Novel evidence that testosterone promotes cell proliferation and differentiation via G protein‐coupled receptors in the rat L6 skeletal muscle myoblast cell line

Abstract

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.

Leukaemia inhibitory factor is expressed in rat gastrocnemius muscle after contusion and increases proliferation of rat L6 myoblasts via c-Myc signalling

“…This cell line, established from the newborn rat thigh muscle, has the characteristics of myogenic precursor cells 26 and has been used as a cell model for proliferation and differentiation by several investigators. 27–29 We found that LIF increased the proliferation of L6 cells and the effects of LIF appeared to be maximal at a concentration of 1 ng/mL. These results are consistent with those of a previous study in C2C12 mouse myoblasts, in which LIF failed to further increase cell number at concentrations higher than 1 or 10 ng/mL.…”

Potential use of adult bovine serum obtained during the slaughtering process as a biological reagent

“…We chose cells that were expected to proliferate better with either female hormones (astrocytes) or male hormones (L6 cells), or that were not expected to be influenced by sex hormones (fibroblasts) (Arnold et al 2008;Fu et al 2012). Although only three types of cells were tested in the experiments, these results indicate that use of FBS is not critical for certain types of cells.…”

“…Data are presented in mean±SEM (***p < 0.001). of the L6 cell line is enhanced by testosterone treatment (Fu et al 2012). In our experiment, however, the L6 cell line showed enhanced proliferation with media supplemented with male or castrated male sera, and even with female serum.…”

Changes in Communication between Muscle Stem Cells and their Environment with Aging

“…The sex-steroid testosterone, secreted by the testis, has been extensively studied in muscle, and can be considered as a double-sided blade, acting both on myoblast proliferation and differentiation. It acts on myoblasts through the androgen receptor localized in the nucleus [73] or through G protein-coupled receptors [74]. It promotes myoblast proliferation through protein kinase C (PKC) signaling [74] - for instance through nPKC δ and extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation [75].…”