RESUMO
The production of type I interferon is essential for viral clearance but is kept under tight control to avoid unnecessary tissue damage from hyperinflammatory responses. Here we found that OASL1 inhibited translation of IRF7, the master transcription factor for type I interferon, and thus negatively regulated the robust production of type I interferon during viral infection. OASL1 inhibited the translation of IRF7 mRNA by binding to the 5' untranslated region (UTR) of IRF7 and possibly by inhibiting scanning of the 43S preinitiation complex along the message. Oasl1-/- mice were resistant to viral infection because of the greater abundance of type I interferon, which suggests that OASL1 could be a potential therapeutic target for boosting the production of type I interferon during viral infection.
Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Regulação da Expressão Gênica , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Biossíntese de Proteínas , 2',5'-Oligoadenilato Sintetase/genética , Regiões 5' não Traduzidas , Animais , Sequência de Bases , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linhagem Celular , Vírus da Encefalomiocardite/imunologia , Homozigoto , Humanos , Indutores de Interferon/administração & dosagem , Indutores de Interferon/farmacologia , Interferon Tipo I/biossíntese , Interferon Tipo I/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Poli I-C/administração & dosagem , Poli I-C/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/metabolismo , Viroses/genética , Viroses/imunologiaRESUMO
BACKGROUND: Macrophages produce many inflammation-associated molecules, released by matrix metalloproteinases, such as adhesion molecules, and cytokines, as well, which play a crucial role in atherosclerosis. In this context, we investigated the relationship between Ninjurin-1 (Ninj1 [nerve injury-induced protein]), a novel matrix metalloproteinase 9 substrate, expression, and atherosclerosis progression. METHODS: Ninj1 expression and atherosclerosis progression were assessed in atherosclerotic aortic tissue and serum samples from patients with coronary artery disease and healthy controls, and atheroprone apolipoprotein e-deficient (Apoe-/-) and wild-type mice, as well. Apoe-/- mice lacking systemic Ninj1 expression (Ninj1-/-Apoe-/-) were generated to assess the functional effects of Ninj1. Bone marrow transplantation was also used to generate low-density lipoprotein receptor-deficient (Ldlr-/-) mice that lack Ninj1 specifically in bone marrow-derived cells. Mice were fed a Western diet for 5 to 23 weeks, and atherosclerotic lesions were investigated. The anti-inflammatory role of Ninj1 was verified by treating macrophages and mice with the peptides Ninj11-56 (ML56) and Ninj126-37 (PN12), which mimic the soluble form of Ninj1 (sNinj1). RESULTS: Our in vivo results conclusively showed a correlation between Ninj1 expression in aortic macrophages and the extent of human and mouse atherosclerotic lesions. Ninj1-deficient macrophages promoted proinflammatory gene expression by activating mitogen-activated protein kinase and inhibiting the phosphoinositide 3-kinase/Akt signaling pathway. Whole-body and bone marrow-specific Ninj1 deficiencies significantly increased monocyte recruitment and macrophage accumulation in atherosclerotic lesions through elevated macrophage-mediated inflammation. Macrophage Ninj1 was directly cleaved by matrix metalloproteinase 9 to generate a soluble form that exhibited antiatherosclerotic effects, as assessed in vitro and in vivo. Treatment with the sNinj1-mimetic peptides, ML56 and PN12, reduced proinflammatory gene expression in human and mouse classically activated macrophages, thereby attenuating monocyte transendothelial migration. Moreover, continuous administration of mPN12 alleviated atherosclerosis by inhibiting the enhanced monocyte recruitment and inflammation characteristics of this disorder in mice, regardless of the presence of Ninj1. CONCLUSIONS: Ninj1 is a novel matrix metalloproteinase 9 substrate in macrophages, and sNinj1 is a secreted atheroprotective protein that regulates macrophage inflammation and monocyte recruitment in atherosclerosis. Moreover, sNinj1-mediated anti-inflammatory effects are conserved in human macrophages and likely contribute to human atherosclerosis.
