This is a competitive renewal of a grant, which has for several years examined the mechanisms of regulation of prostaglandin endoperoxide synthase (PGHS). Glomerular mesangial cells (GMC) play a key role in the genesis of several glomerular diseases such as glomerulosclerosis and proliferative glomerulonephritis, which are characterized by GMC injury and proliferation. Proinflammatory agents can induce cellular synthesis of eicosanoids, namely oxygenated products of arachidonic acid, that can regulate GMC matrix synthesis and proliferation. This application examines the intracellular signaling mechanisms regulating the expression and actions of PGHS, which catalyses prostaglandin production from arachidonic acid. PGHS is of particular interest, being the principal target of NSAIDS. A combination of in vitro and in vivo studies will be used to test the hypothesis that PGHS-2 expression is regulated by mitogen activated protein kinases (MAPKs), and that over expression of PGHS-2 has anti-apoptotic effects mediated through differential gene expression.
Three Specific Aims have been outlined.
Specific Aim 1 will evaluate the hypothesis that activation of MAPKs represents the convergence point of divergent stimuli of PGHS-2 expression, and this is negatively regulated by dual-specificity phosphatases. Here, the effects on PGHS-2 expression after adenovirus mediated transfer of genes encoding wild type and mutant ERK, JNK and p38 MAPKs, and dual-specificity phosphatases will be studied in kidney cells.
Specific Aim 2 will examine the hypothesis that PGHS-2 expression leads to anti-apoptotic effects in several cell types. Here the effect of adenovirus-mediated transfer of PGHS-2 gene into primary cells on apoptotic parameters will be studied. The mechanism of the anti-apoptotic effect will be studied by identifying genes controlled by PGHS-2 expression using cDNA micro-arrays.
In Specific Aim 3, the in vivo relevance of PGHS-2 expression and relation to the MAPK cascade will be evaluated using two rat models of glomerulonephritis (GN). Here, PGHS-2 expression and prostaglandin production will be assessed in rat glomeruli and correlated with MAPK activation and the histopathology of GN at defined time points of the disease.
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