Increased SPHK2 Transcription of Human Colon Cancer Cells in Serum-Depleted Culture: The Involvement of CREB Transcription Factor

Naoki Mizutani, Yukari Omori, Koji Tanaka, Hiromi Ito, Akira Takagi, Tetsuhito Kojima, Masahiro Nakatochi, Hideo Ogiso, Yoshiyuki Kawamoto, Mitsuhiro Nakamura, Motoshi Suzuki, Mamoru Kyogashima, Keiko Tamiya-Koizumi, Yoshinori Nozawa, Takashi Murate

Research output: Contribution to journalArticle

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Abstract

Sphingosine kinases (SPHK) are important to determine cells' fate by producing sphingosine 1-phosphate. Reportedly, exogenous SPHK2 overexpression induces cell cycle arrest or cell death. However, the regulatory mechanism of SPHK2 expression has not been fully elucidated. Here, we analyzed this issue using human colon cancer cell lines under various stress conditions. Serum depletion (FCS(-)) but not hypoxia and glucose depletion increased mRNA, protein and enzyme activity of SPHK2 but not SPHK1. In HCT116 cells mostly used, SPHK2 activity was predominant over SPHK1, and serum depletion increased both nuclear and cytoplasmic SPHK2 activity. Based on previous reports analyzing cellular response after serum depletion, the temporal changes of intracellular signaling molecules and candidate transcription factors for SPHK2 were examined using serum-depleted HCT116 cells, and performed transfection experiments with siRNA or cDNA of candidate transcription factors. Results showed that the rapid and transient JNK activation followed by CREB activation was the major regulator of increased SPHK2 transcription in FCS(-) culture. EMSA and ChIP assay confirmed the direct binding of activated CREB to the CREB binding site of 5′ SPHK2 promoter region. Colon cancer cells examined continued to grow in FCS(-) culture, although mildly, while hypoxia and glucose depletion suppressed cell proliferation or induced cell death, suggesting the different role of SPHK2 in different stress conditions. Because of the unique relationship observed after serum depletion, we examined effects of siRNA for SPHK2, and found the role of SPHK2 as a growth or survival factor but not a cell proliferation inhibitor in FCS(-) culture. J. Cell. Biochem. 116: 2227-2238, 2015.

Original languageEnglish
Pages (from-to)2227-2238
Number of pages12
JournalJournal of Cellular Biochemistry
Volume116
Issue number10
DOIs
Publication statusPublished - 01-10-2015
Externally publishedYes

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Transcription
Cell culture
Colonic Neoplasms
Transcription Factors
Cells
Cell proliferation
Cell death
Small Interfering RNA
HCT116 Cells
Serum
Chemical activation
Glucose
Enzyme activity
Genetic Promoter Regions
Cell Death
Cell Proliferation
Assays
Complementary DNA
Binding Sites
Messenger RNA

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Mizutani, Naoki ; Omori, Yukari ; Tanaka, Koji ; Ito, Hiromi ; Takagi, Akira ; Kojima, Tetsuhito ; Nakatochi, Masahiro ; Ogiso, Hideo ; Kawamoto, Yoshiyuki ; Nakamura, Mitsuhiro ; Suzuki, Motoshi ; Kyogashima, Mamoru ; Tamiya-Koizumi, Keiko ; Nozawa, Yoshinori ; Murate, Takashi. / Increased SPHK2 Transcription of Human Colon Cancer Cells in Serum-Depleted Culture : The Involvement of CREB Transcription Factor. In: Journal of Cellular Biochemistry. 2015 ; Vol. 116, No. 10. pp. 2227-2238.
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abstract = "Sphingosine kinases (SPHK) are important to determine cells' fate by producing sphingosine 1-phosphate. Reportedly, exogenous SPHK2 overexpression induces cell cycle arrest or cell death. However, the regulatory mechanism of SPHK2 expression has not been fully elucidated. Here, we analyzed this issue using human colon cancer cell lines under various stress conditions. Serum depletion (FCS(-)) but not hypoxia and glucose depletion increased mRNA, protein and enzyme activity of SPHK2 but not SPHK1. In HCT116 cells mostly used, SPHK2 activity was predominant over SPHK1, and serum depletion increased both nuclear and cytoplasmic SPHK2 activity. Based on previous reports analyzing cellular response after serum depletion, the temporal changes of intracellular signaling molecules and candidate transcription factors for SPHK2 were examined using serum-depleted HCT116 cells, and performed transfection experiments with siRNA or cDNA of candidate transcription factors. Results showed that the rapid and transient JNK activation followed by CREB activation was the major regulator of increased SPHK2 transcription in FCS(-) culture. EMSA and ChIP assay confirmed the direct binding of activated CREB to the CREB binding site of 5′ SPHK2 promoter region. Colon cancer cells examined continued to grow in FCS(-) culture, although mildly, while hypoxia and glucose depletion suppressed cell proliferation or induced cell death, suggesting the different role of SPHK2 in different stress conditions. Because of the unique relationship observed after serum depletion, we examined effects of siRNA for SPHK2, and found the role of SPHK2 as a growth or survival factor but not a cell proliferation inhibitor in FCS(-) culture. J. Cell. Biochem. 116: 2227-2238, 2015.",
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Mizutani, N, Omori, Y, Tanaka, K, Ito, H, Takagi, A, Kojima, T, Nakatochi, M, Ogiso, H, Kawamoto, Y, Nakamura, M, Suzuki, M, Kyogashima, M, Tamiya-Koizumi, K, Nozawa, Y & Murate, T 2015, 'Increased SPHK2 Transcription of Human Colon Cancer Cells in Serum-Depleted Culture: The Involvement of CREB Transcription Factor', Journal of Cellular Biochemistry, vol. 116, no. 10, pp. 2227-2238. https://doi.org/10.1002/jcb.25173

