Advantages in Wound Healing Process in Female Mice Require Upregulation A2A-Mediated Angiogenesis under the Stimulation of 17?-Estradiol.
ABSTRACT: Estrogenic steroids and adenosine A2A receptors promote the wound healing and angiogenesis processes. However, so far, it is unclear whether estrogen may regulate the expression and pro-angiogenic activity of A2A receptors. Using in vivo analyses, we showed that female wild type (WT) mice have a more rapid wound healing process than female or male A2A-deficient mice (A2AKO) mice. We also found that pulmonary endothelial cells (mPEC) isolated from female WT mice showed higher expression of A2A receptor than mPEC from male WT mice. mPEC from female WT mice were more sensitive to A2A-mediated pro-angiogenic response, suggesting an ER and A2A crosstalk, which was confirmed using cells isolated from A2AKO. In those female cells, 17?-estradiol potentiated A2A-mediated cell proliferation, an effect that was inhibited by selective antagonists of estrogen receptors (ER), ER?, and ER?. Therefore, estrogen regulates the expression and/or pro-angiogenic activity of A2A adenosine receptors, likely involving activation of ER? and ER? receptors. Sexual dimorphism in wound healing observed in the A2AKO mice process reinforces the functional crosstalk between ER and A2A receptors.
Project description:Women have increased prevalence of Th17-mediated autoimmune diseases, including lupus and multiple sclerosis, and severe asthma. While estradiol and progesterone increased IL-17A production in Th17 cells by inhibiting Let7f miRNA expression and increasing IL-23 receptor (IL-23R) expression, it remained unclear how estrogen signaling through the canonical nuclear receptors, estrogen receptor ? (ER?) and/or ER?, regulated this pathway. We hypothesized that estrogen signaling through ER? increased IL-23R expression and IL-17A production from Th17 cells. To test this hypothesis, naïve T cells from WT female, WT male, Esr1 -/- and Esr2 -/- female mice were differentiated into Th17 cells. IL-17A production and IL-23R expression were significantly increased in Th17 cells from WT female mice compared to Th17 cells from WT male mice. Deletion of ER? (Esr1 -/-), but not ER? (Esr2 -/-), significantly decreased IL-17A production and IL-23R expression in Th17 cells by limiting IL-23R expression in a Let-7f dependent manner. ER? deficiency also decreased Th17 cell proliferation as well as decreased T cell metabolism as measured by ATP-linked oxygen consumption rate and proton leakage. Further, we found that Cox20 expression, a protein involved in mitochondrial respiration through assembly of cytochrome c oxidase in the electron transport chain, was increased in Th17 cells from WT female mice compared to Th17 cells from WT male and Esr1 -/- female mice. Inhibition of Cox20 decreased IL-17 production in Th17 cells from WT female mice. Combined these studies showed that ER? signaling increased IL-17A production in Th17 cells by upregulating IL-23R expression and promoting mitochondrial respiration and proliferation.
Project description:Adenomatous polyposis coli (APC)-regulated Wnt and transforming growth factor-? (TGF?) signaling cooperate in the intestine to maintain normal enterocyte functions. Human clinical trials showed that estrogen [17?-estradiol (E2)], the ligand of nuclear receptors estrogen receptor ? (ER?) and ER?, inhibited colorectal cancer (CRC) in women. Consistent with this finding, we reported that E2, ER? and ER? suppressed intestinal tumorigenesis in the C57BL/6J-Min/+ (Min/+) mouse, a CRC model. Here, we extended our results with further comparisons of colon and small intestine from intact female Apc (+/+) (WT), Min/+ and ER-deficient Min/+ mice. In the colon of ER-deficient Min/+ mice, ER loss reduced TGF? signaling in crypt base cells as evidenced by minimal expression of the effectors Smad 2, 3 and 4 in these strains. We also found reduced expression of Indian hedgehog (Ihh), bone morphogenetic protein 4 and hepatocyte nuclear factor 3? or FoxA2, factors needed for paracrine signaling between enterocytes and mesenchyme. In proximal colon, ER loss produced a >10-fold increased incidence of crypt fission, a marker for wound healing and tumor promotion. These data, combined with our previous work detailing the specific roles of E2, ER? and ER? in the colon, suggest that ER activity helps to maintain the intestinal stem cell (ISC) microenvironment by modulating epithelial-stromal crosstalk in ways that regulate cytokine, Wnt and Ihh availability in the extracellular matrix (ECM).
