Endothelin Signaling and Regulation of Protein Kinases

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DESCRIPTION (provided by applicant): Endothelin 1 (ET-1) exerts vasoconstrictor, hypertrophic and mitogenic actions on the renal vasculature and glomerular mesangial cells (GMC). ET-1-dependent contraction of GMC can regulate the glomerular ultrafiltration coefficient and ET-1 induced glomerular hypertrophy and proliferation may contribute to diverse types of proliferative and sclerotic glomerular diseases. Stimulation of GMC with ET-1 evokes a wide variety of signaling events; however, ET-1-induced cell proliferation and hypertrophy occurs primarily via activation of the extracellular signal-regulated kinase (ERK). Contractile responsiveness of GMC was shown to depend on activation of the p38 MAPK. We hypothesize that protein-protein interactions are among crucial components in the endothelin-induced intracellular signaling pathways leading to short- and long-term effects of ET-1 and that protein tyrosine phosphorylation, which often regulates these interactions, may be a principal factor in evolution of renal diseases. Specific Aim 1 will evaluate the putative involvement of tyrosine kinase Pyk2 in ET-1-mediated GMC contractility. We will carry out adenoviral mediated transfer of genes encoding dominant interfering mutant and wild type Pyk2 into rat and human GMC and analyze the effect of inhibition of signaling via Pyk2 upon ET-1 mediated mesangial cell contraction, activation of small GTPases regulating p38 MAPK, activation of MAP kinase-activated protein kinase 2/3 (downstream substrate of p38 MAPK), phosphorylation of HSP 25 and regulation of actin filament dynamics. Specific Aim 2 will evaluate the hypothesis that guanine nucleotide exchange factor Pix mediates stimulation of small GTPase cdc42 by endothelin in GMC. We will study a) the direct interaction of Pix with heterotrimeric G proteins, involved in ET-1 signaling in mesangial cells; b) ability of dominant interfering mutants of Pix to inhibit ET-l-dependent activation of cdc42; c) ability of dominant interfering mutants of Pix to inhibit ET-1- dependent activation of MAPKs. Specific Aim 3 will evaluate the hypothesis that negative regulation of endothelin signaling on the level of intracellular MAPK cascades by dual specificity phosphatases is an essential component of the normal signal transduction by ET-1. We hypothesize that MKP-3 inhibits ET-1 mediated ERK activation, whereas MKP-1 attenuates p38 MAPK and tight regulation of MAPK signaling by MKP-1 and MKP-3 controls expression of genes critical for GMC hypertrophy.