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1.
Mol Cancer Res ; 17(7): 1429-1434, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31043490

RESUMO

Napabucasin is an NAD(P)H:quinone oxidoreductase 1 (NQO1)-bioactivatable small molecule hypothesized to affect multiple oncogenic pathways. In a prespecified, retrospective analysis of the napabucasin phase III CO.23 study, overall survival was longer for napabucasin versus placebo in patients expressing phosphorylated STAT3 (pSTAT3) in tumor cells and cells of the tumor microenvironment (TME). We hypothesized that a connection may exist between NQO1 expression in cancer cells and pSTAT3 in tumor cells and the TME. In 3D spheroid cocultures of cancer cells and cancer-associated fibroblasts, the antitumor activity of napabucasin was NQO1 dependent. The levels of cytokines such as IL6, CXCL10, and GM-CSF were higher in NQO1-positive versus NQO1-deleted cocultures. These differentially secreted cytokines promoted STAT3 phosphorylation in tumor cells and the TME. NQO1-expressing, napabucasin-sensitive tumor cells can modify tumor cells and the TME to promote STAT3 phosphorylation, suggesting that pSTAT3 may be used to identify a subpopulation of patients who would likely respond to napabucasin. IMPLICATIONS: pSTAT3 is a potential biomarker for patient response to the anticancer drug napabucasin.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/7/1429/F1.large.jpg.


Assuntos
Benzofuranos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias Hipofaríngeas/tratamento farmacológico , NAD(P)H Desidrogenase (Quinona)/genética , Naftoquinonas/farmacologia , Fator de Transcrição STAT3/genética , Fibroblastos Associados a Câncer/efeitos dos fármacos , Carcinogênese/efeitos dos fármacos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Linhagem da Célula/efeitos dos fármacos , Técnicas de Cocultura , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Hipofaríngeas/genética , Neoplasias Hipofaríngeas/patologia , Microambiente Tumoral/efeitos dos fármacos
2.
J Biol Chem ; 288(15): 10849-59, 2013 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-23439645

RESUMO

The release of humoral factors between cancer cells and the microenvironmental cells is critical for metastasis; however, the roles of secreted miRNAs in non-cell autonomous cancer progression against microenvironmental cells remain largely unknown. Here, we demonstrate that the neutral sphyngomyelinase 2 (nSMase2) regulates exosomal microRNA (miRNA) secretion and promotes angiogenesis within the tumor microenvironment as well as metastasis. We demonstrate a requirement for nSMase2-mediated cancer cell exosomal miRNAs in the regulation of metastasis through the induction of angiogenesis in inoculated tumors. In addition, miR-210, released by metastatic cancer cells, was shown to transport to endothelial cells and suppress the expression of specific target genes, which resulted in enhanced angiogenesis. These findings suggest that the horizontal transfer of exosomal miRNAs from cancer cells can dictate the microenviromental niche for the benefit of the cancer cell, like "on demand system" for cancer cells.


Assuntos
Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Animais/metabolismo , MicroRNAs/metabolismo , Proteínas de Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , RNA Neoplásico/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Camundongos , MicroRNAs/genética , Metástase Neoplásica , Proteínas de Neoplasias/genética , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , RNA Neoplásico/genética , Esfingomielina Fosfodiesterase/genética
3.
J Biol Chem ; 287(2): 1397-405, 2012 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-22123823

RESUMO

Normal epithelial cells regulate the secretion of autocrine and paracrine factors that prevent aberrant growth of neighboring cells, leading to healthy development and normal metabolism. One reason for tumor initiation is considered to be a failure of this homeostatic cell competitive system. Here we identify tumor-suppressive microRNAs (miRNAs) secreted by normal cells as anti-proliferative signal entities. Culture supernatant of normal epithelial prostate PNT-2 cells attenuated proliferation of PC-3M-luc cells, prostate cancer cells. Global analysis of miRNA expression signature revealed that a variety of tumor-suppressive miRNAs are released from PNT-2 cells. Of these miRNAs, secretory miR-143 could induce growth inhibition exclusively in cancer cells in vitro and in vivo. These results suggest that secretory tumor-suppressive miRNAs can act as a death signal in a cell competitive process. This study provides a novel insight into a tumor initiation mechanism.


