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1.
Zoolog Sci ; 35(2): 109-114, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29623784

RESUMO

Human, mouse, and zebrafish ovarian cancer G protein-coupled receptors (OGR1s) are activated by both metals and extracellular protons. In the present study, we examined whether pig, rat, chicken, and Xenopus OGR1 homologs could sense and be activated by protons and metals. We found that all homologs stimulated serum response element (SRE)-driven promoter activities when they are stimulated by protons. On the other hand, metals differentially activated the homologs. The results using chimeric receptors of human and zebrafish OGR1s indicate that the specificity of the metal-induced activation lies in the extracellular region. These results suggest that protons are an evolutionally conserved agonist of OGR1. However, the types of metals that activated the receptor differed among the homologs.


Assuntos
Galinhas/genética , Metais/administração & dosagem , Prótons , Ratos/genética , Receptores Acoplados a Proteínas G/genética , Sus scrofa/genética , Xenopus/genética , Animais , Galinhas/metabolismo , Feminino , Células HEK293 , Humanos , Neoplasias Ovarianas/genética , Ratos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Elemento de Resposta Sérica/efeitos dos fármacos , Sus scrofa/metabolismo , Xenopus/metabolismo
2.
Bioorg Med Chem Lett ; 27(8): 1744-1749, 2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28285914

RESUMO

We recently reported the development of a novel inhibitor of Rho-mediated gene transcription (1, CCG-203971) that is efficacious in multiple animal models of acute fibrosis, including scleroderma, when given intraperitoneally. The modest in vivo potency and poor pharmacokinetics (PK) of this lead, however, make it unsuitable for long term efficacy studies. We therefore undertook a systematic medicinal chemistry effort to improve both the metabolic stability and the solubility of 1, resulting in the identification of two analogs achieving over 10-fold increases in plasma exposures in mice. We subsequently showed that one of these analogs (8f, CCG-232601) could inhibit the development of bleomycin-induced dermal fibrosis in mice when administered orally at 50mg/kg, an effect that was comparable to what we had observed earlier with 1 at a 4-fold higher IP dose.


Assuntos
Ácidos Nipecóticos/farmacocinética , Ácidos Nipecóticos/uso terapêutico , Fator Rho/antagonistas & inibidores , Escleroderma Sistêmico/tratamento farmacológico , Pele/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Administração Oral , Animais , Modelos Animais de Doenças , Fibrose , Células HEK293 , Humanos , Camundongos , Ácidos Nipecóticos/administração & dosagem , Ácidos Nipecóticos/química , Fator Rho/metabolismo , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/patologia , Elemento de Resposta Sérica/efeitos dos fármacos , Pele/metabolismo , Pele/patologia , Transativadores/antagonistas & inibidores , Transativadores/metabolismo
3.
Can J Physiol Pharmacol ; 95(3): 275-280, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28157379

RESUMO

Lysophosphatidic acid (LPA), one component of oxidized low-density lipoprotein (ox-LDL), is a potent bioactive phospholipid. Our recent data reveal that LPA induces matricellular protein CCN1 (also known as Cyr61) expression in aortic smooth muscle cells (SMCs) and that CCN1 bridges LPA and integrin signaling pathways leading to SMC migration. Whether and how LPA regulates the transcriptional machinery of the CCN1 gene are unknown. In this study, we found that LPA markedly induces CCN1 mRNA expression in SMCs. Using deleting mutation and reporter gene strategies, we demonstrated regions from -2038 to -1787 and from -101 to +63 of the CCN1 promoter contain the essential regulatory elements. The serum response element (SRE) and cyclic AMP-response element (CRE) are located in these regions. LPA induced time-dependent phosphorylation of serum response factor (SRF) and CRE-binding protein (CREB) in mouse SMCs. Luciferase assays of a series of deleted, mutated CCN1 promoter-reporter gene constructs and dominant negative construct revealed the distal SRE and the proximal CRE in the CCN1 promoter are required for LPA-induced CCN1 gene expression. Our results imply that elevated LPA levels may trigger SMC migration and exacerbate restenosis and atherosclerotic lesions through the induced CCN1, which communicates with a set of plasma membrane proteins and intracellular kinases.


