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Altered expression of UPIa, UPIb, UPII, and UPIIIa during urothelial carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine in rats

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Abstract

In normal urothelium, superficial umbrella cells express four major integral membrane proteins, uroplakins UPIa, UPIb, UPII, and UPIIIa, which compose urothelial plaques. In the apical plasma membrane, urothelial plaques form microridges. During neoplastic changes, microridges are replaced by microvilli, while uroplakin expression is retained. We correlated individual uroplakin expression with apical plasma membrane structure, cytokeratin 20 expression, and urothelial cell proliferation (Ki-67). Male Wistar rats were treated with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water, which caused flat hyperplasia with mild dysplasia, low-grade papillary urothelial carcinoma, invasive low- and high-grade papillary urothelial carcinoma and invasive squamous cell carcinoma with extensive keratinization, grade 2. During urothelial carcinogenesis, UPII expression was the most decreased in all urothelial lesions, while UPIa, UPIb, and UPIIIa expression was differently altered in different types of lesions. Superficial cells were covered with microvilli and ropy ridges, while microridges were disappearing. The expression of cytokeratin 20 was decreased and limited to superficial urothelial cells. Proliferation indices were increased, except for invasive squamous cell carcinoma with extensive keratinization. Our results indicate that during urothelial carcinogenesis the expression of UPII is diminished, suggesting that UPIb/UPIIIa heterodimer can still be formed, while heterodimer UPIa/UPII formation is disrupted. Correlation between decreased level of UPII expression and changed apical plasma membrane structure suggests that diminished expression of UPII hinders the urothelial plaque formation.

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References

  1. Negrete HO, Lavelle JP, Berg J, Lewis SA, Zeidel ML (1996) Permeability properties of the intact mammalian bladder epithelium. Am J Physiol 271:F886–F894

    PubMed  CAS  Google Scholar 

  2. Staehelin LA, Chlapowski FJ, Bonneville MA (1972) Lumenal plasma membrane of the urinary bladder. I. Three-dimensional reconstruction from freeze-etch images. J Cell Biol 53:73–91

    Article  PubMed  CAS  Google Scholar 

  3. Koss LG (1969) The asymmetric unit membranes of the epithelium of the urinary bladder of the rat. An electron microscopic study of a mechanism of epithelial maturation and function. Lab Invest 21:154–168

    PubMed  CAS  Google Scholar 

  4. Min G, Zhou G, Schapira M, Sun TT, Kong XP (2003) Structural basis of urothelial permeability barrier function as revealed by Cryo-EM studies of the 16 nm uroplakin particle. J Cell Sci 116:4087–4094

    Article  PubMed  CAS  Google Scholar 

  5. Kachar B, Liang F, Lins U, Ding M, Wu XR, Stoffler D, Aebi U, Sun TT (1999) Three-dimensional analysis of the 16 nm urothelial plaque particle: luminal surface exposure, preferential head-to-head interaction, and hinge formation. J Mol Biol 285:595–608

    Article  PubMed  CAS  Google Scholar 

  6. Romih R, Jezernik K (1996) Reorganization of the urothelial luminal plasma membrane in the cyclophosphamide treated rats. Pflugers Arch Suppl 431:R241–R242

    Article  CAS  Google Scholar 

  7. Romih R, Veranic P, Jezernik K (2002) Appraisal of differentiation markers in urothelial cells. Appl Immunohistochem Mol Morphol 10:339–343

    Article  PubMed  CAS  Google Scholar 

  8. Kreplak L, Wang H, Aebi U, Kong XP (2007) Atomic force microscopy of mammalian urothelial surface. J Mol Biol 374:365–373

    Article  PubMed  CAS  Google Scholar 

  9. Hicks RM, Ketterer B (1969) Hexagonal lattice of subunits in the thick luminal membrane of the rat urinary bladder. Nature 224:1304–1305

    Article  PubMed  CAS  Google Scholar 

  10. Wu XR, Manabe M, Yu J, Sun TT (1990) Large scale purification and immunolocalisation of bovine uroplakins I, II, and III. Molecular markers of urothelial differentiation. J Biol Chem 265:1775–1784

    Google Scholar 

  11. Wu XR, Lin JH, Walz T, Häner M, Yu J, Aebi U, Sun TT (1994) Mammalian uroplakins. A group of highly conserved urothelial differention-related membrane proteins. J Biol Chem 269:13716–13724

    PubMed  CAS  Google Scholar 

  12. Yu J, Lin JH, Wu XR, Sun TT (1994) Uroplakins Ia and Ib, two major differention products of bladder epithelium, belong to a family of four transmembrane domain (4TM) proteins. J Cell Biol 125:171–182

