RESUMO
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.
Assuntos
Estrogênios , Receptores de Estrogênio , Masculino , Feminino , Humanos , Receptores de Estrogênio/metabolismo , Estrogênios/farmacologia , Estrogênios/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Proteínas de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Antígeno de Macrófago 1/metabolismo , Neutrófilos/fisiologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/fisiologia , Animais , Anticorpos Bloqueadores/metabolismo , Antígeno CD11b/imunologia , Antígeno CD11b/metabolismo , Complemento C3/metabolismo , Citotoxicidade Imunológica , Feminino , Interações Hospedeiro-Patógeno , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismo , Caracteres Sexuais , Fatores SexuaisRESUMO
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.
Assuntos
Células Endoteliais/metabolismo , Hipertensão/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Polimorfismo de Nucleotídeo Único , Receptores de Estrogênio/genética , Receptores Acoplados a Proteínas G/genética , Adulto , Antígenos CD/genética , Antígenos CD/metabolismo , Sequência de Bases , Sistemas CRISPR-Cas , Caderinas/genética , Caderinas/metabolismo , Adesão Celular , Diferenciação Celular , Engenharia Celular/métodos , Células Endoteliais/patologia , Feminino , Edição de Genes/métodos , Regulação da Expressão Gênica , Humanos , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Células-Tronco Pluripotentes Induzidas/patologia , Modelos Biológicos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Cultura Primária de Células , Receptores de Estrogênio/deficiência , Receptores Acoplados a Proteínas G/deficiência , Fatores de Risco , Células THP-1 , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismoRESUMO
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.
Assuntos
Retalho Miocutâneo/irrigação sanguínea , Retalho Miocutâneo/patologia , Neovascularização Fisiológica/fisiologia , Traumatismo por Reperfusão/patologia , Caracteres Sexuais , Cicatrização/fisiologia , Animais , Carnitina O-Palmitoiltransferase/genética , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Proteína Forkhead Box O1/genética , Hexoquinase/genética , Fatores de Transcrição Kruppel-Like/genética , Imagem de Contraste de Manchas a Laser , Masculino , Camundongos , Necrose , Neovascularização Fisiológica/genética , Fosfofrutoquinase-2/genética , Receptor Notch1/genética , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Transcriptoma , Fator A de Crescimento do Endotélio Vascular/genética , Cicatrização/genéticaRESUMO
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.
Assuntos
Toxinas Bacterianas/metabolismo , Proteínas Hemolisinas/metabolismo , Infecções Cutâneas Estafilocócicas/microbiologia , Staphylococcus aureus/patogenicidade , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Resistência à Doença , Estrogênios/metabolismo , Feminino , Expressão Gênica , Imunidade Inata , Inflamassomos/metabolismo , Mediadores da Inflamação , Masculino , Camundongos , Viabilidade Microbiana/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Fatores Sexuais , Infecções Cutâneas Estafilocócicas/genética , Infecções Cutâneas Estafilocócicas/imunologia , Infecções Cutâneas Estafilocócicas/metabolismo , Virulência , Fatores de VirulênciaRESUMO
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.
Assuntos
Antineoplásicos/uso terapêutico , Cetorolaco de Trometamina/uso terapêutico , Neoplasias Mamárias Animais/prevenção & controle , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos/métodos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Feminino , Cetorolaco de Trometamina/administração & dosagem , Cetorolaco de Trometamina/farmacologia , Neoplasias Mamárias Animais/patologia , Vírus do Tumor Mamário do Camundongo , Camundongos Transgênicos , PolyomavirusRESUMO
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.
RESUMO
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.
Assuntos
Estrogênios/metabolismo , Receptores de Estrogênio/efeitos dos fármacos , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Animais , Feminino , Humanos , Ligantes , Masculino , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esteroides/farmacologiaRESUMO
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.
Assuntos
Envelhecimento/fisiologia , Endotélio Vascular/fisiologia , NADPH Oxidase 1/fisiologia , Receptores de Estrogênio/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Artéria Renal/fisiologia , Animais , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo , Vasoconstrição , VasodilataçãoRESUMO
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.
