Brought to you by Portland Press Ltd.
Published on behalf of the International Federation for Cell Biology
Cancer Cell death Cell cycle Cytoskeleton Exo/endocytosis Differentiation Division Organelles Signalling Stem cells Trafficking
Cell Biology International (2004) 28, 381–386 (Printed in Great Britain)
Differentiation-dependent localisation of tissue-type plasminogen activator in human bladder urothelium
Jana Makuca*, Andrej Mašerab, Bojan Tršinarc and Kristijan Jezernika
aInstitute of Cell Biology, Medical Faculty, University of Ljubljana, Lipičeva 2, 1000 Ljubljana, Slovenia
bInstitute of Pathology, Medical Faculty, University of Ljubljana, Korytkova 2, 1000 Ljubljana, Slovenia
cDepartment of Urology, University Medical Center, Zaloška 2, 1000 Ljubljana, Slovenia


Human bladder urothelium is able to secrete tissue-type plasminogen activator (tPA). The aim of our study was to analyse localisation of tPA antigen in comparison to differentiation state of cells in samples of histologically normal urothelium and non-invasive tumours of the human urinary bladder. Twenty-five samples of normal urothelium and 31 non-invasive papillary tumours from 36 patients were examined. The presence of tPA antigen was evaluated immunohistochemically. Differentiation of superficial cells was assessed by the presence of urothelial cell differentiation markers, uroplakins (UPs; immunohistochemistry) and cell's apical surface architecture (scanning electron microscopy). All tissue samples stained anti-tPA positive. In normal urothelium, the intensity of anti-tPA staining was the strongest in superficial cells, which were well-differentiated. In tumours, all cell layers stained anti-tPA positive. The intensity of anti-tPA positive reaction in the upper cell layer correlated with the percentage of anti-UP positive superficial cells. Superficial cells showed various differentiation states.

The localisation of tPA antigen in human in vivo tissue is not confined to the well-differentiated superficial cells. Our results suggest a positive correlation between tPA secretion and cell differentiation.

Keywords: Differentiation, Tissue-type plasminogen activator, Urothelium, Papillary tumours, Uroplakins.

*Corresponding author. Tel.: +386-1-543-7696; fax: +386-1-543-7681.

1 Introduction

Tissue-type plasminogen activator (tPA) is a serine protease with molecular mass of 70kDa, involved primarily in intravascular dissolution of blood clots. The main source of plasma tPA is vascular endothelial cells, where tPA is localised in specialized endothelial storage granules and secreted via a regulated pathway (Huber et al., 2002).

Bovine urothelium synthesizes and secretes into urine large amounts of active tPA, and anti-tPA immunostaining was found to be more intensive in well-differentiated superficial cells (Deng et al., 2001). Therefore, tPA is thought to be secreted in a differentiation-dependent manner, although secretory function of well-differentiated superficial urothelial cells seems incompatible with their highly structured apical surface (Deng et al., 2001; Dubeau et al., 1988). In contrast to ruminant tPA that is primarily urothelium derived, mouse and human tPA are mainly kidney derived (Deng et al., 2001). In human tissue, tPA antigen was immunohistochemically located in fetal bladder specimens, where it was confined to superficial cells (Dubeau et al., 1988).

When comparing tPA levels in normal and cancerous human bladder tissue, Hasui et al. (1989) found no significant difference (p>0.005). Human urothelial tumour cell lines, however, express plasminogen activators (PAs) differently from normal urothelium (Dubeau et al., 1988; Hiti et al., 1990) Dubeau et al. (1988) established that they almost exclusively produce urokinase-type PA.

Characteristics of tumour tissue are less-differentiated superficial cells. Differentiation of urothelial cells is characterized by architecture of their apical surface, which reflects the structure of the plasma membrane. In well-differentiated superficial cells, the apical plasma membrane consists of scallop-shaped plaques of asymmetric membrane (AUM), interconnected by hinge regions (Romih et al., 2002; Sun et al., 1996). The thickened outer leaflet of plaques consists of hexagonal protein particles, formed by four uroplakins (UPs): UP Ia, Ib, II, III (Kachar et al., 1999; Liang et al., 2001; Sun et al., 1996; Wu et al., 1990, 1994). Therefore, UPs are used as the major cell differentiation markers.

The aim of our study was to analyse localisation of tPA antigen in comparison to differentiation state of cells in samples of histologically normal urothelium and non-invasive tumours of the human urinary bladder.

2 Patients and methods

2.1 Patients and tissue samples

One female and 35 male patients, 27–88 years old (mean age 65.7), were included in the study. None of the patients suffered from infections or had permanent catheters. A total of 25 histologically normal and 31 tumour samples were obtained by transurethral cold-cup biopsy.

