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1.
Int J Mol Sci ; 24(19)2023 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-37833877

RESUMO

Macrophages undergo different cellular states upon activation that can be hyporesponsive (tolerated) or hyperresponsive (primed or trained) to subsequent stimuli. Epigenetic modifications are known to play key roles in determining these cellular states. However, little is known about the role of signaling pathways that lead to these epigenetic modifications. Here, we examined the effects of various inhibitors targeting key signaling pathways induced by lipopolysaccharide (LPS) on tolerance and priming in murine macrophages. We found that a prolonged inhibition (>18 h) of the mitogen-activated protein kinase (MEK)1/2-extracellular signal-regulated kinase (ERK)1/2 signaling axis reversed tolerance and primed cells in expressing interleukin (IL)-1ß and other inflammatory cytokines such as IL-6, tumor necrosis factor (TNF)α, and CXCL10. The ectopic expression of catalytically active and inactive MEK1 mutants suppressed and enhanced IL-1ß expression, respectively. A transcriptomic analysis showed that cells primed by the MEK1/2 inhibitor U0126 expressed higher levels of gene sets associated with immune responses and cytokine/chemokine production, but expressed lower levels of genes with cell cycle progression, chromosome organization, and heterochromatin formation than non-primed cells. Of interest, the mRNA expressions of the histone 3 lysine 9 (H3K9) methyltransferase Suv39h1 and the H3K9 methylation reader Cbx5 were substantially suppressed, whereas the H3K9 demethylase Kdm7a was enhanced, suggesting a role of the MEK1/2-ERK signaling axis in H3K9 demethylation. The H3K9 trimethylation levels in the genomic regions of IL-1ß, TNFα, and CXCL10 were decreased by U0126. Also, the H3K9 methyltransferase inhibitor BIX01294 mimicked the U0126 training effects and the overexpression of chromobox homolog (CBX)5 prevented the U0126 training effects in both RAW264.7 cells and bone-marrow-derived macrophages. Collectively, these data suggest that the prolonged inhibition of the MEK1/2-ERK signaling axis reverses tolerance and primed macrophages likely through decreasing the H3K9 methylation levels.


Assuntos
Histonas , Lisina , Animais , Camundongos , Histonas/metabolismo , Lisina/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Citocinas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Metiltransferases/metabolismo , Desmetilação , Lipopolissacarídeos/farmacologia
2.
Cells ; 10(5)2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064422

RESUMO

Inhibition of the RAF-MEK1/2-ERK signaling pathway is an ideal strategy for treating cancers with NRAS or BRAF mutations. However, the development of resistance due to incomplete inhibition of the pathway and activation of compensatory cell proliferation pathways is a major impediment of the targeted therapy. The anthrax lethal toxin (LT), which cleaves and inactivates MEKs, is a modifiable biomolecule that can be delivered selectively to tumor cells and potently kills various tumor cells. However, resistance to LT and the mechanism involved are yet to be explored. Here, we show that LT, through inhibiting MEK1/2-ERK activation, inhibits the proliferation of cancer cells with NRAS/BRAF mutations. Among them, the human colorectal tumor HT-29 and murine melanoma B16-BL6 cells developed resistance to LT in 2 to 3 days of treatment. These resistant cells activated AKT through a histone deacetylase (HDAC) 8-dependent pathway. Using an Affymetrix microarray, followed by qPCR validation, we identified that the differential expression of the phospholipase C-ß1 (PLCB1) and squamous cell carcinoma-1 (DESC1) played an important role in HDAC8-mediated AKT activation and resistance to MEK1/2-ERK inhibition. By using inhibitors, small interference RNAs and/or expression vectors, we found that the inhibition of HDAC8 suppressed PLCB1 expression and induced DESC1 expression in the resistant cells, which led to the inhibition of AKT and re-sensitization to LT and MEK1/2 inhibition. These results suggest that targeting PLCB1 and DESC1 is a novel strategy for inhibiting the resistance to MEK1/2 inhibition.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Histona Desacetilases/metabolismo , Proteínas de Membrana/metabolismo , Fosfolipase C beta/metabolismo , Proteínas Repressoras/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antígenos de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células HT29 , Humanos , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Proteínas de Membrana/genética , Camundongos , Fosfolipase C beta/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Serina Endopeptidases/genética
3.
J Biol Chem ; 294(46): 17487-17500, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31586032

