RESUMO
Insulin resistance, hyperlipidemia, and cardiovascular complications are common dysregulations of metabolic syndrome. Transplant patients treated with immunosuppressant drugs such as cyclosporine A (CsA), an inhibitor of calcineurin phosphatase, frequently develop similar metabolic complications. Although calcineurin is known to mediate insulin sensitivity by regulating ß-cell growth and adipokine gene transcription, its role in lipid homeostasis is poorly understood. Here, we examined lipid homeostasis in mice lacking calcineurin Aß (CnAß(-/-)). We show that mice lacking calcineurin Aß are hyperlipidemic and develop age-dependent insulin resistance. Hyperlipidemia found in CnAß(-/-) mice is, in part, due to increased lipolysis in adipose tissues, a process mediated by ß-adrenergic G-protein-coupled receptor signaling pathways. CnAß(-/-) mice also exhibit additional pathophysiological phenotypes caused by the potentiated GPCR signaling pathways. A cell autonomous mechanism with sustained cAMP/PKA activation is found in CnAß(-/-) mice or upon CsA treatment to inhibit calcineurin. Increased PKA activation and cAMP accumulation in CnAß(-/-) mice, however, are sensitive to phosphodiesterase inhibitor. Indeed, we show that calcineurin regulates degradation of phosphodiesterase 3B, in addition to phosphodiesterase 4D. These results establish a role for calcineurin in lipid homeostasis. These data also indicate that potentiated cAMP signaling pathway may provide an alternative molecular pathogenesis for the metabolic complications elicited by CsA in transplant patients.
Assuntos
Calcineurina/deficiência , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Hiperlipidemias/enzimologia , Transdução de Sinais , Envelhecimento/efeitos dos fármacos , Envelhecimento/patologia , Sequência de Aminoácidos , Animais , Células COS , Calcineurina/metabolismo , Chlorocebus aethiops , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/química , Ciclosporina/farmacologia , Embrião de Mamíferos/citologia , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Hiperlipidemias/patologia , Resistência à Insulina , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Inibidores de Fosfodiesterase/farmacologia , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Triglicerídeos/biossínteseRESUMO
IFN-lambda1 (IL-29) plays a novel, emerging role in the inhibition of human Th2 responses. Here, we demonstrate that both naive and memory human CD4(+) T cells express mRNA for the IFN-lambda1-specific receptor, IL-28Ralpha, and are responsive to IFN-lambda1. Expression of Th2 cytokines (IL-4 and IL-13) was suppressed in naive and memory CD4(+) T cells by IFN-lambda1, without affecting their proliferation. Further, acquisition of IL-4Ralpha expression after stimulation was inhibited by IFN-lambda1, as was GATA3 expression. Finally, IFN-lambda1 diminished the change in cell-surface phenotype that accompanies differentiation of "central memory" T cells into "effector memory" T cells. Taken together, our data describe unique immunomodulatory effects of IFN-lambda1 and identify novel mechanisms for the reduction of existing Th2 responses and the regulation of new ones, in circulating naive and memory CD4(+) T cells.
Assuntos
Fator de Transcrição GATA3/metabolismo , Imunidade Inata/imunologia , Memória Imunológica/imunologia , Interleucinas/farmacologia , Células Th2/imunologia , Células Th2/metabolismo , Diferenciação Celular/imunologia , Proliferação de Células , Células Cultivadas , Humanos , Imunidade Inata/efeitos dos fármacos , Memória Imunológica/efeitos dos fármacos , Interleucinas/biossíntese , Receptores de Citocinas/imunologia , Células Th2/citologia , Células Th2/efeitos dos fármacosRESUMO
Compromised immunoregulation contributes to obesity and complications in metabolic pathogenesis. Here, we demonstrate that the nuclear factor of activated T cell (NFAT) group of transcription factors contributes to glucose and insulin homeostasis. Expression of two members of the NFAT family (NFATc2 and NFATc4) is induced upon adipogenesis and in obese mice. Mice with the Nfatc2-/- Nfatc4-/- compound disruption exhibit defects in fat accumulation and are lean. Nfatc2-/- Nfatc4-/- mice are also protected from diet-induced obesity. Ablation of NFATc2 and NFATc4 increases insulin sensitivity, in part, by sustained activation of the insulin signaling pathway. Nfatc2-/- Nfatc4-/- mice also exhibit an altered adipokine profile, with reduced resistin and leptin levels. Mechanistically, NFAT is recruited to the transcription loci and regulates resistin gene expression upon insulin stimulation. Together, these results establish a role for NFAT in glucose/insulin homeostasis and expand the repertoire of NFAT function to metabolic pathogenesis and adipokine gene transcription.
