Due to the complexity of intracellular signaling, the molecular mechanisms underlying skeletal muscle development remain partially understood. It is widely accepted that myogenesis is critically dependent on MRFs, however numerous other signaling pathways have been found to be influential on myogenic differentiation. Therefore, it is important to investigate the signaling pathways that may be involved in the structural remodeling of skeletal muscle. JAK/STAT is now recognized as a critical pathway needed for efficient muscle fibre adaptation. In murine cell models, it was identified that the JAK1/STAT1/STAT3 axis is involved in myoblast proliferation preventing the premature differentiation into myotubes [16
]. However, it is JAK2/STAT2/STAT3 that appears to positively regulate differentiation, indicating that STAT3 elicits specific responses at various times during myogenesis [22
], possibly via JAK/STAT co-operating with various ligands to initiate distinct cellular responses.
Primary human muscle cells provide an appropriate experimental model that has human physiologic relevance. Therefore, using primary human skeletal myoblasts, we sought to corroborate the results obtained in murine cells [16
]. Near confluent myoblasts were exposed to low serum media to induce differentiation for 1, 5 or 10 days. The expression profile of JAK/STAT family members was investigated in conjunction with factors known to regulate myogenesis. As the myoblasts were undergoing differentiation, they displayed a typical genetic expression profile. MRFs are temporally expressed to regulate the proliferation and differentiation of myoblasts. MyoD is expressed in actively proliferating cells prior to differentiation, followed by increased myogenin expression at the beginning of differentiation [4
]. At the onset of differentiation there is also a withdrawal from the cell cycle, represented by changes in expression of cell cycle regulators. As myofibres mature, they begin to fuse to form multinucleated muscle cells which is accompanied by the expression of MHC and α-actin [7
]. In primary human skeletal myoblasts, STAT3 Tyr705 phosphorylation increased during myoblast differentiation, which was accompanied by an apparent elevation in endogenous STAT3, indicating a role during both the proliferative and differentiation phases. STAT3 elevations were associated with unchanged SOCS3 protein expression during the time course. However, SOCS3 mRNA expression was significantly lower at Day 1 of differentiation compared to proliferating and Day 5 differentiated muscle cells, consistent with earlier studies [24
]; further highlighting the importance of STAT3 signaling during the onset of differentiation.
Unlike STAT3, STAT1 Tyr701 phosphorylation was only evident during proliferation despite endogenous STAT1 protein increasing during differentiation. In murine cells, it has been demonstrated that STAT1 is important for proliferation, therefore it was expected that we would observe STAT1 activity only during this stage. However, it has been described that STAT1 activation can often reduce proliferation [25
]. Accordingly, it may be reasonable to assume that STAT1 phosphorylation during proliferation occurs when it complexes with STAT3 to prevent myoblast differentiation [16
]; yet when cells undergo differentiation, STAT1 may be acting alone to inhibit myoblast proliferation [25
Upstream of STATS are gp130, and non-receptor tyrosine kinases, JAK1 and JAK2. Previously it has been demonstrated that JAK1 and JAK2 have differential roles during murine myoblast differentiation despite their high homology; JAK1 is essential for proliferation, while JAK2 is necessary for differentiation [16
]. In this study, JAK1 phosphorylation was only present in proliferating myoblasts; this is consistent with its role in proliferation as described in rodent models [16
]. JAK2 was phosphorylated during each time point, which was unexpected given its reported role in differentiation [22
]. However, this may indicate that there was a population of myoblasts that were spontaneously differentiating, in the proliferating sample. This may also account for the higher than expected expression of MyoD
observed in the proliferating cells.