Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration

TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal... The EMBO Journal (2006) 25, 5826–5839 & 2006 European Molecular Biology Organization All Rights Reserved 0261-4189/06 | | T THE H E www.embojournal.org E E EMB M MB BO O O J JO OURN URN A AL L TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration 1 2 The EMBO Journal (2006) 25, 5826–5839. doi:10.1038/ Mahasweta Girgenrath , Shawn Weng , 1 2 sj.emboj.7601441; Published online 23 November 2006 Christine A Kostek ,BethBrowning , 2 3 Subject Categories: signal transduction; differentiation Monica Wang , Sharron AN Brown , 3 2 & death Jeffrey A Winkles , Jennifer S Michaelson , 2 4 Keywords: Fn14; inflammation; muscle; regeneration; Norm Allaire , Pascal Schneider , Martin 2 2 5 TWEAK L Scott , Yen-ming Hsu , Hideo Yagita , 6 1 Richard A Flavell , Jeffrey Boone Miller , 2 2, Linda C Burkly and Timothy S Zheng * 1 2 Boston Biomedical Research Institute, Watertown, MA, USA, Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA, USA, Departments of Introduction Surgery and Physiology, University of Maryland School of Medicine, Baltimore, MD, USA, Department of Biochemistry, University of Inflammation is the first-line defensive mechanism to noxious Lausanne, Ch. Des Boveresses, Epalinges, Switzerland, Department of insults ranging from microbial infections to physical injury Immunology, Juntendo University School of Medicine, Bunkyo-ku, (Nathan, 2002). Although excessive inflammation can have Tokyo, Japan and Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA devastating consequences, in most cases inflammatory responses lead to effective clearance of pathogens and/or Inflammation participates in tissue repair through multi- healing. In fact, depletion of inflammatory cell types such ple mechanisms including directly regulating the cell fate as macrophages has been shown to result in delayed repair of resident progenitor cells critical for successful regenera- response in injury models of a number of tissues, including tion. Upon surveying target cell types of the TNF ligand muscle (Lescaudron et al, 1999), mesothelium (Mutsaers et al, TWEAK, we observed that TWEAK binds to all progenitor 2002), and both the central (Kotter et al, 2001) and peripheral cells of the mesenchymal lineage and induces NF-jB nerve systems (Luk et al, 2003). It is now well established that activation and the expression of pro-survival, pro-prolif- inflammation contributes to tissue repair by clearing debris, erative and homing receptor genes in the mesenchymal engulfment and digestion of apoptotic/necrotic cell bodies, stem cells, suggesting that this pro-inflammatory cytokine epithelial closure and promoting angiogenesis (Nathan, 2002). may play an important role in controlling progenitor cell Much underappreciated until recently, the various cytokines biology. We explored this potential using both the estab- secreted by inflammatory cells also directly influence the lished C2C12 cell line and primary mouse muscle myo- properties of progenitor cells that reside in tissues undergoing blasts, and demonstrated that TWEAK promoted their repair and regeneration (Duffield, 2003). For example, the proliferation and inhibited their terminal differentiation. macrophage-derived cytokine TNF promotes the proliferation By generating mice deficient in the TWEAK receptor Fn14, of oligodendrocyte progenitors after cuprizone-induced de- we further showed that Fn14-deficient primary myoblasts myelination (Arnett et al, 2001). Similarly, soluble factor(s) displayed significantly reduced proliferative capacity and secreted by macrophages can also drive the proliferation of altered myotube formation. Following cardiotoxin injec- mesothelial cells (Mutsaers et al, 2002) and muscle satellite tion, a known trigger for satellite cell-driven skeletal cells (Merly et al, 1999) during mesothelial and muscle regen- muscle regeneration, Fn14-deficient mice exhibited re- eration, respectively. However, it has also been demonstrated duced inflammatory response and delayed muscle fiber that inflammatory cytokines block neural progenitor differen- regeneration compared with wild-type mice. These results tiation (Ekdahl et al, 2003; Monje et al, 2003), suggesting that indicate that the TWEAK/Fn14 pathway is a novel regu- the biological consequence of these inflammatory mediators lator of skeletal muscle precursor cells and illustrate an on tissue repair and regeneration is likely a complex one. important mechanism by which inflammatory cytokines The largely macrophage-derived cytokine TWEAK and its influence tissue regeneration and repair. Coupled with our receptor Fn14 are relatively new additions to the TNF super- recent demonstration that TWEAK potentiates liver pro- family, which includes well-known modulators of cell pro- genitor cell proliferation, the expression of Fn14 on all liferation, differentiation and apoptosis (Locksley et al, 2001). mesenchymal lineage progenitor cells supports a broad Originally identified as a weak inducer of cell death in tumor involvement of this pathway in other tissue injury and cell lines (Chicheportiche et al, 1997), TWEAK was subse- disease settings. quently shown to exert pleiotropic effects on a variety of cell types in vitro, including pro-angiogenic activities on endothe- *Corresponding author. Biogen Idec Inc., 14 Cambridge Center, lial cells (Lynch et al, 1999; Jakubowski et al, 2002) and pro- Cambridge, MA 02142, USA. Tel.: þ 1 617 679 3348; inflammatory activities on epithelial cells (Chicheportiche Fax: þ 1 617 679 3208; E-mail: [email protected] et al, 1997), astrocytes (Saas et al, 2000), dermal fibroblasts and synoviocytes (Chicheportiche et al, 2002). Conspicuously Received: 4 November 2005; accepted: 17 October 2006; published online: 23 November 2006 absent from the TWEAK-responding cell types are lymphocytes, 5826 The EMBO Journal VOL 25 NO 24 2006 &2006 European Molecular Biology Organization | | TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al and Fn14 expression on these cells has not been demonstrated. mesenchymal lineage, including human primary mesenchy- Interestingly, the TWEAK receptor Fn14 is an FGF-inducible mal stem cells, skeletal muscle myoblasts and preadipocytes gene and is highly upregulated in liver injury and regeneration as well as chondrocyte and osteoblast precursors cultured and arterial wounding (Feng et al, 2000; Wiley et al, 2001), in vitro (Figure 1A). We also demonstrated that the TWEAK suggesting a regulatory role in settings of tissue injury and receptor Fn14 was expressed on these progenitor cells by repair. Recently, we demonstrated that the TWEAK/Fn14 path- staining with the anti-human Fn14 monoclonal antibody way is a potent inducer of liver progenitor cell proliferation in ITEM-4 (Figure 1A). To see if these cells would respond to response to chemical injury in vivo (Jakubowski et al, 2005). TWEAK, we examined NF-kB activation in mesenchymal However, the physiological relevance of the TWEAK/Fn14 stem cells and osteoblast precursors upon TWEAK treatment pathway in other contexts remains elusive. as it has previously been shown that Fn14 signal transduction In this study, we explored the potential role of TWEAK/ is mediated through the TRAF-binding site in its cytoplasmic Fn14 pathway in modulating mesenchymal progenitor cell tail (Brown et al, 2003). As shown in Figure 1B, TWEAK biology in vitro and in vivo in a model of skeletal muscle induced robust NF-kB activation in both cell types as mea- injury and repair. We showed that all progenitor cells of sured by the amount of activated p65 present in the cell lysate mesenchymal lineage express the TWEAK receptor Fn14. following TWEAK stimulation using the TranAM assay We further demonstrated that TWEAK promotes proliferation system, indicating that progenitor cells of the mesenchymal of both an established myoblast cell line and primary muscle lineage are indeed TWEAK-responsive cells. The ability of myoblasts. Finally, by generating mice deficient in Fn14, we TWEAK to induce NF-kB activation in these progenitor established that the TWEAK/Fn14 pathway is required for cells is further confirmed by transcription profiling study of optimal muscle regeneration in vivo. mesenchymal stem cells treated with TWEAK. Even in the high serum culture condition (10% FBS), TWEAK induced robust transcriptional upregulation of well-known NF-kB- Results regulated genes, including TRAF1 and 3, NF-kB2 and RelB, which are regulators of NF-kB pathway themselves, and Mesenchymal progenitor cells are a novel target cell type of TWEAK pro-survival genes such as A20 and c-IAP2, as well as cell In our effort to identify in vivo target cell types for TWEAK, adhesion genes such as ICAM-1 and VCAM-1n (Figure 1C). we found that TWEAK binds to progenitor cells of the Importantly, under low (0.2%) or medium (2%) serum 100 100 100 100 hFc-hTWEAK Item-4 Anti-mouse Fc 80 80 80 80 Anti-human Fc 60 60 60 60 40 40 40 40 20 20 20 20 0 0 0 0 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 FL2-H FL2-H FL2-H FL2-H FL2-H hMesenchymal stem cells hMuscle satellite cells hPreadipocytes hChondrocytes hOsteoblast precursors B CD 1.5 Fold change (+TWEAK) Cell cycle-related genes Fold change (+TWEAK)/LS Genes LS MS HS cdc2 5.8 TRAF1 3.3 3.1 8.0 cdc6 7.0 TRAF3 2.6 3.0 11.0 Replication factor C 4.2 NF-κB 3.1 3.3 9.5 DNA topoisomerase II alpha 28.2 0.5 RelB 3.0 2.7 5.8 cdc46 39.3 A20 5.4 11.1 7.6 Survivin 13.8 c-IAP2 2.8 3.1 5.1 cdc20 7.2 ICAM-1 11.2 7.3 8.0 Cyclin A2 6.5 VCAM-1 6.5 3.9 9.2 MAD2 4.9 MCP-1 2.4 2.6 3.0 Figure 1 Human mesenchymal progenitor cells are a novel target cell type for TWEAK. (A) Human primary mesenchymal stem cells, skeletal muscle myoblasts, preadipocytes, chondrocytes and osteoblast precursors (Cambrex) were cultured according to the manufacturer’s protocols. First-passage cells showed staining for TWEAK binding using Fc-TWEAK and for expression of Fn14 using the anti-hFn14 mAb ITEM-4. Anti- mouse and anti-human Fcs were used as negative controls, (B)NF-kB was activated in human mesenchymal stem cells (hMSCs) and osteoblast precursors (hOsteos) following 2 or 6 h of treatment with 100 ng/m TWEAK (Tw). Activation was measured using the TransAM NF-kB p65 activation assay system with cell lysates from normal and TNF-treated HeLa cells serving as negative and positive controls. The assays were carried out in triplicate and the data shown are representative of three independent experiments. (C) List of representative genes induced by TWEAK (100 ng/ml versus heat-inactivated TWEAK 100 ng/ml) in mesenchymal stem cells in low serum (LS: 0.2% FBS), moderate serum (MS: 2% FBS) and high serum (HS: 10% FBS). (D) List of some cell cycle-related genes induced by TWEAK (versus inactivated TWEAK) in mesenchymal stem cells cultured under low-serum conditions (0.2% FBS). Triplicate samples were analyzed for each condition and the fold changes were calculated using averages from triplicates. All fold changes reached statistical significance (Po0.01). &2006 European Molecular Biology Organization The EMBO Journal VOL 25 NO 24 2006 5827 | | HeLa HeLa+TNF hMSCs hMSCs+Tw (2 h) hMSCs+Tw (6 h) hOsteo hOsteos+Tw (2 h) hOsteo+Tw (6 h) NF-κB p65 activation % of Max (OD nm) % of Max % of Max % of Max % of Max TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al conditions, TWEAK also induced the expression of many body P2D3 also was able to reverse the inhibitory effect cell cycle-related genes including cdc2, cyclin A2, survivin, of TWEAK (Figure 2F), demonstrating that the activity of MAD2, among others (Figure 1D and data not shown). These TWEAK on C2C12 cells was mediated through the Fn14 results therefore indicate that TWEAK may regulate cell fate receptor. As it is well documented that TNF is a potent decisions of progenitor cells. inhibitor of myogenesis in vitro (Szalay et al, 1997; Miller et al, 1988), we decided to investigate if there was any TWEAK promotes proliferation and inhibits terminal potential crosstalk between the TNF and TWEAK pathways myogenesis of C2C12 cells in the C2C12 myogenesis model. Although the soluble TNF To further investigate how TWEAK might influence cell fate receptor mTNFRI-Fc totally reversed the inhibition of C2C12 of progenitor cells, we took advantage of the well-established differentiation by TNF (Figure 2G and H), it had no effect on in vitro cell differentiation model of the mesenchymal the inhibitory activity of TWEAK (Figure 2I). A similar result lineage, C2C12 terminal myogenesis. We first confirmed was also seen using anti-TNF or anti-TNFRI blocking anti- that, similar to human skeletal muscle myoblasts, the murine bodies (data not shown). Conversely, the anti-TWEAK block- C2C12 myoblasts also expressed Fn14 on their surface ing antibody ABG.11 and anti-Fn14 blocking antibody P2D3 (Figure 2A and B). As expected, upon switching from regular both failed to block TNF’s ability to inhibit C2C12 myo- growth medium (GM) (containing 15% FBS) to (DM), (con- genesis (Figure 2J and not shown). Based on these obser- taining 2% horse serum), mononuclear proliferating C2C12 vations, we concluded that although both TWEAK and TNF cells enter into a well-characterized differentiation program exhibit similar potent inhibitory effects on C2C12 differentia- that includes cell cycle arrest, fusion of mononuclear cells tion, these two act independently of each other through and formation of multinucleate myotubes (Figure 2C). In the different receptors. presence of TWEAK, however, C2C12 cells largely remained Terminal myogenic differentiation of C2C12 cells requires as a monolayer of mononuclear cells even when placed in exit from the cell cycle and expression of myogenic transcrip- DM (Figure 2D). Both the anti-TWEAK blocking antibody tion factors such as myogenin, which in turn activate the ABG.11 and the hFn14-hFc-soluble TWEAK receptor effec- transcription of an array of muscle-specific genes. To under- tively reversed the inhibition of myotube formation, confirm- stand the molecular events underlying TWEAK’s inhibitory ing the specificity of our observation (Figure 2E and not effect on C2C12 myogenesis, we performed profiling studies shown). Importantly, addition of the anti-Fn14 blocking anti- comparing global gene expression patterns of C2C12 cells AB 10 100 HSMM C2C12 3 80 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 FL1-H Fn14 Fn14 C D E F DM DM+mTWEAK DM+mTWEAK DM+mTWEAK + + ABG.11 P2D3 G H IJ DM+mTNF DM+mTNF DM+mTWEAK DM+mTNF + + + mTNFRI-Fc mTNFRI-Fc ABG.11 Figure 2 The TWEAK receptor Fn14 is expressed by myoblasts, and TWEAK inhibits differentiation of C2C12 cells. (A) FACS showed that most human skeletal muscle myoblasts (HSMM, from Cambrex) expressed Fn14, as well as the lineage marker CD56, on the cell surface. (B) Myoblasts of the mouse C2C12 line also expressed Fn14 on the surface. (C–J) Myotube formation by C2C12 cells in low-serum differentiation medium (DM) was assessed after addition of the following reagents: (C) no addition, myotube formation was extensive, (D) 100 ng/ml murine recombinant TWEAK, myotube formation inhibited; (E) 100 ng/ml mTWEAKþ 10mg/ml anti-TWEAK mAb ABG.11, normal myotubes; (F) 100ng/ml mTWEAKþ 10mg/ml anti-Fn14 mAb P2D3, normal myotubes; (G) 5 ng/ml mTNF; decreased myotubes; (H) 5 ng/ml mTNFþ 10mg/ml mTNFRI-Fc, normal myotubes; (I) 5 ng/ml mTWEAKþ 10mg/ml mTNFRI-Fc to block TNFR1, decreased myotubes; and (J) 5 ng/ml mTNFþ 10mg/ml anti-TWEAK ABG.11, decreased myotubes. Phase contrast at 5 days after switching to DM with indicated additions; images are representative of at least 10 independent experiments. 