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

Research output: Contribution to journalArticle

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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
Externally publishedYes

Fingerprint

Sphingomyelin Phosphodiesterase
Acute Promyelocytic Leukemia
Tretinoin
Up-Regulation
Cells
Cell Line
Acids
Retinoic Acid Receptors
Luciferases
Genes
Assays
Ceramides
5' Flanking Region
COS Cells
Electrophoretic Mobility Shift Assay
Electrophoresis
Gene expression
Northern Blotting
Leukemia
Proteins

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Murate, Takashi ; Suzuki, Motoshi ; Hattori, Masashi ; Takagi, Akira ; Kojima, Tetsuhito ; Tanizawa, Tomomi ; Asano, Haruhiko ; Hotta, Tomomitsu ; Saito, Hidehiko ; Yoshida, Shonen ; Tamiya-Koizumi, Keiko. / Up-regulation of acid sphingomyelinase during retinoic acid-induced myeloid differentiation of NB4, a human acute promyelocytic leukemia cell line. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 12. pp. 9936-9943.
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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.",
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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, vol. 277, no. 12, pp. 9936-9943. https://doi.org/10.1074/jbc.M111594200

Up-regulation of acid sphingomyelinase during retinoic acid-induced myeloid differentiation of NB4, a human acute promyelocytic leukemia cell line. / Murate, Takashi; Suzuki, Motoshi; Hattori, Masashi; Takagi, Akira; Kojima, Tetsuhito; Tanizawa, Tomomi; Asano, Haruhiko; Hotta, Tomomitsu; Saito, Hidehiko; Yoshida, Shonen; Tamiya-Koizumi, Keiko.

In: Journal of Biological Chemistry, Vol. 277, No. 12, 22.03.2002, p. 9936-9943.

Research output: Contribution to journalArticle

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T1 - Up-regulation of acid sphingomyelinase during retinoic acid-induced myeloid differentiation of NB4, a human acute promyelocytic leukemia cell line

AU - Murate, Takashi

AU - Suzuki, Motoshi

AU - Hattori, Masashi

AU - Takagi, Akira

AU - Kojima, Tetsuhito

AU - Tanizawa, Tomomi

AU - Asano, Haruhiko

AU - Hotta, Tomomitsu

AU - Saito, Hidehiko

AU - Yoshida, Shonen

AU - Tamiya-Koizumi, Keiko

PY - 2002/3/22

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N2 - 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.

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