Focal adhesion kinase regulates smooth muscle cell recruitment to the developing vasculature.

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 fak(loxp) targeted mice with available Cre drivers to deplete FAK in OFT SMC (FAK(wnt) and FAK(nk)) or coronary SMC (FAK(cSMC)). In each case, depletion of FAK led to defective vasculogenesis that was incompatible with postnatal 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, platelet-derived growth factor PDGFBB. FAK depletion resulted in unstable 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 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.

Transient expression of FRNK reveals stage-specific requirement for focal adhesion kinase activity in cardiac growth.

Focal adhesion kinase (FAK) is strongly activated by integrins and growth factors and is essential for embryonic development. We previously showed that the C terminus of FAK is expressed as a separate protein termed FAK-related nonkinase (FRNK) in a smooth muscle cell-selective fashion and that FRNK functions to buffer FAK-dependent signals. We now show that FRNK is also transiently expressed in the neonatal myocardium, with peak levels occurring 5 to 7 days postnatal, just before cell cycle withdrawal. Using novel mouse models, we demonstrate that cardiac-selective expression of FRNK (leading to inhibition of FAK) starting at embryonic day 10.5 leads to a severe ventricular noncompaction defect associated with reduced cardiomyocyte proliferation. Remarkably, postnatal expression of nearly identical levels of FRNK is well tolerated and does not affect viability or anabolic cardiac growth. Nonetheless, FRNK expression in the adult heart does attenuate pathological cardiac hypertrophy following aortic banding, confirming and extending our previous data that this compensatory response is blunted in FAK null hearts. Our mechanistic studies in cultured neonatal cardiomyocytes reveal that FRNK expression induces p38/p27(kip)-dependent cell cycle withdrawal and attenuates extracellular signal-regulated kinase-dependent hypertrophic growth. These findings indicate that dynamic expression of FRNK in the neonatal heart may function to promote cardiomyocyte quiescence in an environment that is particularly rich in growth factors and growth promoting extracellular matrices.

FRNK expression promotes smooth muscle cell maturation during vascular development and after vascular injury.

OBJECTIVE: Smooth muscle cell (SMC) differentiation is a dynamic process that must be tightly regulated for proper vascular development and to control the onset of vascular disease. Our laboratory previously reported that a specific focal adhesion kinase (FAK) inhibitor termed FRNK (FAK Related Non-Kinase) is selectively expressed in large arterioles when SMCs are transitioning from a synthetic to contractile phenotype and that FRNK inhibits FAK-dependent SMC proliferation and migration. Herein, we sought to determine whether FRNK expression modulates SMC phenotypes in vivo. METHODS AND RESULTS: We present evidence that FRNK(-/-) mice exhibit attenuated SM marker gene expression during postnatal vessel growth and after vascular injury. We also show that FRNK expression is regulated by transforming growth factor (TGF)-beta and that forced expression of FRNK in cultured cells induces serum- and TGF-beta-stimulated SM marker gene expression, whereas FRNK deletion or expression of a constitutively activated FAK variant attenuated SM gene transcription. CONCLUSIONS: These data highlight the possibility that extrinsic signals regulate the SMC gene profile, at least in part, by modulating the expression of FRNK and that tight regulation of FAK activity by FRNK is important for proper SMC differentiation during development and after vascular injury.

The LIM protein leupaxin is enriched in smooth muscle and functions as an serum response factor cofactor to induce smooth muscle cell gene transcription.

Leupaxin is a LIM domain-containing adapter protein belonging to the paxillin family that has been previously reported to be preferentially expressed in hematopoietic cells. Herein, we identified leupaxin in a screen for focal adhesion kinase binding partners in aortic smooth muscle, and we show that leupaxin is enriched in human and mouse vascular smooth muscle and that leupaxin expression is dynamically regulated during development. In addition, our studies reveal that leupaxin can undergo cytoplasmic/nuclear shuttling and functions as an serum response factor cofactor in the nucleus. We found that leupaxin forms a complex with serum response factor and associates with CArG-containing regions of smooth muscle promoters and that ectopic expression of leupaxin induces smooth muscle marker gene expression in both 10T1/2 cells and rat aortic smooth muscle cells. Subsequent studies indicated that enhanced focal adhesion kinase activity (induced by fibronectin or expression of constitutively active focal adhesion kinase) attenuates the nuclear accumulation of leupaxin and limits the ability of leupaxin to enhance serum response factor-dependent gene transcription. Thus, these studies indicate that modulation of the subcellular localization of serum response factor cofactors is 1 mechanism by which extracellular matrix-dependent signals may regulate phenotypic switching of smooth muscle cells.