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• W2737205782 abstract "Autosomal recessive polycystic kidney disease (OMIM 263200) is a serious condition of the kidney and liver caused by mutations in a single gene, PKHD1. This gene encodes fibrocystin/polyductin (FPC, PD1), a large protein shown by in vitro studies to undergo Notch-like processing. Its cytoplasmic tail, reported to include a ciliary targeting sequence, a nuclear localization signal, and a polycystin-2 binding domain, is thought to traffic to the nucleus after cleavage. We now report a novel mouse line with a triple HA-epitope “knocked-in” to the C-terminus along with lox P sites flanking exon 67, which encodes most of the C-terminus (Pkhd1Flox67HA). The triple HA-epitope has no functional effect as assayed by phenotype and allows in vivo tracking of Fibrocystin. We used the HA tag to identify previously predicted Fibrocystin cleavage products in tissue. In addition, we found that Polycystin-2 fails to co-precipitate with Fibrocystin in kidney samples. Immunofluorescence studies with anti-HA antibodies demonstrate that Fibrocystin is primarily present in a sub-apical location the in kidney, biliary duct, and pancreatic ducts, partially overlapping with the Golgi. In contrast to previous studies, the endogenous protein in the primary cilia was not detectable in mouse tissues. After Cre-mediated deletion, homozygous Pkhd1Δ67 mice are completely normal. Thus, Pkhd1Flox67HA is a valid model to track Pkhd1-derived products containing the C-terminus. Significantly, exon 67 containing the nuclear localization signal and the polycystin-2 binding domain is not essential for Fibrocystin function in our model. Autosomal recessive polycystic kidney disease (OMIM 263200) is a serious condition of the kidney and liver caused by mutations in a single gene, PKHD1. This gene encodes fibrocystin/polyductin (FPC, PD1), a large protein shown by in vitro studies to undergo Notch-like processing. Its cytoplasmic tail, reported to include a ciliary targeting sequence, a nuclear localization signal, and a polycystin-2 binding domain, is thought to traffic to the nucleus after cleavage. We now report a novel mouse line with a triple HA-epitope “knocked-in” to the C-terminus along with lox P sites flanking exon 67, which encodes most of the C-terminus (Pkhd1Flox67HA). The triple HA-epitope has no functional effect as assayed by phenotype and allows in vivo tracking of Fibrocystin. We used the HA tag to identify previously predicted Fibrocystin cleavage products in tissue. In addition, we found that Polycystin-2 fails to co-precipitate with Fibrocystin in kidney samples. Immunofluorescence studies with anti-HA antibodies demonstrate that Fibrocystin is primarily present in a sub-apical location the in kidney, biliary duct, and pancreatic ducts, partially overlapping with the Golgi. In contrast to previous studies, the endogenous protein in the primary cilia was not detectable in mouse tissues. After Cre-mediated deletion, homozygous Pkhd1Δ67 mice are completely normal. Thus, Pkhd1Flox67HA is a valid model to track Pkhd1-derived products containing the C-terminus. Significantly, exon 67 containing the nuclear localization signal and the polycystin-2 binding domain is not essential for Fibrocystin function in our model. Autosomal recessive polycystic kidney disease (ARPKD) (OMIM 263200) is an often severe disorder that affects 1 in 20,000 live births.1Zerres K. Rudnik-Schoneborn S. Steinkamm C. et al.Autosomal recessive polycystic kidney disease.J Mol Med (Berl). 