Uroplakins Research Articles

Uroplakin 1a Interacts with Regucalcin and Proteasome Subunit Beta 1.

Uroplakins (UPKs) are specialized proteins that plan an important role in protecting the epithelium of the bladder from toxic waste. We recently demonstrated the expression pattern of UPKs in the male reproductive tract and their importance in sperm function in murine models. However, the exact mechanisms through which UPKs affect spermatogenesis are not reported. In this study, using yeast two-hybrid screening was conducted to determine the interaction partners of Uroplakin 1a (UPK1A). Y2H Gold yeast strain overexpressing UPK1A was mated with Y187 yeast strain overexpressing human testis cDNA library and the mutants were plated on SD agar plates containing selection media. Colonies that grew on SD/-Trp, SD/-Leu, SD/-His, and SD/-Ade plates were isolated and evaluated to identify the interacting partners of UPK1A. Regucalcin (RGN) and proteasome subunit beta 1 (PSMB1) were identified as potential interaction partners. Using HEK cells that overexpress UPK1A and RGN or PMSB1, the co-localization and interaction were estimated with high-resolution microscopy and Pearson's coefficient. In light of the fact that UPK1A knockout caused subfertility and that the role of RGN and PSMB1 in spermatogenesis is documented, an interaction between UPK1A and RGN or PSMB1 could be required for spermatogenesis.

Uroplakin 1a Knockout Mice Display Marginal Reduction in Fecundity, Decreased Bacterial Clearance Capacity, and Drastic Changes in the Testicular Transcriptome.

Uroplakins (UPKs) form physical and chemical barriers in the bladder and other urinary tract tissues. We previously reported the identification and localization of UPKs in the male reproductive tract of rat. In this study, we characterized Upk1a knockout mice and report a marginal reduction in fecundity associated with significant decrease in sperm count. Upk1a mice had lower bacterial clearance capacity when challenged with uropathogenic Escherichia coli for 1 to 5days. High-throughput analyses of testicular transcriptome indicated that 1128 genes that are expressed in testis of wild-type mice were completely absent in the knockout, while 2330 genes were found to be expressed only in the testis of knockout mice. Furthermore, differential regulation of 148 (67 upregulated and 81 downregulated) was observed. Gene ontology analyses indicated that processes related to integral components of membrane (plasma membrane), G-protein receptor activity and signaling, olfactory receptor activity and perception of smell, organization of extracellular space/region, immune and inflammatory responses to pathogens, spermatid development, meiotic cell cycle, and formation of synaptonemal complex were affected. Results of this study provide evidence on the possible multi-functional role of Upk1a in male reproductive tract and in other tissues as well.

The Golgi complex: An organelle that determines urothelial cell biology in health and disease

The Golgi complex undergoes considerable structural remodeling during differentiation of urothelial cells in vivo and in vitro. It is known that in a healthy bladder the differentiation from the basal to the superficial cell layer leads to the formation of the tightest barrier in our body, i.e., the blood–urine barrier. In this process, urothelial cells start expressing tight junctional proteins, apical membrane lipids, surface glycans, and integral membrane proteins, the uroplakins (UPs). The latter are the most abundant membrane proteins in the apical plasma membrane of differentiated superficial urothelial cells (UCs) and, in addition to well-developed tight junctions, contribute to the permeability barrier by their structural organization and by hindering endocytosis from the apical plasma membrane. By studying the transport of UPs, we were able to demonstrate their differentiation-dependent effect on the Golgi architecture. Although fragmentation of the Golgi complex is known to be associated with mitosis and apoptosis, we found that the process of Golgi fragmentation is required for delivery of certain specific urothelial differentiation cargoes to the plasma membrane as well as for cell–cell communication. In this review, we will discuss the currently known contribution of the Golgi complex to the formation of the blood–urine barrier in normal UCs and how it may be involved in the loss of the blood–urine barrier in cancer. Some open questions related to the Golgi complex in the urothelium will be highlighted.

MP49-16 IMPAIRED UROTHELIAL BARRIER FUNCTION INDUCED LOWER URINARY TRACT DYSFUNCTION BY PROTEASE-ACTIVATED RECEPTER 2 ACTIVATION

