RESUMO
Feedback regulation of transcription factor NF-kappaB by its inhibitor IkappaBalpha plays an essential role in control of NF-kappaB activity. To understand the biological significance of IkappaBalpha-mediated feedback regulation of NF-kappaB, we generated mice harboring mutated kappaB enhancers in the promoter of the IkappaBalpha gene (IkappaBalpha(M/M)) to inhibit NF-kappaB-regulated IkappaBalpha expression. Here, we report that these mutant mice are defective in NF-kappaB-induced expression of IkappaBalpha. This defective feedback regulation of NF-kappaB by IkappaBalpha not only altered activity of NF-kappaB, but also the expression of NF-kappaB-regulated genes. As a result, IkappaBalpha(M/M), the homozygous knock-in mice with mutated kappaB enhancers in the IkappaBalpha promoter, acquire shorten life span, hypersensitivity to septic shock, abnormal T-cell development and activation, and Sjögren's Syndrome. These findings therefore demonstrate that the IkappaBalpha-mediated feedback regulation of NF-kappaB has an essential role in controlling T-cell development and functions, provide mechanistic insight into the development of Sjögren's Syndrome, and suggest the potential of NF-kappaB signaling as a therapeutic target for Sjögren's Syndrome and other autoimmune diseases.
Assuntos
Proteínas I-kappa B/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Síndrome de Sjogren/genética , Síndrome de Sjogren/metabolismo , Animais , Sequência de Bases , Primers do DNA/genética , Modelos Animais de Doenças , Elementos Facilitadores Genéticos , Retroalimentação Fisiológica , Expressão Gênica , Técnicas de Introdução de Genes , Humanos , Técnicas In Vitro , Ativação Linfocitária , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Inibidor de NF-kappaB alfa , Regiões Promotoras Genéticas , Transdução de Sinais , Síndrome de Sjogren/imunologia , Síndrome de Sjogren/patologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
Multiple myeloma (MM) growth is supported by an immune-tolerant bone marrow microenvironment. Here, we find that loss of Never in mitosis gene A (NIMA)-related kinase 2 (NEK2) in tumor microenvironmental cells is associated with MM growth suppression. The absence of NEK2 leads to both fewer tumor-associated macrophages (TAMs) and inhibitory T cells. NEK2 expression in myeloid progenitor cells promotes the generation of functional TAMs when stimulated with MM conditional medium. Clinically, high NEK2 expression in MM cells is associated with increased CD8+ T effector memory cells, while low NEK2 is associated with an IFN-γ gene signature and activated T cell response. Inhibition of NEK2 upregulates PD-L1 expression in MM cells and myeloid cells. In a mouse model, the combination of NEK2 inhibitor INH154 with PD-L1 blockade effectively eliminates MM cells and prolongs survival. Our results provide strong evidence that NEK2 inhibition may overcome tumor immune escape and support its further clinical development.
Assuntos
Mieloma Múltiplo , Camundongos , Animais , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Antígeno B7-H1/genética , Linfócitos T/metabolismo , Linhagem Celular Tumoral , Células Progenitoras Mieloides/metabolismo , Células Progenitoras Mieloides/patologia , Microambiente TumoralRESUMO
Osteolytic bone disease is a hallmark of multiple myeloma (MM). A significant fraction (~20%) of MM patients do not develop osteolytic lesions (OLs). The molecular basis for the absence of bone disease in MM is not understood. We combined PET-CT and gene expression profiling (GEP) of purified BM CD138+ MM cells from 512 newly diagnosed MM patients to reveal that elevated expression of cystatin M/E (CST6) was significantly associated with the absence of OL in MM. An enzyme-linked immunosorbent assay revealed a strong correlation between CST6 levels in BM serum/plasma and CST6 mRNA expression. Both recombinant CST6 protein and BM serum from patients with high CST6 significantly inhibited the activity of the osteoclast-specific protease cathepsin K and blocked osteoclast differentiation and function. Recombinant CST6 inhibited bone destruction in ex vivo and in vivo myeloma models. Single-cell RNA-Seq showed that CST6 attenuates polarization of monocytes to osteoclast precursors. Furthermore, CST6 protein blocks osteoclast differentiation by suppressing cathepsin-mediated cleavage of NF-κB/p100 and TRAF3 following RANKL stimulation. Secretion by MM cells of CST6, an inhibitor of osteoclast differentiation and function, suppresses osteolytic bone disease in MM and probably other diseases associated with osteoclast-mediated bone loss.
