Helenius M, Hänninen M, Lehtinen S K, Salminen A
University of Jyväskylă, Department of Cell Biology, Finland.
J Mol Cell Cardiol. 1996 Mar;28(3):487-98. doi: 10.1006/jmcc.1996.0045.
The accumulation of lipofuscin to cardiomyocytes is a classical parameter of aging and is believed to reflect oxidative stress. NF-kB transcription factor complex is one of the cellular sensors which responds to oxidative stress and regulates gene expression. Our purpose was to study whether aging affects the level and distribution of DNA binding activities of NF-kB transcription factors both in cardiac sarcoplasm and nuclear extracts. We used electrophoretic mobility shift assays (EMSA) to characterize the DNA binding activities of NF-kB and two other transcription factors. AP-1 and Sp-1, in the myocardium of 4 months and 24 months old male and female NMRI-mice. The protein levels of p50, p52, and p65 components of NF-kB-complex and an inhibitory IkB-alpha/MAD-3 were assayed with Western blots. Surprisingly, aging upregulated by 123% the nuclear NF-kB binding activity in the male and female mice. The sarcoplasmic NF-kB activity, activated by deoxycholate, did not show any change during aging. Aging-induced increase in nuclear NF-kB protein-DNA binding activity was observed both by gel retardation and UV-crosslinking assays. In immunoblotting, the level of p52 component but not those of p50 and p65 components of NF-kB-complex was slightly increased in nuclear fractions. Aging did not affect the sarcoplasmic levels of p50, p52, and p65 proteins. Supershift EMSA assays showed that the nuclear NF-kB complex contained p50, p52, and p65 components. The level of inhibitory IkB-alpha/MAD-3 protein was unaffected by aging both in nuclear and sarcoplasmic fractions. Aging down-regulated the nuclear Sp-1 binding activities but did not affect AP-1 binding activities. Statistically significant sex-related differences did not appear in the aging responses of transcription factors. These results indicate that NF-kB transcription factor pathway is activated during aging in cardiac muscle and could be the signaling route regulating gene expression. However, the activation mechanism of NF-kB during aging whether oxidative stress responsive or not in vivo needs further studies.
脂褐素在心肌细胞中的积累是衰老的一个经典指标，并且被认为反映了氧化应激。NF-κB转录因子复合物是对氧化应激作出反应并调节基因表达的细胞传感器之一。我们的目的是研究衰老是否会影响心肌细胞质和核提取物中NF-κB转录因子的DNA结合活性水平及分布。我们使用电泳迁移率变动分析（EMSA）来表征4个月和24个月大的雄性和雌性NMRI小鼠心肌中NF-κB以及另外两个转录因子AP-1和Sp-1的DNA结合活性。用蛋白质免疫印迹法检测NF-κB复合物的p50、p52和p65成分以及抑制性IkB-α/MAD-3的蛋白质水平。令人惊讶的是，衰老使雄性和雌性小鼠的核NF-κB结合活性上调了123%。由脱氧胆酸盐激活的细胞质NF-κB活性在衰老过程中未显示任何变化。通过凝胶阻滞和紫外线交联分析均观察到衰老诱导的核NF-κB蛋白-DNA结合活性增加。在免疫印迹中，核级分中NF-κB复合物的p52成分水平略有增加，而p50和p65成分的水平未增加。衰老不影响p50、p52和p65蛋白的细胞质水平。超迁移EMSA分析表明，核NF-κB复合物包含p50、p52和p65成分。抑制性IkB-α/MAD-3蛋白的水平在核级分和细胞质级分中均不受衰老影响。衰老下调了核Sp-1结合活性，但不影响AP-1结合活性。转录因子的衰老反应中未出现统计学上显著的性别相关差异。这些结果表明，NF-κB转录因子途径在心肌衰老过程中被激活，并且可能是调节基因表达的信号通路。然而，衰老过程中NF-κB的激活机制在体内是否对氧化应激有反应还需要进一步研究。
