Previous studies by us [20
] and others [36
] have indicated that in the absence of α-syntrophin muscle regeneration is slowed. We postulated that the slow regeneration could be a result of a defect in myoblast migration, an early step of muscle regeneration. Therefore, we designed a series of experiment to investigate how α-syntrophin affects myoblast migration. Our initial experiments showed that while α-syntrophin is diffusely present in the myoblast cytoplasm, it is specifically targeted to trailing edge of myoblasts induced to migrate by treatment with HGF. This localization of syntrophin is similar to that of PTEN, which is restricted to the sides and the rear of migrating cells [30
]. Furthermore, immunoprecipitation experiments demonstrated that a portion of the PTEN and α-syntrophin are in a complex together. This observation suggested that these two proteins are not only co-localized but may also participate in the same signaling pathways.
In the polarization step during initiation of cell migration, the molecular processes at the front and the back of a moving cell are different; Cdc42, Rac, PI3-kinase, PIP3 and microtubules are concentrated in the front of the cells whereas PTEN and myosin II are concentrated in the rear-part of the cells [5
]. Activation of PI3-kinase is required for the HGF-induced adherence junction disassembly, lamellipodia formation, and subsequent migration or scattering of MDCK epithelial cells [37
]. At the trailing edge of the migrating cells, many proteins function by either modulating tension force or rear retraction by disassembly of focal adhesion assemblies. Myosin light chain kinase (MLCK) phosphorylates myosin light chain, which regulates myosin II activity during regulation of the tractional force at the trailing edge [5
]. Recent work identified a novel mechanism of action for PTEN, whereby the C2 domain of PTEN is involved in the inhibition of cell-migration [39
]. The binding of syntrophins with PTEN has not been reported before in spite of the PDZ-binding domain of PTEN [40
]. In this study, we show that syntrophin can interact with PTEN by co-immunoprecipitation and co-immunostaining results (see ). However, the interaction of the two proteins was reduced by HGF treatment in the co-immunoprecipitation assay (see ) (although both proteins moved and concentrated at the trailing edge of the HGF-induced moving cells). Our results do not show whether the binding is direct or indirect. In mature skeletal muscle, α-syntrophin can bind many signaling molecules and kinases including the microtubule-associated serine/threonine kinase (MAST), syntrophin-associated serine/threonine kinase (SAST) [11
], stress-activated protein kinase 3 (SAPK3) [12
], diacylglycerol kinase δ (DGKδ) [13
], neuronal nitric oxide synthase (nNOS) [14
], and water channel aquaporin-4 [15
]. Less is known about the proteins associating with α-syntrophin during muscle development or regeneration. It has been reported that PDZ domains within SAST and MAST can bind to the C-terminal region of PTEN and stabilize its function [40
]. Therefore, α-syntrophin may interact with PTEN indirectly via several other binding proteins, or by direct interaction or even a combination of both.
Cell migration is important for various biological functions of muscle cells including differentiation, muscle development, and muscle regeneration. During skeletal muscle regeneration, satellite cells are activated and migrated by chemotaxis to wounded regions of myofibers, from which HGF is released [41
]. Wounded muscles require regenerating and/or proliferation of cells. HGF inhibits myogenesis and promotes cell proliferation of cultured C2 myoblasts [43
] and chicken skeletal muscle satellite cells [44
]. In addition, HGF plays a crucial role in the regulation of limb myogenic cell migration [45
]. In this study, we also have shown that HGF stimulated migration of C2 myoblasts and primary cultured myoblasts (see and ). The observation that syntrophins concentrate on the rear-lateral part of those migrating cells imply that it may be involved in the cell polarization preceding cell migration. To investigate the role of α-syntrophin in cell migration, we performed experiments using cells with reduced α-syntrophin expression. In both C2 cells and primary cultured myoblasts from transgenic mice, migration could not be stimulated by HGF. Neither α-syntrophin siRNA-transfected C2 cells nor myoblasts from α-syntrophin knockout mice increased to formation of lamellipodia in response to HGF. Based on these results, we conclude that α-syntrophin is required for the HGF-induced migration of cultured myoblasts.
