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1.
PLoS Biol ; 18(9): e3000813, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32991574

RESUMO

Short-chain fatty acids (SCFAs) produced by gastrointestinal microbiota regulate immune responses, but host molecular mechanisms remain unknown. Unbiased screening using SCFA-conjugated affinity nanobeads identified apoptosis-associated speck-like protein (ASC), an adaptor protein of inflammasome complex, as a noncanonical SCFA receptor besides GPRs. SCFAs promoted inflammasome activation in macrophages by binding to its ASC PYRIN domain. Activated inflammasome suppressed survival of Salmonella enterica serovar Typhimurium (S. Typhimurium) in macrophages by pyroptosis and facilitated neutrophil recruitment to promote bacterial elimination and thus inhibit systemic dissemination in the host. Administration of SCFAs or dietary fibers, which are fermented to SCFAs by gut bacteria, significantly prolonged the survival of S. Typhimurium-infected mice through ASC-mediated inflammasome activation. SCFAs penetrated into the inflammatory region of the infected gut mucosa to protect against infection. This study provided evidence that SCFAs suppress Salmonella infection via inflammasome activation, shedding new light on the therapeutic activity of dietary fiber.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Ácidos Graxos Voláteis/metabolismo , Inflamassomos/imunologia , Inflamassomos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Infecções por Salmonella/prevenção & controle , Animais , Proteínas Adaptadoras de Sinalização CARD/genética , Feminino , Microbioma Gastrointestinal/imunologia , Células HEK293 , Humanos , Imunidade Inata/fisiologia , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Receptores Acoplados a Proteínas-G/genética , Infecções por Salmonella/genética , Infecções por Salmonella/imunologia , Infecções por Salmonella/metabolismo , Salmonella typhimurium/imunologia , Células U937
2.
Life Sci ; 259: 118375, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32891612

RESUMO

OBJECTIVE: Short-chain fatty acids were reported to be the precursors of milk fat and can stimulate the de novo synthesis of fatty acids in bovine mammary epithelial cells (bMECs). However, the mechanism has not been elucidated. The purpose of this study was to investigate the effects of sodium butyrate (NaB) on milk fat synthesis in bMECs and explore its potential mechanism. METHODS: Bovine mammary epithelial cells (bMECs) were isolated for subsequent experimental uses. BODIPY staining and triglyceride kit were used to detect the milk fat synthesis in bMECs. Western blotting and RT-PCR assays were performed to detect the expression of related genes in bMECs. Immunoprecipitation was used to detect the acetylation of SREBP1 in bMECs. RESULTS: The results showed that NaB significantly promoted milk fat synthesis, promoted the activity of mechanistic target of rapamycin (mTOR) and S6 kinase (S6K), inhibited the activity of AMP-activated protein kinase (AMPK), and promoted the gene expression of G protein-coupled receptor 41 (GPR41). Knockdown of GPR41 and sterol regulatory element binding protein 1 (SREBP1) and overexpression of sirtuin1 (SIRT1), mTOR inhibitor (rapamycin), and AMPK activator (AICIR) eliminated these effects. These results indicated that NaB increased the nuclear translocation of SREBP1 via the GPR41/AMPK/mTOR/S6K signalling pathway, promoted the acetylation of mature SREBP1a via GPR41/AMPK/SIRT1, and then promoted milk fat synthesis. CONCLUSION: Taken together, these results demonstrated that NaB increased nuclear translocation and acetylation of SREBP1 to promote milk fat synthesis by activating GPR41 and its downstream signalling pathways.


Assuntos
Ácido Butírico/farmacologia , Glicolipídeos/biossíntese , Glicoproteínas/biossíntese , Glândulas Mamárias Animais/efeitos dos fármacos , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Western Blotting , Carbazóis , Bovinos , Células Cultivadas , Feminino , Imunoprecipitação , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Naftalenos , Reação em Cadeia da Polimerase em Tempo Real , Sirtuína 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
3.
Chem Biol Interact ; 330: 109248, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32871113

RESUMO

Dextromethorphan (DM) is a cough suppressant available in many prescribed and over-the-counter medications. Adverse reactions induced by DM have been regularly reported, including allergic skin reactions in some cases. However, the underlying mechanisms of local anaphylaxis induced by DM have not been elucidated. In this study, we found that DM could activate mast cells to increase calcium mobilization and release ß-hexosaminidase, histamine, tumor necrosis factor-α, MCP-1, and IL-8 in a dose-dependent manner. The allergic reactions were confirmed by hind paw swelling and extravasation assay in vivo. Furthermore, DM was revealed to induce local anaphylaxis via MRGPRX2 by the mast cell-deficient kitW-sh/W-sh mice and MRGPRX2 knockdown mast cells. And the MRGPRX2-HEK293/CMC analysis and frontal analysis also showed that DM has a considerable affinity with MRGPRX2. Together, our findings suggest that close monitoring should be drawn on patients with DM for its potential anaphylaxis via MRGPRX2.