You have accessJournal of UrologyCME1 May 2022MP49-16 IMPAIRED UROTHELIAL BARRIER FUNCTION INDUCED LOWER URINARY TRACT DYSFUNCTION BY PROTEASE-ACTIVATED RECEPTER 2 ACTIVATION Hidenori Akaihata, Yusuke Hakozaki, Hiroki Natsuya, Tomoyuki Kumekawa, Shunsuke Yoshioka, Hitomi Imai, Akifumi Onagi, Kanako Matsuoka, Tomoyuki Koguchi, Junya Hata, Yuichi Sato, Masao Kataoka, and Yoshiyuki Kojima Hidenori AkaihataHidenori Akaihata More articles by this author , Yusuke HakozakiYusuke Hakozaki More articles by this author , Hiroki NatsuyaHiroki Natsuya More articles by this author , Tomoyuki KumekawaTomoyuki Kumekawa More articles by this author , Shunsuke YoshiokaShunsuke Yoshioka More articles by this author , Hitomi ImaiHitomi Imai More articles by this author , Akifumi OnagiAkifumi Onagi More articles by this author , Kanako MatsuokaKanako Matsuoka More articles by this author , Tomoyuki KoguchiTomoyuki Koguchi More articles by this author , Junya HataJunya Hata More articles by this author , Yuichi SatoYuichi Sato More articles by this author , Masao KataokaMasao Kataoka More articles by this author , and Yoshiyuki KojimaYoshiyuki Kojima More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002624.16AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Recently, attention has focused on the association between urothelial barrier dysfunction and lower urinary tract dysfunction (LUTD). Uroplakin (UP) is critical to normal urothelial barrier function. Some studies reported that UPs defects lead to abnormal voiding patterns. It was also reported that impairing UP II causes mast cell infiltration in the bladder and that mast cell contributed to bladder hyperactivity. Mast cell tryptase (MCT) can regulate neuronal activity by cleaving protease-activated recepter 2 (PAR2), which is expressed on C-fiber. On the other hands, the mechanisms underlying LUTD caused by chronic ischemia are not completely known. This study aimed to investigate the effects of chronic ischemia on UPs and mast cell in the bladder using a rat model of chronic bladder ischemia (CBI). METHODS: Adult male Sprague-Dawley rats (16 weeks old) were divided into two groups (control and CBI; n=10 each). The CBI group received balloon endothelial injury of bilateral iliac arteries and a 2% cholesterol diet for 8 weeks to induce chronic ischemia. The control group received regular diet for 8 weeks. After monitoring urine output for 24 h, bladders were harvested. Bladder was processed for immunohistochemical staining and methylene blue staining. We used western blotting to measure expressions of UPs(UP Ia, UP Ib, UP II, UP III) in the bladder of this rat model to examine urothelial barrier function. The expression of MCT and PAR2 were also measured to evaluate C-fiber activation by mast cell. RESULTS: Metabolic cage studies showed that mean voided volumes were significantly smaller in the CBI (1.46±0.33 ml) than in the control (1.01±0.21 ml; P=0.001). In western blot analysis, UP II expression was significantly decreased (P=0.011) in the CBI compared with the control. With immunohistochemical staining, UP II-positive cells are located mostly. The percentage of UP II-positive cells on urothelium was significantly lower in the CBI (63.0±0.11%) than in the control (92.1±0.1%, P<0.001). The expression of MCT (P=0.024) and PAR2 (P=0.021) were significantly increased in the CBI compared with the control in western blot analysis. The methylene blue staining showed the number of mast cell was significantly more in the CBI than in the control (P=0.034). CONCLUSIONS: Chronic ischemia might impair urothelial barrier function through reduced expression of UP II in urothelium. Urothelial barrier dysfunction contributed to mast cell infiltration and increased PAR2 on C-fiber, resulting PAR2 activation by MCT. The PAR2 activation may induce LUTD by stimulating C fiber in rat model of chronic bladder ischemia. Source of Funding: None © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e860 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hidenori Akaihata More articles by this author Yusuke Hakozaki More articles by this author Hiroki Natsuya More articles by this author Tomoyuki Kumekawa More articles by this author Shunsuke Yoshioka More articles by this author Hitomi Imai More articles by this author Akifumi Onagi More articles by this author Kanako Matsuoka More articles by this author Tomoyuki Koguchi More articles by this author Junya Hata More articles by this author Yuichi Sato More articles by this author Masao Kataoka More articles by this author Yoshiyuki Kojima More articles by this author Expand All Advertisement PDF DownloadLoading ...

Initial Evaluation of Uroplakins UPIIIa and UPII in Selected Benign Urological Diseases.

Background: Uroplakins (UPs) are glycoproteins that play a specific role in the structure and function of the urothelium. Disorders which affect the normal expression of UPs are associated with the pathogenesis of infections and neoplasms of the urinary tract, primary vesicoureteral reflux, hydronephrosis and renal dysfunction. The appearance of uroplakins in the urine and/or plasma may be of potential importance in the detection of urinary tract dysfunction. The aim of the present study was to investigate uroplakin IIIa (UPIIIa) and uroplakin II (UPII) expression in patients with selected urological diseases. Methods: Plasma and urine from patients with benign prostatic hyperplasia (BPH), urethral stricture (US), urinary tract infection (UTI) and urolithiasis were compared to healthy people without urological disorders. UPs concentrations were measured by the immunoenzymatic method. Results: In patients with BPH and UTI, concentrations of UPIIIa in urine and plasma, as well as UPII in urine, were statistically significantly higher than in the control groups. In the US group, only the plasma UPIIIa concentration differed significantly from the control. Conclusion: The conducted research shows that benign urological diseases may affect the state of the urothelium, as manifested by increased concentrations of both UPs in patients’ urine and plasma, especially in BPH and UTI.

