myoblasts (American Type Culture Col- medium. In addition, we demonstrated that p38β MAPK lection, Manassas, VA, USA) were cultured in growth upregulation of atrogin1/MAFbx is independent of the medium (DMEM supplemented with 10% fetal bovine AKT-FoxO1/3 signaling pathway [17]. Thus, p38β serum) at 37°C under 5% CO . At approximately 85 to emerged as a key mediator and a specific therapeutic 90% confluence, myoblast differentiation was induced by target of cachexia. Notwithstanding, why p38β is incubation for 96 h in differentiation medium (DMEM uniquely capable of activating C/EBPβ among the p38 supplemented with 4% heat-inactivated horse serum) to isoforms is unknown. The current study is designed to form myotubes. Plasmids encoding constitutively active address the mechanism through which p38β specifically p38 isoforms [26] or a C/EBPβ mutant (p3xFlag-CMV- activates C/EBPβ in the context of tumor-induced cach- 10-LAP-T188A) were transfected into C2C12 myoblasts exia. We demonstrate that while p38β shares some of 50% confluence at 1 μg/well in six-well plates using Zhang and Li Skeletal Muscle 2012, 2:20 Page 3 of 9 http://www.skeletalmusclejournal.com/content/2/1/20 deacylated polyethylenimine (PEI) 22000. At 24 h we induced the cells to differentiate by switching them to differentiation medium. When indicated, myotubes were treated with Lewis lung carcinoma cell (National Cancer Institute, Bethesda, MD, USA)-conditioned medium (LCM, 25% final volume) [17] or directly harvested. Cell lysate was prepared using the RIPA buffer for further analyses. Chromosome immunoprecipitation assay Chromosome immunoprecipitation (ChIP) assay was Figure 1 Isolation of C/EBPβ phosphorylated by p38α or p38β performed as previously described [17]. MAPK for tryptic phosphopeptide mapping. HEK293T cells were co-transfected with plasmids encoding C/EBPβ (LAP fused with the FLAG tag) and constitutively active p38α or p38β (fused with the HA Generation of expression vector for a C/EBPβ mutant tag). After 48 h incubation the cells were lysed. Expression of the (LAP-T188A) transfected plasmids was verified by western blot analysis of HA and A plasmid encoding C/EBPβ in which Thr-188 was FLAG. Activity of over-expressed p38α and p38β was evaluated by replaced by alanine in the pcDNA3 vector (a gift from western blot analysis of ATF2 activation (A). Overexpressed C/EBPβ was pulled down with FLAG-M2 magnetic beads and separated by Dr. Qi-Qun Tang of Fudan University, Shanghai, China) SDS-PAGE. The gel was stained with Coommassie Blue R-250 (B). was digested at BamHI and EcoRI restriction sites to re- The C/EBPβ band was then cut out and analyzed by tryptic lease the cDNA insert. That insert was then subcloned phosphopeptide mapping utilizing mass spectrometry for into the p3xFlag-CMV-10 vector (Sigma-Aldrich) at the phosphorylated amino acid residues. MAPK, mitogen-activated same restriction sites to generate plasmid p3xFlag- protein kinase. CMV-10-LAP-T188A. Western blot analysis [28]. While the mice were under anesthesia, plasmids Cell and muscle lysate were prepared and western blot encoding constitutively active p38α or p38β were analysis was carried out as described previously [17]. injected into the tibialis anterior (TA) of the right leg for Antibodies for total and/or phosphorylated ATF2, each mouse (100 μgin50 μl), and the empty vector FoxO1 (Thr-24)/FoxO3a (Thr-32) and C/EBPβ phos- pcDNA3.1 was injected into the left leg as control. Im- phorylated at Thr-188 were from Cell Signaling Tech- mediately after plasmid injection, TA was electroporated nology (Danvers, MA, USA). Antibody for atrogin1/ by applying square-wave electrical pulses (100 V/cm) MAFbx was from ECM Biosciences (Versailles, KY, eight times with an electrical pulse generator (Model USA). Antibodies to C/EBPβ (H-7) were from Santa 830, BTX) at a rate of one pulse per second, with each Cruz Biotechnology (Santa Cruz, CA, USA). Antibody to pulse being 20 ms in duration, through a pair of stainless the HA tag was from Covance (Princeton, NJ, USA). steel needles that were 5 mm apart. The above transfec- Data were normalized to GAPDH. tion procedure was repeated in 7 days. In another 7 days, the mice were sacrificed and TAs were collected Gene knockdown by siRNA for analysis. p38α-specific siRNA (5′-CUCCUUUACUAUCUUUCU CAA-3′) and p38β-specific siRNA (5′-GUCCUGAGGUU Statistical analysis CUAGCAAAdTdT-3′) were synthesized by Sigma-Aldrich Data were analyzed with one-way ANOVA or student t and transfected into C2C12 myoblasts by electroporation test using the SigmaStat software (Systat Software, Point (5 μg/1 × 10 cells) with the Nucleofector system (Lonza, Richmond, CA, USA) as indicated. When applicable, Walkersville, MD, USA), according to the manufacturer’s protocol. Control siRNA was obtained from Ambion Table 1 Expression of constitutively active p38α or p38β (Austin, TX, USA). Differentiation was induced 24 h after resulted in the phosphorylation of diverse amino acid transfection. residues in C/EBPβ Kinase Phosphorylated amino acid residues in C/EBPβ Animal use p38α Ser110, Tyr108 Experimental protocols were approved in advance by the p38β Ser182, Ser183, Ser190, Thr188 institutional Animal Welfare Committee at the Univer- p38α/p38β Ser64, Ser184, Ser222, Ser276 sity of Texas Health Science Center at Houston. C/ −/− The C/EBPβ bands cut out from SDS-PAGE described in Figure 1B were EBPβ mice in C57BL/6 background were bred from analyzed by tryptic phosphopeptide mapping utilizing mass spectrometry. −/+ C/EBPβ mice generated by Dr. Peter Johnson of NCI Identified phosphorylated amino acid residues in C/EBPβ are listed. Zhang and Li Skeletal Muscle 2012, 2:20 Page 4 of 9 http://www.skeletalmusclejournal.com/content/2/1/20 Results p38β MAPK specifically phosphorylates the Thr-188 residue of C/EBPβ C/EBPβ is a transcription factor that is normally repressed due to the intrinsic repression of its DNA- binding and transactivation functions [29,30]. The DNA- binding function of C/EBPβ is activated by sequential phosphorylation of specific amino acid residues by mul- tiple kinases [31-33]. C/EBPβ was previously shown to be a p38 substrate in vitro [34]. Recently, we showed that p38 interacts with and phosphorylates C/EBPβ in C2C12 myotubes. In addition, while the p38α/β inhibitor SB202190 blocks tumor-induced atrogin1/MAFbx upre- gulation, only p38β is capable of upregulating atrogin1/ MAFbx via the activation of C/EBPβ [17]. To investigate why p38β, but not p38α, has the capacity for activating C/EBPβ we set out to investigate the phosphorylation sites in C/EBPβ that are targeted by p38α and p38β. Plasmids encoding C/EBPβ (LAP fused with the FLAG tag) and constitutively active p38α or p38β (fused to HA) were co-transfected into HEK293T cells. At 48 h expression of the transfected plasmids was verified by western blot analysis of HA (p38 MAKPs) and FLAG Figure 2 Expression of constitutively active p38β in C2C12 (LAP) in the cell lysate. Activation of p38 substrate myotubes resulted in specific phosphorylation of C/EBPβ at Thr-188 and upregulation of atrogin1/MAFbx. C2C12 myoblasts ATF2 (via phosphorylation) by expressed p38 MAPKs were transfected with a plasmid encoding constitutively active p38α, was also evaluated by western blot analysis (Figure 1A). p38β or the empty vector (control). The myoblasts were allowed to Overexpressed C/EBPβ was pulled down with FLAG-M2 differentiate for 96 h to form myotubes that were then harvested magnetic beads and separated with SDS-PAGE (Figure 1B). and analyzed for ATF2 activation, Thr-188 phosphorylation in C/EBPβ The C/EBPβ band was cut out from the gel and analyzed and level of atrogin1/MAFbx using western blotting. by tryptic phosphopeptide mapping utilizing mass spec- trometry (for original reports see Additional file 1: Table control samples from independent experiments were S1 and Additional file 2: Table S2). Six phosphorylated normalized to a value of 1 without showing variations amino acid residues were identified in C/EBPβ that was (actual variations were within a normal range). A P value co-expressed with active p38α and eight phosphorylated <0.05 was considered to be statistically significant. Data amino acid residues were identified in C/EBPβ that was are presented as the mean ± S.E. co-expressed with active p38β.The twop38 MAPK Figure 3 p38β is critical to LCM-induced phosphorylation of C/EBPβ at Thr-188 and upregulation of atrogin1/MAFbx. C2C12 myoblasts were transfected with siRNA as indicated. After differentiation, myotubes were treated with LCM or control medium. In 1 h, levels of p38α and p38β,and phosphorylation of C/EBPβ at Thr-188 were evaluated by western blotting (A). In 8 h, levels of atrogin1/MAFbx were evaluated by western blotting (B). Optical density of the bands that represent C/EBP phosphorylated at Thr-188 or atrogin1/MAFbx was analyzed by ANOVA. *denotes a difference from control without LCM treatment and †denotes a difference from control with LCM treatment (P <0.05). LCM, Lewis lung carcinoma cell-conditioned medium. Zhang and Li Skeletal Muscle 2012, 2:20 Page 5 of 9 http://www.skeletalmusclejournal.com/content/2/1/20 isoforms shared four common phosphorylation sites (Ser-64, Ser-184, Ser-222, and Ser-276). On the other hand, p38α uniquely phosphorylated Ser-110 and Tyr-108, while p38β uniquely phosphorylated Ser-182, Ser-183, Ser-190 and Thr-188 (Table 1). Among the