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1.  Alterations in Bladder Function Associated With Urothelial Defects in Uroplakin II and IIIa Knockout Mice 
Neurourology and urodynamics  2009;28(8):1028-1033.
The effects of deleting genes encoding uroplakins II (UPII) and III (UPIIIa) on mouse bladder physiology/ dysfunction were studied in male and female wild type and knockout (KO) mice.
UPII, UPIIIa, and WT mice were catheterized using previously described techniques. Continuous cystometry was conducted in conscious, freely moving animals. Bladder strips were harvested after animal sacrifice and pharmacological studies and EFS were conducted in an organ chamber. Histological studies were also carried on with H&E staining to identify differences among the three mouse types.
These studies have revealed numerous alterations, some of which were apparently gender-specific. Nonvoiding contractions were common in both UPII and UPIIIa KO mice, although more severe in the former. In particular, the increased bladder capacity, micturition pressure and demonstrable nonvoiding contractions observed in the male UPII KO’s, were reminiscent of an obstruction-like syndrome accompanied by evidence of emerging bladder decompensation, as reflected by an increased residual volume. Pharmacological studies revealed a modest, gender-specific reduction in sensitivity of isolated detrusor strips from UPII KO female mice to carbachol-induced contractions. A similar reduction was observed in UPIIIa KO female mice. Histological investigation showed urothelial hyperplasia in both UPII KO and UPIIIa KO mice, although again, apparently more severe in the former.
These results confirm and extend previous work to indicate that urothelial defects due to uroplakin deficiency are associated with significant alterations in bladder function and further highlight the importance of the urothelium to bladder physiology/dysfunction.
PMCID: PMC4048927  PMID: 19267388
BOO; LUTS; urodynamics
2.  Generation of divergent uroplakin tetraspanins and their partners during vertebrate evolution: identification of novel uroplakins 
The recent availability of sequenced genomes from a broad array of chordates (cephalochordates, urochordates and vertebrates) has allowed us to systematically analyze the evolution of uroplakins: tetraspanins (UPK1a and UPK1b families) and their respective partner proteins (UPK2 and UPK3 families).
We report here: (1) the origin of uroplakins in the common ancestor of vertebrates, (2) the appearance of several residues that have statistically significantly positive dN/dS ratios in the duplicated paralogs of uroplakin genes, and (3) the existence of strong coevolutionary relationships between UPK1a/1b tetraspanins and their respective UPK2/UPK3-related partner proteins. Moreover, we report the existence of three new UPK2/3 family members we named UPK2b, 3c and 3d, which will help clarify the evolutionary relationships between fish, amphibian and mammalian uroplakins that may perform divergent functions specific to these different and physiologically distinct groups of vertebrates.
Since our analyses cover species of all major chordate groups this work provides an extremely clear overall picture of how the uroplakin families and their partner proteins have evolved in parallel. We also highlight several novel features of uroplakin evolution including the appearance of UPK2b and 3d in fish and UPK3c in the common ancestor of reptiles and mammals. Additional studies of these novel uroplakins should lead to new insights into uroplakin structure and function.
PMCID: PMC3922775  PMID: 24450554
3.  Uroplakins in Urothelial Biology, Function and Disease 
Kidney international  2009;75(11):1153-1165.
Urothelium covers the inner surfaces of the renal pelvis, ureter, bladder and prostatic urethra. Although morphologically indistinguishable, the urothelia in these anatomic locations differ in their embryonic origin and lineages of cellular differentiation, as reflected in their different uroplakin content, expandability during micturition and susceptibility to chemical carcinogens. Previously thought to be an inert tissue forming a passive barrier between the urine and blood, urothelia have recently been shown to have a secretory activity that actively modifies the urine composition. Urothelial cells express a number of ion channels, receptors and ligands, enabling them to receive and send signals and communicate with adjoining cells and their broader environment. The urothelial surface bears specific receptors that not only allow uropathogenic E. coli to attach to and invade into the bladder mucosa, but also provide a route by which the bacteria ascend via the ureters to the kidney to cause pyelonephritis. Genetic ablation of one or more uroplakin genes in mice causes severe retrograde vesicoureteral reflux, hydronephrosis and renal failure, conditions that mirror certain human congenital diseases. Clearly, abnormalities of the lower urinary tract can impact on the upper tract, and vice versa, through the urothelial connection. In this review, we highlight recent advances in the field of urothelial biology by focusing on the uroplakins, a group of urothelium-specific and differentiation-dependent integral membrane proteins. We discuss these proteins’ biochemistry, structure, assembly, intracellular trafficking and their emerging roles in urothelial biology, function and pathological processes. We also call attention to important areas where greater investigative efforts are warranted.
