RESUMO
GPR34 is a recently identified G-protein coupled receptor, which has an immunomodulatory role and recognizes lysophosphatidylserine (LysoPS) as a putative ligand. Here, we report cryo-electron microscopy structures of human GPR34-Gi complex bound with one of two ligands bound: either the LysoPS analogue S3E-LysoPS, or M1, a derivative of S3E-LysoPS in which oleic acid is substituted with a metabolically stable aromatic fatty acid surrogate. The ligand-binding pocket is laterally open toward the membrane, allowing lateral entry of lipidic agonists into the cavity. The amine and carboxylate groups of the serine moiety are recognized by the charged residue cluster. The acyl chain of S3E-LysoPS is bent and fits into the L-shaped hydrophobic pocket in TM4-5 gap, and the aromatic fatty acid surrogate of M1 fits more appropriately. Molecular dynamics simulations further account for the LysoPS-regioselectivity of GPR34. Thus, using a series of structural and physiological experiments, we provide evidence that chemically unstable 2-acyl LysoPS is the physiological ligand for GPR34. Overall, we anticipate the present structures will pave the way for development of novel anticancer drugs that specifically target GPR34.
Assuntos
Ácidos Graxos , Lisofosfolipídeos , Humanos , Microscopia Crioeletrônica , Ácidos Graxos/metabolismo , Ligantes , Lisofosfolipídeos/metabolismo , Receptores de Lisofosfolipídeos/agonistas , Receptores de Lisofosfolipídeos/metabolismoRESUMO
PURPOSE: G-protein coupled receptor (GPR 34) has been found to play important roles in some cancers and regulates the proliferation, apoptosis, and migration of these cancer cells. However, the mechanisms underlying how GPR34 functions to regulate growth and proliferation of colorectal cancer cells remains to be clarified. METHODS: We employed stable GPR34 knockdown LS174T cell models, GPR34 Mab blocking, a CCK-8 kit, and a colony formation assay to characterize the effect of GPR34 on the proliferation of LS174T in vitro and xenograft tumor growth in vivo. The mRNA level of GPR34 was detected by RT-PCR in tumor tissues and adjacent normal tissues from 34 CRC patients. RESULTS: Based on RT-PCR results, GPR34 exhibited high level in tumor samples compared with adjacent normal samples. Increased expression of GPR34 is more associated with poor prognosis of CRC as shown in The Cancer Genome Atlas (TCGA) dataset by Kaplan-Meier survival analysis. Furthermore, we showed that GPR34 knockdown inhibited the proliferation of LS174T colon cancer cells and related xenograft tumor growth. Searching for the distinct molecular mechanism, we identified several contributors to proliferation of LS174T colon cancer cells: PI3K subunits/PTEN, PDK1/AKT, and Src/Raf/Ras/ERK. GPR34 knockdown inhibited the proliferation of LS174T cells by upregulating expression of PTEN, and downregulating expression of PI3K subunits p110-beta. CONCLUSION: Our findings provide direct evidence that GPR34 regulates the proliferation of LS174T cells and the growth of LS174T tumor xenografts by regulating different pathways. High expression of GPR34 mRNA could then be used to predict poor prognosis of CRC.
