Suppression of COUP-TFII by proinflammatory cytokines contributes to the pathogenesis of endometriosis

Abstract

Context: Endometriosis is one of the most common gynecological diseases in women with a prevalence rate of approximately 10%. Chronic pelvic inflammation has been observed in patients with endometriosis and is associated with disease severity. However, how pelvic inflammation promotes endometriosis progression remains unknown.

Objective: The objective of the study was to investigate the regulatory network of proinflammatory cytokines in endometriosis progression.

Design, settings, and patients: Immunostaining of human endometrial (n = 21) and endometriotic (n = 36) sections, quantitative RT-PCR, Western blotting, chromatin immunoprecipitation, and luciferase reporter assays in primary culture human endometrial stromal cells were performed. Autologous transplantation of uterine endometrium from control chicken ovalbumin upstream promoter-transcription factor II [(COUP-TFII) flox/flox] and uterus-specific COUP-TFII knockout mice was performed.

Results: Expression of COUP-TFII was significantly reduced in endometriotic stroma. Reduction of COUP-TFII in endometriotic stromal cells was mediated by proinflammatory cytokines including IL-1β, TNF-α, and TGF-β1 via a common effector, microRNA-302a. Treatment with these proinflammatory cytokines increased the expression of microRNA-302a, which targets the 3'untranslated region of COUP-TFII to cause its down-regulation. Intriguingly, down-regulation of COUP-TFII in endometrial stromal cells resulted in de-repression of cyclooxygenase-2 (COX-2). Further investigation demonstrated that COUP-TFII directly binds to COX-2 promoter to inhibit its transcription. Forced expression of COUP-TFII inhibited IL-1β-induced COX-2 up-regulation, whereas the knockdown of COUP-TFII augmented this effect.

Conclusion: Because overexpression of COX-2 has been demonstrated to be a master regulator in endometriosis progression, our data demonstrate the critical function of proinflammatory cytokines and the COUP-TFII regulatory gene network in the progression of endometriosis.

Figure 1.

COUP-TFII is down-regulated in endometriotic stromal cells. A, Levels of COUP-TFII and COUP-TFI mRNA in normal endometria (n = 21) and endometriotic tissues (n = 36) determined by quantitative RT-PCR. 18S ribosomal RNA was quantified as an internal control. *, Significant difference from normal at P < .05 by Student's t test. B, Representative Western blot image (upper panel) and quantitative results (lower panel, n = 6) show levels of COUP-TFII protein in eutopic (Eu) and ectopic (Ec) tissues obtained from the same individuals with endometriosis. *, Significant difference from eutopic tissues at P < .05 by Student's t test. C and D, Representative immunohistochemistry pictures (C) and quantitative results (D) show immunoreactivity of COUP-TFII proteins in normal endometria (N, n = 21), eutopic tissue (Eu, n = 36), and ectopic lesion (Ec, n = 36). Staining results were as follows: 0, negative; 1, weak staining; 2, strong staining. E, Representative Western blot (left panel) and quantitative results (right panel, n = 5) show levels of COUP-TFII proteins in paired eutopic endometrial stromal cells (Eu) and ectopic endometriotic stromal cells (Ec) from patients with endometriosis. *, Significant difference from eutopic stromal cells at P < .05 by Student's t test.

Figure 2.

COUP-TFII is down-regulated by proinflammatory cytokines elevated in peritoneal fluid of individuals with endometriosis. A, Representative Western blot (upper panel) and quantitative results (lower panel, n = 4) show levels of COUP-TFII protein in normal endometrial stromal cells treated with peritoneal fluids (PFs) derived from women with [PF(Ec)] and without [PF(N)] endometriosis. *, Significant difference from control (Con). B–D, Representative Western blots (upper panel) and quantitative results (lower panel, n = 4) show levels of COUP-TFII in normal endometrial stromal cells treated with different doses of IL-1β (B), TNF-α (C), or TGF-β1 (D) for 24 hours. *, Significant difference from vehicle controls. E, Representative Western blot (upper panel) and quantitative result (lower panel, n = 4) show levels of COUP-TFII in normal endometrial stromal cells treated with IL-1β (IL; 0.01 ng/mL), TNF-α (TN; 0.1 ng/mL), TGF-β1 (TG; 0.01 ng/mL) or in combination (I/N/G) for 24 hours. *, Significant difference from vehicle control. F, Representative Western blots (upper panel) and quantitative results (low panel, n = 4) show levels of COUP-TFII in normal endometrial stromal cells treated with IL-1β in the presence or absence of recombinant IL-1β receptor antagonist (rIL1-ra; 200 ng/mL) for 24 hours. *, Significant difference from vehicle control (Con); #, significant difference from PBS-treated group. G, Representative Western blots (upper panel) and quantitative results (low panel, n = 4) show levels of COUP-TFII in normal endometrial stromal cells treated with TGF-β1 in the presence or absence of TGF-β receptor inhibitor (SB431542; 10 μM) for 24 hours. *, Significant difference from vehicle control (Con); #, significant difference from PBS-treated group.

Figure 3.

