Androgen receptor expression during C2C12 skeletal muscle cell line differentiation

Abstract

The Androgen Receptor (AR) pathway is involved in the development of skeletal muscle but the molecular basis of androgen-related myogenic enhancement is still unclear. We have investigated AR expression and localization during myoblasts–myotubes differentiation in skeletal muscle cell line C2C12. AR expression increases during proliferation and commitment phase and its levels remain elevated in myotubes. In proliferating and committed cells in the absence of testosterone, AR protein localizes in the nuclei whereas it is almost totally localized in the cytoplasm in myotubes. Low testosterone doses shift the receptor in the nuclei without increasing the amount of total protein. High doses of T induce a significant increase of AR expression during proliferation and differentiation. Little information is available on AR targets that drive the myogenic process. In our study, testosterone induces myogenin, myosin heavy chains (MyHC) and GRIP-1 expression, suggesting that AR and its coregulatory proteins are pivotal factors in skeletal muscle differentiation.

Introduction

The androgen–androgen receptor (AR) signaling pathway is crucial for the determination and differentiation of reproductive organs in men. The activation of this pathway induces important effects in the development of accessory tissues such as brain and skeletal muscle (Herbst and Bhasin, 2004). Strong evidence indicates that testosterone (T), the main circulating androgen secreted by testicular Leydig cells under pulsatile luteinizing hormone stimulation, increases muscle mass and strength, both under physiological and pathological conditions. Physiologic T replacement therapy in healthy young hypogonadal men is associated with significant gains in fat free mass, muscle size and maximal voluntary strength (Bhasin et al., 2000, Snyder et al., 2000, Wang et al., 2000) Furthermore, the effects of androgens in male body composition is established by the use of T supplementation in older men with low levels of circulating hormone, being associated with a significant increase in lean body mass and a reduction in fat mass (Ferrando et al., 1998, Morley et al., 1993, Shi and Garry, 2006, Snyder et al., 1999, Tenover, 1992). The improvement of maximal voluntary strength in men with low T concentrations, is also dependent by T replacement therapy (Bhasin et al., 1997). It has been shown that T supplementation increases muscle mass primarily inducing fiber hypertrophy (Bhasin et al., 1996, Mooradian and Mariash, 1987) due to an increase of protein synthesis (Ferrando et al., 1998, Rooyackers and Nair, 1997, Sheffield-Moore et al., 1999, Urban et al., 1995) and a decrease of muscle protein breakdown (Ferrando et al., 2002). Evidences in ARKO mice, which have deletion of the genomic actions of the androgen receptor, demonstrate that androgens play a physiological role in achieving peak muscle mass in males but not in females (MacLean et al., 2008).

Emerging data show that T–AR complex activates genes involved in myogenesis. AR is a member of the superfamily of ligand-activated transcription factors encoded by a gene located at Xq 11-12, has three functional domains responsible for transactivation, ligand binding and binding to DNA respectively (Gobinet et al., 2002, Kemppainen et al., 1992, Roy et al., 2001). Most of the unliganded AR is localized in the cytoplasmic compartment of target cells, in which it is stored as a multiprotein complex with heat-shock protein and immunophilins. During ligand binding, the receptor dissociates from the multiprotein complex, recruits coactivators and translocates to the nucleus. Subsequently, in concert with co-activators and co-repressors, it binds the promoter region and modulates the expression of androgen-responsive genes that regulate cell fate determination (Gobinet et al., 2002). AR expression has been detected in skeletal muscle cells (Chang et al., 1995, Doumit et al., 1996), in fibroblasts, and in vascular endothelial cells, smooth muscle and mast cells (Sinha-Hikim et al., 2003). Animal studies have shown that fiber hypertrophy is associated with increased expression of AR (Hickson et al., 1983, Hickson et al., 1985); accordingly, clinical reports have indicated that T induces muscle hypertrophy through the expression of its receptor (Bhasin et al., 2001, Sheffield-Moore, 2000). AR has also been shown in satellite cells and in CD34+ stem cells of interstitium (Sinha-Hikim et al., 2004). In young men testosterone-induced fiber hypertrophy is associated with an increase in number and dimension of satellite cells, the major source of myogenic precursors involved in mammalian muscle regeneration (Wozniak et al., 2005), suggesting an intriguing potential role for T in reactivation of the myogenic program (Sinha-Hikim et al., 2003). Moreover, it has been demonstrated that androgens can promote the commitment of mesenchymal pluripotent C3H101T1/2 cells into myogenic lineage, through an androgen receptor-mediated pathway (Singh et al., 2003).

In this study we focused our attention on the expression and the localization of AR during skeletal muscle cells differentiation with or without exogenous T. Using C2C12 cells line we show that AR expression is increased during proliferation and commitment of these cells and that it remains at high level in myotubes. Interestingly, when the cells became differentiated, AR is stored in the cytoplasm and activated only after T treatment.