Assuntos
Neoplasias Colorretais , Fosfatidilinositol 3-Quinases , Receptores de Lisofosfolipídeos , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Lisofosfolipídeos/genética , Receptores de Lisofosfolipídeos/metabolismo , Transdução de Sinais/genéticaRESUMO
GPR34 translocation and mutation are specifically associated with salivary gland MALT lymphoma (SG-MALT-lymphoma). The majority of GPR34 mutations are clustered in its C-terminus, resulting in truncated proteins lacking the phosphorylation motif important for receptor desensitization. It is unclear why GPR34 genetic changes associate with SG-MALT-lymphoma and how these mutations contribute to the development of lymphoma. We generated isogenic Flp-InTRex293 cell lines that stably expressed a single copy of GPR34 or its various mutants and performed a range of in vitro assays. We found that the GPR34 Q340X truncation, but not the R84H and D151A mutants, conferred a significantly increased resistance to apoptosis and greater transforming potential than the GPR34 wild type. The GPR34 truncation mutant had a significantly delayed internalization compared with the wild type after ligand (lysophosphatidylserine) stimulation. Among the 9 signaling pathways examined, the GPR34 Q340X truncation, and to a lesser extent the D151A mutant, significantly activated CRE, NF-κB, and AP1 reporter activities, particularly in the presence of ligand stimulation. We further described the enhanced activities of phospholipase-A1/2 in the culture supernatant of Flp-InTRex293 cells that expressed the GPR34 Q340X mutant, as well as their potential to catalyze the synthesis of lysophosphatidylserine from phosphatidylserine. Importantly, phospholipase-A1 was abundantly expressed in the duct epithelium of salivary glands and those involved in lymphoepithelial lesions (LELs). Our findings advocate a model of paracrine stimulation of malignant B cells via GPR34, in which phospholipase A is released by LELs and hydrolyzes the phosphatidylserine exposed on apoptotic cells, generating lysophosphatidylserine, the ligand for GPR34. Thus, GPR34 activation potentially bridges LELs to genesis of SG-MALT-lymphoma.
Assuntos
Linfoma de Zona Marginal Tipo Células B , Receptores de Lisofosfolipídeos , Humanos , Ligantes , Linfoma de Zona Marginal Tipo Células B/patologia , Fosfatidilserinas , Fosfolipases , Receptores de Lisofosfolipídeos/metabolismo , Glândulas Salivares/metabolismo , Glândulas Salivares/patologiaRESUMO
MicroRNAs (miRNAs) have emerged as the vital post-transcriptional regulators and control the growth and progression of different cancers types. The current study aimed at exploration of the role of microRNA-381 (miRNA-381) in human cervical cancer with emphasis on the evaluation of the underlying molecular mechanism. The results revealed a significant (P < 0.05) downregulation of miRNA-381 was found in cervical cancer tissues and cancer cell lines. Overexpression of miRNA-381 in cervical cancer cells significantly (P < 0.05) inhibited their proliferation through the induction of cell apoptosis which was accompanied by depletion of Bcl-2 and increase in Bax expression. Additionally, the cleavage of caspase-3 and 9 was also activated upon miRNA-381 overexpression. The Overexpression of miRNA-381 further inhibited the migration and invasion of cervical cancer cells. In silico analysis together with dual luciferase assay revealed G protein-Coupled receptor 34 (GPR34) to be the target of miRNA-381. The expression of GPR34 was significantly (P < 0.05) upregulated in the cervical cancer tissues and cell lines. Nonetheless, miRNA-381 overexpression caused a remarkable decrease in the expression of GPR34. The GPR34 knockdown and overexpression proved that the tumor-suppressive effects of miRNA-381 are mediated via GPR34. The study elucidated the essence of miRNA-381/GPR34 molecular regulatory axis in cervical cancer and unraveled the possibility of targeting this molecular axis as an important therapeutic approach against human cervical cancer.
