Aberrant polycystin-1 expression results in modification of activator protein-1 activity, whereas Wnt signaling remains unaffected

Polycystin-1, the polycystic kidney disease 1 gene product, has been implicated in several signaling complexes that are known to regulate essential cellular functions. We investigated the role of polycystin-1 in Wnt signaling and activator protein-1 (AP-1) activation. To this aim, a membrane-targeted construct encoding the conserved C-terminal region of mouse polycystin-1 reported to mediate signal transduction activity was expressed in human embryonic and renal epithelial cells. To ensure specificity and minimal cotransfection effects, we focused our study on the endogenous proteins that actually transduce the signals, beta-catenin and T-cell factor/lymphoid-enhancing factor for Wnt signaling and (phosphorylated) c-Jun, ATF2, and c-Fos for AP-1. Our data indicate that the C-terminal region of polycystin-1 activates AP-1 by inducing phosphorylation and expression of at least c-Jun and ATF2, whereas c-Fos was not affected. Under our experimental conditions, polycystin-1 did not modulate Wnt signaling. AP-1 activity was aberrant in human autosomal dominant polycystic kidney disease (ADPKD) renal cystic epithelial cells and in renal epithelial cells expressing transgenic full-length polycystin-1, resulting in decreased Jun-ATF and increased Jun-Fos activity, whereas Wnt signaling remained unaffected. Since our data indicate that aberrant polycystin-1 expression results in altered AP-1 activity, polycystin-1 may be required for adequate AP-1 activity.     

In vivo interaction of the adapter protein CD2-associated protein with the type 2 polycystic kidney disease protein, polycystin-2

We identified a developmentally regulated gene from mouse kidney whose expression is up-regulated in metanephrogenic mesenchyme cells when they are induced to differentiate to epithelial cells during kidney organogenesis. The deduced 70.5-kDa protein, originally named METS-1 (mesenchyme-to-epithelium transition protein with SH3 domains), has since been cloned as a CD2-associated protein (CD2AP). CD2AP is strongly expressed in glomerular podocytes, and the absence of CD2AP in mice results in congenital nephrotic syndrome. We have found that METS-1/CD2AP (hereafter referred to as CD2AP) is expressed at lower levels in renal tubular epithelial cells in the adult kidney, particularly in distal nephron segments. Independent yeast two-hybrid screens using the COOH-terminal region of either CD2AP or polycystin-2 as bait identified the COOH termini of polycystin-2 and CD2AP, respectively, as strong interacting partners. This interaction was confirmed in cultured cells by co-immunoprecipitation of endogenous polycystin-2 with endogenous CD2AP and vice versa. CD2AP shows a diffuse reticular cytoplasmic and perinuclear pattern of distribution, similar to polycystin-2, in cultured cells, and the two proteins co-localize by indirect double immunofluorescence microscopy. CD2AP is an adapter molecule that associates with a variety of membrane proteins to organize the cytoskeleton around a polarized site. Such a function fits well with that hypothesized for the polycystin proteins in renal tubular epithelial cells, and the present findings suggest that CD2AP has a role in polycystin-2 function.     

Polycystin-2 regulates proliferation and branching morphogenesis in kidney epithelial cells

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of multiple fluid-filled cysts that expand over time and destroy the renal architecture. Loss or mutation of polycystin-1 or polycystin-2, the respective proteins encoded by the ADPKD genes PKD1 and PKD2, is associated with most cases of ADPKD. Thus, the polycystin proteins likely play a role in cell proliferation and morphogenesis. Recent studies indicate that polycystin-1 is involved in these processes, but little is known about the role played by polycystin-2. To address this question, we created a number of related cell lines variable in their expression of polycystin-2. We show that the basal and epidermal growth factor-stimulated rate of cell proliferation is higher in cells that do not express polycystin-2 versus those that do, indicating that polycystin-2 acts as a negative regulator of cell growth. In addition, cells not expressing polycystin-2 exhibit significantly more branching morphogenesis and multicellular tubule formation under basal and hepatocyte growth factor-stimulated conditions than their polycystin-2-expressing counterparts, suggesting that polycystin-2 may also play an important role in the regulation of tubulogenesis. Cells expressing a channel mutant of polycystin-2 proliferated faster than those expressing the wild-type protein, but exhibited blunted tubule formation. Thus, the channel activity of polycystin-2 may be an important component of its regulatory machinery. Finally, we show that polycystin-2 regulation of cell proliferation appears to be dependent on its ability to prevent phosphorylated extracellular-related kinase from entering the nucleus. Our results indicate that polycystin-2 is necessary for the proper growth and differentiation of kidney epithelial cells and suggest a possible mechanism for the cyst formation seen in ADPKD2.     