Tumour samples included papillomas and well-differentiated (grade I) non-invasive (pTa) transitional cell papillary carcinomas.

2.2 Immunohistochemistry

Nineteen normal and 24 tumour samples were fixed in Bouin solution for 24h, dehydrated and embedded in paraffin. Serial sections were cut; the pathologist examined the first section. Immunostaining was performed twice for each specimen.

Mouse monoclonal antibody PAM-3 (American Diagnostica, Greenwich, CT) was used to detect tPA antigen. Immunostaining was performed according to manufacturer's protocol for immunohistochemical staining of uPA in paraffin embedded tissue. Primary antibodies were detected with goat anti-mouse IgG, conjugated with horseradish peroxidase (Dako, Glostrup, Denmark), diluted 1:100. Peroxidase activity was demonstrated with 3,3-diaminobenzidine (Sigma–Aldrich Chemie Gmbh, Steinheim, Germany). Sections were counterstained with haematoxylin. For negative control, the procedure was the same, except primary antibodies were omitted. Bovine urothelium was used as a positive control.

To stain UPs, rabbit polyclonal antibodies raised against highly purified bovine AUM (anti-UP), kindly provided by Prof. Dr. T.-T. Sun, were used. Sections were deparaffinized, rehydrated and rinsed in 0.15M phosphate buffered saline (PBS). Unspecific labeling was blocked by 20% fetal calf serum (FCS; Sigma Chemical Company, St. Louis, MO, USA) in PBS. Sections were incubated overnight at room temperature with primary antisera diluted 1:5000 in 3% bovine serum albumin (BSA; Sigma–Aldrich Chemie GMBH, Steinheim, Germany). Secondary antibodies, goat anti-rabbit IgG, conjugated with 5nm gold particles (Amersham, UK), were diluted 1:100 in 3% BSA. Gold particles were silver enhanced with IntenSe (Amersham, UK). Sections were subsequently washed in PBS and distilled water and counterstained with haematoxylin.

The procedure was the same for negative controls, except primary antibodies were omitted.

2.3 Scanning electron microscopy (SEM)

Six normal and seven tumour samples were fixed in 2.5% glutaraldehyde with 4% paraformaldehyde. After rinsing in 0.1M cacodylate buffer and post-fixing with 1% OsO4, specimens were dehydrated and critical-point dried. Sputter-coated samples were observed with Jeol 840A scanning electron microscope.

2.4 Quantitative and semi-quantitative evaluation

We calculated the percentage of anti-UP positive superficial cells by counting anti-UP positive and negative cells. Each specimen was counted four times: two serial sections containing at least 50 superficial cells were counted on two slides. Individual results represent averages of these counts.

Anti-tPA staining was evaluated semi-quantitatively. Tumour samples were classified into four groups: samples with all cell layers staining equally intensively (group 1), samples with some superficial cells staining more intensively than other cell layers (group 2); samples with superficial cells staining more intensively in parts of tumour (group 3); and samples with most superficial cells staining more intensively (group 4).

3 Results

3.1 Normal urothelium

Anti-tPA immunostaining was intracytoplasmatic, and in normal urothelium, it was most intensive in the superficial cell layer, although usually all cell layers stained positively (Fig. 1A). SEM showed superficial cells that differed in size, shape and surface morphology. Most cells were large, polygonal, with prominent complex ridges (Fig. 2), representing well-differentiated cells. Smaller, less-differentiated cells with low, unlinked ridges and cells with microvilli were rare. Immunostaining indicated that 91.8% (SE: 1.9; range: 65.6–100%) of the superficial cells were anti-UP positive. A much weaker anti-UP positive reaction was observed in the apical region of individual intermediate cells (Fig. 1B).

Fig. 1

Normal urothelium. (A) tPA-immunohistochemistry: anti-tPA staining more intensive in the superficial cell layer. (B) UP-immunohistochemistry: a confluent positive reaction in the upper cell layer and individual intermediate cells (arrows). Bars: 10μm.

Fig. 2

Normal urothelium. SEM micrography of well-differentiated superficial cells: large, polygonal cells with prominent ridges. Bar: 10μm.

3.2 Non-invasive papillary tumours

In tumours, the intensity of anti-tPA positive reaction in the upper cell layer varied. Positive anti-UP staining was noted in 20 (83.3%) samples; 38.9% (SE: 7.5; range: 0–100%) of superficial cells stained positive. All cell layers stained equally intensive for anti-tPA (Fig. 3A) in samples with none or low percentage of anti-UP positive cells (Fig. 3C). Samples with a higher percentage of anti-UP positive superficial cells (Fig. 3D) showed increased intensity of anti-tPA staining in the superficial layer (Fig. 3B). A positive correlation between anti-tPA staining and the percentage of anti-UP positive superficial cells was noted (Fig. 4). SEM revealed diverse cell size and surface morphology (Fig. 5), indicating cells at various differentiation states. Less-differentiated cells bearing uniform microvilli were frequent. Microvilli often appeared in combination with low, unlinked ridges. Cells with complex ridges appeared less often.

