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Feng Qian - One of the best experts on this subject based on the ideXlab platform.
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mtorc1 mediated inhibition of Polycystin 1 expression drives renal cyst formation in tuberous sclerosis complex
Nature Communications, 2016Co-Authors: Monika Pema, Feng Qian, Maddalena Castelli, Marco Chiaravalli, Stefan Somlo, Luca Drusian, Qin Yao, Sara Ricciardi, Stefano Biffo, Alessandra BolettaAbstract:Previous studies report a cross-talk between the polycystic kidney disease (PKD) and tuberous sclerosis complex (TSC) genes. mTOR signalling is upregulated in PKD and rapamycin slows cyst expansion, whereas renal inactivation of the Tsc genes causes cysts. Here we identify a new interplay between the PKD and TSC genes, with important implications for the pathophysiology of both diseases. Kidney-specific inactivation of either Pkd1 or Tsc1 using an identical Cre (KspCre) results in aggressive or very mild PKD, respectively. Unexpectedly, we find that mTORC1 negatively regulates the biogenesis of Polycystin-1 (PC-1) and trafficking of the PC-1/2 complex to cilia. Genetic interaction studies reveal an important role for PC-1 downregulation by mTORC1 in the cystogenesis of Tsc1 mutants. Our data potentially explain the severe renal manifestations of the TSC/PKD contiguous gene syndrome and open new perspectives for the use of mTOR inhibitors in autosomal dominant PKD caused by hypomorphic or missense PKD1 mutations.
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novel functional complexity of Polycystin 1 by gps cleavage in vivo role in polycystic kidney disease
Molecular and Cellular Biology, 2014Co-Authors: Almira Kurbegovic, Robin L. Maser, Alessandra Boletta, Hyunho Kim, Julie Cruanes, Marie Trudel, Feng QianAbstract:Polycystin-1 (Pc1) cleavage at the G protein-coupled receptor (GPCR) proteolytic site (GPS) is required for normal kidney morphology in humans and mice. We found a complex pattern of endogenous Pc1 forms by GPS cleavage. GPS cleavage generates not only the heterodimeric cleaved full-length Pc1 (Pc1(cFL)) in which the N-terminal fragment (NTF) remains noncovalently associated with the C-terminal fragment (CTF) but also a novel (Pc1) form (Pc1(deN)) in which NTF becomes detached from CTF. Uncleaved Pc1 (Pc1(U)) resides primarily in the endoplasmic reticulum (ER), whereas both Pc1(cFL) and Pc1(deN) traffic through the secretory pathway in vivo. GPS cleavage is not a prerequisite, however, for Pc1 trafficking in vivo. Importantly, Pc1(deN) is predominantly found at the plasma membrane of renal epithelial cells. By functional genetic complementation with five Pkd1 mouse models, we discovered that CTF plays a crucial role in Pc1(deN) trafficking. Our studies support GPS cleavage as a critical regulatory mechanism of Pc1 biogenesis and trafficking for proper kidney development and homeostasis.
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Polycystin 1 negatively regulates Polycystin 2 expression via the aggresome autophagosome pathway
Journal of Biological Chemistry, 2014Co-Authors: Valeriu Cebotaru, Feng Qian, Marco Chiaravalli, Alessandra Boletta, Hyunho Kim, Liudmila Cebotaru, William B. GugginoAbstract:Mutations of the PKD1 and PKD2 genes, encoding Polycystin-1 (PC1) and Polycystin-2 (PC2), respectively, lead to autosomal dominant polycystic kidney disease. Interestingly, up-regulation or down-regulation of PKD1 or PKD2 leads to polycystic kidney disease in animal models, but their interrelations are not completely understood. We show here that full-length PC1 that interacts with PC2 via a C-terminal coiled-coil domain regulates PC2 expression in vivo and in vitro by down-regulating PC2 expression in a dose-dependent manner. Expression of the pathogenic mutant R4227X, which lacks the C-terminal coiled-coil domain, failed to down-regulate PC2 expression, suggesting that PC1-PC2 interaction is necessary for PC2 regulation. The proteasome and autophagy are two pathways that control protein degradation. Proteins that are not degraded by proteasomes precipitate in the cytoplasm and are transported via histone deacetylase 6 (HDAC6) toward the aggresomes. We found that HDAC6 binds to PC2 and that expression of full-length PC1 accelerates the transport of the HDAC6-PC2 complex toward aggresomes, whereas expression of the R4227X mutant fails to do so. Aggresomes are engulfed by autophagosomes, which then fuse with the lysosome for degradation; this process is also known as autophagy. We have now shown that PC1 overexpression leads to increased degradation of PC2 via autophagy. Interestingly, PC1 does not activate autophagy generally. Thus, we have now uncovered a new pathway suggesting that when PC1 is expressed, PC2 that is not bound to PC1 is directed to aggresomes and subsequently degraded via autophagy, a control mechanism that may play a role in autosomal dominant polycystic kidney disease pathogenesis.