5828 The EMBO Journal VOL 25 NO 24 2006 &2006 European Molecular Biology Organization | | FL2-H % of Max CD56 TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al cultured in low-serum DM with or without TWEAK, and a are critical for the terminal differentiation of C2C12 cells number of representative genes with significant alteration in and govern the expression of many muscle-specific genes. their expression levels are listed in Figure 3A. Upon analysis, Consequently, the overall expression levels of multiple we noticed that the expression levels of a large number of muscle-related genes, such as the muscle structural proteins genes involved in cell cycle progression (i.e., cyclin D1 and myosin heavy and light chains, as well as the muscle meta- c-myc), DNA/RNA synthesis (i.e., DNA polymerase A and bolic gene carbonic anhydrase, were significantly suppressed TFIIIA) as well as chromosome remodeling (i.e., chromatin by TWEAK treatment (Figure 3A). This suppression of mus- assembly factor) were dramatically increased with TWEAK cle-specific gene expression by TWEAK was accompanied by stimulation, suggesting that these cells were mitotically drastic phenotype differences between the TWEAK-treated active (Figure 3A). Using BrdU labeling, we confirmed that and untreated cells. Whereas the majority of TWEAK-treated most C2C12 cells in DM in the absence of TWEAK are C2C12 cells maintained the undifferentiated mononuclear nonmitotic as expected. However, TWEAK-treated C2C12 morphology with few actin filaments even in low-serum cells were almost all BrdU positive, indicating that TWEAK DM, the untreated C2C12 cells fully differentiated and stimulation potently promoted proliferation of these C2C12 became fused multinucleated myotubes, as revealed by cells even under low-serum condition (Figure 3B). Given that DAPI and phalloidin staining (Figure 3D). withdrawal from cell cycle is a well-established prerequisite for C2C12 terminal differentiation (Walsh and Perlman, Generation of Fn14-deficient mice 1997), it is likely that the perturbation of cell cycle arrest The profound effect of TWEAK on the established myoblast in these cells by TWEAK is responsible for the blockade in line C2C12 suggested that the TWEAK/Fn14 pathway may C2C12 myogenesis. The failure in myogenic progression in indeed be an important pathway regulating progenitor cell the presence of TWEAK was reflected at the molecular level biology. To better understand the physiological relevance of by the decreased expression, based on both DNA array and this pathway, we decided to generate mice deficient in the Western blot analyses (Figure 3A and C), of the muscle- TWEAK receptor Fn14. Using the standard gene-targeting specific transcription factors myogenin and MyoD, which strategy, an B10-kb Kpn1 genomic DNA fragment containing AB Fold change Genes Day 1 s.d. Day 2 s.d. M-ras 8.6 3.3 10.3 1.9 c-myc 8 2.6 5.4 0.3 cdc25c 4.2 1.3 3.4 0.7 cdc45 20 3.7 25 1.3 DM (day 3) DM + Fc-TWEAK (day 3) Cyclin A2 2.8 0.2 3.4 0.8 Cell cycle- Cyclin D1 2.5 0.1 3.2 0.2 related C GM DM (day 5) Ki67 2.9 0.5 2.9 0.4 None hTNFh TWEAK Transcription factor IIIA 4.7 1.0 4.4 1.0 DNA polymerase α 9.6 0.8 3.2 0.6 Chromatin-assembly factor 17.8 3.6 14.3 3.2 Helicase(lsh) 5.7 1.1 5.0 0.1 Myogenin – 3.2 0.6 – 2.8 0.4 Myogenic Myogenin myoD – 3.4 0.2 – 4.2 0.4 TFs Notch-1 – 18.8 2.4 –30 4.7 MHC – 8.5 0.6 –6 0.5 MLC1 –6 0.1 – 3.2 0.1 Muscle- related- α1 skeletal actin –4 0.3 –3 0.1 genes Carbonic anhydrase 3 – 48.2 3.9 – 5 2.5 0.8 IGFII –42 5.3 – 112 6 MCP-1 140 37 187 97 RANTES 75.3 15 202 8.7 Chemo- kines MIP-1 75.1 1.5 109 4.7 MCP-3 64.2 6.3 17.4 2.4 DM (day 5) DM + Fc-TWEAK (day 5) Figure 3 TWEAK blocks the myogenic program and prevents cell cycle arrest in C2C12 cells. (A) TWEAK induced expression level changes in many genes important for cell cycle control and myogenic differentiation,for example, the muscle-specific transcription factors myogenin and MyoD. Fold changes and s.d.s were calculated using data from duplicate samples that passed statistical tests. (B) C2C12 cells continued to proliferate in low-serum DM when treated with TWEAK as shown by the much larger percentage of cells that incorporated BrdU in TWEAK- treated than in untreated cultures. Cells were analyzed after culture in DM for 3 days with or without 100 ng/ml of Fc-TWEAK and after 12 h BrdU labeling. (C) Myogenin protein was much lower after TWEAK treatment as shown by immunoblots of C2C12 cells in DM in the absence (none) or presence of Fc-hTWEAK (hTweak, 100 ng/ml) or hTNFa (10 ng/ml). (D) Staining of actin filaments by fluorescent phalloidin (red) was decreased after TWEAK treatment of C2C12 cells cultured in DM for 5 days with or without 100 ng/ml of Fc-TWEAK as indicated. Nuclei were identified by DAPI stain (blue). &2006 European Molecular Biology Organization The EMBO Journal VOL 25 NO 24 2006 5829 | | TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al the entire mouse Fn14 gene was isolated and a targeting clones appears to be nonspecific, as we could not compete vector was designed to delete the first two exons (encoding it off using the soluble receptor Fn14-Fc (not shown). amino acids 1–66), which contain the entire extracellular ligand-binding domain of Fn14 (Supplementary Figure S1a). TWEAK/Fn14 pathway regulates the proliferative Embryonic stem cell clones undergoing successful homo- potential of primary mouse muscle myoblasts logous recombination (8 out of 136) were identified by both Our in vitro C2C12 results strongly suggested that the genomic Southern blot and PCR analysis (Supplementary TWEAK/Fn14 pathway might also regulate the properties of Figure S1a and data not shown) and injected into the primary myogenic precursor cells such as muscle satellite C57BL/6 blastocysts to generate chimeras. Homozygous cells and their committed myoblast progeny. Following con- Fn14 null mice were obtained through standard breeding firmation that more than 95% of cells in our primary myo- schemes (see Materials and methods). Homozygous blasts preparation do indeed express Fn14 on the surface (not Fn14 mice appear normal and do not have any obvious shown), we examined the direct effect of TWEAK on the developmental defect in muscle or other tissues (Supple- growth and differentiation characteristics of mouse primary mentary Figure S1e and unpublished data). To confirm the myoblasts isolated from the limb muscles of 6 to 8-week-old presence of a null mutation, we derived wild-type, hetero- mice. In GM, addition of TWEAK resulted in an enhanced zygous and homozygous mouse embryonic fibroblast (MEF) proliferation of the primary myoblasts as reflected by the clones and assayed for both Fn14 mRNA and protein surface presence after 3 days of growth of B2 more mononuclear expression. As shown in Supplementary Figure S1b and c, myoblasts in TWEAK-treated than in untreated cultures both Northern blot and RT–PCR analysis clearly indicated (Figure 4A and B). The proliferative effect of TWEAK was the lack of Fn14 mRNA in Fn14 MEF clones. Importantly, specific because the proliferation rate returned to basal level FACS staining also demonstrated that TWEAK no longer upon treatment with the TWEAK-neutralizing reagents anti- bound to individually derived Fn14 clones (Supple- TWEAK antibody or Fn14-Fc (Figure 4C and not shown). mentary Figure S1d), confirming the lack of Fn14 on the Inclusion of TWEAK in the DM of primary myoblasts, on the surface of these cells and that Fn14 is the only receptor other hand, resulted in significantly reduced myotube forma- for TWEAK. The residual low-frequency binding detected tion and the appearance of dying cells (Figure 4D and E). This / þ /þ by Fc-TWEAK in Fn14 (also seen in Fn14 ) MEF cell death response of primary myoblasts was quite different A D F Exp. no. 1 WT 1 and 2 KO 1 and 2 Growth medium Differentiation medium (day 4) 0 5 10 15 8 Exp. no. 2 B E WT 3 and 4 KO 3 and 4 0 5 10 15 Days GM+Fc-TWEAK (100 ng/ml) DM +Fc-TWEAK (day 4) C G H –/– GM+Fc-TWEAK (100 ng/ml) Wild type Fn14 Fn14-Fc Figure 4 The TWEAK/Fn14 pathway regulates proliferation and differentiation of primary muscle myoblasts. TWEAK promoted the growth (A–C) and inhibited the differentiation (D, E) of primary myoblasts from wild-type mice. (A–C) After 3 days of proliferation in growth medium, cultures of primary myoblasts treated with 100 ng/ml Fc-TWEAK (B) containedB2 more cells than untreated cultures (A) or cultures treated with both Fc-TWEAK and Fn14-Fc to block receptor function (C). (D, E) After 4 days in low-serum differentiation medium, untreated cultures of primary myoblasts formed multinucleate myotubes (D), whereas myotubes were rarely found in TWEAK-treated cultures (E). (F) Primary myoblasts isolated from Fn14-deficient mice produced fewer progeny than wild-type myoblasts in culture. Results from two independent experiments (exp. no. 1 and exp. no. 2) are shown. In each experiment, myoblasts were obtained from two individual wild-type (WT) and two individual Fn14 (KO) mice. Cells were seeded at the same initial cell densities. (G, H) Phase-contrast images of myotubes formed by myoblasts isolated from WT and Fn14-deficient mice. 5830 The EMBO Journal VOL 25 NO 24 2006 &2006 European Molecular Biology Organization | | Cell doublings TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al from the response of C2C12 myoblasts, which remained alive damaged areas of wild-type muscles (Figure 5C, I and M) and and capable of proliferation in the TWEAK-containing DM TWEAK mRNA to approximatly the same level in Fn14 as for at least a week. Although we do not yet understand the in wild-type muscles (Figure 5D and M). The similar increase precise reason(s) for the different responses of C2C12 and in TWEAK mRNA in wild-type and Fn14 muscles at 3 primary muscle cells, it is known that immortalized myo- days after cardiotoxin treatment was confirmed by quantita- genic cell lines have mutations in cell cycle regulatory genes tive PCR (not shown). By day 5, TWEAK expression had (Nowak et al, 2004); so it is not surprising that primary cells subsided considerably in wild-type muscles, whereas the would behave differently than an established cell line in both level of Fn14 remained as high at day 5 as at day 3 (Figure proliferative potential and susceptibility to apoptosis. 5E, K and M). The finding that increased TWEAK expression Primary myoblasts isolated from Fn14-deficient mice pro- persisted for a longer period of time in injured muscles of duced fewer viable progeny in culture than did myoblasts Fn14 mice than in wild-type mice (Figure 5E versus F) from wild-type mice (Figure 4F). To rule out the possibility suggests that inactivation of the TWEAK receptor altered the that the proliferation of wild-type myoblasts was ‘artificially’ injury response to cardiotoxin in the Fn14-deficient mice. enhanced by exogenous TWEAK from the chicken extracts Two lines of evidence suggested that macrophages were used in the culture media or TWEAK produced by myoblasts the major source of the increased TWEAK expression in in an autocrine manner, we included the soluble Fn14-Fc injured wild-type and Fn14 muscles. First, bright-field decoy receptor in the wild-type myoblasts culture and analysis revealed a clear association of TWEAK expression observed no reduction in the number of progeny (data not with infiltrating inflammatory cells, whereas Fn14 expression shown). Based on these results, we conclude that the seemed to adopt a much more diffuse pattern (data not TWEAK/Fn14 pathway is intrinsically required for the full shown). Second, using a quantitative PCR assay, we found proliferative potential of mouse myoblasts. Interestingly, that macrophages (i.e., Mac-1-positive cells) isolated from while Fn14-deficient myoblasts retained the ability to differ- both wild-type and Fn14 TA muscles at 3 days after entiate into myotubes when cultured in low-serum DM, we cardiotoxin injection expressed relatively large amounts of have consistently noticed morphological differences in myo- TWEAK mRNA (Figure 5N). In contrast, very little TWEAK tubes fused from wild-type versus Fn14 myoblasts in that mRNA was found in either the non-macrophage cell fraction the Fn14 myoblasts seemed to form thicker myotubes (i.e., Mac-1-negative cells) or in myogenic cells purified by upon fusion (Figure 4G and H). The underlying molecular Percoll gradient fractionation (Figure 5N). basis for this difference is not yet known. To begin to determine if signaling by additional members of the TNF superfamily might also be altered in FN14- deficient compared with wild-type mice, we carried out TWEAK/Fn14 pathway participates in muscle quantitative PCR measurements to measure in regenerating regeneration in vivo Muscle satellite cells are adult muscle precursor cells residing TA muscles (n¼ 3) the relative levels of the mRNAs encoding quiescently under the basal lamina of the muscle fiber. two TNF family ligands, TNFa and EDA (ectodysplasin), and their respective receptors (TNFR1, TNFR2, and XEDAR), as Although not required for early muscle development, these these two TNF ligand/receptor pathways have previously cells are believed to be the main, if not the only, contributor been suggested to play a role in muscle regeneration to muscle regeneration in response to damage or increase in (Guttridge et al, 2000; Newton et al, 2004). As shown in workload. Upon activation, satellite cells are capable of proliferating to produce committed myoblasts as progeny, Figure 5O, we indeed found that the levels of these mRNAs which then repair injured muscle either by fusing with were significantly induced following cardiotoxin-induced in- injured pre-existing myofibers or by forming entirely new jury, but did not differ significantly between the wild-type and Fn14-deficient muscles at 3 days, although there seems myofibers (Hawke and Garry, 2001). Although histologically to be a trend of increased expression in the Fn14 knockout well characterized, the stepwise program of muscle regenera- mice, suggestive of potential compensatory mechanism tion is incompletely understood at the molecular level and (see Discussion). a limited number of regulating factors have been identified to date (Morgan and Partridge, 2003). Based on our culture results, we hypothesized that the TWEAK/Fn14 pathway Defective inflammatory response and muscle might be a novel regulator of muscle regeneration in vivo, regeneration in Fn14 mice following cardiotoxin- and we tested this idea by comparing regeneration of wild- induced muscle injury type versus Fn14 muscle in vivo following injury induced We next assessed muscle regeneration in response to cardio- by the snake venom cardiotoxin (Couteaux et al, 1988). toxin injury in both wild-type and Fn14 knockout mice using We first examined the expression patterns of TWEAK and histological analysis. Similar to previous reports, cardiotoxin Fn14 mRNAs following cardiotoxin injection in the tibialis injection in the TA muscles caused considerable damage anterior (TA) muscle of wild-type and Fn14 mice using to muscle fibers, triggering a robust regenerative process in situ hybrization analysis. The levels of both TWEAK and that resulted in almost complete regeneration of wild-type Fn14 were minimal in the undamaged wild-type muscle muscles by 2 weeks after injury (Figure 6A–D). When mice tissue (Figure 5A and G, quantified in Figure 5M), and deficient in Fn14 were injected with cardiotoxin, however, TWEAK mRNA expression was also low in undamaged regeneration appeared to be delayed compared with that seen Fn14 muscle (Figure 5B). As expected, Fn14 mRNA was in wild-type muscles (Figure 6E–H). For example, large essentially undetectable in FN14 muscles (Figure 5H, J, L numbers of newly formed muscle fibers with their ‘signature’ and M). centrally located nuclei appeared in TA muscles of wild-type Three days after cardiotoxin injury, expression levels of mice at 5 and 7 days following cardiotoxin injection, whereas both TWEAK and Fn14 were significantly increased in the such regenerated myofibers were rare in Fn14 mice at &2006 European Molecular Biology Organization The EMBO Journal VOL 25 NO 24 2006 5831 | | TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al –/– –/– Wild type Fn14 Wild type Fn14 A BH G No inj. C D I J Day 3 EF K L Day 5 TWEAK in situ Fn14 in situ M O TWEAK mRNA Fn14 mRNA 0.0020 TNF 0.00020 EDA 0.0015 0.00015 0.0010 0.00010 0.0005 0.00005 0.0000 0.00000 500 0.075 TNFR1 XEDAR 0.000035 0.000030 0.050 0.000025 0.000020 Days after 03 5 03 5 03 5 0.025 0.000015 injury: –/– 0.000010 Genotype: Wild type Fn14 Wild type 0.000 0.000005 0.000000 No CTX Day 3 N 0.05 0.007 TNFR2 0.006 Macrophages (Mac-1 ) 0.005 –/– 0.04 Myoblasts Fn14 0.004 0.003 Flow-through cells Wild type 0.03 0.002 0.001 0.000 0.02 No CTX Day 3 0.01 ** ** –/– Wild type Fn14 Figure 5 TWEAK and Fn14 mRNAs increased upon injury, but increased TWEAK persists longer in Fn14 than in wild-type muscles and is produced by macrophages. (A–L) In situ hybridzation of TWEAK (A–F) and Fn14 (G–L) mRNAs in control (no inj) and cardiotoxin-treated wild-type and Fn14 muscles at 3 or 5 days post injection as indicated. Scale bar in (A) represents 100mm. (M) At day 3 after cardiotoxin treatment, quantification of grain density showed that TWEAK mRNA was significantly (Po0.01, n¼ 4) increased several-fold in both wild- / / type (gray bars) and Fn14 (blue bars) muscles. At day 5, in constrast, TWEAK persisted at a high level in Fn14 muscles, but returned to near the initial level in wild-type muscles. Fn14 mRNA was also increased by injury at days 3 and 5 in wild-type muscles (green bars, right panel). Error bars¼ s.d. No Fn14 signal above background was found in Fn14 muscles (not shown). (N) Macrophages are the major source of TWEAK expression in regenerating muscles. At 3 days after cardiotoxin, TA muscles (n¼ 3) were dissociated into single-cell suspensions and three cell preparations were made: (i) cells positive for the macrophage marker Mac-1 were purified using Mac-1 magnetic beads (macrophages, blue bars); (ii) myogenic cells were purified on a Percoll gradient (myoblasts, red bars); and (iii) Mac-1-negative cells, which were cells that did not bind to the Mac-1 beads (flow-through, green bars). RNA was isolated from each cell fraction and quantitative PCR was used to measure the amount of TWEAK mRNA in each cell type using an equal amount of input RNA and normalized versus the amount of GAPDH mRNA. TWEAK mRNA expression was significantly higher (*Po0.05, n¼ 3) in macrophages than in myoblasts or flow- through cells. Error bars¼ s.d. (O) mRNA levels of TNF/TNFRs and EDA/XEDAR with and without cardiotoxin in wild-type and Fn14 mice. At 3 days after cardiotoxin (CTX), total RNA was isolated from TA muscles (n¼ 3) and quantitative PCR was used to measure the mRNA levels for TNF, TNFR1, TNFR2, EDA and XEDAR. Statistical significance was not achieved between wild-type and Fn14-deficient mice. 5832 The EMBO Journal VOL 25 NO 24 2006 &2006 European Molecular Biology Organization | | TWEAK mRNA level Signal intensity (normalized) (grains/mm ) 1 TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al –/– –/– Wild type Fn14 Wild type Fn14 A E I J H&E Day 3 (day 5) B F LM eMyHC (day 4) Day 5 Wild type C G 1000 –/– Fn14 ** Day 7 ** D H Day 5 Day 7 Day 14 Day 14 (n = 3) (n = 3) (n = 2) Figure 6 Fn14 mice have delayed muscle regeneration. Cardiotoxin-treated muscles at indicated days after injury of wild-type (A–D) and Fn14 (E–H) mice are shown. Detailed views of B and F are shown in (I) and (J) respectively to show regenerating, central nucleate myotubes, some of which are indicated by arrows. Images are representative of three independent experiments with seven mice per genotype in total. Scale bar: 200mm (A–C, E–G), 100mm (D, H). (K) The number of central nucleated myotubes in Fn14 mice was significantly lower than in wild-type muscles at 5 and 7 days, but not 14 days, after cardiotoxin. Error bars¼ s.d.; **¼ Po0.01 by Student’s t-test; n as indicated. (L) Myofibers that express embryonic myosin heavy chain (eMyHC), an additional marker of regenerating myotubes (dark brown stain), were more abundant in wild-type than in Fn14 muscles at 4 days after injury. these times (Figure 6B, C, F and G). By day 14, the regene- With the interplay between inflammation and tissue rative process appeared to be nearly complete in wild-type regeneration in mind, we explored the inflammatory res- mice as shown by the clearance of inflammation and the ponse following cardiotoxin in wild-type and Fn14 mice. replacement of damaged myofibers by the newly formed Massive infiltration of inflammatory cells, mainly macro- central nucleate fibers. In Fn14 mice, however, signs of phages and neutrophils, is an early hallmark event of cardi- residual muscle damage remained along with regenerated otoxin-induced muscle regeneration which peaks around fibers, and some inflammatory cells also persisted (Figure 6D 3 days after injection (Figure 7A). In Fn14 mice injected and H). with cardiotoxin, H&E staining revealed not only a signifi- To further analyze the delayed regeneration in Fn14 cantly reduced presence of such infiltrates at day 3, but also compared with wild-type muscles, we quantified the number a 2-day delay of peak infiltration in Fn14 muscles to day of central nucleate myofibers and examined expression of the 5 (Figure 6E and F). This observation was consistent with embryonic isoform of myosin heavy chain (eMyHC) at different our in situ analysis, which showed that TWEAK mRNA stages of regeneration. Myofibers in adult animals that have (likely produced by infiltrating inflammatory cells) persisted centrally located nuclei or that express eMyHC are known to be at 5 days post injury in Fn14 but not in wild-type, mice in the process of regeneration (e.g., DiMario et al, 1991; Pavlath (Figure 5E, F and M). et al, 2003). Confirming that regeneration was delayed in the To additionally assess, both qualitatively and quantita- / / Fn14 muscles, central nucleate myofibers were significantly tively, the altered inflammatory response seen in Fn14 less abundant in regenerating Fn14 than in regenerating mice, we dissociated TA muscles using pronase digestion at wild-type muscles at both 5 and 7 days after injury (Figure different time points following cardiotoxin injection, and we 6I–K). At 14 days after injury, in contrast, the Fn14 and carried out FACS analyses to identify inflammatory cells in wild-type muscles had similar numbers of central nucleate the resulting single-cell suspensions. FACS analyses showed fibers. Furthermore, fibers that expressd eMyHC were less that very few inflammatory cells were present in uninjured / / abundant in Fn14 muscles than in wild-type muscles at TA muscles in either wild-type or Fn14 mice, as assessed 4 days after injury (Figure 6L and M) and similar results by double staining with Mac-1 and Gr-1 (Figure 7A), which were seen at 5 and 7 days after injury (not shown). are markers for myeloid lineage inflammatory cells including &2006 European Molecular Biology Organization The EMBO Journal VOL 25 NO 24 2006 5833 | | Central nucleated fibers/mm TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al No injection CTX (day 2) CTX (day 3) 4 4 10 10 10 0.14 0.95 0.12 8.34 0.17 14.6 3 3 10 10 2 2 2 Wild type 10 10 1 1 1 10 10 10 0.41 98.5 81.4 10.1 71.6 13.6 0 0 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 FL2-H FL2-H FL2-H 4 4 4 10 10 10 0.33 1.16 0.19 5.93 0.087 6.48 3 3 10 10 10 –/– 2 2 2 Fn14 10 10 10 1 1 1 10 10 7.99 98 0.49 85.9 81.8 11.7 0 0 0 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 FL2-H FL2-H FL2-H Gr-1 0.045 Fn14 KO F4/80 0.040 Wild type 0.035 B D 0.030 0.025 0.020 0.015 0.010 0.005 Wild type 0.000 0.003 0.002 C E 0.001 0.000 0.015 –/– Fn14 0.010 0.005 0.000 No CTX Day 1 Day 3 Figure 7 Altered inflammatory response in Fn14 muscles in response to cardiotoxin (CTX) injury. (A) FACS based on myeloid markers Mac-1 and Gr-1 showed that inflammatory cells were more abundant in wild-type than in Fn14 muscles at 2 and 3 days post injury. Slightly more cells were consistently recovered from wild-type than Fn14 mice at all time points; the same number of cells (10 000) are shown in each dot plot. For each genotype, cells from two mice were pooled for each time point and the results shown are representative of three independent experiments. (B–E) F4/80 expression (brown staining) of muscle tissue cross-sections shows higher numbers of inflammatory cells in wild-type (B) than in Fn14 (C) muscles on day 3 following cardiotoxin. Detailed views of (B) and (C) are shown in (D) and (E), respectively. Scale bar: 100mm (B, C), 50mm (D, E). (F) mRNA levels of MCP-1, MCP-3 and RANTES with and without cardiotoxin injection in wild-type and Fn14-deficient mice. At 1 and 3 days after cardiotoxin, total RNA was isolated from TA muscles (n¼ 3) and quantitative PCR was used to quantify mRNAs. Asterisk indicates that the difference for MCP-3 expression at day 1 reached statistical significance (Po0.05, by Student’s t-test). monocytes and neutrophils (Lagasse and Weissman, 1996). where the percentage in wild-type TA was more than double Following cardiotoxin injection, wild-type muscles showed that in the Fn14 TA at 3 days after injury (14.6 versus þ þ a very large increase of Mac-1 /Gr-1 infiltrating cells with 6.5%). We further confirmed our observation histologically the percentage increasing from roughly 1% of the cells in by staining with the more specific monocyte/macrophage uninjured muscles to 28% of the cells at day 3 after injury. marker F4/80. On day 3, extensive F4/80 infiltrates were Infiltrating cells also increased in injured Fn14 TA mus- located around the damaged muscle fibers in the wild-type cles, but the increase was smaller than in wild-type muscles, muscles (Figure 7B and D). In Fn14 mice, however, the þ þ þ as Mac-1 /Gr-1 cells accounted for less than 20% of the number of F4/80 infiltrating cells was much less than in total isolated cells on day 3 from injured Fn14 TA muscles. wild-type mice at day 3 (Figure 7C and E). The difference between inflammation in wild-type and Because chemokines play a critical role in regulating the Fn14 muscles was even more pronounced in the Mac- recruitment of inflammatory cells and TWEAK can potently hi þ 1 /Gr-1 subpopulation (marking monocytes/macrophages induce chemokine production from C2C12 cells based on our in a more activated state; Lagasse and Weissman, 1996), profiling study (Figure 3), we conducted quantitative RT–PCR 5834 The EMBO Journal VOL 25 NO 24 2006 &2006 European Molecular Biology Organization | | Mac-1 FL1-H FL1-H FL1-H FL1-H MCP-3 MCP-1 FL1-H FL1-H RANTES TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al experiments to determine the temporal expression of several We found that activation of the TWEAK/Fn14 pathway key chemokines following cardiotoxin injection in both inhibited differentiation and increased proliferation of both wild-type and Fn14 knockout mice. As shown in Figure 7F, the immortalized C2C12 muscle cell line and primary myo- RANTES does not seem to play a key role in the immediate genic cells. Our finding is consistent with previous demon- early phase following cardiotoxin injection, as no increase in stration that activation of the NF-kB pathway could drive cell its expression level was seen after day 1. However, the levels proliferation and inhibit C2C12 differentiation through reg- of MCP-1 were increased comparably in both wild-type and ulation of cyclin D1 (Guttridge et al, 1999). While our paper Fn14 mice, whereas the expression of MCP-3 was signifi- was under initial review, Dogra et al (2006) published their cantly lower in the absence of Fn14 at day 1 post cardiotoxin- study, which also demonstrated that C2C12 differentiation is induced injury, suggesting that MCP-3 might be a key inhibited by TWEAK, and that TWEAK activates the NF-kB mediator for the recruitment of inflammatory infiltrates in pathway and decreases the amount of MyoD. The results of this model. Not surprisingly, the levels of MCP-3, MCP-1 and Dogra et al are consistent with ours, and we additionally RANTES all trended higher in Fn14 mice at day 3 com- demonstrated the effect of TWEAK on different types of pared with wild-type mice, likely contributing to the delayed mesenchymal progenitor cells, identified differences between inflammation in these mice. the responses of C2C12 and primary muscle cells to TWEAK, prepared Fn14 mice to inactivate the TWEAK pathway and identified a significant role for the TWEAK/ Discussion Fn14 pathway in regulating skeletal muscle regeneration after In this study, we identified mesenchymal precursor cells as a injury in vivo. novel target cell type for the inflammatory cytokine TWEAK, Identification of the TWEAK/Fn14 pathway as a regulator showing Fn14 expression on mesenchymal stem cells and all of muscle myoblast cell proliferation and differentiation following muscle injury in vivo is significant. Previously, a progenitor cells of this lineage. Using muscle myoblasts as an number of soluble factors, such as members of the FGF experimental system, we demonstrated that TWEAK stimu- family, PDGF, TGFb, myostatin, IGF, HGF and TNF, have lated proliferation of primary myoblasts and that Fn14- deficient myoblasts exhibited reduced proliferative capacity been implicated in the proliferation and differentiation of in vitro. In addition, TWEAK altered the differentiating capa- muscle precursor cells based on in vitro studies. Studies using city of both C2C12 and primary skeletal myoblast cells. knockout mice, however, have not always provided unequi- vocal support of the in vitro observations, possibly owing to Furthermore, our in vivo studies demonstrated upregulation the presence of redundant mechanisms in vivo. For example, of TWEAK and Fn14 expression within injured muscle as a well-documented cachetic factor on muscle fibers, TNF tissue and delayed muscle regeneration in Fn14-deficient has been reported to inhibit differentiation, promote proli- animals. Thus, the TWEAK/Fn14 pathway is an important modulator of the proliferative and differentiating potentials of feration, and induce apoptosis of myoblast cells in vitro skeletal muscle progenitor cells. In addition, we also defined (Stewart et al, 2004); however, muscle regeneration occurs normally in TNF mice (Collins and Grounds, 2001), a TWEAK/Fn14 as a new biological pathway that regulates the result that is in marked contrast to the delayed regeneration properties of mesenchymal stem cells, herein showing in mice lacking the TWEAK receptor Fn14 (this study). A TWEAK-induced NF-kB activation, as well as expression of further complication comes from the fact that most of these pro-survival, pro-proliferative and cell adhesion genes. The past few years have seen tremendous progress with factors also exert potent effects on many other cell types respect to understanding the potential of mesenchymal including mature muscle fibers, making interpretation of progenitors in tissue repair and regeneration. However, their direct effect on muscle precursor cells from in vivo studies less straightforward. Based on our in situ data, our relatively poor understanding of factors governing their Fn14 expression in quiescent muscle tissue seems to be survival, homing, proliferation and differentiation presents very low, but drastically increases following cardiotoxin- a major challenge for a successful transition into clinical induced muscle damage. Coupled with the observation that applications. In addition to TWEAK’s proliferative effect on muscle myoblasts, we have also observed that TWEAK could no apparent developmental defect in the muscle tissues was potently inhibit the terminal differentiation of human osteo- seen in either the Fn14 knockout mice or TWEAK over- expressing transgenic mice (LC Burkly, unpublished results), blast precursors, as well as adipogenesis and chondrogenesis it appears that TWEAK/Fn14 is a unique pathway that of human mesenchymal stem cells in culture (B Browning becomes specifically engaged in muscle myoblast prolifera- and TS Zheng, unpublished results). Moreover, our profiling tion only during injury settings. Supporting this hypothesis, study indicated that TWEAK could induce the expression of antiapoptotic as well as cell adhesion molecules in mesench- dramatic upregulation of Fn14 mRNA was also observed in ymal stem cells, thus potentially affecting their homing and the denervated muscle tissues of the mutant SOD transgenic model of ALS (J Lincecum and S Perrin, unpublished results). survival capacity in vivo. The ability of the TWEAK/Fn14 Although delayed by several days, regeneration in Fn14 pathway to broadly regulate these critical properties of mice following cardiotoxin injury eventually became as com- mesenchymal precursor cells might therefore present an plete as in wild-type muscles, showing that the TWEAK/Fn14 opportunity of ‘TWEAKing’ existing approaches for cell- based regenerative therapies, particularly in skeletal, cardiac pathway is one of the multiple pathways that likely contri- and joint injury settings (Barry, 2003). This notion is consis- bute to muscle regeneration. In many biological systems, tent with our recent finding that TWEAK also potentiates wholly or partially redundant mechanisms are commonly found. One example is the transcriptional regulation of the proliferation of liver progenitor cells (Fausto, 2005; myogenesis by MyoD and Myf-5 during development Jakubowski et al, 2005), suggesting a fundamental involve- (Rudnicki et al, 1993). Although we clearly demonstrated ment of this pathway in modulating progenitor cell behavior. &2006 European Molecular Biology Organization The EMBO Journal VOL 25 NO 24 2006 5835 | | TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al that TWEAK and TNF could inhibit C2C12 myogenesis in Fn14 mice compared with wild-type mice (data not independent of each other in vitro, it is still tempting to shown) using a pulsing protocol that is reported to label only speculate that TWEAK and TNF could synergize with myoblasts (Yan et al, 2003), we were unable to demonstrate and potentially compensate for each other in vivo, given experimentally that those BrdU-labeled cells were all that they both are potent activators of the NF-kB pathway. myoblasts. As a result, we cannot conclude with absolute In fact, it has been previously shown that TWEAK and TNF certainty that myoblast proliferation was directly affected could synergistically induce the production of chemokines in cardiotoxin-injured Fn14 mice. The second possible and cytokines in dermal fibroblasts (Chicheportiche et al, mechanism for delayed muscle regeneration could be due 2002). The intriguing possibility of such synergism between to the reduced inflammatory response in Fn14 mice, as TNF and TWEAK in regulating muscle regeneration and other inflammation could certainly contribute positively to efficient biological systems in vivo remains to be investigated. In muscle regeneration by a number of means, including addition, although not statistically significant, there seems removal of debris from damaged fibers and secretion of to be some level of compensatory activation of the TNF/ soluble factors that promote muscle regeneration. TNFR and EDA/XEDAR pathways in Fn14 knockout mice Although seemingly independent events, the proliferation/ following cardiotoxin injury according to our quantitative activation of myoblasts and inflammation during muscle RT–PCR analysis, a finding that reinforces the complex regeneration might actually be tightly coupled, as suggested and often redundant nature of many biological responses. by the compelling evidence for active crosstalks between However, our study clearly shows that TWEAK and TNF are muscle precursor cells and inflammatory infiltrates in a two independent ligand/receptor pathways important for the recent study (Chazaud et al, 2003). We believe that the regulation of biological properties of mesenchymal and TWEAK/Fn14 pathway could very well be a mediator of muscle cells; our study is therefore consistent with the such cellular interactions and one can envision a scenario results from a complete survey of binding between TNF of how the TWEAK/Fn14 pathway may function as a positive family receptors and ligands, which showed that TWEAK regulator of muscle precursor cell properties during injury- bound only to Fn14 and TNF bound only to TNFR1 and triggered muscle regeneration (Figure 8). Following cardio- TNFR2 (Bossen et al, 2006). toxin injection, necrotic muscle fibers can release a number The mechanism by which Fn14 deficiency resulted in of soluble factors, including chemokines that will recruit delayed muscle regeneration following cardiotoxin injection neutrophils and macrophages (Tsivitse et al, 2005), as well is likely two-fold. First, based on our in vitro observations as FGFs (D’Amore, 1990), which would induce Fn14 expres- with the C2C12 line and isolated primary muscle myoblasts, sion on muscle precursor cells. As a result, TWEAK produced it is likely that, similar to the reduced proliferative capacity by the early inflammatory infiltrates would engage Fn14- seen with isolated Fn14 myoblasts in culture, the expan- expressing muscle precursor cells, resulting in not only sion of myoblasts triggered by cardiotoxin was less than their proliferation, but also enhanced production of chemo- optimal in Fn14 mice, resulting in delayed muscle regene- kines important for recruitment of additional inflammatory ration. Although we did observe delayed BrdU incorporation cells that are beneficial to the repair response, thus creating Macrophage recruitment Macrophage Myofiber CTX Chemokines TWEAK Chemokines Fn14 FGFs, etc. Quiescent Activated Myoblast muscle satellite cells muscle satellite cells proliferation/activation Figure 8 A working model for regulation of muscle regeneration by the TWEAK/Fn14 pathway. Cardiotoxin-induced injury triggers release of soluble factors that recruit early inflammatory cells and induce Fn14 expression on muscle precursor cells. Secreted by infiltrating inflammatory cells, TWEAK engages Fn14 and promotes the proliferation and activation of myoblasts, which can in turn contribute to a robust inflammatory response by producing additional chemotactant. The TWEAK/Fn14 pathway therefore positively contributes to muscle regeneration by promoting cellular crosstalk between inflammatory cells and muscle precursor cells. 