Fig. 3

Non-invasive papillary tumours. (A) tPA-immunohistochemistry: less-differentiated cells with all cell layers staining equally intensive. (B) tPA-immunohistochemistry: well-differentiated cells, where anti-tPA staining is more intensive in the superficial cell layer. (C) UP-immunohistochemistry: less-differentiated cells. (D) UP-immunohistochemistry: individual UP-positive superficial cells (arrows). Bars: 10μm.

Fig. 4

Percentage of anti-UP positive superficial cells in groups with different anti-tPA staining. 1: all cell layers staining equally intensive; 2: some superficial cells staining more intensive; 3: superficial cells more intensive in parts of tumour; 4: most superficial cells more intensive; 5: normal tissue—most superficial cells staining more intensive. Error: SD.

Fig. 5

Non-invasive papillary tumours. SEM micrography of superficial cells with microvilli, ridges. Bar: 10μm.

4 Discussion

Two patterns of tPA antigen staining in normal urothelium were observed in previous research. In bovine urothelium, anti-tPA immunostaining was more intensive in superficial cells (Deng et al., 2001). In human fetal bladder specimens tPA antigens were located in surface cells close to the bladder lumen, whereas cells near the basement membrane showed no staining (Dubeau et al., 1988). We noticed both the above-described staining patterns in normal samples, suggesting that the localisation of tPA in human in vivo tissue is differentiation-dependent, although not confined to well-differentiated superficial cells.

Research on urothelial tumour tissue or tumour cell lines yield contradictory results. Hasui et al. (1989) found no significant difference between tPA levels in normal and cancerous bladder tissue. On the other hand, urothelial tumour cell lines produced lesser amounts of tPA (Dubeau et al., 1988). This discrepancy in tPA production could be the result of different specimen types (cultured cell lines vs. tumour tissue), as the physiological state of cultured cells can be very different from the in vivo tissue. Surya et al. (1990) suggested that cultured urothelial cells mimic regenerative (or “healing”) urothelium, in which enhancement of the proteolytic activities is frequently noted. When Deng et al. (2001) compared normal bovine urothelium in a short-term organ culture with cultured normal cells, the latter were found to synthesize and secrete relatively more tPA. These differences might be explained by findings from breast cancer diagnosis, where tPA is a prognostic marker and its high levels correlate with a favourable outcome (Duffy, 1996; Duffy et al., 1988; Luqmani et al., 2002). In benign breast tumours, tPA levels are higher (although not significantly, p>0.005) in comparison to levels in malignant samples; therefore, tPA might be a marker for well-differentiated carcinomas (Duffy et al., 1988; O'Grady et al., 1985).

We focused on samples of non-invasive papillary tumours that represent early stages in bladder carcinogenesis. Although all tumours were histologically diagnosed as well-differentiated (grade I), they had great heterogeneity in terms of cytological differentiation of their superficial cells. The percentage of anti-UP positive superficial cells ranged from 0 to 100%. Likewise, cell size and surface morphology varied greatly, revealing cells at diverse differentiation states. Immunolocalisation of tPA antigen also differed in individual samples. This could just mean an abnormal expression of tPA in tumours. Nevertheless, when tPA antigen localisation in individual tumour samples was compared to their superficial cell differentiation, it was again shown to be differentiation-dependent.

The intense staining in the superficial cells possibly corresponds to an accumulation of tPA, since tPA is known to be stored in vesicles and secreted in a polarised fashion via a cAMP- and calcium-regulated pathway (Deng et al., 2001; Huber et al., 2002). The less intense staining in tumour cells could have several reasons. It could be the result of reduced tPA production in less differentiated tumour cells, regulated on a molecular or cellular level. This explanation seems consistent with a reduced amount of tPA, secreted into conditioned medium of cultured tumour cells in comparison to cultured normal cells (Dubeau et al., 1988). Another possibility is a higher tPA-turnover rate in tumour cells, which would lead to a reduced accumulation of tPA within the cells, possibly due to altered mode of secretion—from regulated to constitutive. The previously mentioned reduced amount of secreted tPA in tumour cells does not support this explanation. However, studies also showed that in contrast to normal cells, tumour cells do not seem to regulate appropriately their proteolytic activity in response to growth factor signals, even though some aspects of the signal transduction pathway are apparently intact (Dubeau et al., 1988; Hiti et al., 1990). Because of the demonstrated differences between cultured cells and in vivo tissue, results concerning the amount of tPA in the urine would seem more relevant. Yet, since human tPA is mainly kidney derived (Deng et al., 2001), these results would be difficult to interpret.

