Faculty Bibliography

Although the epithelial lining of much of the mammalian urinary tract is known simply as the urothelium, this epithelium can be divided into at least three lineages of renal pelvis/ureter, bladder/trigone, and proximal urethra based on their embryonic origin, uroplakin content, keratin expression pattern, in vitro growth potential, and propensity to keratinize during vitamin A deficiency. Moreover, these cells remain phenotypically distinct even after they have been serially passaged under identical culture conditions, thus ruling out local mesenchymal influence as the sole cause of their in vivo differences. During vitamin A deficiency, mouse urothelium form multiple keratinized foci in proximal urethra probably originating from scattered K14-positive basal cells, and the keratinized epithelium expands horizontally to replace the surrounding normal urothelium. These data suggest that the urothelium consists of multiple cell lineages, that trigone urothelium is closely related to the urothelium covering the rest of the bladder, and that lineage heterogeneity coupled with cell migration/replacement form the cellular basis for urothelial squamous metaplasia

Urothelial umbrella cells are characterized by apical, rigid membrane plaques, which contain four major uroplakin proteins (UP Ia, Ib, II and III) forming UPIa/UPII and UPIb/UPIII pairs. These integral membrane proteins are thought to play an important role in maintaining the physical integrity and the permeability barrier function of the urothelium. We asked whether the four uroplakins always coexpress in the entire human lower urinary tract. We stained immunohistochemically (ABC-peroxidase method) paraffin sections of normal human ureter (n = 18) and urinary bladder (n = 10) using rabbit antibodies against UPIa, UPIb, UPII and UPIII; a recently raised mouse monoclonal antibody (MAb), AU1, and two new MAbs, AU2 and AU3, all against UPIII; and mouse MAbs against umbrella cell-associated cytokeratins CK18 and CK20. Immunoblotting showed that AU1, AU2 and AU3 antibodies all recognized the N-terminal extracellular domain of bovine UPIII. By immunohistochemistry, we found that in 15/18 cases of human ureter, but in only 2/10 cases of bladder, groups of normal-looking, CK18-positive umbrella cells lacked both UPIII and UPIb immunostaining. The UPIb/UPIII-negative cells showed either normal or reduced amounts of UPIa and UPII staining. These data were confirmed by double immunofluorescence microscopy. The distribution of the UPIb/UPIII-negative umbrella cells was not correlated with localized urothelial proliferation (Ki-67 staining) or with the distribution pattern of CK20. Similar heterogeneities were observed in bovine but not in mouse ureter. We provide the first evidence that urothelial umbrella cells are heterogeneous as some normal-looking umbrella cells can possess only one, instead of two, uroplakin pairs. This heterogeneity seems more prominent in the urothelium of human ureter than that of bladder. This finding may indicate that ureter urothelium is intrinsically different from bladder urothelium. Alternatively, a single lineage of urothelium may exhibit different phenotypes resulting from extrinsic modulations due to distinct mesenchymal influence and different degrees of pressure and stretch in bladder versus ureter. Additional studies are needed to distinguish these two possibilities and to elucidate the physiological and pathological significance of the observed urothelial and uroplakin heterogeneity

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

Our purpose was to determine the clinical relevance of the detection of circulating tumor cells (CTCs) expressing urothelial and epithelial markers in bladder cancer patients. Sixty-two patients who presented to Memorial Sloan-Kettering Cancer Center between July 2000 and September 2001 were studied. Peripheral blood was tested by nested RT-PCR assay for uroplakins (UPs) Ia, Ib, II and III as well as for epidermal growth factor receptor (EGFR). We determined the sensitivity and specificity of each individual marker and the combinations of UPIa/UPII and UPIb/UPIII. The latter strategy was based on our data, which showed that UPIa and UPIb form heterodimers with UPII and UPIII, respectively. Forty patients had clinically advanced bladder cancer and 22 had no evidence of disease at the time of assay. Eight of the 22 patients recurred during the follow-up period. All 8 patients were positive at presentation for UPIa/UPII. The combination of UPIa/UPII provided the best sensitivity (75%) of detecting CTCs, with a specificity of 50%. The combination of UPIb/UPIII was the most specific (79%) but had modest sensitivity (31%). Detection of EGFR-positive cells alone and in combination with UPs was inferior to that for UPIa/UPII. Combinations of urothelial markers are superior to single urothelial or epithelial markers in detecting CTCs in bladder cancer patients. Further efforts are under way to confirm the potential predictive value of these markers in a prospectively designed study of a larger cohort of patients.

Lack of major involvement of human uroplakin genes in vesicoureteral reflux: Implications for disease heterogeneity. Background. Primary vesicoureteral reflux (VUR) is a hereditary disorder characterized by the retrograde flow of urine into the ureters and kidneys. It affects about 1% of the young children and is thus one of the most common hereditary diseases. Its associated nephropathy is an important cause of end-stage renal failure in children and adults. Recent studies indicate that genetic ablation of mouse uroplakin (UP) III gene, which encodes a 47 kD urothelial-specific integral membrane protein forming urothelial plaques, causes VUR and hydronephrosis. Methods. To begin to determine whether mutations in UP genes might play a role in human VUR, we genotyped all four UP genes in 76 patients with radiologically proven primary VUR by polymerase chain reaction (PCR) amplification and sequencing of all their exons plus 50 to 150 bp of flanking intronic sequences. Results. Eighteen single nucleotide polymorphisms (SNPs) were identified, seven of which were missense, with no truncation or frame shift mutations. Since healthy relatives of the VUR probands are not reliable negative controls for VUR, we used a population of 90 race-matched, healthy individuals, unrelated to the VUR patients, as controls to perform an association study. Most of the SNPs were not found to be significantly associated with VUR. However, SNP1 of UP Ia gene affecting a C to T conversion and an Ala7Val change, and SNP7 of UP III affecting a C to G conversion and a Pro154Ala change, were marginally associated with VUR (both P= 0.08). Studies of additional cases yielded a second set of data that, in combination with the first set, confirmed a weak association of UP III SNP7 in VUR (P= 0.036 adjusted for both subsets of cases vs. controls). Conclusion. Such a weak association and the lack of families with simple dominant Mendelian inheritance suggest that missense changes of uroplakin genes cannot play a dominant role in causing VUR in humans, although they may be weak risk factors contributing to a complex polygenic disease. The fact that no truncation or frame shift mutations have been found in any of the VUR patients, coupled with our recent finding that some breeding pairs of UP III knockout mice yield litters that show not only VUR, but also severe hydronephrosis and neonatal death, raises the possibility that major uroplakin mutations could be embryonically or postnatally lethal in humans

