Andrée Krust

Modulation of oestrogen receptor signalling by association with the activated dioxin receptor

Environmental contaminants affect a wide variety of biological events in many species. Dioxins are typical environmental contaminants that exert adverse oestrogen-related effects. Although their anti-oestrogenic actions are well described, dioxins can also induce endometriosis and oestrogen-dependent tumours, implying possible oestrogenic effects. However, the molecular mechanism underlying oestrogen-related actions of dioxins remains largely unknown. A heterodimer of the dioxin receptor (AhR) and Arnt, which are basic helix–loop–helix/PAS-family transcription factors, mediates most of the toxic effects of dioxins. Here we show that the agonist-activated AhR/Arnt heterodimer directly associates with oestrogen receptors ER-α and ER-β. This association results in the recruitment of unliganded ER and the co-activator p300 to oestrogen-responsive gene promoters, leading to activation of transcription and oestrogenic effects. The function of liganded ER is attenuated. Oestrogenic actions of AhR agonists were detected in wild-type ovariectomized mouse uteri, but were absent in AhR-/- or ER-α-/- ovariectomized mice. Our findings suggest a novel mechanism by which ER-mediated oestrogen signalling is modulated by a co-regulatory-like function of activated AhR/Arnt, giving rise to adverse oestrogen-related actions of dioxin-type environmental contaminants.

Estrogen Prevents Bone Loss via Estrogen Receptor α and Induction of Fas Ligand in Osteoclasts

Estrogen prevents osteoporotic bone loss by attenuating bone resorption; however, the molecular basis for this is unknown. Here, we report a critical role for the osteoclastic estrogen receptor alpha (ERalpha) in mediating estrogen-dependent bone maintenance in female mice. We selectively ablated ERalpha in differentiated osteoclasts (ERalpha(DeltaOc/DeltaOc)) and found that ERalpha(DeltaOc/DeltaOc) females, but not males, exhibited trabecular bone loss, similar to the osteoporotic bone phenotype in postmenopausal women. Further, we show that estrogen induced apoptosis and upregulation of Fas ligand (FasL) expression in osteoclasts of the trabecular bones of WT but not ERalpha(DeltaOc/DeltaOc) mice. The expression of ERalpha was also required for the induction of apoptosis by tamoxifen and estrogen in cultured osteoclasts. Our results support a model in which estrogen regulates the life span of mature osteoclasts via the induction of the Fas/FasL system, thereby providing an explanation for the osteoprotective function of estrogen as well as SERMs.

Estrogen Receptor-α Mediates the Protective Effects of Estrogen Against Vascular Injury

Blood vessel cells express the 2 known estrogen receptors, &agr; and &bgr; (ER&agr;, ER&bgr;), which are thought to mediate estrogen inhibition of vascular injury and atherosclerosis, but the relative role of ER&agr; and ER&bgr; in these events is controversial. Estrogen inhibits the vascular injury response to the same extent in ovariectomized female wild-type mice and in the original single gene knockout mice for ER&agr; (ER&agr;KOChapel Hill [ER&agr;KOCH]) and ER&bgr; (ER&bgr;KOChapel Hill [ER&bgr;KOCH]). In double gene knockout mice generated by crossing these animals (ER&agr;,&bgr;KOCH), estrogen no longer inhibits medial thickening after vascular injury, but still inhibits vascular smooth muscle cell proliferation and increases uterine weight. The partial retention of estrogen responsiveness in ER&agr;,&bgr;KOCH mice could be due either to the presence of a novel, unidentified estrogen receptor or to functional expression of an estrogen receptor-&agr; splice variant in the parental ER&agr;KOCH mice. To distinguish between these possibilities, we studied recently generated mice fully null for estrogen receptor &agr; (ER&agr;KOStrasbourg [ER&agr;KOSt]) and examined the effect of estrogen on the response to vascular injury. In the present study, we show that after vascular injury in ovariectomized ER&agr;KOSt mice, estrogen has no detectable effect on any measure of vascular injury, including medial area, proteoglycan deposition, or smooth muscle cell proliferation. These data demonstrate that estrogen receptor-&agr; mediates the protective effects of estrogen on the response to vascular injury.