Assuntos
Proteína Rica em Cisteína 61/genética , Lisofosfolipídeos/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Elemento de Resposta Sérica/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Sítios de Ligação , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteína Rica em Cisteína 61/metabolismo , Genes Reporter , Camundongos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fosforilação , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Resposta Sérica/metabolismo , Fatores de Tempo , Transfecção , Regulação para Cima
4.
Circ Res ; 102(12): 1502-11, 2008 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-18497331

RESUMO

Leupaxin is a LIM domain-containing adapter protein belonging to the paxillin family that has been previously reported to be preferentially expressed in hematopoietic cells. Herein, we identified leupaxin in a screen for focal adhesion kinase binding partners in aortic smooth muscle, and we show that leupaxin is enriched in human and mouse vascular smooth muscle and that leupaxin expression is dynamically regulated during development. In addition, our studies reveal that leupaxin can undergo cytoplasmic/nuclear shuttling and functions as an serum response factor cofactor in the nucleus. We found that leupaxin forms a complex with serum response factor and associates with CArG-containing regions of smooth muscle promoters and that ectopic expression of leupaxin induces smooth muscle marker gene expression in both 10T1/2 cells and rat aortic smooth muscle cells. Subsequent studies indicated that enhanced focal adhesion kinase activity (induced by fibronectin or expression of constitutively active focal adhesion kinase) attenuates the nuclear accumulation of leupaxin and limits the ability of leupaxin to enhance serum response factor-dependent gene transcription. Thus, these studies indicate that modulation of the subcellular localization of serum response factor cofactors is 1 mechanism by which extracellular matrix-dependent signals may regulate phenotypic switching of smooth muscle cells.


Assuntos
Moléculas de Adesão Celular/fisiologia , Miócitos de Músculo Liso/metabolismo , Fosfoproteínas/fisiologia , Elemento de Resposta Sérica/fisiologia , Animais , Aorta/citologia , Aorta/embriologia , Aorta/crescimento & desenvolvimento , Transporte Biológico , Moléculas de Adesão Celular/farmacologia , Diferenciação Celular , Células Cultivadas/efeitos dos fármacos , Vasos Coronários/citologia , Feminino , Quinase 1 de Adesão Focal/fisiologia , Adesões Focais/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Pessoa de Meia-Idade , Miócitos de Músculo Liso/efeitos dos fármacos , Miométrio/citologia , Especificidade de Órgãos , Fosfoproteínas/farmacologia , Mapeamento de Interação de Proteínas , Ratos , Proteínas Recombinantes de Fusão/fisiologia , Elemento de Resposta Sérica/efeitos dos fármacos , Fator de Resposta Sérica/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição , Transcrição Gênica
5.
J Steroid Biochem Mol Biol ; 108(1-2): 137-48, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17942302

RESUMO

Using a luciferase reporter assay we found that human serum transactivated the ileal apical sodium-dependent bile acid transporter (ASBT) promoter three to fourfold. Confirming this effect, addition of human serum to both Caco-2 cells and fresh human ileal biopsies caused an approximate 2.0-fold increase in endogenous ASBT mRNA production. Alteration of non-esterified fatty acid (NEFA) content and cortisol content did not affect the transactivation potential of serum. Site-directed mutagenesis of response elements for corticosteroid, peroxisome proliferation-activated alpha (PPARalpha), hepatocyte nuclear factor 1alpha (HNF1alpha), and retinoic acid (RAR/RXR) did not affect transactivation potential of serum. Three putative serum response elements (SRE) were identified on the promoter, but all were determined inactive using site-directed mutagenesis and electrophoretic mobility shift assay. Promoter deletion analysis demonstrated that >80% of the response to serum was located within the last 273 bp of the 5'-UTR, an area containing one of two activate protein 1 (AP-1) response elements. Site-directed mutagenesis of this downstream AP-1 response element reduced the effect of serum on the promoter by about 50% while full deletion of the response element completely eliminated the effect of serum. These studies demonstrate that one or more constituents of human stimulate ASBT gene expression largely via the down-stream AP-1 response element.


