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Effects of PPAR agonists on proliferation and differentiation in human urothelium
ARTICLE IN PRESS
Experimental and Toxicologic Pathology 60 (2008) 435–441 www.elsevier.de/etp
Effects of PPAR agonists on proliferation and differentiation in human urothelium Claire L. Varley, Jennifer Southgate Jack Birch Unit of Molecular Carcinogenesis, Department of Biology, University of York, York YO10 5YW, UK Received 4 October 2007; accepted 30 April 2008
Abstract Systemic treatment of rats with peroxisome proliferator-activated receptor (PPAR) agonists (mainly of dual a/g activity) has indicated that they may invoke non-genotoxic carcinogenesis in the epithelial lining of the urinary tract (urothelium). Although there is evidence in the male rat to support an indirect effect via a crystaluria-induced urothelial damage response, there is other evidence to indicate a direct signalling effect on the urothelium and hence the full implication for using these drugs in man is unclear. Numerous reports have demonstrated that PPARs are expressed within the urothelium of different species, including man, and from an early developmental stage. We have developed methods to maintain normal human urothelial (NHU) cells in culture, where the cells retain PPAR expression and express a highly proliferative phenotype, mediated via autocrine stimulation of the epidermal growth factor (EGF) receptor. We have shown that specific activation of PPARg results in a programme of gene expression changes associated with late/terminal cytodifferentiation, including induction of cytokeratins CK13 and CK20, tight junction-associated claudin 3, and uroplakins UPK1a and UPK2, but this is dependent upon inhibition of the signalling cascade downstream of the EGF receptor. This indicates a subtle balance in the regulation of proliferation and differentiation in urothelium, with PPARg agonists promoting differentiation. Our data indicate that human urothelium is a target tissue for PPARg signalling, but it has yet to be determined whether dual agonists could have a modulatory effect on the proliferation/differentiation balance. r 2008 Elsevier GmbH. All rights reserved. Keywords: Peroxisome proliferator-activated receptor; Differentiation; Proliferation; Epidermal growth factor; Bladder; Urothelium; Uroplakin; Claudin
Introduction
Abbreviations: AUM, asymmetric unit membrane; CK, cytokeratin; EGF, epidermal growth factor; ERK, extracellular regulated kinase; NHU, normal human urothelial; PPAR, peroxisome proliferatoractivated receptor; RZ, rosiglitazone; TZ, troglitazone; UPK, uroplakin; ZO, zonular occludens. Corresponding author. Tel.: +44 190 432 8706; fax: +44 190 432 8704. E-mail address: [email protected] (C.L. Varley). 0940-2993/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2008.04.009
The urothelium is a potential target tissue of peroxisome proliferator-activated receptor (PPAR) agonists, as all three PPAR subtypes have been reported to be expressed by the urothelium of all mammalian species studied (Guan et al., 1997; Jain et al., 1998; Kawakami et al., 2002) (Fig. 1). However, the roles of the PPARs in the urothelium are still under investigation and here we address the effects of PPAR agonists on proliferation and differentiation in human urothelium.
ARTICLE IN PRESS 436
C.L. Varley, J. Southgate / Experimental and Toxicologic Pathology 60 (2008) 435–441
merise with the retinoid X receptor (RXR) to form transcription factors that bind to peroxisome proliferator response elements (PPREs) in the promoters of target genes (Feige et al., 2006). PPARs are critical modulators of mammalian metabolism, including fatty acid oxidation, and are reported to be activated by naturally occurring or metabolised saturated and unsaturated fatty acids, as well as by fatty acid derivatives such as prostaglandins and leukotrienes (Krey et al., 1997; Berger and Moller, 2002). PPARs are the targets of drugs that are effective in the treatment of a number of diseases including type 2 diabetes, atherosclerosis and obesity (Berger et al., 2005). Therefore, they are important theurapeutic targets and a large number of synthetic agonists have been generated. The fibrates, such as clofibrate and fenofibrate, are anti-atherosclerotic drugs and activate PPARa, whilst the thiazolidinediones, e.g. troglitazone (TZ) and rosiglitazone (RZ), which are used in the treatment of type 2 diabetes, activate PPARg. A number of specific PPARd agonists have been developed, including GW0742 and L165041. Dual agonists activating both PPARa and PPARg have been indicated as effective agents for the treatment of metabolic disease (Berger et al., 2005). However, it has been reported that systemic treatment of rats with dual agonists promotes carcinogenesis in the uro-epithelial lining of the bladder (Cohen, 2005; Dominick et al., 2006; Van Vleet et al., 2007). There is evidence that dual PPARa/g agonists may promote urothelial carcinogenesis indirectly via a crystaluria-induced urothelial damage response (Cohen, 2005), whereas other evidence indicates a direct signalling effect on the urothelium (Egerod et al., 2005; Oleksiewicz et al., 2005). The full implications of using dual PPARa/g agonists in humans remain unclear.
