title: Transcriptional regulation of neutral sphingomyelinase 2 in all-trans retinoic acid-treated human breast cancer cell line, MCF-7.
abstract: Effects of all-trans retinoic acid (ATRA) on sphingomyelinase expression were examined using MCF-7 (ATRA-sensitive) and MDA-MB-231 (ATRA-resistant) breast cancer cells. Increased NSMase activity, NSMase2 mRNA and protein were observed in ATRA-treated MCF-7 but not in ATRA-treated MDA-MB-231. Increased NSMase2 mRNA of ATRA-treated MCF-7 was mostly due to enhanced transcription. Promoter analysis revealed the important 5'-promoter region of NSMase2 between -148 and -42 bp containing three Sp1 sites but no retinoic acid responsive elements. Experiments using mutated Sp1 sites of the NSMase2 promoter, Mithramycin A (a Sp inhibitor) and Sp family over-expression demonstrated the importance of Sp family protein and the three Sp1 sites for ATRA-induced NSMase2 transcription of MCF-7 cells. Although no quantitative change of bound Sp1 on NSMase2 promoter region after ATRA treatment was detected, Sp1 phosphorylation (activation) by ATRA was observed. Interestingly, PKCδ was involved in ATRA-induced increased NSMase2 transcription. ATRA-induced PKCδ phosphorylation and then activated PKCδ phosphorylated Sp1. Chromatin immunoprecipitation (ChIP) assay showed Sp1, RARα and RXRα complex formation in MCF-7 cells regardless of ATRA treatment and ATRA-induced acetylated histone H3 of the 5'-promoter. Thus, NSMase2 mRNA expression enhanced by ATRA was due to increased transcription via phosphorylated Sp1 caused by PKCδ activation, followed by chromatin remodelling with histone H3 acetylation.
authors.affiliation: Department of Medical Technology, Nagoya University School of Health Sciences, Nagoya, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Ito
authors.forename: Kouji
authors.initials: K
authors.lastname: Tanaka
authors.forename: Kazumi
authors.initials: K
authors.lastname: Hagiwara
authors.forename: Misa
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Asuka
authors.initials: A
authors.lastname: Hoshikawa
authors.forename: Naoki
authors.initials: N
authors.lastname: Mizutani
authors.forename: Akira
authors.initials: A
authors.lastname: Takagi
authors.forename: Tetsuhito
authors.initials: T
authors.lastname: Kojima
authors.forename: Sayaka
authors.initials: S
authors.lastname: Sobue
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Ichihara
authors.forename: Motoshi
authors.initials: M
authors.lastname: Suzuki
authors.forename: Keiko
authors.initials: K
authors.lastname: Tamiya-Koizumi
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Nakamura
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Banno
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Nozawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Murate
mesh_terms: D002470:Cell Survival / Q000187:drug effects; D020869:Gene Expression Profiling; D006801:Humans; D061986:MCF-7 Cells; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic / Q000187:drug effects; D014212:Tretinoin / Q000494:pharmacology* / Q000627:therapeutic use
chemical_list: D012333:RNA, Messenger; D014212:Tretinoin; C491484:SMPD3 protein, human; D013108:Sphingomyelin Phosphodiesterase