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

✍ Scribed by 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

Publisher
John Wiley and Sons
Year
2009
Tongue
English
Weight
295 KB
Volume
109
Category
Article
ISSN
0730-2312

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✦ Synopsis

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/EBPb, PLD1, and C/EBPa which is a key transcription factor of late adipocyte differentiation. Among the three inducers, IBMX þ any other inducer induced mild adipocyte differentiation, whereas insulin þ 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 À338 and À231 bp from the first exon as the IBMX responsive elements. Furthermore, overexpression of C/EBPb, but not C/EBPa, increased PLD1 mRNA and PLD1 5 0 promoter activity. EMSA and chromatin immunoprecipitation assay confirmed the direct binding of C/EBPb, but not C/EBPa, to these C/EBP motifs of PLD1 5 0 promoter. Our results show that PLD1 is a target gene of C/EBPb through the increased cellular cAMP during early adipocyte differentiation of 3T3-L1 cells.

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