RESUMO
G Protein Signaling Modulator-3 (GPSM3) is a leukocyte-specific regulator of G protein-coupled receptors (GPCRs), which binds inactivated Gαi·GDP subunits and precludes their reassociation with Gßγ subunits. GPSM3 deficiency protects mice from inflammatory arthritis and, in humans, GPSM3 single-nucleotide polymorphisms (SNPs) are inversely associated with the risk of rheumatoid arthritis development; recently, these polymorphisms were linked to one particular SNP (rs204989) that decreases GPSM3 transcript abundance. However, the precise role of GPSM3 in leukocyte biology is unknown. Here, we show that GPSM3 is induced in the human promyelocytic leukemia NB4 cell line following retinoic acid treatment, which differentiates this cell line into a model of neutrophil physiology (NB4*). Reducing GPSM3 expression in NB4* cells, akin to the effect ascribed to the rs204989 C>T transition, disrupts cellular migration toward leukotriene B4 (LTB4) and (to a lesser extent) interleukin-8 (a.k.a. IL-8 or CXCL8), but not migration toward formylated peptides (fMLP). As the chemoattractants LTB4 and CXCL8 are involved in recruitment of neutrophils to the arthritic joint, our results suggest that the arthritis-protective GPSM3 SNP rs204989 may act to decrease neutrophil chemoattractant responsiveness.
Assuntos
Artrite Reumatoide/genética , Quimiotaxia de Leucócito , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Neutrófilos/metabolismo , Artrite Reumatoide/imunologia , Linhagem Celular Tumoral , Quimiotaxia de Leucócito/genética , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Humanos , Interleucina-8/metabolismo , Leucopoese , Leucotrieno B4/metabolismo , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Polimorfismo de Nucleotídeo Único , Tretinoína/metabolismoRESUMO
G protein signaling modulator 3 (GPSM3) is a regulator of G protein-coupled receptor signaling, with expression restricted to leukocytes and lymphoid organs. Previous genome-wide association studies have highlighted single-nucleotide polymorphisms (SNPs; rs204989 and rs204991) in a region upstream of the GPSM3 transcription start site as being inversely correlated to the prevalence of rheumatoid arthritis (RA)-this association is supported by the protection afforded to Gpsm3-deficient mice in models of inflammatory arthritis. Here, we assessed the functional consequences of these polymorphisms. We collected biospecimens from 50 volunteers with RA diagnoses, 50 RA-free volunteers matched to the aforementioned group and 100 unmatched healthy young volunteers. We genotyped these individuals for GPSM3 (rs204989, rs204991), CCL21 (rs2812378) and HLA gene region (rs6457620) polymorphisms, and found no significant differences in minor allele frequencies between the RA and disease-free cohorts. However, we identified that individuals homozygous for SNPs rs204989 and rs204991 had decreased GPSM3 transcript abundance relative to individuals homozygous for the major allele. In vitro promoter activity studies suggest that SNP rs204989 is the primary cause of this decrease in transcript levels. Knockdown of GPSM3 in THP-1 cells, a human monocytic cell line, was found to disrupt ex vivo migration to the chemokine MCP-1.