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Polycystin 1 regulates the stability and ubiquitination of transcription factor jade 1
Human Molecular Genetics, 2012Co-Authors: Rebecca L Foy, Feng Qian, Alessandra Boletta, Vipul C Chitalia, M V Panchenko, Liling Zeng, Delia Lopez, Jean W Lee, Shaunak V Rana, Leonidas TsiokasAbstract:Autosomal-dominant polycystic kidney disease (ADPKD) and von Hippel-Lindau (VHL) disease lead to large kidney cysts that share pathogenetic features. The Polycystin-1 (PC1) and pVHL proteins may therefore participate in the same key signaling pathways. Jade-1 is a pro-apoptotic and growth suppressive ubiquitin ligase for beta-catenin and transcriptional coactivator associated with histone acetyltransferase activity that is stabilized by pVHL in a manner that correlates with risk of VHL renal disease. Thus, a relationship between Jade-1 and PC1 was sought. Full-length PC1 bound, stabilized and colocalized with Jade-1 and inhibited Jade-1 ubiquitination. In contrast, the cytoplasmic tail or the naturally occurring C-terminal fragment of PC1 (PC1-CTF) promoted Jade-1 ubiquitination and degradation, suggesting a dominant-negative mechanism. ADPKD-associated PC1 mutants failed to regulate Jade-1, indicating a potential disease link. Jade-1 ubiquitination was mediated by Siah-1, an E3 ligase that binds PC1. By controlling Jade-1 abundance, PC1 and the PC1-CTF differentially regulate Jade-1-mediated transcriptional activity. A key target of PC1, the cyclin-dependent kinase inhibitor p21, is also up-regulated by Jade-1. Through Jade-1, PC1 and PC1 cleaved forms may exert fine control of beta-catenin and canonical Wnt signaling, a critical pathway in cystic renal disease. Thus, Jade-1 is a transcription factor and ubiquitin ligase whose activity is regulated by PC1 in a manner that is physiologic and may correlate with disease. Jade-1 may be an important therapeutic target in renal cystogenesis.
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Polycystin 1 is required for stereocilia structure but not for mechanotransduction in inner ear hair cells
The Journal of Neuroscience, 2011Co-Authors: Katherine A Steigelman, Feng Qian, Alessandra Boletta, Klaus Piontek, Claas Wodarczyk, Jiangang Gao, Hyunho Kim, Andrea Lelli, Susan Lin, Gregory G. GerminoAbstract:The polycystic 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 stereocilia in vivo, using a hemagglutinin-tagged PC-1 KI mouse model, and in renal epithelial cell microvilli in 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.
Jing Zhou - One of the best experts on this subject based on the ideXlab platform.
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Loss of Polycystin-1 in Human Cyst-Lining Epithelia Leads
2015Co-Authors: To Ciliary Dysfunction, Jing ZhouAbstract:A “two-hit ” hypothesis predicts a second somatic hit, in addition to the germline mutation, as a prerequisite to cystogenesis and has been proposed to explain the focal nature for renal cyst formation in autosomal dominant polycystic kidney disease (ADPKD). It was reported previously that Pkd1null/nullmouse kidney epithelial cells are unresponsive to flow stimulation. This report shows that Pkd1/null cells are capable of responding to mechanical flow stimulation by changing their intracellular calcium concentration in a manner similar to that of wild-type cells. This paper reports that human renal epithelia require a higher level of shear stress to evoke a cytosolic calcium increase than do mouse renal epithelia. Both immortalized and primary cultured renal epithelial cells that originate from normal and nondilated ADPKD human kidney tubules display normal ciliary expression of the Polycystins and respond to fluid-flow shear stress with the typical change in cytosolic calcium. In contrast, immortalized and primary cultured cyst-lining epithelial cells from ADPKD patients with mutations in PKD1 or with abnormal ciliary expression of Polycystin-1 or-2 were not responsive to fluid shear stress. These data support a two-hit hypothesis as a mechanism of cystogenesis. This report proposes that calcium response to fluid-flow shear stress can be used as a readout of Polycystin function and that loss of mechanosensation in the renal tubular epithelia is a feature of PKD cysts. J Am Soc Nephrol 17: 1015–1025, 2006. doi: 10.1681/ASN.2005080830 A gene mutation can result in disease through direct orindirect mechanisms. For instance, in the gain-of-function mutation, a germline mutant allele confers new or enhanced protein activity with a pathologic function
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growth of cranial synchondroses and sutures requires Polycystin 1
