Marconi Alessandra, Atzei Paola, Panza Cristina, Fila Chiara, Tiberio Rossana, Truzzi Francesca, Wachter Tina, Leverkus Martin, Pincelli Carlo
Institute of Dermatology, University of Modena and Reggio Emilia, Via del Pozzo 71, 41100 Modena, Italy.
J Cell Sci. 2004 Nov 15;117(Pt 24):5815-23. doi: 10.1242/jcs.01490. Epub 2004 Oct 26.
Beta1-integrin protects keratinocyte stem cells (KSC) from cell-detachment apoptosis ('anoikis'). Here we show that caspase-8 active protein is detected in both young transit amplifying (TA) cells and TA cells, but not in KSC. On suspension, caspases are activated earlier in young TA than in KSC, whereas anti-beta1-integrin neutralizing antibody accelerates caspase activation in both KSC and young TA. Caspases 8 and 10 are the first caspases to be activated whereas caspase-8 inhibitor zIETD-fmk delays the activation of Bid, caspase-9 and caspase-3. However, the caspase-9 inhibitor zLEDH-fmk does not block the activation of caspase-8, Bid, caspase-10 and caspase-3. Moreover, caspase-8, but not caspase-9 inhibitor partially prevents keratinocyte anoikis. As FLIP inhibits caspase-8 processing, we retrovirally infected HaCaT keratinocytes with c-FLIP(L). Anti-beta1-integrin fails to activate caspase-8, Bid, caspase-9 and to induce the release of cytochrome c in c-FLIP(L) overexpressing keratinocytes. Finally, overexpression of c-FLIP(L) partially prevents anoikis in both suspended and anti-beta1 integrin-treated cells. Taken together, these results indicate that the extrinsic apoptotic pathway triggered by caspase-8 predominates in keratinocyte anoikis. However, the release of cytochrome c and the later activation of caspase-9 seem to suggest that the intrinsic mitochondrial pathway may intervene as a positive feedback loop of caspase activation.
β1整合素可保护角质形成细胞干细胞（KSC）免受细胞脱离凋亡（“失巢凋亡”）。在此我们发现，在年轻的过渡扩增（TA）细胞和TA细胞中均可检测到半胱天冬酶-8活性蛋白，但在KSC中未检测到。在悬浮状态下，年轻TA细胞中的半胱天冬酶比KSC中更早被激活，而抗β1整合素中和抗体可加速KSC和年轻TA细胞中的半胱天冬酶激活。半胱天冬酶8和10是最早被激活的半胱天冬酶，而半胱天冬酶-8抑制剂zIETD-fmk可延迟Bid、半胱天冬酶-9和半胱天冬酶-3的激活。然而，半胱天冬酶-9抑制剂zLEDH-fmk并不能阻断半胱天冬酶-8、Bid、半胱天冬酶-10和半胱天冬酶-3的激活。此外，半胱天冬酶-8抑制剂而非半胱天冬酶-9抑制剂可部分预防角质形成细胞失巢凋亡。由于FLIP可抑制半胱天冬酶-8的加工过程，我们用c-FLIP(L)逆转录病毒感染了HaCaT角质形成细胞。抗β1整合素未能激活过表达c-FLIP(L)的角质形成细胞中的半胱天冬酶-8、Bid、半胱天冬酶-9，也未能诱导细胞色素c的释放。最后，c-FLIP(L)的过表达可部分预防悬浮细胞和抗β1整合素处理细胞中的失巢凋亡。综上所述，这些结果表明，由半胱天冬酶-8触发的外源性凋亡途径在角质形成细胞失巢凋亡中起主导作用。然而，细胞色素c的释放和半胱天冬酶-9的后期激活似乎表明，内源性线粒体途径可能作为半胱天冬酶激活的正反馈环而发挥作用。
