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.

cAMP/Protein Kinase A Signaling Inhibits Dlx5 Expression via Activation of CREB and Subsequent C/EBPβ Induction in 3T3-L1 Preadipocytes

“…IBMX is an inhibitor of cAMP phosphodiesterase, resulting in accumulation of cAMP and activation of PKA. Therefore, we next examined whether accumulation of cAMP exerts a Dlx5 inhibitory effect using IBMX, a cell-permeable cAMP analogue (8-CPT-cAMP), or the adenylate cyclase activator forskolin [ 8 , 15 , 16 ]. 3T3-L1 cells were incubated for 24 h in the presence of IBMX (0.05, 0.1, or 0.5 mM), forskolin (10 or 50 µM), or 8-CPT-cAMP (0.02, 0.2, or 2 mM), followed by qRT-PCR of the Dlx5 gene.…”

“…Forskolin induces cAMP accumulation by activating the adenylate cyclase. IBMX, prostacyclin, and 8-CPT-cAMP are well known adipogenic inducers through the cAMP induction and PKA activation pathway [ 8 , 15 , 20 ]. When 3T3-L1 preadipocytes were induced with IBMX, forskolin, or 8-CPT-cAMP, Dlx5 expression was suppressed, suggesting that the suppressive effects of IBMX, forskolin, and 8-CPT-cAMP on Dlx5 expression may be ascribed to cAMP-dependent signaling pathways.…”

“…3T3-L1 preadipocytes are a well-established in vitro cell culture model to study adipogenic differentiation [ 6 ]. 3T3-L1 preadipocytes are converted to adipocytes by an induction mixture containing 3-isobutyl-1-methylxanthine (IBMX), insulin, dexamethasone, and indomethacin [ 7 ] that causes induction of transcriptional regulators such as CCAAT/enhancer binding protein (C/EBP) β, δ, and α and PPARγ, leading to differentiation of preadipocytes toward mature adipocytes [ 8 ]. The cAMP-response element binding protein (CREB) is another key transcriptional regulator of adipogenesis [ 9 , 10 , 11 ] that is constitutively expressed in preadipocytes and throughout the differentiation process toward mature adipocytes [ 10 , 12 ].…”

The progesterone receptor as a transcription factor regulates phospholipase D1 expression through independent activation of protein kinase A and Ras during 8-Br-cAMP-induced decidualization in human endometrial stromal cells

“…The region between − 204 and − 74 also contained four binding sites for Sp1 and three binding sites for GR (glucocorticoid receptor). Gao et al [42,43] reported that dexamethasone treatment has no effect on PLD1 promoter activity, but Sp1 is important in Rasinduced PLD1 expression . This result indicated that PR may regulate PLD1 expression with Sp1, not with GR, during decidualization of hES cells.…”