Loss of ERß in Aging LXRαß Knockout Mice Leads to Colitis.

Liver X receptors (LXRα and LXRß) are oxysterol-activated nuclear receptors that play key roles in cholesterol homeostasis, the central nervous system, and the immune system. We have previously reported that LXRαß-deficient mice are more susceptible to dextran sodium sulfate (DSS)-induced colitis than their WT littermates, and that an LXR agonist protects against colitis in mice mainly via the regulation of the immune system in the gut. We now report that both LXRα and LXRß are expressed in the colonic epithelium and that in aging LXRαß-/- mice there is a reduction in the intensity of goblet cells, mucin (MUC2), TFF3, and estrogen receptor ß (ERß) levels. The cytoplasmic compartment of the surface epithelial cells was markedly reduced and there was a massive invasion of macrophages in the lamina propria. The expression and localization of ß-catenin, α-catenin, and E-cadherin were not changed, but the shrinkage of the cytoplasm led to an appearance of an increase in staining. In the colonic epithelium there was a reduction in the expression of plectin, a hemidesmosome protein whose loss in mice leads to spontaneous colitis, ELOVL1, a fatty acid elongase protein coding gene whose overexpression is found in colorectal cancer, and non-neuronal choline acetyltransferase (ChAT) involved in the regulation of epithelial cell adhesion. We conclude that in aging LXRαß-/- mice, the phenotype in the colon is due to loss of ERß expression.

ERß in Granulosa Cell Tumors and Its Clinical Potential.

Granulosa cell tumors (GCTs) are rare ovarian tumors comprising an adult and a juvenile subtype. They have a generally good prognosis, but the survival rate drastically declines in patients with late-stage or recurring tumors. Due to the rarity of GCTs, the tumor type is largely understudied and lacks a specific treatment strategy. Estrogen receptor beta (ERß/ESR2) has been found to be highly expressed in GCTs, which could be of therapeutic importance since it can be targeted with small molecules. However, its role in GCTs is not known. In this review, we summarize the current knowledge about the action of ERß in the ovary and discuss its prospective role in GCTs.

Sex- and Female Age-Dependent Differences in Gene Expression in Diffuse Large B-Cell Lymphoma-Possible Estrogen Effects.

For most lymphomas, including diffuse large B-cell lymphoma (DLBCL), the male incidence is higher, and the prognosis is worse compared to females. The reasons are unclear; however, epidemiological and experimental data suggest that estrogens are involved. With this in mind, we analyzed gene expression data from a publicly available cohort (EGAD00001003600) of 746 DLBCL samples based on RNA sequencing. We found 1293 genes to be differentially expressed between males and females (adj. p-value < 0.05). Few autosomal genes and pathways showed common sex-regulated expression between germinal center B-cell (GCB) and activated B-cell lymphoma (ABC) DLBCL. Analysis of differentially expressed genes between pre- vs. postmenopausal females identified 208 GCB and 345 ABC genes, with only 5 being shared. When combining the differentially expressed genes between females vs. males and pre- vs. postmenopausal females, nine putative estrogen-regulated genes were identified in ABC DLBCL. Two of them, NR4A2 and MUC5B, showed induced and repressed expression, respectively. Interestingly, NR4A2 has been reported as a tumor suppressor in lymphoma. We show that ABC DLBCL females with a high NR4A2 expression showed better survival. Inversely, MUC5B expression causes a more malignant phenotype in several cancers. NR4A2 and MUC5B were confirmed to be estrogen-regulated when the ABC cell line U2932 was grafted to mice. The results demonstrate sex- and female reproductive age-dependent differences in gene expression between DLBCL subtypes, likely due to estrogens. This may contribute to the sex differences in incidence and prognosis.

Harnessing the reverse cholesterol transport pathway to favor differentiation of monocyte-derived APCs and antitumor responses.

