RESUMO
Nuclear factor κB (NF-κB) is a transcription factor important for regulating innate and adaptive immunity, cellular proliferation, apoptosis, and senescence. Dysregulation of NF-κB and its upstream regulator IκB kinase (IKK) contributes to the pathogenesis of multiple inflammatory and degenerative diseases as well as cancer. An 11-amino acid peptide containing the NF-κB essential modulator (NEMO)-binding domain (NBD) derived from the C-terminus of ß subunit of IKK, functions as a highly selective inhibitor of the IKK complex by disrupting the association of IKKß and the IKKγ subunit NEMO. A structure-based pharmacophore model was developed to identify NBD mimetics by in silico screening. Two optimized lead NBD mimetics, SR12343 and SR12460, inhibited tumor necrosis factor α (TNF-α)- and lipopolysaccharide (LPS)-induced NF-κB activation by blocking the interaction between IKKß and NEMO and suppressed LPS-induced acute pulmonary inflammation in mice. Chronic treatment of a mouse model of Duchenne muscular dystrophy (DMD) with SR12343 and SR12460 attenuated inflammatory infiltration, necrosis and muscle degeneration, demonstrating that these small-molecule NBD mimetics are potential therapeutics for inflammatory and degenerative diseases.
Assuntos
Materiais Biomiméticos/farmacologia , Quinase I-kappa B/antagonistas & inibidores , Distrofia Muscular de Duchenne/tratamento farmacológico , Pneumonia/tratamento farmacológico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Materiais Biomiméticos/química , Linhagem Celular , Feminino , Células HEK293 , Humanos , Quinase I-kappa B/química , Quinase I-kappa B/metabolismo , Inflamação/tratamento farmacológico , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Necrose/tratamento farmacológico , Domínios Proteicos , Células RAW 264.7RESUMO
Along with the most common mutation, JAK2V617F, several other acquired JAK2 mutations have now been shown to contribute to the pathogenesis of myeloproliferative neoplasms (MPNs). However, here we describe for the first time a germline mutation that leads to familial thrombocytosis that involves a residue other than Val617. The novel mutation JAK2R564Q, identified in a family with autosomal dominant essential thrombocythemia, increased cell growth resulting from suppression of apoptosis in Ba/F3-MPL cells. Although JAK2R564Q and JAK2V617F have similar levels of increased kinase activity, the growth-promoting effects of JAK2R564Q are much milder than those of JAK2V617F because of at least 2 counterregulatory mechanisms. Whereas JAK2V617F can escape regulation by the suppressor of cytokine signaling 3 and p27/Kip1, JAK2R564Q-expressing cells cannot. Moreover, JAK2R564Q-expressing cells are much more sensitive to the JAK inhibitor, ruxolitinib, than JAK2V617F-expressers, suggesting that lower doses of this drug may be effective in treating patients with MPNs associated with alternative JAK2 mutations, allowing many undesirable adverse effects to be avoided. This work provides a greater understanding of the cellular effects of a non-JAK2V617F, MPN-associated JAK2 mutation; provides insights into new treatment strategies for such patients; and describes the first case of familial thrombosis caused by a JAK2 residue other than Val617.
Assuntos
Mutação em Linhagem Germinativa , Janus Quinase 2/genética , Trombocitemia Essencial/genética , Adulto , Sequência de Aminoácidos , Substituição de Aminoácidos , Arginina/genética , Sequência de Bases , Criança , Feminino , Ácido Glutâmico/genética , Humanos , Janus Quinase 2/química , Masculino , Modelos Moleculares , Dados de Sequência Molecular , LinhagemRESUMO
BACKGROUND: Sepsis is a life-threatening acute inflammatory condition associated with metabolic complications. Accumulation of free fatty acids (FFAs) induces inflammation and causes lipotoxic effects in the liver. Because fatty acid metabolism plays a role in the inflammatory response, we hypothesized that the administration of C75, a fatty acid synthase inhibitor, could alleviate the injury caused by sepsis. METHODS: Male mice were subjected to sepsis by cecal ligation and puncture (CLP). At 4 h after CLP, different doses of C75 (1- or 5-mg/kg body weight) or vehicle (20% dimethyl sulfoxide in saline) were injected intraperitoneally. Blood and liver tissues were collected at 24 h after CLP. RESULTS: C75 treatment with 1- and 5-mg/kg body weight significantly lowered FFA levels in the liver after CLP by 28% and 53%, respectively. Administration of C75 dose dependently reduced serum indexes of organ injury (aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase) and serum levels of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). In the liver, C75 treatment reduced inflammation (TNF-α and IL-6) and oxidative stress (inducible nitric oxide synthase and cyclooxygenase 2) in a dose-dependent manner. The 5-mg dose improved the 10-d survival rate to 85% from that of 55% in the vehicle. In the presence of C75, TNF-α release in RAW 246.7 cells with 4-h lipopolysaccharide stimulation was also significantly reduced. CONCLUSIONS: C75 effectively lowered FFA accumulation in the liver, which was associated with inhibition of inflammation and organ injury as well as improvement in survival rate after CLP. Thus, inhibition of FFA by C75 could ameliorate the hepatic dysfunction seen in sepsis.