PMCID: PMC3717210  PMID: 19340092
uroplakin; urothelium; differentiation; tetraspanin; urinary tract infection; tumorigenesis; permeability barrier; vesicular transport
4.  MAL facilitates the incorporation of exocytic uroplakin-delivering vesicles into the apical membrane of urothelial umbrella cells 
Molecular Biology of the Cell  2012;23(7):1354-1366.
MAL, suggested to play a key role in the apical sorting of membrane proteins, is not involved in the apical sorting of uroplakins. Instead, it plays an important role in facilitating the incorporation of the uroplakin-delivering exocytic vesicles into the apical surface of terminally differentiated urothelial umbrella cells.
The apical surface of mammalian bladder urothelium is covered by large (500–1000 nm) two-dimensional (2D) crystals of hexagonally packed 16-nm uroplakin particles (urothelial plaques), which play a role in permeability barrier function and uropathogenic bacterial binding. How the uroplakin proteins are delivered to the luminal surface is unknown. We show here that myelin-and-lymphocyte protein (MAL), a 17-kDa tetraspan protein suggested to be important for the apical sorting of membrane proteins, is coexpressed with uroplakins in differentiated urothelial cell layers. MAL depletion in Madin–Darby canine kidney cells did not affect, however, the apical sorting of uroplakins, but it decreased the rate by which uroplakins were inserted into the apical surface. Moreover, MAL knockout in vivo led to the accumulation of fusiform vesicles in mouse urothelial superficial umbrella cells, whereas MAL transgenic overexpression in vivo led to enhanced exocytosis and compensatory endocytosis, resulting in the accumulation of the uroplakin-degrading multivesicular bodies. Finally, although MAL and uroplakins cofloat in detergent-resistant raft fractions, they are associated with distinct plaque and hinge membrane subdomains, respectively. These data suggest a model in which 1) MAL does not play a role in the apical sorting of uroplakins; 2) the propensity of uroplakins to polymerize forming 16-nm particles and later large 2D crystals that behave as detergent-resistant (giant) rafts may drive their apical targeting; 3) the exclusion of MAL from the expanding 2D crystals of uroplakins explains the selective association of MAL with the hinge areas in the uroplakin-delivering fusiform vesicles, as well as at the apical surface; and 4) the hinge-associated MAL may play a role in facilitating the incorporation of the exocytic uroplakin vesicles into the corresponding hinge areas of the urothelial apical surface.
PMCID: PMC3315800  PMID: 22323295
5.  The histone deacetylase inhibitor belinostat (PXD101) suppresses bladder cancer cell growth in vitro and in vivo 
Treatment options for patients with recurrent superficial bladder cancer are limited, necessitating aggressive exploration of new treatment strategies that effectively prevent recurrence and progression to invasive disease. We assessed the effects of belinostat (previously PXD101), a novel histone deacetylase inhibitor, on a panel of human bladder cancer cell lines representing superficial and invasive disease, and on a transgenic mouse model of superficial bladder cancer.
Growth inhibition and cell cycle distribution effect of belinostat on 5637, T24, J82, and RT4 urothelial lines were assessed. Ha-ras transgenic mice with established superficial bladder cancer were randomized to receive either belinostat or vehicle alone, and assessed for bladder weight, hematuria, gene expression profiling, and immunohistochemistry (IHC).
Belinostat had a significant linear dose-dependent growth inhibition on all cell lines (IC50 range of 1.0–10.0 μM). The 5637 cell line, which was derived from a superficial papillary tumor, was the most sensitive to treatment. Belinostat (100 mg/kg, intraperitoneal, 5 days each week for 3 weeks) treated mice had less bladder weight (p < 0.05), and no hematuria compared with 6/10 control mice that developed at least one episode. IHC of bladder tumors showed less cell proliferation and a higher expression of p21WAF1 in the belinostat-treated mice. Gene expression profile analysis revealed 56 genes significantly different in the treated group; these included the upregulation of p21WAF1, induction of core histone deacetylase (HDAC), and cell communication genes.
Our data demonstrate that belinostat inhibits bladder cancer and supports the clinical evaluation of belinostat for the treatment of patients with superficial bladder cancer.
PMCID: PMC2100044  PMID: 17935615
6.  Cellular basis of urothelial squamous metaplasia 
The Journal of Cell Biology  2005;171(5):835-844.
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.
PMCID: PMC2171294  PMID: 16330712
7.  Assembly of Urothelial Plaques: Tetraspanin Function in Membrane Protein Trafficking 
Molecular Biology of the Cell  2005;16(9):3937-3950.
