RESUMO
Osteoblasts can synthesize the insulin-like growth factors (IGFs) and the IGF-binding proteins (IGFBPs), which may either enhance or attenuate IGF-stimulated bone cell proliferation. Since estrogen induced osteoblastic differentiation and proliferation through an estrogen-responsive gene in target cells, we investigated the effects of estrogen on IGFBP-6 expression in the human osteoblastic-like cell line SaOS-2. Expressions of IGFBP-6 protein and mRNA increased 2.8 and 2-fold, respectively, in the presence of 17-beta-estradiol (E2) (0.01 to 1 micrometer) and estrogen receptor (ER) in SaOS-2 cells. On the other hand, E2 induced a 2-fold increase in SaOS-2 cell proliferation. To identify genomic sequences associated with estrogen responsiveness, the 5'-promoter region (-44 to +118) of the IGFBP-6 gene was cloned into a chloramphenicol acetyltransferase (CAT) reporter vector. E2 induced a 3-fold increase in CAT activity in SaOS-2 cells transiently transfected with this construct. Identification of the estrogen-responsive element (ERE) [5'-CCTTCA CCTG-3'] (-9 to +1) in this IGFBP-6 gene promoter region was confirmed using electromobility shift assays and deletion analysis. This functional ERE was important for E2-induced trans-activation of the IGFBP-6 gene. These results demonstrate that E2 exhibits a positive effect on IGFBP-6 gene transcription through estrogen-liganded ER binding to the functional ERE in the IGFBP-6 gene promoter in SaOS-2 cells.
Assuntos
Humanos , Western Blotting , Proliferação de Células , Cloranfenicol O-Acetiltransferase/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Estradiol/farmacologia , Receptor alfa de Estrogênio/genética , Estrogênios/farmacologia , Proteína 6 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Osteoblastos/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Elementos de Resposta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional , Células Tumorais CultivadasRESUMO
5'-upstream region of the phospholipase C-beta2 gene, 810 bp, was cloned and characterized. S1 nuclease mapping and primer extension analyses revealed that a single transcriptional start site locates at 284 nucleotides upstream from the beginning of translation. The 5-upstream region lacks both TATA motif and typical initiator sequence, but retains GC-rich segment. Two putative regulatory regions, a negative region (-636/-588) and a positive region (-98/ -13) were identified in the upstream region of PLC-beta2 gene. We suggest that the transcription of PLC-beta2 may be regulated by binding of regulatory proteins to the negative and/or positive regulatory regions located in the upstream of the gene.
Assuntos
Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Sequência de Bases , Células Cultivadas , Cloranfenicol O-Acetiltransferase/metabolismo , Clonagem Molecular , Sequência Conservada , Deleção de Genes , Isoenzimas/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosfolipases Tipo C/química , Regiões Promotoras Genéticas , Ligação Proteica , Transcrição Gênica , TransfecçãoRESUMO
We report here for the first time the structure and function of a promoter from a cestode. The ability of DNA fragments respectively encompassing the 935-bp and 524-bp regions upstream from the ATG codon from the EgactI and EgactII actin genes of Echinococcus granulosus to promote transcription was studied in the NIH3T3 mouse cell line. The results of transfection assays showed that both regions have strong promoter activity in these cells. The fragments were tested in both orientations and the 524-bp fragment of EgactII presented a bidirectional promoter activity. Deletion analysis of EgactI and EgactII promoters indicated the presence of regulatory regions containing putative silencer elements. These results indicate that both EgactI and EgactII promoters are functional and that the preliminary functional evaluation of E. granulosus and possibly of other cestode promoters can be performed in heterologous cell lines
Assuntos
Animais , Camundongos , Células 3T3 , Actinas/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Echinococcus/genética , Regiões Promotoras Genéticas/fisiologia , Sequência de Bases , Técnicas de Cultura de Células , Clonagem Molecular , Expressão Gênica , Genes Reporter , Glicocálix , Regiões Promotoras Genéticas/genética , Transfecção/genéticaRESUMO
The equilibrium between deposition and degradation of extracellular matrix(ECM) is essential to normal tissue development and repair of wound or inflammatory responses. It has recently become apparent that several cytokines and growth factors are capable of modulating fibroblast proliferation and biosynthetic activity. To understand the role of these factors in connective tissue regulation, we examined the effect of interferon-gamma (IFN-gamma) on stromelysin-1 gene expression in cultured human dermal fibroblasts. The steady-state levels of stromelysin-1 mRNA were increased in IFN-gamma treated cultured dermal fibroblasts. In the CAT assay, the stromelysin-1 promoter activity was increased 2.8-fold compared with untreated control. Therefore IFN-gamma stimulates the stromelysin-1 promoter activity, resulting in transcriptional enhancement of gene expression. Transforming growth factor-beta (TGF-beta) showed the antagonistic action to the effects of IFN-gamma in cultured dermal fibroblasts. Furthermore, gel mobility shift assays demonstrated enhanced AP-1 binding activities in nuclear extracts from cells incubated with IFN-gamma. These data suggest that IFN-gamma is an up-regulator and TGF-beta is a down regulator on the stromelysin-1 gene expression, respectively, and the AP-1 binding site may be necessary for gene response.