Characteristics of the urothelium
Fig. 1. Immunofluorescence-labelling of PPAR subtypes in NHU cells. Scale bar, 10 mm.
Peroxisome proliferator-activated receptors The three PPAR subtypes, PPARa, PPARb/d and PPARg, belong to the NRC1 nuclear hormone receptor family. Upon ligand-activation, the PPARs heterodi-
The urothelium is the transitional epithelium that lines the major portion of the lower urinary tract, including the bladder. It is stratified, comprising basal, intermediate and terminally differentiated superficial cell layers. The urothelium is a mitotically quiescent tissue with a very low turnover rate, but has a high regenerative potential in response to injury (Hicks, 1975; Cohen, 1989; Jost et al., 1989; Baskin et al., 1997). The apical surface of the superficial cells is highly specialised, containing multiple thickened plaques of asymmetric unit membrane (AUM), which impart a transcellular urinary barrier function (Hu et al., 2002). The AUM plaques are comprised of the products of the urothelium-specific uroplakin (UPK) genes (Wu et al., 1994), expression of which provides unequivocal markers of terminal differentiation in the urothelium (Wu et al., 1994; Olsburgh et al., 2003).
ARTICLE IN PRESS C.L. Varley, J. Southgate / Experimental and Toxicologic Pathology 60 (2008) 435–441
Other epithelium-associated genes/proteins show characteristic differentiation stage-related expression patterns in urothelium and may also be used as objective markers of differentiation stage. Tight junction proteins control the paracellular permeability of the urothelium and are composed of cytoplasmic plaque proteins (e.g. zonular occludens (ZO)) that link the tight junction to the actin cytoskeleton, and integral transmembrane proteins (e.g. occludin and claudins) that define the properties of the paracellular pore (Schneeberger and Lynch, 2004). By immunohistochemistry, we have shown differentiation stage-associated expression in human urothelium: claudin 3 and ZO1 expressed at the kissing points between superficial cells represent markers of terminal differentiation; claudin 4 and claudin 5 are associated with late/terminal differentiation, whereas occludin and claudin 7 are expressed by intermediate cells (Varley et al., 2006). Cytokeratins (CKs) are cytoskeletal polypeptides expressed by epithelial cells that belong to the intermediate filament family. They exist as obligate heterodimers and form tetrameric structural filaments within the cytoplasm. There are 20 CK isoforms and the specific pattern of expression can be used both to determine differentiation stage and to indicate aberrant changes in the differentiation program, such as squamous metaplasia (reviewed by Southgate et al., 1999). Immunohistochemical analysis of normal human urothelium has shown that CK13 is expressed in basal and intermediate layers, whereas CK20 is restricted to the superficial cells. In squamous metaplasia, there is a switch from CK13 to CK14 expression (Harnden and Southgate, 1997; Varley et al., 2004a).