Lipid and cholesterol metabolism play a crucial role in tumor cell behavior and in shaping the tumor microenvironment. In particular, enzymatic and non-enzymatic cholesterol metabolism, and derived metabolites control dendritic cell (DC) functions, ultimately impacting tumor antigen presentation within and outside the tumor mass, dampening tumor immunity and immunotherapeutic attempts. The mechanisms accounting for such events remain largely to be defined. Here we perturbed (oxy)sterol metabolism genetically and pharmacologically and analyzed the tumor lipidome landscape in relation to the tumor-infiltrating immune cells. We report that perturbing the lipidome of tumor microenvironment by the expression of sulfotransferase 2B1b crucial in cholesterol and oxysterol sulfate synthesis, favored intratumoral representation of monocyte-derived antigen-presenting cells, including monocyte-DCs. We also found that treating mice with a newly developed antagonist of the oxysterol receptors Liver X Receptors (LXRs), promoted intratumoral monocyte-DC differentiation, delayed tumor growth and synergized with anti-PD-1 immunotherapy and adoptive T cell therapy. Of note, looking at LXR/cholesterol gene signature in melanoma patients treated with anti-PD-1-based immunotherapy predicted diverse clinical outcomes. Indeed, patients whose tumors were poorly infiltrated by monocytes/macrophages expressing LXR target genes showed improved survival over the course of therapy. Thus, our data support a role for (oxy)sterol metabolism in shaping monocyte-to-DC differentiation, and in tumor antigen presentation critical for responsiveness to immunotherapy. The identification of a new LXR antagonist opens new treatment avenues for cancer patients.

Immunohistochemical Detection of Estrogen Receptor-Beta (ERß) with PPZ0506 Antibody in Murine Tissue: From Pitfalls to Optimization.

The estrogen receptor beta (ERß) is physiologically essential for reproductive biology and is implicated in various diseases. However, despite more than 20 years of intensive research on ERß, there are still uncertainties about its distribution in tissues and cellular expression. Several studies show contrasts between mRNA and protein levels, and the use of knockout strategies revealed that many commercially available antibodies gave false-positive expression results. Recently, a specific monoclonal antibody against human ERß (PPZ0506) showed cross-reactivity with rodents and was optimized for the detection of rat ERß. Herein, we established an immunohistochemical detection protocol for ERß protein in mouse tissue. Staining was optimized on murine ovaries, as granulosa cells are known to strongly express ERß. The staining results were confirmed by western blot analysis and RT-PCR. To obtain accurate and reliable staining results, different staining conditions were tested in paraffin-embedded tissues. Different pitfalls were encountered in immunohistochemical detection. Strong heat-induced epitope retrieval (HIER) and appropriate antibody dilution were required to visualize specific nuclear expression of ERß. Finally, the specificity of the antibody was confirmed by using ovaries from Esr2-depleted mice. However, in some animals, strong (non-specific) background staining appeared. These signals could not be significantly alleviated with commercially available additional blocking solutions and are most likely due to estrus-dependent expression of endogenous immunoglobulins. In summary, our study showed that the antibody PPZ0506, originally directed against human ERß, is also suitable for reliable detection of murine ERß. An established staining protocol mitigated ambiguities regarding the expression and distribution of ERß in different tissues and will contribute to an improved understanding of its role and functions in murine tissues in the future.

Myeloid LXR (Liver X Receptor) Deficiency Induces Inflammatory Gene Expression in Foamy Macrophages and Accelerates Atherosclerosis.

