| In development and disease, the extracellular matrix (ECM) provides binding sites for cell adhesion and spreading events which alter cell structure and promote changes in phenotype. Here we demonstrate that ECM cues regulate responses of stem cell-derived smooth muscle (SM) progenitor cells to retinoic acid (RA), a key morphogen in SM differentiation. SM progenitor cells plated on fibronectin (FN) or laminin matrices showed markedly enhanced SM differentiation in response to RA as compared to control cells plated on plastic or BSA coated dishes. Cells grown on FN or laminin exhibited increased cell spreading, frequency of vinculin-positive focal adhesions, and coordinate activation of SM differentiation marker genes. Prior studies investigating the role of cell shape or spreading in modulating cell behaviors, including SM myogenesis, failed to separate changes in cell spreading and shape from cell-matrix adhesion. Here, results of quantitative microcontact printing studies involving independently varying cell-matrix adhesion versus cell spreading showed that RA-induced differentiation of SM progenitor cells was dependent on the degree of cell-matrix adhesion and not cell spreading. These effects were Rho and Rho kinase (ROCK) dependent and further found to increase the filamentous: globular (f:g) actin ratio. Changes in this ratio resulted in enhanced nuclear accumulation of myocardin related transcription factor 1, a known SM transcriptional coactivator, which is activated when released from g-actin during filament polymerization. Chromatin immunorecipitation assays also revealed that cell-matrix adhesion to FN or laminin increased histone 4 acetylation of SM gene promoters making DNA more accessible for transcription factor binding. Over expression of histone acetyltransferase p300 with active and wild type Rho showed that the activity of p300 and Rho were interdependent and both necessary for RA-mediated SM differentiation. Lastly, investigation of coronary SM differentiation using explanted murine proepicardial (PE) cells provided results consistent with a model in which RA-induced SM differentiation is dependent on matrix adhesion to ECM proteins FN and laminin. Taken together, these studies demonstrate that cell-matrix adhesion regulates RA induced SM differentiation from progenitor cells and that modulation of this factor and not cell spreading regulates this differentiation response. |