RESUMO

The DNA-binding protein PU.1 is a myeloid lineage-determining and pioneering transcription factor due to its ability to bind "closed" genomic sites and maintain "open" chromatin state for myeloid lineage-specific genes. The precise mechanism of PU.1 in cell type-specific programming is yet to be elucidated. The melanoma cell line B16BL6, although it is nonmyeloid lineage, expressed Toll-like receptors and activated the transcription factor NF-κB upon stimulation by the bacterial cell wall component lipopolysaccharide. However, it did not produce cytokines, such as IL-1ß mRNA. Ectopic PU.1 expression induced remodeling of a novel distal enhancer (located ∼10 kbp upstream of the IL-1ß transcription start site), marked by nucleosome depletion, enhancer-promoter looping, and histone H3 lysine 27 acetylation (H3K27ac). PU.1 induced enhancer-promoter looping and H3K27ac through two distinct PU.1 regions. These PU.1-dependent events were independently required for subsequent signal-dependent and co-dependent events: NF-κB recruitment and further H3K27ac, both of which were required for enhancer RNA (eRNA) transcription. In murine macrophage RAW264.7 cells, these PU.1-dependent events were constitutively established and readily expressed eRNA and subsequently IL-1ß mRNA by lipopolysaccharide stimulation. In summary, this study showed a sequence of epigenetic events in programming IL-1ß transcription by the distal enhancer priming and eRNA production mediated by PU.1 and the signal-dependent transcription factor NF-κB.


Assuntos
Interleucina-1beta/genética , Melanoma Experimental/genética , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , Transativadores/genética , Animais , Linhagem Celular Tumoral , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Camundongos , Regiões Promotoras Genéticas , Células RAW 264.7 , Ativação Transcricional
4.
Sci Rep ; 8(1): 11332, 2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-30054507

RESUMO

Cell death by hypoxia followed by reoxygenation (H/R) is responsible for tissue injury in multiple pathological conditions. Recent studies found that epigenetic reprogramming mediated by histone deacetylases (HDACs) is implicated in H/R-induced cell death. However, among 18 different isoforms comprising 4 classes (I-IV), the role of each HDAC in cell death is largely unknown. This study examined the role of HDAC8, which is the most distinct isoform of class I, in the hypoxia mimetic cobalt- and H/R-induced cytotoxicity of human proximal tubular HK-2 cells. Using the HDAC8-specific activator TM-2-51 (TM) and inhibitor PCI34051, we found that HDAC8 played a protective role in cytotoxicity. TM or overexpression of wild-type HDAC8, but not a deacetylase-defective HDAC8 mutant, prevented mitochondrial fission, loss of mitochondrial transmembrane potential and release of cytochrome C into the cytoplasm. TM suppressed expression of dynamin-related protein 1 (DRP1) which is a key factor required for mitochondrial fission. Suppression of DRP1 by HDAC8 was likely mediated by decreasing the level of acetylated histone H3 lysine 27 (a hallmark of active promoters) at the DRP1 promoter. Collectively, this study shows that HDAC8 inhibits cytotoxicity induced by cobalt and H/R, in part, through suppressing DRP1 expression and mitochondrial fission.


Assuntos
Cobalto/toxicidade , Citoproteção , Histona Desacetilases/metabolismo , Túbulos Renais Proximais/patologia , Dinâmica Mitocondrial , Oxigênio/farmacologia , Proteínas Repressoras/metabolismo , Acetilação , Benzamidas/farmacologia , Morte Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Citoproteção/efeitos dos fármacos , Dinaminas , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Humanos , Lisina/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Feniltioureia/análogos & derivados , Feniltioureia/farmacologia , Regiões Promotoras Genéticas/genética , Quinazolinonas/farmacologia
5.
J Virol Antivir Res ; 7(1)2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29349092

RESUMO

BACKGROUND: Influenza A virus (IAV) is the etiologic agent of the febrile respiratory illness, commonly referred to as 'flu'. The lysosomal protease cathepsin B (CTSB) has shown to be involved in the lifecycle of various viruses. Here, we examined the role of CTSB in the IAV lifecycle. METHODS: CTSB-deficient (CTSB-/-) macrophages and the human lung epithelial cell line A549 cells treated with CA-074Me were infected with the A/Puerto Rico/8/34 strain of IAV (IAV-PR8). Viral entry and propagation were measured through quantitative real-time RT-PCR; production and localization of hemagglutinin (HA) protein in the infected host cells were analysed by Western blots, flow cytometry and confocal microscopy; production of progeny viruses were measured by a hemagglutination assay. RESULTS: CTSB-/- macrophages and CA-074Me-treated A549 cells had no defects in incorporating IAV-PR8 virions and permitting viral RNA synthesis. However, these cells produced significantly lower amounts of HA protein and progeny virions than wild-type or untreated cells. CONCLUSION: These data indicate that CTSB is involved in the expression of IAV-PR8 HA protein and subsequent optimal production of IAV-PR8 progeny virions. Targeting CTSB can be a novel therapeutic strategy for treating IAV infection.

