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

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Sphingomyelin Phosphodiesterase
Tretinoin
Cells
Breast Neoplasms
Cell Line
Transcription
Phosphorylation
MCF-7 Cells
Genetic Promoter Regions
Histones
Messenger RNA
Chromatin
Chemical activation
Acetylation
Chromatin Assembly and Disassembly
Chromatin Immunoprecipitation

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Ito, H., Tanaka, K., Hagiwara, K., Kobayashi, M., Hoshikawa, A., Mizutani, N., ... 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
Ito, Hiromi ; Tanaka, Kouji ; Hagiwara, Kazumi ; Kobayashi, Misa ; Hoshikawa, Asuka ; Mizutani, Naoki ; Takagi, Akira ; Kojima, Tetsuhito ; Sobue, Sayaka ; Ichihara, Masatoshi ; Suzuki, Motoshi ; Tamiya-Koizumi, Keiko ; Nakamura, Mitsuhiro ; Banno, Yoshiko ; Nozawa, Yoshinori ; Murate, Takashi. / Transcriptional regulation of neutral sphingomyelinase 2 in all-trans retinoic acid-treated human breast cancer cell line, MCF-7. In: Journal of Biochemistry. 2012 ; Vol. 151, No. 6. pp. 599-610.
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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.",
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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, vol. 151, no. 6, pp. 599-610. https://doi.org/10.1093/jb/mvs037

Transcriptional regulation of neutral sphingomyelinase 2 in all-trans retinoic acid-treated human breast cancer cell line, MCF-7. / Ito, Hiromi; Tanaka, Kouji; Hagiwara, Kazumi; Kobayashi, Misa; Hoshikawa, Asuka; Mizutani, Naoki; Takagi, Akira; Kojima, Tetsuhito; Sobue, Sayaka; Ichihara, Masatoshi; Suzuki, Motoshi; Tamiya-Koizumi, Keiko; Nakamura, Mitsuhiro; Banno, Yoshiko; Nozawa, Yoshinori; Murate, Takashi.

In: Journal of Biochemistry, Vol. 151, No. 6, 01.06.2012, p. 599-610.

Research output: Contribution to journalArticle

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T1 - Transcriptional regulation of neutral sphingomyelinase 2 in all-trans retinoic acid-treated human breast cancer cell line, MCF-7

AU - Ito, Hiromi

AU - Tanaka, Kouji

AU - Hagiwara, Kazumi

AU - Kobayashi, Misa

AU - Hoshikawa, Asuka

AU - Mizutani, Naoki

AU - Takagi, Akira

AU - Kojima, Tetsuhito

AU - Sobue, Sayaka

AU - Ichihara, Masatoshi

AU - Suzuki, Motoshi

AU - Tamiya-Koizumi, Keiko

AU - Nakamura, Mitsuhiro

AU - Banno, Yoshiko

AU - Nozawa, Yoshinori

AU - Murate, Takashi

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N2 - 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.

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