Estrogen induced downregulation of gene expression and cell biological processes critical for genital tubercle formation via DNA methylation

摘要

BackgroundHypospadias is one of the most common genital birthdefect, characterized by improper closing of the urethralfolds during development and resulting ectopic opening ofthe urethra. Xenoestrogen (XE) exposure increases therate of hypospadias in both animal models and in humans,in association with gene down regulation.[1,2] Whilemany genes (e.g. SHH, WNT5A, CTNNB1, HOXA13) arecritical for genital tubercle (GT) and urethral developmentin genetically deficient models, the role of such genes inresponse to estrogen exposure in GTs has been less studied.While XE exert known epigenetic effects, themechanism involved is not clear. This study examines theepigenetic effects of XE on GT developmental genes, aswell as on DNA methylation enzymes (DNMTs) involvedin epigenetic downregulation.Material and methodsThe BJ normal human foreskin fibroblast cell line wasused to model GT mesenchyme. They were stimulatedevery 24 hours with 100 nM of diethylstibestrol (DES) for6, 24, 48 and 120 hours +/- 2-dexoxy-5-azacytidine (aza), aDNMT inhibitor. Real-time PCR was performed to examineexpression of candidate genes (WNT5A, HoxA13,HoxA10, DNMT-1, -3Aand-3B, and others), normalizedto rpl19 or gapdh.ResultsDNMTs were all temporally upregulated significantly byDES treatment: 75% at 6 hours for DNMT3A (p2 fold at 120 hours for DNMT1 (p5fold at 6, 24 and 120 hours for DNMT3B. Hox andWNT5A gene expression was conversely downregulatedby DES and partially restored by aza. HoxA13was downregulated between 70-92% at 24, 48 and 120hours of DES treatment (p<0.05). Aza recoveredexpression of HoxA13 by 3-fold (p<0.04) by day 5. At48 hours, Wnt5A also showed downregulated expression,which was increased 5.6-fold by aza (p<0.02),though at other time points was not similarly affectedby DES.ConclusionsDES treatment activates the epigenetic machinery, viaincreased DNMT expression. This epigenetic response isaccompanied by a down regulation of WNT5A andHoxA13, genes critical for GT formation. This downregulationis dependent on DNA methylation as DNMT inhibitionwith aza partially restores HoxA13 and WNT5Aexpression. These results suggest that environmental XEmay act epigenetically to induce long-term alterations ingenes crucial for genital development.