Estrogen receptor-α mediates the brain antiinflammatory activity of estradiol

Beyond the key role in reproductive and cognitive functions, estrogens have been shown to protect against neurodegeneration associated with acute and chronic injuries of the adult brain. Current hypotheses reconcile this activity with a direct effect of 17β-estradiol (E2) on neurons. Here we demonstrate that brain macrophages are also involved in E2 action on the brain. Systemic administration of hormone prevents, in a time- and dose-dependent manner, the activation of microglia and the recruitment of peripheral monocytes induced by intraventricular injection of lipopolysaccharide. This effect occurs by limiting the expression of neuroinflammatory mediators, such as the matrix metalloproteinase 9 and lysosomal enzymes and complement C3 receptor, as well as by preventing morphological changes occurring in microglia during the inflammatory response. By injecting lipopolysaccharide in estrogen receptor (ER)-null mouse brains, we demonstrate that hormone action is mediated by activation of ERα but not of ERβ. The specific role of ERα is further confirmed by comparing the effects of ERs on the matrix metalloproteinase 9 promoter activity in transient transfection assays. Finally, we report that genetic ablation of ERα is associated with a spontaneous reactive phenotype of microglia in specific brain regions of adult ERα-null mice. Altogether, these results reveal a previously undescribed function for E2 in brain and provide a mechanism for its beneficial activity on neuroinflammatory pathologies. They also underline the key role of ERα in brain macrophage reactivity and hint toward the usefulness of ERα-specific drugs in hormone replacement therapy of inflammatory diseases.

Cloning of the human oestrogen receptor cDNA

Poly A+ RNA isolated from the human breast cancer cell line MCF-7 was fractionated by sucrose gradient centrifugation and those fractions enriched in oestrogen receptor (ER) mRNA were used to prepare randomly primed cDNA libraries in the lambda gt11 vectors. Clones corresponding to the ER were isolated from both libraries after screening with either ER monoclonal antibodies (lambda gt11) or synthetic oligonucleotide probes designed from two peptide sequences of purified ER (lambda gt10). Five cDNA clones were isolated by antibody screening and five after screening with synthetic oligonucleotides. The two largest ER cDNA clones, lambda OR3 (1.3 kbase) and lambda OR8 (2.1 kbase), isolated using antibodies and oligonucleotides, respectively, were able to enrich selectively for ER mRNA by hybrid-selection. Furthermore, lambda OR8 contains DNA sequences which cross-hybridize with each of the other ER cDNA clones. These results demonstrate that the clones isolated correspond to the ER mRNA sequence. Using lambda OR8 as a hybridization probe revealed a single poly A+ RNA band of approx. 6.2 kbase in the ER containing human breast cancer cell lines MCF-7 and T47D. In contrast, no hybridization was seen in the human ER-cell line HeLa. The same probe hybridizes to a chicken gene which is expressed in oviduct tissue as a 7.5 kbase poly A+ RNA.

Estradiol Accelerates Reendothelialization in Mouse Carotid Artery Through Estrogen Receptor-α but Not Estrogen Receptor-β

Background—The atheroprotective effect of 17β-estradiol (E2) has been suggested in women and clearly demonstrated in animals through both an effect on lipid metabolism and a direct effect on the cells of the arterial wall. It has been shown, for example, that E2 promotes endothelium-dependent relaxation and accelerates reendothelialization in rats. Similar studies have been undertaken in mice to appreciate the molecular mechanism of this process. Methods and Results—We report here a model of electric carotid injury adapted from that described by Carmeliet et al (1997) that allows us to precisely evaluate the reendothelialization process. We demonstrate that E2 accelerates endothelial regeneration in castrated female wild-type mice. In ovariectomized transgenic mice in which either the estrogen receptor (ER)-α or ERβ gene has been disrupted, E2 accelerated reendothelialization in female ERβ knockout mice, whereas this effect was abolished in female ERα knockout mice. Conclusions—This study demonstrates tha...

Increased anxiety and synaptic plasticity in estrogen receptor β-deficient mice

Estrogens are powerful modulators of neuronal physiology and in humans may affect a broad range of functions, including reproductive, emotional, and cognitive behaviors. We studied the contribution of estrogen receptors (ERs) in modulation of emotional processes and analyzed the effects of deleting ERα or ERβ in mice. Behavior consistent with increased anxiety was observed principally in ERβ mutant females and was associated with a reduced threshold for the induction of synaptic plasticity in the basolateral amygdala. Local increase of 5-hydroxytryptamine 1a receptor expression in medial amygdala may contribute to these changes. Our data show that, particularly in females, there is an important role for ERβ-mediated estrogen signaling in the processing of emotional behavior.

