Journal of Pathology J Pathol 2003; 199: 41–49. Published online 18 November 2002 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/path.1252 Original Paper Uroplakin gene expression in normal human tissues and locally advanced bladder cancer Jonathon Olsburgh, 1,2 Patricia Harnden, 1,3 Robert Weeks, 1 Barbara Smith, 4 Adrian Joyce, 2 Geoffrey Hall, 1 Richard Poulsom, 5 Peter Selby 1 and Jennifer Southgate 4 * 1 Imperial Cancer Research Fund Clinical Centre, Leeds, UK 2 Department of Urology, St James’s University Hospital, Leeds, UK 3 Department of Pathology, St James’s University Hospital, Leeds, UK 4 Jack Birch Unit of Molecular Carcinogenesis, University of York, York, UK 5 In Situ Hybridisation Service Imperial Cancer Research Fund, Lincoln’s Inn Fields, London, UK *Correspondence to: Professor Jennifer Southgate, Jack Birch Unit of Molecular Carcinogenesis, University of York, York YO10 5YW, UK. E-mail: js35@york.ac.uk Received: 25 October 2001 Revised: 21 March 2002 Accepted: 8 August 2002 Abstract The uroplakins are widely regarded as urothelium-specific markers of terminal urothelial cytodifferentiation. This study investigated the expression of the four uroplakin genes, UPIa, UPIb, UPII and UPIII, in a wide range of normal human tissues to determine tissue specificity and in advanced transitional cell carcinoma (TCC) to examine gene expression in primary and metastatic disease. In the urinary tract, all four uroplakins were expressed by urothelium and UPIII was also expressed by prostatic glandular epithelium. UPIa and UPII appeared to be urothelium-specific, but UPIb was detected in several non-urothelial tissues, including the respiratory tract, where it was associated with squamous metaplasia of tracheal and bronchial epithelia. The ten cases of primary TCC and corresponding lymph node metastases demonstrated that each uroplakin gene could be expressed at the mRNA level. No single uroplakin gene was expressed in all primary tumours or metastases, but 80% of the primary tumours and 70% of the lymph node metastases expressed at least one uroplakin gene. UPIII mRNA was often expressed in the absence of UPIII protein. These results confirm that in human tissues the expression of UPIa and UPII genes is highly specific to urothelium and suggest that the tight differentiation-restricted expression of uroplakin genes in normal urothelium is lost following malignant transformation. Copyright  2002 John Wiley & Sons, Ltd. Keywords: urothelium; bladder cancer; transitional cell carcinoma; metastasis; differen- tiation; uroplakin; gene expression; in situ hybridization Introduction The uroplakins are four distinct, species-conserved integral membrane proteins that constitute the plaques of the asymmetric unit membrane (AUM) in urothe- lium, present on the apical membrane of the termi- nally-differentiated superficial urothelial cells. The AUM is unique to urothelium, where it is thought to contribute to the specialized barrier and mechan- ical functions of the tissue. Its presence is therefore regarded as an unequivocal marker of terminal urothe- lial cytodifferentiation [1]. Of the uroplakins, UPIa and UPIb are both members of the tetraspanin family of proteins and are thought to form plaques by inter- action with the genetically unrelated UPII and UPIII proteins, respectively [2,3]. The uroplakins are transcribed from four distinct genes that have been described as urothelium-specific in bovine and murine tissues [4–6]. The tissue speci- ficity of the UPII gene has been supported by analysis of transgenic mice in which marker genes expressed from the murine UPII promoter showed a urothelium- restricted pattern of expression [7]. We have previously described the expression pat- terns of the uroplakin genes in human urothelium, where we showed that expression of UPIa and UPII genes was restricted to superficial urothelial cells [8]. By contrast, expression of UPIb was not confined to the superficial cells in normal urothelium and was seen in the intermediate cell layers. In addition, UPIb was highly expressed by normal human urothelial (NHU) cells in culture and by a proportion of invasive and metastatic TCCs [8]. Others have detected the expres- sion of UP transcripts in circulating and metastatic TCC cells [9 – 12]. Preservation of UP gene expression during malignant transformation may be exploited for the diagnosis and monitoring of patients with TCC. Furthermore, if expression of human uroplakin genes is restricted to urothelium, this may open the door to gene targeting strategies through exploitation of their regulatory elements. Although the assembled AUM plaque is consid- ered unique to urothelium, UPIb gene and protein expression has been detected in human corneal epithe- lium [13]. The purpose of this study was to investigate the tissue specificity of the four individual uroplakin Copyright  2002 John Wiley & Sons, Ltd.