A novel mouse model simulating transurethral laser vaporization prostatectomy.

Benign prostatic hyperplasia (BPH) is a common disease in elderly men, and transurethral laser prostatectomy (TULP) has been widely used in the clinic to remove bladder outlet obstruction caused by BPH. Previous animal models for wound repair after prostatectomy have many limitations, and there have been no previous reports of a mouse model of TULP. Therefore, this study aimed to establish a novel mouse model of TULP. Twelve healthy adult Kunming (KM) mice received transurethral laser vaporization prostatectomy with a 200-μm thulium laser. The mice were sacrificed, and wound specimens from the prostatic urethra and bladder neck were harvested at 1 day, 3 days, 5 days, and 7 days after surgery. Hematoxylin-eosin (HE) and immunohistochemistry were applied to confirm the establishment of the mouse TULP model. One day after the surgery, urothelium expressing uroplakin (UPK) was absent in the urethral wound site, and a large number of necrotic tissues were found in the wound site. There was no UPK-positive urothelium in the wound 3 days after surgery. At 5 days after surgery, monolayer urothelium expressing UPK was found in the wound site, indicating that the re-epithelization of the wound had been completed. On the 7th day after surgery, there were multiple layers of urothelium with UPK expression, indicating that the repair was completed. It is feasible to establish a mouse TULP model by using a microcystoscope system and a 200-μm thulium laser.

Combined lectin- and immuno-histochemistry (CLIH) for applications in cell biology and cancer diagnosis: Analysis of human urothelial carcinomas.

Lectin histochemistry (LHC) and immunohistochemistry (IHC), which demonstrate the composition and localisation of sugar residues and proteins in cell membranes, respectively, are generally used separately. Using these two methods, we previously demonstrated that malignant transformation of urothelial cells results in the alterations of protein glycosylation and reduced expression of urothelium-specific integral membrane proteins uroplakins (UPs). However, the correlation between these changes was not studied yet. To evaluate this correlation, we developed innovative method, which we named Combined Lectin- and Immuno-Histochemistry (CLIH). We used human biopsies of 6 normal urothelia and 9 papillary urothelial carcinomas, i.e. 3 papillary urothelial neoplasms of low malignant potential (PUNLMP), 3 non-invasive papillary urothelial carcinomas of low grade (pTa, l.g.), and 3 invasive papillary urothelial carcinomas of high grade (pT1, h.g.). We tested five different protocols (numbered 1-5) of CLIH on paraffin and cryosemithin sections and compared them with LHC and IHC performed separately. Additionally, we carried out western and lectin blotting with antibodies against UPs and lectins Amaranthus caudatus agglutinin (ACA), Datura stramonium agglutinin (DSA), and jacalin, respectively. We showed that incubation with primary antibodies first, followed by the mixture of secondary antibodies and lectins is the most efficient CLIH method (protocol number 5). Additionally, 300 nm thick cryo-semithin sections enabled better resolution of co-localisation between sugar residues and proteins than 5 mm thick paraffin sections. In the normal urothelium, CLIH showed co-localisation of lectins ACA and jacalin with UPs in the apical plasma membrane (PM) of superficial umbrella cells. In papillary urothelial carcinomas, all three lectins (ACA, DSA and jacalin) labelled regions of apical PM, where they occasionally co-localised with UPs. Western and lectin blotting confirmed the differences between normal urothelium and papillary urothelial carcinomas. Our results show that CLIH, when used with various sets of lectins and antigens, is a useful, quick, and reliable method that could be applied for basic cell biology research as well as detailed subtyping of human urothelial carcinomas.

Abstract B12: Carcinogen exposure alters keratin 5 expression and K5-Cre recombination in transgenic mouse urothelium

Abstract In the urinary bladder, the normal urothelium is divided into three subpopulations of cells: superficial umbrella cells, intermediate cells, and a basal layer, each with distinct expression patterns of uroplakins (UP) and keratin 5 (K5). Many current transgenic mouse models (Tg) for urinary bladder cancer rely on uroplakin or keratin promoters to drive inducible Cre-lox recombination in basal or luminal bladder urothelial cells. Further, many models utilize the chemical carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), to drive or accelerate tumorigenesis. A major limitation in Tg model systems where genes are knocked out is the requirement for highly penetrant and reproducible Cre activity and subsequent gene deletion in order to see a phenotype. This problem is compounded in long-term experiments such as BBN carcinogenesis that can run for up to six months after Cre induction. We have previously shown that Trim29 (tripartite motif containing 29) is sufficient to drive invasive bladder cancer formation in Tg models and in human bladder tumors. We have now developed a Tg system utilizing K5-Cre-ERT2 to target Trim29 for deletion in K5-expressing basal urothelial cells. In this model, tamoxifen exposure results in Cre expression and Trim29 deletion in K5 basal cells. Whether this would translate into complete loss of Trim29 in the urothelium over time and what effect BBN might have on tamoxifen induction of K5-Cre and loss of Trim29 were unclear. We found that exposure of K5-Cre-ERT2, Trim29 flox/flox mice to tamoxifen for six weeks resulted in loss of Trim29 expression only in the K5+ basal urothelium and that these K5+ Trim29 -/- basal cells did not give rise to intermediate cells or umbrella cells during the six months of follow-up. In contrast, mice treated with 0.05% BBN in drinking water for six months after induction of Cre produced full thickness of Trim29 knockout (KO) in 50% of mice (8/16 mice). Our results indicate that in a nonstressed state, K5 basal cells give rise to other basal cells but do not repopulate intermediate or umbrella cell layers. Furthermore, exposure to BBN resulted in the proliferation of basal cells and the replacement of intermediate cells by basal precursors in the urothelium. These results suggest that K5-Cre promoters may have different effects on transgene recombination in the bladder in the presence or absence of BBN, which induces more universal expression of Cre and its subsequent recombination events. These findings should be considered when interpreting and developing BBN-induced bladder cancer Tg models. Citation Format: Alan Kelleher, Yin Wang, Luke Broses, Sylvia Zelenka-Wang, Phillip L. Palmbos. Carcinogen exposure alters keratin 5 expression and K5-Cre recombination in transgenic mouse urothelium [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B12.

