TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration
2006; Springer Nature; Volume: 25; Issue: 24 Linguagem: Inglês
10.1038/sj.emboj.7601441
ISSN1460-2075
AutoresMahasweta Girgenrath, Shawn Weng, Christine A. Kostek, Beth Browning, Monica Wang, Sharron A.N. Brown, Jeffrey A. Winkles, Jennifer S. Michaelson, Norm Allaire, Pascal Schneider, Martin Scott, Yen‐Ming Hsu, Hideo Yagita∥, Richard A. Flavell, Jeffrey B. Miller, Linda C. Burkly, Timothy S. Zheng,
Tópico(s)GDF15 and Related Biomarkers
ResumoArticle23 November 2006free access TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration Mahasweta Girgenrath Mahasweta Girgenrath Boston Biomedical Research Institute, Watertown, MA, USA Search for more papers by this author Shawn Weng Shawn Weng Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA, USA Search for more papers by this author Christine A Kostek Christine A Kostek Boston Biomedical Research Institute, Watertown, MA, USA Search for more papers by this author Beth Browning Beth Browning Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA, USA Search for more papers by this author Monica Wang Monica Wang Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA, USA Search for more papers by this author Sharron AN Brown Sharron AN Brown Departments of Surgery and Physiology, University of Maryland School of Medicine, Baltimore, MD, USA Search for more papers by this author Jeffrey A Winkles Jeffrey A Winkles Departments of Surgery and Physiology, University of Maryland School of Medicine, Baltimore, MD, USA Search for more papers by this author Jennifer S Michaelson Jennifer S Michaelson Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA, USA Search for more papers by this author Norm Allaire Norm Allaire Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA, USA Search for more papers by this author Pascal Schneider Pascal Schneider Department of Biochemistry, University of Lausanne, Ch. Des Boveresses, Epalinges, Switzerland Search for more papers by this author Martin L Scott Martin L Scott Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA, USA Search for more papers by this author Yen-ming Hsu Yen-ming Hsu Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA, USA Search for more papers by this author Hideo Yagita Hideo Yagita Department of Immunology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Richard A Flavell Richard A Flavell Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Jeffrey Boone Miller Jeffrey Boone Miller Boston Biomedical Research Institute, Watertown, MA, USA Search for more papers by this author Linda C Burkly Linda C Burkly Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA, USA Search for more papers by this author Timothy S Zheng Corresponding Author Timothy S Zheng Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA, USA Search for more papers by this author Mahasweta Girgenrath Mahasweta Girgenrath Boston Biomedical Research Institute, Watertown, MA, USA Search for more papers by this author Shawn Weng Shawn Weng Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA, USA Search for more papers by this author Christine A Kostek Christine A Kostek Boston Biomedical Research Institute, Watertown, MA, USA Search for more papers by this author Beth Browning Beth Browning Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA, USA Search for more papers by this author Monica Wang Monica Wang Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA, USA Search for more papers by this author Sharron AN Brown Sharron AN Brown Departments of Surgery and Physiology, University of Maryland School of Medicine, Baltimore, MD, USA Search for more papers by this author Jeffrey A Winkles Jeffrey A Winkles Departments of Surgery and Physiology, University of Maryland School of Medicine, Baltimore, MD, USA Search for more papers by this author Jennifer S Michaelson Jennifer S Michaelson Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA, USA Search for more papers by this author Norm Allaire Norm Allaire Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA, USA Search for more papers by this author Pascal Schneider Pascal Schneider Department of Biochemistry, University of Lausanne, Ch. Des Boveresses, Epalinges, Switzerland Search for more papers by this author Martin L Scott Martin L Scott Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA, USA Search for more papers by this author Yen-ming Hsu Yen-ming Hsu Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA, USA Search for more papers by this author Hideo Yagita Hideo Yagita Department of Immunology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Richard A Flavell Richard A Flavell Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Jeffrey Boone Miller Jeffrey Boone Miller Boston Biomedical Research Institute, Watertown, MA, USA Search for more papers by this author Linda C Burkly Linda C Burkly Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA, USA Search for more papers by this author Timothy S Zheng Corresponding Author Timothy S Zheng Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA, USA