Assuntos
4-Butirolactona/análogos & derivados , Inibidores Enzimáticos/uso terapêutico , Insuficiência Hepática/prevenção & controle , Inflamação/prevenção & controle , Lipogênese/efeitos dos fármacos , Sepse/tratamento farmacológico , 4-Butirolactona/farmacologia , 4-Butirolactona/uso terapêutico , Animais , Biomarcadores/metabolismo , Inibidores Enzimáticos/farmacologia , Insuficiência Hepática/etiologia , Insuficiência Hepática/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sepse/complicações , Sepse/metabolismo , Resultado do TratamentoRESUMO
Aging drives progressive loss of the ability of tissues to recover from stress, partly through loss of somatic stem cell function and increased senescent burden. We demonstrate that bone marrow-derived mesenchymal stem cells (BM-MSCs) rapidly senescence and become dysfunctional in culture. Injection of BM-MSCs from young mice prolonged life span and health span, and conditioned media (CM) from young BM-MSCs rescued the function of aged stem cells and senescent fibroblasts. Extracellular vesicles (EVs) from young BM-MSC CM extended life span of Ercc1-/- mice similarly to injection of young BM-MSCs. Finally, treatment with EVs from MSCs generated from human ES cells reduced senescence in culture and in vivo, and improved health span. Thus, MSC EVs represent an effective and safe approach for conferring the therapeutic effects of adult stem cells, avoiding the risks of tumor development and donor cell rejection. These results demonstrate that MSC-derived EVs are highly effective senotherapeutics, slowing the progression of aging, and diseases driven by cellular senescence.
Assuntos
Envelhecimento/metabolismo , Senescência Celular/fisiologia , Vesículas Extracelulares/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Longevidade , Células-Tronco Mesenquimais/citologia , Senoterapia/metabolismo , Animais , Meios de Cultivo Condicionados/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Transdução de Sinais/fisiologiaRESUMO
NF-κB is a transcription factor activated in response to inflammatory, genotoxic and oxidative stress and important for driving senescence and aging. Ataxia-telangiectasia mutated (ATM) kinase, a core component of DNA damage response signaling, activates NF-κB in response to genotoxic and oxidative stress via post-translational modifications. Here we demonstrate that ATM is activated in senescent cells in culture and murine tissues from Ercc1-deficient mouse models of accelerated aging, as well as naturally aged mice. Genetic and pharmacologic inhibition of ATM reduced activation of NF-κB and markers of senescence and the senescence-associated secretory phenotype (SASP) in senescent Ercc1-/- MEFs. Ercc1-/Δ mice heterozygous for Atm have reduced NF-κB activity and cellular senescence, improved function of muscle-derived stem/progenetor cells (MDSPCs) and extended healthspan with reduced age-related pathology especially age-related bone and intervertebral disc pathologies. In addition, treatment of Ercc1-/∆ mice with the ATM inhibitor KU-55933 suppressed markers of senescence and SASP. Taken together, these results demonstrate that the ATM kinase is a major mediator of DNA damage-induced, NF-κB-mediated cellular senescence, stem cell dysfunction and aging and thus represents a therapeutic target to slow the progression of aging.
Assuntos
Envelhecimento/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Senescência Celular/fisiologia , Dano ao DNA/fisiologia , NF-kappa B/metabolismo , Células-Tronco/metabolismo , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Aging is the main risk factor for many chronic degenerative diseases and cancer. Increased senescent cell burden in various tissues is a major contributor to aging and age-related diseases. Recently, a new class of drugs termed senolytics were demonstrated to extending healthspan, reducing frailty and improving stem cell function in multiple murine models of aging. To identify novel and more optimal senotherapeutic drugs and combinations, we established a senescence associated ß-galactosidase assay as a screening platform to rapidly identify drugs that specifically affect senescent cells. We used primary Ercc1 -/- murine embryonic fibroblasts with reduced DNA repair capacity, which senesce rapidly if grown at atmospheric oxygen. This platform was used to screen a small library of compounds that regulate autophagy, identifying two inhibitors of the HSP90 chaperone family as having significant senolytic activity in mouse and human cells. Treatment of Ercc1 -/∆ mice, a mouse model of a human progeroid syndrome, with the HSP90 inhibitor 17-DMAG extended healthspan, delayed the onset of several age-related symptoms and reduced p16INK4a expression. These results demonstrate the utility of our screening platform to identify senotherapeutic agents as well as identified HSP90 inhibitors as a promising new class of senolytic drugs.The accumulation of senescent cells is thought to contribute to the age-associated decline in tissue function. Here, the authors identify HSP90 inhibitors as a new class of senolytic compounds in an in vitro screening and show that administration of a HSP90 inhibitor reduces age-related symptoms in progeroid mice.
