RESUMO
SCOPE: Dendritic cells (DCs) are the most potent antigen-presenting cells that play an important role in the crosstalk between the innate and the adaptive immune response. Quercetin exposure is identified as an effective strategy to suppress the inflammatory response induced by LPS. METHODS AND RESULTS: In this study, using a next-generation sequencing analysis, the effect of quercetin on microRNAs (miRNAs) expression in DCs is examined. A signature of 113 miRNAs that are differentially regulated in LPS-stimulated DCs after quercetin exposure is defined. It is demonstrated that the loss of function of miR-369-3p in LPS-stimulated DCs during quercetin exposure led to an increase of CCAAT/enhancer binding protein ß (C/EBP-ß) mRNA and protein and its downstream targets tumor necrosis factor-α (TNF-α) and interleukin 6 (IL6). Conversely, it is shown that the ectopic induction of miR-369-3p without quercetin suppresses the inflammatory response of LPS reducing C/EBP-ß, TNF-α, and IL6 production. In vivo, oral administration of quercetin in dextran-sulfate-sodium-induced colitis induces miR-369-3p expression. CONCLUSIONS: These findings indicate that quercetin-induced miR-369-3p regulates the inflammatory cascade in chronic inflammatory response and present promising therapeutic implications.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/genética , Células Dendríticas/metabolismo , Expressão Gênica/efeitos dos fármacos , Inflamação/genética , MicroRNAs/fisiologia , Quercetina/farmacologia , Animais , Células da Medula Óssea/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/análise , Células Cultivadas/química , Células Dendríticas/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Interleucina-6/genética , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , RNA Mensageiro/análise , Fator de Necrose Tumoral alfa/genéticaRESUMO
BACKGROUND: Adipocytes behave like a rich source of pro-inflammatory cytokines including monocyte chemoattractant protein-1 (MCP-1). Oxidized low-density lipoprotein (oxLDL) participates in the local chronic inflammatory response, and high-density lipoprotein could counterbalance the proinflammatory function of oxLDL, but the underlying mechanism is not completely understood. This study aimed to evaluate the effect of apolipoprotein A-I mimetic peptide L-4F on the secretion and expression of MCP-1 in fully differentiated 3T3-L1 adipocytes induced by oxLDL and to elucidate the possible mechanisms. METHODS: Fully differentiated 3T3-L1 adipocytes were incubated in the medium containing various concentration of L-4F (0-50 µg/ml) with oxLDL (50 µg/ml) stimulated, with/without protein kinase A (PKA) inhibitor H-89 (10 µmol/L) preincubated. The concentrations of MCP-1 in the supernatant, the mRNA expression of MCP-1, the levels of CCAAT/enhancer binding protein α (C/EBPα), and CCAAT/enhancer binding protein ß (C/EBPß) were evaluated. The monocyte chemotaxis assay was performed by micropore filter method using a modified Boyden chamber. RESULTS: OxLDL stimulation induced a significant increase of MCP-1 expression and secretion in 3T3-L1 adipocytes, which were inhibited by L-4F preincubation in a dose-dependent manner. PKA inhibitor H-89 markedly reduced the oxLDL-induced MCP-1 expression, but no further decrease was observed when H-89 was used in combination with L-4F (50 µg/ml) (P > 0.05). OxLDL stimulation showed no significant effect on C/EBPα protein level but increased C/EBPß protein level in a time-dependent manner. H-89 and L-4F both attenuated C/EBPß protein level in oxLDL-induced 3T3-L1 adipocytes. CONCLUSIONS: OxLDL induces C/EBPß protein synthesis in a time-dependent manner and enhances MCP-1 secretion and expression in 3T3-L1 adipocytes. L-4F dose-dependently counterbalances the pro-inflammatory effect of oxLDL, and cyclic AMP/PKA-C/EBPß signaling pathway may participate in it.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/fisiologia , Quimiocina CCL2/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , AMP Cíclico/fisiologia , Lipoproteínas LDL/antagonistas & inibidores , Peptídeos/farmacologia , Transdução de Sinais/fisiologia , Células 3T3-L1 , Animais , Proteína beta Intensificadora de Ligação a CCAAT/análise , Quimiocina CCL2/genética , Humanos , Lipoproteínas LDL/farmacologia , CamundongosRESUMO
The expression of CCAAT/enhancer-binding protein (C/EBP)ß in the small airway epithelium of COPD is unknown. C/EBPß was assessed in peripheral lung tissue of non-smoking/smoking controls and patients with GOLD I-IV COPD by quantitative immunohistochemistry. The expression of C/EBPß was decreased in smokers compared to never smokers. Furthermore, C/EBPß was significantly elevated in advanced COPD vs. asymptomatic smokers, and the expression correlated to lung function decline. As C/EBPß exerts pro-inflammatory effects in the context of cigarette smoke, the elevated C/EBPß in advanced COPD may be an indication of a breakdown of regulatory mechanisms and excessive inflammation.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/análise , Pulmão/química , Doença Pulmonar Obstrutiva Crônica/metabolismo , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Imuno-Histoquímica , Pulmão/fisiopatologia , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Índice de Gravidade de Doença , Fumar/efeitos adversos , Regulação para CimaRESUMO
Apoptosis is a prominent characteristic in the pathogenesis of liver disease. The mechanism of hepatic apoptosis is not well understood. Hepatic apoptosis alters relative levels of nuclear factors such as Foxa2, NF-κB, C/EBPß, and p53. Regulation of nuclear factors modulates the degree of hepatic apoptosis and the progression of liver disease. Nuclear factors have distinctive mechanisms to mediate hepatic apoptosis. The modification of nuclear factors is a novel therapeutic strategy for liver disease as demonstrated by pre-clinical models and clinical trials.
