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First published online 30 March 2004
doi: 10.1242/jcs.01042

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Role of PPAR and EGFR signalling in the urothelial terminal differentiation programme

¹ Jack Birch Unit of Molecular Carcinogenesis, Department of Biology, University of York YO10 5YW, UK
² Cancer Research UK Clinical Centre, St James's University Hospital, Leeds LS9 7TF, UK
³ Department of Pathology, St James's University Hospital, Leeds LS9 7TF, UK
⁴ GlaxoSmithKline Pharmaceuticals, The Frythe, Welwyn, Hertfordshire, UK

View larger version (70K): [in a new window]   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 PPAR and RXR. (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.

View larger version (26K): [in a new window]   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%.

View larger version (43K): [in a new window]   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).

View larger version (40K): [in a new window]   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 PPAR activation by pre-treatment with the PPAR 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%.

View larger version (54K): [in a new window]   Fig. 5. (A) Effect of PD153035 and TZ on the localisation of PPAR. NHU cells were seeded at 2x105 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 PPAR, 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 PPAR (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 PPAR-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 PPAR was detected using specific antibodies and enhanced chemiluminescence.

View larger version (43K): [in a new window]   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%.