Mechanism of increased PLD1 gene expression during early adipocyte differentiation process of mouse cell line 3T3-L1

Siqiang Gao, Hiromi Ito, Masashi Murakami, Kayo Yoshida, Yoko Tagawa, Kazumi Hagiwara, Akira Takagi, Tetsuhito Kojima, Motoshi Suzuki, Yoshiko Banno, Kenji Ohguchi, Yoshinori Nozawa, Takashi Murate

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

A mouse cell line 3T3-L1 is differentiated into adipocytes when treated with an inducer cocktail (IDX) (insulin, dexametahsone, and a cAMP phosphodiesterase inhibitor of isobutyl-methylxanthine (IBMX)). Here, we report that PLD1, but not PLD2, mRNA and protein increased during the early differentiation process. Our analysis shows that IDX resulted in a sequential induction of C/EBPβ, PLD1, and C/EBPα which is a key transcription factor of late adipocyte differentiation. Among the three inducers, IBMX+any other inducer induced mild adipocyte differentiation, whereas insulin+dexamethasone did not. IBMX increased PLD1 but not PLD2 mRNA. Forskolin, an adenylate cyclase activator, and dbcAMP also increased PLD1 mRNA, suggesting the cellular cAMP as the inducer of both adipocyte differentiation and PLD1 transcription. We focused on the regulatory mechanism of PLD1 transcription during this differentiation process. IDX or a combination of inducers including IBMX increased PLD1 promoter activity, which is consistent with mRNA analysis. Promoter analysis identified two adjacent C/EBP motifs located between -338 and -231 bp from the first exon as the IBMX responsive elements. Furthermore, overexpression of C/EBPβ, but not C/EBPα, increased PLD1 mRNA and PLD1 5′ promoter activity. EMSA and chromatin immunoprecipitation assay confirmed the direct binding of C/EBPβ, but not C/EBPα, to these C/EBP motifs of PLD1 5′ promoter. Our results show that PLD1 is a target gene of C/EBPβ through the increased cellular cAMP during early adipocyte differentiation of 3T3-L1 cells.

Original languageEnglish
Pages (from-to)375-382
Number of pages8
JournalJournal of Cellular Biochemistry
Volume109
Issue number2
DOIs
Publication statusPublished - 01-02-2010
Externally publishedYes

Fingerprint

Adipocytes
Gene expression
Cells
Gene Expression
Cell Line
Messenger RNA
Transcription
Insulin
3T3-L1 Cells
Phosphodiesterase Inhibitors
Chromatin Immunoprecipitation
Colforsin
Adenylyl Cyclases
Dexamethasone
Chromatin
Exons
Assays
Transcription Factors
Genes
methylxanthine

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Gao, Siqiang ; Ito, Hiromi ; Murakami, Masashi ; Yoshida, Kayo ; Tagawa, Yoko ; Hagiwara, Kazumi ; Takagi, Akira ; Kojima, Tetsuhito ; Suzuki, Motoshi ; Banno, Yoshiko ; Ohguchi, Kenji ; Nozawa, Yoshinori ; Murate, Takashi. / Mechanism of increased PLD1 gene expression during early adipocyte differentiation process of mouse cell line 3T3-L1. In: Journal of Cellular Biochemistry. 2010 ; Vol. 109, No. 2. pp. 375-382.
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abstract = "A mouse cell line 3T3-L1 is differentiated into adipocytes when treated with an inducer cocktail (IDX) (insulin, dexametahsone, and a cAMP phosphodiesterase inhibitor of isobutyl-methylxanthine (IBMX)). Here, we report that PLD1, but not PLD2, mRNA and protein increased during the early differentiation process. Our analysis shows that IDX resulted in a sequential induction of C/EBPβ, PLD1, and C/EBPα which is a key transcription factor of late adipocyte differentiation. Among the three inducers, IBMX+any other inducer induced mild adipocyte differentiation, whereas insulin+dexamethasone did not. IBMX increased PLD1 but not PLD2 mRNA. Forskolin, an adenylate cyclase activator, and dbcAMP also increased PLD1 mRNA, suggesting the cellular cAMP as the inducer of both adipocyte differentiation and PLD1 transcription. We focused on the regulatory mechanism of PLD1 transcription during this differentiation process. IDX or a combination of inducers including IBMX increased PLD1 promoter activity, which is consistent with mRNA analysis. Promoter analysis identified two adjacent C/EBP motifs located between -338 and -231 bp from the first exon as the IBMX responsive elements. Furthermore, overexpression of C/EBPβ, but not C/EBPα, increased PLD1 mRNA and PLD1 5′ promoter activity. EMSA and chromatin immunoprecipitation assay confirmed the direct binding of C/EBPβ, but not C/EBPα, to these C/EBP motifs of PLD1 5′ promoter. Our results show that PLD1 is a target gene of C/EBPβ through the increased cellular cAMP during early adipocyte differentiation of 3T3-L1 cells.",
author = "Siqiang Gao and Hiromi Ito and Masashi Murakami and Kayo Yoshida and Yoko Tagawa and Kazumi Hagiwara and Akira Takagi and Tetsuhito Kojima and Motoshi Suzuki and Yoshiko Banno and Kenji Ohguchi and Yoshinori Nozawa and Takashi Murate",
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Gao, S, Ito, H, Murakami, M, Yoshida, K, Tagawa, Y, Hagiwara, K, Takagi, A, Kojima, T, Suzuki, M, Banno, Y, Ohguchi, K, Nozawa, Y & Murate, T 2010, 'Mechanism of increased PLD1 gene expression during early adipocyte differentiation process of mouse cell line 3T3-L1', Journal of Cellular Biochemistry, vol. 109, no. 2, pp. 375-382. https://doi.org/10.1002/jcb.22414