unique amino acid residues phosphorylated by p38β, Thr-188 is known to be crucial for the activation of C/EBPβ binding to its targeted DNA sequence [31-33]. To verify whether p38β specifically mediates the phos- phorylation of Thr-188 of C/EBPβ in muscle cells, we transfected C2C12 myoblasts with plasmids encoding active p38α or active p38β and used empty vector as the control. Although constitutively active p38α appeared to accelerate differentiation during the early stage of differ- entiation, at 96 h of differentiation, there was no visible Figure 4 p38β specifically activates C/EBPβ-binding to the difference in the myotubes formed compared with con- atrogin1/MAFbx promoter. (A) C2C12 myoblasts were transfected trol myotubes and active p38β-expressing myotubes. with a plasmid encoding constitutively active p38α, p38β or the empty vector (control). After differentiation, ChIP assay was carried After differentiation, lysate of myotubes was evaluated out to evaluate C/EBPβ binding in myotubes to a 190-base pair by western blot analysis of expression of the HA tag fragment of the atrogin1/MAFbx promoter that contains the that fused to active p38α or p38β, ATF2 activation and previously identified C/EBPβ-responsive cis-enhancer element [17] C/EBPβ phosphorylation at Thr-188. As shown in Figure 2, using control and the target PCR primers. Pre-immune IgG used as although both of the active p38 isoforms activated ATF2, the control to the antibody against C/EBPβ did not pull down the target fragment (data not shown). (B) C2C12 myoblasts were only the expression of active p38β resulted in C/EBPβ transfected with siRNA as indicated. After differentiation, myotubes phosphorylation at Thr-188 and upregulation of atrogin1/ were treated with LCM or control medium for 1 h and ChIP assay MAFbx. Therefore, the specific phosphorylation of was carried out to evaluate C/EBPβ binding to the atrogin1/MAFbx Thr-188 by p38β may be the key to its specific activation promoter. ChIP, chromosome immunoprecipitation; LCM, Lewis lung of C/EBPβ. carcinoma cell-conditioned medium. Figure 5 C/EBPβ phosphorylation at Thr-188 is crucial for atrogin1/MAFbx upregulation by p38β or LCM. (A) C2C12 myoblasts were transfected with a plasmid encoding constitutively active p38α, p38β or empty vector. A plasmid encoding FLAG-tagged C/EBPβ mutant in which Thr-188 was replaced with alanine (LAP-T188A) or the control vector was co-transfected as indicated. After differentiation, myotubes were lysed and analyzed by western blotting for the expression of LAP-T188A (with antibodies against FLAG and C/EBPβ) and atrogin1/MAFbx. (B) C2C12 myoblasts were transfected with LAP-T188A or the empty vector as control. After differentiation, myotubes were treated with LCM or control medium for 8 h. Lysate of myotubes was analyzed by western blotting for the expression of LAP-T188A (with antibodies against FLAG and C/EBPβ) and atrogin1/MAFbx. Optical density of the bands that represent atrogin1/MAFbx was analyzed by ANOVA. *denotes a difference from control without LCM treatment and †denotes a difference from control with LCM treatment (P <0.05). LCM, Lewis lung carcinoma cell-conditioned medium. Zhang and Li Skeletal Muscle 2012, 2:20 Page 6 of 9 http://www.skeletalmusclejournal.com/content/2/1/20 Previously, we observed that MAPK kinase 6 (MKK6) activates C/EBPβ binding to the C/EBPβ-responsive en- hancer in the atrogin1/MAFbx promoter via the activa- tion of p38 [17]. To investigate whether p38β specifically activates C/EBPβ binding to this C/EBPβ-responsive en- hancer, we conducted the ChIP assay. We observed in C2C12 myotubes that the expression of active p38β, but not active p38α, activated C/EBPβ binding to the atrogin1/MAFbx promoter region containing the C/EBPβ- responsive enhancer (Figure 4A). Conversely, siRNA- mediated knockdown of the p38β gene, but not the p38α gene, blocked LCM-induced activation of C/EBPβ binding to the atrogin1/MAFbx promoter (Figure 4B). Thus, we conclude that p38β specifically activates C/EBPβ binding to its targeted DNA motif. To evaluate whether C/EBPβ phosphorylation at Thr- 188 is critical to p38β-mediated upregulation of atrogin1/ MAFbx, a plasmid encoding a C/EBPβ mutant in which Thr-188 was replaced by alanine (C/EBPβ-T188A) was transfected into C2C12 myoblasts along with a plasmid encoding active p38α or active p38β. After differentiation, levels of atrogin1/MAFbx in myotubes that expressed active p38β,but notactivep38α, were elevated. The elevation of atrogin1/MAFbx was blocked in myotubes co-expressing the C/EBPβ-T188A mutant (Figure 5A). To evaluate whether tumor-induced atrogin1/MAFbx upregulation requires the phosphorylation of C/EBPβ at Thr-188, the plasmid encoding C/EBPβ-T188A was Figure 6 Expression of constitutively active p38β in mouse muscle induces C/EBPβ phosphorylation at Thr-188. Plasmids transfected into C2C12 myoblasts. After differentiation, encoding constitutively active p38α or p38β were transfected into myotubes were treated with LLC cell-conditioned −/− TA of the right leg of wild type or C/EBPβ mice, and the empty medium (LCM). We observed that LCM upregulation of vector into the left leg. In 14 days TA samples were collected, atrogin1/MAFbx was inhibited in myotubes that overex- weighed and lysed. Expression of the p38 isoforms was verified by press the C/EBPβ-T188A mutant (Figure 5B). Therefore, western blot analysis of HA tag expression and ATF2 activation. A representative blot is shown (A). The TA lysate was subjected to p38β-mediated C/EBPβ phosphorylation at Thr-188 is western blot analysis of C/EBPβ phosphorylation at Thr-188 and crucial for atrogin1/MAFbx upregulation by LLC cells. FoxO1/3 activation. Representative blots and densitometry data are shown. Optical density of the bands that represent various proteins p38β MAPK specifically induces atrogin1/MAFbx was measured. Levels of protein phosphorylation were normalized upregulation and muscle mass loss in mice via C/EBPβ to that of total proteins. *denotes a difference (P <0.05) based on Student t test (B). TA, tibialis anterior. We previously showed that C/EBPβ is essential for the atrogin1/MAFbx upregulation and muscle mass loss in Lewis lung carcinoma (LLC)-tumor bearing mice [17]. p38β MAPK-mediated Thr-188 phosphorylation activates To evaluate whether activation of p38β in vivo specif- C/EBPβ binding to the atrogin1/MAFbx promoter and ically induces C/EBPβ phosphorylation at Thr-188, upregulates atrogin1/MAFbx in C2C12 myotubes atrogin1/MAFbx upregulation and muscle mass loss, To verify whether p38β is critical to Thr-188 phosphoryl- the plasmids encoding constitutively active p38α or ation and atrogin1/MAFbx upregulation induced by ca- p38β were transfected into the tibialis anterior (TA) of −/− chectic tumor cells, siRNA-mediated mRNA knockdown wild type or C/EBPβ mice with the empty vector as was carried out. We observed that knockdown of the p38β control. At 14 days, the mice were sacrificed and the expression, but not the p38α expression, blocked Lewis excised TAs were analyzed. Expression of the HA tag lung carcinoma cell-conditioned medium (LCM)-induced that fused to p38α or p38β and activation of the p38 Thr-188 phosphorylation (Figure 3A) and atrogin1/MAFbx substrate ATF2 were evaluated by western blot analysis upregulation (Figure 3B). Therefore, p38β is indeed a key to confirm the expression of active p38α and p38β mediator of Thr-188 phosphorylation and atrogin1/MAFbx (Figure 6A). Expression of active p38β, but not active upregulation by Lewis lung carcinoma. p38α, resulted in phosphorylation of C/EBPβ at Thr- Zhang and Li Skeletal Muscle 2012, 2:20 Page 7 of 9 http://www.skeletalmusclejournal.com/content/2/1/20 188 (Figure 6B). Because the atrogin1/MAFbx pro- 188 in C/EBPβ by ERK1/2 MAPK [31] or cdk2/cyclinA moter also contains FoxO1/3-responsive cis-elements [32] primes C/EBPβ for subsequent phosphorylation on that are regulated by AKT [21], we evaluated whether Ser-184 or Thr-179 by glycogen synthase kinase 3β p38β affected FoxO1/3 activity. Expression of active (GSK3β), which activates the DNA-binding and transacti- p38β did not alter the phosphorylation state of FoxO1/ vation functions of C/EBPβ [33]. Our data presented here 3 (Figure 6B). Therefore, p38β does not affect FoxO1/ demonstrate that p38β has the unique capability of medi- 3 activity. As shown in Figure 7A, expression of active ating dual phosphorylation at both Thr-188 and Ser-184, p38β resulted in atrogin1/MAFbx upregulation in the resulting in the activation of C/EBPβ binding to the atro- TA of wild type mice. However, active p38β failed to gin1/MAFbx promoter. In contrast, p38α that mediates −/− upregulate atrogin1/MAFbx in C/EBPβ mice. Finally, phosphorylation of Ser-184 but not Thr-188 is unable to expression of active p38β, but not active p38α, induced activate C/EBPβ binding to the atrogin1/MAFbx pro- TA weight loss by 14% in wild type mice. In contrast, moter. Because GSK3β was previously shown inactivated active p38β expression did not alter TA weight in C/ by p38 MAPK-mediated phosphorylation [36], it is un- −/− EBPβ mice (Figure 7B). Therefore, in vivo activation likely that GSK3β mediates C/EBPβ phosphorylation at of p38β induces atrogin1/MAFbx upregulation and Ser-184 in response to p38α and p38β activation. muscle mass loss via the activation of C/EBPβ. In the present study we also present the first evidence that