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1.
PLoS One ; 18(5): e0282948, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37141290

RESUMO

ELISA or Western blot is known as a basic technique to be used for measurement of intracellular proteins, but in some cases, they cannot overcome problems such as normalization between samples or extraneous costs for required commercial kits. In order to address this problem, we developed a rapid and effective method (a hybrid of Western blot and ELISA). We use this new hybrid method to detect and normalize trace protein changes in gene expression intracellularly at a lower cost.


Assuntos
Sensibilidade e Especificidade , Ensaio de Imunoadsorção Enzimática/métodos , Western Blotting
2.
Clin Exp Dent Res ; 8(1): 439-448, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34626163

RESUMO

OBJECTIVES: The objective of this study was to introduce the evidence obtained through extensive research that periodontitis increases risk of many systemic diseases. METHOD: Analysis of some oral bacteria (P. gingivalis, T. denticola, T. forsythia, A. actinomycetemcomitans, and F. nucleatum) and its related treatments and mediators by the specific methods (western blot, ELISA, etc). RESULTS: This article reviews in detail the evidence obtained through extensive research that periodontitis increases risk of many systemic diseases, including cardiovascular disease, rheumatoid arthritis, and Alzheimer's disease. These diseases are known to be associated with some certain specific gram-negative bacteria as periodontal pathogens, which induce inflammation and related diseases through TLR receptors, kinases, transcriptional factors and other cytokines. We also reviewed the latest research for inhibitors against inflammation and related diseases that have potential to be further applied clinically. In addition, based on a large amount of research evidence, we draw two tables about the mechanism of disease caused by periodontal bacteria, so that readers can easily search and analyze these research results. DISCUSSION: This review details how the periodontal bacteria and their virulence factors can trigger host immune defense and induce many systemic diseases via inflammation and invasion. This Review also addressed the latest research around inhibitors against inflammation.


Assuntos
Aggregatibacter actinomycetemcomitans , Periodontite , Humanos , Inflamação , Periodontite/complicações , Porphyromonas gingivalis
3.
Hum Cell ; 34(4): 1123-1129, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33909263

RESUMO

It is known that Porphyromonas gingivalis/lipopolysaccharide (P. gingivalis/LPS) induces inflammatory diseases via TNF-α-mediated transcription factors. Our recent data shows that TNFAIP1 (TNF-α induced protein 1) is related to TNF-α. However, little is known regarding how TNFAIP1 is involved in the TNF-α-dependent pathway. We therefore focused on the biological function of TNFAIP1 and examined how TNFAIP1 mediates TNF-α and other genes. We found that TNF-α was upregulated and peaks before the upregulation of apoptotic genes such as Bad, Bcl-x, Caspase 3, Catalase, Claspin, Cytochromic, Ho-1/HMOX1/HSP32, or MCI-1 in our time course with TNFAIP1-treated cells. Our findings here may serve as the foundation for future studies linking regulation of TNFAIP1 and intervention of inflammatory disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Humanos , Inflamação/genética , Lipopolissacarídeos/efeitos adversos , Células THP-1 , Regulação para Cima/genética , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
4.
Rep Pract Oncol Radiother ; 25(3): 323-326, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32194353

RESUMO

AIM: It is well known that inducing hyperthermia is a type of cancer treatment but some research groups indicate that this treatment is not effective. This article finds and explains the mechanism of this treatment and its possible problems. BACKGROUND: Hyperthermia is commonly known as a state when the temperature of the body rises to a level that can threaten one's health. Hyperthermia is a type of cancer treatment in which body tissue is exposed to high temperatures (up to 45 °C). Research has shown that high temperatures can damage and kill cancer cells, usually with minimal injury to normal tissues. However, this mechanism is not known. MATERIALS AND METHODS: We recently treated cancer cells with different temperatures ranging from 37 °C to 47 °C and further measured their caspase 3 secretion by ELISA, western blot and cell survival rate by microscope. RESULTS: We found that most cancer cells are able to resist hyperthermia more than normal cells most likely via non-activation of caspase3. We also found that hyperthermia-treated (≥41°) cancer cells extend a long pseudopod-like extension in comparison to the same cancer cells under normal conditions. CONCLUSION: Our data here indicates that cancer cells have resistance to higher temperatures compared to normal cells via non-activation of caspase 3. This is a significant issue that needs to be brought to attention as the medical community has always believed that a high temperature treatment can selectively kill cancer/tumor cells. Additionally, we believe that the pseudopod-like extensions of hyperthermia-treated cancer cells must be related to its resistance to hyperthermia.

