Materials

Troglitazone was provided as a gift by Parke-Davis Pharmaceutical Research (Ann Arbor, USA) and rosiglitazone and GW9662 were provided as a gift from GlaxoSmithKline (Worthing, UK). The inhibitors PD153035, PD98059, LY294002, U0126 and SB203580 were obtained from Calbiochem-Novabiochem Biosciences (Nottingham, UK). [³²P]CTP was purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK).

Tissues

The collection of surgical specimens was approved by the relevant Local Research Ethics Committees and had full patient consent, as required.

Surgical specimens of normal urothelium were obtained from patients with no history of urothelial dysplasia or malignancy. Tissues were collected in Hanks' balanced salt solution (HBSS) containing 10 mM HEPES pH 7.6 and 20 KIU aprotinin (Trasylol, Bayer plc, Newbury, UK), as described previously (Southgate et al., 1994; Southgate et al., 2002). Representative pieces of each tissue sample were processed into paraffin wax for histology and immunohistochemistry. The remaining sample was cut into approximately 1 cm² pieces, placed into HBSS (Ca²⁺ and Mg²⁺ free) supplemented as above and containing 0.1% (w/v) EDTA, and incubated at 4°C overnight to release pure urothelial cell sheets. The isolated urothelium was used either to establish normal human urothelial (NHU) cell lines (see below) or for RNA or protein extraction.

Cell culture

NHU cell lines were established and maintained in keratinocyte serum-free medium (KSFM) containing bovine pituitary extract and epidermal growth factor at the manufacturer's recommended concentrations (Invitrogen, Paisley, UK) and supplemented with 30 ng/ml cholera toxin (Sigma, Poole, UK). These methods have been described in detail elsewhere (Southgate et al., 1994; Southgate et al., 2002). NHU cell lines were used for these studies between passages 3 and 5. For experiments, cells were seeded at 9×10⁵ cells/ml and allowed to attain approximately 70% confluency before treatment with PPARγ ligands and inhibitors. NHU cultures were maintained at 37°C in a humidified atmosphere of 5% CO₂ in air.

Ribonuclease protection assays

To extract RNA from cell monolayers, cells were solubilised in Trizol™ (Invitrogen, Paisley, UK). Isolation of RNA by chloroform extraction and isopropanol precipitation was performed as recommended by the manufacturer.

Part-length cDNA fragments of the coding region for human UPIa, UPIb, UPII, UPIII and GAPDH genes were cloned into pGEM-T Easy (Promega, Southampton, UK) as described previously (Lobban et al., 1998). GAPDH was used as an internal riboprobe control. ³²P-labelled antisense transcripts of the cDNAs of interest were generated from linearised plasmids using the In Vitro Transcription Kit (Promega), according to the manufacturer's protocol. After DNAse treatment, riboprobes were purified by passage through Chromaspin 30-DEPC columns (BD Biosciences Clontech UK, Oxford, UK).

Ribonuclease protection assays were performed using an RPAIII kit in accordance with the manufacturer's protocols (RPAIII kit, Ambion, UK). Approximately 2 fmol of each riboprobe were mixed and hybridised to 5 μg of total RNA. Products were separated on 5% denaturing polyacrylamide gels (Sequagel from Flowgen, Lichfield, UK), visualised by autoradiography and quantified by means of a phosphorimager (BioRad GS-525 Molecular Imager System, Hemel Hempstead, UK). The quantified uroplakin bands were normalised against the GAPDH signal, which was used to correct for loading efficiency. When there was no signal detected a blank was left when quantifying against control.

Immunofluorescence

Cells grown on slides were fixed in a 1:1 mixture of methanol and acetone, air-dried and incubated with titrated PPARγ antibody for 16 hours at 4°C, before washing and incubation in secondary antibody conjugated to Alexa 488 (Molecular Probes, supplied by Cambridge Bioscience, Cambridge, UK). 0.1 μg/ml Hoechst 33258 (Sigma-Aldrich Ltd., Poole, UK) was included in the penultimate wash in order to visualise nuclei. Slides were observed on an Olympus BX60 microscope under epifluorescence illumination.

