Molecular Mechanism Of The Induction Of Hexose-6-phosphate Dehydrogenase By Cortisol In Human Amnion Fibroblasts

Glucocorticoids are crucial in fetal lung function and the initiation of labor. Human fetal membranes express abundant11β-hydroxysteroid dehydrogenase type1(11β-HSD1) at the end of gestation, which reduces the inactive metabolites cortisone to active glucocorticoids cortisol. The reductase activity of11β-HSD1depends on the availability of the cofactor reduced nicotinamide adenine dinucleotide phosphate (NADPH) derived from the enzymatic activity of hexose-6-phosphate dehydrogenase (H6PD). H6PD catalyzes the first two steps of the pentose phosphate pathway to generate NADPH from NADP+within the endoplasmic reticulum (ER). Lavery et al. showed that H6PD knock-out mice had no11β-HSD1reductase activity, and our previous studies have demonstrated there exists a feed-forward induction of11β-HSD1by cortisol in human fetal membranes. Based on these findings, we hypothesize that there might be a concurrent induction of H6PD and11β-HSD1expression by cortisol in the fetal membranes. To address this hypothesis, we firstly examined the distribution of H6PD in human fetal membranes, and then investigated the role of H6PD in the reductase activity of11β-HSD1, the effect on H6PD expression by cortisol and the related molecular mechanism in cultured primary human amnion fibroblasts.Methods and Results1. Immunohistochemical staining revealed that the distribution of H6PD and11β-HSD1protein was strikingly similar in the fetal membranes. H6PD and11β-HSD41proteins were found in the decidual cells, chorion trophoblasts, and amnion epithelial and fibroblast cells, suggesting that these two proteins are possibly functionally correlated in these cells. When the expression of H6PD protein was knocked-down by H6PD siRNA transfection, the reductase activity of11β-HSD1was significantly attenuated in human amnion fibroblasts. These findings suggested that H6PD protein played a role in the regulation of the reductase activity of11β-HSD1.2. By using quantitative real time-PCR (qt-RT-PCR) and Western blotting, we demonstrated that cortisol increased H6PD mRNA and protein levels in a concentration-dependent manner in human amnion fibroblasts. The induction of H6PD mRNA and protein by cortisol could be obviously attenuated by glucocorticoid receptor (GR) antagonist RU486, indicating GR was involved in the induction of H6PD expression by cortisol. Moreover, the induction of H6PD mRNA by cortisol could be significantly reduced with the protein synthesis inhibitor CHX treatment in human amnion fibroblasts, suggesting that de novo protein synthesis was required in the induction of H6PD mRNA expression by cortisol.3. Further studies with qt-RT-PCR and Western blotting indicated that cortisol increased p300mRNA and protein levels in a dose-dependent manner in human amnion fibroblasts, while this induction could be significantly attenuated by GR antagonist RU486. In addition, the induction of H6PD mRNA and protein by cortisol was significantly attenuated in the presence of histone acetyltransferase (HAT) p300inhibitor C646, suggesting the induction of H6PD expression by cortisol was dependent on the induction of p300.4. We further studied whether GR and p300interact in the induction of H6PD expression by cortisol in human amnion fibroblasts.(1) Coimmunoprecipitation assay showed that GR protein could be detected in the nuclear protein complex precipitated by p300antibody upon the stimulation of human amnion fibroblasts with cortisol incubation, suggesting GR could interact with p300protein in human amnion fibroblasts.(2) Bioinformatic analysis showed there were several putative GREs existed in the proximal promoter region of H6PD.(3) ChIP assay showed that the binding of p300and GR to the H6PD promoter could be significantly increased by cortisol.(4) With the ChIP assay we also found cortisol could induce the enrichment of H3K9ac to the same promoter region that p300and GR binding to which could be blocked by p300HAT inhibitor C646. All these results indicate that GR and p300as well as the acetylation of H3K9by p300are involved in the induction of H6PD gene expression by cortisol.ConclusionsH6PD is expressed in human fetal membranes, and it plays a crucial role in the regulation of the reductase activity of11β-HSD1. The expression of H6PD can be induced by cortisol, and the induction requires the participation of GR and p300as well as the acetylation of H3K9by p300.