Search for more papers by this author Author Information Mahasweta Girgenrath1, Shawn Weng2, Christine A Kostek1, Beth Browning2, Monica Wang2, Sharron AN Brown3, Jeffrey A Winkles3, Jennifer S Michaelson2, Norm Allaire2, Pascal Schneider4, Martin L Scott2, Yen-ming Hsu2, Hideo Yagita5, Richard A Flavell6, Jeffrey Boone Miller1, Linda C Burkly2 and Timothy S Zheng 2 1Boston Biomedical Research Institute, Watertown, MA, USA 2Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA, USA 3Departments of Surgery and Physiology, University of Maryland School of Medicine, Baltimore, MD, USA 4Department of Biochemistry, University of Lausanne, Ch. Des Boveresses, Epalinges, Switzerland 5Department of Immunology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan 6Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA *Corresponding author. Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA 02142, USA. Tel.: +1 617 679 3348; Fax: +1 617 679 3208; E-mail: [email protected] The EMBO Journal (2006)25:5826-5839https://doi.org/10.1038/sj.emboj.7601441 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Inflammation participates in tissue repair through multiple mechanisms including directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand TWEAK, we observed that TWEAK binds to all progenitor cells of the mesenchymal lineage and induces NF-κB activation and the expression of pro-survival, pro-proliferative and homing receptor genes in the mesenchymal stem cells, suggesting that this pro-inflammatory cytokine may play an important role in controlling progenitor cell biology. We explored this potential using both the established C2C12 cell line and primary mouse muscle myoblasts, and demonstrated that TWEAK promoted their proliferation and inhibited their terminal differentiation. By generating mice deficient in the TWEAK receptor Fn14, we further showed that Fn14-deficient primary myoblasts displayed significantly reduced proliferative capacity and altered myotube formation. Following cardiotoxin injection, a known trigger for satellite cell-driven skeletal muscle regeneration, Fn14-deficient mice exhibited reduced inflammatory response and delayed muscle fiber regeneration compared with wild-type mice. These results indicate that the TWEAK/Fn14 pathway is a novel regulator of skeletal muscle precursor cells and illustrate an important mechanism by which inflammatory cytokines influence tissue regeneration and repair. Coupled with our recent demonstration that TWEAK potentiates liver progenitor cell proliferation, the expression of Fn14 on all mesenchymal lineage progenitor cells supports a broad involvement of this pathway in other tissue injury and disease settings. Introduction Inflammation is the first-line defensive mechanism to noxious insults ranging from microbial infections to physical injury (Nathan, 2002). Although excessive inflammation can have devastating consequences, in most cases inflammatory responses lead to effective clearance of pathogens and/or healing. In fact, depletion of inflammatory cell types such as macrophages has been shown to result in delayed repair response in injury models of a number of tissues, including muscle (Lescaudron et al, 1999), mesothelium (Mutsaers et al, 2002), and both the central (Kotter et al, 2001) and peripheral nerve systems (Luk et al, 2003). It is now well established that inflammation contributes to tissue repair by clearing debris, engulfment and digestion of apoptotic/necrotic cell bodies, epithelial closure and promoting angiogenesis (Nathan, 2002). Much underappreciated until recently, the various cytokines secreted by inflammatory cells also directly influence the properties of progenitor cells that reside in tissues undergoing repair and regeneration (Duffield, 2003). For example, the macrophage-derived cytokine TNF promotes the proliferation of oligodendrocyte progenitors after cuprizone-induced demyelination (Arnett et al, 2001). Similarly, soluble factor(s) secreted by macrophages can also drive the proliferation of mesothelial cells (Mutsaers et al, 2002) and muscle satellite cells (Merly et al, 1999) during mesothelial and muscle regeneration, respectively. However, it has also been demonstrated that inflammatory cytokines block neural progenitor differentiation (Ekdahl et al, 2003; Monje et al, 2003), suggesting that the biological consequence of these inflammatory mediators on tissue repair and regeneration is likely a complex one. The largely macrophage-derived cytokine TWEAK and its receptor Fn14 are relatively new additions to the TNF superfamily, which includes well-known modulators of cell proliferation, differentiation and apoptosis (Locksley et al, 2001). Originally identified as a weak inducer of cell death in tumor cell lines (Chicheportiche et al, 1997), TWEAK was subsequently shown to exert pleiotropic effects on a variety of cell types in vitro, including pro-angiogenic activities on endothelial cells (Lynch et al, 1999; Jakubowski et al, 2002) and pro-inflammatory activities on epithelial cells (Chicheportiche et al, 1997), astrocytes (Saas et al, 2000), dermal fibroblasts and synoviocytes (Chicheportiche et al, 2002). Conspicuously absent from the TWEAK-responding cell types are lymphocytes, and Fn14 expression on these cells has not been demonstrated. Interestingly, the TWEAK receptor Fn14 is an FGF-inducible gene and is highly upregulated in liver injury and regeneration and arterial wounding (Feng et al, 2000; Wiley et al, 2001), suggesting a regulatory role in settings of tissue injury and repair. Recently, we demonstrated that the TWEAK/Fn14 pathway is a potent inducer of liver progenitor cell proliferation in response to chemical injury in vivo (Jakubowski et al, 2005). However, the physiological relevance of the TWEAK/Fn14 pathway in other contexts remains elusive. In this study, we explored the potential role of TWEAK/Fn14 pathway in modulating mesenchymal progenitor cell biology in vitro and in vivo in a model of skeletal muscle injury and repair. We showed that all progenitor cells of mesenchymal lineage express the TWEAK receptor Fn14. We further demonstrated that TWEAK promotes proliferation of both an established myoblast cell line and primary muscle myoblasts. Finally, by generating mice deficient in Fn14, we established that the TWEAK/Fn14 pathway is required for optimal muscle regeneration in vivo. Results Mesenchymal progenitor cells are a novel target cell type of TWEAK In our effort to identify in vivo target cell types for TWEAK, we found that TWEAK binds to progenitor cells of the mesenchymal lineage, including human primary mesenchymal stem cells, skeletal muscle myoblasts and preadipocytes as well as chondrocyte and osteoblast precursors cultured in vitro (Figure 1A). We also demonstrated that the TWEAK receptor Fn14 was expressed on these progenitor cells by staining with the anti-human Fn14 monoclonal antibody ITEM-4 (Figure 1A). To see if these cells would respond to TWEAK, we examined NF-κB activation in mesenchymal stem cells and osteoblast precursors upon TWEAK treatment as it has previously been shown that Fn14 signal transduction is mediated through the TRAF-binding site in its cytoplasmic tail (Brown et al, 2003). As shown in Figure 1B, TWEAK induced robust NF-κB activation in both cell types as measured by the amount of activated p65 present in the cell lysate following TWEAK stimulation using the TranAM assay system, indicating that progenitor cells of the mesenchymal lineage are indeed TWEAK-responsive cells. The ability of TWEAK to induce NF-κB activation in these progenitor cells is further confirmed by transcription profiling study of mesenchymal stem cells treated with TWEAK. Even in the high serum culture condition (10% FBS), TWEAK induced robust transcriptional upregulation of well-known NF-κB-regulated genes, including TRAF1 and 3, NF-κB2 and RelB, which are regulators of NF-κB pathway themselves, and pro-survival genes such as A20 and c-IAP2, as well as cell adhesion genes such as ICAM-1 and VCAM-1n (Figure 1C). Importantly, under low (0.2%) or medium (2%) serum conditions, TWEAK also induced the expression of many cell cycle-related genes including cdc2, cyclin A2, survivin, MAD2, among others (Figure 1D and data not shown). These results therefore indicate that TWEAK may regulate cell fate decisions of progenitor cells. 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-κB 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-κB 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 (P<0.01). Download figure Download PowerPoint TWEAK promotes proliferation and inhibits terminal myogenesis of C2C12 cells To further investigate how TWEAK might influence cell fate of progenitor cells, we took advantage of the well-established in vitro cell differentiation model of the mesenchymal lineage, C2C12 terminal myogenesis. We first confirmed that, similar to human skeletal muscle myoblasts, the murine C2C12 myoblasts also expressed Fn14 on their surface (Figure 2A and B). As expected, upon switching from regular growth medium (GM) (containing 15% FBS) to (DM), (containing 2% horse serum), mononuclear proliferating C2C12 cells enter into a well-characterized differentiation program that includes cell cycle arrest, fusion of mononuclear cells and formation of multinucleate myotubes (Figure 2C). In the presence of TWEAK, however, C2C12 cells largely remained as a monolayer of mononuclear cells even when placed in DM (Figure 2D). Both the anti-TWEAK blocking antibody ABG.11 and the hFn14-hFc-soluble TWEAK receptor effectively reversed the inhibition of myotube formation, confirming the specificity of our observation (Figure 2E and not shown). Importantly, addition of the anti-Fn14 blocking antibody P2D3 also was able to reverse the inhibitory effect of TWEAK (Figure 2F), demonstrating that the activity of TWEAK on C2C12 cells was mediated through the Fn14 receptor. As it is well documented that TNF is a potent inhibitor of myogenesis in vitro (Szalay et al, 1997; Miller et al, 1988), we decided to investigate if there was any potential crosstalk between the TNF and TWEAK pathways in the C2C12 myogenesis model. Although the soluble TNF receptor mTNFRI-Fc totally reversed the inhibition of C2C12 differentiation by TNF (Figure 2G and H), it had no effect on the inhibitory activity of TWEAK (Figure 2I). A similar result was also seen using anti-TNF or anti-TNFRI blocking antibodies (data not shown). Conversely, the anti-TWEAK blocking antibody ABG.11 and anti-Fn14 blocking antibody P2D3 both failed to block TNF's ability to inhibit C2C12 myogenesis (Figure 2J and not shown). Based on these observations, we concluded that although both TWEAK and TNF exhibit similar potent inhibitory effects on C2C12 differentiation, these two act independently of each other through different receptors. 