RESUMO
Phosphorylation of G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) desensitizes G-protein signalling and promotes arrestin signalling, which is also modulated by biased ligands1-6. The molecular assembly of GRKs on GPCRs and the basis of GRK-mediated biased signalling remain largely unknown owing to the weak GPCR-GRK interactions. Here we report the complex structure of neurotensin receptor 1 (NTSR1) bound to GRK2, Gαq and the arrestin-biased ligand SBI-5537. The density map reveals the arrangement of the intact GRK2 with the receptor, with the N-terminal helix of GRK2 docking into the open cytoplasmic pocket formed by the outward movement of the receptor transmembrane helix 6, analogous to the binding of the G protein to the receptor. SBI-553 binds at the interface between GRK2 and NTSR1 to enhance GRK2 binding. The binding mode of SBI-553 is compatible with arrestin binding but clashes with the binding of Gαq protein, thus providing a mechanism for its arrestin-biased signalling capability. In sum, our structure provides a rational model for understanding the details of GPCR-GRK interactions and GRK2-mediated biased signalling.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G , Receptores Acoplados a Proteínas G , Transdução de Sinais , Arrestinas/metabolismo , Fosforilação , Receptores Acoplados a Proteínas G/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Quinase 2 de Receptor Acoplado a Proteína G/química , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Ligantes , Ligação Proteica , Receptores de Neurotensina/metabolismoRESUMO
Pulmonary fibrosis is a chronic, progressive, and irreversible interstitial lung disease. Transforming growth factor-ß1 (TGF-ß1) plays a major role in lung fibroblast cell differentiation to myofibroblast cells and production of extracellular matrix, which are hallmarks of pulmonary fibrosis. G protein-coupled receptor kinase-2 (GRK2) has been shown to play controversial roles in TGF-ß1-induced signal transduction in different cell types; however, the role of GRK2 in TGF-ß1-induced activation of lung fibroblast cells and development of pulmonary fibrosis has not been revealed. In this study, we found that GRK2 levels were increased in lungs and isolated fibroblast cells in a murine model of pulmonary fibrosis, as well as TGF-ß1-treated lung fibroblasts. GRK2 levels were not changed in lungs in the injury phase of pulmonary fibrosis. Posttreatment with GRK2 inhibitor reduced extracellular matrix (ECM) accumulation in lungs in bleomycin-challenged mice, suggesting that GRK2 activation contributes to the progressive phase of pulmonary fibrosis. Inhibition or downregulation of GRK2 attenuates fibronectin, collagen, and α-smooth muscle actin expression in TGF-ß1-induced lung fibroblast cells or myofibroblast cells isolated from patients with pulmonary fibrosis. Furthermore, we showed that GRK2 regulates Smad3 expression, indicating that inhibition of GRK2 attenuates ECM accumulation through downregulation of Smad3 expression. This study reveals that GRK2 is a therapeutic target in treating pulmonary fibrosis and inhibition of GRK2 dampens pulmonary fibrosis by suppression of Smad3 expression, eventually attenuating TGF-ß1 signal pathway and ECM accumulation.
Assuntos
Fibroblastos/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Pulmão/metabolismo , Fibrose Pulmonar/metabolismo , Proteína Smad3/biossíntese , Animais , Bleomicina/toxicidade , Linhagem Celular , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Quinase 2 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Quinase 2 de Receptor Acoplado a Proteína G/genética , Expressão Gênica , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Proteína Smad3/antagonistas & inibidores , Proteína Smad3/genéticaRESUMO
Head and neck squamous cell carcinoma (HNSCC) arises from the mucosal lining of the upper aerodigestive tract and display few treatment options in advanced stages. Despite increased knowledge of HNSCC molecular biology, the identification of new players involved in triggering HNSCC recurrence and metastatic disease is needed. We uncover that G-protein-coupled receptor kinase-2 (GRK2) expression is reduced in undifferentiated, high-grade human HNSCC tumors, whereas its silencing in model human HNSCC cells is sufficient to trigger epithelial-to-mesenchymal transition (EMT) phenotypic features, an EMT-like transcriptional program and enhanced lymph node colonization from orthotopic tongue tumors in mice. Conversely, enhancing GRK2 expression counteracts mesenchymal cells traits by mechanisms involving phosphorylation and decreased functionality of the key EMT inducer Snail1. Our results suggest that GRK2 safeguards the epithelial phenotype, whereas its downregulation contributes to the activation of EMT programs in HNSCC.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Neoplasias de Cabeça e Pescoço/enzimologia , Neoplasias de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/enzimologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Animais , Linhagem Celular Tumoral , Progressão da Doença , Regulação para Baixo , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Quinase 2 de Receptor Acoplado a Proteína G/genética , Neoplasias de Cabeça e Pescoço/genética , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Fosforilação , Fatores de Transcrição da Família Snail/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genéticaRESUMO
