Oestradiol decreases colonic permeability through oestrogen receptor beta-mediated up-regulation of occludin and junctional adhesion molecule-A in epithelial cells

Abstract

Oestradiol modulates paracellular permeability and tight junction (TJ) function in endothelia and reproductive tissues, but whether the ovarian hormones and cycle affect the paracellular pathway in the intestinal epithelium remains unclear. Oestrogen receptors (ERs) are expressed in intestinal epithelial cells, and oestradiol regulates epithelium formation. We examined the effects of oestrous cycle stage, oestradiol benzoate (EB), and progesterone (P) on colonic paracellular permeability (CPP) in the female rat, and whether EB affects expression of the TJ proteins in the rat colon and the human colon cell line Caco-2. In cyclic rats, CPP was determined through lumen-to-blood (51)Cr-labelled EDTA clearance, and in Ussing chambers for dextran permeability. CPP was also examined in ovariectomized (OVX) rats treated with P or EB, with and without the ER antagonist ICI 182,780, or with the selective agonists for ER beta (propyl pyrazole triol; PPT) or ER beta (diarylpropionitrile; DPN). In oestrus rats, CPP was reduced (P < 0.01) relative to dioestrus. In OVX rats, EB dose-dependently decreased CPP, an effect mimicked by DPN and blocked by ICI 182,780, whereas P had no effect. Oestradiol increased occludin mRNA and protein in the colon (P < 0.05), but not zona occludens (ZO)-1. Further, EB and DPN enhanced occludin and junctional adhesion molecule (JAM)-A expression in Caco-2 cells without change in ZO-1, an effect blocked by ICI 182,780. These data show that oestrogen reinforces intestinal epithelial barrier through ER beta-mediated up-regulation of the transmembrane proteins occludin and JAM-A determining paracellular spaces. These findings highlight the importance of the ER beta pathway in the control of colonic paracellular transport and mucosal homeostasis.

Figure 1. Effect of oestrous cycle on paracellular permeability

A, in vivo⁵¹Cr-EDTA recovery in urine 24 h after injection into the colon of rats in follicular and luteal phase. B, in vitro paracellular FITC-dextran flux measured for 1 h in Ussing chambers in colon segments from oestrus and dioestrus rats. Bars are means ±s.e.m. Statistical significance was assessed by Student's t-test.

Figure 3. Dose effect of oestradiol on paracellular permeability in OVX rats and involvement of ERα and ERβ

A, dose–response study of oestradiol benzoate (EB, 1 μg to 5 mg (kg BW)⁻¹ day⁻¹ for 5 days, s.c.) on paracellular FITC-dextran flux measured for 1 h in colon segments mounted in Ussing chambers. Values are means ±s.e.m. of triplicate measurements in 5–8 rats per group (P < 0.05, ANOVA). B, effects of a 5 day treatment with the selective ligand agonist for ERα (PPT, 4 mg (kg BW)⁻¹ day⁻¹, s.c.), ERβ (DPN, 4 mg (kg BW)⁻¹ day⁻¹, s.c.) on colonic paracellular permeability in Ussing chambers. OVX rats treated with vehicle DMSO in olive oil (Ve) were used as controls. Values are means ±s.e.m. of triplicate measurements in 7 rats per group. **P < 0.01 and ns (not significant) vs. Ve.

Figure 2. Effect of ovariectomy, progesterone, oestradiol and ICI 182,780 on paracellular permeability

FITC-dextran flux measurements for 1 h in Ussing chambers in colon segments from sham operated rats (sham oestrus), and ovariectomized rats treated for 5 days with empty s.c. implants (OVX) or with implants filled with P (OVX+P) or EB (OVX+EB) or EB with daily s.c. injection of the ER antagonist ICI 182,780 (2 mg (kg BW)⁻¹ day⁻¹) (OVX+EB+ICI). Bars are means ±s.e.m. P < 0.001, ANOVA; *P < 0.05, **P < 0.01 vs. sham oestrus, ^aP < 0.001 vs. OVX or P group of rats; ns: not significant (Bonferroni post hoc test).

Figure 4. Effects of oestradiol on occludin and ZO-1 expression in the colon of OVX rats

A, representative Western blot lanes for occludin and ZO-1 from the same protein extract, and quantitative representation of corresponding protein contents from 7 independent experiments. Note that EB significantly increased occludin protein without change in ZO-1 amount. B, RT-PCR results for occludin, ZO-1, and GAPDH mRNA using total RNA from colon lysates of 6 independent experiments. In OVX rats 8 h after a single EB injection (5 mg kg⁻¹), EB significantly increased occludin mRNA, blocked by ICI 182,780 (EB+ICI), without change in the expression of mRNA for ZO-1. Values are means ±s.e.m. Ve: control vehicle. *P < 0.05 vs. corresponding control (Ve).

Figure 5. Effects of oestradiol, ERα and β agonists, and ICI 182,780 on occludin, JAM-A and ZO-1 expression in Caco-2 cells

The expression of occludin (A), JAM-A (B), and ZO-1 (C) proteins and mRNA were examined by Western blotting (upper panels) and RT-PCR (lower panels), with representative band images and corresponding densitometric analysis. Caco-2 cells were treated for 8 h with ethanol vehicle (C), or with EB (10 nm), DPN (100 nm) or PPT (100 nm), in the presence or absence of ER antagonist ICI 182,780 (ICI, 10 μm). Note that EB like DPN, but not PPT, significantly increased mRNA and protein levels of occludin (A) and JAM-A (B), without effect on ZO-1 expression (C). Both EB and DPN effects on occludin and JAM-A expression were blocked by ICI. Each bar represents mean ±s.e.m. from 6 to 7 independent experiments. P= 0.01, ANOVA; **P < 0.001, *P < 0.05, and ns (not significant) (Bonferroni post hoc test) vs. control cells (C).

Figure 6. Immunofluorescence detection of occludin and JAM-A in Caco-2 cells

Cell monolayers were exposed for 8 h to ethanol vehicle (Control), EB (10 nm), DPN (100 nm), or PPT (100 nm), in the presence or absence of ER antagonist ICI 182,780 (ICI, 10 μm). A, occludin: note that DPN like EB, but not PPT, increases occludin staining at the level of epithelial cell membranes, and ICI added 1 h before EB or DPN treatment blocked this effect. In confocal images in the x–z plane, note that DPN enhances occludin immunostaining at the apical cell-to-cell contact of epithelial cells compared to control cells, and this effect was abrogated in the presence of ICI 182,780. B, JAM-A, representative images of immunostaining with or without DPN treatment and ICI 182,780, and x–z plane confocal images showing modulation of epithelial JAM-A staining at apical sites. Scale bars = 25 μm.