cAMP can be either mitogenic or anti-mitogenic, depending on the cell type. We demonstrated previously that cAMP inhibited the proliferation of normal renal epithelial cells and stimulated the proliferation of cells derived from the cysts of polycystic kidney disease (PKD) patients. The protein products of the genes causing PKD, polycystin-1 and polycystin-2, are thought to regulate intracellular calcium levels, suggesting that abnormal polycystin function may affect calcium signaling and thus cause a switch to the cAMP growth-stimulated phenotype. To test this hypothesis, we disrupted intracellular calcium mobilization by treating immortalized mouse M-1 collecting duct cells and primary cultures of human kidney epithelial cells with calcium channel blockers and by lowering extracellular calcium with EGTA. Calcium restriction for 3-5 h converted both cell types from a normal cAMP growth-inhibited phenotype to an abnormal cAMP growth-stimulated phenotype, characteristic of PKD. In M-1 cells, we showed that calcium restriction was associated with an elevation in B-Raf protein levels and cAMP-stimulated, Ras-dependent activation of B-Raf and ERK. Moreover, the activity of Akt, a negative regulator of B-Raf, was decreased by calcium restriction. Inhibition of Akt or phosphatidylinositol 3-kinase also allowed cAMP-dependent activation of B-Raf and ERK in normal calcium. These results suggest that calcium restriction causes an inhibition of the phosphatidylinositol 3-kinase/Akt pathway, which relieves the inhibition of B-Raf to allow the cAMP growth-stimulated phenotypic switch. Finally, M-1 cells stably overexpressing an inducible polycystin-1 C-terminal cytosolic tail construct were shown to exhibit a cAMP growth-stimulated phenotype involving B-Raf and ERK activation, which was reversed by the calcium ionophore A23187. We conclude that disruption of calcium mobilization in cells that are normally growthinhibited by cAMP can derepress the B-Raf/ERK pathway, thus converting these cells to a phenotype that is growth-stimulated by cAMP.

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Rotaviruses infect epithelial cells of the small intestine, but the pathophysiology of the resulting severe diarrhea is incompletely understood. Histological damage to intestinal epithelium is not a consistent feature, and in vitro studies showed that intestinal cells did not undergo rapid death and lysis during viral replication. We show that rotavirus infection of Caco-2 cells caused disruption of tight junctions and loss of transepithelial resistance (TER) in the absence of cell death. TER declined from 300 to 22 Omega. cm(2) between 8 and 24 h after infection and was accompanied by increased transepithelial permeability to macromolecules of 478 and 4,000 Da. Distribution of tight junction proteins claudin-1, occludin, and ZO-1 was significantly altered during infection. Claudin-1 redistribution was notably apparent at the onset of the decline in TER. Infection was associated with increased production of lactate, decreased mitochondrial oxygen consumption, and reduced cellular ATP (60% of control at 24 h after infection), conditions known to reduce the integrity of epithelial tight junctions. In conclusion, these data show that rotavirus infection of Caco-2 intestinal cells altered tight junction structure and function, which may be a response to metabolic dysfunction.

Regulation of intracellular Ca2؉ mobilization has been associated with the functions of polycystin-1 (PC1) and polycystin-2 (PC2), the protein products of the PKD1 and PKD2 genes. We have now demonstrated that PC1 can activate the calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway through G␣ q -mediated activation of phospholipase C (PLC). Transient transfection of HEK293T cells with an NFAT promoterluciferase reporter demonstrated that membrane-targeted PC1 constructs containing the membrane proximal region of the C-terminal tail, which includes the heterotrimeric G protein binding and activation domain, can stimulate NFAT luciferase activity. Inhibition of glycogen synthase kinase-3␤ by LiCl treatment further increased PC1-mediated NFAT activity. PC1-mediated activation of NFAT was completely inhibited by the calcineurin inhibitor, cyclosporin A. Cotransfection of a construct expressing the G␣ q subunit augmented PC1-mediated NFAT activity, whereas the inhibitors of PLC (U73122) and the inositol trisphosphate and ryanodine receptors (xestospongin and 2-aminophenylborate) and a nonspecific Ca 2؉ channel blocker (gadolinium) diminished PC1-mediated NFAT activity. PC2 was not able to activate NFAT. An NFAT-green fluorescent protein nuclear localization assay demonstrated that PC1 constructs containing the C-tail only or the entire 11-transmembrane spanning region plus C-tail induced NFATgreen fluorescent protein nuclear translocation. NFAT expression was demonstrated in the M-1 mouse cortical collecting duct cell line and in embryonic and adult mouse kidneys by reverse transcriptase-PCR and immunolocalization. These data suggest a model in which PC1 signaling leads to a sustained elevation of intracellular Ca 2؉ mediated by PC1 activation of G␣ q followed by PLC activation, release of Ca 2؉ from intracellular stores, and activation of store-operated Ca 2؉ entry, thus activating calcineurin and NFAT.

Progressive renal enlargement due to the growth of innumerable fluid-filled cysts is a central pathophysiological feature of autosomal dominant polycystic kidney disease (ADPKD). These epithelial neoplasms enlarge slowly and damage noncystic parenchyma by mechanisms that have not been clearly defined. In a microarray analysis of cultured human ADPKD cyst epithelial cells, periostin mRNA was overexpressed 15-fold compared with normal human kidney (NHK) cells. Periostin, initially identified in osteoblasts, is not expressed in normal adult kidneys but is expressed transiently during renal development. We found periostin in cyst-lining cells in situ in the extracellular matrix adjacent to the cysts and within cyst fluid. ADPKD cells secreted periostin across luminal and basolateral plasma membranes. Periostin increased proliferation of cyst epithelial cells 27.9 ± 3.1% ( P < 0.001) above baseline and augmented in vitro cyst growth but did not affect proliferation of normal renal cells. Expression of αV-integrin, a periostin receptor, was ninefold higher in ADPKD cells compared with NHK cells, and antibodies that block αV-integrin inhibited periostin-induced cell proliferation. We conclude that periostin is a novel autocrine mitogen secreted by mural epithelial cells with the potential to accelerate cyst growth and promote interstitial remodeling in ADPKD.