overexpression of active p38β in muscle induces Discussion muscle catabolism, demonstrating a direct effect of p38β The selective activation of substrates by various p38 on muscle catabolism in vivo. Previous studies involving MAPK isoforms was previously attributed to preferential systemic activation of p38 in tumor-bearing mice [17] or activation of the isoforms by specific MAPK kinase as well septic mice [4] did not allow such a conclusion. as compartmentalization of the isoforms. For example, Although p38β is widely distributed in various tissues while p38α is activated by MKK3, MKK6 and MKK4, [37] its function is largely unknown, especially when it is p38β is activated by MKK6 [10,35]. However, these may compared to p38α, which is not only responsible for the not explain the specific activation of C/EBPβ by p38β,be- known roles of p38 in inflammatory responses [38,39] cause both p38α and p38β are present in the nucleus and but also in the regulation of myogenesis [12,13]. The activated by MKK6. The current study demonstrates that present study demonstrates that activation of p38β, not the selective activation of substrates by p38 MAPK iso- p38α, induces atrogin1/MAFbx upregulation and muscle forms is further realized by their recognition of specific mass loss via specific phosphorylation of C/EBPβ, which phosphorylation sites within a substrate. explains at the molecular level why p38 is capable of Particularly, we show that p38β specifically mediates playing the seemingly opposing roles in muscle protein the phosphorylation of C/EBPβ required for the activa- homeostasis (promoting myogenesis versus promoting tion of its binding to the atrogin1/MAFbx promoter. muscle catabolism). Further, these data support p38β Previous studies indicated that phosphorylation of Thr- as a selective therapeutic target of cachexia. Because Figure 7 Expression of constitutively active p38β in mouse muscle induces atrogin1/MAFbx upregulation and muscle mass loss in a C/EBPβ-dependent manner. The TA lysate derived from Figure 6 was further analyzed for level of atrogin1/MAFbx by western blotting. Representative blots and densitometry data are shown (A). The weight of TA transfected with a plasmid encoding an active p38 isoform was compared to that of TA transfected with empty vector (control) from the same mouse (B). *denotes a difference (P <0.05) based on ANOVA. TA, tibialis anterior. Zhang and Li Skeletal Muscle 2012, 2:20 Page 8 of 9 http://www.skeletalmusclejournal.com/content/2/1/20 C/EBPβ is activated by a number of kinases and regu- References 1. Tremblay F, Dubois MJ, Marette A: Regulation of GLUT4 traffic and function by lates a wide variety of genes [40], it may not be suit- insulin and contraction in skeletal muscle. Front Biosci 2003, 8:d1072–d1084. able as a drug target. On the other hand, p38β has few 2. de Alvaro C, Teruel T, Hernandez R, Lorenzo M: Tumor necrosis factor known functions, therefore, specific inhibitors of p38β alpha produces insulin resistance in skeletal muscle by activation of inhibitor kappaB kinase in a p38 MAPK-dependent manner. J Biol Chem MAPK would be highly desirable for the intervention 2004, 279:17070–17078. of cancer cachexia. Unfortunately, only p38α/β-dual or 3. Li YP, Chen Y, John J, Moylan J, Jin B, Mann DL, Reid MB: TNF-alpha acts p38α-specific inhibitors are available at the present time. via p38 MAPK to stimulate expression of the ubiquitin ligase atrogin1/ MAFbx in skeletal muscle. FASEB J 2005, 19:362–370. Because p38β is highly expressed in the heart [37], it 4. Doyle A, Zhang G, Abdel Fattah EA, Eissa NT, Li YP: Toll-like receptor 4 may also regulate the protein homeostasis in heart mediates lipopolysaccharide-induced muscle catabolism via coordinate muscle via influencing C/EBPβ−regulated atrogin1/ activation of ubiquitin-proteasome and autophagy-lysosome pathways. FASEB J 2011, 25:99–110. MAFbx expression. Consistent to this notion, it has been 5. McClung JM, Judge AR, Powers SK, Yan Z: p38 MAPK links oxidative stress shown recently that exercise induces a reduction in C/ to autophagy-related gene expression in cachectic muscle wasting. Am J EBPβ in cardiomyocytes, which mediates cardiomyocyte Physiol Cell Physiol 2010, 298:C542–C549. 6. Long YC, Widegren U, Zierath JR: Exercise-induced mitogen-activated protein hypertrophy [41]. In addition, the transactivation activity kinase signalling in skeletal muscle. Proc Nutr Soc 2004, 63:227–232. of C/EBPβ is suppressed by insulin, an anabolic hor- 7. Jones NC, Tyner KJ, Nibarger L, Stanley HM, Cornelison DD, Fedorov YV, mone [42]. Therefore, diverse signaling pathways that Olwin BB: The p38alpha/beta MAPK functions as a molecular switch to activate the quiescent satellite cell. J Cell Biol 2005, 169:105–116. regulate protein homeostasis in striated muscles may 8. Lluis F, Perdiguero E, Nebreda AR, Munoz-Canoves P: Regulation of skeletal converge upon C/EBPβ. muscle gene expression by p38 MAP kinases. Trends Cell Biol 2006, 16:36–44. 9. Remy G, Risco AM, Inesta-Vaquera FA, Gonzalez-Teran B, Sabio G, Davis RJ, Cuenda A: Differential activation of p38MAPK isoforms by MKK6 and Conclusions MKK3. Cell Signal 2010, 22:660–667. The present study demonstrates that the α and β isoforms 10. Enslen H, Raingeaud J, Davis RJ: Selective activation of p38 mitogen- of p38 MAPK recognize distinct phosphorylation sites in activated protein (MAP) kinase isoforms by the MAP kinase kinases MKK3 and MKK6. J Biol Chem 1998, 273:1741–1748. asubstrate. p38β MAPK has the unique capacity to medi- 11. Loesch M, Chen G: The p38 MAPK stress pathway as a tumor suppressor ate the dual phosphorylation of Thr-188 and Ser-184 in or more? Front Biosci 2008, 13:3581–3593. C/EBPβ, thereby, activating this transcription factor and 12. Perdiguero E, Ruiz-Bonilla V, Gresh L, Hui L, Ballestar E, Sousa-Victor P, Baeza- Raja B, Jardi M, Bosch-Comas A, Esteller M, Caelles C, Serrano AL, Wagner EF, inducing muscle catabolism. Therefore, p38β MAPK Munoz-Canoves P: Genetic analysis of p38 MAP kinases in myogenesis: should be considered as a therapeutic target for cachexia. fundamental role of p38alpha in abrogating myoblast proliferation. EMBO J 2007, 26:1245–1256. 13. Palacios D, Mozzetta C, Consalvi S, Caretti G, Saccone V, Proserpio V, Marquez Additional files VE, Valente S, Mai A, Forcales SV, Sartorelli V, Puri PL: TNF/p38alpha/polycomb signaling to Pax7 locus in satellite cells links inflammation to the epigenetic Additional file 1: C/EBPβ Phosphorylation Sites By p38α. control of muscle regeneration. Cell Stem Cell 2010, 7:455–469. 14. Gillespie MA, Le Grand F, Scime A, Kuang S, von Maltzahn J, Seale V, Additional file 2: C/EBPβ Phosphorylation Sites By p38β. Cuenda A, Ranish JA, Rudnicki MA: p38-{gamma}-dependent gene silencing restricts entry into the myogenic differentiation program. J Cell Abbreviations Biol 2009, 187:991–1005. ChIP: Chromosome immunoprecipitation; COPD: Chronic obstructive 15. Pogozelski AR, Geng T, Li P, Yin X, Lira VA, Zhang M, Chi JT, Yan Z: pulmonary disease; DMEM: Dulbecco’s modified Eagle’s medium; LCM: Lewis p38gamma mitogen-activated protein kinase is a key regulator in lung carcinoma cell-conditioned medium; LLC: Lewis lung carcinoma; skeletal muscle metabolic adaptation in mice. PLoS One 2009, 4:e7934. MAPK: Mitogen-activated protein kinase; MKK6: MAPK kinase 6; 16. Ho RC, Alcazar O, Fujii N, Hirshman MF, Goodyear LJ: p38gamma MAPK NF-κB: Nuclear factor-κB; PCR: Polymerase chain reaction; siRNA: Small regulation of glucose transporter expression and glucose uptake in L6 interfering RNA; TA: Tibialis anterior; TNF-α: tumor necrosis factor-α. myotubes and mouse skeletal muscle. Am J Physiol Regul Integr Comp Physiol 2004, 286:R342–R349. Competing interests 17. Zhang G, Jin B, Li YP: C/EBPbeta mediates tumour-induced ubiquitin The authors declare that they have no competing interests. ligase atrogin1/MAFbx upregulation and muscle wasting. EMBO J 2011, 30:4323–4335. Authors’ contributions 18. Evans WJ, Morley JE, Argiles J, Bales C, Baracos V, Guttridge D, Jatoi A, Kalantar- GZ performed the experiments, analyzed the data, and generated the Zadeh K, Lochs H, Mantovani G, Marks D, Mitch WE, Muscaritoli M, Najand A, figures. Y-PL designed the research and wrote the article. Both authors read Ponikowski P, Rossi Fanelli F, Schambelan M, Schols A, Schuster M, Thomas D, and approved the final manuscript. Wolfe R, Anker SD: Cachexia: a new definition. Clin Nutr 2008, 27:793–799. 19. Tisdale MJ: Mechanisms of cancer cachexia. Physiol Rev 2009, 89:381–410. 20. Lecker SH, Goldberg AL, Mitch WE: Protein degradation by the ubiquitin- Acknowledgements proteasome pathway in normal and disease states. J Am Soc Nephrol This study was supported by an R01 grant from National Institute of Arthritis 2006, 17:1807–1819. and Musculoskeletal and Skin Diseases to Y-P Li (AR052511). We thank Peter −/− 21. Sandri M, Sandri C, Gilbert A, Skurk C, Calabria E, Picard A, Walsh K, Johnson of National Cancer Institute for sharing the C/EBPβ mice, David Schiaffino S, Lecker SH, Goldberg AL: Foxo transcription factors induce the Engelberg of Hebrew University for sharing plasmids encoding the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle constitutively active mutants of p38 MAPK isoforms, and Qi-Qun Tang of atrophy. Cell 2004, 117:399–412. Fudan University for sharing plasmid encoding C/EBPβ mutant pcDNA3-LAP-T188A. 22. Stitt TN, Drujan D, Clarke BA, Panaro F, Timofeyva Y, Kline WO, Gonzalez M, Yancopoulos GD, Glass DJ: The IGF-1/PI3K/Akt pathway prevents expression of Received: 9 August 2012 Accepted: 21 September 2012 muscle atrophy-induced ubiquitin ligases by inhibiting FOXO transcription Published: 9 October 2012 factors. Mol Cell 2004, 14:395–403. Zhang and Li Skeletal Muscle 2012, 2:20 Page 9 of 9 http://www.skeletalmusclejournal.com/content/2/1/20 23. Sacheck JM, Hyatt JP, Raffaello A, Jagoe RT, Roy RR, Edgerton VR, Lecker SH, Goldberg AL: Rapid disuse and denervation atrophy involve transcriptional changes similar to those of muscle wasting during systemic diseases. FASEB J 2007, 21:140–155. 