RESUMO
Protein oligomerization is a common principle of regulating cellular responses. Oligomerization of NLRs is essential for the formation of NLR signaling platforms and can be detected by several biochemical techniques. Some of these biochemical methods can be combined with functional assays, such as caspase-1 activity assay. Size exclusion chromatography (SEC) allows separation of native protein lysates into different sized complexes by FPLC for follow-up analysis. Using co-immunoprecipitation (co-IP), combined with SEC or on its own, enables subsequent antibody-based purification of NLR complexes and associated proteins, which can then be analyzed by immunoblot and/or subjected to functional caspase-1 activity assay. Native gel electrophoresis also allows detection of the NLR oligomerization state by immunoblot. Chemical cross-linking covalently joins two or more molecules, thus capturing the oligomeric state with high sensitivity and stability. ASC oligomerization has been successfully used as readout for NLR/ALR inflammasome activation in response to various PAMPs and DAMPs in human and mouse macrophages and THP-1 cells. Here, we provide a detailed description of the methods used for NLRP7 oligomerization in response to infection with Staphylococcus aureus (S. aureus) in primary human macrophages, co-immunoprecipitation, and immunoblot analysis of NLRP7 and NLRP3 inflammasome complexes as well as caspase-1 activity assays. Also, ASC oligomerization is shown in response to dsDNA, LPS/ATP, and LPS/nigericin in mouse bone marrow-derived macrophages (BMDMs) and/or THP-1 cells or human primary macrophages.
Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Camundongos , Animais , Humanos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Staphylococcus aureus/metabolismo , Lipopolissacarídeos , Cromatografia em Gel , Imunoprecipitação , Caspases/metabolismo , Caspase 1/metabolismo , Interleucina-1beta/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
The efficacy of immune checkpoint blockade (ICB) varies greatly among metastatic non-small cell lung cancer (NSCLC) patients. Loss of heterozygosity at the HLA-I locus (HLA-LOH) has been identified as an important immune escape mechanism. However, despite HLA-I disruptions in their tumor, many patients have durable ICB responses. Here we seek to identify HLA-I-independent features associated with ICB response in NSCLC. We use single-cell profiling to identify tumor-infiltrating, clonally expanded CD4+ T cells that express a canonical cytotoxic gene program and NSCLC cells with elevated HLA-II expression. We postulate cytotoxic CD4+ T cells mediate anti-tumor activity via HLA-II on tumor cells and augment HLA-I-dependent cytotoxic CD8+ T cell interactions to drive ICB response in NSCLC. We show that integrating tumor extrinsic cytotoxic gene expression with tumor mutational burden is associated with longer time to progression in a real-world cohort of 123 NSCLC patients treated with ICB regimens, including those with HLA-LOH.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Biomarcadores Tumorais/genética , Linfócitos T CD8-Positivos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Humanos , Imunoterapia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genéticaRESUMO
BACKGROUND: Colorectal cancer (CRC) consensus molecular subtypes (CMS) have different immunological, stromal cell, and clinicopathological characteristics. Single-cell characterization of CMS subtype tumor microenvironments is required to elucidate mechanisms of tumor and stroma cell contributions to pathogenesis which may advance subtype-specific therapeutic development. We interrogate racially diverse human CRC samples and analyze multiple independent external cohorts for a total of 487,829 single cells enabling high-resolution depiction of the cellular diversity and heterogeneity within the tumor and microenvironmental cells. RESULTS: Tumor cells recapitulate individual CMS subgroups yet exhibit significant intratumoral CMS heterogeneity. Both CMS1 microsatellite instability (MSI-H) CRCs and microsatellite stable (MSS) CRC demonstrate similar pathway activations at the tumor epithelial level. However, CD8+ cytotoxic T cell phenotype infiltration in MSI-H CRCs may explain why these tumors respond to immune checkpoint inhibitors. Cellular transcriptomic profiles in CRC exist in a tumor immune stromal continuum in contrast to discrete subtypes proposed by studies utilizing bulk transcriptomics. We note a dichotomy in tumor microenvironments across CMS subgroups exists by which patients with high cancer-associated fibroblasts (CAFs) and C1Q+TAM content exhibit poor outcomes, providing a higher level of personalization and precision than would distinct subtypes. Additionally, we discover CAF subtypes known to be associated with immunotherapy resistance. CONCLUSIONS: Distinct CAFs and C1Q+ TAMs are sufficient to explain CMS predictive ability and a simpler signature based on these cellular phenotypes could stratify CRC patient prognosis with greater precision. Therapeutically targeting specific CAF subtypes and C1Q + TAMs may promote immunotherapy responses in CRC patients.