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 mTWEAK+10 μg/ml anti-TWEAK mAb ABG.11, normal myotubes; (F) 100ng/ml mTWEAK+10 μg/ml anti-Fn14 mAb P2D3, normal myotubes; (G) 5 ng/ml mTNF; decreased myotubes; (H) 5 ng/ml mTNF+10 μg/ml mTNFRI-Fc, normal myotubes; (I) 5 ng/ml mTWEAK+10 μg/ml mTNFRI-Fc to block TNFR1, decreased myotubes; and (J) 5 ng/ml mTNF+10 μg/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. Download figure Download PowerPoint Terminal myogenic differentiation of C2C12 cells requires exit from the cell cycle and expression of myogenic transcription factors such as myogenin, which in turn activate the transcription of an array of muscle-specific genes. To understand the molecular events underlying TWEAK's inhibitory effect on C2C12 myogenesis, we performed profiling studies comparing global gene expression patterns of C2C12 cells cultured in low-serum DM with or without TWEAK, and a number of representative genes with significant alteration in their expression levels are listed in Figure 3A. Upon analysis, we noticed that the expression levels of a large number of genes involved in cell cycle progression (i.e., cyclin D1 and c-myc), DNA/RNA synthesis (i.e., DNA polymerase A and TFIIIA) as well as chromosome remodeling (i.e., chromatin assembly factor) were dramatically increased with TWEAK stimulation, suggesting that these cells were mitotically active (Figure 3A). Using BrdU labeling, we confirmed that most C2C12 cells in DM in the absence of TWEAK are nonmitotic as expected. However, TWEAK-treated C2C12 cells were almost all BrdU positive, indicating that TWEAK stimulation potently promoted proliferation of these C2C12 cells even under low-serum condition (Figure 3B). Given that withdrawal from cell cycle is a well-established prerequisite for C2C12 terminal differentiation (Walsh and Perlman, 1997), it is likely that the perturbation of cell cycle arrest in these cells by TWEAK is responsible for the blockade in C2C12 myogenesis. The failure in myogenic progression in the presence of TWEAK was reflected at the molecular level by the decreased expression, based on both DNA array and Western blot analyses (Figure 3A and C), of the muscle-specific transcription factors myogenin and MyoD, which are critical for the terminal differentiation of C2C12 cells and govern the expression of many muscle-specific genes. Consequently, the overall expression levels of multiple muscle-related genes, such as the muscle structural proteins myosin heavy and light chains, as well as the muscle metabolic gene carbonic anhydrase, were significantly suppressed by TWEAK treatment (Figure 3A). This suppression of muscle-specific gene expression by TWEAK was accompanied by drastic phenotype differences between the TWEAK-treated and untreated cells. Whereas the majority of TWEAK-treated C2C12 cells maintained the undifferentiated mononuclear morphology with few actin filaments even in low-serum DM, the untreated C2C12 cells fully differentiated and became fused multinucleated myotubes, as revealed by DAPI and phalloidin staining (Figure 3D). 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). Download figure Download PowerPoint Generation of Fn14-deficient mice The profound effect of TWEAK on the established myoblast line C2C12 suggested that the TWEAK/Fn14 pathway may indeed be an important pathway regulating progenitor cell biology. To better understand the physiological relevance of this pathway, we decided to generate mice deficient in the TWEAK receptor Fn14. Using the standard gene-targeting strategy, an ∼10-kb Kpn1 genomic DNA fragment containing the entire mouse Fn14 gene was isolated and a targeting vector was designed to delete the first two exons (encoding amino acids 1–66), which contain the entire extracellular ligand-binding domain of Fn14 (Supplementary Figure S1a). Embryonic stem cell clones undergoing successful homologous recombination (8 out of 136) were identified by both genomic Southern blot and PCR analysis (Supplementary Figure S1a and data not shown) and injected into the C57BL/6 blastocysts to generate chimeras. Homozygous Fn14 null mice were obtained through standard breeding schemes (see Materials and methods). Homozygous Fn14−/− mice appear normal and do not have any obvious developmental defect in muscle or other tissues (Supplementary Figure S1e and unpublished data). To confirm the presence of a null