BACKGROUND: Cholesterol loaded macrophage foam cells are a prominent feature of atherosclerotic plaques. Single-cell RNA sequencing has identified foam cells as TREM2 (triggering receptor expressed on myeloid cells 2) positive populations with low expression of inflammatory genes, resembling the TREM2 positive microglia of neurodegenerative diseases. Cholesterol loading of macrophages in vitro results in activation of LXR (liver X receptor) transcription factors and suppression of inflammatory genes. METHODS: To test the hypothesis that LXRs mediate anti-inflammatory effects in Trem2 expressing atherosclerotic plaque foam cells, we performed RNA profiling on plaque cells from hypercholesterolemic mice with myeloid LXR deficiency. RESULTS: Myeloid LXR deficiency led to a dramatic increase in atherosclerosis with increased monocyte entry, foam cell formation, and plaque inflammation. Bulk cell-RNA profiling of plaque myeloid cells showed prominent upregulation of inflammatory mediators including oxidative, chemokine, and chemotactic genes. Single-cell RNA sequencing revealed increased numbers of foamy TREM2-expressing macrophages; however, these cells had reduced expression of the Trem2 gene expression module, including phagocytic and cholesterol efflux genes, and had switched to a proinflammatory and proliferative phenotype. Expression of Trem2 was suppressed by inflammatory signals but not directly affected by LXR activation in bone marrow-derived macrophages. CONCLUSIONS: Our current studies reveal the key role of macrophage LXRs in promoting the Trem2 gene expression program and in suppressing inflammation in foam cells of atherosclerotic plaques.

Loss of liver X receptor ß in astrocytes leads to anxiety-like behaviors via regulating synaptic transmission in the medial prefrontal cortex in mice.

Astrocytes are integral components of synaptic transmission, and their dysfunction leads to neuropsychiatric disorders such as anxiety and depression. Liver X receptor ß (LXRß) is expressed in astrocytes, and LXRß global knockout mice shows impaired synaptic formation. In order to define the role of LXRß in astrocytes, we used a conditional Cre-loxP system to specifically remove LXRß from astrocytes. We found that this deletion caused anxiety-like but not depressive-like behaviors in adult male mice. This behavioral phenotype could be completely reproduced by selective deletion of LXRß in astrocytes in the medial prefrontal cortex (mPFC). Pyramidal neurons in layer V of mPFC are involved in mood behaviors. We found that there was an increased spontaneous excitatory synaptic transmission in layer V pyramidal neurons of the mPFC of these mice. This was concurrent with increased dendritic complexity, despite normal appearance and number of dendritic spines. In addition, gene ontology analysis of RNA sequencing revealed that deletion of astrocytic LXRß led to the enrichment of the process of synaptic transmission in mPFC. Finally, we also confirmed that renormalized excitatory synaptic transmission in layer V pyramidal neurons alleviated the anxiety in mice with astrocytic LXRß deletion in mPFC. Together, our findings reveal that astrocytic LXRß in mPFC is critical in the regulation of synaptic transmission, and this provides a potential new target for treatment of anxiety-like behavior.

Liver X receptor regulates Th17 and RORγt+ Treg cells by distinct mechanisms.

The gastrointestinal microenvironment, dominated by dietary compounds and the commensal bacteria, is a major driver of intestinal CD4+ T helper (Th) cell differentiation. Dietary compounds can be sensed by nuclear receptors (NRs) that consequently exert pleiotropic effects including immune modulation. Here, we found that under homeostatic conditions the NR Liver X receptor (LXR), a sensor of cholesterol metabolites, regulates RORγt+ CD4 T cells in the intestine draining mesenteric lymph node (MLN). While LXR activation led to a decrease, LXR-deficiency resulted in an increase in MLN Th17 and RORγt+ Tregs. Mechanistically, LXR signaling in CD11c+ myeloid cells was required to control RORγt+ Treg. By contrast, modulation of MLN Th17 was independent of LXR signaling in either immune or epithelial cells. Of note, horizontal transfer of microbiota between LXRα-/- and WT mice was sufficient to only partially increase MLN Th17 in WT mice. Despite LXRα deficiency resulted in an increased abundance of Ruminococcaceae and Lachnospiraceae bacterial families compared to littermate controls, microbiota ablation (including SFB) was not sufficient to dampen LXRα-mediated expansion of MLN Th17. Altogether, our results suggest that LXR modulates RORγt+ Treg and Th17 cells in the MLN through distinct mechanisms.

