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.