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1.
Cytotherapy ; 23(10): 874-885, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34116946

RESUMO

BACKGROUND AIMS: Cell-based regenerative medicine is an innovative field that can potentially alter the overall survival and quality of life of patients with devastating diseases. Several cell therapy products (CTPs) have been approved within the last two decades, and more are under development. The establishment of an effective developmental strategy in accordance with the regulatory bodies of each country/region is crucial for fast delivery of each respective CTP. In particular, facilitating investigational new drug (IND) approval is important for accelerating the transition from non-clinical to clinical research/trial phases. METHODS: Here the authors compared the non-clinical prerequisites for initiating clinical studies in five Asian countries/regions (India, China, Korea, Taiwan and Japan) from an industry viewpoint. The authors first identified the differences and tried to clarify the perspectives/considerations underpinning the different requirements. RESULTS: The authors' findings revealed that differences in regulations and development experiences, especially with CTPs, have led to clear differences in the non-clinical study package and its corresponding study design. CONCLUSIONS: By sharing experiences of the research and development of CTPs among Asian countries/regions and including not only industry but also regulatory authorities, we will be able to expedite cross-border IND approval and eventually contribute to the early delivery of innovative CTPs to many Asian patients.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Qualidade de Vida , Ásia , China , Humanos , Japão
2.
Arthritis Rheumatol ; 72(7): 1192-1202, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32134203

RESUMO

OBJECTIVE: The NLRP3 inflammasome is closely linked to the pathophysiology of a wide range of inflammatory diseases. This study was undertaken to identify small molecules that directly bind to NLRP3 in order to develop pharmacologic interventions for NLRP3-related diseases. METHODS: A structure-based virtual screening analysis was performed with ~62,800 compounds to select efficient NLRP3 inhibitors. The production of caspase 1-p10 and interleukin-1ß (IL-1ß) was measured by immunoblotting and enzyme-linked immunosorbent assay to examine NLRP3 inflammasome activation. Two gouty arthritis models and an air pouch inflammation model induced by monosodium urate monohydrate (MSU) crystal injection were used for in vivo experiments. Primary synovial fluid cells from gout patients were used to determine the relevance of NLRP3 inflammasome inhibition in human gout. RESULTS: Beta-carotene (provitamin A) suppressed the NLRP3 inflammasome activation induced by various activators, including MSU crystals, in mouse bone marrow-derived primary macrophages (P < 0.05). Surface plasmon resonance analysis demonstrated the direct binding of ß-carotene to the pyrin domain (PYD) of NLRP3 (KD = 3.41 × 10-6 ). Molecular modeling and mutation assays revealed the interaction mode between ß-carotene and the NLRP3 PYD. Inflammatory symptoms induced by MSU crystals were attenuated by oral administration of ß-carotene in gouty arthritis mouse models (P < 0.05), correlating with its suppressive effects on the NLRP3 inflammasome in inflamed tissues. Furthermore, ß-carotene reduced IL-1ß secretion from human synovial fluid cells isolated from gout patients (P < 0.05), showing its inhibitory efficacy in human gout. CONCLUSION: Our results present ß-carotene as a selective and direct inhibitor of NLRP3, and the binding of ß-carotene to NLRP3 PYD as a novel pharmacologic strategy to combat NLRP3 inflammasome-driven diseases, including gouty arthritis.


Assuntos
Artrite Gotosa/imunologia , Inflamassomos/antagonistas & inibidores , Inflamação/imunologia , Macrófagos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/efeitos dos fármacos , Provitaminas/farmacologia , beta Caroteno/farmacologia , Animais , Caspase 1/efeitos dos fármacos , Caspase 1/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Gota/imunologia , Humanos , Inflamassomos/imunologia , Inflamassomos/metabolismo , Interleucina-1beta/efeitos dos fármacos , Interleucina-1beta/imunologia , Macrófagos/imunologia , Camundongos , Simulação de Acoplamento Molecular , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Domínio Pirina , Ressonância de Plasmônio de Superfície , Líquido Sinovial/citologia
3.
Mol Carcinog ; 58(1): 88-101, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30230030