Immunoprecipitations

Cells were lysed (50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 50 μg/ml aprotinin and 50 μg/ml leupeptin), extracted and assayed as outlined below. To 200 μg of protein, 10 μg PPARγ-agarose conjugate (Santa Cruz Biotechnology, supplied by Autogen-Bioclear UK, Calne) were added and the samples were rotated overnight at 4°C. Next day, the samples were centrifuged at 1000 g for 5 minutes at 4°C and the pellet retained. The pellet was washed four times in cold PBS, boiled in sample buffer (125 mM Tris, pH 6.8, 4% SDS, 0.01% bromophenol blue and 20% glycerol) before being resolved by SDS-PAGE, as outlined below.

Western blot analysis

Lysates were prepared by treating cells with lysis buffer [25 mM Hepes pH 7.4, 125 mM NaCl, 10 mM NaF, 10 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 0.2% (w/v) SDS, 0.5% (w/v) sodium deoxycholate acid, 1% (w/v) Triton X-100, 1 μg/ml aprotinin, 10 μg/ml leupeptin and 100 μg/ml phenylmethylsulfonyl fluoride]. Lysates were sheared by passing three times through a 21-gauge needle and left on ice for 30 minutes, before microcentrifugation at 10,000 g for 30 minutes at 4°C. The protein concentrations of supernatants were measured by the Bradford assay (Pierce, supplied by Perbio Science UK, Cheshire). Cell extracts were resolved on 10% SDS polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were incubated with primary antibodies for 16 hours at 4°C. Bound antibody was detected with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence using the ECL Detection Kit (Amersham Pharmacia).

Antibodies

Monoclonal antibody against PPARγ (clone E-8) and rabbit polyclonal antibody to RXRα (code D20) were obtained from Santa Cruz Biotechnology (supplied by Autogen-Bioclear UK). Rabbit polyclonal antibody to phospho-extracellular signal-regulated kinase (ERK) was obtained from Cell Signalling Technology (supplied by New England Biolabs UK, Hitchin, UK) and monoclonal anti-total ERK (clone 16) was from Transduction Laboratories (supplied by Becton Dickinson, Oxford, UK). Monoclonal anti-phosphoserine (clone PSR-45) was obtained from Sigma-Aldrich, Poole, UK.

Promoter analysis

The transcriptional start site for each uroplakin gene (NCBI accession numbers: UPIa, NM_007000; UPIb, NM_006952; UPII, AX259986 and UPIII, NM_006953) was retrieved from NCBI (www.ncbi.nlm.nih.gov) or by linking to UCSC Genome Browser (http://genome.ucsc.edu). A 2 kb region upstream of the transcriptional start site for each uroplakin was used to determine if there were any PPAR binding sites. Nine high-affinity PPARγ binding sites (Juge-Aubry et al., 1997) were used to construct a PPARγ-defined PPRE matrix in the MatDef programme of Genomatix suite (Quandt et al., 1995; Wolfertstetter et al., 1996) (Table 1). Using the PPARγ-defined PPRE matrix, the 2 kb regions of the uroplakin genes were analysed for PPRE binding sites in the MatInspector programme of Genomatix suite (Quandt et al., 1995). The outcome was that no PPARγ-PPRE binding sites were predicted on the positive strand of the uroplakins.

Statistical analysis

Comparisons between groups were analysed using a two-tailed Student's t-test. Differences were considered significant when P<0.05.

Expression of PPARγ and RXRα in human urothelial cells

Western blot analysis showed that PPARγ and RXRα proteins were expressed by human urothelium in situ and by all three independent NHU cell lines tested (Fig. 1A).

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Fig. 1.