OBJECTIVE: The aim of the study was to determine whether the inhibition of the G-protein-coupled receptor kinase 2 by adenoviral ßARKct cardiac gene transfer can ameliorate postresuscitation myocardial injury in pigs with cardiac arrest (CA) and explore the mechanism of myocardial protection. METHODS: Male landrace domestic pigs were randomized into the sham group (anesthetized and instrumented, but ventricular fibrillation was not induced) (nâ=â4), control group (ventricular fibrillation 8âmin, nâ=â8), and ßARKct group (ventricular fibrillation 8âmin, nâ=â8). Hemodynamic parameters were monitored continuously. Blood samples were collected at baseline, 30âmin, 2âh, 4âh, and 6âh after the return of spontaneous circulation (ROSC). Left ventricular ejection fraction was assessed by echocardiography at baseline and 6âh after ROSC. These animals were euthanized, and the cardiac tissue was removed for analysis at 6âh after ROSC. RESULTS: Compared with those in the sham group, left ventricular +dp/dtmax, -dp/dtmax, cardiac output (CO), and ejection fraction (EF) in the control group and the ßARKct group were significantly decreased at 6âh after the restoration of spontaneous circulation. However, the ßARKct treatment produced better left ventricular +dp/dtmax, -dp/dtmax, CO, and EF after ROSC. The ßARKct treatment also produced lower serum cardiac troponin I, CK-MB, and lactate after ROSC. Furthermore, the adenoviral ßARKct gene transfer significantly increased ß1 adrenergic receptors, SERCA2a, RyR2 levels, and decreased GRK2 levels compared to control. CONCLUSIONS: The inhibition of GRK2 by adenoviral ßARKct cardiac gene transfer can ameliorate postresuscitation myocardial injury through beneficial effects on restoring the sarcoplasmic reticulum Ca-handling proteins expression and upregulating the ß1-adrenergic receptor level after cardiac arrest.
Assuntos
Adenoviridae , Reanimação Cardiopulmonar , Quinase 2 de Receptor Acoplado a Proteína G , Parada Cardíaca , Traumatismos Cardíacos , Transdução Genética , Animais , Modelos Animais de Doenças , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Quinase 2 de Receptor Acoplado a Proteína G/genética , Parada Cardíaca/genética , Parada Cardíaca/metabolismo , Parada Cardíaca/patologia , Parada Cardíaca/terapia , Traumatismos Cardíacos/genética , Traumatismos Cardíacos/metabolismo , Traumatismos Cardíacos/patologia , Traumatismos Cardíacos/terapia , Masculino , SuínosRESUMO
PURPOSE: Hepatocellular carcinoma (HCC) is a common digestive system malignancy that is associated with a poor prognosis. This study researched the interaction of tumor necrosis factor-α (TNF-α) and angiotensin II (Ang II) in HCC cells proliferation, migration and invasion and examined their influence on the expression of G protein-coupled receptor kinase 2 (GRK2) and relevant receptors. METHODS: Cell Counting Kit-8 and Transwell assays were performed to evaluate the effects of TNF-α and Ang II on HepG2 cells proliferation, migration and invasion. Flow cytometry was used to investigate the expression of tumor necrosis factor receptor 1 (TNFR1), angiotensin II type 1 (AT1R) and type 2 receptors (AT2R) on the surface of HepG2 cells. Additionally, Western blot was performed to assess the modulation of GRK2 expression by TNF-α and Ang II in HepG2 cells. Meanwhile, GRK2 siRNA-transfected HepG2 cells were used to confirm the effects of GRK2, TNF-α and Ang II on the proliferation, migration and invasion of GRK2-knockdown HCC cells. Finally, the expression of TNF-α, Ang II, TNFR1, AT1R, AT2R and GRK2 proteins in HCC, tumor-adjacent and normal liver tissues were tested by immunohistochemistry. RESULTS: The data demonstrated that TNF-α and Ang II can enhance the proliferation, migration and invasion of HepG2 cells through suppressing GRK2 expression but that the two reagents combined did not have synergistic effects. Moreover,overexpression of TNFR1 and AT1R perhaps promoted the formation and progression of HCC, while high AT2R expression had the opposite effect. CONCLUSIONS: This study provides new ideas for the prevention and treatment of HCC by researching the interaction and probable mechanism of different bioactive factors associated with HCC.