6.
Toxins (Basel) ; 9(5)2017 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-28509866

RESUMO

Anthrax lethal toxin (LeTx) is a cytotoxic virulence factor that causes cell cycle arrest and cell death in various cell types. However, susceptibility to the cytotoxic effects varies depending on cell types. In proliferating monocytes, LeTx has only transient cytotoxic effects due to activation of the phosphoinositide 3-kinase (PI3K)-AKT-mediated adaptive responses. To date, the mechanism of LeTx in activating PI3K-AKT signaling axis is unknown. This study shows that the histone deacetylase 8 (HDAC8) is involved in activating PI3K-AKT signaling axis through down-regulating the phosphatase and tensin homolog 1 (PTEN) in human monocytic THP-1 cells. The HDAC8-specific activator TM-2-51 and inhibitor PCI-34051 enhanced and prevented, respectively, AKT activation and cell cycle progression in LeTx-treated cells. Furthermore, HDAC8 induced tri-methylation of histone H3 lysine 27 (H3K27me3), which is known to suppress PTEN expression, through at least in part down-regulating the H3K27me3 eraser Jumonji Domain Containing (JMJD) 3. Importantly, the JMJD3-specific inhibitor GSK-J4 induced AKT activation and protected cell cycle arrest in LeTx-treated cells, regardless the presence of HDAC8 activity. Collectively, this study for the first time demonstrated that HDAC8 activity determines susceptibility to cell cycle arrest induced by LeTx, through regulating the PI3K-PTEN-AKT signaling axis.


Assuntos
Antígenos de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Histona Desacetilases/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Repressoras/metabolismo , Sobrevivência Celular , Inativação Gênica , Histonas/metabolismo , Humanos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Transdução de Sinais , Células THP-1
8.
J Biol Chem ; 291(16): 8745-55, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26912657

RESUMO

Many pathogenic microbes often release toxins that subvert the host's immune responses to render the environment suitable for their survival and proliferation. LeTx is one of the toxins causing immune paralysis by cleaving and inactivating the mitogen-activated protein kinase (MAPK) kinases (MEKs). Here, we show that inhibition of the histone deacetylase 8 (HDAC8) by either the HDAC8-specific inhibitor PCI-34051 or small interference (si)RNAs rendered LeTx-exposed murine macrophages responsive to LPS in pro-IL-1ß production. HDAC8 selectively targeted acetylated histone H3 lysine 27 (H3K27Ac), which is known to associate with active enhancers. LeTx induced HDAC8 expression, in part through inhibiting p38 MAPK, which resulted in a decrease of H3K27Ac levels. Inhibition of HDAC8 increased H3K27Ac levels and enhanced NF-κB-mediated pro-IL-1ß enhancer and messenger RNA production in LeTx-exposed macrophages. Collectively, this study demonstrates a novel role of HDAC8 in LeTx immunotoxicity and regulation of pro-IL-1ß production likely through eRNAs. Targeting HDAC8 could be a strategy for enhancing immune responses in macrophages exposed to LeTx or other toxins that inhibit MAPKs.


Assuntos
Antígenos de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Histona Desacetilases/metabolismo , Interleucina-1beta/biossíntese , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/metabolismo , Acetilação , Animais , Linhagem Celular , Histona Desacetilases/genética , Histonas/genética , Histonas/metabolismo , Interleucina-1beta/genética , Sistema de Sinalização das MAP Quinases/genética , Macrófagos/patologia , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
9.
Cytokine ; 78: 69-78, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26687628