Assuntos
Anti-Inflamatórios/farmacologia , Aterosclerose , Moléculas de Adesão Celular Neuronais , Macrófagos/metabolismo , Fatores de Crescimento Neural , Peptidomiméticos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Aterosclerose/tratamento farmacológico , Aterosclerose/genética , Aterosclerose/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Moléculas de Adesão Celular Neuronais/farmacologia , Feminino , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Knockout para ApoE , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/farmacologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genéticaRESUMO
Disruption of colonic homeostasis caused by aberrant M1/M2 macrophage polarization and dysbiosis contributes to inflammatory bowel disease (IBD) pathogenesis. However, the molecular factors mediating colonic homeostasis are not well characterized. Here, we found that Ninjurin1 (Ninj1) limits colon inflammation by regulating macrophage polarization and microbiota composition under homeostatic conditions and during colitis development. Ninj1 deletion in mice induced hypersusceptibility to colitis, with increased prevalence of colitogenic Prevotellaceae strains and decreased immunoregulatory Lachnospiraceae strains. Upon co-housing (CoH) with WT mice, Ninj1-/- mice showed increased Lachnospiraceae and decreased Prevotellaceae abundance, with subsequent improvement of colitis. Under homeostatic conditions, M1 macrophage frequency was higher in the Ninj1-/- mouse colons than wild-type (WT) mouse colons, which may contribute to increased basal colonic inflammation and microbial imbalance. Following colitis induction, Ninj1 expression was increased in macrophages; meanwhile Ninj1-/- mice showed severe colitis development and impaired recovery, associated with decreased M2 macrophages and escalated microbial imbalance. In vitro, Ninj1 knockdown in mouse and human macrophages activated M1 polarization and restricted M2 polarization. Finally, the transfer of WT macrophages ameliorated severe colitis in Ninj1-/- mice. These findings suggest that Ninj1 mediates colonic homeostasis by modulating M1/M2 macrophage balance and preventing extensive dysbiosis, with implications for IBD prevention and therapy.
Assuntos
Moléculas de Adesão Celular Neuronais/deficiência , Colite/metabolismo , Colite/patologia , Microbioma Gastrointestinal/fisiologia , Macrófagos/metabolismo , Macrófagos/patologia , Fatores de Crescimento Neural/deficiência , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Colite/microbiologia , Colo/metabolismo , Colo/microbiologia , Colo/patologia , Modelos Animais de Doenças , Homeostase/fisiologia , Humanos , Inflamação/metabolismo , Inflamação/microbiologia , Inflamação/patologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/microbiologia , Doenças Inflamatórias Intestinais/patologia , Ativação de Macrófagos/fisiologia , Masculino , Camundongos , Fatores de Crescimento Neural/metabolismo , Células THP-1/metabolismoRESUMO
Early events in atherosclerosis occur in the aortic intima and involve monocytes that become macrophages. We looked for these cells in the steady state adult mouse aorta, and surprisingly, we found a dominance of dendritic cells (DCs) in the intima. In contrast to aortic adventitial macrophages, CD11c(+)MHC II(hi) DCs were poorly phagocytic but were immune stimulatory. DCs were of two types primarily: classical Flt3-Flt3L signaling-dependent, CD103(+)CD11b(-) DCs and macrophage-colony stimulating factor (M-CSF)-dependent, CD14(+)CD11b(+)DC-SIGN(+) monocyte-derived DCs. Both types expanded during atherosclerosis. By crossing Flt3(-/-) to Ldlr(-/-) atherosclerosis-prone mice, we developed a selective and marked deficiency of classical CD103(+) aortic DCs, and they were associated with exacerbated atherosclerosis without alterations in blood lipids. Concomitantly, the Flt3(-/-)Ldlr(-/-) mice had fewer Foxp3(+) Treg cells and increased inflammatory cytokine mRNAs in the aorta. Therefore, functional DCs are dominant in normal aortic intima and, in contrast to macrophages, CD103(+) classical DCs are associated with atherosclerosis protection.
Assuntos
Aterosclerose/imunologia , Células Dendríticas/imunologia , Transdução de Sinais , Tirosina Quinase 3 Semelhante a fms/metabolismo , Animais , Antígenos CD/metabolismo , Aorta/efeitos dos fármacos , Aorta/imunologia , Aterosclerose/genética , Aterosclerose/patologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Regulação da Expressão Gênica/imunologia , Procedimentos de Redução de Leucócitos , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/imunologia , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
RATIONALE: Monocyte infiltration into the subintimal space and its intracellular lipid accumulation are the most prominent features of atherosclerosis. To understand the pathophysiology of atherosclerotic disease, we need to understand the characteristics of lipid-laden foamy macrophages in the subintimal space during atherosclerosis. OBJECTIVE: We sought to examine the transcriptomic profiles of foamy and nonfoamy macrophages isolated from atherosclerotic intima. METHODS AND RESULTS: Single-cell RNA sequencing analysis of CD45+ leukocytes from murine atherosclerotic aorta revealed that there are macrophage subpopulations with distinct differentially expressed genes involved in various functional pathways. To specifically characterize the intimal foamy macrophages of plaque, we developed a lipid staining-based flow cytometric method for analyzing the lipid-laden foam cells of atherosclerotic aortas. We used the fluorescent lipid probe BODIPY493/503 and assessed side-scattered light as an indication of cellular granularity. BODIPYhiSSChi foamy macrophages were found residing in intima and expressing CD11c. Foamy macrophage accumulation determined by flow cytometry was positively correlated with the severity of atherosclerosis. Bulk RNA sequencing analysis showed that compared with nonfoamy macrophages, foamy macrophages expressed few inflammatory genes but many lipid-processing genes. Intimal nonfoamy macrophages formed the major population expressing IL (interleukin)-1ß and many other inflammatory transcripts in atherosclerotic aorta. CONCLUSIONS: RNA sequencing analysis of intimal macrophages from atherosclerotic aorta revealed that lipid-loaded plaque macrophages are not likely the plaque macrophages that drive lesional inflammation.