1998; 76: 303-309Crossref PubMed Scopus (114) Google Scholar It is a complex disease that presents with a wide range of clinical manifestations including enlarged echogenic kidneys primarily due to dilation of collecting ducts, cystic proliferation of biliary ducts, and congenital hepatic fibrosis.1Zerres K. Rudnik-Schoneborn S. Steinkamm C. et al.Autosomal recessive polycystic kidney disease.J Mol Med (Berl). 1998; 76: 303-309Crossref PubMed Scopus (114) Google Scholar, 2Bergmann C. Senderek J. Windelen E. et al.Clinical consequences of PKHD1 mutations in 164 patients with autosomal-recessive polycystic kidney disease (ARPKD).Kidney Int. 2005; 67: 829-848Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 3Guay-Woodford L.M. Desmond R.A. Autosomal recessive polycystic kidney disease: the clinical experience in North America.Pediatrics. 2003; 111: 1072-1080Crossref PubMed Scopus (298) Google Scholar Respiratory failure due to pulmonary hypoplasia is a leading cause of neonatal mortality, affecting 30% to 40%.4Guay-Woodford L.M. Muecher G. Hopkins S.D. et al.The severe perinatal form of autosomal recessive polycystic kidney disease maps to chromosome 6p21.1-p12: implications for genetic counseling.Am J Hum Genet. 1995; 56: 1101-1107PubMed Google Scholar Although congenital hepatic fibrosis is an invariant finding in ARPKD, renal manifestations are highly variable, being most severe in neonatal disease but milder in patients who reach adulthood.2Bergmann C. Senderek J. Windelen E. et al.Clinical consequences of PKHD1 mutations in 164 patients with autosomal-recessive polycystic kidney disease (ARPKD).Kidney Int. 2005; 67: 829-848Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 5Guay-Woodford L.M. Autosomal recessive polycystic kidney disease: the prototype of the hepato-renal fibrocystic diseases.J Pediatr Genet. 2014; 3: 89-101PubMed Google Scholar, 6Gunay-Aygun M. Turkbey I.B. Bryant J. et al.Hepatorenal findings in obligate heterozygotes for autosomal recessive polycystic kidney disease.Mol Genet Metab. 2011; 104: 677-681Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 7Denamur E. Delezoide A.L. Alberti C. et al.Genotype-phenotype correlations in fetuses and neonates with autosomal recessive polycystic kidney disease.Kidney Int. 2010; 77: 350-358Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 8Adeva M. El-Youssef M. Rossetti S. et al.Clinical and molecular characterization defines a broadened spectrum of autosomal recessive polycystic kidney disease (ARPKD).Medicine (Baltimore). 2006; 85: 1-21Crossref PubMed Scopus (189) Google Scholar, 9Bergmann C. Senderek J. Kupper F. et al.PKHD1 mutations in autosomal recessive polycystic kidney disease (ARPKD).Hum Mutat. 2004; 23: 453-463Crossref PubMed Scopus (123) Google Scholar Mutations of a single gene, PKHD1, are responsible for all typical forms of the disease.10Onuchic L.F. Furu L. Nagasawa Y. et al.PKHD1, the polycystic kidney and hepatic disease 1 gene, encodes a novel large protein containing multiple immunoglobulin-like plexin-transcription-factor domains and parallel beta-helix 1 repeats.Am J Hum Genet. 2002; 70: 1305-1317Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 11Ward C.J. Hogan M.C. Rossetti S. et al.The gene mutated in autosomal recessive polycystic kidney disease encodes a large, receptor-like protein.Nat Genet. 2002; 30: 259-269Crossref PubMed Scopus (585) Google Scholar The gene extends more than ∼500 kb, and its longest open reading frame is encoded by a 67-exon transcript that produces a 4074aa protein, fibrocystin/polyductin (FPC). The gene is reported to undergo a complex pattern of tissue-specific splicing with several alternative exons, but the significance of this phenomenon is uncertain because mutations have thus far been exclusively found in the core 67 exons.10Onuchic L.F. Furu L. Nagasawa Y. et al.PKHD1, the polycystic kidney and hepatic disease 1 gene, encodes a novel large protein containing multiple immunoglobulin-like plexin-transcription-factor domains and parallel beta-helix 1 repeats.Am J Hum Genet. 