Transcriptional regulation of neutral sphingomyelinase 2 gene expression of a human breast cancer cell line, MCF-7, induced by the anti-cancer drug, daunorubicin.
Mg(2+)-dependent neutral SMases (NSMases) have emerged as prime candidates for stress-induced ceramide production. Among isoforms identified, previous reports have suggested the importance of NSMase2. However, its activation mechanism has not been precisely reported. Here, we analyzed the mechanism of NSMase2 gene expression by the anti-cancer drug, daunorubicin (DA). DA increased cellular ceramides (C16, C18 and C24) and NSMase activity of a human breast cancer cell line, MCF-7. DA remarkably increased the NSMase2 message and protein, whereas little change in NSMase1 and NSMase3 mRNAs and only a mild increase in acid SMase mRNA were observed. Overexpression and a knock down of NSMase2 indicated that NSMase2 played a role in DA-induced cell death. NSMase2 promoter analysis revealed that three Sp1 motifs located between -148 and -42bp upstream of the first exon were important in basic as well as in DA-induced promoter activity. Consistently, luciferase vectors containing three consensus Sp1-motifs but not its mutated form showed DA-induced transcriptional activation. DA-treated MCF-7 showed increased Sp3 protein. In SL2 cells lacking Sp family proteins, both Sp1 and Sp3 overexpression increased NSMase promoter activity. Increased binding of Sp family proteins by DA to three Sp1 motifs was shown by electrophoresis mobility shift and ChIP assays.
Ito
Hiromi
H
Nagoya University School of Health Sciences, Nagoya, Japan.
Murakami
Masashi
M
Furuhata
Ayako
A
Gao
Siqiang
S
Yoshida
Kayo
K
Sobue
Sayaka
S
Hagiwara
Kazumi
K
Takagi
Akira
A
Kojima
Tetsuhito
T
Suzuki
Motoshi
M
Banno
Yoshiko
Y
Tanaka
Kouji
K
Tamiya-Koizumi
Keiko
K
Kyogashima
Mamoru
M
Nozawa
Yoshinori
Y
Murate
Takashi
T
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002518:Ceramides / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D003630:Daunorubicin / Q000494:pharmacology*; D024202:Electrophoretic Mobility Shift Assay; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D051705:Sp3 Transcription Factor / Q000235:genetics / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D000903:Antibiotics, Antineoplastic; D002518:Ceramides; D034741:RNA, Small Interfering; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D051705:Sp3 Transcription Factor; C491484:SMPD3 protein, human; D013108:Sphingomyelin Phosphodiesterase; C547598:neutral sphingomyelinase 3, human; C519633:SMPD2 protein, human; D003630:Daunorubicin