(A) Western blot analysis of nuclear hormone receptor expression in freshly isolated human urothelium and cultured NHU cell lines. Protein lysates (40 μg/lane) extracted from human urothelium and from three independent NHU cell lines were loaded onto a 10% SDS-polyacrylamide gel, electrophoresed and transferred to nitrocellulose membrane. Membranes were probed with specific antibodies against PPARγ and RXRα. (B) Phase contrast morphology of NHU cells grown to 70% confluence and incubated in the absence or presence of TZ (1 μM or 100 μM, as indicated) for 48 hours. Bar, 100 μm.

Response of cultured NHU cells to troglitazone (TZ)

In normal serum-free growth medium, NHU cells grew as monolayers with a typical epithelioid cobblestone morphology (Fig. 1B). The addition of the PPARγ ligand, troglitazone (TZ) (1 μM) resulted in a dramatic change in morphology, with NHU cells forming rosettes of tear-shaped cells that morphologically resembled transitional epithelial cells in situ (Fig. 1B). At higher concentrations of TZ (>5 μM), extensive cell death was observed, with the cytoplasmic blebbing and nuclear fragmentation characteristic of an apoptotic response (Fig. 1B).

Effect of EGF and PPARγ ligands on uroplakin mRNA gene expression in NHU cells

The expression of the four uroplakin genes by cultured NHU cells was assessed by ribonuclease protection assay (RPA). In agreement with previous findings (Lobban et al., 1998), mRNA for UPIb was expressed by all NHU cell cultures, UPIa was usually negative, although detected weakly in some post-confluent cultures, and neither UPII nor UPIII mRNA were ever detected, irrespective of time in culture or degree of confluency. The presence or absence of exogenous EGF in the medium alone had no effect on the expression of uroplakin transcripts by NHU cells (Fig. 2).

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Fig. 2.

Ribonuclease protection assay (RPA) to quantify the effect of TZ and EGF on uroplakin mRNA expression in NHU cells. NHU cells were treated in the presence or absence of TZ (1 μM) and/or EGF (5 ng/ml) for the times indicated. Total RNA was extracted and 5 μg were hybridised with ³²P-labelled human UPII, UPIb and GAPDH cDNA probes. The samples were electrophoresed and the UP bands were quantified by means of a phosphorimager and normalised against the GAPDH signal, which was used to correct for loading efficiency. Maximum uroplakin expression was taken to be 100%.

The effect of the PPARγ agonist, TZ, was investigated. In the absence of exogenous EGF, expression of UPII mRNA was first detected after 3 days treatment with TZ (Fig. 2). In the presence of EGF (5 ng/ml), TZ induced de novo expression of UPII mRNA, but expression was delayed until 6 days after treatment and the maximum induction was threefold less compared with NHU cells treated with TZ in the absence of EGF (Fig. 2). The effects of TZ and EGF on UPII gene expression were mirrored by the induction of the UPIa gene, although the magnitude of the response was much weaker (data not shown). TZ also upregulated UPIb mRNA expression by up to fourfold above basal by day 3, and expression was enhanced by the absence of EGF (Fig. 2). No UPIII expression was detected under any conditions (data not shown).

Influence of EGFR signalling on PPARγ-mediated response

Although the effects of TZ on uroplakin gene expression were reproduced in at least ten independent NHU cell lines, the extent of the response was variable, even in the absence of exogenous EGF. To determine whether the response of NHU cells to TZ was modulated by autocrine activation of the EGFR, experiments were performed in which cells were treated with TZ in the presence of each of the potent EGFR inhibitors, PD153035 and AG1478. A concentration-dependent enhancement of the TZ-induced expression of UPII and UPIb genes was found with each of the two EGFR inhibitors (Fig. 3A, illustrated with PD153035).

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Fig. 3.

