G Protein-Coupled Estrogen Receptor GPER: Molecular Pharmacology and Therapeutic Applications.

The actions of estrogens and related estrogenic molecules are complex and multifaceted in both sexes. A wide array of natural, synthetic, and therapeutic molecules target pathways that produce and respond to estrogens. Multiple receptors promulgate these responses, including the classical estrogen receptors of the nuclear hormone receptor family (estrogen receptors α and ß), which function largely as ligand-activated transcription factors, and the 7-transmembrane G protein-coupled estrogen receptor, GPER, which activates a diverse array of signaling pathways. The pharmacology and functional roles of GPER in physiology and disease reveal important roles in responses to both natural and synthetic estrogenic compounds in numerous physiological systems. These functions have implications in the treatment of myriad disease states, including cancer, cardiovascular diseases, and metabolic disorders. This review focuses on the complex pharmacology of GPER and summarizes major physiological functions of GPER and the therapeutic implications and ongoing applications of GPER-targeted compounds.

Complement Receptor 3 Contributes to the Sexual Dimorphism in Neutrophil Killing of Staphylococcus aureus.

We previously reported sex differences in innate susceptibility to Staphylococcus aureus skin infection and that bone marrow neutrophils (BMN) from female mice have an enhanced ability to kill S. aureus ex vivo compared with those of male mice. However, the mechanism(s) driving this sex bias in neutrophil killing have not been reported. Given the role of opsonins such as complement, as well as their receptors, in S. aureus recognition and clearance, we investigated their contribution to the enhanced bactericidal capacity of female BMN. We found that levels of C3 in the serum and CR3 (CD11b/CD18) on the surface of BMN were higher in female compared with male mice. Consistent with increased CR3 expression following TNF-α priming, production of reactive oxygen species (ROS), an important bactericidal effector, was also increased in female versus male BMN in response to serum-opsonized S. aureus Furthermore, blocking CD11b reduced both ROS levels and S. aureus killing by murine BMN from both sexes. However, at the same concentration of CD11b blocking Ab, S. aureus killing by female BMN was greatly reduced compared with those from male mice, suggesting CR3-dependent differences in bacterial killing between sexes. Overall, this work highlights the contributions of CR3, C3, and ROS to innate sex bias in the neutrophil response to S. aureus Given that neutrophils are crucial for S. aureus clearance, understanding the mechanism(s) driving the innate sex bias in neutrophil bactericidal capacity could identify novel host factors important for host defense against S. aureus.

Differential effects of CD20+ B cells and PD-L1+ immune cells on pathologic complete response and outcome: comparison between inflammatory breast cancer and locally advanced breast cancer patients.

PURPOSE: This study evaluated epidemiologic and immune factors associated with pathologic complete response (pCR), breast cancer-specific survival (BCSS) and disease-free survival (DFS) outcomes in inflammatory (IBC) and locally advanced breast cancer (LABC) patients. METHODS: Tumor-infiltrating lymphocytes (TILs) and CD20+ B-cell frequencies (CD20+), and PD-L1 expression on tumor (PD-L1+carcinoma cells) and immune (PD-L1+TILs) cells were analyzed by immunohistochemistry along with clinicopathologic factors as modifiers of pCR and outcomes in 221 IBC and 162 LABC patients. Analysis included Kaplan-Meier curves and Cox proportional hazard models. RESULTS: IBC and LABC display similar levels of TILs, CD20+, and combined CD20+ and PD-L1+TILs (CD20+PD-L1+TILs), while LABC contained more PD-L1+TILs and PD-L1+ carcinoma cells. Absence of lymphovascular involvement, high TILs, PD-L1+ carcinoma cells, and combined CD20+ and PD-L1+ carcinoma cells correlated with pCR in IBC and LABC patients. High PD-L1+TILs correlated with pCR only in LABC; less lymph node involvement at diagnosis, CD20+ and CD20+PD-L1+TILs correlated with pCR only in IBC (P < 0.04, all comparisons). Achievement of pCR in IBC and LABC patients correlated with BCSS and DFS (P < 0.02). In multivariate analyses, pCR remained an independent prognostic factor of improved DFS in IBC and LABC patients, but of BCSS in only LABC. CD20+PD-L1+TILs remained an independent prognostic factor of improved DFS and BCSS only in IBC. CONCLUSION: CD20+PD-L1+TILs are an independent prognostic biomarker of improved outcomes in IBC, but not LABC. Selecting IBC patients by CD20 and PD-L1 status could stratify patients and potentially identify those in whom activating CD20 agents and anti-PD-1/PD-L1 therapy could be explored.

