RESUMO
BACKGROUND: Extensive local invasion of glioblastoma (GBM) cells within the central nervous system (CNS) is one factor that severely limits current treatments. The aim of this study was to uncover genes involved in the invasion process, which could also serve as therapeutic targets. For the isolation of invasive GBM cells from non-invasive cells, we used a three-dimensional organotypic co-culture system where glioma stem cell (GSC) spheres were confronted with brain organoids (BOs). Using ultra-low input RNA sequencing (ui-RNA Seq), an invasive gene signature was obtained that was exploited in a therapeutic context. METHODS: GFP-labeled tumor cells were sorted from invasive and non-invasive regions within co-cultures. Ui-RNA sequencing analysis was performed to find a gene cluster up-regulated in the invasive compartment. This gene cluster was further analyzed using the Connectivity MAP (CMap) database. This led to the identification of SKF83566, an antagonist of the D1 dopamine receptor (DRD1), as a candidate therapeutic molecule. Knockdown and overexpression experiments were performed to find molecular pathways responsible for the therapeutic effects of SKF83566. Finally, the effects of SKF83566 were validated in orthotopic xenograft models in vivo. RESULTS: Ui-RNA seq analysis of three GSC cell models (P3, BG5 and BG7) yielded a set of 27 differentially expressed genes between invasive and non-invasive cells. Using CMap analysis, SKF83566 was identified as a selective inhibitor targeting both DRD1 and DRD5. In vitro studies demonstrated that SKF83566 inhibited tumor cell proliferation, GSC sphere formation, and invasion. RNA sequencing analysis of SKF83566-treated P3, BG5, BG7, and control cell populations yielded a total of 32 differentially expressed genes, that were predicted to be regulated by c-Myc. Of these, the UHRF1 gene emerged as the most downregulated gene following treatment, and ChIP experiments revealed that c-Myc binds to its promoter region. Finally, SKF83566, or stable DRD1 knockdown, inhibited the growth of orthotopic GSC (BG5) derived xenografts in nude mice. CONCLUSIONS: DRD1 contributes to GBM invasion and progression by regulating c-Myc entry into the nucleus that affects the transcription of the UHRF1 gene. SKF83566 inhibits the transmembrane protein DRD1, and as such represents a candidate small therapeutic molecule for GBMs.
Assuntos
Antagonistas de Dopamina , Glioblastoma , Glioma , Proteínas Proto-Oncogênicas c-myc , Animais , Humanos , Camundongos , Encéfalo , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Dopamina , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Camundongos Nus , Família Multigênica , Receptores de Dopamina D1/antagonistas & inibidores , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Antagonistas de Dopamina/metabolismo , Antagonistas de Dopamina/farmacologia , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
Prostate cancer (PCa) seriously jeopardizes men's health worldwide. Dihydroartemisinin, which is an effective antimalarial agent, has shown potential anticancer effects in various human cancer cell lines, including PCa cells. However, the mechanisms underlying the anticancer activity of dihydroartemisinin are not fully understood. Ubiquitin-like with plant homeodomain and ring finger domain 1 (UHRF1) is highly expressed in a variety of tumors and is negatively correlated with the prognosis of various tumors. We reported previously that UHRF1 is downregulated during apoptosis induced by dihydroartemisinin in PC-3 PCa cells. In this study, we transfected PC-3 cells with lentiviruses containing UHRF1 or shRNA-UHRF1. Then, the cells were treated with dihydroartemisinin at different concentrations. Our data showed that overexpression of UHRF1 promoted cell proliferation and migration in PC-3 cells, inhibited cell apoptosis, increased cell proportion in G2 phase, increased DNA methyltransferase 1 and decreased p16INK4A expression at mRNA and protein levels. Downregulation of UHRF1 produces the opposite results. Moreover, the phenomena caused by overexpression of UHRF1 were inhibited after dihydroartemisinin treatment. Compared with control cells, cells overexpressing UHRF1 can resist the proapoptotic and antiproliferative effects of dihydroartemisinin to a certain extent. The effects of UHRF1 knockdown were further aggravated by dihydroartemisinin treatment, but no statistically significant effect was observed with increasing drug concentration. Our results suggested that dihydroartemisinin decreases proliferation and migration but enhances apoptosis of PCa cells, likely by downregulating UHRF1 and upregulating p16INK4A.
Assuntos
Artemisininas/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Neoplasias da Próstata/patologia , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferase 1/efeitos dos fármacos , Relação Dose-Resposta a Droga , Técnicas de Silenciamento de Genes , Humanos , MasculinoRESUMO
GSK2606414 is a novel, highly selective inhibitor of protein kinase Rlike endoplasmic reticulum kinase (PERK). GSK2606414 and its analogues have recently been demonstrated to delay tumor growth and prevent neurodegeneration. The present study investigated the effects of GSK2606414 on proliferation, apoptosis, and the expression of activating transcription factor 4 (ATF4), CCAAT/enhancerbinding protein homologous protein (CHOP) and vascular endothelial growth factor (VEGF) in human retinal pigment epithelial (RPE) cells under endoplasmic reticulum (ER) stress. ARPE19 human RPE cells were treated with 0.0150 µM GSK2606414, and ER stress was induced by thapsigargin (TG) treatment. Cell proliferation was assessed using the Cell Counting kit8 cell viability assay. Apoptosis was detected by AnnexinV/propidium iodide double staining using flow cytometry. Western blot analysis was used to measure eukaryotic initiation factor 2α (eIF2α) phosphorylation levels. ATF4, CHOP and VEGF mRNA expression levels were assessed using reverse transcriptionquantitative polymerase chain reaction. GSK2606414 treatment inhibited RPE cell proliferation in a dosedependent manner, however it did not induce apoptosis. In addition, GSK2606414 treatment inhibited eIF2α phosphorylation and reduced CHOP and VEGF mRNA expression levels in RPE cells under TGinduced ER stress. To the best of our knowledge, the present study is the first to demonstrate that GSK2606414 has a potential antiproliferative effect in RPE cells in vitro. This effect appeared to be achieved via inhibition of the PERK/ATF4/CHOP signaling pathway and suppression of VEGF expression levels.