Ribonuclease protection assay (RPA) to quantify the effect of TZ and PD153035 on uroplakin mRNA expression in NHU cells. (A) NHU cells were treated for 24 hours with or without 1 μM TZ, before incubation for 4 days with the EGFR inhibitor PD153035 at the indicated concentrations. Medium containing appropriate inhibitor was replenished every 2 days. Total RNA was extracted and 5 μg were analysed by RPA to assess the relative uroplakin mRNA expression (maximum uroplakin expression assigned 100%), as described in Fig. 2. (B) Concentration-dependent effects of RZ and TZ on uroplakin mRNA expression in EGFR-inhibited NHU cells. NHU cells were pretreated for 24 hours with the indicated concentrations of RZ or TZ, before being incubated in medium containing PD153035 (1 μM). Total RNA was extracted from samples after 4 days and 5 μg were analysed by RPA to assess relative uroplakin mRNA expression, as described in Fig. 2. Maximum uroplakin expression was taken to be 100%. Note that PD153035 alone had no effect on UPII gene expression (B).

Observations were confirmed using a second PPARγ agonist, rosiglitazone (RZ) (Fig. 3B). The induction of uroplakin mRNA by the PPARγ agonists was dose dependent, with maximum induction of UPII mRNA at 0.5 to 1 μM TZ or 1 μM RZ in the presence of PD153035 (Fig. 3B).

Effective period of exposure to TZ

Initial experiments were performed in the constant presence of PPARγ agonist. However, further studies combining PPARγ activation with EGFR inhibition showed that UPII mRNA expression could be induced by just 2 hours exposure to TZ (1 μM), although maximal expression was found when cells were treated with TZ for 24 hours (Fig. 4A).

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Fig. 4.

(A) NHU cells were treated with TZ (1 μM) for the length of time indicated, the medium was changed and the cells were maintained in medium containing PD153035 (1 μM) for 4 days. The RNA was extracted and the RPA was performed and analysed as outlined in Fig. 2. (B) NHU cells were treated for 24 hours with TZ (1 μM) and then maintained in medium in the presence or absence of PD153035 (1 μM) for the times indicated. Uroplakin mRNA expression was quantified by phosphorimager analysis of the RPA hybridisation signal and normalised to the GAPDH signal, to correct for sample loading. Uroplakin expression in TZ-exposed NHU cells treated with PD153035 for 4 days was designated 100%. Diamonds, TZ+PD153035; squares, TZ-PD153035. The data is the mean + s.e.m. of three experiments performed on three independent NHU cell lines.^*P<0.005 TZ compared with TZ+ PD153035. (C) NHU cells were treated in the absence or presence of TZ (1 μM) for 24 hours before the medium was changed and cells were incubated in the presence or absence of PD153035 (1 μM) for 4 days. RPA was performed and quantified as outlined in (A). TZ+PD153035 was taken to be 100% for UPIb. (D) Inhibition of PPARγ activation by pre-treatment with the PPARγ antagonist GW9662. NHU cells were pretreated for 3 hours with GW9662 at the concentrations indicated, before being exposed to TZ (1 μM) for 24 hours and thereafter to PD153035 (1 μM), all in the continued presence of GW9662. After 4 days, total RNA was extracted and 5 μg were analysed by RPA to assess relative uroplakin mRNA expression, as described in (A). TZ+PD153035 was taken to be 100%.

Timecourse studies of NHU cells treated with TZ for 24 hours and maintained for up to 8 days in the presence of PD153035 (1 μM) showed maximal effects on the mRNA expression of UPII, UPIa and UPIb 4 days after treatment with TZ (Fig. 4B).

Using the optimised treatment regime confirmed that neither PD153035 nor TZ alone induced UPII gene transcription, but that NHU cells required both TZ and PD153035 to induce de novo UPII mRNA expression (Fig. 4C). UPIb mRNA expression was upregulated by TZ alone (fivefold), but not by PD153035 alone. UPIb mRNA expression was futher enhanced (twofold above TZ alone) when cells were treated with both TZ and PD153035 (Fig. 4C).

Effect of inhibiting PPARγ on the TZ-induction of UPII expression

To verify that the TZ-mediated induction of UPII expression was mediated by PPARγ activation, NHU cells were pretreated with the PPARγ antagonist, GW9662 (0-5 μM), before exposure to TZ and PD153035. GW9662 inhibited the TZ-mediated induction of UPII mRNA expression in a dose-dependent manner, with 5 μM resulting in 80% inhibition relative to the positive control (Fig. 4D).