24. Penna F, Bonetto A, Muscaritoli M, Costamagna D, Minero VG, Bonelli G, Rossi Fanelli F, Baccino FM, Costelli P: Muscle atrophy in experimental cancer cachexia: is the IGF-1 signaling pathway involved? Int J Cancer 2010, 127(7):1706–1717. 25. Cai D, Frantz JD, Tawa NE Jr, Melendez PA, Oh BC, Lidov HG, Hasselgren PO, Frontera WR, Lee J, Glass DJ, Shoelson SE: IKKbeta/NF-kappaB activation causes severe muscle wasting in mice. Cell 2004, 119:285–298. 26. Askari N, Beenstock J, Livnah O, Engelberg D: p38 is active in vitro and in vivo when monophosphorylated on Thr180. Biochemistry 2009, 48:2497–2504. 27. Thomas M, Lu JJ, Ge Q, Zhang C, Chen J, Klibanov AM: Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung. Proc Natl Acad Sci USA 2005, 102:5679–5684. 28. Sterneck E, Tessarollo L, Johnson PF: An essential role for C/EBPbeta in female reproduction. Genes Dev 1997, 11:2153–2162. 29. Williams SC, Baer M, Dillner AJ, Johnson PF: CRP2 (C/EBP beta) contains a bipartite regulatory domain that controls transcriptional activation, DNA binding and cell specificity. EMBO J 1995, 14:3170–3183. 30. Lee S, Miller M, Shuman JD, Johnson PF: CCAAT/Enhancer-binding protein beta DNA binding is auto-inhibited by multiple elements that also mediate association with p300/CREB-binding protein (CBP). J Biol Chem 2010, 285:21399–21410. 31. Tang QQ, Gronborg M, Huang H, Kim JW, Otto TC, Pandey A, Lane MD: Sequential phosphorylation of CCAAT enhancer-binding protein beta by MAPK and glycogen synthase kinase 3beta is required for adipogenesis. Proc Natl Acad Sci USA 2005, 102:9766–9771. 32. Li X, Kim JW, Gronborg M, Urlaub H, Lane MD, Tang QQ: Role of cdk2 in the sequential phosphorylation/activation of C/EBPbeta during adipocyte differentiation. Proc Natl Acad Sci USA 2007, 104:11597–11602. 33. Kim JW, Tang QQ, Li X, Lane MD: Effect of phosphorylation and S-S bond- induced dimerization on DNA binding and transcriptional activation by C/EBPbeta. Proc Natl Acad Sci USA 2007, 104:1800–1804. 34. Engelman JA, Lisanti MP, Scherer PE: Specific inhibitors of p38 mitogen- activated protein kinase block 3T3-L1 adipogenesis. J Biol Chem 1998, 273:32111–32120. 35. Coulthard LR, White DE, Jones DL, McDermott MF, Burchill SA: p38(MAPK): stress responses from molecular mechanisms to therapeutics. Trends Mol Med 2009, 15:369–379. 36. Thornton TM, Pedraza-Alva G, Deng B, Wood CD, Aronshtam A, Clements JL, Sabio G, Davis RJ, Matthews DE, Doble B, Rincon M: Phosphorylation by p38 MAPK as an alternative pathway for GSK3beta inactivation. Science 2008, 320:667–670. 37. Jiang Y, Chen C, Li Z, Guo W, Gegner JA, Lin S, Han J: Characterization of the structure and function of a new mitogen-activated protein kinase (p38beta). J Biol Chem 1996, 271:17920–17926. 38. O’Keefe SJ, Mudgett JS, Cupo S, Parsons JN, Chartrain NA, Fitzgerald C, Chen SL, Lowitz K, Rasa C, Visco D, Luell S, Carballo-Jane E, Owens K, Zaller DM: Chemical genetics define the roles of p38alpha and p38beta in acute and chronic inflammation. J Biol Chem 2007, 282:34663–34671. 39. Sicard P, Clark JE, Jacquet S, Mohammadi S, Arthur JS, O’Keefe SJ, Marber MS: The activation of p38 alpha, and not p38 beta, mitogen-activated protein kinase is required for ischemic preconditioning. J Mol Cell Cardiol 2010, 48:1324–1328. 40. Ramji DP, Foka P: CCAAT/enhancer-binding proteins: structure, function and regulation. Biochem J 2002, 365:561–575. Submit your next manuscript to BioMed Central 41. Bostrom P, Mann N, Wu J, Quintero PA, Plovie ER, Panakova D, Gupta RK, and take full advantage of: Xiao C, MacRae CA, Rosenzweig A, Spiegelman BM: C/EBPbeta controls exercise-induced cardiac growth and protects against pathological cardiac remodeling. Cell 2010, 143:1072–1083. • Convenient online submission 42. Guo S, Cichy SB, He X, Yang Q, Ragland M, Ghosh AK, Johnson PF, • Thorough peer review Unterman TG: Insulin suppresses transactivation by CAAT/enhancer- • No space constraints or color figure charges binding proteins beta (C/EBPbeta). Signaling to p300/CREB-binding protein by protein kinase B disrupts interaction with the major • Immediate publication on acceptance activation domain of C/EBPbeta. J Biol Chem 2001, 276:8516–8523. • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution doi:10.1186/2044-5040-2-20 Cite this article as: Zhang and Li: p38β MAPK upregulates atrogin1/ MAFbx by specific phosphorylation of C/EBPβ. Skeletal Muscle 2012 2:20. Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

p38β MAPK upregulates atrogin1/MAFbx by specific phosphorylation of C/EBPβ

Skeletal Muscle , Volume 2 (1) – Oct 9, 2012

Loading next page...
 
/lp/springer-journals/p38-mapk-upregulates-atrogin1-mafbx-by-specific-phosphorylation-of-c-UqpiKjEEHp
Publisher
Springer Journals
Copyright
Copyright © 2012 by Zhang and Li; licensee BioMed Central Ltd.
Subject
Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
eISSN
2044-5040
DOI
10.1186/2044-5040-2-20
pmid
23046544
Publisher site
See Article on Publisher Site

Abstract

Background: The p38 mitogen-activated protein kinases (MAPK) family plays pivotal roles in skeletal muscle metabolism. Recent evidence revealed that p38α and p38β exert paradoxical effects on muscle protein homeostasis. However, it is unknown why p38β, but not p38α, is capable of mediating muscle catabolism via selective activation of the C/EBPβ that upregulates atrogin1/MAFbx. Methods: Tryptic phosphopeptide mapping was carried out to identify p38α- and p38β-mediated phosphorylation sites in C/EBPβ. Chromosome immunoprecipitation (ChIP) assay was used to evaluate p38α and p38β effect on C/EBPβ binding to the atrogin1/MAFbx promoter. Overexpression or siRNA-mediated gene knockdown of p38α and p38β, and site-directed mutagenesis or knockout of C/EBPβ, were used to analyze the roles of these kinases in muscle catabolism in C2C12 myotubes and mice. Results: Cellular expression of constitutively active p38α or p38β resulted in phosphorylation of C/EBPβ at multiple serine and threonine residues; however, only p38β phosphorylated Thr-188, which had been known to be critical to the DNA-binding activity of C/EBPβ. Only p38β, but not p38α, activated C/EBPβ-binding to the atrogin1/MAFbx promoter. A C/EBPβ mutant in which Thr-188 was replaced by alanine acted as a dominant-negative inhibitor of atrogin1/MAFbx upregulation induced by either p38β or Lewis lung carcinoma (LLC) cell-conditioned medium (LCM). In addition, knockdown of p38β specifically inhibited C/EBPβ activation and atrogin1/MAFbx upregulation induced by LCM. Finally, expression of active p38β in mouse tibialis anterior specifically induced C/EBPβ phosphorylation at Thr-188, atrogin1/MAFbx upregulation and muscle mass loss, which were blocked in C/EBPβ-null mice. Conclusions: The α and β isoforms of p38 MAPK are capable of recognizing distinct phosphorylation sites in a substrate. The unique capacity of p38β in mediating muscle catabolism is due to its capability in phosphorylating Thr-188 of C/EBPβ. Keywords: Cachexia, E3 protein, Gene regulation, DNA-binding, Thr-188 Background exercise) stimuli [6]. On one hand, p38 stimulates The p38 mitogen-activated protein kinases (MAPK) muscle satellite cell proliferation [7] and differentiation family plays a pivotal role in skeletal muscle by mediat- [8], which increases muscle mass; on the other hand, ing diverse cellular activities, and interestingly, some of p38 stimulates muscle protein degradation leading to which result in paradoxical effects. For example, p38 muscle atrophy [3-5]. Intriguingly, p38 has the capacity mediates both insulin stimulation of glucose uptake [1] to activate different protein substrates depending on the and TNF-α stimulation of insulin resistance [2] in cellular environment [7]. It is of great interest to under- muscle. In the context of skeletal muscle protein homeo- stand how the p38 MAPK family is able to mediate the stasis, p38 responds to both catabolic (lipopolysacchar- discrete and sometimes opposing effects in response to ide, cytokines, and ROS) [3-5] and anabolic (insulin and diverse physiological and pathological stimuli. The fam- ily of MAPK is composed of at least four members (α, β, * Correspondence: yi-ping.li@uth.tmc.edu γ and δ), which enable the transduction of a variety Department of Integrative Biology and Pharmacology, University of Texas of extracellular signals into distinct nuclear responses Health Science Center at Houston, 6431 Fannin Street, Houston, TX 77030, USA © 2012 Zhang and Li; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Zhang and Li Skeletal Muscle 2012, 2:20 Page 2 of 9 http://www.skeletalmusclejournal.com/content/2/1/20 [9-11]. The α, β, and γ isoforms are found in muscle common phosphorylation sites with p38α, it specifically