Assuntos
Reabsorção Óssea , Mieloma Múltiplo , Osteólise , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Diferenciação Celular/fisiologia , Cistatina M/metabolismo , Humanos , Mieloma Múltiplo/complicações , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Osteoclastos/metabolismo , Osteólise/genética , Osteólise/metabolismo , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Ligante RANK/genética , Ligante RANK/metabolismo , Fator 3 Associado a Receptor de TNF/metabolismoRESUMO
Transcription factor nuclear factor-kappaB (NF-kappaB) is constitutively activated in most pancreatic cancer tissues and cell lines but not in normal pancreas nor in immortalized/nontumorigenic human pancreatic ductal epithelial cells. Inhibition of constitutive NF-kappaB activation in pancreatic cancer cell lines suppresses tumorigenesis and tumor metastasis. Recently, we identified autocrine secretion of proinflammatory cytokine interleukin (IL)-1alpha as the mechanism of constitutive NF-kappaB activation in metastatic pancreatic cancer cell lines. However, the role of IL-1alpha in determining the metastatic potential of pancreatic tumor remains to be further investigated. In the current study, we stably expressed IL-1alpha in the nonmetastatic, IL-1alpha-negative MiaPaCa-2 cell lines. Our results showed that the secretion of IL-1alpha in MiaPaCa-2 cells constitutively activated NF-kappaB and increased the expression of NF-kappaB downstream genes involved in the different steps of the metastatic cascade, such as urokinase-type plasminogen activator, vascular endothelial growth factor, and IL-8. MiaPaCa-2/IL-1alpha cells showed an enhanced cell invasion in vitro compared with parental MiaPaCa-2 cells and induced liver metastasis in an orthotopic mouse model. The metastatic phenotype induced by IL-1alpha was inhibited by the expression of phosphorylation-defective IkappaB (IkappaB S32, 36A), which blocked NF-kappaB activation. Consistently, silencing the expression of IL-1alpha by short hairpin RNA in the highly metastatic L3.6pl pancreatic cancer cells completely suppressed their metastatic spread. In summary, these findings showed that IL-1alpha plays key roles in pancreatic cancer metastatic behavior through the constitutive activation of NF-kappaB. Our findings further support the possible link between inflammation and cancer and suggest that IL-1alpha may be a potential therapeutic target for treating pancreatic adenocarcinoma.
Assuntos
Interleucina-1alfa/metabolismo , NF-kappa B/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-1alfa/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos SCID , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Ligação Proteica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Carga TumoralRESUMO
BACKGROUND: Cynomolgus disease models that are similar to the preclinical stage of human type 2 diabetes mellitus (T2DM) were established by feeding middle-aged cynomolgus monkeys different high energy diets to study the differential expression of diabetes-related genes. METHODS: A total of 36 male monkeys were randomly divided into four groups and fed human diets with high sugar, high fat, double high sugar and fat, and a normal diet. The preclinical diabetes phase was determined by monitoring the metabolic characteristic indices and the results of oral glucose tolerance tests (OGTT). The mRNA expression of 45 diabetes-related genes in peripheral blood leukocytes was analyzed using real-time PCR. RESULTS: A total of 22, 25, and 21 genes were significantly up-regulated (P < 0.05) and 5, 7, and 5 genes were significantly down-regulated (P < 0.05) in the above three induced groups, respectively, compared with the control group. Of the 45 tested genes, the expression profiles of 21 genes were consistent. Most of the expression levels in the double high sugar-and-fat individuals were slightly lower than those in the high glucose and high fat groups, although the expression patterns of the three groups were essentially similar. CONCLUSION: The different high energy diets all induced diabetes and shared some phenotypic properties with human T2DM. Most of the expression patterns of the related genes were identical. The gene expression profiles could be used as references for the study of early diagnostic indicators and T2DM pathogenesis.