Assuntos
MicroRNAs , Receptores de Lisofosfolipídeos/genética , Neoplasias do Colo do Útero/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Colo do Útero/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Receptores de Lisofosfolipídeos/metabolismo , Neoplasias do Colo do Útero/patologiaRESUMO
Mucosa-associated lymphoid tissue (MALT) lymphoma originates from a background of diverse chronic inflammatory disorders at various anatomic sites. The genetics underlying its development, particularly in those associated with autoimmune disorders, is poorly characterized. By whole exome sequencing of 21 cases of MALT lymphomas of the salivary gland and thyroid, we have identified recurrent somatic mutations in 2 G-protein coupled receptors (GPR34 and CCR6) not previously reported in human malignancies, 3 genes (PIK3CD, TET2, TNFRSF14) not previously implicated in MALT lymphoma, and a further 2 genes (TBL1XR1, NOTCH1) recently described in MALT lymphoma. The majority of mutations in GPR34 and CCR6 were nonsense and frameshift changes clustered in the C-terminal cytoplasmic tail, and would result in truncated proteins that lack the phosphorylation motif important for ß-arrestin-mediated receptor desensitization and internalization. Screening of these newly identified mutations, together with previously defined genetic changes, revealed distinct mutation profiles in MALT lymphoma of various sites, with those of salivary gland characterized by frequent TBL1XR1 and GPR34 mutations, thyroid by frequent TET2, TNFRSF14 and PIK3CD mutations, and ocular adnexa by frequent TNFAIP3 mutation. Interestingly, in MALT lymphoma of the salivary gland, there was a significant positive association between TBL1XR1 mutation and GPR34 mutation/translocation (P=0.0002). In those of ocular adnexa, TBL1XR1 mutation was mutually exclusive from TNFAIP3 mutation (P=0.049), but significantly associated with IGHV3-23 usage (P=0.03) and PIK3CD mutation (P=0.009). These findings unravel novel insights into the molecular mechanisms of MALT lymphoma and provide further evidence for potential oncogenic co-operation between receptor signaling and genetic changes.
Assuntos
Linfoma de Zona Marginal Tipo Células B/genética , Mutação , Receptores CCR6/genética , Receptores de Lisofosfolipídeos/genética , Perfil Genético , Humanos , Linfoma de Zona Marginal Tipo Células B/patologia , Neoplasias das Glândulas Salivares/genética , Neoplasias da Glândula Tireoide/genética , Sequenciamento do ExomaRESUMO
Research on GPR34, which was discovered in 1999 as an orphan G protein-coupled receptor of the rhodopsin-like class, disclosed its physiologic relevance only piece by piece. Being present in all recent vertebrate genomes analyzed so far it seems to improve the fitness of species although it is not essential for life and reproduction as GPR34-deficient mice demonstrate. However, closer inspection of macrophages and microglia, where it is mainly expressed, revealed its relevance in immune cell function. Recent data clearly demonstrate that GPR34 function is required to arrest microglia in the M0 homeostatic non-phagocytic phenotype. Herein, we summarize the current knowledge on its evolution, genomic and structural organization, physiology, pharmacology and relevance in human diseases including neurodegenerative diseases and cancer, which accumulated over the last 20â¯years.
Assuntos
Receptores de Lisofosfolipídeos/fisiologia , Animais , Evolução Molecular , Genômica , História do Século XX , História do Século XXI , Humanos , Fenótipo , Receptores de Lisofosfolipídeos/química , Receptores de Lisofosfolipídeos/históriaAssuntos
Cromossomos Humanos Par 14 , Cromossomos Humanos X , Genes de Cadeia Pesada de Imunoglobulina , Linfoma de Zona Marginal Tipo Células B/diagnóstico , Linfoma de Zona Marginal Tipo Células B/genética , Receptores de Lisofosfolipídeos/genética , Translocação Genética , Idoso , Anticorpos Monoclonais Murinos/efeitos adversos , Anticorpos Monoclonais Murinos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ciclofosfamida/efeitos adversos , Ciclofosfamida/uso terapêutico , Doxorrubicina/efeitos adversos , Doxorrubicina/uso terapêutico , Éxons , Feminino , Testes Genéticos , Humanos , Linfoma de Zona Marginal Tipo Células B/terapia , Masculino , Gradação de Tumores , Estadiamento de Neoplasias , Prednisona/efeitos adversos , Prednisona/uso terapêutico , Rituximab , Análise de Sequência de DNA , Resultado do Tratamento , Vincristina/efeitos adversos , Vincristina/uso terapêuticoRESUMO