Generation of an all-exon Esr2 deleted mouse line: Effects on fertility.

Estrogen receptor beta (ERß), encoded by the Esr2 gene, is one of two nuclear receptors that mediate the functions of the steroid hormone estradiol. The binding of estradiol to the receptor results in enhanced transcription of many genes that have estrogen response elements in promoter or enhancer regions. Several genetically modified mouse lines with mutations or deletions of exons in the Esr2 gene have been developed and results from analysis of these are not completely consistent, especially regarding ERß's role in fertility. To address these controversies, we have used the CRISPR/Cas9 genome editing system to make a deletion of the entire Esr2 gene in the mouse genome and determined the effect of this mutation on fertility. We show that female Esr2 deleted mice, Esr2ΔE1-10, are subfertile at young age, with fewer litters and smaller litter size, and that they become infertile/have severely reduced fertility at around six months of age, while the male Esr2ΔE1-10 mice are fertile. Ovaries from Esr2ΔE1-10 mice are smaller than those from wild-type littermates and the morphology of the ovary displays very few corpora lutea, indicating a defect in ovulation. We also show that the estradiol levels are reduced at diestrus, the phase in the estrous cycle when levels are expected to start to increase before ovulation. Our results verify that ERß has an important function in female reproduction, likely as a regulator of serum estradiol levels, and that its loss does not affect male reproductive function.

Ventral prostate and mammary gland phenotype in mice with complete deletion of the ERß gene.

Disagreements about the phenotype of estrogen receptor ß (ERß) knockout mouse, created by removing the DNA-binding domain of the ERß gene or interruption of the gene with a neocassette (Oliver Smithies ERß knockout mice [ERßOS-/-]), prompted us to create an ERß knockout mouse by deleting the ERß gene with the use of CRISPR/Cas9 technology. We confirmed that the ERß gene was eliminated from the mouse genome and that no ERß mRNA or protein was detectable in tissues of this mouse. Overall the phenotype of the ventral prostate (VP) and mammary gland (MG) in ERßcrispr-/- mice was similar to, but more severe than, that in the ERßOS-/-mice. In the VP of 6-mo-old ERßcrispr-/- mice there was epithelial hyperplasia, fibroplasia, inflammation, stromal overgrowth, and intraductal cancer-like lesions. This was accompanied by an increase in Ki67 and P63 and loss in DACH1 and PURα, two androgen receptor (AR) repressors. In the MG there was overexpression of estrogen receptor α and progesterone receptor, loss of collagen, increase in proliferation and expression of metalloproteases, and invasive epithelium. Surprisingly, by 18 mo of age, the number of hyperplastic foci was reduced, the ducts of the VP and MG became atrophic, and, in the VP, there was massive immune infiltration and massive desquamation of the luminal epithelial cells. These changes were coincident with reduced levels of androgens in males and estrogens in females. We conclude that ERß is a tumor suppressor gene in the VP and MG where its loss increases the activity AR and ERα, respectively.

Lipidomic analysis of human primary hepatocytes following LXR activation with GW3965 identifies AGXT2L1 as a main target associated to changes in phosphatidylethanolamine.