Assuntos
Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Soro/fisiologia , Simportadores/genética , Ativação Transcricional , Animais , Células CACO-2 , Proteínas de Transporte/farmacologia , Galinhas , Corticosterona , Ácidos Graxos não Esterificados/farmacologia , Genes Reporter/efeitos dos fármacos , Humanos , Hidrocortisona/farmacologia , Íleo/metabolismo , Luciferases/genética , Mifepristona/farmacologia , Mutagênese Sítio-Dirigida , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , RNA Mensageiro/metabolismo , Receptores do Ácido Retinoico/metabolismo , Elemento de Resposta Sérica/efeitos dos fármacos , Elemento de Resposta Sérica/fisiologia , Simportadores/metabolismo , Fator de Transcrição AP-1/metabolismo , Ativação Transcricional/efeitos dos fármacos , Transfecção , Células Tumorais Cultivadas
6.
Biochem Biophys Res Commun ; 355(3): 654-60, 2007 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-17307136

RESUMO

Diseases caused by calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystals occur frequently in osteoarthritic joints. Both crystals induce mitogenesis, metalloproteinase synthesis and secretion by fibroblasts and chondrocytes, promoting degradation of articular tissue. We investigated the mechanism by which BCP activates the c-fos proto-oncogene, which has been shown to activate various matrix metalloproteinases (MMPs). We demonstrate that BCP crystals induce c-fos expression primarily through a Ras/ERK-dependent signaling mechanism targeting two highly conserved regulatory binding sites, the serum response element (SRE) and the cAMP response element (CRE). These results establish a calcium crystal induced, calcium/calmodulin independent, signaling pathway in which BCP crystals activate Ras/MAPK, which can directly target an SRF-containing transcription factor complex, to induce fibroblasts to secrete metalloproteinases.


Assuntos
Fosfatos de Cálcio/toxicidade , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína Oncogênica p21(ras)/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Elemento de Resposta Sérica/efeitos dos fármacos , Animais , Fosfatos de Cálcio/química , Células Cultivadas , Cristalização , AMP Cíclico/metabolismo , Humanos , Camundongos , Osteoartrite/induzido quimicamente , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-fos/genética , Elementos de Resposta/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
7.
J Neurochem ; 97(1): 92-104, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16515541

RESUMO

Tyrosine hydroxylase is the rate-limiting enzyme in the biosynthesis of catecholamines. Expression of the tyrosine hydroxylase gene is regulated at the transcriptional level by extracellular signalling molecules, including epidermal growth factor (EGF), nerve growth factor (NGF) and glucocorticoids. We have analysed the stimulation of tyrosine hydroxylase gene transcription by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in noradrenergic locus coeruleus-like CATH.a cells and observed a striking enhancement of the transcriptional activation potential of the ternary complex factor Ets-like protein-1 (Elk-1), a key transcriptional regulator of serum response element-driven gene transcription. Likewise, TPA strongly up-regulated the biosynthesis of the transcription factor Egr-1 via distal serum response elements within the Egr-1 5'-flanking region. Subsequently, enhancement of the transcriptional activation potential of Egr-1 was observed. Overexpression of Egr-1 was sufficient to activate transcription of a tyrosine hydroxylase promoter/reporter gene, corroborating the view that the tyrosine hydroxylase gene is a target gene of Egr-1. Expression of dominant-negative mutants of Elk-1 or Egr-1 impaired TPA-induced stimulation of a tyrosine hydroxylase promoter/reporter gene transcription. In contrast, dominant-negative mutants of the transcription factors activating transcription factor (ATF)-2, ATF4, cAMP response element-binding protein, c-Jun and CCAAT/enhancer binding protein (C/EBP) did not change TPA-induced tyrosine hydroxylase promoter activity, indicating that these proteins are not part of the TPA-mediated signalling cascade directed towards the tyrosine hydroxylase gene.


Assuntos
Catecolaminas/biossíntese , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Neurônios/enzimologia , Ativação Transcricional/fisiologia , Tirosina 3-Mono-Oxigenase/biossíntese , Proteínas Elk-1 do Domínio ets/metabolismo , Animais , Carcinógenos/farmacologia , Linhagem Celular , Proteína 1 de Resposta de Crescimento Precoce/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Genes Reporter/efeitos dos fármacos , Genes Reporter/fisiologia , Locus Cerúleo/enzimologia , Camundongos , Neurônios/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/fisiologia , Elementos Reguladores de Transcrição/efeitos dos fármacos , Elementos Reguladores de Transcrição/fisiologia , Elemento de Resposta Sérica/efeitos dos fármacos , Elemento de Resposta Sérica/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Proteínas Elk-1 do Domínio ets/efeitos dos fármacos
8.
J Neurochem ; 92(4): 915-24, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15686494

