Androgen signaling through the AR is critical for normal penile development. Diminished androgen signaling results in a spectrum of incompletely virilized external genitalia: complete androgen insensitivity results in external genitalia with a female phenotype and partial insensitivity results in ambiguous genitalia of varying degrees. Originally put forth by Alfred Jost, it has long been held that the female genitalia phenotype is the “default” pathways that occurs in the absent of androgen signaling. Recent studies have refined the Jost hypothesis and suggest that penile development is a balance of androgenic and estrogenic activity (14
). Although the cause of most cases of hypospadias is unknown, disruption of normal androgen signaling is thought to have a significant role. Estrogen is a potential disruptor of androgen signaling as epidemiologic studies have shown that boys who were exposed to elevated levels of estrogen in utero
have increased risk of hypospadias (4
This study has shown that AR is overexpressed in patients with severe hypospadias. We have also demonstrated that ZEB1 binds to the E-box domain of the AR promoter in human foreskin fibroblasts. It has previously been shown that estrogen increases both mRNA and protein levels of ZEB1 and that ZEB1 is overexpressed in patients with severe hypospadias (8
). Graham has shown that ZEB1 binds to the E-box domain on the AR promoter in breast cancer cell lines (13
). The interaction of ZEB1 and the AR reporter in widely disparate models suggests that ZEB1 upregulation of AR is a preserved molecular mechanism through which androgen signaling is regulated. However, to our knowledge, no other studies have examined the role of ZEB1 in androgen signaling.
ZEB1 has been extensively studied as a factor involved in epithelial to mesenchymal transition. It is a key factor in cancer progression during which epithelial cells lose expression of the intercellular molecule E-cadherin and thus become migratory and able to invade. ZEB1 is also a critical molecule during embryogenesis as it is expressed throughout the developing embryo and is necessary for survival (15
). This study has shown that ZEB1 binds to the AR promoter. We previously reported that estrogen increases ZEB1 expression and that ZEB1 is overexpressed in boys with severe hypospadias. Both ZEB1 and AR are expressed in the stratum basale of preputial skin. These results suggest that estrogen, through ZEB1, upregulates AR expression in patients with severe hypospadias.
Estrogen-driven ZEB1 upregulation of AR may provide additional understanding of how hypospadias develops. Hypospadias may arise from abnormal developmental events occurring either during hormone-independent formation of the ambisexual genital tubercle or subsequent hormone-dependent sex differentiation. Environmental exposure to estrogens is widespread and multiple estrogen-responsive genes (ATF3
) have been shown to be upregulated in patients with hypospadias relative to subjects with normal genitalia (7
). It is estimated that 50 million women take oral contraceptives (OCPs); 3–4% continue taking OCPs inadvertently after becoming pregnant (16
). It is unknown what effect this supraphysiologic dose of estrogen may have on the developing fetus; however, it is known that women who maintain soy-based diets, which are rich in phytoestrogens, during pregnancy have increased risk for having a male child with hypospadias (17
). Our finding that ZEB1, which is known to be estrogen-responsive, interacts directly with the AR raises the hypothesis that ZEB1 may be an important mediator of estrogen-driven aberrant androgen signaling in hypospadias.
In our study, AR expression levels increased in Hs68 cells as estrogen concentration increased and AR expression was higher in preputial skin of boys with severe hypospadias compared to control subjects and those with mild hypospadias. It seems counter-intuitive that AR would be upregulated in subjects with hypospadias, which would seem to be associated with decreased rather than overactive androgen signaling. However, male mice exposed prenatally to medroxyprogesterone, a synthetic progesterone, develop hypospadias and have elevated levels of AR (18
). However, neonatal exposure to high levels of estrogen downregulates AR in the testes, prostate, and seminal vesicles, with increasing concentrations of estrogen resulting in corresponding lower levels of AR expression (19
One explanation is that estrogen has differential effects on AR expression depending on tissue type and local environment. Both Hs68 cells and the human subject preputial tissue used in this study are derived from ectoderm, which may have a different response to estrogen stimulation than the mesoderm-derived gonads, prostate, and seminal vesicles. Another explanation is that AR expression is dependent on the timing of estrogen exposure. The studies that reported decreased AR expression after administration of estrogen all used rodent animal models exposed to estrogen after birth (19
). Hypospadias, however, occurs early in gestation as penis development occurs between 9 and 13 wk. It is possible that postnatal estrogen exposure has a suppressive effect on AR expression while prenatal exposure may increase AR, at least in preputial skin.
We believe the most likely explanation for the findings is that there are likely multiple points of interaction between AR-induced signaling and other sex steroid pathways during genital development. In mouse models, AR, estrogen receptor, and progesterone receptors are differentially expressed in the developing mouse genital tubercle and are responsive to exogenous estrogen (22
). Additionally, many of the environmental and pharmacologic compounds that have been associated with hypospadias signal through estrogen receptor and progesterone receptor in addition to or independent of AR (18
). The multiple points through which estrogenic compounds interact in the sex hormone signaling pathways involved in penile development and the emerging evidence that both androgens and estrogens are involved in male genitalia development strengthen the theory that estrogen-induced androgen signaling disruption is a significant molecular mechanism underlying hypospadias.
There are limitations of the Hs68 cell line used in this study. Because Hs68 cells are derived from the foreskin of a human male neonate, they might not reflect the gene expression profile or cellular properties of the developing urethral plate, which is derived from endoderm. Also, Hs68 cells are fibroblasts; hence, we cannot definitively state that estrogen increases ZEB1 expression in human foreskin epithelium in vitro
. However, because ZEB1 levels vary by tissue type, we felt it was of primary importance to perform the in vitro
studies on cells that originated from human neonatal male genital skin (33
). Furthermore, previous studies in which Hs68 cells were exposed to estrogen have supported findings in human tissue samples and mouse models, which is also what we observed in this study (34
Our findings that estrogen upregulates AR in vitro and that AR is overexpressed in patients with severe hypospadias suggest that estrogen-induced abnormal AR expression mediated by ZEB1 may be a mechanism through which estrogen contributes to the development of hypospadias. However, given the complexity of sex steroid signaling involved in penile development, we cannot attribute a single receptor or signaling pathway to be the only factor involved in hypospadias pathogenesis. The interaction of estrogen and progesterone pathways with AR signaling in hypospadias should be further explored. It remains unknown if ZEB1 interacts with these pathways.