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Roles of uroplakins in plaque formation, umbrella cell enlargement, and urinary tract diseases.

Kong XT, Deng FM, Hu P, Liang FX, Zhou G, Auerbach AB, Genieser N, Nelson PK, Robbins ES, Shapiro E, Kachar B, Sun TT - J. Cell Biol. (2004)

Bottom Line: Both knockouts also had small superficial cells, suggesting that continued fusion of uroplakin-delivering vesicles with the apical surface may contribute to umbrella cell enlargement.Both knockouts experienced vesicoureteral reflux, hydronephrosis, renal dysfunction, and, in the offspring of some breeding pairs, renal failure and neonatal death.These results highlight the functional importance of uroplakins and establish uroplakin defects as a possible cause of major urinary tract anomalies and death.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, New York University School of Medicine, New York, NY 10016, USA.

ABSTRACT
The apical surface of mouse urothelium is covered by two-dimensional crystals (plaques) of uroplakin (UP) particles. To study uroplakin function, we ablated the mouse UPII gene. A comparison of the phenotypes of UPII- and UPIII-deficient mice yielded new insights into the mechanism of plaque formation and some fundamental features of urothelial differentiation. Although UPIII knockout yielded small plaques, UPII knockout abolished plaque formation, indicating that both uroplakin heterodimers (UPIa/II and UPIb/III or IIIb) are required for plaque assembly. Both knockouts had elevated UPIb gene expression, suggesting that this is a general response to defective plaque assembly. Both knockouts also had small superficial cells, suggesting that continued fusion of uroplakin-delivering vesicles with the apical surface may contribute to umbrella cell enlargement. Both knockouts experienced vesicoureteral reflux, hydronephrosis, renal dysfunction, and, in the offspring of some breeding pairs, renal failure and neonatal death. These results highlight the functional importance of uroplakins and establish uroplakin defects as a possible cause of major urinary tract anomalies and death.

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Related in: MedlinePlus

Inactivation of the mouse UPII gene (upk2) perturbed the expression of other uroplakins. (A) Alignment of the mouse UPII locus, the targeting vector (V), and the mutated locus (KO). Black boxes, exons; thick lines, mouse genomic sequences included in the vector; crosses, potential crossover sites; PCR1 and 2, PCR products characteristic of the wild-type and KO alleles, respectively; Neo, neomycin-resistant gene; TK, thymidine kinase of the herpes simplex virus; B, BamHI; C, ClaI; E, EcoRI; S, SacI; X, Xhol. (B) PCR analysis of the genomic DNA using a mixture of two pairs of primers that generated 4.0-kb and 3.8-kb PCR products characteristic of the wild-type (+) and KO (−) alleles, respectively. M, +C, and −C denote size markers, positive controls (+/+ genomic DNA or vector used as templates), and a negative control (no template), respectively. (C) Northern blot analysis. 5 μg of total RNA samples was resolved electrophoretically and probed with cDNA of mouse uroplakins Ia, Ib, II, and III, and glyceraldehyde phosphate dehydrogenase (GAPDH) as a loading control. Note, in the −/− animals, the absence of UPII message and the ∼2× and 10–20× increases in UPIa and UPIb message, respectively. (D) Ethidium bromide–stained rRNA of the samples in C serving as a loading control. (E) Immunoblot analysis of mouse uroplakin proteins Ia (27 kD), Ib (28 kD), II (15 kD), and III (47 kD). FG denotes the fast green–stained total mouse urothelial proteins used for immunoblotting. MAPK and tubulin of the same samples were immunoblotted for comparison. Note in the −/− mice the absence of UPII protein, the significant decrease in the levels of the other three uroplakin proteins, and the absence of the highest mol wt (∼30 kD) UPIb species due to defective glycosylation.
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fig1: Inactivation of the mouse UPII gene (upk2) perturbed the expression of other uroplakins. (A) Alignment of the mouse UPII locus, the targeting vector (V), and the mutated locus (KO). Black boxes, exons; thick lines, mouse genomic sequences included in the vector; crosses, potential crossover sites; PCR1 and 2, PCR products characteristic of the wild-type and KO alleles, respectively; Neo, neomycin-resistant gene; TK, thymidine kinase of the herpes simplex virus; B, BamHI; C, ClaI; E, EcoRI; S, SacI; X, Xhol. (B) PCR analysis of the genomic DNA using a mixture of two pairs of primers that generated 4.0-kb and 3.8-kb PCR products characteristic of the wild-type (+) and KO (−) alleles, respectively. M, +C, and −C denote size markers, positive controls (+/+ genomic DNA or vector used as templates), and a negative control (no template), respectively. (C) Northern blot analysis. 5 μg of total RNA samples was resolved electrophoretically and probed with cDNA of mouse uroplakins Ia, Ib, II, and III, and glyceraldehyde phosphate dehydrogenase (GAPDH) as a loading control. Note, in the −/− animals, the absence of UPII message and the ∼2× and 10–20× increases in UPIa and UPIb message, respectively. (D) Ethidium bromide–stained rRNA of the samples in C serving as a loading control. (E) Immunoblot analysis of mouse uroplakin proteins Ia (27 kD), Ib (28 kD), II (15 kD), and III (47 kD). FG denotes the fast green–stained total mouse urothelial proteins used for immunoblotting. MAPK and tubulin of the same samples were immunoblotted for comparison. Note in the −/− mice the absence of UPII protein, the significant decrease in the levels of the other three uroplakin proteins, and the absence of the highest mol wt (∼30 kD) UPIb species due to defective glycosylation.

