PPARgamma-regulated tight junction development during human urothelial cytodifferentiation

Abstract

Urothelial barrier function is maintained by apical membrane plaques and intercellular tight junctions (TJ). Little is known about the composition and regulation of TJ expression in human urothelium. In this study, we have characterised the expression of TJ components in situ and their regulation in an in vitro model of differentiating normal human urothelial (NHU) cells. In normal ureteric urothelium in situ, there was a differentiation-associated profile of claudins 3, 4, 5, 7, ZO1 and occludin proteins. Proliferating NHU cells in vitro expressed predominantly claudin 1 protein and transcripts for claudins 1-5 and 7. Following induction of differentiation by pharmacological activation of PPARgamma and blockade of EGFR, there was de novo expression of claudin 3 mRNA and protein and downregulation of claudin 2 transcription. There was also a massive increase in expression of claudin 4 and 5 proteins which was due to inhibition of proteasomal degradation of claudin 4 and consequential stabilisation of the claudin 5 heterodimerisation partner. NHU cell differentiation was accompanied by relocalisation of TJ proteins to intercellular junctions. The differentiation-associated development of TJ formation in vitro reflected the stage-related TJ expression seen in situ. This was distinct from changes in TJ composition of NHU cells mediated by increasing the calcium concentration of the medium. Our results imply a role for PPARgamma and EGFR signalling pathways in regulating TJ formation in NHU cells and support the hypothesis that TJ development is an integral part of the urothelial differentiation programme.

Fig. 1

A: Immunohistochemical localisation of tight junction proteins in ureteric urothelium. Expression of claudin 3, 4, 5, 7, ZO1, occludin, E-cadherin and UPIIIa was determined by immuno-peroxidase in paraffin wax-embedded sections of normal human ureter. Sections were counterstained with haematoxylin. Scale bar = 100 μm for all except UPIIIa where scale bar = 50 μm. B: Schematic diagram of the localisation of the tight junction proteins in human urothelium.

Fig. 2

RT-PCR of claudin mRNA expression in NHU cells and the effect of TZ and PD153035 on claudin expression. NHU cells were pretreated for 24 h in the absence or presence of TZ (1 μM) and incubated in media with or without PD153035 (1 μM). Media were changed every 3 days with fresh PD153035 added. RNA was exacted at 1, 3 and 6 days, cDNA was generated and RT-PCR was performed as outlined in the methods, using claudin primers and GAPDH as the internal control. The PCR products were electrophoresed on a 2% agarose gel and visualised using ethidium bromide. Similar results were obtained with a second independent NHU cell line.

Fig. 3

Effect of PPARγ ligands (TZ and RZ) and PD153035 on claudin protein expression in NHU cells. NHU cell were treated for 24 h with 1 μM TZ (A) or RZ (B), in the presence or absence of PD153035 (1 μM) and protein was extracted at 0, 1, 3 and 6 days for cells treated with TZ (A) and at 6 days for cell treated with RZ (B). Media were replaced every 3 days with fresh PD153035 added. Cell extracts (20 μg) were resolved on 12.5% SDS–polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were incubated with pre-titrated primary antibodies for 16 h at 4°C, as indicated. Bound antibody was detected with fluorescent-conjugated secondary antibodies and quantified using an infrared imaging system (LI-COR Odyssey). β-actin was used as an internal loading control. Similar results were obtained in three (TZ) and two (RZ) independent NHU cell lines. C: NHU cells were pretreated for 3 h with or without the PPARγ antagonists GW9662 or T0070907 (0.1, 1.0, 5.0 μM) and PD153035 (1 μM) as indicated. Next, cells were treated in the presence or absence of TZ (1 μM) for 24 h, followed by treatment with or without PD153035 (1 μM) for 6 days, all in the presence of PPARγ antagonists (as indicated). After 6 days, protein was extracted and 20 μg was separated per lane on a 12.5% SDS–polyacrylamide gel, transferred onto a nitrocellulose membrane and incubated 16 h at 4°C in the presence of the primary antibody, as indicated, and visualised as above. D: NHU cells were treated with or without TZ (1 μM) for 24 h in the presence of PPARγ siRNA (10, 50 or 100 nM) or GW9662 (5 μM) or T0070907 (5 μM) as indicated. Next, media were replaced with or without PD153035 and GW9662 or T0070907 as indicated and again on day 3. After, 6 days protein was extracted and separated on SDS–PAGE as outlined in (C).

Fig. 4

Influence of TZ and PD153035 on the localisation of the tight junction proteins. NHU cells were seeded at 2 × 10⁵ cells/ml onto glass slides, allowed to adhere and treated with or without TZ (1 μM) for 24 h. Subsequently, cells were treated in the absence or presence of PD153035 (1 μM) and slides were fixed after 1, 3 and 6 days. Media were replaced every 3 days with fresh PD153035 added. Immunofluorescence was performed using the antibodies indicated. The images depicted in the figure are from slides fixed at 6 days. Scale bar, 30 μm. Similar results were obtained in a further two independent NHU cell lines.

Fig. 4

Influence of TZ and PD153035 on the localisation of the tight junction proteins. NHU cells were seeded at 2 × 10⁵ cells/ml onto glass slides, allowed to adhere and treated with or without TZ (1 μM) for 24 h. Subsequently, cells were treated in the absence or presence of PD153035 (1 μM) and slides were fixed after 1, 3 and 6 days. Media were replaced every 3 days with fresh PD153035 added. Immunofluorescence was performed using the antibodies indicated. The images depicted in the figure are from slides fixed at 6 days. Scale bar, 30 μm. Similar results were obtained in a further two independent NHU cell lines.

Fig. 5

Involvement of proteasomes in stabilisation of claudin 4. NHU cells were treated with or without TZ (1 μM) for 24 h followed by treatment in the absence or presence of PD153035 (1 μM) or MG132 (12.5 μM) for 0, 9, 24 and 48 h. Cell extracts (20 μg) were resolved on 10% SDS–polyacrylamide gels and transferred onto nitrocellulose membranes. Membranes were incubated with titrated primary antibodies, fluorescent-conjugated secondary antibodies and the signal quantified by infrared imaging, as described in Figure 3. β-actin was used as an internal loading control. NHU cells treated with TZ, followed by PD153035 for 72 h, were used as a positive control.

Fig. 6

Immunoprecipitation of claudin 4. NHU cells were treated in the presence or absence of TZ (1 μM) for 24 h followed by treatment with or without PD153035 (1 μM) for 6 days. Cell extracts (500 μg) were rotated with an antibody for claudin 4, an irrelevant antibody (ERK), or no antibody on spin-columns, as indicated. As a positive control, 20 μg of whole cell lysate was used. The immunoprecipitated protein was eluted from the column and resolved on SDS–PAGE by Western blot analysis. Membranes were incubated with antibodies against claudin 3, 5 or 7, fluorescent-conjugated secondary antibodies and the signal quantified by infrared imaging, as described in Figure 3.