mutation, we derived wild-type, heterozygous and homozygous mouse embryonic fibroblast (MEF) clones and assayed for both Fn14 mRNA and protein surface expression. As shown in Supplementary Figure S1b and c, both Northern blot and RT–PCR analysis clearly indicated the lack of Fn14 mRNA in Fn14−/− MEF clones. Importantly, FACS staining also demonstrated that TWEAK no longer bound to individually derived Fn14−/− clones (Supplementary Figure S1d), confirming the lack of Fn14 on the surface of these cells and that Fn14 is the only receptor for TWEAK. The residual low-frequency binding detected by Fc-TWEAK in Fn14−/− (also seen in Fn14+/+) MEF clones appears to be nonspecific, as we could not compete it off using the soluble receptor Fn14-Fc (not shown). TWEAK/Fn14 pathway regulates the proliferative potential of primary mouse muscle myoblasts Our in vitro C2C12 results strongly suggested that the TWEAK/Fn14 pathway might also regulate the properties of primary myogenic precursor cells such as muscle satellite cells and their committed myoblast progeny. Following confirmation that more than 95% of cells in our primary myoblasts preparation do indeed express Fn14 on the surface (not shown), we examined the direct effect of TWEAK on the growth and differentiation characteristics of mouse primary myoblasts isolated from the limb muscles of 6 to 8-week-old mice. In GM, addition of TWEAK resulted in an enhanced proliferation of the primary myoblasts as reflected by the presence after 3 days of growth of ∼2 × more mononuclear myoblasts in TWEAK-treated than in untreated cultures (Figure 4A and B). The proliferative effect of TWEAK was specific because the proliferation rate returned to basal level upon treatment with the TWEAK-neutralizing reagents anti-TWEAK antibody or Fn14-Fc (Figure 4C and not shown). Inclusion of TWEAK in the DM of primary myoblasts, on the other hand, resulted in significantly reduced myotube formation and the appearance of dying cells (Figure 4D and E). This cell death response of primary myoblasts was quite different from the response of C2C12 myoblasts, which remained alive and capable of proliferation in the TWEAK-containing DM for at least a week. Although we do not yet understand the precise reason(s) for the different responses of C2C12 and primary muscle cells, it is known that immortalized myogenic cell lines have mutations in cell cycle regulatory genes (Nowak et al, 2004); so it is not surprising that primary cells would behave differently than an established cell line in both proliferative potential and susceptibility to apoptosis. 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) contained ∼2 × 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. Download figure Download PowerPoint Primary myoblasts isolated from Fn14-deficient mice produced fewer viable progeny in culture than did myoblasts from wild-type mice (Figure 4F). To rule out the possibility that the proliferation of wild-type myoblasts was ‘artificially’ enhanced by exogenous TWEAK from the chicken extracts used in the culture media or TWEAK produced by myoblasts in an autocrine manner, we included the soluble Fn14-Fc decoy receptor in the wild-type myoblasts culture and observed no reduction in the number of progeny (data not shown). Based on these results, we conclude that the TWEAK/Fn14 pathway is intrinsically required for the full proliferative potential of mouse myoblasts. Interestingly, while Fn14-deficient myoblasts retained the ability to differentiate into myotubes when cultured in low-serum DM, we have consistently noticed morphological differences in myotubes fused from wild-type versus Fn14−/− myoblasts in that the Fn14−/−myoblasts seemed to form thicker myotubes upon fusion (Figure 4G and H). The underlying molecular basis for this difference is not yet known. TWEAK/Fn14 pathway participates in muscle regeneration in vivo Muscle satellite cells are adult muscle precursor cells residing quiescently under the basal lamina of the muscle fiber. Although not required for early muscle development, these cells are believed to be the main, if not the only, contributor to muscle regeneration in response to damage or increase in workload. Upon activation, satellite cells are capable of proliferating to produce committed myoblasts as progeny, which then repair injured muscle either by fusing with injured pre-existing myofibers or by forming entirely new myofibers (Hawke and Garry, 2001). Although histologically well characterized, the stepwise program of muscle regeneration is incompletely understood at the molecular level and 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 might be a novel regulator of muscle regeneration in vivo, and we tested this idea by comparing regeneration of wild-type versus Fn14−/− muscle in vivo following injury induced by the snake venom cardiotoxin (Couteaux et al, 1988). We first examined the expression patterns of TWEAK and Fn14 mRNAs following cardiotoxin injection in the tibialis anterior (TA) muscle of wild-type and Fn14−/− mi
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