PKD1 inhibits cancer cells migration and invasion via Wnt signaling pathway in vitro

The approximately 14 kb mRNA of the polycystic kidney disease gene PKD1 encodes a large ( approximately 460 kDa) protein, termed polycystin-1 (PC-1), that is responsible for autosomal dominant polycystic kidney disease (ADPKD). The unique organization of its multiple adhesive domains (16 Ig-like domains/PKD domains) suggests that it may play an important role in cell-cell/cell-matrix interactions. Here we demonstrated that PKD1 promoted cell-cell and cell-matrix interactions in cancer cells, indicating that PC-1 is involved in the cell adhesion process. Furthermore in this study, we showed that PKD1 inhibited cancer cells migration and invasion. And we also showed that PC-1 regulated these processes in a process that may be at least partially through the Wnt pathway. Collectively, our data suggest that PKD1 may act as a novel member of the tumor suppressor family of genes.     

Ectodomain shedding of nectin-1alpha by SF/HGF and TPA in MDCK cells

Nectin is a Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecule implicated in the organization of the junctional complex comprised of E-cadherin-based adherens junctions and claudin-based tight junctions in epithelial cells. Scatter factor (SF)/hepatocyte growth factor (HGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumor-promoting phorbol ester, induce cell spreading, followed by cell-cell dissociation and cell scattering, in Madin-Darby canine kidney (MDCK) cells. We found here that SF/HGF and TPA induced proteolytic cleavage of nectin-1alpha in the ectodomain, resulting in generation of the 80-kDa extracellular fragment and the 33-kDa fragment composed of the transmembrane and cytoplasmic domains, in MDCK cells. This shedding of nectin-1alpha was inhibited by metalloprotease inhibitors. These results indicate that SF/HGF and TPA induce the ectodomain shedding of nectin-1alpha presumably by a metalloprotease, and have raised the possibility that this shedding is involved in the SF/HGF- and TPA-induced cell-cell dissociation.     

Inhibition of Pkhd1 impairs tubulomorphogenesis of cultured IMCD cells

Fibrocystin/polyductin (FPC), the gene product of PKHD1, is responsible for autosomal recessive polycystic kidney disease (ARPKD). This disease is characterized by symmetrically large kidneys with ectasia of collecting ducts. In the kidney, FPC predominantly localizes to the apical domain of tubule cells, where it associates with the basal bodies/primary cilia; however, the functional role of this protein is still unknown. In this study, we established stable IMCD (mouse inner medullary collecting duct) cell lines, in which FPC was silenced by short hairpin RNA inhibition (shRNA). We showed that inhibition of FPC disrupted tubulomorphogenesis of IMCD cells grown in three-dimensional cultures. Pkhd1-silenced cells developed abnormalities in cell-cell contact, actin cytoskeleton organization, cell-ECM interactions, cell proliferation, and apoptosis, which may be mediated by dysregulation of extracellular-regulated kinase (ERK) and focal adhesion kinase (FAK) signaling. These alterations in cell function in vitro may explain the characteristics of ARPKD phenotypes in vivo.     

The SGP-2 gene is developmentally regulated in the mouse kidney and abnormally expressed in collecting duct cysts in polycystic kidney disease.

Sulfated glycoprotein-2 (SGP-2) is a secreted, dimeric, glycosylated protein synthesized by a number of different epithelial cell types. Although its function is not yet understood, SGP-2 has been hypothesized to be involved in such diverse processes as the promotion of cell-cell interactions, spermatogenesis, modulation of the complement system, and programmed cell death. We have now found that the SGP-2 gene is developmentally regulated in the mouse kidney. SGP-2 gene expression is first detected in the condensing nephrogenic mesenchyme and is subsequently down-regulated during the maturation of the glomerular epithelia, proximal tubules, and collecting ducts. SGP-2 continues to be expressed in the mature kidney in distal tubules and in the urothelial lining of the calyx and papilla. We have also examined the expression of the SGP-2 gene in polycystic kidneys of the C57BL/6J-cpk mouse, a model of autosomal recessive polycystic kidney disease in which there is development of epithelial-lined cysts arising primarily from the collecting duct system. Abnormally high levels of SGP-2 mRNA were found in the cyst wall epithelium of polycystic kidneys. The expression of the SGP-2 gene in normal development suggests that it plays a role in differentiating epithelial structures; and the abnormally high levels of SGP-2 gene expression in polycystic kidneys suggests that the cells lining cysts are not fully differentiated. It is possible, therefore, that polycystic kidney disease is caused by a defective developmental process in which there is a delay in terminal differentiation.     