5836 The EMBO Journal VOL 25 NO 24 2006 &2006 European Molecular Biology Organization | | TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al supplemented with 2% horse serum). Primary muscle satellite a positive feedback loop promoting muscle regeneration. This cells were isolated from limb muscles of 6- to 8-week-old Fn14 notion is consistent with our C2C12 profiling result demon- mice and wild-type control mice (n¼ 4, for each group) as strating that TWEAK induced drastic upregulation of several described (Dominov et al, 1998, 2001; Girgenrath et al, 2005). chemokines critical for macrophage recruitment including See Supplementary data for additional details. Primary myoblasts were induced to differentiate by replacing GM with (DM that MCP-1, MCP-3, RANTES and MIP-1 (Figure 3A), whose consisted of DMEM without chicken embryo extract and with only expressions are significantly induced following cardiotoxin- 2% serum. Human primary cells were all purchased from Cambrex induced muscle injury (Hirata et al, 2003). In fact, our real- (Walkersville, MD) and cultured according to the supplier’s time PCR analysis confirmed that the initial MCP-3 expres- protocol. sion following cardiotoxin injury was much reduced in the Cardiotoxin injection absence of Fn14, and suggests that MCP-3-mediated chemo- Cardiotoxin injection was performed as before (Yan et al, 2003). taxis is an important downstream trigger for the TWEAK/ Briefly, 50ml of 10 mM cardiotoxin (Sigma) was injected into the TA Fn14-induced inflammatory response amplification loop. muscles of the right legs of wild-type or Fn14-deficient mice of 129/sv background, whereas the left legs were used as uninjected It has traditionally been thought that inflammatory res- control. At the indicated time points, TA muscles were harvested ponses following tissue injury are critical for the clearance of and fixed in 4% paraformaldehyde. For BrdU incorporation, mice damaged tissue debris and removal of necrotic/apoptotic were injected intraperitoneally with 1 mg of BrdU 6 hours before cells. A number of more recent studies, however, provided harvesting the muscles. growing evidence, suggesting that inflammation can also Profiling analysis influence tissue repair by directly acting on the regenerating For transcription profiling of TWEAK’s effect on C2C12 cells, cell pool (Mutsaers et al, 2002; Arnett et al, 2003; Monje et al, 1.2 10 C2C12 cells were seeded in 100-mm cell culture dishes in 2003). In our current study, we demonstrated TWEAK’s GM overnight before being switched to differentiation medium. Total RNA from C2C12 cells cultured in GM and DM at different ability to drive the expansion of muscle myoblasts in vitro time points was extracted using TriZol reagent (Invitrogen) and and apparently in vivo in response to injury, presenting a further purified by RNAeasy (Qiagen). Duplicate samples for each concrete example of how a cytokine derived from inflamma- sample point were subjected to profiling using the Affymetrix tory cells can participate in tissue repair and regeneration. mouse chip MG-U74A according to the protocol recommended by the manufacturer. For profiling of TWEAK’s effect on MSCs, 2 10 Given the broad expression of Fn14 on progenitor cells of the cells were cultured in 100-mm cell culture dishes in regular DMEM mesenchymal lineage and its rapid upregulation in many culture medium containing different serum concentrations (10, 2 or injury settings, we propose that the physiological function 0.2%). After overnight culture, TWEAK (100 ng/ml) or heat- of the TWEAK/Fn14 pathway is to participate in acute phase inactivated TWEAK (100ng/ml) were added in triplicate plates for each treatment condition and cells were harvested 18 h later for tissue repair in multiple biological systems by promoting profiling using the Affymetrix human chip U133. progenitor cell proliferation. On the other hand, we have also observed that TWEAK potently inhibited the terminal FACS differentiation of muscle myoblasts, as well as other mesen- Fc-TWEAK binding to and Fn14 surface expression on primary cells were analyzed by FACS staining according to the standard protocol. chymal progenitor cells. One could therefore envision that Briefly, cells were harvested by incubating in 5 mM EDTA/PBS under pathological conditions where inflammatory responses solution at 371C until the cells detached. Cells were suspended in persist, prolonged presence of TWEAK could potentially FACS buffer (PBS with 1% FBS) and stained with either 100 ng/ml block the differentiation of progenitor cells and impede of Fc-TWEAK or 1 mg/ml of ITEM-4 for 45 min, followed by PE- efficient tissue repair. The complex physiological and patho- conjugated goat anti-human or mouse Fc secondary antibodies. Samples were read on a FACS Calibur and analyzed using FlowJo. logical consequences of TWEAK activity suggest that mani- For FACS of muscle dissociates, TA muscles were dissected, fat and pulation of the TWEAK system may present therapeutic connective tissues were removed, and minced muscles were opportunities for cell-based regenerative therapy and in incubated in 0.2 mg/ml Pronase solution (Roche Diagnostics) at various inflammatory diseases such as arthritis and athero- 371C for 1 h. After terminating digestion by washing with DMEM, the dissociates were recovered by sequentially filtering through sclerosis, where extensive tissue remodeling and regene- 100mm and 40mm cell strainers (BD Labware) and used for staining ration occur. with anti-Mac-1 and anti-Gr-1 antibodies. NF-jB activation assay Materials and methods NF-kB activation was measured (n¼ 3) using the TransAM NF-kB p65 assay system (Active Motif) according to the manufacturer’s Proteins and antibodies protocol. The construct for Fc-TWEAK was generated by replacing the Baff For details on quantitative PCR, generation of Fn14 mice, portion of a previously described Fc-Baff construct with a cDNA in situ hybridization and histological analyses, see Supplementary fragment encoding soluble TWEAK. A stable 293T cell line data. expressing the construct was then generated for protein production. The hamster anti-TWEAK antibody ABG.11 and anti-human Fn14 Supplementary data antibody ITEM-4 were generated as previously described (Jaku- Supplementary data are available at The EMBO Journal Online bowski et al, 2002; Nakayama et al, 2003). The blocking anti-Fn14 (http://www.embojournal.org). antibody P2D3 was generated by immunizing Fn14 knockout mice with Fn14-transfected cells. The following antibodies were pur- chased from the indicated sources: anti-mouse TNF G281-2626, PE- Acknowledgements anti-human CD56, FITC-anti-mouse Mac-1 and PE-anti-mouse Gr-1 (BD Biosciences), anti-myogenin F5D (Santa Cruz Biotechnology). We sincerely thank L Evangelisti and S Shulga-Morskaya for em- bryonic stem cell culture and injection; M Wang and J Lincecum for Cell culture in situ analysis; T Crowell and H Gardner for histological analysis; C2C12 cells (ATCC) were seeded at 2 10 cells/well in six-well D Gong for protein purification; D McCrann, J Shearston, S Perrin plates in growth medium (GM; DMEM supplemented with 15% and S Szak for gene profiling analysis. This work was supported in FBS). The following day, cells were washed with PBS once and part by NIH Grant HL39727 (JAW) and Grants AR49496, ES11384 switched to low-serum differentiation medium (DM: DMEM and HL64641 (JBM). &2006 European Molecular Biology Organization The EMBO Journal VOL 25 NO 24 2006 5837 | | TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al References Arnett HA, Mason J, Marino M, Suzuki K, Matsushima GK, Ting JP Hawke TJ, Garry DJ (2001) Myogenic satellite cells: physiology to (2001) TNF alpha promotes proliferation of oligodendrocyte molecular biology. J Appl Physiol 91: 534–551 progenitors and remyelination. Nat Neurosci 4: 1116–1122 Hirata A, Masuda S, Tamura T, Kai K, Ojima K, Fukase A, Motoyoshi Arnett HA, Wang Y, Matsushima GK, Suzuki K, Ting JP (2003) K, Kamakura K, Miyagoe-Suzuki Y, Takeda S (2003) Expression Functional genomic analysis of remyelination reveals importance profiling of cytokines and related genes in regenerating skeletal of inflammation in oligodendrocyte regeneration. J Neurosci 23: muscle after cardiotoxin injection: a role for osteopontin. Am J 9824–9832 Pathol 163: 203–215 Barry FP (2003) Biology and clinical applications of mesenchymal Jakubowski A, Ambrose C, Parr M, Lincecum JM, Wang MZ, Zheng stem cells. Birth Defects Res Part C Embryo Today 69: 250–256 TS, Browning B, Michaelson JS, Baestcher M, Wang B, Bissell Bossen C, Ingold K, Tardivel A, Bodmer JL, Gaide O, Hertig S, DM, Burkly LC (2005) TWEAK induces liver progenitor cell Ambrose C, Tschopp J, Schneider P (2006) Interactions of tumor proliferation. J Clin Invest 115: 2330–2340 necrosis factor (TNF) and TNF receptor family members in the Jakubowski A, Browning B, Lukashev M, Sizing I, Thompson JS, mouse and human. J Biol Chem 281: 13964–13971 Benjamin CD, Hsu YM, Ambrose C, Zheng TS, Burkly LC Brown SA, Richards CM, Hanscom HN, Feng SL, Winkles JA (2003) (2002) Dual role for TWEAK in angiogenic regulation. J Cell Sci The Fn14 cytoplasmic tail binds tumour-necrosis-factor-receptor- 115: 267–274 associated factors 1, 2, 3 and 5 and mediates nuclear factor- Kotter MR, Setzu A, Sim FJ, Van Rooijen N, Franklin RJ (2001) kappaB activation. Biochem J 371: 395–403 Macrophage depletion impairs oligodendrocyte remyelina- Chazaud B, Sonnet C, Lafuste P, Bassez G, Rimaniol AC, Poron F, tion following lysolecithin-induced demyelination. Glia 35: Authier FJ, Dreyfus PA, Gherardi RK (2003) Satellite cells attract 204–212 monocytes and use macrophages as a support to escape apoptosis Lagasse E, Weissman IL (1996) Flow cytometric identification of and enhance muscle growth. J Cell Biol 163: 1133–1143 murine neutrophils and monocytes. J Immunol Methods 197: Chicheportiche Y, Bourdon PR, Xu H, Hsu YM, Scott H, Hession C, 139–150 Garcia I, Browning JL (1997) TWEAK, a new secreted ligand in Lescaudron L, Peltekian E, Fontaine-Perus J, Paulin D, Zampieri M, the tumor necrosis factor family that weakly induces apoptosis. Garcia L, Parrish E (1999) Blood borne macrophages are essential J Biol Chem 272: 32401–32410 for the triggering of muscle regeneration following muscle trans- Chicheportiche Y, Chicheportiche R, Sizing I, Thompson J, plant. Neuromuscul Disord 9: 72–80 Benjamin CB, Ambrose C, Dayer JM (2002) Proinflammatory Locksley RM, Killeen N, Lenardo MJ (2001) The TNF and TNF activity of TWEAK on human dermal fibroblasts and synovio- receptor superfamilies: integrating mammalian biology. Cell 104: cytes: blocking and enhancing effects of anti-TWEAK monoclonal 487–501 antibodies. Arthritis Res 4: 126–133 Luk HW, Noble LJ, Werb Z (2003) Macrophages contribute to the Collins RA, Grounds MD (2001) The role of tumor necrosis factor- maintenance of stable regenerating neurites following peripheral alpha (TNF-alpha) in skeletal muscle regeneration. Studies nerve injury. J Neurosci Res 73: 644–658 in TNF-alpha(/) and TNF-alpha(/)/LT-alpha(/) mice. Lynch CN, Wang YC, Lund JK, Chen YW, Leal JA, Wiley SR (1999) J Histochem Cytochem 49: 989–1001 TWEAK induces angiogenesis and proliferation of endothelial Couteaux R, Mira JC, d’Albis A (1988) Regeneration of muscles after cells. J Biol Chem 274: 8455–8459 cardiotoxin injury. I. cytological aspects. Biol Cell 62: 171–182 Merly F, Lescaudron L, Rouaud T, Crossin F, Gardahaut MF (1999) D’Amore PA (1990) Modes of FGF release in vivo and in vitro. Macrophages enhance muscle satellite cell proliferation and delay Cancer Metast Rev 9: 227–238 their differentiation. Muscle Nerve 22: 724–732 DiMario JX, Uzman A, Strohman RC (1991) Fiber regeneration is not Miller SC, Ito H, Blau HM, Torti FM (1988) Tumor necrosis persistent in dystrophic (MDX) mouse skeletal muscle. Dev Biol factor inhibits human myogenesis in vitro. Mol Cell Biol 8: 148: 314–321 2295–2301 Dogra C, Changotra H, Mohan S, Kumar A (2006) Tumor necrosis Monje ML, Toda H, Palmer TD (2003) Inflammatory blockade factor-like weak inducer of apoptosis inhibits skeletal myogenesis restores adult hippocampal neurogenesis. Science 302: 1760–1765 through sustained activation of nuclear factor-kappaB and degra- Morgan JE, Partridge TA (2003) Muscle satellite cells. Int J Biochem dation of MyoD protein. J Biol Chem 281: 10327–10336 Cell Biol 35: 1151–1156 Dominov JA, Dunn JJ, Miller JB (1998) Bcl-2 expression identifies Mutsaers SE, Whitaker D, Papadimitriou JM (2002) Stimulation of an early stage of myogenesis and promotes clonal expansion of mesothelial cell proliferation by exudate macrophages enhances muscle cells. J Cell Biol 142: 537–544 serosal wound healing in a murine model. Am J Pathol 160: Dominov JA, Houlihan-Kawamoto CA, Swap CJ, Miller JB (2001) 681–692 Pro- and anti-apoptotic members of the Bcl-2 family in skeletal Nakayama M, Ishidoh K, Kojima Y, Harada N, Kominami E, muscle: a distinct role for Bcl-2 in later stages of myogenesis. Dev Okumura K, Yagita H (2003) Fibroblast growth factor-inducible Dyn 220: 18–26 14 mediates multiple pathways of TWEAK-induced cell death. Duffield JS (2003) The inflammatory macrophage: a story of Jekyll J Immunol 170: 341–348 and Hyde. Clin Sci (London) 104: 27–38 Nathan C (2002) Points of control in inflammation. Nature 420: Ekdahl CT, Claasen JH, Bonde S, Kokaia Z, Lindvall O (2003) 846–852 Inflammation is detrimental for neurogenesis in adult brain. Newton K, French DM, Yan M, Frantz GD, Dixit VM (2004) Proc Natl Acad Sci USA 100: 13632–13637 Myodegeneration in EDA-A2 transgenic mice is prevented by Fausto N (2005) Tweaking liver progenitor cells. Nat Med 11: XEDAR deficiency. Mol Cell Biol 24: 1608–1613 1053–1054 Nowak JA, Malowitz J, Girgenrath M, Kostek CA, Kravetz AJ, Feng SL, Guo Y, Factor VM, Thorgeirsson SS, Bell DW, Testa JR, Dominov JA, Miller JB (2004) Immortalization of myogenic Peifley KA, Winkles JA (2000) The Fn14 immediate-early re- cells does not require loss of p16INK4a, p19ARF, or p53 and is sponse gene is induced during liver regeneration and highly accelerated by inactivation of Bax. BMC Cell Biol 5: 1–14 expressed in both human and murine hepatocellular carcinomas. Pavlath GK, Dominov JA, Kegley KM, Miller JB (2003) Regeneration Am J Pathol 156: 1253–1261 of skeletal muscles with altered timing of expression of the Girgenrath M, Kostek CA, Miller JB (2005) Diseased muscles that myogenic bHLH factor MRF4. Am J Pathol 162: 1685–1691 lack dystrophin or laminin-alpha2 have altered compositions and Rudnicki MA, Schnegelsberg PN, Stead RH, Braun T, Arnold HH, proliferation of mononuclear cell populations. BMC Neurol 5: 7 Jaenisch R (1993) MyoD or Myf-5 is required for the formation of Guttridge DC, Albanese C, Reuther JY, Pestell RG, Baldwin Jr AS skeletal muscle. Cell 75: 1351–1359 (1999) NF-kappaB controls cell growth and differentiation Saas P, Boucraut J, Walker PR, Quiquerez AL, Billot M, Desplat-Jego through transcriptional regulation of cyclin D1. Mol Cell Biol S, Chicheportiche Y, Dietrich PY (2000) TWEAK stimulation 19: 5785–5799 of astrocytes and the proinflammatory consequences. Glia 32: Guttridge DC, Mayo MW, Madrid LV, Wang CY, Baldwin Jr AS 102–107 (2000) NF-kappaB-induced loss of MyoD messenger RNA: Stewart CE, Newcomb PV, Holly JM (2004) Multifaceted roles of possible role in muscle decay and cachexia. Science 289: TNF-alpha in myoblast destruction: a multitude of signal trans- 2363–2366 duction pathways. J Cell Physiol 198: 237–247 5838 The EMBO Journal VOL 25 NO 24 2006 &2006 European Molecular Biology Organization | | TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al Szalay K, Razga Z, Duda E (1997) TNF inhibits myogenesis and Wiley SR, Cassiano L, Lofton T, Davis-Smith T, Winkles JA, Lindner downregulates the expression of myogenic regulatory factors V, Liu H, Daniel TO, Smith CA, Fanslow WC (2001) A novel TNF myoD and myogenin. Eur J Cell Biol 74: 391–398 receptor family member binds TWEAK and is implicated in Tsivitse SK, Mylona E, Peterson JM, Gunning WT, Pizza FX (2005) angiogenesis. Immunity 15: 837–846 Mechanical loading and injury induce human myotubes to release Yan Z, Choi S, Liu X, Zhang M, Schageman JJ, Lee SY, Hart R, Lin L, neutrophil chemoattractants. Am J Physiol Cell Physiol 288: C721–729 Thurmond FA, Williams RS (2003) Highly coordinated gene Walsh K, Perlman H (1997) Cell cycle exit upon myogenic differ- regulation in mouse skeletal muscle regeneration. J Biol Chem entiation. Curr Opin Genet Dev 7: 597–602 278: 8826–8836 &2006 European Molecular Biology Organization The EMBO Journal VOL 25 NO 24 2006 5839 | | http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration

Download PDF
14 pages

Loading next page...