Assuntos
Apoptose , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Fator 3-beta Nuclear de Hepatócito/metabolismo , Hepatopatias/patologia , Fígado/patologia , NF-kappa B/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/análise , Proteína beta Intensificadora de Ligação a CCAAT/genética , Regulação da Expressão Gênica , Fator 3-beta Nuclear de Hepatócito/análise , Fator 3-beta Nuclear de Hepatócito/genética , Humanos , Fígado/metabolismo , Hepatopatias/genética , Hepatopatias/metabolismo , NF-kappa B/análise , NF-kappa B/genética , Proteína Supressora de Tumor p53/análise , Proteína Supressora de Tumor p53/genéticaRESUMO
Our previous study investigated the effects of differentiation inducted by flavonoids derived from aâ¯Chinese herb. In this study, we found that LW-218, aâ¯new synthesized flavonoid, inhibited proliferation and induced differentiation of acute myeloid leukemia cells. The IC50s of LW-218 in HL-60, U937, K562, and NB4 cell lines were all less than 5 µM, suggesting greater capacity than compounds we have reported. LW-218 induced differentiation effects including morphologic changes, NBT reduction, and both of CD11b and CD14 expression. Results of western blots and siRNA transfection revealed that LW-218 increased the LAP/LIP ratio of C/EBPß which regulated monocytic differentiation of leukemia cells. Meanwhile, these differentiation effects could be attenuated by silencing PLSCR1 via siRNA transfection. In addition, regulation on LAP/LIP ratio, of C/EBPß was properly mediated by PLSCR1 which was up-regulated by LW-218. All these results suggested that C/EBPß was involved in regulation of PKCδ/PLSCR1 pathway during flavonoids-induced differentiation. LW-218 was aâ¯prospective differentiation inductor of AML cells and was requisite to proceed further investigation.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/fisiologia , Flavonoides/farmacologia , Leucemia Mieloide Aguda/patologia , Monócitos/citologia , Proteína beta Intensificadora de Ligação a CCAAT/análise , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Leucemia Mieloide Aguda/metabolismo , Proteínas de Transferência de Fosfolipídeos/fisiologia , Fosforilação , Proteína Quinase C-delta/metabolismoRESUMO
Sinonasal inverted papilloma (SIP) is a rare benign tumor featuring increased cell proliferation, a tendency toward squamous differentiation, recurrence and malignant transformation. The CCAAT enhancer binding proteins, C/EBPs, are transcription factors regulating the proliferation and differentiation of various types of cells, including epithelial cells. We prospectively investigated the production of these transcription factors and the related proliferation and differentiation targets, keratin-10, keratin-15 and cyclin-D1, in 26 SIP patients and 8 sinonasal polyposis cases suspected for SIP. Ten of these patients had one or more recurrences over follow-up periods of one to eight years. C/EBP-alpha and C/EBP-beta proteins were not found in normal-looking sinonasal epithelial cells. The proteins and RNAs were detected in SIP and, occasionally, in polyposis tissues. The production of these factors was not significantly correlated with age, sex, site, tumor size or medical history. By contrast, correlations were found between the levels of C/EBP-alpha and keratin-10 levels and between those of C/EBP-beta and keratin-15. C/EBP-alpha levels were also significantly correlated with cyclin-D1 levels. These data suggested that the C/EBPs are implicated in the regulation of cell proliferation and differentiation in SIP. Finally, recurrent SIP produced significantly larger amounts of C/EBP-alpha than non- recurrent tumors. These results implicate CCAAT enhancer binding proteins in the pathogenesis of SIP and highlight the role of C/EBP-alpha as a candidate marker for tumor recurrence.
