p21Cip1 gene expression is modulated by Egr1 -: A novel regulatory mechanism involved in the resveratrol antiproliferative effect
p21Cip1 gene expression is modulated by Egr1 -: A novel regulatory mechanism involved in the resveratrol antiproliferative effect
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DOI：
10.1074/jbc.m300771200
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发表时间：
2003-06-27
影响因子：
4.8
通讯作者：
Zappia, V
中科院分区：
文献类型：
--
作者：
Della Ragione, F;Cucciolla, V;Zappia, V
流行病学观察表明，白藜芦醇是一种天然抗氧化剂二苯乙烯类化合物，具有心脏保护和化学预防作用。此外，该分子还在体外诱导细胞生长抑制和分化。以人红白血病K562细胞为模型系统，我们证实了白藜芦醇诱导了显著的γ-珠蛋白合成，其红系分化与细胞增殖受阻、p21(Cip1)表达增加和CDK2活性抑制有关。放线菌酮抑制了p21(Cip1)转录的上调，表明需要中间蛋白(S)，而中间蛋白反过来又调节基因的表达。对红系相关转录因子GATA-1、GATA-2和Egr1的定量分析表明，白藜芦醇通过ERK1/2依赖的机制选择性上调Egr1。在p21(Cip1)启动子中存在Egr1共有序列，提示该转录因子直接调节CDK抑制物的表达。缺失基因启动子构建体的转染研究以及EMSA、下拉实验和染色质免疫沉淀实验证实了这一观点，表明Egr1在体外和体内都与已确定的p21(Cip1)启动子的共同序列结合。此外，Egr1反义硫代磷酸可抑制p21(Cip1)的积聚和白藜芦醇的抗增殖作用。综上所述，这是首次证明Egr1通过直接与其基因启动子上的特定序列相互作用来控制p21(Cip1)的表达。已知的调控机制也有助于阐明白藜芦醇复杂的化学预防和抗增殖特性。
Epidemiological observations indicate that resveratrol, a natural antioxidant stilbene, exerts cardioprotective and chemopreventive effects. Moreover, the molecule induces in vitro cell growth inhibition and differentiation. Using human erythroleukemic K562 cells as model system, we demonstrated that resveratrol induces a remarkable gamma-globin synthesis, the erythroid differentiation being linked to impairment of cell proliferation, increased p21(Cip1) expression and inhibition of cdk2 activity. The up-regulation of p21(Cip1) transcription is prevented by cycloheximide, indicating the requirement of intermediate protein(s), which, in turn, regulate gene expression. The quantitative analysis of some transcription factors involved in the erythroid lineage, namely GATA-1, GATA-2, and Egr1, indicated that resveratrol selectively up-regulates Egr1 by an Erk1/2-dependent mechanism. The presence of an Egr1 consensus sequence in the p21(Cip1) promoter suggested the hypothesis that this transcription factor directly regulates the expression of the cdk inhibitor. Transfection studies with deleted gene promoter constructs, as well as EMSA, pull-down, and chromatin immunoprecipitation experiments substantiated this view, demonstrating that Egr1 binds in vitro and in vivo to the identified consensus sequence of the p21(Cip1) promoter. Moreover, an Egr1 phosphorothioate antisense hinders p21(Cip1) accumulation and the antiproliferative effects of resveratrol. In conclusion, this is the first demonstration that Egr1 controls p21(Cip1) expression by directly interacting with a specific sequence on its gene promoter. The identified regulatory mechanism also contributes to the clarification of the complex chemopreventive and antiproliferative properties of resveratrol.