Assuntos
Proliferação de Células , MicroRNAs/metabolismo , Neoplasias da Próstata/metabolismo , Linhagem Celular Tumoral , Humanos , Masculino , Neoplasias da Próstata/patologia
4.
Commun Integr Biol ; 3(5): 478-81, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21057646

RESUMO

The physiological role of microRNAs (miRNAs) is widely appreciated as a fine-tuner to post-transcriptionally regulate the expression of multiple genes in the cells of origin. Here, we highlight two significant characteristics of miRNAs: (1) they are secreted from the producing cells and (2) they can deliver the gene silencing signals between living cells in vitro and in vivo. The circulation of miRNAs in human body fluids can be provided with a logical explanation by our discovery that the release of miRNAs is actively controlled through a ceramide-dependent machinery associated with exosome secretion. This finding can contribute to the development of circulating miRNAs as diagnostic biomarkers for a variety of diseases. We also demonstrated that secretory miR-16 was transferred into prostate cancer PC-3M cells subcutaneously xenografted in nude mice, resulting in the suppression of its target gene. This result suggests that faithfully to their primary role, secretory miRNAs can function as a translational inhibitor in recipient cells as well. In conclusion, miRNAs are liberated from their incipient cells, whereby they can exert their full potentials as a silence master of gene expressions.

5.
Curr Drug Discov Technol ; 7(2): 95-105, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20836759

RESUMO

Over the past several years, microRNAs (miRNAs) have been identified as a fine-tuner in a wide array of biological processes, including development, cell growth and metabolism. Recent studies have shown that many kinds of miRNAs act as oncomirs or tumor suppressors in tumors where the miRNA genes are up- or down- regulated, respectively. These dysregulations occur through a variety of mechanisms, such as genetic alterations, epigenetic repression or altered expression of transcription factors which target miRNAs. The aberrant expressions of miRNAs are observed not only in tumor lesions but also in plasma and serum of cancer patients. These characteristics of miRNAs have created extensive interest in tapping into them for diagnosis and prognosis as well as drug discovery in cancer therapy. In this literature, the significance of miRNAs in tumor initiation and development is first reviewed. Second topic is extracellular miRNAs as biomarkers for cancer classification and prediction. Further, we focus on secretory machinery of miRNAs and share new evidence suggesting that extracellular miRNAs can play biological roles beyond mere biomarkers. Extending this concept, our hypothetical model that extracellular miRNAs may function as a signaling molecule in a crosstalk between cancer cells and their surrounding cells is presented. Finally, we discuss the potential of miRNAs for therapeutic applications in clinical oncology.


Assuntos
Antineoplásicos/farmacologia , MicroRNAs/metabolismo , Neoplasias/tratamento farmacológico , Animais , Biomarcadores Tumorais/metabolismo , Desenho de Fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/administração & dosagem , Neoplasias/diagnóstico , Neoplasias/genética , Prognóstico
6.
Cancer Sci ; 101(10): 2087-92, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20624164

RESUMO

In the past several years, the importance of microRNA (miRNA) in cancer cells has been recognized. Proper control of miRNA expression is essential for maintaining a steady state of the cellular machinery. Recently, it was discovered that extracellular miRNAs circulate in the blood of both healthy and diseased patients, although ribonuclease is present in both plasma and serum. Most of the circulating miRNAs are included in lipid or lipoprotein complexes, such as apoptotic bodies, microvesicles, or exosomes, and are, therefore, highly stable. The existence of circulating miRNAs in the blood of cancer patients has raised the possibility that miRNAs may serve as a novel diagnostic marker. However, the secretory mechanism and biological function, as well as the meaning of the existence of extracellular miRNAs, remain largely unclear. In this review, we summarize the usefulness of circulating miRNA for cancer diagnosis, prognosis, and therapeutics. Furthermore, we propose a mechanism for the secretion and incorporation of miRNA into the cells.