A hypertension patient-derived iPSC model demonstrates a role for G protein-coupled estrogen receptor in hypertension risk and development.

Hypertension (HTN) is a polyfactorial disease that can manifest severe cardiovascular pathologies such as heart failure or stroke. Genome-wide association studies (GWAS) of HTN indicate that single-nucleotide polymorphisms (SNPs) contribute to increased risk for HTN and resistance to some HTN drug regimens (Hiltunen TP et al., J Am Heart Assoc 4: e001521, 2015; Le MT et al., PLoS One 8: e52062, 2013; McDonough CW et al., J Hypertens 31: 698-704, 2013; Vandell AG et al., Hypertension 60: 957-964, 2012). However, cellular mechanistic insights of such SNPs remain largely unknown. Using a bank of induced pluripotent stem cells (iPSCs) derived from patients with HTN and CRISPR/Cas9-mediated gene-editing approach, we investigated the effects of a female HTN risk-associated SNP (rs1154431) of the G protein-coupled estrogen receptor (GPER) (Bassuk SS, Manson JE., Clin Chem 60: 68-77, 2014) in vascular endothelial cells. Although GPER1 deletion reduced endothelial nitric oxide synthase (eNOS) activation in iPSC-derived endothelial cells (iECs), the polymorphism itself did not significantly affect eNOS and NO production in a comparison of isogenic hemizygous iECs expressing either normal (P16) or HTN-associated (L16) GPER. Interestingly, we demonstrate for the first time that GPER plays a role in regulation of adhesion molecule expression and monocyte adhesion to iECs. Moreover, the L16 iECs had higher expression of inflammation genes than P16 iECs, implying that the risk variant may affect carrier individuals through increased inflammatory activity. This study further indicates that iPSCs are a useful platform for exploring mechanistic insights underlying hypertension GWAS endeavors.

Sex differences in murine myocutaneous flap revascularization.

Sex differences in susceptibility to ischemia/reperfusion injury have been documented in humans. Premenopausal women have a lower risk of ischemic heart disease than age-matched men, whereas after menopause, the risk is similar or even higher in women. However, little is known about the effects of sex on myocutaneous ischemia/reperfusion. To explore sex differences in wound revascularization, we utilized a murine myocutaneous flap model of graded ischemia. A cranial-based, peninsular-shaped, myocutaneous flap was surgically created on the dorsum of male and female mice. Physiological, pathological, immunohistochemical, and molecular parameters were analyzed. Flaps created on female mice were re-attached to the recipient site resulting in nearly complete viability at post-operative day 10. In contrast, distal full-thickness myocutaneous necrosis was evident at 10 days post-surgery in male mice. Over the 10 day study interval, laser speckle imaging documented functional revascularization in all flap regions in female mice, but minimal distal flap reperfusion in male mice. Day 10 immunostained histologic sections confirmed significant increases in distal flap vessel count and vascular surface area in female compared to male mice. RT-PCR demonstrated significant differences in growth factor and metabolic gene expression between female and male mice at day 10. In conclusion, in a graded-ischemia wound healing model, flap revascularization was more effective in female mice. The recognition and identification of sex-specific wound healing differences may lead to a better understanding of the underlying mechanisms of myocutaneous revascularization and drive novel discovery to improve soft tissue wound healing following tissue transfer for traumatic injury and cancer resection.

Innate Sex Bias of Staphylococcus aureus Skin Infection Is Driven by α-Hemolysin.