Assuntos
Biomarcadores Tumorais/análise , Proteína alfa Estimuladora de Ligação a CCAAT/análise , Proteína beta Intensificadora de Ligação a CCAAT/análise , Papiloma Invertido/metabolismo , Neoplasias dos Seios Paranasais/metabolismo , Adolescente , Adulto , Idoso , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/metabolismo , Papiloma Invertido/patologia , Neoplasias dos Seios Paranasais/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
Intramuscular fat content is increased by feeding of low lysine diets in pigs. Reduction in dietary lysine intake results in low plasma lysine concentration and low cytosolic lysine concentration in skeletal muscles. From these observations, we hypothesized that low plasma lysine concentration in pigs fed on low lysine diets reduced supply of lysine from blood circulation to preadipocytes, and this limited supply of lysine might promote adipocyte differentiation in porcine muscles. In order to verify the hypothesis, we investigated the effects of low concentrations of lysine in culture medium on differentiation of 3T3-L1 preadipocytes. Low concentration of lysine suppressed lipid accumulation and messenger RNA (mRNA) expression and enzyme activity of fatty acid synthase. mRNA expressions of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) were lower in cells cultured in low lysine medium. On the other hand, mRNA and protein expressions of C/EBPß and C/EBPδ were not inhibited by low concentrations of lysine in culture medium. These results indicate that low lysine concentrations in culture medium inhibit differentiation of 3T3-L1 preadipocytes through inhibiting the mRNA expressions of PPARγ and C/EBPα.
Assuntos
Adipócitos/citologia , Diferenciação Celular/efeitos dos fármacos , Lisina/administração & dosagem , Suínos/fisiologia , Células 3T3-L1 , Animais , Western Blotting , Proteína alfa Estimuladora de Ligação a CCAAT/análise , Proteína beta Intensificadora de Ligação a CCAAT/análise , Proteína delta de Ligação ao Facilitador CCAAT/análise , Células Cultivadas , Meios de Cultura , Lipídeos/análise , Lisina/farmacologia , Camundongos , PPAR gama/análise , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Histiocytic and interdigitating dendritic cell sarcomas are rare tumors originating from bone marrow-derived myeloid stem cells. Recent studies have shown evidence of cross-lineage transdifferentiation of B cells in follicular lymphoma to histiocytic and dendritic cell sarcomas. In this study, we report the morphologic, molecular and cytogenetic analysis of seven cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) associated with histiocytic and dendritic cell sarcomas. All seven patients were elderly males (median age 71 years). The B-cell neoplasms preceded the development of the histiocytic and dendritic cell sarcomas in six of seven patients, and one patient had both tumors diagnosed at the same time. The tumors included four interdigitating dendritic cell sarcomas: one Langerhans cell sarcoma, one histiocytic sarcoma and one immature neoplasm with evidence of histiocytic origin. Laser-capture microdissection and PCR analysis showed identical clonal immunoglobulin gene rearrangements in the two phenotypically distinct components in all cases. There was a preferential usage of IGHV4-39 by the V-D-J gene rearrangement. By fluorescence in situ hybridization (FISH) analysis, two cases showed deletion 17p in both components, whereas four cases had normal cytogenetic findings by FISH in the CLL/SLL cells, but acquired cytogenetic abnormalities in the corresponding histiocytic and dendritic tumors. Chromosome 17p abnormalities were the most common cytogenetic abnormality detected in the sarcomas, seen in five of six cases studied. Compared with the CLL/SLL cells, the histiocytic/dendritic cells were largely negative for PAX5, but showed strong expression of PU.1 and variable and weak expression of CEBPß. Our study provides evidence for transdifferentiation of CLL/SLL B cells to tumors of dendritic and less often histiocytic lineage, and suggests that secondary genetic events may play a role in this phenomenon.