RESUMO
Nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1) transcription factors regulate many important biological and pathological processes. Activation of NF-kappaB is regulated by the inducible phosphorylation of NF-kappaB inhibitor IkappaB by IkappaB kinase. In contrast, Fos, a key component of AP-1, is primarily transcriptionally regulated by serum responsive factors (SRFs) and ternary complex factors (TCFs). Despite these different regulatory mechanisms, there is an intriguing possibility that NF-kappaB and AP-1 may modulate each other, thus expanding the scope of these two rapidly inducible transcription factors. To determine whether NF-kappaB activity is involved in the regulation of fos expression in response to various stimuli, we analyzed activity of AP-1 and expression of fos, fosB, fra-1, fra-2, jun, junB, and junD, as well as AP-1 downstream target gene VEGF, using MDAPanc-28 and MDAPanc-28/IkappaBalphaM pancreatic tumor cells and wild-type, IKK1-/-, and IKK2-/- murine embryonic fibroblast cells. Our results show that elk-1, a member of TCFs, is one of the NF-kappaB downstream target genes. Inhibition of NF-kappaB activity greatly decreased expression of elk-1. Consequently, the reduced level of activated Elk-1 protein by extracellular signal-regulated kinase impeded constitutive, serum-, and superoxide-inducible c-fos expression. Thus, our study revealed a distinct and essential role of NF-kappaB in participating in the regulation of elk-1, c-fos, and VEGF expression.
Assuntos
Regulação da Expressão Gênica , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Fator de Transcrição AP-1/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Doxiciclina/metabolismo , Humanos , Quinase I-kappa B , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células-Tronco/citologia , Células-Tronco/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Elk-1 do Domínio etsRESUMO
The Rel/NF-kappaB transcription factors play a key role in the regulation of apoptosis and in tumorigenesis by controlling the expressions of specific genes. To determine the role of the constitutive activity of RelA in tumorigenesis, we generated pancreatic tumor cell lines that express a dominant negative mutant of IkappaBalpha (IkappaBalphaM). In this report, we show that the inhibition of constitutive NF-kappaB activity, either by ectopic expression of IkappaBalphaM or by treating the cells with a proteasome inhibitor PS-341 which blocks intracellular degradation of IkappaBalpha proteins, downregulates the expression of bcl-xl. We identified two putative NF-kappaB binding sites (kappaB/A and B) in the bcl-xl promoter and found that these two sites interact with different NF-kappaB proteins. p65/p50 heterodimer interacts with kappaB/A site whereas p50/p50 homodimer interacts with kappaB/B. The bcl-xl promoter reporter gene assays reveal that NF-kappaB dependent transcriptional activation is mainly mediated by kappaB/A site, indicating that bcl-xl is one of the downstream target genes regulated by RelA/p50. Both IkappaBalphaM and PS-341 completely abolish NF-kappaB DNA binding activity; however, PS-341, but not ectopic expression of IkappaBalphaM, sensitized cells to apoptosis induced by Taxol. This is due to the Taxol-mediated reactivation of RelA through phosphorylation and degradation of IkappaBbeta and the re-expression of NF-kappaB regulated bcl-xl gene in these cancer cells as ectopic expression of the bcl-xl gene confers resistance to Taxol-induced apoptosis in PS-341 sensitized cells. These results demonstrate the important function of various NF-kappaB/IkappaB complexes in regulating anti-apoptotic genes in response to apoptotic stimuli, and they raise the possibility that NF-kappaB : IkappaBalpha and NF-kappaB : IkappaBbeta complexes are regulated by different upstream activators, and that NF-kappaB plays a key role in pancreatic tumorigenesis.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Proteínas de Ligação a DNA/fisiologia , Resistencia a Medicamentos Antineoplásicos , Proteínas I-kappa B , NF-kappa B/fisiologia , Paclitaxel/farmacologia , Neoplasias Pancreáticas/metabolismo , Inibidores de Proteases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Pirazinas/farmacologia , Bortezomib , Diferenciação Celular/genética , Divisão Celular/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Neoplasias Pancreáticas/patologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/farmacologia , Pirofosfatases/metabolismo , Ativação Transcricional , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Regulação para Cima , Proteína bcl-XRESUMO