Numerous studies have reported sex bias in infectious diseases, with bias direction dependent on pathogen and site of infection. Staphylococcus aureus is the most common cause of skin and soft tissue infections (SSTIs), yet sex bias in susceptibility to S. aureus SSTI has not been described. A search of electronic health records revealed an odds ratio of 2.4 for S. aureus SSTI in males versus females. To investigate the physiological basis of this bias, we compared outcomes between male and female mice in a model of S. aureus dermonecrosis. Consistent with the epidemiological data, female mice were better protected against SSTI, with reduced dermonecrosis followed later by increased bacterial clearance. Protection in females was disrupted by ovariectomy and restored by short-term estrogen administration. Importantly, this sex bias was mediated by a sex-specific response to the S. aureus-secreted virulence factor α-hemolysin (Hla). Infection with wild-type S. aureus suppressed inflammatory cytokine production in the skin of female, but not male, mice when compared with infection with an isogenic hla deletion mutant. This differential response was conserved following injection with Hla alone, demonstrating a direct response to Hla independent of bacterial burden. Additionally, neutrophils, essential for clearing S. aureus, demonstrated sex-specific S. aureus bactericidal capacity ex vivo. This work suggests that sex-specific skin innate responsiveness to Hla and neutrophil bactericidal capacity play important roles in limiting S. aureus SSTI in females. Understanding the molecular mechanisms controlling this sex bias may reveal novel targets to promote host innate defense against S. aureus skin infection.

The R-Enantiomer of Ketorolac Delays Mammary Tumor Development in Mouse Mammary Tumor Virus-Polyoma Middle T Antigen (MMTV-PyMT) Mice.

Epidemiologic studies report improved breast cancer survival in women who receive ketorolac (Toradol) for postoperative pain relief compared with other analgesic agents. Ketorolac is a racemic drug. The S-enantiomer inhibits cyclooxygenases; R-ketorolac is a selective inhibitor of the small GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42), which are signaling molecules up-regulated during breast cancer progression and metastasis. The goal of this study was to determine whether R-ketorolac altered breast cancer development in the mouse mammary tumor virus-polyoma middle T-antigen model. Mice were administered ketorolac orally at 1 mg/kg twice daily to approximate the typical human dose. Mammary glands were analyzed for tumor number and immunohistochemical markers of proliferation and differentiation. R-ketorolac treatment significantly reduced mammary epithelial proliferation, based on Ki67 staining, and suppressed tumor development. Proliferative mammary epithelium from R-ketorolac-treated mice displayed greater differentiation, based on significantly higher total E-cadherin and decreased keratin 5 staining than epithelium of placebo-treated mice. No differences were detected in estrogen receptor, progesterone receptor, ß-catenin, or vimentin expression between placebo and R-ketorolac treatment groups. These findings indicate that R-ketorolac treatment slows tumor progression in an aggressive model of breast cancer. R-ketorolac may thus represent a novel therapeutic approach for breast cancer prevention or treatment based on its pharmacologic activity as a Rac1 and Cdc42 inhibitor.

Towards a transcriptome-based theranostic platform for unfavorable breast cancer phenotypes.

Inflammatory breast carcinoma (IBC) is one of the most lethal forms of human breast cancer, and effective treatment for IBC is an unmet clinical need in contemporary oncology. Tumor-targeted theranostic approaches are emerging in precision medicine, but only a few specific biomarkers are available. Here we report up-regulation of the 78-kDa glucose-regulated protein (GRP78) in two independent discovery and validation sets of specimens derived from IBC patients, suggesting translational promise for clinical applications. We show that a GRP78-binding motif displayed on either bacteriophage or adeno-associated virus/phage (AAVP) particles or loop-grafted onto a human antibody fragment specifically targets orthotopic IBC and other aggressive breast cancer models in vivo. To evaluate the theranostic value, we used GRP78-targeting AAVP particles to deliver the human Herpes simplex virus thymidine kinase type-1 (HSVtk) transgene, obtaining simultaneous in vivo diagnosis through PET imaging and tumor treatment by selective activation of the prodrug ganciclovir at tumor sites. Translation of this AAVP system is expected simultaneously to image, monitor, and treat the IBC phenotype and possibly other aggressive (e.g., invasive and/or metastatic) subtypes of breast cancer, based on the inducible cell-surface expression of the stress-response chaperone GRP78, and possibily other cell-surface receptors in human tumors.

Breast cancer survival, survival disparities, and guideline-based treatment.