RESUMO

Ras/Raf/MEKs/ERKs and PI3 K/Akt/mTOR signaling pathways have key roles in cancer development and growth processes, as well as in cancer malignance and chemoresistance. In this study, we screened the therapeutic potential of magnolin using 15 human cancer cell lines and combined magnolin sensitivity with the CCLE mutaome analysis for relevant mutation information. The results showed that magnolin efficacy on cell proliferation inhibition were lower in TOV-112D ovarian cancer cells than that in SKOV3 cells by G1 and G2/M cell cycle phase accumulation. Notably, magnolin suppressed colony growth of TOV-112D cells in soft agar, whereas colony growth of SKOV3 cells in soft agar was not affected by magnolin treatment. Interestingly, phospho-protein profiles in the MAPK and PI3 K signaling pathways indicated that SKOV3 cells showed marked increase of Akt phosphorylation at Thr308 and Ser473 and very weak ERK1/2 phosphorylation levels by EGF stimulation. The phospho-protein profiles in TOV-112D cells were the opposite of those of SKOV3 cells. Importantly, magnolin treatment suppressed phosphorylation of RSKs in TOV-112D, but not in SKOV3 cells. Moreover, magnolin increased SA-ß-galactosidase-positive cells in a dose-dependent manner in TOV-112D cells, but not in SKOV3 cells. Notably, oral administration of Shin-Yi fraction 1, which contained magnolin approximately 53%, suppressed TOV-112D cell growth in athymic nude mice by induction of p16Ink4a and p27Kip1 . Taken together, targeting of ERK1 and ERK2 is suitable for the treatment of ovarian cancer cells that do not harbor the constitutive active P13 K mutation and the loss-of-function mutations of the p16 and/or p53 tumor suppressor proteins.


Assuntos
Proliferação de Células/efeitos dos fármacos , Senescência Celular , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Lignanas/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Neoplasias Ovarianas/patologia , Animais , Apoptose , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Masculino , Camundongos , Camundongos Nus , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Nutr Rev ; 69(6): 310-20, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21631512

RESUMO

Emerging evidence reveals that pattern-recognition receptors (PRRs), Toll-like receptors (TLRs), and nucleotide-binding oligomerization domain proteins (NODs) mediate both infection-induced and sterile inflammation by recognizing pathogen-associated molecular patterns and endogenous molecules, respectively. PRR-mediated chronic inflammation is a determinant for the development and progression of chronic diseases including cancer, atherosclerosis, and insulin resistance. Recent studies demonstrated that certain phytochemicals inhibit PRR-mediated pro-inflammation. Curcumin, helenalin, and cinnamaldehyde with α, ß-unsaturated carbonyl groups, or sulforaphane with an isothiocyanate group, inhibit TLR4 activation by interfering with cysteine residue-mediated receptor dimerization, while resveratrol, with no unsaturated carbonyl group, did not. Similarly, curcumin, parthenolide, and helenalin, but not resveratrol and (-)-epigallocatechin-3-gallate (EGCG), also inhibit NOD2 activation by interfering with NOD2 dimerization. In contrast, resveratrol, EGCG, luteolin, and structural analogs of luteolin specifically inhibit TLR3 and TLR4 signaling by targeting TANK binding kinase 1 (TBK1) and receptor interacting protein 1 (RIP1) in Toll/IL-1 receptor domain-containing adaptor inducing IFN-ß (TRIF) complex. Together, these results suggest that PRRs and downstream signaling components are molecular targets for dietary strategies to reduce PRR-mediated chronic inflammation and consequent risks of chronic diseases.


Assuntos
Anti-Inflamatórios não Esteroides/metabolismo , Dieta , Inflamação/prevenção & controle , Plantas Comestíveis/química , Receptores de Reconhecimento de Padrão/antagonistas & inibidores , Transdução de Sinais , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Humanos , Inflamação/dietoterapia , Proteínas Adaptadoras de Sinalização NOD/antagonistas & inibidores , Receptores Toll-Like/antagonistas & inibidores
5.
Nutr Rev ; 68(1): 38-61, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20041999

RESUMO

Chronic inflammation is known to promote the development of many chronic diseases. Pattern recognition receptors (PRRs), Toll-like receptors (TLRs), and nucleotide-binding oligomerization domain proteins (NODs) mediate both infection-induced inflammation and sterile inflammation by recognizing pathogen- associated molecular patterns and endogenous molecules, respectively. PRR-mediated inflammation is an important determinant in altering the risk of many chronic diseases. Saturated fatty acids (SFAs) can activate PRRs, leading to enhanced expression of pro-inflammatory target gene products. However, n-3 polyunsaturated fatty acids (PUFAs) inhibit agonist-induced activation of PRRs. These results suggest that SFAs and n-3 PUFAs can reciprocally modulate PRR-mediated inflammation, and that PRRs and their downstream signaling components are molecular targets for dietary strategies to reduce chronic inflammation and subsequent risk of chronic diseases. This advancement in knowledge provides a new paradigm for understanding the mechanism by which different dietary fatty acids modify risk of chronic diseases including insulin resistance, atherosclerosis, and cancer.


