title: Mechanism of increased PLD1 gene expression during early adipocyte differentiation process of mouse cell line 3T3-L1.
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/EBPbeta, PLD1, and C/EBPalpha 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/EBPbeta, but not C/EBPalpha, increased PLD1 mRNA and PLD1 5' promoter activity. EMSA and chromatin immunoprecipitation assay confirmed the direct binding of C/EBPbeta, but not C/EBPalpha, to these C/EBP motifs of PLD1 5' promoter. Our results show that PLD1 is a target gene of C/EBPbeta through the increased cellular cAMP during early adipocyte differentiation of 3T3-L1 cells.
authors: Department of Medical Technology, Nagoya University Graduate School of Health Sciences, Nagoya, Japan.
authors: Siqiang
authors: S
authors: Gao
authors: Hiromi
authors: H
authors: Ito
authors: Masashi
authors: M
authors: Murakami
authors: Kayo
authors: K
authors: Yoshida
authors: Yoko
authors: Y
authors: Tagawa
authors: Kazumi
authors: K
authors: Hagiwara
authors: Akira
authors: A
authors: Takagi
authors: Tetsuhito
authors: T
authors: Kojima
authors: Motoshi
authors: M
authors: Suzuki
authors: Yoshiko
authors: Y
authors: Banno
authors: Kenji
authors: K
authors: Ohguchi
authors: Yoshinori
authors: Y
authors: Nozawa
authors: Takashi
authors: T
authors: Murate
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology*; D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism; D050156:Adipogenesis / Q000235:genetics*; D000818:Animals; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism*; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D007328:Insulin / Q000494:pharmacology; D051379:Mice; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010739:Phospholipase D / Q000235:genetics* / Q000378:metabolism; D020218:Response Elements
chemical_list: D022763:CCAAT-Enhancer-Binding Protein-alpha; D022782:CCAAT-Enhancer-Binding Protein-beta; D007328:Insulin; D010726:Phosphodiesterase Inhibitors; D003907:Dexamethasone; D000242:Cyclic AMP; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1; D015056:1-Methyl-3-isobutylxanthine