PURPOSE: The tumor suppressor gene Smad4/DPC4, a key transcription factorin transforming growth factor beta (TGF-beta) signaling cascades,is inactivated in 50% of pancreatic adenocarcinomas. We seek to determine the role of Smad4/DPC4 in the suppression of tumor cell growth and in the regulation of TGF-beta-mediated expression of cell-cycle regulatory genes p15(ink4b) and p21(waf1). EXPERIMENTAL DESIGN: Smad4/DPC4 is overexpressed by adenoviral infection in CFPac-1 pancreatic cancer cells, in which the Smad4/DPC4 is homozygously deleted, and in Capan-1 pancreatic cancer cells, in which Smad4/DPC4 is not expressed. Expression of the TGF-beta downstream target gene p21(waf1), regulation of the p15(ink4b) promoter, anchorage-independent growth, and tumorigenesis were examined. RESULTS: We demonstrate that expression of Smad4/DPC4 in Capan-1 cells reduced anchorage-independent growth by more than 50%, and inhibited xenograft tumor growth. However, overexpression of Smad4/DPC4 did not inhibit CFPac-1 cell growth. Interestingly, Smad4/DPC4 induced expression of p15(ink4b), p21(waf1), and TGF-beta-responsive reporter gene in Capan-1 but not in CFPac-1 cells. Furthermore, we found a previously unidentified Smad4 binding element (SBE) located in the region between -356 and -329 bp of the p15(ink4b) promoter. The p15(ink4b) promoter reporter gene assays revealed that Smad4-dependent transcriptional activation is mediated by this SBE, which indicates that p15(ink4b) is one of the downstream target genes regulated by Smad/DPC4. CONCLUSION: These results explain the role of Smad4/DPC4 in TGF-beta-mediated inhibition of cell proliferation in vitro and in vivo. Moreover, these results suggest that Smad4/DPC4-mediated tumor suppression and induction of TGF-beta-regulated cell-cycle-inhibitory genes may depend on additional factors that are absent in CFPac-1 cells.
Assuntos
Proteínas de Ciclo Celular/farmacologia , Inibidor p16 de Quinase Dependente de Ciclina/farmacologia , Proteínas de Ligação a DNA/metabolismo , Neoplasias Pancreáticas/metabolismo , Transativadores/metabolismo , Proteínas Supressoras de Tumor , Adenoviridae/genética , Animais , Northern Blotting , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Núcleo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p15 , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Deleção de Genes , Genes Reporter , Homozigoto , Humanos , Immunoblotting , Luciferases/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Regiões Promotoras Genéticas , Transdução de Sinais , Proteína Smad4 , Fatores de Tempo , Ativação Transcricional , Transfecção , Fator de Crescimento Transformador beta/metabolismo , Células Tumorais CultivadasRESUMO
Human connexin 26 gene (Cx26) has been considered a candidate suppressor gene in mammary epithelial cells. To explain regulatory mechanism of the Cx26, a DNase-1 hypersensitive 1.6 kb fragment upstream of the 5'-terminus of the gene was sequenced and assayed with CAT reporter system. The results showed that the 1.6 kb sequence had powerful promoter activity. The fragment contains two GT boxes (centering at -6158 and -6213 bp), and a TATA-less TTAAAA box (-6237/-6232 bp) which is the new promoter region of human Cx26.
RESUMO
Expression of embryonic stem cells (ESCs) markers (SOX2, OCT4, Nanog and Nestin) is crucial for progression of various human malignancies. The purpose of this study was to investigate the expression and prognostic impact of these molecules in nasopharyngeal carcinoma (NPC) patients by immunohistochemistry and immunofluorescence. In the present study, we found that the expression levels of SOX2, OCT4 and Nanog were highly expressed in NPC compared with the non-tumorous tissues. Furthermore, these proteins correlated significantly with several clinicalpathological factors and epithelial-mesenchymal transition (EMT)-associated indicators (E-cadherin/N-cadherin and Snail). In multivariate analyses, high expression of OCT4 (Pâ=â0.013) and Nanog (Pâ=â0.040), but not that of SOX2, was associated with worse survival and had strongly independent prognostic effects. Of note, OCT4 and Nanog were more frequently located at the invasive front of tumors, and correlated significantly with various aggressive behaviors including T classification, N classification, M classification and clinical stage. Furthermore, patients with co-expression of OCT4 and Nanog in the invasive front had significantly worse survival (Pâ=â0.005). Interestingly, at the invasive front, these molecules correlated significantly with Nestin expression in endothelial cells (P<0.001). These findings provide evidence that ESCs biomarkers OCT4 and Nanog serves as independent prognostic factors for NPC. Additionally, cancer cells in the invasive front of NPC acquiring ESCs-like features should be maintained by vascular niches.