Assuntos
Gorduras na Dieta , Ácidos Graxos , Inflamação/fisiopatologia , Receptores de Reconhecimento de Padrão/fisiologia , Animais , Doença Crônica/prevenção & controle , Ácidos Graxos/metabolismo , Ácidos Graxos/fisiologia , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-3/fisiologia , Humanos , Proteínas Adaptadoras de Sinalização NOD/agonistas , Proteínas Adaptadoras de Sinalização NOD/antagonistas & inibidores , Proteínas Adaptadoras de Sinalização NOD/fisiologia , Receptores de Reconhecimento de Padrão/agonistas , Receptores de Reconhecimento de Padrão/antagonistas & inibidores , Fatores de Risco , Receptores Toll-Like/agonistas , Receptores Toll-Like/antagonistas & inibidores , Receptores Toll-Like/fisiologia
6.
Biochem Pharmacol ; 75(7): 1515-25, 2008 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-18243161

RESUMO

Nucleotide-binding oligomerization domain containing proteins (Nods) are intracellular pattern recognition receptors (PRRs) that recognize conserved moieties of bacterial peptidoglycan and activate downstream signaling pathways, including NF-kappaB pathway. Here, we show that Nod2 agonist muramyldipeptide (MDP) induces Akt phosphorylation in time and dose-dependent manner. The pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) (wortmannin) and dominant-negative forms of p85 (the regulatory subunit of PI3K) or Akt enhance, while constitutive active forms of p110 (the catalytic subunit of PI3K) or Akt inhibit, NF-kappaB activation and the target gene interleukin (IL)-8 induced by MDP. In addition, the pharmacological inhibitors of PI3K (wortmannin and LY294002) enhance phosphorylation of NF-kappaB p65 on Ser529 and Ser536 residues, which result in enhanced p65 transactivation activity. Furthermore, we show that the inhibition of PI3K by the pharmacological inhibitors prevent the inactivation of glycogen synthase kinase (GSK)-3beta, suggesting that the negative regulation of PI3K/Akt on MDP-induced NF-kappaB activation is at least in part mediated through inactivation of GSK-3beta. Taken together, our results demonstrate that PI3K/Akt pathway is activated by Nod2 agonist MDP and negatively regulates NF-kappaB pathway downstream of Nod2 activation. Our results suggest that PI3K/Akt pathway may involve in the resolution of inflammatory responses induced by Nod2 activation.


Assuntos
NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Humanos , NF-kappa B/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD2/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Transdução de Sinais/efeitos dos fármacos
7.
Biochem Pharmacol ; 75(2): 494-502, 2008 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-17920563

RESUMO

Toll-like receptors (TLRs) play a critical role in induction of innate immune and inflammatory responses by recognizing invading pathogens or non-microbial endogenous molecules. TLRs have two major downstream signaling pathways, MyD88- and TRIF-dependent pathways leading to the activation of NFkappaB and IRF3 and the expression of inflammatory mediators. Deregulation of TLR activation is known to be closely linked to the increased risk of many chronic diseases. Cinnamaldehyde (3-phenyl-2-propenal) has been reported to inhibit NFkappaB activation induced by pro-inflammatory stimuli and to exert anti-inflammatory and anti-bacterial effects. However, the underlying mechanism has not been clearly identified. Our results showed that cinnamaldehyde suppressed the activation of NFkappaB and IRF3 induced by LPS, a TLR4 agonist, leading to the decreased expression of target genes such as COX-2 and IFNbeta in macrophages (RAW264.7). Cinnamaldehyde did not inhibit the activation of NFkappaB or IRF3 induced by MyD88-dependent (MyD88, IKKbeta) or TRIF-dependent (TRIF, TBK1) downstream signaling components. However, oligomerization of TLR4 induced by LPS was suppressed by cinnamaldehyde resulting in the downregulation of NFkappaB activation. Further, cinnamaldehyde inhibited ligand-independent NFkappaB activation induced by constitutively active TLR4 or wild-type TLR4. Our results demonstrated that the molecular target of cinnamaldehyde in TLR4 signaling is oligomerization process of receptor, but not downstream signaling molecules suggesting a novel mechanism for anti-inflammatory activity of cinnamaldehyde.


Assuntos
Acroleína/análogos & derivados , Anti-Inflamatórios/farmacologia , Receptor 4 Toll-Like/antagonistas & inibidores , Acroleína/farmacologia , Animais , Linhagem Celular , Humanos , Fator Regulador 3 de Interferon/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Camundongos , NF-kappa B/antagonistas & inibidores , Receptor 4 Toll-Like/química , Receptor 4 Toll-Like/fisiologia
8.
J Biol Chem ; 282(16): 11618-28, 2007 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-17303577

