Insulin resistance (IR) disrupts ovarian functions in polycystic ovary syndrome (PCOS). The contributing factors remains elusive. High mobility group box 1 (HMGB1), a damage‐associated molecular pattern molecule, has been shown to be related to IR and autophagy, respectively, in peripheral tissues. Here, we investigated whether increased HMGB1 contributes to IR in granulosa cells of PCOS patients via induction of aberrant autophagy. Results showed that HMGB1 abundance in the follicular fluid was significantly increased with enhanced autophagy in granulosa cells in PCOS patients with IR. HMGB1 exacerbated autophagy in granulosa cells as evinced by increased LC3B II/I ratio and ATG7 as well as decreased p62, the markers for autophagy. Concurrently, HMGB1 impaired insulin sensitivities by attenuating the abundance of insulin receptor substrate‐1, Akt phosphorylation, GLUT4 translocation, and glucose uptake in granulosa cells, which were reversed by blocking autophagy pathways with siRNA‐mediated knockdown of ATG7 or with chloroquine and bafilomycin A1, the lysosome inhibitors. In conclusion, our results indicate that increased HMGB1 contributes to IR development in granulosa cells of PCOS patients, which is associated with exacerbation of autophagy by HMGB1. Control of HMGB1 production may be benefical for the improvement of insulin sensitivity in granulosa cells in PCOS.

Glucocorticoids and proinflammatory cytokines may be involved in parturition by stimulation of prostaglandin production in the fetal membranes. The actions of glucocorticoids on the fetal membranes are amplified by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which converts biologically inactive cortisone into active cortisol. Whether glucocorticoids and proinflammatory cytokines regulate the expression of 11beta-HSD1 in the major prostaglandin-producing tissue, amnion, thus further increasing prostaglandin production, is not known. In this study, we found that term amnion fibroblasts had higher 11beta-HSD1 mRNA and activity per cell than amnion epithelial cells. Both isoforms of glucocorticoid receptor (alpha and beta) were expressed in amnion fibroblasts and epithelial cells. Quantitative real-time PCR showed that dexamethasone (0.01-1 microm) dose-dependently induced 11beta-HSD1 mRNA expression only in amnion fibroblasts but not in amnion epithelial cells. The induction of 11beta-HSD1 mRNA expression by dexamethasone was blocked by glucocorticoid receptor antagonist RU486. Although only a modest increase or no change in 11beta-HSD1 mRNA expression and activity was observed with IL-1beta (10 ng/ml) or TNFalpha (10 ng/ml) treatment, respectively, in amnion fibroblasts, combination of dexamethasone with either IL-1beta or TNFalpha significantly enhanced the induction of 11beta-HSD1 mRNA expression and activity, as compared with dexamethasone treatment alone. With prior induction of 11beta-HSD1 expression by dexamethasone, cortisone caused more prostaglandin E2 production in the amnion fibroblast. This study suggests that glucocorticoids can positively induce 11beta-HSD1 expression in amnion fibroblasts, an effect further strengthened by proinflammatory cytokines.

This study examines the regulation of major enzymes in prostaglandin E₂ (PGE₂) synthesis by glucocorticoids in separate cultures of human amnion epithelial and fibroblast cells at term. Cytosolic phospholipase A₂ (cPLA₂), cytosolic PGES (cPGES), and microsomal PGES (mPGES) mRNA were expressed at similar levels in both cell types, whereas a greater prostaglandin H synthase type 2 (PGHS-2) mRNA expression was observed in amnion fibroblasts than in epithelial cells. Amnion fibroblasts produced 50-fold more PGE₂ per cell than epithelial cells. Dexamethasone (0.01–1 μm) increased PGE₂ production in amnion fibroblasts in a concentration-dependent manner but did not affect PGE₂ production in amnion epithelial cells. Both mRNA and protein expression of cPLA₂ and PGHS-2 but not cPGES and mPGES were increased in a dose-dependent manner by dexamethasone (0.01–1 μm) in amnion fibroblasts. Induction of cPLA₂ and PGHS-2 mRNA by dexamethasone was blocked by RU486. Dexamethasone did not affect PGHS-2, cPGES, and mPGES mRNA expression in amnion epithelial cells. In conclusion, amnion fibroblasts express a higher level of PGHS-2 mRNA and produced more PGE₂ per cell than amnion epithelial cells at term of human pregnancy. Glucocorticoids increase PGE₂ production only in the amnion fibroblasts mainly through induction of cPLA₂ and PGHS-2 expression.

The amount of cortisol available to its receptors is increased by the pre-receptor enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) which converts cortisone to cortisol. We examined the molecular mechanisms of the feedback effect of cortisol on 11β-HSD1 mRNA expression in human amnion fibroblasts. Our data showed that cortisol-induced 11β-HSD1 mRNA expression dose dependently in amnion fibroblasts, which could be completely blocked both by the mRNA transcription inhibitor 5,6-dichlorobenzimidazole riboside and by the glucocorticoid receptor (GR) antagonist RU486, and partially blocked by global inhibition of CCAAT/enhancer-binding proteins (C/EBPs) with transfection of C/EBP-specific dominant-negative expression CMV500 plasmid (AC/EBP) into the cells. Likewise, the induction of the promoter activity by cortisol could also be completely blocked by RU486 and partially by AC/EBP transfection. Progressive 5′ deletion of the 11β-HSD1promoter located the region responsible for cortisol’s induction within −204 bp upstream to the transcription start site. Specific nucleotide mutations of the putative glucocorticoid responsive element or CCAAT in this promoter region attenuated the induction by cortisol. Moreover, chromatin immunoprecipitation assay and electrophoretic mobility shift assay showed that GR and C/EBPα but not C/EBPβ could bind this promoter region upon cortisol stimulation of amnion fibroblasts. In conclusion, we demonstrated that GR and C/EBPα were involved in cortisol-induced 11β-HSD1 mRNA expression via binding to 11β-HSD1 promoter in amnion fibroblasts, which may cast a feed-forward production of cortisol in the fetal membranes at the end of gestation.