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The interplay between immune checkpoints KLRB1 and CLEC2D is crucial for tumor progression and immune evasion, yet the interaction dynamics are not fully understood. This study aims to elucidate the interaction across various cancers and identify small molecule inhibitors that can disrupt it. We perform a comprehensive pan-cancer analysis of the KLRB1-CLEC2D pair, including mRNA expression patterns, pathological stages, survival outcomes, and single-cell omics, immune infiltration, copy number variations, and DNA methylation profiles. Our findings reveal a consistently higher CLEC2D/KLRB1 ratio in most cancer types compared to normal tissues, and this ratio also increased with advancing pathological stages. Lower KLRB1 expression correlated with higher mortality in most cancers, opposite to CLEC2D. Expression variations were attributed to differential lymphocyte infiltration, CNV, and DNA methylation. Structure-based virtual screening analysis identified compounds including forsythiaside A and RGD peptides as effective inhibitors of the KLRB1-CLEC2D interaction, validated through microscale thermophoresis. This research advances understanding of the KLRB1-CLEC2D interaction within the tumor microenvironment and introduces novel therapeutic strategies to modulate this interaction.
Assuntos
Lectinas Tipo C , Neoplasias , Microambiente Tumoral , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Lectinas Tipo C/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/antagonistas & inibidores , Microambiente Tumoral/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Variações do Número de Cópias de DNA , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/metabolismo , Receptores Imunológicos/genéticaRESUMO
During chronic infection, virus-specific CD8+ cytotoxic T lymphocytes (CTLs) progressively lose their ability to mount effective antiviral responses. This "exhaustion" is coupled to persistent upregulation of inhibitory receptor programmed death-1 (PD-1) (Pdcd1)-key in suppressing antiviral CTL responses. Here, we investigate allelic Pdcd1 subnuclear localization and transcription during acute and chronic lymphocytic choriomeningitis virus (LCMV) infection in mice. Pdcd1 alleles dissociate from transcriptionally repressive chromatin domains (lamin B) in virus-specific exhausted CTLs but not in naive or effector CTLs. Relative to naive CTLs, nuclear positioning and Pdcd1-lamina dissociation in exhausted CTLs reflect loss of Pdcd1 promoter methylation and greater PD-1 upregulation, although a direct correlation is not observed in effector cells, 8 days post-infection. Genetic deletion of B lymphocyte-induced maturation protein 1 (Blimp-1) enhances Pdcd1-lamina dissociation in effector CTLs, suggesting that Blimp-1 contributes to maintaining Pdcd1 localization to repressive lamina. Our results identify mechanisms governing Pdcd1 subnuclear localization and the broader role of chromatin dynamics in T cell exhaustion.
Assuntos
Coriomeningite Linfocítica , Vírus da Coriomeningite Linfocítica , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1 , Linfócitos T Citotóxicos , Animais , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/genética , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Núcleo Celular/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Doença Crônica , Regiões Promotoras Genéticas/genética , Loci GênicosRESUMO
Protein arginine methylation is a common post-translational modification involved in the regulation of various cellular functions. Coactivator-associated arginine methyltransferase 1 (CARM1) is a protein arginine methyltransferase that asymmetrically dimethylates histone H3 and non-histone proteins to regulate gene transcription. CARM1 has been found to play important roles in cell differentiation and development, cell cycle progression, autophagy, metabolism, pre-mRNA splicing and transportation, and DNA replication. In this review, we describe the molecular characteristics of CARM1 and summarize its roles in the regulation of cell differentiation and development in mammals.