Abundance of G protein-coupled receptor 34 (GPR34) mRNA is greater in granulosa cells (GCs) of cystic vs normal follicles of cattle. The present experiments were designed to determine if GPR34 mRNA in granulosa cell [GC] changes during selection and growth of dominant follicles in cattle as well as to investigate the hormonal regulation of GPR34 mRNA in bovine GC in vitro. In Exp. 1, estrous cycles of nonlactating cows were synchronized and then ovariectomized on either day 3-4 or 5-6 after ovulation. GPR34 mRNA abundance in GC was 2.8- to 3.8-fold greater (P < 0.05) in small (1-5 mm) and large (≥8 mm) estrogen-inactive dominant follicles than in large estrogen-active follicles. Also, GPR34 mRNA tended to be greater (P < 0.10) in F2 than F1 follicles on day 3-4 postovulation. In Exp. 2-7, ovaries were collected at an abattoir and GC were isolated and treated in vitro. Expression of GPR34 was increased (P < 0.05) 2.2-fold by IGF1. Tumor necrosis factor (TNF)-α decreased (P < 0.05) the IGF1-induced GPR34 mRNA abundance in small-follicle GC, whereas IGF1 decreased (P < 0.05) GPR34 expression by 45% in large-follicle GC. Treatment of small-follicle GC with either IL-2, prostaglandin E2 or angiogenin decreased (P < 0.05) GPR34 expression, whereas FSH, cortisol, wingless 3A, or hedgehog proteins did not affect (P > 0.10) GPR34 expression. In Exp. 6 and 7, 2 presumed ligands of GPR34, L-a-lysophosphatidylserine (LPPS) and LPP-ethanolamine, increased (P < 0.05) GC numbers and estradiol production by 2-fold or more in small-follicle GC, and this response was only observed in IGF1-treated GC. In conclusion, GPR34 is a developmentally and hormonally regulated gene in GC, and its presumed ligands enhance IGF1-induced proliferation and steroidogenesis of bovine GC.
Assuntos
Bovinos/fisiologia , Células da Granulosa/metabolismo , Folículo Ovariano/crescimento & desenvolvimento , Receptores de Lisofosfolipídeos/metabolismo , Animais , Células Cultivadas , Citocinas/farmacologia , Feminino , Regulação da Expressão Gênica/fisiologia , Células da Granulosa/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Lisofosfolipídeos/genéticaRESUMO
Protein folding, topogenesis and intracellular targeting of G protein-coupled receptors (GPCRs) must be precisely coordinated to ensure correct receptor localization. To elucidate how different steps of GPCR biosynthesis work together, we investigated the process of membrane topology determination and how it relates to the acquisition of cell surface trafficking competence in human GPR34. By monitoring a fused FLAG-tag and a conformation-sensitive native epitope during the expression of GPR34 mutant panel, a tri-basic motif in the first intracellular loop was identified as the key topogenic signal that dictates the orientation of transmembrane domain-1 (TM1). Charge disruption of the motif perturbed topogenic processes and resulted in the conformational epitope loss, post-translational processing alteration, and trafficking arrest in the Golgi. The placement of a cleavable N-terminal signal sequence as a surrogate topogenic determinant overcame the effects of tri-basic motif mutations and rectified the TM1 orientation; thereby restored the conformational epitope, post-translational modifications, and cell surface trafficking altogether. Progressive N-tail truncation and site-directed mutagenesis revealed that a proline-rich segment of the N-tail and all four cysteines individually located in the four separate extracellular regions must simultaneously reside in the ER lumen to muster the conformational epitope. Oxidation of all four cysteines was necessary for the epitope formation, but the cysteine residues themselves were not required for the trafficking event. The underlying biochemical properties of the conformational epitope was therefore the key to understand mechanistic processes propelled by positive-inside rule that simultaneously regulate the topogenesis and intracellular trafficking of GPR34.
