Identification of a Polycystin-1 Cleavage Product, P100, That Regulates Store Operated Ca2+ Entry through Interactions with STIM1

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a genetic disorder resulting in large kidney cysts and eventual kidney failure. Mutations in either the PKD1 or PKD2/TRPP2 genes and their respective protein products, polycystin-1 (PC1) and polycystin-2 (PC2) result in ADPKD. PC2 is known to function as a non-selective cation channel, but PC1's function and the function of PC1 cleavage products are not well understood. Here we identify an endogenous PC1 cleavage product, P100, a 100 kDa fragment found in both wild type and epitope tagged PKD1 knock-in mice. Expression of full length human PC1 (FL PC1) and the resulting P100 and C-Terminal Fragment (CTF) cleavage products in both MDCK and CHO cells significantly reduces the store operated Ca2+ entry (SOCE) resulting from thapsigargin induced store depletion. Exploration into the roles of P100 and CTF in SOCE inhibition reveal that P100, when expressed in Xenopus laevis oocytes, directly inhibits the SOCE currents but CTF does not, nor does P100 when containing the disease causing R4227X mutation. Interestingly, we also found that in PC1 expressing MDCK cells, translocation of the ER Ca2+ sensor protein STIM1 to the cell periphery was significantly altered. In addition, P100 Co-immunoprecipitates with STIM1 but CTF does not. The expression of P100 in CHO cells recapitulates the STIM1 translocation inhibition seen with FL PC1. These data describe a novel polycystin-1 cleavage product, P100, which functions to reduce SOCE via direct inhibition of STIM1 translocation; a function with consequences for ADPKD

Establishing of ES cell lines for a predictable autoregulated expression of transgenes in mice

Die meisten derzeit angewandten Verfahren zur Etablierung transgener Mäuse erfordern ein weitläufiges Screening von Tieren, da die zufällige Integration eines Transgens zu einer, vom Integrationsort abhängigen und daher nicht vorhersagbaren Transgen-Expression führt. Die Wiederverwendung von geeigneten Genorten in ES-Zellen zur Etablierung transgener Mäuse scheint daher sinnvoll. Zu diesem Zweck wurden autoregulierte Expressionskassetten, basierend auf dem TetOff-, TetOn-, E.REXOff- und PIPOff-System, konstruiert, die eine Identifizierung regulierbarer Genorte im Mausgenom und deren Wiederverwendung für eine vorhersagbare, regulierte Expression eines beliebigen Transgens ermöglichen sollten. Die Expressionskapazitäten der Systeme wurden in verschiedenen ES-Zelllinien analysiert. Dabei zeigte sich, dass das TetOn-System ein sehr hohes, das TetOff-System jedoch nur ein niedriges Expressionspotential besitzt. In NIH3T3- und in in vitro differenzierten Zellen wurden jedoch hohe Expressionen mit dem TetOff-System erzielt. Auf der Basis Flp-vermittelter Rekombination wurde eine Kassettenaustauschstrategie entwickelt, die eine Integration eines Transgen-exprimierenden Konstruktes in bereits charakterisierte Genorte, die mit heterospezifischen FRT-Sites markiert sind, erlauben sollte. Diese Strategie wurde in NIH3T3-Zellen getestet und führte zu Zelllinien mit homogener, vorhersagbarer Transgen-Expression.Most of the current procedures for generating transgenic mice require a large scale screening of animals, since random integration of the transgene into the mouse genome results in integration site specific and thus unpredictable transgene expression. The reuse of appropriate integration sites in mouse ES cells for creation of transgenic mice seems to overcome these problems. For this purpose, autoregulated expression cassettes, based on the TetOff, TetOn, E.REXOff and PIPOff system, were designed for screening the mouse genome for inducible chromosomal sites and subsequent reuse of these sites by Flp-mediated recombination for a predictable and regulatable expression of any gene. The expression capacities of these systems were analyzed in different ES cell lines resulting in the observation that the TetOn systems has a very high and the TetOff system only a poor expression potential. However, with the TetOff system high expressions were realized in NIH3T3 cells and in in vitro differentiated cells. For the reuse of transgenic cells a cassette exchange strategy was evolved which should allow the efficient integration of a target construct in a precharacterized locus tagged with heterospecific FRT sites. This strategy was testet in NIH3T3 cells leading to cell lines with homogenous, predictable transgene expression

A novel mouse model reveals that polycystin-1 deficiency in ependyma and choroid plexus results in dysfunctional cilia and hydrocephalus.

Polycystin-1 (PC-1), the product of the PKD1 gene, mutated in the majority of cases of Autosomal Dominant Polycystic Kidney Disease (ADPKD), is a very large (approximately 520 kDa) plasma membrane receptor localized in several subcellular compartments including cell-cell/matrix junctions as well as cilia. While heterologous over-expression systems have allowed identification of several of the potential biological roles of this receptor, its precise function remains largely elusive. Studying PC-1 in vivo has been a challenging task due to its complexity and low expression levels. To overcome these limitations and facilitate the study of endogenous PC-1, we have inserted HA- or Myc-tag sequences into the Pkd1 locus by homologous recombination. Here, we show that our approach was successful in generating a fully functional and easily detectable endogenous PC-1. Characterization of PC-1 distribution in vivo showed that it is expressed ubiquitously and is developmentally-regulated in most tissues. Furthermore, our novel tool allowed us to investigate the role of PC-1 in brain, where the protein is abundantly expressed. Subcellular localization of PC-1 revealed strong and specific staining in ciliated ependymal and choroid plexus cells. Consistent with this distribution, we observed hydrocephalus formation both in the ubiquitous knock-out embryos and in newborn mice with conditional inactivation of the Pkd1 gene in the brain. Both choroid plexus and ependymal cilia were morphologically normal in these mice, suggesting a role for PC-1 in ciliary function or signalling in this compartment, rather than in ciliogenesis. We propose that the role of PC-1 in the brain cilia might be to prevent hydrocephalus, a previously unrecognized role for this receptor and one that might have important implications for other genetic or sporadic diseases

Polycystin-1 Is Required for Stereocilia Structure But Not for Mechanotransduction in Inner Ear Hair Cells

Thepolycystic kidney disease-1(Pkd1) gene encodes a large transmembrane protein (polycystin-1, or PC-1) that is reported to function as a fluid flow sensor in the kidney. As a member of the transient receptor potential family, PC-1 has also been hypothesized to play a role in the elusive mechanoelectrical transduction (MET) channel in inner ear hair cells. Here, we analyze two independent mouse models of PC-1, a knock-in (KI) mutant line and a hair cell-specific inducible Cre-mediated knock-out line. Both models exhibit normal MET channel function at neonatal ages despite hearing loss and ultrastructural abnormalities of sterecilia that remain properly polarized at adult ages. These findings demonstrate that PC-1 plays an essential role in stereocilia structure and maintenance but not directly in MET channel function or planar cell polarity. We also demonstrate that PC-1 is colocalized with F-actin in hair cell stereociliain vivo, using a hemagglutinin-tagged PC-1 KI mouse model, and in renal epithelial cell microvilliin vitro. These results not only demonstrate a novel role for PC-1 in the cochlea, but also suggest insight into the development of polycystic kidney disease.</jats:p