Assuntos
Anafilaxia/induzido quimicamente , Dextrometorfano/efeitos adversos , Mastócitos/efeitos dos fármacos , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Antitussígenos/efeitos adversos , Células Cultivadas , Dextrometorfano/toxicidade , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Receptores de Neuropeptídeos/metabolismo
4.
Adv Exp Med Biol ; 1274: 223-258, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32894513

RESUMO

G protein-coupled receptors (GPCRs) have seven transmembrane spanning domains and comprise the largest superfamily with ~800 receptors in humans. GPCRs are attractive targets for drug discovery because they transduce intracellular signaling in response to endogenous ligands via heterotrimeric G proteins or arrestins, resulting in a wide variety of physiological and pathophysiological responses. The endogenous ligands for GPCRs are highly chemically diverse and include ions, biogenic amines, nucleotides, peptides, and lipids. In this review, we follow the KonMari method to better understand druggable lipid GPCRs. First, we have a comprehensive tidying up of lipid GPCRs including receptors for prostanoids, leukotrienes, specialized pro-resolving mediators (SPMs), lysophospholipids, sphingosine 1-phosphate (S1P), cannabinoids, platelet-activating factor (PAF), free fatty acids (FFAs), and sterols. This tidying up consolidates 46 lipid GPCRs and declutters several perplexing lipid GPCRs. Then, we further tidy up the lipid GPCR-directed drugs from the literature and databases, which identified 24 clinical drugs targeting 16 unique lipid GPCRs available in the market and 44 drugs under evaluation in more than 100 clinical trials as of 2019. Finally, we introduce drug designs for GPCRs that spark joy, such as positive or negative allosteric modulators (PAM or NAM), biased agonism, functional antagonism like fingolimod, and monoclonal antibodies (MAbs). These strategic drug designs may increase the efficacy and specificity of drugs and reduce side effects. Technological advances will help to discover more endogenous lipid ligands from the vast number of remaining orphan GPCRs and will also lead to the development novel lipid GPCR drugs to treat various diseases.


Assuntos
Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Arrestinas/metabolismo , Doença , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Lipídeos/farmacologia , Lipídeos/uso terapêutico , Receptores Acoplados a Proteínas-G/metabolismo
5.
Nat Commun ; 11(1): 4457, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32901017

RESUMO

Innate lymphoid cells (ILCs) and CD4+ T cells produce IL-22, which is critical for intestinal immunity. The microbiota is central to IL-22 production in the intestines; however, the factors that regulate IL-22 production by CD4+ T cells and ILCs are not clear. Here, we show that microbiota-derived short-chain fatty acids (SCFAs) promote IL-22 production by CD4+ T cells and ILCs through G-protein receptor 41 (GPR41) and inhibiting histone deacetylase (HDAC). SCFAs upregulate IL-22 production by promoting aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1α (HIF1α) expression, which are differentially regulated by mTOR and Stat3. HIF1α binds directly to the Il22 promoter, and SCFAs increase HIF1α binding to the Il22 promoter through histone modification. SCFA supplementation enhances IL-22 production, which protects intestines from inflammation. SCFAs promote human CD4+ T cell IL-22 production. These findings establish the roles of SCFAs in inducing IL-22 production in CD4+ T cells and ILCs to maintain intestinal homeostasis.


Assuntos
Ácidos Graxos Voláteis/imunologia , Microbioma Gastrointestinal/imunologia , Imunidade Inata , Interleucinas/biossíntese , Animais , Butiratos/imunologia , Butiratos/metabolismo , Butiratos/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Citrobacter rodentium , Colite/imunologia , Colite/microbiologia , Colite/prevenção & controle , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/prevenção & controle , Ácidos Graxos Voláteis/metabolismo , Ácidos Graxos Voláteis/farmacologia , Microbioma Gastrointestinal/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Técnicas In Vitro , Interleucinas/deficiência , Interleucinas/genética , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Linfócitos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Regiões Promotoras Genéticas , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo
6.
Nat Commun ; 11(1): 4752, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958754