Developmental Biology, 2008Co-Authors: Elona Kolpakovahart, Jing Zhou, Naomi Fukai, Bo Hou, Brandeis Mcbratneyowen, Claudia Nicolae, Bjorn OlsenAbstract:Abstract In vertebrates, coordinated embryonic and postnatal growth of the craniofacial bones and the skull base is essential during the expansion of the rostrum and the brain. Identification of molecules that regulate skull growth is important for understanding the nature of craniofacial defects and for development of non-invasive biologically based diagnostics and therapies. Here we report on spatially restricted growth defects at the skull base and in craniofacial sutures of mice deficient for Polycystin-1 (Pkd1). Mutant animals reveal a premature closure of both presphenoid and sphenooccipital synchondroses at the cranial base. Furthermore, knockout mice lacking Pkd1 in neural crest cells are characterized by impaired postnatal growth at the osteogenic fronts in craniofacial sutures that are subjected to tensile forces. Our data suggest that Polycystin-1 is required for proliferation of subpopulations of cranial osteochondroprogenitor cells of both mesodermal and neural crest origin during skull growth. However, the Erk1/2 signalling pathway is up-regulated in the Pkd1-deficient skeletal tissue, similarly to that previously reported for polycystic kidney.
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endothelial cilia are fluid shear sensors that regulate calcium signaling and nitric oxide production through Polycystin 1
Circulation, 2008Co-Authors: Surya M Nauli, Donald E Ingber, Yoshifumi Kawanabe, John J Kaminski, William J Pearce, Jing ZhouAbstract:Methods and Results—Cells with the Pkd1 null/null or Tg737 orpk/orpk mutation encoded for Polycystin-1 or polaris, respectively, are unable to transmit extracellular shear stress into intracellular calcium signaling and biochemical nitric oxide synthesis. Cytosolic calcium and nitric oxide recordings further show that fluid shear sensing is a cilia-specific mechanism because other mechanical or pharmacological stimulation does not abolish calcium and nitric oxide signaling in Polycystin-1 and polaris mutant endothelial cells. Polycystin-1 localized in the basal body of Tg737 orpk/orpk endothelial cells is insufficient for a fluid shear stress response. Furthermore, the optimal shear stress to which the cells respond best does not alter the apical cilia structure but modifies the responsiveness of cells to higher shear stresses through proteolytic modification of Polycystin-1. Conclusions—We demonstrate for the first time that Polycystin-1 (required for cilia function) and polaris (required for cilia structure) are crucial mechanosensitive molecules in endothelial cells. We propose that a distinctive communication with the extracellular microenvironment depends on the proper localization and function of Polycystin-1 in cilia. (Circulation. 2008;117:1161-1171.)
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Nek8 Regulates the Expression and Localization of Polycystin-1 and Polycystin-2
Journal of the American Society of Nephrology : JASN, 2008Co-Authors: Eisei Sohara, Ying Luo, Jingjing Zhang, Danielle K. Manning, David R. Beier, Jing ZhouAbstract:Nek8 is a serine/threonine kinase that is mutated in the jck (juvenile cystic kidneys) mouse, a model of autosomal recessive juvenile polycystic kidney disease, but its function is poorly understood. We used the jck mouse to study the functional relationship between Nek8 and other proteins that have been implicated in polycystic kidney diseases. In the collecting tubules and collecting ducts of wild-type mice, we found that Nek8 was localized to the proximal portion of primary cilia and was weakly detected in the cytosol. In the jck mutant, however, Nek8 was found along the entire length of cilia. Coimmunoprecipitation experiments demonstrated that Nek8 interacted with Polycystin-2, but not with Polycystin-1, and that the jck mutation did not affect this interaction. Western blot analysis and real-time reverse transcriptase PCR revealed that the protein and mRNA expression of Polycystin-1 (PC1) and Polycystin-2 (PC2) were increased in jck mouse kidneys. The jck mutation also led to abnormal phosphorylatin of PC2, and this was associated with longer cilia and ciliary accumulation of PC1 and PC2. Our data suggests that Nek8 interacts with the signal transduction pathways of the Polycystins and may control the targeting of these ciliary proteins. Dysfunction Nek8 may lead to cystogenesis by altering the structure and function of cilia in the distal nephron.