cells. Recently, it became clear that p38α MAPK plays phosphorylates the Thr-188 residue of C/EBPβ, which an essential role in myogenic differentiation [12,13]. activates C/EBPβ binding to the atrogin1/MAFbx pro- On the other hand, p38γ MAPK appears to regulate moter and upregulates this gene in response to a tumor. the expansion of myogenic precursor cells [14], endur- ance exercise-induced mitochondrial biogenesis and Methods angiogenesis [15], as well as glucose uptake [16]. But, Tryptic phosphopeptide mapping was carried out to little was known about the function of p38β until our identify p38α- and p38β-mediated phosphorylation sites most recent discovery of its role in regulating the in C/EBPβ. ChIP assay was used to evaluate p38α and atrogin1/MAFbx gene [17]. p38β effect on C/EBPβ-binding to the atrogin1/MAFbx Cachexia, a wasting disease characterized by loss of promoter. Overexpression or siRNA-mediated gene muscle mass with or without loss of fat mass, is frequently knockdown of p38α and p38β, and site-directed muta- associated with such diseases as cancer, sepsis, AIDS, con- genesis or knockout of C/EBPβ, were used to analyze gestive heart failure, diabetes, chronic renal failure and the roles of these kinases in muscle catabolism in C2C12 chronic obstructive pulmonary disease (COPD). Cachexia myotubes and mice. is distinct from starvation-, disuse-, aging-, primary de- pression-, malabsorption- and hyperthyroidism-induced Tryptic phosphopeptide mapping muscle mass loss and is associated with increased morbid- HEK293T cells (American Type Culture Collection, ity and mortality [18,19]. The prominent clinical feature of Manassas, VA, USA) cultured in 150 mm culture plates cachexia is weight loss with anorexia, inflammation, in- that were ~50% confluent were co-transfected with a sulin resistance, and increased muscle protein break- plasmid encoding LAP with a FLAG tag (Addgene) and down. Increased muscle protein breakdown in cachexia a plasmid encoding constitutively active p38α or p38β is at least partially due to accelerated muscle proteoly- [26] (10 μg each) using deacylated polyethylenimine sis by the ubiquitin-proteasome pathway, a common (PEI) 22000 [27], a gift from Dr. Guangwei Du (Univer- pathway of muscle mass loss due to pathological as sity of Texas Health Science Center at Houston, Hous- well as physiological causes [20]. However, the signal- ton, TX, USA). The cell culture medium was replaced ing mechanism of the activation of the ubiquitin- with fresh medium at 24 h. Cells were lysed in RIPA buf- proteasome pathway in cachexia appears to be different fer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 2 mM from that of physiological muscle atrophy. It is well EDTA, 1% NP-40, 0.1% SDS, 2 mM phenylmethylsul- established that a depression in AKT activity activates phonylfluoride (PMSF), 0.5% sodium deoxycholate, 1 FoxO1/3 transcription factors, which upregulates two mM NaF, 1/100 protease inhibitor cocktail, and 1/100 key ubiquitin ligases (E3 proteins), atrogin1/MAFbx and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MuRF1, in animal models of physiological muscle atro- MO, USA) after an additional incubation of 24 h. LAP phy caused by fasting, denervation and disuse [21-23]. in cell lysates was precipitated using FLAG-M2 magnetic In animal models of cachexia, however, AKT is often beads (Sigma-Aldrich) and subjected to 10% SDS-PAGE. activated, which leads to the inactivation of FoxO1/3 The gel was then stained with Coommassie Blue R-250. [4,5,24]. In fact, it has been shown in animal models of The LAP band was cut out and subjected to tryptic cachexia that upregulation of MuRF1 is mediated by phosphopeptide mapping conducted by Taplin Mass NF-κB [25], and upregulation of atrogin1/MAFbx is Spectrometry Facility at Harvard Medical School using mediated by p38 MAPK [4,5]. an LTQ-Orbitrap mass spectrometer (Thermo Electron, We showed most recently that among the known p38 West Palm Beach, FL, USA). MAPK isoforms only p38β MAPK is capable of upregu- lating atrogin1/MAFbx via the activation of transcription Myogenic cell culture and transfection factor C/EBPβ in response to tumor cell-conditioned Murine C2C12 myoblasts (American Type Culture Col- medium. In addition, we demonstrated that p38β MAPK lection, Manassas, VA, USA) were cultured in growth upregulation of atrogin1/MAFbx is independent of the medium (DMEM supplemented with 10% fetal bovine AKT-FoxO1/3 signaling pathway [17]. Thus, p38β serum) at 37°C under 5% CO . At approximately 85 to emerged as a key mediator and a specific therapeutic 90% confluence, myoblast differentiation was induced by target of cachexia. Notwithstanding, why p38β is incubation for 96 h in differentiation medium (DMEM uniquely capable of activating C/EBPβ among the p38 supplemented with 4% heat-inactivated horse serum) to isoforms is unknown. The current study is designed to form myotubes. Plasmids encoding constitutively active address the mechanism through which p38β specifically p38 isoforms [26] or a C/EBPβ mutant (p3xFlag-CMV- activates C/EBPβ in the context of tumor-induced cach- 10-LAP-T188A) were transfected into C2C12 myoblasts exia. We demonstrate that while p38β shares some of 50% confluence at 1 μg/well in six-well plates using Zhang and Li Skeletal Muscle 2012, 2:20 Page 3 of 9 http://www.skeletalmusclejournal.com/content/2/1/20 deacylated polyethylenimine (PEI) 22000. At 24 h we induced the cells to differentiate by switching them to differentiation medium. When indicated, myotubes were treated with Lewis lung carcinoma cell (National Cancer Institute, Bethesda, MD, USA)-conditioned medium (LCM, 25% final volume) [17] or directly harvested. Cell lysate was prepared using the RIPA buffer for further analyses. Chromosome immunoprecipitation assay Chromosome immunoprecipitation (ChIP) assay was Figure 1 Isolation of C/EBPβ phosphorylated by p38α or p38β performed as previously described [17]. MAPK for tryptic phosphopeptide mapping. HEK293T cells were co-transfected with plasmids encoding C/EBPβ (LAP fused with the FLAG tag) and constitutively active p38α or p38β (fused with the HA Generation of expression vector for a C/EBPβ mutant tag). After 48 h incubation the cells were lysed. Expression of the (LAP-T188A) transfected plasmids was verified by western blot analysis of HA and A plasmid encoding C/EBPβ in which Thr-188 was FLAG. Activity of over-expressed p38α and p38β was evaluated by replaced by alanine in the pcDNA3 vector (a gift from western blot analysis of ATF2 activation (A). Overexpressed C/EBPβ was pulled down with FLAG-M2 magnetic beads and separated by Dr. Qi-Qun Tang of Fudan University, Shanghai, China) SDS-PAGE. The gel was stained with Coommassie Blue R-250 (B). was digested at BamHI and EcoRI restriction sites to re- The C/EBPβ band was then cut out and analyzed by tryptic lease the cDNA insert. That insert was then subcloned phosphopeptide mapping utilizing mass spectrometry for into the p3xFlag-CMV-10 vector (Sigma-Aldrich) at the phosphorylated amino acid residues. MAPK, mitogen-activated same restriction sites to generate plasmid p3xFlag- protein kinase. CMV-10-LAP-T188A. Western blot analysis [28]. While the mice were under anesthesia, plasmids Cell and muscle lysate were prepared and western blot encoding constitutively active p38α or p38β were analysis was carried out as described previously [17]. injected into the tibialis anterior (TA) of the right leg for Antibodies for total and/or phosphorylated ATF2, each mouse (100 μgin50 μl), and the empty vector FoxO1 (Thr-24)/FoxO3a (Thr-32) and C/EBPβ phos- pcDNA3.1 was injected into the left leg as control. Im- phorylated at Thr-188 were from Cell Signaling Tech- mediately after plasmid injection, TA was electroporated nology (Danvers, MA, USA). Antibody for atrogin1/ by applying square-wave electrical pulses (100 V/cm) MAFbx was from ECM Biosciences (Versailles, KY, eight times with an electrical pulse generator (Model USA). Antibodies to C/EBPβ (H-7) were from Santa 830, BTX) at a rate of one pulse per second, with each Cruz Biotechnology (Santa Cruz, CA, USA). Antibody to pulse being 20 ms in duration, through a pair of stainless the HA tag was from Covance (Princeton, NJ, USA). steel needles that were 5 mm apart. The above transfec- Data were normalized to GAPDH. tion procedure was repeated in 7 days. In another 7 days, the mice were sacrificed and TAs were collected Gene knockdown by siRNA for analysis. p38α-specific siRNA (5′-CUCCUUUACUAUCUUUCU CAA-3′) and p38β-specific siRNA (5′-GUCCUGAGGUU Statistical analysis CUAGCAAAdTdT-3′) were synthesized by Sigma-Aldrich Data were analyzed with one-way ANOVA or student t and transfected into C2C12 myoblasts by electroporation test using the SigmaStat software (Systat Software, Point (5 μg/1 × 10 cells) with the Nucleofector system (Lonza, Richmond, CA, USA) as indicated. When applicable, Walkersville, MD, USA), according to the manufacturer’s protocol. Control siRNA was obtained from Ambion Table 1 Expression of constitutively active p38α or p38β (Austin, TX, USA). Differentiation was induced 24 h after resulted in the phosphorylation of diverse amino acid transfection. residues in C/EBPβ Kinase Phosphorylated amino acid residues in C/EBPβ Animal use p38α Ser110, Tyr108 Experimental protocols were approved in advance by the p38β Ser182, Ser183, Ser190, Thr188 institutional Animal Welfare Committee