Assuntos
Adenina/análogos & derivados , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Indóis/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Adenina/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Fosforilação/efeitos dos fármacos , RNA Mensageiro/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tapsigargina/farmacologia , Fator de Transcrição CHOP/efeitos dos fármacos , Fator de Transcrição CHOP/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Bisphenol A diglycidyl ether (BADGE), a PPARγ2 antagonist, has been shown to inhibit marrow adipogenesis and promote bone formation in intact animals. We investigated the impact of BADGE on a new and more clinically relevant physiological model, the ovariectomized (OVX) rat model. Forty female Wistar rats were divided into four treatment groups for 12 wk (n = 10/group): sham+vehicle, sham+BADGE, OVX+vehicle, and OVX+BADGE. Postmortem analyses included MRI, micro-CT, serological test, histomorphometry, biomechanical tests, RT-PCR, and Western blot. Overall, OVX induced a sequential marrow fat expansion accompanied by bone deterioration. Compared with OVX controls, BADGE reduced fat fraction of the distal femur by 36.3%, adipocyte density by 33.0%, adipocyte size by 28.6%, adipocyte volume percentage by 57.8%, and adipogenic markers PPARγ2 and C/EBPα by â¼50% in OVX rats. Similar results were observed in sham rats vs. vehicle. BADGE could promote bone quality in sham rats; however, BADGE did not significantly improve trabecular microarchitecture, biomechanical strength, and dynamic histomorphometric parameters except for trabecular separation in OVX rats. We concluded that early BADGE treatment at a dose of 30 mg/kg attenuates marrow adiposity in ovary-intact and OVX rats and stimulates bone formation in ovary-intact rats but does not significantly rescue bone quality in OVX rats.
Assuntos
Adipócitos/efeitos dos fármacos , Adiposidade/efeitos dos fármacos , Compostos Benzidrílicos/farmacologia , Medula Óssea/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Carcinógenos/farmacologia , Compostos de Epóxi/farmacologia , Adipócitos/patologia , Adipogenia/efeitos dos fármacos , Animais , Medula Óssea/patologia , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Contagem de Células , Tamanho Celular , Subunidade alfa 1 de Fator de Ligação ao Core/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Feminino , Osteocalcina/efeitos dos fármacos , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , Ovariectomia , PPAR gama/efeitos dos fármacos , PPAR gama/metabolismo , Ratos , Ratos Wistar , Microtomografia por Raio-XRESUMO
MEK/ERK signal pathway was required for the differentiation of granulocytes, megakaryocytes and erythrocytes. Recently, MEK/ERK cascade was reported to be involved in all-trans retinoic acid (ATRA) induced differentiation in acute promyelocytic leukemia (APL) cells. However, the upstream and downstream molecules of MEK/ERK signal pathway in this cell model remains to be elucidated. In this work, we showed that RAF-1 was activated and the blockade of RAF-1 activation attenuated MEK/ERK activation as well as ATRA-induced differentiation. ATRA-enhanced protein levels of C/EBPß, C/EBPε and PU.1, which were required for differentiation in APL cells, were suppressed by the specific inhibitor of MEK. However, MEK inhibition had no effect on the degradation of PML-RARα fusion protein or the restoration of PML nuclear bodies by ATRA treatment. Taken together, our study suggested that RAF-1/MEK/ERK cascade was involved in ATRA-induced differentiation in APL cells through enhancing the protein level of C/EBPß, C/EBPε and PU.1.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Leucemia Promielocítica Aguda/patologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Proto-Oncogênicas c-raf/fisiologia , Tretinoína/farmacologia , Antineoplásicos/farmacologia , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Proteína beta Intensificadora de Ligação a CCAAT/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Linhagem Celular Tumoral , Granulócitos/efeitos dos fármacos , Granulócitos/patologia , Humanos , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Transativadores/biossíntese , Transativadores/efeitos dos fármacosRESUMO
BACKGROUND: The accumulation of advanced glycation end products (AGEs) appears to be the main factor responsible for modulating periodontal inflammation in diabetes. The aim of this study is to examine the effects of AGEs on inflammation in human periodontal ligament cells and to investigate the mechanism with a specific emphasis on the role of endoplasmic reticulum (ER) stress-induced nuclear factor-kappa B (NF-κB) pathway. METHODS: Cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The protein expressions of ER markers and NF-κB were examined by Western blot analysis. The translocation of NF-κB was observed by immunofluorescence assay. Proinflammatory chemokine production was determined by enzyme-linked immunosorbent assay. RESULTS: Treatment with AGEs reduced cell viability in a concentration- and time-dependent manner. AGEs induced ER stress, as evidenced by survival molecules, such as glucose-regulated protein 78 (GRP78), double-stranded RNA-activated protein kinase-like ER kinase (PERK), and activating transcription factor 6 (ATF-6), and apoptotic molecules, such as CCAAT/enhancer binding protein homologous protein (CHOP) and caspase 12. AGEs upregulated the nucleoprotein expression of NF-κB, enhanced translocation of NF-κB from the cytoplasm to the nucleus, and increased the production of proinflammatory chemokines interleukin-6 and interleukin-8. CONCLUSION: AGEs mediate inflammation of human periodontal ligament cells via the ER stress-induced NF-κB pathway.
Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Produtos Finais de Glicação Avançada/farmacologia , Ligamento Periodontal/citologia , Fator 6 Ativador da Transcrição/efeitos dos fármacos , Adolescente , Adulto , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Caspase 12/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Quimiocinas/efeitos dos fármacos , Relação Dose-Resposta a Droga , Chaperona BiP do Retículo Endoplasmático , Feminino , Proteínas de Choque Térmico/efeitos dos fármacos , Humanos , Cadeias Pesadas de Imunoglobulinas/efeitos dos fármacos , Mediadores da Inflamação/análise , Interleucina-6/análise , Interleucina-8/efeitos dos fármacos , Masculino , NF-kappa B/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Fator de Transcrição CHOP/efeitos dos fármacos , Adulto Jovem , eIF-2 Quinase/efeitos dos fármacosRESUMO
BACKGROUND AND OBJECTIVE: Tobacco smoking is considered to be one of the major risk factors for periodontitis. For example, about half the risk of periodontitis can be attributable to smoking in the USA. It is evident that smokers have greater bone loss, greater attachment loss and deeper periodontal pockets than nonsmoking patients. It has recently been reported that endoplasmic reticulum (ER) stress markers are upregulated in periodontitis patients; however, the direct effects of nicotine on ER stress in regard to extracellular matrix (ECM) degradation are unclear. The purpose of this study was to examine the effects of nicotine on cytotoxicity and expression of ER stress markers, selected ECM molecules and MMPs, and to identify the underlying mechanisms in human periodontal ligament cells. We also examined whether ER stress was responsible for the nicotine-induced cytotoxicity and ECM degradation. MATERIAL AND METHODS: Cytotoxicity and cell death were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay and flow cytometric annexin V and propidium iodide staining. The mRNA and protein expressions of MMPs and ER markers were examined by RT-PCR and western blot analysis. RESULTS: Treatment with nicotine reduced cell viability and increased the proportion of annexin V-negative, propidium iodide-positive cells, an indication of cell death. Nicotine induced ER stress, as evidenced by survival molecules, such as phosphorylated protein kinase-like ER-resident kinase, phosphorylated eukaryotic initiation factor-2α and glucose-regulated protein-78, and apoptotic molecules, such as CAAT/enhancer binding protein homologous protein (CHOP). Nicotine treatment led to the downregulation of ECM molecules, including collagen type I, elastin and fibronectin, and upregulation of MMPs (MMP-1, MMP-2, MMP-8 and MMP-9). Inhibition of ER stress by salubrinal and transfection of CHOP small interfering RNA attenuated the nicotine-induced cell death, ECM degradation and production of MMPs. Salubrinal and CHOP small interfering RNA inhibited the effects of nicotine on the activation of Akt, JNK and nuclear factor-κB. CONCLUSION: These results indicate that nicotine-induced cell death is mediated by the ER stress pathway, involving ECM degradation by MMPs, in human periodontal ligament cells.
Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Nicotina/toxicidade , Ligamento Periodontal/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cinamatos/farmacologia , Colágeno Tipo I/efeitos dos fármacos , Elastina/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Chaperona BiP do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Fator de Iniciação 2 em Eucariotos/efeitos dos fármacos , Matriz Extracelular/enzimologia , Fibronectinas/efeitos dos fármacos , Proteínas de Choque Térmico/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 1 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 8 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Metaloproteinases da Matriz/efeitos dos fármacos , NF-kappa B/efeitos dos fármacos , Nicotina/antagonistas & inibidores , Ligamento Periodontal/citologia , Ligamento Periodontal/enzimologia , Proteínas Quinases/análise , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Tioureia/análogos & derivados , Tioureia/farmacologia , Fator de Transcrição CHOP/efeitos dos fármacos , Fator de Transcrição CHOP/genéticaRESUMO
In this paper, we review the current literature about the UHRF family that in particular includes the UHRF1 and UHRF2 genes. Its members play a fundamental role in cell proliferation through different structural domains. These domains include a ubiquitin-like domain (NIRF_N), a plant homeodomain (PHD) domain, a SRA domain and a RING domain. The SRA domain has only been observed in this family probably conferring unique properties to it. The unique enzymatic activity so far identified in this family involves the RING finger that contains a ubiquitin E3 ligase activity toward, for instance, histones. The physiological roles played by the UHRF family are most likely exerted during embryogenic development and when proliferation is required in adults. Interestingly, UHRF members are putative oncogenes regulated by tumor suppressor genes, but they exert also a feedback control on these latter. Finally, we propose some new roles for this family, including regulation and/or inheritance of the epigenetic code. Alteration of these regulatory mechanisms, such as those occurring in cancer cells, may be involved in carcinogenesis. The reasons why the UHRF family could be an interesting target for developing anticancer drugs is also developed.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Neoplasias/tratamento farmacológico , Ubiquitina-Proteína Ligases/metabolismo , Animais , Antineoplásicos/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Proliferação de Células , Sistemas de Liberação de Medicamentos , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor/fisiologia , Humanos , Camundongos , Neoplasias/genética , Neoplasias/fisiopatologia , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Oncogenes/efeitos dos fármacos , Ubiquitina-Proteína Ligases/efeitos dos fármacosRESUMO