Normal human urothelial cell culture To address the effects of PPAR agonists on proliferation and differentiation in human urothelium, we used our well-characterised normal human urothelial (NHU) cell culture system (Southgate et al., 1994, 2002). Normal human urothelial tissues were obtained during surgical procedures from patients with no history of urothelial cancer; all specimens were obtained with informed patient consent and were covered by relevant Research Ethics Committee approvals. The urothelium was separated from the stroma and treated with collagenase, before seeding into tissue culture flasks in a low-calcium keratinocyte serum-free medium (KSFM) supplemented with bovine pituitary extract, epidermal growth factor (EGF) and cholera toxin, as described (Southgate et al., 1994, 2002). All experiments were performed on a minimum of three independent cell lines. When established using these procedures, NHU cells show a regenerative phenotype and proliferate rapidly, enabling them to be maintained through serial passages
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as finite cell lines. Both exogenous and autocrine mechanisms are responsible for driving proliferation, which can be blocked using inhibitors to the EGF receptor and may be monitored by western blot analysis of phospho-extracellular regulated kinase (ERK) as a downstream indicator of EGF receptor activity (Varley et al., 2005; MacLaine et al., 2008) (Table 1). Based on immunocytochemical analysis of CK (e.g. CK7+, CK8+, CK17+, CK18+, CK19+) and junctional protein (E-cadherinlo, occludin+, claudin 4+, claudin 7+) expression, it is apparent that the phenotype of NHU cell cultures most closely resembles basal/intermediate urothelial cells in situ (Table 2). However, there is some evidence of squamous metaplasia, with expression of CK14 rather than the transitional differentiation marker, CK13. Cultured NHU cells do not differentiate spontaneously in culture and, with the exception of UPK1b, are negative for expression of markers associated with late/terminal urothelial differentiation in situ, including UPK1a, UPK2, UPK3a, CK13, CK20, claudin 3 and claudin 5 (Lobban et al., 1998; Varley et al., 2006).
PPAR agonist effects on proliferation of NHU cells To assess the effects of PPAR agonists on the proliferation of NHU cells, NHU cells were treated with PPARa (fenofibrate), PPARd (GW0742 and L165041) or PPARg (TZ) agonists (10 nM–100 mM) for up to 6 days, before the biomass of the culture was determined by methylthiazolyldiphenyl-tetrazolium bromide colorimetric assay. When cultures were treated with p5 mM PPAR agonists there was no significant difference in population growth, except with PPARg agonists, which showed a small growth inhibition. When cultures were treated with 45 mM of PPAR agonists, the PPARd and PPARg agonists caused cell death.
PPAR agonist effects on differentiation of NHU cells The differentiation potential of PPAR agonists on NHU cells was assessed by treating cells with PPARa (clofibrate) or PPARg (TZ and RZ) agonists (0.1–5 mM) for 24 h and determining expression of differentiationassociated markers at 6 days. In some experiments, the autocrine EGF receptor loop was inhibited by the use of specific inhibitors against the EGF receptor tyrosine kinase (PD153035 1 mM) or downstream signalling cascades including ERK (UO126 5 mM; PD98059
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Table 1.
C.L. Varley, J. Southgate / Experimental and Toxicologic Pathology 60 (2008) 435–441
Expression of differentiation-associated markers by NHU cell cultures
Marker
Control
TZ/PD153035
0.001870.0001 0.002870.0004
0.204070.0026 0.436370.0363
Phospho_ERK
CK13
CK20
Claudin 3
UPK1a UPK2
NHU cells treated with TZ and the EGF receptor inhibitor, PD153035, for 6 days, as previously described (Varley et al., 2004b) expressed a more differentiated phenotype. This is illustrated by indirect immunofluorescence for phospho-ERK, CK13, CK20 and claudin 3 and by quantitative realtime PCR for UPK1a and UPK2 transcripts (mean of n ¼ 3, 7S.D. relative to GAPDH (Southgate et al., 2007)).
10 mM) or PI3-kinase (LY294002 1 mM) (Varley et al., 2005). We found that the PPARa agonist, clofibrate, did not induce differentiation in NHU cells. However, when NHU cells were treated with the PPARg agonists TZ or RZ (1 mM), there was a switch from a non-differentiated squamous phenotype (CK14+, CK13, CK20) to a terminally differentiated transitional phenotype (CK14, CK13+, CK20+) (Varley et al., 2004a) (Table 1). For effective induction of differentiation by PPARg agonists, it was mandatory that EGF receptor signalling was blocked. In this context, we demonstrated that PPARg was phosphorylated on a serine residue by ERK, resulting in inhibition of PPARg translocation to the nucleus (Varley et al., 2004b). This is in agreement with previous observations (Adams et al., 1997; Camp and Tafuri, 1997). Similar experiments were carried out to assess the effect of PPAR agonists on the expression of UPKs and claudins. PPARg activation by TZ or RZ induced expression of UPK1a and UPK2 genes when the EGF receptor was inhibited (Varley et al., 2004b) (Table 1).