    Article  PubMed  CAS  Google Scholar 

  13. Wu XR, Sun TT (1993) Molecular cloning of a 47 kDa tissue-specific and differentiation-dependent urothelial cell glycoprotein. J Cell Sci 106:31–43

    PubMed  CAS  Google Scholar 

  14. Lin JH, Wu XR, Kreibich G, Sun TT (1994) Precursor sequence, processing, and urothelium specific expression of a major 15-kDa protein subunit of asymmetric unit membrane. J Biol Chem 269:1775–1784

    PubMed  CAS  Google Scholar 

  15. Wu XR, Medina JJ, Sun TT (1995) Selective interactions of UPIa and UPIb, two members of the transmembrane 4 superfamily, with distinct single transmembrane-domained proteins in differentiated urothelial cells. J Biol Chem 270:29752–29759

    Article  PubMed  CAS  Google Scholar 

  16. Liang FX, Riedel I, Deng FM, Zhou G, Xu C, Wu XR, Kong XP, Moll R, Sun TT (2001) Organisation of uroplakin subunits: transmembrane topology, pair formation and plaque composition. Biochem J 355:13–18

    Article  PubMed  CAS  Google Scholar 

  17. Tu L, Sun TT, Kreibich G (2002) Specific heterodimer formation is a prerequisite for uroplakins to exit from the endoplasmic reticulum. Mol Biol Cell 13:4221–4230

    Article  PubMed  CAS  Google Scholar 

  18. Hu P, Deng FM, Liang FX, Hu CM, Auerbach A, Shapiro E, Wu XR, Kachar B, Sun TT (2000) Ablation of uroplakin III gene results in small urothelial plaques, urothelial leakage, and vesicoureteral reflux. J Cell Biol 151:961–972

    Article  PubMed  CAS  Google Scholar 

  19. Kong XT, Deng FM, Hu P, Liang FX, Zhou G, Auerbach AB, Genieser N, Nelson PK, Robbins ES, Shapiro E, Kachar B, Sun TT (2004) Roles of uroplakins in plaque formation, umbrella cell enlargement, and urinary tract diseases. J Cell Biol 167:1195–1204

    Article  PubMed  CAS  Google Scholar 

  20. Sun TT (2006) Altered phenotype of cultured urothelial and other stratified epithelial cells: implications for wound healing. Am J Physiol Ren Physiol 291:F9–F21

    Article  CAS  Google Scholar 

  21. Kreft ME, Romih R, Kreft M, Jezernik K (2009) Endocytotic activity of bladder superficial urothelial cells is inversely related to their differentiation stage. Differentiation 77:48–59

    Article  PubMed  CAS  Google Scholar 

  22. Ogawa K, St John M, Luiza de Oliveira M, Arnold L, Shirai T, Sun TT, Cohen SM (1999) Comparison of uroplakin expression during urothelial carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine in rats and mice. Toxicol Pathol 27:645–651

    Article  PubMed  Google Scholar 

  23. Huang HY, Shariat SF, Sun TT, Lepor H, Shapiro E, Hsieh JT, Ashfaq R, Lotan Y, Wu XR (2007) Persistent uroplakin expressin in advanced urothelial carcinomas: implications in urothelial tumors progression and clinical outcome. Hum Pathol 38:1703–1713

    Article  PubMed  CAS  Google Scholar 

  24. Ramos-Vara JA, Miller MA, Boucher M, Roudabush A, Johnson GC (2003) Immunohistochemical detection of uroplakin III, cytokeratin 7, and cytokeratin 20 in canine urothelial tumors. Vet Pathol 40:55–62

    Article  PubMed  CAS  Google Scholar 

  25. Ambrosio V, Borzacchiello G, Bruno F, Galati P, Roperto F (2001) Uroplakin expression in the urothelial tumors of cows. Vet Pathol 38:657–660

    Article  PubMed  CAS  Google Scholar 

  26. Jacobs JB, Arai M, Cohen SM, Friedell GH (1977) A long-term study of reversibile and progressive urinary bladder cancer lesions in rats fed N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide. Cancer Res 37:2817–2821

    PubMed  CAS  Google Scholar 

  27. Fukushima S, Cohen SM (1980) Saccharin-induced hyperplasia of the rat urinary bladder. Cancer Res 40:734–736

    PubMed  CAS  Google Scholar 

  28. Pauli BU, Alroy J, Weinstein RS (1983) The ultrastructure and pathobiology of urinary bladder cancer. In: Bryan GT, Cohen SM (eds) The pathology of bladder cancer, vol 2. CRC Press, Boca Raton, FL, pp 41–140

