Id3 induces a caspase-3- and -9-dependent apoptosis and mediates UVB sensitization of HPV16 E6/7 immortalized human keratinocytes
Id3 induces a caspase-3- and -9-dependent apoptosis and mediates UVB sensitization of HPV16 E6/7 immortalized human keratinocytes
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DOI：
10.1038/sj.onc.1209407
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发表时间：
2006-06-22
期刊：
影响因子：
8
通讯作者：
Rosenthal, D. S.
中科院分区：
文献类型：
--
作者：
Simbulan-Rosenthal, C. M.;Daher, A.;Rosenthal, D. S.
分化抑制剂/DNA结合(Id)蛋白包含一类参与增殖、分化、细胞凋亡和癌发生的螺旋-环-螺旋转录因子。我们已经证明，虽然 Id2 在原代角质形成细胞中由 UVB 诱导，但 Id3 仅在永生化细胞中上调。我们现在已经确定，永生化角质形成细胞和原代角质形成细胞之间 Id3 蛋白异位表达的后果显着不同。膜联蛋白 V 阳性以及永生化细胞中 caspase-3 的蛋白水解加工表明，Id3 的过度表达会诱导凋亡细胞显着增加，但在原代角质形成细胞中则不然。与 Id3-GFP 阴性细胞相比，Id3-绿色荧光蛋白 (GFP) 阳性细胞的凋亡核碎片增加了五倍。这些细胞凋亡反应伴随着 caspase-3 的激活，如活性 caspase-3 抗体的免疫细胞化学染色所示。使用针对 caspase-9 活性形式以及 Bax 活性形式的抗体进行的免疫染色进一步揭示，Id3 过度表达的角质形成细胞中的细胞凋亡诱导是通过线粒体 caspase-9 介导的途径发生的。显性失活 caspase-9 与 Id3 的共表达显着抑制了细胞凋亡核断裂，表明 caspase-9 激活对于 Id3 诱导的细胞死亡至关重要。与 Bax 拮抗剂抗凋亡蛋白 Bcl-2 共表达也显着减弱了这种反应，证实了 Bax 激活在这种凋亡反应中的作用。 Id3 诱导的 Bax 激活可能是由于 Bax 蛋白表达增加所致。此外，小干扰RNA减少Id3表达消除了这些细胞中UVB诱导的caspase-3的蛋白水解激活。这些数据共同表明，UVB 诱导的永生化角质形成细胞凋亡至少部分归因于这些细胞中 Id3 的上调。
Inhibitor of differentiation/DNA binding ( Id) proteins comprise a class of helix-loop-helix transcription factors involved in proliferation, differentiation, apoptosis, and carcinogenesis. We have shown that while Id2 is induced by UVB in primary keratinocytes, Id3 is upregulated only in immortalized cells. We have now determined that the consequences of ectopic expression of Id3 protein are strikingly different between immortalized and primary keratinocytes. Overexpression of Id3 induces a significant increase in apoptotic cells as revealed by Annexin V positivity as well as proteolytic processing of caspase-3 in immortalized, but not in primary keratinocytes. Id3-green fluorescent protein ( GFP)-positive cells exhibited a fivefold increase in apoptotic nuclear fragmentation compared to Id3-GFP-negative cells. These apoptotic responses were accompanied by activation of caspase-3, as shown by immunocytochemical staining with antibodies to active caspase-3. Immunostaining with antibodies to the active form of caspase-9 as well as to the active form of Bax further revealed that induction of apoptosis in Id3-overexpressing keratinocytes occurred via a mitochondrial-caspase-9-mediated pathway. Coexpression of dominant-negative caspase-9 with Id3 significantly suppressed apoptotic nuclear fragmentation, indicating that caspase-9 activation is essential for Id3-induced cell death. This response was also markedly attenuated by coexpression with the Bax antagonist antiapoptotic protein Bcl-2, confirming a role for Bax activation in this apoptotic response. Id3-induced Bax activation may result from increased expression of Bax protein. Furthermore, reduction of Id3 expression by small interfering RNAs abrogated the UVB-induced proteolytic activation of caspase- 3 in these cells. These data together suggest that UVB-induced apoptosis of immortalized keratinocytes is at least in part due to Id3 upregulation in these cells.