RESUMEN
G protein-coupled receptor 30 (GPR30), also named G protein-coupled estrogen receptor (GPER), and the ß1-adrenergic receptor (ß1AR) are G protein-coupled receptors (GPCR) that are implicated in breast cancer progression. Both receptors contain PSD-95/Discs-large/ZO-1 homology (PDZ) motifs in their C-terminal tails through which they interact in the plasma membrane with membrane-associated guanylate kinase (MAGUK) scaffold proteins, and in turn protein kinase A anchoring protein (AKAP) 5. GPR30 constitutively and PDZ-dependently inhibits ß1AR-mediated cAMP production. We hypothesized that this inhibition is a consequence of a plasma membrane complex of these receptors. Using co-immunoprecipitation, confocal immunofluorescence microscopy, and bioluminescence resonance energy transfer (BRET), we show that GPR30 and ß1AR reside in close proximity in a plasma membrane complex when transiently expressed in HEK293. Deleting the GPR30 C-terminal PDZ motif (-SSAV) does not interfere with the receptor complex, indicating that the complex is not PDZ-dependent. MCF7 breast cancer cells express GPR30, ß1AR, MAGUKs, and AKAP5 in the plasma membrane, and co-immunoprecipitation revealed that these proteins exist in close proximity also under native conditions. Furthermore, expression of GPR30 in MCF7 cells constitutively and PDZ-dependently inhibits ß1AR-mediated cAMP production. AKAP5 also inhibits ß1AR-mediated cAMP production, which is not additive with GPR30-promoted inhibition. These results argue that GPR30 and ß1AR form a PDZ-independent complex in MCF7 cells through which GPR30 constitutively and PDZ-dependently inhibits ß1AR signaling via receptor interaction with MAGUKs and AKAP5.
Asunto(s)
Neoplasias de la Mama , Proteínas Quinasas Dependientes de AMP Cíclico , Femenino , Humanos , Proteínas de Anclaje a la Quinasa A/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Guanilato-Quinasas , Células HEK293 , Células MCF-7 , Receptores Adrenérgicos/metabolismo , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
BACKGROUND: Breast-conserving surgery followed by radiotherapy is part of standard treatment for early-stage breast cancer. Hypoxia is common in cancer and may affect the benefit of radiotherapy. Cells adapt to hypoxic stress largely via the transcriptional activity of hypoxia-inducible factor (HIF)-1α. Here, we aim to determine whether tumour HIF-1α-positivity and hypoxic gene-expression signatures associated with the benefit of radiotherapy, and outcome. METHODS: Tumour HIF-1α-status and expression of hypoxic gene signatures were retrospectively analysed in a clinical trial where 1178 women with primary T1-2N0M0 breast cancer were randomised to receive postoperative radiotherapy or not and followed 15 years for recurrence and 20 years for breast cancer death. RESULTS: The benefit from radiotherapy was similar in patients with HIF-1α-positive and -negative primary tumours. Both ipsilateral and any breast cancer recurrence were more frequent in women with HIF-1α-positive primary tumours (hazard ratio, HR0-5 yrs1.9 [1.3-2.9], p = 0.003 and HR0-5 yrs = 2.0 [1.5-2.8], p < 0.0001). Tumour HIF-1α-positivity is also associated with increased breast cancer death (HR0-10 years 1.9 [1.2-2.9], p = 0.004). Ten of the 11 investigated hypoxic gene signatures correlated positively to HIF-1α-positivity, and 5 to increased rate/risk of recurrence. CONCLUSIONS: The benefit of postoperative radiotherapy persisted in patients with hypoxic primary tumours. Patients with hypoxic primary breast tumours had an increased risk of recurrence and breast cancer death.
Asunto(s)
Neoplasias de la Mama , Mastectomía Segmentaria , Neoplasias de la Mama/genética , Neoplasias de la Mama/radioterapia , Neoplasias de la Mama/cirugía , Femenino , Estudios de Seguimiento , Humanos , Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Recurrencia Local de Neoplasia/radioterapia , Pronóstico , Estudios RetrospectivosRESUMEN
G protein-coupled receptor 30 (GPR30) is a membrane receptor reported to bind 17ß-estradiol (E2) and mediate rapid nongenomic estrogen responses, hence also named G protein-coupled estrogen receptor. G-1 is a proposed GPR30-specific agonist that has been used to implicate the receptor in several pathophysiological events. However, controversy surrounds the role of GPR30 in G-1 and E2 responses. We investigated GPR30 activity in the absence and presence of G-1 and E2 in several eukaryotic systems ex vivo and in vitro in the absence and presence of the receptor. Ex vivo activity was addressed using the caudal artery from wild-type (WT) and GPR30 knockout (KO) mice, and in vitro activity was addressed using a HeLa cell line stably expressing a synthetic multifunctional promoter (nuclear factor κB, signal transducer and activator of transcription, activator protein 1)-luciferase construct (HFF11 cells) and a human GPR30-inducible T-REx system (T-REx HFF11 cells), HFF11 and human embryonic kidney 293 cells transiently expressing WT GPR30 and GPR30 lacking the C-terminal PDZ (postsynaptic density-95/discs-large /zonula occludens-1 homology) motif SSAV, and yeast Saccharomyces cerevisiae transformed to express GPR30. WT and KO arteries exhibited similar contractile responses to 60 mM KCl and 0.3 µM cirazoline, and G-1 relaxed both arteries with the same potency and efficacy. Furthermore, expression of GPR30 did not introduce any responses to 1 µM G-1 and 0.1 µM E2 in vitro. On the other hand, receptor expression caused considerable ligand-independent activity in vitro, which was receptor PDZ motif-dependent in mammalian cells. We conclude from these results that GPR30 exhibits ligand-independent activity in vitro but no G-1- or E2-stimulated activity in any of the systems used. SIGNIFICANCE STATEMENT: Much controversy surrounds 17ß-estradiol (E2) and G-1 as G protein-coupled receptor 30 (GPR30) agonists. We used several recombinant eukaryotic systems ex vivo and in vitro with and without GPR30 expression to address the role of this receptor in responses to these proposed agonists. Our results show that GPR30 exhibits considerable ligand-independent activity in vitro but no G-1- or E2-stimulated activity in any of the systems used. Thus, classifying GPR30 as an estrogen receptor and G-1 as a specific GPR30 agonist is unfounded.
