Androgens are known to modulate the skeletal muscle proliferation and differentiation processes. Recent in vitro studies have shown that dihydrotestosterone and anabolic steroids have functions in promoting the proliferation and differentiation of the mouse skeletal muscle myoblast C2C12 cell line through the classical androgen receptor (AR) signaling pathway. But there are contradictory reports that androgen plays its roles through the membrane signaling pathways. In the present study, we show that there is no expression of the classical AR in L6 cells both at gene and protein levels. We then investigated the effects of testosterone (T) on L6 cell proliferation and differentiation. The results show that T promotes L6 cell proliferation after a 24 h treatment, which followed by enhancing L6 cell differentiation, but these effects are not inhibited by flutamide (F), an antagonist of intracellular AR. Further, we tested the effect of testosterone covalently bounding to albumin (T-BSA), which does not cross the plasma membrane. The results demonstrate that T-BSA and free T have similar effects on L6 cell proliferation and differentiation, and that these effects involve G protein-coupled receptors and different downstream pathways. The L6 cell proliferation induced by T involves PKC and ERK1/2 signaling pathways and cell differentiation happens via the PKA signaling pathway. These results suggest that T promotes cell proliferation and differentiation via G protein-coupled receptors and different downstream pathways in the L6 cell line, although the related molecular mechanisms need to be elucidated in future studies.
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