Cells respond to and bind countin,a component of a multisubunit cell number counting factor

In Dictyostelium discoideum counting factor (CF), a secreted approximately 450-kDa complex of polypeptides, inhibits group and fruiting body size. When the gene encoding countin (a component of CF) was disrupted, cells formed large groups. We find that recombinant countin causes developing cells to form small groups, with an EC(50) of approximately 3 ng/ml, and affects cAMP signal transduction in the same manner as semipurified CF. Recombinant countin increases cell motility, decreases cell-cell adhesion, and regulates gene expression in a manner similar to the effect of CF. However, countin does not decrease adhesion or group size to the extent that semipurified CF does. A 1-min exposure of developing cells to countin causes an increase in F-actin polymerization and myosin phosphorylation and a decrease in myosin polymerization, suggesting that countin activates a rapid signal transduction pathway. (125)I-Labeled countin has countin bioactivity, and binding experiments suggest that vegetative and developing cells have approximately 53 cell-surface sites that bind countin with a K(D) of approximately 1.5 ng/ml or 60 pm. We hypothesize that countin regulates cell development through the same pathway as CF and that other proteins within the complex may modify the activity of countin and/or have independent size-regulating activities.     

The relationship of polysialic acid and the neural cell adhesion molecule N-CAM in Wilms tumor and their subcellular distributions

In previous studies we have reported that polysialic acid is an oncodevelopmental antigen in human kidney but its relationship to the neural cell adhesion molecule (N-CAM) remained undefined. In the present study, we showed by the combination of immunoprecipitation and immunoblotting that renal polysialic acid is a structural component of N-CAM polypeptide and that two highly sialylated N-CAM isoforms of approximately 120 kDa and 140 kDa existed in Wilms tumor. The presence of a cell surface coat composed of polysialic acid and N-CAM was revealed by immunoelectron microscopy, and morphological evidence for its involvement in modulating cell-cell adhesion has been provided. Furthermore, highly sialylated N-CAM was detectable extracellularly. N-CAM immunolabeling was present in compartments from the nuclear envelope to the plasma membrane. However, polysialic acid was only detectable at the cell surface suggesting that in Wilms tumor cells sialyl polymer synthesis may occur partially or exclusively at this site.     

Down-regulation of Pkd2 by siRNAs suppresses cell–cell adhesion in the mouse melanoma cells

The Pkd2 gene encodes an integral protein (~130 kDa), named polycystin-2 (PC-2). PC-2 is mainly involved in autosomal dominant polycystic kidney disease. Recently, polycystin-1/polycystin-2 complex has been shown to act as an adhesion complex mediating or regulating cell–cell or cell–matrix adhesion, suggesting that PC-2 may play a role in cell–cell/cell–matrix interactions. Here, we knocked down the expression of Pkd2 gene with small interfering RNAs (siRNAs) in the mouse melanoma cells (B16 cells), indicating that the cells transfected with the targeted siRNAs significantly suppressed cell–cell adhesion, but not cell–matrix adhesion, compared to the cells transfected with non-targeted control (NC) siRNA. This study provides the first directly functional evidence that PC-2 mediates cell–cell adhesion. Furthermore, we demonstrated that PC-2 modulated cell–cell adhesion may be, at least partially, associated with E-cadherin. Collectively, these findings for the first time showed that PC-2 may mediate cell–cell adhesion, at least partially, through E-cadherin.     

JACOP, a novel plaque protein localizing at the apical junctional complex with sequence similarity to cingulin

The apical junctional complex is composed of various cell adhesion molecules and cytoplasmic plaque proteins. Using a monoclonal antibody that recognizes a chicken 155-kDa cytoplasmic antigen (p155) localizing at the apical junctional complex, we have cloned a cDNA of its mouse homologue. The full-length cDNA of mouse p155 encoded a 148-kDa polypeptide containing a coiled-coil domain with sequence similarity to cingulin, a tight junction (TJ)-associated plaque protein. We designated this protein JACOP (junction-associated coiled-coil protein). Immunofluorescence staining showed that JACOP was concentrated in the junctional complex in various types of epithelial and endothelial cells. Furthermore, in the liver and kidney, JACOP was also distributed along non-junctional actin filaments. Upon immunoelectron microscopy, JACOP was found to be localized to the undercoat of TJs in the liver, but in some tissues, its distribution was not restricted to TJs but extended to the area of adherens junctions. Overexpression studies have revealed that JACOP was recruited to the junctional complex in epithelial cells and to cell-cell contacts and stress fibers in fibroblasts. These findings suggest that JACOP is involved in anchoring the apical junctional complex, especially TJs, to actin-based cytoskeletons.     