Assuntos
Glucose/metabolismo , Homeostase , Insulina/metabolismo , Fatores de Transcrição NFATC/metabolismo , Proteínas Quinases Ativadas por AMP , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipócitos/patologia , Animais , Diferenciação Celular , Linhagem Celular , Chlorocebus aethiops , Gorduras na Dieta/farmacologia , Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Complexos Multienzimáticos/metabolismo , Fatores de Transcrição NFATC/deficiência , Fatores de Transcrição NFATC/genética , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Resistina/metabolismo , Sensibilidade e Especificidade , Transdução de SinaisRESUMO
Charcot-Marie-Tooth (CMT) disease is an inherited neurological disorder. Mutations in the small integral membrane protein of the lysosome/late endosome (SIMPLE) account for the rare autosomal-dominant demyelination in CMT1C patients. Understanding the molecular basis of CMT1C pathogenesis is impeded, in part, by perplexity about the role of SIMPLE, which is expressed in multiple cell types. Here we show that SIMPLE resides within the intraluminal vesicles of multivesicular bodies (MVBs) and inside exosomes, which are nanovesicles secreted extracellularly. Targeting of SIMPLE to exosomes is modulated by positive and negative regulatory motifs. We also find that expression of SIMPLE increases the number of exosomes and secretion of exosome proteins. We engineer a point mutation on the SIMPLE allele and generate a physiological mouse model that expresses CMT1C-mutated SIMPLE at the endogenous level. We find that CMT1C mouse primary embryonic fibroblasts show decreased number of exosomes and reduced secretion of exosome proteins, in part due to improper formation of MVBs. CMT1C patient B cells and CMT1C mouse primary Schwann cells show similar defects. Together the data indicate that SIMPLE regulates the production of exosomes by modulating the formation of MVBs. Dysregulated endosomal trafficking and changes in the landscape of exosome-mediated intercellular communications may place an overwhelming burden on the nervous system and account for CMT1C molecular pathogenesis.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Exossomos/metabolismo , Sistema Nervoso/metabolismo , Proteínas Nucleares/genética , Mutação Puntual , Fatores de Transcrição/genética , Alelos , Motivos de Aminoácidos , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Sequência de Bases , Transporte Biológico , Comunicação Celular , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Proteínas de Ligação a DNA , Modelos Animais de Doenças , Embrião de Mamíferos , Exossomos/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Corpos Multivesiculares/metabolismo , Corpos Multivesiculares/patologia , Sistema Nervoso/patologia , Proteínas Nucleares/metabolismo , Células de Schwann/metabolismo , Células de Schwann/patologia , Fatores de Transcrição/metabolismoRESUMO
Calcineurin is a widely expressed and highly conserved Ser/Thr phosphatase. Calcineurin is inhibited by the immunosuppressant drug cyclosporine A (CsA) or tacrolimus (FK506). The critical role of CsA/FK506 as an immunosuppressant following transplantation surgery provides a strong incentive to understand the phosphatase calcineurin. Here we uncover a novel regulatory pathway for cyclic AMP (cAMP) signaling by the phosphatase calcineurin which is also evolutionarily conserved in Caenorhabditis elegans. We found that calcineurin binds directly to and inhibits the proteosomal degradation of cAMP-hydrolyzing phosphodiesterase 4D (PDE4D). We show that ubiquitin conjugation and proteosomal degradation of PDE4D are controlled by a cullin 1-containing E(3) ubiquitin ligase complex upon dual phosphorylation by casein kinase 1 (CK1) and glycogen synthase kinase 3beta (GSK3beta) in a phosphodegron motif. Our findings identify a novel signaling process governing G-protein-coupled cAMP signal transduction-opposing actions of the phosphatase calcineurin and the CK1/GSK3beta protein kinases on the phosphodegron-dependent degradation of PDE4D. This novel signaling system also provides unique functional insights into the complications elicited by CsA in transplant patients.
