ABSTRACT: Hypospadias is a penile developmental abnormality that may partly result from in utero exposure to estrogenic compounds. Expression of activating transcription factor 3 (ATF3) is elevated in human hypospadic tissue, and in utero exposure to ethinyl estradiol (17-EE) causes ATF3 upregulation in a hypospadias mouse model. We investigated the effects of in vitro exposure to EE on ATF3 expression and promoter activity in human foreskin fibroblasts using immuncytochemistry, quantitative PCR, Western blot, and the luciferase activity assay. In addition, we compared ATF3 expression in a human fetus with penoscrotal hypospadias with expression in a fetus without hypospadias. Immunocytochemistry showed peak positive staining at 2 hours after 0.5-3 hours of EE treatment (10-7 M). Western blot showed significantly increased ATF3 protein expression (p=0.006) after EE exposure. ATF3 mRNA, as evaluated using RT-PCR and TaqMan Realtime PCR, also increased (p=0.146). In addition, the luciferase activity assay showed that ATF3 promoter activity was significantly enhanced after 1 hour EE exposure (p<0.0001). Urethral tissue from the fetus with hypospadias was positive for ATF3; tissue from the normal genital tubercle was not. Thus, EE upregulates ATF3 expression in fibroblasts in vitro, consistent with our previous results with human tissue and in vivo mouse models. ATF3 is involved in the TGF-beta epithelial-mesenchymal signaling pathway, and its involvement in hypospadias suggests that ATF3 plays a role in development of this anomaly as a result of exposure to estrogenic compounds. Its potential involvement in other manifestations of developmental endocrine disruption are worth investigating.
Pediatric and Developmental Pathology 04/2007; · 0.99 Impact Factor