RESUMO

Growth hormone (GH) is a key modulator of growth and GH over-secretion can lead to gigantism. One form is X-linked acrogigantism (X-LAG), in which infants develop GH-secreting pituitary tumors over-expressing the orphan G-protein coupled receptor, GPR101. The role of GPR101 in GH secretion remains obscure. We studied GPR101 signaling pathways and their effects in HEK293 and rat pituitary GH3 cell lines, human tumors and in transgenic mice with elevated somatotrope Gpr101 expression driven by the rat Ghrhr promoter (GhrhrGpr101). Here, we report that Gpr101 causes elevated GH/prolactin secretion in transgenic GhrhrGpr101 mice but without hyperplasia/tumorigenesis. We show that GPR101 constitutively activates not only Gs, but also Gq/11 and G12/13, which leads to GH secretion but not proliferation. These signatures of GPR101 signaling, notably PKC activation, are also present in human pituitary tumors with high GPR101 expression. These results underline a role for GPR101 in the regulation of somatotrope axis function.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Gigantismo/metabolismo , Hormônio do Crescimento/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Acromegalia/metabolismo , Acromegalia/patologia , Animais , Composição Corporal , Linhagem Celular , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Gigantismo/patologia , Adenoma Hipofisário Secretor de Hormônio do Crescimento/genética , Adenoma Hipofisário Secretor de Hormônio do Crescimento/metabolismo , Adenoma Hipofisário Secretor de Hormônio do Crescimento/patologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Hipófise/metabolismo , Proteína Quinase C/metabolismo , Ratos , Receptores Acoplados a Proteínas-G/genética
7.
Nat Commun ; 11(1): 4121, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807782

RESUMO

Vasoactive intestinal polypeptide receptor (VIP1R) is a widely expressed class B G protein-coupled receptor and a drug target for the treatment of neuronal, metabolic, and inflammatory diseases. However, our understanding of its mechanism of action and the potential of drug discovery targeting this receptor is limited by the lack of structural information of VIP1R. Here we report a cryo-electron microscopy structure of human VIP1R bound to PACAP27 and Gs heterotrimer, whose complex assembly is stabilized by a NanoBiT tethering strategy. Comparison with other class B GPCR structures reveals that PACAP27 engages VIP1R with its N-terminus inserting into the ligand binding pocket at the transmembrane bundle of the receptor, which subsequently couples to the G protein in a receptor-specific manner. This structure has provided insights into the molecular basis of PACAP27 binding and VIP receptor activation. The methodology of the NanoBiT tethering may help to provide structural information of unstable complexes.


Assuntos
Microscopia Crioeletrônica/métodos , Proteínas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Difusão Dinâmica da Luz , Humanos , Microscopia Eletrônica , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo
8.
Int J Mol Sci ; 21(15)2020 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-32731360

RESUMO

Along with cancer, cardiovascular and cerebrovascular diseases remain by far the most common causes of death. Heart attacks and strokes are diseases in which platelets play a role, through activation on ruptured plaques and subsequent thrombus formation. Most platelet agonists activate platelets via G protein-coupled receptors (GPCRs), which make these receptors ideal targets for many antiplatelet drugs. However, little is known about the mechanisms that provide feedback regulation on GPCRs to limit platelet activation. Emerging evidence from our group and others strongly suggests that GPCR kinases (GRKs) are critical negative regulators during platelet activation and thrombus formation. In this review, we will summarize recent findings on the role of GRKs in platelet biology and how one specific GRK, GRK6, regulates the hemostatic response to vascular injury. Furthermore, we will discuss the potential role of GRKs in thrombotic disorders, such as thrombotic events in COVID-19 patients. Studies on the function of GRKs during platelet activation and thrombus formation have just recently begun, and a better understanding of the role of GRKs in hemostasis and thrombosis will provide a fruitful avenue for understanding the hemostatic response to injury. It may also lead to new therapeutic options for the treatment of thrombotic and cardiovascular disorders.


Assuntos
Quinases de Receptores Acoplados a Proteína G/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Trombose/metabolismo , Plaquetas/metabolismo , Humanos , Ativação Plaquetária
9.
PLoS One ; 15(8): e0227157, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32817719