COUP-TFII is down-regulated by miR-302a. A, Representative Western blot images show levels of COUP-TFII in normal endometrial stromal cells treated with IL-1β (upper panel), TNF-α (middle panel), and TGF-β1 (lower panel) in the presence or absence of actinomycin D (Act D; 1 μM) or cycloheximide (CHX; 1 μM) for 24 hours. These experiments were repeated three times using different batches of cells. B, Representative Western blot (upper panel) and quantitative result (lower panel) show levels of COUP-TFII in normal endometrial stromal cells treated with miR-302a mimics (Mimic). *, Significant difference from control. C, Schematic drawing (upper panel) shows construct of luciferase reporter system containing COUP-TFII 3′UTR. The two seed regions of miR-302a (WT) and mutated sequences (Mut1, Mut2, and Mut1+2) are shown. Lower panel shows relative luciferase activity in endometrial stromal cells transfected with wild-type or miR-302a seed sequence-mutated reporter constructs cotransfected with control miRNA (Con) or miR-302a. *, Significant difference from control miRNAs; #, significant difference compared with wild-type constructs treated with miR-302a. D and E, Quantitative results show miR-302a levels in endometrial stromal cells treated with different doses of IL-1β (D) or 1 ng/ml IL-1β for different lengths of time (E). *, Significant difference from control. F, Quantitative results show levels of miR-302a in normal endometrial stromal cells (Nor; n = 21) and endometriotic stromal cells (Endo; n = 36). *, Significant difference from normal cells by a Student's t test.

Figure 4.

Expression of COX-2 is inhibited by COUP-TFII. A, Representative Western blot (left panel) and quantitative results (middle and right panel) show levels of COX-2 protein in paired eutopic and ectopic endometrial stromal cells from patients with endometriosis (n = 6). *, Significant difference from eutopic endometrial stromal cells. B, Representative Western blot (left panel) shows level of COUP-TFII protein in normal endometrial stromal cells treated with siRNA against COUP-TFII (si-COUP-TFII) or scrambled siRNA. Right panel shows quantitative result of COX-2 mRNA quantified by quantitative RT-PCR (n = 5). *, Significant difference from scrambled control. C, Representative Western blot (left panel) shows level of COUP-TFII and COX-2 proteins in ectopic endometriotic stromal cells transfected with human COUP-TFII plasmid or empty vector. Right panel shows quantitative result of COX-2 protein (n = 4). *, Significant difference from empty vector control. D, Quantitative result shows COX-2 promoter activities in normal endometrial stromal cells transfected with empty vector or COUP-TFII containing plasmids (n = 5). pXP1, basic vector without human COX-2 promoter (−916 to +23); Wt, Cox-2 promoter with wild-type COUP-TFII binding site; Mut, COX-2 promoter with COUP-TFII binding site mutated. Wild-type (Wt) and mutated (Mut) sequences of COUP-TFII binding region in the COX-2 promoter were shown at the bottom of the figure. E, Representative picture (left panel) and quantitative result (right panel) show results of COUP-TFII binding to COX-2 promoter determined by ChIP assay. COX-2, PCR products amplified by specific primer; Distal, PCR products amplified by primer pairs 2000 bp upstream of predicted COUP-TFII binding site; aromatase, PCR products amplified by aromatase primers as a positive control. *, Significant difference from nonspecific control using mouse IgG.

Figure 5.

COUP-TFII negatively regulates COX-2 promoter activity. A, Representative Western blot (upper panel) and quantitative results (lower panel) show levels of COX-2 protein in COUP-TFII-specific siRNA (siCII#1 and siCII#2) or scrambled siRNA transfected normal endometrial stromal cells treated with or without IL-1β for 24 hours. *, Significant difference between groups as indicated. B, Representative Western blot (upper panel) and quantitative results (lower panel) show levels of COX-2 protein in COUP-TFII plasmids or empty vector-transfected ectopic endometriotic stromal cells treated with or without IL-1β for 24 hours. *, Significant difference between groups as indicated.

Figure 6.

miR-302a mediates IL-1β-induced COUP-TFII down-regulation and COX-2 up-regulation. A, Representative Western blot (left panel) and quantitative results (middle and right panels, n = 5) show levels of COUP-TFII and COX-2 proteins in normal endometrial stromal cells treated with miR-302a mimics (mimic) or scrambled miRNA (NC). *, Significant difference compared with control. B, Quantitative results show ratios of COX-2, COUP-TFII, and miR-302a in six pairs of endometrial stromal cells and endometriotic stromal cells from patients with endometriosis. Levels of COX-2, COUP-TFII, and miR-302a in eutopic endometrial stromal cells were set as 0.0 after logarithmic transformation. C, Representative Western blot (upper panel) and quantitative results (lower panels, n = 5) show levels of COUP-TFII and COX-2 proteins in normal endometrial stromal cells treated with IL-1β with or without pretreatment with recombinant IL-1β receptor antagonist (rIL-1ra). *, Significant difference from control; #, significant difference from PBS-treated cells. D, Representative Western blot (upper panel) and quantitative results (lower panels, n = 3) show levels of COUP-TFII and COX-2 proteins in normal endometrial stromal cells treated with IL-1β with or without pretreatment with miR-302a inhibitor (302a-Inh). *, Significant difference from control; #, significant difference from IL-1β-treated cells. E, Representative immunohistochemistry pictures show levels of COX-2 in donor endometrial tissues (donor) and endometriotic lesions (Endo) after injection of COUP-TFII-knockout or intact endometrial tissues into the peritoneal cavity of receipt mice for 4 weeks. COUP-TFII F/F, wild-type COUP-TFII; COUP-TFII −/−, uterine stroma-specific COUP-TFII knockout; E, epithelial cells; S, stromal cells.