Identification and characterization of polycystin-2, the PKD2 gene product.

PKD2, the second gene for the autosomal dominant polycystic kidney disease (ADPKD), encodes a protein, polycystin-2, with predicted structural similarity to cation channel subunits. However, the function of polycystin-2 remains unknown. We used polyclonal antisera specific for the intracellular NH(2) and COOH termini to identify polycystin-2 as an approximately 110-kDa integral membrane glycoprotein. Polycystin-2 from both native tissues and cells in culture is sensitive to Endo H suggesting the continued presence of high-mannose oligosaccharides typical of pre-middle Golgi proteins. Immunofluorescent cell staining of polycystin-2 shows a pattern consistent with localization in the endoplasmic reticulum. This finding is confirmed by co-localization with protein-disulfide isomerase as determined by double indirect immunofluorescence and co-distribution with calnexin in subcellular fractionation studies. Polycystin-2 translation products truncated at or after Gly(821) retain their exclusive endoplasmic reticulum localization while products truncated at or before Glu(787) additionally traffic to the plasma membrane. Truncation mutants that traffic to the plasma membrane acquire Endo H resistance and can be biotinylated on the cell surface in intact cells. The 34-amino acid region Glu(787)-Ser(820), containing two putative phosphorylation sites, is responsible for the exclusive endoplasmic reticulum localization of polycystin-2 and is the site of specific interaction with an as yet unidentified protein binding partner for polycystin-2. The localization of full-length polycystin-2 to intracellular membranes raises the possibility that the PKD2 gene product is a subunit of intracellular channel complexes.     

The N-terminal extracellular domain is required for polycystin-1-dependent channel activity

Autosomal dominant polycystic kidney disease (PKD) is caused by mutation of polycystin-1 or polycystin-2. Polycystin-2 is a Ca(2+)-permeable cation channel. Polycystin-1 is an integral membrane protein of less defined function. The N-terminal extracellular region of polycystin-1 contains potential motifs for protein and carbohydrate interaction. We now report that expression of polycystin-1 alone in Chinese hamster ovary (CHO) cells and in PKD2-null cells can confer Ca(2+)-permeable non-selective cation currents. Co-expression of a loss-of-function mutant of polycystin-2 in CHO cells does not reduce polycystin-1-dependent channel activity. A polycystin-1 mutant lacking approximately 2900 amino acids of the extracellular region is targeted to the cell surface but does not produce current. Extracellular application of antibodies against the immunoglobulin-like PKD domains reduces polycystin-1-dependent current. These results support the hypothesis that polycystin-1 is a surface membrane receptor that transduces the signal via changes in ionic currents.     

Polycystin-1 distribution is modulated by polycystin-2 expression in mammalian cells

Mutations in PKD1 and PKD2, the genes that encode polycystin-1 and polycystin-2 respectively, account for almost all cases of autosomal dominant polycystic kidney disease. Although the polycystins are believed to interact in vivo, the two proteins often display dissimilar patterns and gradients of expression during development. In an effort to understand this apparent discrepancy, we investigated how changes in polycystin-2 expression can affect the subcellular localization of polycystin-1. We show that, when polycystin-1 is expressed alone in a PKD2 null cell line, it localizes to the cell surface, as well as to the endoplasmic reticulum. When co-expressed with polycystin-2, however, polycystin-1 is not seen at the cell surface and co-localizes completely with polycystin-2 in the endoplasmic reticulum. The localization of a polycystin-1 fusion protein was similarly affected by changes in its level of expression relative to that of polycystin-2. This phenomenon was observed in populations as well as in individual COS-7 cells. Our data suggest that the localization of polycystin-1 can be regulated via the relative expression level of polycystin-2 in mammalian cells. This mechanism may help to explain the divergent patterns and levels of expression observed for the polycystins, and may provide clues as to how the function of these two proteins are regulated during development.     

Polycystin-2 accelerates Ca2+ release from intracellular stores in Caenorhabditis elegans

Polycystin-2, a member of the TRP family of calcium channels, is encoded by the human PKD2 gene. Mutations in that gene can lead to swelling of nephrons into the fluid-filled cysts of polycystic kidney disease. In addition to expression in tubular epithelial cells, human polycystin-2 is found in muscle and neuronal cells, but its cell biological function has been unclear. A homologue in Caenorhabditis elegans is necessary for male mating behavior. We compared the behavior, calcium signaling mechanisms, and electrophysiology of wild-type and pkd-2 knockout C. elegans. In addition to characterizing PKD-2-mediated aggregation and mating behaviors, we found that polycystin-2 is an intracellular Ca(2+) release channel that is required for the normal pattern of Ca(2+) responses involving IP(3) and ryanodine receptor-mediated Ca(2+) release from intracellular stores. Activity of polycystin-2 creates brief cytosolic Ca(2+) transients with increased amplitude and decreased duration. Polycystin-2, along with the IP(3) and ryanodine receptors, acts as a major calcium-release channel in the endoplasmic reticulum in cells where rapid calcium signaling is required, and polycystin-2 activity is essential in those excitable cells for rapid responses to stimuli.     