Assuntos
Angiotensina II/farmacologia , Antineoplásicos/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Fator de Necrose Tumoral alfa/farmacologia , Angiotensina II/uso terapêutico , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Citometria de Fluxo , Quinase 2 de Receptor Acoplado a Proteína G/genética , Técnicas de Silenciamento de Genes , Humanos , Invasividade Neoplásica/patologia , RNA Interferente Pequeno/genética , Receptor Tipo 1 de Angiotensina/biossíntese , Receptor Tipo 2 de Angiotensina/biossíntese , Receptores Tipo I de Fatores de Necrose Tumoral/biossíntese , Fator de Necrose Tumoral alfa/uso terapêuticoAssuntos
Glândulas Suprarrenais/metabolismo , Catecolaminas/metabolismo , Células Cromafins/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Insuficiência Cardíaca/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Glândulas Suprarrenais/patologia , Animais , Células Cultivadas , Células Cromafins/patologia , Insuficiência Cardíaca/patologia , Humanos , Transdução de SinaisRESUMO
T cell infiltration to synovial tissue is an early pathogenic mechanism of rheumatoid arthritis (RA). In the present work, we reveal that G protein coupled receptor kinase 2 (GRK2) is abundantly expressed in T cells of collagen-induced arthritis (CIA). A GRK2 inhibitor, paroxetine protects the joints from inflammation and destruction, primarily through inhibition of both CD4+ helper T (Th) cell and CD8+ cytotoxic T (Tc) cell migration to synovial tissue. Meanwhile, paroxetine restores the balance of Th/Tc, effector Th (Theff)/ naïve Th (Thnaive) and effector Tc (Tceff)/ naïve Tc (Tcnaive) to equilibrium by elevating the frequency of Thnaive, Tcnaive and regulatory Th cells; reducing the increased Theff, activated Th and Tceff, having a similar effect as methotrexate (MTX). In addition, both serum and synovial IL-1ß, TNF-α and CX3CL1 expression was effectively inhibited in treated rats. In vitro assay confirmed that paroxetine inhibits CX3CL1-induced T cell migration through blocking the activity of GRK2. Among three MAPK families, paroxetine was found to be able to decrease the phosphorylation of ERK. This study elucidates that paroxetine attenuates the symptoms of CIA rats due to its inhibitory effect on T cell activation and infiltration to synovial tissue via suppression of ERK pathway.
Assuntos
Anti-Inflamatórios/farmacologia , Artrite Experimental/imunologia , Quinase 2 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Paroxetina/farmacologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/patologia , Movimento Celular/efeitos dos fármacos , Quimiocina CX3CL1/sangue , Modelos Animais de Doenças , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Imunossupressores/farmacologia , Interleucina-1beta/sangue , Ativação Linfocitária/efeitos dos fármacos , Masculino , Metotrexato/farmacologia , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/sangueRESUMO
The objective of this study was to explore the potential role of G-protein-coupled receptor kinase 2 (GRK2) in the progression of cannabinoid 2 receptor (CB2) agonist-induced analgesic effects of bone cancer pain. Female Sprague-Dawley rats, weighing 160-180 g, were utilized to establish a model of bone cancer pain induced by intra-tibia inoculation of Walker 256 mammary gland carcinoma cells. JWH-015, a selective CB2 agonist, was injected intrathecally or intraperitoneally on postoperative day 10. Bone cancer-induced pain behaviors-mechanical allodynia and ambulatory pain-were assessed on postoperative days -1 (baseline), 4, 7, and 10 and at post-treatment hours 2, 6, 24, 48, and 72. The expressions of spinal CB2 and GRK2 protein were detected by Western Blotting on postoperative days -1 (baseline), 4, 7, and 10 and at post-treatment hours 6, 24, and 72. The procedure produced prolonged mechanical allodynia, ambulatory pain, and different changes in spinal CB2 and GRK2 expression levels. Intrathecal or intraperitoneal administration of JWH-015 alleviated the induced mechanical allodynia and ambulatory pain, and inhibited the downregulation of spinal GRK2 expression. These effects were in a time-dependent manner and reversed by pretreatment of CB2 selective antagonist AM630. The results affirmed CB2 receptor agonists might serve as new treatment targets for bone cancer pain. Moreover, spinal GRK2 was an important regulator of CB2 receptor agonist-analgesia pathway.