RESUMO

Nucleotide-binding oligomerization domain-containing proteins (Nods) are intracellular pattern recognition receptors recognizing conserved moieties of bacterial peptidoglycan through their leucine-rich repeats domain. The agonists for Nods activate proinflammatory signaling pathways, including NF-kappaB pathways. The results from our previous studies showed that the activation of TLR4 and TLR2, leucine-rich repeat-containing pattern recognition receptors, were differentially modulated by saturated and n-3 polyunsaturated fatty acids in macrophages and dendritic cells. Here, we show the differential modulation of NF-kappaB activation and interleukin-8 (IL-8) expression in colonic epithelial cells HCT116 by saturated and unsaturated fatty acids mediated through Nods proteins. Lauric acid (C12:0) dose dependently activated NF-kappaB and induced IL-8 expression in HCT116 cells, which express both Nod1 and Nod2, but not detectable amounts of TLR2 and TLR4. These effects of lauric acid were inhibited by dominant negative forms of Nod1 or Nod2, but not by dominant negative forms of TLR2, TLR4, and TLR5. The effects of lauric acid were also attenuated by small RNA interference targeting Nod1 or Nod2. In contrast, polyunsaturated fatty acids, especially n-3 polyunsaturated fatty acids, inhibited the activation of NF-kappaB and IL-8 expression induced by lauric acid or known Nods ligands in HCT116. Furthermore, lauric acid induced, but docosahexaenoic acid inhibited lauric acid- or Nod2 ligand MDP-induced, Nod2 oligomerization in HEK293T cells transfected with Nod2. Together, these results provide new insights into the role of dietary fatty acids in modulating inflammation in colon epithelial cells. The results suggest that Nods may be involved in inducing sterile inflammation, one of the key etiological conditions in the development of many chronic inflammatory diseases.


Assuntos
Colo/citologia , Células Epiteliais/citologia , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica , Transdução de Sinais , Linhagem Celular , Humanos , Inflamação/patologia , Interleucina-8/biossíntese , Interleucina-8/metabolismo , Ácidos Láuricos/metabolismo , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Estrutura Terciária de Proteína , Ativação Transcricional
9.
Immunology ; 119(3): 421-9, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17067317

RESUMO

Glucocorticoid-induced tumour necrosis factor receptor family related protein (GITR) is the 18th member of the tumour necrosis factor receptor superfamily (TNFRSF18) and is known to interact with its cognate ligand GITRL (TNFSF18). We investigated the potential role of GITR in the pro-inflammatory activation of macrophages. Immunohistochemistry and in situ hybridization analyses of human atherosclerotic plaques demonstrated that GITR and its ligand are expressed mainly in lipid-rich macrophages. We then investigated the role of GITR in human and mouse monocyte/macrophage functions. Stimulation of GITR caused nuclear factor (NF)-kappaB-dependent activation of matrix metalloproteinase-9 (MMP-9) and pro-inflammatory cytokine expression in both the human and mouse monocytic/macrophage cell lines, THP-1 and RAW264.7, respectively. These cellular responses were also observed when the THP-1 cells were treated with phorbol-12 myristate-13 acetate (PMA), which is known to induce macrophage differentiation. To demonstrate that these responses are not restricted to cultured cell lines, we tested primary macrophages. Both peritoneal and bone marrow-derived macrophages responded to GITR stimulation with induction of MMP-9 and tumour necrosis factor-alpha (TNF-alpha). Furthermore, the GITR staining pattern overlapped with those of MMP-9 and TNF-alpha in atherosclerotic plaques. These data indicate that GITR-mediated macrophage activation may promote atherogenesis via the induction of pro-atherogenic cytokines/chemokines, and destabilize the atherosclerotic plaques via the induction of the matrix-degrading enzyme, MMP-9.


Assuntos
Aterosclerose/imunologia , Proteína Relacionada a TNFR Induzida por Glucocorticoide/imunologia , Ativação de Macrófagos/imunologia , Idoso , Idoso de 80 Anos ou mais , Animais , Aterosclerose/patologia , Estenose das Carótidas/imunologia , Estenose das Carótidas/patologia , Células Cultivadas , Citocinas/metabolismo , Ativação Enzimática , Proteína Relacionada a TNFR Induzida por Glucocorticoide/metabolismo , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
10.
Biochem Biophys Res Commun ; 350(4): 866-71, 2006 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-17034761

RESUMO

Toll-like receptors (TLRs), which are activated by invading microorganisms or endogenous molecules, evoke immune and inflammatory responses. TLR activation is closely linked to the development of many chronic inflammatory diseases including rheumatoid arthritis. Auranofin, an Au(I) compound, is a well-known and long-used anti-rheumatic drug. However, the mechanism as to how auranofin relieves the symptom of rheumatoid arthritis has not been fully clarified. Our results demonstrated that auranofin suppressed TLR4-mediated activation of transcription factors, NF-kappaB and IRF3, and expression of COX-2, a pro-inflammatory enzyme. This suppression was well correlated with the inhibitory effect of auranofin on the homodimerization of TLR4 induced by an agonist. Furthermore, auranofin inhibited NF-kappaB activation induced by MyD88-dependent downstream signaling components of TLR4, MyD88, IKKbeta, and p65. IRF3 activation induced by MyD88-independent signaling components, TRIF and TBK1, was also downregulated by auranofin. Our results first demonstrate that auranofin suppresses the multiple steps in TLR4 signaling, especially the homodimerization of TLR4. The results suggest that the suppression of TLR4 activity by auranofin may be the molecular mechanism through which auranofin exerts anti-rheumatic activity.