RESUMO

Granulocyte colony-stimulating factor (G-CSF) is a pleiotropic cytokine best known for its role in promoting the generation and function of neutrophils. G-CSF is also found to be involved in macrophage generation and immune regulation; however, its in vivo role in immune homeostasis is largely unknown. Here, we examined the role of G-CSF in dextran sulfate sodium (DSS)-induced acute colitis using G-CSF receptor-deficient (G-CSFR(-/-)) mice. Mice were administered with 1.5% DSS in drinking water for 5days, and the severity of colitis was measured for the next 5days. GCSFR(-/-) mice were more susceptible to DSS-induced colitis than G-CSFR(+/+) or G-CSFR(-/+) mice. G-CSFR(-/-) mice harbored less F4/80(+) macrophages, but a similar number of neutrophils, in the intestine. In vitro, bone marrow-derived macrophages prepared in the presence of both G-CSF and macrophage colony-stimulating factor (M-CSF) (G-BMDM) expressed higher levels of regulatory macrophage markers such as programmed death ligand 2 (PDL2), CD71 and CD206, but not in arginase I, transforming growth factor (TGF)-ß, Ym1 (chitinase-like 3) and FIZZ1 (found in inflammatory zone 1), and lower levels of inducible nitric oxide synthase (iNOS), CD80 and CD86 than bone marrow-derived macrophages prepared in the presence of M-CSF alone (BMDM), in response to interleukin (IL)-4/IL-13 and lipopolysaccharide (LPS)/interferon (IFN)-γ, respectively. Adoptive transfer of G-BMDM, but not BMDM, protected G-CSFR(-/-) mice from DSS-induced colitis, and suppressed expression of tumor necrosis factor (TNF)-α, IL-1ß and iNOS in the intestine. These results suggest that G-CSF plays an important role in preventing colitis, likely through populating immune regulatory macrophages in the intestine.


Assuntos
Colite/imunologia , Colite/prevenção & controle , Fator Estimulador de Colônias de Granulócitos/fisiologia , Homeostase , Intestinos/imunologia , Macrófagos/fisiologia , Transferência Adotiva , Animais , Células Cultivadas , Colite/induzido quimicamente , Sulfato de Dextrana , Interleucina-13/imunologia , Interleucina-1beta/metabolismo , Intestinos/citologia , Intestinos/fisiologia , Lipopolissacarídeos/imunologia , Fator Estimulador de Colônias de Macrófagos/imunologia , Macrófagos/imunologia , Camundongos , Óxido Nítrico Sintase Tipo II/metabolismo , Receptores de Fator Estimulador de Colônias de Granulócitos/deficiência , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Fator de Necrose Tumoral alfa/metabolismo
10.
BMC Microbiol ; 15: 238, 2015 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-26502905

RESUMO

BACKGROUND: Different species and strains of probiotic bacteria confer distinct immunological responses on immune cells. Lactobacillus rhamnosus GR-1 (GR-1) is a probiotic bacterial strain found in both the intestinal and urogenital tracts, and has immunomodulatory effects on several cell types including macrophages. However, detailed immunological responses and the signaling mechanism involved in the response are largely unknown. RESULTS: We examined the production of GR-1-induced cytokines/chemokines and signaling events in macrophages. Among 84 cytokines and chemokines examined, GR-1 discretely induced granulocyte colony-stimulating factor (G-CSF) mRNA at highest levels (>60-fold) without inducing other cytokines such as IL-1α, IL-1ß, IL-6 and TNF-α (<5-fold). The toll-like receptor (TLR) 2/6-agonist PAM2CSK4, TLR2/1-agonist PAM3CSK4 and TLR4-agonist lipopolysaccharide induced all of these inflammatory cytokines at high levels (>50-fold). The TLR2 ligand lipoteichoic acid activated all mitogen-activated kinases, Akt and NF-κB; whereas, GR-1 selectively activated extracellular regulated kinases and p38, NF-κB and Akt, but not c-Jun N-terminal kinases (JNKs) in a TLR2-dependent manner. Using specific inhibitors, we demonstrated that lack of JNKs activation by GR-1 caused inefficient production of pro-inflammatory cytokines but not G-CSF production. A secreted heat-labile protein-like molecule, 30-100 kDa in size, induced the preferential production of G-CSF. CONCLUSION: This study elucidated unique signaling events triggered by GR-1, resulting in selective production of the immunomodulatory cytokine G-CSF in macrophages.