Liver X receptor (LXR) agonists have the potential to alleviate obesity related diseases, particularly atherosclerosis. However, LXRs are transcriptional regulators that induce de novo lipogenesis and lipid accumulation in hepatocytes which represents a serious adverse effect. In this work, we sought to characterize the LXR agonist GW3965 effects on fatty acid (FA) and phospholipid (PL) remodelling and the correlation with gene expression in order to better understand the underlying effects leading to hepatic pathology upon LXR activation. Human primary hepatocytes treated for 48 h with GW3965 were analysed for changes in lipid metabolism gene expression by qPCR, variations in the FA profile was evaluated by GC-FID and in PL profiles using thin layer chromatography, ESI-MS and MS/MS analysis. Changes in cell membrane biochemical properties were studied using bilayer models generated with CHARMM-GUI. ELOLV6 and SCD1 mRNA increase was consistent with higher C16:1 and C18:1n9 at the expense of C16:0 and C18:0. The reduction of C18:2n6 and increase in C20:2n6 was in agreement with ELOVL5 upregulation. Phosphatydilethanolamine (PE) levels tended to decrease and phosphatidylinositol to increase; although differences did not reach significance, they correlated with changes in AGXT2L1, CDS1 and LPIN1 mRNA levels that were increased. The overall effect of GW3965 on PEs molecular profiles was an increase of long-chain polyunsaturated FA chains and a decrease of C16/C18 saturated and monounsaturated FAs chains. Additionally, PC (32:1) and PC (34:2) were decreased, and PC (36:1) and PC (34:1) were increased. AGXT2L1 is an enzyme with strict substrate specificity for phosphoethanolamine, which is converted into ammonia in GW3965-treated hepatocytes and could explain the PE reduction. In summary, LXR activation by GW3965 targets PE biosynthesis and FA elongation/desaturation, which tends to decrease PE in relation to total PL levels, and remodelling of PC and PE molecular species. We identified the human AGXT2L1 gene as induced by LXR activation by both synthetic and endogenous agonist treatment. The increase in acetaldehyde-induced oxidative stress, and in the lipid species identified have the potential to enhance the inflammatory process and impair membrane function. Future studies should focus on inhibition of AGXT2L1 activity with the aim of reverting the steatosis induced by LXR activation.

Correction: Estrogen Signalling and the Metabolic Syndrome: Targeting the Hepatic Estrogen Receptor Alpha Action.

[This corrects the article DOI: 10.1371/journal.pone.0057458.].

LXR Suppresses Inflammatory Gene Expression and Neutrophil Migration through cis-Repression and Cholesterol Efflux.

The activation of liver X receptor (LXR) promotes cholesterol efflux and repression of inflammatory genes with anti-atherogenic consequences. The mechanisms underlying the repressive activity of LXR are controversial and have been attributed to cholesterol efflux or to transrepression of activator protein-1 (AP-1) activity. Here, we find that cholesterol efflux contributes to LXR repression, while the direct repressive functions of LXR also play a key role but are independent of AP-1. We use assay for transposase-accessible chromatin using sequencing (ATAC-seq) to show that LXR reduces chromatin accessibility in cis at inflammatory gene enhancers containing LXR binding sites. Targets of this repressive activity are associated with leukocyte adhesion and neutrophil migration, and LXR agonist treatment suppresses neutrophil recruitment in a mouse model of sterile peritonitis. These studies suggest a model of repression in which liganded LXR binds in cis to canonical nuclear receptor binding sites and represses pro-atherogenic leukocyte functions in tandem with the induction of LXR targets mediating cholesterol efflux.

Molecular and functional heterogeneity of IL-10-producing CD4+ T cells.

IL-10 is a prototypical anti-inflammatory cytokine, which is fundamental to the maintenance of immune homeostasis, especially in the intestine. There is an assumption that cells producing IL-10 have an immunoregulatory function. However, here we report that IL-10-producing CD4+ T cells are phenotypically and functionally heterogeneous. By combining single cell transcriptome and functional analyses, we identified a subpopulation of IL-10-producing Foxp3neg CD4+ T cells that displays regulatory activity unlike other IL-10-producing CD4+ T cells, which are unexpectedly pro-inflammatory. The combinatorial expression of co-inhibitory receptors is sufficient to discriminate IL-10-producing CD4+ T cells with regulatory function from others and to identify them across different tissues and disease models in mice and humans. These regulatory IL-10-producing Foxp3neg CD4+ T cells have a unique transcriptional program, which goes beyond the regulation of IL-10 expression. Finally, we found that patients with Inflammatory Bowel Disease demonstrate a deficiency in this specific regulatory T-cell subpopulation.