Assuntos
Membrana Celular/metabolismo , Receptores de Lisofosfolipídeos/metabolismo , Motivos de Aminoácidos , Anticorpos Monoclonais/imunologia , Retículo Endoplasmático/metabolismo , Epitopos , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Mutação , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Transporte Proteico , Receptores de Lisofosfolipídeos/química , Receptores de Lisofosfolipídeos/genética , Receptores de Lisofosfolipídeos/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , TransfecçãoRESUMO
Dendritic cells (DCs) are specifically equipped with the G protein-coupled receptor 34 (GPR34). Tight regulation of GPR34 gene expression seems highly important for proper immunological functions, because the absence of this receptor leads to an alteration of the immune response, whereas overexpression was reported to be involved in neuroinflammation. However, the regulatory mechanism of GPR34 expression has not yet been investigated. Whole-transcriptome RNA sequencing analysis from spleens and DCs of GPR34 knockout and wild-type mice, combined with protein-protein interaction data, revealed functional modules affected by the absence of this receptor. Among these, NF-κB, MAPK, and apoptosis-signaling pathways showed high significance. Using murine DCs we experimentally show that NF-κB and MAPK pathways are involved in the downregulation of GPR34. DCs lacking GPR34 have a higher caspase-3/7 activity and increased apoptosis levels. Our study reveals a novel role of GPR34 in the fate of DCs and identifies a regulatory mechanism that could be relevant for treatment of GPR34-overexpressing pathologies, such as neuroinflammatory or cancer conditions.
Assuntos
Apoptose/imunologia , Células Dendríticas/imunologia , Receptores de Lisofosfolipídeos/imunologia , Animais , Western Blotting , Citometria de Fluxo , Regulação da Expressão Gênica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Over the past decade the importance of lipids for cancer cell metabolism and cancer-related processes such as proliferation, metastasis and chemotherapy resistance has become more apparent. The mechanisms by which lipid signals are transduced are poorly understood, but frequently involve G-protein Coupled Receptors (GPCRs), which can be explored as druggable targets. Here, we discuss how GPCRs recognize four classes of cancer-relevant lipids (lysophospholipids, phospholipids, fatty acids and eicosanoids). We compare the ligand-binding properties of >50 lipid receptors, we examine how their dysregulation contributes to tumorigenesis and how they may be therapeutically exploited.
Assuntos
Lipídeos/fisiologia , Neoplasias/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Animais , Eicosanoides/metabolismo , Endocanabinoides/fisiologia , Ácidos Graxos/metabolismo , Humanos , Lisofosfolipídeos/química , Lisofosfolipídeos/fisiologia , Neoplasias/etiologia , Receptores de Leucotrienos/fisiologia , Receptores de Lisofosfolipídeos/fisiologia , Receptores de Tromboxanos/fisiologia , Transdução de SinaisAssuntos
Lisofosfolipídeos/fisiologia , Neoplasias/metabolismo , Esfingosina/análogos & derivados , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Transformação Celular Neoplásica/metabolismo , Ceramidas/fisiologia , Humanos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptores de Lisofosfolipídeos/metabolismo , Transdução de Sinais , Esfingosina/fisiologiaRESUMO
BACKGROUND: Overexpression of G-protein coupled receptor 34 (GPR34) affects the progression and prognosis of human gastric adenocarcinoma, however, the role of GPR34 in gastric cancer development and progression has not been well-determined. The current study aimed to investigate the effect of GPR34 knockdown on the proliferation, migration, and apoptosis of HGC-27 gastric cancer cells and the underlying mechanisms. METHODS: The expression of GPR34 in gastric cancer cell line HGC-27 was detected by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. HGC-27 cells were employed to construct the stable GPR34 knockdown cell model in this study. Real-time RT-PCR and Western blotting were applied to validate the effect of short hairpin RNA (ShRNA) on the expression of GPR34 in HGC-27 gastric cells. The proliferation, migration of these cells were examined by Cell Counting Kit-8 and transwell. We also measured expression profile of PI3K/PDK1/AKT and ERK using Western blotting. RESULTS: The ShRNA directed against GPR34 effectively inhibited both endogenous mRNA and protein expression levels of GPR34, and significantly down-regulated the expression of PIK3CB (P < 0.01), PIK3CD (P < 0.01), PDK1 (P < 0.01), phosphorylation of PDK1 (P < 0.01), Akt (P < 0.01), and ERK (P < 0.01). Furthermore, GPR34 knockdown resulted in an obvious reduction in HGC-27 cancer cell proliferation and migration activity (P < 0.01). CONCLUSIONS: GPR34 knockdown impairs the proliferation and migration of HGC-27 gastric cancer cells in vitro and provides a potential implication for therapy of gastric cancer.
