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

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Abstract

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 α (RARα) gene. We found that ATRA induced the 4-fold elevation of acid sphingomyelinase (ASMase) 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 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 RARα or the PML/RARα 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.

Original languageEnglish
Pages (from-to)9936-9943
Number of pages8
JournalJournal of Biological Chemistry
Volume277
Issue number12
DOIs
Publication statusPublished - 22-03-2002

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Murate, T., Suzuki, M., Hattori, M., Takagi, A., Kojima, T., Tanizawa, T., Asano, H., Hotta, T., Saito, H., Yoshida, S., & Tamiya-Koizumi, K. (2002). Up-regulation of acid sphingomyelinase during retinoic acid-induced myeloid differentiation of NB4, a human acute promyelocytic leukemia cell line. Journal of Biological Chemistry, 277(12), 9936-9943. https://doi.org/10.1074/jbc.M111594200