Assuntos
Neoplasias Ósseas/metabolismo , Dor do Câncer/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Indóis/administração & dosagem , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/metabolismo , Animais , Neoplasias Ósseas/tratamento farmacológico , Dor do Câncer/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Injeções Intraperitoneais , Injeções Espinhais , Medição da Dor/efeitos dos fármacos , Medição da Dor/métodos , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
AIMS: G protein-coupled receptor kinase 2 (GRK2), a cytosolic enzyme desensitizing G protein-couple receptors (e.g., ß-adrenergic receptors [ß-ARs]), is involved in regulation of hypertension, congestive heart failure, and inflammatory response. Since cellular GRK2 levels change quickly in response to exogenous/endogenous stimuli, this study examined whether GRK2 levels in human peripheral blood mononuclear cells (PBMCs) would increase during acute aerobic exercise and be associated with plasma IL-6 and cardiorespiratory fitness levels. MAIN METHODS: Eighteen subjects (8 men and 10 women), ages 18 to 30 years, were recruited to perform a 30-minute bout of acute aerobic exercise at 75% VO2max. KEY FINDINGS: Our results demonstrated that women exhibited significantly greater exercise-induced GRK2 expression in PBMCs compared to men. IL-6 modulation is independent of GRK2 expression. Furthermore, the percent change in GRK2 expression was negatively correlated with cardiorespiratory fitness levels (relative VO2max), but not plasma IL-6. SIGNIFICANCE: Acute aerobic exercise induces a greater GRK2 expression in women than men, while increased cardiorespiratory fitness is associated with exercise-induced GRK2 expression in PBMCs. Gender could be a contributor to regulate this GRK2 responsiveness to acute aerobic exercise.
Assuntos
Exercício Físico/fisiologia , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , Leucócitos Mononucleares/enzimologia , Caracteres Sexuais , Adolescente , Adulto , Feminino , Humanos , Interleucina-6/sangue , Leucócitos Mononucleares/citologia , MasculinoRESUMO
We have previously demonstrated the involvement of milk fat globule-epidermal growth factor-factor 8 (MFGE8) in reducing neutrophil infiltration in a murine model of acute lung injury (ALI). In the present study, we aimed to delineate the mechanisms through which MFGE8 attenuates neutrophil migration. Recombinant human MFGE8 (rhMFGE8) was expressed and purified in our facility. The human differentiated neutrophil cell line, dHL60, was treated with rhMFGE8 and cell migration assay was performed in a Boyden chamber using recombinant interleukin8 (IL8) as the chemoattractant. Surface CXCR2 and intracellular G proteincoupled receptor kinase 2 (GRK2) levels were evaluated by flow cytometry or western blot analysis. The levels of mitogenactivated protein (MAP) kinases were determined by western blot analysis. Treatment with rhMFGE8 resulted in a significant inhibition of dHL60 cell migration in a dosedependent manner. There was a 46% decrease in CXCR2 expression in the rhMFGE8treated dHL60 cells, which was associated with a 32% increase in GRK2 expression. In the dHL60 cells, treatment with rhMFGE8 promoted the phosphorylation of p38 and extracellular signal-regulated kinase (ERK) within 1030 min. The use of SB203580, a p38 inhibitor, and PD98059, an ERK inhibitor, resulted in the restoration of dHL60 cell migration which was significantly inhibited treatment with rhMFGE8. Furthermore, blocking the MFGE8 receptors, αvß3/αvß5integrins, by antiαvintegrin neutralizing antibody (Ab) inhibited the activation of p38 and ERK, and reversed the rhMFGE8induced inhibition of dHL60 cell migration. Finally, treatment of the dHL60 cells with SB203580 and PD98059 neutralized the rhMFGE8induced downregulation of CXCR2 expression and upregulation of GRK2 expression, as well as the inhibitory effects on cell migration. Our findings reveal a novel mechanism of action of MFGE8 through which it inhibits neutrophil migration through αvß3-integrin-dependent MAP kinase activation.
Assuntos
Antígenos de Superfície/farmacologia , Movimento Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Integrina alfaVbeta3/antagonistas & inibidores , Proteínas do Leite/farmacologia , Neutrófilos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Flavonoides , Citometria de Fluxo , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Células HL-60 , Humanos , Imidazóis/farmacologia , Integrina alfaVbeta3/metabolismo , Interleucina-8/imunologia , Infiltração de Neutrófilos/efeitos dos fármacos , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Receptores de Interleucina-8B/biossíntese , Receptores de Interleucina-8B/metabolismo , Receptores de Vitronectina/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Hedgehog (Hh) signaling is essential for normal growth, patterning, and homeostasis of many tissues in diverse organisms, and is misregulated in a variety of diseases including cancer. Cytoplasmic Hedgehog signaling is activated by multisite phosphorylation of the seven-pass transmembrane protein Smoothened (Smo) in its cytoplasmic C-terminus. Aside from a short membrane-proximal stretch, the sequence of the C-terminus is highly divergent in different phyla, and the evidence suggests that the precise mechanism of Smo activation and transduction of the signal to downstream effectors also differs. To clarify the conserved role of G-protein-coupled receptor kinases (GRKs) in Smo regulation, we mapped four clusters of phosphorylation