Figure 1.
Effect of SEGRAs on GR binding and nuclear translocation.
(A) Immunofluorescence analysis for GR location in Caco-2 cells. Cells were treated for 3 h with Dex [1 µM], CpdA or ZK216348 [10 µM]. DAPI staining was used for visualisation of the cell nuclei. (B) 100 cells were randomly chosen, analysed and the percentage of nuclear GR calculated. Western blot analysis for translocation of GR in (C) Caco-2/GR and (D) HeLa cell lysates after 3 h cultivation with Dex or SEGRAs. One representative blot of three is shown. Densitometric analysis of GR is normalised to β-actin (cytoplasmic extract) or lamin (nuclear extract), respectively. Bars represent mean ± S.E.M., n = 3, *P<0.05; **P<0.01 relative to vehicle.
Figure 2.
Effect of SEGRAs on transcriptional activity of GR.
Cells were pre-treated with Dex [0.1–1 µM], CpdA or ZK216348 [1–10 µM] for 1 h before cultivation in the co-presence or absence of TNF-α/IL-1β [0.5 nM] and harvested for cytoplasmic and nuclear protein extract preparations. (A) The activity of NF-κB in nuclear extracts of IEC-6 cells was analysed by EMSA using an end-labelled NF-κB consensus oligonucleotide. The EMSA is representative for two experiments giving similar results. (B) NF-κB activity in nuclear extracts of Caco-2 cells was measured by transcription factor assay for p65. (C) Western blot analysis for nuclear p65, or cytoplasmic Iκ-Bα expression in TNF-α-/IL-1β-stimulated Caco-2 cell extracts. One representative blot of three is shown. (D) IL-8 content of cell culture supernatants was quantified by ELISA. Caco-2/GR cells were pre-treated with Dex [0.1–1 µM], CpdA or ZK216348 [1–10 µM] for 1 h before cultivation for 16–24 h in the co-presence or absence of TNF-α/IL-1β [0.5 nM]. Bars indicate mean ± S.E.M., n = 4, *P≤0.05; **P≤0.01; ***P≤0.001 relative to TNF-α/IL-1β treatment. (E) Relative Luciferase Activity of Caco-2/GR cells transfected with the glucocorticoid response element (GRE)-driven luciferase construct (pGRE-luc) and pSV-40 Renilla after 24 h of treatment with or without Dex [1 µM], CpdA or ZK216348 [1–10 µM] or co-presence of the GR agonist RU-486 [10 µM] given as 1 h pre-treatment. Bars indicate mean ± S.E.M., n = 3, ***P≤0.001 relative to vehicle, ###P≤0.001 relative to Dex- or ZK216348-treatment, respectively.
Figure 3.
Effect of SEGRAs on cell apoptosis.
Apoptotic effects of (A) Dex- [0.1–10 µM] or (B) CpdA- and ZK216348- [1–20 µM] treatment on IEC-6 cells was evaluated after 24 h by Annexin-/7-AAD staining and flow cytometric analysis. (C) Cytotoxicity of CpdA or ZK216348 [1–25 µM] treatment on Caco-2 cells was tested by Cytotoxicity Detection (LDH release) kit. Bars indicate mean ± S.E.M., n = 3, *P≤0.05, **P≤0.01, ***P≤0.001 relative to vehicle. (D) Activation of Caspase-3 in Caco-2 cells after 24 h of incubation with Dex, CpdA and ZK216348 [1–25 µM]. Results are expressed as a percentage of control. Bars indicate mean ± S.E.M., n = 3, *P≤0.05, **P≤0.01, ***P≤0.001 relative to vehicle.
Figure 4.
Effect of SEGRAs on intestinal epithelial cell restitution and proliferation.
IEC-6 cells were wounded and cultured in the presence of (A) Dex [0.1–1 µM] or (B) CpdA or (C) ZK216348 [1–20 µM] for 24 h. Cell migration was assessed using an in vitro migration assay. Bars indicate mean values of remaining wounded area ± S.E.M., n = 3, *P≤0.05, **P≤0.01 relative to control. (D) BrdU incorporation assay was used for determination of IEC-6 cell proliferation after 24 h incubation with Dex or SEGRAs [1–20 µM]. Results indicate mean ± S.E.M., n = 3, ***P≤0.001 relative to vehicle. (E) Representative experiments illustrating the effects of Dex and SEGRAs on intestinal epithelial cell restitution. Cntr (0 h) represents cells immediately after wounding, picture cntr (24 h) and others show wounds 24 h after cultivation with Dex, CpdA or ZK216348. Dotted line indicates original margin of wound (Magnification ×100).
Table 1.
Effect of SEGRAs on HaCaT cell restitution.
Figure 5.
Effect of SEGRAs on TGF-β - mediated intestinal epithelial cell restitution.
Wounded IEC-6 cells were cultured for 24 h in the presence of Dex, CpdA or ZK216348 with or without co-presence of (A) TGF-β or (B) selective inhibitor of activin receptor-like kinase (ALK) receptor SB431542. Cell migration was assessed using an in vitro migration assay. Bars indicate mean values of remaining wounded area ± S.E.M., n = 3, *P≤0.05, **P≤0.01, ***P≤0.001 relative to vehicle, ###P≤0.001 relative to Dex (C) Relative luciferase activity of HEK293T cells transfected with the reporter gene construct pSBE4-luc construct after 24 h incubation with TGF-β (5 ng/ml), Dex or SEGRAs. IEC-6 cells were treated with Dex, CpdA or ZK216348 for the indicated time periods. (D) TGF-β peptide levels in cell culture supernatants were determined by ELISA. (E) TGF-β mRNA expression was monitored by qPCR and normalised against β-actin. Bars represent mean ± S.E.M., n = 3, *P≤0.05 relative to vehicle.
Figure 6.
Effect of SEGRAs on EGF/ERK1/2/MAPK pathway - mediated intestinal epithelial restitution.
Wounded IEC-6 cells were cultured for 24 h in the presence of Dex [1 µM], CpdA or ZK216348 [10 µM] with or without co-presence of (A) EGF (5 nM) or (B) specific inhibitor of MEK1/2 phosphorylation PD98059 [50 µM]. Cell migration was assessed using an in vitro migration assay. Bars indicate mean values of remaining wounded area ± S.E.M., n = 3, *P≤0.05, **P≤0.01, ***P≤0.001 relative to vehicle, ###P≤0.001 relative to Dex. (C) Western blot analysis for pERK1/2 and ERK2 in Caco-2/GR cell lysates treated with Dex [1 µM], CpdA or ZK216348 [10 µM] for 1 h. One representative blot of three is shown and densitometric analysis of pERK1/2 is normalised to ERK2. (D) MKP-1 or Annexin-1 mRNA expression in Caco-2/GR cells after 6 h of treatment was monitored by qPCR and normalized against β-Actin. Western blot analysis for (E) Annexin-1 or (F) EGF-R protein expression in Caco-2/GR cells treated for 24 h or 1 h respectively with Dex or SEGRAs in the presence or absence of RU-486 [10 µM]. One representative blot of three is shown. Bars indicate mean ± S.E.M., n = 3, *P≤0.05, **P≤0.01, ***P≤0.001 relative to vehicle.