Asunto(s)
Ciclopentanos/farmacología , Estradiol/farmacología , Quinolinas/farmacología , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Arterias/efectos de los fármacos , Línea Celular , Homólogo 4 de la Proteína Discs Large/metabolismo , Femenino , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Relajación Muscular/efectos de los fármacos , Dominios PDZ/genética , Receptores de Estrógenos/efectos de los fármacos , Receptores de Estrógenos/genética , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores Acoplados a Proteínas G/genética , Saccharomyces cerevisiae/genéticaRESUMEN
BACKGROUND: The NaCl cotransporter NCC in the kidney distal convoluted tubule (DCT) regulates urinary NaCl excretion and BP. Aldosterone increases NaCl reabsorption via NCC over the long-term by altering gene expression. But the acute effects of aldosterone in the DCT are less well understood. METHODS: Proteomics, bioinformatics, and cell biology approaches were combined with animal models and gene-targeted mice. RESULTS: Aldosterone significantly increases NCC activity within minutes in vivo or ex vivo. These effects were independent of transcription and translation, but were absent in the presence of high potassium. In vitro, aldosterone rapidly increased intracellular cAMP and inositol phosphate accumulation, and altered phosphorylation of various kinases/kinase substrates within the MAPK/ERK, PI3K/AKT, and cAMP/PKA pathways. Inhibiting GPR30, a membrane-associated receptor, limited aldosterone's effects on NCC activity ex vivo, and NCC phosphorylation was reduced in GPR30 knockout mice. Phosphoproteomics, network analysis, and in vitro studies determined that aldosterone activates EGFR-dependent signaling. The EGFR immunolocalized to the DCT and EGFR tyrosine kinase inhibition decreased NCC activity ex vivo and in vivo. CONCLUSIONS: Aldosterone acutely activates NCC to modulate renal NaCl excretion.
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Aldosterona/farmacología , Túbulos Renales Distales/metabolismo , Transducción de Señal , Tiazidas/farmacología , Aldosterona/metabolismo , Animales , Presión Sanguínea , Calcio/metabolismo , Línea Celular , Membrana Celular/metabolismo , Biología Computacional , AMP Cíclico/metabolismo , Receptores ErbB/metabolismo , Síndrome de Gitelman/metabolismo , Riñón/metabolismo , Masculino , Ratones , Mineralocorticoides/metabolismo , Fosforilación , Proteómica , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Cloruro de Sodio/metabolismo , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismoRESUMEN
PURPOSE: According to the 2017 St Gallen surrogate definitions of the intrinsic subtypes, Ki67, progesterone receptor (PR) and Nottingham histological grade (NHG) are used for prognostic classification of estrogen receptor (ER) positive/HER2-negative breast cancer into luminal A- or luminal B-like. The aim of the present study was to investigate if additional biomarkers, related to endocrine signaling pathways, e.g., amplified in breast cancer 1 (AIB1), androgen receptor (AR), and G protein-coupled estrogen receptor (GPER), can provide complementary prognostic information in a subset of ER-positive/HER-negative invasive lobular carcinoma (ILC). METHODS: Biomarkers from 224 patients were analyzed immunohistochemically on tissue microarray. The primary endpoint was breast cancer mortality (BCM), analyzed with 10- and 25-year follow-up (FU). In addition, the prognostic value of gene expression data for these biomarkers was analyzed in three publicly available ILC datasets. RESULTS: AIB1 (high vs. low) was associated to BCM in multivariable analysis (adjusted for age, tumor size, nodal status, NHG, Ki67, luminal-like classification, and adjuvant systemic therapy) with 10-year FU (HR 6.8, 95% CI 2.3-20, P = 0.001) and 25-year FU (HR 3.0, 95% CI 1.1-7.8, P = 0.03). The evidence of a prognostic effect of AIB1 could be confirmed by linking gene expression data to outcome in independent publicly available ILC datasets. AR and GPER were neither associated to BCM with 10-year nor with 25-year FU (P > 0.33). Furthermore, Ki67 and NHG were prognostic for BCM at both 10-year and 25-year FU, whereas PR was not. CONCLUSIONS: AIB1 is a new putative prognostic biomarker in ER-positive/HER2-negative ILC.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias de la Mama/terapia , Carcinoma Lobular/terapia , Coactivador 3 de Receptor Nuclear/genética , Adulto , Anciano , Anciano de 80 o más Años , Mama/patología , Mama/cirugía , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinoma Lobular/genética , Carcinoma Lobular/patología , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Metástasis Linfática/genética , Metástasis Linfática/patología , Mastectomía , Persona de Mediana Edad , Pronóstico , Receptores Androgénicos/genética , Receptores de Estrógenos/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Progesterona/genéticaRESUMEN