5.
J Cell Biochem ; 119(7): 6072-6079, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29637600

RESUMO

TNF-a is an important cytokine mediator of inflammation which suggests that inhibition of TNF activity may provide potential for clinical application. Recent data indicated that treatment of both human and mouse cells with Kavain significantly modulates P. gingivalis- and LPS-induced TNF-α expression. In order to obtain a selective analog with optimized biological activity and structural physico-chemical properties of Kavain, Kavain analogs were designed and synthesized and found one Kavain analogue (named Kav001) that is similar to Kavain but soluble and does not induce a significant toxicity. Both studies in vitro and in vivo treatment by Kav001 showed stronger biological function as compared to Kavain. Furthermore, most mouse bone marrow macrophages up-regulated Bcl-6 while down-regulating LITAF expression after treatment with Kav001 for 36 h. Consequently, this led to an extension of macrophage pseudopods due to its immune response to P.g. infection/LPS stimulation.


Assuntos
Artrite Experimental/tratamento farmacológico , Inflamação/tratamento farmacológico , Lipopolissacarídeos/toxicidade , Macrófagos Peritoneais/efeitos dos fármacos , Porphyromonas gingivalis/patogenicidade , Pironas/farmacologia , Animais , Anticonvulsivantes/farmacologia , Artrite Experimental/etiologia , Artrite Experimental/patologia , Infecções por Bacteroidaceae/tratamento farmacológico , Infecções por Bacteroidaceae/microbiologia , Citocinas/metabolismo , Inflamação/etiologia , Inflamação/patologia , Macrófagos Peritoneais/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pironas/química , Fator de Necrose Tumoral alfa/metabolismo
6.
Anal Biochem ; 544: 93-97, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29305095

RESUMO

Adherent cells such as mouse RAW cells or human cancer U2OS cells are beneficial to DNA transfection, with 20%-60% transfection efficiency. However, this DNA transfection is rarely used on suspension cells due to its low transfection efficiency (≤5%). We recently found a new DNA transfection method to increase the efficiency up to 13.5% in suspension cells without PMA treatment. We also found that DNA transfection of human TNFAIP1 or CXCL1 recombinant plasmid DNA in THP-1 cells induces a high level of TNF-α protein. Overall, this new method is simple yet efficient and can be used for the overexpression of DNA in suspension cells.


Assuntos
Transfecção , Células Cultivadas , DNA/genética , Humanos , Plasmídeos/genética , Células THP-1 , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologia
7.
J Cell Biochem ; 117(10): 2272-80, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-26917453

RESUMO

Kavain, a compound extracted from the Kava plant, Piper methysticum, is found to be involved in TNF-α expression in human and mouse cells via regulation of transcriptional factors such as NF-kB and LITAF. LITAF is known to activate the transcription of more than 20 cytokines that are involved in a variety of cellular processes and is associated with many inflammatory diseases, including angiogenesis, cancer, arthritis, and more. The modulation of LITAF is expected to positively affect cytokine-mediated diseases. Thus, intensive efforts have been deployed in search of LITAF inhibitors. In this work, we found that, in vitro, Kavain reduced LPS- induced TNF-α secretion in mouse macrophages, mouse bone marrow macrophages (BMM), and human peripheral blood mononuclear cells (HPBMC). We also found that Kavain treatment in RAW264.7 cells deactivated MyD88 and Akt, inhibited LITAF, and reduced the production of TNF-α, IL-27, and MIG in response to LPS. Similarly, it had a significant in vivo anti-inflammatory effect on wild-type (WT) mice that developed Collagen Antibody Induced Arthritis (CAIA). Overall, MyD88 was found to be an important mediator of the LPS-induced inflammatory response that can be distinguished from the NF-κB pathway. We also found that MyD88 is involved in the pathway linking LPS/LITAF to TNF-α. Therefore, given that Kavain modulates LPS-induced signaling pathways leading to cytokine expression, therapeutic interventions involving Kavain in inflammatory diseases are warranted. J. Cell. Biochem. 117: 2272-2280, 2016. © 2016 Wiley Periodicals, Inc.


