Estrogen-treated Mice Research Articles

GPER expression prevents estrogen-induced urinary retention in obese mice

Long-term administration of exogenous estrogen is known to cause urinary retention and marked, often fatal, bladder distention in both male and female mice. Estrogen-treated mice have increased bladder pressure and decreased urine flow, suggesting that urinary retention in estrogen-treated mice is due to infravesicular obstruction to urine outflow. Thus, the condition is commonly referred to as bladder outlet obstruction (BOO). Obesity can also lead to urinary retention. As the effects of estrogen are mediated by multiple receptors, including estrogen receptors ERα and ERβ and the G protein-coupled estrogen receptor (GPER), we sought to determine whether GPER plays a role in estrogen-induced BOO, particularly in the context of obesity. Wild type and GPER knockout (KO) mice fed a high-fat diet were ovariectomized or left ovary-intact (sham surgery) and supplemented with slow-release estrogen or vehicle-only pellets. Supplementing both GPER KO and wild type obese mice with estrogen for 8 weeks resulted in weight loss, splenic enlargement, and thymic atrophy, as expected. However, estrogen-treated obese GPER KO mice developed abdominal distension, debilitation, and ulceration of the skin surrounding the urogenital opening. At necropsy, these mice had prominently distended bladders and hydronephrosis. In contrast, estrogen-treated obese wild type mice only rarely displayed these signs. Our results suggest that, under conditions of obesity, estrogen induces BOO as a result of ERα-driven pathways and that GPER expression is protective against BOO.

Estrogen alleviates acute and chronic itch in mice.

Itching is associated with various skin diseases, including atopic dermatitis and allergic dermatitis, and leads to repeated scratching behavior and unpleasant sensation. Although clinical and laboratory research data have shown that estrogen is involved in regulating itch, the molecular and cellular basis of estrogen in itch sensation remains elusive. In the present study, it was found that estrogen-treated mice exhibited reduced scratching bouts when challenged with histamine, chloroquine, the proteinase-activated receptor-2 activating peptide SLIGRL-NH2 (SLIGRL), compound 48/80, and 5-hydroxytryptamine when compared with mice in the placebo group. Moreover, estrogen also suppressed scratching bouts in the mouse model of chronic itch induced by acetone-ether-water treatment. Notably, consistent with the behavioral tests, the present RNA-seq analysis showed that estrogen treatment caused significantly reduced expression levels of itch-related molecules such as Mas-related G-protein coupled receptor member A3, neuromedin B and natriuretic polypeptide b. In addition, estradiol attenuated histamine-induced and chloroquine-induced calcium influx in dorsal root ganglion neurons. Collectively, the data of the present study suggested that estrogen modulates the expression of itch-related molecules and suppresses both acute and chronic itch in mice.

Stress keratin 17 and estrogen support viral persistence and modulate the immune environment during cervicovaginal murine papillomavirus infection

A murine papillomavirus, MmuPV1, infects both cutaneous and mucosal epithelia of laboratory mice and can be used to model high-risk human papillomavirus (HPV) infection and HPV-associated disease. We have shown that estrogen exacerbates papillomavirus-induced cervical disease in HPV-transgenic mice. We have also previously identified stress keratin 17 (K17) as a host factor that supports MmuPV1-induced cutaneous disease. Here, we sought to test the role of estrogen and K17 in MmuPV1 infection and associated disease in the female reproductive tract. We experimentally infected wild-type and K17 knockout (K17KO) mice with MmuPV1 in the female reproductive tract in the presence or absence of exogenous estrogen for 6 mon. We observed that a significantly higher percentage of K17KO mice cleared the virus as opposed to wild-type mice. In estrogen-treated wild-type mice, the MmuPV1 viral copy number was significantly higher compared to untreated mice by as early as 2 wk postinfection, suggesting that estrogen may help facilitate MmuPV1 infection and/or establishment. Consistent with this, viral clearance was not observed in either wild-type or K17KO mice when treated with estrogen. Furthermore, neoplastic disease progression and cervical carcinogenesis were supported by the presence of K17 and exacerbated by estrogen treatment. Subsequent analyses indicated that estrogen treatment induces a systemic immunosuppressive state in MmuPV1-infected animals and that both estrogen and K17 modulate the local intratumoral immune microenvironment within MmuPV1-induced neoplastic lesions. Collectively, these findings suggest that estrogen and K17 act at multiple stages of papillomavirus-induced disease at least in part via immunomodulatory mechanisms.

