Requirement of AP-1 for Ceramide-induced Apoptosis in Human Leukemia HL-60 Cells

Hirofumi Sawai,Toshiro Okazaki,Hirotaka Yamamoto,Hakuro Okano,Yasushi Takeda,Masaro Tashima,Hiroyoshi Sawada,Minoru Okuma,Hiroto Ishikura,Hisanori Umehara,Naochika Domae

doi:10.1074/jbc.270.45.27326

Abstract

Ceramide has emerged as a novel lipid mediator in cell proliferation, differentiation, and apoptosis. In this work, we demonstrate that the levels of c-jun mRNA, c-Jun protein, and DNA binding activity of a nuclear transcription factor AP-1 to 12-o-tetradecanoylphorbol 13-acetate responsive elements all increased following treatment with the cell-permeable ceramide, N-acetylsphingosine in human leukemia HL-60 cells. N-Acetylsphingosine (1-10 microM) increased the levels of c-jun mRNA in a dose-dependent manner, and maximal expression was achieved 1 h after treatment. Increase of c-jun expression treated with 5 microM N-acetyldihydrosphingosine, which could not induce apoptosis, was one third of that with 5 microM N-acetylsphingosine. Ceramide-induced growth inhibition and DNA fragmentation were both prevented by treatment with curcumin, 1,7-bis[4-hydroxy-3-methoxy-phenyl]-1,6-heptadiene-3,5-dione (an inhibitor of AP-1 activation), or antisense oligonucleotides for c-jun. These results suggest that the transcription factor AP-1 is critical for apoptosis in HL-60 cells and that an intracellular sphingolipid mediator, ceramide, modulates a signal transduction inducing apoptosis through AP-1 activation.

Highlights

Ceramide has emerged as a novel lipid mediator in cell proliferation, differentiation, and apoptosis
We examined whether ceramide induces c-jun/ AP-1 activation and whether its activation is required for the induction of apoptosis by ceramide in human leukemia HL-60 cells, because c-jun/AP-1 activation seems to play a role in inducing apoptosis as well as other early responsive genes such as NF-␬B and c-myc
The sphingomyelin cycle was discovered in HL-60 human myelogenous leukemia cells in response to 1␣,25-dihydroxyvitamin D3 [3, 4]

Summary

EXPERIMENTAL PROCEDURES

Materials—Human leukemia HL-60 cells were kindly provided by Dr M. Electrophoretic Mobility Shift Assay—Nuclear extract (6 ␮g) was mixed with 4 ␮g of poly(dI1⁄7dC)1⁄7poly(dI1⁄7dC) (Pharmacia Biotech Inc.) in a binding buffer (5 mM HEPES, pH 7.9, 5 mM MgCl2, 50 mM KCl, 1 mM dithiothreitol, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride; final volume, 20 ␮l) for 10 min on ice. AP-1 consensus oligonucleotide (5ЈCGCTTGATGACTCAGCCGGAA-3Ј) (Santa Cruz Biotechnology, Inc.) was 5Ј-end labeled as described above. The labeled probe (2.5 ϫ 104 cpm) was added, and the reaction mixture was incubated for 30 min on ice. the samples were separated by 4% native polyacrylamide gel electrophoresis in 1 ϫ TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA). Analysis of DNA Fragmentation—DNA was isolated by using a GENOME kit (Bio 101, CA), electrophoresed through 3% NuSieve agarose (FMC BioProducts) mini-gel in 1 ϫ TAE buffer at 50 V for 1.5 h and visualized under UV light after ethidium bromide staining

RESULTS

Findings

DISCUSSION