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.

Integrin receptors play a central role in cell migration through their roles as adhesive receptors for both other cells and extracellular matrix components. In this study, we demonstrate that integrin and cadherin receptors coordinately regulate contact-mediated inhibition of cell migration. In addition to promoting proliferation (Sastry, S., M. Lakonishok, D. Thomas, J. Muschler, and A. Horwitz. 1996. J. Cell Biol. 133:169–184), ectopic expression of the α5 integrin in cultures of primary quail myoblasts promotes a striking contact-mediated inhibition of cell migration. Myoblasts ectopically expressing α5 integrin (α5 myoblasts) move normally when not in contact, but upon contact, they show inhibition of migration and motile activity (i.e., extension and retraction of membrane protrusions). As a consequence, these cells tend to grow in aggregates and do not migrate to close a wound. This phenotype is also seen with ectopic expression of β1 integrin, paxillin, or activated FAK (CD2 FAK) and therefore appears to result from enhanced integrin-mediated signaling. The contact inhibition observed in the α5 myoblasts is mediated by N-cadherin, whose expression is upregulated more than fivefold. Perturbation studies using low calcium conditions, antibody inhibition, and ectopic expression of wild-type and mutant N-cadherins all implicate N-cadherin in the contact inhibition of migration. Ectopic expression of N-cadherin also produces cells that show inhibited migration upon contact; however, they do not show suppressed motile activity, suggesting that integrins and cadherins coordinately regulate motile activity. These observations have potential importance to normal and pathologic processes during embryonic development and tumor metastasis.

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Use of experimentally derived induced pluripotent stem cells (iPSCs) has led to the development of cell models for differentiation, drug testing and understanding disease pathogenesis. For these models to be informative, reprogrammed cell lines need to be adequately characterized and shown to preserve all of the critical characteristics of pluripotency and differentiation. Here, we report a detailed protocol for the generation of iPSCs from human fibroblasts containing mutations in COL3A1 using a Sendai virus mediated integration-free reprogramming approach. We describe how to characterize the putative iPSCs in vivo and in vitro to ensure potency and differentiation potential. As an example of how these mutations may affect cell surface and extracellular matrix (ECM) interactions, we provide protocols for the differentiation of these cells into smooth muscle cells to illustrate how different cell types may display cell autonomous differences in collagen receptors that may affect their phenotype. These cells, when applied to mechanical model systems (see Chapter 18 by Bose et al.) facilitate an assessment of stiffness and stress-strain relationships useful for understanding how extracellular matrix dysfunction and its interactions with surface proteins contribute to disease processes.