 
/lp/springer-journals/tweak-via-its-receptor-fn14-is-a-novel-regulator-of-mesenchymal-PiwqXrP0rr

References (58)

Publisher
Springer Journals
Copyright
Copyright © European Molecular Biology Organization 2006
ISSN
0261-4189
eISSN
1460-2075
DOI
10.1038/sj.emboj.7601441
Publisher site
See Article on Publisher Site

Abstract

The EMBO Journal (2006) 25, 5826–5839 & 2006 European Molecular Biology Organization All Rights Reserved 0261-4189/06 | | T THE H E www.embojournal.org E E EMB M MB BO O O J JO OURN URN A AL L TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration 1 2 The EMBO Journal (2006) 25, 5826–5839. doi:10.1038/ Mahasweta Girgenrath , Shawn Weng , 1 2 sj.emboj.7601441; Published online 23 November 2006 Christine A Kostek ,BethBrowning , 2 3 Subject Categories: signal transduction; differentiation Monica Wang , Sharron AN Brown , 3 2 & death Jeffrey A Winkles , Jennifer S Michaelson , 2 4 Keywords: Fn14; inflammation; muscle; regeneration; Norm Allaire , Pascal Schneider , Martin 2 2 5 TWEAK L Scott , Yen-ming Hsu , Hideo Yagita , 6 1 Richard A Flavell , Jeffrey Boone Miller , 2 2, Linda C Burkly and Timothy S Zheng * 1 2 Boston Biomedical Research Institute, Watertown, MA, USA, Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA, USA, Departments of Introduction Surgery and Physiology, University of Maryland School of Medicine, Baltimore, MD, USA, Department of Biochemistry, University of Inflammation is the first-line defensive mechanism to noxious Lausanne, Ch. Des Boveresses, Epalinges, Switzerland, Department of insults ranging from microbial infections to physical injury Immunology, Juntendo University School of Medicine, Bunkyo-ku, (Nathan, 2002). Although excessive inflammation can have Tokyo, Japan and Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA devastating consequences, in most cases inflammatory responses lead to effective clearance of pathogens and/or Inflammation participates in tissue repair through multi- healing. In fact, depletion of inflammatory cell types such ple mechanisms including directly regulating the cell fate as macrophages has been shown to result in delayed repair of resident progenitor cells critical for successful regenera- response in injury models of a number of tissues, including tion. Upon surveying target cell types of the TNF ligand muscle (Lescaudron et al, 1999), mesothelium (Mutsaers et al, TWEAK, we observed that TWEAK binds to all progenitor 2002), and both the central (Kotter et al, 2001) and peripheral cells of the mesenchymal lineage and induces NF-jB nerve systems (Luk et al, 2003). It is now well established that activation and the expression of pro-survival, pro-prolif- inflammation contributes to tissue repair by clearing debris, erative and homing receptor genes in the mesenchymal engulfment and digestion of apoptotic/necrotic cell bodies, stem cells, suggesting that this pro-inflammatory cytokine epithelial closure and promoting angiogenesis (Nathan, 2002). may play an important role in controlling progenitor cell Much underappreciated until recently, the various cytokines biology. We explored this potential using both the estab- secreted by inflammatory cells also directly influence the lished C2C12 cell line and primary mouse muscle myo- properties of progenitor cells that reside in tissues undergoing blasts, and demonstrated that TWEAK promoted their repair and regeneration (Duffield, 2003). For example, the proliferation and inhibited their terminal differentiation. macrophage-derived cytokine TNF promotes the proliferation By generating mice deficient in the TWEAK receptor Fn14, of oligodendrocyte progenitors after cuprizone-induced de- we further showed that Fn14-deficient primary myoblasts myelination (Arnett et al, 2001). Similarly, soluble factor(s) displayed significantly reduced proliferative capacity and secreted by macrophages can also drive the proliferation of altered myotube formation. Following cardiotoxin injec- mesothelial cells (Mutsaers et al, 2002) and muscle satellite tion, a known trigger for satellite cell-driven skeletal cells (Merly et al, 1999) during mesothelial and muscle regen- muscle regeneration, Fn14-deficient mice exhibited re- eration, respectively. However, it has also been demonstrated duced inflammatory response and delayed muscle fiber that inflammatory cytokines block neural progenitor differen- regeneration compared with wild-type mice. These results tiation (Ekdahl et al, 2003; Monje et al, 2003), suggesting that indicate that the TWEAK/Fn14 pathway is a novel regu- the biological consequence of these inflammatory mediators lator of skeletal muscle precursor cells and illustrate an on tissue repair and regeneration is likely a complex one. important mechanism by which inflammatory cytokines The largely macrophage-derived cytokine TWEAK and its influence tissue regeneration and repair. Coupled with our receptor Fn14 are relatively new additions to the TNF super- recent demonstration that TWEAK potentiates liver pro- family, which includes well-known modulators of cell pro- genitor cell proliferation, the expression of Fn14 on all liferation, differentiation and apoptosis (Locksley et al, 2001). mesenchymal lineage progenitor cells supports a broad Originally identified as a weak inducer of cell death in tumor involvement of this pathway in other tissue injury and cell lines (Chicheportiche et al, 1997), TWEAK was subse- disease settings. quently shown to exert pleiotropic effects on a variety of cell types in vitro, including pro-angiogenic activities on endothe- *Corresponding author. Biogen Idec Inc., 14 Cambridge Center, lial cells (Lynch et al, 1999; Jakubowski et al, 2002) and pro- Cambridge, MA 02142, USA. Tel.: þ 1 617 679 3348; inflammatory activities on epithelial cells (Chicheportiche Fax: þ 1 617 679 3208; E-mail: [email protected] et al, 1997), astrocytes (Saas et al, 2000), dermal fibroblasts and synoviocytes (Chicheportiche et al, 2002). Conspicuously Received: 4 November 2005; accepted: 17 October 2006; published online: 23 November 2006 absent from the TWEAK-responding cell types are lymphocytes, 5826 The EMBO Journal VOL 25 NO 24 2006 &2006 European Molecular Biology Organization | | TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al and Fn14 expression on these cells has not been demonstrated. mesenchymal lineage, including human primary mesenchy- Interestingly, the TWEAK receptor Fn14 is an FGF-inducible mal stem cells, skeletal muscle myoblasts and preadipocytes gene and is highly upregulated in liver injury and regeneration as well as chondrocyte and osteoblast precursors cultured and arterial wounding (Feng et al, 2000; Wiley et al, 2001), in vitro (Figure 1A). We also demonstrated that the TWEAK suggesting a regulatory role in settings of tissue injury and receptor Fn14 was expressed on these progenitor cells by repair. Recently, we demonstrated that the TWEAK/Fn14 path- staining with the anti-human Fn14 monoclonal antibody way is a potent inducer of liver progenitor cell proliferation in ITEM-4 (Figure 1A). To see if these cells would respond to response to chemical injury in vivo (Jakubowski et al, 2005). TWEAK, we examined NF-kB activation in mesenchymal However, the physiological relevance of the TWEAK/Fn14 stem cells and osteoblast precursors upon TWEAK treatment pathway in other contexts remains elusive. as it has previously been shown that Fn14 signal transduction In this study, we explored the potential role of TWEAK/ is mediated through the TRAF-binding site in its cytoplasmic Fn14 pathway in modulating mesenchymal progenitor cell tail (Brown et al, 2003). As shown in Figure 1B, TWEAK biology in vitro and in vivo in a model of skeletal muscle induced robust NF-kB activation in both cell types as mea- injury and repair. We showed that all progenitor cells of sured by the amount of activated p65 present in the cell lysate mesenchymal lineage express the TWEAK receptor Fn14. following TWEAK stimulation using the TranAM assay We further demonstrated that TWEAK promotes proliferation system, indicating that progenitor cells of the mesenchymal of both an established myoblast cell line and primary muscle lineage are indeed TWEAK-responsive cells. The ability of myoblasts. Finally, by generating mice deficient in Fn14, we TWEAK to induce NF-kB activation in these progenitor established that the TWEAK/Fn14 pathway is required for cells is further confirmed by transcription profiling study of optimal muscle regeneration in vivo. mesenchymal stem cells treated with TWEAK. Even in the high serum culture condition (10% FBS), TWEAK induced robust transcriptional upregulation of well-known NF-kB- Results regulated genes, including TRAF1 and 3, NF-kB2 and RelB, which are regulators of NF-kB pathway themselves, and Mesenchymal progenitor cells are a novel target cell type of TWEAK pro-survival genes such as A20 and c-IAP2, as well as cell In our effort to identify in vivo target cell types for TWEAK, adhesion genes such as ICAM-1 and VCAM-1n (Figure 1C). we found that TWEAK binds to progenitor cells of the Importantly, under low (0.2%) or medium (2%) serum 100 100 100 100 hFc-hTWEAK Item-4 Anti-mouse Fc 80 80 80 80 Anti-human Fc 60 60 60 60 40 40 40 40 20 20 20 20 0 0 0 0 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 FL2-H FL2-H FL2-H FL2-H FL2-H hMesenchymal stem cells hMuscle satellite cells hPreadipocytes hChondrocytes hOsteoblast precursors B CD 1.5 Fold change (+TWEAK) Cell cycle-related genes Fold change (+TWEAK)/LS Genes LS MS HS cdc2 5.8 TRAF1 3.3 3.1 8.0 cdc6 7.0 TRAF3 2.6 3.0 11.0 Replication factor C 4.2 NF-κB 3.1 3.3 9.5 DNA topoisomerase II alpha 28.2 0.5 RelB 3.0 2.7 5.8 cdc46 39.3 A20 5.4 11.1 7.6 Survivin 13.8 c-IAP2 2.8 3.1 5.1 cdc20 7.2 ICAM-1 11.2 7.3 8.0 Cyclin A2 6.5 VCAM-1 6.5 3.9 9.2 MAD2 4.9 MCP-1 2.4 2.6 3.0 Figure 1 Human mesenchymal progenitor cells are a novel target cell type for TWEAK. (A) Human primary mesenchymal stem cells, skeletal muscle myoblasts, preadipocytes, chondrocytes and osteoblast precursors (Cambrex) were cultured according to the manufacturer’s protocols. First-passage cells showed staining for TWEAK binding using Fc-TWEAK and for expression of Fn14 using the anti-hFn14 mAb ITEM-4. Anti- mouse and anti-human Fcs were used as negative controls, (B)NF-kB was activated in human mesenchymal stem cells (hMSCs) and osteoblast precursors (hOsteos) following 2 or 6 h of treatment with 100 ng/m TWEAK (Tw). Activation was measured using the TransAM NF-kB p65 activation assay system with cell lysates from normal and TNF-treated HeLa cells serving as negative and positive controls. The assays were carried out in triplicate and the data shown are representative of three independent experiments. (C) List of representative genes induced by TWEAK (100 ng/ml versus heat-inactivated TWEAK 100 ng/ml) in mesenchymal stem cells in low serum (LS: 0.2% FBS), moderate serum (MS: 2% FBS) and high serum (HS: 10% FBS). (D) List of some cell cycle-related genes induced by TWEAK (versus inactivated TWEAK) in mesenchymal stem cells cultured under low-serum conditions (0.2% FBS). Triplicate samples were analyzed for each condition and the fold changes were calculated using averages from triplicates. All fold changes reached statistical significance (Po0.01). &2006 European Molecular Biology Organization The EMBO Journal VOL 25 NO 24 2006 5827 | | HeLa HeLa+TNF hMSCs hMSCs+Tw (2 h) hMSCs+Tw (6 h) hOsteo hOsteos+Tw (2 h) hOsteo+Tw (6 h) NF-κB p65 activation % of Max (OD nm) % of Max % of Max % of Max % of Max TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al conditions, TWEAK also induced the expression of many body P2D3 also was able to reverse the inhibitory effect cell cycle-related genes including cdc2, cyclin A2, survivin, of TWEAK (Figure 2F), demonstrating that the activity of MAD2, among others (Figure 1D and data not shown). These TWEAK on C2C12 cells was mediated through the Fn14 results therefore indicate that TWEAK may regulate cell fate receptor. As it is well documented that TNF is a potent decisions of progenitor cells. inhibitor of myogenesis in vitro (Szalay et al, 1997; Miller et al, 1988), we decided to investigate if there was any TWEAK promotes proliferation and inhibits terminal potential crosstalk between the TNF and TWEAK pathways myogenesis of C2C12 cells in the C2C12 myogenesis model. Although the soluble TNF To further investigate how TWEAK might influence cell fate receptor mTNFRI-Fc totally reversed the inhibition of C2C12 of progenitor cells, we took advantage of the well-established differentiation by TNF (Figure 2G and H), it had no effect on in vitro cell differentiation model of the mesenchymal the inhibitory activity of TWEAK (Figure 2I). A similar result lineage, C2C12 terminal myogenesis. We first confirmed was also seen using anti-TNF or anti-TNFRI blocking anti- that, similar to human skeletal muscle myoblasts, the murine bodies (data not shown). Conversely, the anti-TWEAK block- C2C12 myoblasts also expressed Fn14 on their surface ing antibody ABG.11 and anti-Fn14 blocking antibody P2D3 (Figure 2A and B). As expected, upon switching from regular both failed to block TNF’s ability to inhibit C2C12 myo- growth medium (GM) (containing 15% FBS) to (DM), (con- genesis (Figure 2J and not shown). Based on these obser- taining 2% horse serum), mononuclear proliferating C2C12 vations, we concluded that although both TWEAK and TNF cells enter into a well-characterized differentiation program exhibit similar potent inhibitory effects on C2C12 differentia- that includes cell cycle arrest, fusion of mononuclear cells tion, these two act independently of each other through and formation of multinucleate myotubes (Figure 2C). In the different receptors. presence of TWEAK, however, C2C12 cells largely remained Terminal myogenic differentiation of C2C12 cells requires as a monolayer of mononuclear cells even when placed in exit from the cell cycle and expression of myogenic transcrip- DM (Figure 2D). Both the anti-TWEAK blocking antibody tion factors such as myogenin, which in turn activate the ABG.11 and the hFn14-hFc-soluble TWEAK receptor effec- transcription of an array of muscle-specific genes. To under- tively reversed the inhibition of myotube formation, confirm- stand the molecular events underlying TWEAK’s inhibitory ing the specificity of our observation (Figure 2E and not effect on C2C12 myogenesis, we performed profiling studies shown). Importantly, addition of the anti-Fn14 blocking anti- comparing global gene expression patterns of C2C12 cells AB 10 100 HSMM C2C12 3 80 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 FL1-H Fn14 Fn14 C D E F DM DM+mTWEAK DM+mTWEAK DM+mTWEAK + + ABG.11 P2D3 G H IJ DM+mTNF DM+mTNF DM+mTWEAK DM+mTNF + + + mTNFRI-Fc mTNFRI-Fc ABG.11 Figure 2 The TWEAK receptor Fn14 is expressed by myoblasts, and TWEAK inhibits differentiation of C2C12 cells. (A) FACS showed that most human skeletal muscle myoblasts (HSMM, from Cambrex) expressed Fn14, as well as the lineage marker CD56, on the cell surface. (B) Myoblasts of the mouse C2C12 line also expressed Fn14 on the surface. (C–J) Myotube formation by C2C12 cells in low-serum differentiation medium (DM) was assessed after addition of the following reagents: (C) no addition, myotube formation was extensive, (D) 100 ng/ml murine recombinant TWEAK, myotube formation inhibited; (E) 100 ng/ml mTWEAKþ 10mg/ml anti-TWEAK mAb ABG.11, normal myotubes; (F) 100ng/ml mTWEAKþ 10mg/ml anti-Fn14 mAb P2D3, normal myotubes; (G) 5 ng/ml mTNF; decreased myotubes; (H) 5 ng/ml mTNFþ 10mg/ml mTNFRI-Fc, normal myotubes; (I) 5 ng/ml mTWEAKþ 10mg/ml mTNFRI-Fc to block TNFR1, decreased myotubes; and (J) 5 ng/ml mTNFþ 10mg/ml anti-TWEAK ABG.11, decreased myotubes. Phase contrast at 5 days after switching to DM with indicated additions; images are representative of at least 10 independent experiments. 