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loss of Polycystin 1 in human cyst lining epithelia leads to ciliary dysfunction
Journal of The American Society of Nephrology, 2006Co-Authors: Surya M Nauli, Peter C Harris, Sandro Rossetti, Robert J Kolb, Francis J Alenghat, Mark B Consugar, Donald E Ingber, Mahmoud Loghmanadham, Jing ZhouAbstract:A “two-hit” hypothesis predicts a second somatic hit, in addition to the germline mutation, as a prerequisite to cystogenesis and has been proposed to explain the focal nature for renal cyst formation in autosomal dominant polycystic kidney disease (ADPKD). It was reported previously that Pkd1 null/null mouse kidney epithelial cells are unresponsive to flow stimulation. This report shows that Pkd1 +/null cells are capable of responding to mechanical flow stimulation by changing their intracellular calcium concentration in a manner similar to that of wild-type cells. This paper reports that human renal epithelia require a higher level of shear stress to evoke a cytosolic calcium increase than do mouse renal epithelia. Both immortalized and primary cultured renal epithelial cells that originate from normal and nondilated ADPKD human kidney tubules display normal ciliary expression of the Polycystins and respond to fluid-flow shear stress with the typical change in cytosolic calcium. In contrast, immortalized and primary cultured cyst-lining epithelial cells from ADPKD patients with mutations in PKD1 or with abnormal ciliary expression of Polycystin-1 or -2 were not responsive to fluid shear stress. These data support a two-hit hypothesis as a mechanism of cystogenesis. This report proposes that calcium response to fluid-flow shear stress can be used as a readout of Polycystin function and that loss of mechanosensation in the renal tubular epithelia is a feature of PKD cysts.
Peter C Harris - One of the best experts on this subject based on the ideXlab platform.
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Polycystin 1 maturation requires Polycystin 2 in a dose dependent manner
Journal of Clinical Investigation, 2015Co-Authors: Vladimir G Gainullin, Christopher J. Ward, Katharina Hopp, Cynthia J Hommerding, Peter C HarrisAbstract:Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited nephropathy responsible for 4%–10% of end-stage renal disease cases. Mutations in the genes encoding Polycystin-1 (PC1, PKD1) or Polycystin-2 (PC2, PKD2) cause ADPKD, and PKD1 mutations are associated with more severe renal disease. PC1 has been shown to form a complex with PC2, and the severity of PKD1-mediated disease is associated with the level of the mature PC1 glycoform. Here, we demonstrated that PC1 and PC2 first interact in the ER before PC1 cleavage at the GPS/GAIN site and determined that PC2 acts as an essential chaperone for PC1 maturation and surface localization. The chaperone function of PC2 was dependent on the presence of the distal coiled-coil domain and was disrupted by pathogenic missense mutations. In Pkd2–/– mice, complete loss of PC2 prevented PC1 maturation. In Pkd2 heterozygotes, the 50% PC2 reduction resulted in a nonequimolar reduction (20%–25%) of the mature PC1 glycoform. Interbreeding between various Pkd1 and Pkd2 models revealed that animals with reduced levels of functional PC1 and PC2 in the kidney exhibited severe, rapidly progressive disease, illustrating the importance of complexing of these proteins for function. Our results indicate that PC2 regulates PC1 maturation; therefore, mature PC1 levels are a determinant of disease severity in PKD2 as well as PKD1.
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A Telomerase Immortalized Human Proximal Tubule Cell Line with a Truncation Mutation (Q4004X) in Polycystin-1
PloS one, 2013Co-Authors: Brittney-shea Herbert, Peter C Harris, Christopher J. Ward, Sandro Rossetti, Brenda Grimes, Michael Werner, Elsa Bello-reuss, Heather H. Ward, Caroline MillerAbstract:Autosomal dominant polycystic kidney disease (ADPKD) is associated with a variety of cellular phenotypes in renal epithelial cells. Cystic epithelia are secretory as opposed to absorptive, have higher proliferation rates in cell culture and have some characteristics of epithelial to mesenchymal transitions [1], [2]. In this communication we describe a telomerase immortalized cell line that expresses proximal tubule markers and is derived from renal cysts of an ADPKD kidney. These cells have a single detectable truncating mutation (Q4004X) in Polycystin-1. These cells make normal appearing but shorter cilia and fail to assemble Polycystin-1 in the cilia, and less uncleaved Polycystin-1 in membrane fractions. This cell line has been maintained in continuous passage for over 35 passages without going into senescence. Nephron segment specific markers suggest a proximal tubule origin for these cells and the cell line will be useful to study mechanistic details of cyst formation in proximal tubule cells.