Bactericidal/permeability-increasing protein (BPI) neutralizes the proinflammatory effects of lipopolysaccharide and is of potential clinical use in the treatment of fulminant Gram-negative infections. BPI is a cationic protein with antibacterial activity stored in azurophil (primary) granules of neutrophil granulocytes. However, the absence of BPI in patients with specific granule deficiency indicates a transcriptional control of BPI, which is distinct from that of other azurophil granule proteins. Accordingly, we demonstrate in vivo that the BPI mRNA level peaks, together with mRNA for specific granule proteins, during the myelocytic and metamyelocytic stage of granulocytic maturation. The human promyelocytic cell line NB4 expresses several azurophil granule proteins, but expression of BPI is undetectable. We show that treatment of NB4 cells with all-trans retinoic acid (ATRA) induces BPI expression at mRNA and at protein level. The induction is dependent on de novo protein synthesis, as judged by sensitivity to cycloheximide. Previous investigations have indicated a potential role of CCAAT/enhancer-binding protein (C/EBP) transcription factors in the regulation of BPI expression. Here, we show that induction of NB4 cells with ATRA correlates to direct binding of C/EBPbeta and C/EBPepsilon to the proximal BPI promoter, as determined by electrophoretic mobility shift analysis and chromatin immunoprecipitation. The dependency on C/EBPbeta and C/EBPepsilon provides an explanation for delayed BPI mRNA expression, as compared with mRNA of other azurophil granule proteins.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Membrana/metabolismo , Células Mieloides/efeitos dos fármacos , Regiões Promotoras Genéticas/fisiologia , Tretinoína/farmacologia , Peptídeos Catiônicos Antimicrobianos/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos/genética , Sítios de Ligação , Proteínas Sanguíneas/efeitos dos fármacos , Proteínas Sanguíneas/genética , Proteína beta Intensificadora de Ligação a CCAAT/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Humanos , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/genética , Células Mieloides/metabolismo , Neutrófilos/citologia , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade , Relação Estrutura-AtividadeRESUMO
The high-cholesterol/high-fat Western diet has abetted an epidemic of atherosclerotic cardiovascular disease, the leading cause of death in industrialized nations. Liver X receptors (LXRs) are oxysterol sensors that are required for normal cholesterol and triglyceride homeostasis, yet synthetic LXR agonists produce undesirable hypertriglyceridemia. Here we report a previously unrecognized role for hepatic LXRalpha in the links between diet, serum lipids, and atherosclerosis. A modest increase in hepatic LXRalpha worsened serum lipid profiles in LDL-receptor null mice fed normal chow but had the opposite effect on lipids and afforded strong protection against atherosclerosis on a Western diet. The beneficial effect of hepatic LXRalpha was abrogated by a synthetic LXR agonist, which activated SREBP-1c and its target genes. Thus, the interplay between diet and hepatic LXRalpha is a critical determinant of serum lipid profiles and cardiovascular risk, and selective modulation of LXR target genes in liver can ameliorate hyperlipidemia and cardiovascular disease.
Assuntos
Doenças Cardiovasculares/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dieta , Metabolismo dos Lipídeos , Fígado/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Anticolesterolemiantes/farmacologia , Arteriosclerose/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA/agonistas , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Feminino , Regulação da Expressão Gênica , Humanos , Hidrocarbonetos Fluorados , Lipídeos/sangue , Receptores X do Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores Nucleares Órfãos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/genética , Proteína de Ligação a Elemento Regulador de Esterol 1 , Sulfonamidas , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The isoforms of sterol regulatory element-binding proteins (SREBP) (1a, 1c, and 2) are key transcriptional regulators of lipid biosynthesis. We examined their regulation by gonadotropin and insulin in human granulosa cells. After removal of leukocytes, granulosa cells were exposed to hormonal additions for 16 h starting on d 2 of culture. Progesterone, lactate, and IGF binding protein-1 were measured in culture medium and cellular mRNA measured by competitive RT-PCR. Addition of human chorionic gonadotropin (hCG) (100 ng/ml) stimulated progesterone production (7.0-fold, P < 0.001 vs. control), whereas lactate was increased by hCG (1.6-fold, P < 0.001) and insulin (1.4-fold, P < 0.001; 1000 ng/ml). Insulin decreased IGF binding protein-1 production by 85% (P < 0.001). There were no significant effects on the expression of SREBP-1a but significant increases in mRNA for SREBP-1c with insulin (6.3-fold), hCG (10.4-fold) and in combination (15.2-fold; P < 0.01 for all comparisons). No consistent effects on SREBP-2 were observed. The expression of mRNA for fatty acid synthase, a target gene for SREBP-1c, was increased by hCG (24-fold, P = 0.006) and insulin (19-fold, P = 0.024), which also increased the level of cellular, total fatty acid (1.34-fold; P = 0.03). Thus, hCG and insulin cause a switch toward expression of the SREBP-1c isoform with consequent effects on fatty acid synthesis. We suggest that high circulating insulin, associated with clinically defined insulin resistance, may up-regulate SREBP-1c expression in the ovary.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Gonadotropina Coriônica/farmacologia , Proteínas de Ligação a DNA/genética , Células da Granulosa/metabolismo , Insulina/farmacologia , Fatores de Transcrição/genética , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Células Cultivadas , Primers do DNA , DNA Complementar/genética , Proteínas de Ligação a DNA/efeitos dos fármacos , Ácido Graxo Sintases/genética , Ácidos Graxos não Esterificados/metabolismo , Feminino , Fertilização in vitro , Células da Granulosa/efeitos dos fármacos , Humanos , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Progesterona/metabolismo , RNA Mensageiro/genética , Proteína de Ligação a Elemento Regulador de Esterol 1 , Proteína de Ligação a Elemento Regulador de Esterol 2 , Fatores de Transcrição/efeitos dos fármacosRESUMO
All-trans retinoic acid (ATRA) potently inhibits the differentiation of porcine preadipocytes in primary culture; however, the mechanism by which ATRA exerts this effect in pigs is poorly understood. The objective of this study was to use retinoid receptor-specific ligands to investigate the mechanism underlying the antiadipogenic action of retinoids in cultured pig preadipocytes by identifying the retinoid receptor mediating this action and examining the effect of retinoids on the expression of key adipogenic transcription factors. Stromal-vascular cells were harvested from porcine adipose tissue and cultured in serum-free medium. Glycerol-3-phoshphate dehydrogenase (GPDH) activity, a late marker of preadipocyte differentiation, was decreased (P < 0.01) by the addition of 0 to 10 microM of either ATRA, a nonspecific agonist for both the retinoic acid receptor (RAR) and the retinoid X receptor (RXR) or the selective RAR agonist, 4-(E-2-[5,6,7,8-tet-rahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl) benzoic acid (TTNPB). Addition of increasing amounts of Ro-61, a RAR-specific antagonist (0 to 10 microM) prevented ATRA and TTNBP from decreasing GPDH activity. Addition of methoprene acid, an RXR-specific agonist, increased (P < 0.01) GPDH activity. Preadipocytes were then continuously treated with 10 nM of TTNPB in the presence or absence of 1 microM Ro-61, and mRNA was isolated on d 2 and 8. Addition of TTNPB decreased (P < 0.001) the expression of peroxisome proliferator-activated receptor gamma (PPARgamma), sterol regulatory element-binding protein-1c (SREBP-1c), retinoid X receptor alpha (RXRalpha), and adipocyte fatty acid binding protein (aP2) mRNA transcripts, whereas these effects were prevented by the presence of Ro-61. Interestingly, TTNBP increased (P < 0.001) the mRNA abundance of the orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1), whereas Ro-61 prevented this increase. These changes were independent of alterations in the mRNA abundances of the retinoic acid receptor alpha, and CCAAT/enhancer binding protein alpha and beta (C/EBPbeta; C/EBPalpha) genes. These results indicate that retinoic acid inhibits porcine preadipocyte differentiation by a mechanism that involves activation of the RAR and downregulation of PPARgamma, RXRalpha, and SREBP-1C mRNA. This mechanism is independent of changes in C/EBPbeta and C/EBPalpha mRNA abundance and may involve COUP-TF.
Assuntos
Adipócitos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Receptores do Ácido Retinoico/fisiologia , Suínos/fisiologia , Tretinoína/farmacologia , Adipócitos/citologia , Animais , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Fator I de Transcrição COUP/efeitos dos fármacos , Células Cultivadas , Primers do DNA/química , Expressão Gênica/efeitos dos fármacos , Glicerolfosfato Desidrogenase/efeitos dos fármacos , Glicerolfosfato Desidrogenase/metabolismo , PPAR gama/efeitos dos fármacos , Receptores do Ácido Retinoico/agonistas , Receptores do Ácido Retinoico/antagonistas & inibidores , Proteína de Ligação a Elemento Regulador de Esterol 1/efeitos dos fármacos , Fatores de Tempo , Fatores de Transcrição/efeitos dos fármacosRESUMO