Assuntos
Metabolismo Energético , Estrogênios/metabolismo , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Fármacos Antiobesidade/uso terapêutico , Peso Corporal , Diabetes Mellitus/epidemiologia , Diabetes Mellitus/metabolismo , Diabetes Mellitus/fisiopatologia , Diabetes Mellitus/prevenção & controle , Metabolismo Energético/efeitos dos fármacos , Feminino , Homeostase , Humanos , Hipoglicemiantes/uso terapêutico , Masculino , Obesidade/epidemiologia , Obesidade/metabolismo , Obesidade/fisiopatologia , Obesidade/prevenção & controle , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Receptores de Estrogênio/efeitos dos fármacos , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Caracteres Sexuais , Fatores Sexuais , Transdução de SinaisAssuntos
Insulina , Ilhotas Pancreáticas , Animais , Feminino , Masculino , Ratos , Ratos Wistar , Estresse Psicológico , DesmameRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Complemento C5a/metabolismo , Hidrolases/metabolismo , Porphyromonas gingivalis/enzimologia , Arginina/metabolismo , Proteínas de Bactérias/genética , Cálcio/metabolismo , Membrana Celular/enzimologia , Movimento Celular , Células Cultivadas , Quimiotaxia , Citrulina/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Hidrolases/genética , Mutação , Neutrófilos/citologia , Neutrófilos/metabolismo , Porphyromonas gingivalis/genética , Desiminases de Arginina em Proteínas , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/metabolismo , Vesículas Transportadoras/enzimologia , Células U937RESUMO
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.
Assuntos
Neoplasias da Mama/genética , Estrogênios/metabolismo , Fatores de Transcrição Forkhead/genética , Receptores de Estrogênio/biossíntese , Receptores Acoplados a Proteínas G/biossíntese , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Estrogênios/administração & dosagem , Feminino , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores de Transcrição Forkhead/biossíntese , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , RNA Interferente Pequeno , Cloridrato de Raloxifeno/administração & dosagem , Receptores de Estrogênio/genética , Receptores Acoplados a Proteínas G/genética , Tamoxifeno/administração & dosagemRESUMO
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.
Assuntos
Neoplasias da Mama/diagnóstico , Hormônio Liberador de Gonadotropina/química , Hormônio Liberador de Gonadotropina/metabolismo , Fenilalanina/química , Fenilalanina/metabolismo , Receptores LHRH/metabolismo , Animais , Ligação Competitiva , Cromatografia Líquida de Alta Pressão , Feminino , Xenoenxertos , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Nus , Estrutura Molecular , Imagem Óptica , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
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.
Assuntos
Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide , Toxinas Bacterianas , Proteínas Hemolisinas , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Receptores de Estrogênio , Receptores Acoplados a Proteínas G , Staphylococcus aureus , Proteína ADAM10/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Humanos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Hemolisinas/metabolismo , Inflamassomos/metabolismo , Toxinas Bacterianas/metabolismo , Células THP-1 , Receptores de Estrogênio/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/agonistas , Caspase 1/metabolismo , Trifosfato de Adenosina/metabolismo , Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/microbiologia , Dipeptídeos , Ácidos HidroxâmicosRESUMO
Estrogens regulate numerous physiological and pathological processes, including wide-ranging effects in wound healing. The effects of estrogens are mediated through multiple estrogen receptors (ERs), including the classical nuclear ERs (ERα and ER ß ), that typically regulate gene expression, and the 7-transmembrane G protein-coupled estrogen receptor (GPER), that predominantly mediates rapid "non-genomic" signaling. Estrogen modulates the expression of various genes involved in epidermal function and regeneration, inflammation, matrix production, and protease inhibition, all critical to wound healing. Our previous work demonstrated improved myocutaneous wound healing in female mice compared to male mice. In the current study, we employed male and female GPER knockout mice to investigate the role of this estrogen receptor in wound revascularization and tissue viability. Using a murine myocutaneous flap model of graded ischemia, we measured real-time flap perfusion via laser speckle perfusion imaging. We conducted histologic and immunohistochemical analyses to assess skin and muscle viability, microvascular density and vessel morphology. Our results demonstrate that GPER is crucial in wound healing, mediating effects that are both dependent and independent of sex. Lack of GPER expression is associated with increased skin necrosis, reduced flap perfusion and altered vessel morphology. These findings contribute to understanding GPER signaling in wound healing and suggest possible therapeutic opportunities by targeting GPER.