Changes in uroplakin expression in the urothelium of patients with ulcerative interstitial cystitis/bladder pain syndrome.

PurposeWe evaluated changes in the expression of uroplakin (UP) in the urothelium of patients with ulcerative interstitial cystitis/bladder pain syndrome (IC/BPS).Materials and MethodsBladder samples were collected from 19 patients with ulcerative IC/BPS who were treated with augmentation ileocystoplasty and from 5 control patients. Frequency-volume charts, the pain visual analogue scale (VAS), and the O'Leary-Sant interstitial cystitis symptom index (ICSI) and problem index (ICPI) were used to evaluate the patients' symptoms preoperatively. The expression levels of UP-Ib and UP-III in the urothelium were compared between the IC/BPS patients and control patients.ResultsSixteen women and three men with IC/BPS were evaluated. Their values for preoperative mean voiding frequency, number of nocturia episodes, and functional bladder capacity as recorded in frequency-volume charts were 21.1±12.8, 5.9±4.2, and 151.1±62.7 mL, respectively. The mean pain VAS, ICSI, and ICPI scores were 8.4±1.3, 17.7±2.2, and 14.7±1.8, respectively. Immunofluorescence staining showed that UP-Ib and UP-III were localized in the urothelium. Upon Western blot analysis, the expression of UP-III was significantly increased in the IC/BPS group compared with the control group. However, expression of UP-Ib did not differ significantly between the IC/BPS and control groups.ConclusionsUP-III was significantly upregulated in patients with ulcerative IC/BPS. UP-III is a potential biomarker for the diagnosis of ulcerative IC/BPS.

Uroplakin expression in the male reproductive tract of rat

Uroplakins (UPKs) play an important role in the normal and pathophysiology of the urothelium. They protect the urothelium and play a crucial role during urothelial infections by Uropathogenic E. coli. However, their functions beyond this organ system remain unexplored. A wide variety of proteins secreted in the male reproductive tract tissues contribute to spermatogenesis, sperm maturation, fertilization and innate immunity. However, the presence of UPKs and their possible contribution to the male reproductive tract physiology is not yet reported. Hence, in this study, we characterized UPKs in the male reproductive tract of rats. To the best of our knowledge, for the first time, we report the expression of UPKs in the male reproductive system. Upk1a, Upk1b, Upk2 and Upk3b mRNA and their corresponding proteins were abundantly expressed in the caput, cauda, testis, seminal vesicles and the prostate. Their expression was not developmentally regulated. UPK protein expression was also localized on the spermatozoa, suggesting a role for these proteins in sperm function. To study the role of UPKs in innate immunity, Upk mRNA expression in response to endotoxin challenge was evaluated in vitro and in vivo. In the rat testicular and epididymal cell lines, Upk mRNA levels increased in response to lipopolysaccharide challenge. However, in the caput, cauda, testes, seminal vesicle and prostate obtained from LPS treated rats, Upk mRNA expression was significantly reduced. Results of this study indicate a role for UPKs in male reproductive physiology and innate immune responses.

A non-canonical autophagy-dependent role of the ATG16L1T300A variant in urothelial vesicular trafficking and uropathogenic Escherichia coli persistence

