RESUMO
This report describes the isolation and characterization of the 5' flanking region of the murine transforming growth factor beta-2 (TGF-beta2) gene. A genomic clone containing the promoter region of the gene was isolated after screening a bacteriophage P1 genomic library. The resulting clone was sequenced and compared to promoters for the human and chicken TGF-beta2 genes. The sequence located near the transcription start site is highly conserved. It includes a TATA box, an E-box, and a largely conserved CRE/ATF site. A series of murine TGF-beta2 promoter/reporter constructs was generated to identify regulatory regions of the gene. As in the case of the human TGF-beta2 gene, sequences just upstream of the TATA box, including the CRE/ATF site, actively stimulate the murine TGF-beta2 promoter. However, unlike the human TGF-beta2 gene, the 5' flanking region of the murine TGF-beta2 gene contains a long alternating purine/pyrimidine repeat that unexpectedly exerts a strong positive effect on its promoter. This is of particular interest since alternating purine/pyrimidine repeats in other promoters have been observed to be inhibitory.
Assuntos
Regiões Promotoras Genéticas/genética , Fator de Crescimento Transformador beta/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , Galinhas , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , DNA/química , DNA/genética , DNA/isolamento & purificação , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Deleção de Sequência , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta2 , Células Tumorais CultivadasRESUMO
The objective of this experiment was to determine the dose response of dexamethasone (DEX) on C2C12 myogenic cells and to examine the effects of the anabolic compounds estradiol (E), testosterone (T), and dihydrotestosterone (D) alone and in combination with DEX on proliferation and protein turnover in cultured C2C12 myogenic cells. In the first study, cells were treated with seven concentrations (0, 25, 50, 75, 100, 150, or 200 nM) of DEX in medium with or without 5% horse serum (HS) for the determination of protein synthesis and degradation, and six concentrations (0, 50, 100, 150, 200, or 250 nM) of DEX in medium with 5% fetal bovine serum for cell proliferation measurements. Proliferation of myoblasts decreased (P < .05) with DEX. As DEX concentration increased, protein degradation in myotubes increased (P < .05) up to 100 nM, then declined. Protein synthesis decreased linearly (P < .01) as DEX concentration increased. The presence of HS in the medium decreased (P < .01) protein degradation by 32% as compared with no HS and increased (P < .05) protein synthesis. In the second study, cells were treated with E, T, or D at four concentrations (0, 100, 500, or 1,000 nM) in medium containing 0 or 100 nM DEX. Cells were assayed for protein synthesis or protein degradation. Synthesis decreased (P < .01) and degradation increased (P < .01) with DEX. No differences (P > .05) were found between E, T, or D hormone treatments or concentrations. To measure proliferation, myoblasts were treated 1 d after plating with the same anabolic hormone treatments in medium containing 0 to 100 nM DEX. Cells were grown to confluence and assayed for proliferation. Proliferation decreased (P < .01) in the presence of DEX in each treatment compared with controls. Cells treated with E had significantly lower (P < .05) proliferation rates than cells treated with T and D. The presence of concentrations of DEX at 100 nM inhibited proliferation and protein synthesis and increased protein degradation. Anabolic agents at pharmacological doses do not inhibit the DEX effects on C2C12 myogenic cells, nor do they directly affect proliferation or protein turnover.