Assuntos
Neoplasias Colorretais , Complemento C1q , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Complemento C1q/genética , Complemento C1q/uso terapêutico , Humanos , Instabilidade de Microssatélites , Transcriptoma , Microambiente Tumoral/genéticaRESUMO
Hepatic steatosis is a metabolic disease, characterized by selective and progressive accumulation of lipids in liver, leading to progressive non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and cirrhosis. The existing in vitro models of hepatic steatosis to elucidate the molecular mechanisms behind the onset of hepatic steatosis and to profile small molecule modulators uses lipid loaded primary hepatocytes, and cell lines like HepG2. The limitation of these models includes high variability between the different donor samples, reproducibility, and translatability to physiological context. An in vitro human hepatocyte derived model that mimics the pathophysiological changes seen in hepatic steatosis may provide an alternative tool for pre-clinical drug discovery research. We report the development of an in vitro experimental model of hepatic steatosis using human induced pluripotent stem cell (iPSC) derived hepatocytes like cells (HLC), loaded with lipids. Our data suggests that HLC carry some of the functional characteristics of primary hepatocytes and are amenable for development of an in vitro steatosis model using lipid loading method. The in vitro experimental model of hepatic steatosis was further characterized using biomarker analysis and validated using telmisartan. With some refinement and additional validation, our in vitro steatosis model system may be useful for profiling small molecule inhibitors and studying the mechanism of action of new drugs.
RESUMO
Parasitic worms are receiving much attention as a potential new therapeutic approach to treating autoimmune and allergic conditions but concerns remain regarding their safety. As an alternative strategy, we have focused on the use of defined parasitic worm products and recently taken this one step further by designing drug-like small molecule analogues of one such product, ES-62, which is anti-inflammatory by virtue of covalently attached phosphorylcholine moieties. Previously, we have shown that ES-62 mimics are efficacious in protecting against disease in mouse models of rheumatoid arthritis, systemic lupus erythematosus and skin and lung allergy. Given the potential role of chronic inflammation in fibrosis, in the present study we have focused our attention on lung fibrosis, a debilitating condition for which there is no cure and which in spite of treatment slowly gets worse over time. Two mouse models of fibrosis - bleomycin-induced and LPS-induced - in which roles for inflammation have been implicated were adopted. Four ES-62 analogues were tested - 11a and 12b, previously shown to be active in mouse models of allergic and autoimmune disease and 16b and AIK-29/62 both of which are structurally related to 11a. All four compounds were found to significantly reduce disease development in both fibrosis models, as shown by histopathological analysis of lung tissue, indicating their potential as treatments for this condition.
