Regulation of cell proliferation and apoptosis through fibrocystin-prosaposin interaction

Mutations in PKHD1 (polycystic kidney and hepatic disease gene 1) gene cause the autosomal recessive polycystic kidney disease (ARPKD). It is widely accepted that cystogenesis is owing to aberrant cell proliferation and apoptosis, increased fluid secretion, and extracellular matrix abnormality. Fibrocystin/polyductin (FPC), the encoded protein product by PKHD1, is a single transmembrane protein and believed to be a novel receptor-like molecule. FPC has been located mainly on the plasma membrane and cilium/basal body. However, its biological functions remain poorly understood. To investigate the roles of FPC in the pathogenesis of ARPKD, we searched for FPC-interacting proteins by yeast two-hybrid assay, and found a novel partner, prosaposin. Prosaposin is a glycoprotein with multiple functions. With GST pull-down assay and co-immunoprecipitation, we confirmed the interaction between FPC and prosaposin. In order to study the effects of FPC-prosaposin interaction on cell proliferation and apoptosis, we have made stable cell lines in which FPC was overexpressed or knocked down alone or in combination with prosaposin overexpression. By MTT assay, we found that FPC knockdown and prosaposin overexpression increased cell proliferation, respectively, while overexpression of FPC C-tail did the opposite. With apoptosis assay, we found that overexpression of FPC C-tail promoted cell apoptosis. However, overexpression of prosaposin significantly enhanced cell survival in FPC knockdown cells. All these findings indicated that FPC and prosaposin may play significant roles in regulation of cell proliferation and apoptosis. Taken together, we have disclosed a novel signaling pathway of FPC, which may be important for the pathogenesis of ARPKD.     

The genetics of Autosomal Recessive Polycystic Kidney Disease (ARPKD)

ARPKD is a genetically inherited kidney disease that manifests by bilateral enlargement of cystic kidneys and liver fibrosis. It shows a range of severity, with 30% of individuals dying early on and the majority having good prognosis if they survive the first year of life. The reasons for this variability remain unclear. Two genes have been shown to cause ARPKD when mutated, PKHD1, mutations in which lead to most of ARPKD cases and DZIP1L, which is associated with moderate ARPKD. This mini review will explore the genetics of ARPKD and discuss potential genetic modifiers and phenocopies that could affect diagnosis.     

Cystogenesis in ARPKD results from increased apoptosis in collecting duct epithelial cells of Pkhd1 mutant kidneys

Mutations in the PKHD1 gene result in autosomal recessive polycystic kidney disease (ARPKD) in humans. To determine the molecular mechanism of the cystogenesis in ARPKD, we recently generated a mouse model for ARPKD that carries a targeted mutation in the mouse orthologue of human PKHD1. The homozygous mutant mice display hepatorenal cysts whose phenotypes are similar to those of human ARPKD patients. By littermates of this mouse, we developed two immortalized renal collecting duct cell lines with Pkhd1 and two without. Under nonpermissive culture conditions, the Pkhd1^−/− renal cells displayed aberrant cell–cell contacts and tubulomorphogenesis. The Pkhd1^−/− cells also showed significantly reduced cell proliferation and elevated apoptosis. To validate this finding in vivo, we examined proliferation and apoptosis in the kidneys of Pkhd1^−/− mice and their wildtype littermates. Using proliferation (PCNA and Histone-3) and apoptosis (TUNEL and caspase-3) markers, similar results were obtained in the Pkhd1^−/− kidney tissues as in the cells. To identify the molecular basis of these findings, we analyzed the effect of Pkhd1 loss on multiple putative signaling regulators. We demonstrated that the loss of Pkhd1 disrupts multiple major phosphorylations of focal adhesion kinase (FAK), and these disruptions either inhibit the Ras/C-Raf pathways to suppress MEK/ERK activity and ultimately reduce cell proliferation, or suppress PDK1/AKT to upregulate Bax/caspase-9/caspase-3 and promote apoptosis. Our findings indicate that apoptosis may be a major player in the cyst formation in ARPKD, which may lead to new therapeutic strategies for human ARPKD.     