2002; 70: 1305-1317Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 12Boddu R. Yang C. O'Connor A.K. et al.Intragenic motifs regulate the transcriptional complexity of Pkhd1/PKHD1.J Mol Med (Berl). 2014; 92: 1045-1056Crossref PubMed Scopus (24) Google Scholar, 13Sharp A.M. Messiaen L.M. Page G. et al.Comprehensive genomic analysis of PKHD1 mutations in ARPKD cohorts.J Med Genet. 2005; 42: 336-349Crossref PubMed Scopus (81) Google Scholar Mutations have been identified across the length of the gene, with 2 truncating mutations associated with a more severe phenotype than other mutational patterns.2Bergmann C. Senderek J. Windelen E. et al.Clinical consequences of PKHD1 mutations in 164 patients with autosomal-recessive polycystic kidney disease (ARPKD).Kidney Int. 2005; 67: 829-848Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 9Bergmann C. Senderek J. Kupper F. et al.PKHD1 mutations in autosomal recessive polycystic kidney disease (ARPKD).Hum Mutat. 2004; 23: 453-463Crossref PubMed Scopus (123) Google Scholar, 13Sharp A.M. Messiaen L.M. Page G. et al.Comprehensive genomic analysis of PKHD1 mutations in ARPKD cohorts.J Med Genet. 2005; 42: 336-349Crossref PubMed Scopus (81) Google Scholar, 14Furu L. Onuchic L.F. Gharavi A. et al.Milder presentation of recessive polycystic kidney disease requires presence of amino acid substitution mutations.J Am Soc Nephrol. 2003; 14: 2004-2014Crossref PubMed Scopus (97) Google Scholar, 15Gunay-Aygun M. Tuchman M. Font-Montgomery E. et al.PKHD1 sequence variations in 78 children and adults with autosomal recessive polycystic kidney disease and congenital hepatic fibrosis.Mol Genet Metab. 2010; 99: 160-173Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar FPC is an ∼500-kDa protein with a long extracellular N-terminal region, a single transmembrane span and an intracellular cytoplasmic domain (ICD).10Onuchic L.F. Furu L. Nagasawa Y. et al.PKHD1, the polycystic kidney and hepatic disease 1 gene, encodes a novel large protein containing multiple immunoglobulin-like plexin-transcription-factor domains and parallel beta-helix 1 repeats.Am J Hum Genet. 2002; 70: 1305-1317Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 11Ward C.J. Hogan M.C. Rossetti S. et al.The gene mutated in autosomal recessive polycystic kidney disease encodes a large, receptor-like protein.Nat Genet. 2002; 30: 259-269Crossref PubMed Scopus (585) Google Scholar, 16Nagasawa Y. Matthiesen S. Onuchic L.F. et al.Identification and characterization of Pkhd1, the mouse orthologue of the human ARPKD gene.J Am Soc Nephrol. 2002; 13: 2246-2258Crossref PubMed Scopus (88) Google Scholar It appears to be a ciliary protein, although it also has been localized to the basal body and the plasma membrane.17Ward C.J. Yuan D. Masyuk T.V. et al.Cellular and subcellular localization of the ARPKD protein; fibrocystin is expressed on primary cilia.Hum Mol Genet. 2003; 12: 2703-2710Crossref PubMed Scopus (260) Google Scholar, 18Menezes L.F. Cai Y. Nagasawa Y. et al.Polyductin, the PKHD1 gene product, comprises isoforms expressed in plasma membrane, primary cilium, and cytoplasm.Kidney Int. 2004; 66: 1345-1355Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 19Wang S. Luo Y. Wilson P.D. et al.The autosomal recessive polycystic kidney disease protein is localized to primary cilia, with concentration in the basal body area.J Am Soc Nephrol. 