Characterization of the uterine phenotype of non-classical ERα (estrogen receptor α) signaling in vivo.

Abstract

Determine the non-ERE (estrogen response element) dependent actions of ERα on growth, differentiation, and developmental gene expression in the female reproductive tract. Experimental laboratory study. Morphometric analysis, immunohistochemistry, and quantitative real-time PCR. Previous work from our laboratory demonstrated that the mutation of residues 207 and 208 of ERα (within the proximal P-box of the first zinc finger of the DNA binding domain) to alanines abolished classical ERα signaling via ERE. We generated a knock-in/knockout mouse (E207A/G208A, ERAA/-) lacking classical ERα signaling by crossbreeding ERAA/WTto estrogen receptor-α knockout (αERKO) animals. The ERAA/- animals signal via the non-classical ERα pathway. The non-classical pathway involves a ligand bound ERα that interacts with transcription factors such as AP1 and Sp1. Animals were sacrificed at 8 weeks of age. The presence of a single ERAA/- allele (n = 8) supported significant uterine growth compared with αERKO animals (n = 6). The uterine radius of ERAA/-animals was larger than age matched αERKO mice (321 μm ± 58 vs. 170 ± 62, p < .01). The width of the inner circular muscle (ICM) layer of ERAA/- mice was also increased (54 μm ± 34 vs. 38 ± 15, p < .05). The ERAA/-uterus also exhibited altered glandular development. The endometrial glands were more numerous but remained organized in clusters in the stroma. The mesenchymal differentiation characteristic of wild-type (WT) mice was restored with positive ICM coexpression for vimentin and α-actin in all ERAA/-animals. In contrast, the αERKO uterus lacked cytodifferentiation with no positive staining of the ICM for vimentin or α-actin. The expression of the Wnt genes (Wnt-7a, Wnt-5a, and Wnt-4a) and EGFR (epidermal growth factor receptor) did not differ between ERAA/-(n = 8) and αERKO (n = 7). However, the uterine expression of cyclin D2 (which lacks an ERE) increased 2.6-fold in ERAA/- compared to αERKO. The expression of PR (progesterone receptor isoform A and B) was down-regulated significantly in ERAA/- compared to αERKO (-2.5-fold, p < .05). Non-classical ERα signaling via the presence of a single ERAA/-allele is sufficient to restore many features of growth and differentiation of the murine uterus. In addition, glandulargenesis progresses beyond that observed in the αERKO uterus suggesting that non-classical signaling is involved in the epithelial-mesenchyme interactions required for complex gland formation. Developmental gene expression is not altered by the restoration of non-classical ERα signaling. The up-regulation of cyclin D2 and the loss of progesterone receptor expression provide a potential mechanism for the observed phenotype.