The proliferation of glomerular mesangial cells and expansion of mesangial extracellular matrix appear to be common denominators in many types of glomerular disease. This application seeks to determine how mesangial cells grow in the context of signals mediated by SPARC, a secreted protein that modulates the behavior of endothelial cells and fibroblasts. We propose three Aims to determine how this Ca+2-and Cu+2- binding protein controls the shape an proliferation of mesangial cells. Specific activities of SPARC have been assigned to distinct domains of the molecule through studies with synthetic peptides and domain specific antibodies. An important extension of these peptide studies is the use of recombinant mutations to identify the active sites of SPARC. It is our hypothesis that SPARC is required for mesangial cell growth and that an internal, cationic Cu+2-binding domain contains bioactive elements necessary for mesangial proliferation. We will test our hypothesis by the assessment of the biological activities of specific mutants. Mutants will be engineered by in vitro oligonucleotide-directed mutagenesis, expressed in E. coli, and purified by nickel-chelate affinity chromatography. Recombinant SPARC, mutants and peptide fragments will be assayed for their ability to bind Cu+2 and to influence the phenotype and progression of the mesangial cell cycle. Proliferation will be measured by the incorporation of [3H]-thymidine into the cellular DNA of mesangial cells from rat glomeruli. Cellular spreading will be quantified by a Rounding index. We also hypothesize that SPARC can influence the morphology and attachment of mesangial cells through Ca+2-dependent activities. We propose that two Ca+2-binding regions are responsible for the anti- adhesive activity of SPARC. We will test this hypothesis by the construction of mutants that lack one or the other of these Ca+2-binding domains and by the determination of the extent to which these mutants bind Ca+2 and affect cell shape and cellular spreading. Through these Aims, we hope to acquire useful insights that define that active sites of SPARC, an extracellular protein with the potential to play a dynamic role in the regulations of mesangial cell proliferation and in the interaction between cells and their extracellular matrix. Insights derived from this project will be applied to studies of the renal pathophysiology of SPARC in project #2.
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