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
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 , TransfecçãoAssuntos
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
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
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.
Assuntos
Cromossomos Humanos Par 14 , Cromossomos Humanos X , Linfoma de Zona Marginal Tipo Células B/genética , Receptores de Lisofosfolipídeos/genética , Translocação Genética , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Pontos de Quebra do Cromossomo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Ordem dos Genes , Células HeLa , Humanos , Linfoma de Zona Marginal Tipo Células B/metabolismo , Linfoma não Hodgkin/genética , Linfoma não Hodgkin/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Dados de Sequência Molecular , Receptores de Lisofosfolipídeos/metabolismo , Fatores de Transcrição SOXC/genética , Transdução de SinaisRESUMO
Gastric cancer (GC) is one of the most common causes of cancer-related deaths worldwide. We investigated the differential expression and putative tumor biological significance of five G-protein-coupled receptors (GPCRs) in GC, i.e., LGR4, LGR6, GPR34, GPR160, and GPR171. Based on our previous microarray analyses, we identified five candidate genes in human GC samples. Real-time RT-PCR was carried out to validate their expression in malignant and non-malignant tissues on an independent collective comprising 32 GC patients with and without lymph node metastases. Selected protein targets LGR4 and LGR6 were further validated on paraffin-embedded sections of ten intestinal and ten poorly cohesive (diffuse)-type GCs and their corresponding non-malignant tissue using immunohistochemistry. Additionally, the putative tumor biological significance of LGR4 and LGR6 was studied using tissue microarrays obtained from a cohort of 481 GC patients. On transcriptional level, GPR34, GPR160, and GPR171 were not differentially expressed in GC compared with non-neoplastic mucosa. LGR4 and LGR6 were up-regulated on transcriptional (real-time RT-PCR) and translational (immunohistochemistry) levels in GC. Furthermore, in tissue microarray analysis, LGR6 expression was significantly associated with local tumor growth (T-category; p = 0.04) and correlated with patient survival. LGR4 expression was significantly correlated with nodal spread (N-category; p = 0.025). Our systematic analysis indicates that LGR4 and LGR6 may play a role in GC biology. Future studies will have to demonstrate whether these are also putative diagnostic, prognostic, and/or therapeutic targets for GC.
Assuntos
Adenocarcinoma/metabolismo , Biomarcadores Tumorais/metabolismo , Mucosa Gástrica/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias Gástricas/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Idoso , Idoso de 80 Anos ou mais , Biópsia , Estudos de Casos e Controles , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática/patologia , Masculino , Pessoa de Meia-Idade , Receptores de Lisofosfolipídeos/metabolismo , Estômago/patologia , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Taxa de Sobrevida , Regulação para CimaRESUMO
The discovery of lysophospholipid (LP) 7-transmembrane, G protein-coupled receptors (GPCRs) that began in the 1990s, together with research into the functional roles of the major LPs known as lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), have opened new research avenues into their biological processes and mechanisms. Major examples of LP signalling effects include embryogenesis, nervous system development, vascular development, uterine implantation, immune cell trafficking, and inflammatory reactions. LP signalling also influences the pathophysiology of many diseases including cancer, autoimmune and inflammatory diseases, which indicate that LP receptors may be attractive targets for pharmacological therapies. A key example of such a therapeutic agent is the S1P receptor modulator FTY720, which upon phosphorylation and continued drug exposure, acts as an S1P receptor functional antagonist. This compound (also known as fingolimod or Gilenya) has recently been approved by the FDA for the treatment of relapsing forms of multiple sclerosis. Continued basic and translational research on LP signalling should provide novel insights into both basic biological mechanisms, as well as novel therapeutic approaches to combat a range of human diseases.