Genomics of sex hormone receptor signaling in hepatic sexual dimorphism.

The liver plays a crucial role in a variety of physiological processes. Sexual dimorphism is markedly defined in liver disorders, such as fatty liver diseases and liver cancer, but barely addressed in the normal liver. Distinct sex hormone signaling between male and female livers is the major driving factor for hepatic sexual dimorphism. Over 6000 genes are differently expressed between male and female livers in mice. Here we address how sex hormone receptors, estrogen receptor alpha (ERα) and androgen receptor (AR), mediate sexually dimorphic gene expression in mouse livers. We identified 5192 ERα target genes and 4154 AR target genes using ChIP-Seq. Using liver-specific ERα or AR knockout mice, we further identified direct and functional target genes of ERα (123 genes) and AR (151 genes) that contribute to hepatic sexual dimorphism. We also found that the most significant sexually dimorphic gene expression was initiated at birth by comparing hepatic gene expression data from the embryonic stage E10.5 to the postnatal stage P60 during liver development. Overall, our study indicates that sex hormone receptor signaling drives sexual dimorphism of hepatic gene expression throughout liver development.

Role of estrogen receptor beta in neural differentiation of mouse embryonic stem cells.

The ability to propagate mature cells and tissue from pluripotent stem cells offers enormous promise for treating many diseases, including neurodegenerative diseases. Before such cells can be used successfully in neurodegenerative diseases without causing unwanted cell growth and migration, genes regulating growth and migration of neural stem cells need to be well characterized. Estrogen receptor beta (ERß) is essential for migration of neurons and glial cells in the developing mouse brain. To examine whether ERß influences differentiation of mouse embryonic stem cells (mESC) into neural lineages, we compared control and ERß knockout (BERKO) mESCs at defined stages of neural development and examined the effects of an ERß-selective ligand (LY3201) with a combination of global and targeted gene-expression profiling and the expression of key pluripotency markers. We found that ERß was induced in embryoid bodies (EBs) and neural precursor cells (NPCs) during development. Proliferation was higher in BERKO NPCs and was inhibited by LY3201. Neurogenesis was reduced in BERKO ES cells, and oligodendrogliogenesis was enhanced. BERKO EBs expressed higher levels of key ectodermal and neural progenitor markers and lower levels of markers for mesoderm and endoderm lineages. ERß-regulated factors are involved in cell adhesion, axon guidance, and signaling of Notch and GABA receptor pathways, as well as factors important for the differentiation of neuronal precursors into dopaminergic neurons (Engrailed 1) and for the oligodendrocyte fate acquisition (Olig2). Our data suggest that ERß is an important component for differentiation into midbrain neurons as well as for preventing precocious oligodendrogliogenesis.

Estrogen receptor ß, a regulator of androgen receptor signaling in the mouse ventral prostate.

As estrogen receptor ß-/- (ERß-/-) mice age, the ventral prostate (VP) develops increased numbers of hyperplastic, fibroplastic lesions and inflammatory cells. To identify genes involved in these changes, we used RNA sequencing and immunohistochemistry to compare gene expression profiles in the VP of young (2-mo-old) and aging (18-mo-old) ERß-/- mice and their WT littermates. We also treated young and old WT mice with an ERß-selective agonist and evaluated protein expression. The most significant findings were that ERß down-regulates androgen receptor (AR) signaling and up-regulates the tumor suppressor phosphatase and tensin homolog (PTEN). ERß agonist increased expression of the AR corepressor dachshund family (DACH1/2), T-cadherin, stromal caveolin-1, and nuclear PTEN and decreased expression of RAR-related orphan receptor c, Bcl2, inducible nitric oxide synthase, and IL-6. In the ERß-/- mouse VP, RNA sequencing revealed that the following genes were up-regulated more than fivefold: Bcl2, clusterin, the cytokines CXCL16 and -17, and a marker of basal/intermediate cells (prostate stem cell antigen) and cytokeratins 4, 5, and 17. The most down-regulated genes were the following: the antioxidant gene glutathione peroxidase 3; protease inhibitors WAP four-disulfide core domain 3 (WFDC3); the tumor-suppressive genes T-cadherin and caveolin-1; the regulator of transforming growth factor ß signaling SMAD7; and the PTEN ubiquitin ligase NEDD4. The role of ERß in opposing AR signaling, proliferation, and inflammation suggests that ERß-selective agonists may be used to prevent progression of prostate cancer, prevent fibrosis and development of benign prostatic hyperplasia, and treat prostatitis.