IL-33 induces NF-κB activation in ILC2 that can be suppressed by in vivo and ex vivo 17β-estradiol.

Asthmatic women tend to develop severe airway disease in their reproductive years, and 30%-40% of asthmatic women have peri-menstrual worsening of asthma symptoms. This indicates that fluctuations in ovarian hormones are involved in advancement of asthmatic disease and exacerbation of symptoms. Group 2 innate lymphoid cells, or ILC2, are readily detected in allergic conditions, such as rhinosinusitis, in individuals that develop nasal polyps do to allergen exposures, and in allergic asthma. ILC2 are airway localized immune cells activated by IL-33, an innate cytokine that perpetuates allergic inflammation by driving the production of IL-5 and IL-13. We have previously shown that ILC2 are highly activated in naïve and ovalbumin (OVA) challenged, female BALB/c mice in comparison to male mice following stimulation with IL-33. Here, we investigated the effect of steady-state ovarian hormones on ILC2 and the NF-κB signaling pathway following OVA sensitization and challenge. We found that estrogen-treated ovariectomized mice (OVX-E2) that had been challenged with OVA had reduced IL-5 and IL-13 production by lung ILC2 as compared to lung ILC2 isolated from intact male and female sham-operated controls that had been treated with OVA. ILC2 were isolated from untreated animals and co-cultured ex vivo with and without estrogen plus IL-33. Those estrogen-treated ILC2 similarly produced less IL-5 and IL-13 in comparison to untreated, and had reduced NF-κB activation. Single-cell RNA sequencing showed that 120 genes were differentially expressed in male and female ILC2, and Nfkb1 was found among top-ranked regulatory interactions. Together, these results provide new insight into the suppressive effect of estrogen on ILC2 which may be protective in female asthmatics. Understanding further how estrogen modulates ILC2 may provide therapeutic targets for the treatment of allergic diseases.

Estrogen mediates inflammatory role of mast cells in endometriosis pathophysiology.

Endometriosis is an estrogen dependent, chronic inflammatory disease characterized by the growth of endometrial lining outside of the uterus. Mast cells have emerged as key players in regulating not only allergic responses but also other mechanisms such as angiogenesis, fibrosis, and pain. The influence of estrogen on mast cell function has also been recognized as a potential factor driving disease pathophysiology in number of allergic and chronic inflammatory conditions. However, precise information is lacking on the cross talk between endocrine and immune factors within the endometriotic lesions and whether that contributes to the involvement of mast cells with disease pathophysiology. In this study, we observed a significant increase in mast cell numbers within endometriotic lesions compared to matched eutopic endometrium from the same patients. Compared to eutopic endometrium, endometriotic lesions had significantly higher levels of stem cell factor (SCF), a potent growth factor critical for mast cell expansion, differentiation, and survival for tissue resident mast cells. Targeted mRNA Q-PCR array revealed that the endometriotic lesions harbour microenvironment (upregulation of CPA3, VCAM1, CCL2, CMA1, CCR1, and KITLG) that is conducive to mast cells recruitment and subsequent differentiation. To examine cross-talk of mast cells within the endometriotic lesion microenvironment, endometriotic epithelial cells (12Z) and endometrial stromal cells (hESC) incubated with mast cell-conditioned media showed significantly increased production of pro-inflammatory and chemokinetic cytokines. To further understand the impact of estrogen on mast cells in endometriosis, we induced endometriosis in C57BL/6 mice. Mature mast cells were significantly higher in peritoneal fluid of estrogen-treated mice compared to untreated mice within the sham operated groups. Mouse endometriotic lesion tissue revealed several genes (qRT-PCR) relevant in mast cell biology significantly upregulated in the estrogen treated, endometriosis-induced group compared to control endometrium. The endometriotic lesions from estrogen treated mice also had significantly higher density of Alcian blue stained mast cells compared to untreated lesions or control endometrium. Collectively, these findings suggest that endometriotic lesions provide a microenvironment necessary for recruitment and differentiation of mast cells. In turn, mast cells potentially release pro-inflammatory mediators that contribute to chronic pelvic pain and endometriosis disease progression.

Breast Cancer Endocrine Therapy Promotes Weight Gain With Distinct Adipose Tissue Effects in Lean and Obese Female Mice.

