Androgen receptor is overexpressed in boys with severe hypospadias, and ZEB1 regulates androgen receptor expression in human foreskin cells

INTRODUCTION: ZEB1 is overexpressed in patients with severe hypospadias. We examined the interaction between ZEB1 and the androgen receptor (AR) in vitro and the expression of AR in boys with hypospadias. RESULTS: ZEB1 and AR colocalize to the nucleus. Estrogen upregulated ZEB1 and AR expression. Chromatin immunoprecipitation (ChIP) demonstrated that ZEB1 binds to an E-box sequence in the AR gene promoter. AR expression is higher in subjects with severe hypospadias than those with mild hypospadias and control subjects (P < 0.05). ZEB1 physically interacts with AR in human foreskin cells. DISCUSSION: AR is overexpressed in patients with severe hypospadias. Environmental estrogenic compounds may increase the risk of hypospadias by facilitating the interaction between ZEB1 and AR. METHODS: Hs68 cells, a fibroblast cell line derived from neonatal human foreskin, were exposed to 0, 10, and 100 nmol/l of estrogen, after which the cellular localization of ZEB1 and AR was assessed using immunocytochemistry. To determine if ZEB1 interacted with the AR gene, ChIP was performed using ZEB1 antibody and polymerase chain reaction (PCR) for AR. Second, AR expression was quantified using real-time PCR and western blot in normal subjects (n = 32), and subjects with mild (n = 16) and severe hypospadia (n = 16).