Assuntos
Receptores de Lisofosfolipídeos/metabolismo , Animais , Expressão Gênica , Humanos , Lisofosfolipídeos/metabolismo , Receptores de Lisofosfolipídeos/genéticaRESUMO
Genetic events underlying pathogenesis of nodal and extranodal marginal zone lymphoma are not completely understood. We report here a novel t(X;14)(p11.4;q32.33) identified in 4 lymphoma cases: 2 with a mucosa-associated lymphoid tissue lymphoma, one with a nodal marginal zone lymphoma and one with gastric diffuse large B-cell lymphoma. In all cases, lymphoma evolved from a previous auto-immune disorder. Fluorescence in situ hybridization and molecular studies showed that t(X;14), which is mediated by immunoglobulin heavy chain locus, targets the GPR34 gene at Xp11.4. Upregulation of GPR34 mRNA and aberrant expression of GPR34 protein has been demonstrated in 3 presented cases by quantitative real-time polymerase chain reaction and immunohistochemistry, respectively. GPR34 belongs to the largest family of cell surface molecules involved in signal transmission that play important roles in many physiological and pathological processes, including tumorigenesis. Although functional consequences of t(X;14) have not been identified, our studies suggest that up-regulated GPR34 activate neither nuclear factor-κB nor ELK-related tyrosine kinase.
Assuntos
Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 14/genética , Linfoma de Zona Marginal Tipo Células B/genética , Receptores de Lisofosfolipídeos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Linfoma de Zona Marginal Tipo Células B/metabolismo , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Masculino , Receptores de Lisofosfolipídeos/genética , Regulação para CimaRESUMO
The X-chromosomal GPR34 gene encodes an orphan G(i) protein-coupled receptor that is highly conserved among vertebrates. To evaluate the physiological relevance of GPR34, we generated a GPR34-deficient mouse line. GPR34-deficient mice were vital, reproduced normally, and showed no gross abnormalities in anatomical, histological, laboratory chemistry, or behavioral investigations under standard housing. Because GPR34 is highly expressed in mononuclear cells of the immune system, mice were specifically tested for altered functions of these cell types. Following immunization with methylated BSA, the number of granulocytes and macrophages in spleens was significantly lower in GPR34-deficient mice as in wild-type mice. GPR34-deficient mice showed significantly increased paw swelling in the delayed type hypersensitivity test and higher pathogen burden in extrapulmonary tissues after pulmonary infection with Cryptococcus neoformans compared with wild-type mice. The findings in delayed type hypersensitivity and infection tests were accompanied by significantly different basal and stimulated TNF-α, GM-CSF, and IFN-γ levels in GPR34-deficient animals. Our data point toward a functional role of GPR34 in the cellular response to immunological challenges.
Assuntos
Criptococose/imunologia , Cryptococcus neoformans/imunologia , Granulócitos/imunologia , Hipersensibilidade Tardia/imunologia , Macrófagos/metabolismo , Pneumonia/imunologia , Receptores de Lisofosfolipídeos/imunologia , Animais , Bovinos , Criptococose/metabolismo , Citocinas/biossíntese , Citocinas/genética , Citocinas/imunologia , Granulócitos/metabolismo , Hipersensibilidade Tardia/genética , Hipersensibilidade Tardia/metabolismo , Imunização , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Pneumonia/metabolismo , Receptores de Lisofosfolipídeos/genética , Receptores de Lisofosfolipídeos/metabolismo , Soroalbumina Bovina/imunologia , Soroalbumina Bovina/farmacologia , Cromossomo X/genética , Cromossomo X/imunologia , Cromossomo X/metabolismoRESUMO
Lysophospholipid (LP) research has experienced a period of renaissance with the discovery of the lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) receptors in the late 1990s. Vertebrate LP receptors regulate embryogenesis, vascular development, neurogenesis, uterine development, oocyte survival, immune cell trafficking and inflammatory reactions. LP signaling is important in cancer, autoimmunity and inflammatory diseases. Research on LP biology has contributed to the development of a first-generation S1P receptor modulator that has entered phase III clinical trials for the treatment of multiple sclerosis. Further basic research on LP signaling is anticipated to lead to novel therapeutic tools to combat various human diseases.
Assuntos
Receptores de Lisofosfolipídeos/metabolismo , Vertebrados/metabolismo , Animais , Sistema Cardiovascular/metabolismo , Doença , Humanos , Sistema Nervoso/metabolismo , Receptores de Lisofosfolipídeos/imunologia , Reprodução , Vertebrados/imunologiaRESUMO
Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are potent biologically active lipid mediators that exert a wide range of cellular effects through specific G protein-coupled receptors. To date, four LPA receptors and five S1P receptors have been identified. These receptors are expressed in a large number of tissues and cell types, allowing for a wide variety of cellular responses to lysophospholipid signaling, including cell adhesion, cell motility, cytoskeletal changes, proliferation, angiogenesis, process retraction, and cell survival. In addition, recent studies in mice show that specific lysophospholipid receptors are required for proper cardiovascular, immune, respiratory, and reproductive system development and function. Lysophospholipid receptors may also have specific roles in cancer and other diseases. This review will cover identification and expression of the lysophospholipid receptors, as well as receptor signaling properties and function. Additionally, phenotypes of mice deficient for specific lysophospholipid receptors will be discussed to demonstrate how these animals have furthered our understanding of the role lysophospholipids play in normal biology and disease.