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homophilic and heterophilic Polycystin 1 interactions regulate e cadherin recruitment and junction assembly in mdck cells
Journal of Cell Science, 2009Co-Authors: Andrew J. Streets, Peter C Harris, Christopher J. Ward, Bart E Wagner, Albert C.m. OngAbstract:Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited human renal disease and is caused by mutations in two genes, PKD1 (85%) and PKD2 (15%). Cyst epithelial cells are characterised by a complex cellular phenotype including changes in proliferation, apoptosis, basement membrane composition and apicobasal polarity. Since Polycystin 1 (PC1), the PKD1 protein, has been located in the basolateral membrane of kidney epithelial cells, we hypothesised that it might have a key role in mediating or stabilising cell-cell interactions. In non-ciliated L929 cells, stable or transient surface expression of the PC1 extracellular domain was sufficient to confer an adhesive phenotype and stimulate junction formation. In MDCK cells, we found that PC1 was recruited to the lateral membranes coincident with E-cadherin within 30 minutes after a `calcium switch'. Recruitment of both proteins was significantly delayed when cells were treated with a PC1 blocking antibody raised to the PKD domains. Finally, PC1 and E-cadherin could be coimmunoprecipitated together from MDCK cells. We conclude that PC1 has a key role in initiating junction formation via initial homophilic interactions and facilitates junction assembly and the establishment of apicobasal polarity by E-cadherin recruitment.
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novel role for Polycystin 1 in modulating cell proliferation through calcium oscillations in kidney cells
Cell Proliferation, 2008Co-Authors: Gianluca Aguiari, Peter C Harris, Viky Trimi, Marco Bogo, Alessandra Mangolini, Gyorgy Szabadkai, Paolo Pinton, Ralph Witzgall, Pier Andrea Borea, Rosario RizzutoAbstract:Objectives: Polycystin-1 (PC1), a signalling receptor regulating Ca2+-permeable cation channels, is mutated in autosomal dominant polycystic kidney disease, which is typically characterized by increased cell proliferation. However, the precise mechanisms by which PC1 functions on Ca2+ homeostasis, signalling and cell proliferation remain unclear. Here, we investigated the possible role of PC1 as a modulator of non-capacitative Ca2+ entry (NCCE) and Ca2+ oscillations, with downstream effects on cell proliferation. Results and discussion: By employing RNA interference, we show that depletion of endogenous PC1 in HEK293 cells leads to an increase in serum-induced Ca2+ oscillations, triggering nuclear factor of activated T cell activation and leading to cell cycle progression. Consistently, Ca2+ oscillations and cell proliferation are increased in PC1-mutated kidney cystic cell lines, but both abnormal features are reduced in cells that exogenously express PC1. Notably, blockers of the NCCE pathway, but not of the CCE, blunt abnormal oscillation and cell proliferation. Our study therefore provides the first demonstration that PC1 modulates Ca2+ oscillations and a molecular mechanism to explain the association between abnormal Ca2+ homeostasis and cell proliferation in autosomal dominant polycystic kidney disease.
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loss of Polycystin 1 in human cyst lining epithelia leads to ciliary dysfunction
Journal of The American Society of Nephrology, 2006Co-Authors: Surya M Nauli, Peter C Harris, Sandro Rossetti, Robert J Kolb, Francis J Alenghat, Mark B Consugar, Donald E Ingber, Mahmoud Loghmanadham, Jing ZhouAbstract:A “two-hit” hypothesis predicts a second somatic hit, in addition to the germline mutation, as a prerequisite to cystogenesis and has been proposed to explain the focal nature for renal cyst formation in autosomal dominant polycystic kidney disease (ADPKD). It was reported previously that Pkd1 null/null mouse kidney epithelial cells are unresponsive to flow stimulation. This report shows that Pkd1 +/null cells are capable of responding to mechanical flow stimulation by changing their intracellular calcium concentration in a manner similar to that of wild-type cells. This paper reports that human renal epithelia require a higher level of shear stress to evoke a cytosolic calcium increase than do mouse renal epithelia. Both immortalized and primary cultured renal epithelial cells that originate from normal and nondilated ADPKD human kidney tubules display normal ciliary expression of the Polycystins and respond to fluid-flow shear stress with the typical change in cytosolic calcium. In contrast, immortalized and primary cultured cyst-lining epithelial cells from ADPKD patients with mutations in PKD1 or with abnormal ciliary expression of Polycystin-1 or -2 were not responsive to fluid shear stress. These data support a two-hit hypothesis as a mechanism of cystogenesis. This report proposes that calcium response to fluid-flow shear stress can be used as a readout of Polycystin function and that loss of mechanosensation in the renal tubular epithelia is a feature of PKD cysts.