Breast cancer survivors treated with tamoxifen and aromatase inhibitors report weight gain and have an elevated risk of type 2 diabetes, especially if they have obesity. These patient experiences are inconsistent with, preclinical studies using high doses of tamoxifen which reported acute weight loss. We investigated the impact of breast cancer endocrine therapies in a preclinical model of obesity and in a small group of breast adipose tissue samples from women taking tamoxifen to understand the clinical findings. Mature female mice were housed at thermoneutrality and fed either a low-fat/low-sucrose (LFLS) or a high-fat/high-sucrose (HFHS) diet. Consistent with the high expression of Esr1 observed in mesenchymal stem cells from adipose tissue, endocrine therapy was associated with adipose accumulation and more preadipocytes compared with estrogen-treated control mice but resulted in fewer adipocyte progenitors only in the context of HFHS. Analysis of subcutaneous adipose stromal cells revealed diet- and treatment-dependent effects of endocrine therapies on various cell types and genes, illustrating the complexity of adipose tissue estrogen receptor signaling. Breast cancer therapies supported adipocyte hypertrophy and associated with hepatic steatosis, hyperinsulinemia, and glucose intolerance, particularly in obese females. Current tamoxifen use associated with larger breast adipocyte diameter only in women with obesity. Our translational studies suggest that endocrine therapies may disrupt adipocyte progenitors and support adipocyte hypertrophy, potentially leading to ectopic lipid deposition that may be linked to a greater type 2 diabetes risk. Monitoring glucose tolerance and potential interventions that target insulin action should be considered for some women receiving life-saving endocrine therapies for breast cancer.

17β-estradiol alters mRNA co-expression after murine muscle injury and mild hypobaria.

Here, we assessed the effects of 17β-estradiol exposure on mRNA co-expression patterns of muscle tissue during recovery in a closed muscle crush injury and hypobaria exposure murine model. Eighteen ovariectomized placebo-treated and 18 ovariectomized 17β-estradiol-treated female mice underwent closed muscle crush injury and hypobaric simulated flight. The mice recovered for 32, 96, or 192 h, and then were euthanized. Their harvested injured lateral gastrocnemius muscles underwent microarray analysis. We used weighted gene co-expression network analysis to construct a co-expression network for the control mice, and then applied the same network to the estrogen-treated mice. We compared the relationships between co-expression in gene modules over time between the two experimental groups. Enriched functional cluster analyses of significant co-expression network modules document a variety of different pathways of interest. Some of the functional cluster enrichments within several of the significantly correlated modules are related to the formation and function of microtubules. Our findings demonstrate that following a closed muscle crush injury in a murine model, the presence of 17β-estradiol alters mRNA co-expression patterns over time. It appears that estrogen promotes the expression of mRNA related to microtubule activity within the cytoskeleton of myofibers and in movement of organelles and receptors. Further study is needed, but the enrichment of these microtubule-related pathways may be integral in the muscle tissue regeneration process, and thus suggests that the presence of estrogen may promote muscle recovery through the work of the microtubules. Impact statement This study uses a murine model to address the clinical situation of transporting soldiers or civilians who have sustained skeletal muscle trauma by air. Our findings show that crush-injured muscle tissue of ovariectomized, 17β-estradiol-treated mice exposed to mild hypobaric hypoxia exhibited mRNA co-expression patterns among pathways associated with microtubule-dependent processes. Palmitoylation and other pathways necessary for movement of estrogen receptors to the cell membrane were also differentially enriched in the estrogen-treated mice. These first findings reframe the discussion regarding estrogen effects during muscle recovery from an inflammation-oriented inquiry to that of a structural, cytoskeletal inquiry and support additional research to understand the non-inflammation-related influences of estrogen during muscle recovery. Also, these results may suggest a role for estrogen or estrogen-like substances to treat muscle trauma.

Low-dose inoculation of Escherichia coli achieves robust vaginal colonization and results in ascending infection accompanied by severe uterine inflammation in mice.

Escherichia coli infection of the female reproductive tract is a significant cause of disease in humans and animals, but simple animal models are lacking. Here we report that vaginal inoculation of uropathogenic E. coli strains UTI89 and CFT073 in non-pregnant, estrogen-treated mice resulted in robust colonization of the vagina and uterine horns, whereas titers of the lab strain MG1655 were significantly lower. Non-estrogenized mice also became colonized, but there was more variation in titers. A dose of 104 colony-forming units (CFU) UTI89 was sufficient to result in colonization in all estrogenized mice, and we also observed bacterial transfer between inoculated and uninoculated estrogenized cage mates. UTI89 infection led to inflammation and leukocyte infiltration into the uterine horns as evidenced by tissue histology. Flow cytometry experiments revealed that neutrophil, monocyte and eosinophil populations were significantly increased in infected uterine horns. This model is a simple way to study host-pathogen interactions in E. coli vaginal colonization and uterine infection. There are immediate implications for investigators studying urinary tract infection using mouse models, as few E. coli are required to achieve reproductive colonization, resulting in an additional, underappreciated mucosal reservoir.

