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1.
Dev Dyn ; 240(6): 1493-501, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21465620

ABSTRACT

Polycystic kidney diseases (PKD) are inherited disorders characterized by fluid-filled cysts primarily in the kidneys. We previously reported differences between the expression of Cux1, p21, and p27 in the cpk and Pkd1 null mouse models of PKD. Embryonic lethality of Pkd1 null mice limits its study to early stages of kidney development. Therefore, we examined mice with a collecting duct specific deletion in the Pkd1 gene. Cux1 was ectopically expressed in the cyst lining epithelial cells of newborn, P7 and P15 Pkd1(CD) mice. Cux1 expression correlated with cell proliferation in early stages of cystogenesis, however, as the disease progressed, fewer cyst lining cells showed increased cell proliferation. Rather, Cux1 expression in late stage cystogenesis was associated with increased apoptosis. Taken together, our results suggest that increased Cux1 expression associated with apoptosis is a common feature of late stage cyst progression in both the cpk and Pkd1(CD) mouse models of PKD.


Subject(s)
Apoptosis/genetics , Cyclin-Dependent Kinase Inhibitor p27/genetics , Homeodomain Proteins/genetics , Kidney Diseases, Cystic/genetics , Nuclear Proteins/genetics , Repressor Proteins/genetics , TRPP Cation Channels/genetics , Animals , Animals, Newborn , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Disease Progression , Down-Regulation , Enzyme Activation/genetics , Female , Gene Expression Regulation , Gene Silencing , Kidney Diseases, Cystic/metabolism , Kidney Diseases, Cystic/pathology , Kidney Tubules, Collecting/metabolism , Male , Mice , Mice, Transgenic , TRPP Cation Channels/metabolism , Transfection , Up-Regulation/genetics
2.
Biochemistry ; 50(3): 349-55, 2011 Jan 25.
Article in English | MEDLINE | ID: mdl-21142036

ABSTRACT

Polycystin-1 (PC1), the product of the polycystic kidney disease-1 (PKD1) gene, has a number of reported missense mutations whose pathogenicity is indeterminate. Previously, we utilized N-linked glycosylation reporter tags along with membrane insertion and topology assays to define the 11 membrane-spanning domains (I-XI) of PC1. In this report, we utilize glycosylation assays to determine whether two reported human polymorphisms/missense mutations within transmembrane (TM) domains VI and X affect the membrane topology of PC1. M3677T within TM VI had no effect on the topology of this TM domain as shown by the ability of two native N-linked glycosylation sites within the extracellular loop following TM VI to be glycosylated. In contrast, G4031D, within TM X, decreased the glycosylation of TM X reporter constructs, demonstrating that the substitution affected the C-terminal translocating activity of TM X. Furthermore, G4031D reduced the membrane association of TM X and XI together. These results suggest that G4031D affects the membrane insertion and topology of the C-terminal portion of polycystin-1 and represents a bona fide pathogenic mutation.


Subject(s)
Cell Membrane/metabolism , Polycystic Kidney, Autosomal Dominant/genetics , TRPP Cation Channels/genetics , TRPP Cation Channels/metabolism , Glycosylation , HEK293 Cells , Humans , Mutation, Missense , Polymorphism, Genetic , Protein Conformation , TRPP Cation Channels/chemistry
3.
Gene ; 439(1-2): 87-94, 2009 Jun 15.
Article in English | MEDLINE | ID: mdl-19332113

ABSTRACT

The homeodomain protein Cux1 is highly expressed in the nephrogenic zone of the developing kidney where it functions to regulate cell proliferation. Here we show that Cux1 directly interacts with the co-repressor Grg4 (Groucho 4), a known effector of Notch signaling. Promoter reporter based luciferase assays revealed enhanced repression of p27(kip1) promoter activity by Cux1 in the presence of Grg4. Chromatin immunoprecipitation (ChIP) assays demonstrated the direct interaction of Cux1 with p27(kip1) in newborn kidney tissue in vivo. ChIP assays also identified interactions of Cux1, Grg4, HDAC1, and HDAC3 with p27(kip1) at two separate sites in the p27(kip1) promoter. DNAse1 footprinting experiments revealed that Cux1 binds to the p27(kip1) promoter on the sequence containing two Sp1 sites and a CCAAT box approximately 500 bp from the transcriptional start site, and to an AT rich sequence approximately 1.5 kb from the transcriptional start site. Taken together, these results identify Grg4 as an interacting partner for Cux1 and suggest a mechanism of p27(kip1) repression by Cux1 during kidney development.


