PRODUCTION & FUNCTION OF PDGF LIKE MOLECULES

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Recent evidence suggests that a platelet-derived growth factor-like molecule (PDGFc) is produced by several normal cell types under circumstances which are associated with cell proliferation in vivo. This project will investigate the nature, regulation, and possible roles of PDGFc. Specifically, the project will address the following questions: (1) Does PDGFc differ from PDGF from platelets? PDGFc will be compared with PDGF by SDS-polyacrylaminde gel electrophoresis and isoelectric focusing followed by detection of receptor-binding activity after gel elution and of antigenic reactivity by immunoblotting with anti-PDGF antisera. (2) What cells produce PDGFc and under what circumstances? Using cultured cells, initial observations of developmentally regulated production of PDGFc production by rat aortic smooth muscle cells (SMC), and of thrombin-stimulated production of PDGFc by vascular endothelial cells, will be further investigated and extended to other species and cell types. Biochemical and histochemical techniques will be used to investigate PDGFc production in vivo. (3) Is production of PDGFc regulated at the level of transcription, translation, post-translational processing, or secretion? This will be investigated in culture using thrombin stimulation of endothelial cells and developmentally regulated differences between pup and adult rat SMC as model systems. mRNA levels will be determined by hybridization with sis cDNA probes; potential protein precursors will be identified by immunoprecipitation of [35]cysteine-labeled intracellular proteins and by immunoblotting with peptide-specific antisera; active PDGFc will be quantitated by radioreceptor (RRA). (4) Is the reduction in the number of PDGF receptors on cells which produce PDGFc a consequence of decreased receptor synthesis or of downregulation by endogenously synthesized PDGFc? If the latter, where does the PDGFc-receptor interaction occur? Techniques will be developed to study solubilized receptors and PDGF-receptor complexes, and to determine the rate of accumulation of complexes, the locus of formation, and the physiological consequences of formation of cell surface vs intracellular complexes. (5) Does PDGFc production play an important role in determining the phenotype of a PDGF-responsive cell, or is it just an epiphenomenon? The importance of the extracellular phase of autocrine exposure to PDGFc will be evaluated by incubating the test cells with antibodies to PDGF or with inhibitors of PDGF binding. The effects of specifically terminating PDGFc synthesis will be evaluated by transfecting PDGFc-producing cells with a construct expressing anti-sense mRNA under the control of an inducible promotor.