RESUMO

In mice, experimental influenza virus infection stimulates CD8 T cell infiltration of the airways. Virus is cleared by day 9, and between days 8 and 9 there is an abrupt change in CD8 T cell motility behavior transitioning from low velocity and high confinement on day 8, to high velocity with continued high confinement on day 9. We hypothesized that loss of virus and/or antigen signals in the context of high chemokine levels drives the T cells into a rapid surveillance mode. Virus infection induces chemokine production, which may change when the virus is cleared. We therefore sought to examine this period of rapid changes to the T cell environment in the tissue and seek evidence on the roles of peptide-MHC and chemokine receptor interactions. Experiments were performed to block G protein coupled receptor (GPCR) signaling with Pertussis toxin (Ptx). Ptx treatment generally reduced cell velocities and mildly increased confinement suggesting chemokine mediated arrest (velocity <2 µm/min) (Friedman RS, 2005), except on day 8 when velocity increased and confinement was relieved. Blocking specific peptide-MHC with monoclonal antibody unexpectedly decreased velocities on days 7 through 9, suggesting TCR/peptide-MHC interactions promote cell mobility in the tissue. Together, these results suggest the T cells are engaged with antigen bearing and chemokine producing cells that affect motility in ways that vary with the day after infection. The increase in velocities on day 9 were reversed by addition of specific peptide, consistent with the idea that antigen signals become limiting on day 9 compared to earlier time points. Thus, antigen and chemokine signals act to alternately promote and restrict CD8 T cell motility until the point of virus clearance, suggesting the switch in motility behavior on day 9 may be due to a combination of limiting antigen in the presence of high chemokine signals as the virus is cleared.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Movimento Celular/fisiologia , Vírus da Influenza A/imunologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/fisiologia , Movimento Celular/efeitos dos fármacos , Quimiocinas/imunologia , Vírus da Influenza A/patogenicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Orthomyxoviridae , Infecções por Orthomyxoviridae/imunologia , Toxina Pertussis/metabolismo , Toxina Pertussis/farmacologia , Receptores de Quimiocinas , Receptores Acoplados a Proteínas-G/metabolismo
10.
Nat Commun ; 11(1): 4137, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811827

RESUMO

The class B secretin GPCR (SecR) has broad physiological effects, with target potential for treatment of metabolic and cardiovascular disease. Molecular understanding of SecR binding and activation is important for its therapeutic exploitation. We combined cryo-electron microscopy, molecular dynamics, and biochemical cross-linking to determine a 2.3 Å structure, and interrogate dynamics, of secretin bound to the SecR:Gs complex. SecR exhibited a unique organization of its extracellular domain (ECD) relative to its 7-transmembrane (TM) core, forming more extended interactions than other family members. Numerous polar interactions formed between secretin and the receptor extracellular loops (ECLs) and TM helices. Cysteine-cross-linking, cryo-electron microscopy multivariate analysis and molecular dynamics simulations revealed that interactions between peptide and receptor were dynamic, and suggested a model for initial peptide engagement where early interactions between the far N-terminus of the peptide and SecR ECL2 likely occur following initial binding of the peptide C-terminus to the ECD.


Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Simulação de Dinâmica Molecular , Receptores Acoplados a Proteínas-G/química , Receptores dos Hormônios Gastrointestinais/química , Secretina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Linhagem Celular , Cricetinae , Microscopia Crioeletrônica , Cristalografia por Raios X , Cisteína/química , Cisteína/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/ultraestrutura , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Insetos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos/genética , Estrutura Secundária de Proteína , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Acoplados a Proteínas-G/ultraestrutura , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores dos Hormônios Gastrointestinais/ultraestrutura , Secretina/metabolismo
11.
Ecotoxicol Environ Saf ; 204: 111068, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32745784

RESUMO

Herein, eight common endocrine disrupting chemicals (EDCs) were exposed to zebrafish (Danio rerio) to investigate the relationship between different EDCs and their activated estrogen receptors. Under acute exposure, we identified five major malformation types whose incidence and deformity modes differed among EDCs. Luciferase analysis divided the EDC receptors into four categories: (i) triclosan (TCS), 17ß-estradiol (E2) and estriol (E3) mainly activated GPER expression; (ii) bisphenol A (BPA), p-(tert-octyl) phenol (POP), 17α-ethynylestradiol (EE2), E2 and E3 activated ERß expression; (iii) E2 and E3 acted on both GPER and ERß; and (iv) estrone (E1) and 9,9-bis(4-hydroxyphenyl)fluorene (BHPF) had little effect on the two receptors. In vivo immunofluorescence experiments on 96-hpf larvae provided evidence that TCS and POP acted on GPER and ERß, respectively, while E2 acted on the two receptors simultaneously. Luciferase activities in the promoter regions of gper (-986 to -488) and erß (-1998 to -1496) were higher than those in other regions, identifying these key regions as targets for transcription activity. TCS promoted GPER expression by acting on the JUND transcription factor, while POP promoted ERß expression by activating the Foxl1 transcription factor. In contrast, E2 mainly regulated transcription of GPER and ERß by Arid3a. These findings provide compelling evidence that different EDCs possess varying estrogen receptors, leading to differential regulatory pathways and abnormality symptoms. These results offer an experimental strategy and fundamental information to assess the molecular mechanisms of EDC-induced estrogen effects.