    Google Scholar 

  29. Arai M, Kani T, Sugihara S, Matsumura K, Miyata Y, Shinohara Y, Ito N (1974) Scanning and transmission electron microscopy of changes in the urinary bladder in rats treated with N-butyl-N-(4-hydroxy-butyl)nitrosamine. Gann 65:529–540

    PubMed  CAS  Google Scholar 

  30. Jacobs JB, Arai M, Cohen SM, Friedell GH (1976) Early lesions in experimental bladder cancer: scanning electron microscopy of cell surface markers. Cancer Res 36:2512–2517

    PubMed  CAS  Google Scholar 

  31. Moll R, Löwe A, Laufer J, Franke WW (1992) Cytokeratin 20 in human carcinomas. Am J Pathol 140:427–447

    PubMed  CAS  Google Scholar 

  32. Parker DC, Folpe AL, Bell J, Oliva E, Young RH, Cohen C, Amin MB (2003) Potential utility of uroplakin III, thrombomodulin, high molecular weight cytokeratin, and cytokeratin 20 in noninvasive, invasive, and metastatic urothelial (transitional cell) carcinomas. Am J Surg Pathol 27:1–10

    Article  PubMed  Google Scholar 

  33. Stavropoulos NE, Ioackim-Velogianni E, Hastazeris K, Kitsiou E, Stefanaki S, Agnantis N (1993) Growth fractions in bladder cancer defined by Ki67: association with cancer grade, category and recurrence rate of superficial lesions. Br J Urol 72:736–739

    Article  PubMed  CAS  Google Scholar 

  34. Margulis V, Lotan Y, Karakiewicz PI, Fradet Y, Ashfaq R, Capitanio U, Montorsi F, Bastian PJ, Nielsen ME, Müller SC, Rigaud J, Heukamp LC, Netto G, Lerner SP, Sagalowsky AI, Shariat SF (2009) Multi-institutional validation of the predictive value if Ki-67 labeling index in patients with urinary bladder cancer. J Natl Cancer Inst 101:114–119

    Article  PubMed  Google Scholar 

  35. Eble JN, Sauter G, Epstein J, Sesterhenn IA (2004) World Health Organization Classification of Tumours: Tumours of the Urinary System and Male Genital Organs. IARC Press, Lyon, France

    Google Scholar 

  36. Zupančič D, Jezernik K, Vidmar G (2008) Effect of melatonin on apoptosis, proliferation and differentiation of urothelial cells after cyclophosphamide treatment. J Pineal Res 44:299–306

    Article  PubMed  Google Scholar 

  37. Ito N (1976) Early changes caused by N-butyl-N-(4-hydroxybutyl)nitrosamine in the bladder epithelium of different animal species. Cancer Res 36:2528–2531

    PubMed  CAS  Google Scholar 

  38. Gofrit ON, Birman T, Dinaburg A, Ayesh S, Ohana P, Hochberg A (2006) Chemically induced bladder cancer—a sonographic and morphological description. Urology 68:231–235

    Article  PubMed  Google Scholar 

  39. Kunze E, Schauer A (1977) Morphology classification, and histogenesis of N-butyl-N-(4-hydroxybutyl)nitrosamine in rats. Z Krebsforsch Klin Onkol Cancer Res 88:273–289

    Article  Google Scholar 

  40. Ariel I, Ayesh S, Gofrit O, Ayesh B, Abdul-Ghani R, Pizov G, Smith Y, Sidi AA, Birman T, Schneider T, de Groot N, Hochberg A (2004) Gene expression in the bladder carcinoma rat model. Mol Carcinog 41:69–76

    Article  PubMed  CAS  Google Scholar 

  41. Hu CC, Liang FX, Zhou G, Tu L, Tang CH, Zhou J, Kreibich G, Sun TT (2005) Assembly of urothelial plaques: tetraspanin function in membrane protein trafficking. Mol Biol Cell 16:3937–3950

    Article  PubMed  CAS  Google Scholar 

  42. Surya B, Yu J, Manabe M, Sun TT (1990) Assessing the differentiation state of cultured bovine urothelial cells: elevated synthesis of stratification-related K5 and K6 keratins and persistent expression of uroplakin I. J Cell Sci 97:419–432

    PubMed  CAS  Google Scholar 

  43. Kreft ME, Sterle M, Veranic P, Jezernik K (2005) Urothelial injuries and the early wound healing response: tight junctions and urothelial cytodifferentiation. Histochem Cell Biol 123:529–539

    Article  PubMed  CAS  Google Scholar 

  44. Kreft ME, Sterle M, Jezernik K (2006) Distribution of junction- and differentiation-related proteins in urothelial cells at the leading edge of primary explant outgrowths. Histochem Cell Biol 125:475–485