PURPOSE: The role of appropriate therapy in breast cancer survival and survival disparities by race/ethnicity has not been fully elucidated. We investigated whether lack of guideline-recommended therapy contributed to survival differences overall and among Hispanics relative to non-Hispanic white (NHW) women in a case-cohort study. METHODS: The study included a 15% random sample of female invasive breast cancer patients diagnosed from 1997 to 2009 in 6 New Mexico counties and all deaths due to breast cancer-related causes. Information was obtained from comprehensive medical chart reviews. National Comprehensive Cancer Network (NCCN®) guideline-recommended treatment was assessed among white women aged < 70 who were free of contraindications for recommended therapy, had stage I-III tumors, and survived ≥ 12 months. Hazard ratios (HRs) and 95% confidence intervals (CIs) for breast cancer death were estimated using Cox proportional hazards models. RESULTS: Included women represented 4635 patients and 449 breast cancer deaths. Women who did not receive radiotherapy (HR 2.3; 95% CI 1.2-4.4) or endocrine therapy (HR 2.0; 95% CI 1.0-4.0) as recommended by guidelines had an increased risk of breast cancer death, relative to those treated appropriately. Receipt of guideline-recommended therapy did not differ between Hispanic and NHW women for chemotherapy (84.2% vs. 81.3%, respectively), radiotherapy (89.2% vs. 91.1%), or endocrine therapy (89.2% vs. 85.8%), thus did not influence Hispanic survival disparities. CONCLUSIONS: Lack of guideline-recommended radiotherapy or endocrine therapy contributed to survival as strongly as other established prognostic indicators. Hispanic survival disparities in this population do not appear to be attributable to treatment differences.

Female Sex Hormone Receptor Profiling in Uterine Adenosarcomas.

OBJECTIVE: This study aimed to identify the hormonal receptor status in uterine adenosarcoma (AS) and uterine AS with sarcomatous overgrowth (AS + SO), including those with high-grade histologic features (nuclear pleomorphism, atypical mitoses, necrosis), with or without heterologous elements. Estrogen receptor (ER) status, including estrogen receptor α (ERα), estrogen receptor ß (ERß), and G protein-coupled estrogen receptor (GPER), and progesterone receptor (PgR) status were examined. METHODS: From August 2001 to November 2013, 11 patients with histologic diagnosis of uterine AS were identified. Tumor tissue sections were stained for ERα, ERß, GPER, and PgR and examined both for percentage of overall cells stained and for intensity of staining. Descriptive statistics were calculated using clinicopathologic data abstracted from the medical record. RESULTS: Eight cases of AS and 3 cases of AS with high-grade features were identified. Seven of 8 tumor samples of AS showed strong or moderate intensity immunostaining for ERα; all AS + SO tumor samples showed minimal to no immunoreactivity for ERα. There was a significant decrease in ERα H scores in high-grade tumors when compared with AS (P = 0.01). Lower PgR H scores were observed in high-grade tumors compared with those in AS (P = 0.04). Estrogen receptor ß immunostaining was variable, and GPER immunostaining was absent in the majority of tumor samples. CONCLUSIONS: Higher expression of ERα and PgR was observed in AS when compared with those with AS + SO and high-grade features. Both tumor subtypes showed similar levels of ERß and GPER expression, although significant differences in ERß and GPER expression were not detected. In contrast to our previous findings in uterine carcinosarcoma, ERs ERß and GPER do not seem to play a significant role in AS in this study.

International Union of Basic and Clinical Pharmacology. XCVII. G Protein-Coupled Estrogen Receptor and Its Pharmacologic Modulators.

