Polycystin-1 induces cell migration by regulating phosphatidylinositol 3-kinase-dependent cytoskeletal rearrangements and GSK3beta-dependent cell cell mechanical adhesion.

Polycystin-1 (PC-1) is a large plasma-membrane receptor encoded by the PKD1 gene mutated in autosomal dominant polycystic kidney disease (ADPKD). Although the disease is thought to be recessive on a molecular level, the precise mechanism of cystogenesis is unclear, although cytoarchitecture defects seem to be the most likely initiating events. Here we show that PC-1 regulates the actin cytoskeleton in renal epithelial cells (MDCK) and induces cell scattering and cell migration. All of these effects require phosphatidylinositol 3-kinase (PI3-K) activity. Consistent with these observations Pkd1-/- mouse embryonic fibroblasts (MEFs) have reduced capabilities to migrate compared with controls. PC-1 overexpressing MDCK cells are able to polarize normally with proper adherens and tight junctions formation, but show quick reabsorption of ZO-1, E-cadherin, and beta-catenin upon wounding of a monolayer and a transient epithelial-to-mesenchymal transition (EMT) that favors a rapid closure of the wound and repolarization. Finally, we show that PC-1 is able to control the turnover of cytoskeletal-associated beta-catenin through activation of GSK3beta. Expression of a nondegradable form of beta-catenin in PC-1 MDCK cells restores strong cell-cell mechanical adhesion. We propose that PC-1 might be a central regulator of epithelial plasticity and its loss results in impaired normal epithelial homeostasis.

Apoptosis-related gene expression affected by a GnRH analogue without induction of programmed cell death in LNCaP cells.

BACKGROUND: In this study we confirmed the ability of a Gonadotropin Releasing Hormone (GnRH) agonist, leuprorelin acetate (LA), to counteract or even suppress the 5alpha-dihydrotestosterone (DHT)-stimulated growth of androgen-sensitive prostate cancer cells (LNCaP). Since the cellular mechanisms mediating this effect are not well defined, we investigated the activity of LA, also in combination with DHT or with cyproterone acetate (CA), on the expression of genes (bcl-2, bax, c-myc) which may contribute to the proliferative behaviour of LNCaP cells. In addition, experiments aimed to evaluate the action of the analogue on apoptosis were performed. MATERIALS AND METHODS: Gene expression was evaluated by RT-PCR and Western blotting on cells treated with LA (10(-11) or 10(-6) M), alone or combined with 10(-9) M DHT or 10(-7) M CA. The occurrence of apoptosis following treatment with LA (10(-11), 10(-6) or 10(-5) M), alone or combined with 10(-9) M DHT, was assessed by DNA fragmentation analysis. RESULTS: Both the mRNA and protein of the anti-apoptotic gene bcl-2 were induced (30-125%) by DHT after 24-144 h. LA decreased bcl-2 mRNA (10-40%), while it did not unequivocally affect protein expression. The analogue always reduced (13-74%) both mRNA and protein levels obtained under DHT treatment. The mRNA and protein of the pro-apoptotic gene bax were down-regulated by DHT (15-40%), while LA generally induced bax protein but not its mRNA. LA counteracted DHT activity, even increasing bax protein levels over the controls. c-myc mRNA and protein were enhanced by DHT (15-45%) but down-regulated by LA (10-40%). Once more, the androgen effect was antagonized by LA, sometimes reducing c-myc content below the controls. CA produced the most similar effects to those triggered by DHT. The hormonal treatment did not induce any DNA fragmentation. CONCLUSION: In spite of gene modulation, apoptosis was not observed under LA treatment, in agreement with the lack of a cell growth effect when the analogue was used alone. Nevertheless, the observed changes in gene expression may be directly or indirectly involved in the antiproliferative effect of LA on androgen-stimulated cells.

Polycystin-1 binds Par3/aPKC and controls convergent extension during renal tubular morphogenesis.

Several organs, including the lungs and kidneys, are formed by epithelial tubes whose proper morphogenesis ensures correct function. This is best exemplified by the kidney, where defective establishment or maintenance of tubular diameter results in polycystic kidney disease, a common genetic disorder. Most polycystic kidney disease cases result from loss-of-function mutations in the PKD1 gene, encoding Polycystin-1, a large receptor of unknown function. Here we demonstrate that PC-1 has an essential role in the establishment of correct tubular diameter during nephron development. Polycystin-1 associates with Par3 favouring the assembly of a pro-polarizing Par3/aPKC complex and it regulates a programme of cell polarity important for oriented cell migration and for a convergent extension-like process during tubular morphogenesis. Par3 inactivation in the developing kidney results in defective convergent extension and tubular morphogenesis, and in renal cyst formation. Our data define Polycystin-1 as central to cell polarization and to epithelial tube morphogenesis and homeostasis.