Polycystin-1 and polycystin-2 regulate the cell cycle through the helix-loop-helix inhibitor Id2

Autosomal-dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and is characterized by progressive cyst formation and ultimate loss of renal function. Increased cell proliferation is a key feature of the disease. Here, we show that the ADPKD protein polycystin-2 (PC2) regulates the cell cycle through direct interaction with Id2, a member of the helix-loop-helix (HLH) protein family that is known to regulate cell proliferation and differentiation. Id2 expression suppresses the induction of a cyclin-dependent kinase inhibitor, p21, by either polycystin-1 (PC1) or PC2. The PC2-Id2 interaction is regulated by PC1-dependent phosphorylation of PC2. Enhanced Id2 nuclear localization is seen in human and mouse cystic kidneys. Inhibition of Id2 expression by RNA interference corrects the hyperproliferative phenotype of PC1 mutant cells. We propose that Id2 has a crucial role in cell-cycle regulation that is mediated by PC1 and PC2.     

Induction of gene expression by exon 2 of the major E1A proteins of adenovirus type 5.

We have constructed an adenovirus type 5 (Ad5) E1A mutant, dl1119/520, that produces essentially only exon 2 of the major E1A proteins. In infected primary baby rat kidney cells, this mutant induced expression of the E1B 55-kDa protein, and in infected human KB cells, it induced expression of this protein, the E2A 72-kDa protein, and hexon. In KB cells, this mutant grew substantially better than Ad5 dl312, which lacks E1A, and as well as Ad5 dl520, an E1A mutant producing only the 243-residue protein. These results suggest that exon 2 of E1A proteins on its own was able to activate gene expression. We also constructed mutants of dl1119/520, containing small deletions in regions of exon 2 that others found to be associated with effects on the properties of E1A transformants. None of these deletions destroyed gene activation completely, indicating that there may be some redundancy among sequences in exon 2 for inducing gene expression. The two deletions that decreased induction the most, residues 224 to 238 and 255 to 270, were in regions reported to be associated with the expression of a metalloprotease and with enhanced transformation, suggesting that exon 2 may regulate expression of genes governing cell growth. It is remarkable that all sections of E1A proteins, exon 1, the unique region, and exon 2, have now been found to affect gene expression.     

Tuberin-dependent membrane localization of polycystin-1: a functional link between polycystic kidney disease and the TSC2 tumor suppressor gene

The PKD1 gene accounts for 85% of autosomal dominant polycystic kidney disease (ADPKD), the most common human genetic disorder. Rats with a germline inactivation of one allele of the Tsc2 tumor suppressor gene developed early onset severe bilateral polycystic kidney disease, with similarities to the human contiguous gene syndrome caused by germline codeletion of PKD1 and TSC2 genes. Polycystic rat renal cells retained two normal Pkd1 alleles but were null for Tsc2 and exhibited loss of lateral membrane-localized polycystin-1. In tuberin-deficient cells, intracellular trafficking of polycystin-1 was disrupted, resulting in sequestration of polycystin-1 within the Golgi and reexpression of Tsc2 restored correct polycystin-1 membrane localization. These data identify tuberin as a determinant of polycystin-1 functional localization and, potentially, ADPKD severity.     

Polycystin-1 and polycystin-2 are both required to amplify inositol-trisphosphate-induced Ca2+ release