Mentions: After transfecting embryonic stem (ES) cells (from 129/SvEv mice) with a vector designed to delete the first four exons and a part of the fifth exon of the mouse uroplakin II gene (Fig. 1 A), we screened 320 ES cell colonies and found 4 that were harboring the correct homologous recombination events as determined by long template PCR (Fig. 1 B) and Southern blot (not depicted). 18 chimeric mice from three ES cell lines were germline transmitting and were bred with black Swiss Webster mice to yield homozygous UPII knockout mice. Northern (Fig. 1, C and D) and Western (Fig. 1 E) blotting confirmed the absence of UPII in homozygous uroplakin II–deficient (−/−) mice. The protein levels of uroplakin Ia, UPII's partner, and of uroplakins Ib and III (of the uroplakin Ib/III pair) were reduced 10–20-fold (on a per total cellular protein basis) when compared with those of the normal control mice (+/+; Fig. 1 E). The mRNA levels of UPIa and UPIII were slightly up-regulated (about twofold), whereas that of UPIb was drastically up-regulated by 10–20-fold (Fig. 1 C; see Discussion).


Roles of uroplakins in plaque formation, umbrella cell enlargement, and urinary tract diseases.

Kong XT, Deng FM, Hu P, Liang FX, Zhou G, Auerbach AB, Genieser N, Nelson PK, Robbins ES, Shapiro E, Kachar B, Sun TT - J. Cell Biol. (2004)

Inactivation of the mouse UPII gene (upk2) perturbed the expression of other uroplakins. (A) Alignment of the mouse UPII locus, the targeting vector (V), and the mutated locus (KO). Black boxes, exons; thick lines, mouse genomic sequences included in the vector; crosses, potential crossover sites; PCR1 and 2, PCR products characteristic of the wild-type and KO alleles, respectively; Neo, neomycin-resistant gene; TK, thymidine kinase of the herpes simplex virus; B, BamHI; C, ClaI; E, EcoRI; S, SacI; X, Xhol. (B) PCR analysis of the genomic DNA using a mixture of two pairs of primers that generated 4.0-kb and 3.8-kb PCR products characteristic of the wild-type (+) and KO (−) alleles, respectively. M, +C, and −C denote size markers, positive controls (+/+ genomic DNA or vector used as templates), and a negative control (no template), respectively. (C) Northern blot analysis. 5 μg of total RNA samples was resolved electrophoretically and probed with cDNA of mouse uroplakins Ia, Ib, II, and III, and glyceraldehyde phosphate dehydrogenase (GAPDH) as a loading control. Note, in the −/− animals, the absence of UPII message and the ∼2× and 10–20× increases in UPIa and UPIb message, respectively. (D) Ethidium bromide–stained rRNA of the samples in C serving as a loading control. (E) Immunoblot analysis of mouse uroplakin proteins Ia (27 kD), Ib (28 kD), II (15 kD), and III (47 kD). FG denotes the fast green–stained total mouse urothelial proteins used for immunoblotting. MAPK and tubulin of the same samples were immunoblotted for comparison. Note in the −/− mice the absence of UPII protein, the significant decrease in the levels of the other three uroplakin proteins, and the absence of the highest mol wt (∼30 kD) UPIb species due to defective glycosylation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172608&req=5