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CD8 cytotoxic T cells are a potent line of defense against invading pathogens. To aid in curtailing aberrant immune responses, the activation status of CD8 T cells is highly regulated. One mechanism in which CD8 T cell responses are dampened is via signaling through the immune-inhibitory receptor Programmed Cell Death Protein-1, encoded by Pdcd1. Pdcd1 expression is regulated through engagement of the TCR, as well as by signaling from extracellular cytokines. Understanding such pathways has influenced the development of numerous clinical treatments. In this study, we showed that signals from the cytokine IL-6 enhanced Pdcd1 expression when paired with TCR stimulation in murine CD8 T cells. Mechanistically, signals from IL-6 were propagated through activation of the transcription factor STAT3, resulting in IL-6-dependent binding of STAT3 to Pdcd1 cis-regulatory elements. Intriguingly, IL-6 stimulation overcame B Lymphocyte Maturation Protein 1-mediated epigenetic repression of Pdcd1, which resulted in a transcriptionally permissive landscape marked by heightened histone acetylation. Furthermore, in vivo-activated CD8 T cells derived from lymphocytic choriomeningitis virus infection required STAT3 for optimal Programmed Cell Death Protein-1 surface expression. Importantly, STAT3 was the only member of the STAT family present at Pdcd1 regulatory elements in lymphocytic choriomeningitis virus Ag-specific CD8 T cells. Collectively, these data define mechanisms by which the IL-6/STAT3 signaling axis can enhance and prolong Pdcd1 expression in murine CD8 T cells.
Assuntos
Linfócitos T CD8-Positivos , Interleucina-6 , Receptor de Morte Celular Programada 1 , Animais , Camundongos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Interleucina-6/metabolismo , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Receptor de Morte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Vírus da Coriomeningite Linfocítica/imunologiaRESUMO
Programmed cell death-1 (PD-1) is a co-inhibitory receptor that plays important roles in regulating T cell immunity and peripheral tolerance. PD-1 signaling prevents T cells from overactivation during acute infections, but it maintains T cell exhaustion during chronic infections. Tumor cells can exploit the PD-1 signaling pathway to evade antitumor immune responses. The PD-1 signaling pathway is also essential for maintaining peripheral tolerance and prevention of autoimmunity. PD-1 expression is strictly and differentially regulated by diverse mechanisms in immune cells. It is activated and repressed by distinct transcription factors in different circumstances. Moreover, epigenetic mechanisms are also involved in regulating PD-1 expression. In this review, we summarize the knowledge of the transcriptional and epigenetic regulation of PD-1 expression during different immune responses.
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Autoimunidade , Epigênese Genética , Regulação da Expressão Gênica , Receptor de Morte Celular Programada 1/metabolismo , Animais , Humanos , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Transdução de SinaisRESUMO
During prolonged exposure to Ags, such as chronic viral infections, sustained TCR signaling can result in T cell exhaustion mediated in part by expression of programmed cell death-1 (PD-1) encoded by the Pdcd1 gene. In this study, dynamic changes in histone H3K4 modifications at the Pdcd1 locus during ex vivo and in vivo activation of CD8 T cells suggested a potential role for the histone H3 lysine 4 demethylase LSD1 in regulating PD-1 expression. CD8 T cells lacking LSD1 expressed higher levels of Pdcd1 mRNA following ex vivo stimulation as well as increased surface levels of PD-1 during acute, but not chronic, infection with lymphocytic choriomeningitis virus (LCMV). Blimp-1, a known repressor of PD-1, recruited LSD1 to the Pdcd1 gene during acute, but not chronic, LCMV infection. Loss of DNA methylation at Pdcd1's promoter-proximal regulatory regions is highly correlated with its expression. However, following acute LCMV infection, in which PD-1 expression levels return to near baseline, LSD1-deficient CD8 T cells failed to remethylate the Pdcd1 locus to the levels of wild-type cells. Finally, in a murine melanoma model, the frequency of PD-1-expressing tumor-infiltrating LSD1-deficient CD8 T cells was greater than in wild type. Thus, LSD1 is recruited to the Pdcd1 locus by Blimp-1, downregulates PD-1 expression by facilitating the removal of activating histone marks, and is important for remethylation of the locus. Together, these data provide insight into the complex regulatory mechanisms governing T cell immunity and regulation of a critical T cell checkpoint gene.