A role for both Ets and C/EBP transcription factors and mRNA stabilization in the MAPK-dependent increase in p21Cip1/WAF1/mda6 protein levels in primary hepatocytes
A role for both Ets and C/EBP transcription factors and mRNA stabilization in the MAPK-dependent increase in p21Cip1/WAF1/mda6 protein levels in primary hepatocytes
复制标题
DOI：
10.1091/mbc.11.9.2915
复制
发表时间：
2000-09-01
影响因子：
3.3
通讯作者：
Dent, P
中科院分区：
文献类型：
--
作者：
Park, JS;Qiao, L;Dent, P
在原代肝细胞和HepG 2肝癌细胞中，p42/44丝裂原活化蛋白激酶（MAPK）通路的长期激活与DNA合成减少有关，这是由细胞周期蛋白依赖性激酶抑制剂蛋白p21（Cip-1/WAF 1/mda 6）（p21）表达增加介导的。在野生型或p21缺失肝细胞中MAPK途径的延长激活分别引起DNA合成的大幅减少和增加。在野生型或p21反义HepG 2细胞中MAPK途径的长期激活也分别导致DNA合成的大幅减少和增加。MAPK信号增加了转录因子Ets 2、C/EBP α和C/EBP β的磷酸化，并通过增强子区域内的多个Ets和C/EBP元件快速增加了从p21启动子的转录。MAPK激活后5小时，C/EBP β或Ets 2功能的丧失显著降低了MAPK刺激的p21启动子转录，并消除了p21蛋白表达的增加。此时，MAPK信号增加了p21 mRNA和p21蛋白的稳定性，这也被证明是p21蛋白水平显著增加所必需的。MAPK激活36小时后，在没有C/EBP β或Ets 2功能的细胞中，p21启动子的转录仍然显著减少;然而，这些细胞现在能够表现出p21蛋白表达的部分增加，相反，C/EBP α功能的丧失适度降低MAPK-刺激p21启动子的转录，但强烈抑制延长MAPK活化以增加p21蛋白水平的能力。可能在转录后控制下。与这一假设相一致，与8小时的时间点相比，延长MAPK信号传导在36小时进一步增加了p21 mRNA的稳定性。我们的数据表明，MAPK信号通过多种转录因子增加p21启动子活性，这些转录因子本身不足以使原代肝细胞中p21蛋白水平长期稳定增加，从而增加p21。蛋白水平也需要增强p21 mRNA和p21蛋白的稳定性。总的来说，这些数据表明，转录因子和mRNA/蛋白质稳定功能的丧失与MAPK信号传导不能引起原代肝细胞生长停滞与增殖相关。
In primary hepatocytes and HepG2 hepatoma cells, prolonged activation of the p42/44 mitogen-activated protein kinase (MAPK) pathway is associated with a reduction in DNA synthesis, mediated by increased expression of the cyclin-dependent kinase inhibitor protein p21 (Cip-1/WAF1/mda6) (p21) This study was performed to evaluate the contribution of transcriptional and post-transcriptional regulation in this response. prolonged activation of the MAPK pathway in wild-type or p21 null hepatocytes caused a large decrease and increase, respectively, in DNA synthesis. Prolonged activation of the MAPK pathway in either wild-type or p21 antisense HepG2 cells also caused large decreases and increases, respectively, in DNA synthesis. MAPK signaling increased the phosphorylation of the transcription factors Ets2, C/EBP alpha and C/EBP beta, and rapidly increased transcription from the p21 promoter via multiple Ets- and C/EBP-elements within the enhancer region. Fight hours after MAPK activation, loss of C/EBP beta or Ets2 function significantly reduced MAPK-stimulated transcription from the p21 promoter and abolished increased p21 protein expression. At this time, MAPK signaling increased both p21 mRNA and p21 protein stabilities that were also demonstrated to be essential for a profound increase in p21 protein levels. Thirty-six hours after MAPK activation, transcription from the p21 promoter was still significantly reduced in cells without either C/EBP beta or Ets2 function; however, these cells were now capable of exhibiting a partial increase in p21 protein expression, in contrast, loss of C/EBP alpha function modestly reduced MAPK-stimulated transcription from the p21 promoter but strongly inhibited the ability of prolonged MAPK activation to increase protein levels of p21 This data suggested that prolonged enhancement of p21 protein levels may be under posttranscriptional control. In agreement with this hypothesis, prolonged MAPK signaling further increased p21 mRNA stability at 36 h, compared with the 8-h time point. Our data argue that MAPK signaling increased p21 promoter activity via multiple transcription factors, which alone were insufficient for a robust prolonged increase in p21 protein levels in primary hepatocytes, and that to increase p21. protein levels also required enhanced stabilization of p21 mRNA and p21 protein. Collectively, these data suggest that loss of transcription factor and mRNA/protein stabilization functions correlates with an inability of MAPK signaling to cause growth arrest versus proliferation in primary hepatocytes.