Estrogens are critical mediators of multiple and diverse physiologic effects throughout the body in both sexes, including the reproductive, cardiovascular, endocrine, nervous, and immune systems. As such, alterations in estrogen function play important roles in many diseases and pathophysiological conditions (including cancer), exemplified by the lower prevalence of many diseases in premenopausal women. Estrogens mediate their effects through multiple cellular receptors, including the nuclear receptor family (ERα and ERß) and the G protein-coupled receptor (GPCR) family (GPR30/G protein-coupled estrogen receptor [GPER]). Although both receptor families can initiate rapid cell signaling and transcriptional regulation, the nuclear receptors are traditionally associated with regulating gene expression, whereas GPCRs are recognized as mediating rapid cellular signaling. Estrogen-activated pathways are not only the target of multiple therapeutic agents (e.g., tamoxifen, fulvestrant, raloxifene, and aromatase inhibitors) but are also affected by a plethora of phyto- and xeno-estrogens (e.g., genistein, coumestrol, bisphenol A, dichlorodiphenyltrichloroethane). Because of the existence of multiple estrogen receptors with overlapping ligand specificities, expression patterns, and signaling pathways, the roles of the individual receptors with respect to the diverse array of endogenous and exogenous ligands have been challenging to ascertain. The identification of GPER-selective ligands however has led to a much greater understanding of the roles of this receptor in normal physiology and disease as well as its interactions with the classic estrogen receptors ERα and ERß and their signaling pathways. In this review, we describe the history and characterization of GPER over the past 15 years focusing on the pharmacology of steroidal and nonsteroidal compounds that have been employed to unravel the biology of this most recently recognized estrogen receptor.

Estrogen receptor quantitative measures and breast cancer survival.

PURPOSE: While the estrogen receptor (ER) is the single most widely used biomarker to evaluate breast cancer outcomes, aspects of ER marker biology remain poorly understood. We sought to determine whether quantitative measures of ER, such as protein expression and intensity, were associated with survival, or with survival disparities experienced by Hispanic women. METHODS: A case-cohort study included a 15% random sample of invasive breast cancer cases diagnosed from 1997 to 2009 in six New Mexico counties and all deaths due to breast cancer-related causes. Pathology reports and tissue microarrays served as sources of ER information. Analyses were restricted to women with ≥1% ER immunohistochemical staining. Hazard ratios (HR) and 95% confidence intervals (CI) for breast cancer death were estimated using Cox proportional hazards models. RESULTS: Included women represented 4336 ER+ breast cancer cases and 448 deaths. Median follow-up was 93 months. ER percent expression was not associated with breast cancer survival after adjustment for standard prognostic factors (p trend = 0.76). ER intensity remained a strong and independent risk factor for breast cancer survival in multivariate analyses: Women whose tumors expressed ER at intensity = 2 (HR 0.6; 95% CI 0.4-1.0) or 3 (HR 0.5; 95% CI 0.2-0.9) had a reduced risk of breast cancer mortality, compared to ER intensity = 1 (p trend = 0.02). Neither ER protein expression nor intensity influenced Hispanic survival disparities. CONCLUSIONS: Estrogen receptor percent positive staining is not independently related to breast cancer survival after adjustment for other survival-related factors. ER intensity, in contrast, demonstrates promise for prognostic utility.

GPER Mediates Functional Endothelial Aging in Renal Arteries.

Aging is associated with impaired renal artery function, which is partly characterized by arterial stiffening and a reduced vasodilatory capacity due to excessive generation of reactive oxygen species by NADPH oxidases (Nox). The abundance and activity of Nox depends on basal activity of the heptahelical transmembrane receptor GPER; however, whether GPER contributes to age-dependent functional changes in renal arteries is unknown. This study investigated the effect of aging and Nox activity on renal artery tone in wild-type and GPER-deficient (Gper-/-) mice (4 and 24 months old). In wild-type mice, aging markedly impaired endothelium-dependent, nitric oxide (NO)-mediated relaxations to acetylcholine, which were largely preserved in renal arteries of aged Gper-/- mice. The Nox inhibitor gp91ds-tat abolished this difference by greatly enhancing relaxations in wild-type mice, while having no effect in Gper-/- mice. Contractions to angiotensin II and phenylephrine in wild-type mice were partly sensitive to gp91ds-tat but unaffected by aging. Again, deletion of GPER abolished effects of Nox inhibition on contractile responses. In conclusion, basal activity of GPER is required for the age-dependent impairment of endothelium-dependent, NO-mediated relaxation in the renal artery. Restoration of relaxation by a Nox inhibitor in aged wild-type but not Gper-/- mice strongly supports a role for Nox-derived reactive oxygen species as the underlying cause. Pharmacological blockers of GPER signaling may thus be suitable to inhibit functional endothelial aging of renal arteries by reducing Nox-derived oxidative stress and, possibly, the associated age-dependent deterioration of kidney function.

