CALCIUM REGULATION OF cAMP-DEPENDENT PROLIFERATION

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Polycystic kidney diseases (PKDs) are lethal, hereditary disorders characterized by hyperplasia of the tubule epithelium, cyst formation and massive kidney enlargement. cAMP agonists, including arginine vasopressin, accelerate the proliferation of epithelial cells from PKD cysts but not from normal human kidneys (NHK). We discovered that cAMP activates extracellular signal-regulated kinases1/2[2] (ERK) in human PKD, but inhibits ERK activation in NHK cells. The molecular mechanisms underlying this phenotypic difference between PKD and NHK cells are linked to cAMP/ protein kinase A-dependent B-Raf activation of MEK and ERK, leading to increased cell proliferation. Recent studies in animals with four different genetic types of PKD showed that inhibition of renal cAMP production by vasopressin V2 receptor antagonist OPC-31260 dramatically halted cyst and kidney enlargement, demonstrating a central role for cAMP in renal cystic disease. Mutated gene products of hereditary cystic disorders are thought to cause abnormal Ca2+ levels in renal tubule cells. Recently, we found that inhibition of Ca2+ entry in mouse cortical collecting duct cells (M-1) with channel blockers or reduced extracellular [Ca2+] caused a phenotypic switch in the proliferation response to cAMP. cAMP inhibited the proliferation of M-1 cells with normal intracellular [Ca2+]; however, in M-1 cells with reduced [Ca2+] cAMP stimulated B-Raf, the MEK-ERK pathway and cell proliferation, mimicking the PKD phenotype. The central hypothesis is that in human ADPKD and ARPKD, dysfunctional Ca2+ metabolism by renal epithelial cells induces and maintains a "phenotypic switch" that uncovers a common cellular pathway leading to cAMP-dependent activation of B-Raf and ERK, and increased cell proliferation. The strength of this proposal is the use of cyst epithelial cells from two different types of human hereditary disease, ADPKD and ARPKD, to address the following specific aims: Aim 1: Determine if [Ca2+]i modifies cAMP-dependent B-Raf signaling through the MEK-ERK pathway and contributes to the phenotypic difference in between PKD and NHK cells in the cAMP mitogenic response. Aim 2. Elucidate mechanisms by which vasopressin V2 receptor agonists and antagonists adjust intracellular Ca2+ and modulate cAMP-dependent B-Raf activation and the proliferation of human PKD cells. Aim 3. Determine if selective reduction of B-Raf abundance and inhibition of B-Raf kinase activity diminish cAMP-dependent ERK activation and cell proliferation in PKD cells. The results from these studies will provide fundamentally new opportunities for developing novel small molecule therapies to slow, and possibly halt the progression of PKD in patients.