Increased SPHK2 Transcription of Human Colon Cancer Cells in Serum-Depleted Culture : The Involvement of CREB Transcription Factor. / Mizutani, Naoki; Omori, Yukari; Tanaka, Koji; Ito, Hiromi; Takagi, Akira; Kojima, Tetsuhito; Nakatochi, Masahiro; Ogiso, Hideo; Kawamoto, Yoshiyuki; Nakamura, Mitsuhiro; Suzuki, Motoshi; Kyogashima, Mamoru; Tamiya-Koizumi, Keiko; Nozawa, Yoshinori; Murate, Takashi.

In: Journal of Cellular Biochemistry, Vol. 116, No. 10, 01.10.2015, p. 2227-2238.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Increased SPHK2 Transcription of Human Colon Cancer Cells in Serum-Depleted Culture

T2 - The Involvement of CREB Transcription Factor

AU - Mizutani, Naoki

AU - Omori, Yukari

AU - Tanaka, Koji

AU - Ito, Hiromi

AU - Takagi, Akira

AU - Kojima, Tetsuhito

AU - Nakatochi, Masahiro

AU - Ogiso, Hideo

AU - Kawamoto, Yoshiyuki

AU - Nakamura, Mitsuhiro

AU - Suzuki, Motoshi

AU - Kyogashima, Mamoru

AU - Tamiya-Koizumi, Keiko

AU - Nozawa, Yoshinori

AU - Murate, Takashi

PY - 2015/10/1

Y1 - 2015/10/1

N2 - Sphingosine kinases (SPHK) are important to determine cells' fate by producing sphingosine 1-phosphate. Reportedly, exogenous SPHK2 overexpression induces cell cycle arrest or cell death. However, the regulatory mechanism of SPHK2 expression has not been fully elucidated. Here, we analyzed this issue using human colon cancer cell lines under various stress conditions. Serum depletion (FCS(-)) but not hypoxia and glucose depletion increased mRNA, protein and enzyme activity of SPHK2 but not SPHK1. In HCT116 cells mostly used, SPHK2 activity was predominant over SPHK1, and serum depletion increased both nuclear and cytoplasmic SPHK2 activity. Based on previous reports analyzing cellular response after serum depletion, the temporal changes of intracellular signaling molecules and candidate transcription factors for SPHK2 were examined using serum-depleted HCT116 cells, and performed transfection experiments with siRNA or cDNA of candidate transcription factors. Results showed that the rapid and transient JNK activation followed by CREB activation was the major regulator of increased SPHK2 transcription in FCS(-) culture. EMSA and ChIP assay confirmed the direct binding of activated CREB to the CREB binding site of 5′ SPHK2 promoter region. Colon cancer cells examined continued to grow in FCS(-) culture, although mildly, while hypoxia and glucose depletion suppressed cell proliferation or induced cell death, suggesting the different role of SPHK2 in different stress conditions. Because of the unique relationship observed after serum depletion, we examined effects of siRNA for SPHK2, and found the role of SPHK2 as a growth or survival factor but not a cell proliferation inhibitor in FCS(-) culture. J. Cell. Biochem. 116: 2227-2238, 2015.

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