Canine PKD1 is a single-copy gene: genomic organization and comparative analysis

Most cases of autosomal dominant polycystic kidney disease are caused by mutations in the gene PKD1, encoding polycystin-1. To gain insight into the role of polycystin-1 in tubulogenesis and cystogenesis using the well-characterized canine kidney epithelial cell line MDCK, we have now cloned and characterized the exon/intron structure of the canine gene PKD1. FISH analysis showed that the dog genome lacks the multiple PKD1 homologs present in human. Intron 21 of dog PKD1 lacked the polypyrimidine tract characteristic of the human gene, whereas pyrimidine-rich elements were identified in canine intron 30. Canine polycystin-1 showed a higher degree of homology with the human counterpart and lower homology with mouse and rat. A striking degree of conservation (97% identity) was determined for the leucine-rich repeat domain between dog and human. Also, the homology analysis indicated that 4 of 16 Ig-like repeats (IgIII, IgVII, IgX, and IgXV) are likely to be functionally significant. This is particularly important in light of our recent findings demonstrating that Iglike domains form strong homophilic interactions and can mediate cell-cell adhesion. These data enable detailed analysis of the role of polycystin-1 in cystogenesis and tubulogenesis using the canine MDCK cell line.     

Folliculin,the Product of the Birt-Hogg-Dube Tumor Suppressor Gene,Interacts with the Adherens Junction Protein p0071 to Regulate Cell-Cell Adhesion

Birt-Hogg-Dube (BHD) is a tumor suppressor gene syndrome associated with fibrofolliculomas, cystic lung disease, and chromophobe renal cell carcinoma. In seeking to elucidate the pathogenesis of BHD, we discovered a physical interaction between folliculin (FLCN), the protein product of the BHD gene, and p0071, an armadillo repeat containing protein that localizes to the cytoplasm and to adherens junctions. Adherens junctions are one of the three cell-cell junctions that are essential to the establishment and maintenance of the cellular architecture of all epithelial tissues. Surprisingly, we found that downregulation of FLCN leads to increased cell-cell adhesion in functional cell-based assays and disruption of cell polarity in a three-dimensional lumen-forming assay, both of which are phenocopied by downregulation of p0071. These data indicate that the FLCN-p0071 protein complex is a negative regulator of cell-cell adhesion. We also found that FLCN positively regulates RhoA activity and Rho-associated kinase activity, consistent with the only known function of p0071. Finally, to examine the role of Flcn loss on cell-cell adhesion in vivo, we utilized keratin-14 cre-recombinase (K14-cre) to inactivate Flcn in the mouse epidermis. The K14-Cre-Bhd^flox/flox mice have striking delays in eyelid opening, wavy fur, hair loss, and epidermal hyperplasia with increased levels of mammalian target of rapamycin complex 1 (mTORC1) activity. These data support a model in which dysregulation of the FLCN-p0071 interaction leads to alterations in cell adhesion, cell polarity, and RhoA signaling, with broad implications for the role of cell-cell adhesion molecules in the pathogenesis of human disease, including emphysema and renal cell carcinoma.     

OFD1 and Flotillins Are Integral Components of a Ciliary Signaling Protein Complex Organized by Polycystins in Renal Epithelia and Odontoblasts

Mutation of the X-linked oral-facial-digital syndrome type 1 (OFD1) gene is embryonic lethal in males and results in craniofacial malformations and adult onset polycystic kidney disease in females. While the OFD1 protein localizes to centriolar satellites, centrosomes and basal bodies, its cellular function and how it relates to cystic kidney disease is largely unknown. Here, we demonstrate that OFD1 is assembled into a protein complex that is localized to the primary cilium and contains the epidermal growth factor receptor (EGFR) and domain organizing flotillin proteins. This protein complex, which has similarity to a basolateral adhesion domain formed during cell polarization, also contains the polycystin proteins that when mutant cause autosomal dominant polycystic kidney disease (ADPKD). Importantly, in human ADPKD cells where mutant polycystin-1 fails to localize to cilia, there is a concomitant loss of localization of polycystin-2, OFD1, EGFR and flotillin-1 to cilia. Together, these data suggest that polycystins are necessary for assembly of a novel flotillin-containing ciliary signaling complex and provide a molecular rationale for the common renal pathologies caused by OFD1 and PKD mutations.     