Assuntos
Biomarcadores Tumorais/metabolismo , Células-Tronco Embrionárias/metabolismo , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Adolescente , Adulto , Idoso , Caderinas/metabolismo , Carcinoma , Feminino , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Masculino , Pessoa de Meia-Idade , Proteína Homeobox Nanog , Carcinoma Nasofaríngeo , Proteínas do Tecido Nervoso/metabolismo , Nestina , Fator 3 de Transcrição de Octâmero/metabolismo , Transporte Proteico , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição da Família Snail , Análise de Sobrevida , Fatores de Transcrição/metabolismo , Adulto JovemRESUMO
To explore pathological alteration of T2DM in cynomolgus monkeys, gene expression profiles of peripheral blood leukocytes from spontaneous and diet-induced T2DM models was analyzed using quantitative real-time PCR. Among 36 T2DM associated genes tested, 19 genes (including G6PC, CCR2B, CTLA4) displayed a similar expression pattern in both spontaneous and diet-induced T2DM models and were significantly up-regulated or down-regulated compared to controls. Interestingly, expression abundance of all up-regulated genes in the diet-induced T2DM was stronger, although not significantly, than spontaneous models, indicating diet-induced T2DM in monkeys should be a reliable research model for changes in gene expression. The characteristic gene expression pattern obtained here may be useful for the clinical diagnosis of T2DM.
Assuntos
Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Dieta Hiperlipídica/efeitos adversos , Gorduras na Dieta/efeitos adversos , Modelos Animais de Doenças , Macaca fascicularis , Animais , Diabetes Mellitus Tipo 2/etiologia , Gorduras na Dieta/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Macaca fascicularis/genética , Macaca fascicularis/metabolismoRESUMO
Constitutive Kras and NF-κB activation is identified as signature alterations in pancreatic ductal adenocarcinoma (PDAC). However, how NF-κB is activated in PDAC is not yet understood. Here, we report that pancreas-targeted IKK2/ß inactivation inhibited NF-κB activation and PDAC development in Kras(G12D) and Kras(G12D);Ink4a/Arf(F/F) mice, demonstrating a mechanistic link between IKK2/ß and Kras(G12D) in PDAC inception. Our findings reveal that Kras(G12D)-activated AP-1 induces IL-1α, which, in turn, activates NF-κB and its target genes IL-1α and p62, to initiate IL-1α/p62 feedforward loops for inducing and sustaining NF-κB activity. Furthermore, IL-1α overexpression correlates with Kras mutation, NF-κB activity, and poor survival in PDAC patients. Therefore, our findings demonstrate the mechanism by which IKK2/ß/NF-κB is activated by Kras(G12D) through dual feedforward loops of IL-1α/p62.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Quinase I-kappa B/metabolismo , Interleucina-1alfa/metabolismo , NF-kappa B/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Proteínas ras/fisiologia , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Interleucina-1alfa/genética , Camundongos , Mutação , NF-kappa B/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Proteína Sequestossoma-1 , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Type 2 diabetes mellitus (T2DM) is a metabolic disease with a strong genetic component that is very prevalent in the world. The aim of this study is to investigate the association of a set of six obesity-related genes with the different disease phases of T2DM in a model using middle or aged cynomogus monkeys. A total of 25 male monkeys were used and fed with high-fat diet (15% lard). The disease development and progression of T2DM were monitored through the levels of plasma glucose and lipid. The mRNA expression of 6 genes was evaluated using real-time PCR on monocyte isolated from monkey peripheral blood. The 2-hour plasma glucose levels followed oral glucose tolerance test (OGTT) were (11.06+/-6.05) mmol/L and (13.12+/-2.89) mmol/L respectively (P<0.01), and the fasting plasma glucose level was (7.58+/-1.56) mmol/L (vs controls, P<0.01), indicating that we developed successful the models of pre-diabetic and diabetic disease in the cynomolgus monkey. Of the six tested genes, CDKN2B, IGF2BP2, and FTO