Helenius M, Hänninen M, Lehtinen S K, Salminen A
Department of Neuroscience and Neurology, University of Kuopio, Finland.
Biochem J. 1996 Sep 1;318 ( Pt 2)(Pt 2):603-8. doi: 10.1042/bj3180603.
Both the aging of animals and the senescence of cultured cells involve an altered pattern of gene expression, suggesting changes in transcription factor regulation. We studied age-related changes in transcription factors nuclear factor (NF)-kappa B, activator protein factor-1 (AP-1) and Sp-1 by using electrophoretic mobility shift binding assays; we also analysed changes in the protein components of NF-kappa B complex with Western blot assays. Nuclear and cytoplasmic extracts were prepared from heart, liver, kidney and brain of young adult and old NMRI mice and Wistar rats as well as from presenescent, senescent and simian virus 40-immortalized human WI-38 fibroblasts. Aging of both mice and rats induced a strong and consistent increase in the nuclear binding activity of NF-kappa B factor in all tissues studied, whereas those of AP-1 and Sp-1 decreased, e.g. in liver. Protein levels of p50, p52 and p65 components of the NF-kappa B complex did not show any age-associated changes in the cytoplasmic fraction but in the nuclear fraction the level of p52 strongly increased in heart and liver during aging. The protein levels of inhibitory I kappa B-alpha and Bcl-3 components were not affected by aging in any of the tissues studied. Replicative cellular senescence of human WI-38 fibroblasts induced a strong decrease in nuclear NF-kappa B, AP-1 and Sp-1 binding activities. Protein levels of p50 and p52 components of NF-kappa B complex were decreased in the nuclear fraction of senescent WI-38 fibroblasts but in the cytoplasm of senescent fibroblasts the level of p65 protein was increased. Cellular senescence also slightly decreased the protein levels of I kappa B-alpha and Bcl-3. Transfection assays with NF-kappa B-enhancer-driven chloramphenicol acetyltransferase reporter gene showed a significant down-regulation of NF-kappa B promoter activity in senescent WI-38 fibroblasts. Results suggest that the aging process might be regulated differently in tissues and cultured fibroblasts, perhaps reflecting differences between mitotic and post-mitotic cells. In tissues, aging seems to involve specific changes in the regulation of NF-kappa B components and perhaps also changes in the DNA-binding affinities of the NF-kappa B complex.
动物衰老和培养细胞衰老均涉及基因表达模式的改变，提示转录因子调控发生变化。我们通过电泳迁移率变动结合分析研究了转录因子核因子（NF）-κB、活化蛋白因子-1（AP-1）和Sp-1与年龄相关的变化；我们还通过蛋白质印迹分析检测了NF-κB复合物蛋白质成分的变化。从年轻成年和老年NMRI小鼠及Wistar大鼠的心脏、肝脏、肾脏和大脑以及衰老前、衰老和猴病毒40永生化的人WI-38成纤维细胞中制备核提取物和细胞质提取物。小鼠和大鼠的衰老均导致所研究的所有组织中NF-κB因子的核结合活性显著且持续增加，而AP-1和Sp-1的核结合活性则降低，例如在肝脏中。NF-κB复合物的p50、p52和p65成分的蛋白质水平在细胞质部分未显示出任何与年龄相关的变化，但在核部分，衰老过程中心脏和肝脏中p52的水平显著增加。在所研究的任何组织中，抑制性IκB-α和Bcl-3成分的蛋白质水平均不受衰老影响。人WI-38成纤维细胞的复制性细胞衰老导致核NF-κB、AP-1和Sp-1结合活性显著降低。NF-κB复合物的p50和p52成分的蛋白质水平在衰老的WI-38成纤维细胞核部分降低，但在衰老成纤维细胞的细胞质中p65蛋白水平增加。细胞衰老也使IκB-α和Bcl-3的蛋白质水平略有降低。用NF-κB增强子驱动的氯霉素乙酰转移酶报告基因进行的转染分析显示，衰老的WI-38成纤维细胞中NF-κB启动子活性显著下调。结果表明，衰老过程在组织和培养的成纤维细胞中的调控方式可能不同，这可能反映了有丝分裂细胞和有丝分裂后细胞之间的差异。在组织中，衰老似乎涉及NF-κB成分调控的特定变化，也许还涉及NF-κB复合物与DNA结合亲和力的变化。
