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1.
Trans Am Clin Climatol Assoc ; 130: 88-99, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31516171

RESUMO

We have found that calcium calmodulin kinase IV is increased in T cells, podocytes, and mesangial cells from patients with systemic lupus erythematosus, as well as in lupus-prone mice, podocytes of patients with focal segmental glomerulosclerosis, and in mice injected with doxorubicin. We showed that this accounts for aberrant T cell function and glomerular damage. Using nanoparticles (nlg) loaded with a small drug inhibitor of calcium calmodulin kinase IV and tagged with antibodies directed to CD4 we have been able to show inhibition of autoimmunity and lupus nephritis. Also, using nlg tagged with antibodies to nephrin, we showed suppression of nephritis in lupus-prone mice and of glomerular damage in mice exposed to doxorubicin. We propose the development of approaches to deliver drugs to cells in a targeted and precise manner.


Assuntos
Benzilaminas/administração & dosagem , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Glomerulosclerose Segmentar e Focal/tratamento farmacológico , Nefrite Lúpica/tratamento farmacológico , Nanopartículas , Inibidores de Proteínas Quinases/administração & dosagem , Sulfonamidas/administração & dosagem , Linfócitos T/imunologia , Animais , Antibióticos Antineoplásicos/toxicidade , Benzilaminas/uso terapêutico , Antígenos CD4 , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/imunologia , Metilação de DNA , Modelos Animais de Doenças , Doxorrubicina/toxicidade , Sistemas de Liberação de Medicamentos , Glomerulosclerose Segmentar e Focal/induzido quimicamente , Glomerulosclerose Segmentar e Focal/imunologia , Humanos , Lúpus Eritematoso Sistêmico , Nefrite Lúpica/imunologia , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos MRL lpr , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/uso terapêutico , Sulfonamidas/uso terapêutico , Linfócitos T Reguladores/imunologia , Células Th17/imunologia
2.
J Clin Invest ; 128(8): 3445-3459, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29985166

RESUMO

Podocyte malfunction occurs in autoimmune and nonautoimmune kidney disease. Calcium signaling is essential for podocyte injury, but the role of Ca2+/calmodulin-dependent kinase (CaMK) signaling in podocytes has not been fully explored. We report that podocytes from patients with lupus nephritis and focal segmental glomerulosclerosis and lupus-prone and lipopolysaccharide- or adriamycin-treated mice display increased expression of CaMK IV (CaMK4), but not CaMK2. Mechanistically, CaMK4 modulated podocyte motility by altering the expression of the GTPases Rac1 and RhoA and suppressed the expression of nephrin, synaptopodin, and actin fibers in podocytes. In addition, it phosphorylated the scaffold protein 14-3-3ß, which resulted in the release and degradation of synaptopodin. Targeted delivery of a CaMK4 inhibitor to podocytes preserved their ultrastructure, averted immune complex deposition and crescent formation, and suppressed proteinuria in lupus-prone mice and proteinuria in mice exposed to lipopolysaccharide-induced podocyte injury by preserving nephrin/synaptopodin expression. In animals exposed to adriamycin, podocyte-specific delivery of a CaMK4 inhibitor prevented and reversed podocyte injury and renal disease. We conclude that CaMK4 is pivotal in immune and nonimmune podocyte injury and that its targeted cell-specific inhibition preserves podocyte structure and function and should have therapeutic value in lupus nephritis and podocytopathies, including focal segmental glomerulosclerosis.