Assuntos
Auranofina/administração & dosagem , Linfócitos B/metabolismo , Lipopolissacarídeos/administração & dosagem , Receptor 4 Toll-Like/metabolismo , Animais , Antirreumáticos/administração & dosagem , Linfócitos B/efeitos dos fármacos , Linhagem Celular , Dimerização , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Ouro/administração & dosagem , Camundongos
11.
Biochem Pharmacol ; 72(7): 850-9, 2006 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-16890209

RESUMO

Toll-like receptors (TLRs) play an important role in recognition of microbial components and induction of innate immunity. The microbial components trigger the activation of two downstream signaling pathways of TLRs; MyD88- and/or TRIF-dependent pathways leading to activation of NF-kappaB. (-)-Epigallocatechin-3-gallate (EGCG), a flavonoid found in green tea, is known to inhibit NF-kappaB activation induced by many pro-inflammatory stimuli. EGCG was shown to inhibit the activity of IKKbeta which is the key kinase in the canonical pathway for NF-kappaB activation in MyD88-dependent pathway of TLRs. However, it is not known whether EGCG inhibits TRIF-dependent pathway through which more than 70% of lipopolysaccharide (LPS)-induced genes are regulated. Therefore, we attempted to identify the molecular target of EGCG in TRIF-dependent pathways of TLR3 and TLR4. EGCG inhibited the activation of IFN regulatory factor 3 (IRF3) induced by LPS, poly[I:C], or the overexpression of TRIF. The inhibition of IRF3 activation by EGCG was mediated through the suppression of the kinase activity of TBK1. However, EGCG did not inhibit activation of IRF3 induced by overexpression of constitutively active IRF3. These results suggest that the molecular target of EGCG is TBK1 in TRIF-dependent signaling pathways of TLR3 and TLR4. Therefore, our results suggest that green tea flavonoids can modulate both MyD88- and TRIF-dependent signaling pathways of TLRs and subsequent inflammatory target gene expression.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Catequina/análogos & derivados , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/fisiologia , Animais , Catequina/química , Catequina/farmacologia , Linhagem Celular , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Flavonoides/química , Flavonoides/farmacologia , Expressão Gênica/genética , Humanos , Quinase I-kappa B/metabolismo , Interferon gama/genética , Luciferases/genética , Luciferases/metabolismo , Camundongos , Fator 88 de Diferenciação Mieloide , NF-kappa B/genética , NF-kappa B/metabolismo , Fenóis/química , Fenóis/farmacologia , Polifenóis , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Chá/química , Receptor 2 Toll-Like/fisiologia , Receptor 4 Toll-Like/fisiologia , Transfecção , Quinase Induzida por NF-kappaB
12.
Mol Cells ; 21(2): 174-85, 2006 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-16682810

RESUMO

Toll-like receptors (TLRs) were evolved to detect invading pathogens and to induce innate immune responses in order to mount host defense mechanisms. It becomes apparent that the activation of certain TLRs is also modulated by endogenous molecules including lipid components, fatty acids. Results from epidemiological and animal studies demonstrated that saturated and polyunsaturated dietary fatty acids can differentially modify the risk of development of many chronic diseases. Inflammation is now recognized as an important underlying etiologic condition for the pathogenesis of many chronic diseases. Therefore, if the activation of TLRs and consequent inflammatory and immune responses are differentially modulated by types of lipids in vivo, this would suggest that the risk of the development of chronic inflammatory diseases and the host defense against microbial infection may be modified by the types of dietary fat consumed.


Assuntos
Regulação da Expressão Gênica , Inflamação/genética , Lipídeos , Receptores Toll-Like/metabolismo , Animais , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Humanos , Transdução de Sinais/fisiologia
13.
Biochem Pharmacol ; 70(8): 1231-40, 2005 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-16140274

RESUMO

Bacterial lipopolysaccharide (LPS) activates Toll-like receptor 4 (TLR4) leading to the expression of inflammatory gene products. Src-family tyrosine kinases (STKs) are known to be activated by LPS in monocytes/macrophages. Therefore, we determined the role of STKs in TLR4 signaling pathways and target gene expression in macrophages. The activation of NFkappaB, and p38 MAPK, and the expression of inducible nitric oxide synthase (iNOS) induced by LPS were not affected in macrophages deficient in three STKs (Lyn, Hck, and Fgr). These results suggest that the deletion of the three STKs among possibly nine STKs is not sufficient to abolish total activity of STKs possibly due to the functional redundancy of other STKs present in macrophages. However, two structurally unrelated pan-inhibitors of STKs, PP1 and SU6656, suppressed LPS-induced iNOS expression in MyD88-knockout as well as wild-type macrophages. The suppression of iNOS expression by the inhibitors was correlated with the downregulation of IFNbeta (a MyD88-independent gene) expression and subsequent decrease in STAT1 phosphorylation. Moreover, PP1 suppressed the expression of IFNbeta and iNOS induced by TRIF, a MyD88-independent adaptor of TLR4. PP1 suppressed STAT1 phosphorylation induced by LPS, but not by IFNbeta suggesting that STKs are involved in the primary downstream signaling pathways of TLR4, but not the secondary signaling pathways downstream of IFNbeta receptor. Together, these results demonstrate that STKs play a positive regulatory role in TLR4-mediated iNOS expression in a MyD88-independent (TRIF-dependent) manner. These results provide new insight in understanding the role of STKs in TLR4 signaling pathways and inflammatory target gene expression.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos de Diferenciação/metabolismo , Receptores Imunológicos/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Quinases da Família src/metabolismo , Animais , Sequência de Bases , Western Blotting , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Fator 88 de Diferenciação Mieloide , Fosforilação , Quinases da Família src/antagonistas & inibidores
14.
J Immunol ; 175(5): 3339-46, 2005 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16116226