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Histona Desmetilases/metabolismo , Coriomeningite Linfocítica/metabolismo , Vírus da Coriomeningite Linfocítica/fisiologia , Melanoma/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Acetilação , Doença Aguda , Animais , Doença Crônica , Regulação da Expressão Gênica , Histona Desmetilases/genética , Histonas/metabolismo , Ativação Linfocitária/genética , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Experimentais , Receptor de Morte Celular Programada 1/genética , Transdução de SinaisRESUMO
The incidence of vascular dementia (VaD) has rapidly increased over the past few decades. Although officially approved medications for VaD remain limited, cerebrolysin (CBL) had preventive and treatment effects on VaD in some clinical trials. However, the underlying mechanisms have not been determined. The aim of this study was to determine whether CBL protects against cognitive deficits in a rat model of VaD induced by chronic cerebral hypoperfusion by increasing the levels of plasticity-related proteins and decreasing the levels of apoptosis-related proteins. In our study, adult male Sprague-Dawley rats were subjected to bilateral common carotid artery occlusion (BCCAO) surgery. The animals were randomly divided into four groups after the operation: Sham, Vehicle, L-CBL (2.5ml/kg), and H-CBL (5ml/kg). CBL was administered after the operation daily for 28 days. The CBL treatment significantly decreased the escape latency and increased the percentage of time the rat spent in the target quadrant of the Morris water maze (MWM) task. Pathological changes in the hippocampus, such as reduced cell count numbers and obvious pyknosis, were observed using haematoxylin-eosin (HE) staining. Furthermore, CBL significantly increased the expression of plasticity-related synaptic proteins, such as postsynaptic density protein 95 (PSD-95), protein kinase C subunit gamma (PKCγ), phosphorylated cAMP response element binding protein (p-CREB), and decreased the expression of apoptosis-related proteins in the hippocampus. In summary, CBL likely protects against cognitive deficits by improving synaptic plasticity and decreasing apoptosis.
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Aminoácidos/administração & dosagem , Apoptose/efeitos dos fármacos , Demência Vascular/tratamento farmacológico , Demência Vascular/psicologia , Hipocampo/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Fármacos Neuroprotetores/administração & dosagem , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Demência Vascular/metabolismo , Demência Vascular/patologia , Proteína 4 Homóloga a Disks-Large/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Masculino , Ratos Sprague-Dawley , Aprendizagem Espacial/efeitos dos fármacos , Memória Espacial/efeitos dos fármacosRESUMO
BACKGROUND: Asia will soon have the majority of demented patients in the world. OBJECTIVE: To assess dementia using a uniform data system to update the current status of dementia in Asia. METHODS: A uniformed data set was administered in Taiwan, China, Hong Kong, Korea, Japan, Philippines, Thailand, Singapore, and Indonesia to gather data with regard to Alzheimer's disease (AD) and its related issues for these countries. RESULTS: In total, 2,370 AD patients and their caregivers were recruited from 2011 to 2014. The demographic characteristics of these patients and the relationships between patients and caregivers were different among individuals in these countries (p < 0.001). Of note, the family history for having dementia was 8.2% for females in contrast to 3.2% for males. CONCLUSION: Our study highlighted the differences in dementia assessment and care in developing versus developed countries. Greater effort with regard to studying dementia, especially in developing countries, is necessary.