Project description:<h4>Objective</h4>Erythropoietin (EPO), the cytokine required for erythropoiesis, contributes to metabolic regulation of fat mass and glycemic control. EPO treatment in mice on high-fat diets (HFD) improved glucose tolerance and decreased body weight gain via reduced fat mass in males and ovariectomized females. The decreased fat accumulation with EPO treatment during HFD in ovariectomized females was abrogated with estradiol supplementation, providing evidence for estrogen-related gender-specific EPO action in metabolic regulation. In this study, we examined the cross-talk between estrogen mediated through estrogen receptor ? (ER?) and EPO for the regulation of glucose metabolism and fat mass accumulation.<h4>Methods</h4>Wild-type (WT) mice and mouse models with ER? knockout (ER?-/-) and targeted deletion of ER? in adipose tissue (ER?<sup>adipoKO</sup>) were used to examine EPO treatment during high-fat diet feeding and after diet-induced obesity.<h4>Results</h4>ER?-/- mice on HFD exhibited increased fat mass and glucose intolerance. EPO treatment on HFD reduced fat accumulation in male WT and ER?-/- mice and female ER?-/- mice but not female WT mice. EPO reduced HFD increase in adipocyte size in WT mice but not in mice with deletion of ER? independent of EPO-stimulated reduction in fat mass. EPO treatment also improved glucose and insulin tolerance significantly greater in female ER?-/- mice and female ER?<sup>adipoKO</sup> compared with WT controls. Increased metabolic activity by EPO was associated with browning of white adipocytes as shown by reductions in white fat-associated genes and induction of brown fat-specific uncoupling protein 1 (UCP1).<h4>Conclusions</h4>This study clearly identified the role of estrogen signaling in modifying EPO regulation of glucose metabolism and the sex-differential EPO effect on fat mass regulation. Cross-talk between EPO and estrogen was implicated for metabolic homeostasis and regulation of body mass in female mice.
Project description:Estrogen receptors (ERs) are expressed in leukocytes and in every ocular tissue. However, sex-specific differences and the role of estradiol in ocular inflammatory-reparative responses are not well understood. We found that female mice exhibited delayed corneal epithelial wound closure and attenuated polymorphonuclear (PMN) leukocyte responses, a phenotype recapitulated by estradiol treatment both in vivo (topically in male mice) and in vitro (corneal epithelial cell wound healing). The cornea expresses 15-lipoxygenase (15-LOX) and receptors for lipoxin A(4) (LXA(4)), which have been implicated in an intrinsic lipid circuit that regulates corneal inflammation and wound healing. Delayed epithelial wound healing correlated with lower expression of 15-LOX in the regenerated epithelium of female mice. Estradiol in vitro and in vivo down-regulated epithelial 15-LOX expression and LXA(4) formation, while estradiol abrogation of epithelial wound healing was completely reversed by treatment with LXA(4). More important, ER? and ER? selectively regulated epithelial wound healing, PMN cell recruitment, and activity of the intrinsic 15-LOX/LXA(4) circuit. Our results demonstrate for the first time a sex-specific difference in the corneal reparative response, which is mediated by ER? and ER? selective regulation of the epithelial and PMN 15-LOX/LXA(4) circuit. These findings may provide novel insights into the etiology of sex-specific ocular inflammatory diseases.
