Up-regulation of Acid Sphingomyelinase during Retinoic Acid-induced Myeloid Differentiation of NB4, a Human Acute Promyelocytic Leukemia Cell Line

Takashi Murate, Motoshi Suzuki, Masashi Hattori, Akira Takagi, Tetsuhito Kojima, Tomomi Tanizawa, Haruhiko Asano, Tomomitsu Hotta, Hidehiko Saito, Shonen Yoshida, Keiko Tamiya-Koizumi
2002 Journal of Biological Chemistry  
All-trans-retinoic acid (ATRA) induces myeloid differentiation of a human promyelocytic leukemia cell line, NB4, but does not affect its subclone NB4/RA harboring a point-mutated ligand-binding domain (AF2) in retinoic acid receptor ␣ (RAR␣) gene. We found that ATRA induced the 4-fold elevation of acid sphingomyelinase (AS-Mase) activity 24 h after treatment in NB4 cells, but not in NB4/RA cells. ATRA did not affect neutral sphingomyelinase activity in either NB4 or NB4/RA. Upon treatment with
more » ... pon treatment with ATRA, ceramide, the product of an ASMase reaction, accumulated in NB4 cells. Northern blot analysis showed a marked elevation of the ASMase mRNA 8 h after ATRA treatment, reaching a plateau at 24 h. Regulation of ASMase gene expression was studied by a promoter analysis using luciferase reporter assay. The 5-upstream flanking region of human ASMase gene (؊519/؉300) conjugated with the luciferase gene was introduced into COS-7 cells. Luciferase activity in transformed cells markedly increased in response to ATRA stimulation when the wild type RAR␣ or the PML/RAR␣ hybrid protein was co-expressed. Deletion experiments revealed that a short sequence at the 5-end (؊519/؊485) was indispensable for the ATRA response. Within this short region, two retinoic acid-responsive element-like motifs (TGCCCG and TCTCCT) and one AP2-like motif (CCCTTCCC) were identified. Deletion and base-substitution experiments showed that all three motifs are required for the full expression induced by ATRA. Electrophoresis mobility shift assays with the nuclear extract of ATRA-treated NB4 cells showed that proteins were bound specifically to the probe being mediated by all three motifs in the promoter sequence. 1 The abbreviations used are: ASMase, acid sphingomyelinase; NS-Mase, neutral sphingomyelinase; APL, acute promyelocytic leukemia;
doi:10.1074/jbc.m111594200 pmid:11788605 fatcat:ku43tnpv65bbxhihvdedszuudi