Response of adult mouse uterus to early disruption of estrogen receptor-alpha signaling is influenced by Krüppel-like factor 9

Abstract

Inappropriate early exposure of the hormone-responsive uterus to estrogenic compounds is associated with increased risk for adult reproductive diseases including endometrial cancers. While the dysregulation of estrogen receptor-alpha (ESR1) signaling is well acknowledged to mediate early events in tumor initiation, mechanisms contributing to sustained ESR1 activity later in life and leading to induction of oncogenic pathways remain poorly understood. We had shown previously that the transcription factor Krüppel-like factor 9 (KLF9) represses ESR1 expression and activity in Ishikawa endometrial glandular epithelial cells. We hypothesized that KLF9 functions as a tumor suppressor, and that loss of its expression enhances ESR1 signaling. Here, we evaluated the contribution of KLF9 to early perturbations in uterine ESR1 signaling pathways elicited by the administration of synthetic estrogen diethylstilbestrol (DES) to wild-type (WT) and Klf9 null (KO) mice on postnatal days (PNDs) 1-5. Uterine tissues collected at PND84 were subjected to histological, immunological, and molecular analyses. Compared with WT mice, KO mice demonstrated larger endometrial glands and lower endometrial gland numbers; DES exposure exacerbated these differences. Loss of KLF9 expression resulted in increased glandular ESR1 immunoreactivity with DES, without effects on serum estradiol levels. Quantitative RT-PCR analyses indicated altered expression of uterine genes commonly dysregulated in endometrial cancers (Akt1, Mmp9, Slpi, and Tgfbeta1) and of those involved in growth regulation (Fos, Myc, Tert, and Syk), with loss of Klf9, alone or in concert with DES. Our data support a molecular network between KLF9 and ESR1 in the uterus, and suggest that silencing of KLF9 may contribute to endometrial dysfunctions initiated by aberrant estrogen action.

Figure 1

Experimental design using Klf9 WT and null mutant mice according to the DES model of endometrial carcinoma. PND=postnatal day.

Figure 2

Body and gonadal fat pad weights of control (Oil) and DES-treated Klf9 WT and null mutant mice at postnatal day (PND) 21 (A) and 84 (B and C). Data are mean±SEM. Means with different superscripts differed at P<0.05 by Two-Way ANOVA followed by Tukey Test.

Figure 3

Uterine and ovarian indices of control (Oil) and DES-treated Klf9 WT and null mutant mice at PND84. Data are mean±SEM. Means with different superscripts differed at P<0.05 by Two-Way ANOVA followed by Tukey Test.

Figure 4

Temporal expression of uterine Klf9. (A) Uterine X-gal staining demonstrating absence of Klf9 gene expression at PND5 and PND11 and predominantly stromal Klf9 expression (arrow) at PND21 and PND28, using Klf9 (+/−) mice. Images were taken at 100X (upper panels; scale bar, 40 μm) and 400X (lower panels; scale bar, 10 μm). (B) Uterine Klf9 mRNA expression was quantified by QPCR and normalized to 18S mRNA for Oil and DES-treated Klf9 WT mice at PND84. Data (mean±SEM) are expressed as fold change relative to WT Oil treatment group (n=4–8 mice per treatment per genotype). (C) Western blot analyses for KLF9 protein levels in Oil and DES-treated Klf9 WT mice at PND84. Data (mean±SEM; n=2 independent analyses) are expressed relative to loading control, a non-specific (<17 kDa) protein detected in the same blot.

Figure 5

Endometrial stromal and glandular epithelial ESR1 expression levels as a function of Klf9 genotype and DES exposure. (A) Representative images of ESR1 immunostaining in stromal and glandular epithelial cells of Oil-treated PND84 Klf9 WT and KO mice. Images are at 400X magnification; scale bar, 10μm. Control (NTC) represents tissue section from Klf9 mutant mice, in the absence of primary antibody. Arrows indicate nuclear staining. PND84 stromal (B) and glandular epithelial (C) ESR1 quantification (mean±SEM) for n=3–4 mice per genotype per treatment (Oil or DES). Means with different superscripts differed at P<0.05 by Two-Way ANOVA followed by Tukey Test.

Figure 6

Uterine expression of selected genes as a function of Klf9 genotype and DES exposure. mRNA levels were quantified by QPCR and normalized to GAPDH (for Klf13) or 18S mRNA (for Pten and Slpi) for Oil and DES-treated Klf9 WT and null mutant mice at PND84. Data (mean±SEM) are expressed as fold change relative to WT Oil treatment group (n=4–8 mice per treatment per genotype). Means with different superscripts differed at P<0.05 by Two-Way ANOVA followed by Tukey Test.

Figure 7

Differential expression of uterine genes identified from Focused QPCR Array. Uterine mRNA expression at PND84 (A) and PND28 (B) was quantified by QPCR and normalized to 18S mRNA for Oil and DES-treated Klf9 WT and null mice. Data (mean±SEM) are expressed as fold change relative to WT Oil treatment group (n=4–8 mice per treatment per genotype). Means with different superscripts differed at P<0.05 by Two-Way ANOVA followed by Tukey Test. For Myc (A) and Akt1 (B), additional analyses by Students t-test between bracketed groups indicated significance difference (asterisk).