Alessandra Boletta - One of the best experts on this subject based on the ideXlab platform.
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mtorc1 mediated inhibition of Polycystin 1 expression drives renal cyst formation in tuberous sclerosis complex
Nature Communications, 2016Co-Authors: Monika Pema, Feng Qian, Maddalena Castelli, Marco Chiaravalli, Stefan Somlo, Luca Drusian, Qin Yao, Sara Ricciardi, Stefano Biffo, Alessandra BolettaAbstract:Previous studies report a cross-talk between the polycystic kidney disease (PKD) and tuberous sclerosis complex (TSC) genes. mTOR signalling is upregulated in PKD and rapamycin slows cyst expansion, whereas renal inactivation of the Tsc genes causes cysts. Here we identify a new interplay between the PKD and TSC genes, with important implications for the pathophysiology of both diseases. Kidney-specific inactivation of either Pkd1 or Tsc1 using an identical Cre (KspCre) results in aggressive or very mild PKD, respectively. Unexpectedly, we find that mTORC1 negatively regulates the biogenesis of Polycystin-1 (PC-1) and trafficking of the PC-1/2 complex to cilia. Genetic interaction studies reveal an important role for PC-1 downregulation by mTORC1 in the cystogenesis of Tsc1 mutants. Our data potentially explain the severe renal manifestations of the TSC/PKD contiguous gene syndrome and open new perspectives for the use of mTOR inhibitors in autosomal dominant PKD caused by hypomorphic or missense PKD1 mutations.
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novel functional complexity of Polycystin 1 by gps cleavage in vivo role in polycystic kidney disease
Molecular and Cellular Biology, 2014Co-Authors: Almira Kurbegovic, Robin L. Maser, Alessandra Boletta, Hyunho Kim, Julie Cruanes, Marie Trudel, Feng QianAbstract:Polycystin-1 (Pc1) cleavage at the G protein-coupled receptor (GPCR) proteolytic site (GPS) is required for normal kidney morphology in humans and mice. We found a complex pattern of endogenous Pc1 forms by GPS cleavage. GPS cleavage generates not only the heterodimeric cleaved full-length Pc1 (Pc1(cFL)) in which the N-terminal fragment (NTF) remains noncovalently associated with the C-terminal fragment (CTF) but also a novel (Pc1) form (Pc1(deN)) in which NTF becomes detached from CTF. Uncleaved Pc1 (Pc1(U)) resides primarily in the endoplasmic reticulum (ER), whereas both Pc1(cFL) and Pc1(deN) traffic through the secretory pathway in vivo. GPS cleavage is not a prerequisite, however, for Pc1 trafficking in vivo. Importantly, Pc1(deN) is predominantly found at the plasma membrane of renal epithelial cells. By functional genetic complementation with five Pkd1 mouse models, we discovered that CTF plays a crucial role in Pc1(deN) trafficking. Our studies support GPS cleavage as a critical regulatory mechanism of Pc1 biogenesis and trafficking for proper kidney development and homeostasis.
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Polycystin 1 negatively regulates Polycystin 2 expression via the aggresome autophagosome pathway
Journal of Biological Chemistry, 2014Co-Authors: Valeriu Cebotaru, Feng Qian, Marco Chiaravalli, Alessandra Boletta, Hyunho Kim, Liudmila Cebotaru, William B. GugginoAbstract:Mutations of the PKD1 and PKD2 genes, encoding Polycystin-1 (PC1) and Polycystin-2 (PC2), respectively, lead to autosomal dominant polycystic kidney disease. Interestingly, up-regulation or down-regulation of PKD1 or PKD2 leads to polycystic kidney disease in animal models, but their interrelations are not completely understood. We show here that full-length PC1 that interacts with PC2 via a C-terminal coiled-coil domain regulates PC2 expression in vivo and in vitro by down-regulating PC2 expression in a dose-dependent manner. Expression of the pathogenic mutant R4227X, which lacks the C-terminal coiled-coil domain, failed to down-regulate PC2 expression, suggesting that PC1-PC2 interaction is necessary for PC2 regulation. The proteasome and autophagy are two pathways that control protein degradation. Proteins that are not degraded by proteasomes precipitate in the cytoplasm and are transported via histone deacetylase 6 (HDAC6) toward the aggresomes. We found that HDAC6 binds to PC2 and that expression of full-length PC1 accelerates the transport of the HDAC6-PC2 complex toward aggresomes, whereas expression of the R4227X mutant fails to do so. Aggresomes are engulfed by autophagosomes, which then fuse with the lysosome for degradation; this process is also known as autophagy. We have now shown that PC1 overexpression leads to increased degradation of PC2 via autophagy. Interestingly, PC1 does not activate autophagy generally. Thus, we have now uncovered a new pathway suggesting that when PC1 is expressed, PC2 that is not bound to PC1 is directed to aggresomes and subsequently degraded via autophagy, a control mechanism that may play a role in autosomal dominant polycystic kidney disease pathogenesis.
