C/EBPalpha activates the transcription of triacylglycerol hydrolase in 3T3-L1 adipocytes

Abstract

TGH (triacylglycerol hydrolase) catalyses the lipolysis of intracellular stored triacylglycerol. To explore the mechanisms that regulate TGH expression in adipose tissue, we studied the expression of TGH during the differentiation of 3T3-L1 adipocytes. TGH mRNA and protein levels increased dramatically in 3T3-L1 adipocytes compared with pre-adipocytes. Electrophoretic mobility shift assays demonstrated enhanced binding of nuclear proteins of adipocytes to the distal murine TGH promoter region (-542/-371 bp), yielding one adipocyte-specific migrating complex. Competitive and supershift assays demonstrated that the distal TGH promoter fragment bound C/EBPalpha (CCAAT/enhancer-binding protein alpha). Transient transfections of different mutant TGH promoter-luciferase constructs into 3T3-L1 adipocytes and competitive electromobility shift assays showed that the C/EBP-binding elements at positions -470/-459 bp and -404/-390 bp are important for transcriptional activation. Co-transfection with C/EBPalpha cDNA and TGH promoter constructs in 3T3-L1 pre-adipocytes demonstrated that C/EBPalpha increased TGH promoter activity. Ectopic expression of C/EBPalpha in NIH 3T3 cells activated TGH mRNA expression without causing differentiation into adipocytes. These experiments directly link increased TGH expression in adipocytes to transcriptional regulation by C/EBPalpha. This is the first evidence that C/EBPalpha participates directly in the regulation of an enzyme associated with lipolysis.

Figure 1. TGH expression and activity are induced during adipocyte differentiation

NIH 3T3-L1 fibroblasts were induced to differentiate into adipocytes. Day 0 represents 2 days post-confluence and the starting point of the differentiation programme. Cells were harvested at the indicated times, and RT-PCR analysis of TGH mRNA was performed. Total RNA (2 μg) was reverse-transcribed to cDNA, and TGH, aP2 and cyclophilin (CYC) mRNAs were detected by PCR. Results are representative of three independent experiments.

Figure 2. Binding of nuclear extracts to the TGH promoter (−542/+112)

Cell nuclear extracts were prepared from 3T3-L1 pre-adipocytes (P; day 0) and adipocytes (A; day 5) and used in binding assays with each of three ³²P-labelled promoter fragments. Protein–DNA complexes were resolved by non-denaturing 4%-PAGE with Tris/borate/EDTA buffer and were detected by autoradiography. The arrow shows the specific DNA–protein complex. These results are representative of three independent experiments, each using freshly isolated nuclear extracts.

Figure 3. Tissue-specific activity of the TGH promoter

(A) Schematic representation of the different truncated reporter constructs (−542 Luc, −370 Luc and pGL3-Basic) used in transfection assays. Transfections were performed as described in the Materials and methods section. Luciferase activity in 3T3-L1 pre-adipocytes and adipocytes is shown. Values are means±S.E.M. of three independent transfections, normalized to cotransfected β-galactosidase. (B) Luciferase activity in monkey Cos-7 cells. Each value is the mean±S.E.M. of three experiments.

Figure 4. Analysis of protein–DNA interactions using competitive EMSA

Band-shift assays were performed with nuclear extracts from 3T3-L1 pre-adipocytes (P; day 0) and adipocytes (A; day 5) using the distal (−542/−371 bp) promoter fragment as a probe. Unlabelled oligonucleotides (400 pmol) against CREB, Stat 5, Sp1 or C/EBP consensus sites were added to the binding assays as competitors, as indicated. The arrow shows the specific DNA–protein complex. The results are representative of three independent experiments.

Figure 5. EMSA-supershift analysis of proteins binding to the TGH promoter

Cell nuclear extracts were prepared from 3T3-L1 preadipocytes (P; day 0) and adipocytes (A; day 5) and used in binding assays with the ³²P-labelled distal (−542/−371 bp) promoter fragment with (+) or without (−) antibodies against C/EBPα, C/EBPβ or C/EBPδ. The arrow shows the specific DNA–protein complex. The results are representative of three independent experiments.

Figure 6. Analysis of C/EBPβ, C/EBPα and TGH proteins using immunoblotting

NIH 3T3-L1 fibroblasts were induced to differentiate into adipocytes. Day 0 represents 2 days post-confluence and the starting point of the differentiation programme. Cells were harvested at the indicated times. Proteins of cell homogenates (50 μg) were resolved on SDS/10%-polyacrylamide gels, electroblotted on to nitrocellulose membranes, and probed with antibodies against C/EBPβ, C/EBPα, TGH or protein disulphide isomerase (PDI). The arrows show three isoforms of C/EBPβ (40, 35 and 20 kDa) and two isoforms of C/EBPα (42 and 30 kDa) separately [22,23]. The results are representative of two independent experiments.

Figure 7. Ectopic expression of C/EBPα stimulates TGH promoter activity and mRNA expression

(A) The luciferase activity of the −542 Luc reporter vector is induced by C/EBPα in a dose-dependent fashion in 3T3-L1 pre-adipocytes. The TGH reporter plasmid −542 Luc (4 μg) was co-transfected into 3T3-L1 pre-adipocytes with pSV-β-galactosidase vector (4 μg) and the indicated concentrations of C/EBPα. Luciferase activity was measured relative to β-galactosidase activity. Values are means±S.E.M. of three experiments. (B) C/EBPα induces TGH mRNA expression in NIH 3T3 cells. C/EBPα or control vector pCDNA3.1(+) (2 μg) was transiently transfected into NIH 3T3 cells. After 48 h, cell homogenates were extracted and used to analyse C/EBPα and protein disulphide isomerase (PDI) expression by immunoblot analysis. TGH and cyclophilin (CYC) mRNAs were detected by RT-PCR. The results are representative of two independent experiments.

Figure 8. C/EBP-binding sites (−470 to −459 bp and −404 to −390 bp) in the distal fragment are important for TGH promoter activity

(A) EMSA was performed with nuclear extracts from 3T3-L1 adipocytes (day 5). A ³²P-labelled C/EBP consensus oligonucleotide was used as a probe. Unlabelled oligonucleotides (50×, 100×, 200× or 400×) derived from the TGH promoter (−470/−459 bp, 5′-TCTAAAGGTTGGTAAGTGTGAGTT-3′; −404/−390 bp, 5′-CAGGAAGGCTGTGAAATGTGTCCG-3′) or the C/EBP consensus [5′-TCTAAT(GT)(AGCT)(AGCT)G(AGCT)AA(GT)GAGTTC-3′] were added to the binding assays as competitors. The arrow shows the specific DNA–protein complex. The results are representative of two independent experiments. (B) Schematic representation of the different mutated reporter constructs (−542 Luc, −542 M1, −542 M2 and pGL3-Basic) used in transfection assays. Also shown are the results of trans-activation assays with different mutated TGH promoter–reporter constructs co-transfected with C/EBPα in 3T3-L1 pre-adipocytes. Values are means±S.E.M. of three independent transfections, normalized to co-transfected β-galactosidase. (C) Trans-activation assays of different mutated TGH promoter–reporter constructs transfected into 3T3-L1 adipocytes. Values are means±S.E.M. of three experiments.