G protein-coupled receptor 30 (GPR30), also called G protein-coupled estrogen receptor 1 (GPER1), is thought to play important roles in breast cancer and cardiometabolic regulation, but many questions remain about ligand activation, effector coupling, and subcellular localization. We showed recently that GPR30 interacts through the C-terminal type I PDZ motif with SAP97 and protein kinase A (PKA)-anchoring protein (AKAP) 5, which anchor the receptor in the plasma membrane and mediate an apparently constitutive decrease in cAMP production independently of Gi/o Here, we show that GPR30 also constitutively increases ERK1/2 activity. Removing the receptor PDZ motif or knocking down specifically AKAP5 inhibited the increase, showing that this increase also requires the PDZ interaction. However, the increase was inhibited by pertussis toxin as well as by wortmannin but not by AG1478, indicating that Gi/o and phosphoinositide 3-kinase (PI3K) mediate the increase independently of epidermal growth factor receptor transactivation. FK506 and okadaic acid also inhibited the increase, implying that a protein phosphatase is involved. The proposed GPR30 agonist G-1 also increased ERK1/2 activity, but this increase was only observed at a level of receptor expression below that required for the constitutive increase. Furthermore, deleting the PDZ motif did not inhibit the G-1-stimulated increase. Based on these results, we propose that GPR30 increases ERK1/2 activity via two Gi/o-mediated mechanisms, a PDZ-dependent, apparently constitutive mechanism and a PDZ-independent G-1-stimulated mechanism.
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Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos Moleculares , Fosfatidilinositol 3-Quinasa/metabolismo , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Anclaje a la Quinasa A/antagonistas & inhibidores , Proteínas de Anclaje a la Quinasa A/genética , Proteínas de Anclaje a la Quinasa A/metabolismo , Sustitución de Aminoácidos , Ciclopentanos/farmacología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/química , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/química , Proteína Quinasa 3 Activada por Mitógenos/genética , Mutación , Dominios PDZ , Fosfatidilinositol 3-Quinasa/química , Fosfatidilinositol 3-Quinasa/genética , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Quinolinas/farmacología , Interferencia de ARN , Ensayo de Unión Radioligante , Receptores de Estrógenos/química , Receptores de Estrógenos/genética , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The kinin system is activated during vasculitis and may contribute to chronic inflammation. C1-inhibitor is the main inhibitor of the kinin system. In this study, we investigated the presence of the kinin B1 receptor on endothelial microvesicles and its contribution to the inflammatory process. Compared with controls (n=15), patients with acute vasculitis (n=12) had markedly higher levels of circulating endothelial microvesicles, identified by flow cytometry analysis, and significantly more microvesicles that were positive for the kinin B1 receptor (P<0.001). Compared with microvesicles from wild-type cells, B1 receptor-positive microvesicles derived from transfected human embryonic kidney cells induced a significant neutrophil chemotactic effect, and a B1 receptor antagonist blocked this effect. Likewise, patient plasma induced neutrophil chemotaxis, an effect decreased by reduction of microvesicle levels and by blocking the B1 receptor. We used a perfusion system to study the effect of patient plasma (n=6) and control plasma (n=6) on the release of microvesicles from glomerular endothelial cells. Patient samples induced the release of significantly more B1 receptor-positive endothelial microvesicles than control samples, an effect abrogated by reduction of the microvesicles in the perfused samples. Perfusion of C1-inhibitor-depleted plasma over glomerular endothelial cells promoted excessive release of B1 receptor-positive endothelial microvesicles compared with normal plasma, an effect significantly decreased by addition of C1-inhibitor or B1 receptor-antagonist. Thus, B1 receptor-positive endothelial microvesicles may contribute to chronic inflammation by inducing neutrophil chemotaxis, and the reduction of these microvesicles by C1-inhibitor should be explored as a potential treatment for neutrophil-induced inflammation.