Assuntos
Transdiferenciação Celular , Sarcoma de Células Dendríticas Interdigitantes , Sarcoma Histiocítico , Sarcoma de Células de Langerhans , Leucemia Linfocítica Crônica de Células B , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Biomarcadores Tumorais/análise , Proteína beta Intensificadora de Ligação a CCAAT/análise , Aberrações Cromossômicas , Cromossomos Humanos Par 17 , Sarcoma de Células Dendríticas Interdigitantes/genética , Sarcoma de Células Dendríticas Interdigitantes/imunologia , Sarcoma de Células Dendríticas Interdigitantes/patologia , Rearranjo Gênico do Linfócito B , Sarcoma Histiocítico/genética , Sarcoma Histiocítico/imunologia , Sarcoma Histiocítico/patologia , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Sarcoma de Células de Langerhans/genética , Sarcoma de Células de Langerhans/imunologia , Sarcoma de Células de Langerhans/patologia , Microdissecção e Captura a Laser , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Maryland , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fator de Transcrição PAX5/análise , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas/análise , Transativadores/análise , Recombinação V(D)JRESUMO
BACKGROUND: The transcription factor CCAAT/enhancer-binding protein (C/EBP) beta is a critical mediator of murine endometrial function during embryo implantation. Our objective is to characterize changes in C/EBP beta mRNA abundance and protein localization over the normal human menstrual cycle. METHODS: Fifty normally cycling volunteers without reproductive disorders were randomized to undergo endometrial sampling on a specific cycle day, with secretory phase samples timed using urinary LH surge. Samples were assessed for relative C/EBP beta mRNA expression using quantitative real-time RT-PCR and for C/EBP beta protein localization using immunohistochemistry. The semiquantitative histologic scoring (HSCORE) system was used to compare staining intensity in each tissue compartment between each cycle phase. RESULTS: C/EBP beta mRNA expression by whole endometrium peaks in the late secretory phase and is significantly higher than that in the proliferative and mid-secretory phases. A marked increase in nuclear C/EBP beta protein immunostaining is seen in stromal cells beginning about cycle day 20, coincident with the start of endometrial receptivity. This increased staining continues for the remainder of the cycle. CONCLUSION: In the normal human menstrual cycle, C/EBP beta mRNA and protein expression also change, with increased nuclear immunostaining in the mid-secretory phase, suggesting a possible role for C/EBP beta in human endometrial receptivity.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/genética , Endométrio/metabolismo , Regulação da Expressão Gênica , Adulto , Proteína beta Intensificadora de Ligação a CCAAT/análise , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proliferação de Células , Feminino , Humanos , Imuno-Histoquímica , RNA Mensageiro/metabolismoRESUMO
Rehmannia glutinosa, a Traditional Chinese Medicine (TCM), has been used to increase physical strength. Here, we report that Rehmannia glutinosa extract (RE) inhibits adipocyte differentiation and adipogenesis. RE impairs differentiation of 3T3-L1 preadipocytes in a dose-dependent manner. At the molecular level, treatment with RE inhibits expression of the key adipocyte differentiation regulator C/EBPbeta, as well as C/EBPalpha and the terminal marker protein 422/aP2, during differentiation of preadipocytes into adipocytes. Additionally, RE inhibits the mitotic clonal expansion (MCE) process of adipocyte differentiation, and RE prevents localization of C/EBPbeta to the centromeres. RE also prevents high fat diet (HFD) induced weight gain and adiposity in rats. Taken together, our results indicate that Rehmannia glutinosa extract inhibits preadipocyte differentiation and adipogenesis in cultured cells and in rodent models of obesity.
Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Proteína alfa Estimuladora de Ligação a CCAAT/antagonistas & inibidores , Proteína beta Intensificadora de Ligação a CCAAT/antagonistas & inibidores , Obesidade/prevenção & controle , Extratos Vegetais/farmacologia , Células 3T3-L1 , Adipócitos/metabolismo , Adipogenia/genética , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/análise , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/análise , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Centrômero/química , Centrômero/metabolismo , Dieta , Modelos Animais de Doenças , Gorduras/administração & dosagem , Gorduras/efeitos adversos , Proteínas de Ligação a Ácido Graxo/antagonistas & inibidores , Proteínas de Ligação a Ácido Graxo/metabolismo , Masculino , Camundongos , Mitose/efeitos dos fármacos , Obesidade/induzido quimicamente , Obesidade/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
CONTEXT: Control of aromatase expression in uterine leiomyoma has significant clinical implications because aromatase inhibitors reduce tumor growth and associated irregular uterine bleeding. The mechanisms that regulate aromatase expression in leiomyoma are unknown. OBJECTIVES: We previously demonstrated that the cAMP-responsive proximal promoters I.3 and II regulate aromatase expression in vivo in uterine leiomyoma tissue. Here, we investigated the cellular and molecular mechanisms responsible for promoter I.3/II usage. RESULTS: In smooth muscle cells isolated from leiomyoma (LSMCs), dibutyryl cAMP significantly induced aromatase mRNA and enzyme activity. Reporter constructs of promoter I.3/II deletion and site-directed mutants with selective disruption of cis-regulatory elements in the -517/-16 bp region revealed that five out of seven elements, including three CCAAT/enhancer binding protein (C/EBP) binding sites and two cAMP response elements, were essential for cAMP-induced promoter activity. EMSAs demonstrated that nuclear extracts from LSMCs contain complexes assembled on four of the five cis-elements, with C/EBP binding sites, including a novel -245/-231 bp sequence, clearly associating with C/EBPbeta. Chromatin immunoprecipitation assays revealed that C/EBPbeta binds specifically to the promoter I.3/II region in intact cells. Dibutyryl cAMP significantly induced nuclear C/EBPbeta protein levels in LSMCs in a time-dependent manner. Conversely, knockdown of C/EBPbeta dramatically suppressed cAMP-induced aromatase mRNA and enzyme activity. CONCLUSIONS: C/EBPbeta, which binds to multiple cis-regulatory elements in promoter I.3/II, is a key factor in the transcriptional complex controlling aromatase expression in uterine leiomyoma cells. Definition of this mechanism further may assist in designing inhibitors of aromatase specific for leiomyoma tissue.