Our results confirm that the localisation of tPA antigen in human in vivo tissue is not confined to the well-differentiated superficial cells. They suggest a positive correlation between tPA secretion and cell differentiation. Since anti-tPA staining is more intense in well-differentiated cells, tumours containing more well-differentiated cells should produce higher amounts of this enzyme. To establish a correlation between tPA content and tumour diagnosis and prognosis, further studies are required.


We thank Prof. Dr. T.-T. Sun and Dr. R. Romih for helpful suggestions and critical remarks. Prof. Dr. T.-T. Sun also provided the anti-UP antibodies. We also thank the surgeons: D. Cotič, G. Homan, M. Lovšin, B. Sedmak, B. Štrus, A. Vrhovec, and M. Žumer-Pregelj from the Department of Urology, University Medical Center, Ljubljana, Slovenia, for providing the tissue samples used in this study.


Deng F-M, Ding, M, Lavker, RM, Sun, T-T. Urothelial function reconsidered: a role in urinary protein secretion. Proc Nat Acad Sci 2001:98:1:154-9
Crossref   Medline   

Dubeau L, Jones, PA, Rideout, WM, Laug, WE. Differential regulation of plasminogen activators by epidermal growth factor in normal and neoplastic human urothelium. Cancer Res 1988:48:5552-6

Duffy MJ. Proteases as prognostic markers in cancer. Clin Cancer Res 1996:2:613-8

Duffy MJ, O'Grady, P, Devaney, D, O'Siorain, L, Fennelly, JJ, Lijnen, HR. Tissue-type plasminogen activator, a new prognostic marker in breast cancer. Cancer Res 1988:48:1348-9

Hasui Y, Suzumiya, J, Marutsuka, K, Sumiyoshi, A, Hashida, S, Ishikawa, E. Comparative study of plasminogen activators in cancers and normal mucosae of human urinary bladder. Cancer Res 1989:49:1067-70

Hiti AL, Rideout, WM, Laug, WE, Jones, PA. Plasminogen activator regulation by transforming growth factor-β in normal and neoplastic human urothelium. Cancer Commun 1990:2:3:123-8

Huber D, Cramer, EM, Kaufmann, JE, Meda, P, Massé, J-M, Kruithof, EKO. Tissue-type plasminogen activator (t-PA) is stored in Weibel-Palade bodies in human endothelial cells both in vitro and in vivo. Blood 2002:99:10:3637-45
Crossref   Medline   

Kachar B, Liang, F, Lins, U, Ding, M, Wu, X-R, Stoffler, D. Three-dimensional analysis of the 16nm urothelial plaque particle: luminal surface exposure, preferential head-to-head interaction, and hinge formation. J Mol Biol 1999:285:595-608
Crossref   Medline   

Liang F-X, Riedel, I, Deng, F-M, Zhou, G, Xu, C, Wu, X-R. Organization of uroplakin subunits: transmembrane topology, pair formation and plaque composition. Biochem J 2001:355:13-8
Crossref   Medline   

Luqmani YA, Temmim, L, Parkar, AH, Mathew, M. Clinical implications of urokinase and tissue type plasminogen activators and their inhibitor (PAI-1) in breast cancer tissue. Oncol Rep 2002:9:645-51

O'Grady P, Lijnen, HR, Duffy, MJ. Multiple forms of plasminogen activator in human breast tumors. Cancer Res 1985:45:6216-8

Romih R, Veranic, P, Jezernik, K. Appraisal of differentiation markers in urothelial cells. Appl Immunohisto Mol Morphol 2002:10:4:339-43

Sun T-T, Zhao, H, Provet, J, Aebi, U, Wu, X-R. Formation of asymmetric unit membrane during urothelial differentiation. Mol Biol Rep 1996:23:3-11
Crossref   Medline   

Surya B, Yu, J, Manabe, M, Sun, T-T. 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 1990:97:3:419-32

Wu X-R, Manabe, M, Yu, J, Sun, T-T. Large scale purification and immunolocalization of bovine uroplakins I, II and III. J Biol Chem 1990:265:19170-9

Wu X-R, Lin, J-H, Walz, T, Häner, M, Yu, J, Aebi, U. Mammalian uroplakins. J Biol Chem 1994:269:18:13716-24

Received 21 October 2003/16 February 2004; accepted 15 March 2004


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)