DES inhibits activation of TP63 and induces adenosis through ERα within vaginal or cervical epithelial cells

The development of cervical and vaginal adenosis as a result of diethylstilbestrol exposure in utero

2012 Study Abstract

Exposure to exogenous hormones during development can result in permanent health problems. In utero exposure to diethylstilbestrol (DES) is probably the most well documented case in human history. DES, an orally active synthetic estrogen, was believed to prevent adverse pregnancy outcome and thus was routinely given to selected pregnant women from the 1940s to the 1960s. It has been estimated that 5 million pregnant women worldwide were prescribed DES during this period. In the early 1970s, vaginal clear cell adenocarcinomas (CCACs) were diagnosed in daughters whose mother took DES during pregnancy (known as DES daughters). Follow-up studies demonstrated that exposure to DES in utero causes a spectrum of congenital anomalies in female reproductive tracts and CCACs. Among those, cervical and vaginal adenoses are most commonly found, which are believed to be the precursors of CCACs. Transformation related protein 63 (TRP63/p63) marks the cell fate decision of Müllerian duct epithelium (MDE) to become squamous epithelium in the cervix and vagina. DES disrupts the TRP63 expression in mice and induces adenosis lesions in the cervix and vagina. This review describes mouse models that can be used to study the development of DES-induced anomalies, focusing on cervical and vaginal adenoses, and discusses their molecular pathogenesis.

Inhibition of TRP63 by DES in the developing cervix and vagina

The transcription factor transformation related protein 63 (TRP63/p63) was highly expressed in the squamous epithelia of the cervix and vagina, but not in the columnar epithelium of the uterus. In mice, TRP63 was undetectable in the vagina and cervix until just before birth, suggesting that TRP63 may be involved in squamous differentiation of the MDE. Since Trp63-null mice died at birth due to defects in skin development, their female reproductive tracts could not be directly studied. Therefore, anlagen of uterus, cervix and vagina were developed into mature organs by grafting them under the kidney capsule of mature female mice. In such grafts, the Trp63-null uterine epithelium appeared normal, but the entire Trp63-null vagina and cervix were lined with columnar epithelium and contained gland-like structures instead of normal squamous epithelium, demonstrating that the developmental loss of Trp63 is the cause of adenosis. When wild-type newborn mice were exposed to DES, expression of Trp63 in MDE was inhibited and TRP63-negative MDE differentiated into uterine-like glandular epithelium within the cervix and vagina, as seen in Trp63-null mice. However, once MDE cells became positive for TRP63, expression was not affected by DES exposure, suggesting that DES inhibited induction, but not maintenance of Trp63 expression in MDE. Hence, the timing of exposure was critical for the pathogenesis of DES-induced adenosis. See study Roles of p63 in differentiation of Müllerian duct epithelial cells and study Roles of p63 in the diethylstilbestrol-induced cervicovaginal adenosis.

The expression pattern of tumor protein 63 (TP63/p63), the ortholog of mouse TRP63, has been studied in the female reproductive tracts of human fetuses. See study Differential expression of p63 isoforms in female reproductive organs. TP63 expression was greater in the caudal and lesser or absent in the cranial cells of the MDE in the human fetus at PLM week 12 – 13, which is equivalent to the TRP63 expression in mice at embryonic day (E) 16 – postnatal day (P) 1. The human specimens at PLM week 16 – 18 contained both TP63-positive and negative cells within the stratified epithelium of ectocervical or vaginal anlagen, and the concentration of TP63-positive cells increased caudally to cranially as the fetus developed. The expression pattern of TP63 in the vagina of PLM 18 week fetuses appeared to be comparable to the expression in P2 – 3 mice. While most epithelial cells were positive for TRP63 in the caudal (lower vaginal) regions, the cranial (upper vaginal and cervical) regions contained substantial TRP63-negative epithelial cells. Like P2 – 3 is the last point of greatest susceptibility to DES-induced adenosis in mice, exposure to DES in humans within the first 18 weeks of pregnancy resulted in a higher incidence of vaginal epithelial changes. This suggests that DES induces vaginal or cervical adenosis in human fetuses by blocking the expression of TP63 in MDE, as in mice.