Assuntos
Aromatase/genética , Proteína beta Intensificadora de Ligação a CCAAT/fisiologia , Regulação Enzimológica da Expressão Gênica , Leiomioma/enzimologia , Neoplasias Uterinas/enzimologia , Proteína beta Intensificadora de Ligação a CCAAT/análise , Linhagem Celular Tumoral , AMP Cíclico/fisiologia , CMP Cíclico/análogos & derivados , CMP Cíclico/farmacologia , Feminino , Humanos , Leiomioma/patologia , Fosforilação , Regiões Promotoras Genéticas , Neoplasias Uterinas/patologiaRESUMO
GH activates the c-fos promoter by regulating multiple transcription factors. This study adds to our understanding of GH-regulated transcription by demonstrating that GH regulates the c-fos cAMP-response element (CRE) and its binding protein, CREB. Activation of the c-fos promoter by GH is impaired by expression of dominant-negative A-CREB. GH stimulates rapid and transient phosphorylation of CREB at Ser 133 (P-CREB), a critical site for transactivation by CREB, in 3T3-F442A preadipocytes. Mutation of this residue impairs GH-induced c-fos expression, suggesting that phosphorylation of CREB at Ser 133 contributes to GH-induced c-fos activation. The MEK inhibitor UO126 impaired the phosphorylation of CREB and that of C/EBPbeta, suggesting that ERKs mediate the phosphorylation of both proteins. UO126, but not the protein kinase A inhibitor H89, blocked GH-induced c-fos mRNA expression. A combination of CREB and C/EBPbeta enhanced c-fos promoter activation, and mutation of the CRE impaired the enhancement, as well as GH-stimulated c-fos activation. GH treatment increased the occupancy of both endogenous phospho-CREB and phospho-C/EBPbeta on the c-fos promoter. The increases were impaired by UO126. The active P-CREB and P-C/EBPbeta are induced by GH to occupy the same c-fos promoter DNA, suggesting that they may participate in a GH-regulated complex on c-fos. These findings suggest that coordinated phosphorylation of CREB and C/EBPbeta in response to GH is mediated by ERK1/2, and that the phosphorylated proteins are part of a regulatory complex that occupies c-fos in vivo to regulate c-fos transcription cooperatively in response to GH.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Expressão Gênica/efeitos dos fármacos , Genes fos/genética , Hormônio do Crescimento/farmacologia , Células 3T3 , Adipócitos/metabolismo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/análise , Células CHO , Cricetinae , Cricetulus , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/análise , DNA/metabolismo , Genes fos/efeitos dos fármacos , Humanos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/análise , Receptores da Somatotropina/fisiologia , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
BACKGROUND: Proliferative inflammatory atrophy (PIA) in the prostate has been proposed to be a precursor to prostate cancer. CCAAT/enhancer-binding protein beta (C/EBPbeta) is an important transcription factor involved in cellular proliferation and differentiation. Activation of C/EBPbeta plays a crucial role during the initial stage of cyclo-oxygenase 2 (COX-2) induction by proinflammatory mediators. Overexpression of C/EBPbeta has been reported in several human tumors. Nevertheless, the C/EBPbeta expression and functions in human prostate tissue are basically unknown. METHODS: C/EBPbeta immunohistochemical staining was performed on 45 benign prostate hyperplasia (BPH) samples. The expression of C/EBPbeta in PIA lesions and normal-appearing acini was analyzed. In addition, by using double-IHC staining, C/EBPbeta expression and the association with chronic inflammatory cell density, co-expression of COX-2 and androgen receptor (AR) were also investigated. RESULTS: C/EBPbeta was occasionally observed in normal-appearing prostate