Endometrial Epithelial Proliferation Research Articles

Hyperinsulinemia-induced PAX6 expression promotes endometrial epithelial cell proliferation via negatively modulating p27 signaling

Polycystic ovarian syndrome (PCOS) is thought to involve hyperinsulinism (insulin resistance, IR) and high prevalence of endometrial epithelial hyperplasia, but how these two pathologies are synergistically regulated in endometrial epithelial cells remains largely unknown. Here, we report a key role for the transcription factor PAX6 in the modulation of PCOS-induced endometrial epithelial proliferation. PAX6 was significantly induced in the endometrial tissues from PCOS patients, and this induction, regulated upstream by high levels of insulin, was closely correlated to the pathogenesis of IR in endometrial epithelial cells. Overexpression of the exogenous PAX6 potentiated the insulin-elicited accumulation of S phase in endometrial epithelial cells and thereby promoted endometrial epithelial proliferation. In parallel, by using luciferase reporter and ChIP assay, we found that PAX6 directly bound to the promoter of CDKN1B gene (the gene encoding p27 protein, a negative regulator of cell cycle) and inhibited CDKN1B transcription in the insulin-stimulated endometrial epithelial cells. We conclude that excessive PAX6 expression in insulin-challenged endometrial epithelial cells may contribute to the uncontrollable endometrial epithelial proliferation. Our results also provide a mechanistic explanation for the functional link between hyperinsulinemia and p27 loss in the pathogenesis of endometrial epithelial hyperplasia in PCOS patients.

0178 : The activation function-1 (AF1) of estrogen receptor α prevents arterial neointima through a direct effect on smooth muscle cells

Estradiol (E2) regulates gene transcription through nuclear Estrogen Receptor alpha (ERα) thanks to two Activation Functions, AF-1 and AF- 2, but can activate membrane ERαand thereby rapid « non genomic » signals. Some tissue-specific stimulatory roles of these ERα functions have already been described: for instance ERα AF1 and membrane ERα activation are each necessary and sufficient to induce endometrial epithelial proliferation and to accelerate endothelial healing respectively in response to E2. E2 can also inhibit vascular smooth muscle cell proliferation, but the mechanisms of this action are poorly recognized. Using a mouse model of femoral artery injury, we found that 1) E2 prevents intimal smooth muscle hyperplasia. 2) Selective inactivation of ERαin smooth muscle cells abrogates this protective action, whereas its endothelial and hematopoietic inactivation did not alter this effect. 3) Estrogen dendrimer conjugate, a selective activator of membrane ERa, fail to prevent intimal hyperplasia. 4) E2 does not inhibit post-injury arterial smooth muscle cell proliferation in mice selectively deficient in ERαAF1 (through deletion of the N-terminal A/B domain which harbors this function), demonstrating that ERαAF1 is necessary to mediate this effect, 5) Finally, tamoxifen, a selective partial AF-1 activator of ERα, is sufficient to prevent the hyperplasia. Altogether, we demonstrate here, for the first time, that ERαAF1 activation is both necessary and sufficient to prevent post-injury arterial smooth muscle cell proliferation, in striking contrast to its stimulatory action on the endometrial epithelium. This underlines the exquisite tissue-specific actions of ERαsubfunctions as well as of selective ER modulators, and shed some light on one of the most intriguing mysteries of biology of estrogens.

Mutation of palmitoylation site of Estrogen Receptor ER alpha in vivo reveals tissue-specific roles for membrane versus genomic effects of estrogens