The apical surface of mammalian urothelium is covered by 16-nm protein particles packed hexagonally to form 2D crystals of asymmetric unit membranes (AUM) that contribute to the remarkable permeability barrier function of the urinary bladder. We have shown previously that bovine AUMs contain four major integral membrane proteins, i.e., uroplakins Ia, Ib, II, and IIIa, and that UPIa and Ib (both tetraspanins) form heterodimers with UPII and IIIa, respectively. Using a panel of antibodies recognizing different conformational states of uroplakins, we demonstrate that the UPIa-dependent, furin-mediated cleavage of the prosequence of UPII leads to global conformational changes in mature UPII and that UPIb also induces conformational changes in its partner UPIIIa. We further demonstrate that tetraspanins CD9, CD81, and CD82 can stabilize their partner protein CD4. These results indicate that tetraspanin uroplakins, and some other tetraspanin proteins, can induce conformational changes leading to the ER-exit, stabilization, and cell surface expression of their associated, single-transmembrane-domained partner proteins and thus can function as “maturation-facilitators.” We propose a model of AUM assembly in which conformational changes in integral membrane proteins induced by uroplakin interactions, differentiation-dependent glycosylation, and the removal of the prosequence of UPII play roles in regulating the assembly of uroplakins to form AUM.
PMCID: PMC1196309  PMID: 15958488
8.  Roles of uroplakins in plaque formation, umbrella cell enlargement, and urinary tract diseases 
The Journal of Cell Biology  2004;167(6):1195-1204.
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.
PMCID: PMC2172608  PMID: 15611339
9.  Uroplakin IIIb, a urothelial differentiation marker, dimerizes with uroplakin Ib as an early step of urothelial plaque assembly 
The Journal of Cell Biology  2002;159(4):685-694.
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.
PMCID: PMC2173100  PMID: 12446744
bladder; urothelium; tetraspanin; membrane; DNA mismatch repair enzyme
10.  Retinoid-signaling in progenitors controls specification and regeneration of the urothelium 
Developmental cell  2013;26(5):469-482.
The urothelium is a stratified epithelium that prevents exchange of water and toxic substances between the urinary tract and blood. It is composed of Keratin-5-expressing-basal-cells (K5-BCs), intermediate cells and superficial cells specialized for synthesis and transport of uroplakins that assemble into the apical barrier. K5-BCs are considered to be a progenitor cell type in the urothelium and other stratified epithelia. Fate mapping studies however, reveal that P-cells, a transient population, are urothelial progenitors in the embryo, intermediate cells are superficial cell progenitors in the adult regenerating urothelium, and K5-BCs are a distinct lineage. Our studies indicate that retinoids, potent regulators of ES cells and other progenitors, are also required in P-cells and intermediate cells for their specification. These observations have important implications for tissue engineering and repair, and ultimately, may lead to treatments that prevent loss of the urothelial barrier, a major cause of voiding dysfunction and bladder pain syndrome.
PMCID: PMC4024836  PMID: 23993789
11.  SNX31: A Novel Sorting Nexin Associated with the Uroplakin-Degrading Multivesicular Bodies in Terminally Differentiated Urothelial Cells 
PLoS ONE  2014;9(6):e99644.
Uroplakins (UP), a group of integral membrane proteins, are major urothelial differentiation products that form 2D crystals of 16-nm particles (urothelial plaques) covering the apical surface of mammalian bladder urothelium. They contribute to the urothelial barrier function and, one of them, UPIa, serves as the receptor for uropathogenic Escherichia coli. It is therefore important to understand the mechanism by which these surface-associated uroplakins are degraded. While it is known that endocytosed uroplakin plaques are targeted to and line the multivesicular bodies (MVBs), it is unclear how these rigid-looking plaques can go to the highly curved membranes of intraluminal vesicles (ILVs). From a cDNA subtraction library, we identified a highly urothelium-specific sorting nexin, SNX31. SNX31 is expressed, like uroplakins, in terminally differentiated urothelial umbrella cells where it is predominantly associated with MVBs. Apical membrane proteins including uroplakins that are surface biotin-tagged are endocytosed and targeted to the SNX31-positive MVBs. EM localization demonstrated that SNX31 and uroplakins are both associated not only with the limiting membranes of MVBs containing uroplakin plaques, but also with ILVs. SNX31 can bind, on one hand, the PtdIns3P-enriched lipids via its N-terminal PX-domain, and, on the other hand, it binds uroplakins as demonstrated by co-immunoprecipitation and proximity ligation assay, and by its reduced membrane association in uroplakin II-deficient urothelium. The fact that in urothelial umbrella cells MVBs are the only major intracellular organelles enriched in both PtdIns3P and uroplakins may explain SNX31's MVB-specificity in these cells. However, in MDCK and other cultured cells transfected SNX31 can bind to early endosomes possibly via lipids. These data support a model in which SNX31 mediates the endocytic degradation of uroplakins by disassembling/collapsing the MVB-associated uroplakin plaques, thus enabling the uroplakin-containing (but ‘softened’) membranes to bud and form the ILVs for lysosomal degradation and/or exosome formation.
