Abstract-The nonreceptor tyrosine kinase focal adhesion kinase (FAK) is a point of convergence for signals from extracellular matrix, soluble factors, and mechanical stimuli. Targeted disruption of the fak gene in mice leads to death at embryonic day 8.5 (E8.5). FAK Ϫ/Ϫ embryos have severely impaired blood vessel development. Gene expression and in vitro differentiation studies revealed that endothelial cell differentiation was comparable in FAK Ϫ/Ϫ and wild-type E8.5 embryos. We examined the role of FAK in blood vessel morphogenesis using an in vitro tubulogenesis assay and three different culture systems: FAK Key Words: focal adhesion kinase Ⅲ vasculogenesis Ⅲ angiogenesis Ⅲ fibronectin Ⅲ endothelial cell differentiation D uring embryogenesis, endothelial cells differentiate from mesodermal blood islands, proliferate, and form new blood vessels throughout embryonic and early postnatal life. The developing embryo first forms a primary vascular plexus by a process termed "vasculogenesis." These vessels are remodeled, and further expansion of the vasculature occurs by both sprouting and nonsprouting angiogenesis, leading to development of a functional circulatory system. Proliferation of endothelial cells virtually stops at that point and is very low in the adult. It resumes under certain physiological (eg, acute wound healing, cycling endometrium, pregnancy) and pathological (eg, tumor growth, rheumatoid arthritis) conditions. 1 Major roles have been described for angiogenic growth factors, extracellular matrix (ECM) components, and integrin ECM receptors in the processes of vasculogenesis and angiogenesis. However, it is still unclear how proangiogenic factors and integrin-mediated events cooperate in forming the vasculature. Gene targeting studies in mice have led to an appreciation of the stepwise process involved in forming functional blood vessels. In the most severe phenotype, deletion of the vascular endothelial growth factor receptor 2 (VEGFR-2) results in the absence of the hemangioblast precursor of both endothelial cells and hematopoietic stem cells. 2 Deletion of various proangiogenic growth factors and/or their receptors allows for formation of endothelial cells and initial vasculogenesis but interferes with later steps of vessel remodeling and sprouting. 1 Deletion of fibronectin (FN) or the ␣ 5 subunit of its receptor, ␣ 5  1 integrin, permits formation of endothelial cells and blood islands but interferes with later steps of forming a functional circulation. [3][4][5] Interestingly, other ECM components, notably thrombospondins 1 and 2, as well as fragments of ECM components or matrix remodeling enzymes, inhibit angiogenesis. 6 -10 Because integrins do not posses intrinsic enzymatic (ie, kinase) activity, signals from FN and other ECM components must be transduced indirectly to the signaling machinery of the cell. Integrins accomplish this by recruiting multimolecular complexes of cytoskeletal and signaling molecules at focal adhesion sites. FAK was the first known nonreceptor protein tyrosine...
Targeted disruption of the focal adhesion kinase (FAK) gene in mice is lethal at embryonic day 8.5 (E8.5). Vascular defects in FAK-/- mice result from the inability of FAK-deficient endothelial cells to organize themselves into vascular network. We found that, although fibronectin (FN) levels were similar, its organization was less fibrillar in both FAK-/- endothelial cells and mesoderm of E8.5 FAK-/- embryos, as well as in mouse embryonic fibroblasts isolated from mutant embryos. FAK catalytic activity, proline-rich domains, and location in focal contacts were all required for proper allocation and patterning of FN matrix. Cells lacking FAK in focal adhesions fail to translocate supramolecular complexes of integrin-bound FN and focal adhesion proteins along actin filaments to form mature fibrillar adhesions. Taken together, our data suggest that proper FN allocation and organization are dependent on FAK-mediated remodeling of focal adhesions.
Focal adhesion kinase (FAK) is a critical component in transducing signals downstream of both integrins and growth factor receptors. To determine how the loss of FAK affects the epidermis in vivo, we have generated a mouse model with a keratinocyte-restricted deletion of fak (FAK K5 KO mice). FAK K5 KO mice displayed three major phenotypes -irregularities of hair cycle, sebaceous glands hypoplasia, and a thinner epidermis -pointing to defects in the proliferative capacity of multipotent stem cells found in the bulge. FAK-null keratinocytes in conventional primary culture undergo massive apoptosis hindering further analyses, whereas the defects observed in vivo do not shorten the mouse lifespan. These results suggest that the structure and the signaling environment of the native tissue may overcome the lack of signaling through FAK. Our findings point to the importance of in vivo and threedimensional in vitro models in analyses of cell migration, proliferation, and survival. Surprisingly, the difference between FAK loxP/ þ and FAK K5 KO mice in wound closure was not statistically significant, suggesting that in vivo loss of FAK does not affect migration/proliferation of basal keratinocytes in the same way as it affects multipotent stem cells of the skin.
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