Epithelial-to-mesenchymal transition in cyst lining epithelial cells in an orthologous PCK rat model of autosomal-recessive polycystic kidney disease

In polycystic kidney disease (PKD), cyst lining cells show polarity abnormalities. Recent studies have demonstrated loss of cell contact in cyst cells, suggesting induction of epithelial-to-mesenchymal transition (EMT). Recently, EMT has been implicated in the pathogenesis of PKD. To explore further evidence of EMT in PKD, we examined age- and segment-specific expression of adhesion molecules and mesenchymal markers in PCK rats, an orthologous model of human autosomal-recessive PKD. Kidneys from 5 male PCK and 5 control rats each at 0 days, 1, 3, 10, and 14 wk, and 4 mo of age were serially sectioned and stained with segment-specific markers and antibodies against E-cadherin, Snail1, β-catenin, and N-cadherin. mRNAs for E-cadherin and Snail1 were quantified by real-time PCR. Vimentin, fibronectin, and α-smooth muscle actin (α-SMA) expressions were assessed as mesenchymal markers. E-cadherin expression pattern was correlated with the disease pathology in that tubule segments showing the highest expression in control had much severer cyst formation in PCK rats. In PCK rats, E-cadherin and β-catenin in cystic tubules was attenuated and localized to lateral areas of cell-cell contact, whereas nuclear expression of Snail1 increased in parallel with cyst enlargement. Some epithelial cells in large cysts derived from these segments, especially in adjacent fibrotic areas, showed positive immunoreactivity for vimentin and fibronectin. In conclusion, these findings suggest that epithelial cells in cysts acquire mesenchymal features in response to cyst enlargement and participate in progressive renal fibrosis. Our study clarified the nephron segment-specific cyst profile related to EMT in PCK rats. EMT may play a key role in polycystic kidney disease.     

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.     

Nephrocystin-conserved domains involved in targeting to epithelial cell-cell junctions,interaction with filamins,and establishing cell polarity

Nephrocystin is the protein product of the gene mutated in juvenile nephronophthisis, an autosomal recessive cystic kidney disease afflicting children and young adults. Because the normal cellular function of nephrocystin is largely unknown, the molecular defects underlying disease pathogenesis remain obscure. Analysis of nephrocystin amino acid sequences from human and other species revealed three distinct conserved domains including Src homology 3 and coil-coil domains in the N-terminal region, as well as a large highly conserved C-terminal region bearing no obvious homology to other proteins and hence referred to as the "nephrocystin homology domain" (NHD). The objective of this study was to gain insight into nephrocystin function by defining functional properties of the conserved domains. We analyzed a series of nephrocystin deletion mutants expressed in Madin-Darby canine kidney and COS-7 cells. This analysis revealed previously unrecognized functional attributes of the NHD, including abilities to promote both self-association and epithelial cell-cell junctional targeting. We further observed that Madin-Darby canine kidney cell lines stably expressing a nephrocystin mutant with a deletion of the Src homology 3 domain have reduced ability to establish tight junctions as measured by transepithelial electrical resistance. Finally, from a two-hybrid screen and coimmunoprecipitation studies we identified members of the filamin family of actin-binding proteins as having the capacity to interact with the NHD. These findings support a functional role for nephrocystin as a docking protein involved in organizing a protein complex to regulate the actin cytoskeleton at sites of epithelial cell-cell adhesion and further suggest that these properties are important for establishing epithelial cell polarity.     

Homo- and heterodimerization of APP family members promotes intercellular adhesion

The amyloid precursor protein (APP) plays a central role in Alzheimer's disease, but its physiological function and that of its mammalian paralogs, the amyloid precursor-like proteins 1 and 2 (APLPs), is still poorly understood. APP has been proposed to form dimers, a process that could promote cell adhesion via trans-dimerization. We investigated the dimerization and cell adhesion properties of APP/APLPs and provide evidence that all three paralogs are capable of forming homo- and heterocomplexes. Moreover, we show that trans-interaction of APP family proteins promotes cell-cell adhesion in a homo- and heterotypic fashion and that endogenous APLP2 is required for cell-cell adhesion in mouse embryonic fibroblasts. We further demonstrate interaction of all the three APP family members in mouse brain, genetic interdependence, and molecular interaction of APP and APLPs in synaptically enriched membrane compartments. Together, our results provide evidence that homo- and heterocomplexes of APP/APLPs promote trans-cellular adhesion in vivo.