Akirin2 could promote the proliferation but not the differentiation of duck myoblasts via the activation of the mTOR/p70S6K signaling pathway

Highlights

• Avian Akirin gene family comprises only one gene (Akirin2).

• Duck Akirin2 does not seem to be capable of regulating myoblast differentiation.

• Duck Akirin2 could promote myoblast proliferation.

• Duck Akirin2 could activate mTOR/S6K signaling pathway.

• A novel insight into the function of Akirin gene family was presented.

Abstract

The Akirin gene family normally contains two members that are essential to myoblast differentiation. Noticeably, the avian Akirin gene family comprises only one gene (Akirin2), However, it remains unknown whether avian Akirin gene family still has the function of Akirin1; moreover, it is still unclear whether and how Akirin2 plays a role in myoblast proliferation and differentiation. Interestingly, the unexpected functions of duck Akirin2 were revealed in the present study. The Real-time PCR results showed that between 12 and 48 h during the process of duck myoblasts differentiation, the overexpression of Akirin2 did not significantly increase the expression of myogenic regulatory factors. Flow cytometry analysis revealed that the cell cycle transition was accelerated by Akirin2 overexpression. Moreover, the overexpression of Akirin2 did not influence the myotube formation. Strikingly, when duck myoblasts were cultured in the growth medium, the overexpression of Akirin2 significantly enhanced cell viability. Although the expression of cyclin-dependent proteins did not significantly increase after transfection, the expression of the mammalian targets of rapamycin (mTOR) and p70 S6 kinase (p70S6K) increased. Furthermore, the protein expression of phospho-p70S6K (Ser 417) also increased. However, when rapamycin and pEGFP-N1-Akirin2 plasmids were added together to the growth medium, the positive impact of Akirin2 on cell viability and the mRNA expression of mTOR and p70S6K were significantly blocked. Furthermore, the expression of phospho-mTOR (Ser 2448) and phospho-p70S6K (Ser 417) were also blocked. Taken together, these results could suggest that duck Akirin2 could promote myoblast proliferation via the activation of the mTOR/p70S6K signaling pathway.

Introduction

The Akirin gene family, which normally contains two homologues, Akirin1 and Akirin2, is conserved in vertebrates (Goto, 2008, Macqueen and Johnston, 2009). Homology analysis reveals that the conserved region of the Akirin protein occurs at the N-terminal and C-terminal domains, which generally involve some functional elements, such as nuclear localization signals (NLS) (Chen et al., 2012, Macqueen et al., 2010), suggesting that these two genes may have similar functions. Previous studies showed that both Akirin1 and Akirin2 are expressed in skeletal muscle and especially highly expressed at the early stage of skeletal muscle development (Ma et al., 2015, Macqueen et al., 2010, Marshall et al., 2008, Wanxia et al., 2010), which suggests that both Akirin1 and Akirin2 may be essential to early skeletal muscle growth. It is well known that early skeletal muscle growth mainly involves myoblast proliferation and differentiation (Picard et al., 2002). Thus, the Akirin gene family may play some roles in myoblast proliferation and differentiation.

A previous study demonstrated that murine Akirin1 is highly expressed in differentiating myoblasts and could promote myoblast differentiation in many ways. The overexpression of murine Akirin1 increases the cell cycle arrest and the expression of several myogenic regulatory factors (MRFs) (Marshall et al., 2008). In comparison with Akirin1, studies on Akirin2 have mostly focused on the innate immune response (Akiyama et al., 2013, Bonnay et al., 2014, Komiya et al., 2014, Tartey et al., 2014). However, a current study showed that porcine Akirin2 could also promote myoblast differentiation by mediating the expression of myocyte enhancer factor-2 (MEF2) and myotube formation (Chen et al., 2015). In summary, these reports suggested that both mammalian Akirin1 and Akirin2 could regulate myoblast differentiation, but perhaps rely on different methods.

Interestingly, in the avian animal model (red jungle fowl, Gallus gallus), no akirin1 ortholog was detected by genome assembly. Furthermore, no such data existed among ∼600,000 GenBank Gallus gallus ESTs, despite the presence of multiple positive akirin2 hits (Macqueen and Johnston, 2009). Although the avian Akirin gene family only comprises a single gene (Akirin2), relatively high expression at the early stage of skeletal muscle development was found (Wanxia et al., 2010). This finding suggests that the avian Akirin2 gene may be essential to myoblast proliferation and differentiation. However, it remains unknown whether avian Akirin gene family still has the function of Akirin1; moreover, it is still unclear whether and how Akirin2 plays a role in myoblast proliferation and differentiation.

Therefore, based on overexpression experiments of duck Akirin2 in duck myoblasts, our study deeply investigated the molecular regulatory mechanisms of duck Akirin2 on myoblast proliferation and differentiation from several aspects, such as the expression of the MRFs, the cell-cycle variability, the formation of myotubes and the metabolism of intracellular proteins.