Assuntos
Macrófagos/metabolismo , Placa Aterosclerótica/metabolismo , Transcriptoma , Animais , Aorta/metabolismo , Aorta/patologia , Células Cultivadas , Humanos , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Placa Aterosclerótica/patologiaRESUMO
RATIONALE: Circulating CTRP1 (C1q/TNF-α [tumor necrosis factor-α]-related protein 1) levels are increased in hypertensive patients compared with those in healthy subjects. Nonetheless, little is known about the molecular and physiological function of CTRP1 in blood pressure (BP) regulation. OBJECTIVE: To investigate the physiological/pathophysiological role of CTRP1 in BP regulation. METHODS AND RESULTS: CTRP1 production was increased to maintain normotension under dehydration conditions, and this function was impaired in inducible CTRP1 KO (knockout) mice (CTRP1 ΔCAG). The increase in CTRP1 under dehydration conditions was mediated by glucocorticoids, and the antagonist mifepristone prevented the increase in CTRP1 and attenuated BP recovery. Treatment with a synthetic glucocorticoid increased the transcription, translation, and secretion of CTRP1 from skeletal muscle cells. Functionally, CTRP1 increases BP through the stimulation of the AT1R (Ang II [angiotensin II] receptor 1)-Rho (Ras homolog gene family)/ROCK (Rho kinase)-signaling pathway to induce vasoconstriction. CTRP1 promoted AT1R plasma membrane trafficking through phosphorylation of AKT and AKT substrate of 160 kDa (AS160). In addition, the administration of an AT1R blocker, losartan, recovered the hypertensive phenotype of CTRP1 TG (transgenic) mice. CONCLUSIONS: For the first time, we provide evidence that CTRP1 contributes to the regulation of BP homeostasis by preventing dehydration-induced hypotension.
Assuntos
Adipocinas/metabolismo , Pressão Sanguínea , Desidratação/metabolismo , Hipotensão/metabolismo , Adipocinas/genética , Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Animais , Linhagem Celular , Células Cultivadas , Desidratação/complicações , Desidratação/fisiopatologia , Feminino , Glucocorticoides/metabolismo , Humanos , Hipotensão/tratamento farmacológico , Hipotensão/etiologia , Hipotensão/fisiopatologia , Losartan/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Vasoconstrição , Quinases Associadas a rho/metabolismoRESUMO
Ischemic myocardial injury results in sterile cardiac inflammation that leads to tissue repair, two processes controlled by mononuclear phagocytes. Despite global burden of cardiovascular diseases, we do not understand the functional contribution to pathogenesis of specific cardiac mononuclear phagocyte lineages, in particular dendritic cells. To address this limitation, we used detailed lineage tracing and genetic studies to identify bona fide murine and human CD103+ conventional dendritic cell (cDC)1s, CD11b+ cDC2s, and plasmacytoid DCs (pDCs) in the heart of normal mice and immunocompromised NSG mice reconstituted with human CD34+ cells, respectively. After myocardial infarction (MI), the specific depletion of cDCs, but not pDCs, improved cardiac function and prevented adverse cardiac remodeling. Our results showed that fractional shortening measured after MI was not influenced by the absence of pDCs. Interestingly, however, depletion of cDCs significantly improved reduction in fractional shortening. Moreover, fibrosis and cell areas were reduced in infarcted zones. This correlated with reduced numbers of cardiac macrophages, neutrophils, and T cells, indicating a blunted inflammatory response. Accordingly, mRNA levels of proinflammatory cytokines IL-1ß and IFN-γ were reduced. Collectively, our results demonstrate the unequivocal pathological role of cDCs following MI.