Insights into the pharmacology of GPER/GPR30 and its involvement in gallstone formation

The prevalence of gallstone disease is a major public health issue globally, and due to complex etiology of gallstones, there are a limited number of pharmaceuticals available. While the G protein‐coupled estrogen receptor (GPR30/GPER) has been shown to induce gallstone formation, little is known about its chemical biology and pharmacology. Building upon previous computational modeling of GPER, a novel series of antagonists were designed and synthesized. These novel compounds were assessed for their therapeutic value via calcium mobilization, cyclic AMP, and estrogen receptor a and β binding assays. From this series of compounds, one compound, SG‐1, exhibited superior antagonism and selectivity for GPER. The potential therapeutic value of SG‐1 was further validated in an in vivo mouse model of gallstone formation. Data revealed that SG‐1 treatment results in a dose‐dependent reduction in gallstone prevalence in estrogen‐treated ovariectomized mice.Support or Funding InformationSupported by startup funds from Saint Louis University (C.A.) and this work was supported in part by research grants DK73917 and DK101793 (to D.W.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Direct Inoculation and Human Transferrin Supplementation Increases the Survivability of Neisseria gonorrhoeae in the Upper Reproductive Tract of Estrogen‐Treated Female BALB/c Mice

ObjectiveThis study intended to investigate methods of increasing Neisseria gonorrhoeae (Gonococcus; Gc) survivability in the upper reproductive tract (URT) of female mice with the aim of establishing a previously undescribed murine model of Gc URT infections ‐ also known as pelvic inflammatory disease (PID) ‐ in women. Such a model would allow for further investigations into the natural history and pathogenesis of ascending Gc infections.MethodsTwo main experimental arms were used in this study: 1. Gc‐infected mice treated with estrogen alone vs estrogen and human transferrin (hTF) and 2. Gc‐infected mice treated with long‐acting progesterone (Medroxyprogesterone) alone vs progesterone and hTF. Appropriate uninfected, negative controls were used in all experiments. All mice were pre‐treated with antibiotics to suppress commensal flora and then given either estrogen or progesterone prior to inoculation, according to their study groups. Diestrous or anestrous stage BALB/c mice were then inoculated with Neisseria gonorrhoeae (strain FA1090) using a novel, trans‐cervical catheterization technique and the appropriate groups were treated with hTF up to 5 days post‐inoculation. Daily vaginal swabs were taken for polymorphonuclear white blood cell (PMN WBC) enumeration and quantitative Gc cultures up to day 5. In addition, part of each study group was sacrificed at days 3 and 5 post‐inoculation for Gc quantitative cultures of endometrial scrapings. Lastly, whole URTs from a portion of each group were fixed and stained for WBCs and Gc on days 3 and 5 post‐inoculation.ResultsDirect inoculation via trans‐cervical catheter proved successful in introducing and establishing Gc infection in the URT of all groups of estrogen‐treated mice for at least five days as determined by culture of endometrial scrapings and Gc‐specific IHC staining at day 5 post‐inoculation. Mice treated with progesterone showed no infection at any time point by either culture of daily vaginal swabs or of endometrial scrapings. Higher numbers of Gc were recovered from vaginal swabs (p<.05) and endometrial tissue on day 3 and 5 post‐inoculation from mice treated with estrogen and hTF compared to those treated with estrogen alone (comparisons made by geometric mean of Gc colonies recovered on each day).Conclusions and Future DirectionsThis project established a novel method of direct, trans‐cervical inoculation of Gc into the female mouse URT thereby creating a usable model of Gc URT infection (PID) in women for further study. Future projects will utilize this model to better understand the natural history of Gc infections and to investigate potential methods for treating these infections. Currently, this laboratory is investigating host immune responses by measuring serum antibodies and vaginal chemokines/cytokines using this model. In addition, GFP‐labeled FA1090 strain Gc have been created and will be used for better visualization of Gc in histologic samples, which in turn could be used to track Gc movement through the mouse URT, as well as cellular responses to infection using sequentially fixed tissue samples.Support or Funding InformationThe Uniformed Services University of the Health Sciences and NIH/NIAID grant # R01 AI42053.

