Kondo T, Matsuda T, Kitano T, Takahashi A, Tashima M, Ishikura H, Umehara H, Domae N, Uchiyama T, Okazaki T
Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawaramachi, Sakyo-ku, Kyoto 606-8507, Japan.
J Biol Chem. 2000 Mar 17;275(11):7668-76. doi: 10.1074/jbc.275.11.7668.
Ceramide has emerged as a lipid mediator in apoptosis induced by a variety of stresses. As we previously showed that the activation of AP-1, a nuclear transcription factor was indispensable to ceramide-induced apoptosis in human leukemia HL-60 cells (Sawai, H., Okazaki, T., Yamamoto, H., Okano, H., Takeda, Y., Tashima, M., Sawada, H., Okuma, M., Ishikura, H., Umehara, H., and Domae, N. (1995) J. Biol. Chem. 270, 27326-27331), the role and mechanism of heat shock (HS)-increased c-jun expression in apoptosis was here investigated. HS increased morphological changes compatible with apoptosis in human leukemia HL-60 cells, and induced ceramide generation and sphingomyelin hydrolysis with an increase of neutral magnesium-dependent sphingomyelinase activity. When HS failed to induce apoptosis in HS-resistant HL-60 cells, ceramide generation was not detected, suggesting that ceramide was involved in downstream signals required for HS-induced apoptosis. Both HS and N-acetylsphingosine (C(2)-ceramide) increased the expression of c-jun/c-fos mRNAs with the peak 2 h after treatment. When we examined whether the inhibition of c-jun expression by its antisense oligodeoxynucleotides (AS) blocked HS- or C(2)-ceramide-induced apoptosis, AS of c-jun gene inhibited apoptotic morphological changes and DNA fragmentation whereas did not sense oligodeoxynucleotides. Moreover, a synthetic tetrapeptide, acetyl-Asp-Met-Gln-Asp-aldehyde (DMQD-CHO), which inhibited the formation of active form of caspase-3 more efficiently than those of caspase-4, -6, -7, and -8, blocked both caspase-3 like activity, c-jun expression and apoptosis induced by HS or C(2)-ceramide, although DMQD-CHO did not affect HS-induced ceramide generation. These results suggested that the ceramide was generated through sphingomyelin hydrolysis by HS-activated neutral, magnesium-dependent sphingomyelinase and that subsequent c-jun expression through activation of caspase-3 played a role in HS-induced HL-60 cell apoptosis.
神经酰胺已成为多种应激诱导凋亡过程中的脂质介质。正如我们之前所表明的，核转录因子AP-1的激活对于人白血病HL-60细胞中神经酰胺诱导的凋亡是不可或缺的（泽井，H.，冈崎，T.，山本，H.，冈野，H.，武田，Y.，田岛，M.，泽田，H.，奥久马，M.，石仓，H.，梅原，H.，和堂前，N.（1995年）《生物化学杂志》270，27326 - 27331），在此研究了热休克（HS）增加的c-jun表达在凋亡中的作用及机制。热休克增加了人白血病HL-60细胞中与凋亡相符的形态学变化，并诱导了神经酰胺的生成和鞘磷脂的水解，同时中性镁依赖性鞘磷脂酶活性增加。当热休克未能在热休克抗性HL-60细胞中诱导凋亡时，未检测到神经酰胺的生成，这表明神经酰胺参与了热休克诱导凋亡所需的下游信号传导。热休克和N-乙酰鞘氨醇（C2 - 神经酰胺）均增加了c-jun/c-fos mRNA的表达，在处理后2小时达到峰值。当我们检测其反义寡脱氧核苷酸（AS）对c-jun表达的抑制是否能阻断热休克或C2 - 神经酰胺诱导的凋亡时，c-jun基因的反义寡脱氧核苷酸抑制了凋亡形态学变化和DNA片段化，而正义寡脱氧核苷酸则无此作用。此外，一种合成四肽，乙酰 - 天冬氨酸 - 蛋氨酸 - 谷氨酰胺 - 醛（DMQD - CHO），其比半胱天冬酶 - 4、 - 6、 - 7和 - 8更有效地抑制了活性形式的半胱天冬酶 - 3的形成，阻断了热休克或C2 - 神经酰胺诱导的半胱天冬酶 - 3样活性、c-jun表达和凋亡，尽管DMQD - CHO不影响热休克诱导的神经酰胺生成。这些结果表明，神经酰胺是通过热休克激活的中性镁依赖性鞘磷脂酶水解鞘磷脂产生的，随后通过激活半胱天冬酶 - 3导致的c-jun表达在热休克诱导的HL-60细胞凋亡中起作用。