Assuntos
Fator Estimulador de Colônias de Granulócitos/biossíntese , Lacticaseibacillus rhamnosus/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Transdução de Sinais , Receptor 2 Toll-Like/metabolismo , Fatores de Virulência/imunologia , Animais , Western Blotting , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Perfilação da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase , Fatores de Virulência/metabolismo
11.
J Leukoc Biol ; 96(4): 549-61, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24981628

RESUMO

The G-CSF is best known for its activity in the generation and activation of neutrophils. In addition, studies on G-CSF(-/-) or G-CSFR(-/-) mice and BMC cultures suggested a role of G-CSF in macrophage generation. However, our understanding on the role of G-CSF in macrophage development is limited. Here, using in vitro BMC models, we demonstrated that G-CSF promoted the generation of Gr-1(high)/F4/80(+) macrophage-like cells in M-BMCs, likely through suppressing cell death and enhancing generation of Gr-1(high)/F4/80(+) macrophage-like cells. These Gr-1(high) macrophage-like cells produced "M2-like" cytokines and surface markers in response to LPS and IL-4/IL-13, respectively. Adoptive transfer of EGFP-expressing (EGFP(+)) M-BMCs showed a dominant, gut-homing phenotype. The small intestinal lamina propria of G-CSFR(-/-) mice also harbored significantly reduced numbers of Gr-1(high)/F4/80(+) macrophages compared with those of WT mice, but levels of Gr-1(+)/F4/80(-) neutrophil-like cells were similar between these mice. Collectively, these results suggest a novel function of G-CSF in the generation of gut-homing, M2-like macrophages.


Assuntos
Trato Gastrointestinal/citologia , Trato Gastrointestinal/imunologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Antígenos Ly/metabolismo , Antígenos de Superfície/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Feminino , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos/farmacologia , Imunofenotipagem , Macrófagos/citologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Receptores de Superfície Celular/genética
12.
J Immunol ; 193(3): 1333-43, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24973453

RESUMO

Macrophages pre-exposed to a sublethal dose of anthrax lethal toxin (LeTx) are refractory to subsequent high cytolytic doses of LeTx, termed toxin-induced resistance (TIR). A small population of TIR cells (2-4%) retains TIR characteristics for up to 5-6 wk. Through studying these long-term TIR cells, we found that a high level of histone deacetylase (HDAC)8 expression was crucial for TIR. Knocking down or inhibition of HDAC8 by small interfering RNAs or the HDAC8-specific inhibitor PCI-34051, respectively, induced expression of the mitochondrial death genes Bcl2 adenovirus E1B 19 kDa-interacting protein 3 (BNIP3), BNIP3-like and metastatic lymph node 64, and resensitized TIR cells to LeTx. Among multiple histone acetylations, histone H3 lysine 27 (H3K27) acetylation was most significantly decreased in TIR cells in an HDAC8-dependent manner, and the association of H3K27 acetylation with the genomic regions of BNIP3 and metastatic lymph node 64, where HDAC8 was recruited to, was diminished in TIR cells. Furthermore, overexpression of HDAC8 or knocking down the histone acetyltransferase CREB-binding protein/p300, known to target H3K27, rendered wild-type cells resistant to LeTx. As in RAW264.7 cells, primary bone marrow-derived macrophages exposed to a sublethal dose of LeTx were resistant to LeTx in an HDAC8-dependent manner. Collectively, this study demonstrates that epigenetic reprogramming mediated by HDAC8 plays a key role in determining the susceptibility of LeTx-induced pyroptosis in macrophages.


Assuntos
Antígenos de Bactérias/toxicidade , Apoptose/imunologia , Toxinas Bacterianas/toxicidade , Epigenômica/métodos , Histona Desacetilases/genética , Macrófagos/imunologia , Proteínas Repressoras/genética , Animais , Apoptose/efeitos dos fármacos , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Linhagem Celular , Testes Imunológicos de Citotoxicidade/métodos , Citotoxicidade Imunológica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos da Linhagem 129 , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/imunologia , Panobinostat , Proteínas Repressoras/antagonistas & inibidores
13.
PLoS One ; 8(2): e57138, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23437331