BACKGROUND AND OBJECTIVES: C/EBP homologous protein (CHOP) is an endoplasmic reticulum (ER) stress inducible transcription factor involved in the development of apoptosis, growth arrest, and differentiation. CHOP deficient (chop - / - ) mouse embryonic fibroblasts (MEFs) exposed to ER stresses such as tunicamycin exhibit decreased apoptosis and reduced toxicity when compared to chop + / + control cells. Overexpression of the 70 kDa heat shock stress protein (HSP-70) can inhibit apoptotic pathways. The biological significance of photodynamic therapy (PDT) protocols that induce cellular damage resulting in differential CHOP and stress protein expression patterns was examined. STUDY DESIGN/MATERIALS AND METHODS: Wild type mouse radiation induced fibrosarcoma (RIF) cells as well as MEFs with chop + / + and chop - / - genotypes were used with either a mitochondrial and ER localizing porphyrin (PH) photosensitizer or a lysosomal localizing chlorin (NPe6) photosensitizer. PDT induced cytotoxicity, apoptosis, and stress protein expression patterns were determined as a function of cell type and photosensitizer. RESULTS: PH mediated PDT induced expression of CHOP and 78 kDa glucose regulated protein (GRP-78), but not HSP-70 while NPe6 mediated PDT induced protein expression of HSP-70 but did not activate CHOP or GRP-78 expression. Enhanced apoptosis and toxicity were observed in chop + / + cells following exposure to tunicamycin or PH mediated PDT when compared to identical treatments in chop - / - cells. NPe6 mediated PDT induced minimally detectable apoptosis in both chop + / + and chop - / - cells and only a modest increase in survival for chop - / - cells. CONCLUSIONS: These results demonstrate that PDT activation of CHOP, GRP-78, and HSP-70 varied as a function of photosensitizer subcellular localization and that a single oxidative stress response was not observed following PDT. We also show that CHOP expression increased apoptosis following PH mediated PDT and that increased CHOP expression is associated with enhanced PDT photosensitization.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Fatores de Transcrição/efeitos dos fármacos , Animais , Apoptose/fisiologia , Proteínas Estimuladoras de Ligação a CCAAT/biossíntese , Técnicas de Cultura de Células , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP70/efeitos dos fármacos , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/efeitos dos fármacos , Camundongos , Chaperonas Moleculares/biossíntese , Chaperonas Moleculares/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Porfirinas/farmacologia , Fator de Transcrição CHOP , Fatores de Transcrição/biossínteseRESUMO
Activation of beta-adrenergic receptor (betaAR) increases the synthesis of nerve growth factor (NGF) in the brain and in C6-2B glioma cells. However, in the brain, the betaAR-mediated increase in NGF expression appears to require the presence of glucocorticoids, suggesting that NGF promoter may be sensitive to cAMP and glucocorticoid-dependent transcription factors. We tested this hypothesis by exposing C6-2B glioma cells to dexamethasone (DEX) in combination with agents that increase cAMP levels and examining the DNA binding activity of two cAMP-dependent transcription factors that regulate NGF expression: cAMP responsive element binding protein (CREB) and CCAAT/enhancer binding protein delta (C/EBPdelta). Electrophoretic mobility shift assays revealed that the beta(2)AR agonist clenbuterol (CLE) or high levels of cAMP elicited a time-dependent increase in C/EBPdelta binding activity as well as phosphorylated CREB (P-CREB). When DEX, which per se showed little effect on these transcription factors, was combined with CLE, dibutyryl cAMP or isoproterenol, enhanced induction of P-CREB and C/EBP binding activity as well as NGF mRNA was observed. Moreover, the increase in NGF mRNA in the presence of DEX was prolonged compared to that obtained by CLE or other cAMP inducing agents alone. In fact, NGF mRNA levels remained significantly elevated at least for 24 h. These studies suggest that the synergistic effect of DEX on the induction of NGF mRNA may include the ability of this glucocorticoid to potentiate the betaAR-mediated induction of transcription factors.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Dexametasona/farmacologia , Fator de Crescimento Neural/genética , Neurônios/metabolismo , Receptores Adrenérgicos beta/metabolismo , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Sinergismo Farmacológico , Glucocorticoides/metabolismo , Glucocorticoides/farmacologia , Neurônios/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Receptores Adrenérgicos beta/efeitos dos fármacos , Fatores de Tempo , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Adipogenesis is dependent on the sequential activation of transcription factors including the CCAAT/enhancer-binding proteins (C/EBP), peroxisome proliferator-activated receptor gamma (PPARgamma), and steroid regulatory element-binding protein (SREBP). We show that the mood stabilizing drug valproic acid (VPA; 0.5-2 mm) inhibits mouse 3T3 L1 and human preadipocyte differentiation, likely through its histone deacetylase (HDAC) inhibitory properties. The HDAC inhibitor trichostatin A (TSA) also inhibited adipogenesis, whereas the VPA analog valpromide, which does not possess HDAC inhibitory effects, did not prevent adipogenesis. Acute or chronic VPA treatment inhibited differentiation yet did not affect mitotic clonal expansion. VPA (1 mm) inhibited PPARgamma induced differentiation but does not activate a PPARgamma reporter gene, suggesting that it is not a PPARgamma ligand. VPA or TSA treatment reduced mRNA and protein levels of PPARgamma and SREBP1a. TSA reduced C/EBPalpha mRNA and protein levels, whereas VPA only produced a decrease in C/EBPalpha protein expression. Overall our results highlight a role for HDAC activity in adipogenesis that can be blocked by treatment with VPA.