Assuntos
Receptores de Lisofosfolipídeos/metabolismo , Neoplasias Gástricas/metabolismo , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/fisiologia , Humanos , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Lisofosfolipídeos/genética , Neoplasias Gástricas/genéticaRESUMO
Tyrosine 177 and the Src homology 2 (SH2) domain play important roles in linking p185Bcr-Abl to downstream pathways critical for cell growth and survival. However, a mutant p185(Y177FR552L) (p185(YR)), in which tyrosine 177 and arginine 552 in the SH2 domain are mutated, is still capable of transforming hematopoietic cells in vitro. Transplant of these cells into syngeneic mice also leads to leukemogenesis, albeit with a phenotype distinct from that produced by wild-type p185Bcr-Abl (p185(wt))-transformed cells. Here we show that G-protein coupled receptor 34 (Gpr34) expression is markedly up-regulated in p185(YR)-transformed cells compared to those transformed by p185(wt). Knockdown of Gpr34 in p185(YR) cells is sufficient to suppress growth factor-independent proliferation and survival in vitro and attenuate leukemogenesis in vivo. The Erk and phosphatidylinositol 3-kinase/Akt pathways are activated in p185(YR) cells and the activation is dependent on Gpr34 expression. These studies identify Gpr34 as an alternative pathway that may mediate p185Bcr-Abl-induced transformation and leukemogenesis.
Assuntos
Transformação Celular Neoplásica/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Fusão bcr-abl/metabolismo , Leucemia/patologia , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Lisofosfolipídeos/metabolismo , Adulto , Animais , Apoptose , Western Blotting , Ciclo Celular , Proliferação de Células , Transformação Celular Neoplásica/genética , Resistencia a Medicamentos Antineoplásicos , MAP Quinases Reguladas por Sinal Extracelular/genética , Feminino , Proteínas de Fusão bcr-abl/genética , Humanos , Técnicas Imunoenzimáticas , Leucemia/genética , Leucemia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Mutação/genética , Fosfatidilinositol 3-Quinase/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Lisofosfolipídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Células Tumorais CultivadasRESUMO
GPR34 is a Gi/o protein-coupled receptor (GPCR) of the nucleotide receptor P2Y12 -like group. This receptor is highly expressed in microglia, however, the functional relevance of GPR34 in these glial cells is unknown. Previous results suggested an impaired immune response in GPR34-deficient mice infected with Cryptococcus neoformans. Here we show that GPR34 deficiency results in morphological changes in retinal and cortical microglia. RNA sequencing analysis of microglia revealed a number of differentially expressed transcripts involved in cell motility and phagocytosis. We found no differences in microglial motility after entorhinal cortex lesion and in response to laser lesion. However, GPR34-deficient microglia showed reduced phagocytosis activity in both retina and acutely isolated cortical slices. Our study identifies GPR34 as an important signaling component controlling microglial function, morphology and phagocytosis.