PMCID: PMC4051706  PMID: 24914955
12.  Assembly of a membrane receptor complex: roles of the uroplakin II prosequence in regulating uroplakin bacterial receptor oligomerization 
The Biochemical journal  2008;414(2):195-203.
The apical surface of the mammalian urothelium is almost completely covered by two-dimensional protein crystals (known as urothelial plaques) of hexagonally packed 16 nm particles consisting of two UP (uroplakin) heterodimers, i.e. UPs Ia/II and Ib/III pairs. UPs are functionally important as they contribute to the urothelial permeability barrier function, and UPIa may serve as the receptor for the uropathogenic Escherichia coli that causes over 90% of urinary tract infections. We study here how the UP proteins are assembled and targeted to the urothelial apical surface, paying special attention to the roles of the prosequence of UPII in UP oligomerization. We show that (i) the formation of the UPIa/UPII heterodimer, necessary for ER (endoplasmic reticulum) exit, requires disulfide formation in the prosequence domain of proUPII (the immature form of UPII still containing its prosequence); (ii) differentiation-dependent N-glycosylation of the prosequence leads to UP stabilization; (iii) a failure to form tetramers in cultured urothelial cells, in part due to altered glycosylation of the prosequence, may block two-dimensional crystal formation; and (iv) the prosequence of UPII remains attached to the mature protein complex on the urothelial apical surface even after it has been cleaved by the trans-Golgi-network-associated furin. Our results indicate that proper secondary modifications of the prosequence of UPII play important roles in regulating the oligomerization and function of the UP protein complex.
PMCID: PMC4048928  PMID: 18481938
disulfide formation; glycosylation; integral membrane protein; prosequence; protein assembly; uroplakin
13.  Uroplakins Do Not Restrict CO2 Transport through Urothelium* 
The Journal of Biological Chemistry  2012;287(14):11011-11017.
Background: The tightness of various membrane barriers to CO2 is of unknown molecular origin.
Results: The bladder tissue lacks carbonic anhydrase. The resulting low intra-epithelial CO2 concentration gives rise to the apparent CO2 impermeability.
Conclusion: Uroplakins do not act to decrease transepithelial CO2 flux.
Significance: Enzymatic regulation of CO2 abundance rules out that aquaporins significantly contribute to the maintenance of acid base homeostasis.
Lipid bilayers and biological membranes are freely permeable to CO2, and yet partial CO2 pressure in the urine is 3–4-fold higher than in blood. We hypothesized that the responsible permeability barrier to CO2 resides in the umbrella cell apical membrane of the bladder with its dense array of uroplakin complexes. We found that disrupting the uroplakin layer of the urothelium resulted in water and urea permeabilities (P) that were 7- to 8-fold higher than in wild type mice with intact urothelium. However, these interventions had no impact on bladder PCO2 (∼1.6 × 10−4 cm/s). To test whether the observed permeability barrier to CO2 was due to an unstirred layer effect or due to kinetics of CO2 hydration, we first measured the carbonic anhydrase (CA) activity of the bladder epithelium. Finding none, we reduced the experimental system to an epithelial monolayer, Madin-Darby canine kidney cells. With CA present inside and outside the cells, we showed that PCO2 was unstirred layer limited (∼7 × 10−3 cm/s). However, in the total absence of CA activity PCO2 decreased 14-fold (∼ 5.1 × 10−4 cm/s), indicating that now CO2 transport is limited by the kinetics of CO2 hydration. Expression of aquaporin-1 did not alter PCO2 (and thus the limiting transport step), which confirmed the conclusion that in the urinary bladder, low PCO2 is due to the lack of CA. The observed dependence of PCO2 on CA activity suggests that the tightness of biological membranes to CO2 may uniquely be regulated via CA expression.
PMCID: PMC3322830  PMID: 22315218
Aquaporin; Carbon Dioxide; Membrane Biophysics; Permeability; Physiology; Scanning Electrochemical Microscopy; Unstirred Layers
14.  Deficiency of pRb Family Proteins and p53 in Invasive Urothelial Tumorigenesis 
Cancer research  2009;69(24):9413-9421.
