The long term objective of this project is a combined biochemical and genetic analysis of the hormonal regulation of cell membrane characteristics relevant to neoplasia. These studies have focused on the glucocorticoid and cyclic nucleotide regulation of the serine protease, tissue-type plasminogen activator (tPA), in HTC rat hepatoma cells in culture. The synthetic glucocorticoid dexamethasone rapidly inhibits tPA activity, whereas cyclic nucleotides dramatically stimulate tPA activity; paradoxically, dexamethasone enhances this stimulation. We have demonstrated that the glucocorticoid inhibition of tPA activity is secondary to the induction of a specific PA-inhibitor, PAI-1. We have purified and characterized the HTC PAI-1, and have cloned and characterized the cDNA for human endothelial cell PAI-1. The objective of the current proposal is to extend our studies on the hormonal regulation of tPA and PAI-1 to the molecular level. Techniques for the measurement of PA and PAI activity, antigen and mRNA are available and in use in our laboratory. We plan to compare the glucocorticoid and cyclic nucleotide regulation of tPA and PAI-1 in primary rat hepatocyte cultures with that in HTC rat hepatoma cells, examining the regulation of activity, antigen and mRNA levels for these two proteins and the mechanisms by which these levels are controlled. We will clone and characterize the cDNA and gemonic DNA for rat PAI-1, and clone and characterize the putative 5' regulatory regions of the rat tPA gene. Finally, we will analyze the nucleotide sequence requirements for the glucocorticoid and cyclic nucleotide regulation of rat tPA and PAI-1 gene expression with particular emphasis on combinatorial regulation by these agents. These studies will utilize DNAse hypersensitivity assays, nuclease protection techniques, and construction of chimeric genes containing wild-type and mutant regulatory elements of the tPA and PAI-1 genes and characterization of their hormonal regulation in transfected cells. Limited proteolysis mediated by the plasminogen activator- plasmin cascade plays a significant role in the behavior of transformed cells and in many normal processes. These studies should increase our understanding of the hormonal regulation of this important cell membrane property and of the biology of neoplastic cells.
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