5828 The EMBO Journal VOL 25 NO 24 2006 &2006 European Molecular Biology Organization | | FL2-H % of Max CD56 TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al cultured in low-serum DM with or without TWEAK, and a are critical for the terminal differentiation of C2C12 cells number of representative genes with significant alteration in and govern the expression of many muscle-specific genes. their expression levels are listed in Figure 3A. Upon analysis, Consequently, the overall expression levels of multiple we noticed that the expression levels of a large number of muscle-related genes, such as the muscle structural proteins genes involved in cell cycle progression (i.e., cyclin D1 and myosin heavy and light chains, as well as the muscle meta- c-myc), DNA/RNA synthesis (i.e., DNA polymerase A and bolic gene carbonic anhydrase, were significantly suppressed TFIIIA) as well as chromosome remodeling (i.e., chromatin by TWEAK treatment (Figure 3A). This suppression of mus- assembly factor) were dramatically increased with TWEAK cle-specific gene expression by TWEAK was accompanied by stimulation, suggesting that these cells were mitotically drastic phenotype differences between the TWEAK-treated active (Figure 3A). Using BrdU labeling, we confirmed that and untreated cells. Whereas the majority of TWEAK-treated most C2C12 cells in DM in the absence of TWEAK are C2C12 cells maintained the undifferentiated mononuclear nonmitotic as expected. However, TWEAK-treated C2C12 morphology with few actin filaments even in low-serum cells were almost all BrdU positive, indicating that TWEAK DM, the untreated C2C12 cells fully differentiated and stimulation potently promoted proliferation of these C2C12 became fused multinucleated myotubes, as revealed by cells even under low-serum condition (Figure 3B). Given that DAPI and phalloidin staining (Figure 3D). withdrawal from cell cycle is a well-established prerequisite for C2C12 terminal differentiation (Walsh and Perlman, Generation of Fn14-deficient mice 1997), it is likely that the perturbation of cell cycle arrest The profound effect of TWEAK on the established myoblast in these cells by TWEAK is responsible for the blockade in line C2C12 suggested that the TWEAK/Fn14 pathway may C2C12 myogenesis. The failure in myogenic progression in indeed be an important pathway regulating progenitor cell the presence of TWEAK was reflected at the molecular level biology. To better understand the physiological relevance of by the decreased expression, based on both DNA array and this pathway, we decided to generate mice deficient in the Western blot analyses (Figure 3A and C), of the muscle- TWEAK receptor Fn14. Using the standard gene-targeting specific transcription factors myogenin and MyoD, which strategy, an B10-kb Kpn1 genomic DNA fragment containing AB Fold change Genes Day 1 s.d. Day 2 s.d. M-ras 8.6 3.3 10.3 1.9 c-myc 8 2.6 5.4 0.3 cdc25c 4.2 1.3 3.4 0.7 cdc45 20 3.7 25 1.3 DM (day 3) DM + Fc-TWEAK (day 3) Cyclin A2 2.8 0.2 3.4 0.8 Cell cycle- Cyclin D1 2.5 0.1 3.2 0.2 related C GM DM (day 5) Ki67 2.9 0.5 2.9 0.4 None hTNFh TWEAK Transcription factor IIIA 4.7 1.0 4.4 1.0 DNA polymerase α 9.6 0.8 3.2 0.6 Chromatin-assembly factor 17.8 3.6 14.3 3.2 Helicase(lsh) 5.7 1.1 5.0 0.1 Myogenin – 3.2 0.6 – 2.8 0.4 Myogenic Myogenin myoD – 3.4 0.2 – 4.2 0.4 TFs Notch-1 – 18.8 2.4 –30 4.7 MHC – 8.5 0.6 –6 0.5 MLC1 –6 0.1 – 3.2 0.1 Muscle- related- α1 skeletal actin –4 0.3 –3 0.1 genes Carbonic anhydrase 3 – 48.2 3.9 – 5 2.5 0.8 IGFII –42 5.3 – 112 6 MCP-1 140 37 187 97 RANTES 75.3 15 202 8.7 Chemo- kines MIP-1 75.1 1.5 109 4.7 MCP-3 64.2 6.3 17.4 2.4 DM (day 5) DM + Fc-TWEAK (day 5) Figure 3 TWEAK blocks the myogenic program and prevents cell cycle arrest in C2C12 cells. (A) TWEAK induced expression level changes in many genes important for cell cycle control and myogenic differentiation,for example, the muscle-specific transcription factors myogenin and MyoD. Fold changes and s.d.s were calculated using data from duplicate samples that passed statistical tests. (B) C2C12 cells continued to proliferate in low-serum DM when treated with TWEAK as shown by the much larger percentage of cells that incorporated BrdU in TWEAK- treated than in untreated cultures. Cells were analyzed after culture in DM for 3 days with or without 100 ng/ml of Fc-TWEAK and after 12 h BrdU labeling. (C) Myogenin protein was much lower after TWEAK treatment as shown by immunoblots of C2C12 cells in DM in the absence (none) or presence of Fc-hTWEAK (hTweak, 100 ng/ml) or hTNFa (10 ng/ml). (D) Staining of actin filaments by fluorescent phalloidin (red) was decreased after TWEAK treatment of C2C12 cells cultured in DM for 5 days with or without 100 ng/ml of Fc-TWEAK as indicated. Nuclei were identified by DAPI stain (blue). &2006 European Molecular Biology Organization The EMBO Journal VOL 25 NO 24 2006 5829 | | TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al the entire mouse Fn14 gene was isolated and a targeting clones appears to be nonspecific, as we could not compete vector was designed to delete the first two exons (encoding it off using the soluble receptor Fn14-Fc (not shown). amino acids 1–66), which contain the entire extracellular ligand-binding domain of Fn14 (Supplementary Figure S1a). TWEAK/Fn14 pathway regulates the proliferative Embryonic stem cell clones undergoing successful homo- potential of primary mouse muscle myoblasts logous recombination (8 out of 136) were identified by both Our in vitro C2C12 results strongly suggested that the genomic Southern blot and PCR analysis (Supplementary TWEAK/Fn14 pathway might also regulate the properties of Figure S1a and data not shown) and injected into the primary myogenic precursor cells such as muscle satellite C57BL/6 blastocysts to generate chimeras. Homozygous cells and their committed myoblast progeny. Following con- Fn14 null mice were obtained through standard breeding firmation that more than 95% of cells in our primary myo- schemes (see Materials and methods). Homozygous blasts preparation do indeed express Fn14 on the surface (not Fn14 mice appear normal and do not have any obvious shown), we examined the direct effect of TWEAK on the developmental defect in muscle or other tissues (Supple- growth and differentiation characteristics of mouse primary mentary Figure S1e and unpublished data). To confirm the myoblasts isolated from the limb muscles of 6 to 8-week-old presence of a null mutation, we derived wild-type, hetero- mice. In GM, addition of TWEAK resulted in an enhanced zygous and homozygous mouse embryonic fibroblast (MEF) proliferation of the primary myoblasts as reflected by the clones and assayed for both Fn14 mRNA and protein surface presence after 3 days of growth of B2 more mononuclear expression. As shown in Supplementary Figure S1b and c, myoblasts in TWEAK-treated than in untreated cultures both Northern blot and RT–PCR analysis clearly indicated (Figure 4A and B). The proliferative effect of TWEAK was the lack of Fn14 mRNA in Fn14 MEF clones. Importantly, specific because the proliferation rate returned to basal level FACS staining also demonstrated that TWEAK no longer upon treatment with the TWEAK-neutralizing reagents anti- bound to individually derived Fn14 clones (Supple- TWEAK antibody or Fn14-Fc (Figure 4C and not shown). mentary Figure S1d), confirming the lack of Fn14 on the Inclusion of TWEAK in the DM of primary myoblasts, on the surface of these cells and that Fn14 is the only receptor other hand, resulted in significantly reduced myotube forma- for TWEAK. The residual low-frequency binding detected tion and the appearance of dying cells (Figure 4D and E). This / þ /þ by Fc-TWEAK in Fn14 (also seen in Fn14 ) MEF cell death response of primary myoblasts was quite different A D F Exp. no. 1 WT 1 and 2 KO 1 and 2 Growth medium Differentiation medium (day 4) 0 5 10 15 8 Exp. no. 2 B E WT 3 and 4 KO 3 and 4 0 5 10 15 Days GM+Fc-TWEAK (100 ng/ml) DM +Fc-TWEAK (day 4) C G H –/– GM+Fc-TWEAK (100 ng/ml) Wild type Fn14 Fn14-Fc Figure 4 The TWEAK/Fn14 pathway regulates proliferation and differentiation of primary muscle myoblasts. TWEAK promoted the growth (A–C) and inhibited the differentiation (D, E) of primary myoblasts from wild-type mice. (A–C) After 3 days of proliferation in growth medium, cultures of primary myoblasts treated with 100 ng/ml Fc-TWEAK (B) containedB2 more cells than untreated cultures (A) or cultures treated with both Fc-TWEAK and Fn14-Fc to block receptor function (C). (D, E) After 4 days in low-serum differentiation medium, untreated cultures of primary myoblasts formed multinucleate myotubes (D), whereas myotubes were rarely found in TWEAK-treated cultures (E). (F) Primary myoblasts isolated from Fn14-deficient mice produced fewer progeny than wild-type myoblasts in culture. Results from two independent experiments (exp. no. 1 and exp. no. 2) are shown. In each experiment, myoblasts were obtained from two individual wild-type (WT) and two individual Fn14 (KO) mice. Cells were seeded at the same initial cell densities. (G, H) Phase-contrast images of myotubes formed by myoblasts isolated from WT and Fn14-deficient mice. 5830 The EMBO Journal VOL 25 NO 24 2006 &2006 European Molecular Biology Organization | | Cell doublings TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al from the response of C2C12 myoblasts, which remained alive damaged areas of wild-type muscles (Figure 5C, I and M) and and capable of proliferation in the TWEAK-containing DM TWEAK mRNA to approximatly the same level in Fn14 as for at least a week. Although we do not yet understand the in wild-type muscles (Figure 5D and M). The similar increase precise reason(s) for the different responses of C2C12 and in TWEAK mRNA in wild-type and Fn14 muscles at 3 primary muscle cells, it is known that immortalized myo- days after cardiotoxin treatment was confirmed by quantita- genic cell lines have mutations in cell cycle regulatory genes tive PCR (not shown). By day 5, TWEAK expression had (Nowak et al, 2004); so it is not surprising that primary cells subsided considerably in wild-type muscles, whereas the would behave differently than an established cell line in both level of Fn14 remained as high at day 5 as at day 3 (Figure proliferative potential and susceptibility to apoptosis. 5E, K and M). The finding that increased TWEAK expression Primary myoblasts isolated from Fn14-deficient mice pro- persisted for a longer period of time in injured muscles of duced fewer viable progeny in culture than did myoblasts Fn14 mice than in wild-type mice (Figure 5E versus F) from wild-type mice (Figure 4F). To rule out the possibility suggests that inactivation of the TWEAK receptor altered the that the proliferation of wild-type myoblasts was ‘artificially’ injury response to cardiotoxin in the Fn14-deficient mice. enhanced by exogenous TWEAK from the chicken extracts Two lines of evidence suggested that macrophages were used in the culture media or TWEAK produced by myoblasts the major source of the increased TWEAK expression in in an autocrine manner, we included the soluble Fn14-Fc injured wild-type and Fn14 muscles. First, bright-field decoy receptor in the wild-type myoblasts culture and analysis revealed a clear association of TWEAK expression observed no reduction in the number of progeny (data not with infiltrating inflammatory cells, whereas Fn14 expression shown). Based on these results, we conclude that the seemed to adopt a much more diffuse pattern (data not TWEAK/Fn14 pathway is intrinsically required for the full shown). Second, using a quantitative PCR assay, we found proliferative potential of mouse myoblasts. Interestingly, that macrophages (i.e., Mac-1-positive cells) isolated from while Fn14-deficient myoblasts retained the ability to differ- both wild-type and Fn14 TA muscles at 3 days after entiate into myotubes when cultured in low-serum DM, we cardiotoxin injection expressed relatively large amounts of have consistently noticed morphological differences in myo- TWEAK mRNA (Figure 5N). In contrast, very little TWEAK tubes fused from wild-type versus Fn14 myoblasts in that mRNA was found in either the non-macrophage cell fraction the Fn14 myoblasts seemed to form thicker myotubes (i.e., Mac-1-negative cells) or in myogenic cells purified by upon fusion (Figure 4G and H). The underlying molecular Percoll gradient fractionation (Figure 5N). basis for this difference is not yet known. To begin to determine if signaling by additional members of the TNF superfamily might also be altered in FN14- deficient compared with wild-type mice, we carried out TWEAK/Fn14 pathway participates in muscle quantitative PCR measurements to measure in regenerating regeneration in vivo Muscle satellite cells are adult muscle precursor cells residing TA muscles (n¼ 3) the relative levels of the mRNAs encoding quiescently under the basal lamina of the muscle fiber. two TNF family ligands, TNFa and EDA (ectodysplasin), and their respective receptors (TNFR1, TNFR2, and XEDAR), as Although not required for early muscle development, these these two TNF ligand/receptor pathways have previously cells are believed to be the main, if not the only, contributor been suggested to play a role in muscle regeneration to muscle regeneration in response to damage or increase in (Guttridge et al, 2000; Newton et al, 2004). As shown in workload. Upon activation, satellite cells are capable of proliferating to produce committed myoblasts as progeny, Figure 5O, we indeed found that the levels of these mRNAs which then repair injured muscle either by fusing with were significantly induced following cardiotoxin-induced in- injured pre-existing myofibers or by forming entirely new jury, but did not differ significantly between the wild-type and Fn14-deficient muscles at 3 days, although there seems myofibers (Hawke and Garry, 2001). Although histologically to be a trend of increased expression in the Fn14 knockout well characterized, the stepwise program of muscle regenera- mice, suggestive of potential compensatory mechanism tion is incompletely understood at the molecular level and (see Discussion). a limited number of regulating factors have been identified to date (Morgan and Partridge, 2003). Based on our culture results, we hypothesized that the TWEAK/Fn14 pathway Defective inflammatory response and muscle might be a novel regulator of muscle regeneration in vivo, regeneration in Fn14 mice following cardiotoxin- and we tested this idea by comparing regeneration of wild- induced muscle injury type versus Fn14 muscle in vivo following injury induced We next assessed muscle regeneration in response to cardio- by the snake venom cardiotoxin (Couteaux et al, 1988). toxin injury in both wild-type and Fn14 knockout mice using We first examined the expression patterns of TWEAK and histological analysis. Similar to previous reports, cardiotoxin Fn14 mRNAs following cardiotoxin injection in the tibialis injection in the TA muscles caused considerable damage anterior (TA) muscle of wild-type and Fn14 mice using to muscle fibers, triggering a robust regenerative process in situ hybrization analysis. The levels of both TWEAK and that resulted in almost complete regeneration of wild-type Fn14 were minimal in the undamaged wild-type muscle muscles by 2 weeks after injury (Figure 6A–D). When mice tissue (Figure 5A and G, quantified in Figure 5M), and deficient in Fn14 were injected with cardiotoxin, however, TWEAK mRNA expression was also low in undamaged regeneration appeared to be delayed compared with that seen Fn14 muscle (Figure 5B). As expected, Fn14 mRNA was in wild-type muscles (Figure 6E–H). For example, large essentially undetectable in FN14 muscles (Figure 5H, J, L numbers of newly formed muscle fibers with their ‘signature’ and M). centrally located nuclei appeared in TA muscles of wild-type Three days after cardiotoxin injury, expression levels of mice at 5 and 7 days following cardiotoxin injection, whereas both TWEAK and Fn14 were significantly increased in the such regenerated myofibers were rare in Fn14 mice at &2006 European Molecular Biology Organization The EMBO Journal VOL 25 NO 24 2006 5831 | | TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al –/– –/– Wild type Fn14 Wild type Fn14 A BH G No inj. C D I J Day 3 EF K L Day 5 TWEAK in situ Fn14 in situ M O TWEAK mRNA Fn14 mRNA 0.0020 TNF 0.00020 EDA 0.0015 0.00015 0.0010 0.00010 0.0005 0.00005 0.0000 0.00000 500 0.075 TNFR1 XEDAR 0.000035 0.000030 0.050 0.000025 0.000020 Days after 03 5 03 5 03 5 0.025 0.000015 injury: –/– 0.000010 Genotype: Wild type Fn14 Wild type 0.000 0.000005 0.000000 No CTX Day 3 N 0.05 0.007 TNFR2 0.006 Macrophages (Mac-1 ) 0.005 –/– 0.04 Myoblasts Fn14 0.004 0.003 Flow-through cells Wild type 0.03 0.002 0.001 0.000 0.02 No CTX Day 3 0.01 ** ** –/– Wild type Fn14 Figure 5 TWEAK and Fn14 mRNAs increased upon injury, but increased TWEAK persists longer in Fn14 than in wild-type muscles and is produced by macrophages. (A–L) In situ hybridzation of TWEAK (A–F) and Fn14 (G–L) mRNAs in control (no inj) and cardiotoxin-treated wild-type and Fn14 muscles at 3 or 5 days post injection as indicated. Scale bar in (A) represents 100mm. (M) At day 3 after cardiotoxin treatment, quantification of grain density showed that TWEAK mRNA was significantly (Po0.01, n¼ 4) increased several-fold in both wild- / / type (gray bars) and Fn14 (blue bars) muscles. At day 5, in constrast, TWEAK persisted at a high level in Fn14 muscles, but returned to near the initial level in wild-type muscles. Fn14 mRNA was also increased by injury at days 3 and 5 in wild-type muscles (green bars, right panel). Error bars¼ s.d. No Fn14 signal above background was found in Fn14 muscles (not shown). (N) Macrophages are the major source of TWEAK expression in regenerating muscles. At 3 days after cardiotoxin, TA muscles (n¼ 3) were dissociated into single-cell suspensions and three cell preparations were made: (i) cells positive for the macrophage marker Mac-1 were purified using Mac-1 magnetic beads (macrophages, blue bars); (ii) myogenic cells were purified on a Percoll gradient (myoblasts, red bars); and (iii) Mac-1-negative cells, which were cells that did not bind to the Mac-1 beads (flow-through, green bars). RNA was isolated from each cell fraction and quantitative PCR was used to measure the amount of TWEAK mRNA in each cell type using an equal amount of input RNA and normalized versus the amount of GAPDH mRNA. TWEAK mRNA expression was significantly higher (*Po0.05, n¼ 3) in macrophages than in myoblasts or flow- through cells. Error bars¼ s.d. (O) mRNA levels of TNF/TNFRs and EDA/XEDAR with and without cardiotoxin in wild-type and Fn14 mice. At 3 days after cardiotoxin (CTX), total RNA was isolated from TA muscles (n¼ 3) and quantitative PCR was used to measure the mRNA levels for TNF, TNFR1, TNFR2, EDA and XEDAR. Statistical significance was not achieved between wild-type and Fn14-deficient mice. 5832 The EMBO Journal VOL 25 NO 24 2006 &2006 European Molecular Biology Organization | | TWEAK mRNA level Signal intensity (normalized) (grains/mm ) 1 TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al –/– –/– Wild type Fn14 Wild type Fn14 A E I J H&E Day 3 (day 5) B F LM eMyHC (day 4) Day 5 Wild type C G 1000 –/– Fn14 ** Day 7 ** D H Day 5 Day 7 Day 14 Day 14 (n = 3) (n = 3) (n = 2) Figure 6 Fn14 mice have delayed muscle regeneration. Cardiotoxin-treated muscles at indicated days after injury of wild-type (A–D) and Fn14 (E–H) mice are shown. Detailed views of B and F are shown in (I) and (J) respectively to show regenerating, central nucleate myotubes, some of which are indicated by arrows. Images are representative of three independent