Introduction Estradiol (E2) binds to Estrogen Receptor alpha (ERα) activation functions (AF-1 and -2) and regulates gene transcription. A fraction of ERa is targeted to plasma membrane and elicits membrane-initiated steroid signalling (MISS) but its physiological role has never been directly investigated in vivo. Methods We thus generated a mouse mutated for the palmitoylation site (C451A-ERα) by homologous recombination, leading to an abrogation of membrane localization of ERα. Results Surprisingly, this single mutation leads to total female infertility with abnormal ovaries lacking corpus luteum and hence low progesterone production. However, E2 proliferative action was completely preserved in the uterus of C451A-ERa mutant mice and endometrial epithelial proliferation was similar to wild-type. On the opposite, E2 vascular effects, such as rapid dilatation, acceleration of endothelial healing and endothelial NO synthase phosphorylation were abrogated in arteries of C451A-ERa mice. In striking contrast, in mice inactivated for genomic effects (ERa-AF2°), acceleration of re-endothelialization using membrane-selective activator estrogen-dendrimer conjugate (EDC) was preserved, demonstrating the integrity of the MISS actions in ERa-AF2°. Using a large scale analysis of uterine gene expression, almost all uterine E2-dependent gene expression was abrogated in ERa-AF2°, whereas in C451A mice, gene regulation was essentially similar to wild-type, revealing that this uterine transcriptional response depends primarily on genomic/nuclear functions of ERα. Conclusion These models provide evidence for the first time of the physiological role of MISS effects of ERα in vivo and delineate tissue specific roles of membrane and nuclear actions of ERα.

0053: The estrogen receptor alpha C451 palmitoylation site is absolutely required for vascular membrane-initiated action of estrogens in mice

Estrogen Receptor alpha (ERα) activation functions AF-1 and AF-2 classically mediate gene transcription in response to estradiol (E2). A fraction of ERα is targeted to plasma membrane and elicits membrane-initiated steroid signalling (MISS), but the physiological roles of MISS in vivo are poorly understood. We therefore generated a mouse with a point mutation of the palmitoylation site of ERα (C451A-ERα) to obtain membrane-specific loss-of-function of ERα. The abrogation of membrane localization of ERα in vivo was confirmed in primary hepatocytes, and it resulted in female infertility with abnormal ovaries lacking corpora lutea and increase in luteinizing hormone levels. In contrast, E2 action in the uterus was preserved in C451A-ERα mice and endometrial epithelial proliferation was similar to wild-type. However, E2 vascular actions such as rapid dilatation, the acceleration of endothelial repair and endothelial NO synthase phosphorylation were abrogated in C451A-ERα mice. A complementary mutant mouse lacking the transactivation function AF-2 of ER (ERα-AF2°) provided selective loss-of-function of nuclear ERα actions. In ERα-AF2°, the acceleration of endothelial repair in response to estrogen-dendrimer conjugate, which is a membrane-selective ER ligand, was unaltered, demonstrating integrity of MISS actions. In genome-wide analysis of uterine gene expression, the vast majority of E2-dependent gene regulation was abrogated in ERα-AF2° whereas in C451A-ERα it was nearly fully preserved, indicating that membrane-to-nuclear receptor crosstalk in vivo is modest in the uterus. Thus, this work is the first to genetically segregate membrane versus nuclear actions of a steroid hormone receptor and to demonstrate their in vivo tissue-specific roles.

Prospective Multicenter Randomized Intermediate Biomarker Study of Oral Contraceptive versus Depo-Provera for Prevention of Endometrial Cancer in Women with Lynch Syndrome

Women with Lynch syndrome have a 40% to 60% lifetime risk for developing endometrial cancer, a cancer associated with estrogen imbalance. The molecular basis for endometrial-specific tumorigenesis is unclear. Progestins inhibit estrogen-driven proliferation, and epidemiologic studies have shown that progestin-containing oral contraceptives (OCP) reduce the risk of endometrial cancer by 50% in women at general population risk. It is unknown whether they are effective in women with Lynch syndrome. Asymptomatic women ages 25 to 50 with Lynch syndrome were randomized to receive the progestin compounds Depo-Provera (depo-MPA) or OCP for three months. An endometrial biopsy and transvaginal ultrasound were conducted before and after treatment. Endometrial proliferation was evaluated as the primary endpoint. Histology and a panel of surrogate endpoint biomarkers were evaluated for each endometrial biopsy as secondary endpoints. A total of 51 women were enrolled, and 46 completed treatment. Two of the 51 women had complex hyperplasia with atypia at the baseline endometrial biopsy and were excluded from the study. Overall, both depo-MPA and OCP induced a dramatic decrease in endometrial epithelial proliferation and microscopic changes in the endometrium characteristic of progestin action. Transvaginal ultrasound measurement of endometrial stripe was not a useful measure of endometrial response or baseline hyperplasia. These results show that women with Lynch syndrome do show an endometrial response to short-term exogenous progestins, suggesting that OCP and depo-MPA may be reasonable chemopreventive agents in this high-risk patient population.