Autosomal dominant polycystic kidney disease is caused by loss-of-function mutations in the PKD1 or PKD2 genes encoding respectively polycystin-1 and polycystin-2. Polycystin-2 stimulates the inositol trisphosphate (IP(3)) receptor (IP(3)R), a Ca(2+)-release channel in the endoplasmic reticulum (ER). The effect of ER-located polycystin-1 is less clear. Polycystin-1 has been reported both to stimulate and to inhibit the IP(3)R. We now studied the effect of polycystin-1 and of polycystin-2 on the IP(3)R activity under conditions where the cytosolic Ca(2+) concentration was kept constant and the reuptake of released Ca(2+) was prevented. We also studied the interdependence of the interaction of polycystin-1 and polycystin-2 with the IP(3)R. The experiments were done in conditionally immortalized human proximal-tubule epithelial cells in which one or both polycystins were knocked down using lentiviral vectors containing miRNA-based short hairpins. The Ca(2+) release was induced in plasma membrane-permeabilized cells by various IP(3) concentrations at a fixed Ca(2+) concentration under unidirectional (45)Ca(2+)-efflux conditions. We now report that knock down of polycystin-1 or of polycystin-2 inhibited the IP(3)-induced Ca(2+) release. The simultaneous presence of the two polycystins was required to fully amplify the IP(3)-induced Ca(2+) release, since the presence of polycystin-1 alone or of polycystin-2 alone did not result in an increased Ca(2+) release. These novel findings indicate that ER-located polycystin-1 and polycystin-2 operate as a functional complex. They are compatible with the view that loss-of-function mutations in PKD1 and in PKD2 both cause autosomal dominant polycystic kidney disease.     

An ever-expanding story of cyst formation

Autosomal-dominant polycystic kidney disease represents one of the most common monogenetic human disorders. The cloning of the PKD1 and PKD2 genes, which are mutated in far more than 90% of the patients affected by this disease, has generated high hopes for a quick understanding of the pathogenesis of cyst formation. However, these expectations have not yet been fulfilled, since the function of both polycystin-1 and polycystin-2, the two proteins encoded by PKD1 and PKD2, still remains a puzzle. In this review, we will highlight some of the characteristics of polycystic kidney disease, briefly touch on polycystin-1, and then go on to describe recent results of experiments with polycystin-2, since the latter is the major focus of our work. We will discuss new evidence which suggests that autosomal-dominant polycystic kidney disease actually behaves recessively on a cellular level. Finally, a model will be presented that tries to explain the available data.     

PKD1 induces p21(waf1) and regulation of the cell cycle via direct activation of the JAK-STAT signaling pathway in a process requiring PKD2

Autosomal dominant polycystic kidney disease is characterized by cyst formation in the kidney and other organs and results from mutations of PKD1 or PKD2. Previous studies suggest that their gene products have an important role in growth regulation. We now show that expression of polycystin-1 activates the JAK-STAT pathway, thereby upregulating p21(waf1) and inducing cell cycle arrest in G0/G1. This process requires polycystin-2, a channel protein, as an essential cofactor. Mutations that disrupt polycystin-1/2 binding prevent activation of the pathway. Mouse embryos lacking Pkd1 have defective STAT1 phosphorylation and p21(waf1) induction. These results suggest that one function of the polycystin-1/2 complex is to regulate the JAK/STAT pathway and explain how mutations of either gene can result in dysregulated growth.     

Identification, characterization, and localization of a novel kidney polycystin-1-polycystin-2 complex

The functions of the two proteins defective in autosomal dominant polycystic kidney disease, polycystin-1 and polycystin-2, have not been fully clarified, but it has been hypothesized that they may heterodimerize to form a "polycystin complex" involved in cell adhesion. In this paper, we demonstrate for the first time the existence of a native polycystin complex in mouse kidney tubular cells transgenic for PKD1, non-transgenic kidney cells, and normal adult human kidney. Polycystin-1 is heavily N-glycosylated, and several glycosylated forms of polycystin-1 differing in their sensitivity to endoglycosidase H (Endo H) were found; in contrast, native polycystin-2 was fully Endo H-sensitive. Using highly specific antibodies to both proteins, we show that polycystin-2 associates selectively with two species of full-length polycystin-1, one Endo H-sensitive and the other Endo H-resistant; importantly, the latter could be further enriched in plasma membrane fractions and co-immunoprecipitated with polycystin-2. Finally, a subpopulation of this complex co-localized to the lateral cell borders of PKD1 transgenic kidney cells. These results demonstrate that polycystin-1 and polycystin-2 interact in vivo to form a stable heterodimeric complex and suggest that disruption of this complex is likely to be of primary relevance to the pathogenesis of cyst formation in autosomal dominant polycystic kidney disease.     

A modified mammalian tandem affinity purification procedure to prepare functional polycystin-2 channel

The tandem affinity purification (TAP) procedure was initially developed as a tool for rapid purification of native protein complexes expressed at their natural levels in yeast cells. This purification procedure was also applied to study interactions between soluble proteins in mammalian cells. In order to apply this procedure to mammalian membrane proteins, we created a modified TAP tag expression vector and fused with the PKD2 gene, encoding a membrane cation channel protein, polycystin-2, mutated in 15% of autosomal dominant polycystic kidney disease. We generated epithelial Madin-Darby canine kidney cell line stably expressing TAP-tagged polycystin-2, improved the subsequent steps for membrane protein release and stability, and succeeded in purifying this protein. Using patch clamp electrophysiology, we detected specific polycystin-2 channel activities when the purified protein was reconstituted into a lipid bilayer system. Thus, this modified TAP procedure provides a powerful alternative to functionally characterize membrane proteins, such as ion channels, transporters and receptors, using cell-free system derived from mammalian cells.     

