Role of metalloprotease disintegrin ADAM12 in determination of quiescent reserve cells during myogenic differentiation in vitro.

Development and regeneration of skeletal muscle are tightly coupled to the cell cycle machinery (32, 62). During myogenic differentiation, proliferating myoblasts pause in G1, then permanently withdraw from the cell cycle, differentiate, and fuse into multinucleated myotubes (1, 45, 50, 68). In adult skeletal muscle, ≈5% of the nuclei belong to undifferentiated, G0-arrested cells (satellite cells) that play an essential role during muscle regeneration, muscle hypertrophy, and postnatal muscle growth (23, 54, 55). In response to injury, stretch, or exercise, satellite cells become activated, reenter the cell cycle, and start to proliferate. The progeny of activated satellite cells can either differentiate and fuse with adjacent muscle fibers, effectively replacing the damaged fibers (4, 21), or reenter G0, remain undifferentiated, and replenish the satellite cell compartment (55). Mouse myoblastic cell line C2 and its subclone C2C12 have been used frequently as model systems of myogenic differentiation in vitro. Differentiation of myoblasts cultured in growth factor-rich medium (growth medium) is induced by transfer of cells to medium containing low concentration of mitogens (differentiation medium). Mitogen depletion leads to one of the following outcomes: a subpopulation of cells undergoes terminal differentiation and forms myotubes; another subpopulation called reserve cells remains undifferentiated and shares many characteristics with muscle satellite cells (3, 10, 18, 67); and a subset of cells undergoes apoptosis (62, 63). Reserve cells are arrested at the G0 phase of the cycle, have increased levels of the retinoblastoma-related protein p130, a quiescent cell marker (10), and decreased expression of myogenic transcription factor MyoD (31, 67). Interestingly, forced expression of p130 in myoblasts inhibits MyoD gene expression and its ability to transactivate muscle genes (10). Therefore, it has been proposed that induction of p130 is a part of a specific pathway that defines the pool of reserve cells during differentiation of myoblasts in vitro (10). The mechanisms involved in generation of reserve cells and upregulation of p130 in a fraction of myogenic cells upon mitogen deprivation remain, however, unknown. ADAMs (proteins containing a disintegrin and metalloprotease domain) are cell surface receptors that are involved in cell-cell adhesion, surface proteolysis, and transmembrane signaling (5, 53, 60). A typical ADAM consists of a prodomain, metalloprotease, disintegrin-like and cysteine-rich domains, and, in most cases, epidermal growth factor-like, transmembrane, and cytoplasmic domains. ADAM12 has been implicated in skeletal muscle development and regeneration (6, 19, 20, 66), but its exact function in muscle is not clear. ADAM12 mRNA is detected predominantly in cardiac and smooth muscle (20), placenta (20), and neonatal skeletal muscle (66). In adult skeletal muscle, the expression level of ADAM12 is very low in both differentiated muscle fibers and quiescent satellite cells (6, 20, 66). The amount of ADAM12 protein increases dramatically in regenerating muscle (19), and its mRNA is readily detected in satellite cells following their activation (6). During differentiation of C2C12 cells, ADAM12 mRNA and protein are present in undifferentiated cells, and during early stages of differentiation, and they both decrease to low levels as differentiation proceeds (8, 19, 66). Since differentiating C2C12 cultures are heterogeneous and contain both differentiated myotubes and undifferentiated reserve cells, we examined the distribution of ADAM12 between these two populations of cells to gain new insight into the function of ADAM12 during myoblast differentiation. We found that expression of ADAM12 is decreased in myotubes and preserved in reserve cells. Although inhibition of the expression of endogenous ADAM12 by small interfering RNA (siRNA) is accompanied by lower expression levels of both quiescence and differentiation markers, enhanced expression of ADAM12 induces a quiescent cell-like phenotype and does not stimulate differentiation. Our results suggest a novel role for ADAM12 in the establishment of the pool of reserve cells during myoblast differentiation.