Kapoor Gurpreet S, Golden Carmen, Atkins Brett, Mehta Kamal D
Department of Molecular and Cellular Biochemistry, The Ohio State University College of Medicine and Public Health, 464 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210, USA.
J Lipid Res. 2003 Mar;44(3):584-93. doi: 10.1194/jlr.M200302-JLR200. Epub 2003 Jan 1.
We have previously shown that different extracellular stimuli require signaling through the Raf/MEK/p42/44MAPK cascade to induce LDL receptor expression. The present studies were designed to delineate the molecular mechanisms underlying p42/44MAPK-induced LDL receptor transcription in HepG2-Delta Raf-1:ER cells, a modified HepG2 cell line in which the Raf-1/MEK/p42/44MAPK cascade can be specifically activated by anti-estradiol ICI182,780 in an agonist-specific manner. Using these cells, we show that: a) LDL receptor induction was reduced in reporter constructs containing mutation in either Sp1 or sterol-regulatory element-1 (SRE-1) sites, whereas inactivation of both sites abolished the induction; b) E1A, which inhibits CREB binding protein (CBP), a common activator of SRE-1 binding protein and Sp1, strongly repressed the induction; c) intracellular inhibition of the 90 kDa ribosomal S6 kinase (pp90RSK) cascade reduced LDL receptor induction; d) highly selective protein kinase C (PKC) inhibitors effectively abrogated the induction without affecting activation of pp90RSK; and e) overexpression of PKC beta significantly induced LDL receptor promoter activity. Taken together, these results demonstrate that pp90RSK and PKC beta are downstream effectors and Sp1, SRE-1 binding protein, and CBP are part of the transcriptional complex resulting in induction of LDL receptor expression in response to activation of the Raf/MEK/p42/44MAPK cascade. These findings identify for the first time a role for PKC beta in determining the specificity of p42/44MAPK signaling by participating with pp90RSK in regulating gene expression.
我们之前已经表明，不同的细胞外刺激需要通过Raf/MEK/p42/44MAPK级联进行信号传导，以诱导低密度脂蛋白（LDL）受体表达。本研究旨在阐明在HepG2-Delta Raf-1:ER细胞中p42/44MAPK诱导LDL受体转录的分子机制，HepG2-Delta Raf-1:ER细胞是一种经过修饰的HepG2细胞系，其中Raf-1/MEK/p42/44MAPK级联可以通过抗雌激素ICI182,780以激动剂特异性方式被特异性激活。利用这些细胞，我们发现：a）在含有Sp1或固醇调节元件-1（SRE-1）位点突变的报告基因构建体中，LDL受体诱导减少，而两个位点的失活则消除了诱导；b）E1A抑制CREB结合蛋白（CBP），CBP是SRE-1结合蛋白和Sp1的共同激活剂，强烈抑制诱导；c）细胞内抑制90 kDa核糖体S6激酶（pp90RSK）级联减少LDL受体诱导；d）高度选择性的蛋白激酶C（PKC）抑制剂有效消除诱导，而不影响pp90RSK的激活；e）PKCβ的过表达显著诱导LDL受体启动子活性。综上所述，这些结果表明pp90RSK和PKCβ是下游效应器，Sp1、SRE-1结合蛋白和CBP是转录复合物的一部分，导致在Raf/MEK/p42/44MAPK级联激活时诱导LDL受体表达。这些发现首次确定了PKCβ在通过与pp90RSK共同参与调节基因表达来决定p42/44MAPK信号特异性方面的作用。