Assuntos
Anti-Inflamatórios/uso terapêutico , Proteínas de Helminto/uso terapêutico , Fibrose Pulmonar/tratamento farmacológico , Animais , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Inflammasomes are protein platforms linking recognition of microbe, pathogen-associated and damage-associated molecular patterns by cytosolic sensory proteins to caspase-1 activation. Caspase-1 promotes pyroptotic cell death and the maturation and secretion of interleukin (IL)-1ß and IL-18, which trigger inflammatory responses to clear infections and initiate wound-healing; however, excessive responses cause inflammatory disease. Inflammasome assembly requires the PYRIN domain (PYD)-containing adaptor ASC, and depends on PYD-PYD interactions. Here we show that the PYD-only protein POP2 inhibits inflammasome assembly by binding to ASC and interfering with the recruitment of ASC to upstream sensors, which prevents caspase-1 activation and cytokine release. POP2 also impairs macrophage priming by inhibiting the activation of non-canonical IκB kinase É and IκBα, and consequently protects from excessive inflammation and acute shock in vivo. Our findings advance our understanding of the complex regulatory mechanisms that maintain a balanced inflammatory response and highlight important differences between individual POP members.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Caspase 1/metabolismo , Inflamassomos/metabolismo , Proteínas Nucleares/metabolismo , Domínio Pirina , Animais , Citocinas/metabolismo , Ativação Enzimática , Citometria de Fluxo , Humanos , Quinase I-kappa B/metabolismo , Inflamação , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , PiroptoseRESUMO
Oligomerization of nod-like receptors (NLRs) can be detected by several biochemical techniques dependent on the stringency of protein-protein interactions. Some of these biochemical methods can be combined with functional assays, such as caspase-1 activity assay. Size exclusion chromatography (SEC) allows separation of native protein lysates into different sized complexes by fast protein liquid chromatography (FPLC) for follow-up analysis. Using co-immunoprecipitation (co-IP), combined with SEC or on its own, enables subsequent antibody-based purification of NLR complexes and associated proteins, which can then be analyzed by immunoblot and/or subjected to functional caspase-1 activity assay. Chemical cross-linking covalently joins two or more molecules, thus capturing the oligomeric state with high sensitivity and stability. Apoptosis-associated speck-like protein containing a caspase activation domain (ASC) oligomerization has been successfully used as readout for NLR or AIM2-like receptor (ALR) inflammasome activation in response to various pathogen- or damage-associated molecular patterns (PAMPs or DAMPs) in human and mouse macrophages and THP-1 cells. Here, we provide a detailed description of the methods used for NLRP7 oligomerization in response to infection with Staphylococcus aureus (S. aureus) in primary human macrophages, co-immunoprecipitation and immunoblot analysis of NLRP7 and NLRP3 inflammasome complexes, as well as caspase-1 activity assays. Also, ASC oligomerization is shown in response to dsDNA, LPS/ATP, and LPS/nigericin in mouse bone marrow-derived macrophages (BMDMs) and/or THP-1 cells or human primary macrophages.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Sinalização CARD/química , Proteína 3 que Contém Domínio de Pirina da Família NLR/química , Infecções Estafilocócicas/metabolismo , Animais , Cromatografia em Gel , Cromatografia Líquida , Reagentes de Ligações Cruzadas , Humanos , Imunoprecipitação , Macrófagos/citologia , Camundongos , Multimerização Proteica , Staphylococcus aureus/fisiologia , Células THP-1RESUMO
INTRODUCTION: Although caspase-8 is a well-established initiator of apoptosis and suppressor of necroptosis, recent evidence suggests that this enzyme maintains functions beyond its role in cell death. As cells of the innate immune system, and in particular macrophages, are now at the forefront of autoimmune disease pathogenesis, we examined the potential involvement of caspase-8 within this population. METHODS: Cre (LysM) Casp8 (fl/fl) mice were bred via a cross between Casp8 (fl/fl) mice and Cre (LysM) mice, and RIPK3 (-/-) Cre (LysM) Casp8 (fl/fl) mice were generated to assess the contribution of receptor-interacting serine-threonine kinase (RIPK)3. Immunohistochemical and immunofluorescence analyses were used to examine renal damage. Flow cytometric analysis was employed to characterize splenocyte distribution and activation. Cre (LysM) Casp8 (fl/fl) mice were treated with either Toll-like receptor (TLR) agonists or oral antibiotics to assess their response to TLR activation or TLR agonist removal. Luminex-based assays and enzyme-linked immunosorbent assays were used to measure cytokine/chemokine and immunoglobulin levels in serum and cytokine levels in cell culture studies. In vitro cell culture was used to assess macrophage response to cell death stimuli, TLR activation, and M1/M2 polarization. Data were compared using the Mann-Whitney U test. RESULTS: Loss of caspase-8 expression in macrophages promotes onset of a mild systemic inflammatory disease, which is preventable by the deletion of RIPK3. In vitro cell culture studies reveal that caspase-8-deficient macrophages are prone to a caspase-independent death in response to death receptor ligation; yet, caspase-8-deficient macrophages are not predisposed to unchecked survival, as analysis of mixed bone marrow chimeric mice demonstrates that caspase-8 deficiency does not confer preferential expansion of myeloid populations. Loss of caspase-8 in macrophages dictates the response to TLR activation, as injection of TLR ligands upregulates expression of costimulatory CD86 on the Ly6C(high)CD11b(+)F4/80(+) splenic cells, and oral antibiotic treatment to remove microbiota prevents splenomegaly and lymphadenopathy in Cre (LysM) Casp8 (fl/fl) mice. Further, caspase-8-deficient macrophages are hyperresponsive to TLR activation and exhibit aberrant M1 macrophage polarization due to RIPK activity. CONCLUSIONS: These data demonstrate that caspase-8 functions uniquely in macrophages by controlling the response to TLR activation and macrophage polarization in an RIPK-dependent manner.