Asunto(s)
Micropartículas Derivadas de Células/fisiología , Proteínas Inactivadoras del Complemento 1/fisiología , Vasculitis/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Quimiotaxis , Niño , Proteína Inhibidora del Complemento C1 , Endotelio Vascular/citología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
During vasculitis, activation of the kinin system induces inflammation, whereby the kinin B1-receptor is expressed and activated after ligand binding. Additionally, activated blood cells release microvesicles into the circulation. Here we determined whether leukocyte-derived microvesicles bear B1-kinin receptors during vasculitis, and if microvesicles transfer functional B1-receptors to recipient cells, thus promoting inflammation. By flow cytometry, plasma from patients with vasculitis were found to contain high levels of leukocyte-derived microvesicles bearing B1-receptors. Importantly, renal biopsies from two patients with vasculitis showed leukocyte-derived microvesicles bearing B1-receptors docking on glomerular endothelial cells providing in vivo relevance. Microvesicles derived from B1-receptor-transfected human embryonic kidney cells transferred B1-receptors to wild-type human embryonic kidney cells, lacking the receptor, and to glomerular endothelial cells. The transferred B1-receptors induced calcium influx after B1-receptor agonist stimulation: a response abrogated by a specific B1-receptor antagonist. Microvesicles derived from neutrophils also transferred B1-receptors to wild-type human embryonic kidney cells and induced calcium influx after stimulation. Thus, we found a novel mechanism by which microvesicles transfer functional receptors and promote kinin-associated inflammation.
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Bradiquinina/metabolismo , Micropartículas Derivadas de Células/metabolismo , Inflamación/metabolismo , Riñón/metabolismo , Receptor de Bradiquinina B1/metabolismo , Vasculitis/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Calcio , Línea Celular , Niño , Células Endoteliales/metabolismo , Femenino , Citometría de Flujo , Humanos , Riñón/citología , Cininas , Leucocitos/metabolismo , Masculino , Persona de Mediana Edad , Neutrófilos/metabolismo , Receptor de Bradiquinina B1/sangre , Receptor de Bradiquinina B1/genética , Transfección , Adulto JovenRESUMEN
Bradykinin (BK) and des-Arg9-BK are pro-inflammatory mediators acting via B2 (B2R) and B1 (B1R) receptors, respectively. We investigated the role of B2R and B1R in lipopolysaccharide (LPS)-induced hypothalamo-pituitary-adrenal (HPA) axis activation in SD rats. LPS given intraperitoneally (ip) up-regulated B1R mRNA in the hypothalamus, both B1R and B2R were up-regulated in pituitary and adrenal glands. Receptor localization was performed using immunofluorescence staining. B1R was localized in the endothelial cells, nucleus supraopticus (SON), adenohypophysis and adrenal cortex. B2R was localized nucleus paraventricularis (PVN) and SON, pituitary and adrenal medulla. Blockade of B1R prior to LPS further increased ACTH release and blockade of B1R 1 h after LPS decreased its release. In addition, we evaluated if blockade of central kinin receptors influence the LPS-induced stimulation of hypothalamic neurons. Blockade of both B1R and B2R reduced the LPS-induced c-Fos immunoreactivity in the hypothalamus. Our data demonstrate that a single injection of LPS induced a differential expression pattern of kinin B1R and B2R in the HPA axis. The tissue specific cellular localization of these receptors indicates that they may play a crucial role in the maintenance of body homeostasis during endotoxemia.
Asunto(s)
Endotoxemia/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Sistema Hipófiso-Suprarrenal/metabolismo , Receptor de Bradiquinina B1/biosíntesis , Receptor de Bradiquinina B2/biosíntesis , Enfermedad Aguda , Animales , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotoxemia/inducido químicamente , Homeostasis/efectos de los fármacos , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/farmacología , Masculino , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptor de Bradiquinina B1/análisis , Receptor de Bradiquinina B1/metabolismo , Receptor de Bradiquinina B2/análisis , Receptor de Bradiquinina B2/metabolismoRESUMEN
GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17ß-estradiol (E2), couple to the G proteins Gs and Gi/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E2 and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of Gi/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIß regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of Gi/o and retains receptors in the plasma membrane.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , AMP Cíclico/biosíntesis , Guanilato-Quinasas/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Estrógenos/química , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Anclaje a la Quinasa A/química , Proteínas de Anclaje a la Quinasa A/genética , Animales , Células CHO , Línea Celular , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Perros , Técnicas de Silenciamiento del Gen , Guanilato-Quinasas/química , Guanilato-Quinasas/genética , Células HEK293 , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Modelos Biológicos , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Dominios PDZ , Receptores de Estrógenos/genética , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de SeñalRESUMEN
