Focal adhesion kinase and its endogenous inhibitor, FRNK, in vascular development and injur

Development and maintenance of a circulatory system is required for transport of essential gases and nutrients in vertebrates. Within this work, I have aimed to elucidate the role of a certain family of adhesion signaling molecules in regulating the responses of vascular smooth muscle cells (VSMC) that enable circulatory vessels to form and maintain a differentiated phenotype. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that coordinates signaling from integrins, growth factor receptors, and Gprotein coupled receptors. While FAK is ubiquitously expressed, its variant FAK-related non kinase (FRNK) is selectively expressed in smooth muscle containing tissues. Our group has recently demonstrated that over-expression of FRNK in vascular smooth muscle cells (VSMC) can attenuate PDGF-stimulated proliferation and migration of vascular smooth muscle cells. FRNK is highly expressed during early postnatal development (particularly in large arteries) and two weeks following endovascular injury in the rat femoral artery. Regulation of FRNK in smooth muscles cells is unique in that FRNK is expressed as a gene within a gene under the control of non-coding sequence within a FAK intron. This FRNK promoter sequence is void of CArG boxes, making transcription independent of serum response factor (SRF). Germline deletion of FAK causes mesodermal defects resulting in embryonic lethality, implicating FAK as a significant regulator of vascular development. Since FAK-/- embryos die prior to complete formation of the vasculature, we used a conditional approach to delete FAK specifically in cardiac neural-crest derived cells. The goals of this work were to (1) determine the factors that regulate SRF-independent smooth muscle-specific expression of FRNK, (2) use a gene ablation model to elucidate an in vivo function for FRNK, and (3) use SMC-specific conditional knockout approach to examine a role for FAK in vascular development. Herein we found that FRNK expression is regulated by TGF-beta and extracellular matrix components. Conservation analysis revealed two ∼100-150 bp regions of the FRNK promoter that are over 75% conserved between mouse, rat and human. Although we found that FRNK-/- mice exhibit increased phosphorylation of FAK Y397 and proliferation our most striking observation was that deletion of FRNK causes decreased expression of smooth muscle alpha-actin, smooth muscle myosin heavy chain, and smoothelin during postnatal development and in the neointima following carotid artery ligation. Likewise, overexpression of FRNK stimulated smooth muscle promoter activity in serum and enhanced TGF-beta mediated increases in smooth muscle promoter activity. To study the role of FAK in development of cardiac neural crest-derived smooth muscle cells, we used a previously described Wnt-1-Cre expressing mouse to conditionally delete FAK in this cell population. We found that deletion of FAK resulted in malformation of the aorticopulmonary septum and that animals did not survive to adulthood. Additional in vivo and in vitro studies revealed that FAK activity downregulates activity of smooth muscle specific genes. The studies described herein indicate that the adhesion signaling molecule FAK and its truncated family member, FRNK, regulate smooth muscle cell phenotype.