Fibroblast growth factor-inducible 14 (Fn14), distantly related to tumor necrosis factor receptor superfamily and a receptor for TWEAK cytokine, has been implicated in several biological responses. In this study, we have investigated the role of Fn14 in skeletal muscle formation in vitro. Flow cytometric and Western blot analysis revealed that Fn14 is highly expressed on myoblastic cell line C2C12 and mouse primary myoblasts. The expression of Fn14 was decreased upon differentiation of myoblasts into myotubes. Suppression of Fn14 expression using RNA interference inhibited the myotube formation in both C2C12 and primary myoblast cultures. Fn14 was required for the transactivation of skeletal -actin promoter and the expression of specific muscle proteins such as myosin heavy chain fast type and creatine kinase. RNA interference-mediated knockdown of Fn14 receptor in C2C12 myoblasts decreased the levels of myogenic regulatory factors MyoD and myogenin upon induction of differentiation. Conversely, overexpression of MyoD increased differentiation in Fn14-knockdown C2C12 cultures. Suppression of Fn14 expression in C2C12 myoblasts also inhibited the differentiation-associated increase in the activity of serum response factor and RhoA GTPase. In addition, our data suggest that the role of Fn14 during myogenic differentiation could be independent of TWEAK cytokine. Collectively, our study suggests that the Fn14 receptor is required for the expression of myogenic regulatory factors and differentiation of myoblasts into myotubes.

Glucocorticoids inhibit bone formation by inhibiting osteoblast proliferation and differentiation, and stimulating osteoblast apoptosis. Glucocorticoids inhibit expression of cytokines and growth factors, including IGF I and IGF binding protein-5 (IGFBP-5). IGFBP-5 enhances IGF activities on osteoblasts in many conditions, stimulates bone formation in vivo, and may function in part as a growth factor that affects cells by IGF-independent mechanisms. To investigate glucocorticoid inhibition of IGFBP-5 expression, we identified sequences in the proximal human IGFBP-5 gene promoter that mediate Dexamethasone inhibition of transcription. Promoter deletion and mutation constructs in pGL3 luciferase reporter were tested by transient transfection of human U2-OS and TE-85 osteosarcoma cells. An activator protein-2 (AP-2) binding site sequence contributed to dexamethasone inhibition of transcription. AP-2a bound to this sequence and overexpression of AP-2a protein in TE-85 cells increased promoter activity and prevented dexamethasone inhibition. Dexamethasone induced glucocorticoid receptor (GF) binding to AP-2a as determined by GST-GR fusion protein pull down, co-immune precipitation, and mammalian two-hybrid interaction assay. GR did not bind directly to a promoter DNA sequence containing the AP-2 site, or prevent AP-2 from binding. Results suggest that glucocorticoids induce GR binding to AP-2 and inhibit human IGFBP-5 transcription in part by decreasing AP-2 transactivation of the promoter.