Assuntos
Movimento Celular/imunologia , Células Dendríticas/imunologia , Infarto do Miocárdio/imunologia , Animais , Movimento Celular/genética , Células Dendríticas/patologia , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Monócitos/imunologia , Monócitos/patologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Neutrófilos/imunologia , Neutrófilos/patologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
CpG, 5'-C-phosphate-G-3', islands (CGIs) have long been known for their association with enhancers, silencers, and promoters, and for their epigenetic signatures. They are maintained in embryonic stem cells (ESCs) in a poised but inactive state via the formation of bivalent chromatin containing both active and repressive marks. CGIs also occur within coding sequences, where their functional role has remained obscure. Intragenic CGIs (iCGIs) are largely absent from housekeeping genes, but they are found in all genes associated with organ development and cell lineage control. In this paper, we investigated the epigenetic status of iCGIs and found that they too reside in bivalent chromatin in ESCs. Cell type-specific DNA methylation of iCGIs in differentiated cells was linked to the loss of both the H3K4me3 and H3K27me3 marks, and disruption of physical interaction with promoter regions, resulting in transcriptional activation of key regulators of differentiation such as PAXs, HOXs, and WNTs. The differential epigenetic modification of iCGIs appears to be mediated by cell type-specific transcription factors distinct from those bound by promoter, and these transcription factors may be involved in the hypermethylation of iCGIs upon cell differentiation. iCGIs thus play a key role in the cell type-specific regulation of transcription.
Assuntos
Diferenciação Celular/genética , Ilhas de CpG/genética , Metilação de DNA/genética , Epigênese Genética/genética , Linhagem da Célula/genética , Cromatina/genética , Células-Tronco Embrionárias/citologia , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Humanos , Regiões Promotoras GenéticasRESUMO
The role of peroxisome proliferator-activated receptor α (PPAR-α) in innate host defense is largely unknown. In this study, we show that PPAR-α is essential for antimycobacterial responses via activation of transcription factor EB (TFEB) transcription and inhibition of lipid body formation. PPAR-α deficiency resulted in an increased bacterial load and exaggerated inflammatory responses during mycobacterial infection. PPAR-α agonists promoted autophagy, lysosomal biogenesis, phagosomal maturation, and antimicrobial defense against Mycobacterium tuberculosis or M. bovis bacillus Calmette-Guérin. PPAR-α agonists regulated multiple genes involved in autophagy and lysosomal biogenesis, including Lamp2, Rab7, and Tfeb in bone marrow-derived macrophages. Silencing of TFEB reduced phagosomal maturation and antimicrobial responses, but increased macrophage inflammatory responses during mycobacterial infection. Moreover, PPAR-α activation promoted lipid catabolism and fatty acid ß-oxidation in macrophages during mycobacterial infection. Taken together, our data indicate that PPAR-α mediates antimicrobial responses to mycobacterial infection by inducing TFEB and lipid catabolism.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/imunologia , Imunidade Inata/imunologia , Metabolismo dos Lipídeos/imunologia , Infecções por Mycobacterium/imunologia , PPAR alfa/imunologia , Animais , Autofagia/fisiologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Immunoblotting , Imuno-Histoquímica , Gotículas Lipídicas/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium , PPAR alfa/metabolismo , Reação em Cadeia da PolimeraseRESUMO
Defects in the renal fatty acid oxidation (FAO) pathway have been implicated in the development of renal fibrosis. Although, compared with young kidneys, aged kidneys show significantly increased fibrosis with impaired kidney function, the mechanisms underlying the effects of aging on renal fibrosis have not been investigated. In this study, we investigated peroxisome proliferator-activated receptor α (PPARα) and the FAO pathway as regulators of age-associated renal fibrosis. The expression of PPARα and the FAO pathway-associated proteins significantly decreased with the accumulation of lipids in the renal tubular epithelial region during aging in rats. In particular, decreased PPARα protein expression associated with increased expression of PPARα-targeting microRNAs. Among the microRNAs with increased expression during aging, miR-21 efficiently decreased PPARα expression and impaired FAO when ectopically expressed in renal epithelial cells. In cells pretreated with oleic acid to induce lipid stress, miR-21 treatment further enhanced lipid accumulation. Furthermore, treatment with miR-21 significantly exacerbated the TGF-ß-induced fibroblast phenotype of epithelial cells. We verified the physiologic importance of our findings in a calorie restriction model. Calorie restriction rescued the impaired FAO pathway during aging and slowed fibrosis development. Finally, compared with kidneys of aged littermate controls, kidneys of aged PPARα-/- mice showed exaggerated lipid accumulation, with decreased activity of the FAO pathway and a severe fibrosis phenotype. Our results suggest that impaired renal PPARα signaling during aging aggravates renal fibrosis development, and targeting PPARα is useful for preventing age-associated CKD.