at the Univer- p38α/p38β Ser64, Ser184, Ser222, Ser276 sity of Texas Health Science Center at Houston. C/ −/− The C/EBPβ bands cut out from SDS-PAGE described in Figure 1B were EBPβ mice in C57BL/6 background were bred from analyzed by tryptic phosphopeptide mapping utilizing mass spectrometry. −/+ C/EBPβ mice generated by Dr. Peter Johnson of NCI Identified phosphorylated amino acid residues in C/EBPβ are listed. Zhang and Li Skeletal Muscle 2012, 2:20 Page 4 of 9 http://www.skeletalmusclejournal.com/content/2/1/20 Results p38β MAPK specifically phosphorylates the Thr-188 residue of C/EBPβ C/EBPβ is a transcription factor that is normally repressed due to the intrinsic repression of its DNA- binding and transactivation functions [29,30]. The DNA- binding function of C/EBPβ is activated by sequential phosphorylation of specific amino acid residues by mul- tiple kinases [31-33]. C/EBPβ was previously shown to be a p38 substrate in vitro [34]. Recently, we showed that p38 interacts with and phosphorylates C/EBPβ in C2C12 myotubes. In addition, while the p38α/β inhibitor SB202190 blocks tumor-induced atrogin1/MAFbx upre- gulation, only p38β is capable of upregulating atrogin1/ MAFbx via the activation of C/EBPβ [17]. To investigate why p38β, but not p38α, has the capacity for activating C/EBPβ we set out to investigate the phosphorylation sites in C/EBPβ that are targeted by p38α and p38β. Plasmids encoding C/EBPβ (LAP fused with the FLAG tag) and constitutively active p38α or p38β (fused to HA) were co-transfected into HEK293T cells. At 48 h expression of the transfected plasmids was verified by western blot analysis of HA (p38 MAKPs) and FLAG Figure 2 Expression of constitutively active p38β in C2C12 (LAP) in the cell lysate. Activation of p38 substrate myotubes resulted in specific phosphorylation of C/EBPβ at Thr-188 and upregulation of atrogin1/MAFbx. C2C12 myoblasts ATF2 (via phosphorylation) by expressed p38 MAPKs were transfected with a plasmid encoding constitutively active p38α, was also evaluated by western blot analysis (Figure 1A). p38β or the empty vector (control). The myoblasts were allowed to Overexpressed C/EBPβ was pulled down with FLAG-M2 differentiate for 96 h to form myotubes that were then harvested magnetic beads and separated with SDS-PAGE (Figure 1B). and analyzed for ATF2 activation, Thr-188 phosphorylation in C/EBPβ The C/EBPβ band was cut out from the gel and analyzed and level of atrogin1/MAFbx using western blotting. by tryptic phosphopeptide mapping utilizing mass spec- trometry (for original reports see Additional file 1: Table control samples from independent experiments were S1 and Additional file 2: Table S2). Six phosphorylated normalized to a value of 1 without showing variations amino acid residues were identified in C/EBPβ that was (actual variations were within a normal range). A P value co-expressed with active p38α and eight phosphorylated <0.05 was considered to be statistically significant. Data amino acid residues were identified in C/EBPβ that was are presented as the mean ± S.E. co-expressed with active p38β.The twop38 MAPK Figure 3 p38β is critical to LCM-induced phosphorylation of C/EBPβ at Thr-188 and upregulation of atrogin1/MAFbx. C2C12 myoblasts were transfected with siRNA as indicated. After differentiation, myotubes were treated with LCM or control medium. In 1 h, levels of p38α and p38β,and phosphorylation of C/EBPβ at Thr-188 were evaluated by western blotting (A). In 8 h, levels of atrogin1/MAFbx were evaluated by western blotting (B). Optical density of the bands that represent C/EBP phosphorylated at Thr-188 or atrogin1/MAFbx was analyzed by ANOVA. *denotes a difference from control without LCM treatment and †denotes a difference from control with LCM treatment (P <0.05). LCM, Lewis lung carcinoma cell-conditioned medium. Zhang and Li Skeletal Muscle 2012, 2:20 Page 5 of 9 http://www.skeletalmusclejournal.com/content/2/1/20 isoforms shared four common phosphorylation sites (Ser-64, Ser-184, Ser-222, and Ser-276). On the other hand, p38α uniquely phosphorylated Ser-110 and Tyr-108, while p38β uniquely phosphorylated Ser-182, Ser-183, Ser-190 and Thr-188 (Table 1). Among the unique amino acid residues phosphorylated by p38β, Thr-188 is known to be crucial for the activation of C/EBPβ binding to its targeted DNA sequence [31-33]. To verify whether p38β specifically mediates the phos- phorylation of Thr-188 of C/EBPβ in muscle cells, we transfected C2C12 myoblasts with plasmids encoding active p38α or active p38β and used empty vector as the control. Although constitutively active p38α appeared to accelerate differentiation during the early stage of differ- entiation, at 96 h of differentiation, there was no visible Figure 4 p38β specifically activates C/EBPβ-binding to the difference in the myotubes formed compared with con- atrogin1/MAFbx promoter. (A) C2C12 myoblasts were transfected trol myotubes and active p38β-expressing myotubes. with a plasmid encoding constitutively active p38α, p38β or the empty vector (control). After differentiation, ChIP assay was carried After differentiation, lysate of myotubes was evaluated out to evaluate C/EBPβ binding in myotubes to a 190-base pair by western blot analysis of expression of the HA tag fragment of the atrogin1/MAFbx promoter that contains the that fused to active p38α or p38β, ATF2 activation and previously identified C/EBPβ-responsive cis-enhancer element [17] C/EBPβ phosphorylation at Thr-188. As shown in Figure 2, using control and the target PCR primers. Pre-immune IgG used as although both of the active p38 isoforms activated ATF2, the control to the antibody against C/EBPβ did not pull down the target fragment (data not shown). (B) C2C12 myoblasts were only the expression of active p38β resulted in C/EBPβ transfected with siRNA as indicated. After differentiation, myotubes phosphorylation at Thr-188 and upregulation of atrogin1/ were treated with LCM or control medium for 1 h and ChIP assay MAFbx. Therefore, the specific phosphorylation of was carried out to evaluate C/EBPβ binding to the atrogin1/MAFbx Thr-188 by p38β may be the key to its specific activation promoter. ChIP, chromosome immunoprecipitation; LCM, Lewis lung of C/EBPβ. carcinoma cell-conditioned medium. Figure 5 C/EBPβ phosphorylation at Thr-188 is crucial for atrogin1/MAFbx upregulation by p38β or LCM. (A) C2C12 myoblasts were transfected with a plasmid encoding constitutively active p38α, p38β or empty vector. A plasmid encoding FLAG-tagged C/EBPβ mutant in which Thr-188 was replaced with alanine (LAP-T188A) or the control vector was co-transfected as indicated. After differentiation, myotubes were lysed and analyzed by western blotting for the expression of LAP-T188A (with antibodies against FLAG and C/EBPβ) and atrogin1/MAFbx. (B) C2C12 myoblasts were transfected with LAP-T188A or the empty vector as control. After differentiation, myotubes were treated with LCM or control medium for 8 h. Lysate of myotubes was analyzed by western blotting for the expression of LAP-T188A (with antibodies against FLAG and C/EBPβ) and atrogin1/MAFbx. Optical density of the bands that represent atrogin1/MAFbx was analyzed by ANOVA. *denotes a difference from control without LCM treatment and †denotes a difference from control with LCM treatment (P <0.05). LCM, Lewis lung carcinoma cell-conditioned medium. Zhang and Li Skeletal Muscle 2012, 2:20 Page 6 of 9 http://www.skeletalmusclejournal.com/content/2/1/20 Previously, we observed that MAPK kinase 6 (MKK6) activates C/EBPβ binding to the C/EBPβ-responsive en- hancer in the atrogin1/MAFbx promoter via the activa- tion of p38 [17]. To investigate whether p38β specifically activates C/EBPβ binding to this C/EBPβ-responsive en- hancer, we conducted the ChIP assay. We observed in C2C12 myotubes that the expression of active p38β, but not active p38α, activated C/EBPβ binding to the atrogin1/MAFbx promoter region containing the C/EBPβ- responsive enhancer (Figure 4A). Conversely, siRNA- mediated knockdown of the p38β gene, but not the p38α gene, blocked LCM-induced activation of C/EBPβ binding to the atrogin1/MAFbx promoter (Figure 4B). Thus, we conclude that p38β specifically activates C/EBPβ binding to its targeted DNA motif. To evaluate whether C/EBPβ phosphorylation at Thr- 188 is critical to p38β-mediated upregulation of atrogin1/ MAFbx, a plasmid encoding a C/EBPβ mutant in which Thr-188 was replaced by alanine (C/EBPβ-T188A) was transfected into C2C12 myoblasts along with a plasmid encoding active p38α or active p38β. After differentiation, levels of atrogin1/MAFbx in myotubes that expressed active p38β,but notactivep38α, were elevated. The elevation of atrogin1/MAFbx was blocked in myotubes co-expressing the C/EBPβ-T188A mutant (Figure 5A). To evaluate whether tumor-induced atrogin1/MAFbx upregulation requires the phosphorylation of C/EBPβ at Thr-188, the plasmid encoding C/EBPβ-T188A was Figure 6 Expression of constitutively active p38β in mouse muscle induces C/EBPβ phosphorylation at Thr-188. Plasmids transfected into C2C12 myoblasts. After differentiation, encoding constitutively active p38α or p38β were transfected into myotubes were treated with LLC cell-conditioned −/− TA of the right leg of wild type or C/EBPβ mice, and the empty medium (LCM). We observed that LCM upregulation of vector into the left leg. In 14 days TA samples were collected, atrogin1/MAFbx