Molecular and cellular pathophysiology of autosomal recessive polycystic kidney disease (ARPKD)

Autosomal recessive polycystic kidney disease (ARPKD) belongs to a group of congenital hepatorenal fibrocystic syndromes characterized by dual renal and hepatic involvement of variable severity. Despite the wide clinical spectrum of ARPKD (MIM 263200), genetic linkage studies indicate that mutations at a single locus, PKHD1 (polycystic kidney and hepatic disease 1), located on human chromosome region 6p21.1–p12, are responsible for all phenotypes of ARPKD. Identification of cystic disease genes and their encoded proteins has provided investigators with critical tools to begin to unravel the molecular and cellular mechanisms of PKD. PKD cystic epithelia share common phenotypic abnormalities despite the different genetic mutations that underlie the disease. Recent studies have shown that many cyst-causing proteins are expressed in multimeric complexes at distinct subcellular locations within epithelia. This co-expression of cystoproteins suggests that cyst formation, regardless of the underlying disease gene, results from perturbations in convergent and/or integrated signal transduction pathways. To date, no specific therapies are in clinical use for ameliorating cyst growth in ARPKD. However, studies noted in this review suggest that therapeutic targeting of the cAMP and epidermal growth factor receptor (EGFR)-axis abnormalities in cystic epithelia may translate into effective therapies for ARPKD and, by analogy, autosomal dominant polycystic kidney disease (ADPKD). A particularly promising approach appears to be the targeting of downstream intermediates of both the cAMP and EGFR axis. This review focuses on ARPKD and presents a concise summary of the current understanding of the molecular genetics and cellular pathophysiology of this disease. It also highlights phenotypic and mechanistic similarities between ARPKD and ADPKD.The authors are supported by the National Institutes of Health (grant no. 1-P50-DK57306), the PKD Foundation (grant no. 76a2r), and the Children’s Research Institute, Children’s Hospital of Wisconsin.     

A novel mutation identified in PKHD1 by targeted exome sequencing: Guiding prenatal diagnosis for an ARPKD family

Autosomal recessive polycystic kidney disease (ARPKD) is a rare hereditary renal cystic disease involving multiple organs, mainly the kidney and liver. Parents who had an affected child with ARPKD are in strong demand for an early and reliable prenatal diagnosis to guide the future pregnancies. Here we provide an example of prenatal diagnosis of an ARPKD family where traditional antenatal ultrasound examinations failed to produce conclusive results till 26th week of gestation. Compound heterozygous mutations c.274C>T (p.Arg92Trp) and c.9059T>C (p.Leu3020Pro) were identified using targeted exome sequencing in the patient and confirmed by Sanger sequencing. Further, the mother and father were revealed to be carriers of heterozygous c.274C>T and c.9059T>C mutations, respectively. Molecular prenatal diagnosis was performed for the current pregnancy by direct sequencing plus linkage analysis. Two mutations identified in the patient were both found in the fetus. In conclusion, compound heterozygous PKHD1 mutations were elucidated to be the molecular basis of the patient with ARPKD. The newly identified c.9059T>C mutation in the patient expands mutation spectrum in PKHD1 gene. For those ultrasound failed to provide clear diagnosis, we propose the new prenatal diagnosis procedure: first, screening underlying mutations in PKHD1 gene in the proband by targeted exome sequencing; then detecting causative mutations by direct sequencing in the fetal DNA and confirming results by linkage analysis.     

SEC14 and Spectrin Domains 1 (Sestd1) and Dapper Antagonist of Catenin 1 (Dact1) Scaffold Proteins Cooperatively Regulate the Van Gogh-like 2 (Vangl2) Four-pass Transmembrane Protein and Planar Cell Polarity (PCP) Pathway during Embryonic Development in Mice

