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-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.
Kondo
T
T
Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawaramachi, Sakyo-ku, Kyoto 606-8507, Japan.
Matsuda
T
T
Kitano
T
T
Takahashi
A
A
Tashima
M
M
Ishikura
H
H
Umehara
H
H
Domae
N
N
Uchiyama
T
T
Okazaki
T
T
D017209:Apoptosis; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002455:Cell Division; D002518:Ceramides / Q000378:metabolism*; D004789:Enzyme Activation; D018922:HL-60 Cells; D018869:Heat-Shock Response / Q000502:physiology*; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000378:metabolism* / Q000628:therapy; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D012334:RNA, Neoplasm / Q000096:biosynthesis; D015398:Signal Transduction; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism
D061945:Caspase Inhibitors; D002518:Ceramides; D020319:Oligodeoxyribonucleotides, Antisense; D009842:Oligopeptides; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D018808:Transcription Factor AP-1; C110922:acetyl-aspartyl-methionyl-glutaminyl-aspartyl-aldehyde; D013108:Sphingomyelin Phosphodiesterase; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases