Up-regulation of acid sphingomyelinase during retinoic acid-induced myeloid differentiation of NB4, a human acute promyelocytic leukemia cell line.
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 alpha (RARalpha) 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 RARalpha or the PML/RARalpha 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.
Murate
Takashi
T
Nagoya University School of Health Science, Nagoya 466-8550, Japan. murate@met.nagoya-u.ac.jp
Suzuki
Motoshi
M
Hattori
Masashi
M
Takagi
Akira
A
Kojima
Tetsuhito
T
Tanizawa
Tomomi
T
Asano
Haruhiko
H
Hotta
Tomomitsu
T
Saito
Hidehiko
H
Yoshida
Shonen
S
Tamiya-Koizumi
Keiko
K
D020816:Amino Acid Motifs; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D019556:COS Cells; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D002518:Ceramides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D017930:Genes, Reporter; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000201:enzymology*; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018168:Receptors, Retinoic Acid / Q000235:genetics; D000072482:Retinoic Acid Receptor alpha; D020133:Reverse Transcriptase Polymerase Chain Reaction; D021241:Spectrometry, Mass, Electrospray Ionization; D013108:Sphingomyelin Phosphodiesterase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D014212:Tretinoin / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D015854:Up-Regulation; D001616:beta-Galactosidase / Q000378:metabolism
D002518:Ceramides; C000607071:RARA protein, human; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D014212:Tretinoin; D008156:Luciferases; C112980:acid sphingomyelinase-1; D013108:Sphingomyelin Phosphodiesterase; D001616:beta-Galactosidase