Dean D C, McQuillan J J, Weintraub S
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1990 Feb 25;265(6):3522-7.
Fibronectin (FN) mRNA levels increased when quiescent cells (serum starved) were stimulated to undergo the G0/G1 transition by the addition of 20% given fetal calf serum to the media. The 5'-flanking region of the FN gene (position +69 to -510 base pairs (bp] was fused to the coding region of the chloramphenicol acetyltransferase (CAT), and the fusion gene was used in transfection assays. Expression of FNCAT increased on serum treatment indicating that the region of the FN gene between positions +69 and -510 bp mediated serum responsiveness. Deletion of FN gene 5'-flanking sequences from position -510 to -122 bp eliminated serum responsiveness suggesting that an element between these positions was mediating the effect. Sequences between positions -122 and -510 bp of the FN gene were able to confer serum responsiveness on a herpes virus thymidine kinase promoter-CAT fusion gene (TKCAT) when the FN gene sequences were cloned upstream of TKCAT. The ability to confer serum responsiveness on TKCAT was retained with a smaller 100-bp sequence (position -122 to -222 bp). Both a cAMP response element (position -170 bp) and a nuclear factor-1 binding site (position -155 bp) have been identified within this sequence (Dean, D. C., Blakeley, M. S., Newby, R. F., Ghazal, P., Hennighausen, L., and Bourgeois, S. (1989) Mol. Cell. Biol. 9, 1498-1506). The cAMP response element was serum-responsive when cloned upstream of TKCAT or a minimal FN promoter (deleted to position -56 bp) while the nuclear factor-1 binding site was unresponsive. Therefore, the cAMP regulatory element (CRE) is the serum-responsive element between position -122 and -222 bp. Serum-induced binding of proteins to the CRE was detected in gel retardation assays with extracts from cell lines where FN expression was serum-responsive. However, no serum-induced binding was detected with extracts from the JEG-3 cell line where FN expression was not serum-responsive. Serum-induced binding occurred rapidly, within 15 min, and did not require protein synthesis. The decay of serum-induced binding was relatively slow as increased binding was still detectable 24 h after removal of serum. The CRE also mediates transcriptional stimulation by cAMP, but unlike serum stimulation increased CRE binding activity was not detectable in extracts from cAMP-treated cells (Dean, D. C., Blakeley, M. S., Newby, R. F., Ghazal, P., Hennighausen, L., and Bourgeois, S. (1989) Mol. Cell. Biol. 9, 1498-1506).(ABSTRACT TRUNCATED AT 400 WORDS)
当向培养基中添加20%的胎牛血清刺激静止细胞（血清饥饿）进入G0/G1期转变时，纤连蛋白（FN）的mRNA水平升高。将FN基因的5'侧翼区（第+69至-510碱基对（bp））与氯霉素乙酰转移酶（CAT）的编码区融合，并将融合基因用于转染实验。血清处理后FNCAT的表达增加，表明FN基因位于+69和-510 bp之间的区域介导血清反应性。从-510至-122 bp缺失FN基因的5'侧翼序列消除了血清反应性，提示这些位置之间的一个元件介导了该效应。当FN基因序列克隆到疱疹病毒胸苷激酶启动子-CAT融合基因（TKCAT）上游时，FN基因-122至-510 bp之间的序列能够赋予TKCAT血清反应性。赋予TKCAT血清反应性的能力在一个较小的100 bp序列（-122至-222 bp）中得以保留。在该序列中已鉴定出一个cAMP反应元件（-170 bp位置）和一个核因子-1结合位点（-155 bp位置）（迪恩，D.C.，布莱克利，M.S.，纽比，R.F.，加扎尔，P.，亨尼豪森，L.，和布尔乔亚，S.（1989年）《分子与细胞生物学》9，1498 - 1506）。当克隆到TKCAT或最小的FN启动子（缺失至-56 bp位置）上游时，cAMP反应元件对血清有反应，而核因子-1结合位点无反应。因此，cAMP调节元件（CRE）是-122至-222 bp之间的血清反应元件。在凝胶阻滞实验中，用FN表达对血清有反应的细胞系提取物检测到血清诱导的蛋白质与CRE结合。然而，用FN表达对血清无反应的JEG - 3细胞系提取物未检测到血清诱导的结合。血清诱导的结合迅速发生，在15分钟内，且不需要蛋白质合成。血清诱导结合的衰减相对较慢，因为在去除血清24小时后仍可检测到结合增加。CRE也介导cAMP的转录刺激，但与血清刺激不同，在cAMP处理的细胞提取物中未检测到CRE结合活性增加（迪恩，D.C.，布莱克利，M.S.，纽比，R.F.，加扎尔，P.，亨尼豪森，L.，和布尔乔亚，S.（1989年）《分子与细胞生物学》9，1498 - 1506）。（摘要截断于400字）
