Transcriptional regulation of neutral sphingomyelinase 2 in all-trans retinoic acid-treated human breast cancer cell line, MCF-7

Hiromi Ito, Kouji Tanaka, Kazumi Hagiwara, Misa Kobayashi, Asuka Hoshikawa, Naoki Mizutani, Akira Takagi, Tetsuhito Kojima, Sayaka Sobue, Masatoshi Ichihara, Motoshi Suzuki, Keiko Tamiya-Koizumi, Mitsuhiro Nakamura, Yoshiko Banno, Yoshinori Nozawa, Takashi Murate

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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.

Original languageEnglish
Pages (from-to)599-610
Number of pages12
JournalJournal of Biochemistry
Volume151
Issue number6
DOIs
Publication statusPublished - 01-06-2012
Externally publishedYes

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Ito, H., Tanaka, K., Hagiwara, K., Kobayashi, M., Hoshikawa, A., Mizutani, N., Takagi, A., Kojima, T., Sobue, S., Ichihara, M., Suzuki, M., Tamiya-Koizumi, K., Nakamura, M., Banno, Y., Nozawa, Y., & Murate, T. (2012). Transcriptional regulation of neutral sphingomyelinase 2 in all-trans retinoic acid-treated human breast cancer cell line, MCF-7. Journal of Biochemistry, 151(6), 599-610. https://doi.org/10.1093/jb/mvs037