G-Protein-Coupled Estrogen Receptor (GPER) and Sex-Specific Metabolic Homeostasis.

Obesity and metabolic syndrome display disparate prevalence and regulation between males and females. Human, as well as rodent, females with regular menstrual/estrous cycles exhibit protection from weight gain and associated chronic diseases. These beneficial effects are predominantly attributed to the female hormone estrogen, specifically 17ß-estradiol (E2). E2 exerts its actions via multiple receptors, nuclear and extranuclear estrogen receptor (ER) α and ERß, and the G-protein-coupled estrogen receptor (GPER, previously termed GPR30). The roles of GPER in metabolic homeostasis are beginning to emerge but are complex and remain unclear. The discovery of GPER-selective pharmacological agents (agonists and antagonists) and the availability of GPER knockout mice have significantly enhanced our understanding of the functions of GPER in normal physiology and disease. GPER action manifests pleiotropic effects in metabolically active tissues such as the pancreas, adipose, liver, and skeletal muscle. Cellular and animal studies have established that GPER is involved in the regulation of body weight, feeding behavior, inflammation, as well as glucose and lipid homeostasis. GPER deficiency leads to increased adiposity, insulin resistance, and metabolic dysfunction in mice. In contrast, pharmacologic stimulation of GPER in vivo limits weight gain and improves metabolic output, revealing a promising novel therapeutic potential for the treatment of obesity and diabetes.

Peptidyl arginine deiminase from Porphyromonas gingivalis abolishes anaphylatoxin C5a activity.

Evasion of killing by the complement system, a crucial part of innate immunity, is a key evolutionary strategy of many human pathogens. A major etiological agent of chronic periodontitis, the Gram-negative bacterium Porphyromonas gingivalis, produces a vast arsenal of virulence factors that compromise human defense mechanisms. One of these is peptidylarginine deiminase (PPAD), an enzyme unique to P. gingivalis among bacteria, which converts Arg residues in polypeptide chains into citrulline. Here, we report that PPAD citrullination of a critical C-terminal arginine of the anaphylatoxin C5a disabled the protein function. Treatment of C5a with PPAD in vitro resulted in decreased chemotaxis of human neutrophils and diminished calcium signaling in monocytic cell line U937 transfected with the C5a receptor (C5aR) and loaded with a fluorescent intracellular calcium probe: Fura-2 AM. Moreover, a low degree of citrullination of internal arginine residues by PPAD was also detected using mass spectrometry. Further, after treatment of C5 with outer membrane vesicles naturally shed by P. gingivalis, we observed generation of C5a totally citrullinated at the C-terminal Arg-74 residue (Arg74Cit). In stark contrast, only native C5a was detected after treatment with PPAD-null outer membrane vesicles. Our study suggests reduced antibacterial and proinflammatory capacity of citrullinated C5a, achieved via lower level of chemotactic potential of the modified molecule, and weaker cell activation. In the context of previous studies, which showed crosstalk between C5aR and Toll-like receptors, as well as enhanced arthritis development in mice infected with PPAD-expressing P. gingivalis, our findings support a crucial role of PPAD in the virulence of P. gingivalis.

Estrogen-mediated inactivation of FOXO3a by the G protein-coupled estrogen receptor GPER.