acini (4.9% +/- 6.7%, Mean +/- SD) but was clearly overexpressed in PIA lesions (81.8% +/- 16.4%) (P < 0.0001). Atrophic glands with T-lymphocyte and macrophage inflammation expressed higher level of C/EBPbeta. Furthermore, C/EBPbeta correlated significantly with COX-2 expression. Downregulation of the AR was common in PIA and was also related to the C/EBPbeta overexpression. CONCLUSIONS: The data demonstrated that chronic inflammation appeared to play roles in the induction of C/EBPbeta expression in prostate epithelium, which was in turn associated with increased COX-2 expression and AR downregulation. In combining with other molecular alteration in the epithelium of PIA, it is suggested that these cells might be a kind of intermediate cells and involved in the pathogenesis of prostate cancer.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/análise , Ciclo-Oxigenase 2/análise , Prostatite/metabolismo , Prostatite/patologia , Receptores Androgênicos/análise , Idoso , Atrofia , Biomarcadores/análise , Epitélio/patologia , Humanos , Imuno-Histoquímica , Inflamação , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/patologiaRESUMO
Geranylgeranylacetone (GGA) effectively protects the gastric mucosa against noxious agents. The precise mechanisms underlying the gastroprotective actions of GGA are not known. To elucidate the precise mechanism of GGA, the effect of GGA treatment on COX-2 expression in rat gastric epithelial (RGM1) cells was investigated. We used a prostaglandin E2 (PGE2) enzyme-linked immunoassay kit and Western blot analysis to measure PGE2 production and COX-2 induction by GGA treatment in serum-starved RGM1 cells. Gel-shift assay, Western blot analysis, and a reporter assay were performed to determine which COX-2 promoter was involved in GGA-induced COX-2 expression. GGA treatment dose dependently increased COX-2 expression and PGE2 production. The nuclear factor (NF)-kappaB sites of the COX-2 gene promoter were critical for GGA-mediated COX-2 expression. GGA induces COX-2 expression and increases PGE2 production in serum-starved RGM1 cells via activation of the NF-kappaB sites of COX-2 gene promoters.
Assuntos
Antiulcerosos/farmacologia , Ciclo-Oxigenase 2/biossíntese , Diterpenos/farmacologia , Mucosa Gástrica/efeitos dos fármacos , Animais , Western Blotting , Proteína beta Intensificadora de Ligação a CCAAT/análise , Células Cultivadas , Ciclo-Oxigenase 1/análise , Dinoprostona/biossíntese , Indução Enzimática , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Mucosa Gástrica/enzimologia , Proteínas de Membrana/análise , NF-kappa B/análise , RatosRESUMO
Early inflammatory changes in psoriatic plaques were investigated immunohistochemically by studying the normal-appearing skin adjacent to the plaques (perilesional skin), lesional skin, and distant uninvolved skin from psoriasis patients. Perilesional epidermis contained numerous CD1a-positive Langerhans cells, some of which expressed HLA-DR, CD83, CD80, and CD86, at the same time expressing Langerin. There were also numerous CD83-positive, CD11c-positive, Langerin-negative dendritic cells (DCs) in the epidermal-dermal junction of perilesional skin. CD3-positive T lymphocytes were sparse in the perilesional skin. Perilesional epidermis expressed keratin K6 and K16, inflammatory keratins, and C/EBPbeta, a transcription factor related to inflammatory cytokines. Our results demonstrated the abundant distribution of activated DCs in the perilesional skin of psoriatic plaques, where early inflammatory changes occur in the epidermal keratinocytes, which suggests their involvement in the provocation of epidermal inflammation in the perilesional epidermis and further pathogenic roles in the formation of psoriatic plaques.