Defects in pRb tumor suppressor pathway occur in ~50% of the deadly muscle-invasive urothelial carcinomas in humans and urothelial carcinoma is the most prevalent epithelial cancer in long-term survivors of hereditary retinoblastomas caused by loss-of-function RB1 mutations. Here we show that conditional inactivation of both RB1 alleles in mouse urothelium failed to accelerate urothelial proliferation. Instead, it profoundly activated the p53 pathway, leading to extensive apoptosis, and selectively induced pRb family member p107. Thus, pRb loss triggered multiple failsafe mechanisms whereby urothelial cells evade tumorigenesis. Additional loss of p53 in pRb-deficient urothelial cells removed these p53-dependent tumor barriers, resulting in late-onset hyperplasia, umbrella cell nuclear atypia and rare-occurring low-grade, superficial papillary bladder tumors, without eliciting invasive carcinomas. Importantly, mice deficient in both pRb and p53, but not those deficient in either protein alone, were highly susceptible to sub-threshold carcinogen exposure and developed invasive urothelial carcinomas that strongly resembled the human counterparts. The invasive lesions had a marked reduction of p107, but not p130, of the pRb family. Our data provide compelling evidence indicating that urothelium, one of the slowest cycling epithelia, is remarkably resistant to transformation by pRb or p53 deficiency; that concurrent loss of these two tumor suppressors is necessary but insufficient to initiate urothelial tumorigenesis along the invasive pathway; that p107 may play a critical role in suppressing invasive urothelial tumor formation; and that replacing/restoring the function of pRb, p107 or p53 could be explored as a potential therapeutic strategy to block urothelial tumor progression.
PMCID: PMC2794922  PMID: 19951992
bladder cancer; tumor suppressor gene; conditional knockout; tumor progression; uroplakin
15.  Uropathogenic E. coli adhesin-induced host cell receptor conformational changes: implications in transmembrane signaling transduction 
Journal of molecular biology  2009;392(2):352-361.
Urinary tract infection (UTI) is the second most common infectious disease, and is caused predominantly by type 1-fimbriated uropathogenic E. coli (UPEC). UPEC initiates infection by attaching to uroplakin Ia, its urothelial surface receptor, via the FimH adhesins capping the distal end of its fimbriae. Uroplakin Ia, together with uroplakins Ib, II and IIIa, forms a 16 nm receptor complex that is assembled into hexagonally packed two-dimensional crystals (urothelial plaques) covering >90% of the urothelial apical surface. Recent studies indicate that FimH is the invasin of UPEC as its attachment to the urothelial surface can induce cellular signaling events including calcium elevation and the phosphorylation of the uroplakin IIIa cytoplasmic tail, leading to cytoskeletal rearrangements and bacterial invasion. However, it remains unknown how the binding of FimH to the uroplakin receptor triggers a signal that can be transmitted through the highly impermeable urothelial apical membrane. We show here by cryo-electron microscopy that FimH-binding to the extracellular domain of UPIa induces global conformational changes in the entire uroplakin receptor complex, including a coordinated movement of the tightly bundled transmembrane helices. This movement of the transmembrane helix bundles can cause a corresponding lateral translocation of the uroplakin cytoplasmic tails, which can be sufficient to trigger downstream signaling events. Our results suggest a novel pathogen-induced transmembrane signal transduction mechanism that plays a key role in the initial stages of UPEC invasion and receptor-mediated bacterial invasion in general.
PMCID: PMC2755582  PMID: 19577575
bacterial adhesin; tetraspanins; transmembrane signaling; urinary tract infections; uroplakins
16.  Persistent Uroplakin Expression in Advanced Urothelial Carcinomas: Implications in Urothelial Tumor Progression and Clinical Outcome 
Human pathology  2007;38(11):1703-1713.
As the terminal differentiation products of human urothelium, uroplakins (UPs) would be expected to diminish during urothelial tumorigenesis. Surprisingly, recent studies found UPs to be retained even by well-advanced urothelial carcinomas, suggesting that the loss of UPs does not strictly parallel urothelial transformation. Little is known, however, about whether the status of UPs is associated with a particular pathological parameter, tumor’s biological behavior or patient outcome. Here we assessed UP expression by immunohistochemistry on tissue arrays from 285 patients with bladder urothelial carcinomas or non-tumor conditions. UPs were expressed in all 9 normal urothelial specimens, 63/74 (85%) patients with non-muscle-invasive urothelial carcinomas on transurethral resection, 104/202 (51.5%) patients who underwent radical cystectomy for advanced urothelial carcinomas, and 33/50 (66%) lymph node metastases. Normally associated with urothelial apical surface, UPs were localized aberrantly in tumors, including micro-luminal, basal-laminal, cytoplasmic or uniform patterns. In non-muscle-invasive diseases, there was no association between UP expression and disease recurrence, progression or mortality. In contrast, in invasive diseases, absent UP expression was significantly associated with advanced pathologic stage, lymph node metastases, disease recurrence and bladder cancer-specific mortality (p=0.042, p=0.035, p=0.023 and p=0.022, respectively) in univariate analyses. Furthermore, UP status was independent of key cell-cycle regulators, including p53, pRb, p27 and cyclin D1, thus excluding a functional link between these two groups of proteins. Our data demonstrate for the first time that persistent UP expression is associated with a favorable clinical outcome and that UPs may be used as adjunct markers for predicting the prognoses of patients with invasive and metastatic bladder carcinomas. Our results also suggest that UP-positive and –negative carcinomas have different clonal origins or may be derived from different cancer stem cells.