genes were significantly up-regulated with disease progression in T2DM. We found that the expression of IGF2BP2 and FTO increased 65.92 and 4.30 folds in the developed T2DM. We conclude that the genes of CDKN2B, IGF2BP2, and FTO can be used as early diagnostic and prognostic biomarkers in type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/genética , Modelos Animais de Doenças , Expressão Gênica , Macaca fascicularis , Obesidade/genética , Animais , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Teste de Tolerância a Glucose , Humanos , Metabolismo dos Lipídeos , Macaca fascicularis/genética , Macaca fascicularis/metabolismo , Masculino , Obesidade/metabolismoRESUMO
A total of 138 middle and aged cynomolgus monkeys (Macaca fascicularis) (above 10 years) were classified into three groups based on fasting plasma glucose (FPG) values, specifically low FPG, normal FPG, and high FPG group. Total cholesterol (TCHO), triglycerides (TG), high density lipoprotein cholesterol (HDL-C), and low density lipoprotein cholesterol (LDL-C) were detected in blood by automatic biochemical analyzer. The mRNA expressions of 37 diabetes-associated genes were analyzed with Real-time PCR in monocytes isolated from monkey peripheral blood. No significant correlation between the four serum lipid indictors HDL-C, LDL-C, TCHO, TG and FPG (P>0.05) were found. However, the expressions of ACE, ACLY, PRKCB1, SLC2A4, SNAP23, VAPA, IGF2BP2, and IFNG were significantly enhanced when FPG increased (P<0.05).
Assuntos
Diabetes Mellitus Tipo 2/genética , Regulação da Expressão Gênica , Macaca fascicularis , Fatores Etários , Animais , Glicemia/análise , HDL-Colesterol/sangue , Diabetes Mellitus Tipo 2/sangue , Modelos Animais de Doenças , Feminino , Humanos , Macaca fascicularis/genética , Macaca fascicularis/metabolismo , Masculino , Triglicerídeos/sangueRESUMO
To screen spontaneous diabetic mellitus and explore methods for its rapid identification, the basal and inferred levels of blood glucose of 440 overweight, middle- and old-aged cynomolgus monkeys were analyzed. Diagnostic diabetes was further validated by the oral glucose tolerance test (OGTT) and urine glucose. The average level of blood glucose of these cynomolgus monkeys was (3.88±0.98) mmol/L, which was lower than the level for suspected diabetes (5.0 mmol/L). Of them, 56 (12.72%) monkeys were identified with levels of blood glucose greater than 5.0 mmol/L and diagnosed as the diabetic subjects. This population showed impaired glucose tolerance using the OGTT and 39 of the 56 (69.23%) had glucose positive urine. The methods for screening diabetic mellitus used in this study were simple, quick, and limited the harm to animals. However, the incidence of diabetes was higher in these tested monkeys than in the regular human population in China (9.7%), suggesting that these methods are useful for screening diabetic disease in a large population but not suitable for all cynomolgus monkeys.
Assuntos
Diabetes Mellitus/metabolismo , Macaca fascicularis/metabolismo , Fatores Etários , Animais , Glicemia/análise , Feminino , Teste de Tolerância a Glucose , MasculinoRESUMO
The correlation between cardiovascular risk factors and cardiovascular disease related genes plays an important role in early-warning risk and early diagnosis. Thirty middle-aged male crab-eating macaques were fed a moderately atherogenic diet (0.053 mg cholesterol/kJ and 40% of calories as saturated fat) for twelve months. According to cardiovascular risk factors, we selected low-risk and high-risk crab-eating macaques, then analyzed the expression of 113 cardiovascular related genes by real-time PCR. A total of 65 genes were detected in peripheral blood leukocytes by real-time PCR. Sixteen up-regulated genes and nineteen down-regulated genes were detected in low-risk and high-risk crab-eating macaques compared to normal crab-eating macaques (P<0.05), in addition to fifteen genes that showed unique expression patterns (P<0.05). We also detected 42 genes in human peripheral blood leukocytes. The expression patterns of 22 genes were consistent between human and crab-eating macaques. These results narrowed the scope of genes for further research.