RESUMO

Pathological conditions such as ischaemic stroke and inflammatory disorders cause c-fos activation in the brain. This activation contributes to the initiation of the brain's inflammatory response, orchestrated by activated glial cells. The inflammatory signalling cascades leading to c-fos activation in glial cells are not well characterized. Thus, we have attempted a detailed analysis of the cis-acting elements, transcription factors and upstream kinase pathways involved in the activation of c-fos by lipopolysaccharide (LPS) in primary rat cortical glial cells. We found that (1) LPS-induced c-fos mRNA levels were sensitive to p38 mitogen-activated protein kinase (MAPK) inhibitors but not to mitogen-activated/extracellular signal-regulated kinase (ERK) or calcium-calmodulin-dependent kinase inhibitors, (2) LPS activated both serum response element (SRE) and cyclic AMP/calcium response element (CRE)-driven luciferase reporters in transient transfection assays, (3) LPS induced the phosphorylation of Elk1 CRE-binding protein (CREB)/activated transcription factor-1 (ATF-1) and the activation of GAL4-Elk1 and GAL4-CREB chimeric proteins, and (4) mutation of both SRE and CRE elements was necessary and sufficient to completely abolish LPS induction of a rat c-fos proximal promoter-luciferase reporter. Thus, c-fos activation by LPS in glial cells occurs via the SRE or CRE in an independent manner, and involves the Elk1 or CREB/ATF-1 transcription factors. Elk1-mediated transactivation was dependent on p38 MAPK, suggesting a crucial role of these factors in mediating inflammatory responses in the CNS.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Regulação da Expressão Gênica/fisiologia , Genes fos/fisiologia , Lipopolissacarídeos/farmacologia , Neuroglia/enzimologia , Elemento de Resposta Sérica/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Animais , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Genes fos/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Ratos , Elemento de Resposta Sérica/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/genética
9.
Eur J Endocrinol ; 151(5): 619-28, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15538941

RESUMO

BACKGROUND AND METHODS: The majority of human endometrial (>80%), ovarian (>80%) and breast (>50%) cancers express GnRH receptors. Their spontaneous and epidermal growth-factor-induced proliferation is dose- and time-dependently reduced by treatment with GnRH and its agonists. In this study, we demonstrate that the GnRH agonist triptorelin inhibits estradiol (E2)-induced cancer cell proliferation. RESULTS: The proliferation of quiescent estrogen receptor alpha (ER alpha)-/ER beta-positive, but not of ER alpha-negative/ER beta-positive endometrial, ovarian and breast cancer cell lines, was significantly stimulated (P<0.001) (ANOVA) after treatment with E2 (10(-8) M). This effect was time- and dose-dependently antagonized by simultaneous treatment with triptorelin. The inhibitory effect was maximal at 10(-5) M concentration of triptorelin (P<0.001). In addition, we could show that, in ER alpha-/ER beta-positive cell lines, E2 induces activation of serum response element (SRE) and expression of the immediate early-response gene c-fos. These effects were blocked by triptorelin (P<0.001). E2-induced activation of estrogen-response element (ERE) was not affected by triptorelin. CONCLUSIONS: The transcriptional activation of SRE by E2 is due to ER alpha activation of the mitogen-activated protein kinase (MAPK) pathway. This pathway is impeded by GnRH, resulting in a reduction of E2-induced SRE activation and, in consequence, a reduction of E2-induced c-fos expression. This causes downregulation of E2-induced cancer cell proliferation.


Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/metabolismo , Estradiol/farmacologia , Neoplasias dos Genitais Femininos/metabolismo , Proteínas Proto-Oncogênicas c-fos/antagonistas & inibidores , Elemento de Resposta Sérica/efeitos dos fármacos , Pamoato de Triptorrelina/farmacologia , Antineoplásicos Hormonais/administração & dosagem , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Estrogênios/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Genes fos , Neoplasias dos Genitais Femininos/patologia , Hormônio Liberador de Gonadotropina/agonistas , Humanos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Elementos de Resposta/efeitos dos fármacos , Pamoato de Triptorrelina/administração & dosagem
10.
Arterioscler Thromb Vasc Biol ; 24(8): 1384-90, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15217807