RESUMO

TLRs can activate two distinct branches of downstream signaling pathways. MyD88 and Toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF) pathways lead to the expression of proinflammatory cytokines and type I IFN genes, respectively. Numerous reports have demonstrated that resveratrol, a phytoalexin with anti-inflammatory effects, inhibits NF-kappaB activation and other downstream signaling pathways leading to the suppression of target gene expression. However, the direct targets of resveratrol have not been identified. In this study, we attempted to identify the molecular target for resveratrol in TLR-mediated signaling pathways. Resveratrol suppressed NF-kappaB activation and cyclooxygenase-2 expression in RAW264.7 cells following TLR3 and TLR4 stimulation, but not TLR2 or TLR9. Further, resveratrol inhibited NF-kappaB activation induced by TRIF, but not by MyD88. The activation of IFN regulatory factor 3 and the expression of IFN-beta induced by LPS, poly(I:C), or TRIF were also suppressed by resveratrol. The suppressive effect of resveratrol on LPS-induced NF-kappaB activation was abolished in TRIF-deficient mouse embryonic fibroblasts, whereas LPS-induced degradation of IkappaBalpha and expression of cyclooxygenase-2 and inducible NO synthase were still inhibited in MyD88-deficient macrophages. Furthermore, resveratrol inhibited the kinase activity of TANK-binding kinase 1 and the NF-kappaB activation induced by RIP1 in RAW264.7 cells. Together, these results demonstrate that resveratrol specifically inhibits TRIF signaling in the TLR3 and TLR4 pathway by targeting TANK-binding kinase 1 and RIP1 in TRIF complex. The results raise the possibility that certain dietary phytochemicals can modulate TLR-derived signaling and inflammatory target gene expression and can alter susceptibility to microbial infection and chronic inflammatory diseases.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/antagonistas & inibidores , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Glicoproteínas de Membrana/fisiologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptores de Superfície Celular/fisiologia , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/fisiologia , Transdução de Sinais/efeitos dos fármacos , Estilbenos/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Animais , Antígenos de Diferenciação/fisiologia , Linhagem Celular , Ciclo-Oxigenase 2 , Proteínas Ativadoras de GTPase/fisiologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Fator 88 de Diferenciação Mieloide , NF-kappa B/metabolismo , Poli I-C/farmacologia , Prostaglandina-Endoperóxido Sintases/biossíntese , Proteínas Serina-Treonina Quinases/fisiologia , Resveratrol , Receptor 2 Toll-Like , Receptor 3 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-Like
15.
J Immunol ; 174(9): 5390-7, 2005 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-15843537

RESUMO

TLRs provide critical signals to induce innate immune responses in APCs such as dendritic cells (DCs) that in turn link to adaptive immune responses. Results from our previous studies demonstrated that saturated fatty acids activate TLRs, whereas n-3 polyunsaturated fatty acids inhibit agonist-induced TLR activation. These results raise a significant question as to whether fatty acids differentially modulate immune responses mediated through TLR activation. The results presented in this study demonstrate that the saturated fatty acid, lauric acid, up-regulates the expression of costimulatory molecules (CD40, CD80, and CD86), MHC class II, and cytokines (IL-12p70 and IL-6) in bone marrow-derived DCs. The dominant negative mutant of TLR4 or its downstream signaling components inhibits lauric acid-induced expression of a CD86 promoter-reporter gene. In contrast, an n-3 polyunsaturated fatty acid, docosahexaenoic acid, inhibits TLR4 agonist (LPS)-induced up-regulation of the costimulatory molecules, MHC class II, and cytokine production. Similarly, DCs treated with lauric acid show increased T cell activation capacity, whereas docosahexaenoic acid inhibits T cell activation induced by LPS-treated DCs. Together, our results demonstrate that the reciprocal modulation of both innate and adaptive immune responses by saturated fatty acid and n-3 polyunsaturated fatty acid is mediated at least in part through TLRs. These results imply that TLRs are involved in sterile inflammation and immune responses induced by nonmicrobial endogenous molecules. These results shed new light in understanding how types of dietary fatty acids differentially modulate immune responses that could alter the risk of many chronic diseases.


Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Láuricos/farmacologia , Receptores Imunológicos/fisiologia , Transdução de Sinais/imunologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígeno B7-2 , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Antígeno CD11c/biossíntese , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Células Cultivadas , Citocinas/antagonistas & inibidores , Citocinas/biossíntese , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Knockout , Dados de Sequência Molecular , Regiões Promotoras Genéticas/efeitos dos fármacos , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Linfócitos T/imunologia , Receptor 4 Toll-Like , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia
16.
J Biol Chem ; 279(17): 16971-9, 2004 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-14966134

RESUMO

Toll-like receptor 4 (TLR4) and TLR2 agonists from bacterial origin require acylated saturated fatty acids in their molecules. Previously, we reported that TLR4 activation is reciprocally modulated by saturated and polyunsaturated fatty acids in macrophages. However, it is not known whether fatty acids can modulate the activation of TLR2 or other TLRs for which respective ligands do not require acylated fatty acids. A saturated fatty acid, lauric acid, induced NFkappaB activation when TLR2 was co-transfected with TLR1 or TLR6 in 293T cells, but not when TLR1, 2, 3, 5, 6, or 9 was transfected individually. An n-3 polyunsaturated fatty acid (docosahexaenoic acid (DHA)) suppressed NFkappaB activation and cyclooxygenase-2 expression induced by the agonist for TLR2, 3, 4, 5, or 9 in a macrophage cell line (RAW264.7). Because dimerization is considered one of the potential mechanisms for the activation of TLR2 and TLR4, we determined whether the fatty acids modulate the dimerization. However, neither lauric acid nor DHA affected the heterodimerization of TLR2 with TLR6 as well as the homodimerization of TLR4 as determined by co-immunoprecipitation assays in 293T cells in which these TLRs were transiently overexpressed. Together, these results demonstrate that lauric acid activates TLR2 dimers as well as TLR4 for which respective bacterial agonists require acylated fatty acids, whereas DHA inhibits the activation of all TLRs tested. Thus, responsiveness of different cell types and tissues to saturated fatty acids would depend on the expression of TLR4 or TLR2 with either TLR1 or TLR6. These results also suggest that inflammatory responses induced by the activation of TLRs can be differentially modulated by types of dietary fatty acids.


Assuntos
Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos/metabolismo , Glicoproteínas de Membrana/química , Receptores de Superfície Celular/química , Animais , Medula Óssea/metabolismo , Linhagem Celular , Ciclo-Oxigenase 2 , Dimerização , Genes Dominantes , Humanos , Immunoblotting , Isoenzimas/biossíntese , Ácidos Láuricos/metabolismo , Ligantes , Luciferases/metabolismo , Macrófagos/metabolismo , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C3H , NF-kappa B/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Plasmídeos/metabolismo , Testes de Precipitina , Prostaglandina-Endoperóxido Sintases/biossíntese , Receptores de Superfície Celular/agonistas , Receptores de Superfície Celular/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Receptor 1 Toll-Like , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptor 6 Toll-Like , Receptores Toll-Like , Transfecção
17.
J Biol Chem ; 278(39): 37041-51, 2003 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-12865424

RESUMO

Toll-like receptor-4 (TLR4) can be activated by nonbacterial agonists, including saturated fatty acids. However, downstream signaling pathways activated by nonbacterial agonists are not known. Thus, we determined the downstream signaling pathways derived from saturated fatty acid-induced TLR4 activation. Saturated fatty acid (lauric acid)-induced NFkappaB activation was inhibited by a dominant-negative mutant of TLR4, MyD88, IRAK-1, TRAF6, or IkappaBalpha in macrophages (RAW264.7) and 293T cells transfected with TLR4 and MD2. Lauric acid induced the transient phosphorylation of AKT. LY294002, dominant-negative (DN) phosphatidylinositol 3-kinase (PI3K), or AKT(DN) inhibited NFkappaB activation, p65 transactivation, and cyclooxygenase-2 (COX-2) expression induced by lauric acid or constitutively active (CA) TLR4. AKT(DN) blocked MyD88-induced NFkappaB activation, suggesting that AKT is a MyD88-dependent downstream signaling component of TLR4. AKT(CA) was sufficient to induce NFkappaB activation and COX-2 expression. These results demonstrate that NFkappaB activation and COX-2 expression induced by lauric acid are at least partly mediated through the TLR4/PI3K/AKT signaling pathway. In contrast, docosahexaenoic acid (DHA) inhibited the phosphorylation of AKT induced by lipopolysaccharide or lauric acid. DHA also suppressed NFkappaB activation induced by TLR4(CA), but not MyD88(CA) or AKT(CA), suggesting that the molecular targets of DHA are signaling components upstream of MyD88 and AKT. Together, these results suggest that saturated and polyunsaturated fatty acids reciprocally modulate the activation of TLR4 and its downstream signaling pathways involving MyD88/IRAK/TRAF6 and PI3K/AKT and further suggest the possibility that TLR4-mediated target gene expression and cellular responses are also differentially modulated by saturated and unsaturated fatty acids.