Assuntos
Lisofosfolipídeos , Receptores de Lisofosfolipídeos , Esfingosina/análogos & derivados , Animais , Membrana Celular/metabolismo , Humanos , Lisofosfolipídeos/biossíntese , Lisofosfolipídeos/metabolismo , Lisofosfolipídeos/fisiologia , Receptores de Lisofosfolipídeos/biossíntese , Receptores de Lisofosfolipídeos/metabolismo , Receptores de Lisofosfolipídeos/fisiologia , Transdução de Sinais , Esfingosina/biossíntese , Esfingosina/metabolismo , Esfingosina/fisiologiaRESUMO
Abundant evidence documents the highly proinflammatory actions of lysophosphatidylcholine (LPC). Further, LPC, found in high amounts in oxidized low-density lipoprotein (LDL), is implicated as an atherogenic factor. In endothelial cells, LPC impairs endothelial barrier function through GPR4, a novel receptor hypothesized to be sensitive to LPC and protons. The authors investigated the stimulation by LPC or low pH of GPR4 in human brain microvascular endothelial cells (HBMECs) and whether the activated GPR4 regulates in vitro monocyte transmigration. The results indicated that HBMECs stimulated by LPC (5 microM), but not low pH, showed a twofold increase in monocyte transmigration. Using retroviruses containing siRNA to GPR4, a > 60% reduction of GPR4 expression resulted in blockade of the LPC-stimulated transmigration. The inhibited response was restored by co-expression with an small interference RNA (siRNA)-resistant, but functional, GPR4 mutant construct. To investigate potential signaling mechanisms, the siRNA-mediated knockdown of GPR4 also prevented LPC-induced RhoA activation. C3 transferase, a Rho inhibitor, prevented approximately approximately 65% of the LPC-stimulated transmigration. LPC also increased MLC phosphorylation by 5 min, which was inhibited by the Rho kinase inhibitor, Y-27632 (10 microM) or ML-7 (myosin light chain kinase (MLCK) inhibitor). The findings indicate that the proinflammatory and atherogenic LPC stimulated endothelial GPR4, which promoted monocyte transmigration through a RhoA-dependent pathway.
Assuntos
Encéfalo/irrigação sanguínea , Células Endoteliais/metabolismo , Monócitos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Lisofosfolipídeos/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/citologia , Técnica Indireta de Fluorescência para Anticorpo , Regulação da Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Medições Luminescentes , Lisofosfatidilcolinas/farmacologia , Mutação , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores de Lisofosfolipídeos/genética , Retroviridae/genéticaRESUMO
The lysophospholipids, sphingosine-1-phosphate (S1P), lysophosphatidic acid (LPA), sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC), activate diverse groups of G-protein-coupled receptors that are widely expressed and regulate decisive cellular functions. Receptors of the endothelial differentiation gene family are activated by S1P (S1P(1-5)) or LPA (LPA(1-3)); two more distantly related receptors are activated by LPA (LPA(4/5)); the GPR(3/6/12) receptors have a high constitutive activity but are further activated by S1P and/or SPC; and receptors of the OGR1 cluster (OGR1, GPR4, G2A, TDAG8) appear to be activated by SPC, LPC, psychosine and/or protons. G-protein-coupled lysophospholipid receptors regulate cellular Ca(2+) homoeostasis and the cytoskeleton, proliferation and survival, migration and adhesion. They have been implicated in development, regulation of the cardiovascular, immune and nervous systems, inflammation, arteriosclerosis and cancer. The availability of S1P and LPA at their G-protein-coupled receptors is regulated by enzymes that generate or metabolize these lysophospholipids, and localization plays an important role in this process. Besides FTY720, which is phosphorylated by sphingosine kinase-2 and then acts on four of the five S1P receptors of the endothelial differentiation gene family, other compounds have been identified that interact with more ore less selectivity with lysophospholipid receptors.