Arlt Alexander, Kruse Marie-Luise, Breitenbroich Maike, Gehrz Andre, Koc Bülent, Minkenberg Jörg, Fölsch Ulrich R, Schäfer Heiner
Laboratory of Molecular Gastroenterology & Hepatology, 1st Department of Medicine, Christian-Albrechts University of Kiel, Schittenhelmstr. 12, D-24105 Kiel, Germany.
Oncogene. 2003 May 22;22(21):3343-51. doi: 10.1038/sj.onc.1206524.
The early response gene IEX-1 is involved in the regulation of cellular growth and survival, and its expression is related to stress-, growth- and death-inducing signals. Addressing the role of IEX-1 in the promotion of apoptosis, we investigated the effect of IEX-1 on nuclear factor-kappaB (NF-kappaB) activation. Stably transfected HEK-293 cells conditionally overexpressing IEX-1 exhibit decreased levels of NF-kappaB activity, either basal or TNFalpha induced, as shown by gel-shift and luciferase reporter gene assay. Furthermore, activated p65 accumulated in the nuclei of 293 cells to a lower degree, if IEX-1 expression was increased. This inhibited NF-kappaB activation was preceded by an altered turnover of IkappaBalpha and phospho-IkappaBalpha. In addition, IEX-1 expression also inhibited the activity of the 26S-proteasome, as shown by a fluorometric proteasome assay. Conversely, disruption of IEX-1 expression in 293 cells by stable transfection with specific anti-IEX-1 hammerhead ribozymes increased NF-kappaB activity, and accelerated the degradation of IkappaBalpha. Along with these opposite effects of IEX-1 expression and IEX-1 disruption on NF-kappaB activation, the sensitivity of 293 cells towards various apoptotic stimuli also changed. In contrast to ribozyme-transduced 293 cells that were significantly less sensitive to apoptosis, this sensitivity was enhanced if IEX-1 expression was increased. Our data suggest that IEX-1 - itself an NF-kappaB target gene - inhibits the activation of this transcription factor, and hereby may counteract the antiapoptotic potential of NF-kappaB.
早期反应基因IEX - 1参与细胞生长和存活的调控，其表达与应激、生长及死亡诱导信号相关。为了研究IEX - 1在促进细胞凋亡中的作用，我们调查了IEX - 1对核因子-κB（NF - κB）激活的影响。如凝胶迁移和荧光素酶报告基因检测所示，稳定转染条件性过表达IEX - 1的HEK - 293细胞，其基础或肿瘤坏死因子α诱导的NF - κB活性水平均降低。此外，如果IEX - 1表达增加，激活的p65在293细胞核中的积累程度会降低。这种对NF - κB激活的抑制作用之前伴随着IκBα和磷酸化IκBα周转的改变。另外，如荧光蛋白酶体检测所示，IEX - 1表达也抑制了26S蛋白酶体的活性。相反，用特异性抗IEX - 1锤头状核酶稳定转染293细胞以破坏IEX - 1的表达，会增加NF - κB活性，并加速IκBα的降解。随着IEX - 1表达和IEX - 1破坏对NF - κB激活产生的这些相反作用，293细胞对各种凋亡刺激的敏感性也发生了变化。与对凋亡显著不敏感的核酶转导293细胞相反，如果IEX - 1表达增加，这种敏感性会增强。我们的数据表明，IEX - 1本身作为NF - κB的靶基因，抑制了该转录因子的激活，从而可能抵消NF - κB的抗凋亡潜能。