Subject(s)
Cyclin-Dependent Kinase Inhibitor p27/metabolism , Homeodomain Proteins/metabolism , Kidney/metabolism , Nuclear Proteins/metabolism , Repressor Proteins/metabolism , Animals , Animals, Newborn , Binding Sites , CCAAT-Enhancer-Binding Proteins/genetics , CCAAT-Enhancer-Binding Proteins/metabolism , Cell Line , Chromatin Immunoprecipitation , Cyclin-Dependent Kinase Inhibitor p27/genetics , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Humans , Kidney/growth & development , Mice , Mice, Inbred C57BL , Nuclear Proteins/genetics , Promoter Regions, Genetic , Protein Binding , Repressor Proteins/genetics
4.
Am J Physiol Renal Physiol ; 295(6): F1725-34, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18829740

ABSTRACT

Polycystic kidney diseases (PKD) are inherited as autosomal dominant (ADPKD) or autosomal recessive (ARPKD) traits and are characterized by progressive enlargement of renal cysts. Aberrant cell proliferation is a key feature in the progression of PKD. Cux1 is a homeobox gene that is related to Drosophila cut and is the murine homolog of human CDP (CCAAT Displacement Protein). Cux1 represses the cyclin kinase inhibitors p21 and p27, and transgenic mice ectopically expressing Cux1 develop renal hyperplasia. However, Cux1 transgenic mice do not develop PKD. Here, we show that a 246 amino acid deletion in Cux1 accelerates PKD progression in cpk mice. Cystic kidneys isolated from 10-day-old cpk/Cux1 double mutant mice were significantly larger than kidneys from 10-day-old cpk mice. Moreover, renal function was significantly reduced in the Cux1 mutant cpk mice, compared with cpk mice. The mutant Cux1 protein was ectopically expressed in cyst-lining cells, where expression corresponded to increased cell proliferation and apoptosis, and a decrease in expression of the cyclin kinase inhibitors p27 and p21. While the mutant Cux1 protein altered PKD progression, kidneys from mice carrying the mutant Cux1 protein alone were phenotypically normal, suggesting the Cux1 mutation modifies PKD progression in cpk mice. During cell cycle progression, Cux1 is proteolytically processed by a nuclear isoform of the cysteine protease cathepsin-L. Analysis of the deleted sequences reveals that a cathepsin-L processing site in Cux1 is deleted. Moreover, nuclear cathepsin-L is significantly reduced in both human ADPKD cells and in Pkd1 null kidneys, corresponding to increased levels of Cux1 protein in the cystic cells and kidneys. These results suggest a mechanism in which reduced Cux1 processing by cathepsin-L results in the accumulation of Cux1, downregulation of p21/p27, and increased cell proliferation in PKD.


Subject(s)
Nuclear Proteins/deficiency , Polycystic Kidney Diseases/genetics , Polycystic Kidney Diseases/physiopathology , Animals , Cathepsin L , Cathepsins/genetics , Crosses, Genetic , Cysteine Endopeptidases/genetics , Disease Progression , Homeodomain Proteins/genetics , Humans , Mice , Mice, Knockout , Mutation , Nuclear Proteins/genetics , Organ Size , Polycystic Kidney Diseases/pathology , Polycystic Kidney, Autosomal Dominant/genetics , Repressor Proteins/genetics , Sequence Deletion
5.
Biochemistry ; 42(44): 13035-48, 2003 Nov 11.
Article in English | MEDLINE | ID: mdl-14596619