Assuntos
Biomarcadores Tumorais/sangue , MicroRNAs/sangue , Neoplasias/diagnóstico , Humanos , MicroRNAs/metabolismo , Neoplasias/mortalidade , Prognóstico
7.
J Biol Chem ; 285(23): 17442-52, 2010 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-20353945

RESUMO

The existence of circulating microRNAs (miRNAs) in the blood of cancer patients has raised the possibility that miRNAs may serve as a novel diagnostic marker. However, the secretory mechanism and biological function of extracellular miRNAs remain unclear. Here, we show that miRNAs are released through a ceramide-dependent secretory machinery and that the secretory miRNAs are transferable and functional in the recipient cells. Ceramide, whose biosynthesis is regulated by neutral sphingomyelinase 2 (nSMase2), triggers secretion of small membrane vesicles called exosomes. The decreased activity of nSMase2 with a chemical inhibitor, GW4869, and a specific small interfering RNA resulted in the reduced secretion of miRNAs. Complementarily, overexpression of nSMase2 increased extracellular amounts of miRNAs. We also revealed that the endosomal sorting complex required for transport system is unnecessary for the release of miRNAs. Furthermore, a tumor-suppressive miRNA secreted via this pathway was transported between cells and exerted gene silencing in the recipient cells, thereby leading to cell growth inhibition. Our findings shed a ray of light on the physiological relevance of secretory miRNAs.


Assuntos
MicroRNAs/metabolismo , Compostos de Anilina/farmacologia , Animais , Compostos de Benzilideno/farmacologia , Transporte Biológico , Biomarcadores Tumorais/metabolismo , Células COS , Chlorocebus aethiops , Meios de Cultivo Condicionados/metabolismo , Exossomos/metabolismo , Inativação Gênica , Humanos , Neoplasias/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Esfingomielina Fosfodiesterase/metabolismo
8.
Gan To Kagaku Ryoho ; 37(3): 389-95, 2010 Mar.
Artigo em Japonês | MEDLINE | ID: mdl-20332672

RESUMO

MicroRNAs (miRNAs) are small 20-22 nucleotide-long members of the non-protein-coding RNA family and cause an inhibition of translation and some degree of degradation of the target messenger RNAs (mRNAs) through binding to partially complementary sites, usually in the 3' untranslated regions of the target mRNAs. Therefore, miRNAs play pivotal roles as negative regulators of gene expression in a wide array of physiological processes. Recent observations reveal that many miRNAs have been implicated in various human cancers. Both losses and gains of miRNA function have been shown to contribute to cancer development through a variety of mechanisms. Over the past few years, miRNAs have been reported in human bodily fluids and represent new effective biomarkers. Particularly, in oncology, detection and monitoring of tumors are now becoming possible by the evaluation of tumor-derived secretory miRNAs. However, the secretory mechanism and biological function of extra cellular miRNAs remain unclear. Our ongoing studies show that secretory miRNAs in exosomes can be transferred and exert their function in living cells, suggesting that extra cellular miRNAs may mediate intercellular communication between cancer cells and their surrounding cells in a cancer microenvironment. The fact that several miRNA genes are dysregulated in multiple types of cancer indicates that significant pathways involved in tumorigenesis may have miRNAs as downstream targets. Thus, in tumors where miRNA genes are lost or amplified, miRNA mimetics or antagomirs, respectively, are considered as promising drugs to induce apoptosis and/or cell cycle arrest in cancer cells that depend on miRNA dysregulation for growth and survival. There is growing evidence that miRNA therapy could be a potent means to curtail tumor growth.


Assuntos
MicroRNAs/genética , Neoplasias/tratamento farmacológico , Animais , Desenho de Fármacos , Humanos , MicroRNAs/agonistas , MicroRNAs/antagonistas & inibidores
9.
Biochem Biophys Res Commun ; 381(4): 537-43, 2009 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-19233136