Assuntos
Comunicação Celular , Células Dendríticas/fisiologia , Queratinócitos/fisiologia , Psoríase/patologia , Pele/patologia , Adulto , Idoso , Antígenos CD/análise , Antígenos CD1/análise , Antígeno B7-1/análise , Antígeno B7-2/análise , Proteína beta Intensificadora de Ligação a CCAAT/análise , Complexo CD3/análise , Citocinas/biossíntese , Feminino , Antígenos HLA-DR/análise , Humanos , Imunoglobulinas/análise , Células de Langerhans/fisiologia , Masculino , Glicoproteínas de Membrana/análise , Pessoa de Meia-Idade , Psoríase/imunologia , Pele/imunologia , Antígeno CD83RESUMO
Pulmonary fibrosis is characterized by inflammation, genesis of myofibroblasts, and abnormal tissue repair. Despite extensive research, its pathogenesis remains incompletely understood. Previously, the transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) was found to be a key regulator of myofibroblast differentiation in vitro, and to be involved in the acute phase and inflammatory responses. In an attempt to test the role of C/EBPbeta in the development of pulmonary fibrosis, experiments using C/EBPbeta null mice and their wild-type littermates were conducted. Our findings indicated that, compared to wild-type mice, animals deficient in C/EBPbeta showed significantly reduced fibrotic lesions and collagen deposition in the lung upon endotracheal injection of bleomycin. Further studies on the mechanisms by which C/EBPbeta regulates fibrosis indicated that knockout of C/EBPbeta attenuates inflammatory cytokine expression in bleomycin-treated mice. The reduced alpha-smooth muscle actin gene expression in either isolated lung fibroblasts or lung tissue from bleomycin or saline-treated C/EBPbeta deficient mice suggests that C/EBPbeta regulates myofibroblast differentiation during fibrosis. Consistent with this finding, cells from C/EBPbeta deficient mice exhibited higher proliferative rates than those from wild-type mice. These data suggest that C/EBPbeta plays an essential role in pulmonary fibrosis and that this role appears to be multifactorial with respect to cytokine expression, cell differentiation, and proliferation.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/análise , Fibrose Pulmonar/metabolismo , Actinas , Animais , Antibióticos Antineoplásicos , Bleomicina , Proteína beta Intensificadora de Ligação a CCAAT/deficiência , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Células Cultivadas , Colágeno/análise , Citocinas/análise , Fibroblastos/fisiologia , Expressão Gênica , Genótipo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso/metabolismo , Fibrose Pulmonar/patologia , Fibrose Pulmonar/fisiopatologia , RNA Mensageiro/análiseRESUMO
BACKGROUND: The brain is considered to be a reservoir of latent human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). We examined the mechanism by which innate immune responses contribute to the establishment of this reservoir. METHODS: Gene-specific RNA and DNA were quantitated using real-time reverse-transcription polymerase chain reaction (RT-PCR). Protein expression was examined using Western blot analysis. Binding to and regulation of the SIV long terminal repeat (LTR) was examined using electrophoretic mobility shift assay, luciferase reporter constructs, and chromatin immunoprecipitation assay. RESULTS: Interferon-beta (IFN-beta) and myxovirus A (MxA) mRNA are produced in the brain during acute SIV infection. IFN-beta both suppresses SIV LTR activity and induces expression of the dominant-negative isoform of CCAAT/enhancer-binding protein-beta (C/EBP-beta). C/EBP-beta and its dominant-negative isoform respectively enhance and suppress histone acetylation at the SIV LTR and are present at the SIV LTR in vivo. SIV DNA persists when viral RNA is undetectable in the brain, and activation of the LTR is suppressed at the level of histone acetylation. CONCLUSION: Innate immune responses to virus infection that suppress acute virus replication in the brain also facilitate transcriptional latency of SIV. These data provide the first mechanistic model of HIV latency in the brain.
Assuntos
Encéfalo/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologia , Latência Viral/fisiologia , Animais , Western Blotting , Proteína beta Intensificadora de Ligação a CCAAT/análise , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular , Imunoprecipitação da Cromatina , DNA Viral/análise , Ensaio de Desvio de Mobilidade Eletroforética , Expressão Gênica , Humanos , Imunidade Inata , Interferon beta/genética , Interferon beta/imunologia , Macaca nemestrina , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia , Sequências Repetidas Terminais , Proteínas Virais/análiseRESUMO
C/EBP beta (CCAAT/enhancer binding protein beta) is a transcriptional factor that belongs to the basic region-leucine zipper class DNA-binding proteins and plays a role in cell differentiation and inflammatory reactions. Although high tissue levels of inflammatory cytokines, such as interleukin (IL)-6, IL-8 and transforming growth factor-beta, have been observed in glioma patients, the mechanisms underlying this phenomenon remain to be elucidated. C/EBP beta induces a variety of cytokines and thus may play a role in the pathogenesis of glioma. In this study, we investigated the relationship between C/EBP beta expression, tumor histology, and prognosis in glioma. The expression of C/EBP beta mRNA was examined with quantitative real-time PCR and protein expression was examined with immunohistochemical techniques in 47 glioma tissue samples. Expression of C/EBP beta mRNA and protein was markedly increased in high grade glioma compared with low grade glioma. Patients whose expression of C/EBP beta mRNA and protein in tumor tissue was lower survived longer than those whose expressions were higher. In vitro, C/EBP beta siRNA inhibited glioma cell proliferation and invasion. Moreover, IL-8 production by glioma cells was inhibited by C/EBP beta siRNA transfection. These data suggest that increased expression of C/EBP beta may contribute to the promotion of tumor invasiveness and progression. The data imply that the comparison of C/EBP beta expression could be a prognostic marker for patients with glioma.