Assuntos
Regulação da Expressão Gênica/genética , Microglia/fisiologia , Fagocitose/genética , Receptores de Lisofosfolipídeos/deficiência , Animais , Encéfalo/citologia , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Receptor 1 de Quimiocina CX3C , Movimento Celular/genética , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Receptores de Lisofosfolipídeos/genética , Retina/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Lysophosphatidylserine (lysoPS) is a type of lysophospholipid mediator, which is involved in allergic conditions and tumor progression. We investigated the physiological function of lysoPS on colorectal cancer (CRC) cell lines, as well as the involved receptor and signaling pathways. MATERIALS AND METHODS: Expression of lysoPS receptors on six cell lines was examined by reverse transcription-polymerase chain reaction (RT-PCR). The physiological functions of lysoPS were investigated, and experiments using small interfering RNA (siRNA) or inhibitors of the signaling pathways were conducted. RESULTS: Among the three lysoPS receptors, GPR34 was highly expressed on all cell lines. LysoPS stimulated the chemotactic migratory ability. Wortmannin inhibited the migratory ability, as well as the GPR34 knock-down, strongly suggestive of the involvement of this receptor in the PI3K/Akt pathway. CONCLUSION: The involved receptor and pathways in the migratory ability in response to lysoPS was demonstrated, which opens premises for targeting as a new strategy for prevention and treatment of colorectal cancer.
Assuntos
Movimento Celular/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Lisofosfolipídeos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Lisofosfolipídeos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Proteínas da Matriz Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Receptores de Lisofosfolipídeos/genéticaRESUMO
G-protein-coupled receptors (GPCRs) that recognize the lysophospholipids (LPLs) are grouped into two phylogenetically distinct families: the endothelial differentiation gene (Edg) and non-Edg GPCRs. Owing to their more recent identification, and hindered by a lack of selective pharmacological tools, our understanding of the functions and signaling pathways of the non-Edg GPCRs is still in its infancy. Targeting the non-conserved allosteric binding sites of the LPL GPCRs shows particular promise for the development of selective modulators by structure-based drug design. However, only one Edg GPCR (S1PR1) structure has been determined to date, and it has low sequence identity with the non-Edg GPCRs (<20%). Thus, a representative structure of a non-Edg GPCR remains a pressing objective for selective structure-based drug design. Obtaining selective modulators targeting the non-Edg receptors would help to unravel the biology behind these novel GPCRs and potentially will support therapeutic treatment of diseases such as cancer, inflammation, and neuropsychiatric disorders.
Assuntos
Receptores de Lisofosfolipídeos/agonistas , Receptores de Lisofosfolipídeos/antagonistas & inibidores , Sítios de Ligação , Sistemas de Liberação de Medicamentos , Humanos , Modelos Moleculares , Receptores de Lisofosfolipídeos/química , Receptores de Lisofosfolipídeos/metabolismoRESUMO
PURPOSE: G-protein coupled receptor 34 (GPR34), which belongs to the G-protein coupled receptors superfamily, is reportedly expressed highly in the spread of several solid tumors. However, its expression in gastric primary tumor and potential role in gastric cancer development and progression have not been determined. METHODS: Immunohistochemistry, real-time RT-PCR and western blot methods were used to determine GPR34 expression in human gastric cancer tissues/cell lines and matched adjacent tissues/ normal mucosal cell line. A statistical analysis was performed to establish the potential correlation between GPR34 expression and the patients' clinicopathological characteristics, tumor progression, and prognosis. Stably transfected NCI-N87 cell lines with either GPR34 over-expression or knock-down were constructed to determine the effect of GPR34 on gastric cancer cell invasion and migration, and to explain the preliminary molecular mechanism of GPR34 in gastric cancer metastasis. RESULTS: GPR34 is up-regulated in primary gastric cancer tissues/cell lines compared with matched adjacent tissues/normal mucosal cell line, and when the relationship between GPR34 expression and the the clinicopathological characteristics was analyzed, it was shown that GPR34 expression is significantly correlated with tumor differentiation, infiltration depth, and lymph node status and had a significant influence on prognosis. Furthermore, GPR34-overexpression increased while GPR34-knockdown inhibited NCI-N87 cell invasion in vitro by PI3K/PDK1/AKT pathway. CONCLUSIONS: Taken together, up-regulation of GPR34 expression in human gastric carcinoma may play a critical role in tumor progression and in determining patient prognosis. GPR34 may be a useful diagnostic or prognostic molecular biomarker, and a potential target for therapeutic intervention.