Mechanism of increased PLD1 gene expression during early adipocyte differentiation process of mouse cell line 3T3-L1. / Gao, Siqiang; Ito, Hiromi; Murakami, Masashi; Yoshida, Kayo; Tagawa, Yoko; Hagiwara, Kazumi; Takagi, Akira; Kojima, Tetsuhito; Suzuki, Motoshi; Banno, Yoshiko; Ohguchi, Kenji; Nozawa, Yoshinori; Murate, Takashi.

In: Journal of Cellular Biochemistry, Vol. 109, No. 2, 01.02.2010, p. 375-382.

Research output: Contribution to journalArticle

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T1 - Mechanism of increased PLD1 gene expression during early adipocyte differentiation process of mouse cell line 3T3-L1

AU - Gao, Siqiang

AU - Ito, Hiromi

AU - Murakami, Masashi

AU - Yoshida, Kayo

AU - Tagawa, Yoko

AU - Hagiwara, Kazumi

AU - Takagi, Akira

AU - Kojima, Tetsuhito

AU - Suzuki, Motoshi

AU - Banno, Yoshiko

AU - Ohguchi, Kenji

AU - Nozawa, Yoshinori

AU - Murate, Takashi

PY - 2010/2/1

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N2 - A mouse cell line 3T3-L1 is differentiated into adipocytes when treated with an inducer cocktail (IDX) (insulin, dexametahsone, and a cAMP phosphodiesterase inhibitor of isobutyl-methylxanthine (IBMX)). Here, we report that PLD1, but not PLD2, mRNA and protein increased during the early differentiation process. Our analysis shows that IDX resulted in a sequential induction of C/EBPβ, PLD1, and C/EBPα which is a key transcription factor of late adipocyte differentiation. Among the three inducers, IBMX+any other inducer induced mild adipocyte differentiation, whereas insulin+dexamethasone did not. IBMX increased PLD1 but not PLD2 mRNA. Forskolin, an adenylate cyclase activator, and dbcAMP also increased PLD1 mRNA, suggesting the cellular cAMP as the inducer of both adipocyte differentiation and PLD1 transcription. We focused on the regulatory mechanism of PLD1 transcription during this differentiation process. IDX or a combination of inducers including IBMX increased PLD1 promoter activity, which is consistent with mRNA analysis. Promoter analysis identified two adjacent C/EBP motifs located between -338 and -231 bp from the first exon as the IBMX responsive elements. Furthermore, overexpression of C/EBPβ, but not C/EBPα, increased PLD1 mRNA and PLD1 5′ promoter activity. EMSA and chromatin immunoprecipitation assay confirmed the direct binding of C/EBPβ, but not C/EBPα, to these C/EBP motifs of PLD1 5′ promoter. Our results show that PLD1 is a target gene of C/EBPβ through the increased cellular cAMP during early adipocyte differentiation of 3T3-L1 cells.

AB - A mouse cell line 3T3-L1 is differentiated into adipocytes when treated with an inducer cocktail (IDX) (insulin, dexametahsone, and a cAMP phosphodiesterase inhibitor of isobutyl-methylxanthine (IBMX)). Here, we report that PLD1, but not PLD2, mRNA and protein increased during the early differentiation process. Our analysis shows that IDX resulted in a sequential induction of C/EBPβ, PLD1, and C/EBPα which is a key transcription factor of late adipocyte differentiation. Among the three inducers, IBMX+any other inducer induced mild adipocyte differentiation, whereas insulin+dexamethasone did not. IBMX increased PLD1 but not PLD2 mRNA. Forskolin, an adenylate cyclase activator, and dbcAMP also increased PLD1 mRNA, suggesting the cellular cAMP as the inducer of both adipocyte differentiation and PLD1 transcription. We focused on the regulatory mechanism of PLD1 transcription during this differentiation process. IDX or a combination of inducers including IBMX increased PLD1 promoter activity, which is consistent with mRNA analysis. Promoter analysis identified two adjacent C/EBP motifs located between -338 and -231 bp from the first exon as the IBMX responsive elements. Furthermore, overexpression of C/EBPβ, but not C/EBPα, increased PLD1 mRNA and PLD1 5′ promoter activity. EMSA and chromatin immunoprecipitation assay confirmed the direct binding of C/EBPβ, but not C/EBPα, to these C/EBP motifs of PLD1 5′ promoter. Our results show that PLD1 is a target gene of C/EBPβ through the increased cellular cAMP during early adipocyte differentiation of 3T3-L1 cells.

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