G protein-coupled estrogen receptor (GPER), or GPR30, is a membrane receptor reported to mediate non-genomic estrogen responses. Tamoxifen is a partial agonist at GPER in vitro. Here, we investigated if GPER expression is prognostic in primary breast cancer, if the receptor is treatment-predictive for adjuvant tamoxifen, and if receptor subcellular localization has any impact on the prognostic value. Total and plasma membrane (PM) GPER expression was analyzed by immunohistochemistry in breast tumors from 742 postmenopausal lymph node-negative patients subsequently randomized for tamoxifen treatment for 2-5 years versus no systemic treatment, regardless of estrogen receptor (ER) status, and with a median follow-up of 17 years for patients free of event. PM GPER expression was a strong independent prognostic factor for poor prognosis in breast cancer without treatment-predictive information for tamoxifen. In the tamoxifen-treated ER-positive and progesterone receptor (PgR)-positive patient subgroup, the absence of PM GPER (53 % of all ER-positive tumors) predicted 91 % 20-year distant disease-free survival, compared to 73 % in the presence of GPER (p = 0.001). Total GPER expression showed positive correlations with ER and PgR and negative correlation with histological grade, but the correlations were biphasic. On the other hand, PM GPER expression showed strong negative correlations with ER and PgR, and strong positive correlation with HER2 overexpression and high histological grade. GPER overexpression and PM localization are critical events in breast cancer progression, and lack of GPER in the PM is associated with excellent long-term prognosis in ER-positive and PgR-positive tamoxifen-treated primary breast cancer.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias de la Mama/metabolismo , Membrana Celular/metabolismo , Receptores de Estrógenos/biosíntesis , Receptores Acoplados a Proteínas G/biosíntesis , Antineoplásicos Hormonales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/mortalidad , Membrana Celular/química , Supervivencia sin Enfermedad , Femenino , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Pronóstico , Modelos de Riesgos Proporcionales , Receptores de Estrógenos/análisis , Receptores Acoplados a Proteínas G/análisis , Tamoxifeno/uso terapéutico , Análisis de Matrices Tisulares , Resultado del TratamientoRESUMEN
Kinins are potent pro-inflammatory peptides that act through two G protein-coupled receptor subtypes, B1 (B1R) and B2 (B2R). Kinin-stimulated B2R signaling is often transient, whereas B1R signaling is sustained. This was confirmed by monitoring agonist-stimulated intracellular Ca(2+) mobilization in A10 smooth muscle cells expressing human wild-type B2R and B1R. We further studied the role of receptor membrane trafficking in receptor-mediated phosphoinositide (PI) hydrolysis in model HEK293 cell lines stably expressing the receptors. Treatment of cells with brefeldin A, to inhibit maturation of de novo synthesized receptors, or hypertonic sucrose, to inhibit receptor endocytosis, showed that the basal cell surface receptor turnover was considerably faster for B1R than for B2R. Inhibition of endocytosis, which stabilized B1R on the cell surface, inhibited B1R signaling, whereas B2R signaling was not perturbed. Signaling by a B1R construct in which the entire C-terminal domain was deleted remained sensitive to inhibition of receptor endocytosis, whereas signaling by a B1R construct in which this domain was substituted with the corresponding domain in B2R was not sensitive. B2R and B1R co-expression, which also appeared to stabilize B1R on the cell surface, presumably by receptor hetero-dimerization, also inhibited B1R signaling, whereas B2R signaling was slightly enhanced. Furthermore, the B2R-specific agonist bradykinin (BK) directed both receptors through a common endocytic pathway, whereas the B1R-specific agonist Lys-desArg(9)-BK was unable to do so. These results suggest that B1R-mediated PI hydrolysis depends on a step in receptor endocytosis, whereas B2R-mediated PI hydrolysis does not. We propose that B1R uses at least part of the endocytic machinery to sustain agonist-promoted signaling.
Asunto(s)
Bradiquinina/farmacología , Endocitosis/fisiología , Receptor de Bradiquinina B1/metabolismo , Receptor de Bradiquinina B2/metabolismo , Transducción de Señal/fisiología , Relación Dosis-Respuesta a Droga , Endocitosis/efectos de los fármacos , Células HEK293 , Humanos , Receptor de Bradiquinina B1/agonistas , Receptor de Bradiquinina B2/agonistas , Transducción de Señal/efectos de los fármacosRESUMEN
The G protein-coupled estrogen receptor GPER1/GPR30 is implicated in blood pressure regulation but the mechanisms are not identified. Here, we hypothesize that GPER1 controls blood pressure by regulating vascular smooth muscle cell Ca(2+) handling. Treatment with the GPER1 agonist G-1 (in the µM concentration range) acutely reduced spontaneous and synchronous Ca(2+) spike activity in A7r5 vascular smooth muscle cells expressing mRNA for GPER1. Furthermore, G-1 (1 µM) attenuated the thromboxane A2 analogue U46619-stimulated Ca(2+) spike activity but had no effect on the U46619-induced increase in the basal level of Ca(2+). The voltage-sensitive L-type Ca(2+) channel blocker nifedipine (100 nM) reduced Ca(2+) spike activity similar to G-1. Pharmacological, but not physiological, concentrations of the estrogen 17ß-estradiol reduced Ca(2+) spike activity. The GPER1 antagonist G-15 blocked G-1-induced downregulation of Ca(2+) spike activity, supporting a GPER1-dependent mechanism. G-1 (1 µM) and nifedipine (100 nM) attenuated the 30-mM KCl-evoked rise in intracellular Ca(2+) concentration, suggesting that G-1 blocks inflow of Ca(2+) via voltage-sensitive Ca(2+) channels. In conclusion, we demonstrate that the GPER1 agonist G-1 regulates vascular smooth muscle cell Ca(2+) handling by lowering Ca(2+) spike activity, suggesting a role for this mechanism in GPER1-mediated control of blood pressure. © 2013 S. Karger AG, Basel.