Assuntos
Envelhecimento/metabolismo , Ácidos Graxos/metabolismo , Rim/patologia , PPAR alfa/metabolismo , Envelhecimento/patologia , Animais , Restrição Calórica , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Fibrose , Regulação da Expressão Gênica , Rim/metabolismo , Camundongos , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/farmacologia , Ácido Oleico/farmacologia , Oxirredução , PPAR alfa/deficiência , PPAR alfa/genética , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta/fisiologiaRESUMO
S-palmitoylation is an important post-translational modification that affects the translocation and the activity of target proteins in a variety of cell types including cardiomyocytes. Since endothelial nitric oxide synthase (eNOS) is known to be palmitoylated and the activity of eNOS is essential in fatty acid-dependent ß-oxidation in muscle, we aimed to test whether palmitoylation of eNOS is involved in palmitic acid (PA) regulation of left ventricular (LV) myocyte contraction from healthy (sham) and hypertensive (HTN) rats. Our results showed that PA, a predominant metabolic substrate for cardiac ß-oxidation, significantly increased contraction and oxygen consumption rate (OCR) in LV myocytes from sham. Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) or eNOS gene deletion prevented PA regulation of the myocyte contraction or OCR, indicating the pivotal role of eNOS in mediating the effects of PA in cardiac myocytes. PA increased the palmitoylation of eNOS in LV myocytes and depalmitoylation with 2-bromopalmitate (2BP; 100 µM) abolished the increment. Furthermore, although PA did not increase eNOS-Ser1177, 2BP reduced eNOS-Ser1177 with and without PA. Intriguingly, PA-induced increases in contraction and OCR were unaffected by 2BP treatment. In HTN, PA did not affect eNOS palmitoylation, eNOS-Ser1177, or myocyte contraction. However, 2BP diminished eNOS palmitoylation and eNOS-Ser1177 in the presence and absence of PA but did not change myocyte contraction. Collectively, our results confirm eNOS palmitoylation in LV myocytes from sham and HTN rats and its upregulation by PA in sham. However, such post-transcriptional modification plays negligible role in PA regulation of myocyte contraction and mitochondrial activity in sham and HTN.
Assuntos
Ácidos Graxos/metabolismo , Ventrículos do Coração/metabolismo , Hipertensão/metabolismo , Miócitos Cardíacos/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Animais , Arginina/análogos & derivados , Arginina/metabolismo , Modelos Animais de Doenças , Masculino , Miocárdio/metabolismo , NG-Nitroarginina Metil Éster/metabolismo , Consumo de Oxigênio/fisiologia , Ácido Palmítico/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Penile erection is a neurovascular phenomenon, and erectile dysfunction (ED) is caused mainly by vascular risk factors or diseases, neurologic abnormalities, and hormonal disturbances. Men with diabetic ED often have severe endothelial dysfunction and peripheral nerve damage, which result in poor response to oral phosphodiesterase-5 inhibitors. Nerve injury-induced protein 1 (Ninjurin 1, Ninj1) is known to be involved in neuroinflammatory processes and to be related to vascular regression during the embryonic period. Here, we demonstrate in streptozotocin-induced diabetic mice that inhibition of the Ninj1 pathway by administering Ninj1-neutralizing antibody (Ninj1-Ab) or by using Ninj1-knockout mice successfully restored erectile function through enhanced penile angiogenesis and neural regeneration. Angiopoietin-1 (Ang1) expression was down-regulated and angiopoietin-2 expression was up-regulated in the diabetic penis compared with that in controls, and these changes were reversed by treatment with Ninj1-Ab. Ninj1 blockade-mediated penile angiogenesis and neural regeneration as well as recovery of erectile function were abolished by inhibition of Ang1-Tie2 (tyrosine kinase with Ig and epidermal growth factor homology domain-2) signaling with soluble Tie2 antibody or Ang1 siRNA. The present results suggest that inhibition of the Ninj1 pathway will be a novel therapeutic strategy for treating ED.