2004; 15: 592-602Crossref PubMed Scopus (126) Google Scholar Although FPC’s structure and its extracellular motifs suggest that it functions as a receptor, its ligand(s) remains unknown. Previous in vitro studies of overexpressed recombinant, epitope-tagged human FPC showed that it undergoes Notch-like processing with multiple posttranslational proteolytic steps.20Kaimori J.Y. Nagasawa Y. Menezes L.F. et al.Polyductin undergoes notch-like processing and regulated release from primary cilia.Hum Mol Genet. 2007; 16: 942-956Crossref PubMed Scopus (81) Google Scholar, 21Hiesberger T. Gourley E. Erickson A. et al.Proteolytic cleavage and nuclear translocation of fibrocystin is regulated by intracellular Ca2+ and activation of protein kinase C.J Biol Chem. 2006; 281: 34357-34364Crossref PubMed Scopus (63) Google Scholar The polyductin extracellular domain (PECD) is first cleaved by a likely proprotein convertase, and it then remains tethered to the stalk by disulfide bridges. The entire ectodomain undergoes regulated shedding, reportedly mediated by a metalloprotease, with γ-secretase–dependent release of an ICD that translocates to the nucleus where it may regulate transcriptional pathways.20Kaimori J.Y. Nagasawa Y. Menezes L.F. et al.Polyductin undergoes notch-like processing and regulated release from primary cilia.Hum Mol Genet. 2007; 16: 942-956Crossref PubMed Scopus (81) Google Scholar, 21Hiesberger T. Gourley E. Erickson A. et al.Proteolytic cleavage and nuclear translocation of fibrocystin is regulated by intracellular Ca2+ and activation of protein kinase C.J Biol Chem. 2006; 281: 34357-34364Crossref PubMed Scopus (63) Google Scholar The ICD, encoded mostly by Pkhd1 exon 67, is reported to have several functional motifs including a ciliary targeting sequence, a nuclear localization signal, and a polycystin-2 (PC2) binding domain.21Hiesberger T. Gourley E. Erickson A. et al.Proteolytic cleavage and nuclear translocation of fibrocystin is regulated by intracellular Ca2+ and activation of protein kinase C.J Biol Chem. 2006; 281: 34357-34364Crossref PubMed Scopus (63) Google Scholar, 22Wu Y. Dai X.Q. Li Q. et al.Kinesin-2 mediates physical and functional interactions between polycystin-2 and fibrocystin.Hum Mol Genet. 2006; 15: 3280-3292Crossref PubMed Scopus (99) Google Scholar, 23Wang S. Zhang J. Nauli S.M. et al.Fibrocystin/polyductin, found in the same protein complex with polycystin-2, regulates calcium responses in kidney epithelia.Mol Cell Biol. 2007; 27: 3241-3252Crossref PubMed Scopus (143) Google Scholar, 24Follit J.A. Li L. Vucica Y. Pazour G.J. The cytoplasmic tail of fibrocystin contains a ciliary targeting sequence.J Cell Biol. 2010; 188: 21-28Crossref PubMed Scopus (117) Google Scholar, 25Kim I. Fu Y. Hui K. et al.Fibrocystin/polyductin modulates renal tubular formation by regulating polycystin-2 expression and function.J Am Soc Nephrol. 2008; 19: 455-468Crossref PubMed Scopus (95) Google Scholar, 26Kim I. Li C. Liang D. et al.Polycystin-2 expression is regulated by a PC2-binding domain in the intracellular portion of fibrocystin.J Biol Chem. 2008; 283: 31559-31566Crossref PubMed Scopus (56) Google Scholar PC2 is the protein encoded by the PKD2 gene, which is linked to the less common form of human autosomal dominant polycystic kidney disease.27Mochizuki T. Wu G. Hayashi T. et al.PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein.Science. 1996; 272: 1339-1342Crossref PubMed Scopus (1175) Google Scholar, 28Torres V.E. Harris P.C. Autosomal dominant polycystic kidney disease: the last 3 years.Kidney Int. 2009; 76: 149-168Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar The interaction between FPC and PC2 is reported to regulate the latter’s channel activity.22Wu Y. Dai X.Q. Li Q. et al.Kinesin-2 mediates physical and functional interactions between polycystin-2 and fibrocystin.Hum Mol Genet. 