RESUMO

The transcriptional regulator peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) controls mitochondrial biogenesis and energy homeostasis. Although physical exercise induces PGC-1alpha expression in muscle, the underlying mechanism of this effect has remained incompletely understood. We recently identified a novel muscle-enriched isoform of PGC-1alpha transcript (designated PGC-1alpha-b) that is derived from a previously unidentified first exon. We have now cloned and characterized the human PGC-1alpha-b promoter. The muscle-specific transcription factors MyoD and MRF4 transactivated this promoter through interaction with a proximal E-box motif. Furthermore, either forced expression of Ca(2+)- and calmodulin-dependent protein kinase IV (CaMKIV), calcineurin A, or the p38 mitogen-activated protein kinase (p38 MAPK) kinase MKK6 or the intracellular accumulation of cAMP activated the PGC-1alpha-b promoter in cultured myoblasts through recruitment of cAMP response element (CRE)-binding protein (CREB) to a putative CRE located downstream of the E-box. Our results thus reveal a potential molecular basis for isoform-specific regulation of PGC-1alpha expression in contracting muscle.


Assuntos
Proteínas de Choque Térmico/genética , Contração Muscular/genética , Músculo Esquelético/fisiologia , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Transcrição Gênica , Ativação Transcricional , Sequência de Bases , Cálcio/farmacologia , AMP Cíclico/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Humanos , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fatores de Transcrição de p300-CBP/metabolismo
10.
J Biol Chem ; 282(26): 19052-61, 2007 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-17412698

RESUMO

Sex-determining region Y-box (SOX) 6 negatively regulates glucose-stimulated insulin secretion from beta-cells and is a down-regulated transcription factor in the pancreatic islet cells of hyperinsulinemic obese mice. To determine the contribution of SOX6 to insulin resistance, we analyzed the effects of SOX6 on cell proliferation. Small interfering RNA-mediated attenuation of SOX6 expression stimulated the proliferation of insulinoma INS-1E and NIH-3T3 cells, whereas retroviral overexpression resulted in inhibition of cell growth. Quantitative real time-PCR analysis revealed that the levels of cyclin D1 transcripts were markedly decreased by SOX6 overexpression. Luciferase-reporter assay with beta-catenin showed that SOX6 suppresses cyclin D1 promoter activities. In vitro binding experiments showed that the LZ/Q domain of SOX6 physically interacts with armadillo repeats 1-4 of beta-catenin. Furthermore, chromatin immunoprecipitation assay revealed that increased SOX6 expression significantly reduced the levels of acetylated histones H3 and H4 at the cyclin D1 promoter. By using a histone deacetylase (HDAC) inhibitor and co-immunoprecipitation analysis, we showed that SOX6 suppressed cyclin D1 activities by interacting withbeta-catenin and HDAC1. The data presented suggest that SOX6 may be an important factor in obesity-related insulin resistance.


Assuntos
Ciclina D1/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Histona Desacetilases/metabolismo , Células Secretoras de Insulina/fisiologia , Fatores de Transcrição/metabolismo , beta Catenina/metabolismo , Animais , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Ciclina D1/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Regulação para Baixo/fisiologia , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Grupo de Alta Mobilidade/genética , Histona Desacetilase 1 , Histonas/metabolismo , Humanos , Hiperinsulinismo/metabolismo , Hiperinsulinismo/fisiopatologia , Resistência à Insulina/fisiologia , Células Secretoras de Insulina/citologia , Insulinoma , Rim/citologia , Zíper de Leucina/fisiologia , Camundongos , Células NIH 3T3 , Obesidade/metabolismo , Obesidade/fisiopatologia , Neoplasias Pancreáticas , Regiões Promotoras Genéticas/fisiologia , Estrutura Terciária de Proteína , Ratos , Fatores de Transcrição SOXD , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transdução Genética
12.
Proc Natl Acad Sci U S A ; 103(19): 7438-43, 2006 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-16648250

RESUMO

Here, we report the isolation and characterization of an endogenous peptide ligand of GPR103 from rat brains. The purified peptide was found to be the 43-residue RF-amide peptide QRFP. We also describe two mouse homologues of human GPR103, termed mouse GPR103A and GPR103B. QRFP binds and activates the human GPR103, as well as mouse GPR103A and GPR103B, with nanomolar affinities in transfected cells. Systematic in situ hybridization analysis in mouse brains showed that QRFP is expressed exclusively in the periventricular and lateral hypothalamus, whereas the two receptor mRNAs are distinctly localized in various brain areas without an overlap to each other. When administered centrally in mice, QRFP induced feeding behavior, accompanied by increased general locomotor activity and metabolic rate. QRFP-induced food intake was abolished by preadministration of BIBP3226, a specific antagonist for the Y1 neuropeptide Y receptor. Hypothalamic prepro-QRFP mRNA expression was up-regulated upon fasting and in genetically obese ob/ob and db/db mice. Central QRFP administration also evoked highly sustained elevation of blood pressure and heart rate. Our findings suggest that QRFP and GPR103A/B may regulate diverse neuroendocrine and behavioral functions and implicate this neuropeptide system in metabolic syndrome.