ABSTRACT50% of Caucasians carry a Thr300Ala variant (T300A) in the protein encoded by the macroautophagy/autophagy gene ATG16L1. Here, we show that the T300A variant confers protection against urinary tract infections (UTIs), the most common infectious disease in women. Using knockin mice carrying the human T300A variant, we show that the variant limits the UTI-causing bacteria, uropathogenic Escherichia coli (UPEC), from establishing persistent intracellular reservoirs, which can seed UTI recurrence. This phenotype is recapitulated in mice lacking Atg16l1 or Atg7 exclusively in the urothelium. We further show that mice with the T300A variant exhibit urothelial cellular abnormalities, including vesicular congestion and aberrant accumulation of UPK (uroplakin) proteins. Importantly, presence of the T300A variant in humans is associated with similar urothelial architectural abnormalities, indicating an evolutionarily conserved impact. Mechanistically, we show that the reduced bacterial persistence is independent of basal autophagic flux or proinflammatory cytokine responses and does not involve Atg14 or Epg5. However, the T300A variant is associated with increased expression of the small GTPase Rab33b; RAB33B interacts with ATG16L1, as well as other secretory RABs, RAB27B and RAB11A, important for UPEC exocytosis from the urothelium. Finally, inhibition of secretory RABs in bladder epithelial cells increases intracellular UPEC load. Together, our results reveal that UPEC selectively utilize genes important for autophagosome formation to persist in the urothelium, and that the presence of the T300A variant in ATG16L1 is associated with changes in urothelial vesicle trafficking, which disrupts the ability of UPEC to persist, thereby limiting the risk of recurrent UTIs.Abbreviations: 3-PEHPC: 3-pyridinyl ethylidene hydroxyl phosphonocarboxylate; ATG: autophagy; ATG16L1: autophagy related 16 like 1; BECs: bladder epithelial cells; dpi: days post infection; hpi: hours post infection; IF: immunofluorescence; IL1B: interleukin 1 beta; IL6: interleukin 6; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MVB: multivesicular bodies; T300A: Thr300Ala; TNF: tumor necrosis factor; QIR(s): quiescent intracellular reservoir(s); siRNA: short interfering RNA; UPEC: uropathogenic Escherichia coli; UTI(s): urinary tract infection(s); TEM: transmission electron microscopy; WT: wild type

Uroplakins play conserved roles in egg fertilization and acquired additional urothelial functions during mammalian divergence.

Uroplakin (UP) tetraspanins and their associated proteins are major mammalian urothelial differentiation products that form unique two-dimensional crystals of 16-nm particles (“urothelial plaques”) covering the apical urothelial surface. Although uroplakins are highly expressed only in mammalian urothelium and are often referred to as being urothelium specific, they are also expressed in several mouse nonurothelial cell types in stomach, kidney, prostate, epididymis, testis/sperms, and ovary/oocytes. In oocytes, uroplakins colocalize with CD9 on cell-surface and multivesicular body-derived exosomes, and the cytoplasmic tail of UPIIIa undergoes a conserved fertilization-dependent, Fyn-mediated tyrosine phosphorylation that also occurs in Xenopus laevis eggs. Uroplakin knockout and antibody blocking reduce mouse eggs’ fertilization rate in in vitro fertilization assays, and UPII/IIIa double-knockout mice have a smaller litter size. Phylogenetic analyses showed that uroplakin sequences underwent significant mammal-specific changes. These results suggest that, by mediating signal transduction and modulating membrane stability that do not require two-dimensional-crystal formation, uroplakins can perform conserved and more ancestral fertilization functions in mouse and frog eggs. Uroplakins acquired the ability to form two-dimensional-crystalline plaques during mammalian divergence, enabling them to perform additional functions, including umbrella cell enlargement and the formation of permeability and mechanical barriers, to protect/modify the apical surface of the modern-day mammalian urothelium.

Porous poly-l-lactide-co-ɛ-caprolactone scaffold: a novel biomaterial for vaginal tissue engineering.

The surgical reconstruction of functional neovagina is challenging and susceptible to complications. Therefore, developing tissue engineering-based treatment methods for vaginal defects is important. Our aim was to develop and test a novel supercritical carbon dioxide foamed poly-l-lactide-co-ɛ-caprolactone (scPLCL) scaffold for vaginal reconstruction. The scaffolds were manufactured and characterized for porosity (65 ± 4%), pore size (350 ± 150 µm) and elastic modulus (2.8 ± 0.4 MPa). Vaginal epithelial (EC) and stromal cells (SC) were isolated, expanded and characterized with flow cytometry. Finally, cells were cultured with scPLCL scaffolds in separate and/or co-cultures. Their attachment, viability, proliferation and phenotype were analysed. Both cell types strongly expressed cell surface markers CD44, CD73 and CD166. Strong expression of CD326 was detected with ECs and CD90 and CD105 with SCs. Both ECs and SCs attached and maintained viability on scPLCL. Further, scPLCL supported the proliferation of especially ECs, which also maintained epithelial phenotype (cytokeratin expression) during 14-day assessment period. Interestingly, ECs expressed uroplakin (UP) Ia, UPIb and UPIII markers; further, UPIa and UPIII expression was significantly higher on ECs cultured on scPLCL than on cell culture plastic. In conclusion, the scPLCL is potential scaffold for vaginal tissue engineering and the results of this study further illustrate the excellent biocompatibility of PLCL.