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Doença de Alzheimer/epidemiologia , Idoso , Idoso de 80 Anos ou mais , Ásia/epidemiologia , Coleta de Dados , Bases de Dados Factuais , Países Desenvolvidos , Países em Desenvolvimento , Feminino , Humanos , MasculinoRESUMO
Programmed cell death-1 (PD-1) is responsible for T cell exhaustion during chronic viral infections and is expressed on a variety of immune cells following activation. Despite its importance, the mechanisms that regulate PD-1 in cell types other than CD8 T cells are poorly defined. In this study, the molecular mechanisms for inducing PD-1 expression in CD4 T cells, macrophages, and B cells were explored. In CD4 T cells, PD-1 induction following TCR stimulation required NFAT, as the calcineurin/NFAT pathway inhibitor cyclosporin A was able to block PD-1 induction in a manner similar to that seen in CD8 T cells. In contrast, LPS but not PMA and ionomycin stimulation was able to induce PD-1 expression in macrophages in a manner insensitive to cyclosporin A-mediated inhibition. B cells could use both pathways, although the levels of PD-1 expression were highest with PMA and ionomycin. An NF-κB binding site located upstream of the gene in conserved region C was required for NF-κB-dependent PD-1 gene activation in macrophages. Chromatin immunoprecipitation showed NF-κB p65 binding to this region following stimulation of macrophages with LPS. PD-1 induction was associated with histone modifications characteristic of accessible chromatin; however, in contrast to CD8 T cells, conserved region B in macrophages did not lose CpG methylation upon stimulation and PD-1 expression. The linkage of TLR/NF-κB signaling to the induction of PD-1 suggests the possibility of an opportunistic advantage to microbial infections in manipulating immune inhibitory responses.
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Regulação da Expressão Gênica , Macrófagos/metabolismo , NF-kappa B/metabolismo , Receptor de Morte Celular Programada 1/genética , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Sítios de Ligação , Linhagem Celular , Sequência Conservada , Metilação de DNA , Ligantes , Macrófagos/imunologia , Camundongos , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Motivos de Nucleotídeos , Receptor de Morte Celular Programada 1/metabolismo , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico , Linfócitos T/imunologia , Linfócitos T/metabolismo , Receptores Toll-Like/metabolismoRESUMO
Lithium is a renowned pharmacological treatment for mood disorders. Recent studies suggest that lithium chloride (LiCl) performs neuroprotective effects on cerebrovascular diseases. The present study is to investigate the protective effects of LiCl treatment on the hippocampus of mice with repeated cerebral ischemia-reperfusion (IR). Mice were subjected to IR through repeated bilateral common carotid artery occlusion. LiCl (2 mmol/kg) was administered daily postoperative until the mice were sacrificed. Swimming time was prolonged and error count increased in the model group through learning and memory tests. Pathological changes such as reduction in cell count and obvious pyknosis were seen in haematoxylin-eosin staining, and apoptosis was detected by TUNEL staining in hippocampal CA1 regions in the model group. The model animals exhibited more phospho-Akt Ser473 and phospho-GSK3ß Ser9 than the sham group when measured by Western blot. LiCl treatment mitigated the prolonged swimming time and the increased error count compared with NaCl-treated group and improved the pathological changes. Meanwhile, LiCl further up-regulated phospho-Akt Ser473 and phospho-GSK3ß Ser9 expression. The highest level of diversity was at 4 weeks postoperative. Therefore, repeated IR can severely damage the hippocampus and decrease the learning and memory functions in mice. Changes in the Akt and GSK3ß protein activity were involved in the IR process. LiCl treatment exerted a neuroprotective effect on learning and memory by potentiating the Akt/GSK3ß cell-signaling pathway.