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Polycystin 1 binds par3 apkc and controls convergent extension during renal tubular morphogenesis
Nature Communications, 2013Co-Authors: Maddalena Castelli, Manila Boca, Marco Chiaravalli, Harini Ramalingam, Isaline Rowe, Gianfranco Distefano, Thomas J Carroll, Alessandra BolettaAbstract:Loss-of-function mutations in PKD1, the gene encoding the plasma membrane receptor Polycystin-1, lead to renal cyst formation in polycystic kidney disease. Here, Castelli et al. show that Polycystin-1 interacts with the Par3 polarity complex and has a role in the morphogenesis of kidney tubules during mouse development.
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Polycystin 1 binds par3 apkc and controls convergent extension during renal tubular morphogenesis
Nature Communications, 2013Co-Authors: Maddalena Castelli, Manila Boca, Marco Chiaravalli, Harini Ramalingam, Isaline Rowe, Gianfranco Distefano, Thomas J Carroll, Alessandra BolettaAbstract: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.
Michael J. Caplan - One of the best experts on this subject based on the ideXlab platform.
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Polycystin 1 regulates bone development through an interaction with the transcriptional coactivator taz
Human Molecular Genetics, 2019Co-Authors: David Merrick, Kavita Mistry, Nikolay Gresko, Julie E Baggs, John B Hogenesch, Zhaoxia Sun, Michael J. CaplanAbstract:Polycystin-1 (PC1), encoded by the PKD1 gene that is mutated in the autosomal dominant polycystic kidney disease, regulates a number of processes including bone development. Activity of the transcription factor RunX2, which controls osteoblast differentiation, is reduced in Pkd1 mutant mice but the mechanism governing PC1 activation of RunX2 is unclear. PC1 undergoes regulated cleavage that releases its C-terminal tail (CTT), which translocates to the nucleus to modulate transcriptional pathways involved in proliferation and apoptosis. We find that the cleaved CTT of PC1 (PC1-CTT) stimulates the transcriptional coactivator TAZ (Wwtr1), an essential coactivator of RunX2. PC1-CTT physically interacts with TAZ, stimulating RunX2 transcriptional activity in pre-osteoblast cells in a TAZ-dependent manner. The PC1-CTT increases the interaction between TAZ and RunX2 and enhances the recruitment of the p300 transcriptional co-regulatory protein to the TAZ/RunX2/PC1-CTT complex. Zebrafish injected with morpholinos directed against pkd1 manifest severe bone calcification defects and a curly tail phenotype. Injection of messenger RNA (mRNA) encoding the PC1-CTT into pkd1-morphant fish restores bone mineralization and reduces the severity of the curly tail phenotype. These effects are abolished by co-injection of morpholinos directed against TAZ. Injection of mRNA encoding a dominant-active TAZ construct is sufficient to rescue both the curly tail phenotype and the skeletal defects observed in pkd1-morpholino treated fish. Thus, TAZ constitutes a key mechanistic link through which PC1 mediates its physiological functions.
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newly synthesized Polycystin 1 takes different trafficking pathways to the apical and ciliary membranes
Traffic, 2018Co-Authors: Allison Louise Gilder, Vanathy Rajendran, Hannah C. Chapin, Valeria Padovano, Christina L Hueschen, Michael J. CaplanAbstract:Mutations in the genes encoding Polycystin-1 (PC1) and Polycystin 2 (PC2) cause autosomal dominant polycystic kidney disease. These transmembrane proteins colocalize in the primary cilia of renal epithelial cells, where they may participate in sensory processes. PC1 is also found in the apical membrane when expressed in cultured epithelial cells. PC1 undergoes autocatalytic cleavage, producing an extracellular N-terminal fragment that remains noncovalently attached to the transmembrane C-terminus. Exposing cells to alkaline solutions elutes the N-terminal fragment while the C-terminal fragment is retained in the cell membrane. Utilizing this observation, we developed a "strip-recovery" synchronization protocol to study PC1 trafficking in polarized LLC-PK1 renal epithelial cells. Following alkaline strip, a new cohort of PC1 repopulates the cilia within 30 minutes, while apical delivery of PC1 was not detectable until 3 hours. Brefeldin A (BFA) blocked apical PC1 delivery, while ciliary delivery of PC1 was BFA insensitive. Incubating cells at 20°C to block trafficking out of the trans-Golgi network also inhibits apical but not ciliary delivery. These results suggest that newly synthesized PC1 takes distinct pathways to the ciliary and apical membranes. Ciliary PC1 appears to by-pass BFA sensitive Golgi compartments, while apical delivery of PC1 traverses these compartments.
