Tnfsf14 may be an attractive candidate to pursue in the search for such a multitasking treatment. Materials and Methods Antibodies and other reagents Anti-MHC (MF20) and Ad5-luciferase adenovirus were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD, National Institutes of Health and maintained by the University of Iowa, Department of Biological Sciences. may be involved in regulating cell migration during Pravastatin sodium myogenesis.9 However, functions Pravastatin sodium of the newly identified muscle-secreted cytokines are mostly unexplored. Using RNAi, we conducted the first functional screen of cytokines for their impact on myogenic differentiation in C2C12 myoblasts, which allowed us to identify potential regulators of myogenesis in distinct functional groups.12 These results suggest the intriguing possibility that muscle cell-secreted proteins have a previously under-appreciated role in modulating muscle development and regeneration. The function of cytokines in myogenesis is relevant to our understanding of Pravastatin sodium not only basic muscle physiology, but also the diseases that negatively affect the health of muscle tissue, such as cachexia. Cachexia is characterized by extreme wasting of lean body mass and often occurs with an underlying chronic illness, such as cancer or congestive cardiac failure.13 Muscle atrophy during cachexic states ultimately stems from ubiquitin-mediated breakdown of myofibrils.14 Significantly, a well-documented association exists between cachexia and the dysregulation of cytokines, most notably the pro-inflammatory RAB11B cytokines tumor necrosis factor alpha (TNFreceptor (LTsystem to study myoblast differentiation, as well as the effects of gene knockdown by lentivirus-delivered shRNA. We found that knockdown of Tnfsf14 (Figure 1a) significantly impaired C2C12 myotube formation, as indicated by myosin heavy chain (MHC) staining of the myocytes and quantification of the fusion index (Figure 1b). Two independent shRNAs yielded similar results, confirming the specificity of RNAi targeting. Moreover, Tnfsf14 depletion reduced the expression of muscle differentiation-specific proteins (Figure 1c), including the early myogenic markers MEF2A, p21 and myogenin. This result suggests that Tnfsf14 functions during the early stages of differentiation. Open in a separate window Figure 1 Pravastatin sodium Tnfsf14 is a positive regulator of myoblast differentiation. (a) C2C12 cells were infected with lentiviruses expressing shTnfsf14 or shScramble (negative control), selected for 2 days, followed by cell lysis and western analysis (data demonstrating that Tnfsf14 functions as pro-survival factor in myogenic cells (Figures 2a and c; Supplementary Figure S2A). Taken together, our observations provide strong evidence that Tnfsf14 is necessary for muscle regeneration post-injury. Tnfsf14 overexpression enhances skeletal muscle regeneration via activation of Akt Next, we examined the effect of Tnfsf14 overexpression on muscle regeneration. Adenovirus expressing mTnfsf14 was co-injected with BaCl2 into TA muscles. Remarkably, significantly larger regenerating myofibers were observed on day 7 and day 14 AI in mTnfsf14 adenovirus-injected muscles compared with muscles injected with a control adenovirus (Figure 6a). To test whether this regeneration-promoting effect of Tnfsf14 was through activation of Akt, we repeated this experiment in the presence of the Akt inhibitor triciribine. As shown in Figure 6b, daily injections of triciribine abrogated the effect of mTnfsf14 and the regenerating myofibers returned to a normal size. Therefore, overexpression of Tnfsf14 can enhance normal muscle regeneration post-injury in an Akt-dependent manner. Open in a separate window Figure 6 Local overexpression of Tnfsf14 enhances skeletal muscle regeneration in an Akt-dependent manner. (a) TA muscles were co-injected with BaCl2 and adenoviruses expressing mTnfsf14 (Ad-mTnfsf14) or luciferase (Ad-luc), and isolated on days 5, 7 and 14 AI. Upon cryosection, H&E staining was performed, and regenerating myofiber CSA was quantified. For each time point, five to seven mice were analyzed. (b) Pravastatin sodium The procedure described in a was repeated, and the animals received daily intraperitoneal injection of 100?l triciribine (0.26?were not muscle cell specific. Thus, although muscle cells should be the prevailing cell type present during intramuscular injection, it is conceivable that other cell types (e.g., infiltrating macrophages).