The goal of this proposal is to characterize the molecular mechanism by which extracellular matrix (ECM) molecules induce expression of growth- associated genes and control cell cycle progression in capillary endothelial (CE) cells. The design of this APPLICATION is based on the our recent results which show that ECM molecules, such as fibronectin (FN), induce CE cells to pass through the G0/G1 transition by clustering cell surface integrin receptors, activating the cell surface Na+/H+ antiporter, and inducing expression of early growth response genes including c-fos, c- jun, and c-myc. However, while these chemical signaling events are required for growth, they are not sufficient. CE cells must also change shape and physically extend in order to progress further through G1 and enter S phase. We propose to first fully characterize the time course of gene expression and other biochemical regulatory events that mediate of cell cycle progression in CE cells. Northern analysis and nuclear run on assays will be used to identify early, mid, and late growth-associated genes (e.g., c-myb, p53, c-ras, ornithine decarboxylase, thymidine kinase, histone 3.2, and cyclins D, E, & A) that become transcriptionally activated in response to cell binding to FN. Western blots, immunoprecipitations, and in vitro kinase assays will be carried out to analyze effects on activation of cyclin-dependent kinases and Retinoblastoma protein phosphorylation. We will also analyze the mechanism by which adhesion to FN and associated Na+/H+ antiporter stimulation result in transcriptional activation of growth-associated genes. Specifically, c-jun promoter constructs with CAT-reporter genes will be transiently expressed in CE cells. Soluble RGD-peptides and anti-integrin antibodies will be used to confirm that gene activation is mediated by ligation of integrin receptors and to identify gene regulatory elements that mediate this response. Amiloride analogues (hexamethylene amiloride, ethylisopropyl amiloride) that specifically inhibit Na+/H+ antiporter function will be used in conjunction with manipulation of medium conditions (pH, [Na+]0) to identify antiporter-sensitive response elements. We will use similar methods to dissect the molecular basis of regulation of other FN-sensitive genes, once they are identified. Finally, we will combine the above methodologies with a newly devised method for fabricating ECM-coated adhesive islands that promote integrin binding but prevent cell spreading to identify growth response genes and other cell cycle-related regulatory events that require cell spreading for their activation. Computerized image analysis will be used to quantitate changes in cell size and shape and to relate changes in cell structure and function. This experimental approach will facilitate analysis of the chemical signaling cascade that begins with integrin binding and ends with activation of specific growth response genes. It will also enable us to begin to translate the long recognized coupling between cell shape and growth into molecular terms. In this manner, we hope to better understand how capillary growth is controlled during angiogenesis, a process which is rate-limiting for the growth of solid tumors.
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