2006; 15: 3280-3292Crossref PubMed Scopus (99) Google Scholar However, the functional relationship between PC2 and FPC has been questioned given the very different phenotypes associated with loss of either protein.29Kaimori J.Y. Germino G.G. ARPKD and ADPKD: first cousins or more distant relatives?.J Am Soc Nephrol. 2008; 19: 416-418Crossref PubMed Scopus (14) Google Scholar Previous studies, however, have reported a genetic interaction in mice between Pkhd1 and either Pkd1 or Pkd2 suggesting that FPC and the polycystins may cooperatively modulate signaling pathways.25Kim I. Fu Y. Hui K. et al.Fibrocystin/polyductin modulates renal tubular formation by regulating polycystin-2 expression and function.J Am Soc Nephrol. 2008; 19: 455-468Crossref PubMed Scopus (95) Google Scholar, 30Garcia-Gonzalez M.A. Menezes L.F. Piontek K.B. et al.Genetic interaction studies link autosomal dominant and recessive polycystic kidney disease in a common pathway.Hum Mol Genet. 2007; 16: 1940-1950Crossref PubMed Scopus (88) Google Scholar, 31Fedeles S.V. Tian X. Gallagher A.R. et al.A genetic interaction network of five genes for human polycystic kidney and liver diseases defines polycystin-1 as the central determinant of cyst formation.Nat Genet. 2011; 43: 639-647Crossref PubMed Scopus (176) Google Scholar Whether Notch-like processing of FPC occurs in vivo is still uncertain. Bakeberg et al.32Bakeberg J.L. Tammachote R. Woollard J.R. et al.Epitope-tagged Pkhd1 tracks the processing, secretion, and localization of fibrocystin.J Am Soc Nephrol. 2011; 22: 2266-2277Crossref PubMed Scopus (51) Google Scholar have reported detection of a 450kDa product in exosome-like vesicles that decreases to ∼390 kDa after deglycosylation, which they conclude is likely the shed ectodomain. However, biochemical analyses of samples from both wild-type mice and a mouse line with a double V5-tag knocked into exon 3 suggest that the uncleaved 500-kDa product is the predominant form present in vivo.17Ward C.J. Yuan D. Masyuk T.V. et al.Cellular and subcellular localization of the ARPKD protein; fibrocystin is expressed on primary cilia.Hum Mol Genet. 2003; 12: 2703-2710Crossref PubMed Scopus (260) Google Scholar, 18Menezes L.F. Cai Y. Nagasawa Y. et al.Polyductin, the PKHD1 gene product, comprises isoforms expressed in plasma membrane, primary cilium, and cytoplasm.Kidney Int. 2004; 66: 1345-1355Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 32Bakeberg J.L. Tammachote R. Woollard J.R. et al.Epitope-tagged Pkhd1 tracks the processing, secretion, and localization of fibrocystin.J Am Soc Nephrol. 2011; 22: 2266-2277Crossref PubMed Scopus (51) Google Scholar None of the studies report detection of the cleaved N-terminal fragment tethered to a C-terminal stalk in vivo or shorter C-terminal fragments. Genetically modified mice are invaluable tools for elucidating the function of a gene and the protein it encodes in a physiologic setting. To better track the C-terminal ICD fragment of FPC and to determine its physiologic role in vivo, we targeted exon 67 and the 3′ UTR of the Pkhd1 gene. We introduced Lox P sites into intron 66 and adjacent to the 3′ UTR. In addition, we knocked a triple hemagglutinin (HA) epitope into the C-terminus of FPC. Using this model, we find that the C-terminus of FPC can be detected in vivo. We also unexpectedly discovered that mice lacking exon 67, which encodes the nuclear localization signal and PC2 binding domain, are completely normal. To investigate the functional role of the C-terminal tail of FPC, we used a homologous recombination gene–targeting strategy to flox exon 67 and to introduce a triple HA epitope-tag into the mouse Pkhd1 gene locus (Pkhd1Flox67HA) (Figure 1a–e). To make the