RESUMO

OBJECTIVE: Transforming growth factor-beta1 (TGFbeta1) and fibroblast growth factor (FGF) families play a pivotal role during vascular development and in the pathogenesis of vascular disease. However, the interaction of intracellular signaling evoked by each of these growth factors is not well understood. The present study was undertaken to examine the molecular mechanisms that mediate the effects of TGFbeta1 and basic FGF (bFGF) on smooth muscle cell (SMC) gene expression. METHODS AND RESULTS: TGFbeta1 induction of SMC gene expression, including smooth muscle protein 22-alpha (SM22alpha) and smooth muscle alpha-actin, was examined in the pluripotent 10T1/2 cells. Marked increase in these mRNA levels by TGFbeta1 was inhibited by c-Src-tyrosine kinase inhibitors and protein synthesis inhibitor cycloheximide. Functional studies with deletion and site-directed mutation analysis of the SM22alpha promoter demonstrated that TGFbeta1 activated the SM22alpha promoter through a CC(A/T-rich)6GG (CArG) box, which serves as a serum response factor (SRF)-binding site. TGFbeta1 increased SRF expression through an increase in transcription of the SRF gene. In the presence of bFGF, TGFbeta1 induction of SMC marker gene expression was significantly attenuated. Transient transfection assays showed that bFGF significantly suppressed induction of the SM22alpha promoter-driven luciferase activity by TGFbeta1, whereas bFGF had no effects on the TGFbeta1-mediated increase in SRF expression and SRF:DNA binding activity. Mitogen-activated protein kinase kinase-1 (MEK1) inhibitor PD98059 abrogated the bFGF-mediated suppression of TGFbeta1-induced SMC gene expression. CONCLUSIONS: Our data suggest that bFGF-induced MEK/extracellular signal-regulated kinase signaling plays an antagonistic role in TGFbeta1-induced SMC gene expression through suppression of the SRF function. These data indicate that opposing effects of bFGF and TGFbeta1 on SMC gene expression control the phenotypic plasticity of SMCs.


Assuntos
Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/antagonistas & inibidores , Quinases da Família src/fisiologia , Actinas/biossíntese , Actinas/genética , Animais , Aorta , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/genética , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Cicloeximida/farmacologia , DNA/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/fisiologia , Camundongos , Camundongos Endogâmicos C3H , Proteínas dos Microfilamentos/biossíntese , Proteínas dos Microfilamentos/genética , Proteínas Musculares/biossíntese , Proteínas Musculares/genética , Mutagênese Sítio-Dirigida , Miócitos de Músculo Liso/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Proteínas Recombinantes/farmacologia , Elemento de Resposta Sérica/efeitos dos fármacos , Fator de Resposta Sérica/metabolismo , Fator de Resposta Sérica/farmacologia , Transcrição Gênica/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1 , Quinases da Família src/antagonistas & inibidores , Calponinas
11.
J Bone Miner Res ; 19(5): 862-9, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15068510

RESUMO

UNLABELLED: Defining the molecular target for strontium in osteoblasts is important for understanding the anabolic effects of this cation on bone. The current studies demonstrate that a G-protein-mediated response to strontium persists in osteoblasts that lack CASR, suggesting a predominant role for a novel cation-sensing receptor in mediating the osseous response to strontium. INTRODUCTION: Strontium has anabolic effects on bone and is currently being developed for the treatment of osteoporosis. The molecular target for strontium in osteoblasts has not been determined, but the existence of CASR, a G-protein-coupled receptor calcium-sensing receptor, raises the possibility that strontium actions on bone are mediated through this or a related receptor. MATERIALS AND METHODS: We used activation of a transfected serum response element (SRE)-luciferase reporter in HEK-293 cells to determine if CASR is activated by strontium. In addition, we examined strontium-mediated responses in MC3T3-E1 osteoblasts and osteoblasts derived from wild-type and CASR null mice to determine if other cation-sensing mechanisms are present in osteoblasts. RESULTS AND CONCLUSIONS: We found that strontium stimulated SRE-luc activity in HEK-293 cells transfected with full-length CASR but not in cells expressing the alternatively spliced CASR construct lacking exon 5. In contrast, we found that MC3T3-E1 osteoblasts that lack CASR as well as osteoblasts derived from CASR null mice respond to millimolar concentrations of strontium. The response to strontium in osteoblasts was nonadditive to a panel of extracellular cations, including aluminum, gadolinium, and calcium, suggesting a common mechanism of action. In contrast, neither the CASR agonist magnesium nor the calcimimetic NPS-R568 activated SRE activity in osteoblasts, but the response to these agonists was imparted by transfection of CASR into these osteoblasts, consistent with the presence of distinct cation-sensing mechanisms. Co-expression of the dominant negative Galphaq(305-359) minigene also inhibited cation-stimulated SRE activity in osteoblasts lacking known CASR. These findings are consistent with strontium activation of a novel Galphaq-coupled extracellular cation-sensing receptor in osteoblasts with distinct cation specificity.