Assuntos
Artrite Experimental/tratamento farmacológico , Leucócitos Mononucleares/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Pironas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Anticonvulsivantes/farmacologia , Artrite Experimental/etiologia , Artrite Experimental/patologia , Western Blotting , Células Cultivadas , Citocinas/metabolismo , Humanos , Leucócitos Mononucleares/citologia , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
8.
Toxicol Res (Camb) ; 5(1): 188-196, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26918116

RESUMO

Kavain, an extract from the shrub Piper Methysticum, was recently reported to modulate TNF-α expression in both human and mouse cells via regulation of LPS-Induced TNF-Alpha Factor (LITAF). The purpose of the present study was to define the molecular pathway(s) associated with Kavain effects on TNF modulation. In vitro studies using WT mouse primary macrophages showed that Kavain significantly reduced E.coli LPS-induced TNF-α production but this effect was almost abrogated in LITAF-/- and ERK2-/- cells. Therefore we reintroduced the ERK2 gene in ERK2-/- cells and partially restored E.coli LPS-induced LITAF-mediated TNF-α production. The translocation of LITAF into to nucleus was found to be dependent on ERK2 S206 residue. Kavain inhibits LITAF/TNF-α expression via dephosphorylation of ERK2 in response to E.coli LPS. Finally, in vivo, Kavain had a significant anti-inflammatory effect on wild type mice that developed Collagen Antibody Induced Arthritis (CAIA), but only a minor effect in ERK2-/- mice also affected by CAIA. Based on these findings, we concluded that ERK2 may be the kinase upstream of LITAF with its Serine residue 206 being crucial for the regulation of LPS-induced TNF-α.

9.
Tumour Biol ; 36(4): 2801-8, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25492482

RESUMO

Chemokine (C-C motif) ligand 2 (CCL2) has recently been found to be a key player in the pathology of many human glomerular and tubulointerstitial diseases. CCL2 has also been found to be expressed in various cancers, including human hepatoma cells, human cancer progression, and human multiple myeloma cells. Thus, the inhibition of elevated CCL2 production may provide a new avenue for therapeutic intervention in CCL2-mediated cancer diseases. A previous study has indicated that knockdown of human p53 has a strong negative impact on CCL2 induction. We therefore are interested in how p53 regulates CCL2 gene expression. In the following study, our findings indicate that p53 binds to CCL2, consequently significantly downregulating CCL2 promoter activity. Furthermore, injection of CCL2-promoting cancer cells (CCL2/A549) in p53-deficient mice for 3 weeks strongly induced subcutaneous xenograft tumor growth compared with the control. Overall, the research results support the novel role of p53 in suppression of chemokine (such as CCL2)-mediated cancer diseases.


Assuntos
Quimiocina CCL2/biossíntese , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Proteína Supressora de Tumor p53/genética , Animais , Apoptose/genética , Linhagem Celular Tumoral , Quimiocina CCL2/genética , Xenoenxertos , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Ligação Proteica , Proteína Supressora de Tumor p53/metabolismo
10.
J Cell Mol Med ; 17(3): 386-97, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23414097

RESUMO

Vascular endothelial growth factor (VEGF) is a critical angiogenic factor affecting endothelial cells, inflammatory cells and neuronal cells. In addition to its well-defined positive role in wound healing, pathological roles for VEGF have been described in cancer and inflammatory diseases (i.e. atherosclerosis, rheumatoid arthritis, inflammatory bowel disease and osteoarthritis). Recently, we showed that transcription factors LITAF and STAT6B affected the inflammatory response. This study builds upon our previous results in testing the role of mouse LITAF and STAT6B in the regulation of VEGF-mediated processes. Cells cotransfected with a series of VEGF promoter deletions along with truncated forms of mLITAF and/or mSTAT6B identified a DNA binding site (between -338 and -305 upstream of the transcription site) important in LITAF and/or STAT6B-mediated transcriptional regulation of VEGF. LITAF and STAT6B corresponding protein sites were identified. In addition, siRNA-mediated knockdown of mLITAF and/or mSTAT6B leads to significant reduction in VEGF mRNA levels and inhibits LPS-induced VEGF secretion in mouse RAW 264.7 cells. Furthermore, VEGF treatment of mouse macrophage or endothelial cells induces LITAF/STAT6B nuclear translocation and cell migration. To translate these observations in vivo, VEGF164-soaked matrigel were implanted in whole-body LITAF-deficient animals (TamLITAF(-/-) ), wild-type mice silenced for STAT6B, and in respective control animals. Vessel formation was found significantly reduced in TamLITAF(-/-) as well as in STAT6B-silenced wild-type animals compared with control animals. The present data demonstrate that VEGF regulation by LITAF and/or STAT6B is important in angiogenesis signalling pathways and may be a useful target in the treatment of VEGF diseases.