Assuntos
Adenocarcinoma/metabolismo , Receptores de Lisofosfolipídeos/biossíntese , Transdução de Sinais/fisiologia , Neoplasias Gástricas/metabolismo , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Western Blotting , Progressão da Doença , Elafina/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/patologia , Regulação para Cima , Adulto JovemRESUMO
This review presents a summary of what is known about the G-protein coupled receptors GPR35 and GPR55 and their potential characterization as lysophospholipid or cannabinoid receptors, respectively. Both GPR35 and GPR55 have been implicated as important targets in pain and cancer, and additional diseases as well. While kynurenic acid was suggested to be an endogenous ligand for GPR35, so was 2-arachidonoyl lysophosphatidic acid (LPA). Similarly, GPR55 has been suggested to be a cannabinoid receptor, but is quite clearly also a receptor for lysophosphatidylinositol. Interestingly, 2-arachidonyl glycerol (2-AG), an endogenous ligand for cannabinoid receptors, can be metabolized to 2-arachidonoyl LPA through the action of a monoacylglycerol kinase; the reverse reaction has also been demonstrated. Thus, it appears that mutual interconversion is possible between 2-arachidonoyl LPA and 2-AG within a cell, though the direction of the reaction may be site-dependent. The GPR55 natural ligand, 2-arachidonoyl LPI, can be degraded either to 2-AG by phospholipase C or to 2-arachidonoyl LPA by phospholipase D. Thus, GPR35, GPR55 and CB receptors are linked together through their natural ligand conversions. Additional agonists and antagonists have been identified for both GPR35 and GPR55, which will facilitate the future study of these receptors with respect to their physiological function. Potential therapeutic targets include pain, cancer, metabolic diseases and drug addiction.
Assuntos
Receptores de Canabinoides/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Receptores de Lisofosfolipídeos/fisiologia , Animais , Canabinoides/farmacologia , Canabinoides/uso terapêutico , Expressão Gênica , Humanos , Ligantes , Receptores de Canabinoides/efeitos dos fármacos , Receptores de Canabinoides/genética , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Receptores Acoplados a Proteínas G/genética , Receptores de Lisofosfolipídeos/efeitos dos fármacos , Receptores de Lisofosfolipídeos/genéticaRESUMO
Genetic aberrations, including trisomies 3 and 18, and well-defined IGH translocations, have been described in marginal zone lymphomas (MZLs); however, these known genetic events are present in only a subset of cases. Here, we report the cloning of an IGH translocation partner on chromosome X, t(X;14)(p11.4;q32) that deregulates expression of an poorly characterized orphan G-protein-coupled receptor, GPR34. Elevated GPR34 gene expression was detected independent of the translocation in multiple subtypes of non-Hodgkin lymphoma and distinguished a unique molecular subtype of MZL. Increased expression of GPR34 was also detected in tissue from brain tumors and surface expression of GPR34 was detected on human MZL tumor cells and normal immune cells. Overexpression of GPR34 in lymphoma and HeLa cells resulted in phosphorylation of ERK, PKC, and CREB; induced CRE, AP1, and NF-κB-mediated gene transcription; and increased cell proliferation. In summary, these results are the first to identify a role for a GPR34 in lymphoma cell growth, provide insight into GPR34-mediated signaling, identify a genetically unique subset of MZLs that express high levels of GPR34, and suggest that MEK inhibitors may be useful for treatment of GPR34-expressing tumors.