sites in the membrane-proximal C-terminus of Drosophila Smo that are phosphorylated by Gprk2, one of the two fly GRKs. Phosphorylation at these sites enhances Smo dimerization and increases but is not essential for Smo activity. Three of these clusters overlap with regulatory phosphorylation sites in mouse Smo and are highly conserved throughout the bilaterian lineages, suggesting that they serve a common function. Consistent with this, we find that a C-terminally truncated form of Drosophila Smo consisting of just the highly conserved core, including Gprk2 regulatory sites, can recruit the downstream effector Costal-2 and activate target gene expression, in a Gprk2-dependent manner. These results indicate that GRK phosphorylation in the membrane proximal C-terminus is an evolutionarily ancient mechanism of Smo regulation, and point to a higher degree of similarity in the regulation and signaling mechanisms of bilaterian Smo proteins than has previously been recognized.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Quinase 2 de Receptor Acoplado a Proteína G/genética , Regulação da Expressão Gênica no Desenvolvimento , Receptores Acoplados a Proteínas G/metabolismo , Animais , Proteínas de Drosophila/biossíntese , Drosophila melanogaster/crescimento & desenvolvimento , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Cinesinas/metabolismo , Camundongos , Fosforilação/genética , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/genética , Receptor SmoothenedRESUMO
Heart failure (HF) causes a tremendous burden on the worldwide healthcare system, affecting >23 million people. There are many cardiovascular disorders that contribute to the development of HF and multiple risk factors that accelerate its occurrence, but regardless of its underlying cause, HF is characterized by a marked decrease in myocardial contractility and loss of pump function. One biomarker molecule consistently shown to be upregulated in human HF and several animal models is G protein-coupled receptor kinase-2 (GRK2), a kinase originally discovered to be involved in G protein-coupled receptor desensitization, especially ß-adrenergic receptors. Higher levels of GRK2 can impair ß-adrenergic receptor-mediated inotropic reserve and its inhibition, or molecular reduction has shown to improve pump function in several animal models including a preclinical pig model of HF. Recently, nonclassical roles for GRK2 in cardiovascular disease have been described, including negative regulation of insulin signaling, a role in myocyte cell survival and apoptotic signaling, and it has been shown to be localized in/on mitochondria. These new roles of GRK2 suggest that GRK2 may be a nodal link in the myocyte, influencing both cardiac contractile function and cell metabolism and survival and contributing to HF independent of its canonical role in G protein-coupled receptor desensitization. In this review, classical and nonclassical roles for GRK2 will be discussed, focusing on recently discovered roles for GRK2 in cardiomyocyte metabolism and the effects that these roles may have on myocardial contractile function and HF development.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/fisiologia , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/fisiopatologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos/enzimologia , Animais , Biomarcadores/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Insuficiência Cardíaca/patologia , Humanos , Miócitos Cardíacos/patologia , Miócitos Cardíacos/fisiologiaRESUMO
OBJECTIVE: In the acute stage of infectious diseases such as pneumonia and sepsis, sequelae hypercytokinemia and cytokine storm are often observed simultaneously. During bacterial infections, activated polymorphonuclear leukocytes (PMNs) cause inflammation and organ dysfunction in severely ill patients. Gene expression of the triggering receptor on myeloid cells (TREM)-1 and G-coupled-protein receptor kinase (GRK)-2 in PMNs isolated from patients was analysed to identify genes correlated with the severity of pathophysiological conditions. METHODS: mRNA levels of TREM1 and GRK2 in the PMNs from 26 patients (13 with pneumonia, 5 with severe sepsis, and 8 with septic shock) were analysed by using quantitative real-time PCR. The synthesised soluble form (s)TREM-1 was incubated with normal PMNs to investigate its biological functions in vitro. RESULTS: Copies of TREM1 transcript were 0.7- to 2.1-fold higher in patients with pneumonia compared to those of normal subjects; the average fold-change was 1.1-fold. The mRNA levels of patients suffering from severe sepsis and septic shock were 0.34- and 0.33-fold lower compared to those of healthy subjects, respectively. TREM1 mRNA levels in 5 of 26 patients in convalescent stages recovered to normal levels. The mRNA levels of GRK2 in the PMNs of patients were also downregulated. The synthesised sTREM-1 upregulated the mRNA levels of TREM1 in normal PMNs. CONCLUSIONS: TREM1 mRNA levels were inversely correlated with the severity of pathophysiological conditions in acute bacterial infections. The gene expression levels of TREM1 in PMNs isolated from patients with bacterial infections may be used as a surrogate biomarker for determining the severity.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Glicoproteínas de Membrana/biossíntese , Neutrófilos/metabolismo , Pneumonia/metabolismo , Receptores Imunológicos/biossíntese , Sepse/metabolismo , Idoso , Infecções Bacterianas/metabolismo , Infecções Bacterianas/patologia , Biomarcadores/metabolismo , Feminino , Quinase 2 de Receptor Acoplado a Proteína G/genética , Expressão Gênica , Humanos , Inflamação/metabolismo , Inflamação/fisiopatologia , Masculino , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , Neutrófilos/patologia , Pneumonia/patologia , RNA Mensageiro/biossíntese , Receptores Imunológicos/genética , Sepse/fisiopatologia , Receptor Gatilho 1 Expresso em Células MieloidesRESUMO