Assuntos
Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , Glomerulosclerose Segmentar e Focal/enzimologia , Glomérulos Renais/enzimologia , Nefrite Lúpica/enzimologia , Transdução de Sinais , Animais , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/imunologia , Linhagem Celular Transformada , Feminino , Glomerulosclerose Segmentar e Focal/imunologia , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Glomérulos Renais/imunologia , Glomérulos Renais/patologia , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos MRL lpr , Camundongos Knockout , Proteinúria/enzimologia , Proteinúria/imunologia , Proteinúria/patologia
3.
J Immunol ; 193(5): 2405-15, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25070845

RESUMO

Autophagy, an evolutionarily conserved homeostasis process regulating biomass quantity and quality, plays a critical role in the host response to sepsis. Recent studies show its calcium dependence, but the calcium-sensitive regulatory cascades have not been defined. In this study, we describe a novel mechanism in which calcium/calmodulin-dependent protein kinase IV (CaMKIV), through inhibitory serine phosphorylation of GSK-3ß and inhibition of FBXW7 recruitment, prevents ubiquitin proteosomal degradation of mammalian target of rapamycin (mTOR) and thereby augments autophagy in both the macrophage and the kidney. Under the conditions of sepsis studied, mTOR expression and activity were requisite for autophagy, a paradigm countering the current perspective that prototypically, mTOR inhibition induces autophagy. CaMKIV-mTOR-dependent autophagy was fundamentally important for IL-6 production in vitro and in vivo. Similar mechanisms were operant in the kidney during endotoxemia and served a cytoprotective role in mitigating acute kidney injury. Thus, CaMKIV-mTOR-dependent autophagy is conserved in both immune and nonimmune/parenchymal cells and is fundamental for the respective functional and adaptive responses to septic insult.


Assuntos
Injúria Renal Aguda/imunologia , Autofagia/efeitos dos fármacos , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Macrófagos/imunologia , Serina-Treonina Quinases TOR/imunologia , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/genética , Injúria Renal Aguda/patologia , Animais , Autofagia/genética , Autofagia/imunologia , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/genética , Linhagem Celular , Proteínas F-Box/genética , Proteínas F-Box/imunologia , Proteína 7 com Repetições F-Box-WD , Regulação da Expressão Gênica/imunologia , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/imunologia , Glicogênio Sintase Quinase 3 beta , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Serina-Treonina Quinases TOR/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia
4.
Autoimmunity ; 47(7): 445-50, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24829059

RESUMO

OBJECTIVE: Foxp3(+) regulatory T cells (Treg) are pivotal for the maintenance of peripheral tolerance and prevent development of autoimmune diseases. We have reported that calcium/calmodulin-dependent protein kinase IV (CaMK4) deficient MRL/lpr mice display less disease activity by promoting IL-2 production and increasing the activity of Treg cells. To further define the mechanism of CaMK4 on Treg cells in systemic lupus erythematosus (SLE), we used the Foxp3-GFP reporter mice and treated them with KN-93, an inhibitor of CaMK4. METHODS: We generated MRL/lpr Foxp3-GFP mice to record Treg cells; stimulated naïve CD4(+) T cells from MRL/lpr Foxp3-GFP mice under Treg polarizing conditions in the absence or presence of KN-93; evaluated the number of GFP positive cells in lymphoid organs and examined skin and kidney pathology at 16 weeks of age. We also examined the infiltration of cells and recruitment of Treg cells in the kidney. RESULTS: We show that culture of MRL/lpr Foxp3-GFP T cells in the presence of KN-93 promotes Treg differentiation in a dose-dependent manner. Treatment of MRL/lpr Foxp3-GFP mice with KN-93 results in a significant induction of Treg cells in the spleen, peripheral lymph nodes and peripheral blood and this is accompanied by decreased skin and kidney damage. Notably, KN-93 clearly diminishes the accumulation of inflammatory cells along with reciprocally increased Treg cells in target organ. CONCLUSION: Our results indicate that KN-93 treatment enhances the generation of Treg cells in vitro and in vivo highlighting its potential therapeutic use for the treatment of human autoimmune diseases.