Uroplakin traffic through the Golgi apparatus induces its fragmentation: new insights from novel in vitro models

Uroplakins (UPs) play an essential role in maintaining an effective urothelial permeability barrier at the level of superficial urothelial cell (UC) layer. Although the organization of UPs in the apical plasma membrane (PM) of UCs is well known, their transport in UCs is only partially understood. Here, we dissected trafficking of UPs and its differentiation-dependent impact on Golgi apparatus (GA) architecture. We demonstrated that individual subunits UPIb and UPIIIa are capable of trafficking from the endoplasmic reticulum to the GA in UCs. Moreover, UPIb, UPIIIa or UPIb/UPIIIa expressing UCs revealed fragmentation and peripheral redistribution of Golgi-units. Notably, expression of UPIb or UPIb/UPIIIa triggered similar GA fragmentation in MDCK and HeLa cells that do not express UPs endogenously. The colocalization analysis of UPIb/UPIIIa-EGFP and COPI, COPII or clathrin suggested that UPs follow constitutively the post-Golgi route to the apical PM. Depolymerisation of microtubules leads to complete blockade of the UPIb/UPIIIa-EGFP post-Golgi transport, while disassembly of actin filaments shows significantly reduced delivery of UPIb/UPIIIa-EGFP to the PM. Our findings show the significant effect of the UPs expression on the GA fragmentation, which enables secretory Golgi-outpost to be distributed as close as possible to the sites of cargo delivery at the PM.

Serous Carcinoma Mimicking Primary Urothelial Carcinoma on Clinical Evaluation and Pathology: A Potential Diagnostic Pitfall.

- Serous carcinoma of the gynecologic tract often involves the external bladder wall and can occasionally mimic primary urothelial carcinoma of the bladder. - To define the spectrum of morphologic and immunohistochemical features that characterize serous carcinoma involving the bladder wall and its distinction from urothelial carcinoma. - We reviewed all cases of serous carcinoma secondarily involving the bladder wall from the University of California San Diego and Polytechnic Institute for histopathologic and immunohistochemical features. - We identified 20 cases of Müllerian high-grade serous carcinoma involving the bladder wall. Five cases were clinical mimics of urothelial carcinoma, including 2 cases that presented as a large, transmural, primary bladder mass without precedent gynecologic history in women younger than 60 years, and 3 cases presumed to be new bladder carcinoma occurring distant to a serous carcinoma diagnosis. A subset of cases were morphologic mimics of urothelial carcinoma, which showed nested growth patterns (2 of 20; 10%), squamouslike foci (2 of 20; 10%), spindled/sarcomatoid growth (2 of 20; 10%), basaloid morphology (3 of 20; 15%), and syncytial growth patterns (1 of 20; 5%). Immunohistochemical stains in 17 cases showed immunoreactivity for CK7 (17 of 17; 100%), WT1 (17 of 17; 100%), uroplakin (UP) II (1 of 17; 6%), p63 (2 of 17; 12%), GATA3 (2 of 17; 12%), and PAX8 (17 of 17; 100%). - A subset of serous carcinomas involving the bladder wall can mimic urothelial carcinoma. Awareness of this mimicker and use of an immunohistochemical panel that includes CK7, WT1, UPII, PAX8, p63, and GATA3 can be helpful in confirming the diagnosis.

Urothelial proliferation and regeneration after spinal cord injury

The basal, intermediate, and superficial cell layers of the urothelium undergo rapid and complete recovery following acute injury; however, the effects of chronic injury on urothelial regeneration have not been well defined. To address this discrepancy, we employed a mouse model to explore urothelial changes in response to spinal cord injury (SCI), a condition characterized by life-long bladder dysfunction. One day post SCI there was a focal loss of umbrella cells, which are large cells that populate the superficial cell layer and normally express uroplakins (UPKs) and KRT20, but not KRT5, KRT14, or TP63. In response to SCI, regions of urothelium devoid of umbrella cells were replaced with small superficial cells that lacked KRT20 expression and appeared to be derived in part from the underlying intermediate cell layer, including cells positive for KRT5 and TP63. We also observed KRT14-positive basal cells that extended thin cytoplasmic extensions, which terminated in the bladder lumen. Both KRT14-positive and KRT14-negative urothelial cells proliferated 1 day post SCI, and by 7 days, cells in the underlying lamina propria, detrusor, and adventitia were also dividing. At 28 days post SCI, the urothelium appeared morphologically patent, and the number of proliferative cells decreased to baseline levels; however, patches of small superficial cells were detected that coexpressed UPKs, KRT5, KRT14, and TP63, but failed to express KRT20. Thus, unlike the rapid and complete restoration of the urothelium that occurs in response to acute injuries, regions of incompletely differentiated urothelium were observed even 28 days post SCI.

Sequential and compartmentalized action of Rabs, SNAREs, and MAL in the apical delivery of fusiform vesicles in urothelial umbrella cells.