Assuntos
Movimento Celular , Regulação da Expressão Gênica , Inflamação/genética , Neovascularização Patológica , Proteínas Nucleares/fisiologia , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Apoptose , Western Blotting , Adesão Celular , Proliferação de Células , Colágeno/metabolismo , Proteínas de Ligação a DNA , Combinação de Medicamentos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunoprecipitação , Inflamação/metabolismo , Inflamação/patologia , Laminina/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Knockout , Proteína Quinase 14 Ativada por Mitógeno , Fosforilação , Transporte Proteico , Proteoglicanas/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética
11.
PLoS One ; 6(9): e25083, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21980379

RESUMO

Inflammation is a multifaceted process: beneficial as a defense mechanism but also detrimental depending on its severity and duration. At the site of injury, inflammatory cells are activated by a cascade of mediators, one of which is LITAF, a transcription regulator known to upregulate TNF-α. We previously showed that human LITAF forms a complex with human STAT6B, which translocates into the nucleus to upregulate cytokine transcription. To dissect the molecular implications of this complex, a murine model was developed and interactions between mouse STAT6B (mSTAT6B) and mouse LITAF (mLITAF) were analyzed. Both mLITAF and mSTAT6B expression were MyD88- and TLR ligand-dependent. Furthermore, mLITAF was found to mediate LPS-induced CCL2 gene transcription with the cooperation of mSTAT6B leading to CCL2 protein expression. In LITAF-deficient mice, mLITAF-mediated CCL2 production in macrophages was significantly reduced compared to the wild-type control animals. Mice knockdown for mSTAT6B by 6BsiRNA1 tail vein injection resulted in a decrease in serum TNF-α and CCL2 production. mLITAF/mSTAT6B complex is proposed to play a role in LPS-induced CCL2 expression and possibly other cytokines.


Assuntos
Quimiocina CCL2/metabolismo , Proteínas Nucleares/metabolismo , Fator de Transcrição STAT6/metabolismo , Fatores de Transcrição/metabolismo , Animais , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Quimiocina CCL2/genética , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas Nucleares/genética , Ligação Proteica/genética , Ligação Proteica/fisiologia , RNA Interferente Pequeno , Fator de Transcrição STAT6/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Fatores de Transcrição/genética , Fator de Necrose Tumoral alfa/metabolismo
12.
J Innate Immun ; 2(1): 43-55, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20375622

RESUMO

Lipopolysaccharide (LPS) stimulation of macrophages initiates intracellular signaling pathways leading to activation of MAPK and its subsequent influence on cytokine production. We recently identified a LITAF-STAT6(B) complex regulated by p38 MAPK in response to LPS stimulation. However, the LPS-induced cascade in the p38/LITAF/TNF signaling pathway remains unclear. Here, we identified PTP4A3, a protein tyrosine phosphotase, as a novel negative regulator of LPS-induced LITAF/TNF-alpha production. PTP4A3 exerts its negative role by dephosphorylating p38 alpha MAPK in response to LPS stimulation of primary macrophages. PTP4A3 expression is upregulated in primary macrophages. Further structure-function analysis revealed that a unique short peptide (PIMAP39) derived from PTP4A3 is capable of mimicking the functionality of full-length PTP4A3 to selectively dephosphorylate p38 alpha and indirectly suppress LPS-induced LITAF-STAT6B complex when it is translocated from the cytoplasmic region to the nucleus of the cell. Treatment of mice with PIMAP39 significantly attenuates the severity of adverse host responses to LPS stimulation, and in some cases provides complete resistance to a lethal dose of LPS due to suppression of TNF-alpha production. All together, these results reveal a previously unrecognized role for the PTP4A3 pathway in response to LPS.