Assuntos
Benzilaminas/farmacologia , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Lúpus Eritematoso Sistêmico , Inibidores de Proteínas Quinases/farmacologia , Sulfonamidas/farmacologia , Linfócitos T Reguladores/imunologia , Animais , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/imunologia , Contagem de Células , Fatores de Transcrição Forkhead , Interleucina-2 , Lúpus Eritematoso Sistêmico/enzimologia , Lúpus Eritematoso Sistêmico/imunologia , Camundongos Endogâmicos MRL lpr , Recuperação de Função Fisiológica/efeitos dos fármacos , Linfócitos T Reguladores/enzimologia
5.
J Clin Invest ; 124(5): 2234-45, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24667640

RESUMO

Tissue inflammation in several autoimmune diseases, including SLE and MS, has been linked to an imbalance of IL-17-producing Th (Th17) cells and Tregs; however, the factors that promote Th17-driven autoimmunity are unclear. Here, we present evidence that the calcium/calmodulin-dependent protein kinase IV (CaMK4) is increased and required during Th17 cell differentiation. Isolation of naive T cells from a murine model of lupus revealed increased levels of CaMK4 following stimulation with Th17-inducing cytokines but not following Treg, Th1, or Th2 induction. Furthermore, naive T cells from mice lacking CaMK4 did not produce IL-17. Genetic or pharmacologic inhibition of CaMK4 decreased the frequency of IL-17-producing T cells and ameliorated EAE and lupus-like disease in murine models. Inhibition of CaMK4 reduced Il17 transcription through decreased activation of the cAMP response element modulator α (CREM-α) and reduced activation of the AKT/mTOR pathway, which is known to enhance Th17 differentiation. Importantly, silencing CaMK4 in T cells from patients with SLE and healthy individuals inhibited Th17 differentiation through reduction of IL17A and IL17F mRNA. Collectively, our results suggest that CaMK4 inhibition has potential as a therapeutic strategy for Th17-driven autoimmune diseases.


Assuntos
Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/imunologia , Modulador de Elemento de Resposta do AMP Cíclico/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Esclerose Múltipla/imunologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Serina-Treonina Quinases TOR/imunologia , Células Th17/imunologia , Animais , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Modulador de Elemento de Resposta do AMP Cíclico/genética , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Feminino , Humanos , Interleucina-17/genética , Interleucina-17/imunologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Lúpus Eritematoso Sistêmico/terapia , Masculino , Camundongos , Camundongos Knockout , Esclerose Múltipla/genética , Esclerose Múltipla/patologia , Esclerose Múltipla/terapia , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Serina-Treonina Quinases TOR/genética , Células Th17/patologia
6.
J Biol Chem ; 288(42): 30752-30762, 2013 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-24014023

RESUMO

miR-185 is a microRNA (miR) that targets Bruton's tyrosine kinase in B cells, with reductions in miR-185 linked to B cell autoantibody production. In hippocampal neurons, miR-185 targets both sarcoplasmic/endoplasmic reticulum calcium ATPase 2 and a novel Golgi inhibitor. This miR is haploinsufficient in 90-95% of individuals with chromosome 22q11.2 deletion syndrome, patients who can present with immune, cardiac, and parathyroid problems, learning disorders, and a high incidence of schizophrenia in adults. The reduced levels of miR-185 in neurons cause presynaptic neurotransmitter release. Many of the 22q11.2 deletion syndrome patients have a thymic hypoplasia, which results in a peripheral T cell lymphopenia and unusual T helper cell skewing. The molecular targets of miR-185 in thymocytes are unknown. Using an miR-185 T cell transgenic approach, increasing levels of miR-185 attenuated T cell development at the T cell receptor ß (TCRß) selection checkpoint and during positive selection. This caused a peripheral T cell lymphopenia. Mzb1, Nfatc3, and Camk4 were identified as novel miR-185 targets. Elevations in miR-185 enhanced TCR-dependent intracellular calcium levels, whereas a knockdown of miR-185 diminished these calcium responses. These effects concur with reductions in Mzb1, an endoplasmic reticulum calcium regulator. Consistent with their haploinsufficiency of miR-185, Mzb1 levels were elevated in thymocyte extracts from several 22q11.2 deletion syndrome patients. Our findings indicate that miR-185 regulates T cell development through its targeting of several mRNAs including Mzb1.