Project description:Female sex is a risk factor for lupus. Sex hormones, sex chromosomes and hormone receptors are implicated in the pathogenic pathways in lupus. Estrogen receptor alpha (ER?) knockout (KO) mice are used for defining hormone receptor effects in lupus. Prior studies of ER? KO in lupus have conflicting results, likely due to sex hormone levels, different lupus strains and different ER? KO constructs. Our objective was to compare a complete KO of ER? vs. the original functional KO of ER? (expressing a short ER?) on disease expression and immune phenotype, while controlling sex hormone levels. We studied female lupus prone NZM2410 WT and ER? mutant mice. All mice (n = 44) were ovariectomized (OVX) for hormonal control. Groups of each genotype were estrogen (E2)-repleted after OVX. We found that OVXed NZM mice expressing the truncated ER? (ER? short) had significantly reduced nephritis and prolonged survival compared to both wildtype and the complete ER?KO (ER? null) mice, but surprisingly only if E2-repleted. ER? null mice were not protected regardless of E2 status. We observed significant differences in splenic B cells and dendritic cells and a decrease in cDC2 (CD11b+CD8-) dendritic cells, without a concomitant decrease in cDC1 (CD11b-CD8a+) cells comparing ER? short to ER? null or WT mice. Our data support a protective role for the ER? short protein. ER? short is similar to an endogenously expressed ER? variant (ER?46). Modulating its expression/activity represents a potential approach for treating female-predominant autoimmune diseases.
Project description:Progranulin (PGRN) is a multifunctional growth factor with functions in neuroprotection, anti-inflammation, and neural progenitor cell proliferation. These functions largely overlap with the actions of estrogen in the brain. Indeed, we have previously shown that PGRN mediates the functions of estrogen, such as masculinizing the rodent brain and promoting adult neurogenesis. To evaluate the underlying mechanism of PGRN in mediating the actions of estrogen, the localization of estrogen receptor ? (ER?) in the brains of wild-type (WT) and PGRN-deficient (KO) mice was investigated. First, double-labeling immunofluorescence was performed for ER? with neuronal nuclei (NeuN), ionized calcium-binding adaptor molecule 1 (Iba1), and glial fibrillary acidic protein (GFAP), as markers for neurons, microglia, and astrocytes, respectively, in female mice in diestrous and estrous stages. ER?-immunoreactive (IR) cells were widespread and co-localized with NeuN in brain sections analyzed (bregma -1.06 to -3.16 mm) of both WT and KO mice. In contrast, expression of ER? was not observed in Iba1-IR cells from both genotypes. Interestingly, although ER? was co-localized with GFAP in WT mice, virtually no ER? expression was discernible in GFAP-IR cells in KO mice. Next, the brains of ovariectomized adult female, adult male, and immature female mice were subjected to immunostaining for ER? and GFAP. Again, co-localization of ER? with GFAP was observed in WT mice, whereas this co-localization was not detected in KO mice. These results suggest that PGRN plays a crucial role in the expression of ER? in astrocytes regardless of the estrous cycle stage, sex, and maturity.
Project description:Experimental studies showed that 17?-estradiol (E2) and activated Estrogen Receptors (ER) protect the heart from ischemic injury. However, the underlying molecular mechanisms are not well understood. To investigate the role of ER-alpha (ER?) in cardiomyocytes in the setting of myocardial ischemia, we generated transgenic mice with cardiomyocyte-specific overexpression of ER? (ER?-OE) and subjected them to Myocardial Infarction (MI). At the basal level, female and male ER?-OE mice showed increased Left Ventricular (LV) mass, LV volume and cardiomyocyte length. Two weeks after MI, LV volume was significantly increased and LV wall thickness decreased in female and male WT-mice and male ER?-OE, but not in female ER?-OE mice. ER?-OE enhanced expression of angiogenesis and lymphangiogenesis markers (Vegf, Lyve-1), and neovascularization in the peri-infarct area in both sexes. However, attenuated level of fibrosis and higher phosphorylation of JNK signaling pathway could be detected only in female ER?-OE after MI. In conclusion, our study indicates that ER? protects female mouse cardiomyocytes from the sequelae of ischemia through induction of neovascularization in a paracrine fashion and impaired fibrosis, which together may contribute to the attenuation of cardiac remodelling.