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Isquemia Encefálica/tratamento farmacológico , Região CA1 Hipocampal/efeitos dos fármacos , Cloreto de Lítio/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Isquemia Encefálica/psicologia , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/patologia , Modelos Animais de Doenças , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Memória/efeitos dos fármacos , Memória/fisiologia , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Distribuição Aleatória , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Traumatismo por Reperfusão/psicologia , Natação , Fatores de Tempo , Resultado do TratamentoRESUMO
Programmed death-1 (PD-1) is a crucial negative regulator of CD8 T cell development and function, yet the mechanisms that control its expression are not fully understood. Through a nonbiased DNase I hypersensitivity assay, four novel regulatory regions within the Pdcd1 locus were identified. Two of these elements flanked the locus, bound the transcriptional insulator protein CCCTC-binding factor, and interacted with each other, creating a potential regulatory compartmentalization of the locus. In response to T cell activation signaling, NFATc1 bound to two of the novel regions that function as independent regulatory elements. STAT binding sites were identified in these elements as well. In splenic CD8 T cells, TCR-induced PD-1 expression was augmented by IL-6 and IL-12, inducers of STAT3 and STAT4 activity, respectively. IL-6 or IL-12 on its own did not induce PD-1. Importantly, STAT3/4 and distinct chromatin modifications were associated with the novel regulatory regions following cytokine stimulation. The NFATc1/STAT regulatory regions were found to interact with the promoter region of the Pdcd1 gene, providing a mechanism for their action. Together these data add multiple novel distal regulatory regions and pathways to the control of PD-1 expression and provide a molecular mechanism by which proinflammatory cytokines, such as IL-6 or IL-12, can augment PD-1 expression.
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Linfócitos T CD8-Positivos/imunologia , Fatores de Transcrição NFATC/imunologia , Receptor de Morte Celular Programada 1/imunologia , Proteínas Repressoras/imunologia , Elementos de Resposta/imunologia , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT4/imunologia , Animais , Fator de Ligação a CCCTC , Linfócitos T CD8-Positivos/citologia , Linhagem Celular Tumoral , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Camundongos , Fatores de Transcrição NFATC/genética , Receptor de Morte Celular Programada 1/genética , Proteínas Repressoras/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT4/genéticaRESUMO
Programmed cell death 1 (PD-1) is an inhibitory immune receptor that regulates T cell function, yet the molecular events that control its expression are largely unknown. We show here that B lymphocyte-induced maturation protein 1 (Blimp-1)-deficient CD8 T cells fail to repress PD-1 during the early stages of CD8 T cell differentiation after acute infection with lymphocytic choriomeningitis virus (LCMV) strain Armstrong. Blimp-1 represses PD-1 through a feed-forward repressive circuit by regulating PD-1 directly and by repressing NFATc1 expression, an activator of PD-1 expression. Blimp-1 binding induces a repressive chromatin structure at the PD-1 locus, leading to the eviction of NFATc1 from its site. These data place Blimp-1 at an important phase of the CD8 T cell effector response and provide a molecular mechanism for its repression of PD-1.
Assuntos
Infecções por Arenaviridae/imunologia , Linfócitos T CD8-Positivos/metabolismo , Regulação da Expressão Gênica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Fatores de Transcrição/imunologia , Animais , Sítios de Ligação/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Primers do DNA/genética , Luciferases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição NFATC/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
OBJECTIVE: To investigate the relationship between cognitive impairment and somatosensory evoked magnetic field and auditory evoked magnetic field changes in elderly male patients with subcortical ischemic vascular dementia (SIVD). METHODS: Magnetoencephalography (MEG) was used to record evoked magnetic field changes from 4 SIVD patients (76-88 years), 3 patients with vascular cognitive impairment with no dementia (VCI-ND; 74-87 years), and 6 healthy volunteers (72-85 years). Latency peaks, equivalent current dipole (ECD) strength, and bilateral ECD position were recorded. The MEG data were superimposed on magnetic resonance imaging to produce magnetic source imaging. RESULTS: Compared to controls, SIVD patients showed increased M20 latency and ECD strength. There were no significant differences in M20 inter-hemispheric positions across diagnostic categories. At M100, SIVD patients showed delayed auditory evoked magnetic field latency compared to controls. However, ECD strength and 3-dimensional inter-hemispheric differences were similar across the groups at the M100 measurement. CONCLUSIONS: Changes in somatosensory and auditory evoked magnetic field changes correlated with cognitive impairment in SIVD patients. Magnetic field latency measures may provide an objective and sensitive index for early dementia detection and monitoring of cognitive function.