Assuntos
Proteínas Imediatamente Precoces/imunologia , Proteínas Nucleares/imunologia , Proteínas Tirosina Fosfatases/imunologia , Choque Séptico/imunologia , Fatores de Transcrição/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Proteínas de Ligação a DNA , Humanos , Proteínas Imediatamente Precoces/genética , Lipopolissacarídeos/imunologia , Ativação de Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 14 Ativada por Mitógeno/imunologia , Proteínas Nucleares/genética , Peptídeos/imunologia , Proteínas Tirosina Fosfatases/genética , Choque Séptico/enzimologia , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Regulação para Cima
13.
Chem Biol Drug Des ; 74(2): 121-8, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19538508

RESUMO

There is a substantial unmet need for new classes of drugs that block TNF-alpha-mediated inflammation, and particularly for small molecule agents that can be taken orally. We have screened a library of natural products against an assay measuring TNF-alpha secretion in lipopolysaccharide-stimulated THP-1 cells, seeking compounds capable of interfering with the TNF-alpha-inducing transcription factor lipopolysaccharide-induced TNF-alpha factor. Among the active compounds were several produced by the kava plant (Piper mysticum), extracts of which have previously been linked to a range of therapeutic effects. When tested in vivo, a representative of these compounds, kavain, was found to render mice immune to lethal doses of lipopolysaccharide. Kavain displays promising pharmaceutical properties, including good solubility and high cell permeability, but pharmacokinetic experiments in mice showed relatively rapid clearance. A small set of kavain analogs was synthesized, resulting in compounds of similar or greater potency in vitro compared with kavain. Interestingly, a ring-opened analog of kavain inhibited TNF-alpha secretion in the cell-based assay and suppressed lipopolysaccharide-induced TNF-alpha factor expression in the same cells, whereas the other compounds inhibited TNF-alpha secretion without affecting lipopolysaccharide-induced TNF-alpha factor levels, indicating a potential divergence in mechanism of action.


Assuntos
Anti-Inflamatórios/farmacologia , Kava/química , Pironas/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacocinética , Linhagem Celular Tumoral , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Extratos Vegetais/química , Pironas/química , Pironas/farmacocinética , Fator de Necrose Tumoral alfa/antagonistas & inibidores
14.
Cancer Res ; 67(3): 1308-16, 2007 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-17283168

RESUMO

The p53 protein is a sequence-specific DNA-binding factor that can induce apoptosis or activate genes whose dysregulation is involved in cancer. By using serial analysis of gene expression technique, p53-induced genes (PIGs) have been identified, one of which was lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-alpha) factor (LITAF/PIG7). LITAF regulates the transcription of cytokines such as TNF-alpha. To further elucidate the role of p53 in LITAF expression, LITAF promoter activity was carefully dissected. In this study, we found that the element required for transcriptional activity is mainly located in the region from -990 to -500 of the LITAF promoter; the specific site required for p53 protein-DNA binding is located between -550 and -500. We also found that transient transfection of either a p53 short DNA sequence, called p53LFB12, or its corresponding 7-amino-acid synthetic peptide from amino acids 164 to 170 (K164Q165S166Q167H168M169T170), named p53pep164, significantly reduced LITAF promoter activity to 15% in p53-null H1299 cells. Transfection of p53pep164 into H1299 cells significantly down-regulated LPS-induced LITAF expression as well. Furthermore, transfection of p53pep164 into human monocytes resulted in down-regulation of nine proinflammatory cytokines, including TNF-alpha. We also found that the LPS-activated p53 is a short-lived protein, and that p53-orchestrated apoptosis occurs shortly after the initiation stage following LPS stimulation and lasts a short time. Once p53 levels return to baseline, the p53-mediated inhibition of LITAF is released, and LITAF-mediated cytokine production can proceed. The present finding proposes a novel link between p53 and the inflammatory processes and highlights potential interventional approaches to control p53-associated inflammatory processes.