We also showed that these same experimental conditions induced expression of the tight junction proteins claudin 3, claudin 4 and claudin 5. In all cases, maximal effect was achieved when NHU cells were treated in the presence of TZ and PD153035 (Varley et al., 2006). These effects were dependent on PPARg activation, as confirmed by the abrogation of CK13, UPK2, and claudins 3, 4 and 5 induction when experiments were performed in the presence of PPARg antagonists (1 mM GW9662 or 1 mM T0070907) and/or PPARg siRNA (Varley et al., 2004a, 2006). This suggests that PPARg agonists activate a programme of gene expression changes associated with urothelial terminal cytodifferentiation.
Regulation of PPARs in the urothelium We postulated that natural PPAR agonist(s) would be excreted in the urine to induce terminal differentiation in the superficially exposed cells of the urothelium. To address this hypothesis, a study was performed using a cohort of patients with end-stage renal disease who
ARTICLE IN PRESS C.L. Varley, J. Southgate / Experimental and Toxicologic Pathology 60 (2008) 435–441
Table 2.
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Summary of expression of differentiation markers in human urothelium and NHU cells
Cl ¼ claudin, OD ¼ occludin, CK ¼ cytokeratin, UPK ¼ uroplakin, IHC ¼ immunohistochemistry.
excreted very little, if any, urine. If the hypothesis was correct there should be low/no expression of urothelial differentiation markers in these anuric patients. We demonstrated that the anuric patients expressed a normal intensity and distribution of urothelial differentiation-associated markers (Stahlschmidt et al., 2005). This suggested that the natural PPARg agonist was not secreted in the urine, but is potentially produced endogenously by the urothelium or is circulated via the serum. In the latter case, this could lead to sequestering of high concentrations of systemic PPAR agonists in the urothelium.
Concluding remarks Our studies have demonstrated that high concentrations of PPARd and PPARg agonists are growth
inhibitory and induce cell death in NHU cells, but these pro-apoptotic effects are likely to be mediated via PPAR-independent mechanisms (Chopra et al., submitted). Of the three PPAR isotypes expressed in human urothelium, it appears that PPARg plays a critical role in mediating urothelial cytodifferentiation, whereas the functions of PPARa and PPARd remain to be elucidated. Cross-talk between PPARg and the signalling pathways downstream of autocrine EGF receptor activation may have a role in regulating the switch from a differentiated urinary barrier to a highly regenerative epithelial tissue; this provides a potential mechanism in urothelial cell carcinogenesis. Further work is required to identify the nature and origin of the natural agonist and to determine whether PPARa/g dual agonists might modulate the proliferation/differentiation balance.
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Acknowledgements The authors are grateful to York Against Cancer for funding.
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Experimental and Toxicologic Pathology 60 (2008) 435–441 www.elsevier.de/etp
Effects of PPAR agonists on proliferation and differentiation in human urothelium Claire L. Varley, Jennifer Southgate Jack Birch Unit of Molecular Carcinogenesis, Department of Biology, University of York, York YO10 5YW, UK Received 4 October 2007; accepted 30 April 2008
Abstract Systemic treatment of rats with peroxisome proliferator-activated receptor (PPAR) agonists (mainly of dual a/g activity) has indicated that they may invoke non-genotoxic carcinogenesis in the epithelial lining of the urinary tract (urothelium). Although there is evidence in the male rat to support an indirect effect via a crystaluria-induced urothelial damage response, there is other evidence to indicate a direct signalling effect on the urothelium and hence the full implication for using these drugs in man is unclear. Numerous reports have demonstrated that PPARs are expressed within the urothelium of different species, including man, and from an early developmental stage. We have developed methods to maintain normal human urothelial (NHU) cells in culture, where the cells retain PPAR expression and express a highly proliferative phenotype, mediated via autocrine stimulation of the epidermal growth factor (EGF) receptor. We have shown that specific activation of PPARg results in a programme of gene expression changes associated with late/terminal cytodifferentiation, including induction of cytokeratins CK13 and CK20, tight junction-associated claudin 3, and uroplakins UPK1a and UPK2, but this is dependent upon inhibition of the signalling cascade downstream of the EGF receptor. This indicates a subtle balance in the regulation of proliferation and differentiation in urothelium, with PPARg agonists promoting differentiation. Our data indicate that human urothelium is a target tissue for PPARg signalling, but it has yet to be determined whether dual agonists could have a modulatory effect on the proliferation/differentiation balance. r 2008 Elsevier GmbH. All rights reserved. Keywords: Peroxisome proliferator-activated receptor; Differentiation; Proliferation; Epidermal growth factor; Bladder; Urothelium; Uroplakin; Claudin
Introduction
Abbreviations: AUM, asymmetric unit membrane; CK, cytokeratin; EGF, epidermal growth factor; ERK, extracellular regulated kinase; NHU, normal human urothelial; PPAR, peroxisome proliferatoractivated receptor; RZ, rosiglitazone; TZ, troglitazone; UPK, uroplakin; ZO, zonular occludens. Corresponding author. Tel.: +44 190 432 8706; fax: +44 190 432 8704. E-mail address: [email protected] (C.L. Varley). 0940-2993/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2008.04.009
The urothelium is a potential target tissue of peroxisome proliferator-activated receptor (PPAR) agonists, as all three PPAR subtypes have been reported to be expressed by the urothelium of all mammalian species studied (Guan et al., 1997; Jain et al., 1998; Kawakami et al., 2002) (Fig. 1). However, the roles of the PPARs in the urothelium are still under investigation and here we address the effects of PPAR agonists on proliferation and differentiation in human urothelium.