Assuntos
Adipócitos/citologia , Histona Desacetilases/fisiologia , Ácido Valproico/farmacologia , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Humanos , Camundongos , RNA Mensageiro/análise , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Proteína de Ligação a Elemento Regulador de Esterol 1 , Fatores de Tempo , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/fisiologiaRESUMO
Adrenoleukodystrophy protein (ABCD1), a peroxisomal membrane protein, is mutated in patients affected by X-linked adrenoleukodystrophy (X-ALD). Adrenoleukodystrophy-related protein (ABCD2) is the closest relative of ABCD1. Pharmacological induction of ABCD2 gene expression has been proposed as a novel therapy strategy for X-ALD. Fibrates induce peroxisome proliferation and Abcd2 expression in rodent liver. Here we evaluate the possibility of using peroxisome proliferator-activated receptor alpha (PPARalpha) agonists for pharmacological induction of ABCD2 expression. In the liver of PPARalpha-deficient mice, both the constitutive and the fenofibrate-inducible Abcd2 gene expression was found to be PPARalpha-dependent. In the brain, PPARalpha-deficiency has no effect on Abcd2 expression. In mice orally treated with the novel, highly selective, and potent PPARalpha agonists GW 7647, GW 6867, and tetradecylthioacetic acid, Abcd2 expression was induced in liver and adrenal glands, but not in brain and testis. None of four putative PPREs identified in the 5(')-flanking DNA and in intron 1 of the Abcd2 gene conferred fibrate response in luciferase reporter assays. Thus, although fibrate-mediated Abcd2 induction is PPARalpha-dependent, it appears to be an indirect mechanism. Within the mouse Abcd2 promoter, a putative sterol regulatory element (SRE) similar in sequence and position to the characterized SRE sequence of the human ABCD2 promoter, was identified. A PPARalpha dependent induction of the sterol regulatory-binding protein 2 (SREBP2) and a down-regulation of SREBP1c mRNA levels could be demonstrated after fenofibrate treatment of mice. Our results suggest that the PPARalpha agonist-mediated induction of Abcd2 expression seems to be indirect and possibly mediated by SREBP2.
Assuntos
Adrenoleucodistrofia/tratamento farmacológico , Receptores Citoplasmáticos e Nucleares/agonistas , Fatores de Transcrição/agonistas , Subfamília D de Transportador de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Butiratos/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Íntrons , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Dados de Sequência Molecular , Compostos de Fenilureia/farmacologia , Receptores Citoplasmáticos e Nucleares/deficiência , Elementos de Resposta/efeitos dos fármacos , Elementos de Resposta/genética , Proteína de Ligação a Elemento Regulador de Esterol 1 , Proteína de Ligação a Elemento Regulador de Esterol 2 , Esteróis/metabolismo , Sulfetos/farmacologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismoRESUMO
AIM: To explore the mechanisms of effects of oil A on apoptosis of human pancreatic cancer cells. METHODS: Cellular DNA content was analyzed by flow cytometry. Western blotting was used for caspase-3 and PARP, caspase-7, caspase-9, cytochrome c, Bcl-2, Bax, Mcl-1, cyclinA, cyclin B1, cyclin D1, cyclin E, CDK2, CDK4, CDK6, P21, P27, GADD45, GADD153. RESULTS: The caspase-3, caspase-7, and caspase-9 activities were significantly increased as well as the cleavage of caspase-3, downstream substrate poly-ADP ribose polymerase (PARP) was induced. The amount of cytochrome c in the cytosolic fraction was increased, while the amount of cytochrome c in the mitochondrial fraction was decreased after oil A treatment. The anti-apoptosis proteins Bcl-2 and Mcl-1 were decreased in parallel and Bax increased, indicating that Bcl-2 family proteins-mitochondria-caspase cascade was responsible for oil-induced apoptosis. The proportion of cells in the G0/G1 decreased in MiaPaCa-2 and AsPC-1 cells after the treatment of oil A for 24 hours. The number of cells in S phase was increased in two cancer cell lines at 24 hours. Therefore, cells were significantly accumulated in G2/M phase. The cells with a sub-G0/G1 DNA content, a hallmark of apoptosis, were seen at 24 hours both in MiaPaCa-2 and AsPC-1 cells following exposure to oil A. The expression of cyclin A and cyclin B1 was slightly decreased and cyclin D1 levels were markedly lowered in MiaPaCa-2 cells. The expression of cyclin A and cyclin B1 was markedly decreased and cyclin D1 levels were slightly lowered in AsPC-1 cells, while cyclin E was not affected and the levels of CDK2, CDK4, and CDK6 were unchanged in MiaPaCa-2 and AsPC-1 cells. In response to oil A, P21 expression was increased, but P27 expression was not affected. The expression of both GADD45 and GADD153 was increased in two cell lines following oil A treatment. CONCLUSION: Oil A induces apoptosis of pancreatic cancer cells via activating caspase cascade, modifying cell cycle progress and changing cell cycle-regulating proteins and GADD expression.
Assuntos
Anticarcinógenos/farmacologia , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Lipídeos/farmacologia , Neoplasias Pancreáticas/patologia , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Caspases/efeitos dos fármacos , Ciclinas/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , DNA de Neoplasias/efeitos dos fármacos , Citometria de Fluxo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Mitocôndrias/fisiologia , Neoplasias Pancreáticas/genética , Proteínas/efeitos dos fármacos , Proteínas/metabolismo , Fator de Transcrição CHOP , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Proteínas GADD45RESUMO
OBJECTIVES: This study was designed to investigate the effect of hibiscus (Hibiscus sabdariffa) on adipogenic differentiation of 3T3-L1 cells at the cellular and molecular levels. DESIGN: Various concentrations of hibiscus extract were added to confluent 3T3-L1 preadipocytes at the outset of the differentiation program and further incubated for 36 hours. Cells were maintained in postdifferentiation medium containing insulin with hibiscus extract in complete culture medium. RESULTS: Hibiscus extract inhibited the adipocyte differentiation of 3T3-L1 preadipocytes induced by insulin, dexamethasone, and isobutylmethylxanthine (IBMX) in a dose-dependent manner. Hibiscus blocked the cytoplasmic lipid accumulation when administered at the onset of differentiation and 4 days after induction of differentiation. The inhibitory effect of hibiscus on adipogenic lipid accumulation of preadipocytes was significant (p < 0.01) between control cells and cells treated with hibiscus. Hibiscus extract significantly attenuated the expression of key adipogenic transcription factors, including CCAAT element binding protein (C/EBP)alpha and peroxisome proliferator-activated receptor (PPAR)gamma at protein levels. CONCLUSION: These results suggest that hibiscus extract blocks adipogenesis, in part, by its suppression on the expression of adipogenic transcription factors, including C/EBPalpha and PPARgamma.
