Abstract. The tyrosine kinase called pp125 FAK is believed to play an important role in integrin-mediated signal transduction, pp125 FAK is associated both functionally and spatially with integrins, which are the cell surface receptors for extracellular matrix components. Although the precise function of pp125 FAK is not known, two possibilities have been proposed: pp125 yAK may regulate the assembly of focal adhesions in spreading or migrating cells, or pp125 FAK may participate in a signal transduction cascade to inform the nucleus that the cell is anchored. To test these models in living cells, a peptide representing the focal adhesion kinase (FAK)-binding site of the 131 tail was coupled to carrier protein and injected into cultured cells to competitively inhibit the binding of pp125 FAK to endogenous integrin, thus inhibiting activation of pp125 FAK on a cell-by-cell basis. In addition, an antibody directed against an epitope adjacent to the focal adhesion targeting sequence on pp125 FAK was microinjected, as an alternative means of inhibiting pp125 FAK activation. It was observed that when rounded cells were injected with either the integrin peptide or the anti-FAK antibody, the cells rapidly began to apoptose, within 4 h after injection. These results indicate that pp125 FA~ may play a critical role in suppressing apoptosis in fibroblasts.

The building of the vessel wall from its cellular and extracellular matrix (ECM) components is a critical event in the development and maturation of the cardiovascular system. However, little is known about the events that occur after the initial vascular network, a nascent endothelium, is established. The proper recruitment of vascular smooth muscle cells (VSMCs) to the endothelium is one such critical event. Although the majority of VSMCs are of mesodermal origin, it is not understood which populations of embryonic cells are capable of following the VSMC differentiation pathway. Previous studies, which have focused on the VSMC component of vessel wall development, have been limited by the use of markers that are not smooth muscle specific, or have focused on events that occur after a multilayered wall has been established. Therefore, the initial goal of this study was to define when overtly identifiable VSMCs were first associated with the vascular endothelium. Monoclonal antibodies (MAbs) were generated from embryonic vessel wall antigens in order to circumvent problems of cell specificity associated with the use of previously available markers to VSMCs. Critical to this study is our MAb, 1E12, which unlike other antibody markers, is smooth muscle specific. Using 1E12, we defined a pattern for recruitment and differentiation of the VSMC component of the descending aorta in stage 12 to stage 20 (Hamburger and Hamilton, 1951) quail embryos. Immunofluorescent labeling of quail embryos with 1E12 and a MAb to smooth muscle alpha-actin (SM alpha A) shows that the first mesodermally derived cells to associate with the aortic endothelium do so at the ventral surface. Recruitment of these cells, which we believe to be primordial VSMCs, proceeds in a ventral to dorsal direction along the aorta and in a radial direction, emanating from the endothelium. Additionally, we have determined the distribution of several ECM proteins, during the initial events of vessel wall development. Our studies show that fibulin-1 is expressed surrounding the primordial VSMCs of the vessel wall before elastin precursors are present and suggest that differential expression of the JB3 antigen (Wunsch et al., 1994) may be indicative of early diversity among embryonic VSMCs.

—The smooth muscle myosin heavy chain (SM-MHC) gene encodes a major contractile protein whose expression exclusively marks the smooth muscle cell (SMC) lineage. To better understand smooth muscle differentiation at the transcriptional level, we have initiated studies to identify those DNA sequences critical for expression of the SM-MHC gene. Here we report the identification of an SM-MHC promoter-intronic DNA fragment that directs smooth muscle–specific expression in transgenic mice. Transgenic mice harboring an SM-MHC- lac Z reporter construct containing ≈16 kb of the SM-MHC genomic region from −4.2 to +11.6 kb (within the first intron) expressed the lac Z transgene in all smooth muscle tissue types. The inclusion of the intronic sequence was required for transgene expression, since 4.2 kb of the 5′-flanking region alone was not sufficient for expression. In the adult mouse, transgene expression was observed in both arterial and venous smooth muscle, in airway smooth muscle of the trachea and bronchi, and in the smooth muscle layers of all abdominal organs, including the stomach, intestine, ureters, and bladder. During development, transgene expression was first detected in airway SMCs at embryonic day 12.5 and in vascular and visceral SMC tissues by embryonic day 14.5. Of interest, expression of the SM-MHC transgene was markedly reduced or absent in some SMC tissues, including the pulmonary circulation. Moreover, the transgene exhibited a heterogeneous pattern between individual SMCs within a given tissue, suggesting the possibility of the existence of different SM-MHC gene regulatory programs between SMC subpopulations and/or of episodic rather than continuous expression of the SM-MHC gene. To our knowledge, results of these studies are the first to identify a promoter region that confers complete SMC specificity in vivo, thus providing a system with which to define SMC-specific transcriptional regulatory mechanisms and to design vectors for SMC-specific gene targeting.