Assuntos
Artrite Reumatoide/genética , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genética , Adulto , Idoso , Alelos , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Estudos de Casos e Controles , Linhagem Celular , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiotaxia , Feminino , Expressão Gênica , Frequência do Gene , Genótipo , Inibidores de Dissociação do Nucleotídeo Guanina/antagonistas & inibidores , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Monócitos/patologia , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
Probably the foremost hypothesis of depression is the 5-hydroxytryptamine (5-HT, serotonin) deficiency hypothesis. Accordingly, anomalies in putative 5-HT biomarkers have repeatedly been reported in depression patients. However, whether such anomalies in fact reflect deficient central 5-HT neurotransmission remains unresolved. We employed a naturalistic model of 5-HT deficiency, the tryptophan hydroxylase 2 (Tph2) R439H knockin mouse, to address this question. We report that Tph2 knockin mice have reduced basal and stimulated levels of extracellular 5-HT (5-HT(Ext)). Interestingly, cerebrospinal fluid (CSF) 5-hydroxyindoleacetic acid (5-HIAA) and fenfluramine-induced plasma prolactin levels are markedly diminished in the Tph2 knockin mice. These data seemingly confirm that low CSF 5-HIAA and fenfluramine-induced plasma prolactin reflects chronic, endogenous central nervous system (CNS) 5-HT deficiency. Moreover, 5-HT(1A) receptor agonist-induced hypothermia is blunted and frontal cortex 5-HT(2A) receptors are increased in the Tph2 knockin mice. These data likewise parallel core findings in depression, but are usually attributed to anomalies in the respective receptors rather than resulting from CNS 5-HT deficiency. Further, 5-HT(2A) receptor function is enhanced in the Tph2 knockin mice. In contrast, 5-HT(1A) receptor levels and G-protein coupling is normal in Tph2 knockin mice, indicating that the blunted hypothermic response relates directly to the low 5-HT(Ext). Thus, we show that not only low CSF 5-HIAA and a blunted fenfluramine-induced prolactin response, but also blunted 5-HT(1A) agonist-induced hypothermia and increased 5-HT(2A) receptor levels are bona fide biomarkers of chronic, endogenous 5-HT deficiency. Potentially, some of these biomarkers could identify patients likely to have 5-HT deficiency. This could have clinical research utility or even guide pharmacotherapy.
Assuntos
Depressão/sangue , Ácido Hidroxi-Indolacético/líquido cefalorraquidiano , Receptor 5-HT2A de Serotonina/metabolismo , Neurônios Serotoninérgicos/fisiologia , Serotonina/deficiência , Transmissão Sináptica/fisiologia , Triptofano Hidroxilase/fisiologia , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Biomarcadores/metabolismo , Corticosterona/sangue , Depressão/líquido cefalorraquidiano , Depressão/genética , Modelos Animais de Doenças , Líquido Extracelular/metabolismo , Feminino , Fenfluramina/farmacologia , Lobo Frontal/metabolismo , Técnicas de Introdução de Genes/métodos , Técnicas de Introdução de Genes/psicologia , Hipocampo/metabolismo , Hipotermia/induzido quimicamente , Hipotermia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Prolactina/sangue , Receptor 5-HT1A de Serotonina/genética , Receptor 5-HT1A de Serotonina/metabolismo , Receptor 5-HT2A de Serotonina/genética , Neurônios Serotoninérgicos/efeitos dos fármacos , Neurônios Serotoninérgicos/enzimologia , Serotonina/metabolismo , Agonistas do Receptor 5-HT1 de Serotonina/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética , Triptofano Hidroxilase/genéticaRESUMO
Caveolin-1 (Cav-1) is a scaffolding protein important for regulating receptor signaling cascades by partitioning signaling molecules into membrane microdomains. Disruption of the CAV1 gene has recently been identified as a rare structural variant associated with schizophrenia. Although Cav-1 knockout (KO) mice displayed no baseline behavioral disruptions, Cav-1 KO mice, similar to schizophrenic individuals, exhibited increased sensitivity to the psychotomimetic N-methyl-D-aspartate receptor antagonist phencyclidine (PCP). Thus, PCP disruption of prepulse inhibition (PPI) and PCP-induced mouse locomotor activity were both enhanced by genetic deletion of Cav-1. Interestingly, genetic deletion of Cav-1 rendered the atypical antipsychotics clozapine and olanzapine and the 5-HT(2A)-selective antagonist M100907 ineffective at normalizing PCP-induced disruption of PPI. We also discovered that genetic deletion of Cav-1 attenuated 5-HT(2A)-induced c-Fos and egr-1 expression in mouse frontal cortex and also reduced 5-HT(2A)-mediated Ca(2+) mobilization in primary cortical neuronal cultures. The behavioral effects of the 5-HT(2A) agonist (2,5-dimethoxy-4-iodoamphetamine) including head twitch responses and disruption of PPI were also attenuated by genetic deletion of Cav-1, indicating that Cav-1 is required for both inverse agonist (that is, atypical antipsychotic drug) and agonist actions at 5-HT(2A) receptors. This study demonstrates that disruption of the CAV1 gene--a rare structural variant associated with schizophrenia--is not only pro-psychotic but also attenuates atypical antipsychotic drug actions.