Asunto(s)
Calcio/metabolismo , Ciclopentanos/farmacología , Receptor alfa de Estrógeno/efectos de los fármacos , Quinolinas/farmacología , Receptores Acoplados a Proteínas G/fisiología , Animales , Benzodioxoles/farmacología , Línea Celular , Estradiol/farmacología , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Nifedipino/farmacología , Ratas , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidoresRESUMEN
The G protein-coupled oestrogen receptor GPER1, also known as GPR30, has been implicated in oestrogen signalling, but the physiological importance of GPER1 is not fully understood. The GPER1 agonist G-1 has become an important tool to assess GPER1-mediated cellular effects. Here, we report that this substance, besides acting via GPER1, affects the microtubule network in endothelial cells. Treatment with G-1 (3 µM) for 24 h reduced DNA synthesis by about 60 % in mouse microvascular endothelial bEnd.3 cells. Treatment with 3 µM G-1 prevented outgrowth of primary endothelial cells from mouse aortic explants embedded in Matrigel. Treatment with G-1 (0.3-3 µM) for 24 h disrupted bEnd.3 cell and HUVEC microtubule structure in a concentration-dependent manner as assessed by laser-scanning confocal immunofluorescence microscopy. G-1-induced (3 µM) disruption of microtubule was observed also after acute (3 and 6 h) treatment and in the presence of the protein synthesis inhibitor cycloheximide. Disruption of microtubules by 3 µM G-1 was observed in aortic smooth muscle cells obtained from both GPER1 knockout and wild-type mice, suggesting that G-1 influences microtubules through a mechanism independent of GPER1. G-1 dose dependently (10-50 µM) stimulated microtubule assembly in vitro. On the other hand, microtubules appeared normal in the presence of 10-50 µM G-1 as determined by electron microscopy. We suggest that G-1-promoted endothelial cell anti-proliferation is due in part to alteration of microtubule organization through a mechanism independent of GPER1. This G-1-promoted mechanism may be used to block unwanted endothelial cell proliferation and angiogenesis such as that observed in, e.g. cancer.
Asunto(s)
Ciclopentanos/farmacología , Células Endoteliales/metabolismo , Microtúbulos/efectos de los fármacos , Quinolinas/farmacología , Receptores de Estrógenos/agonistas , Receptores Acoplados a Proteínas G/agonistas , Moduladores de Tubulina/farmacología , Animales , Aorta/citología , Aorta/efectos de los fármacos , Proliferación Celular , Células Cultivadas , ADN Polimerasa Dirigida por ADN/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Células Endoteliales/ultraestructura , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Cinética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microtúbulos/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/ultraestructura , Multimerización de Proteína/efectos de los fármacos , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Técnicas de Cultivo de Tejidos , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
G protein-coupled receptor 30 [G protein-coupled estrogen receptor 1 (GPER1)], has been introduced as a membrane estrogen receptor and a candidate cancer biomarker and therapeutic target. However, several questions surround the subcellular localization and signaling of this receptor. In native cells, including mouse myoblast C(2)C(12) cells, Madin-Darby canine kidney epithelial cells, and human ductal breast epithelial tumor T47-D cells, G-1, a GPER1 agonist, and 17ß-estradiol stimulated GPER1-dependent cAMP production, a defined plasma membrane (PM) event, and recruitment of ß-arrestin2 to the PM. Staining of fixed and live cells showed that GPER1 was localized both in the PM and on intracellular structures. One such intracellular structure was identified as cytokeratin (CK) intermediate filaments, including those composed of CK7 and CK8, but apparently not endoplasmic reticulum, Golgi, or microtubules. Reciprocal coimmunoprecipitation of GPER1 and CKs confirmed an association of these proteins. Live staining also showed that the PM receptors constitutively internalize apparently to reach CK filaments. Receptor localization was supported using FLAG- and hemagglutinin-tagged GPER1. We conclude that GPER1-mediated stimulation of cAMP production and ß-arrestin2 recruitment occur in the PM. Furthermore, the PM receptors constitutively internalize and localize intracellularly on CK. This is the first observation that a G protein-coupled receptor is capable of associating with intermediate filaments, which may be important for GPER1 regulation in epithelial cells and the relationship of this receptor to cancer.