RESUMO

NOD2 is a cytosolic pattern-recognition receptor that senses muramyl dipeptide of peptidoglycan that constitutes the bacterial cell wall, and plays an important role in maintaining immunological homeostasis in the intestine. To date, multiple molecules have shown to be involved in regulating NOD2 signaling cascades. p62 (sequestosome-1; SQSTM1) is a multifaceted scaffolding protein involved in trafficking molecules to autophagy, and regulating signal cascades activated by Toll-like receptors, inflammasomes and several cytokine receptors. Here, we show that p62 positively regulates NOD2-induced NF-κB activation and p38 MAPK, and subsequent production of cytokines IL-1ß and TNF-α. p62 associated with the nucleotide binding domain of NOD2 through a bi-directional interaction mediated by either TRAF6-binding or ubiquitin-associated domains. NOD2 formed a large complex with p62 in an electron-dense area of the cytoplasm, which increased its signaling cascade likely through preventing its degradation. This study for the first time demonstrates a novel role of p62 in enhancing NOD2 signaling effects.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Citocinas/biossíntese , Proteína Adaptadora de Sinalização NOD2/metabolismo , Multimerização Proteica , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Citoplasma/metabolismo , Células HEK293 , Humanos , Macrófagos/metabolismo , Proteína Adaptadora de Sinalização NOD2/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Transporte Proteico , Proteína Sequestossoma-1 , Fator 6 Associado a Receptor de TNF/metabolismo
14.
Mol Cell Biol ; 32(23): 4846-60, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23028046

RESUMO

Cellular adaptation to different stresses related to survival and function has been demonstrated in several cell types. Anthrax lethal toxin (LeTx) induces rapid cell death, termed "pyroptosis," by activating NLRP1b/caspase-1 in murine macrophages. We and others (S. D. Ha et al., J. Biol. Chem. 282:26275-26283, 2007; I. I. Salles et al., Proc. Natl. Acad. Sci. U. S. A. 100:12426 -12431, 2003) have shown that RAW264.7 cells preexposed to sublethal doses of LeTx become resistant to subsequent high cytolytic doses of LeTx, termed toxin-induced resistance (TIR). To date, the cellular mechanisms of pyroptosis and TIR are largely unknown. We found that LeTx caused NLRP1b/caspase-1-dependent mitochondrial dysfunction, including hyperpolarization and generation of reactive oxygen species, which was distinct from that induced by stimuli such as NLRP3-activating ATP. In TIR cells, these mitochondrial events were not detected, although caspase-1 was activated, in response to LeTx. We identified that downregulation of the late endosomal cholesterol-transferring protein MLN64 in TIR cells was involved in TIR. The downregulation of MLN64 in TIR cells was at least in part due to DNA methyltransferase 1-mediated DNA methylation. In wild-type RAW264.7 cells and primary bone marrow-derived macrophages, LeTx caused NLRP1b/caspase-1-dependent mitochondrial translocation of MLN64, resulting in cholesterol enrichment, membrane hyperpolarization, reactive oxygen species (ROS) generation, and depletion of free glutathione (GSH). This study demonstrates for the first time that MLN64 plays a key role in LeTx/caspase-1-induced mitochondrial dysfunction.


Assuntos
Antraz/imunologia , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Toxinas Bacterianas/imunologia , Colesterol/imunologia , Macrófagos/virologia , Mitocôndrias/virologia , Fosfoproteínas/genética , Animais , Antraz/genética , Antraz/virologia , Caspase 1/imunologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Metilação de DNA , Regulação para Baixo , Glutationa/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/imunologia , Fosfoproteínas/análise , Fosfoproteínas/imunologia , Espécies Reativas de Oxigênio/imunologia , beta-Ciclodextrinas/farmacologia
15.
Antiviral Res ; 93(1): 175-84, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22138708

RESUMO

Human immunodeficiency virus type 1 (HIV-1) egresses from infected cells through utilizing the host membrane budding mechanisms. Assembly of HIV-1 Gag particles occurs on membranes where the Gag multimers subsequently bud off and form enveloped viral particles. In certain cell types such as macrophages, HIV-1 Gag particles have shown to be released into intracellular virus containing compartments (VCC) such as late endosomes, multivesicular bodies (MVBs) or invaginated plasma membrane pockets. Here, we showed that macrophages or HEK293T cells treated with the cathepsin B (CTSB)-specific inhibitor CA-074Me or cells deficient in CTSB failed to release HIV-1 Gag pseudoparticles into the extracellular environment. Based on immunofluorescence and electron microscopy, these cells retained the pseudoparticles in heterogeneous intracellular VCC. CA-074Me was also able to inhibit propagation of two enveloped viruses, herpes simplex virus and influenza A virus, but not non-enveloped enterovirus. These results suggest that CTSB is required for the efficient release of HIV-1 Gag pseudoparticles and targeting CTSB can be a new therapeutic strategy for inhibiting egress of HIV-1 and other enveloped viruses.