PMCID: PMC2778836  PMID: 17707461
bladder cancer; uroplakin; progression; prognosis; transitional cell carcinoma
17.  Bacteria-Induced Uroplakin Signaling Mediates Bladder Response to Infection 
PLoS Pathogens  2009;5(5):e1000415.
Urinary tract infections are the second most common infectious disease in humans and are predominantly caused by uropathogenic E. coli (UPEC). A majority of UPEC isolates express the type 1 pilus adhesin, FimH, and cell culture and murine studies demonstrate that FimH is involved in invasion and apoptosis of urothelial cells. FimH initiates bladder pathology by binding to the uroplakin receptor complex, but the subsequent events mediating pathogenesis have not been fully characterized. We report a hitherto undiscovered signaling role for the UPIIIa protein, the only major uroplakin with a potential cytoplasmic signaling domain, in bacterial invasion and apoptosis. In response to FimH adhesin binding, the UPIIIa cytoplasmic tail undergoes phosphorylation on a specific threonine residue by casein kinase II, followed by an elevation of intracellular calcium. Pharmacological inhibition of these signaling events abrogates bacterial invasion and urothelial apoptosis in vitro and in vivo. Our studies suggest that bacteria-induced UPIIIa signaling is a critical mediator of bladder responses to insult by uropathogenic E. coli.
Author Summary
Urinary tract infections (UTI) are the second most common infectious disease in humans and are predominantly caused by uropathogenic E. coli (UPEC). In vitro and in vivo studies have demonstrated that UPEC induce several responses in the bladder, including inflammation, rapid onset of bladder cell death, and bacterial invasion of bladder cells. This last event, invasion, is now also thought to underlie recurrent UTI. Although members of the highly-expressed “uroplakin” proteins serve as bladder receptors for UPEC binding, it was unclear how UPEC binding to uroplakin receptors caused signals within bladder cells that mediate rapid cell death and bacterial invasion. Here, we show that another uroplakin, uroplakin III, which is associated with the receptor transduces signals within the cell in response to UPEC binding. UPEC causes elevated calcium within bladder cells, and this elevation requires phosphorylation of uroplakin III by a specific kinase. Blocking these events blocks both bladder cell death and bacterial invasion of bladder cells by UPEC. Thus, uroplakin III is the mediator of key events in UTI pathogenesis.
PMCID: PMC2669708  PMID: 19412341
18.  Analysis of Electroblotted Proteins by Mass Spectrometry: Protein Identification after Western Blotting 
We describe a new approach for the identification and characterization by mass spectrometry of proteins that have been electroblotted onto nitrocellulose. Using this method (Blotting And Removal of Nitrocellulose, or BARN), proteins can be analyzed either as intact proteins for molecular weight determination or as peptides generated by on-membrane proteolysis. Acetone is used to dissolve the nitrocellulose and to precipitate the adsorbed proteins/peptides, thus removing the nitrocellulose which can interfere with mass spectrometry analysis. This method offers improved protein coverage, especially for membrane proteins such as uroplakins, since the extraction step after in-gel digestion is avoided. Moreover, removal of nitrocellulose from the sample solution allows sample analysis by both MALDI- and (LC) ESI-based mass spectrometers. Finally, we demonstrate the utility of BARN for the direct identification of soluble and membrane proteins after Western blotting, obtaining comparable or better results than with in-gel digestion.
PMCID: PMC2667373  PMID: 17938404
19.  Intron Evolution: Testing Hypotheses of Intron Evolution Using the Phylogenomics of Tetraspanins 
PLoS ONE  2009;4(3):e4680.
Although large scale informatics studies on introns can be useful in making broad inferences concerning patterns of intron gain and loss, more specific questions about intron evolution at a finer scale can be addressed using a gene family where structure and function are well known. Genome wide surveys of tetraspanins from a broad array of organisms with fully sequenced genomes are an excellent means to understand specifics of intron evolution. Our approach incorporated several new fully sequenced genomes that cover the major lineages of the animal kingdom as well as plants, protists and fungi. The analysis of exon/intron gene structure in such an evolutionary broad set of genomes allowed us to identify ancestral intron structure in tetraspanins throughout the eukaryotic tree of life.