Assuntos
Proteínas de Caenorhabditis elegans , Calcineurina/genética , Calcineurina/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Evolução Molecular , Sistemas do Segundo Mensageiro/fisiologia , Motivos de Aminoácidos , Animais , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Inibidores de Calcineurina , Linhagem Celular , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Ciclosporina/metabolismo , Inibidores Enzimáticos/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismoRESUMO
Diffuse large B-cell lymphoma (DLBCL) consists of at least 2 phenotypic subtypes; that is, the germinal center B-cell-like (GCB-DLBCL) and the activated B-cell-like (ABC-DLBCL) groups. It has been shown that GCB-DLBCL responds favorably to chemotherapy and expresses high levels of BCL6, a transcription repressor known to play a causative role in lymphomagenesis. In comparison, ABC-DLBCL has lower levels of BCL6, constitutively activated nuclear factor-kappaB, and tends to be refractory to chemotherapy. Here, we report that the STAT3 gene is a transcriptional target of BCL6. As a result, high-level STAT3 expression and activation are preferentially detected in ABC-DLBCL and BCL6-negative normal germinal center B cells. Most importantly, inactivating STAT3 by either AG490 or small interference RNA in ABC-DLBCL cells inhibits cell proliferation and triggers apoptosis. These phenotypes are accompanied by decreased expression of several known STAT3 target genes, including c-Myc, JunB, and Mcl-1, and increased expression of the cell- cycle inhibitor p27. In addition to identifying STAT3 as a novel BCL6 target gene, our results define a second oncogenic pathway, STAT3 activation, which operates in ABC-DLBCL, suggesting that STAT3 may be a new therapeutic target in these aggressive lymphomas.
Assuntos
Ativação Linfocitária/imunologia , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/metabolismo , Fator de Transcrição STAT3/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Centro Germinativo/imunologia , Humanos , Linfoma Difuso de Grandes Células B/classificação , Linfoma Difuso de Grandes Células B/patologia , Mutação/genética , Proteínas Proto-Oncogênicas c-bcl-6 , Interferência de RNA , RNA Mensageiro/genética , Fator de Transcrição STAT3/genética , Transcrição Gênica/genética , Tirfostinas/farmacologiaRESUMO
The target of rapamycin (TOR) signaling regulates the nucleocytoplasmic shuttling of transcription factors in yeast. Whether the mammalian counterpart of TOR (mTOR) also regulates nucleocytoplasmic shuttling is not known. Using a phospho-specific monoclonal antibody, we demonstrate that mTOR phosphorylates Ser(168,170) of endogenous NFATc4, which are conserved gate-keeping Ser residues that control NFAT subcellular distribution. The mTOR acts as a basal kinase during the resting state to maintain NFATc4 in the cytosol. Inactivation and nuclear export of NFATc4 are mediated by rephosphorylation of Ser(168,170), which can be a nuclear event. Kinetic analyses demonstrate that rephosphorylation of Ser(168,170) of endogenous NFATc4 is mediated by mTOR and, surprisingly, by extracellular signal-regulated kinase 5 (ERK5) mitogen-activated protein kinase as well. Ablation of ERK5 in the Erk5(-/-) cells ascertains defects in NFATc4 rephosphorylation and nucleocytoplasmic shuttling. In addition, phosphorylation of NFATc4 by ERK5 primes subsequent phosphorylation mediated by CK1alpha. These results demonstrate that distinct protein kinases are integrated to phosphorylate the gate-keeping residues Ser(168,170) of NFATc4, to regulate subcellular distribution. These data also expand the repertoire of physiological substrates of mTOR and ERK5.
Assuntos
Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Fatores de Transcrição NFATC/metabolismo , Proteínas Quinases/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Células COS , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Camundongos , Camundongos Knockout , Proteína Quinase 7 Ativada por Mitógeno/deficiência , Proteína Quinase 7 Ativada por Mitógeno/genética , Modelos Biológicos , Fatores de Transcrição NFATC/química , Fatores de Transcrição NFATC/deficiência , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/imunologia , Fosforilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Serina-Treonina Quinases TOR , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
ADP-ribosylation is a reversible posttranslational modification mediated by poly-ADP-ribose polymerase (PARP). The results of recent studies demonstrate that ADP-ribosylation contributes to transcription regulation. Here, we report that transcription factor NFAT binds to and is ADP-ribosylated by PARP-1 in an activation-dependent manner. Mechanistically, ADP-ribosylation increases NFAT DNA binding. Functionally, NFAT-mediated interleukin-2 (IL-2) expression was reduced in T cells upon genetic ablation or pharmacological inhibition of PARP-1. Parp-1(-/-) T cells also exhibit reduced expression of other NFAT-dependent cytokines, such as IL-4. Together, these results demonstrate that ADP-ribosylation mediated by PARP-1 provides a molecular switch to positively regulate NFAT-dependent cytokine gene transcription. These results also imply that, similar to the effect of calcineurin inhibition, PARP-1 inhibition may be beneficial in modulating immune functions.