was inhibited in myotubes that overex- weighed and lysed. Expression of the p38 isoforms was verified by press the C/EBPβ-T188A mutant (Figure 5B). Therefore, western blot analysis of HA tag expression and ATF2 activation. A representative blot is shown (A). The TA lysate was subjected to p38β-mediated C/EBPβ phosphorylation at Thr-188 is western blot analysis of C/EBPβ phosphorylation at Thr-188 and crucial for atrogin1/MAFbx upregulation by LLC cells. FoxO1/3 activation. Representative blots and densitometry data are shown. Optical density of the bands that represent various proteins p38β MAPK specifically induces atrogin1/MAFbx was measured. Levels of protein phosphorylation were normalized upregulation and muscle mass loss in mice via C/EBPβ to that of total proteins. *denotes a difference (P <0.05) based on Student t test (B). TA, tibialis anterior. We previously showed that C/EBPβ is essential for the atrogin1/MAFbx upregulation and muscle mass loss in Lewis lung carcinoma (LLC)-tumor bearing mice [17]. p38β MAPK-mediated Thr-188 phosphorylation activates To evaluate whether activation of p38β in vivo specif- C/EBPβ binding to the atrogin1/MAFbx promoter and ically induces C/EBPβ phosphorylation at Thr-188, upregulates atrogin1/MAFbx in C2C12 myotubes atrogin1/MAFbx upregulation and muscle mass loss, To verify whether p38β is critical to Thr-188 phosphoryl- the plasmids encoding constitutively active p38α or ation and atrogin1/MAFbx upregulation induced by ca- p38β were transfected into the tibialis anterior (TA) of −/− chectic tumor cells, siRNA-mediated mRNA knockdown wild type or C/EBPβ mice with the empty vector as was carried out. We observed that knockdown of the p38β control. At 14 days, the mice were sacrificed and the expression, but not the p38α expression, blocked Lewis excised TAs were analyzed. Expression of the HA tag lung carcinoma cell-conditioned medium (LCM)-induced that fused to p38α or p38β and activation of the p38 Thr-188 phosphorylation (Figure 3A) and atrogin1/MAFbx substrate ATF2 were evaluated by western blot analysis upregulation (Figure 3B). Therefore, p38β is indeed a key to confirm the expression of active p38α and p38β mediator of Thr-188 phosphorylation and atrogin1/MAFbx (Figure 6A). Expression of active p38β, but not active upregulation by Lewis lung carcinoma. p38α, resulted in phosphorylation of C/EBPβ at Thr- Zhang and Li Skeletal Muscle 2012, 2:20 Page 7 of 9 http://www.skeletalmusclejournal.com/content/2/1/20 188 (Figure 6B). Because the atrogin1/MAFbx pro- 188 in C/EBPβ by ERK1/2 MAPK [31] or cdk2/cyclinA moter also contains FoxO1/3-responsive cis-elements [32] primes C/EBPβ for subsequent phosphorylation on that are regulated by AKT [21], we evaluated whether Ser-184 or Thr-179 by glycogen synthase kinase 3β p38β affected FoxO1/3 activity. Expression of active (GSK3β), which activates the DNA-binding and transacti- p38β did not alter the phosphorylation state of FoxO1/ vation functions of C/EBPβ [33]. Our data presented here 3 (Figure 6B). Therefore, p38β does not affect FoxO1/ demonstrate that p38β has the unique capability of medi- 3 activity. As shown in Figure 7A, expression of active ating dual phosphorylation at both Thr-188 and Ser-184, p38β resulted in atrogin1/MAFbx upregulation in the resulting in the activation of C/EBPβ binding to the atro- TA of wild type mice. However, active p38β failed to gin1/MAFbx promoter. In contrast, p38α that mediates −/− upregulate atrogin1/MAFbx in C/EBPβ mice. Finally, phosphorylation of Ser-184 but not Thr-188 is unable to expression of active p38β, but not active p38α, induced activate C/EBPβ binding to the atrogin1/MAFbx pro- TA weight loss by 14% in wild type mice. In contrast, moter. Because GSK3β was previously shown inactivated active p38β expression did not alter TA weight in C/ by p38 MAPK-mediated phosphorylation [36], it is un- −/− EBPβ mice (Figure 7B). Therefore, in vivo activation likely that GSK3β mediates C/EBPβ phosphorylation at of p38β induces atrogin1/MAFbx upregulation and Ser-184 in response to p38α and p38β activation. muscle mass loss via the activation of C/EBPβ. In the present study we also present the first evidence that overexpression of active p38β in muscle induces Discussion muscle catabolism, demonstrating a direct effect of p38β The selective activation of substrates by various p38 on muscle catabolism in vivo. Previous studies involving MAPK isoforms was previously attributed to preferential systemic activation of p38 in tumor-bearing mice [17] or activation of the isoforms by specific MAPK kinase as well septic mice [4] did not allow such a conclusion. as compartmentalization of the isoforms. For example, Although p38β is widely distributed in various tissues while p38α is activated by MKK3, MKK6 and MKK4, [37] its function is largely unknown, especially when it is p38β is activated by MKK6 [10,35]. However, these may compared to p38α, which is not only responsible for the not explain the specific activation of C/EBPβ by p38β,be- known roles of p38 in inflammatory responses [38,39] cause both p38α and p38β are present in the nucleus and but also in the regulation of myogenesis [12,13]. The activated by MKK6. The current study demonstrates that present study demonstrates that activation of p38β, not the selective activation of substrates by p38 MAPK iso- p38α, induces atrogin1/MAFbx upregulation and muscle forms is further realized by their recognition of specific mass loss via specific phosphorylation of C/EBPβ, which phosphorylation sites within a substrate. explains at the molecular level why p38 is capable of Particularly, we show that p38β specifically mediates playing the seemingly opposing roles in muscle protein the phosphorylation of C/EBPβ required for the activa- homeostasis (promoting myogenesis versus promoting tion of its binding to the atrogin1/MAFbx promoter. muscle catabolism). Further, these data support p38β Previous studies indicated that phosphorylation of Thr- as a selective therapeutic target of cachexia. Because Figure 7 Expression of constitutively active p38β in mouse muscle induces atrogin1/MAFbx upregulation and muscle mass loss in a C/EBPβ-dependent manner. The TA lysate derived from Figure 6 was further analyzed for level of atrogin1/MAFbx by western blotting. Representative blots and densitometry data are shown (A). The weight of TA transfected with a plasmid encoding an active p38 isoform was compared to that of TA transfected with empty vector (control) from the same mouse (B). *denotes a difference (P <0.05) based on ANOVA. TA, tibialis anterior. Zhang and Li Skeletal Muscle 2012, 2:20 Page 8 of 9 http://www.skeletalmusclejournal.com/content/2/1/20 C/EBPβ is activated by a number of kinases and regu- References 1. Tremblay F, Dubois MJ, Marette A: Regulation of GLUT4 traffic and function by lates a wide variety of genes [40], it may not be suit- insulin and contraction in skeletal muscle. Front Biosci 2003, 8:d1072–d1084. able as a drug target. On the other hand, p38β has few 2. de Alvaro C, Teruel T, Hernandez R, Lorenzo M: Tumor necrosis factor known functions, therefore, specific inhibitors of p38β alpha produces insulin resistance in skeletal muscle by activation of inhibitor kappaB kinase in a p38 MAPK-dependent manner. J Biol Chem MAPK would be highly desirable for the intervention 2004, 279:17070–17078. of cancer cachexia. Unfortunately, only p38α/β-dual or 3. Li YP, Chen Y, John J, Moylan J, Jin B, Mann DL, Reid MB: TNF-alpha acts p38α-specific inhibitors are available at the present time. via p38 MAPK to stimulate expression of the ubiquitin ligase atrogin1/ MAFbx in skeletal muscle. FASEB J 2005, 19:362–370. Because p38β is highly expressed in the heart [37], it 4. Doyle A, Zhang G, Abdel Fattah EA, Eissa NT, Li YP: Toll-like receptor 4 may also regulate the protein homeostasis in heart mediates lipopolysaccharide-induced muscle catabolism via coordinate muscle via influencing C/EBPβ−regulated atrogin1/ activation of ubiquitin-proteasome and autophagy-lysosome pathways. FASEB J 2011, 25:99–110. MAFbx expression. Consistent to this notion, it has been 5. McClung JM, Judge AR, Powers SK, Yan Z: p38 MAPK links oxidative stress shown recently that exercise induces a reduction in C/ to autophagy-related gene expression in cachectic muscle wasting. Am J EBPβ in cardiomyocytes, which mediates cardiomyocyte Physiol Cell Physiol 2010, 298:C542–C549. 6. Long YC, Widegren U, Zierath JR: Exercise-induced mitogen-activated protein hypertrophy [41]. In addition, the transactivation activity kinase signalling in skeletal muscle. Proc Nutr Soc 2004, 63:227–232. of C/EBPβ is suppressed by insulin, an anabolic hor- 7. Jones NC, Tyner KJ, Nibarger L, Stanley HM, Cornelison DD, Fedorov YV, mone [42]. Therefore, diverse signaling pathways that Olwin BB: The p38alpha/beta MAPK functions as a molecular switch to activate the quiescent satellite cell. J Cell Biol 2005, 169:105–116. regulate protein homeostasis in striated muscles may 8. Lluis F, Perdiguero E, Nebreda AR, Munoz-Canoves P: Regulation of skeletal converge upon C/EBPβ. muscle gene expression by p38 MAP kinases. Trends Cell Biol 2006, 16:36–44. 