Estrogen Receptor-α Knockout Mice.

Tissue specific knockout mice are valuable tools to study gene function in vivo. The method uses the Cre/loxP system in which loxP sites are cloned into the genome surrounding one or more exons of a gene and the targeted exon(s) are deleted when the Cre enzyme is expressed. Mouse lines that are prepared for the generation of knockout ERα mice have been developed independently by many research groups and the number of available transgenic mouse lines that express Cre under tissue specific promoters is large. Here, we describe how tissue specific ERα knockout mice are generated.

Identification of proteins highly expressed in uterine fluid from mice with hydrometra.

Estrogen receptor alpha (ERα) is an important regulator of the estrous cycle and mice with global ERα deletion, as well as some conditional knockout mouse lines, have an interruption in the estrous cycle. In this study we observed that conditional ERα knockout mice where the Cre gene is regulated by the rat insulin promoter (RIP), RIP-Cre/ERα(KO) mice, have a 3.7-fold increase in serum 17ß-estradiol levels, blocked estrous cycle, and develop a fluid-filled uterus (hydrometra). Using a proteomics approach, we identified three proteins, lactoferrin, complement C3 and chitinase 3-like protein 1 (CHI3L1), as highly expressed proteins in hydrometra fluid. The mRNA levels of the corresponding genes were more than 50-fold higher in RIP-Cre/ERα(KO) uterus compared to controls. High expression of CHI3L1 in the uterine fluid was not reflected as elevated levels in the serum. The high expression of lactoferrin, complement C3 and CHI3L1 in the uterine fluid, in association with elevated estrogen levels, prompted us to address if the expression of these genes is related to reproduction. However, gonadotropin treatment of mice reduced the uterine expression of these genes in a model of in vitro fertilization. Our findings identify lactoferrin, complement C3 and CHI3L1 as highly expressed proteins in hydrometra fluid in association with chronically elevated serum estradiol levels.

Estrogen receptor ß exon 3-deleted mouse: The importance of non-ERE pathways in ERß signaling.

In 1998, an estrogen receptor ß (ERß) knockout (KO) mouse was created by interrupting the gene at the DNA binding domain (DBD) with a neocassette. The mutant females were subfertile and there were abnormalities in the brain, prostate, lung, colon, and immune system. In 2008, another ERß mutant mouse was generated by deleting ERß exon 3 which encodes the first zinc finger in the DBD. The female mice of this strain were unable to ovulate but were otherwise normal. The differences in the phenotypes of the two KO strains, have led to questions about the physiological function of ERß. In the present study, we created an ERß exon 3-deleted mouse (ERß-Δex3) and confirmed that the only observable defect was anovulation. Despite the two in-frame stop codons introduced by splicing between exons 2 and 4, an ERß protein was expressed in nuclei of prostate epithelial cells. Using two different anti-ERß antibodies, we showed that an in-frame ligand binding domain and C terminus were present in the ERß-Δex3 protein. Moreover, with nuclear extracts from ERß-Δex3 prostates, there was an ERß-dependent retardation of migration of activator protein-1 response elements in EMSA. Unlike the original knockout mouse, expression of Ki67, androgen receptor, and Dachshund-1 in prostate epithelium was not altered in the ERß-Δex3 mouse. We conclude that very little of ERß transcriptional activity depends on binding to classical estrogen response elements (EREs).