RESUMO
Regulation of acute-phase serum amyloid A (A-SAA) synthesis by proinflammatory cytokines and steroid hormones in human aortic smooth muscle cells (HASMCs) is distinct from that in HepG2 cells. To study the cis- and trans-activating promoter element involved in the SAA1 gene expression by HASMCs and HepG2 cells, we constructed plasmid vectors for luciferase reporter gene assay with varying lengths of SAA1 upstream regulatory region (up to 1431 bp), and examined their response to proinflammatory cytokines and/or steroid hormones. The corresponding vectors with the SAA4 upstream regulatory region served as controls. The presence of proposed transcriptional regulatory factors binding to these regions was confirmed immunohistochemically. The sequences of 1478 and 1836 bp of the SAA1 and SAA4 5'-flanking regions were determined, respectively. SAA1 promoter transcription in cultured HASMCs was upregulated not by proinflammatory cytokines, but rather by glucocorticoids. This differed from HepG2 cells, in which SAA1 promoter transcription was upregulated synergistically by proinflammatory cytokines and glucocorticoids. The promoter activity of a series of truncated SAA1 promoter constructs measured using the reporter gene assay showed that the 5'-region from -252 to -175, containing a consensus site for CCAAT/enhancer binding proteins alpha,beta (C/EBPalpha,beta), was essential for SAA1 induction in HASMCs. In HepG2 cells, the 5'-region from -119 to -79, containing a nuclear factor kappa-B (NFkappaB) consensus sequence, was essential for the induction. The functional significance of the C/EBP site as indicated by the immunohistochemical result was that in HASMCs anti-C/EBPbeta reactivity was shifted from the cytoplasm to the nuclei. We have, therefore, demonstrated that the region containing the C/EBPalpha,beta consensus binding site between the bases -252 and -175 is important for the glucocorticoid-induced SAA1 gene expression in HASMCs but not in HepG2 cells.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Músculo Liso Vascular/metabolismo , Proteína Amiloide A Sérica/genética , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Citocinas/farmacologia , DNA/genética , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Dados de Sequência Molecular , Músculo Liso Vascular/efeitos dos fármacos , NF-kappa B/metabolismo , Esteroides/farmacologia , Fator de Transcrição AP-1/metabolismoRESUMO
3,4-Dihydro-6-[4-(3,4- dimethoxybenzoyl )-piperazinyl]-2(1H)- quin olinone ( OPC -8212) is a new positive inotropic agent. In studies designed to elucidate information concerning its mechanism of action, its effect on Ca+ transport and related enzyme activities in sarcolemma, sarcoplasmic reticulum and mitochondria were studied. OPC -8212 was found to have little or no effect on these processes. The mechanism of action therefore is clearly different from other known positive inotropic drugs.