Assuntos
Nível de Alerta/fisiologia , Comportamento Animal/fisiologia , Neuropeptídeos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Ração Animal , Animais , Pressão Sanguínea/efeitos dos fármacos , Encéfalo/metabolismo , Humanos , Ligantes , Locomoção/efeitos dos fármacos , Camundongos , Neuropeptídeos/administração & dosagem , Neuropeptídeos/química , Neuropeptídeos/genética , RNA Mensageiro/genética , Ratos , Regulação para Cima
13.
J Biol Chem ; 280(45): 37669-80, 2005 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-16148004

RESUMO

In obesity-related insulin resistance, pancreatic islets compensate for insulin resistance by increasing secretory capacity. Here, we report the identification of sex-determining region Y-box 6 (SOX6), a member of the high mobility group box superfamily of transcription factors, as a co-repressor for pancreatic-duodenal homeobox factor-1 (PDX1). SOX6 mRNA levels were profoundly reduced by both a long term high fat feeding protocol in normal mice and in genetically obese ob/ob mice on a normal chow diet. Interestingly, we show that SOX6 is expressed in adult pancreatic insulin-producing beta-cells and that overexpression of SOX6 decreased glucose-stimulated insulin secretion, which was accompanied by decreased ATP/ADP ratio, Ca(2+) mobilization, proinsulin content, and insulin gene expression. In a complementary fashion, depletion of SOX6 by small interfering RNAs augmented glucose-stimulated insulin secretion in insulinoma mouse MIN6 and rat INS-1E cells. These effects can be explained by our mechanistic studies that show SOX6 acts to suppress PDX1 stimulation of the insulin II promoter through a direct protein/protein interaction. Furthermore, SOX6 retroviral expression decreased acetylation of histones H3 and H4 in chromatin from the promoter for the insulin II gene, suggesting that SOX6 may decrease PDX1 stimulation through changes in chromatin structure at specific promoters. These results suggest that perturbations in transcriptional regulation that are coordinated through SOX6 and PDX1 in beta-cells may contribute to the beta-cell adaptation in obesity-related insulin resistance.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Glucose/farmacologia , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Homeodomínio/antagonistas & inibidores , Hiperinsulinismo/metabolismo , Insulina/metabolismo , Obesidade/metabolismo , Transativadores/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Acetilação , Trifosfato de Adenosina/metabolismo , Animais , Movimento Celular , Cromatina/metabolismo , Dieta , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/farmacologia , Glucose/metabolismo , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Hiperinsulinismo/genética , Insulina/genética , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Obesos , Mitocôndrias/metabolismo , Obesidade/genética , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOXD , Transativadores/metabolismo , Transcrição Gênica
14.
Biochem Biophys Res Commun ; 333(3): 778-86, 2005 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-15967412

RESUMO

The glucose-induced insulin secretion is fine-tuned by numerous factors. To systematically identify insulinotropic factors, we optimized a primary beta-cell-based functional assay to monitor intracellular Ca2+ flux ([Ca2+]i). By this assay system, we successfully identified several insulinotropic peptides including cholecystokinin, gastrin releasing peptide, vasopressin, and oxytocin from tissue extracts. Screening of an assortment of chemical compounds, we determined three novel insulin secretagogues: N-arachidonylglycine (NAGly), 3beta-(2-diethylamino-ethoxy) androstenone hydrochloride (U18666A), and 4-androstene-3,17-dione. The NAGly increased [Ca2+]i through stimulation of the voltage-dependent Ca2+ channels and it was dependent on extracellular glucose level. On the other hand, U18666A and 4-androstene-3,17-dione increased [Ca2+]i in the presence of K ATP channel opener diazoxide while it was inhibited by the presence of Ca2+ channel blocker nitrendipine, suggesting that their effects are independent of K ATP channel. These unique features will be useful for further development of insulinotropic factors and drugs for treating type 2 diabetes.