Assuntos
Anticorpos Neutralizantes/farmacologia , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Complicações do Diabetes/tratamento farmacológico , Disfunção Erétil/tratamento farmacológico , Neovascularização Fisiológica/fisiologia , Fatores de Crescimento Neural/antagonistas & inibidores , Regeneração Nervosa/fisiologia , Ereção Peniana/fisiologia , Análise de Variância , Angiopoietina-1/metabolismo , Animais , Western Blotting , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/imunologia , Primers do DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Neovascularização Fisiológica/efeitos dos fármacos , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/imunologia , Regeneração Nervosa/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Ereção Peniana/efeitos dos fármacos , Receptor TIE-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
We previously reported that hypoxia augments α-adrenergic contraction (hypoxic vasoconstriction, HVC) of skeletal arteries in rats. The underlying mechanism may involve hypoxic inhibition of endothelial nitric oxide synthase (eNOS) expressed in skeletal arterial myocytes (16). To further explore the novel role of muscular eNOS in the skeletal artery, we compared HVC in femoral arteries (FAs) from eNOS knockout (KO) mice with that from wild-type (WT) and heterozygous (HZ) mice. Immunohistochemical assays revealed that, in addition to endothelia, eNOS is also expressed in the medial layer of FAs, albeit at a much lower level. However, the medial eNOS signal was not evident in HZ FAs, despite strong expression in the endothelium; similar observations were made in WT carotid arteries (CAs). The amplitude of contraction induced by 1 µM phenylephrine (PhE) was greater in HZ than in WT FAs. Hypoxia (3% Po2) significantly augmented PhE-induced contraction in WT FAs but not in HZ or KO FAs. No HVC was observed in PhE-pretreated WT CAs. The NOS inhibitor nitro-l-arginine methyl ester (0.1 mM) also augmented PhE contraction in endothelium-denuded WT FAs but not in WT CAs. Inhibitors specific to neuronal NOS and inducible NOS did not augment PhE-induced contraction of WT FAs. NADPH oxidase 4 (NOX4) inhibitor (GKT137831, 5 µM), but not NOX2 inhibitor (apocynin, 100 µM), suppressed HVC. Consistent with the role of reactive oxygen species (ROS), HVC was also inhibited by pretreatment with tiron or polyethylene glycol-catalase. Taken together, these data suggest that the eNOS expressed in smooth muscle cells in FAs attenuates α-adrenergic vasoconstriction; this suppression is alleviated under hypoxia, which potentiates vasoconstriction in a NOX4/ROS-dependent mechanism.
Assuntos
Artérias Carótidas/metabolismo , Endotélio/fisiologia , Artéria Femoral/metabolismo , Músculo Liso Vascular/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Vasoconstrição/fisiologia , Animais , Arginina/análogos & derivados , Arginina/metabolismo , Artérias Carótidas/efeitos dos fármacos , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Inibidores Enzimáticos/farmacologia , Artéria Femoral/efeitos dos fármacos , Artéria Femoral/fisiologia , Camundongos , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , NADPH Oxidase 4 , NADPH Oxidases/metabolismo , Fenilefrina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Vasoconstrição/efeitos dos fármacosRESUMO
Insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2), one of the most abundant circulating IGFBPs, is known to attenuate the biological action of IGF-1. Although the effect of IGFBP-2 in preventing metabolic disorders is well known, its regulatory mechanism remains unclear. In the present study, we demonstrated the transcriptional regulation of the Igfbp-2 gene by peroxisome-proliferator-activated receptor (PPAR) α in the liver. During fasting, both Igfbp-2 and PPARα expression levels were increased. Wy14643, a selective PPARα agonist, significantly induced Igfbp-2 gene expression in primary cultured hepatocytes. However, Igfbp-2 gene expression in Pparα null mice was not affected by fasting or Wy14643. In addition, through transient transfection and chromatin immunoprecipitation assay in fasted livers, we determined that PPARα bound to the putative PPAR-responsive element between -511 bp and -499 bp on the Igfbp-2 gene promoter, indicating that the Igfbp-2 gene transcription is activated directly by PPARα. To explore the role of PPARα in IGF-1 signalling, we treated primary cultured hepatocytes with Wy14643 and observed a decrease in the number of IGF-1 receptors (IGF-1Rs) and in Akt phosphorylation. No inhibition was observed in the hepatocytes isolated from Pparα null mice. These results suggest that PPARα controls IGF-1 signalling through the up-regulation of hepatic Igfbp-2 transcription during fasting and Wy14643 treatment.