The c-Met, receptor tyrosine kinase is the receptor of HGF and it is also called HGF-Receptor. c-Met is a disulfide-linked heterodimer composed of an α subunit (50 kDa) and β subunit (145 kDa) with tyrosine-kinase activity [47
]. On binding of HGF, c-Met forms a dimer and is induced to autophosphorylate tyrosine residues generating protein docking sites [37
]. Both Ras and PI3-kinase/Akt pathways are important signaling pathways in HGF-induced cell migration [35
]. The phosphorylation of tyrosine 1349, 1356, and 1365 in C-terminal on c-Met makes docking sites for interaction with scaffolding and signaling molecules such as PI3-kinase, Ras and PLC-γ [49
], while the phosphorylation of tyrosine 1234 and 1235 are related with c-Met kinase activity [51
]. We initially examined whether the phosphorylation of c-Met on tyrosine 1234 and 1235 is altered by HGF treatment of C2 myoblasts. Using western blotting with the phosphorylation specific antibody, we could not detect a significant difference on the level of phosphorylation on tyrosine 1234 and 1235 with treatment of HGF (see Supp Fig. 2
). We subsequently turned our attention to the effect of α-syntrophin on the PI3-kinase/Akt signaling pathway because the phosphorylation of c-Met on tyrosine 1349 was increased by HGF treatment in C2 myoblasts (see Supp Fig. 2
). First, we demonstrated that inhibitors of PI3-kinase blocked the HGF-induced lamellipodium formation (see ). The phosphorylation of Akt (Ser 473), a downstream effector of PI3-kinase, increases with HGF incubation (see ). Interestingly, when C2 cells are transfected with α-syntrophin-specific siRNA, phosphorylation of Akt was dramatically reduced regardless of HGF stimulation. In the α-syntrophin knock-down C2 cells, the distribution of PTEN in the rear-lateral part of the cells was disrupted even with HGF treatment (see ). In this case, it could be that PTEN dephosphorylates PIP3, which inhibits phosphorylation of Akt. These results suggest that syntrophin is involved in the PI3-kinase/Akt pathway in the HGF-induced lamellipodium formation.
α-Syntrophin knockout mice do not have specific symptoms for muscular dystrophy although they do have aberrant neuromuscular junctions [22
]. Previously, we have shown that both differentiation of α-syntrophin siRNA C2 cells and the regeneration of muscle in the α-syntrophin knock-out mouse is delayed compared to controls [20
]. In this study, we show that both α-syntrophin siRNA C2 cells and the myoblasts from the α-syntrophin knockout mice do not respond to HGF stimulation to initiate cell migration (see and ). These results suggest that deficiency of α-syntrophin results in insensitivity to HGF, which may cause delayed differentiation and regeneration.
Based on our observation that syntrophin is concentrated at the trailing edge of migrating myoblasts, we can propose mechanisms for syntrophin function. Intracellular calcium levels are implicated in the disassembly of adhesions at the trailing edge [52
]. Important targets of calcium are the calcium-regulated phosphatase, calcineurin [53
] and calcium-activated protease, calpain [54
], which is involved in adhesion disassembly. Syntrophin can bind to calmodulin via C-terminal 24 amino acids in the SU domain in a Ca2+
-dependent manner and α-syntrophin also can bind to plasma membrane Ca2+
/calmodulin-dependent ATPase (PMCA) [55
]. Both syntrophin and dystrophin are phosphorylated in vivo
and in vitro
/calmodulin dependent kinase II (CamKII) [57
] which is also required for the migration of PDGF-stimulated vascular smooth muscle cell [58
]. Together these reports suggest that syntrophins have a role in trailing edge retraction in a calcium-dependent manner. The PH1 domain of α-syntrophin also binds phosphoinositol 4, 5 bisphosphate (PtdIns(4,5)P2
] which is formed by phosphatidylinositol phosphate 5 kinase or PTEN. PtdIns(4, 5) P2
is involved in actin organization and focal adhesion formation [61
]. In addition, the heterotrimeric Gαβγ is bound by syntrophin in a laminin-dependent manner [63
]. It is likely that syntrophins function by linking these diverse signaling molecules to form a functional complex at the trailing edge that can modulate cell migration.