Assuntos
Compostos Benzidrílicos/toxicidade , Disruptores Endócrinos/toxicidade , Receptor beta de Estrogênio/metabolismo , Fenóis/toxicidade , Receptores Acoplados a Proteínas-G/metabolismo , Poluentes Químicos da Água/toxicidade , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Compostos Benzidrílicos/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Disruptores Endócrinos/metabolismo , Larva/efeitos dos fármacos , Larva/metabolismo , Fenóis/metabolismo , Poluentes Químicos da Água/metabolismo
12.
Gene ; 763: 145067, 2020 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-32827681

RESUMO

BACKGROUND: rs2274911 (Pro91Ser, G > A) is a missense mutation located on the second exon of the GPRC6A gene. Increasing evidence revealed a significant association between the A allele of rs2274911 and male diseases, such as oligospermia, cryptorchidism, and prostate tumor. However, the function of rs2274911 in healthy males is unclear. SUBJECTS AND METHODS: A total of 1742 healthy men were selected from the Fangchenggang Area Male Health and Examination Survey (FAMHES). The association between rs2274911 and phenotype was evaluated. The cell characteristics of rs2274911 mutation (mu), wild-type GPRC6A (WT), and RFP control in human embryonic kidney (293T) and human prostate cancer (PC3) cells were analyzed. RNA sequencing was performed on PC3 cells. RESULTS: E2 and PSA serum levels increased with the accumulation of the A allele (E2: G vs. A, -0.029 [-0.050, -0.008], P < 0.01, P trend = 0.027; PSA: G vs. A, -0.040 [-0.079, 0.000], P < 0.05, P trend = 0.048). rs2274911 enhanced the proliferation and invasion ability of PC3 or 293T cells and activated the ERK pathway. The genes were identified as rs2274911 mu-affected genes through RNA sequential analysis of rs2274911 mu, GPRC6A WT, and RFP control of PC3 cells. Most of these genes were related to cancer development processes, cAMP, and the ERK cell signaling pathway. CONCLUSION: This project represents that rs2274911 is associated with E2 and PSA serum levels in Southern Chinese men. Rs2274991 mutation promotes 293T and PC3 cell proliferation in vitro. These results suggest that rs2274911 is a functional variant of GPRC6A.


Assuntos
Antígeno 12E7/sangue , Polimorfismo de Nucleotídeo Único , Antígeno Prostático Específico/sangue , Receptores Acoplados a Proteínas-G/genética , Adulto , Proliferação de Células , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Pessoa de Meia-Idade , Células PC-3 , Receptores Acoplados a Proteínas-G/metabolismo
13.
Arterioscler Thromb Vasc Biol ; 40(9): 2070-2083, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32762445

RESUMO

OBJECTIVE: Emerging evidence suggests that C3aR (C3a anaphylatoxin receptor) signaling has protective roles in various inflammatory-related diseases. However, its role in atherosclerosis has been unknown. The purpose of the study was to investigate the possible protective role of C3aR in aortic atherosclerosis and explore molecular and cellular mechanisms involved in the protection. Approach and Results: C3ar-/-/Apoe-/- mice were generated by cross-breeding of atherosclerosis-prone Apoe-/- mice and C3ar-/- mice. C3ar-/-/Apoe-/- mice and Apoe-/- mice (as a control) underwent high-fat diet for 16 weeks were assessed for (1) atherosclerotic plaque burden, (2) aortic tissue inflammation, (3) recruitment of CD11b+ leukocytes into atherosclerotic lesions, and (4) systemic inflammatory responses. Compared with Apoe-/- mice, C3ar-/-/Apoe-/- mice developed more severe atherosclerosis. In addition, C3ar-/-/Apoe-/- mice have increased local production of proinflammatory mediators (eg, CCL2 [chemokine (C-C motif) ligand 2], TNF [tumor necrosis factor]-α) and infiltration of monocyte/macrophage in aortic tissue, and their lesional macrophages displayed an M1-like phenotype. Local pathological changes were associated with enhanced systemic inflammatory responses (ie, elevated plasma levels of CCL2 and TNF-α, increased circulating inflammatory cells). In vitro analyses using peritoneal macrophages showed that C3a stimulation resulted in upregulation of M2-associated signaling and molecules, but suppression of M1-associated signaling and molecules, supporting the roles of C3a/C3aR axis in mediating anti-inflammatory response and promoting M2 macrophage polarization. CONCLUSIONS: Our findings demonstrate a protective role for C3aR in the development of atherosclerosis and suggest that C3aR confers the protection through C3a/C3aR axis-mediated negative regulation of proinflammatory responses and modulation of macrophage toward the anti-inflammatory phenotype.