Assuntos
Jejum/metabolismo , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Animais , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , PPAR alfa/deficiência , PPAR alfa/genética , PPAR gama/agonistas , Proliferadores de Peroxissomos/farmacologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rosiglitazona , Transdução de Sinais , Tiazolidinedionas/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Ninjurin1 is a homotypic adhesion molecule that contributes to leukocyte trafficking in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, in vivo gene deficiency animal studies have not yet been done. Here, we constructed Ninjurin1 knock-out (KO) mice and investigated the role of Ninjurin1 on leukocyte trafficking under inflammation conditions such as EAE and endotoxin-induced uveitis. Ninjurin1 KO mice attenuated EAE susceptibility by reducing leukocyte recruitment into the injury regions of the spinal cord and showed less adhesion of leukocytes on inflamed retinal vessels in endotoxin-induced uveitis mice. Moreover, the administration of a custom-made antibody (Ab26-37) targeting the Ninjurin1 binding domain ameliorated the EAE symptoms, showing the contribution of its adhesion activity to leukocyte trafficking. In addition, we addressed the transendothelial migration (TEM) activity of bone marrow-derived macrophages and Raw264.7 cells according to the expression level of Ninjurin1. TEM activity was decreased in Ninjurin1 KO bone marrow-derived macrophages and siNinj1 Raw264.7 cells. Consistent with this, GFP-tagged mNinj1-overexpressing Raw264.7 cells increased their TEM activity. Taken together, we have clarified the contribution of Ninjurin1 to leukocyte trafficking in vivo and delineated its direct functions to TEM, emphasizing Ninjurin1 as a beneficial therapeutic target against inflammatory diseases such as multiple sclerosis.
Assuntos
Células da Medula Óssea/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Movimento Celular , Encefalomielite Autoimune Experimental/metabolismo , Macrófagos/metabolismo , Fatores de Crescimento Neural/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Células da Medula Óssea/patologia , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Moléculas de Adesão Celular Neuronais/genética , Linhagem Celular , Suscetibilidade a Doenças , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/terapia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Esclerose Múltipla/terapia , Fatores de Crescimento Neural/antagonistas & inibidores , Fatores de Crescimento Neural/genéticaRESUMO
Ninjurin1 is involved in the pathogenesis of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, by mediating leukocyte extravasation, a process that depends on homotypic binding. However, the precise regulatory mechanisms of Ninjurin1 during inflammation are largely undefined. We therefore examined the pro-migratory function of Ninjurin1 and its regulatory mechanisms in macrophages. Interestingly, Ninjurin1-deficient bone marrow-derived macrophages exhibited reduced membrane protrusion formation and dynamics, resulting in the impairment of cell motility. Furthermore, exogenous Ninjurin1 was distributed at the membrane of filopodial structures in Raw264.7 macrophage cells. In Raw264.7 cells, RNA interference of Ninjurin1 reduced the number of filopodial projections, whereas overexpression of Ninjurin1 facilitated their formation and thus promoted cell motility. Ninjurin1-induced filopodial protrusion formation required the activation of Rac1. In Raw264.7 cells penetrating an MBEC4 endothelial cell monolayer, Ninjurin1 was localized to the membrane of protrusions and promoted their formation, suggesting that Ninjurin1-induced protrusive activity contributed to transendothelial migration. Taking these data together, we conclude that Ninjurin1 enhances macrophage motility and consequent extravasation of immune cells through the regulation of protrusive membrane dynamics. We expect these findings to provide insight into the understanding of immune responses mediated by Ninjurin1.