The planar cell polarity (PCP) pathway is a conserved non-canonical (β-catenin-independent) branch of Wnt signaling crucial to embryogenesis, during which it regulates cell polarity and polarized cell movements. Disruption of PCP components in mice, including Vangl2 and Dact1, results in defective neural tube closure and other developmental defects. Here, we show that Sestd1 is a novel binding partner of Vangl2 and Dact1. The Sestd1-Dact1 interface is formed by circumscribed regions of Sestd1 (the carboxyl-terminal region) and Dact1 (the amino-terminal region). Remarkably, we show that loss of Sestd1 precisely phenocopies loss of Dact1 during embryogenesis in mice, leading to a spectrum of birth malformations, including neural tube defects, a shortened and/or curly tail, no genital tubercle, blind-ended colons, hydronephrotic kidneys, and no bladder. Moreover, as with Dact1, a knock-out mutation at the Sestd1 locus exhibits reciprocal genetic rescue interactions during development with a semidominant mutation at the Vangl2 locus. Consistent with this, examination of Wnt pathway activities in Sestd1 mutant mouse embryonic tissue reveals disrupted PCP pathway biochemistry similar to that characterized in Dact1 mutant embryos. The Sestd1 protein is a divergent member of the Trio family of GTPase regulatory proteins that lacks a guanine nucleotide exchange factor domain. Nonetheless, in cell-based assays the Sestd1-Dact1 interaction can induce Rho GTPase activation. Together, our data indicate that Sestd1 cooperates with Dact1 in Vangl2 regulation and in the PCP pathway during mammalian embryonic development.     

Down-regulation of PKHD1 induces cell apoptosis through PI3K and NF-κB pathways

Mutations in PKHD1 (polycystic kidney and hepatic disease gene 1) gene cause the autosomal recessive polycystic kidney disease (ARPKD). Fibrocystin/polyductin (FPC), encoded by PKHD1, is a membrane-associated receptor-like protein. Although it is widely accepted that cystogenesis is mostly due to aberrant cell proliferation and apoptosis, it is still unclear how apoptosis is regulated. The aim of this study is to analyze the relationship among apoptosis, phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor κB (NF-κB) in FPC knockdown kidney cells. We show that PKHD1-silenced HEK293 cells demonstrate a higher PI3K/Akt activity. Selective inhibition of PI3K/Akt using LY294002 or wortmannin in these cells increases serum starvation-induced HEK293 cell apoptosis with a concomitant decrease in cell proliferation and higher caspase-3 activity. PI3K/Akt inhibition also leads to increased NF-κB activity in these cells. We conclude that the PI3K/Akt pathway is involved in apoptotic function in PKHD1-silenced cells, and PI3K/Akt inhibition correlates with upregulation of NF-κB activity. These observations provide a potential platform for determining FPC function and therapeutic investigation of ARPKD.     

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 Wnt Coreceptor Ryk Regulates Wnt/Planar Cell Polarity by Modulating the Degradation of the Core Planar Cell Polarity Component Vangl2

The Wnt signaling pathways control many critical developmental and adult physiological processes. In vertebrates, one fundamentally important function of Wnts is to provide directional information by regulating the evolutionarily conserved planar cell polarity (PCP) pathway during embryonic morphogenesis. However, despite the critical roles of Wnts and PCP in vertebrate development and disease, little is known about the molecular mechanisms underlying Wnt regulation of PCP. Here, we have found that the receptor-like tyrosine kinase (Ryk), a Wnt5a-binding protein required in axon guidance, regulates PCP signaling. We show that Ryk interacts with Vangl2 genetically and biochemically, and such interaction is potentiated by Wnt5a. Loss of Ryk in a Vangl2^+/− background results in classic PCP defects, including open neural tube, misalignment of sensory hair cells in the inner ear, and shortened long bones in the limbs. Complete loss of both Ryk and Vangl2 results in more severe phenotypes that resemble the Wnt5a^−/− mutant in many aspects such as shortened anterior-posterior body axis, limb, and frontonasal process. Our data identify the Wnt5a-binding protein Ryk as a general regulator of the mammalian Wnt/PCP signaling pathway. We show that Ryk transduces Wnt5a signaling by forming a complex with Vangl2 and that Ryk regulates PCP by at least in part promoting Vangl2 stability. As human mutations in WNT5A and VANGL2 are found to cause Robinow syndrome and neural tube defects, respectively, our results further suggest that human mutations in RYK may also be involved in these diseases.     