Assuntos
Antipsicóticos/farmacologia , Comportamento Animal/efeitos dos fármacos , Caveolina 1/genética , Esquizofrenia/etiologia , Esquizofrenia/genética , Animais , Antipsicóticos/administração & dosagem , Comportamento Animal/fisiologia , Caveolina 1/deficiência , Caveolina 1/fisiologia , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Receptor 5-HT2A de Serotonina/genética , Esquizofrenia/fisiopatologiaRESUMO
The 5-HT(2A)-serotonin receptor is a major molecular target for most atypical antipsychotic drugs as well as most hallucinogens, which can exacerbate psychotic symptoms. In this study, we examined whether random sequence variations in the gene (single nucleotide polymorphisms, SNPs) encoding the 5-HT(2A)-serotonin receptor could explain inter-individual variability in atypical antipsychotic and agonist drug response. We examined the in vitro pharmacology of four non-synonymous SNPs, which give rise to T25N, I197V, A447V, and H452Y variant 5-HT(2A)-serotonin receptors. Our data indicate that these non-synonymous SNPs exert statistically significant, although modest, effects on the affinity and functional effects of several currently approved atypical antipsychotics (aripiprazole, clozapine, olanzapine, quetiapine, risperidone, and ziprasidone). Also, the 5-HT(2A) receptor SNPs slightly altered the potency and relative efficacy of a small number of selected agonists (2,5-dimethoxy-4-iodoamphetamine, tryptamine, 5-hydroxytryptamine, m-chlorophenylpiperazine, and 5-methoxy-N, N-dimethyltryptamine). In all, our results show that the in vitro pharmacological effects of the SNPs are drug specific.
Assuntos
Antipsicóticos/farmacologia , Polimorfismo de Nucleotídeo Único , Receptor 5-HT2A de Serotonina/genética , Agonistas do Receptor de Serotonina/farmacologia , Ligação Competitiva , Linhagem Celular , Clonagem Molecular , Relação Dose-Resposta a Droga , Humanos , Ligantes , Ensaio Radioligante , Receptor 5-HT2A de Serotonina/biossíntese , Agonistas do Receptor 5-HT2 de Serotonina , TransfecçãoRESUMO
An examination of several amine-substituted analogs of N(1)-benzenesulfonylindoles reveals that although they bind at human 5-HT(6) serotonin receptors with high affinity, they are likely to bind in a dissimilar manner.
Assuntos
Indóis/química , Receptores de Serotonina/química , Aminas/química , Humanos , Ligação ProteicaRESUMO
We investigated the regulation of beta(2)-adrenergic receptors (beta(2)AR) by protein kinase C (PKC) in rat C6 glioma cells at the levels of receptor activity, protein expression and gene expression. Cells exposed to 4beta-phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, exhibited a time- and concentration-dependent decrease in beta(2)AR binding activity. Maximum down-regulation was approximately 50% by 24 h and western blot analysis revealed a parallel decrease in beta(2)AR protein. In addition, PMA treatment resulted in an acute desensitization of beta(2)AR-stimulated cyclic AMP response prior to any reduction in receptor levels. PMA exposure also affected steady-state beta(2)AR mRNA levels in a time-dependent, biphasic manner. During the first 4 h, levels decreased by approximately 60% and then slowly recovered to approximately 75% of control by 24 h. As the reduction in receptor mRNA was not due to a decrease in its stability, we examined beta(2)AR gene transcription by nuclear run-on assays. Transcriptional activity in nuclei from C6 cells treated with PMA for 2 h was reduced by 70% compared to controls. Thus PKC can regulate beta(2)AR at least two levels: the first being an acute desensitization of receptor function, and the second being a more prolonged repression of receptor gene transcription that in turn results in decreased receptor expression.