    Article  PubMed  CAS  Google Scholar 

  45. Veranic P, Romih R, Jezernik K (2004) What determines differentiation of urothelial umbrella cells? Eur J Cell Biol 83:27–34

    Article  PubMed  Google Scholar 

  46. Yin H, Leong AS (2004) Histologic grading of noninvasive papillary urothelial tumors: validation of the 1998 WHO/ISUP system by immunophenotyping and follow-up. Am J Clin Pathol 121:679–687

    Article  PubMed  Google Scholar 

  47. Serio G (1994) PCNA/cyclin expression in transitional cell carcinomas of the human bladder: its correlation with Ki-67 and epidermal growth factor receptor immunostainings. Pathologica 86:161–166

    PubMed  CAS  Google Scholar 

  48. Mallofré C, Castillo M, Morente V, Solé M (2003) Immunohistochemical expression of CK20, p53, and Ki-67 as objective markers of urothelial dysplasia. Mod Pathol 16:187–191

    Article  PubMed  Google Scholar 

  49. Friedell GH, Nagy GK, Cohen SM (1983) Pathology of human bladder cancer and related lesions. In: Bryan GT, Cohen SM (eds) The pathology of bladder cancer, vol 2. CRC Press, Boca Raton, FL, pp 11–42

    Google Scholar 

  50. Celis JE, Rasmussen HH, Vorum H, Madsen P, Honoré B, Wolf H, Orntoft TF (1996) Bladder squamous cell carcinomas express psoriasin and externalize it to the urine. J Urol 155:2105–2112

    Article  PubMed  CAS  Google Scholar 

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Acknowledgment

We are grateful to Prof. T.T. Sun for careful reading of the manuscript and valuable suggestions. We thank Nada Pavlica, Sabina Železnik, Sanja Čabraja, and Linda Štrus for their technical assistance. This study was supported by a grand from Ministry of Higher Education, Science and Technology, Republic of Slovenia (P3-0108).

Conflict of interests

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Institute of Cell Biology, Faculty of Medicine, Lipičeva 2, 1000, Ljubljana, Slovenia

    Daša Zupančič & Rok Romih

  2. Institute of Pathology, Faculty of Medicine, Korytkova 2, 1000, Ljubljana, Slovenia

    Zdenka Ovčak

  3. University Rehabilitation Institute, Linhartova 51, 1000, Ljubljana, Slovenia

    Gaj Vidmar

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  1. Daša Zupančič
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  2. Zdenka Ovčak
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  4. Rok Romih
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Correspondence to Daša Zupančič.

Electronic supplementary material

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Online Resource 1

Summary of statistical analyses of data from scanning electron microscopy (PDF 23 kb)

Online Resource 2

Ki-67 immunohistochemistry. A Ki-67-negative normal urothelium of control animal B Ki-67-positive cells (brown nuclei) in the basal and intermediate layers of the low-grade papillary urothelial carcinoma (10 weeks of BBN administration). C Ki-67-positive cells (brown nuclei) in the basal, intermediate, and superficial layers of the invasive high-grade papillary urothelial carcinoma (20 weeks of BBN administration followed by 15 weeks of tap water) (TIFF 6.74 mb)

Online Resource 3

Distribution of proliferation index across groups. In the boxplots, thick line denotes median, box denotes interquartile range (IQR, from first to third quartile), whiskers denote non-outlier range (largest/smallest observation within 1.5 IQR from the third/first quartile), and circles denote outliers (more than 1.5 IQR above/below third/first quartile). In the control group (C), median, IQR, and non-outlier range limits all equal zero (TIFF 3.61 mb)

Online Resource 4

Mean proliferation index as function of lesion severity. Error bars represent estimated 95% confidence interval (TIFF 3.09 mb)

Online Resource 5

Statistics for CK 20 staining data by group (a) and by lesion severity (b) (PDF 25 kb)

Online Resource 6

UPII immunohistochemistry with different dilutions of anti-UPII antibody (TIFF 7.17 mb)

Online Resource 7

Statistics for UPs immunohistochemistry data by group (PDF 26 kb)

Online Resource 8

Correlations (Spearman Rho; p values in parentheses) between UP expression, Ki-67 proliferation index, and CK 20 expression in BBN-treated animals. CK 20 expression in intermediate cells is excluded since its median value was 1 (i.e., no expression) in all animals (PDF 24 kb)

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Zupančič, D., Ovčak, Z., Vidmar, G. et al. Altered expression of UPIa, UPIb, UPII, and UPIIIa during urothelial carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine in rats. Virchows Arch 458, 603–613 (2011). https://doi.org/10.1007/s00428-011-1045-6

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  • DOI: https://doi.org/10.1007/s00428-011-1045-6

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