Assuntos
Proteínas de Ciclo Celular , Osteoblastos/metabolismo , Estrôncio/farmacologia , Processamento Alternativo , Alumínio/metabolismo , Alumínio/farmacologia , Compostos de Anilina/farmacologia , Animais , Cálcio/metabolismo , Cátions , Linhagem Celular , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Gadolínio/farmacologia , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Magnésio/metabolismo , Magnésio/farmacologia , Camundongos , Camundongos Knockout , Fenetilaminas , Propilaminas , Ratos , Receptores de Detecção de Cálcio/agonistas , Receptores de Detecção de Cálcio/genética , Receptores de Detecção de Cálcio/metabolismo , Proteína A6 Ligante de Cálcio S100 , Proteínas S100/metabolismo , Elemento de Resposta Sérica/efeitos dos fármacos , Elemento de Resposta Sérica/genética , Estrôncio/metabolismo , Transfecção
12.
Mol Cell Neurosci ; 25(2): 345-54, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15019950

RESUMO

Bone morphogenetic proteins (BMPs) 4 and 6 as well as MEK inhibitors PD98059 and U0126 potentiate neurotrophin 3 (NT3)- and neurturin (NTN)-induced neurite outgrowth and survival of peripheral neurons from the E9 chicken embryo. Preexposure to BMP4 or PD98059 was sufficient to prime the potentiation of subsequently added NT3. Phosphorylation of Erk2, induced by NT3, was reduced by MEK inhibition but unaffected by BMP signaling. Real-time PCR showed that neither BMP stimulation nor MEK inhibition increased Trk receptor expression and that the BMP-induced genes Smad6 and Id1 were not upregulated by PD98059. In contrast, both MEK inhibition and BMP signaling suppressed transcription of the serum-response element (SRE)-driven Egr1 gene. A reporter assay using NGF-stimulated PC12 cells demonstrated that MEK/Erk/Elk-driven transcriptional activity was inhibited by Smad1/5 and by PD98059. Thus, suppression of SRE-controlled transcription represents a likely convergence point for pathways regulating neurotrophic responses.


Assuntos
Diferenciação Celular/fisiologia , Gânglios/enzimologia , Sistema de Sinalização das MAP Quinases/fisiologia , Fatores de Crescimento Neural/metabolismo , Neurônios/enzimologia , Sistema Nervoso Periférico/enzimologia , Fatores de Transcrição , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Embrião de Galinha , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/farmacologia , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Gânglios/citologia , Gânglios/crescimento & desenvolvimento , Genes Reguladores/efeitos dos fármacos , Genes Reguladores/genética , MAP Quinase Quinase 1 , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fatores de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Neuritos/enzimologia , Neuritos/ultraestrutura , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurotrofina 3/metabolismo , Neurotrofina 3/farmacologia , Células PC12 , Sistema Nervoso Periférico/citologia , Sistema Nervoso Periférico/crescimento & desenvolvimento , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Ratos , Elemento de Resposta Sérica/efeitos dos fármacos , Elemento de Resposta Sérica/genética , Proteínas Smad , Proteína Smad1 , Transativadores/metabolismo , Transativadores/farmacologia , Proteínas Elk-1 do Domínio ets
13.
J Natl Cancer Inst ; 95(10): 733-40, 2003 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-12759391