Assuntos
Regulação para Baixo , Expressão Gênica , Glioma/metabolismo , Proteína Quinase C/metabolismo , Receptores Adrenérgicos beta 2/genética , Animais , Western Blotting , AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Cinética , RNA Mensageiro/análise , Ratos , Receptores Adrenérgicos beta 2/análise , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica , Células Tumorais CultivadasRESUMO
The subcellular localization of the survival motor neuron (SMN) protein, encoded by the spinal muscular atrophy determining gene, was investigated in motor neurons of the developing and adult rat spinal cord by light and electron microscopy immunocytochemistry. The experiments were carried out with a panel of anti-SMN antibodies, all recognizing an SMN-specific protein band at 39 kDa in HeLa cells and rat spinal cord protein extracts. SMN protein expression decreased during postnatal spinal cord development, but it remained unchanged in distribution and intensity in motor neurons at all ages examined. SMN protein was mainly organized in immunoreactive aggregates sparse in the nucleoplasm and cytoplasm of both mature and developing motor neurons, and it was more rarely localized within the endoplasmic reticulum and in apposition to the external mitochondrial membrane. Most strikingly, the SMN protein was found in association with cytoskeletal elements in spinal dendrites and axons, where it was particularly evident during postnatal development. The present findings suggest that SMN protein may be transported via axoplasmic flow in maturing neurons. Given the RNA-binding activity of SMN, the SMN protein could be involved in the transport of specific mRNAs in axons and dendrites of motor neurons. The reduced transport of specific mRNAs within motor neurons during development could play a role in the motoneuronal degeneration and impaired axonal sprouting observed in spinal muscular atrophy.
Assuntos
Axônios/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismo , Animais , Anticorpos/análise , Axônios/ultraestrutura , Transporte Biológico , Western Blotting , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Células HeLa/imunologia , Células HeLa/metabolismo , Humanos , Imuno-Histoquímica , Microscopia/métodos , Microscopia Eletrônica , Neurônios Motores/metabolismo , Neurônios Motores/ultraestrutura , Proteínas do Tecido Nervoso/imunologia , Proteínas de Ligação a RNA , Ratos , Ratos Sprague-Dawley , Proteínas do Complexo SMN , Medula Espinal/ultraestrutura , Frações Subcelulares , Proteína 1 de Sobrevivência do Neurônio MotorRESUMO
We report the discovery, tissue distribution and pharmacological characterization of a novel receptor, which we have named H4. Like the three histamine receptors reported previously (H1, H2, and H3), the H4 receptor is a G protein-coupled receptor and is most closely related to the H3 receptor, sharing 58% identity in the transmembrane regions. The gene encoding the H4 receptor was discovered initially in a search of the GenBank databases as sequence fragments retrieved in a partially sequenced human genomic contig mapped to chromosome 18. These sequences were used to retrieve a partial cDNA clone and, in combination with genomic fragments, were used to determine the full-length open reading frame of 390 amino acids. Northern analysis revealed a 3.0-kb transcript in rat testis and intestine. Radioligand binding studies indicated that the H4 receptor has a unique pharmacology and binds [(3)H]histamine (K(d) = 44 nM) and [(3)H]pyrilamine (K(d) = 32 nM) and several psychoactive compounds (amitriptyline, chlorpromazine, cyproheptadine, mianserin) with moderate affinity (K(i) range of 33-750 nM). Additionally, histamine induced a rapid internalization of HA-tagged H4 receptors in transfected human embryonic kidney 293 cells.