Zhang Tong, Zhuang Shunhui, Casteel Darren E, Looney David J, Boss Gerry R, Pilz Renate B
Department of Medicine, University of California, San Diego, California, 92093.
Department of Medicine, University of California, San Diego, California, 92093; Veterans Administration Medical Center, La Jolla, California 92161.
J Biol Chem. 2007 Nov 16;282(46):33367-33380. doi: 10.1074/jbc.M707186200. Epub 2007 Sep 18.
Vascular smooth muscle cells (VSMCs) undergo phenotypic modulation, changing from a differentiated, contractile to a de-differentiated, synthetic phenotype; the change is associated with decreased expression of smooth muscle (SM)-specific genes and loss of cGMP-dependent protein kinase (PKG), but transfection of PKG into de-differentiated VSMCs restores SM-specific gene expression. We show that small interference RNA-mediated down-regulation or pharmacologic inhibition of PKG reduced SM-specific gene expression in differentiated VSMCs and provide a mechanism for cGMP/PKG regulation of SM-specific genes involving the cysteine-rich LIM-only protein CRP4. PKG associated with CRP4 and phosphorylated the protein in intact cells. CRP4 had no intrinsic transcriptional activity, but exhibited adaptor function, because it acted synergistically with serum response factor (SRF) and GATA6 to activate the SM-alpha-actin promoter. cGMP stimulation of the promoter required PKG and CRP4 co-expression with SRF and GATA6. A phosphorylation-deficient mutant CRP4 and a CRP4 deletion mutant deficient in PKG binding did not support cGMP/PKG stimulation of the SM-alpha-actin promoter. In the presence of wild-type but not mutant CRP4, cGMP/PKG enhanced SRF binding to a probe encoding the distal SM-alpha-actin promoter CArG (CC(AT)(6)GG) element. CRP4 and SRF associated with CArG elements of endogenous SM-specific genes in intact chromatin. Small interference RNA-mediated down-regulation of CRP4 prevented the positive effects of cGMP/PKG on SM-specific gene expression. In the presence of CRP4, cGMP/PKG increased SRF- and GATA6-dependent expression of endogenous SM-specific genes in pluripotent 10T1/2 cells. Thus, CRP4 mediates cGMP/PKG stimulation of SM-specific gene expression, and PKG plays an important role in regulating the phenotype of VSMCs.
血管平滑肌细胞（VSMCs）会发生表型调节，从分化的收缩型转变为去分化的合成型；这种变化与平滑肌（SM）特异性基因表达降低以及环鸟苷酸依赖性蛋白激酶（PKG）缺失有关，但将PKG转染到去分化的VSMCs中可恢复SM特异性基因表达。我们发现，小干扰RNA介导的PKG下调或药物抑制会降低分化的VSMCs中SM特异性基因的表达，并提供了一种cGMP/PKG对涉及富含半胱氨酸的仅含LIM结构域蛋白CRP4的SM特异性基因进行调控的机制。PKG与CRP4结合并在完整细胞中使该蛋白磷酸化。CRP4没有内在的转录活性，但具有衔接子功能，因为它与血清反应因子（SRF）和GATA6协同作用以激活SM-α-肌动蛋白启动子。启动子的cGMP刺激需要PKG和CRP4与SRF和GATA6共同表达。磷酸化缺陷型突变体CRP4和缺乏PKG结合的CRP4缺失突变体不支持cGMP/PKG对SM-α-肌动蛋白启动子的刺激。在存在野生型而非突变型CRP4的情况下，cGMP/PKG增强了SRF与编码远端SM-α-肌动蛋白启动子CArG（CC(AT)(6)GG）元件的探针的结合。CRP4和SRF与完整染色质中内源性SM特异性基因的CArG元件相关联。小干扰RNA介导的CRP4下调阻止了cGMP/PKG对SM特异性基因表达的积极作用。在存在CRP4的情况下，cGMP/PKG增加了多能10T1/2细胞中内源性SM特异性基因的SRF和GATA6依赖性表达。因此，CRP4介导cGMP/PKG对SM特异性基因表达的刺激，并且PKG在调节VSMCs的表型中起重要作用。