Assuntos
Envelhecimento/fisiologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Benzoquinonas/farmacologia , Bioensaio , Biomarcadores/metabolismo , Senescência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Endonucleases/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Lactamas Macrocíclicas/farmacologia , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
We have previously demonstrated the involvement of milk fat globule-epidermal growth factor-factor 8 (MFGE8) in reducing neutrophil infiltration in a murine model of acute lung injury (ALI). In the present study, we aimed to delineate the mechanisms through which MFGE8 attenuates neutrophil migration. Recombinant human MFGE8 (rhMFGE8) was expressed and purified in our facility. The human differentiated neutrophil cell line, dHL60, was treated with rhMFGE8 and cell migration assay was performed in a Boyden chamber using recombinant interleukin8 (IL8) as the chemoattractant. Surface CXCR2 and intracellular G proteincoupled receptor kinase 2 (GRK2) levels were evaluated by flow cytometry or western blot analysis. The levels of mitogenactivated protein (MAP) kinases were determined by western blot analysis. Treatment with rhMFGE8 resulted in a significant inhibition of dHL60 cell migration in a dosedependent manner. There was a 46% decrease in CXCR2 expression in the rhMFGE8treated dHL60 cells, which was associated with a 32% increase in GRK2 expression. In the dHL60 cells, treatment with rhMFGE8 promoted the phosphorylation of p38 and extracellular signal-regulated kinase (ERK) within 1030 min. The use of SB203580, a p38 inhibitor, and PD98059, an ERK inhibitor, resulted in the restoration of dHL60 cell migration which was significantly inhibited treatment with rhMFGE8. Furthermore, blocking the MFGE8 receptors, αvß3/αvß5integrins, by antiαvintegrin neutralizing antibody (Ab) inhibited the activation of p38 and ERK, and reversed the rhMFGE8induced inhibition of dHL60 cell migration. Finally, treatment of the dHL60 cells with SB203580 and PD98059 neutralized the rhMFGE8induced downregulation of CXCR2 expression and upregulation of GRK2 expression, as well as the inhibitory effects on cell migration. Our findings reveal a novel mechanism of action of MFGE8 through which it inhibits neutrophil migration through αvß3-integrin-dependent MAP kinase activation.
Assuntos
Antígenos de Superfície/farmacologia , Movimento Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Integrina alfaVbeta3/antagonistas & inibidores , Proteínas do Leite/farmacologia , Neutrófilos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Flavonoides , Citometria de Fluxo , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Células HL-60 , Humanos , Imidazóis/farmacologia , Integrina alfaVbeta3/metabolismo , Interleucina-8/imunologia , Infiltração de Neutrófilos/efeitos dos fármacos , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Receptores de Interleucina-8B/biossíntese , Receptores de Interleucina-8B/metabolismo , Receptores de Vitronectina/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Renal ischemia-reperfusion (IR) injury (IRI) after shock states or transplantation causes tissue damage and delayed graft function, respectively. The Wnt/ß-catenin signaling pathway plays a critical role in nephrogenesis. We therefore hypothesized that pharmacological activation of the Wnt/ß-catenin signaling by the Wnt agonist, a synthetic pyrimidine, could protect kidneys from IRI. Adult male rats were subjected to bilateral clamping of the renal pedicles with microvascular clips for 60 min, followed by reperfusion. The Wnt agonist (5 mg/kg body weight) or vehicle (20% dimethyl sulfoxide in saline) was administered intravenously 1 h before ischemia. Blood and renal tissues were collected 24 h after IR for evaluation. Renal IR caused a significant reduction of ß-catenin and its downstream target gene cyclin D1 by 65% and 39%, respectively, compared with the sham, whereas the Wnt agonist restored them to sham levels. The number and intensity of cells staining with the proliferation marker Ki67 in ischematized kidneys were enhanced by the Wnt agonist. The integrity of the renal histological architecture in the Wnt agonist group was better preserved than the vehicle group. The Wnt agonist significantly lowered serum levels of creatinine, aspartate aminotransferase, and lactate dehydrogenase and inhibited the production of interleukin 6 and interleukin 1ß and myeloperoxidase activities. Lastly, the Wnt agonist reduced inducible nitric oxide synthase, nitrotyrosine proteins, and 4-hydroxynonenal in the kidneys by 60%, 47%, and 21%, respectively, compared with the vehicle. These results indicate that the Wnt agonist improves renal regeneration and function while attenuating inflammation and oxidative stress in the kidneys after IR. Thus, pharmacologic stimulation of the Wnt/ß-catenin signaling provides a beneficial effect on the prevention of renal IRI.