RESUMO

BACKGROUND: Lysophosphatidic acid (LPA), at concentrations present in ascitic fluid, indirectly stimulates the growth of malignant ovarian tumors by increasing the expression of vascular endothelial growth factor (VEGF) in ovarian cancer cells. We investigated whether LPA could also directly promote ovarian tumor growth by increasing the level of cyclin D1, a key G1-phase checkpoint regulator, which thereby increases cell proliferation. METHODS: Expression of cyclin D1 and LPA receptors (EDG4 and EDG7) was determined in six ovarian cancer cell lines (including OVCAR-3 cells) and immortalized ovarian surface epithelial cells (IOSE-29). Cyclin D1 promoter activity was measured in LPA-treated OVCAR-3 cells cotransfected with cyclin D1 promoter-driven luciferase constructs and cDNA expression plasmids for IkappaBalphaM (a nuclear factor kappaB [NFkappaB] super-repressor). RESULTS: Four of six cancer cell lines, including OVCAR-3, overexpressed cyclin D1 protein relative to levels in IOSE-29 cells. LPA treatment increased cyclin D1 protein in a dose- and time-dependent manner in OVCAR-3 cells but not in IOSE-29 cells. LPA stimulated cyclin D1 promoter activity (3.0-fold, 95% confidence interval [CI] = 2.7-fold to 3.3-fold). Mutation of the NFkappaB-binding site in the cyclin D1 promoter to block NFkappaB binding and expression of IkappaBalphaM, which binds NFkappaB and inhibits its binding to the promoter, markedly diminished LPA stimulation of cyclin D1 promoter activity (activity stimulated only 1.4-fold, 95% CI = 1.1-fold to 1.7-fold, and 0.7-fold, 95% CI = 0.6-fold to 0.8-fold, respectively). EDG4 was overexpressed in all cancer cell lines studied relative to that in IOSE-29 cells, but EDG7 was overexpressed in only two lines. CONCLUSIONS: Dual mechanisms are probably involved in LPA stimulation of ovarian tumor growth in vivo. In addition to the previously characterized indirect mechanism that increases angiogenesis via VEGF, LPA may directly increase the level of cyclin D1 in ovarian cancer cells, increasing their proliferation.


Assuntos
Carcinoma/metabolismo , Ciclina D1/metabolismo , Lisofosfolipídeos/metabolismo , Neoplasias Ovarianas/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G , Northern Blotting , Western Blotting , Divisão Celular/efeitos dos fármacos , Ciclina D1/efeitos dos fármacos , Ciclina D1/genética , Relação Dose-Resposta a Droga , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas I-kappa B/genética , Luciferases/metabolismo , Lisofosfolipídeos/farmacologia , Mutação , NF-kappa B/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Receptores de Ácidos Lisofosfatídicos , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Elemento de Resposta Sérica/efeitos dos fármacos , Fatores de Tempo , Transfecção , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacos
14.
Am J Physiol Cell Physiol ; 285(2): C370-6, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12711593

RESUMO

Emerging evidence suggests that tumor necrosis factor (TNF)-alpha plays a role in muscle repair. To determine whether TNF-alpha modulates satellite cell proliferation, the current study evaluated TNF-alpha effects on DNA synthesis in primary myoblasts and on satellite cell activation in adult mouse muscle. Exposure to recombinant TNF-alpha increased total DNA content in rat primary myoblasts dose-dependently over a 24-h period and increased the number of primary myoblasts incorporating 5-bromo-2'-deoxyuridine (BrdU) during a 30-min pulse labeling. Systemic injection of TNF-alpha stimulated BrdU incorporation by satellite cells in muscles of adult mice, whereas no BrdU was incorporated by satellite cells in control mice. TNF-alpha stimulated serum response factor (SRF) binding to the serum response element (SRE) present in the c-fos gene promoter and stimulated reporter gene expression controlled by the same element. Our data suggest that TNF-alpha activates satellite cells to enter the cell cycle and accelerates G1-to-S phase transition, and these actions may involve activation of early response genes via SRF.


Assuntos
Divisão Celular/fisiologia , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Fator de Resposta Sérica/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Animais Recém-Nascidos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , DNA/biossíntese , DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Genes Reporter/efeitos dos fármacos , Genes Reporter/genética , Masculino , Camundongos , Camundongos Endogâmicos ICR , Músculo Esquelético/efeitos dos fármacos , Mioblastos/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-fos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/genética , Ratos , Regeneração/efeitos dos fármacos , Regeneração/fisiologia , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Elemento de Resposta Sérica/efeitos dos fármacos , Elemento de Resposta Sérica/genética , Fator de Resposta Sérica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia
15.
Blood ; 100(13): 4454-61, 2002 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-12393577

