Objective The goal of the present study was to identify novel mechanisms that regulate SMC differentiation marker gene expression. Approach and Results We demonstrate that the CArG-containing regions of many SMC-specific promoters are imbedded within CpG islands. A previously identified GC repressor element in the SM MHC promoter was highly methylated in cultured aortic SMC but not in the aorta and this difference was inversely correlated with SM MHC expression. Using an affinity chromatography/mass spectroscopy-based approach, we identified the multifunctional Notch transcription factor, RBPJ/CSL1, as a methylated GC-repressor binding protein. RBPJ protein levels and binding to the endogenous SM MHC GC repressor were enhanced by PDGF-BB treatment. A methylation mimetic mutation to the GC repressor that facilitated RBPJ binding inhibited SM MHC promoter activity as did over-expression of RBPJ. Consistent with this, knockdown of RBPJ in phenotypically modulated human aortic SMC enhanced endogenous SMC marker gene expression, an effect likely mediated by increased recruitment of SRF and Pol II to the SMC-specific promoters. In contrast, depletion of RBPJ in differentiated TGF-β-treated SMC inhibited SMC-specific gene activation supporting the idea that the effects of RBPJ/Notch signaling are context dependent. Conclusions Our results indicate that methylation-dependent binding of RBPJ to a GC repressor element can negatively regulate SM MHC promoter activity and that RBPJ can inhibit SMC marker gene expression in phenotypically modulated SMC. These results will have important implications on the regulation of SMC phenotype and on Notch-dependent transcription.

Objective The investment of newly formed endothelial cell tubes with differentiated smooth muscle cells (SMC) is critical for appropriate vessel formation, but the underlying mechanisms remain unknown. We previously showed that depletion of focal adhesion kinase (FAK) in the nkx2.5 expression domain led to aberrant outflow tract (OFT) morphogenesis and strove herein to determine the cell types and mechanisms involved. Methods and Results We crossed fakloxp targeted mice with available Cre drivers to deplete FAK in OFT SMC (FAKwnt and FAKnk) or coronary SMC (FAKcSMC). In each case, depletion of FAK led to defective vasculogenesis that was incompatible with post-natal life. Immunohistochemical analysis of the mutant vascular structures revealed that FAK was not required for progenitor cell proliferation, survival, or differentiation into SMC, but was necessary for subsequent SMC recruitment to developing vasculature. Using a novel FAK-null SMC culture model, we found that depletion of FAK did not influence SMC growth or survival, but blocked directional SMC motility and invasion toward the potent endothelial-derived chemokine, PDGFBB. FAK depletion resulted in un-stable lamellipodial protrusions due to defective spatial-temporal activation of the small GTPase, Rac-1 and lack of Rac1-dependent recruitment of cortactin (an actin stabilizing protein) to the leading edge. Moreover, FAK null SMC exhibited a significant reduction in PDGF-stimulated extracellular matrix degradation. Conclusions FAK drives PDGFBB-stimulated SMC chemotaxis/invasion and is essential for SMC to appropriately populate the aorticopulmonary septum and the coronary vascular plexus.