Chronic pain is a major clinical problem, yet the mechanisms underlying the transition from acute to chronic pain remain poorly understood. In mice, reduced expression of GPCR kinase 2 (GRK2) in nociceptors promotes cAMP signaling to the guanine nucleotide exchange factor EPAC1 and prolongs the PGE2-induced increase in pain sensitivity (hyperalgesia). Here we hypothesized that reduction of GRK2 or increased EPAC1 in dorsal root ganglion (DRG) neurons would promote the transition to chronic pain. We used 2 mouse models of hyperalgesic priming in which the transition from acute to chronic PGE2-induced hyperalgesia occurs. Hyperalgesic priming with carrageenan induced a sustained decrease in nociceptor GRK2, whereas priming with the PKCε agonist ΨεRACK increased DRG EPAC1. When either GRK2 was increased in vivo by viral-based gene transfer or EPAC1 was decreased in vivo, as was the case for mice heterozygous for Epac1 or mice treated with Epac1 antisense oligodeoxynucleotides, chronic PGE2-induced hyperalgesia development was prevented in the 2 priming models. Using the CFA model of chronic inflammatory pain, we found that increasing GRK2 or decreasing EPAC1 inhibited chronic hyperalgesia. Our data suggest that therapies targeted at balancing nociceptor GRK2 and EPAC1 levels have promise for the prevention and treatment of chronic pain.
Assuntos
Dor Crônica/prevenção & controle , Quinase 2 de Receptor Acoplado a Proteína G/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Hiperalgesia/fisiopatologia , Animais , Carragenina/toxicidade , Bovinos , Dor Crônica/etiologia , Dor Crônica/genética , Dor Crônica/fisiopatologia , AMP Cíclico/fisiologia , Dinoprostona/fisiologia , Feminino , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Quinase 2 de Receptor Acoplado a Proteína G/genética , Gânglios Espinais/patologia , Regulação da Expressão Gênica , Inativação Gênica , Terapia Genética , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Fatores de Troca do Nucleotídeo Guanina/genética , Membro Posterior/inervação , Hiperalgesia/induzido quimicamente , Hiperalgesia/genética , Hiperalgesia/terapia , Injeções Espinhais , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Nociceptores/enzimologia , Nociceptores/fisiologia , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/farmacologia , Oligopeptídeos/toxicidade , Proteínas Recombinantes de Fusão/genética , Nervo Isquiático/patologia , Sistemas do Segundo Mensageiro , Células Receptoras Sensoriais/enzimologia , Células Receptoras Sensoriais/fisiologiaRESUMO
RATIONALE: G protein-coupled receptor kinase 2 (GRK2) is abundantly expressed in the heart, and its expression and activity are increased in injured or stressed myocardium. This upregulation has been shown to be pathological. GRK2 can promote cell death in ischemic myocytes, and its inhibition by a peptide comprising the last 194 amino acids of GRK2 (known as carboxyl-terminus of ß-adrenergic receptor kinase [bARKct]) is cardioprotective. OBJECTIVE: The aim of this study was to elucidate the signaling mechanism that accounts for the prodeath signaling seen in the presence of elevated GRK2 and the cardioprotection afforded by the carboxyl-terminus of ß-adrenergic receptor kinase. METHODS AND RESULTS: Using in vivo mouse models of ischemic injury and also cultured myocytes, we found that GRK2 localizes to mitochondria, providing novel insight into GRK2-dependent pathophysiological signaling mechanisms. Mitochondrial localization of GRK2 in cardiomyocytes was enhanced after ischemic and oxidative stress, events that induced prodeath signaling. Localization of GRK2 to mitochondria was dependent on phosphorylation at residue Ser670 within its extreme carboxyl-terminus by extracellular signal-regulated kinases, resulting in enhanced GRK2 binding to heat shock protein 90, which chaperoned GRK2 to mitochondria. Mechanistic studies in vivo and in vitro showed that extracellular signal-regulated kinase regulation of the C-tail of GRK2 was an absolute requirement for stress-induced, mitochondrial-dependent prodeath signaling, and blocking this led to cardioprotection. Elevated mitochondrial GRK2 also caused increased Ca(2+)-induced opening of the mitochondrial permeability transition pore, a key step in cellular injury. CONCLUSIONS: We identify GRK2 as a prodeath kinase in the heart, acting in a novel manner through mitochondrial localization via extracellular signal-regulated kinase regulation.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Proteínas de Choque Térmico HSP90/fisiologia , Mitocôndrias Cardíacas/metabolismo , Isquemia Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Animais Recém-Nascidos , Bovinos , Células Cultivadas , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Quinase 2 de Receptor Acoplado a Proteína G/genética , Células HEK293 , Proteínas de Choque Térmico HSP90/biossíntese , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/genética , Isquemia Miocárdica/enzimologia , Isquemia Miocárdica/patologia , Miócitos Cardíacos/patologia , Estresse Oxidativo/genética , Ratos , Transdução de Sinais/fisiologiaRESUMO