Assuntos
Androstenodiona/farmacologia , Androstenos/farmacologia , Ácidos Araquidônicos/farmacologia , Glicina/análogos & derivados , Insulina/metabolismo , Animais , Cálcio/metabolismo , Glicina/farmacologia , Secreção de Insulina , Masculino , Camundongos , Camundongos Endogâmicos ICR , Ratos , Ratos Wistar
15.
J Biol Chem ; 280(13): 12867-75, 2005 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15664998

RESUMO

Krüppel-like zinc finger transcription factors (KLFs) play diverse roles during cell differentiation and development in mammals. We have now shown by microarray analysis that expression of the KLF15 gene is markedly up-regulated during the differentiation of 3T3-L1 preadipocytes into adipocytes. Inhibition of the function of KLF15, either by expression of a dominant negative mutant or by RNA interference, both reduced the expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and blocked adipogenesis in 3T3-L1 preadipocytes exposed to inducers of adipocyte differentiation. However, the dominant negative mutant of KLF15 did not affect the expression of CCAAT/enhancer-binding protein beta (C/EBPbeta) elicited by inducers of differentiation in 3T3-L1 preadipocytes. In addition, ectopic expression of KLF15 in NIH 3T3 or C2C12 cells triggered both lipid accumulation and the expression of PPARgamma in the presence of inducers of adipocyte differentiation. Ectopic expression of C/EBPbeta, C/EBPdelta, or C/EBPalpha in NIH 3T3 cells also elicited the expression of KLF15 in the presence of inducers of adipocyte differentiation. Moreover, KLF15 and C/EBPalpha acted synergistically to increase the activity of the PPARgamma2 gene promoter in 3T3-L1 adipocytes. Our observations thus demonstrate that KLF15 plays an essential role in adipogenesis in 3T3-L1 cells through its regulation of PPAR gamma expression.


Assuntos
Adipócitos/citologia , PPAR gama/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Northern Blotting , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular , Linhagem Celular , Proteínas de Ligação a DNA , Fibroblastos/metabolismo , Genes Dominantes , Vetores Genéticos , Fatores de Transcrição Kruppel-Like , Luciferases/metabolismo , Camundongos , Modelos Biológicos , Mutação , Células NIH 3T3 , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , Retroviridae/genética , Fatores de Tempo , Regulação para Cima
16.
Biochem Biophys Res Commun ; 316(2): 490-500, 2004 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-15020244

RESUMO

Bone morphogenetic proteins (BMPs) play critical roles in cellular proliferation, differentiation, and programmed cell death in multiple tissues. An increasing body of recent evidence has suggested that classes of molecules collectively termed BMP antagonists play important roles for the local regulation of BMP actions by binding BMPs and neutralizing their activities. Uterine sensitization-associated gene-1 (USAG-1) was previously reported as a gene of unknown function, preferentially expressed in sensitized endometrium of the rat uterus. Here, we show that USAG-1 is abundantly expressed in the kidney and functions as a BMP antagonist. Recombinant USAG-1 binds directly to BMPs and antagonizes the BMP-mediated induction of alkaline phosphatase in C2C12 cells. USAG-1 also induces formation of secondary axis and/or hyperdorsalization when its mRNA is injected to Xenopus embryos. In the early stage of mouse embryogenesis, USAG-1 is expressed in the first and second branchial arches and in metanephros, while in later stages the expression is confined to renal tubules and ameloblasts of teeth. Postnatally, the expression is further restricted to distal tubules of kidney, in a pattern similar to the localization of BMP-7, which has been shown to be important in the development of kidney and preservation of adult renal functions under pathological stresses. Collectively, we suggest that USAG-1 is a BMP antagonist that interacts with BMP-7 in the developing and adult kidney.