fig1: Inactivation of the mouse UPII gene (upk2) perturbed the expression of other uroplakins. (A) Alignment of the mouse UPII locus, the targeting vector (V), and the mutated locus (KO). Black boxes, exons; thick lines, mouse genomic sequences included in the vector; crosses, potential crossover sites; PCR1 and 2, PCR products characteristic of the wild-type and KO alleles, respectively; Neo, neomycin-resistant gene; TK, thymidine kinase of the herpes simplex virus; B, BamHI; C, ClaI; E, EcoRI; S, SacI; X, Xhol. (B) PCR analysis of the genomic DNA using a mixture of two pairs of primers that generated 4.0-kb and 3.8-kb PCR products characteristic of the wild-type (+) and KO (−) alleles, respectively. M, +C, and −C denote size markers, positive controls (+/+ genomic DNA or vector used as templates), and a negative control (no template), respectively. (C) Northern blot analysis. 5 μg of total RNA samples was resolved electrophoretically and probed with cDNA of mouse uroplakins Ia, Ib, II, and III, and glyceraldehyde phosphate dehydrogenase (GAPDH) as a loading control. Note, in the −/− animals, the absence of UPII message and the ∼2× and 10–20× increases in UPIa and UPIb message, respectively. (D) Ethidium bromide–stained rRNA of the samples in C serving as a loading control. (E) Immunoblot analysis of mouse uroplakin proteins Ia (27 kD), Ib (28 kD), II (15 kD), and III (47 kD). FG denotes the fast green–stained total mouse urothelial proteins used for immunoblotting. MAPK and tubulin of the same samples were immunoblotted for comparison. Note in the −/− mice the absence of UPII protein, the significant decrease in the levels of the other three uroplakin proteins, and the absence of the highest mol wt (∼30 kD) UPIb species due to defective glycosylation.
Mentions: After transfecting embryonic stem (ES) cells (from 129/SvEv mice) with a vector designed to delete the first four exons and a part of the fifth exon of the mouse uroplakin II gene (Fig. 1 A), we screened 320 ES cell colonies and found 4 that were harboring the correct homologous recombination events as determined by long template PCR (Fig. 1 B) and Southern blot (not depicted). 18 chimeric mice from three ES cell lines were germline transmitting and were bred with black Swiss Webster mice to yield homozygous UPII knockout mice. Northern (Fig. 1, C and D) and Western (Fig. 1 E) blotting confirmed the absence of UPII in homozygous uroplakin II–deficient (−/−) mice. The protein levels of uroplakin Ia, UPII's partner, and of uroplakins Ib and III (of the uroplakin Ib/III pair) were reduced 10–20-fold (on a per total cellular protein basis) when compared with those of the normal control mice (+/+; Fig. 1 E). The mRNA levels of UPIa and UPIII were slightly up-regulated (about twofold), whereas that of UPIb was drastically up-regulated by 10–20-fold (Fig. 1 C; see Discussion).

Bottom Line: Both knockouts also had small superficial cells, suggesting that continued fusion of uroplakin-delivering vesicles with the apical surface may contribute to umbrella cell enlargement.Both knockouts experienced vesicoureteral reflux, hydronephrosis, renal dysfunction, and, in the offspring of some breeding pairs, renal failure and neonatal death.These results highlight the functional importance of uroplakins and establish uroplakin defects as a possible cause of major urinary tract anomalies and death.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, New York University School of Medicine, New York, NY 10016, USA.

ABSTRACT
The apical surface of mouse urothelium is covered by two-dimensional crystals (plaques) of uroplakin (UP) particles. To study uroplakin function, we ablated the mouse UPII gene. A comparison of the phenotypes of UPII- and UPIII-deficient mice yielded new insights into the mechanism of plaque formation and some fundamental features of urothelial differentiation. Although UPIII knockout yielded small plaques, UPII knockout abolished plaque formation, indicating that both uroplakin heterodimers (UPIa/II and UPIb/III or IIIb) are required for plaque assembly. Both knockouts had elevated UPIb gene expression, suggesting that this is a general response to defective plaque assembly. Both knockouts also had small superficial cells, suggesting that continued fusion of uroplakin-delivering vesicles with the apical surface may contribute to umbrella cell enlargement. Both knockouts experienced vesicoureteral reflux, hydronephrosis, renal dysfunction, and, in the offspring of some breeding pairs, renal failure and neonatal death. These results highlight the functional importance of uroplakins and establish uroplakin defects as a possible cause of major urinary tract anomalies and death.

Show MeSH
Related in: MedlinePlus