targeting vector, 3 HA tags were cloned in the frame at the end of Pkhd1 exon 67, just before the stop codon (Figure 1a and b). The HA tags allow in vivo tracking of full-length FPC (FPC-HA) and any cleavage fragments containing the C-terminus of the protein. A neomycin cassette, flanked by 2 FLP recombinase target (FRT) sites, was added after the 3′ UTR and LoxP sites were inserted into intron 66 and adjacent to the neomycin cassette (Figure 1b and d). This design allows Cre-mediated deletion of the C-terminal 137 amino acids of FPC (Figure 1a). This segment harbors the nuclear localization signal and the PC2 binding domain and is a site for pathogenic mutations in humans (Figure 1a and c)7Denamur E. Delezoide A.L. Alberti C. et al.Genotype-phenotype correlations in fetuses and neonates with autosomal recessive polycystic kidney disease.Kidney Int. 2010; 77: 350-358Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 9Bergmann C. Senderek J. Kupper F. et al.PKHD1 mutations in autosomal recessive polycystic kidney disease (ARPKD).Hum Mutat. 2004; 23: 453-463Crossref PubMed Scopus (123) Google Scholar, 33Bergmann C. Senderek J. Sedlacek B. et al.Spectrum of mutations in the gene for autosomal recessive polycystic kidney disease (ARPKD/PKHD1).J Am Soc Nephrol. 2003; 14: 7689Crossref Scopus (157) Google Scholar and RWTH Aachen University PKHD1 mutation database, http://www.humgen.rwth-aachen.de/index.php). Once we obtained germline transmission of the targeted allele, we deleted Pkhd1 exon 67 by breeding Pkhd1Flox67HA mice with a Meox2-Cre transgenic line (Meox2tm1(cre)Sor/J) to produce Pkhd1Δ67 offspring.34Tallquist M.D. Soriano P. Epiblast-restricted Cre expression in MORE mice: a tool to distinguish embryonic vs. extra-embryonic gene function.Genesis. 2000; 26: 113-115Crossref PubMed Scopus (290) Google Scholar Cre-recombinase–mediated deletion also results in the loss of the HA tag. We devised a single polymerase chain reaction (PCR) genotyping strategy that allowed us to distinguish 3 products corresponding to the untagged, wild-type Pkhd1 (188 bp, Pkd1wt), HA-tagged Pkhd1 (320 bp, Pkhd1Flox67HA), and exon 67–deleted Pkhd1 (268 bp, Pkhd1Δ67) (Figure 1e). Pkhd1Flox67HA homozygous mice were viable and fertile with normal behavior throughout postnatal and adult life (Supplementary Table S1). Renal, hepatic, and pancreatic histology from adult Pkhd1Flox67HA mice was indistinguishable from that of Pkd1wt control littermates (Figure 2a–d), suggesting that epitope tagging of FPC at its C-terminus does not have overt functional consequences. We took advantage of the C-terminal HA tag to survey FPC expression. We prepared total lysates from murine tissues and probed Western blots with anti-HA antibody. In neonatal and adult kidney as well as in adult pancreas, we detected a band of ∼500 kDa, compatible with the predicted size of full-length tagged FPC (FPC-HA) as well as a few fainter, less consistently detected high molecular weight bands ranging in size from 180 kDa to just less than 500 kDa in P0 kidney (Figure 2e–g). Anti-HA did not detect tagged FPC in negative controls lacking HA, including wild-type (Pkhd1wt) and Pkhd1Δ67 mice in which Cre recombinase deleted the HA tag. A new rat monoclonal antibody generated against the C-terminal region of FPC (mFPC-ct) recognized a ∼500-kDa protein, expressed at comparable levels, in Pkhd1wt and Pkhd1Flox67HA kidneys (Figure 2e and f). The mFPC-ct antibody did not detect FPC in either Pkhd1Δ67 or Pkhd1LSL kidneys, indicating that this antibody is specific (Figure 2f). Pkhd1LSL is a previously described null allele that produces no Pkhd1 mRNA.32Bakeberg J.L. Tammachote R. Woollard J.R. et al.Epitope-tagged Pkhd1 tracks the processing, secretion, and localization of fibrocystin.J Am Soc Nephrol. 