ARTICLE IN PRESS 436
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merise with the retinoid X receptor (RXR) to form transcription factors that bind to peroxisome proliferator response elements (PPREs) in the promoters of target genes (Feige et al., 2006). PPARs are critical modulators of mammalian metabolism, including fatty acid oxidation, and are reported to be activated by naturally occurring or metabolised saturated and unsaturated fatty acids, as well as by fatty acid derivatives such as prostaglandins and leukotrienes (Krey et al., 1997; Berger and Moller, 2002). PPARs are the targets of drugs that are effective in the treatment of a number of diseases including type 2 diabetes, atherosclerosis and obesity (Berger et al., 2005). Therefore, they are important theurapeutic targets and a large number of synthetic agonists have been generated. The fibrates, such as clofibrate and fenofibrate, are anti-atherosclerotic drugs and activate PPARa, whilst the thiazolidinediones, e.g. troglitazone (TZ) and rosiglitazone (RZ), which are used in the treatment of type 2 diabetes, activate PPARg. A number of specific PPARd agonists have been developed, including GW0742 and L165041. Dual agonists activating both PPARa and PPARg have been indicated as effective agents for the treatment of metabolic disease (Berger et al., 2005). However, it has been reported that systemic treatment of rats with dual agonists promotes carcinogenesis in the uro-epithelial lining of the bladder (Cohen, 2005; Dominick et al., 2006; Van Vleet et al., 2007). There is evidence that dual PPARa/g agonists may promote urothelial carcinogenesis indirectly via a crystaluria-induced urothelial damage response (Cohen, 2005), whereas other evidence indicates a direct signalling effect on the urothelium (Egerod et al., 2005; Oleksiewicz et al., 2005). The full implications of using dual PPARa/g agonists in humans remain unclear.
Characteristics of the urothelium
Fig. 1. Immunofluorescence-labelling of PPAR subtypes in NHU cells. Scale bar, 10 mm.
Peroxisome proliferator-activated receptors The three PPAR subtypes, PPARa, PPARb/d and PPARg, belong to the NRC1 nuclear hormone receptor family. Upon ligand-activation, the PPARs heterodi-
The urothelium is the transitional epithelium that lines the major portion of the lower urinary tract, including the bladder. It is stratified, comprising basal, intermediate and terminally differentiated superficial cell layers. The urothelium is a mitotically quiescent tissue with a very low turnover rate, but has a high regenerative potential in response to injury (Hicks, 1975; Cohen, 1989; Jost et al., 1989; Baskin et al., 1997). The apical surface of the superficial cells is highly specialised, containing multiple thickened plaques of asymmetric unit membrane (AUM), which impart a transcellular urinary barrier function (Hu et al., 2002). The AUM plaques are comprised of the products of the urothelium-specific uroplakin (UPK) genes (Wu et al., 1994), expression of which provides unequivocal markers of terminal differentiation in the urothelium (Wu et al., 1994; Olsburgh et al., 2003).