Assuntos
Sinalização do Cálcio , Citocinas/biossíntese , MicroRNAs/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Timócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Cálcio/imunologia , Cálcio/metabolismo , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/imunologia , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , Citocinas/genética , Citocinas/imunologia , Humanos , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , MicroRNAs/imunologia , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/imunologia , Fatores de Transcrição NFATC/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/imunologia , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Timócitos/citologia , Timócitos/imunologia , Transgenes/genética , Transgenes/imunologia
7.
Blood ; 119(6): 1390-8, 2012 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-22174157

RESUMO

Effector CD8(+) T cells are believed to be terminally differentiated cells having cytotoxic activity and the ability to produce effector cytokines such as INF-γ and TNF-α. We investigated the difference between CXCR1(+) and CXCR1(-) subsets of human effector CD27(-)CD28(-)CD8(+) T cells. The subsets expressed cytolytic molecules similarly and exerted substantial cytolytic activity, whereas only the CXCR1(-) subset had IL-2 productivity and self-proliferative activity and was more resistant to cell death than the CXCR1(+) subset. These differences were explained by the specific up-regulation of CAMK4, SPRY2, and IL-7R in the CXCR1(-) subset and that of pro-apoptotic death-associated protein kinase 1 (DAPK1) in the CXCR1(+) subset. The IL-2 producers were more frequently found in the IL-7R(+) subset of the CXCR1(-) effector CD8(+) T cells than in the IL-7R(-) subset. IL-7/IL-7R signaling promoted cell survival only in the CXCR1(-) subset. The present study has highlighted a novel subset of effector CD8(+) T cells producing IL-2 and suggests the importance of this subset in the homeostasis of effector CD8(+) T cells.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica/imunologia , Interleucina-2/imunologia , Receptores de Interleucina-8A/imunologia , Adulto , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/imunologia , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/imunologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Citotoxicidade Imunológica/genética , Proteínas Quinases Associadas com Morte Celular , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Interleucina-2/genética , Interleucina-2/metabolismo , Interleucina-7/genética , Interleucina-7/imunologia , Interleucina-7/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/imunologia , Receptores de Interleucina-7/metabolismo , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8A/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
8.
Blood ; 111(2): 723-31, 2008 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-17909078

RESUMO

Microbial products, including lipopolysaccharide (LPS), an agonist of Toll-like receptor 4 (TLR4), regulate the lifespan of dendritic cells (DCs) by largely undefined mechanisms. Here, we identify a role for calcium-calmodulin-dependent kinase IV (CaMKIV) in this survival program. The pharmacologic inhibition of CaMKs as well as ectopic expression of kinase-inactive CaMKIV decrease the viability of monocyte-derived DCs exposed to bacterial LPS. The defect in TLR4 signaling includes a failure to accumulate the phosphorylated form of the cAMP response element-binding protein (pCREB), Bcl-2, and Bcl-xL. CaMKIV null mice have a decreased number of DCs in lymphoid tissues and fail to accumulate mature DCs in spleen on in vivo exposure to LPS. Although isolated Camk4-/- DCs are able to acquire the phenotype typical of mature cells and release normal amounts of cytokines in response to LPS, they fail to accumulate pCREB, Bcl-2, and Bcl-xL and therefore do not survive. The transgenic expression of Bcl-2 in CaMKIV null mice results in full recovery of DC survival in response to LPS. These results reveal a novel link between TLR4 and a calcium-dependent signaling cascade comprising CaMKIV-CREB-Bcl-2 that is essential for DC survival.


Assuntos
Sinalização do Cálcio/fisiologia , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/imunologia , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/metabolismo , Células Dendríticas/metabolismo , Monócitos/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/imunologia , Proteína de Ligação a CREB/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células Dendríticas/citologia , Células Dendríticas/imunologia , Humanos , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Knockout , Monócitos/citologia , Monócitos/imunologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Baço/citologia , Baço/imunologia , Baço/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Proteína bcl-X/genética , Proteína bcl-X/imunologia , Proteína bcl-X/metabolismo
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