Asunto(s)
Filamentos Intermedios/metabolismo , Queratinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Arrestinas/metabolismo , AMP Cíclico/biosíntesis , Perros , Células HeLa , Humanos , Inmunoprecipitación , Ratones , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa , Receptores de Estrógenos/metabolismo , Células Tumorales Cultivadas , beta-ArrestinasRESUMEN
G protein-coupled receptor 30 (GPR30) or G protein-coupled estrogen receptor 1 (GPER1) is expressed in the vasculature, but the importance of vascular GPER1 remains to be clarified. Here we investigate effects of the GPER1 agonist G-1 on endothelial cell proliferation using mouse microvascular endothelial bEnd.3 cells. The bEnd.3 cells express mRNA for GPER1. The bEnd.3 cells expressed both ERα and ERß immunoreactivities. Treatment with G-1 reduced DNA synthesis and cell number with IC(50) values of about 2 µM. GPER1 siRNA prevented G-1-induced attenuation of DNA synthesis. G-1 accumulated cells in S and G2 phases of the cell cycle, suggesting that G-1 blocks transition between G2 and M. G-1 had no effect on DNA synthesis in COS-7 cells only weakly expressing GPER1 mRNA. 17ß-Estradiol had no effect on DNA synthesis in physiological concentrations (nM). The ER blocker ICI182780 reduced DNA synthesis with similar potency as G-1. Treatment with the ERK/MAP kinase inhibitor PD98059 had no effect on G-1-induced attenuation of DNA synthesis. G-1- induced antiproliferation was observed not only in bEnd.3 cells but also in human umbilical vein endothelial cells and HMEC-1 endothelial cells. We conclude that the GPER1 agonist G-1 attenuates endothelial cell proliferation via inhibition of DNA synthesis and by accumulation of cells in S and G2.
Asunto(s)
Ciclopentanos/farmacología , ADN/biosíntesis , Células Endoteliales/efectos de los fármacos , Fase G2/efectos de los fármacos , Quinolinas/farmacología , Receptores Acoplados a Proteínas G/agonistas , Fase S/efectos de los fármacos , Animales , Células COS , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Chlorocebus aethiops , Células Endoteliales/fisiología , Humanos , Ratones , Receptores de Estrógenos/análisis , Receptores Acoplados a Proteínas G/análisis , Receptores Acoplados a Proteínas G/fisiologíaRESUMEN
The kinin-forming pathway is activated on endothelial cells and neutrophils when high-molecular weight kininogen (HK) is cleaved by plasma kallikrein liberating bradykinin, a potent mediator of inflammation. Kinins are released during inflammatory conditions such as vasculitis, associated with neutrophil influx around blood vessels. Some patients with vasculitis have elevated plasma levels of neutrophil-derived proteinase 3 (PR3) and anti-PR3 Abs. This study investigated if neutrophil-derived PR3 could induce activation of the kinin pathway. PR3 incubated with HK, or a synthetic peptide derived from HK, induced breakdown and release of a novel tridecapeptide termed PR3-kinin, NH(2)-MKRPPGFSPFRSS-COOH, consisting of bradykinin with two additional amino acids on each terminus. The reaction was specific and inhibited by anti-PR3 and alpha(1)-antitrypsin. Recombinant wild-type PR3 incubated with HK induced HK breakdown, whereas mutated PR3, lacking enzymatic activity, did not. PR3-kinin bound to and activated human kinin B(1) receptors, but did not bind to B(2) receptors, expressed by transfected HEK293 cells in vitro. In human plasma PR3-kinin was further processed to the B(2) receptor agonist bradykinin. PR3-kinin exerted a hypotensive effect in vivo through both B(1) and B(2) receptors as demonstrated using wild-type and B(1) overexpressing rats as well as wild-type and B(2) receptor knockout mice. Neutrophil extracts from vasculitis patients and healthy controls contained comparable amounts of PR3 and induced HK proteolysis, an effect that was abolished when PR3 was immunoadsorbed. Neutrophil-derived PR3 can proteolyze HK and liberate PR3-kinin, thereby initiating kallikrein-independent activation of the kinin pathway.
Asunto(s)
Cininas/metabolismo , Mieloblastina/metabolismo , Neutrófilos/enzimología , Adolescente , Anciano , Anciano de 80 o más Años , Animales , Bradiquinina/sangre , Niño , Humanos , Calicreínas/metabolismo , Quininógenos/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Mieloblastina/genética , Unión Proteica , RatasRESUMEN
BACKGROUND: G protein-coupled estrogen receptor (GPER), or G protein-coupled receptor 30 (GPR30), is reported to mediate non-genomic estrogen signaling. GPR30 associates with breast cancer (BC) outcome and may contribute to tamoxifen resistance. We investigated the expression and prognostic significance of GPR30 in metachronous contralateral breast cancer (CBC) as a model of tamoxifen resistance. METHODS: Total GPR30 expression (GPR30TOT) and plasma membrane-localized GPR30 expression (GPR30PM) were analyzed by immunohistochemistry in primary (BC1; nBC1 = 559) and contralateral BC (BC2; nBC2 = 595), and in lymph node metastases (LGL; nLGL1 = 213; nLGL2 = 196). Death from BC (BCD), including BC death or death after documented distant metastasis, was used as primary end-point. RESULTS: GPR30PM in BC2 and LGL2 were associated with increased risk of BCD (HRBC2 = 1.7, p = 0.03; HRLGL2 = 2.0; p = 0.02). In BC1 and BC2, GPR30PM associated with estrogen receptor (ER)-negativity (pBC1<0.0001; pBC2<0.0001) and progesterone receptor (PR)-negativity (pBC1 = 0.0007; pBC2<0.0001). The highest GPR30TOT and GPR30PM were observed in triple-negative BC. GPR30PM associated with high Ki67 staining in BC1 (p<0.0001) and BC2 (p<0.0001). GPR30TOT in BC2 did not associate with tamoxifen treatment for BC1. However, BC2 that were diagnosed during tamoxifen treatment were more likely to express GPR30PM than BC2 diagnosed after treatment completion (p = 0.01). Furthermore, a trend was observed that patients with GPR30PM in an ER-positive BC2 had greater benefit from tamoxifen treatment. CONCLUSION: PM-localized GPR30 staining is associated with increased risk of BC death when expressed in BC2 and LGL2. Additionally, PM-localized GPR30 correlates with prognostic markers of worse outcome, such as high Ki67 and a triple-negative subtype. Therefore, PM-localized GPR30 may be an interesting new target for therapeutic exploitation. We found no clear evidence that total GPR30 expression is affected by tamoxifen exposure during development of metachronous CBC, or that GPR30 contributes to tamoxifen resistance.