ß-Adrenergic receptor (ßAR) dysfunction in acute myocardial infarction (MI) is associated with elevated levels of the G-protein-coupled receptor kinase-2 (GRK2), which plays a key role in heart failure progression. Inhibition of GRK2 via expression of a peptide ßARKct transferred by molecular cardiac surgery with recirculating delivery (MCARD) may be a promising intervention. Five sheep underwent scAAV6-mediated MCARD delivery of ßARKct, and five received no treatment (control). After a 3-week period, the branch of the circumflex artery (OM1) was ligated. Quantitative PCR data showed intense ßARKct expression in the left ventricle (LV). Circumferential fractional shortening was 23.4 ± 7.1 % (baseline) vs. -2.9 ± 5.2 % (p < 0.05) in the control at 10 weeks. In the MCARD-ßARKct group, this parameter was close to baseline. The same trend was observed with LV wall thickening. Cardiac index fully recovered in the MCARD-ßARKct group. LV end-diastolic volume and LV end-diastolic pressure did not differ in both groups. MCARD-mediated ßARKct gene expression results in preservation of regional and global systolic function after acute MI without arresting progressive ventricular remodeling.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/genética , Técnicas de Transferência de Genes , Terapia Genética , Infarto do Miocárdio/terapia , Miocárdio/enzimologia , Fragmentos de Peptídeos/genética , Animais , Cardiomegalia/enzimologia , Cardiomegalia/genética , Cardiomegalia/patologia , Cardiomegalia/terapia , Dependovirus/genética , Modelos Animais de Doenças , Quinase 2 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Regulação da Expressão Gênica , Vetores Genéticos , Imageamento por Ressonância Magnética , Masculino , Contração Miocárdica , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Fragmentos de Peptídeos/biossíntese , Receptores Adrenérgicos beta/metabolismo , Recuperação de Função Fisiológica , Ovinos , Volume Sistólico , Sístole , Fatores de Tempo , Função Ventricular Esquerda , Pressão Ventricular , Remodelação VentricularRESUMO
In heterozygous mice, attenuation of G-protein-coupled receptor kinase 2 (GRK2) level in nociceptors is associated with enhanced and prolonged inflammatory hyperalgesia. To further elucidate the role of GRK2 in nociceptor function we reversibly decreased GRK2 expression using intrathecal antisense oligodeoxynucleotide (AS-ODN). GRK2 AS-ODN administration led to an enhanced and prolonged hyperalgesia induced by prostaglandin E(2), epinephrine and carrageenan. Moreover, this effect persisted unattenuated 2weeks after the last dose of antisense, well after GRK2 protein recovered, suggesting that transient attenuation of GRK2 produced neuroplastic changes in nociceptor function. Unlike hyperalgesic priming induced by transient activation of protein kinase C epsilon (PKCε), (Aley et al., 2000; Parada et al., 2003b), the enhanced and prolonged hyperalgesia following attenuation of GRK2 is PKCε- and cytoplasmic polyadenylation element binding protein (CPEB)-independent and is protein kinase A (PKA)- and Src tyrosine kinase (Src)-dependent. Finally, rats treated with GRK2 AS-ODN exhibited enhanced and prolonged hyperalgesia induced by direct activation of second messengers, adenyl cyclase, Epac or PKA, suggesting changes downstream of G-protein-coupled receptors. Because inflammation can produce a decrease in GRK2, such a mechanism could help explain a predilection to develop chronic pain, after resolution of acute inflammation.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/genética , Inflamação/genética , Nociceptores/metabolismo , Dor/genética , Animais , Western Blotting , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Hiperalgesia/genética , Hiperalgesia/psicologia , Inflamação/complicações , Masculino , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Dor/etiologia , Limiar da Dor , Fosfolipase C beta/biossíntese , Fosfolipase C beta/genética , Proteína Quinase C-épsilon/fisiologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Sistemas do Segundo Mensageiro/fisiologiaRESUMO
BACKGROUND: The activation of innate immune responses by Plasmodium vivax results in activation of effector cells and an excessive production of pro-inflammatory cytokines that may culminate in deleterious effects. Here, we examined the activation and function of neutrophils during acute episodes of malaria. MATERIALS AND METHODS: Blood samples were collected from P. vivax-infected patients at admission (day 0) and 30-45 days after treatment with chloroquine and primaquine. Expression of activation markers and cytokine levels produced by highly purified monocytes and neutrophils were measured by the Cytometric Bead Assay. Phagocytic activity, superoxide production, chemotaxis and the presence of G protein-coupled receptor (GRK2) were also evaluated in neutrophils from malaria patients. PRINCIPAL FINDINGS: Both monocytes and neutrophils from P. vivax-infected patients were highly activated. While monocytes were found to be the main source of cytokines in response to TLR ligands, neutrophils showed enhanced phagocytic activity and superoxide production. Interestingly, neutrophils from the malaria patients expressed high levels of GRK2, low levels of CXCR2, and displayed impaired chemotaxis towards IL-8 (CXCL8). CONCLUSION: Activated neutrophils from malaria patients are a poor source of pro-inflammatory cytokines and display reduced chemotactic activity, suggesting a possible mechanism for an enhanced susceptibility to secondary bacterial infection during malaria.
