Response of adult mouse uterus to early disruption of estrogen receptor-α 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-a (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 Tgfb1) 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.

The Reproductive Phenotype of Mice Null for Transcription Factor Krüppel-Like Factor 13 Suggests Compensatory Function of Family Member Krüppel-Like Factor 9 in the Peri-Implantation Uterus1

“…Specifically, both Klf9 [22] and Klf13 (present study) null females exhibit normal steroid hormone production, as evidenced by their ability to normally cycle and achieve pregnancy. Although our results are not supported by the recent report that KLF9 and KLF13 are transcriptional activators of the steroidogenic genes LDLR, StAR, and CYP11A in ovarian granulosa cells [44], suggesting that loss of their respective expression should lead to reduced steroidogenesis, it is possible that the KLF9-regulated gene KLF4 [23] may take over the roles of KLF9 and KLF13 in a compensatory manner, as demonstrated for members of this family [25,45] (present study). Alternatively, KLF9 and KLF13 may compensate for each other's function in the context of the ovary.…”

“…The extent to which KLF9 plays a role in uterine function was previously demonstrated from our studies of female Klf9 null mice. We showed that Klf9 null mutation resulted in smaller litters due to reduced numbers of implanted embryos, a shift in the normal window of embryo implantation, partial uterine P 4 resistance, and enhanced uterine estrogen sensitivity [20,22,23]. Klf9 null mutation also dramatically influenced LE proliferative, apoptotic, and PGR expression status [22,24], consistent with LE as a target of stromal KLF9 through paracrine signaling.…”

“…Our studies provide a novel mechanism (i.e., increased nuclear localization/retention) by which KLF9 may take over the function of KLF13 to maintain the appropriate expression of PGR and Wnt signaling components. Given the lack of a uterine phenotype with loss of Klf13 expression and that KLF4 is a target of KLF9 [23] but not KLF13 regulation, we propose that KLF9 occupies a higher position relative to KLF13 in the hierarchy of PGR transactivators to mediate uterine function for optimal embryo implantation.…”

“…Sections (thickness, 5 mm) were mounted on poly-L-lysinecoated slides (Fisher Scientific) and processed for hematoxylin/eosin staining following standard protocols [23,24]. LE height was quantified using the Axiovert 200M microscope equipped with a Axiocam HRc camera and Axiovision software (Carl Zeiss, Inc.).…”

Krüppel-Like Factor 9 Loss-of-Expression in Human Endometrial Carcinoma Links Altered Expression of Growth-Regulatory Genes with Aberrant Proliferative Response to Estrogen1

“…Our laboratory has identified the transcription factor Krüppel-like factor (KLF) 9, an Sp-family member [9], as a transcriptional coregulator of ESR1 and PGR-B signaling in uterine cells [10][11][12][13][14][15]. KLF9 is expressed in uterine endometrial stroma and glandular, but not luminal, epithelium in mice [16], a finding recently confirmed for human endometrial cells [17].…”