Assuntos
Moléculas de Adesão Celular Neuronais/fisiologia , Movimento Celular/fisiologia , Macrófagos/fisiologia , Fatores de Crescimento Neural/fisiologia , Animais , Adesão Celular/fisiologia , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/genética , Linhagem Celular , Membrana Celular/fisiologia , Células Cultivadas , Células Endoteliais/fisiologia , Técnicas de Silenciamento de Genes , Inflamação/etiologia , Inflamação/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Fatores de Crescimento Neural/deficiência , Fatores de Crescimento Neural/genética , Neuropeptídeos/metabolismo , Pseudópodes/fisiologia , Interferência de RNA , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
CD137 (4-1BB), a member of the tumor necrosis factor receptor superfamily, has been reported to be expressed in atherosclerotic plaques, and to promote lesion formation. However, the role of CD137 in mediating atherosclerotic plaque stability and the possible underlying molecular and cellular mechanisms are poorly understood. Here, apolipoprotein E-deficient (ApoE(-/-)) and CD137-deficient ApoE(-/-) (ApoE(-/-)CD137(-/-)) mice fed a chow diet for 66 wk were used. CD137 induces plaque instability, which is characterized by increased plaque necrosis, decreased collagen content, decreased vascular smooth muscle cell (VSMC) content, and increased macrophage infiltration. CD137 also increases the infiltration of effector T (Teff) cells into plaque lesion sites, resulting in increased interferon-γ (IFN-γ) expression. Interestingly, Teff-cell-derived IFN-γ inhibits collagen synthesis in atherosclerotic plaques. Furthermore, CD137 activation increases the apoptosis of VSMCs, possibly by decreasing the antiapoptotic regulator, Bcl-2, and subsequently up-regulating cleaved caspase-3. In macrophages, activation of CD137 signaling boosted the oxidized low density lipoprotein-induced expression of matrix metalloproteinase 9 via the p38 mitogen-activated protein kinase and extracellular signal-regulated kinase1/2 signaling pathways. In summary, activation of CD137 signaling decreases the stability of advanced atherosclerotic plaques via its combined effects on Teff cells, VSMCs, and macrophages.
Assuntos
Ligante 4-1BB/imunologia , Aterosclerose/metabolismo , Hiperlipidemias/metabolismo , Macrófagos/metabolismo , Placa Aterosclerótica/metabolismo , Linfócitos T/metabolismo , Animais , Apoptose/efeitos dos fármacos , Aterosclerose/imunologia , Interferon gama/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Placa Aterosclerótica/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologiaRESUMO
Cilia in ciliated cells consist of protruding structures that sense mechanical and chemical signals from the extracellular environment. Cilia are assembled with variety molecules via a process known as intraflagellar transport (IFT). What controls the length of cilia in ciliated cells is critical to understand ciliary disease such as autosomal dominant polycystic kidney disease, which involves abnormally short cilia. But this control mechanism is not well understood. Previously, multiple tubular cysts have been observed in the kidneys of max-interacting protein 1 (Mxi1)-deficient mice aged 6 months or more. Here, we clarified the relationship between Mxi1 inactivation and cilia disassembly. Cilia phenotypes were observed in kidneys of Mxi1-deficient mice using scanning electron microscopy to elucidate the effect of Mxi1 on renal cilia phenotype, and cilia disassembly was observed in Mxi1-deficient kidney. In addition, genes related to cilia were validated in vitro and in vivo using quantitative PCR, and Ift20 was selected as a candidate gene in this study. The length of cilium decreased, and p-ERK level induced by a cilia defect increased in kidneys of Mxi1-deficient mice. Ciliogenesis of Mxi1-deficient mouse embryonic fibroblasts (MEFs) decreased, and this abnormality was restored by Mxi1 transfection in Mxi1-deficient MEFs. We confirmed that ciliogenesis and Ift20 expression were regulated by Mxi1 in vitro. We also determined that Mxi1 regulates Ift20 promoter activity via Ets-1 binding to the Ift20 promoter. These results indicate that inactivating Mxi1 induces ciliary defects in polycystic kidney.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Transporte/biossíntese , Rim/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Transporte/genética , Células Cultivadas , Cílios/metabolismo , Cílios/ultraestrutura , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/ultraestrutura , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Regulação da Expressão Gênica/genética , Rim/ultraestrutura , Camundongos , Camundongos Mutantes , Microscopia Eletrônica de Varredura , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , Reação em Cadeia da Polimerase , Proteína Proto-Oncogênica c-ets-1/genética , Proteína Proto-Oncogênica c-ets-1/metabolismo , Elementos de Resposta/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Flo8 is a transcriptional activator essential for the inducible expression of a set of target genes such as STA1, FLO11, and FLO1 encoding an extracellular glucoamylase and two cell surface proteins, respectively. However, the molecular mechanism of Flo8-mediated transcriptional activation remains largely elusive. By generating serial deletion constructs, we revealed here that a novel transcriptional activation domain on its extreme C-terminal region plays a crucial role in activating transcription. On the other hand, the N-terminal LisH motif of Flo8 appears to be required for its physical interaction with another transcriptional activator, Mss11, for their cooperative transcriptional regulation of the shared targets. Additionally, GST pull-down experiments uncovered that Flo8 and Mss11 can directly form either a heterodimer or a homodimer capable of binding to DNA, and we also showed that this formed complex of two activators interacts functionally and physically with the Swi/Snf complex. Collectively, our findings provide valuable clues for understanding the molecular mechanism of Flo8-mediated transcriptional control of multiple targets.