Assuntos
Catepsina B/antagonistas & inibidores , Catepsina B/deficiência , HIV-1/metabolismo , Macrófagos/virologia , Vírion/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Animais , Linhagem Celular , Dipeptídeos/farmacologia , Enterovirus/efeitos dos fármacos , Células HEK293 , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Vírus da Influenza A/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transporte Proteico/efeitos dos fármacos , Simplexvirus/efeitos dos fármacos , Tetraspanina 30/metabolismo , Vírion/efeitos dos fármacos , Vírion/ultraestrutura , Montagem de Vírus/efeitos dos fármacos , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética
16.
Protein Cell ; 2(7): 564-72, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21822801

RESUMO

The responses of macrophages to Bacillus anthracis infection are important for the survival of the host, since macrophages are required for the germination of B. anthracis spores in lymph nodes, and macrophage death exacerbates anthrax lethal toxin (LeTx)-induced organ collapse. To elucidate the mechanism of macrophage cell death induced by LeTx, we performed a genetic screen to search for genes associated with LeTx-induced macrophage cell death. RAW264.7 cells, a macrophage-like cell line sensitive to LeTx-induced death, were randomly mutated and LeTx-resistant mutant clones were selected. AMP deaminase 3 (AMPD3), an enzyme that converts AMP to IMP, was identified to be mutated in one of the resistant clones. The requirement of AMPD3 in LeTx-induced cell death of RAW 264.7 cells was confirmed by the restoration of LeTx sensitivity with ectopic reconstitution of AMPD3 expression. AMPD3 deficiency does not affect LeTx entering cells and the cleavage of mitogen-activated protein kinase kinase (MKK) by lethal factor inside cells, but does impair an unknown downstream event that is linked to cell death. Our data provides new information regarding LeTx-induced macrophage death and suggests that there is a key regulatory site downstream of or parallel to MKK cleavage that controls the cell death in LeTx-treated macrophages.


Assuntos
AMP Desaminase , Antraz/patologia , Antígenos de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Morte Celular , Exotoxinas/toxicidade , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , AMP Desaminase/genética , Animais , Sequência de Bases , Western Blotting , Morte Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Macrófagos/citologia , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase
17.
J Leukoc Biol ; 89(6): 907-15, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21385950

RESUMO

MΦs are important sensory cells of the innate immune system and regulate immune responses through releasing different combinations of cytokines. In this study, we examined whether cytokines released by MΦs in response to the probiotic bacterial strain GR-1 modulate the responses of DCs. The cytokine profile released by GR-1-treated MΦs was characterized by low levels of TNF-α, GM-CSF, IL-6, and IL-12 but very high levels of G-CSF. GR-1 CM did not induce expression of the shared p40 subunit of IL-12 and IL-23 and costimulatory molecules CD80 or CD86 or increase T cell stimulatory capacity in DCs. However, in G-CSFR-deficient DCs or after antibody-mediated neutralization of G-CSF, GR-1 CM induced IL-12/23 p40 production significantly, indicating that G-CSF within the GR-1 CM inhibits IL-12/23 p40 production induced by other CM components. GR-1 CM and rG-CSF also inhibited LPS-induced IL-12 production at the mRNA and protein levels. The inhibition of IL-12 production by G-CSF was at least in part mediated through inhibition of JNK activation. Finally, splenic DCs of GR-1-injected mice produced less IL-12/23 p40 than those of PBS-injected mice in response to LPS ex vivo, and this was at least partially dependent on exposure to GR-1-induced G-CSF in vivo. Altogether, these results suggest that G-CSF modulates the IL-12/23 p40 response of DCs in the context of the probiotic GR-1 through MΦ-DC crosstalk.