ABSTRACT

Polycystin-1, the protein product of the polycystic kidney disease-1 (PKD1) gene, was originally predicted to be an integral membrane glycoprotein with 11 transmembrane (TM) domains (TM 1-11). Subsequent comparative sequence analyses led to a revision of the original model, which retained the overall topology and 11 TM segments (TM I-XI) but dropped 3 of the original domains and introduced 3 new TM domains. The membrane-spanning potential and the orientation of each of the proposed TM domains following the extracellular REJ domain (TM I-XI and TM 11) have now been tested. Using a series of N-terminal polycystin TM-glycosylation reporter gene fusions expressed in vivo, we assayed N-linked glycosylation of the C-terminal glycosylation reporter as an indicator of TM domain presence and orientation. This approach has clearly demonstrated that 7 of the 12 TM domains tested function as membrane-spanning domains. In vitro analysis of the topogenic potential of the five remaining TM domains revealed that four of these also function as membrane-spanning domains, thus supporting an 11 TM structure for polycystin-1 comprised of TM domains I-XI. In addition, these studies suggest that the membrane insertion of TM domains I-IX occurs in a cotranslational and sequential manner, while multiple topogenic determinants appear to be required for the integration of the C-terminal-most TM segments of polycystin-1.


Subject(s)
Models, Molecular , Polycystic Kidney, Autosomal Dominant/genetics , Proteins/chemistry , Proteins/genetics , Animals , Cell Line , Clone Cells , Gene Expression Regulation , Genes, Reporter , Genetic Vectors , Glycosylation , Humans , Mice , Models, Genetic , Peptide Fragments/biosynthesis , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Polycystic Kidney, Autosomal Dominant/metabolism , Protein Biosynthesis , Protein Structure, Tertiary/genetics , Proteins/metabolism , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , TRPP Cation Channels , Transfection
6.
J Am Soc Nephrol ; 13(4): 991-999, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11912258

ABSTRACT

Oxidative stress has been implicated in the pathogenesis of both acquired and hereditary polycystic kidney disease. Mechanisms of oxidant injury in C57BL/6J-cpk mice and Han:SPRD-Cy rats with rapidly or slowly progressive polycystic kidney disease were explored. Expression of heme oxygenase-1 mRNA, an inducible marker of oxidative stress, was shown to be increased in cystic kidneys of mice and rats in a pattern that reflected disease severity. By contrast, there was a decrease in mRNA expression of the antioxidant enzymes extracellular glutathione peroxidase, superoxide dismutase, catalase, and glutathione S-transferase during disease progression. Renal mRNA levels of these enzymes were strikingly reduced in rapidly progressive disease in homozygous cystic mice and rats. In slowly progressive disease in heterozygous rats, renal antioxidant mRNA levels were decreased to a greater extent in cystic males than in the less severely affected females. Protein levels for extracellular glutathione peroxidase were also reduced in plasma and in cystic kidneys of mice and rats. Plasma extracellular glutathione peroxidase enzymatic activity was also decreased, whereas the lipid peroxidation products malondialdehyde and 4-hydroxy-2(E)-nonenal were increased in kidneys and blood plasma of cystic mice. Reduced antioxidant enzyme protection and increased oxidative damage represent general mechanisms in the pathogenesis of polycystic kidney disease.


Subject(s)
Oxidative Stress , Oxidoreductases/metabolism , Polycystic Kidney Diseases/metabolism , Aldehydes/metabolism , Animals , Blood/metabolism , Disease Progression , Heme Oxygenase (Decyclizing)/genetics , Heme Oxygenase-1 , Heterozygote , Kidney/metabolism , Malondialdehyde/metabolism , Membrane Proteins , Mice , Mice, Inbred C57BL , Oxidoreductases/genetics , Polycystic Kidney Diseases/genetics , RNA, Messenger/metabolism , Rats , Rats, Inbred Strains , Sex Characteristics
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