Assuntos
Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Rim/metabolismo , Proteínas/fisiologia , Proteínas de Peixe-Zebra , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Proteínas Morfogenéticas Ósseas/química , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/metabolismo , Embrião não Mamífero , Desenvolvimento Embrionário e Fetal , Feto/química , Humanos , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Dados de Sequência Molecular , Proteínas/química , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , RNA Mensageiro/análise , Ratos , Alinhamento de Sequência , Distribuição Tecidual , Proteínas Wnt , Xenopus
17.
J Biol Chem ; 279(17): 16954-62, 2004 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-14960588

RESUMO

Acetyl-CoA synthetase 2 (AceCS2) produces acetyl-CoA for oxidation through the citric acid cycle in the mitochondrial matrix. AceCS2 is highly expressed in the skeletal muscle and is robustly induced by fasting. Quantification of AceCS2 transcripts both in C2C12 and human myotubes indicated that fasting-induced AceCS2 gene expression appears to be independent on insulin action. Characterization of 5'-flanking region of the mouse AceCS2 gene demonstrates that Krüppel-like factor 15 (KLF15) plays a key role in the trans-activation of the AceCS2 gene. Deletion and mutation analyses of AceCS2 promoter region revealed that the most proximal KLF site is a curtail site for the trans-activation of the AceCS2 gene by KLF15. Using Sp-null Drosophila SL2 cells, we showed that the combination of KLF15 and Sp1 resulted in a synergistic activation of the AceCS2 promoter. Mutation analyses of three GC-boxes in the AceCS2 promoter indicated that the GC-box, located 8 bases downstream of the most proximal KLF15 site, is the most important GC-box in the synergistic trans-activation of the AceCS2 gene by KLF15 and Sp1. GST pull-down assays showed that KLF15 interacts with Sp1 in vitro. Quantification of various KLF transcripts revealed that 48 h fasting robustly induced the KLF15 transcripts in the skeletal muscle. Together with the trans-activation of the AceCS2 promoter, it is suggested that fasting-induced AceCS2 expression is largely contributed by KLF15. Furthermore, KLF15 overexpression induced the levels of AceCS2 transcripts both in myoblasts and in myotubes, indicating that AceCS2 gene expression in vivo is indeed induced by KLF15.


Assuntos
Acetato-CoA Ligase/genética , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Ativação Transcricional , Acetato-CoA Ligase/metabolismo , Motivos de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Ciclo do Ácido Cítrico , Clonagem Molecular , Análise Mutacional de DNA , DNA Complementar/metabolismo , Proteínas de Ligação a DNA , Drosophila , Deleção de Genes , Genes Reporter , Glutationa Transferase/metabolismo , Humanos , Insulina/metabolismo , Fatores de Transcrição Kruppel-Like , Masculino , Camundongos , Camundongos Endogâmicos ICR , Modelos Genéticos , Dados de Sequência Molecular , Células Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteínas Nucleares/genética , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Fator de Transcrição Sp1/metabolismo , Fatores de Tempo , Fatores de Transcrição/genética , Transfecção
18.
Nat Med ; 10(2): 168-74, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14716305

RESUMO

The transcription factor, signal transducer and activator of transcription-3 (STAT-3) contributes to various physiological processes. Here we show that mice with liver-specific deficiency in STAT-3, achieved using the Cre-loxP system, show insulin resistance associated with increased hepatic expression of gluconeogenic genes. Restoration of hepatic STAT-3 expression in these mice, using adenovirus-mediated gene transfer, corrected the metabolic abnormalities and the alterations in hepatic expression of gluconeogenic genes. Overexpression of STAT-3 in cultured hepatocytes inhibited gluconeogenic gene expression independently of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha), an upstream regulator of gluconeogenic genes. Liver-specific expression of a constitutively active form of STAT-3, achieved by infection with an adenovirus vector, markedly reduced blood glucose, plasma insulin concentrations and hepatic gluconeogenic gene expression in diabetic mice. Hepatic STAT-3 signaling is thus essential for normal glucose homeostasis and may provide new therapeutic targets for diabetes mellitus.