Sex Hormone Contributes to Sexually Dimorphic Susceptibility in CVB3-Induced Viral Myocarditis via Modulating IFN-γ+ NK Cell Production

BackgroundViral myocarditis is a widespread cardiac disease associated with inflammation and myocardial injury and is predominantly caused by coxsackievirus B3 (CVB3) infection in humans as well as in mice. CVB3-induced myocarditis shows sexually dimorphic sensitivity and is more prevalent in male mice. Our previous studies showed that natural killer (NK) cells played an indispensable role in CVB3-induced myocarditis, and female mice exhibited less pathological cardiac interferon gamma (IFN-γ)+ NK cell infiltration than did male mice. However, the precise mechanisms were not well elucidated. MethodsWe investigated the influence of estrogen on cardiac IFN-γ+ NK cell enrichment in CVB3-induced myocarditis and explored the underlying molecular mechanism. ResultsIn this study, we found that CVB3 stimulation could clearly induce IFN-γ expression by NK cells; however, this trend could be blunted by estrogen treatment. Consistently, ovariectomized female mice with decreased estrogen levels exhibited substantially increased enrichment of cardiac IFN-γ+ NK cells and displayed significantly aggravated myocarditis. Similarly, estrogen-treated male mice showed less cardiac IFN-γ+ NK cell infiltration, accompanied by significantly alleviated viral myocarditis. In sharp contrast, sexually immature female and male mice (with similar estrogen levels) showed comparable levels of cardiac IFN-γ+ NK cell infiltration and similar levels of myocarditis severity. Upon further exploration of the underlying mechanisms, we found that estrogen could downregulate expression of Th1-specific T box transcription factor (T-bet), the key transcription factor associated with IFN-γ production, in CVB3-stimulated NK cells. ConclusionsOverall, this study might help us understand the mechanism of increased cardiac infiltration by IFN-γ+ NK cells in CVB3-infected male mice compared with that in female mice and might provide new clues for the sex bias in CVB3-induced myocarditis.

Human papillomavirus oncogenes reprogram the cervical cancer microenvironment independently of and synergistically with estrogen

High-risk human papillomaviruses (HPVs) infect epithelial cells and are causally associated with cervical cancer, but HPV infection is not sufficient for carcinogenesis. Previously, we reported that estrogen signaling in the stromal tumor microenvironment is associated with cervical cancer maintenance and progression. We have now determined how HPV oncogenes and estrogen treatment affect genome-wide host gene expression in laser-captured regions of the cervical epithelium and stroma of untreated or estrogen-treated nontransgenic and HPV-transgenic mice. HPV oncogene expression in the cervical epithelium elicited significant gene-expression changes in the proximal stromal compartment, and estrogen treatment uniquely affected gene expression in the cervical microenvironment of HPV-transgenic mice compared with nontransgenic mice. Several potential estrogen-induced paracrine-acting factors were identified in the expression profile of the cervical tumor microenvironment. The microenvironment of estrogen-treated HPV-transgenic mice was significantly enriched for chemokine/cytokine activity and inflammatory and immune functions associated with carcinogenesis. This inflammatory signature included several proangiogenic CXCR2 receptor ligands. A subset of the same CXCR2 ligands was likewise increased in cocultures of early-passage cells from human cervical samples, with levels highest in cocultures of cervical fibroblasts and cancer-derived epithelial cells. Our studies demonstrate that high-risk HPV oncogenes profoundly reprogram the tumor microenvironment independently of and synergistically with estrogen. These observations illuminate important means by which HPVs can cause cancer through alterations in the tumor microenvironment.

Neutrophils and neutrophil serine proteases are increased in the spleens of estrogen-treated C57BL/6 mice and several strains of spontaneous lupus-prone mice.