Uroplakins (UPs) are major differentiation products of urothelial umbrella cells and play important roles in forming the permeability barrier and in the expansion/stabilization of the apical membrane. Further, UPIa serves as a uropathogenic Escherichia coli receptor. Although it is understood that UPs are delivered to the apical membrane via fusiform vesicles (FVs), the mechanisms that regulate this exocytic pathway remain poorly understood. Immunomicroscopy of normal and mutant mouse urothelia show that the UP-delivering FVs contained Rab8/11 and Rab27b/Slac2-a, which mediate apical transport along actin filaments. Subsequently a Rab27b/Slp2-a complex mediated FV-membrane anchorage before SNARE-mediated and MAL-facilitated apical fusion. We also show that keratin 20 (K20), which forms a chicken-wire network ∼200 nm below the apical membrane and has hole sizes allowing FV passage, defines a subapical compartment containing FVs primed and strategically located for fusion. Finally, we show that Rab8/11 and Rab27b function in the same pathway, Rab27b knockout leads to uroplakin and Slp2-a destabilization, and Rab27b works upstream from MAL. These data support a unifying model in which UP cargoes are targeted for apical insertion via sequential interactions with Rabs and their effectors, SNAREs and MAL, and in which K20 plays a key role in regulating vesicular trafficking.

Abstract 808: Cystine stones as a potential cause of bladder cancer in Slc3a1 knockout male mice

Abstract Bladder cancer is the sixth leading cause of cancer in the United States. Patients with bladder stones are at an increased risk for developing bladder cancer. Bladder stones are an established cause of bladder irritation and inflammation. Proliferative and phenotypic changes induced by chronic inflammation are the proposed etiology for the development of bladder cancer in individuals with chronically irritated bladders. The purpose of this study was to identify the early genetic changes associated with bladder inflammation that may be associated with the development of bladder cancer. Slc3a1 knockout male mice develop cystine bladder stones from the second month of life. Prior investigations demonstrated bladder inflammation in these mice, which may closely mimic inflammation inducing the earliest stages of bladder cancer in humans. To investigate changes in cell structure, proliferation, and differentiation, we performed PCR expression arrays on tissue from bladders of Slc3a1 knockout mice and compared them to wild-type mice. We tested genes in the uroplakin (UP), cytokeratin (CK), and tissue inhibitor of mellatoprotease (TIMP) families of proteins, as well as genes involved in EMT. We localized expression of selected markers by immunofluorescence. Results demonstrated pronounced alterations in expression of E-cadherin, TIMP-1, TIMP-2, UP1A, UP2, UP3A, UP3B and CK 14 and CK17. E-cadherin and TIMP proteins are involved in cell motility underpinning epithelial-mesenchymal transition. CKs are structural proteins believed to be indicative of the stem-cell population in urothelial cancers. Metastatic potential and stem-cell behavior are key features of cancer cells, and expression changes in genes underpinning these functions may represent the earliest steps in the progression to bladder cancer. Prior studies had shown UP2 to be up-regulated in bladder cancer, suggesting that it could serve as a marker for the disease. We found up-regulation of UP2, as well as up-regulation of UP1A, UP3A, and UP3B in our model, indicating that changes in expression of these genes may occur before the disease is clinically evident. Thus, they have the potential to serve as biomarkers for this disease in its early, silent stages. Genetic changes discovered in Slc3a1 knockout mice may closely mirror the early cellular changes seen in bladder cancer. Our study has provided insight into the earliest genes exhibiting altered expression by inflammation-induced changes in the bladder, including a set of potential biomarkers for early detection of the disease. These genes require in-depth analysis to identify the mechanistic contribution of each one to the development and progression of bladder cancer in the hopes of generating molecular targets for future therapy. Citation Format: Kathleen M. Capaccione, Min Yang, Lourdes Serrano, Jay A. Tishfield, Amrik Sahota. Cystine stones as a potential cause of bladder cancer in Slc3a1 knockout male mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 808. doi:10.1158/1538-7445.AM2015-808

Uroplakin II is a more sensitive immunohistochemical marker than uroplakin III in urothelial carcinoma and its variants.

Uroplakin (UP) II and UPIII are highly specific immunohistochemical markers for urothelial differentiation. Here we studied the sensitivity of UPII and UPIII in conventional and variant urothelial carcinomas (UCs). Immunohistochemical staining for UPII and UPIII was performed on tissue microarray slides, including 105 conventional bladder UCs (BUCs), 90 upper urinary tract UCs (UUTUCs), and 47 micropapillary, 16 plasmacytoid, 22 small cell carcinoma, and 41 sarcomatoid UC variants. UPII expression was significantly higher than UPIII expression in conventional BUC (44% vs 17%, P < .001) and UUTUC (67% vs 46%, P = .045). UPIII expression was significantly higher in UUTUC than in BUC (P < .001). In UC variants, UPII expression was significantly higher than UPIII expression in micropapillary (91% vs 25%, P < .001), plasmacytoid (63% vs 6%, P < .001), and sarcomatoid (29% vs 5%, P = .032) variants. Only rare cases of the small cell carcinoma variant had focal UPII and UPIII expression. Compared with conventional UC, the sarcomatoid variant had significantly lower UPII expression, whereas the micropapillary variant had significantly higher UPII expression (P < .001). UPII demonstrates a significantly higher sensitivity than UPIII in conventional and variant UCs. Thus, UPII is a more valuable marker than UPIII in immunohistochemical analyses for confirming the urothelial origin of carcinomas.