Effects of EGFR signalling on PPARγ in NHU cells

When NHU cells were maintained in the presence of exogenous EGF, localisation of PPARγ was predominantly perinuclear and excluded from the nuclei (Fig. 5A). Treatment of NHU cells with PD153035 resulted in the translocation of PPARγ to the nucleus, irrespective of whether cells were treated with TZ. When cells were treated with TZ, without inhibition of EGFR signalling, the majority of cells showed perinuclear localisation of PPARγ. In cultures not treated with PD153035, occasional cells were observed with nuclear PPARγ localisation; this probably reflected downregulation of EGFR signalling in these cells by other mechanisms (e.g. contact inhibition).

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Fig. 5.

(A) Effect of PD153035 and TZ on the localisation of PPARγ. NHU cells were seeded at 2×10⁵ cells/ml onto glass slides, allowed to attach and treated for 4 hours with or without PD153035 (1 μM) in the presence or absence of TZ (1 μM). The slides were fixed and immunofluorescence was performed for PPARγ, with nuclei counterstained using Hoechst 33258. Bar, 100 μm. Western blot analysis was used to show the effect of EGFR inhibition on the phosphorylation of ERK (B) or PPARγ (C). NHU cells were treated with (+) or without (–) PD153035 (1 μM) for 4 hours. (B) Protein lysate (40 μg) from each sample was used to analyse phospho- and total ERK, as described in Materials and Methods. The data are representative of three separate experiments. (C) Protein lysate (200 μg) was used to immunoprecipitate with PPARγ-agarose conjugate (10 μg), as outlined in the Materials and Methods, before being resolved on an 10% SDS-PAGE and transferred to nitrocellulose; phospho-serine or PPARγ was detected using specific antibodies and enhanced chemiluminescence.

It is well known that EGF binding to its cognate receptors activates several signalling cascades, including the mitogen-activated protein kinase (MAPK), ERK. There was an 80% inhibition of phosphorylated ERK after 4 hours treatment of NHU cells with PD153035 (Fig. 5B). Immunoprecipitation showed that PPARγ was dephosphorylated by 70% within 4 hours of treatment with PD153035 (Fig. 5C).

Downstream EGFR signalling pathways

We used a range of kinase inhibitors to identify the EGFR signalling pathways involved in inhibiting PPARγ-mediated expression of the uroplakin genes (Fig. 6). In the presence of the MAPK kinase inhibitors PD98059 and U0126, or the phosphoinositide 3-kinase inhibitor LY294002, TZ induced UPII mRNA to a similar extent as the induction caused by TZ treated with PD153035. By contrast, there was no TZ-induced UPII mRNA expression in the presence of the p38 kinase inhibitor, SB203580. No additive effect was found on the induction of UPII mRNA levels when the cells were treated with TZ together with both PD98059 and LY294002, suggesting that both pathways acted on the same target phosphoprotein. These results suggest that the ERK and phosphoinositide 3-kinase pathways inhibit the ability of TZ to induce UPII gene expression (Fig. 6).

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Fig. 6.

Effect of kinase inhibitors on UPII mRNA expression. NHU cells were pretreated for 1 hour with an inhibitor: PD153035 (1 μM), PD98059 (5, 10, 25 μM), U0126 (2, 5, 10 μM), SB203580 (10 μM) or LY294002 (1 μM) and then for a further 24 hours with or without TZ (1 μM) and inhibitors, as indicated. The medium was replaced with inhibitors alone and replenished every 2 days. After 4 days, total RNA was extracted and 5 μg were analysed by RPA to assess relative UPII mRNA expression, as described in Fig. 2. The data are representative of three independent experiments. TZ+PD153035 was taken to be 100%.

Other inhibitors, including those against protein kinase C (bisindolylmaleimide I) and protein kinase A (H-89) had no effect on UPII mRNA induction by TZ (data not shown).