Assuntos
DNA/metabolismo , Proteínas Nucleares/biossíntese , Fragmentos de Peptídeos/metabolismo , Fatores de Transcrição/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Proteína Supressora de Tumor p53/metabolismo , Apoptose/genética , Sequência de Bases , Linhagem Celular Tumoral , Expressão Gênica/efeitos dos fármacos , Humanos , Lipopolissacarídeos/farmacologia , Dados de Sequência Molecular , Proteínas Nucleares/genética , Fragmentos de Peptídeos/genética , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição/genética , Transfecção , Fator de Necrose Tumoral alfa/genética , Proteína Supressora de Tumor p53/genética
15.
Proc Natl Acad Sci U S A ; 103(37): 13777-82, 2006 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-16954198

RESUMO

Previously we identified a transcription factor, LPS-Induced TNF-alpha Factor (LITAF), mediating inflammatory cytokine expression in LPS-induced processes. To characterize the role of LITAF in vivo, we generated a macrophage-specific LITAF-deficient mouse (macLITAF(-/-)). Our data demonstrate that in macrophages (i) several cytokines (such as TNF-alpha, IL-6, sTNF-RII, and CXCL16) are induced at lower levels in macLITAF(-/-) compared with LITAF(+/+) control macrophages; (ii) macLITAF(-/-) mice are more resistant to LPS-induced lethality. To further identify LITAF signaling pathways, we tested mouse TLR-2(-/-), -4(-/-), and -9(-/-) and WT peritoneal macrophages exposed to LPS. Using these cells, we now show that LITAF expression can be induced after challenge either with LPS from Porphyromonas gingivalis via agonism at TLR-2, or with LPS from Escherichia coli via agonism at TLR-4, both requiring functional MyD88. We also show that, in response to LPS, the MyD88-dependent LITAF pathway differs from the NF-kappaB pathway. Furthermore, using a kinase array, p38alpha was found to mediate LITAF phosphorylation and the inhibition of p38alpha with a p38-specific inhibitor (SB203580) blocked LITAF nuclear translocation and reduced LPS-induced TNF-alpha protein levels. Finally, macLITAF(-/-) macrophages rescued by LITAF cDNA transfection restored levels of TNF-alpha similar to those observed in WT cells. We conclude that LITAF is an important mediator of the LPS-induced inflammatory response that can be distinguished from NF-kappaB pathway and that p38alpha is the specific kinase involved in the pathway linking LPS/MyD88/LITAF to TNF.


Assuntos
Citocinas/metabolismo , Macrófagos/imunologia , Proteínas Nucleares/fisiologia , Choque Séptico/genética , Receptores Toll-Like/metabolismo , Fatores de Transcrição/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ligação a DNA , Regulação para Baixo , Escherichia coli/imunologia , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Mutantes , Fator 88 de Diferenciação Mieloide , NF-kappa B/metabolismo , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Fosforilação , Porphyromonas gingivalis/imunologia , Transporte Proteico , Choque Séptico/imunologia , Transdução de Sinais , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
16.
Proc Natl Acad Sci U S A ; 102(14): 5132-7, 2005 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-15793005

RESUMO

TNF-alpha is a pivotal cytokine whose overproduction can be lethal. Previously, we identified a transcription factor, LPS-induced TNF-alpha factor (LITAF), that regulates TNF-alpha transcription. We now report the discovery and characterization of a regulatory cofactor that we call signal transducer and activator of transcription (STAT) 6(B) because of its considerable homology to STAT6 [here referred to as STAT6(A)]. The STAT6(B) gene expression was found to be activated by LPS. Furthermore, we show that cotransfection of STAT6(B) and LITAF induces an interaction between the two proteins, consequently forming a complex that subsequently translocates into the nucleus and up-regulates the transcription of cytokines. The effect of the complex on a panel of cytokines was tested. In addition, the specific role of LITAF in this complex was established with experiments, including RNA interference technology. Overall, these findings describe roles for LITAF, STAT6(B), and the LITAF-STAT6(B) complex in the regulation of inflammatory cytokines in response to LPS stimulation in mammalian cells.


Assuntos
Citocinas/metabolismo , Lipopolissacarídeos/farmacologia , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Sequência de Bases , Linhagem Celular , DNA Complementar/genética , Feminino , Inativação Gênica , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multiproteicos , Proteínas Nucleares/genética , Interferência de RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Transcrição STAT6 , Transativadores/genética , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido
17.
J Biol Chem ; 279(51): 53317-22, 2004 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-15485814