Assuntos
Aorta/metabolismo , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Inflamação/prevenção & controle , Macrófagos Peritoneais/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Aorta/imunologia , Aorta/patologia , Doenças da Aorta/imunologia , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Aterosclerose/imunologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Células Cultivadas , Quimiocina CCL2/metabolismo , Quimiotaxia , Modelos Animais de Doenças , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , NF-kappa B/metabolismo , Fenótipo , Placa Aterosclerótica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Acoplados a Proteínas-G/deficiência , Receptores Acoplados a Proteínas-G/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
14.
Int J Mol Sci ; 21(15)2020 Jul 28.
Artigo em Inglês | MEDLINE | ID: covidwho-680188

RESUMO

Along with cancer, cardiovascular and cerebrovascular diseases remain by far the most common causes of death. Heart attacks and strokes are diseases in which platelets play a role, through activation on ruptured plaques and subsequent thrombus formation. Most platelet agonists activate platelets via G protein-coupled receptors (GPCRs), which make these receptors ideal targets for many antiplatelet drugs. However, little is known about the mechanisms that provide feedback regulation on GPCRs to limit platelet activation. Emerging evidence from our group and others strongly suggests that GPCR kinases (GRKs) are critical negative regulators during platelet activation and thrombus formation. In this review, we will summarize recent findings on the role of GRKs in platelet biology and how one specific GRK, GRK6, regulates the hemostatic response to vascular injury. Furthermore, we will discuss the potential role of GRKs in thrombotic disorders, such as thrombotic events in COVID-19 patients. Studies on the function of GRKs during platelet activation and thrombus formation have just recently begun, and a better understanding of the role of GRKs in hemostasis and thrombosis will provide a fruitful avenue for understanding the hemostatic response to injury. It may also lead to new therapeutic options for the treatment of thrombotic and cardiovascular disorders.


Assuntos
Quinases de Receptores Acoplados a Proteína G/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Trombose/metabolismo , Plaquetas/metabolismo , Humanos , Ativação Plaquetária
15.
J Cancer Res Clin Oncol ; 146(10): 2547-2557, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32671503

RESUMO

INTRODUCTION: Colorectal cancer (CRC) constitutes one of the most prevalent malignancies in the world. Recent research suggests that cancer stem cells (CSCs) are responsible for tumor cell's malignant behavior in CRC. This study has been designed to determinate clinical implications of CSC markers: CD44, DCLK1, Lgr5, and ANXA2 in CRC. MATERIALS AND METHODS: The study was performed on tissue samples which were collected from 89 patients undergoing colectomy. Formalin-fixed paraffin-embedded tissue blocks with representative tumor areas were identified and corded. Immunohistochemical staining was performed using anti-CD44, anti-LGR5, anti-ANXA2, and anti-DCLK1 antibodies. The H-score system was utilized to determine the immunointensity of CRC cells. RESULTS: The lower expression of Lgr5 was significantly correlated with the presence of lymph-node metastases (p = 0.011), while high expression of Lgr5 was statistically significant in vascular invasion in examined cancer tissue samples (p = 0.027). Moreover, a high H-score value of Lgr5 expression was significantly related to a reduced overall survival rate (p = 0.043). CONCLUSION: Our results suggest a strong relationship between CSC marker Lgr5 and vascular invasion, presence of lymph-node metastasis, and overall poor survival. The presence of Lgr5 might be an unfavorable prognostic factor, and its high level in cancer tissue is related to an aggressive course. This marker could also be used to access the effectiveness of the treatment.


Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Receptores Acoplados a Proteínas-G/metabolismo , Idoso , Anexina A2/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/irrigação sanguínea , Progressão da Doença , Feminino , Humanos , Receptores de Hialuronatos/metabolismo , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Prognóstico , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Acoplados a Proteínas-G/biossíntese , Análise Serial de Tecidos
16.
Nat Commun ; 11(1): 3547, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669546

RESUMO

Neutrophils provide first line of host defense against bacterial infections utilizing glycolysis for their effector functions. How glycolysis and its major byproduct lactate are triggered in bone marrow (BM) neutrophils and their contribution to neutrophil mobilization in acute inflammation is not clear. Here we report that bacterial lipopolysaccharides (LPS) or Salmonella Typhimurium triggers lactate release by increasing glycolysis, NADPH-oxidase-mediated reactive oxygen species and HIF-1α levels in BM neutrophils. Increased release of BM lactate preferentially promotes neutrophil mobilization by reducing endothelial VE-Cadherin expression, increasing BM vascular permeability via endothelial lactate-receptor GPR81 signaling. GPR81-/- mice mobilize reduced levels of neutrophils in response to LPS, unless rescued by VE-Cadherin disrupting antibodies. Lactate administration also induces release of the BM neutrophil mobilizers G-CSF, CXCL1 and CXCL2, indicating that this metabolite drives neutrophil mobilization via multiple pathways. Our study reveals a metabolic crosstalk between lactate-producing neutrophils and BM endothelium, which controls neutrophil mobilization under bacterial infection.


Assuntos
Células da Medula Óssea/imunologia , Ácido Láctico/metabolismo , Neutrófilos/imunologia , Receptores Acoplados a Proteínas-G/metabolismo , Infecções por Salmonella/imunologia , Animais , Medula Óssea/irrigação sanguínea , Células da Medula Óssea/metabolismo , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Feminino , Humanos , Lipopolissacarídeos/imunologia , Masculino , Camundongos , Camundongos Knockout , Neutrófilos/metabolismo , Receptores Acoplados a Proteínas-G/genética , Infecções por Salmonella/microbiologia , Salmonella typhimurium/imunologia , Transdução de Sinais/imunologia
17.
J Immunol ; 205(5): 1198-1206, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32680957

RESUMO

Fever in infections correlates with inflammation, macrophage infiltration into the affected organ, macrophage activation, and release of cytokines involved in immune response, hematopoiesis, and homeostatic processes. Angiotensin-converting enzyme 2 (ACE2) is the canonical cell surface receptor for SARS-CoV-2. ACE2 together with angiotensin receptor types 1 and 2 and ACE2 are components of the renin-angiotensin system (RAS). Exacerbated production of cytokines, mainly IL-6, points to macrophages as key to understand differential COVID-19 severity. SARS-CoV-2 may modulate macrophage-mediated inflammation events by altering the balance between angiotensin II, which activates angiotensin receptor types 1 and 2, and angiotensin 1-7 and alamandine, which activate MAS proto-oncogene and MAS-related D receptors, respectively. In addition to macrophages, lung cells express RAS components; also, some lung cells are able to produce IL-6. Addressing how SARS-CoV-2 unbalances RAS functionality via ACE2 will help design therapies to attenuate a COVID-19-related cytokine storm.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/imunologia , Interleucina-6/biossíntese , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/imunologia , Sistema Renina-Angiotensina , Angiotensina I/metabolismo , Animais , Betacoronavirus/imunologia , Infecções por Coronavirus/virologia , Humanos , Inflamação/imunologia , Macrófagos/imunologia , Pandemias , Fragmentos de Peptídeos/metabolismo , Pneumonia Viral/virologia , Proteínas Proto-Oncogênicas/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Virais/metabolismo
18.
J Immunol ; 205(5): 1198-1206, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: covidwho-654195

RESUMO

Fever in infections correlates with inflammation, macrophage infiltration into the affected organ, macrophage activation, and release of cytokines involved in immune response, hematopoiesis, and homeostatic processes. Angiotensin-converting enzyme 2 (ACE2) is the canonical cell surface receptor for SARS-CoV-2. ACE2 together with angiotensin receptor types 1 and 2 and ACE2 are components of the renin-angiotensin system (RAS). Exacerbated production of cytokines, mainly IL-6, points to macrophages as key to understand differential COVID-19 severity. SARS-CoV-2 may modulate macrophage-mediated inflammation events by altering the balance between angiotensin II, which activates angiotensin receptor types 1 and 2, and angiotensin 1-7 and alamandine, which activate MAS proto-oncogene and MAS-related D receptors, respectively. In addition to macrophages, lung cells express RAS components; also, some lung cells are able to produce IL-6. Addressing how SARS-CoV-2 unbalances RAS functionality via ACE2 will help design therapies to attenuate a COVID-19-related cytokine storm.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/imunologia , Interleucina-6/biossíntese , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/imunologia , Sistema Renina-Angiotensina , Angiotensina I/metabolismo , Animais , Betacoronavirus/imunologia , Infecções por Coronavirus/virologia , Humanos , Inflamação/imunologia , Macrófagos/imunologia , Pandemias , Fragmentos de Peptídeos/metabolismo , Pneumonia Viral/virologia , Proteínas Proto-Oncogênicas/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Virais/metabolismo
19.
Anticancer Res ; 40(6): 3097-3108, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32487604