Assuntos
Citocinas/biossíntese , Malária Vivax/imunologia , Malária Vivax/patologia , Neutrófilos/imunologia , Neutrófilos/parasitologia , Plasmodium vivax/imunologia , Plasmodium vivax/patogenicidade , Adolescente , Adulto , Idoso , Quimiotaxia , Feminino , Citometria de Fluxo , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Humanos , Malária Vivax/complicações , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/parasitologia , Fagocitose , Superóxidos/metabolismo , Adulto JovemRESUMO
Obesity is a major health problem and an important risk factor for the development of multiple disorders. Previous studies in our laboratory have revealed that down-regulation of GRK2 decreases age-related adiposity, but the physiological and molecular mechanisms underlying this outcome remain unclear. We evaluate whether the lean phenotype results from a direct effect of GRK2 on energy homeostasis. The study of white adipose tissue (WAT) in wild-type (WT) and GRK2(+/-) littermates showed a reduced expression of lipogenic enzymes and enhanced lipolytic rate in adult GRK2(+/-) mice. Moreover, hemizygous mice display higher energy expenditure and lower respiratory exchange ratio. Analysis of brown adipose tissue (BAT) from adult GRK2(+/-) mice showed a less deteriorated morphology associated with age compared to WT, which is correlated with a higher basal core temperature. BAT from young GRK2(+/-) mice showed an increase in gene expression of thermogenesis-related genes. Accordingly, hemizygous mice displayed better thermogenic capacity and exhibited a more oxidative phenotype in both BAT and WAT than WT littermates. Overexpression of GRK2 in brown adipocytes corroborated the negative effect of this kinase in BAT function and differentiation. Collectively, our data point to GRK2 inhibition as a potential tool for the enhancement of brown fat activity, which may have important therapeutic implications for the treatment of obesity and associated metabolic disorders.
Assuntos
Tecido Adiposo Marrom/fisiologia , Metabolismo Energético/fisiologia , Quinase 2 de Receptor Acoplado a Proteína G/fisiologia , Obesidade/genética , Tecido Adiposo Branco/metabolismo , Envelhecimento/fisiologia , Animais , Diferenciação Celular , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Quinase 2 de Receptor Acoplado a Proteína G/genética , Hemizigoto , Camundongos , Termogênese/fisiologiaRESUMO
Beta-adrenergic receptor- (ß-AR) mediated vasorelaxation declines with age. This change is likely related to receptor desensitization, rather than down regulation. One kinase responsible for desensitization is G protein receptor kinase 2 (GRK2). We have shown that GRK expression and activity increases with age in Fischer 344 rat aorta. In this study we validated that carotid arteries have similar age-related changes in the ß-AR signaling axis as aorta. This finding allowed use of in vivo infection and delivery of two adenovirus vectors to carotid arteries of 2-month-old (2M) and 12-month-old (12M) male Fischer 344 rats. Adeno-GRK2 was used to overexpress GRK2, and adeno-ß-ARK-ct was used to inhibit GRK2 function. Following a five-day infection, vessels were collected and ex vivo tissue bath was used to evaluate vasoreactivity. We used KCl contracted segments, and determined that overexpression of GRK2 significantly impaired isoproterenol (ISO)-mediated vasorelaxation in both age groups. Maximum relaxation (MAX) to ISO in vessels from 2M decreased from 44% to 21%. MAX to ISO in vessels from 12M decreased from 12% to 6%. Sensitivity (ED50) in vessels from 2M and 12M was also impaired 57%, and 30% respectively. We also determined that expression of adeno-ß-ARK-ct significantly improved ISO-mediated vasorelaxation in both age groups. MAX in vessels from 2M increased from 44% to 58%. MAX in vessels from 12M increased from 15% to 69%. ED50 in vessels from 2M and 12M was also improved 46%, and 50% respectively. These findings further implicate age-related increases in GRK2 expression as an important regulator of the age-related decline in ß-AR-mediated vasorelaxation.