C2C12 myoblastoma cell differentiation and proliferation is stimulated by androgens and associated with a modulation of myostatin and Pax7 expression

Abstract

Androgens are modulators of skeletal muscle adaptation and regeneration processes. The control of satellite cell activity is a key mechanism during this process. In this study, we analyzed the ability of dihydrotestosterone (DHT) and anabolic steroids to induce and modulate the differentiation of C2C12 myoblastoma cells toward myotubes. C2C12 cells were dose-dependently treated with DHT and anabolic steroids. The time-dependent effects on differentiation were measured and correlated with the expression of genes involved in the regulation of satellite cell activity. The distribution of C2C12 cells within the cell cycle was measured by flow cytometry and differentiation by creatine kinase (CK) activity. Gene expression was analyzed using quantitative real-time PCR and confocal microscopy. The treatment with DHT and anabolic steroids resulted in a stimulation of C2C12 cell proliferation and CK activity. The antiandrogen flutamide was able to antagonize this effect. The expression of the androgen receptor, SOX8, SOX9, Delta, Notch, myostatin, and paired box gene7 (Pax7) was modulated by androgens. The treatment with DHT and anabolic steroids resulted in a strong stimulation of myostatin expression not only in undifferentiated cells but also in myotubes. The stimulation could be antagonized by flutamide. The expression of Pax7 was detectable in C2C12 cells early after treatment with DHT. Our results demonstrate that the key mechanisms of satellite cell differentiation are modulated by androgens. Androgens stimulate the proliferation of C2C12 cells, accelerate the process of differentiation, and increase the expression of myostatin in undifferentiated and differentiated cells. Our findings may have implications not only for the treatment of muscular diseases but also for the improvement of doping analytical methods.

The ubiquitin proteasome system in atrophying skeletal muscle: roles and regulation

“…Testosterone can stimulate satellite cell proliferation (41), but its effects on differentiation are still unclear (17,20,113). The roles of estrogen in skeletal muscle are more pleiotropic and complex.…”

Muscle-specific androgen receptor deletion shows limited actions in myoblasts but not in myofibers in different muscles in vivo

“…Adult skeletal muscle fibers are terminally differentiated, and muscle repair and hypertrophy can occur either through an increase in the number of myonuclei, via activation of satellite cells (muscle stem cells) to proliferate as myoblasts, differentiate into myotubes and fuse with myofibers, or via an increase in protein content of postproliferative myofibers. While some studies have shown that androgens increase myoblast proliferation rate and/or delay differentiation (Powers & Florini 1975, Doumit et al 1996, Sinha-Hikim et al 2006, Diel et al 2008, others suggest the predominant actions are on protein accretion in myofibers (Ferrando et al 1998, Chen et al 2008. Maximal hypertrophic response to muscle overloading requires myofiber AR, but also involves other target cells (Ferry et al 2014).…”

The transcriptomic signature of myostatin inhibitory influence on the differentiation of mouse C2C12 myoblasts

“…Another response to GDF8 treatment was the increased expression of Pax7 and Sox8. The down-regulation of Pax7 by constant MyoD expression is described to be the initial signal for differentiation of the cells (Diel et al 2008). All these transcriptomic changes indicate that GDF8-treated myoblasts did not reach the status of differentiated muscle cells.…”