Prostaglandin E2 via steroidogenic factor-1 coordinately regulates transcription of steroidogenic genes necessary for estrogen synthesis in endometriosis

Abstract

Context: Products of at least five specific steroidogenic genes, including steroidogenic acute regulatory protein (StAR), which facilitates the entry of cytosolic cholesterol into the mitochondrion, side chain cleavage P450 enzyme, 3beta-hydroxysteroid-dehydrogenase-2, 17-hydroxylase/17-20-lyase, and aromatase, which catalyzes the final step, are necessary for the conversion of cholesterol to estrogen. Expression and biological activity of StAR and aromatase were previously demonstrated in endometriosis but not in normal endometrium. Prostaglandin E2 (PGE2) induces aromatase expression via the transcriptional factor steroidogenic factor-1 (SF1) in endometriosis, which is opposed by chicken-ovalbumin upstream-transcription factor (COUP-TF) and Wilms' tumor-1 (WT1) in endometrium.

Objective: The aim of the study was to demonstrate a complete steroidogenic pathway leading to estrogen biosynthesis in endometriotic cells and the transcriptional mechanisms that regulate basal and PGE2-stimulated estrogen production in endometriotic cells and endometrium.

Results: Compared with normal endometrial tissues, mRNA levels of StAR, side chain cleavage P450, 3beta-hydroxysteroid-dehydrogenase-2, 17-hydroxylase/17-20-lyase, aromatase, and SF1 were significantly higher in endometriotic tissues. PGE2 induced the expression of all steroidogenic genes; production of progesterone, estrone, and estradiol; and StAR promoter activity in endometriotic cells. Overexpression of SF1 induced, whereas COUP-TFII or WT1 suppressed, StAR promoter activity. PGE2 induced coordinate binding of SF1 to StAR and aromatase promoters but decreased COUP-TFII binding in endometriotic cells. COUP-TFII or WT1 binding to both promoters was significantly higher in endometrial compared with endometriotic cells.

Conclusion: Endometriotic cells contain the full complement of steroidogenic genes for de novo synthesis of estradiol from cholesterol, which is stimulated by PGE2 via enhanced binding of SF1 to promoters of StAR and aromatase genes in a synchronous fashion.

Figure 1

In vivo (tissue) mRNA levels of steroidogenic genes (real-time PCR). mRNA levels of the steroidogenic genes including the mitochondrial cholesterol transporter, StAR (STAR); the enzymes P450scc (CYP11A1), HSD3B2, P450c17 (CYP17A1), and P450arom (CYP19A1); and the transcription factor SF1 were found to be strikingly higher in tissues of endometriosis (n = 13) compared with endometrium (n = 13). On the other hand, mRNA levels of the SF1 homolog gene, LRH1, were higher in endometrial tissue. E-IUM, Endometrium; E-OSIS, endometriosis.

Figure 2

mRNA levels of steroidogenic genes in stromal cells isolated from normal eutopic endometrium vs. endometriosis incubated in the absence or presence of PGE₂ (10⁻⁷ m). PGE₂ significantly stimulated mRNA levels of each steroidogenic gene except for HSD3B1 in endometriotic stromal cells (E-OSIS; n = 6). The mRNA levels of these genes in normal endometrium (E-IUM; n = 6) were very low regardless of the absence or presence of PGE₂.

Figure 3

Hormone concentrations in media conditioned by stromal cells isolated from endometrium (n = 3) or endometriosis (n = 3) incubated in the absence or presence of PGE₂ (10⁻⁷ m), 22(R)-OH-cholesterol (22R; 10 μm), or LDL (50 μg/ml). 22(R)-OH-cholesterol can diffuse into the mitochondria without a requirement for StAR. LDL was added in an attempt to increase the availability of cholesterol. PGE₂ stimulated the levels progesterone (A), estrone (B), and estradiol (C) in media from endometriosis-derived stromal cells. The addition of 22R-OH-cholesterol or LDL did not modify this effect of PGE₂ significantly, except for the enhancing effects of LDL on estrone levels.

Figure 4

Time and dose-dependent induction of StAR mRNA and protein by PGE₂ in endometriotic stromal cells. PGE₂ stimulated StAR mRNA (A) and protein levels (B) in a time- and dose-dependent manner in endometriotic stromal cells. StAR protein in the last lane in the upper panel B was detected in endometriotic stromal cells treated for 48 h with dibutyryl cAMP. mRNA or protein levels of the house-keeping genes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and actin, were used as loading controls.

Figure 5

Regulation of StAR promoter activity in endometriotic and endometrial cells. A, PGE₂ stimulated the activity of the −885 bp StAR promoter fused to the luciferase (LUC) reporter gene in both endometriotic and endometrial cells, whereas it did not stimulate luciferase activity of the empty vector. *, P value for endometriotic cells; **, P value for endometrial cells. B, A vector overexpressing a transcription factor was cotransfected together with the −885 bp StAR-luciferase (LUC) into endometriotic or endometrial stromal cells. C/EBPβ, C/EBPα, SF1, or LRH1 induced StAR promoter activity in both cell types. On the other hand, WT1 or COUP-TFII inhibited StAR promoter activity. *, P value for endometriotic cells comparing an expression vector with the empty vector; **, P value for endometrial cells.

Figure 6

ChIP assay demonstrating coordinate binding activity of transcription factors to StAR and P450arom promoters. In endometriotic (E-OSIS) stromal cells, PGE₂ induced binding of SF1 to both StAR and P450arom promoters, whereas very low SF1 binding was observed in endometrial (E-IUM) stromal cells. On the other hand, the inhibitory transcription factors COUP-TFII and WT1 were associated with StAR and P450arom promoters more avidly in endometrial cells compared with endometriotic cells. Note that PGE₂ reduced binding of COUP-TFII or WT1 in endometriotic or endometrial cells. The association of the control protein, nonacetylated histone H3, to chromatin did not vary with cell type or treatment. No binding activity was observed after chromatin was immunoprecipitated with nonspecific IgG.