2011; 22: 2266-2277Crossref PubMed Scopus (51) Google Scholar We used immunoprecipitation with anti-HA affinity agarose beads followed by immunoblotting with anti-HA to enhance detection of FPC-HA. Using this enrichment strategy, we not only identified FPC-HA in the pancreas and kidney but also in the liver (Figure 2h and Supplementary Figure S1A and B). Low hepatic FPC levels are likely due to restricted expression of FPC in cholangiocytes, which make up a small fraction of the cell types found in the liver. FPC-HA could not be detected in adult heart or lung, indicating that FPC is either absent or present in very low abundance (Supplementary Figure S1A). In some gels, FPC resolved as a doublet, as previously reported (Figure 2h and Supplementary Figure S1A and B).32Bakeberg J.L. Tammachote R. Woollard J.R. et al.Epitope-tagged Pkhd1 tracks the processing, secretion, and localization of fibrocystin.J Am Soc Nephrol. 2011; 22: 2266-2277Crossref PubMed Scopus (51) Google Scholar Immunoprecipitation with anti-HA was negative in tissues from all mice lacking FPC-HA (Figure 2h and Supplementary Figure S1A and B). Several studies have demonstrated that FPC undergoes proteolytic cleavage when overexpressed in cell culture (Figure 3a).20Kaimori J.Y. Nagasawa Y. Menezes L.F. et al.Polyductin undergoes notch-like processing and regulated release from primary cilia.Hum Mol Genet. 2007; 16: 942-956Crossref PubMed Scopus (81) Google Scholar, 21Hiesberger T. Gourley E. Erickson A. et al.Proteolytic cleavage and nuclear translocation of fibrocystin is regulated by intracellular Ca2+ and activation of protein kinase C.J Biol Chem. 2006; 281: 34357-34364Crossref PubMed Scopus (63) Google Scholar However, FPC protein processing has never been confirmed in vivo due to a dearth of antibodies targeting the C-terminus of the protein. We prepared lysates from freshly isolated renal tubules and total kidney using a sucrose-containing lysis buffer. Western blots were probed with anti-HA or mFPC-ct antisera (Figure 3b–d). Both antibodies detected a specific band of ∼55 kDa in Pkhd1Flox67HA renal tissue that was absent in the appropriate negative control samples (Figure 3b and c). This band corresponds to the predicted polyductin transmembrane fragment (PTM) that was reported to result from cleavage at a proprotein convertase site. The PTM detected by mFPC-ct in Pkhd1wt tubules is slightly smaller (∼51 kDa) due to the absence of the 3X-HA tag. We did not observe a PTM fragment in the pancreas. To resolve small fragments, we ran total lysates on 4% to 12% Bis-Tris acetate gels. Anti-HA detected a fragment of ∼17 kDa in total kidney and in Dolichos biflorus agglutinin (DBA)–positive cells isolated from Pkhd1Flox67HA kidneys (Figure 3d and Supplementary Figure S1C). This band was not found in either Pkhd1wt or Pkhd1Δ67 tissue lacking FPC-HA. This band is similar in size to the ICD fragment that has been previously observed in overexpression systems and that is thought to be the result of γ-secretase–dependent cleavage.20Kaimori J.Y. Nagasawa Y. Menezes L.F. et al.Polyductin undergoes notch-like processing and regulated release from primary cilia.Hum Mol Genet. 2007; 16: 942-956Crossref PubMed Scopus (81) Google Scholar, 21Hiesberger T. Gourley E. Erickson A. et al.Proteolytic cleavage and nuclear translocation of fibrocystin is regulated by intracellular Ca2+ and activation of protein kinase C.J Biol Chem. 2006; 281: 34357-34364Crossref PubMed Scopus (63) Google Scholar We found that detection of PTM and ICD was highly dependent on the conditions that were used for tissue preparation, including the use of freshly isolated tissue and the composition of lysis buffer. The PTM was best visualized when tissues were lysed in a sucrose-based buffer, whereas the ICD could best be resolved when a triton-based buffer was used. It was previously reported that the FPC extracellular PECD fragment is present in whole urine and urinary exosome-like vesicles (ELVs).32Bakeberg J.L. Tammachote R. Woollard J.R. et al.Epitope-tagged Pkhd1 tracks the processing, secretion, and localization of fibrocystin.J Am Soc Nephrol. 