Simbulan-Rosenthal C M, Daher A, Trabosh V, Chen W-C, Gerstel D, Soeda E, Rosenthal D S
Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, DC 20007, USA.
Oncogene. 2006 Jun 22;25(26):3649-60. doi: 10.1038/sj.onc.1209407. Epub 2006 Jan 30.
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 Bcl2, 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.
分化抑制因子/DNA结合（Id）蛋白是一类螺旋-环-螺旋转录因子，参与细胞增殖、分化、凋亡和致癌作用。我们已经表明，虽然Id2在原代角质形成细胞中由紫外线B（UVB）诱导产生，但Id3仅在永生化细胞中上调。我们现在已经确定，Id3蛋白异位表达的后果在永生化和原代角质形成细胞之间存在显著差异。如膜联蛋白V阳性以及永生化而非原代角质形成细胞中半胱天冬酶-3的蛋白水解加工所揭示的，Id3的过表达导致凋亡细胞显著增加。与Id3-绿色荧光蛋白（GFP）阴性细胞相比，Id3-GFP阳性细胞的凋亡核碎片化增加了五倍。这些凋亡反应伴随着半胱天冬酶-3的激活，如用活性半胱天冬酶-3抗体进行免疫细胞化学染色所示。用活性形式的半胱天冬酶-9以及活性形式的Bax抗体进行免疫染色进一步表明，Id3过表达的角质形成细胞中凋亡的诱导是通过线粒体-半胱天冬酶-9介导的途径发生的。显性负性半胱天冬酶-9与Id3共表达显著抑制凋亡核碎片化，表明半胱天冬酶-9的激活对于Id3诱导的细胞死亡至关重要。与Bax拮抗剂抗凋亡蛋白Bcl2共表达也显著减弱了这种反应，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降低Id3表达消除了这些细胞中UVB诱导的半胱天冬酶-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是由于这些细胞中Id3上调所致。
