Wassmann Sven, Wassmann Kerstin, Jung Alexander, Velten Markus, Knuefermann Pascal, Petoumenos Vasileios, Becher Ulrich, Werner Christian, Mueller Cornelius, Nickenig Georg
Medizinische Klinik und Poliklinik II, Universitätsklinikum Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany.
J Mol Cell Cardiol. 2007 Sep;43(3):301-7. doi: 10.1016/j.yjmcc.2007.06.001. Epub 2007 Jun 16.
Proliferation of vascular smooth muscle cells (VSMC) plays an important role in the pathogenesis of atherosclerosis and restenosis. Recent studies have demonstrated that the transcription factor gut-enriched Krüppel-like factor (GKLF, KLF4) is involved in redox-sensitive growth arrest of VSMC. We investigated the role of GKLF in VSMC proliferation and differentiation and the potentially important interaction with the tumor suppressor gene p53. Cultured rat aortic VSMC were transfected with GKLF sense and antisense constructs by electroporation. GKLF enhanced the mRNA expression of the differentiation marker SM22-alpha, but had no effect on the expression of alpha-smooth muscle actin (real-time RT-PCR, Western blot). Overexpression of GKLF significantly reduced VSMC proliferation (cell count, BrdU FACS analysis). Because p53 is essential for proliferation processes, the effect of GKLF on p53 gene expression was investigated. GKLF overexpression led to an enhanced p53 promoter activity and increased p53 mRNA and protein expression (luciferase reporter assay, real-time PCR, Western blot). Consistently, GKLF overexpression induced an enhanced expression of the p53 target genes p21(WAF1/Cip1) and Mdm2. Co-transfection experiments revealed that the growth arrest induced by GKLF sense transfection was completely abolished by co-transfection of p53 antisense constructs, whereas the reduced proliferation exerted by p53 sense transfection was not inhibited by GKLF antisense transfection, suggesting that p53 induction is essential for the interference of GKLF with VSMC proliferation. Finally, stimulation of VSMC with hydroxyl radicals increased expression of GKLF and p53 and reduced cell proliferation. The transcription factor GKLF induces inhibition of proliferation of VSMC which is mechanistically linked to a GKLF-induced enhancement of the expression of the tumor suppressor gene p53.
血管平滑肌细胞（VSMC）的增殖在动脉粥样硬化和再狭窄的发病机制中起重要作用。最近的研究表明，转录因子肠道富集型Krüppel样因子（GKLF，KLF4）参与VSMC的氧化还原敏感生长停滞。我们研究了GKLF在VSMC增殖和分化中的作用以及与肿瘤抑制基因p53潜在的重要相互作用。通过电穿孔将GKLF正义和反义构建体转染培养的大鼠主动脉VSMC。GKLF增强了分化标志物SM22-α的mRNA表达，但对α-平滑肌肌动蛋白的表达没有影响（实时RT-PCR，蛋白质印迹法）。GKLF的过表达显著降低了VSMC增殖（细胞计数，BrdU FACS分析）。由于p53对增殖过程至关重要，因此研究了GKLF对p53基因表达的影响。GKLF过表达导致p53启动子活性增强以及p53 mRNA和蛋白质表达增加（荧光素酶报告基因检测，实时PCR，蛋白质印迹法）。一致地，GKLF过表达诱导了p53靶基因p21（WAF1/Cip1）和Mdm2的表达增强。共转染实验表明，p53反义构建体的共转染完全消除了GKLF正义转染诱导的生长停滞，而p53正义转染所产生的增殖减少并未被GKLF反义转染所抑制，这表明p53的诱导对于GKLF干扰VSMC增殖至关重要。最后，用羟基自由基刺激VSMC增加了GKLF和p53的表达并降低了细胞增殖。转录因子GKLF诱导VSMC增殖的抑制，其机制与GKLF诱导的肿瘤抑制基因p53表达增强有关。