The Trp63 gene is transcribed into two alternative splice variants producing isoforms with and without the transactivation domain (TA). The isoforms containing or missing a TA domain are called the TA and ΔN forms, respectively, and each of them has three alternative splice variants of the carboxy terminus, identified as α, β and γ forms. In mice, the ΔNα form (ΔNp63α), lacking the transactivating domain and containing the sterile alpha motif domain, is the dominant form of TRP63 expressed in the neonatal and adult cervix and vagina. This is also true for the vaginal epithelium of the human fetus. These commonalities confirm the rationale for utilization of mouse models to study the molecular pathogenesis of cervical and vaginal adenoses in DES daughters.

ESR1-mediated DES action in vaginal epithelium

Estrogen receptor alpha (ERα) knockout mice (Esr1-null) exhibit resistance to neonatal DES exposure, indicating that ERα is essential for DES-induced urogenital abnormalities. However, whether ERα mediated DES action in the pathogenesis of vaginal adenosis was not fully established due to a low prevalence of DES-induced adenosis in the wild-type mice. The essential role of ERα in DES-induced vaginal adenosis was later established by analysis of TRP63 expression in DES-treated Esr1-null mice. In the study, neonatal DES treatment inhibited TRP63 expression in the vaginal epithelium of wild-type but not Esr1-null mice. In developing vaginae, ERα is expressed in both epithelium and mesenchyme. Therefore, the role of epithelial versus mesenchymal ERα in DES-induced vaginal adenosis was further clarified through tissue-recombination experiments. In this study, MDE from Esr1-null (ERα negative) and wild-type (ERα positive) mice was combined with Esr1-null or wild-type vaginal mesenchyme. While DES failed to induce adenosis in Esr1-null MDE, DES completely blocked TRP63 expression in wild-type MDE. Expression of ERα in MDE was the single determinant if DES inhibited expression of TRP63, and the presence or absence of mesenchymal ERα did not affect the result. Therefore, DES acts through ERα within MDE and induces vaginal adenosis by blocking expression of TRP63.

A recent study suggested that Activin signaling from the stroma is the major factor regulating proliferation and differentiation of vaginal epithelial cells. The study further proposed that DES induces vaginal adenosis through disruption of this signaling, because DES reduced Acvr2b and Inhba mRNA by 20% and 40%, respective, and upregulated Inhbb mRNA 20 fold, as normalized to the level of Ppia mRNA. However, Ppia is upregulated by estrogen in mouse uterus, thus the analysis may have been skewed by the effect of DES on the Ppia level. The authors also demonstrated reduced Activin signaling by immunofluorescence analysis of phospho-SMAD2. Although the overall signal level for phospho-SMAD2 was reduced in the entire vagina of neonatally DES-treated mice, the epithelial tissue that contained reduced phosphorylated-SMAD2 was fully differentiated as squamous stratified epithelium, indicating that reduced Activin A signaling is not necessarily associated with adenosis. While Activin A signaling may play a role in vaginal epithelial differentiation or proliferation, it has been shown that the underlying pathway through which DES causes adenosis is mediated by intrinsic epithelial ERα signaling. Therefore, alterations in pathways through the stroma, such as down-regulation of Activin ligand, would not play a role in the pathogenesis. How DES disrupts expression of TRP63 through ERα within epithelial cells has yet to be elucidated. Recently, an essential role of androgen receptor (AR) binding to a regulatory element of Trp63 gene was demonstrated in developing mouse epididymal epithelium. Likewise, ERα may directly bind to the regulatory elements of Trp63 to block expression.

Sources and more information
  • Full text (free access) : The Development of Cervical and Vaginal Adenosis as a Result of Diethylstilbestrol Exposure In Utero, Journal List, HHS Author Manuscripts, NCBI PubMed PMC3443265, 2012 Jun 6.
  • DES inhibits activation of TP63 and induces adenosis through ERα within vaginal or cervical epithelial cells featured image credit PMC3443265/figure/F5.

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