Assuntos
Antígenos de Diferenciação/fisiologia , Ácidos Graxos/farmacologia , Glicoproteínas de Membrana/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/fisiologia , Receptores de Superfície Celular/fisiologia , Receptores Imunológicos/fisiologia , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linhagem Celular , Ciclo-Oxigenase 2 , Ácidos Docosa-Hexaenoicos/farmacologia , Humanos , Quinases Associadas a Receptores de Interleucina-1 , Isoenzimas/biossíntese , Proteínas de Membrana , Camundongos , Fator 88 de Diferenciação Mieloide , NF-kappa B/metabolismo , Fosforilação , Prostaglandina-Endoperóxido Sintases/biossíntese , Proteínas/fisiologia , Proteínas Proto-Oncogênicas c-akt , Fator 6 Associado a Receptor de TNF , Receptor 4 Toll-Like , Receptores Toll-Like
18.
J Lipid Res ; 44(3): 479-86, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12562875

RESUMO

Human subjects consuming fish oil showed a significant suppression of cyclooxygenase-2 (COX-2) expression in blood monocytes when stimulated in vitro with lipopolysaccharide (LPS), an agonist for Toll-like receptor 4 (TLR4). Results with a murine monocytic cell line (RAW 264.7) stably transfected with COX-2 promoter reporter gene also demonstrated that LPS-induced COX-2 expression was preferentially inhibited by docosahexaenoic acid (DHA, C22:6n-3) and eicosapentaenoic acid (EPA, C20:5n-3), the major n-3 polyunsaturated fatty acids (PUFAs) present in fish oil. Additionally, DHA and EPA significantly suppressed COX-2 expression induced by a synthetic lipopeptide, a TLR2 agonist. These results correlated with the preferential suppression of LPS- or lipopeptide-induced NF kappa B activation by DHA and EPA. The target of inhibition by DHA is TLR itself or its associated molecules, but not downstream signaling components. In contrast, COX-2 expression by TLR2 or TRL4 agonist was potentiated by lauric acid, a saturated fatty acid. These results demonstrate that inhibition of COX-2 expression by n-3 PUFAs is mediated through the modulation of TLR-mediated signaling pathways. Thus, the beneficial or detrimental effects of different types of dietary fatty acids on the risk of the development of many chronic inflammatory diseases may be in part mediated through the modulation of TLRs.


Assuntos
Ácidos Graxos Insaturados/farmacologia , Óleos de Peixe/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Isoenzimas/genética , Glicoproteínas de Membrana/antagonistas & inibidores , Prostaglandina-Endoperóxido Sintases/genética , Receptores de Superfície Celular/antagonistas & inibidores , Triglicerídeos/farmacologia , Animais , Linhagem Celular , Ciclo-Oxigenase 2 , Suplementos Nutricionais , Ácidos Graxos Ômega-3 , Humanos , Ácido Linoleico , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , NF-kappa B/metabolismo , Regiões Promotoras Genéticas/genética , Receptores de Superfície Celular/agonistas , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-Like
19.
Biochem Biophys Res Commun ; 298(5): 667-74, 2002 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-12419306

RESUMO

Fatty acids are generally considered as agonists for peroxisome proliferator-activated receptors (PPARs). Fatty acids have been shown to bind to and transactivate PPARs; it is not known whether fatty acids act as generalized agonists for PPARs in different cell types, and thus, stimulate the expression of PPAR-regulated target genes. Here, we investigated the potency of unsaturated fatty acids on transactivation of PPRE, DNA-binding activity of PPARs, and the expression of a PPAR-regulated gene product, CD36. Docosahexaenoic acid (DHA) suppressed the basal and PPAR agonist-induced transactivation of PPRE, and DNA binding of PPARs in colon tumor cells (HCT116). The suppression of PPAR transactivation by DHA leads to reduced expression of CD36 in HCT116 cells and human monocytic cells (THP-1) as determined by promoter reporter gene assay and flow cytometric analysis. Our results demonstrate that DHA and other unsaturated fatty acids act as antagonists instead of agonists for transactivation of PPRE and PPAR-regulated gene expression in the cell lines tested. These results suggest that PPAR-mediated gene expression and cellular responses can be dynamically modulated by different types of dietary fatty acids consumed.


Assuntos
Ácidos Docosa-Hexaenoicos/farmacologia , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Sítios de Ligação/genética , Antígenos CD36/genética , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , DNA/genética , DNA/metabolismo , Gorduras Insaturadas na Dieta/farmacologia , Ácidos Graxos Insaturados/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Ácido Linoleico/farmacologia , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/genética , Ativação Transcricional/efeitos dos fármacos , Células Tumorais Cultivadas
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