The terminally differentiated umbrella cells of bladder epithelium contain unique cytoplasmic organelles, the fusiform vesicles, which deliver preassembled crystalline arrays of uroplakin proteins to the apical cell surface of urothelial umbrella cells. We have investigated the possible role of Rab proteins in this delivery process, and found Rab27b to be expressed at an extraordinary high level (0.1% of total protein) in urothelium, whereas Rab27b levels were greatly reduced (to <5% of normal urothelium) in cultured urothelial cells, which synthesized only small amounts of uroplakins and failed to form fusiform vesicles. Immuno-electron microscopy showed that Rab27b was associated with the cytoplasmic face of the fusiform vesicles, but not with that of the apical plasma membrane. The association of Rab27b with fusiform vesicles and its differentiation-dependent expression suggest that this Rab protein plays a role in regulating the delivery of fusiform vesicles to the apical plasma membrane of umbrella cells

Human tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz-type serine proteinase inhibitor that is secreted by all cells of the vasculature, and presumably plays a role in the regulation of plasmin-mediated matrix remodeling. In this report, we describe the cloning and expression of a full-length cDNA for bovine TFPI-2 that exhibits 72% sequence identity with that of human TFPI-2. Following a 22 residue signal peptide, the mature protein contains 212 amino acids with 18 cysteines, three putative N-glycosylation sites, and one putative O-glycosylation site. The deduced sequence of mature bovine TFPI-2 revealed a short acidic amino-terminal region, three tandem Kunitz-type domains, and a carboxy-terminal tail highly enriched in basic amino acids. Recombinant bovine TFPI-2 was expressed in HEK 293 cells and resolved into two isoforms, designated as alpha-TFPI-2 (M(r) 33 kDa) and beta-TFPI-2 (M(r) 31 kDa), which presumably represent differentially glycosylated forms of the inhibitor. Similar to human TFPI-2, both bovine TFPI-2 isoforms exhibited strong inhibitory activity towards trypsin and plasmin, and weak inhibitory activity towards the factor VIIa-tissue factor complex.

Although water, small nonelectrolytes, and gases are freely permeable through most biological membranes, apical membranes of certain barrier epithelia exhibit extremely low permeabilities to these substances. The role of integral membrane proteins in this barrier function has been unclear. To study this problem, we have ablated the mouse gene encoding uroplakin III (UPIII), one of the major protein subunits in urothelial apical membranes, and measured the permeabilities of these membranes. Ablation of the UPIII gene greatly diminishes the amounts of uroplakins on the apical urothelial membrane (Hu P, Deng FM, Liang FX, Hu CM, Auerbach AB, Shapiro E, Wu XR, Kachar B, and Sun TT. J Cell Biol 151: 961-972, 2000). Our results indicate that normal mouse urothelium exhibits high transepithelial resistance and low urea and water permeabilities. The UPIII-deficient urothelium exhibits a normal transepithelial resistance (normal 2,024 +/- 122, knockout 2,322 +/- 114 Omega. cm(2); P > 0.5). However, the UPIII-deficient apical membrane has a significantly elevated water permeability (normal 0.91 +/- 0.06, knockout 1.83 +/- 0.14 cm/s x 10(-5); P < 0.05). The urea permeability of the UPIII-deficient membrane also increased, although to a lesser extent (normal 2.22 +/- 0.24, knockout 2.93 +/- 0.31 cm/s x 10(-6); P = 0.12). These results indicate that reduced targeting of uroplakins to the apical membrane does not significantly alter the tight junctional barrier but does double the water permeability. We provide the first demonstration that integral membrane proteins contribute to the apical membrane permeability barrier function of urothelium

Urothelial plaques consist of four major uroplakins (Ia, Ib, II, and III) that form two-dimensional crystals covering the apical surface of urothelium, and provide unique opportunities for studying membrane protein assembly. Here, we describe a novel 35-kD urothelial plaque-associated glycoprotein that is closely related to uroplakin III: they have a similar overall type 1 transmembrane topology; their amino acid sequences are 34% identical; they share an extracellular juxtamembrane stretch of 19 amino acids; their exit from the ER requires their forming a heterodimer with uroplakin Ib, but not with any other uroplakins; and UPIII-knockout leads to p35 up-regulation, possibly as a compensatory mechanism. Interestingly, p35 contains a stretch of 80 amino acid residues homologous to a hypothetical human DNA mismatch repair enzyme-related protein. Human p35 gene is mapped to chromosome 7q11.23 near the telomeric duplicated region of Williams-Beuren syndrome, a developmental disorder affecting multiple organs including the urinary tract. These results indicate that p35 (uroplakin IIIb) is a urothelial differentiation product structurally and functionally related to uroplakin III, and that p35-UPIb interaction in the ER is an important early step in urothelial plaque assembly