experiments with seven mice per genotype in total. Scale bar: 200mm (A–C, E–G), 100mm (D, H). (K) The number of central nucleated myotubes in Fn14 mice was significantly lower than in wild-type muscles at 5 and 7 days, but not 14 days, after cardiotoxin. Error bars¼ s.d.; **¼ Po0.01 by Student’s t-test; n as indicated. (L) Myofibers that express embryonic myosin heavy chain (eMyHC), an additional marker of regenerating myotubes (dark brown stain), were more abundant in wild-type than in Fn14 muscles at 4 days after injury. these times (Figure 6B, C, F and G). By day 14, the regene- With the interplay between inflammation and tissue rative process appeared to be nearly complete in wild-type regeneration in mind, we explored the inflammatory res- mice as shown by the clearance of inflammation and the ponse following cardiotoxin in wild-type and Fn14 mice. replacement of damaged myofibers by the newly formed Massive infiltration of inflammatory cells, mainly macro- central nucleate fibers. In Fn14 mice, however, signs of phages and neutrophils, is an early hallmark event of cardi- residual muscle damage remained along with regenerated otoxin-induced muscle regeneration which peaks around fibers, and some inflammatory cells also persisted (Figure 6D 3 days after injection (Figure 7A). In Fn14 mice injected and H). with cardiotoxin, H&E staining revealed not only a signifi- To further analyze the delayed regeneration in Fn14 cantly reduced presence of such infiltrates at day 3, but also compared with wild-type muscles, we quantified the number a 2-day delay of peak infiltration in Fn14 muscles to day of central nucleate myofibers and examined expression of the 5 (Figure 6E and F). This observation was consistent with embryonic isoform of myosin heavy chain (eMyHC) at different our in situ analysis, which showed that TWEAK mRNA stages of regeneration. Myofibers in adult animals that have (likely produced by infiltrating inflammatory cells) persisted centrally located nuclei or that express eMyHC are known to be at 5 days post injury in Fn14 but not in wild-type, mice in the process of regeneration (e.g., DiMario et al, 1991; Pavlath (Figure 5E, F and M). et al, 2003). Confirming that regeneration was delayed in the To additionally assess, both qualitatively and quantita- / / Fn14 muscles, central nucleate myofibers were significantly tively, the altered inflammatory response seen in Fn14 less abundant in regenerating Fn14 than in regenerating mice, we dissociated TA muscles using pronase digestion at wild-type muscles at both 5 and 7 days after injury (Figure different time points following cardiotoxin injection, and we 6I–K). At 14 days after injury, in contrast, the Fn14 and carried out FACS analyses to identify inflammatory cells in wild-type muscles had similar numbers of central nucleate the resulting single-cell suspensions. FACS analyses showed fibers. Furthermore, fibers that expressd eMyHC were less that very few inflammatory cells were present in uninjured / / abundant in Fn14 muscles than in wild-type muscles at TA muscles in either wild-type or Fn14 mice, as assessed 4 days after injury (Figure 6L and M) and similar results by double staining with Mac-1 and Gr-1 (Figure 7A), which were seen at 5 and 7 days after injury (not shown). are markers for myeloid lineage inflammatory cells including &2006 European Molecular Biology Organization The EMBO Journal VOL 25 NO 24 2006 5833 | | Central nucleated fibers/mm TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al No injection CTX (day 2) CTX (day 3) 4 4 10 10 10 0.14 0.95 0.12 8.34 0.17 14.6 3 3 10 10 2 2 2 Wild type 10 10 1 1 1 10 10 10 0.41 98.5 81.4 10.1 71.6 13.6 0 0 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 FL2-H FL2-H FL2-H 4 4 4 10 10 10 0.33 1.16 0.19 5.93 0.087 6.48 3 3 10 10 10 –/– 2 2 2 Fn14 10 10 10 1 1 1 10 10 7.99 98 0.49 85.9 81.8 11.7 0 0 0 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 FL2-H FL2-H FL2-H Gr-1 0.045 Fn14 KO F4/80 0.040 Wild type 0.035 B D 0.030 0.025 0.020 0.015 0.010 0.005 Wild type 0.000 0.003 0.002 C E 0.001 0.000 0.015 –/– Fn14 0.010 0.005 0.000 No CTX Day 1 Day 3 Figure 7 Altered inflammatory response in Fn14 muscles in response to cardiotoxin (CTX) injury. (A) FACS based on myeloid markers Mac-1 and Gr-1 showed that inflammatory cells were more abundant in wild-type than in Fn14 muscles at 2 and 3 days post injury. Slightly more cells were consistently recovered from wild-type than Fn14 mice at all time points; the same number of cells (10 000) are shown in each dot plot. For each genotype, cells from two mice were pooled for each time point and the results shown are representative of three independent experiments. (B–E) F4/80 expression (brown staining) of muscle tissue cross-sections shows higher numbers of inflammatory cells in wild-type (B) than in Fn14 (C) muscles on day 3 following cardiotoxin. Detailed views of (B) and (C) are shown in (D) and (E), respectively. Scale bar: 100mm (B, C), 50mm (D, E). (F) mRNA levels of MCP-1, MCP-3 and RANTES with and without cardiotoxin injection in wild-type and Fn14-deficient mice. At 1 and 3 days after cardiotoxin, total RNA was isolated from TA muscles (n¼ 3) and quantitative PCR was used to quantify mRNAs. Asterisk indicates that the difference for MCP-3 expression at day 1 reached statistical significance (Po0.05, by Student’s t-test). monocytes and neutrophils (Lagasse and Weissman, 1996). where the percentage in wild-type TA was more than double Following cardiotoxin injection, wild-type muscles showed that in the Fn14 TA at 3 days after injury (14.6 versus þ þ a very large increase of Mac-1 /Gr-1 infiltrating cells with 6.5%). We further confirmed our observation histologically the percentage increasing from roughly 1% of the cells in by staining with the more specific monocyte/macrophage uninjured muscles to 28% of the cells at day 3 after injury. marker F4/80. On day 3, extensive F4/80 infiltrates were Infiltrating cells also increased in injured Fn14 TA mus- located around the damaged muscle fibers in the wild-type cles, but the increase was smaller than in wild-type muscles, muscles (Figure 7B and D). In Fn14 mice, however, the þ þ þ as Mac-1 /Gr-1 cells accounted for less than 20% of the number of F4/80 infiltrating cells was much less than in total isolated cells on day 3 from injured Fn14 TA muscles. wild-type mice at day 3 (Figure 7C and E). The difference between inflammation in wild-type and Because chemokines play a critical role in regulating the Fn14 muscles was even more pronounced in the Mac- recruitment of inflammatory cells and TWEAK can potently hi þ 1 /Gr-1 subpopulation (marking monocytes/macrophages induce chemokine production from C2C12 cells based on our in a more activated state; Lagasse and Weissman, 1996), profiling study (Figure 3), we conducted quantitative RT–PCR 5834 The EMBO Journal VOL 25 NO 24 2006 &2006 European Molecular Biology Organization | | Mac-1 FL1-H FL1-H FL1-H FL1-H MCP-3 MCP-1 FL1-H FL1-H RANTES TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al experiments to determine the temporal expression of several We found that activation of the TWEAK/Fn14 pathway key chemokines following cardiotoxin injection in both inhibited differentiation and increased proliferation of both wild-type and Fn14 knockout mice. As shown in Figure 7F, the immortalized C2C12 muscle cell line and primary myo- RANTES does not seem to play a key role in the immediate genic cells. Our finding is consistent with previous demon- early phase following cardiotoxin injection, as no increase in stration that activation of the NF-kB pathway could drive cell its expression level was seen after day 1. However, the levels proliferation and inhibit C2C12 differentiation through reg- of MCP-1 were increased comparably in both wild-type and ulation of cyclin D1 (Guttridge et al, 1999). While our paper Fn14 mice, whereas the expression of MCP-3 was signifi- was under initial review, Dogra et al (2006) published their cantly lower in the absence of Fn14 at day 1 post cardiotoxin- study, which also demonstrated that C2C12 differentiation is induced injury, suggesting that MCP-3 might be a key inhibited by TWEAK, and that TWEAK activates the NF-kB mediator for the recruitment of inflammatory infiltrates in pathway and decreases the amount of MyoD. The results of this model. Not surprisingly, the levels of MCP-3, MCP-1 and Dogra et al are consistent with ours, and we additionally RANTES all trended higher in Fn14 mice at day 3 com- demonstrated the effect of TWEAK on different types of pared with wild-type mice, likely contributing to the delayed mesenchymal progenitor cells, identified differences between inflammation in these mice. the responses of C2C12 and primary muscle cells to TWEAK, prepared Fn14 mice to inactivate the TWEAK pathway and identified a significant role for the TWEAK/ Discussion Fn14 pathway in regulating skeletal muscle regeneration after In this study, we identified mesenchymal precursor cells as a injury in vivo. novel target cell type for the inflammatory cytokine TWEAK, Identification of the TWEAK/Fn14 pathway as a regulator showing Fn14 expression on mesenchymal stem cells and all of muscle myoblast cell proliferation and differentiation following muscle injury in vivo is significant. Previously, a progenitor cells of this lineage. Using muscle myoblasts as an number of soluble factors, such as members of the FGF experimental system, we demonstrated that TWEAK stimu- family, PDGF, TGFb, myostatin, IGF, HGF and TNF, have lated proliferation of primary myoblasts and that Fn14- deficient myoblasts exhibited reduced proliferative capacity been implicated in the proliferation and differentiation of in vitro. In addition, TWEAK altered the differentiating capa- muscle precursor cells based on in vitro studies. Studies using city of both C2C12 and primary skeletal myoblast cells. knockout mice, however, have not always provided unequi- vocal support of the in vitro observations, possibly owing to Furthermore, our in vivo studies demonstrated upregulation the presence of redundant mechanisms in vivo. For example, of TWEAK and Fn14 expression within injured muscle as a well-documented cachetic factor on muscle fibers, TNF tissue and delayed muscle regeneration in Fn14-deficient has been reported to inhibit differentiation, promote proli- animals. Thus, the TWEAK/Fn14 pathway is an important modulator of the proliferative and differentiating potentials of feration, and induce apoptosis of myoblast cells in vitro skeletal muscle progenitor cells. In addition, we also defined (Stewart et al, 2004); however, muscle regeneration occurs normally in TNF mice (Collins and Grounds, 2001), a TWEAK/Fn14 as a new biological pathway that regulates the result that is in marked contrast to the delayed regeneration properties of mesenchymal stem cells, herein showing in mice lacking the TWEAK receptor Fn14 (this study). A TWEAK-induced NF-kB activation, as well as expression of further complication comes from the fact that most of these pro-survival, pro-proliferative and cell adhesion genes. The past few years have seen tremendous progress with factors also exert potent effects on many other cell types respect to understanding the potential of mesenchymal including mature muscle fibers, making interpretation of progenitors in tissue repair and regeneration. However, their direct effect on muscle precursor cells from in vivo studies less straightforward. Based on our in situ data, our relatively poor understanding of factors governing their Fn14 expression in quiescent muscle tissue seems to be survival, homing, proliferation and differentiation presents very low, but drastically increases following cardiotoxin- a major challenge for a successful transition into clinical induced muscle damage. Coupled with the observation that applications. In addition to TWEAK’s proliferative effect on muscle myoblasts, we have also observed that TWEAK could no apparent developmental defect in the muscle tissues was potently inhibit the terminal differentiation of human osteo- seen in either the Fn14 knockout mice or TWEAK over- expressing transgenic mice (LC Burkly, unpublished results), blast precursors, as well as adipogenesis and chondrogenesis it appears that TWEAK/Fn14 is a unique pathway that of human mesenchymal stem cells in culture (B Browning becomes specifically engaged in muscle myoblast prolifera- and TS Zheng, unpublished results). Moreover, our profiling tion only during injury settings. Supporting this hypothesis, study indicated that TWEAK could induce the expression of antiapoptotic as well as cell adhesion molecules in mesench- dramatic upregulation of Fn14 mRNA was also observed in ymal stem cells, thus potentially affecting their homing and the denervated muscle tissues of the mutant SOD transgenic model of ALS (J Lincecum and S Perrin, unpublished results). survival capacity in vivo. The ability of the TWEAK/Fn14 Although delayed by several days, regeneration in Fn14 pathway to broadly regulate these critical properties of mice following cardiotoxin injury eventually became as com- mesenchymal precursor cells might therefore present an plete as in wild-type muscles, showing that the TWEAK/Fn14 opportunity of ‘TWEAKing’ existing approaches for cell- based regenerative therapies, particularly in skeletal, cardiac pathway is one of the multiple pathways that likely contri- and joint injury settings (Barry, 2003). This notion is consis- bute to muscle regeneration. In many biological systems, tent with our recent finding that TWEAK also potentiates wholly or partially redundant mechanisms are commonly found. One example is the transcriptional regulation of the proliferation of liver progenitor cells (Fausto, 2005; myogenesis by MyoD and Myf-5 during development Jakubowski et al, 2005), suggesting a fundamental involve- (Rudnicki et al, 1993). Although we clearly demonstrated ment of this pathway in modulating progenitor cell behavior. &2006 European Molecular Biology Organization The EMBO Journal VOL 25 NO 24 2006 5835 | | TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al that TWEAK and TNF could inhibit C2C12 myogenesis in Fn14 mice compared with wild-type mice (data not independent of each other in vitro, it is still tempting to shown) using a pulsing protocol that is reported to label only speculate that TWEAK and TNF could synergize with myoblasts (Yan et al, 2003), we were unable to demonstrate and potentially compensate for each other in vivo, given experimentally that those BrdU-labeled cells were all that they both are potent activators of the NF-kB pathway. myoblasts. As a result, we cannot conclude with absolute In fact, it has been previously shown that TWEAK and TNF certainty that myoblast proliferation was directly affected could synergistically induce the production of chemokines in cardiotoxin-injured Fn14 mice. The second possible and cytokines in dermal fibroblasts (Chicheportiche et al, mechanism for delayed muscle regeneration could be due 2002). The intriguing possibility of such synergism between to the reduced inflammatory response in Fn14 mice, as TNF and TWEAK in regulating muscle regeneration and other inflammation could certainly contribute positively to efficient biological systems in vivo remains to be investigated. In muscle regeneration by a number of means, including addition, although not statistically significant, there seems removal of debris from damaged fibers and secretion of to be some level of compensatory activation of the TNF/ soluble factors that promote muscle regeneration. TNFR and EDA/XEDAR pathways in Fn14 knockout mice Although seemingly independent events, the proliferation/ following cardiotoxin injury according to our quantitative activation of myoblasts and inflammation during muscle RT–PCR analysis, a finding that reinforces the complex regeneration might actually be tightly coupled, as suggested and often redundant nature of many biological responses. by the compelling evidence for active crosstalks between However, our study clearly shows that TWEAK and TNF are muscle precursor cells and inflammatory infiltrates in a two independent ligand/receptor pathways important for the recent study (Chazaud et al, 2003). We believe that the regulation of biological properties of mesenchymal and TWEAK/Fn14 pathway could very well be a mediator of muscle cells; our study is therefore consistent with the such cellular interactions and one can envision a scenario results from a complete survey of binding between TNF of how the TWEAK/Fn14 pathway may function as a positive family receptors and ligands, which showed that TWEAK regulator of muscle precursor cell properties during injury- bound only to Fn14 and TNF bound only to TNFR1 and triggered muscle regeneration (Figure 8). Following cardio- TNFR2 (Bossen et al, 2006). toxin injection, necrotic muscle fibers can release a number The mechanism by which Fn14 deficiency resulted in of soluble factors, including chemokines that will recruit delayed muscle regeneration following cardiotoxin injection neutrophils and macrophages (Tsivitse et al, 2005), as well is likely two-fold. First, based on our in vitro observations as FGFs (D’Amore, 1990), which would induce Fn14 expres- with the C2C12 line and isolated primary muscle myoblasts, sion on muscle precursor cells. As a result, TWEAK produced it is likely that, similar to the reduced proliferative capacity by the early inflammatory infiltrates would engage Fn14- seen with isolated Fn14 myoblasts in culture, the expan- expressing muscle precursor cells, resulting in not only sion of myoblasts triggered by cardiotoxin was less than their proliferation, but also enhanced production of chemo- optimal in Fn14 mice, resulting in delayed muscle regene- kines important for recruitment of additional inflammatory ration. Although we did observe delayed BrdU incorporation cells that are beneficial to the repair response, thus creating Macrophage recruitment Macrophage Myofiber CTX Chemokines TWEAK Chemokines Fn14 FGFs, etc. Quiescent Activated Myoblast muscle satellite cells muscle satellite cells proliferation/activation Figure 8 A working model for regulation of muscle regeneration by the TWEAK/Fn14 pathway. Cardiotoxin-induced injury triggers release of soluble factors that recruit early inflammatory cells and induce Fn14 expression on muscle precursor cells. Secreted by infiltrating inflammatory cells, TWEAK engages Fn14 and promotes the proliferation and activation of myoblasts, which can in turn contribute to a robust inflammatory response by producing additional chemotactant. The TWEAK/Fn14 pathway therefore positively contributes to muscle regeneration by promoting cellular crosstalk between inflammatory cells and muscle precursor cells. 