Estrogen, a natural immunomodulator, regulates the development and function of diverse immune cell types. There is now renewed attention on neutrophils and neutrophil serine proteases (NSPs) such as neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CG) in inflammation and autoimmunity. In this study, we found that although estrogen treatment significantly reduced total splenocytes number, it markedly increased the splenic neutrophil absolute numbers in estrogen-treated C57BL/6 (B6) mice when compared to placebo controls. Concomitantly, the levels of NSPs and myeloperoxidase (MPO) were highly upregulated in the splenocytes from estrogen-treated mice. Despite the critical role of NSPs in the regulation of non-infectious inflammation, by employing NE-/-/PR3-/-/CG-/- triple knock out mice, we demonstrated that the absence of NSPs affected neither estrogen’s ability to increase splenic neutrophils nor the induction of inflammatory mediators (IFNγ, IL-1β, IL-6, TNFα, MCP-1, and NO) from ex vivo activated splenocytes. Depletion of neutrophils in vitro in splenocytes with anti-Ly6G antibody also had no obvious effect on NSP expression or LPS-induced IFNγ and MCP-1. These data suggest that estrogen augments NSPs, which appears to be independent of enhancing ex vivo inflammatory responses. Since estrogen has been implicated in regulating several experimental autoimmune diseases, we extended our observations in estrogen-treated B6 mice to spontaneous autoimmune-prone female MRL-lpr, B6-lpr and NZB/WF1 mice. There was a remarkable commonality with regards to the increase of neutrophils and concomitant increase of NSPs and MPO in the splenic cells of different strains of autoimmune-prone mice and estrogen-treated B6 mice. Collectively, since NSPs and neutrophils are involved in diverse pro-inflammatory activities, these data suggest a potential pathologic implication of increased neutrophils and NSPs that merits further investigation.

Klotho/fibroblast growth factor 23- and PTH-independent estrogen receptor-α-mediated direct downregulation of NaPi-IIa by estrogen in the mouse kidney.

Estrogen treatment causes renal phosphate (Pi) wasting and hypophosphatemia in rats and humans; however, the signaling mechanisms mediating this effect are still poorly understood. To determine the specific roles of estrogen receptor isoforms (ERα and ERβ) and the Klotho pathway in mediating these effects, we studied the effects of estrogen on renal Pi handling in female mice with null mutations of ERα or ERβ or Klotho and their wild type (WT) using balance studies in metabolic cages. Estrogen treatment of WT and ERβ knockout (KO) mice caused a significant reduction in food intake along with increased renal phosphate wasting. The latter resulted from a significant downregulation of NaPi-IIa and NaPi-IIc protein abundance. The mRNA expression levels of both transporters were unchanged in estrogen-treated mice. These effects on both food intake and renal Pi handling were absent in ERα KO mice. Estrogen treatment of Klotho KO mice or parathyroid hormone (PTH)-depleted thyroparathyroidectomized mice exhibited a significant downregulation of NaPi-IIa with no change in the abundance of NaPi-IIc. Estrogen treatment of a cell line (U20S) stably coexpressing both ERα and ERβ caused a significant downregulation of NaPi-IIa protein when transiently transfected with a plasmid containing full-length or open-reading frame (ORF) 3'-untranslated region (UTR) but not 5'-UTR ORF of mouse NaPi-IIa transcript. In conclusion, estrogen causes phosphaturia and hypophosphatemia in mice. These effects result from downregulation of NaPi-IIa and NaPi-IIc proteins in the proximal tubule through the activation of ERα. The downregulation of NaPi-IIa by estrogen involves 3'-UTR of its mRNA and is independent of Klotho/fibroblast growth factor 23 and PTH signaling pathways.

Secreted Phosphoprotein 1 and Sex-Specific Differences in Silica-Induced Pulmonary Fibrosis in Mice.

Background:Fibrotic lung diseases occur predominantly in males, and reports describe better survival in affected females. Male mice are more sensitive to silica-induced lung fibrosis than silica-treated female mice. Secreted phosphoprotein 1 (SPP1, also known as osteopontin) increases in pulmonary fibrosis, and Spp1 transcription may be regulated by estrogen or estrogen receptor–related receptors.Objective:We determined whether differences in silica-induced SPP1 levels contribute to sex differences in lung fibrosis.Methods:Male and female mice were treated with 0.2 g/kg intratracheal silica, and lung injury was assessed 1, 3, or 14 days post-exposure. Gene-targeted (Spp1–/–) mice, control Spp1+/+ (C57BL/6J) mice, ovariectomized (OVX) female mice, and estrogen-treated male mice were treated with silica, and lung injury was assessed.Results:Silica-induced SPP1 in lung tissue, bronchoalveolar lavage, and serum increased more in male than in female mice. Following silica treatment, bronchoalveolar lavage cell infiltrates decreased in female Spp1–/– mice compared with female Spp1+/+ mice, and lung hydroxyproline decreased in male Spp1–/– mice compared with male Spp1+/+ mice. OVX female mice had increased lung SPP1 expression in response to silica compared with silica-treated sham female mice. Silica-induced lung collagen and hydroxyproline (markers of fibrosis), and SPP1 levels decreased in estrogen-treated males compared with untreated males.Conclusion:These findings suggest that sex-specific differences in SPP1 levels contribute to the differential sensitivity of male and female mice to the development of silica-induced fibrosis.Citation:Latoche JD, Ufelle AC, Fazzi F, Ganguly K, Leikauf GD, Fattman CL. 2016. Secreted phosphoprotein 1 and sex-specific differences in silica-induced pulmonary fibrosis in mice. Environ Health Perspect 124:1199–1207; http://dx.doi.org/10.1289/ehp.1510335

