The progesterone receptor coactivator Hic-5 is involved in the pathophysiology of endometriosis

Abstract

Endometriosis is an estrogen-dependent disorder primarily associated with pelvic pain and infertility in up to 10% of women of reproductive age. Recent studies suggest that resistance to progesterone action may contribute to the development and pathophysiology of this disorder. In this study we examined the in vivo and in vitro expression and function of one progesterone receptor (PR) coactivator, Hic-5, in human endometrium and endometrial stromal fibroblasts (hESFs) from 29 women with and 30 (control) women without endometriosis. Hic-5 was highly expressed in stromal, but not epithelial, cells in women without endometriosis, in a cycle-dependent manner. In contrast, Hic-5 expression was not regulated during the menstrual cycle in hESFs from women with endometriosis and was significantly reduced in hESFs from women with vs. without disease. Hic-5 mRNA expression throughout the cycle in endometrium from control women, but not those with endometriosis, correlated with expression of PR. Hic-5 mRNA in hESFs was significantly up-regulated in control but not endometriosis hESFs after treatment in vitro with 8-bromoadenosine-cAMP for 96 h but only modestly after 14 d of progesterone treatment. Hic-5 silencing did not influence cAMP-regulated gene expression but affected genes regulated solely by progesterone (e.g. DKK1 and calcitonin). Together the data suggest that the proposed progesterone resistance in endometrium from women with endometriosis derives, in part, from impaired expression of the PR coactivator, Hic-5, in endometrial tissue and cultured endometrial stromal fibroblasts.

Figure 1

Relative expression of Hic-5 mRNA in human endometrial tissue throughout the menstrual cycle in women without (white bars) and with (shaded bars) endometriosis. Same letters indicate statistical significance: a, P = 0.02; b, P = 0.03; c, P = 0.01. Significance accepted at P ≤ 0.05.

Figure 2

Immunofluorescent analysis of Hic-5 protein in human endometrium throughout the menstrual cycle. A, Samples from women without endometriosis, B, samples from women with endometriosis. S, Stroma; M, myometrium. Magnification, ×200.

Figure 3

A, Relative expression of mRNA for IGFBP1, PRL, FOXO1A, and calcitonin in eutopic endometrium from women with (shaded bars) and without (white bars) endometriosis. B, Relative expression of mRNA for PRA/B and PRB in eutopic endometrium from women with and without endometriosis. Gene expression is normalized to expression of the house keeping gene, RPL19, mRNA. Same letters (a–d) indicate statistical significance. Significance accepted at P ≤ 0.05. Note different scale on y-axes (different relative expression of different genes).

Figure 4

Expression of Hic-5 mRNA in hESFs decidualized with 10 nm E₂ per 1 μm P₄ for 14 d, normalized to 14 d control (A) or with 0.5 mm cAMP for 96 h, normalized to 96 h control (B). Significance accepted at P ≤ 0.05.

Figure 5

Effects of silencing Hic-5 mRNA on select gene expression in hESFs treated for 96 h with or without 0.5 mm 8-bromoadenosine-cAMP. A, Hic-5 mRNA. B, Paxillin mRNA. C, IGFBP1 mRNA. D, PRL mRNA. E, FOXO1A mRNA. F, PRA/B mRNA. G, PRB mRNA. H, SST mRNA. I, DKK1 mRNA. J, Calcitonin mRNA. siHic-5, hESFs transfected with Hic-5 siRNA; siNeg, hESFs transfected with scrambled siRNA (negative control). All experiments were done with four subject samples, in triplicates. Data are expressed as fold change to siNeg vehicle (sterile distilled H₂O) treated for 96 h. Same letters (a–c) indicate statistical significance. Significance accepted at P ≤ 0.05.

Figure 6

Effects of silencing Hic-5 mRNA on DKK1 (A) and calcitonin (B) gene expression in hESFs treated with 10 nm E₂ per 1 μm P₄ for 48 h. Insert, Analysis of Hic-5 mRNA demonstrating transfection efficacy. *, Significance accepted at P ≤ 0.05. siHic-5, hESFs transfected with Hic-5 siRNA; siNeg, hESFs transfected with scrambled siRNA (negative control).