Evolving practices of diagnostic immunohistochemistry.

It has been more than 6 years since the Archives of Pathology & Laboratory Medicine published the special issue on diagnostic immunohistochemistry (IHC) consisting of a series of review articles edited by Jaishree Jagirdar, MD, which was a great, concise, and yet comprehensive review and update on important IHC markers, panels, and diagnostic strategies. It has been my great honor and immense privilege to organize this new special issue on IHC, contributed to by pathologists from Geisinger Medical Center (Danville, Pennsylvania) and many expert pathologists from others medical centers. This 2-part special issue features 14 review articles with an attempt to cover IHC automation, standardization of diagnostic IHC, and the role of IHC in diagnosing tumors in major organs and tumors of unknown primary. This series begins with an article emphasizing standardization of diagnostic IHC in the preanalytic, analytic, and postanalytic phases, with a specific focus on (1) newly proposed guidelines on antibody validation from the College of American Pathologists Pathology and Laboratory Quality Center, (2) testing/optimizing a new antibody and troubleshooting, (3) interpreting and reporting IHC assay results, (4) continuing quality improvement programs, and (5) developing and implementing the concept of best practices in IHC. When it comes to the question of how to implement best practices, emphasis is placed on the evidence-based application of IHC markers to practical scenarios, which includes eliminating unnecessary markers, starting with a small IHC panel and continuing with a second panel only when the first panel leads to an inconclusive result. Two questions should be asked before ordering any IHC assay: ‘‘Why should I order this marker?’’ and ‘‘Does it change my diagnosis and patient care?’’ In addition, some external quality control programs and College of American Pathologists checklists for inspecting a clinical IHC laboratory are outlined. The role of digital pathology in the field of diagnostic IHC is briefly discussed as well. The second review article compares and contrasts the recent technological advances in new generation automated IHC platforms from all major companies. Some advantages over the prior automated platforms are stressed, including complete walkaway automation, faster speed, use of less reagents, user friendliness, better integration with laboratory information systems, effective waste control, capability for multiplexing, and more reliable and reproducible results. One of the most frequent and important applications of diagnostic IHC is to assist in working up an undifferentiated neoplasm/tumor of uncertain primary site. The third review article comprehensively reviews some diagnostic strategies and algorithms, updates many recently described diagnostic markers such as GATA binding protein 3 (GATA3), placental S100 (S100P), mammary serine protease inhibitor (maspin), von Hippel-Lindau tumor suppressor gene protein (pVHL), paired box gene (PAX) 8, ETS-related gene (ERG), sal-like protein 4 (SALL4), sex-determining region Y box (SOX) 10, arginase-1, napsin A, special AT-rich sequence-binding protein 2 (SATB2), and cadherin-17, and refines the diagnostic IHC panels frequently used in daily practice. The remaining 11 review articles are devoted to organspecific diagnostic IHC. In the lung and pleural review article, p40, desmoglein-3, desmocollin-3, and cytokeratin (CK) 5 are described as the most effective panel of IHC markers for squamous cell carcinoma; hepatocyte nuclear factor 4 a (HNF4a) is demonstrated to be a marker for invasive mucinous adenocarcinoma of the lung; and glucose transporter 1 (Glut1), insulin-like growth factor II messenger RNA–binding protein (IMP3), and cluster of differentiation (CD) 146 are shown to be helpful in differentiating reactive conditions from malignant mesothelial proliferations. In breast pathology, GATA3 has been reported in several publications to be expressed in most breast ductal and lobular carcinomas, including more than 50% of ERnegative breast carcinomas and metaplastic carcinomas. Ankyrin repeat domain 30A (NY-BR-1) is another newly described marker useful in identifying a breast primary, although in limited publications. Numerous predictive biomarkers have been evaluated, and additional studies are needed to confirm the initial findings. In genitourinary pathology, PAX8 and PAX2 have proven to be important diagnostic markers for identifying renal cell carcinoma when working on a tumor of unknown primary. ERG is a specific but not sensitive marker for prostatic adenocarcinoma. In contrast, NK3 homeobox 1 (NKX3.1) may become a highly sensitive and specific marker for prostatic adenocarcinoma if additional publications substantiate the initial reports. In limited studies, uroplakin (UP) II has proven to be a more sensitive marker than UPIII for identifying urothelial carcinoma. GATA3 and S100P are useful markers for urothelial carcinoma. A new generation Accepted for publication August 18, 2014. From the Department of Laboratory Medicine, Geisinger Medical Center, Danville, Pennsylvania. The author has no relevant financial interest in the products or companies described in this article. doi: 10.5858/arpa.2014-0294-ED Reprints: Fan Lin, MD, PhD, Department of Laboratory Medicine. Geisinger Medical Center, MC 01–31, 100 N Academy Ave, Danville, PA 17822 (e-mail: flin1@geisinger.edu).