2011; 22: 2266-2277Crossref PubMed Scopus (51) Google Scholar To better define which FPC fragments are found in urine, we prepared urinary Western blots from several FPC mouse models (Supplementary Figure S1D and E). We confirmed that FPC PECD was detected by anti-V5 in unprocessed urine as well as in ELVs from mice bearing a 5’-V5 tag (Pkhd1Pk(+)). In contrast, anti-HA detected a clear PTM band only in ELVs and not in unprocessed urine from Pkhd1Flox67HA mice (Supplementary Figure S1D and E). These results suggest that there are 2 different classes of FPC molecules in urine: a highly abundant free PECD and a second less abundant PECD that is either tethered to or shed with the PTM in ELVs. To localize endogenous FPC, we costained frozen kidney sections from neonatal and adult Pkhd1Flox67HA mice and controls with anti-HA antibody and segment-specific markers (Figure 4a–h, Supplementary Figures S2 and S3 and Supplementary Movie S1). In neonatal mice (P0), we observed robust anti-HA staining in DBA–positive tubules (Supplementary Figure S3A–D) but also faint staining in some Lotus Tetragonolobus Lectin (LTL)– (Vector Laboratories, Burlingame, CA) positive renal segments (Supplementary Figure S2E–H). In adult mice, however, we detected anti-HA staining only in DBA- and Na-K-Cl cotransporter 2–positive segments (Figure 4a–h) but not in LTL-expressing tubules (Supplementary Figure S2A–D). Taken together, our data show that FPC is expressed primarily in the distal tubule and thick ascending limb of the loop of Henle, but there appears to be low-level patchy expression in the proximal tubule before renal development is complete at P0. In the liver (Figure 4i–l and Supplementary Figure S3E–H) and the pancreas (Figure 4m–p and Supplementary Figure S3I–K), FPC-HA was identified only in cells expressing cytokeratin 19, which is a ductule-specific marker.35Zong Y. Panikkar A. Xu J. et al.Notch signaling controls liver development by regulating biliary differentiation.Development. 2009; 136: 1727-1739Crossref PubMed Scopus (351) Google Scholar The distribution of FPC within cells appeared to be primarily in cystoplasmic subapical vesicle-like structures (Figure 4c, d, g, h, k, l, o, and p and Supplementary Movie S1). Anti-HA staining was negative in all control tissue (Pkhd1wt and Pkhd1Δ67), indicating that antibody staining is specific (Figure 4b, f, j, and n and Supplementary Movie S1). Next, we examined the subcellular localization of FPC-HA in adult tissue from Pkhd1Flox67HA mice (Figure 5a–f and Supplementary Figure S4A–D). We found that FPC-HA colocalized with the endoplasmic reticulum marker calnexin and with the Golgi marker Golph4 in renal tubular epithelial cells (Figure 5a and d and Supplementary Figure S4A–D) and in hepatic cholangiocytes and pancreatic ducts (Figure 5b, c, e, and f). We were unable to detect FPC-HA in the cilia of any cell types that we tested including renal tubule epithelial cells (Figure 6a–d and Supplementary Figure S5), DBA-positive cells isolated from Pkhd1Flox67HA kidneys (Figure 6e–h), or in duct-lining cells of the pancreas (Figure 6i–l) and liver (Figure 6m–p).Figure 6Fibrocystin (FPC)-hemagglutinin (HA) is n" @default.
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