Project description:OBJECTIVE:Hepatocyte deletion of estrogen receptor alpha (LKO-ER?) worsens fatty liver, dyslipidemia, and insulin resistance in high-fat diet fed female mice. However, whether or not hepatocyte ER? regulates reverse cholesterol transport (RCT) in mice has not yet been reported. METHODS AND RESULTS:Using LKO-ER? mice and wild-type (WT) littermates fed a Western-type diet, we found that deletion of hepatocyte ER? impaired in vivo RCT measured by the removal of 3H-cholesterol from macrophages to the liver, and subsequently to feces, in female mice but not in male mice. Deletion of hepatocyte ER? decreased the capacity of isolated HDL to efflux cholesterol from macrophages and reduced the ability of isolated hepatocytes to accept cholesterol from HDL ex vivo in both sexes. However, only in female mice, LKO-ER? increased serum cholesterol levels and increased HDL particle sizes. Deletion of hepatocyte ER? increased adiposity and worsened insulin resistance to a greater degree in female than male mice. All of the changes lead to a 5.6-fold increase in the size of early atherosclerotic lesions in female LKO-ER? mice compared to WT controls. CONCLUSIONS:Estrogen signaling through hepatocyte ER? plays an important role in RCT and is protective against lipid retention in the artery wall during early stages of atherosclerosis in female mice fed a Western-type diet.
Project description:Hydrogen sulfide (H(2)S) can be endogenously generated from cystathionine gamma-lyase (CSE) in cardiovascular system, offering a cardiovascular protection. It is also known that the lower risk of cardiovascular diseases in female is partially attributed to the protective effect of estrogen. The current study explores the interaction of H(2)S and estrogen on smooth muscle cell (SMC) growth. In the present study, we found that the proliferation of cultured vascular SMCs isolated from wild-type mice (WT-SMCs) was inhibited, but that from CSE gene knockout mice (CSE-KO-SMCs) increased, by estrogen treatments. The expression of estrogen receptor ? (ER?), but not ER?, was significantly decreased in CSE-KO-SMCs compared with that in WT-SMCs. Exogenously applied H(2)S markedly increased ER? but not ER? expression. In addition, the inhibition of ER activation and knockdown of ER? expression in WT-SMCs or the overexpression of ER? in CSE-KO-SMCs reversed the respective effects of estrogen on cell proliferation. The expression of cyclin D1 was reduced in WT-SMCs but increased in CSE-KO-SMCs after estrogen treatments, which was reversed by knockdown of ER? in WT-SMCs or overexpression of ER? in CSE-KO-SMCs, respectively. The overexpression of cyclin D1 in WT-SMCs or knockdown of cyclin D1 expression in CSE-KO-SMCs reversed the effects of estrogen on cell proliferation. These results suggest that H(2)S mediates estrogen-inhibited proliferation of SMCs via selective activation of ER?/cyclin D1 pathways.
Project description:The estrogen receptor (ER) is a ligand-dependent transcription factor containing two transcriptional activation domains. AF-1 is in the N terminus of the receptor protein and AF-2 activity is dependent on helix 12 of the C-terminal ligand-binding domain. Two point mutations of leucines 543 and 544 to alanines (L543A, L544A) in helix 12 minimized estrogen-dependent transcriptional activation and reversed the activity of the estrogen antagonists ICI182780 (ICI) and tamoxifen (TAM) into agonists in a similar manner that TAM activated WT ER? through AF-1 activation. To evaluate the physiological role of AF-1 and AF-2 for the tissue-selective function of TAM, we generated an AF-2-mutated ER? knock-in (AF2ERKI) mouse model. AF2ERKI homozygote female mice have hypoplastic uterine tissue and rudimentary mammary glands similar to ER?-KO mice. Female mice were infertile as a result of anovulation from hemorrhagic cystic ovaries and elevated serum LH and E2 levels, although the mutant ER? protein is expressed in the AF2ERKI model. The AF2ERKI phenotype suggests that AF-1 is not activated independently, even with high serum E2 levels. ICI and TAM induced uterotropic and ER-mediated gene responses in ovariectomized AF2ERKI female mice in the same manner as in TAM- and E2-treated WT mice. In contrast, ICI and TAM did not act as agonists to regulate negative feedback of serum LH or stimulate pituitary prolactin gene expression in a different manner than TAM- or E2-treated WT mice. The functionality of the mutant ER? in the pituitary appears to be different from that in the uterus, indicating that ER? uses AF-1 differently in the uterus and the pituitary for TAM action.