Fig 1: Epithelial GR modulates gene expression in intestinal mucosa from DSS-treated mice. (A) Top five upstream regulators and canonical pathways from gene expression microarray in GRiKO DSS vs. GRflox DSS (Comparison 1), GRiKO DSS vs. GRiKO Ctol (Comparison 2), and GRflox DSS vs. GRflox Ctol (Comparison 3) using Ingenuity Pathway Software (IPA); (B) Venn diagram of the overlap in significant gene probes determined from 3 comparisons; (C) Number of gene probes either induced (up) or repressed (down) by each comparison; (D) Gene probes that showed statistically significant differences for any of the three comparisons and that affected immune response and steroidogenesis pathway were selected and grouped into five categories: (1) steroidogenesis, (2) cytokines, (3) M1-like markers, (4) M2-like markers and (5) re-epithelialization. The heatmap indicates increased (red/upregulated) or decreased (green/downregulated) expression levels. Two-way ANOVA (p < 0.05); (E) Nr5a2 transcript (relative to Ppib) from colonic samples from each mice group. one-way ANOVA with Tukey’s post-test.** p < 0.01, *** p < 0.001, **** p < 0.0001.
Fig 2: LRH-1 and GRβ are upregulated in intestinal mucosa from patients with impaired GC response. (A) Transcript levels (relative to PPIB) of NR5A2, NR3C1 and NR3C1 isoform β in intestinal mucosa from healthy controls (H), responders (R), dependent (D) and refractory (Rf) UC patients by RTqPCR. Transcript levels (relative to PPIB) of NR5A2, NR3C1 and NR3C1 isoform β; differences between group medians were assessed using Kruskal–Wallis with Dunn’s post-test and Pearson correlation performed. Each point represents an individual value: non-inflamed (circles), responders (squares), dependents (triangles) and refractory (diamonds), * p < 0.05; ** p < 0.01. (B) Representative immunoreactivity of LRH-1, GR and GRβ isoform (red) in paraffin-embedded PFA-fixed sections of intestinal mucosa biopsies taken from healthy individuals and active UC patients according to their GC response by tissue array. E-cadherin (Ecad) co-localization (green) was used for epithelium recognition. In the zoomed image indicated with dashed line and yellow arrow: positive nuclear stain; solid line: negative nuclear stain. Objective 60×, scale bar 30 μm.
Fig 3: Dexamethasone induces cortisol and LRH-1, enhancing GR-binding to the NR5A2 gene promoter in colonocytes. (A) The CCD841CoN colonocyte cell line was stimulated with dexamethasone (100 nM) for 3, 6 and 24 h, and cortisol levels were determined in supernatants by ELISA; (B) Cells upon Dex and/or GR antagonist RU-486 (10 µM) treatment for 8 h were used to evaluate mature (left) and precursor (right) NR5A2 mRNA by RT-qPCR, and protein content by immunoblot; (C) Band quantification was performed by densitometry analysis and normalized to β-actin protein (% of control); (D) Cells stimulated with Dex for 2 h were analyzed for binding of GR to the NR5A2 promoter by ChIP-qPCR; For each immunoprecipitated sample, the statistical analyses were performed with respect to an unrelated region from the GAPDH gene promoter. Results are expressed as % input ± SEM. One-way ANOVA with Tukey post-test was performed. ** p < 0.01, *** p < 0.001, **** p < 0.0001. C: control, Dex: dexamethasone, RU: RU-486, GRE: glucocorticoid responsive element, IgG: immunoglobulin G, nt: nucleotides; n = 4.
Fig 4: LRH-1 upregulated in intestinal mucosa lamina propria from DSS-treated GRiKO mice. (A) Representative images of LRH-1 and total GR (red) immunofluorescent staining with co-localization of E-cadherin (green) as epithelial marker in intestinal mucosa from vehicle and DSS-treated GRflox and GRiKO mice. Hoechst was used for nuclear counterstaining. In the LRH-1 zoomed image: dashed line and/or yellow arrows: positive nuclear stain; solid line: negative nuclear stain. In the GR zoomed image, yellow arrows: positive nuclear stain; dashed line: epithelial outline. Objective 60×. Scale bar 30 μm. (vehicle n = 4 GRflox and 3 GRiKO, DSS-treated n = 4 GRflox and 6 GRiKO); (B) Integrated density from immunofluorescence images calculated from epithelial crypts; and (C) LP cells from intestinal mucosa of vehicle and DSS-treated GRflox and GRiKO mice, with (D) a representative image showing LRH-1 stain in LP from DSS-treated groups. Dashed line: epithelial outline. * p < 0.05, ** p < 0.01.
Fig 5: Dexamethasone induces LRH-1 in primary human intestinal organoids. Colonic organoids from healthy tissue were stimulated with 100 nM Dex for 24 h. (A) Organoids under light microscopy 4×. Scale bar 1000 μm. (B) Organoids were analyzed for TSC22D3 (left), NR5A2 (center) and NR3C1 (right) transcript levels, relative to 18s rRNA. Paired t-test, * p < 0.05; ** p < 0.01; n = 3. Representative immunoreactivity of (C) LRH-1 and (D) GR with corresponding (E) IgG isotype control in paraffin-embedded PFA-fixed sections of colonic organoids stimulated with 100 nM Dex for 24 h. Percentage of positive and negative nuclear staining for (C, bottom) LRH-1 and (D, bottom) GR. E-cadherin (E-cad, green) was used for epithelium counterstain and Hoescht as nuclear stain, **** p < 0.0001. In zoomed image: whole organoid for context purposes. Dashed line: positive nuclear stain; dotted line: negative nuclear stain. Objective 40×. Scale bar 20 μm.
Supplier Page from Novus Biologicals, a Bio-Techne Brand for Recombinant Human LRH-1/NR5A2 GST (N-Term) Protein