Assuntos
Caspase 8/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Animais , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Antígenos Ly/imunologia , Antígenos Ly/metabolismo , Antígeno B7-2/imunologia , Antígeno B7-2/metabolismo , Western Blotting , Antígeno CD11b/imunologia , Antígeno CD11b/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Células Cultivadas , Feminino , Citometria de Fluxo , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Macrófagos/enzimologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Muramidase/genética , Muramidase/imunologia , Muramidase/metabolismo , Células Mieloides/enzimologia , Células Mieloides/imunologia , Células Mieloides/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Baço/imunologia , Baço/metabolismo , Baço/patologia , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismoRESUMO
In response to infections and tissue damage, ASC-containing inflammasome protein complexes are assembled that promote caspase-1 activation, IL-1ß and IL-18 processing and release, pyroptosis, and the release of ASC particles. However, excessive or persistent activation of the inflammasome causes inflammatory diseases. Therefore, a well-balanced inflammasome response is crucial for the maintenance of homeostasis. We show that the PYD-only protein POP1 inhibited ASC-dependent inflammasome assembly by preventing inflammasome nucleation, and consequently interfered with caspase-1 activation, IL-1ß and IL-18 release, pyroptosis, and the release of ASC particles. There is no mouse ortholog for POP1, but transgenic expression of human POP1 in monocytes, macrophages, and dendritic cells protected mice from systemic inflammation triggered by molecular PAMPs, inflammasome component NLRP3 mutation, and ASC danger particles. POP1 expression was regulated by TLR and IL-1R signaling, and we propose that POP1 provides a regulatory feedback loop that shuts down excessive inflammatory responses and thereby prevents systemic inflammation.
Assuntos
Síndromes Periódicas Associadas à Criopirina/imunologia , Células Dendríticas/imunologia , Inflamassomos/metabolismo , Macrófagos Peritoneais/imunologia , Monócitos/imunologia , Peritonite/imunologia , Ribonucleoproteínas/metabolismo , Animais , Apoptose/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Caspase 1/metabolismo , Linhagem Celular , Feminino , Regulação da Expressão Gênica/genética , Homeostase , Humanos , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Peritonite/induzido quimicamente , Multimerização Proteica/genética , RNA Interferente Pequeno/genética , Ribonucleoproteínas/genéticaRESUMO
Nucleotide-binding oligimerization domain (NOD)-like receptors (NLRs) are pattern recognition receptors (PRRs) involved in innate immune responses. NLRs encode a central nucleotide-binding domain (NBD) consisting of the NAIP, CIITA, HET-E and TP1 (NACHT) domain and the NACHT associated domain (NAD), which facilitates receptor oligomerization and downstream inflammasome signaling. The NBD contains highly conserved regions, known as Walker motifs, that are required for nucleotide binding and hydrolysis. The NLR containing a PYRIN domain (PYD) 7 (NLRP7) has been recently shown to assemble an ASC and caspase-1-containing high molecular weight inflammasome complex in response to microbial acylated lipopeptides and Staphylococcus aureus infection. However, the molecular mechanism responsible for NLRP7 inflammasome activation is still elusive. Here we demonstrate that the NBD of NLRP7 is an ATP binding domain and has ATPase activity. We further show that an intact nucleotide-binding Walker A motif is required for NBD-mediated nucleotide binding and hydrolysis, oligomerization, and NLRP7 inflammasome formation and activity. Accordingly, THP-1 cells expressing a mutated Walker A motif display defective NLRP7 inflammasome activation, interleukin (IL)-1ß release and pyroptosis in response to acylated lipopeptides and S. aureus infection. Taken together, our results provide novel insights into the mechanism of NLRP7 inflammasome assembly.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Trifosfato de Adenosina/metabolismo , Inflamassomos/metabolismo , Lipopeptídeos/farmacologia , Acilação , Proteínas Adaptadoras de Transdução de Sinal/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Células HEK293 , Humanos , Hidrólise , Interleucina-1beta/metabolismo , Dados de Sequência Molecular , Monócitos/metabolismo , Monócitos/microbiologia , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica , Estrutura Terciária de Proteína , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacosRESUMO
The innate immune system responds to infection and tissue damage by activating cytosolic sensory complexes called 'inflammasomes'. Cytosolic DNA is sensed by AIM2-like receptors (ALRs) during bacterial and viral infections and in autoimmune diseases. Subsequently, recruitment of the inflammasome adaptor ASC links ALRs to the activation of caspase-1. A controlled immune response is crucial for maintaining homeostasis, but the regulation of ALR inflammasomes is poorly understood. Here we identified the PYRIN domain (PYD)-only protein POP3, which competes with ASC for recruitment to ALRs, as an inhibitor of DNA virus-induced activation of ALR inflammasomes in vivo. Data obtained with a mouse model with macrophage-specific POP3 expression emphasize the importance of the regulation of ALR inflammasomes in monocytes and macrophages.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Infecções por Vírus de DNA/imunologia , Vírus de DNA/imunologia , Inflamassomos/metabolismo , Macrófagos/imunologia , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Caspase 1/metabolismo , Proteínas de Ligação a DNA , Células HEK293 , Humanos , Imunidade/genética , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Interferon gama/genética , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Alinhamento de Sequência , Transgenes/genética , Proteínas Virais/genética , Homólogo LST8 da Proteína Associada a mTORRESUMO
Cytosolic pathogen- and damage-associated molecular patterns are sensed by pattern recognition receptors, including members of the nucleotide-binding domain and leucine-rich repeat-containing gene family (NLR), which cause inflammasome assembly and caspase-1 activation to promote maturation and release of the inflammatory cytokines interleukin-1ß (IL-1ß) and IL-18 and induction of pyroptosis. However, the contribution of most of the NLRs to innate immunity, host defense, and inflammasome activation and their specific agonists are still unknown. Here we describe identification and characterization of an NLRP7 inflammasome in human macrophages, which is induced in response to microbial acylated lipopeptides. Activation of NLRP7 promoted ASC-dependent caspase-1 activation, IL-1ß and IL-18 maturation, and restriction of intracellular bacterial replication, but not caspase-1-independent secretion of the proinflammatory cytokines IL-6 and tumor necrosis factor-α. Our study therefore increases our currently limited understanding of NLR activation, inflammasome assembly, and maturation of IL-1ß and IL-18 in human macrophages.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Inflamassomos/imunologia , Lipopeptídeos/imunologia , Macrófagos/imunologia , Infecções Bacterianas/imunologia , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Transporte/imunologia , Caspase 1/metabolismo , Proteínas do Citoesqueleto/imunologia , Humanos , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Macrófagos/microbiologia , Complexos Multiproteicos/imunologia , Mycoplasma/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The rhs genes are a family of enigmatic composite genes, widespread among Gram-negative bacteria. In this study, we characterized rhsT, a Pseudomonas aeruginosa rhs gene that encodes a toxic protein. Expression of rhsT was induced upon contact with phagocytic cells. The RhsT protein was exposed on the bacterial surface and translocated into phagocytic cells; these cells subsequently underwent inflammasome-mediated death. Moreover, RhsT enhanced host secretion of the potent proinflammatory cytokines IL-1ß and IL-18 in an inflammasome-dependent manner. In a mouse model of acute pneumonia, infection with a P. aeruginosa strain lacking rhsT was associated with less IL-18 production, fewer recruited leukocytes, reduced pulmonary bacterial load, and enhanced animal survival. Thus, rhsT encodes a virulence determinant that activates the inflammasome.
