Quantitative Changes in Integrin and Focal Adhesion Signaling Regulate Myoblast Cell Cycle Withdrawal

Abstract

We previously demonstrated contrasting roles for integrin α subunits and their cytoplasmic domains in controlling cell cycle withdrawal and the onset of terminal differentiation (Sastry, S., M. Lakonishok, D. Thomas, J. Muschler, and A.F. Horwitz. 1996. J. Cell Biol. 133:169–184). Ectopic expression of the integrin α5 or α6A subunit in primary quail myoblasts either decreases or enhances the probability of cell cycle withdrawal, respectively. In this study, we addressed the mechanisms by which changes in integrin α subunit ratios regulate this decision. Ectopic expression of truncated α5 or α6A indicate that the α5 cytoplasmic domain is permissive for the proliferative pathway whereas the COOH-terminal 11 amino acids of α6A cytoplasmic domain inhibit proliferation and promote differentiation. The α5 and α6A cytoplasmic domains do not appear to initiate these signals directly, but instead regulate β1 signaling. Ectopically expressed IL2R-α5 or IL2R-α6A have no detectable effect on the myoblast phenotype. However, ectopic expression of the β1A integrin subunit or IL2R-β1A, autonomously inhibits differentiation and maintains a proliferative state. Perturbing α5 or α6A ratios also significantly affects activation of β1 integrin signaling pathways. Ectopic α5 expression enhances expression and activation of paxillin as well as mitogen-activated protein (MAP) kinase with little effect on focal adhesion kinase (FAK). In contrast, ectopic α6A expression suppresses FAK and MAP kinase activation with a lesser effect on paxillin. Ectopic expression of wild-type and mutant forms of FAK, paxillin, and MAP/erk kinase (MEK) confirm these correlations. These data demonstrate that (a) proliferative signaling (i.e., inhibition of cell cycle withdrawal and the onset of terminal differentiation) occurs through the β1A subunit and is modulated by the α subunit cytoplasmic domains; (b) perturbing α subunit ratios alters paxillin expression and phosphorylation and FAK and MAP kinase activation; (c) quantitative changes in the level of adhesive signaling through integrins and focal adhesion components regulate the decision of myoblasts to withdraw from the cell cycle, in part via MAP kinase.

TGF-β1 up-regulates paxillin protein expression in malignant astrocytoma cells: requirement for a fibronectin substrate

“…In our experiments, we found similar levels of paxillin in the malignant astrocytoma cells plated onto ®bronectin, laminin, or vitronectin for 5 h. Thus, although our experiments are not directly comparable to those reported in myoblasts (Sastry et al, 1999), our data demonstrate that engagement of the a5b1 integrin by plating on a ®bronectin substrate does not in itself result in increased paxillin protein expression in U-251MG cells, as compared to engagement of the a6b1 laminin receptor integrin and the avb3/avb5 vitronectin receptor integrins. However, our data do suggest that there is a cooperation of TGF-b1 with the a5b1 ®bronectin receptor integrin in the malignant astrocytoma cells, as TGF-b1 failed to increase the expression of paxillin protein when the cells were plated onto other substrates (laminin, collagen, or vitronectin).…”

“…Other investigators have reported previously that transfection of the a5 subunit of the ®bronectin receptor integrin into myoblasts promoted cell proliferation and increased paxillin protein expression (Sastry et al, 1999). In our experiments, we found similar levels of paxillin in the malignant astrocytoma cells plated onto ®bronectin, laminin, or vitronectin for 5 h. Thus, although our experiments are not directly comparable to those reported in myoblasts (Sastry et al, 1999), our data demonstrate that engagement of the a5b1 integrin by plating on a ®bronectin substrate does not in itself result in increased paxillin protein expression in U-251MG cells, as compared to engagement of the a6b1 laminin receptor integrin and the avb3/avb5 vitronectin receptor integrins.…”

Paxillin: Adapting to Change

“…Forced overexpression of paxillin in cultured quail myoblasts blocks differentiation, instead promoting continued cell proliferation and signaling through the MAP kinase pathway (227). Phosphorylation of paxillin on amino acids Y31/118 and S188/190, which have each been shown to be regulated via integrin signaling, is necessary to maintain the undifferentiated phenotype (227).…”

Integrins in the mouse myotome: Developmental changes and differences between the epaxial and hypaxial lineage

“…In vitro studies have shown that ␣6␤1 is able to inhibit proliferation and stimulate differentiation of myoblasts (Sastry et al, 1996(Sastry et al, , 1999 and laminin promotes myoblast differentiation in vitro, inducing them to become postmitotic, fusion-capable, and myosin-and desmin-positive (von der Mark and Ocalan, 1989). In fact, it has been demonstrated recently that the ECM is necessary for skeletal muscle differentiation, independently of MRF expression, suggesting that, upon binding to the ECM, integrins generate signals that drive skeletal muscle differentiation (Osses and Brandan, 2002).…”