de Moissac D, Mustapha S, Greenberg A H, Kirshenbaum L A
The Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba, R2H 2A6.
J Biol Chem. 1998 Sep 11;273(37):23946-51. doi: 10.1074/jbc.273.37.23946.
Nuclear factor kappaB (NFkappaB) is a ubiquitously expressed transcription factor that is regulated by the cytoplasmic inhibitor protein IkappaBalpha. Biological agents such as tumor necrosis factor alpha (TNFalpha), which activate NFkappaB, result in the rapid degradation of IkappaBalpha. Adenoviral-mediated gene transfer of Bcl-2 prevents apoptosis of neonatal ventricular myocytes induced by TNFalpha. In view of the growing evidence that NFkappaB may play an important role in regulating apoptosis, we determined whether TNFalpha and Bcl-2 could modulate the activity of NFkappaB in ventricular myocytes. Stimulation of myocytes with TNFalpha resulted in a 2.1-fold increase (p < 0.001) in NFkappaB-dependent gene transcription and nuclear DNA binding. Similarly, a 1.9-fold increase (p < 0.0002) in NFkappaB-dependent gene transcription was observed in myocytes expressing Bcl-2. Nuclear DNA binding activity of NFkappaB was significantly increased in myocytes expressing Bcl-2, with a concomitant reduction in IkappaBalpha protein level. The Bcl-2-mediated loss of IkappaBalpha could be prevented by the proteasome inhibitor lactacystin, consistent with the notion that the targeted degradation of IkappaBalpha consequent to overexpression of Bcl-2 utilizes the ubiquitin-proteasome pathway. This was further tested in human 293 cells in which the N-terminal region of IkappaBalpha was identified to be an important regulatory site for Bcl-2. Deletion of this region or a serine to alanine substitution mutant at amino acids 32 and 36, which are defective for both phosphorylation and degradation, were more resistant than wild type IkappaBalpha to the inhibitory effects of Bcl-2. To our knowledge, this provides the first evidence for the regulation of IkappaBalpha by Bcl-2 and suggests a link between Bcl-2 and the NFkappaB signaling pathway in the suppression of apoptosis.
核因子κB（NFκB）是一种普遍表达的转录因子，受细胞质抑制蛋白IκBα调控。诸如肿瘤坏死因子α（TNFα）等激活NFκB的生物制剂会导致IκBα迅速降解。腺病毒介导的Bcl-2基因转移可防止TNFα诱导的新生心室肌细胞凋亡。鉴于越来越多的证据表明NFκB可能在调节细胞凋亡中起重要作用，我们确定了TNFα和Bcl-2是否能调节心室肌细胞中NFκB的活性。用TNFα刺激心肌细胞导致NFκB依赖性基因转录和核DNA结合增加2.1倍（p < 0.001）。同样，在表达Bcl-2的心肌细胞中观察到NFκB依赖性基因转录增加1.9倍（p < 0.0002）。表达Bcl-2的心肌细胞中NFκB的核DNA结合活性显著增加，同时IκBα蛋白水平降低。蛋白酶体抑制剂乳胞素可阻止Bcl-2介导的IκBα缺失，这与Bcl-2过表达导致IκBα靶向降解利用泛素-蛋白酶体途径的观点一致。这在人293细胞中进一步得到验证，其中IκBα的N端区域被确定为Bcl-2的重要调控位点。该区域的缺失或第32和36位氨基酸处丝氨酸到丙氨酸的替代突变体，由于磷酸化和降解均有缺陷，比野生型IκBα对Bcl-2的抑制作用更具抗性。据我们所知，这为Bcl-2对IκBα的调节提供了首个证据，并表明Bcl-2与NFκB信号通路在抑制细胞凋亡方面存在联系。