ARTICLE IN PRESS C.L. Varley, J. Southgate / Experimental and Toxicologic Pathology 60 (2008) 435–441
Other epithelium-associated genes/proteins show characteristic differentiation stage-related expression patterns in urothelium and may also be used as objective markers of differentiation stage. Tight junction proteins control the paracellular permeability of the urothelium and are composed of cytoplasmic plaque proteins (e.g. zonular occludens (ZO)) that link the tight junction to the actin cytoskeleton, and integral transmembrane proteins (e.g. occludin and claudins) that define the properties of the paracellular pore (Schneeberger and Lynch, 2004). By immunohistochemistry, we have shown differentiation stage-associated expression in human urothelium: claudin 3 and ZO1 expressed at the kissing points between superficial cells represent markers of terminal differentiation; claudin 4 and claudin 5 are associated with late/terminal differentiation, whereas occludin and claudin 7 are expressed by intermediate cells (Varley et al., 2006). Cytokeratins (CKs) are cytoskeletal polypeptides expressed by epithelial cells that belong to the intermediate filament family. They exist as obligate heterodimers and form tetrameric structural filaments within the cytoplasm. There are 20 CK isoforms and the specific pattern of expression can be used both to determine differentiation stage and to indicate aberrant changes in the differentiation program, such as squamous metaplasia (reviewed by Southgate et al., 1999). Immunohistochemical analysis of normal human urothelium has shown that CK13 is expressed in basal and intermediate layers, whereas CK20 is restricted to the superficial cells. In squamous metaplasia, there is a switch from CK13 to CK14 expression (Harnden and Southgate, 1997; Varley et al., 2004a).
Normal human urothelial cell culture To address the effects of PPAR agonists on proliferation and differentiation in human urothelium, we used our well-characterised normal human urothelial (NHU) cell culture system (Southgate et al., 1994, 2002). Normal human urothelial tissues were obtained during surgical procedures from patients with no history of urothelial cancer; all specimens were obtained with informed patient consent and were covered by relevant Research Ethics Committee approvals. The urothelium was separated from the stroma and treated with collagenase, before seeding into tissue culture flasks in a low-calcium keratinocyte serum-free medium (KSFM) supplemented with bovine pituitary extract, epidermal growth factor (EGF) and cholera toxin, as described (Southgate et al., 1994, 2002). All experiments were performed on a minimum of three independent cell lines. When established using these procedures, NHU cells show a regenerative phenotype and proliferate rapidly, enabling them to be maintained through serial passages
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as finite cell lines. Both exogenous and autocrine mechanisms are responsible for driving proliferation, which can be blocked using inhibitors to the EGF receptor and may be monitored by western blot analysis of phospho-extracellular regulated kinase (ERK) as a downstream indicator of EGF receptor activity (Varley et al., 2005; MacLaine et al., 2008) (Table 1). Based on immunocytochemical analysis of CK (e.g. CK7+, CK8+, CK17+, CK18+, CK19+) and junctional protein (E-cadherinlo, occludin+, claudin 4+, claudin 7+) expression, it is apparent that the phenotype of NHU cell cultures most closely resembles basal/intermediate urothelial cells in situ (Table 2). However, there is some evidence of squamous metaplasia, with expression of CK14 rather than the transitional differentiation marker, CK13. Cultured NHU cells do not differentiate spontaneously in culture and, with the exception of UPK1b, are negative for expression of markers associated with late/terminal urothelial differentiation in situ, including UPK1a, UPK2, UPK3a, CK13, CK20, claudin 3 and claudin 5 (Lobban et al., 1998; Varley et al., 2006).
PPAR agonist effects on proliferation of NHU cells To assess the effects of PPAR agonists on the proliferation of NHU cells, NHU cells were treated with PPARa (fenofibrate), PPARd (GW0742 and L165041) or PPARg (TZ) agonists (10 nM–100 mM) for up to 6 days, before the biomass of the culture was determined by methylthiazolyldiphenyl-tetrazolium bromide colorimetric assay. When cultures were treated with p5 mM PPAR agonists there was no significant difference in population growth, except with PPARg agonists, which showed a small growth inhibition. When cultures were treated with 45 mM of PPAR agonists, the PPARd and PPARg agonists caused cell death.
PPAR agonist effects on differentiation of NHU cells The differentiation potential of PPAR agonists on NHU cells was assessed by treating cells with PPARa (clofibrate) or PPARg (TZ and RZ) agonists (0.1–5 mM) for 24 h and determining expression of differentiationassociated markers at 6 days. In some experiments, the autocrine EGF receptor loop was inhibited by the use of specific inhibitors against the EGF receptor tyrosine kinase (PD153035 1 mM) or downstream signalling cascades including ERK (UO126 5 mM; PD98059
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Table 1.