Methodology/Principal Findings
We performed a phylogenomic analysis of the intron/exon structure of the tetraspanin protein family. In addition, to the already characterized tetraspanin introns numbered 1 through 6 found in animals, three additional ancient, phase 0 introns we call 4a, 4b and 4c were found. These three novel introns in combination with the ancestral introns 1 to 6, define three basic tetraspanin gene structures which have been conserved throughout the animal kingdom. Our phylogenomic approach also allows the estimation of the time at which the introns of the 33 human tetraspanin paralogs appeared, which in many cases coincides with the concomitant acquisition of new introns. On the other hand, we observed that new introns (introns other than 1–6, 4a, b and c) were not randomly inserted into the tetraspanin gene structure. The region of tetraspanin genes corresponding to the small extracellular loop (SEL) accounts for only 10.5% of the total sequence length but had 46% of the new animal intron insertions.
Our results indicate that tests of intron evolution are strengthened by the phylogenomic approach with specific gene families like tetraspanins. These tests add to our understanding of genomic innovation coupled to major evolutionary divergence events, functional constraints and the timing of the appearance of evolutionary novelty.
PMCID: PMC2650405  PMID: 19262691
20.  Compositional Differences between Infant and Adult Human Corneal Basement Membranes 
Adult human corneal epithelial basement membrane (EBM) and Descemet's membrane (DM) components exhibit heterogeneous distribution. The purpose of the study was to identify changes of these components during postnatal corneal development.
Thirty healthy adult corneas and 10 corneas from 12-day- to 3-year-old children were studied by immunofluorescence with antibodies against BM components.
Type IV collagen composition of infant corneal central EBM over Bowman's layer changed from α1-α2 to α3-α4 chains after 3 years of life; in the adult, α1-α2 chains were retained only in the limbal BM. Laminin α2 and β2 chains were present in the adult limbal BM where epithelial stem cells are located. By 3 years of age, β2 chain appeared in the limbal BM. In all corneas, limbal BM contained laminin γ3 chain. In the infant DM, type IV collagen α1-α6 chains, perlecan, nidogen-1, nidogen-2, and netrin-4 were found on both faces, but they remained only on the endothelial face of the adult DM. The stromal face of the infant but not the adult DM was positive for tenascin-C, fibrillin-1, SPARC, and laminin-332. Type VIII collagen shifted from the endothelial face of infant DM to its stromal face in the adult. Matrilin-4 largely disappeared after the age of 3 years.
The distribution of laminin γ3 chain, nidogen-2, netrin-4, matrilin-2, and matrilin-4 is described in the cornea for the first time. The observed differences between adult and infant corneal BMs may relate to changes in their mechanical strength, corneal cell adhesion and differentiation in the process of postnatal corneal maturation.
PMCID: PMC2151758  PMID: 17962449
21.  Use of Nitrocellulose Membranes for Protein Characterization by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry 
Analytical chemistry  2006;78(14):5102-5108.
We present an improved method for MALDI-MS analysis of proteins that have been electroblotted onto a nitrocellulose (NC) membrane. With this approach, electroblotted proteins can be analyzed directly for intact molecular weight determination or after on-membrane digestion by dissolution of the nitrocellulose in MALDI matrix solution containing 70% acetonitrile and 30% methanol. This solution helps maintain solubility of proteins and peptides while dissolving the NC membrane, which is dissolved by 100% acetone in other protocols. On-membrane tryptic digestion using this method requires half the time of in-gel digestion, and results in fewer missed cleavages and better protein coverage. For the membrane proteins studied, bovine uroplakins II and III, the protein coverage was almost twice that provided by conventional in-gel digestion, and the transmembrane domains of both uroplakins were detected only after on-membrane digestion. We also demonstrated the compatibility with MALDI-MS of a new dye, MemCode™, which is specifically designed for staining NC membrane-immobilized proteins and is faster and more sensitive than Ponceau-S. Our improved on-membrane digestion protocol greatly improves the study of soluble and, particularly strikingly, integral membrane proteins by mass spectrometry.
PMCID: PMC2538422  PMID: 16841935
22.  EEDA: A Protein Associated with an Early Stage of Stratified Epithelial Differentiation 
Journal of cellular physiology  2006;206(1):103-111.
Using suppressive subtractive hybridization, we have identified a novel gene, which we named EEDA (early epithelial differentiation- associated), which is uniquely associated with an early stage of stratified epithelial differentiation. In epidermis, esophageal epithelium, and tongue epithelium, EEDA mRNA and antigen was abundant in suprabasal cells, but was barely detectable in more differentiated cells. Consistent with the limbal location of corneal epithelial stem cells, EEDA was expressed in basal corneal epithelial cells that are out of the stem cell compartment, as well as the suprabasal corneal epithelial cells. The strongest EEDA expression occurred in suprabasal precortical cells of mouse, bovine and human anagen follicles. Developmental studies showed that the appearance of EEDA in embryonic mouse epidermis (E 15.5) coincided with morphological keratinization. Interestingly, EEDA expression is turned off when epithelia were perturbed by wounding and by cultivation under both low and high Ca2+ conditions. Our results indicate that EEDA is involved in the early stages of normal epithelial differentiation, and that EEDA is important for the “normal” differentiation pathway in a wide range of stratified epithelia.
