Two sterol regulatory element-like sequences mediate up-regulation of caveolin gene transcription in response to low density lipoprotein free cholesterol
Two sterol regulatory element-like sequences mediate up-regulation of caveolin gene transcription in response to low density lipoprotein free cholesterol
复制标题
DOI：
10.1073/pnas.94.20.10693
复制
发表时间：
1997-09-30
影响因子：
11.1
通讯作者：
Fielding, CJ
中科院分区：
文献类型：
--
作者：
Bist, A;Fielding, PE;Fielding, CJ
小凹是细胞内游离胆固醇(FC)转运的主要途径，随着低密度脂蛋白(LDL)摄取增加，人皮肤成纤维细胞中Caveolin基因表达水平上调，而FC诱导的上调与可检测到的mRNA稳定性变化无关，表明小窝基因的表达是在基因转录水平上调节的。启动子序列包括3个富含G+C的潜在类固醇调节元件(SRE)、1个CAAT序列和1个Sp1共有序列。单个SRE样序列的缺失突变表明，这两个序列(-646和-395bp)对于低密度脂蛋白-Fc介导的转录速率的提高是必不可少的，而第三个序列是无关紧要的。从核提取液中提取的蛋白质与这些小窝蛋白启动子DNA序列结合的凝胶位移分析，以及DNase I足迹分析，证实核蛋白与SRE样元件结合是对低密度脂蛋白-Fc转录反应的一部分，与SRE结合蛋白1抗体(SPEBP-1)的超位移表明，该蛋白在-395bp处结合，在-395bp处与抗SPL抗体没有反应，在-646bp处也没有与两种抗体发生反应。半胱氨酸蛋白酶抑制剂N-乙酰-亮氨酸-去甲亮氨酸(ALLN)可抑制SREBP的分解代谢，但对小窝蛋白基因表达水平的抑制作用与低密度脂蛋白无关。这一结果表明，SREBP抑制小窝蛋白基因转录，而对其他启动子的刺激作用相反。本研究结果与小窝蛋白作为静止期外周细胞FC动态平衡调节器的假设一致，并与SREBP对FC流入和流出的协调调节一致。
Caveolae form the terminus for a major pathway of intracellular free cholesterol (FC) transport, Caveolin mRNA levels in confluent human skin fibroblasts were up-regulated following increased uptake of low density lipoprotein (LDL) FC, The increase induced by FC was not associated with detectable change in mRNA stability, indicating that caveolin mRNA levels were mediated at the level of gene transcription, A total of 924 bp of 5' flanking region of the caveolin gene were cloned and sequenced. The promoter sequence included three G+C-rich potential sterol regulatory elements (SREs), a CAAT sequence and a Sp1 consensus sequence. Deletional mutagenesis of individual SRE-like sequences indicated that of these two (at -646 and -395 bp) were essential for the increased transcription rates mediated by LDL-FC, whereas the third was inconsequential. Gel shift analysis of protein binding from nuclear extracts to these caveolin promoter DNA sequences, together with DNase I footprinting, confirmed nucleoprotein binding to the SRE-like elements as part of the transcriptional response to LDL-FC, A supershift obtained with antibody to SRE-binding protein 1 (SPEBP-1) indicated that this protein binds at -395 bp, There was no reaction at -395 bp with anti-Spl antibody nor with either antibody at -646 bp. The cysteine protease inhibitor N-acetyl-leu-leu-norleucinal (ALLN), which inhibits SREBP catabolism, superinhibited caveolin mRNA levels regardless of LDL-FC, This finding suggests that SREBP inhibits caveolin gene transcription in contrast to its stimulating effect on other promoters, The findings of this study are consistent with the postulated role for caveolin as a regulator of cellular FC homeostasis in quiescent peripheral cells, and the coordinate regulation by SREBP of FC influx and efflux.