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Expression of differentiation-associated markers by NHU cell cultures
Marker
Control
TZ/PD153035
0.001870.0001 0.002870.0004
0.204070.0026 0.436370.0363
Phospho_ERK
CK13
CK20
Claudin 3
UPK1a UPK2
NHU cells treated with TZ and the EGF receptor inhibitor, PD153035, for 6 days, as previously described (Varley et al., 2004b) expressed a more differentiated phenotype. This is illustrated by indirect immunofluorescence for phospho-ERK, CK13, CK20 and claudin 3 and by quantitative realtime PCR for UPK1a and UPK2 transcripts (mean of n ¼ 3, 7S.D. relative to GAPDH (Southgate et al., 2007)).
10 mM) or PI3-kinase (LY294002 1 mM) (Varley et al., 2005). We found that the PPARa agonist, clofibrate, did not induce differentiation in NHU cells. However, when NHU cells were treated with the PPARg agonists TZ or RZ (1 mM), there was a switch from a non-differentiated squamous phenotype (CK14+, CK13, CK20) to a terminally differentiated transitional phenotype (CK14, CK13+, CK20+) (Varley et al., 2004a) (Table 1). For effective induction of differentiation by PPARg agonists, it was mandatory that EGF receptor signalling was blocked. In this context, we demonstrated that PPARg was phosphorylated on a serine residue by ERK, resulting in inhibition of PPARg translocation to the nucleus (Varley et al., 2004b). This is in agreement with previous observations (Adams et al., 1997; Camp and Tafuri, 1997). Similar experiments were carried out to assess the effect of PPAR agonists on the expression of UPKs and claudins. PPARg activation by TZ or RZ induced expression of UPK1a and UPK2 genes when the EGF receptor was inhibited (Varley et al., 2004b) (Table 1).
We also showed that these same experimental conditions induced expression of the tight junction proteins claudin 3, claudin 4 and claudin 5. In all cases, maximal effect was achieved when NHU cells were treated in the presence of TZ and PD153035 (Varley et al., 2006). These effects were dependent on PPARg activation, as confirmed by the abrogation of CK13, UPK2, and claudins 3, 4 and 5 induction when experiments were performed in the presence of PPARg antagonists (1 mM GW9662 or 1 mM T0070907) and/or PPARg siRNA (Varley et al., 2004a, 2006). This suggests that PPARg agonists activate a programme of gene expression changes associated with urothelial terminal cytodifferentiation.
Regulation of PPARs in the urothelium We postulated that natural PPAR agonist(s) would be excreted in the urine to induce terminal differentiation in the superficially exposed cells of the urothelium. To address this hypothesis, a study was performed using a cohort of patients with end-stage renal disease who
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Table 2.
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Summary of expression of differentiation markers in human urothelium and NHU cells
Cl ¼ claudin, OD ¼ occludin, CK ¼ cytokeratin, UPK ¼ uroplakin, IHC ¼ immunohistochemistry.
excreted very little, if any, urine. If the hypothesis was correct there should be low/no expression of urothelial differentiation markers in these anuric patients. We demonstrated that the anuric patients expressed a normal intensity and distribution of urothelial differentiation-associated markers (Stahlschmidt et al., 2005). This suggested that the natural PPARg agonist was not secreted in the urine, but is potentially produced endogenously by the urothelium or is circulated via the serum. In the latter case, this could lead to sequestering of high concentrations of systemic PPAR agonists in the urothelium.
Concluding remarks Our studies have demonstrated that high concentrations of PPARd and PPARg agonists are growth
inhibitory and induce cell death in NHU cells, but these pro-apoptotic effects are likely to be mediated via PPAR-independent mechanisms (Chopra et al., submitted). Of the three PPAR isotypes expressed in human urothelium, it appears that PPARg plays a critical role in mediating urothelial cytodifferentiation, whereas the functions of PPARa and PPARd remain to be elucidated. Cross-talk between PPARg and the signalling pathways downstream of autocrine EGF receptor activation may have a role in regulating the switch from a differentiated urinary barrier to a highly regenerative epithelial tissue; this provides a potential mechanism in urothelial cell carcinogenesis. Further work is required to identify the nature and origin of the natural agonist and to determine whether PPARa/g dual agonists might modulate the proliferation/differentiation balance.
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Acknowledgements The authors are grateful to York Against Cancer for funding.
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