Assuntos
Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/genética , Modelos Animais de Doenças , Macaca fascicularis , Animais , Doenças Cardiovasculares/metabolismo , Colesterol na Dieta/efeitos adversos , Colesterol na Dieta/metabolismo , Dieta/efeitos adversos , Dieta Aterogênica , Regulação da Expressão Gênica , Humanos , Macaca fascicularis/genética , Macaca fascicularis/metabolismo , Masculino , Fatores de RiscoRESUMO
Keratinocyte growth factor (KGF)/fibroblast growth factor-7 (FGF-7) is a paracrine- and epithelium-specific growth factor produced by cells of mesenchymal origin. It acts exclusively through FGF-7 receptor (FGFR2/IIIb), which is expressed predominantly by epithelial cells, but not by fibroblasts, suggesting that it might function as a paracrine mediator of mesenchymal-epithelial interactions. KGF/FGF-7 plays an essential role in the growth of epithelial cells and is frequently overexpressed in cancers of epithelial origin such as pancreatic cancer, switching paracrine stimulation of KGF/FGF-7 to an autocrine loop. Less is known, however, about the signaling pathways by which KGF/FGF-7 regulates the response of epithelial cells. To delineate the signaling pathways activated by KGF/FGF-7 and examine cellular response to KGF/FGF-7 stimulation, we performed functional analysis of KGF/FGF-7 action. In this report, we show that KGF/FGF-7 activated nuclear factor kappaB (NF-kappaB), which in turn induced expression of VEGF, MMP-9, and urokinase-type plasminogen activator and increased migration and invasion of KGF/FGF-7-stimulated human pancreatic ductal epithelial cells. Expression of phosphorylation-defective IkappaBalpha (IkappaBalphaS32A,S36A), which blocked NF-kappaB activation, inhibited KGF/FGF-7-induced gene expression and cell migration and invasion. Our results demonstrate for the first time that KGF/FGF-7 induces NF-kappaB activation and that NF-kappaB plays an essential role in regulation of KGF/FGF-7-inducible gene expression and KGF/FGF-7-initiated cellular responses. Thus, these findings identify one signaling pathway for KGF/FGF-7-regulated cell migration and invasion and suggest that paracrine sources of KGF/FGF-7 are one of the malignancy-contributing factors from tumor stroma.
Assuntos
Movimento Celular/efeitos dos fármacos , Fator 7 de Crescimento de Fibroblastos/fisiologia , NF-kappa B/metabolismo , Animais , Células Cultivadas , Células Epiteliais/citologia , Fator 7 de Crescimento de Fibroblastos/genética , Fator 7 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica , Humanos , Camundongos , Comunicação Parácrina , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
BACKGROUND: Constitutive activation of nuclear factor-kappaB (NF-kappaB) is a frequent molecular alteration in pancreatic cancer and a number of studies have suggested that constitutive NF-kappaB activity plays a key role in the aggressive behavior of this disease. In an attempt to identify an effective therapeutic agent for pancreatic cancer, the authors studied the role of FUT-175, a synthetic serine protease inhibitor, in the inhibition of NF-kappaB activation and the induction of apoptotic responses. METHODS: To examine the effect of FUT-175 on the inhibition of NF-kappaB and the induction of apoptosis in pancreatic cancer cell lines, Western and Northern blot analyses, electromobility shift (EMSA), luciferase reporter gene, DNA fragmentation, immunoprecipitation, in vitro kinase, small interfering RNA (siRNA), and chromatin immunoprecipitation (ChIP) assays were performed. RESULTS: In a time-dependent and dose-dependent manner, FUT-175 inhibited IkappaBalpha phosphorylation and NF-kappaB activation, thereby inhibiting the antiapoptotic activity of NF-kappaB. Simultaneously, FUT-175 up-regulated the expression of tumor necrosis factor receptor-1 (TNFR1), which in turn activated the proapoptotic caspase-8 and Bid pathways and induced apoptosis in pancreatic cancer cells. FUT-175-induced activation of Fas-associated death domain (FADD) and caspase-8 was suppressed by RNA interference-mediated inhibition of TNFR1 expression. Furthermore, expression of the transcription factor PEA3 was up-regulated by FUT-175 and was involved in FUT-175-mediated TNFR1 expression. CONCLUSIONS: These results suggested a possible mechanism by which FUT-175 may disrupt interconnected signaling pathways by both suppressing the NF-kappaB antiapoptotic activity and inducing TNFR-mediated apoptosis. Supported by this unique function as a NF-kappaB inhibitor and apoptosis inducer, this well-established synthetic serine protease inhibitor with as-of-yet poorly understood mechanisms of actions appears to be a potentially therapeutic agent for pancreatic cancer.