5836 The EMBO Journal VOL 25 NO 24 2006 &2006 European Molecular Biology Organization | | TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al supplemented with 2% horse serum). Primary muscle satellite a positive feedback loop promoting muscle regeneration. This cells were isolated from limb muscles of 6- to 8-week-old Fn14 notion is consistent with our C2C12 profiling result demon- mice and wild-type control mice (n¼ 4, for each group) as strating that TWEAK induced drastic upregulation of several described (Dominov et al, 1998, 2001; Girgenrath et al, 2005). chemokines critical for macrophage recruitment including See Supplementary data for additional details. Primary myoblasts were induced to differentiate by replacing GM with (DM that MCP-1, MCP-3, RANTES and MIP-1 (Figure 3A), whose consisted of DMEM without chicken embryo extract and with only expressions are significantly induced following cardiotoxin- 2% serum. Human primary cells were all purchased from Cambrex induced muscle injury (Hirata et al, 2003). In fact, our real- (Walkersville, MD) and cultured according to the supplier’s time PCR analysis confirmed that the initial MCP-3 expres- protocol. sion following cardiotoxin injury was much reduced in the Cardiotoxin injection absence of Fn14, and suggests that MCP-3-mediated chemo- Cardiotoxin injection was performed as before (Yan et al, 2003). taxis is an important downstream trigger for the TWEAK/ Briefly, 50ml of 10 mM cardiotoxin (Sigma) was injected into the TA Fn14-induced inflammatory response amplification loop. muscles of the right legs of wild-type or Fn14-deficient mice of 129/sv background, whereas the left legs were used as uninjected It has traditionally been thought that inflammatory res- control. At the indicated time points, TA muscles were harvested ponses following tissue injury are critical for the clearance of and fixed in 4% paraformaldehyde. For BrdU incorporation, mice damaged tissue debris and removal of necrotic/apoptotic were injected intraperitoneally with 1 mg of BrdU 6 hours before cells. A number of more recent studies, however, provided harvesting the muscles. growing evidence, suggesting that inflammation can also Profiling analysis influence tissue repair by directly acting on the regenerating For transcription profiling of TWEAK’s effect on C2C12 cells, cell pool (Mutsaers et al, 2002; Arnett et al, 2003; Monje et al, 1.2 10 C2C12 cells were seeded in 100-mm cell culture dishes in 2003). In our current study, we demonstrated TWEAK’s GM overnight before being switched to differentiation medium. Total RNA from C2C12 cells cultured in GM and DM at different ability to drive the expansion of muscle myoblasts in vitro time points was extracted using TriZol reagent (Invitrogen) and and apparently in vivo in response to injury, presenting a further purified by RNAeasy (Qiagen). Duplicate samples for each concrete example of how a cytokine derived from inflamma- sample point were subjected to profiling using the Affymetrix tory cells can participate in tissue repair and regeneration. mouse chip MG-U74A according to the protocol recommended by the manufacturer. For profiling of TWEAK’s effect on MSCs, 2 10 Given the broad expression of Fn14 on progenitor cells of the cells were cultured in 100-mm cell culture dishes in regular DMEM mesenchymal lineage and its rapid upregulation in many culture medium containing different serum concentrations (10, 2 or injury settings, we propose that the physiological function 0.2%). After overnight culture, TWEAK (100 ng/ml) or heat- of the TWEAK/Fn14 pathway is to participate in acute phase inactivated TWEAK (100ng/ml) were added in triplicate plates for each treatment condition and cells were harvested 18 h later for tissue repair in multiple biological systems by promoting profiling using the Affymetrix human chip U133. progenitor cell proliferation. On the other hand, we have also observed that TWEAK potently inhibited the terminal FACS differentiation of muscle myoblasts, as well as other mesen- Fc-TWEAK binding to and Fn14 surface expression on primary cells were analyzed by FACS staining according to the standard protocol. chymal progenitor cells. One could therefore envision that Briefly, cells were harvested by incubating in 5 mM EDTA/PBS under pathological conditions where inflammatory responses solution at 371C until the cells detached. Cells were suspended in persist, prolonged presence of TWEAK could potentially FACS buffer (PBS with 1% FBS) and stained with either 100 ng/ml block the differentiation of progenitor cells and impede of Fc-TWEAK or 1 mg/ml of ITEM-4 for 45 min, followed by PE- efficient tissue repair. The complex physiological and patho- conjugated goat anti-human or mouse Fc secondary antibodies. Samples were read on a FACS Calibur and analyzed using FlowJo. logical consequences of TWEAK activity suggest that mani- For FACS of muscle dissociates, TA muscles were dissected, fat and pulation of the TWEAK system may present therapeutic connective tissues were removed, and minced muscles were opportunities for cell-based regenerative therapy and in incubated in 0.2 mg/ml Pronase solution (Roche Diagnostics) at various inflammatory diseases such as arthritis and athero- 371C for 1 h. After terminating digestion by washing with DMEM, the dissociates were recovered by sequentially filtering through sclerosis, where extensive tissue remodeling and regene- 100mm and 40mm cell strainers (BD Labware) and used for staining ration occur. with anti-Mac-1 and anti-Gr-1 antibodies. NF-jB activation assay Materials and methods NF-kB activation was measured (n¼ 3) using the TransAM NF-kB p65 assay system (Active Motif) according to the manufacturer’s Proteins and antibodies protocol. The construct for Fc-TWEAK was generated by replacing the Baff For details on quantitative PCR, generation of Fn14 mice, portion of a previously described Fc-Baff construct with a cDNA in situ hybridization and histological analyses, see Supplementary fragment encoding soluble TWEAK. A stable 293T cell line data. expressing the construct was then generated for protein production. The hamster anti-TWEAK antibody ABG.11 and anti-human Fn14 Supplementary data antibody ITEM-4 were generated as previously described (Jaku- Supplementary data are available at The EMBO Journal Online bowski et al, 2002; Nakayama et al, 2003). The blocking anti-Fn14 (http://www.embojournal.org). antibody P2D3 was generated by immunizing Fn14 knockout mice with Fn14-transfected cells. The following antibodies were pur- chased from the indicated sources: anti-mouse TNF G281-2626, PE- Acknowledgements anti-human CD56, FITC-anti-mouse Mac-1 and PE-anti-mouse Gr-1 (BD Biosciences), anti-myogenin F5D (Santa Cruz Biotechnology). We sincerely thank L Evangelisti and S Shulga-Morskaya for em- bryonic stem cell culture and injection; M Wang and J Lincecum for Cell culture in situ analysis; T Crowell and H Gardner for histological analysis; C2C12 cells (ATCC) were seeded at 2 10 cells/well in six-well D Gong for protein purification; D McCrann, J Shearston, S Perrin plates in growth medium (GM; DMEM supplemented with 15% and S Szak for gene profiling analysis. This work was supported in FBS). The following day, cells were washed with PBS once and part by NIH Grant HL39727 (JAW) and Grants AR49496, ES11384 switched to low-serum differentiation medium (DM: DMEM and HL64641 (JBM). &2006 European Molecular Biology Organization The EMBO Journal VOL 25 NO 24 2006 5837 | | TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al References Arnett HA, Mason J, Marino M, Suzuki K, Matsushima GK, Ting JP Hawke TJ, Garry DJ (2001) Myogenic satellite cells: physiology to (2001) TNF alpha promotes proliferation of oligodendrocyte molecular biology. J Appl Physiol 91: 534–551 progenitors and remyelination. Nat Neurosci 4: 1116–1122 Hirata A, Masuda S, Tamura T, Kai K, Ojima K, Fukase A, Motoyoshi Arnett HA, Wang Y, Matsushima GK, Suzuki K, Ting JP (2003) K, Kamakura K, Miyagoe-Suzuki Y, Takeda S (2003) Expression Functional genomic analysis of remyelination reveals importance profiling of cytokines and related genes in regenerating skeletal of inflammation in oligodendrocyte regeneration. J Neurosci 23: muscle after cardiotoxin injection: a role for osteopontin. Am J 9824–9832 Pathol 163: 203–215 Barry FP (2003) Biology and clinical applications of mesenchymal Jakubowski A, Ambrose C, Parr M, Lincecum JM, Wang MZ, Zheng stem cells. Birth Defects Res Part C Embryo Today 69: 250–256 TS, Browning B, Michaelson JS, Baestcher M, Wang B, Bissell Bossen C, Ingold K, Tardivel A, Bodmer JL, Gaide O, Hertig S, DM, Burkly LC (2005) TWEAK induces liver progenitor cell Ambrose C, Tschopp J, Schneider P (2006) Interactions of tumor proliferation. J Clin Invest 115: 2330–2340 necrosis factor (TNF) and TNF receptor family members in the Jakubowski A, Browning B, Lukashev M, Sizing I, Thompson JS, mouse and human. J Biol Chem 281: 13964–13971 Benjamin CD, Hsu YM, Ambrose C, Zheng TS, Burkly LC Brown SA, Richards CM, Hanscom HN, Feng SL, Winkles JA (2003) (2002) Dual role for TWEAK in angiogenic regulation. J Cell Sci The Fn14 cytoplasmic tail binds tumour-necrosis-factor-receptor- 115: 267–274 associated factors 1, 2, 3 and 5 and mediates nuclear factor- Kotter MR, Setzu A, Sim FJ, Van Rooijen N, Franklin RJ (2001) kappaB activation. Biochem J 371: 395–403 Macrophage depletion impairs oligodendrocyte remyelina- Chazaud B, Sonnet C, Lafuste P, Bassez G, Rimaniol AC, Poron F, tion following lysolecithin-induced demyelination. Glia 35: Authier FJ, Dreyfus PA, Gherardi RK (2003) Satellite cells attract 204–212 monocytes and use macrophages as a support to escape apoptosis Lagasse E, Weissman IL (1996) Flow cytometric identification of and enhance muscle growth. J Cell Biol 163: 1133–1143 murine neutrophils and monocytes. J Immunol Methods 197: Chicheportiche Y, Bourdon PR, Xu H, Hsu YM, Scott H, Hession C, 139–150 Garcia I, Browning JL (1997) TWEAK, a new secreted ligand in Lescaudron L, Peltekian E, Fontaine-Perus J, Paulin D, Zampieri M, the tumor necrosis factor family that weakly induces apoptosis. Garcia L, Parrish E (1999) Blood borne macrophages are essential J Biol Chem 272: 32401–32410 for the triggering of muscle regeneration following muscle trans- Chicheportiche Y, Chicheportiche R, Sizing I, Thompson J, plant. Neuromuscul Disord 9: 72–80 Benjamin CB, Ambrose C, Dayer JM (2002) Proinflammatory Locksley RM, Killeen N, Lenardo MJ (2001) The TNF and TNF activity of TWEAK on human dermal fibroblasts and synovio- receptor superfamilies: integrating mammalian biology. Cell 104: cytes: blocking and enhancing effects of anti-TWEAK monoclonal 487–501 antibodies. Arthritis Res 4: 126–133 Luk HW, Noble LJ, Werb Z (2003) Macrophages contribute to the Collins RA, Grounds MD (2001) The role of tumor necrosis factor- maintenance of stable regenerating neurites following peripheral alpha (TNF-alpha) in skeletal muscle regeneration. Studies nerve injury. J Neurosci Res 73: 644–658 in TNF-alpha(/) and TNF-alpha(/)/LT-alpha(/) mice. Lynch CN, Wang YC, Lund JK, Chen YW, Leal JA, Wiley SR (1999) J Histochem Cytochem 49: 989–1001 TWEAK induces angiogenesis and proliferation of endothelial Couteaux R, Mira JC, d’Albis A (1988) Regeneration of muscles after cells. J Biol Chem 274: 8455–8459 cardiotoxin injury. I. cytological aspects. Biol Cell 62: 171–182 Merly F, Lescaudron L, Rouaud T, Crossin F, Gardahaut MF (1999) D’Amore PA (1990) Modes of FGF release in vivo and in vitro. Macrophages enhance muscle satellite cell proliferation and delay Cancer Metast Rev 9: 227–238 their differentiation. Muscle Nerve 22: 724–732 DiMario JX, Uzman A, Strohman RC (1991) Fiber regeneration is not Miller SC, Ito H, Blau HM, Torti FM (1988) Tumor necrosis persistent in dystrophic (MDX) mouse skeletal muscle. Dev Biol factor inhibits human myogenesis in vitro. Mol Cell Biol 8: 148: 314–321 2295–2301 Dogra C, Changotra H, Mohan S, Kumar A (2006) Tumor necrosis Monje ML, Toda H, Palmer TD (2003) Inflammatory blockade factor-like weak inducer of apoptosis inhibits skeletal myogenesis restores adult hippocampal neurogenesis. Science 302: 1760–1765 through sustained activation of nuclear factor-kappaB and degra- Morgan JE, Partridge TA (2003) Muscle satellite cells. Int J Biochem dation of MyoD protein. J Biol Chem 281: 10327–10336 Cell Biol 35: 1151–1156 Dominov JA, Dunn JJ, Miller JB (1998) Bcl-2 expression identifies Mutsaers SE, Whitaker D, Papadimitriou JM (2002) Stimulation of an early stage of myogenesis and promotes clonal expansion of mesothelial cell proliferation by exudate macrophages enhances muscle cells. J Cell Biol 142: 537–544 serosal wound healing in a murine model. Am J Pathol 160: Dominov JA, Houlihan-Kawamoto CA, Swap CJ, Miller JB (2001) 681–692 Pro- and anti-apoptotic members of the Bcl-2 family in skeletal Nakayama M, Ishidoh K, Kojima Y, Harada N, Kominami E, muscle: a distinct role for Bcl-2 in later stages of myogenesis. Dev Okumura K, Yagita H (2003) Fibroblast growth factor-inducible Dyn 220: 18–26 14 mediates multiple pathways of TWEAK-induced cell death. Duffield JS (2003) The inflammatory macrophage: a story of Jekyll J Immunol 170: 341–348 and Hyde. Clin Sci (London) 104: 27–38 Nathan C (2002) Points of control in inflammation. Nature 420: Ekdahl CT, Claasen JH, Bonde S, Kokaia Z, Lindvall O (2003) 846–852 Inflammation is detrimental for neurogenesis in adult brain. Newton K, French DM, Yan M, Frantz GD, Dixit VM (2004) Proc Natl Acad Sci USA 100: 13632–13637 Myodegeneration in EDA-A2 transgenic mice is prevented by Fausto N (2005) Tweaking liver progenitor cells. Nat Med 11: XEDAR deficiency. Mol Cell Biol 24: 1608–1613 1053–1054 Nowak JA, Malowitz J, Girgenrath M, Kostek CA, Kravetz AJ, Feng SL, Guo Y, Factor VM, Thorgeirsson SS, Bell DW, Testa JR, Dominov JA, Miller JB (2004) Immortalization of myogenic Peifley KA, Winkles JA (2000) The Fn14 immediate-early re- cells does not require loss of p16INK4a, p19ARF, or p53 and is sponse gene is induced during liver regeneration and highly accelerated by inactivation of Bax. BMC Cell Biol 5: 1–14 expressed in both human and murine hepatocellular carcinomas. Pavlath GK, Dominov JA, Kegley KM, Miller JB (2003) Regeneration Am J Pathol 156: 1253–1261 of skeletal muscles with altered timing of expression of the Girgenrath M, Kostek CA, Miller JB (2005) Diseased muscles that myogenic bHLH factor MRF4. Am J Pathol 162: 1685–1691 lack dystrophin or laminin-alpha2 have altered compositions and Rudnicki MA, Schnegelsberg PN, Stead RH, Braun T, Arnold HH, proliferation of mononuclear cell populations. BMC Neurol 5: 7 Jaenisch R (1993) MyoD or Myf-5 is required for the formation of Guttridge DC, Albanese C, Reuther JY, Pestell RG, Baldwin Jr AS skeletal muscle. Cell 75: 1351–1359 (1999) NF-kappaB controls cell growth and differentiation Saas P, Boucraut J, Walker PR, Quiquerez AL, Billot M, Desplat-Jego through transcriptional regulation of cyclin D1. Mol Cell Biol S, Chicheportiche Y, Dietrich PY (2000) TWEAK stimulation 19: 5785–5799 of astrocytes and the proinflammatory consequences. Glia 32: Guttridge DC, Mayo MW, Madrid LV, Wang CY, Baldwin Jr AS 102–107 (2000) NF-kappaB-induced loss of MyoD messenger RNA: Stewart CE, Newcomb PV, Holly JM (2004) Multifaceted roles of possible role in muscle decay and cachexia. Science 289: TNF-alpha in myoblast destruction: a multitude of signal trans- 2363–2366 duction pathways. J Cell Physiol 198: 237–247 5838 The EMBO Journal VOL 25 NO 24 2006 &2006 European Molecular Biology Organization | | TWEAK/Fn14 pathway and muscle regeneration M Girgenrath et al Szalay K, Razga Z, Duda E (1997) TNF inhibits myogenesis and Wiley SR, Cassiano L, Lofton T, Davis-Smith T, Winkles JA, Lindner downregulates the expression of myogenic regulatory factors V, Liu H, Daniel TO, Smith CA, Fanslow WC (2001) A novel TNF myoD and myogenin. Eur J Cell Biol 74: 391–398 receptor family member binds TWEAK and is implicated in Tsivitse SK, Mylona E, Peterson JM, Gunning WT, Pizza FX (2005) angiogenesis. Immunity 15: 837–846 Mechanical loading and injury induce human myotubes to release Yan Z, Choi S, Liu X, Zhang M, Schageman JJ, Lee SY, Hart R, Lin L, neutrophil chemoattractants. Am J Physiol Cell Physiol 288: C721–729 Thurmond FA, Williams RS (2003) Highly coordinated gene Walsh K, Perlman H (1997) Cell cycle exit upon myogenic differ- regulation in mouse skeletal muscle regeneration. J Biol Chem entiation. Curr Opin Genet Dev 7: 597–602 278: 8826–8836 &2006 European Molecular Biology Organization The EMBO Journal VOL 25 NO 24 2006 5839 | |

Journal

The EMBO Journal – Springer Journals

Published: Dec 13, 2006

Keywords: Fn14; inflammation; muscle; regeneration; TWEAK

There are no references for this article.