Estradiol enhances primary antigen-specific CD4 T cell responses and Th1 development in vivo. Essential role of estrogen receptor α expression in hematopoietic cells

It is widely accepted that females have superior immune responses than males, but the ways by which sex hormones may enhance T cell responses are still poorly understood. In the present study,we analyzed the effect of estrogens on CD4 T cell activation and differentiation after immunization with exogenous antigens. We show that administration of low doses of 17ß‐estradiol (E2) to castrated female mice results in a striking increase of antigen‐specific CD4 T cell responses and in the selective development of IFN‐γ‐producing cells. Quantitative assessment of the frequency of T cells bearing a public TCR ß chain CDR3 motif demonstrated that the clonal size of primary antigen‐specific CD4 T cells was dramatically increased in immune lymph nodes from E2‐treated mice. By usingmice with disrupted estrogen receptor (ER) α or ß genes, we show that ERα, but not ERβ, was necessary for the enhanced E2‐driven Th1 cell responsiveness. Furthermore, ERα expressionin hematopoietic cells was essential, since E2 effects on Th1 responses were only observed in mice reconstituted with bone marrow cells from ERα+/+, but not ERα‐deficient mice. These results demonstrate that estrogen administration promotes strong antigen‐specific Th1 cell responses in a mechanism that requires functional expression of ERα in hematopoietic cells.

The AF-1 activation-function of ERα may be dispensable to mediate the effect of estradiol on endothelial NO production in mice

Two isoforms of estrogen receptor (ER) have been described: ERα and ERβ. The initial gene targeting of ERα, consisting in the introduction of a Neo cassette in exon 1 [αERKO, hereafter called ERα-Neo KO (knockout)], was reported in 1993. More recently, another mouse deficient in ERα because of the deletion of exon 2 (ERαKO, hereafter called ERα-Δ2 KO) was generated. In ovariectomized ERα-wild-type mice, estradiol (E2) increases uterine weight and basal production of endothelial nitric oxide (NO). Both of these effects are abolished in ERα-Δ2 KO mice. In contrast, we show here that both of these effects of E2 are partially (uterine weight) or totally (endothelial NO production) preserved in ERα-Neo KO. We also confirm the presence of two ERα mRNA splice variants in uterus and aorta from ERα-Neo KO mice. One of them encodes a chimeric ERα protein (ERα55), partially deleted in the A/B domain, that was detected in both uterus and aorta by Western blot analysis. The other ERα mRNA splice variant codes for an isoform deleted for the A/B domain (ERα46), which was detected in uterus of ERα-Neo KO, and wild-type mice. This protein isoform was not detected in aorta. The identification of these two N-terminal modified isoforms in uterus, and at least one of them in aorta, probably explains the persistence of the E2 effects in ERα-Neo KO mice. Furthermore, ERα-Neo KO mice may help in the elucidation of the specific functions of full-length ERα (ERα66) and ERα46, both shown to be physiologically generated in vivo.

Sterility and absence of histopathological defects in nonreproductive organs of a mouse ERβ-null mutant

Estrogen signaling is mediated by estrogen receptors α (ERα) and β (ERβ). Although a consensus has now been reached concerning many physiological functions of ERα, those of ERβ are still controversial: When housed and examined in two distant laboratories, mice originating from the same initial ERβ mutant exhibited widely different phenotypes, which were themselves different from the phenotype of another ERβ mutant previously generated in our laboratory. Because, in addition to a knockout insertion in exon 3, all these mouse mutants displayed alternative splicing transcripts, we have now constructed a ERβ mouse mutant (ERβSTL−/L−) in which exon 3 was cleanly deleted by Cre/LoxP-mediated excision and was devoid of any transcript downstream of exon 3. Both females and males were sterile. The histology of the ovary was mildly affected, and no histological defects were detected in other organs, neither in females nor in males. Our present results, which are in contrast with previously published data, suggest that, with the notable exception of male and female reproduction, ERβ is not required in the mouse for the development and homeostasis of the major body systems.