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Polycystin-1 cleavage and the regulation of transcriptional pathways
Pediatric Nephrology, 2014Co-Authors: David Merrick, Veronique Chauvet, Claudia A. Bertuccio, Hannah C. Chapin, Michael J. CaplanAbstract:Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of end-stage renal disease, affecting approximately 1 in 1,000 people. The disease is characterized by the development of numerous large fluid-filled renal cysts over the course of decades. These cysts compress the surrounding renal parenchyma and impair its function. Mutations in two genes are responsible for ADPKD. The protein products of both of these genes, Polycystin-1 and Polycystin-2, localize to the primary cilium and participate in a wide variety of signaling pathways. Polycystin-1 undergoes several proteolytic cleavages that produce fragments which manifest biological activities. Recent results suggest that the production of Polycystin-1 cleavage fragments is necessary and sufficient to account for at least some, although certainly not all, of the physiological functions of the parent protein.
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Polycystin 1 c terminus cleavage and its relation with Polycystin 2 two proteins involved in polycystic kidney disease
Medicina-buenos Aires, 2013Co-Authors: Claudia A. Bertuccio, Michael J. CaplanAbstract:Autosomal dominant polycystic kidney disease (ADPKD), a most common genetic cause of chronic renal failure, is characterized by the progressive development and enlargement of cysts in kidneys and other organs. The cystogenic process is highly complex and involves a high proliferative rate, increased apoptosis, altered protein sorting, changed secretory characteristics, and disorganization of the extracellular matrix. ADPKD is caused by mutations in the genes encoding Polycystin-1 (PC-1) or Polycystin-2 (PC-2). PC-1 undergoes multiple cleavages that intervene in several signaling pathways involved in cellular proliferation and differentiation mechanisms. One of these cleavages releases the cytoplasmic C-terminal tail of PC-1. In addition, the C-terminal cytoplasmic tails of PC-1 and PC-2 interact in vitro and in vivo. The purpose of this review is to summarize recent literature that suggests that PC-1 and PC-2 may function through a common signaling pathway necessary for normal tubulogenesis. We hope that a better understanding of PC-1 and PC-2 protein function will lead to progress in diagnosis and treatment for ADPKD.
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Polycystin 1 c terminal cleavage is modulated by Polycystin 2 expression
Journal of Biological Chemistry, 2009Co-Authors: Claudia Bertuccio, Yiqiang Cai, Veronique Chauvet, Hannah C. Chapin, Kavita Mistry, Stefan Somlo, Michael J. CaplanAbstract:Autosomal dominant polycystic kidney disease is caused by mutations in the genes encoding Polycystin-1 (PC-1) and Polycystin-2 (PC-2). PC-1 cleavage releases its cytoplasmic C-terminal tail (CTT), which enters the nucleus. To determine whether PC-1 CTT cleavage is influenced by PC-2, a quantitative cleavage assay was utilized, in which the DNA binding and activation domains of Gal4 and VP16, respectively, were appended to PC-1 downstream of its CTT domain (PKDgalvp). Cells cotransfected with the resultant PKDgalvp fusion protein and PC-2 showed an increase in luciferase activity and in CTT expression, indicating that the C-terminal tail of PC-1 is cleaved and enters the nucleus. To assess whether CTT cleavage depends upon Ca2+ signaling, cells transfected with PKDgalvp alone or together with PC-2 were incubated with several agents that alter intracellular Ca2+ concentrations. PC-2 enhancement of luciferase activity was not altered by any of these treatments. Using a series of PC-2 C-terminal truncated mutations, we identified a portion of the PC-2 protein that is required to stimulate PC-1 CTT accumulation. These data demonstrate that release of the CTT from PC-1 is influenced and stabilized by PC-2. This effect is independent of Ca2+ but is regulated by sequences contained within the PC-2 C-terminal tail, suggesting a mechanism through which PC-1 and PC-2 may modulate a novel signaling pathway.