Asunto(s)
Neoplasias de la Mama/metabolismo , Membrana Celular/metabolismo , Neoplasias Primarias Secundarias/metabolismo , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Estudios de Cohortes , Femenino , Células HeLa , Humanos , Incidencia , Células MCF-7 , Persona de Mediana Edad , Neoplasias Primarias Secundarias/tratamiento farmacológico , Neoplasias Primarias Secundarias/patología , Pronóstico , Factores de Riesgo , Tamoxifeno/uso terapéutico , Resultado del TratamientoRESUMEN
G protein-coupled receptor 30 (GPR30), or G protein-coupled estrogen receptor (GPER), is a G protein-coupled receptor (GPCR) that is currently attracting considerable attention in breast cancer and cardiometabolic regulation. The receptor was reported to be a novel membrane estrogen receptor mediating rapid non-genomic responses. However, questions remain about both the cognate ligand and the subcellular localization of receptor activity. Here, we used human embryonic kidney (HEK) 293 (HEK293) cells ectopically expressing N-terminally FLAG-tagged human GPR30 and three unique antibodies (Ab) specifically targetting the receptor N-terminal domain (N-domain) to investigate the role of N-glycosylation in receptor maturation and activity, the latter assayed by constitutive receptor-stimulated extracellular-regulated protein kinase (ERK) 1/2 (ERK1/2) activity. GPR30 expression was complex with receptor species spanning from approximately 40 kDa to higher molecular masses and localized in the endoplasmatic reticulum (ER), the plasma membrane (PM), and endocytic vesicles. The receptor contains three conserved asparagines, Asn25, Asn32, and Asn44, in consensus N-glycosylation motifs, all in the N-domain, and PNGase F treatment showed that at least one of them is N-glycosylated. Mutating Asn44 to isoleucine inactivated the receptor, yielding a unique receptor species at approximately 20 kDa that was recognized by Ab only in a denatured state. On the other hand, mutating Asn25 or Asn32 either individually or in combination, or truncating successively N-domain residues 1-42, had no significant effect either on receptor structure, maturation, or activity. Thus, Asn44 in the GPR30 N-domain is required for receptor structure and activity, whereas N-domain residues 1-42, including specifically Asn25 and Asn32, do not play any major structural or functional role(s).
Asunto(s)
Asparagina/metabolismo , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Secuencia de Aminoácidos , Asparagina/análisis , Glicosilación , Células HEK293 , Humanos , Proteína Quinasa 1 Activada por Mitógenos/análisis , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/análisis , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Conformación Proteica , Dominios Proteicos , Receptores de Estrógenos/análisis , Receptores Acoplados a Proteínas G/análisisRESUMEN
Kinins are potent proinflammatory peptides that are produced extracellularly and are rapidly degraded by extracellular peptidases and by intracellular peptidases accessed by kinins via receptor-mediated endocytosis. In this study, we developed model cell systems expressing the kinin B(2) receptor (B(2)R) and the metalloendopeptidase thimet oligopeptidase (EC 3.4.24.15; EP24.15) either individually or together to address 1) the cellular and functional relationship between these proteins and 2) the participation of EP24.15 in the metabolism of bradykinin (BK) after BK internalization via B(2)R. B(2)R was localized almost exclusively in the plasma membrane, whereas EP24.15 was localized both intracellularly and on the cell surface and secreted in the media. Intracellular EP24.15 was present throughout the cell, both cytosolic and particulate, with less nuclear localization and no colocalization with either the endoplasmic reticulum marker calnexin or the Golgi marker GM130. No direct colocalization of B(2)R and EP24.15 was observed using immunofluorescence microscopy. However, the two proteins coimmunoprecipitated specifically, and EP24.15 attenuated maximal B(2)R responsiveness without influencing the potency of BK to stimulate phosphoinositide hydrolysis and intracellular Ca(2+) mobilization. Cell surface-bound BK remained intact in cells overexpressing EP24.15 but was degraded intracellularly in an EP24.15-dependent manner upon B(2)R-mediated endocytosis. These results show that EP24.15 acts to negatively regulate B(2)R responsiveness, and it serves as an intracellular peptidase in the degradation of BK specifically internalized via this receptor.