Abstract P5-04-01: ERβ elicits tumor suppressive effects in triple negative breast cancer through the induction of cystatins and suppression of TGFβ signaling

Abstract Background: Triple negative breast cancer (TNBC) accounts for approximately 20% of all breast cancer diagnoses. Clinical management of TNBC is limited to surgery, chemotherapy and radiation due to lack of estrogen receptor alpha and HER2 expression. Recently, we have shown that approximately 40% of TNBCs express estrogen receptor beta (ERβ) and have begun to explore the possibility that this receptor could be utilized as a novel therapeutic target for this disease. Methods: To examine the biological functions of ERβ in TNBC, novel ERβ expressing TN cell lines (MDA-MB-231 and Hs578T) were developed. In vitro experiments were employed to determine alterations in the global gene expression profiles, biological pathways, proliferation rates, and cell cycle progression following estrogen or ERβ-specific agonist treatment. Cell line xenografts were also established in athymic ovariectomized nude mice to examine tumoral responses to ERβ targeting agents and to investigate gene and protein expression patterns as well as potential serum biomarkers indicative of therapeutic response. Additionally, using the resources of the Mayo Clinic Breast Cancer Genome Guided Therapy Study (BEAUTY), we have identified, and begun to analyze, ERβ+ and ERβ- patient derived xenografts (PDX) established from women with TNBC. Results: Our studies have revealed that both estrogen and multiple ERβ-specific agonists elicit significant anti-proliferative effects in ERβ+ TNBC cells primarily through a G1/S phase cell cycle arrest. These anti-proliferative effects appear to be mediated by cystatins, a family of small secreted cysteine protease inhibitors which are highly induced following estrogen and ERβ-specific agonist treatment. Conditioned media isolated from estrogen or ERβ-specific agonist treated cells decreased the proliferation rates of multiple non-ERβ expressing cell lines; effects that were completely reversed when cystatins were depleted from the media. In addition, we have shown that activation of ERβ, and the subsequent induction of cystatin gene expression, leads to suppression of canonical TGFβ signaling through multiple mechanisms including suppression of TGFβR2 expression, induction of Smad7 expression and blockade of TGFβ ligand-mediated activation of this pathway both in vitro and in vivo. Finally, ERβ+ TNBC PDXs exhibit significantly decreased tumor growth rates in estrogen-treated mice compared to ERβ- TN breast tumors. Conclusions: Our in vitro and in vivo data show that estrogen and ERβ-specific agonists elicit anti-cancer effects in ERβ+ TNBC. These effects appear to be mediated, in part, by cystatins through their inhibitory effects on canonical TGFB signaling, a pathway known to drive TNBC progression. Importantly, these data lay the foundation for studies aimed at examining the ability to therapeutically target ERβ in TNBC patients. Citation Format: Reese J, Bruinsma E, Subramaniam M, Suman V, Pitel K, Kalari K, Yu J, Wang L, Goetz M, Ingle J, Hawse J. ERβ elicits tumor suppressive effects in triple negative breast cancer through the induction of cystatins and suppression of TGFβ signaling. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-01.

Sexual maturation protects against development of lung inflammation through estrogen.

Increasing levels of estrogen and progesterone are suggested to play a role in the gender switch in asthma prevalence during puberty. We investigated whether the process of sexual maturation in mice affects the development of lung inflammation in adulthood and the contributing roles of estrogen and progesterone during this process. By inducing ovalbumin-induced lung inflammation in sexually mature and immature (ovariectomized before sexual maturation) adult mice, we showed that sexually immature adult mice developed more eosinophilic lung inflammation. This protective effect of "puberty" appears to be dependent on estrogen, as estrogen supplementation at the time of ovariectomy protected against development of lung inflammation in adulthood whereas progesterone supplementation did not. Investigating the underlying mechanism of estrogen-mediated protection, we found that estrogen-treated mice had higher expression of the anti-inflammatory mediator secretory leukoprotease inhibitor (SLPI) and lower expression of the proasthmatic cytokine IL-33 in parenchymal lung tissue and that their expressions colocalized with type II alveolar epithelial cells (AECII). Treating AECII directly with SLPI significantly inhibited IL-33 production upon stimulation with ATP. Our data suggest that estrogen during puberty has a protective effect on asthma development, which is accompanied by induction of anti-inflammatory SLPI production and inhibition of proinflammatory IL-33 production by AECII.

