Role of c-jun expression increased by heat shock- and ceramide-activated caspase-3 in HL-60 cell apoptosis -: Possible involvement of ceramide in heat shock-induced apoptosis

Ceramide has emerged as a lipid mediator in apoptosis induced by a variety of stresses. As we previously showed that the activation of AP-I, 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, M., 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. KS 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 MS failed to induce apoptosis in MS-resistant HL-60 cells, ceramide generation was not detected, suggesting that ceramide was involved in downstream signals required for MS-induced apoptosis. Both MS 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-CRO), 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 MS or C,-ceramide, although DMQD-CHO did not affect HS-induced ceramide generation. These results suggested that the ceramide was generated through sphingomyelin hydrolysis by MS-activated neutral, magnesiun-dependent sphingomyelinase and that subsequent c-jun expression through activation of caspase-3 played a role in MS-induced ML-60 cell apoptosis.