Assuntos
Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Hibiscus , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Células 3T3/efeitos dos fármacos , Animais , Western Blotting , Fator de Ligação a CCAAT/efeitos dos fármacos , Fator de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Hibiscus/metabolismo , Humanos , Camundongos , Extratos Vegetais , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Fator de Transcrição CHOPRESUMO
Oltipraz, a cancer chemopreventive agent, induces CYP1A1 to a certain extent by transactivation of the gene via the Ah receptor (AhR)-xenobiotic response element (XRE) pathway. Previously, we showed that oltipraz promoted CCAAT/enhancer binding proteinbeta (C/EBPbeta) activation, which leads to the induction of glutathione S-transferase. Given that oltipraz activates C/EBPbeta for gene transactivation and that the putative C/EBP binding site is located in the CYP1A1 promoter region, this study investigated the effect of oltipraz on CYP1A1 induction by 3-methylcholanthrene (3-MC). 3-MC induced CYP1A1 in H4IIE cells in a time- and concentration-dependent manner. Gel shift analysis showed that 3-MC increased the band intensity of protein binding to the XRE. Immunocompetition analysis verified the specificity of AhR-XRE binding. Oltipraz (30 microM) induced CYP1A1 and the CYP1A1 promoter-luciferase gene and increased AhR DNA binding activity, which was 10-20% of those in 3-MC (100 nM)-treated cells. However, AhR-XRE binding was not increased after 10 microM oltipraz treatment. Oltipraz (10 microM) significantly inhibited CYP1A1 and CYP1A1-luciferase gene induction by 3-MC with no increase in AhR DNA binding. Oltipraz enhanced protein binding to the C/EBP binding site in the gene promoter and the binding complex comprised of C/EBPbeta and partly C/EBPdelta. Overexpression of dominant-negative mutant C/EBP significantly abolished the ability of oltipraz to suppress 3-MC-inducible CYP1A1 and the CYP1A1 reporter gene expression. Consistently, C/EBPbeta overexpression blocked CYP1A1 reporter gene induction by 3-MC. These results provide evidence that oltipraz suppresses 3-MC induction of CYP1A1 gene expression and that activation of C/EBPbeta by oltipraz contributes to suppression of 3-MC-inducible AhR-mediated CYP1A1 expression.
Assuntos
Anticarcinógenos/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Citocromo P-450 CYP1A1/biossíntese , Metilcolantreno/farmacologia , Pirazinas/farmacologia , Animais , Sítios de Ligação , Western Blotting , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular , Citocromo P-450 CYP1A1/genética , DNA/metabolismo , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Hepatócitos , Luciferases/genética , Mutação , Regiões Promotoras Genéticas/genética , Ratos , Receptores de Hidrocarboneto Arílico/genética , Proteínas Recombinantes de Fusão , Tionas , Tiofenos , Ativação Transcricional/efeitos dos fármacos , TransfecçãoRESUMO
Liver X receptors (LXRs) and peroxisome proliferator-activated receptors (PPARs) are members of nuclear receptors that form obligate heterodimers with retinoid X receptors (RXRs). These nuclear receptors play crucial roles in the regulation of fatty acid metabolism: LXRs activate expression of sterol regulatory element-binding protein 1c (SREBP-1c), a dominant lipogenic gene regulator, whereas PPARalpha promotes fatty acid beta-oxidation genes. In the current study, effects of PPARs on the LXR-SREBP-1c pathway were investigated. Luciferase assays in human embryonic kidney 293 cells showed that overexpression of PPARalpha and gamma dose-dependently inhibited SREBP-1c promoter activity induced by LXR. Deletion and mutation studies demonstrated that the two LXR response elements (LXREs) in the SREBP-1c promoter region are responsible for this inhibitory effect of PPARs. Gel shift assays indicated that PPARs reduce binding of LXR/RXR to LXRE. PPARalpha-selective agonist enhanced these inhibitory effects. Supplementation with RXR attenuated these inhibitions by PPARs in luciferase and gel shift assays, implicating receptor interaction among LXR, PPAR, and RXR as a plausible mechanism. Competition of PPARalpha ligand with LXR ligand was observed in LXR/RXR binding to LXRE in gel shift assay, in LXR/RXR formation in nuclear extracts by coimmunoprecipitation, and in gene expression of SREBP-1c by Northern blot analysis of rat primary hepatocytes and mouse liver RNA. These data suggest that PPARalpha activation can suppress LXR-SREBP-1c pathway through reduction of LXR/RXR formation, proposing a novel transcription factor cross-talk between LXR and PPARalpha in hepatic lipid homeostasis.