Nephrocystin-1 forms a complex with polycystin-1 via a polyproline motif/SH3 domain interaction and regulates the apoptotic response in mammals.

Mutations in PKD1, the gene encoding for the receptor Polycystin-1 (PC-1), cause autosomal dominant polycystic kidney disease (ADPKD). The cytoplasmic C-terminus of PC-1 contains a coiled-coil domain that mediates an interaction with the PKD2 gene product, Polycystin-2 (PC-2). Here we identify a novel domain in the PC-1 C-terminal tail, a polyproline motif mediating an interaction with Src homology domain 3 (SH3). A screen for interactions using the PC-1 C-terminal tail identified the SH3 domain of nephrocystin-1 (NPHP1) as a potential binding partner of PC-1. NPHP1 is the product of a gene that is mutated in a different form of renal cystic disease, nephronophthisis (NPHP). We show that in vitro pull-down assays and NMR structural studies confirmed the interaction between the PC-1 polyproline motif and the NPHP1 SH3 domain. Furthermore, the two full-length proteins interact through these domains; using a recently generated model system allowing us to track endogenous PC-1, we confirm the interaction between the endogenous proteins. Finally, we show that NPHP1 trafficking to cilia does not require PC-1 and that PC-1 may require NPHP1 to regulate resistance to apoptosis, but not to regulate cell cycle progression. In line with this, we find high levels of apoptosis in renal specimens of NPHP patients. Our data uncover a link between two different ciliopathies, ADPKD and NPHP, supporting the notion that common pathogenetic defects, possibly involving de-regulated apoptosis, underlie renal cyst formation.

Polycystin-1 regulates extracellular signal-regulated kinase-dependent phosphorylation of tuberin to control cell size through mTOR and its downstream effectors S6K and 4EBP1.

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease characterized by bilateral renal cyst formation. Both hyperproliferation and hypertrophy have been previously observed in ADPKD kidneys. Polycystin-1 (PC-1), a large orphan receptor encoded by the PKD1 gene and mutated in 85% of all cases, is able to inhibit proliferation and apoptosis. Here we show that overexpression of PC-1 in renal epithelial cells inhibits cell growth (size) in a cell cycle-independent manner due to the downregulation of mTOR, S6K1, and 4EBP1. Upregulation of the same pathway leads to increased cell size, as found in mouse embryonic fibroblasts derived from Pkd1-/- mice. We show that PC-1 controls the mTOR pathway in a Tsc2-dependent manner, by inhibiting the extracellular signal-regulated kinase (ERK)-mediated phosphorylation of tuberin in Ser664. We provide a detailed molecular mechanism by which PC-1 can inhibit the mTOR pathway and regulate cell size.

Polycystin-1 induces resistance to apoptosis through the phosphatidylinositol 3-kinase/Akt signaling pathway.

Polycystin-1 (PC-1), the PKD1 gene product, is a large receptor whose expression in renal epithelial cells results in resistance to apoptosis and tubulogenesis, a model consistent with the phenotype observed in patients. This study links PC-1 expression to a signaling pathway that is known to be both antiapoptotic and important for normal tubulogenesis. This study found that PC-1 expression results in phosphorylation of Akt and downstream effectors and that phosphatidylinositol 3-kinase (PI3-K) inhibitors prevent this process. In addition, it is shown that dominant negative Akt can revert PC-1-induced protection from apoptosis. Furthermore, it was observed that increased PI3-K beta activity in PC-1-expressing MDCK cells seems to be dependent on both tyrosine-kinase activity and heterotrimeric G proteins. It also was found that PC-1-induced tubulogenesis is inhibited by PI3-K inhibitors. Taken together, these data suggest that the PI3-K/Akt cascade may be a central modulator of PC-1 function and that its deregulation might be important in autosomal dominant polycystic kidney disease.

Cyclooxygenase-2 and caspase 3 expression in trimethyltin-induced apoptosis in the mouse hippocampus.

The neurotoxicant trimethyltin (TMT) induces massive neuronal loss in vivo in the hippocampus of rodents, accompanied by behavioral alterations. The present study investigates the pattern of cell death after in vivo administration of TMT to adult mice. In the granular cell layer of the Dentate Gyrus, TUNEL staining detected DNA fragmentation, and apoptotic bodies were also evident. In addition, a ladder pattern of internucleosomal DNA fragmentation was shown in agarose gel electrophoresis. We show that activated caspase-3, which is known to play a pivotal role in apoptotic processes, is clearly expressed by degenerating neurons. Inducible cyclooxygenase is also expressed at cytoplasmic level by degenerating granular neurons, suggesting that this enzyme may participate in TMT-induced neurodegeneration.