The PTK7 and ROR2 Protein Receptors Interact in the Vertebrate WNT/Planar Cell Polarity (PCP) Pathway

The non-canonical WNT/planar cell polarity (WNT/PCP) pathway plays important roles in morphogenetic processes in vertebrates. Among WNT/PCP components, protein tyrosine kinase 7 (PTK7) is a tyrosine kinase receptor with poorly defined functions lacking catalytic activity. Here we show that PTK7 associates with receptor tyrosine kinase-like orphan receptor 2 (ROR2) to form a heterodimeric complex in mammalian cells. We demonstrate that PTK7 and ROR2 physically and functionally interact with the non-canonical WNT5A ligand, leading to JNK activation and cell movements. In the Xenopus embryo, Ptk7 functionally interacts with Ror2 to regulate protocadherin papc expression and morphogenesis. Furthermore, we show that Ptk7 is required for papc activation induced by Wnt5a. Interestingly, we find that Wnt5a stimulates the release of the tagged Ptk7 intracellular domain, which can translocate into the nucleus and activate papc expression. This study reveals novel molecular mechanisms of action of PTK7 in non-canonical WNT/PCP signaling that may promote cell and tissue movements.     

Expression of Wnt signaling components during Xenopus pronephros development

Background

The formation of the vertebrate kidney is tightly regulated and relies on multiple evolutionarily conserved inductive events. These are present in the complex metanephric kidney of higher vertebrates, but also in the more primitive pronephric kidney functional in the larval stages of amphibians and fish. Wnts have long been viewed as central in this process. Canonical β-Catenin-dependent Wnt signaling establishes kidney progenitors and non-canonical β-Catenin-independent Wnt signaling participate in the morphogenetic processes that form the highly sophisticated nephron structure. While some individual Wnt signaling components have been studied extensively in the kidney, the overall pathway has not yet been analyzed in depth.

Methodology/Principal Findings

Here we report a detailed expression analysis of all Wnt ligands, receptors and several downstream Wnt effectors during pronephros development in Xenopus laevis using in situ hybridization. Out of 19 Wnt ligands, only three, Wnt4, Wnt9a and Wnt11, are specifically expressed in the pronephros. Others such as Wnt8a are present, but in a broader domain comprising adjacent tissues in addition to the kidney. The same paradigm is observed for the Wnt receptors and its downstream signaling components. Fzd1, Fzd4, Fzd6, Fzd7, Fzd8 as well as Celsr1 and Prickle1 show distinct expression domains in the pronephric kidney, whereas the non-traditional Wnt receptors, Ror2 and Ryk, as well as the majority of the effector molecules are rather ubiquitous. In addition to this spatial regulation, the timing of expression is also tightly regulated. In particular, non-canonical Wnt signaling seems to be restricted to later stages of pronephros development.

Conclusion/Significance

Together these data suggest a complex cross talk between canonical and non-canonical Wnt signaling is required to establish a functional pronephric kidney.     

Secreted Frizzled-related Protein 1 (Sfrp1) Regulates the Progression of Renal Fibrosis in a Mouse Model of Obstructive Nephropathy

Renal fibrosis is responsible for progressive renal diseases that cause chronic renal failure. Sfrp1 (secreted Frizzled-related protein 1) is highly expressed in kidney, although little is known about connection between the protein and renal diseases. Here, we focused on Sfrp1 to investigate its roles in renal fibrosis using a mouse model of unilateral ureteral obstruction (UUO). In wild-type mice, the expression of Sfrp1 protein was markedly increased after UUO. The kidneys from Sfrp1 knock-out mice showed significant increase in expression of myofibrobast markers, α-smooth muscle actin (αSMA). Sfrp1 deficiency also increased protein levels of the fibroblast genes, vimentin, and decreased those of the epithelial genes, E-cadherin, indicated that enhanced epithelial-to-mesenchymal transition. There was no difference in the levels of canonical Wnt signaling; rather, the levels of phosphorylated c-Jun and JNK were more increased in the Sfrp1^−/− obstructed kidney. Moreover, the apoptotic cell population was significantly elevated in the obstructed kidneys from Sfrp1^−/− mice following UUO but was slightly increased in those from wild-type mice. These results indicate that Sfrp1 is required for inhibition of renal damage through the non-canonical Wnt/PCP pathway.