Klotho/fgf23‐Independent and ERα Mediated Downregulation of NaPi‐IIa and NaPi‐IIc by Estrogen in the Mouse Kidney Proximal Tubule

In these studies, we examined the effect of estrogen on renal inorganic phosphate (Pi) transport and the roles of its receptor isoforms (ERα and ERβ) and Klotho/FGF23 pathway in this effect in mice. Mice with null mutations (KO) of ERα or ERβ or Klotho and their wild‐type (WT) were placed in metabolic cages and injected daily with 17β‐estradiol or vehicle for 3 days. Food intake, urine volume and Pi excretion were measured daily. The results indicate that estrogen‐treated WT mice exhibited a significant increase in renal Pi wasting despite a reduction in food intake. Molecular studies showed a significant downregulation of NaPi‐IIa and NaPi‐IIc proteins with no change in their mRNA expression levels in the kidney. Estrogen‐induced reduction in food intake, phosphaturia and downregulation of NaPi‐IIa and NaPi‐IIc proteins were observed in ERβ KO but not in ERα KO mice. Further, Klotho KO mice exhibited a significant downregulation of NaPi‐IIa and hypophosphatemia in response to estrogen. In a cell line stably co‐expressing both ERα and ERβ, 24h of estrogen downregulated NaPi‐IIa protein, when cells are transiently transfected with a plasmid containing ORF‐3'UTR but not 5'UTR‐ORF of the mouse NaPi‐IIa transcript. In conclusion, estrogen causes phosphaturia in mice through the downregulation NaPi‐IIa and NaPi‐IIc protein abundance in the kidney. This effect is independent of Klotho/FGF23 pathway and is mediated through the activation of ERα. 3'UTR region of mouse NaPi‐IIa mRNA likely plays an important role in the inhibition of its translation by estrogen.

Ovarian Hormones Regulate SP-D Expression in the Mouse Uterus During Estrous Cycle and Early Pregnancy.

Differential expression of SP-D in the cycling human and mouse endometrium suggests its regulation by ovarian hormones. SP-D expression in the mouse uterus was analyzed across the estrous cycle and during early pregnancy. Effect of exogenous ovarian hormones on the uterine expression of SP-D was analyzed. SP-D expression varied across the estrous cycle and peaked in the estrous phase. SP-D transcript levels increased by fourfold in the uteri of estrogen-treated mice while co-administration of estrogen and progesterone enhanced SP-D levels by ninefold. However, treatment with progesterone alone significantly downregulated SP-D expression. Diethylstilbestrol enhanced SP-D transcript levels in the uteri of immature mice by 10-fold. During pregnancy, SP-D levels declined rapidly from 0.5 dpc to 6.5 dpc. In silico analysis predicted the presence of two potential ERE and 1 PRE in the mouse SP-D gene promoter region. Estrogen positively regulates expression of SP-D in the mouse uterus. Progesterone, along with estrogen synergizes SP-D expression, however, when administered alone results in negative regulation.

Female resistance to pneumonia identifies lung macrophage nitric oxide synthase-3 as a therapeutic target.

To identify new approaches to enhance innate immunity to bacterial pneumonia, we investigated the natural experiment of gender differences in resistance to infections. Female and estrogen-treated male mice show greater resistance to pneumococcal pneumonia, seen as greater bacterial clearance, diminished lung inflammation, and better survival. In vitro, lung macrophages from female mice and humans show better killing of ingested bacteria. Inhibitors and genetically altered mice identify a critical role for estrogen-mediated activation of lung macrophage nitric oxide synthase-3 (NOS3). Epidemiologic data show decreased hospitalization for pneumonia in women receiving estrogen or statins (known to activate NOS3). Pharmacologic targeting of NOS3 with statins or another small-molecule compound (AVE3085) enhanced macrophage bacterial killing, improved bacterial clearance, and increased host survival in both primary and secondary (post-influenza) pneumonia. The data identify a novel mechanism for host defense via NOS3 and suggest a potential therapeutic strategy to reduce secondary bacterial pneumonia after influenza.