RESUMO

Thrombin signaling in endothelial cells provides an important link between coagulation and inflammation. We report here that thrombin induces endogenous Egr-1 mRNA and Egr-1 promoter activity in primary human endothelial cells by approximately 6-fold and 3-fold, respectively. In transient transfection assays, deletion of the 3' cluster of serum response elements (SREs), but not the 5' cluster of SREs, resulted in a loss of thrombin response. When coupled to a heterologous core promoter, a region spanning the 3' SRE cluster contained information for thrombin response, whereas a region spanning the 5' SRE cluster had no such effect. A point mutation of the most proximal SRE (SRE-1), but not of the proximal Ets motif or upstream SREs, abrogated the response to thrombin. In electrophoretic mobility shift assays, nuclear extracts from thrombin-treated cells displayed increased binding of total and phosphorylated serum response factor (SRF) to SRE-1. Thrombin-mediated induction of Egr-1 was blocked by inhibitors of MEK1/2, but not by inhibitors of protein kinase C, phosphatidylinositol 3-kinase, or p38 mitogen-activated protein kinase (MAPK). Taken together, these data suggest that thrombin induces Egr-1 expression in endothelial cells by a MAPK-dependent mechanism that involves an interaction between SRF and SRE-1.


Assuntos
Proteínas de Ligação a DNA/genética , Endotélio Vascular/efeitos dos fármacos , Proteínas Imediatamente Precoces , Elemento de Resposta Sérica/efeitos dos fármacos , Fator de Resposta Sérica/farmacologia , Trombina/farmacologia , Fatores de Transcrição/genética , Motivos de Aminoácidos , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Meios de Cultura Livres de Soro/farmacologia , Proteínas de Ligação a DNA/biossíntese , Proteína 1 de Resposta de Crescimento Precoce , Ensaio de Desvio de Mobilidade Eletroforética , Endotélio Vascular/citologia , Inibidores Enzimáticos/farmacologia , Genes Reporter , Genes Sintéticos , Humanos , Luciferases/biossíntese , Luciferases/genética , MAP Quinase Quinase 1 , MAP Quinase Quinase 2 , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Mutação Puntual , Ligação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Transporte Proteico/efeitos dos fármacos , Proteínas Tirosina Quinases/antagonistas & inibidores , RNA Mensageiro/biossíntese , Deleção de Sequência , Fatores de Transcrição/biossíntese , Ativação Transcricional/efeitos dos fármacos , Transfecção
16.
Biochem J ; 368(Pt 2): 665-72, 2002 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-12169092

RESUMO

Interaction of the neuropeptide substance P (SP) and its neurokinin-1 receptor (NK-1R) plays an important role in the pathophysiology of intestinal inflammation. SP is known to stimulate production of interleukin (IL)-6 and IL-8 in the U-373-MG human astrocytoma cell line via activation of p38 MAPK (mitogen-activated protein kinase) and nuclear factor (NF)-kappaB, respectively. However, the signalling mechanisms by which SP-NK-1R interaction induces NF-kappaB activation and IL-8 expression are still not clear. In this study we demonstrate that SP stimulates IL-8 secretion and IL-8 promoter activity in the NCM460 non-transformed human colonic epithelial cell line transfected with NK-1R cDNA. Our results indicate that inhibition of endogenous Rho family proteins (RhoA, Rac1 and Cdc42) by their respective dominant negative mutants significantly decreases SP-induced IL-8 secretion and IL-8 promoter activity. We also demonstrate that SP rapidly activates RhoA, Rac1 and Cdc42 and that co-expression of the dominant negative mutants of RhoA, Rac1 and Cdc42 in NK-1R cDNA-transfected NCM460 cells significantly inhibits SP-induced NF-kappaB-dependent gene expression. These results demonstrate that Rho family small GTPases RhoA, Rac1 and Cdc42 are novel signal transducers for SP-stimulated IL-8 expression.


Assuntos
Colo/metabolismo , Interleucina-8/metabolismo , Substância P/farmacologia , Proteína rhoA de Ligação ao GTP/metabolismo , Células Cultivadas , Colo/citologia , Colo/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Genes Dominantes , Humanos , Interleucina-8/genética , NF-kappa B/efeitos dos fármacos , NF-kappa B/genética , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Receptores da Neurocinina-1/efeitos dos fármacos , Receptores da Neurocinina-1/genética , Receptores da Neurocinina-1/metabolismo , Elemento de Resposta Sérica/efeitos dos fármacos , Transdução de Sinais , Substância P/metabolismo , Proteína cdc42 de Ligação ao GTP/efeitos dos fármacos , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/efeitos dos fármacos , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/genética
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