9. Remy G, Risco AM, Inesta-Vaquera FA, Gonzalez-Teran B, Sabio G, Davis RJ, Cuenda A: Differential activation of p38MAPK isoforms by MKK6 and Conclusions MKK3. Cell Signal 2010, 22:660–667. The present study demonstrates that the α and β isoforms 10. Enslen H, Raingeaud J, Davis RJ: Selective activation of p38 mitogen- of p38 MAPK recognize distinct phosphorylation sites in activated protein (MAP) kinase isoforms by the MAP kinase kinases MKK3 and MKK6. J Biol Chem 1998, 273:1741–1748. asubstrate. p38β MAPK has the unique capacity to medi- 11. Loesch M, Chen G: The p38 MAPK stress pathway as a tumor suppressor ate the dual phosphorylation of Thr-188 and Ser-184 in or more? Front Biosci 2008, 13:3581–3593. C/EBPβ, thereby, activating this transcription factor and 12. Perdiguero E, Ruiz-Bonilla V, Gresh L, Hui L, Ballestar E, Sousa-Victor P, Baeza- Raja B, Jardi M, Bosch-Comas A, Esteller M, Caelles C, Serrano AL, Wagner EF, inducing muscle catabolism. Therefore, p38β MAPK Munoz-Canoves P: Genetic analysis of p38 MAP kinases in myogenesis: should be considered as a therapeutic target for cachexia. fundamental role of p38alpha in abrogating myoblast proliferation. EMBO J 2007, 26:1245–1256. 13. Palacios D, Mozzetta C, Consalvi S, Caretti G, Saccone V, Proserpio V, Marquez Additional files VE, Valente S, Mai A, Forcales SV, Sartorelli V, Puri PL: TNF/p38alpha/polycomb signaling to Pax7 locus in satellite cells links inflammation to the epigenetic Additional file 1: C/EBPβ Phosphorylation Sites By p38α. control of muscle regeneration. Cell Stem Cell 2010, 7:455–469. 14. Gillespie MA, Le Grand F, Scime A, Kuang S, von Maltzahn J, Seale V, Additional file 2: C/EBPβ Phosphorylation Sites By p38β. Cuenda A, Ranish JA, Rudnicki MA: p38-{gamma}-dependent gene silencing restricts entry into the myogenic differentiation program. J Cell Abbreviations Biol 2009, 187:991–1005. ChIP: Chromosome immunoprecipitation; COPD: Chronic obstructive 15. Pogozelski AR, Geng T, Li P, Yin X, Lira VA, Zhang M, Chi JT, Yan Z: pulmonary disease; DMEM: Dulbecco’s modified Eagle’s medium; LCM: Lewis p38gamma mitogen-activated protein kinase is a key regulator in lung carcinoma cell-conditioned medium; LLC: Lewis lung carcinoma; skeletal muscle metabolic adaptation in mice. PLoS One 2009, 4:e7934. MAPK: Mitogen-activated protein kinase; MKK6: MAPK kinase 6; 16. Ho RC, Alcazar O, Fujii N, Hirshman MF, Goodyear LJ: p38gamma MAPK NF-κB: Nuclear factor-κB; PCR: Polymerase chain reaction; siRNA: Small regulation of glucose transporter expression and glucose uptake in L6 interfering RNA; TA: Tibialis anterior; TNF-α: tumor necrosis factor-α. myotubes and mouse skeletal muscle. Am J Physiol Regul Integr Comp Physiol 2004, 286:R342–R349. Competing interests 17. Zhang G, Jin B, Li YP: C/EBPbeta mediates tumour-induced ubiquitin The authors declare that they have no competing interests. ligase atrogin1/MAFbx upregulation and muscle wasting. EMBO J 2011, 30:4323–4335. Authors’ contributions 18. Evans WJ, Morley JE, Argiles J, Bales C, Baracos V, Guttridge D, Jatoi A, Kalantar- GZ performed the experiments, analyzed the data, and generated the Zadeh K, Lochs H, Mantovani G, Marks D, Mitch WE, Muscaritoli M, Najand A, figures. Y-PL designed the research and wrote the article. Both authors read Ponikowski P, Rossi Fanelli F, Schambelan M, Schols A, Schuster M, Thomas D, and approved the final manuscript. Wolfe R, Anker SD: Cachexia: a new definition. Clin Nutr 2008, 27:793–799. 19. Tisdale MJ: Mechanisms of cancer cachexia. Physiol Rev 2009, 89:381–410. 20. Lecker SH, Goldberg AL, Mitch WE: Protein degradation by the ubiquitin- Acknowledgements proteasome pathway in normal and disease states. J Am Soc Nephrol This study was supported by an R01 grant from National Institute of Arthritis 2006, 17:1807–1819. and Musculoskeletal and Skin Diseases to Y-P Li (AR052511). We thank Peter −/− 21. Sandri M, Sandri C, Gilbert A, Skurk C, Calabria E, Picard A, Walsh K, Johnson of National Cancer Institute for sharing the C/EBPβ mice, David Schiaffino S, Lecker SH, Goldberg AL: Foxo transcription factors induce the Engelberg of Hebrew University for sharing plasmids encoding the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle constitutively active mutants of p38 MAPK isoforms, and Qi-Qun Tang of atrophy. Cell 2004, 117:399–412. Fudan University for sharing plasmid encoding C/EBPβ mutant pcDNA3-LAP-T188A. 22. Stitt TN, Drujan D, Clarke BA, Panaro F, Timofeyva Y, Kline WO, Gonzalez M, Yancopoulos GD, Glass DJ: The IGF-1/PI3K/Akt pathway prevents expression of Received: 9 August 2012 Accepted: 21 September 2012 muscle atrophy-induced ubiquitin ligases by inhibiting FOXO transcription Published: 9 October 2012 factors. Mol Cell 2004, 14:395–403. Zhang and Li Skeletal Muscle 2012, 2:20 Page 9 of 9 http://www.skeletalmusclejournal.com/content/2/1/20 23. Sacheck JM, Hyatt JP, Raffaello A, Jagoe RT, Roy RR, Edgerton VR, Lecker SH, Goldberg AL: Rapid disuse and denervation atrophy involve transcriptional changes similar to those of muscle wasting during systemic diseases. FASEB J 2007, 21:140–155. 24. Penna F, Bonetto A, Muscaritoli M, Costamagna D, Minero VG, Bonelli G, Rossi Fanelli F, Baccino FM, Costelli P: Muscle atrophy in experimental cancer cachexia: is the IGF-1 signaling pathway involved? Int J Cancer 2010, 127(7):1706–1717. 25. Cai D, Frantz JD, Tawa NE Jr, Melendez PA, Oh BC, Lidov HG, Hasselgren PO, Frontera WR, Lee J, Glass DJ, Shoelson SE: IKKbeta/NF-kappaB activation causes severe muscle wasting in mice. Cell 2004, 119:285–298. 26. Askari N, Beenstock J, Livnah O, Engelberg D: p38 is active in vitro and in vivo when monophosphorylated on Thr180. Biochemistry 2009, 48:2497–2504. 27. Thomas M, Lu JJ, Ge Q, Zhang C, Chen J, Klibanov AM: Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung. Proc Natl Acad Sci USA 2005, 102:5679–5684. 28. Sterneck E, Tessarollo L, Johnson PF: An essential role for C/EBPbeta in female reproduction. Genes Dev 1997, 11:2153–2162. 29. Williams SC, Baer M, Dillner AJ, Johnson PF: CRP2 (C/EBP beta) contains a bipartite regulatory domain that controls transcriptional activation, DNA binding and cell specificity. EMBO J 1995, 14:3170–3183. 30. Lee S, Miller M, Shuman JD, Johnson PF: CCAAT/Enhancer-binding protein beta DNA binding is auto-inhibited by multiple elements that also mediate association with p300/CREB-binding protein (CBP). J Biol Chem 2010, 285:21399–21410. 31. Tang QQ, Gronborg M, Huang H, Kim JW, Otto TC, Pandey A, Lane MD: Sequential phosphorylation of CCAAT enhancer-binding protein beta by MAPK and glycogen synthase kinase 3beta is required for adipogenesis. Proc Natl Acad Sci USA 2005, 102:9766–9771. 32. Li X, Kim JW, Gronborg M, Urlaub H, Lane MD, Tang QQ: Role of cdk2 in the sequential phosphorylation/activation of C/EBPbeta during adipocyte differentiation. Proc Natl Acad Sci USA 2007, 104:11597–11602. 33. Kim JW, Tang QQ, Li X, Lane MD: Effect of phosphorylation and S-S bond- induced dimerization on DNA binding and transcriptional activation by C/EBPbeta. Proc Natl Acad Sci USA 2007, 104:1800–1804. 34. Engelman JA, Lisanti MP, Scherer PE: Specific inhibitors of p38 mitogen- activated protein kinase block 3T3-L1 adipogenesis. J Biol Chem 1998, 273:32111–32120. 35. Coulthard LR, White DE, Jones DL, McDermott MF, Burchill SA: p38(MAPK): stress responses from molecular mechanisms to therapeutics. Trends Mol Med 2009, 15:369–379. 36. Thornton TM, Pedraza-Alva G, Deng B, Wood CD, Aronshtam A, Clements JL, Sabio G, Davis RJ, Matthews DE, Doble B, Rincon M: Phosphorylation by p38 MAPK as an alternative pathway for GSK3beta inactivation. Science 2008, 320:667–670. 37. Jiang Y, Chen C, Li Z, Guo W, Gegner JA, Lin S, Han J: Characterization of the structure and function of a new mitogen-activated protein kinase (p38beta). J Biol Chem 1996, 271:17920–17926. 38. O’Keefe SJ, Mudgett JS, Cupo S, Parsons JN, Chartrain NA, Fitzgerald C, Chen SL, Lowitz K, Rasa C, Visco D, Luell S, Carballo-Jane E, Owens K, Zaller DM: Chemical genetics define the roles of p38alpha and p38beta in acute and chronic inflammation. J Biol Chem 2007, 282:34663–34671. 39. Sicard P, Clark JE, Jacquet S, Mohammadi S, Arthur JS, O’Keefe SJ, Marber MS: The activation of p38 alpha, and not p38 beta, mitogen-activated protein kinase is required for ischemic preconditioning. J Mol Cell Cardiol 2010, 48:1324–1328. 40. Ramji DP, Foka P: CCAAT/enhancer-binding proteins: structure, function and regulation. Biochem J 2002, 365:561–575. Submit your next manuscript to BioMed Central 41. Bostrom P, Mann N, Wu J, Quintero PA, Plovie ER, Panakova D, Gupta RK, and take full advantage of: Xiao C, MacRae CA, Rosenzweig A, Spiegelman BM: C/EBPbeta controls exercise-induced cardiac growth and protects against pathological cardiac remodeling. Cell 2010, 143:1072–1083. • Convenient online submission 42. Guo S, Cichy SB, He X, Yang Q, Ragland M, Ghosh AK, Johnson PF, • Thorough peer review Unterman TG: Insulin suppresses transactivation by CAAT/enhancer- • No space constraints or color figure charges binding proteins beta (C/EBPbeta). Signaling to p300/CREB-binding protein by protein kinase B disrupts interaction with the major • Immediate publication on acceptance activation domain of C/EBPbeta. J Biol Chem 2001, 276:8516–8523. • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution doi:10.1186/2044-5040-2-20 Cite this article as: Zhang and Li: p38β MAPK upregulates atrogin1/ MAFbx by specific phosphorylation of C/EBPβ. Skeletal Muscle 2012 2:20. Submit your manuscript at www.biomedcentral.com/submit

Journal

Skeletal MuscleSpringer Journals

Published: Oct 9, 2012

References