PMCID: PMC1523255  PMID: 15920738
epidermis; corneal epithelium; hair follicle; wound repair
23.  Hyperactivation of Ha-ras oncogene, but not Ink4a/Arf deficiency, triggers bladder tumorigenesis 
Journal of Clinical Investigation  2007;117(2):314-325.
Although ras is a potent mitogenic oncogene, its tumorigenicity depends on cellular context and cooperative events. Here we show that low-level expression of a constitutively active Ha-ras in mouse urothelium induces simple urothelial hyperplasia that is resistant to progression to full-fledged bladder tumors even in the absence of Ink4a/Arf. In stark contrast, doubling of the gene dosage of the activated Ha-ras triggered early-onset, rapidly growing, and 100% penetrant tumors throughout the urinary tract. Tumor initiation required superseding a rate-limiting step between simple and nodular hyperplasia, the latter of which is marked by the emergence of mesenchymal components and the coactivation of AKT and STAT pathways as well as PTEN inactivation. These results indicate that overactivation of Ha-ras is both necessary and sufficient to induce bladder tumors along a low-grade, noninvasive papillary pathway, and they shed light on the recent findings that ras activation, via point mutation, overexpression, or intensified signaling from FGF receptor 3, occurs in 70%–90% of these tumors in humans. Our results highlight the critical importance of the dosage/strength of Ha-ras activation in dictating its tumorigenicity — a mechanism of oncogene activation not fully appreciated to date. Finally, our results have clinical implications, as inhibiting ras and/or its downstream effectors, such as AKT and STAT3/5, could provide alternative means to treat low-grade, superficial papillary bladder tumors, the most common tumor in the urinary system.
PMCID: PMC1770948  PMID: 17256055
24.  Structural basis for tetraspanin functions as revealed by the cryo-EM structure of uroplakin complexes at 6-Å resolution 
The Journal of Cell Biology  2006;173(6):975-983.
Tetraspanin uroplakins (UPs) Ia and Ib, together with their single-spanning transmembrane protein partners UP II and IIIa, form a unique crystalline 2D array of 16-nm particles covering almost the entire urothelial surface. A 6 Å–resolution cryo-EM structure of the UP particle revealed that the UP tetraspanins have a rod-shaped structure consisting of four closely packed transmembrane helices that extend into the extracellular loops, capped by a disulfide-stabilized head domain. The UP tetraspanins form the primary complexes with their partners through tight interactions of the transmembrane domains as well as the extracellular domains, so that the head domains of their tall partners can bridge each other at the top of the heterotetramer. The secondary interactions between the primary complexes and the tertiary interaction between the 16-nm particles contribute to the formation of the UP tetraspanin network. The rod-shaped tetraspanin structure allows it to serve as stable pilings in the lipid sea, ideal for docking partner proteins to form structural/signaling networks.
PMCID: PMC2063921  PMID: 16785325
25.  Specific Heterodimer Formation Is a Prerequisite for Uroplakins to Exit from the Endoplasmic Reticulum 
Molecular Biology of the Cell  2002;13(12):4221-4230.
Much of the lower urinary tract, including the bladder, is lined by a stratified urothelium forming a highly differentiated, superficial umbrella cell layer. The apical plasma membrane as well as abundant cytoplasmic fusiform vesicles of the umbrella cells is covered by two-dimensional crystals that are formed by four membrane proteins named uroplakins (UPs) Ia, Ib, II, and III. UPs are synthesized on membrane-bound polysomes, and after several co- and posttranslational modifications they assemble into planar crystals in a post-Golgi vesicular compartment. Distension of the bladder may cause fusiform vesicles to fuse with the apical plasma membrane. We have investigated the early stages of uroplakin assembly by expressing the four uroplakins in 293T cells. Transfection experiments showed that, when expressed individually, only UPIb can exit from the endoplasmic reticulum (ER) and move to the plasma membrane, whereas UPII and UPIII reach the plasma membrane only when they form heterodimeric complexes with UPIa and UPIb, respectively. Heterodimer formation in the ER was confirmed by pulse-chase experiment followed by coimmunoprecipitation. Our results indicate that the initial building blocks for the assembly of crystalline uroplakin plaques are heterodimeric uroplakin complexes that form in the ER.
PMCID: PMC138628  PMID: 12475947

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