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Isquemia Encefálica/complicações , Transtornos Cognitivos/fisiopatologia , Transtornos Cognitivos/psicologia , Demência Vascular/fisiopatologia , Demência Vascular/psicologia , Campos Magnéticos , Estimulação Acústica , Idoso , Idoso de 80 Anos ou mais , Córtex Auditivo/fisiopatologia , Estudos de Casos e Controles , Cérebro/fisiopatologia , Demência Vascular/etiologia , Estimulação Elétrica , Humanos , Magnetoencefalografia , Masculino , Córtex Somatossensorial/fisiopatologiaRESUMO
Functionally exhausted T cells have high expression of the PD-1 inhibitory receptor, and therapies that block PD-1 signaling show promise for resolving chronic viral infections and cancer. By using human and murine systems of acute and chronic viral infections, we analyzed epigenetic regulation of PD-1 expression during CD8(+) T cell differentiation. During acute infection, naive to effector CD8(+) T cell differentiation was accompanied by a transient loss of DNA methylation of the Pdcd1 locus that was directly coupled to the duration and strength of T cell receptor signaling. Further differentiation into functional memory cells coincided with Pdcd1 remethylation, providing an adapted program for regulation of PD-1 expression. In contrast, the Pdcd1 regulatory region was completely demethylated in exhausted CD8(+) T cells and remained unmethylated even when virus titers decreased. This lack of DNA remethylation leaves the Pdcd1 locus poised for rapid expression, potentially providing a signal for premature termination of antiviral functions.
Assuntos
Antígenos CD/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Linfócitos T CD8-Positivos/imunologia , Metilação de DNA , Regiões Promotoras Genéticas , Viroses/patologia , Animais , Antígenos CD/genética , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Proteínas Reguladoras de Apoptose/genética , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/patologia , Diferenciação Celular , Células Cultivadas , Doença Crônica , Epigenômica , Humanos , Memória Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1 , Transdução de SinaisRESUMO
The level of TGF-ß/bone morphogenetic protein (BMP) signaling through Smad is tightly regulated to ensure proper embryonic patterning and homeostasis. Here we show that Smad activation by TGF-ß/BMP is blocked by a highly conserved phosphorylation event in the α-helix 1 region of Smad [T312 in Drosophila Smad1 (MAD)]. α-helix 1 phosphorylation reduces Smad interaction with TGF-ß/BMP receptor kinase and affects all receptor-activated Smads except Smad3. Tissue culture and transgenic studies in Drosophila further demonstrate that the biological activity of MAD is repressed by T312 phosphorylation in vivo. Through RNAi screening of the kinome, we have identified Misshapen (Msn) and the mammalian orthologs TNIK, MINK1, and MAP4K4 as the kinases responsible for α-helix 1 phosphorylation. Targeted expression of an active form of Msn in the wing imaginal disk disrupted activation of endogenous MAD by Dpp and expression of the Dpp/MAD target gene. Msn kinases belong to the Ste20 kinase family that has been shown to act as MAP kinase kinase kinase kinase (MAP4K). Our findings thus reveal a function of Msn independent of its impact on MAP kinase cascades. This Smad inhibition mechanism by Msn likely has important implications for development and disease.