Mammary gland and endometrial effects of testosterone in combination with oral estradiol and progesterone.

The goal of this pilot study was to evaluate the effects of testosterone (T) cotherapy on mammary gland and endometrial measures in a postmenopausal primate model. Twenty-five surgically postmenopausal cynomolgus monkeys were randomized by social group to receive daily treatment with (1) placebo, (2) oral micronized 17beta-estradiol (1 mg/d equivalent in women) + progesterone (200 mg/d equivalent in women) (E + P), or (3) E + P with T administered via subcutaneous pellets for 8 weeks at a high dose (15 mg) followed by 8 weeks at a low dose (1.5 mg) (E + P + T). The main outcome measures were breast and endometrial epithelial proliferation, as measured by Ki67/MIB1 immunolabeling. Intralobular breast proliferation did not differ significantly among groups after 8 weeks of treatment but was marginally higher (P = 0.03) in the E + P + T group after 16 weeks of treatment. No significant increase in proliferation was seen for E + P alone. Comparable changes in mammary gland markers of estrogen-receptor activity were seen for the E + P and E + P + T groups. In the endometrium, the addition of T did not increase endometrial glandular proliferation or estrogen-receptor activity or result in any distinct histologic changes. The findings of this study do not support the idea that T antagonizes the effects of combined hormone therapy on breast proliferation or markers of estrogen-receptor activity. Overall, the short-term effects of T cotherapy on the mammary gland and endometrium were minimal.

Nuclear receptor co-regulator Krüppel-like factor 9 and prohibitin 2 expression in estrogen-induced epithelial cell proliferation in the mouse uterus

Estrogen, acting through its cognate receptor estrogen receptor-alpha (ESR1), is a critical regulator of uterine endometrial epithelial proliferation. Although the dynamic communication between endometrial stromal (ST) and epithelial cells is considered to be an important component in this process, key molecular players in particular compartments remain poorly defined. Here, we used mice null for Krüppel-like factor 9 (KLF9) to evaluate the contribution of this nuclear protein in ST-epithelial interactions underlying proliferative effects of estrogen. We found that in ovariectomized mice administered estradiol-17beta (E(2)) for 24 h, Klf9 null mutation resulted in lack of E(2)-induced proliferative response in all endometrial compartments. We demonstrated a negative association between Klf9 expression and nuclear levels of ESR1 transcriptional corepressor prohibitin (PHB) 2 in uterine ST and epithelial cells of E(2)-treated wild-type (WT) and Klf9 null mice. In early pregnancy uteri of WT mice, the temporal pattern of Klf9 transcript levels was inversely associated with that of Phb2. Deletion of Klf9 up-regulated uterine Phb2 expression and increased PHB2 nuclear localization in endometrial ST and epithelial cells, with no effects on the expression of the related Phb1. In the human endometrial ST cell line treated with E(2) for 24 h, Klf9 siRNA targeting augmented PHB2 transcript and increased nuclear PHB2 protein levels, albeit this effect was not to the extent seen in vivo with Klf9 null mutants. Our findings suggest a novel mechanism for control of estrogen-induced luminal epithelial proliferation involving ST KLF9 regulation of paracrine factor(s) to repress epithelial expression of corepressor PHB2.