Assuntos
Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Lacticaseibacillus rhamnosus/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Receptores de Fator Estimulador de Colônias de Granulócitos/fisiologia , Animais , Western Blotting , Meios de Cultivo Condicionados/farmacologia , Células Dendríticas/citologia , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Interleucina-12/metabolismo , Interleucina-23/metabolismo , Lacticaseibacillus rhamnosus/crescimento & desenvolvimento , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Macrófagos/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
18.
Protein Cell ; 1(2): 161-73, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21088703

RESUMO

Metaxin, a mitochondrial outer membrane protein, is critical for TNF-induced cell death in L929 cells. Its deficiency, caused by retroviral insertion-mediated mutagenesis, renders L929 cells resistance to TNF killing. In this study, we further characterized metaxin deficiency-caused TNF resistance in parallel with Bcl-X(L) overexpression-mediated death resistance. We did not find obvious change in mitochondria membrane potential in metaxin-deficient (Met(mut)) and Bcl-X(L)-overexpressing cells, but we did find an increase in the release rate of the mitochondrial membrane potential probe rhodamine 123 (Rh123) that was preloaded into mitochondria. In addition, overexpression of a function-interfering mutant of metaxin (MetaΔTM/C) or Bcl-X(L) in MCF-7.3.28 cells also resulted in an acquired resistance to TNF killing and a faster rate of Rh123 release, indicating a close correlation between TNF resistance and higher rates of the dye release from the mitochondria. The release of Rh123 can be controlled by the mitochondrial membrane permeability transition (PT) pore, as targeting an inner membrane component of the PT pore by cyclosporin A (CsA) inhibited Rh123 release. However, metaxin deficiency and Bcl-X(L) overexpression apparently affect Rh123 release from a site(s) different from that of CsA, as CsA can overcome their effect. Though both metaxin and Bcl-X(L) appear to function on the outer mitochondrial membrane, they do not interact with each other. They may use different mechanisms to increase the permeability of Rh123, since previous studies have suggested that metaxin may influence certain outer membrane porins while Bcl-X(L) may form pores on the outer membrane. The alteration of the mitochondrial outer membrane properties by metaxin deficiency and Bcl-X(L) overexpression, as indicated by a quicker Rh123 release, may be helpful in maintaining mitochondrial integrity.


Assuntos
Apoptose , Permeabilidade da Membrana Celular , Membranas Mitocondriais/metabolismo , Proteínas/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Proteína bcl-X/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Poro de Transição de Permeabilidade Mitocondrial , Mutação , Necrose , Proteínas/genética , Espécies Reativas de Oxigênio/metabolismo , Rodamina 123/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
19.
Methods Mol Biol ; 634: 331-42, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20676994

RESUMO

In vitro random mutagenesis, followed by phenotype screening, provides a rapid and convenient tool for identifying novel genes involved in the phenotype of interest. However, the forward mutagenic approach in mammalian somatic cells is seriously limited by the diploidic nature of the genome. To overcome this impediment, we developed a method that allows functional screening for both haploid insufficient and sufficient genes involved in the phenotype of interest, utilizing a retrovirus gene trap mutagenesis in chemical mutagen-generated quasi-haploid cells. This method was used to identify novel host genes that are required for macrophage sensitivity to anthrax lethal toxin.


Assuntos
Macrófagos/metabolismo , Retroviridae/genética , Animais , Linhagem Celular , Haploidia , Camundongos
20.
J Biol Chem ; 285(3): 2120-9, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19858192

RESUMO

Anthrax lethal toxin (LeTx) is a virulence factor secreted by Bacillus anthracis and has direct cytotoxic effects on most cells once released into the cytoplasm. The cytoplasmic delivery of the proteolytically active component of LeTx, lethal factor (LF), is carried out by the transporter component, protective antigen, which interacts with either of two known surface receptors known as anthrax toxin receptor (ANTXR) 1 and 2. We found that the cytoplasmic delivery of LF by ANTXR2 was mediated by cathepsin B (CTSB) and required lysosomal fusion with LeTx-containing endosomes. Also, binding of protective antigen to ANXTR1 or -2 triggered autophagy, which facilitated the cytoplasmic delivery of ANTXR2-associated LF. We found that whereas cells treated with the membrane-permeable CTSB inhibitor CA074-Me- or CTSB-deficient cells had no defect in fusion of LC3-containing autophagic vacuoles with lysosomes, autophagic flux was significantly delayed. These results suggested that the ANTXR2-mediated cytoplasmic delivery of LF was enhanced by CTSB-dependent autophagic flux.


Assuntos
Antígenos de Bactérias/metabolismo , Autofagia , Toxinas Bacterianas/metabolismo , Catepsina B/metabolismo , Citoplasma/metabolismo , Endocitose , Receptores de Peptídeos/metabolismo , Animais , Catepsina B/antagonistas & inibidores , Linhagem Celular , Citoplasma/efeitos dos fármacos , Dipeptídeos/farmacologia , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Inibidores de Proteases/farmacologia
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