Basic helix-loop-helix protein E47-mediated p21Waf1/Cip1 gene expression regulates apoptosis of intestinal epithelial cells
Basic helix-loop-helix protein E47-mediated p21Waf1/Cip1 gene expression regulates apoptosis of intestinal epithelial cells
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DOI：
10.1042/bj20070293
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发表时间：
2007-10-15
影响因子：
4.1
通讯作者：
Johnson, Leonard R.
中科院分区：
文献类型：
--
作者：
Bhattacharya, Sujoy;Guo, Huazhang;Johnson, Leonard R.
用DFMO （α -二氟甲基鸟氨酸）抑制鸟氨酸脱羧酶和随后的多胺消耗增加p21Cip1蛋白，诱导细胞周期阻滞，并对肠上皮细胞凋亡具有抗性。然而，多胺调节p21Cip1表达和细胞凋亡的机制尚不清楚。在p21Cip1作为抗凋亡蛋白参与的基础上，我们测试了p21Cip1在提供细胞凋亡保护中的作用。同时，我们研究了基本螺旋-环-螺旋蛋白E47在p21Cip1基因转录调控中的作用。基因特异性siRNA（小干扰RNA）降低E47蛋白水平，增加p21Cip1启动子活性和蛋白水平，保护细胞免受TNF α（肿瘤坏死因子α）诱导的凋亡。siRNA敲低p21Cip1蛋白导致细胞更容易凋亡。相反，表皮生长因子（EGF）刺激p21Cip1 mRNA和蛋白水平，使细胞免于凋亡。在细胞凋亡过程中，E47 mRNA水平升高，导致p21Cip1 mRNA和蛋白水平降低。多胺缺失降低了E47 mRNA水平和细胞存活率。Caspase 3介导的p130Cas的裂解与p21Cip1的转录有关。凋亡的进展导致p130Cas的caspase 3依赖性切割，产生一个31 kDa的片段，该片段易位到细胞核，与核E47相关，并抑制p21Cip1的转录。多胺缺失抑制了所有这些作用。31 kDa片段的瞬时表达阻止了p21Cip1蛋白的表达，增加了细胞凋亡。这些结果暗示p21Cip1是一种抗凋亡蛋白，并提示多胺通过转录抑制因子E47调控p21Cip1。caspase介导的p130Cas的切割产生一个31 kDa片段，抑制p21Cip1的转录，并作为凋亡信号传导的放大器。
Inhibition of ornithine decarboxylase by DFMO (alpha-difluro-methylornithine) and subsequent polyamine depletion increases p21Cip1 protein, inducescell cycle arrest and confers resistance to apoptosis on intestinal epithelial cells. However, the mechanism by which polyamines regulate p21Cip1 expression and apoptosis is unknown. On the basis of the involvement of p21Cip1 as an anti-apoptotic protein, we tested the role of p21Cip1 in providing protection from apoptosis. Simultaneously, we investigated the role of E47, a basic helix-loop-helix protein, in the regulation of p21Cip1 gene transcription. Gene-specific siRNA (small interfering RNA) decreased E47 protein levels, increased p21Cip1 promoter activity and protein levels and protected cells from TNF alpha (tumour necrosis factor alpha)-induced apoptosis. Knockdown of p21Cip1 protein by siRNA resulted in cells becoming more susceptible to apoptosis. In contrast, incubation with EGF (epidermal growth factor) stimulated p21Cip1 mRNA and protein levels and rescued cells from apoptosis. During apoptosis, the level of E47 mRNA increased, causing a concomitant decrease in p21Cip1 mRNA and protein levels. Polyamine depletion decreased E47 mRNA levels and cell survival. Caspase 3-mediated cleavage of p130Cas has been implicated in p21Cip1 transcription. The progression of apoptosis led to a caspase 3-dependent cleavage of p130Cas and generated a 31 kDa fragment, which translocated to the nucleus, associated with nuclear E47 and inhibited p21Cip1 transcription. Polyamine depletion inhibited all these effects. Transient expression of the 31 kDa fragment prevented the expression of p21Cip1 protein and increased apoptosis. These results implicate p21Cip1 as an anti-apoptotic protein and suggest a role for polyamines in the regulation of p21Cip1 via the transcription repressor E47. Caspase-mediated cleavage of p130Cas generates a 31 kDa fragment, inhibits p21Cip1 transcription and acts as an amplifier of apoptotic signalling.