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Proteínas de Drosophila/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Smad/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Genes de Insetos , Humanos , Dados de Sequência Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Estrutura Secundária de Proteína , Interferência de RNA , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Proteínas Smad/química , Proteínas Smad/genética , Proteínas Smad/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Oxidative stress induces neuronal apoptosis and is implicated in cerebral ischemia, head trauma, and age-related neurodegenerative diseases. An early step in this process is the loss of intracellular K(+) via K(+) channels, and evidence indicates that K(v)2.1 is of particular importance in this regard, being rapidly inserted into the plasma membrane in response to apoptotic stimuli. An additional feature of neuronal oxidative stress is the up-regulation of the inducible enzyme heme oxygenase-1 (HO-1), which catabolizes heme to generate biliverdin, Fe(2+), and carbon monoxide (CO). CO provides neuronal protection against stresses such as stroke and excitotoxicity, although the underlying mechanisms are not yet elucidated. Here, we demonstrate that CO reversibly inhibits K(v)2.1. Channel inhibition by CO involves reactive oxygen species and protein kinase G activity. Overexpression of K(v)2.1 in HEK293 cells increases their vulnerability to oxidant-induced apoptosis, and this is reversed by CO. In hippocampal neurons, CO selectively inhibits K(v)2.1, reverses the dramatic oxidant-induced increase in K(+) current density, and provides marked protection against oxidant-induced apoptosis. Our results provide a novel mechanism to account for the neuroprotective effects of CO against oxidative apoptosis, which has potential for therapeutic exploitation to provide neuronal protection in situations of oxidative stress.
Assuntos
2,2'-Dipiridil/análogos & derivados , Apoptose/efeitos dos fármacos , Monóxido de Carbono/farmacologia , Dissulfetos/farmacologia , Oxidantes/farmacologia , Canais de Potássio Shab/metabolismo , 2,2'-Dipiridil/farmacologia , Animais , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular , Eletrofisiologia , Células HEK293 , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Ratos , Ratos WistarRESUMO
Ahead of Print article withdrawn by publisher.
RESUMO
Hox proteins form complexes with Pbx and Meis cofactors to control gene expression, but the role of Meis is unclear. We demonstrate that Hoxb1-regulated promoters are highly acetylated on histone H4 (AcH4) and occupied by Hoxb1, Pbx, and Meis in zebrafish tissues where these promoters are active. Inhibition of Meis blocks gene expression and reduces AcH4 levels at these promoters, suggesting a role for Meis in maintaining AcH4. Within Hox transcription complexes, Meis binds directly to Pbx and we find that this binding displaces histone deacetylases (HDACs) from Hoxb1-regulated promoters in zebrafish embryos. Accordingly, Pbx mutants that cannot bind Meis act as repressors by recruiting HDACs and reducing AcH4 levels, while Pbx mutants that bind neither HDAC nor Meis are constitutively active and recruit CBP to increase AcH4 levels. We conclude that Meis acts, at least in part, by controlling access of HDAC and CBP to Hox-regulated promoters.
Assuntos
Proteína de Ligação a CREB/metabolismo , Embrião não Mamífero/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Histona Desacetilases/genética , Proteínas de Homeodomínio/genética , Regiões Promotoras Genéticas/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Acetilação , Animais , Western Blotting , Proteína de Ligação a CREB/genética , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário , Histona Desacetilase 1 , Histona Desacetilases/metabolismo , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Imunoprecipitação , Luciferases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/metabolismoAssuntos
Demência Vascular/metabolismo , Demência Vascular/fisiopatologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hipocampo/citologia , Neurônios/metabolismo , Animais , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Masculino , Camundongos , Neurônios/fisiologia , Distribuição AleatóriaRESUMO
Nuclear translocation of Smad proteins is a critical step in signal transduction of transforming growth factor beta (TGF-beta) and bone morphogenetic proteins (BMPs). Using nuclear accumulation of the Drosophila Smad Mothers against Decapentaplegic (Mad) as the readout, we carried out a whole-genome RNAi screening in Drosophila cells. The screen identified moleskin (msk) as important for the nuclear import of phosphorylated Mad. Genetic evidence in the developing eye imaginal discs also demonstrates the critical functions of msk in regulating phospho-Mad. Moreover, knockdown of importin 7 and 8 (Imp7 and 8), the mammalian orthologues of Msk, markedly impaired nuclear accumulation of Smad1 in response to BMP2 and of Smad2/3 in response to TGF-beta. Biochemical studies further suggest that Smads are novel nuclear import substrates of Imp7 and 8. We have thus identified new evolutionarily conserved proteins that are important in the signal transduction of TGF-beta and BMP into the nucleus.