Assuntos
Neoplasias Encefálicas/patologia , Proteína beta Intensificadora de Ligação a CCAAT/genética , Glioma/patologia , Adolescente , Adulto , Idoso , Astrocitoma/genética , Astrocitoma/metabolismo , Astrocitoma/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/análise , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Criança , Feminino , Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioma/genética , Glioma/metabolismo , Humanos , Imuno-Histoquímica , Interleucina-8/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Interferência de RNA/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sobrevida , TransfecçãoRESUMO
Many studies examine the molecular genetics of gastric cancer, but few look at young patients in particular and there is no comparison of molecular expression between early-onset gastric cancer (< or = 45 years old) and conventional gastric cancers. Expression of cycloxygenase-2 (COX-2) is elevated in gastric adenocarcinomas compared to non-neoplastic mucosa, and in light of studies showing reduced risk of gastric cancer in nonsteroidal anti-inflammatory drug users, we have chosen to investigate the expression of COX-2 and related molecules in 113 early-onset gastric cancers and compare it with 91 conventional gastric cancers, using tissue microarrays. These markers include molecules known to be important in conventional gastric carcinogenesis, such as E-Cadherin, p53, COX-2, Trefoil Factor-1 (TFF1), beta-catenin, p16 and c-myc; as well as molecules not yet described as being important in gastric cancer, such as the transcription factor c-jun, the COX-2 mRNA stabilizer HuR, and C/EBP-beta, a transcription factor for COX-2. All markers showed a statistically significant difference between early-onset gastric cancers and conventional gastric cancers, using a chi2 test. In particular, early-onset gastric cancers displayed a COX-2 Low, TFF1-expressing phenotype, whereas COX-2 overexpression and loss of TFF1 was found in conventional cancers, and this difference between early-onset gastric cancers and conventional cancers remained statistically significant when adjusted for location and histology (P<0.0001 and P = 0.002 respectively). We found that COX-2 overexpression correlates significantly with loss of TFF1 (P = 0.001), overexpression of C/EBP-beta (P<0.001) and cytoplasmic HuR (P = 0.016). COX-2 was significantly associated with p53 positivity (P = 0.003). Abnormalities in E-Cadherin correlated significantly with diffuse phenotype, whereas high expression of COX-2, loss of TFF1 and overexpression of C/EBP-beta correlated with the intestinal phenotype. Our results provide further evidence that early-onset gastric cancer exhibits a distinctive expression profile that may have practical implications.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias Gástricas/patologia , Adulto , Idade de Início , Antígenos de Superfície/análise , Proteína beta Intensificadora de Ligação a CCAAT/análise , Caderinas/análise , Distribuição de Qui-Quadrado , Ciclo-Oxigenase 2/análise , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Humanos , Imuno-Histoquímica , Proteínas de Membrana/análise , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-myb/análise , Proteínas de Ligação a RNA/análise , Neoplasias Gástricas/epidemiologia , Neoplasias Gástricas/metabolismo , Fator Trefoil-1 , Proteína Supressora de Tumor p53/análise , Proteínas Supressoras de Tumor/análise , beta Catenina/análiseRESUMO
CCAAT element binding protein beta (C/EBPbeta) is an important regulator of cell growth, differentiation and in promoting tumor invasiveness. C/EBPbeta is located on chromosome 20q, which is amplified in many solid tumors including gastric cancers (GC). We sought to characterize the status of C/EBPbeta expression in GCs, which was recently found to repres TFF1 gene. Microarray analysis revealed overexpression of C/EBPbeta in 25 of 27 (93%) GC when compared to 12 normal gastric tissue samples. RT-PCR analysis confirmed the overexpression of C/EBPbeta transcripts in 54 of 59 (91%) GC. In total, 15 of 18 gastric tumors exhibited at least fivefold higher C/EBPbeta transcript levels compared to their corresponding adjacent normal gastric tissue samples. Moreover, immunohistochemistry analysis demonstrated increased nuclear staining of C/EBPbeta in 10 of 13 GC and at least fourfold overexpression of C/EBPbeta in three primary GC compared to adjacent normal gastric tissue. Furthermore, a striking correlation of decreased TFF1 expression with increased C/EBPbeta was observed in the gastric tumors studied. Microarray analysis demonstrated a loss of TFF1 expression in all 27 GC cases examined, of which 25 exhibited high C/EBPbeta expression compared to normal gastric tissue. RT-PCR analysis revealed loss of TFF1 expression in 56 of 59 gastric tumors in which 54 of these tumors exhibited overexpression of C/EBPbeta. Immunohistochemical analysis revealed overexpression of C/EBPbeta in 10 of 13 gastric tumors that exhibited low expression of TFF1 at the protein level. Thus, overexpression of the transcription factor C/EBPbeta in the majority of GCs is a novel finding.