Assuntos
Camundongos Knockout , Neovascularização Fisiológica , Receptores de Estrogênio , Receptores Acoplados a Proteínas G , Cicatrização , Animais , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores de Estrogênio/metabolismo , Receptores de Estrogênio/genética , Masculino , Camundongos , Feminino , Pele/metabolismo , Pele/irrigação sanguínea , Isquemia/metabolismo , Retalhos CirúrgicosRESUMO
The coexistence of brown adipocytes with low and high thermogenic activity is a fundamental feature of brown adipose tissue heterogeneity and plasticity. However, the mechanisms that govern thermogenic adipocyte heterogeneity and its significance in obesity and metabolic disease remain poorly understood. Here we show that in male mice, a population of transcription factor jun-B (JunB)-enriched (JunB+) adipocytes within the brown adipose tissue exhibits lower thermogenic capacity compared to high-thermogenic adipocytes. The JunB+ adipocyte population expands in obesity. Depletion of JunB in adipocytes increases the fraction of adipocytes exhibiting high thermogenic capacity, leading to enhanced basal and cold-induced energy expenditure and protection against diet-induced obesity and insulin resistance. Mechanistically, JunB antagonizes the stimulatory effects of PPARγ coactivator-1α on high-thermogenic adipocyte formation by directly binding to the promoter of oestrogen-related receptor alpha, a PPARγ coactivator-1α downstream effector. Taken together, our study uncovers that JunB shapes thermogenic adipocyte heterogeneity, serving a critical role in maintaining systemic metabolic health.
Assuntos
Resistência à Insulina , Camundongos , Masculino , Animais , PPAR gama/metabolismo , Adipócitos Marrons/metabolismo , Obesidade/etiologia , Obesidade/metabolismo , Dieta Hiperlipídica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In view of recent findings on the anatomic heterogeneity of rapid vasodilation via estrogen receptor (ER)-dependent mechanisms, it is obvious that with regard to human physiology and disease much of it is still unknown, and research in this area is urgently needed. This is also important because chronic drug therapy with estrogens in women systemically affects the circulation and may affect certain arterial beds but not others. It is conceivable that the presence of any vascular disease (as was the case for coronary and carotid atherosclerosis in many of the patients in the large randomized controlled trials HERS and WHI) is likely to affect vascular responses to estrogens as well, and that any beneficial effects may be attenuated or even completely lost. Further work is required to decipher the mechanisms of vasodilation brought about by estrogens in humans and experimental animals, whether anatomic heterogeneity exists with regard to vascular beds and individual estrogen receptors, and how vascular disease (atherosclerosis in particular) affects responsiveness. Also, pharmacologcial tools for newly identified ERs are now available. The hypothesis that disease may modify or even abrogate estrogen-dependent or ER-selective vasodilation should also be tested. Finally, given that certain clinically approved drugs such as SERM or SERDs (thought only to block or downregulate nuclear ERs) actually cause vasodilation through GPER and have been shown in recent clinical studies to provide cardiovascular protection in postmenopausal women, we may have to rethink our current understanding, concepts, and strategies of how to interfere with the increased risk of vascular disease in women with estrogen deficiency or after menopause.
Assuntos
Receptores de Estrogênio/fisiologia , Vasodilatação/fisiologia , Doenças Cardiovasculares/prevenção & controle , Estrogênios/deficiência , Estrogênios/farmacologia , Feminino , Humanos , Masculino , Moduladores Seletivos de Receptor Estrogênico/farmacologiaRESUMO
Three new DOTA-conjugated GnRH peptides with various hydrocarbon linkers were synthesized to evaluate the influences of the linkers on their receptor binding affinities. The hydrocarbon linker displayed a profound impact on the receptor binding affinities of DOTA-conjugated GnRH peptides. The Aun linker was better than Gaba, Ahx and Aoc linkers in retaining strong receptor binding affinity of the GnRH peptide. DOTA-Aun-(D-Lys(6)-GnRH) displayed 22.8 nM GnRH receptor binding affinity. (111)In-DOTA-Aun-(D-Lys(6)-GnRH) exhibited fast tumor uptake and urinary clearance in MDA-MB-231 human breast cancer-xenografted nude mice. The cellular and biological results provided an insight into the design of new GnRH peptides in the future.