Regulation of Myogenesis by Fibroblast Growth Factors Requires Beta-Gamma Subunits of Pertussis Toxin-Sensitive G Proteins

Of the soluble growth factors thought to play critical roles in the development of skeletal muscle, fibroblast growth factors (FGFs), Sonic hedgehog, scatter factor/hepatocyte growth factor, and transforming growth factor β are thought to be required for skeletal muscle development in vivo (2, 5, 16, 23, 48). We are attempting to delineate the signaling pathways utilized by FGFs that regulate the proliferation and differentiation of skeletal muscle cells. Previous studies performed by other groups as well as data obtained in our laboratory have demonstrated that (i) distinct FGF pathways are involved in regulating MM14 myoblast growth and differentiation (37), (ii) FGF signaling pathways cannot be replaced by stimulation of other growth factor receptors (36, 38, 39), and (iii) FGFs stimulate activation of mitogen-activated protein kinase (MAPK) pathways (8, 36, 45, 47). Of the four identified FGF receptor tyrosine kinases, only one, FGF receptor-1, is detectably expressed in MM14 cells (37, 63); it is required for FGF-mediated repression of terminal differentiation (22). Additionally, high-affinity binding and subsequent signaling events require that FGFs bind to both the tyrosine kinase and a heparan sulfate proteoglycan (49, 53, 54). Pertussis toxin-sensitive, G-protein-coupled mechanisms have been reported to affect myoblast differentiation and proliferation, although the mechanisms involved have not been investigated (30, 67). Pertussis toxin (PT), a protein virulence factor produced by Bordetella pertussis, is composed of an A protomer and a B oligomer. The A protomer consists of a single peptide that ADP-ribosylates specific eucaryotic G proteins (Gi/o), locking the G protein in the GDP-bound state and preventing dissociation of Gα and Gβγ subunits, thus leading to inactivation of the G-protein signal. The B oligomer binds to cell surface receptor proteoglycans and transfers the A protomer to the interior of the cell (29). The heterotrimeric G proteins are composed of distinct α, β, and γ subunits, and all three can participate in signal transduction. Following receptor activation by agonist, Gα subunits of PT-sensitive proteins transmit signals to adenylyl cyclase and other effector molecules (66). The Gβγ heterodimer, released upon activation of PT-sensitive G proteins, activates K+ channels (35), mediates the translocation of the β-adrenergic receptor kinase 1 (βARK1) (64), regulates specific isoforms of adenylyl cyclase (62) and phospholipase C (PLC) (9), and stimulates the MAPKs (12, 15, 41). Stimulation of MAPK activity by the insulin-like growth factor 1 (IGF-1) receptor tyrosine kinase depends on participation of Gβγ subunits derived from PT-sensitive G proteins (41). As for the G-protein-coupled receptor-mediated pathways, IGF-1 signaling can be inhibited by PT treatment or by a Gβγ subunit inhibitor (41). A large number of polypeptide growth factor receptors stimulate activation of MAPKs (4, 46, 55). A few reports have demonstrated that MAPK stimulation is PT sensitive. Among the receptor tyrosine kinases, PT interferes with the activation of MAPK by epidermal growth factor in hepatocytes (20) and by IGF-1 in Rat-1 fibroblasts (66). Activation of MAPKs is known to occur via the Ras/Raf/MKK1/2 pathway (27, 60). Recently, MAPKs were reported to be activated by Ras-independent mechanisms that include c-Src protein tyrosine kinase (18) and protein kinase C (7, 44, 46, 65) pathways. Additional complexity in these signaling pathways is suggested by the existence of MAPK kinase kinases other than Raf (3). G-protein-dependent signaling can be coupled to the MAPK cascade through release of free βγ subunits, which is linked to activation of a Ras-dependent pathway (32), or through activation of MAPK by PT-sensitive Gα subunits (31, 64). Here we report that PT stimulates myogenic differentiation in the presence of FGF-2, inhibits FGF-induced proliferation of MM14 cells, and blocks FGF-2-stimulated MAPK activity. In addition, FGF-2 signaling can be blocked by inhibitors of Gβγ subunits. Expression of the free Gβγ dimer suppresses PT-stimulated differentiation and mimics the effect of FGF-2 on MM14 cells. Thus, we demonstrate for the first time that signaling pathways regulated by binding of FGF-2 to FGF receptor-1 can be mediated by Gβγ subunits of PT-sensitive heterotrimeric G proteins.