RESUMO

Retinoblastoma (Rb) protein is a paradigm of tumor suppressors. Inactivation of Rb plays a critical role in the development of human malignancies. MDM2, an oncogene frequently found amplified and overexpressed in a variety of human tumors and cancers, directly interacts and inhibits the p53 tumor suppressor protein. In addition, MDM2 has been shown to stimulate E2F transactivation activity and promote S-phase entry independent of p53, yet the mechanism of which is still not fully understood. In this study, we demonstrate that MDM2 specifically binds to Rb C-pocket and that the central acidic domain of MDM2 is essential for Rb interaction. In addition, we show that overexpression of MDM2 reduces Rb-E2F complexes in vivo. Moreover, the ectopic expression of the wild type MDM2, but not mutant MDM2 defective in Rb interaction, stimulates E2F transactivation activity and inhibits Rb growth suppression function. Taken together, these results suggest that MDM2-mediated inhibition of Rb likely contributes to MDM2 oncogenic activity.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ligação a DNA/química , Proteínas Nucleares/química , Proteínas Proto-Oncogênicas/química , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/química , Sequência de Aminoácidos , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Linhagem Celular Tumoral , Proliferação de Células , Cromatografia Líquida de Alta Pressão , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição E2F , Genes Reporter , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutação , Fosforilação , Plasmídeos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-mdm2 , Proto-Oncogenes , Proteínas Recombinantes de Fusão/metabolismo , Proteína do Retinoblastoma/química , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/metabolismo , Ativação Transcricional , Transfecção
18.
Proc Natl Acad Sci U S A ; 100(7): 4096-101, 2003 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-12655064

RESUMO

Transcription of the tumor necrosis factor (TNF) gene is rapidly and transiently induced by lipopolysaccharide in cells of monocytemacrophage lineage. Previous studies have suggested that in the mouse, multiple NF-kappaBRel-binding sites contribute to the TNF transcriptional response to LPS. But the role of these regulatory elements in transcriptional activation of the TNF-alpha gene in human monocytes remains unclear. Previously, a transcription factor, termed lipopolysaccharide-induced TNF-alpha factor (LITAF), was found to regulate TNF-alpha gene expression. However, the specific protein domain(s) of human (h)LITAF that interact with the hTNF-alpha promoter had not been identified. In this study, we identify by footprinting a sequence motif, CTCCC (-515 to -511), within the TNF-alpha promoter that binds to hLITAF. We also identify the region of hLITAF (amino acids 165-180) that was named peptide B and specifically mediates binding to the hTNF-alpha promoter. When THP-1 cells were stimulated with this peptide B, it was sufficient to induce TNF-alpha secretion. Induction of TNF-alpha transcription by LPS or peptide B depended on the presence of the -515 to -511 promoter region, which was found to be essential for hLITAF binding. Together, these findings help to clarify the mechanism of hLITAFhTNF-alpha interaction and the manner by which hLITAF contributes to hTNF-alpha regulation in an attempt to design new pharmacological interventions to address TNF-related diseases.


Assuntos
Regiões Promotoras Genéticas/genética , Fator de Necrose Tumoral alfa/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Clonagem Molecular , Pegada de DNA , Primers do DNA , Escherichia coli/genética , Escherichia coli/imunologia , Humanos , Lipopolissacarídeos/farmacologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/efeitos dos fármacos , TATA Box
19.
Nature ; 419(6909): 849-53, 2002 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-12397361

RESUMO

p53 is activated in response to various genotoxic stresses resulting in cell cycle arrest or apoptosis. It is well documented that DNA damage leads to phosphorylation and activation of p53 (refs 1-3), yet how p53 is activated is still not fully understood. Here we report that DNA damage specifically induces p53 phosphorylation on Ser/Thr-Pro motifs, which facilitates its interaction with Pin1, a member of peptidyl-prolyl isomerase. Furthermore, the interaction of Pin1 with p53 is dependent on the phosphorylation that is induced by DNA damage. Consequently, Pin1 stimulates the DNA-binding activity and transactivation function of p53. The Pin1-mediated p53 activation requires the WW domain, a phosphorylated Ser/Thr-Pro motif interaction module, and the isomerase activity of Pin1. Moreover, Pin1-deficient cells are defective in p53 activation and timely accumulation of p53 protein, and exhibit an impaired checkpoint control in response to DNA damage. Together, these data suggest a mechanism for p53 regulation in cellular response to genotoxic stress.


Assuntos
Dano ao DNA , Proteínas Nucleares , Peptidilprolil Isomerase/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Motivos de Aminoácidos , Animais , Ciclo Celular , Fibroblastos , Deleção de Genes , Humanos , Camundongos , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/genética , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , Ativação Transcricional , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/química
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