RESUMO

BACKGROUND/AIM: C-C motif chemokine ligand 18 (CCL18) is overexpressed in the microenvironment of tumors, promotes invasion and metastasis and is thus important for the therapeutic outcome of many tumor entities. The Gs-coupled seven-transmembrane receptor GPR30 is known as both a CCL18 and an estrogen receptor; its activation by estradiol leads to a transactivation of membrane-tethered pro-heparin-binding EGF-like growth factor and the MAPK/ERK pathway. We examined whether this signaling pathway remains the same under CCL18 stimulation, as opposed to estradiol stimulation. MATERIALS AND METHODS: We investigated the effects of CCL18 on the lung cancer cell line A549, that show low GPR30 expression and the breast cancer cell lines MCF-7, that has high GPR30 expression and MDA-MB-231. These cells were stimulated in different media with CCL18 and then analyzed by qPCR, In-Cell Western®, western blot and ELISA. RESULTS: Many similarities on the effect of CCL18 on the already known estradiol-activated signaling pathway via the G protein-coupled estrogen receptor GPR30 were identified. GPR30 is involved in the expression of matrix metalloproteinases (MMPs), which may play a role in the transactivation of ERK-1/-2 via the cleavage of membrane-bound HB-EGF, via Src-related tyrosine kinases and Gßγ-subunits. With increasing CCL18 concentration, the expression of MMP7 decreased in A549 cells. With decreasing estrogen content of the medium, there was an increasing effect of CCL18 on the inhibition of the relative expression of MMP7. Inhibition of GPR30 with G15 also resulted in a decrease in the relative expression of MMP7, irrespective of the subsequent stimulation with CCL18. This is a rather unexpected result, because the estrogen estradiol and CCL18 both activate GPR30. MCF-7 cells which express more GPR30 did not show any dependence of the relative MMP7 expression on CCL18 except in estrogen-free FCS medium. CCL18 induced an increased relative ERK activation in In-Cell western (ICW) at A549 cells. Stimulation with CCL18 caused decreased ERK activation with simultaneous inhibition of adenylate cyclase in MCF-7. However, stimulation with CCL18 and simultaneous inhibition of cyclooxygenase in MCF-7 resulted in increased ERK activation. In A549, stimulation with CCL18 and co-incubation with dbcAMP resulted in decreased ERK activation in both ICW and Western blot. CONCLUSION: In summary, the Gs-coupled receptor GPR30 plays an important role in the signaling pathway of CCL18. CCL18 and estradiol may not lead to the same signaling pathway after activating GPR30.


Assuntos
Quimiocinas CC/metabolismo , Estradiol/metabolismo , Metaloproteinase 7 da Matriz/metabolismo , Receptores Estrogênicos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Células A549 , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Quimiocinas CC/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Células MCF-7 , Metaloproteinase 7 da Matriz/biossíntese , Metaloproteinase 7 da Matriz/genética , Fosforilação , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/genética , Receptores Estrogênicos/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Transdução de Sinais
20.
Nat Rev Drug Discov ; 19(6): 389-413, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32494050

RESUMO

Dysregulation of peptide-activated pathways causes a range of diseases, fostering the discovery and clinical development of peptide drugs. Many endogenous peptides activate G protein-coupled receptors (GPCRs) - nearly 50 GPCR peptide drugs have been approved to date, most of them for metabolic disease or oncology, and more than 10 potentially first-in-class peptide therapeutics are in the pipeline. The majority of existing peptide therapeutics are agonists, which reflects the currently dominant strategy of modifying the endogenous peptide sequence of ligands for peptide-binding GPCRs. Increasingly, novel strategies are being employed to develop both agonists and antagonists, to both introduce chemical novelty and improve drug-like properties. Pharmacodynamic improvements are evolving to allow biasing ligands to activate specific downstream signalling pathways, in order to optimize efficacy and reduce side effects. In pharmacokinetics, modifications that increase plasma half-life have been revolutionary. Here, we discuss the current status of the peptide drugs targeting GPCRs, with a focus on evolving strategies to improve pharmacokinetic and pharmacodynamic properties.


Assuntos
Desenho de Fármacos , Peptídeos , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Ensaios Clínicos como Assunto , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligantes , Terapia de Alvo Molecular , Biblioteca de Peptídeos , Peptídeos/farmacocinética , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Ligação Proteica , Transdução de Sinais
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