Assuntos
Metabolismo dos Carboidratos , Proteínas de Ligação a DNA/metabolismo , Gluconeogênese/genética , Fígado/fisiologia , Transativadores/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Glicemia/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/genética , Diabetes Mellitus/metabolismo , Gorduras na Dieta , Técnicas de Transferência de Genes , Gluconeogênese/fisiologia , Hepatócitos/citologia , Hepatócitos/metabolismo , Insulina/metabolismo , Fígado/citologia , Fígado/patologia , Masculino , Camundongos , Camundongos Knockout , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores para Leptina , Fator de Transcrição STAT3 , Transativadores/genética , Fatores de Transcrição/metabolismo
19.
Proc Natl Acad Sci U S A ; 100(26): 15924-9, 2003 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-14676330

RESUMO

In this study, we defined the role of peroxisome proliferator-activated receptor beta/delta (PPARdelta) in metabolic homeostasis by using subtype selective agonists. Analysis of rat L6 myotubes treated with the PPARdelta subtype-selective agonist, GW501516, by the Affymetrix oligonucleotide microarrays revealed that PPARdelta controls fatty acid oxidation by regulating genes involved in fatty acid transport, beta-oxidation, and mitochondrial respiration. Similar PPARdelta-mediated gene activation was observed in the skeletal muscle of GW501516-treated mice. Accordingly, GW501516 treatment induced fatty acid beta-oxidation in L6 myotubes as well as in mouse skeletal muscles. Administration of GW501516 to mice fed a high-fat diet ameliorated diet-induced obesity and insulin resistance, an effect accompanied by enhanced metabolic rate and fatty acid beta-oxidation, proliferation of mitochondria, and a marked reduction of lipid droplets in skeletal muscles. Despite a modest body weight change relative to vehicle-treated mice, GW501516 treatment also markedly improved diabetes as revealed by the decrease in plasma glucose and blood insulin levels in genetically obese ob/ob mice. These data suggest that PPARdelta is pivotal to control the program for fatty acid oxidation in the skeletal muscle, thereby ameliorating obesity and insulin resistance through its activation in obese animals.


Assuntos
Ácidos Graxos não Esterificados/metabolismo , Resistência à Insulina/fisiologia , Síndrome Metabólica/prevenção & controle , Músculo Esquelético/fisiologia , Receptores Citoplasmáticos e Nucleares/agonistas , Fatores de Transcrição/agonistas , Animais , Dimetil Sulfóxido/farmacologia , Enzimas/genética , Metabolismo dos Lipídeos , Fígado/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Músculo Esquelético/efeitos dos fármacos , Oxirredução , Ratos , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Tiazóis/farmacologia , Fatores de Transcrição/efeitos dos fármacos
20.
J Clin Invest ; 112(6): 935-44, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12975478

RESUMO

PKClambda is implicated as a downstream effector of PI3K in insulin action. We show here that mice that lack PKClambda specifically in the liver (L-lambdaKO mice), produced with the use of the Cre-loxP system, exhibit increased insulin sensitivity as well as a decreased triglyceride content and reduced expression of the sterol regulatory element-binding protein-1c (SREBP-1c) gene in the liver. Induction of the hepatic expression of Srebp1c and of its target genes involved in fatty acid/triglyceride synthesis by fasting and refeeding or by hepatic expression of an active form of PI3K was inhibited in L-lambdaKO mice compared with that in control animals. Expression of Srebp1c induced by insulin or by active PI3K in primary cultured rat hepatocytes was inhibited by a dominant-negative form of PKClambda and was mimicked by overexpression of WT PKClambda. Restoration of PKClambda expression in the liver of L-lambdaKO mice with the use of adenovirus-mediated gene transfer corrected the metabolic abnormalities of these animals. Hepatic PKClambda is thus a determinant of hepatic lipid content and whole-body insulin sensitivity.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA/metabolismo , Insulina/metabolismo , Metabolismo dos Lipídeos , Fígado/fisiologia , Proteína Quinase C/metabolismo , Animais , Glicemia/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Isoenzimas , Fígado/química , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Proteína Quinase C/genética , Ratos , Proteína de Ligação a Elemento Regulador de Esterol 1 , Distribuição Tecidual , Fatores de Transcrição/metabolismo
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