Transcriptional regulation of serum amyloid A1 gene expression in human aortic smooth muscle cells involves CCAAT/enhancer binding proteins (C/EBP) and is distinct from HepG2 cells.
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.
Kumon
Y
Y
Second Department of Internal Medicine, Kochi Medical School, Kohasu Okoh-Cho, Nankoku, Kochi, Japan. kumony@kochi-ms.ac.jp
Suehiro
T
T
Faulkes
D J
DJ
Hosakawa
T
T
Ikeda
Y
Y
Woo
P
P
Sipe
J D
JD
Hashimoto
K
K
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism*; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D002460:Cell Line; D016207:Cytokines / Q000494:pharmacology; D004247:DNA / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D000685:Serum Amyloid A Protein / Q000235:genetics*; D013256:Steroids / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000378:metabolism
D022763:CCAAT-Enhancer-Binding Protein-alpha; D022782:CCAAT-Enhancer-Binding Protein-beta; D016207:Cytokines; D016328:NF-kappa B; C094771:SAA4 protein, human; D000685:Serum Amyloid A Protein; D013256:Steroids; D018808:Transcription Factor AP-1; D003907:Dexamethasone; D004247:DNA