Assuntos
Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Guanidinas/farmacologia , Neoplasias Pancreáticas/patologia , Inibidores de Serina Proteinase/farmacologia , Benzamidinas , Caspase 8/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Proteína de Domínio de Morte Associada a Fas/metabolismo , Humanos , Proteínas I-kappa B/metabolismo , NF-kappa B/efeitos adversos , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Inhibition of peroxisome proliferator-activated receptor gamma (PPARgamma) function by TNF-alpha contributes to glucose and fatty acid metabolic disorders in inflammation and cancer, although the molecular mechanism is not fully understood. In this study, we demonstrate that nuclear translocation of HDAC3 is regulated by TNF-alpha, and this event is required for inhibition of transcriptional activity of PPARgamma by TNF-alpha. HDAC3 is associated with IkappaBalpha in the cytoplasm. After IkappaBalpha degradation in response to TNF-alpha, HDAC3 is subject to nuclear translocation, leading to an increase in HDAC3 activity in the nucleus. This event leads to subcellular redistribution of HDAC3. Knock-out of IkappaBalpha, but not p65 or p50, leads to disappearance of HDAC3 in the cytoplasm, which is associated with HDAC3 enrichment in the nucleus. These data suggest that inhibition of PPARgamma by TNF-alpha is not associated with a reduction in the DNA binding activity of PPARgamma. Rather, these results suggest that IkappaBalpha-dependent nuclear translocation of HDAC3 is responsible for PPARgamma inhibition by TNF-alpha.
Assuntos
Núcleo Celular/metabolismo , Histona Desacetilases/metabolismo , Proteínas I-kappa B/fisiologia , PPAR gama/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologia , Células 3T3-L1 , Transporte Ativo do Núcleo Celular , Animais , Humanos , Metabolismo dos Lipídeos , Camundongos , Inibidor de NF-kappaB alfa , PPAR gama/fisiologia , Transporte ProteicoRESUMO
RelA, the p65 subunit of NF-kappaB transcription factors, plays a key role in regulation of antiapoptotic and proapoptotic responses. However, the downstream target genes regulated by RelA-NF-kappaB in the initiation of proapoptotic signaling were not identified. We previously showed that RelA-NF-kappaB functioned as a proapoptotic factor by activating the p53-signaling pathway in response to doxycycline-induced superoxide. In the present study, we demonstrate that the ability of doxycycline/superoxide to induce expression of polo-like kinase 3 (Plk3) depends on NF-kappaB activity. We identified a kappaB binding site in the promoter of Plk3, and this kappaB site is directly involved in its induction by the RelA-NF-kappaB complex. Plk3 formed a complex with p53 and was involved in the phosphorylation of p53 on Ser-20 in response to superoxide. Inhibition of Plk3 expression by Plk3 small interfering RNA suppressed the doxycycline/superoxide-mediated apoptosis. Overexpression of wild-type Plk3 in HCT116 p53+/+ cells induced rapid apoptosis, whereas overexpression of wild-type Plk3 in HCT116 p53-/- cells and the kinase-defective mutant Plk3(K91R) in p53+/+ cells induced delayed onset of apoptosis. Furthermore, mutagenesis of Plk3 showed that the N-terminal domain (amino acids 1-26) is essential for the induction of delay onset of apoptosis. These data show that Plk3 is a RelA-NF-kappaB-regulated gene that induces apoptosis in both p53-dependent and -independent signaling pathways, suggesting a possible mechanism for RelA-NF-kappaB-regulated proapoptotic responses.