BACKGROUND: Estrogen (17ß-estradiol) promotes the survival and proliferation of breast cancer cells and its receptors represent important therapeutic targets. The cellular actions of estrogen are mediated by the nuclear estrogen receptors ERα and ERß as well as the 7-transmembrane spanning G protein-coupled estrogen receptor (GPER). We previously reported that estrogen activates the phosphoinositide 3-kinase (PI3Kinase) pathway via GPER, resulting in phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production within the nucleus of breast cancer cells; however, the mechanisms and consequences of this activity remained unclear. METHODS: MCF7 breast cancer cells were transfected with GFP-fused Forkhead box O3 (FOXO3) as a reporter to assess localization in response to estrogen stimulation. Inhibitors of PI3Kinases and EGFR were employed to determine the mechanisms of estrogen-mediated FOXO3a inactivation. Receptor knockdown with siRNA and the selective GPER agonist G-1 elucidated the estrogen receptor(s) responsible for estrogen-mediated FOXO3a inactivation. The effects of selective estrogen receptor modulators and downregulators (SERMs and SERDs) on FOXO3a in MCF7 cells were also determined. Cell survival (inhibition of apoptosis) was assessed by caspase activation. RESULTS: In the estrogen-responsive breast cancer cell line MCF7, FOXO3a inactivation occurs on a rapid time scale as a result of GPER, but not ERα, stimulation by estrogen, established by the GPER-selective agonist G-1 and knockdown of GPER and ERα. GPER-mediated inactivation of FOXO3a is effected by the p110α catalytic subunit of PI3Kinase as a result of transactivation of the EGFR. The SERMs tamoxifen and raloxifene, as well as the SERD ICI182,780, were active in mediating FOXO3a inactivation in a GPER-dependent manner. Additionally, estrogen-and G-1-mediated stimulation of MCF7 cells results in a decrease in caspase activation under proapoptotic conditions. CONCLUSIONS: Our results suggest that non-genomic signaling by GPER contributes, at least in part, to the survival of breast cancer cells, particularly in the presence of ER-targeted therapies involving SERMs and SERDs. Our results further suggest that GPER expression and FOXO3a localization could be utilized as prognostic markers in breast cancer therapy and that GPER antagonists could promote apoptosis in GPER-positive breast cancers, particularly in combination with chemotherapeutic and ER-targeted drugs, by antagonizing estrogen-mediated FOXO3a inactivation.

Introduction of D-phenylalanine enhanced the receptor binding affinities of gonadotropin-releasing hormone peptides.

The purpose of this study was to examine whether the introduction of D-Phe could improve the GnRH receptor binding affinities of DOTA-conjugated D-Lys(6)-GnRH peptides. Building upon the construct of DOTA-Ahx-(D-Lys(6)-GnRH1) we previously reported, an aromatic amino acid of D-Phe was inserted either between the DOTA and Ahx or between the Ahx and D-Lys(6) to generate new DOTA-D-Phe-Ahx-(D-Lys(6)-GnRH) or DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) peptides. Compared to DOTA-Ahx-(D-Lys(6)-GnRH1) (36.1 nM), the introduction of D-Phe improved the GnRH receptor binding affinities of DOTA-D-Phe-Ahx-(D-Lys(6)-GnRH) (16.3 nM) and DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) (7.6 nM). The tumor targeting and pharmacokinetic properties of (111)In-DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) was determined in MDA-MB-231 human breast cancer-xenografted nude mice. Compared to (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1), (111)In-DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) exhibited comparable tumor uptake with faster renal and liver clearance. The MDA-MB-231 human breast cancer-xenografted tumors were clearly visualized by single photon emission computed tomography (SPECT) using (111)In-DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) as an imaging probe, providing a new insight into the design of new GnRH peptides in the future.

G protein-coupled estrogen receptor agonist G-1 decreases ADAM10 levels and NLRP3-inflammasome component activation in response to Staphylococcus aureus alpha-hemolysin.

The G protein-coupled estrogen receptor, also known as GPER1 or originally GPR30, is found in various tissues, indicating its diverse functions. It is typically present in immune cells, suggesting its role in regulating immune responses to infectious diseases. Our previous studies have shown that G-1, a selective GPER agonist, can limit the pathogenesis mediated by Staphylococcus aureus alpha-hemolysin (Hla). It aids in clearing bacteria in a mouse skin infection model and restricts the surface display of the Hla receptor, ADAM10 (a disintegrin and metalloprotease 10) in HaCaT keratinocytes. In this report, we delve into the modulation of GPER in human immune cells in relation to the NLRP3 inflammasome. We used macrophage-like differentiated THP-1 cells for our study. We found that treating these cells with G-1 reduces ATP release, decreases the activity of the caspase-1 enzyme, and lessens cell death following Hla intoxication. This is likely due to the reduced levels of ADAM10 and NLRP3 proteins, as well as the decreased display of the ADAM10 receptor in the G-1-treated THP-1 cells. Our studies, along with our previous work, suggest the potential therapeutic use of G-1 in reducing Hla susceptibility in humans. This highlights the importance of GPER in immune regulation and its potential as a therapeutic target.