Assuntos
Toxinas Bacterianas/genética , Inflamassomos/metabolismo , Pneumonia/imunologia , Pseudomonas aeruginosa/genética , Fatores de Virulência/genética , Animais , Linhagem Celular , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Immunoblotting , Peptídeos e Proteínas de Sinalização Intercelular , Interleucina-1beta/metabolismo , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Fagócitos/metabolismo , Pneumonia/microbiologia , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas em Tandem , TransfecçãoRESUMO
The innate immune system relies on the recognition of pathogens by pattern recognition receptors as a first line of defense and to initiate the adaptive immune response. Substantial progress has been made in defining the role of Nod (nucleotide-binding oligimerization domain)-like receptors and AIM2 (absent in melanoma 2) as pattern recognition receptors that activate inflammasomes in macrophages. Inflammasomes are protein platforms essential for the activation of inflammatory caspases and subsequent maturation of their pro-inflammatory cytokine substrates and induction of pyroptosis. This paper summarizes recent developments regarding the function of Nod-like receptors in immunity and disease.
Assuntos
Inflamação/imunologia , Proteínas Adaptadoras de Sinalização NOD/imunologia , Animais , Autoimunidade , Citocinas/imunologia , Humanos , Estresse FisiológicoRESUMO
Peroxisome proliferators activate nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha) and enhance the transcription of several genes in liver. We report here that synthetic PPARalpha ligands Wy-14,643, ciprofibrate, clofibrate, and others induce the nuclear translocation of constitutive androstane receptor (CAR) in mouse liver cells in vivo. Adenoviral-enhanced green fluorescent protein-CAR expression demonstrated that PPARalpha synthetic ligands drive CAR into the hepatocyte nucleus in a PPARalpha- and PPARbeta-independent manner. This translocation is dependent on the transcription coactivator PPAR-binding protein but independent of coactivators PRIP and SRC-1. PPARalpha ligand-induced nuclear translocation of CAR is not associated with induction of Cyp2b10 mRNA in mouse liver. PPARalpha ligands interfered with coactivator recruitment to the CAR ligand binding domain and reduced the constitutive transactivation of CAR. Both Wy-14,643 and ciprofibrate occupied the ligand binding pocket of CAR and adapted a binding mode similar to that of the CAR inverse agonist androstenol. These observations, therefore, provide information for the first time to indicate that PPARalpha ligands not only serve as PPARalpha agonists but possibly act as CAR antagonists.
Assuntos
Núcleo Celular/metabolismo , Hepatócitos/metabolismo , PPAR alfa/agonistas , PPAR alfa/metabolismo , Proliferadores de Peroxissomos/farmacologia , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Androstenóis/farmacologia , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Linhagem Celular Tumoral , Receptor Constitutivo de Androstano , Família 2 do Citocromo P450 , Indução Enzimática/efeitos dos fármacos , Humanos , Ligantes , Fígado/metabolismo , Camundongos , Camundongos Knockout , PPAR beta/agonistas , PPAR beta/metabolismo , Receptores Citoplasmáticos e Nucleares/agonistas , Esteroide Hidroxilases/metabolismo , Fatores de Transcrição/agonistasRESUMO
There is no report on the gene expression profile of retinoblastoma (Rb). We analyzed the gene expression profile of Rb by the microarray technique. One thousand four genes were upregulated and 481 genes were downregulated. Microarray data were confirmed by semiquantitative RT-PCR for 5 genes in Rb samples: CDC25A, C17orf75, ERBB3, LATS2, and CHFR. Clusters of differentially expressed genes were identified on chromosomes 1, 16, and 17. Based on the expression profile, we hypothesized that the PI3K/AKT/mTOR (insulin signaling) pathway might be dysregulated in Rb. Our semiquantitative RT-PCR analysis of the PIK3CA, AKT1, FRAP1, and RPS6KB1 genes in Rb samples supported this hypothesis. We suggest that known inhibitors of this pathway could be evaluated for the treatment of Rb.