Annexin A5 interacts with polycystin-1 and interferes with the polycystin-1 stimulated recruitment of E-cadherin into adherens junctions

Polycystin-1 is the gene product of PKD1, the first gene identified to be causative for the condition of autosomal dominant polycystic kidney disease (ADPKD). Mutations in PKD1 are responsible for the majority of ADPKD cases worldwide. Polycystin-1 is a protein of the transient receptor potential channels superfamily, with 11 transmembrane spans and an extracellular N-terminal region of approximately 3109 amino acid residues, harboring multiple putative ligand binding domains. We demonstrate here that annexin A5 (ANXA5), a Ca(2+) and phospholipid binding protein, interacts with the N-terminal leucine-rich repeats of polycystin-1, in vitro and in a cell culture model. This interaction is direct and specific and involves a conserved sequence of the ANXA5 N-terminal domain. Using Madin-Darby canine kidney cells expressing polycystin-1 in an inducible manner we also show that polycystin-1 colocalizes with E-cadherin at cell-cell contacts and accelerates the recruitment of intracellular E-cadherin to reforming junctions. This polycystin-1 stimulated recruitment is significantly delayed by extracellular annexin A5.     

The sequence, expression, and chromosomal localization of a novel polycystic kidney disease 1-like gene, PKD1L1, in human

Polycystin-1 and polycystin-2 are the products of PKD1 and PKD2, genes that are mutated in most cases of autosomal dominant polycystic kidney disease. Since the first two polycystins were cloned, three new members, polycystin-L, -2L2, and -REJ, have been identified. In this study, we describe a sixth member of the family, polycystin-1L1, encoded by PKD1L1 in human. The full-length cDNA sequence of PKD1L1, determined from human testis cDNA, encodes a 2849-amino-acid protein and 58 exons in a 187-kb genomic region. The deduced amino acid sequence of polycystin-1L1 has significant homology with all known polycystins, but the longest stretches of homology were found with polycystin-1 and -REJ over the 1453- and 932-amino-acid residues, respectively. Polycystin-1L1 is predicted to have two Ig-like PKD, a REJ, a GPS, a LH2/PLAT, a coiled-coil, and 11 putative transmembrane domains. Several rhodopsin-like G-protein-coupled receptor (GPCR) signatures are also found in polycystin-1L1. Dot-blot analysis and RT-PCR revealed that human PKD1L1 is expressed in testis and in fetal and adult heart. In situ hybridization analysis showed that the most abundant and specific expression of Pkd1l1 was found in Leydig cells, a known source of testosterone production, in mouse testis. We have assigned PKD1L1 to the short arm of human chromosome 7 in bands p12--p13 and Pkd1l1 to mouse chromosome 11 in band A2 by fluorescence in situ hybridization. We hypothesize a role for polycystin-1L1 in the heart and in the male reproductive system.     

Altered distribution and co-localization of polycystin-2 with polycystin-1 in MDCK cells after wounding stress

Polycystin-1 and -2 are integral membrane glycoproteins defective in autosomal dominant polycystic kidney disease (ADPKD). Recent studies showed a coupled polycystin-1 and -2 action in cell signaling and channel activation suggesting an important biological role for the two proteins at the plasma membrane. To gain a better understanding about the (co)-distribution and dynamics of the polycystin-1 and -2 complex under stress conditions, we used a wound-healing model of Madine Darby canine kidney (MDCK) renal epithelial cells. In this model, cells near the wound edge undergo a process of reorganization to active migration, while cells further from the edge are unaffected and remain confluent. For the first time, endogenous polycystin-1 and -2 were found to partly co-localize in the plasma membrane of confluent monolayers, and both proteins co-localized in the primary cilium. Upon wound healing, the association of polycystin-2 to the membrane was greatly reduced at the wound edge and the submarginal cells. Polycystin-1 remained incorporated to the membrane at the edge of the cell sheet at all time points, although strongly reduced in lamellipodia-forming cells. Adherens junctions and desmosomes, and respective connected actin and keratin cytoskeleton were also disturbed in lamellipodia-forming cells. We propose that altered subcellular localization of polycystin-1 and -2 as a result of stress will affect signaling and other cellular processes mediated by these proteins.