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.2004 Mar 19;279(12):11711-8.
doi: 10.1074/jbc.M310240200. Epub 2003 Dec 30.

Role of the POZ zinc finger transcription factor FBI-1 in human and murine adipogenesis

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Role of the POZ zinc finger transcription factor FBI-1 in human and murine adipogenesis

Matthias Laudes et al. J Biol Chem..

Abstract

Poxvirus zinc finger (POZ) zinc finger domain transcription factors have been shown to play a role in the control of growth arrest and differentiation in several types of mesenchymal cells but not, as yet, adipocytes. We found that a POZ domain protein, factor that binds to inducer of short transcripts-1 (FBI-1), was induced during both murine and human preadipocyte differentiation with maximal expression levels seen at days 2-4. FBI-1 mRNA was expressed in human adipose tissue with the highest levels found in samples from morbidly obese subjects. Murine cell lines constitutively expressing FBI-1 showed evidence for accelerated adipogenesis with earlier induction of markers of differentiation and enhanced lipid accumulation, suggesting that FBI-1 may be an active participant in the differentiation process. Consistent with the properties of this family of proteins in other cell systems, 3T3L1 cells stably overexpressing FBI-1 showed reduced DNA synthesis and reduced expression of cyclin A, cyclin-dependent kinase 2, and p107, proteins known to be involved in the regulation of mitotic clonal expansion. In addition, FBI-1 reduced the transcriptional activity of the cyclin A promoter. Thus, FBI-1, a POZ zinc finger transcription factor, is induced during the early phases of human and murine preadipocyte differentiation where it may contribute to adipogenesis through influencing the switch from cellular proliferation to terminal differentiation.

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Figures

Fig. 1
Fig. 1. Oligonucleotide microarray experiment
A, scatter plot of the average difference fluorescence intensity for each gene represented on the vehicle array and rosiglitazone array. The average difference fluorescence intensity is the average difference in fluorescence intensity between the perfect match oligonucleotides and the mismatch oligonucleotides for each gene. The average difference fluorescence intensities for those genes that were called “absent” on both arrays are not shown.B, confirmation of microarray results for two up-regulated (FABP-4 and FBI-1) and a down-regulated gene (ISGF-3) by semiquantitative RT-PCR. GAPDH expression was used as loading control.
Fig. 2
Fig. 2. FBI-1 expression in human adipose tissue and adipogenesis
A, after induction of differentiation of human preadipocytesin vitro, RNA was extracted at the given time points, and semiquantitative RT-PCR for FBI-1 was performed. A 2% agarose gel with ethidium bromide staining is shown. Data were normalized using the GAPDH control.B andC, real time PCR for FBI-1 expression in the stromovascular fractionversus mature adipocytes (B) and in whole adipose tissue of subjects with different body mass index (BMI) (C). Data are mean ± S.E. *,p < 0.05; **,p < 0.01; ***,p < 0.001.
Fig. 3
Fig. 3. FBI-1 expression in 3T3-L1 adipogenesis
A andB, semiquantitative RT-PCR for the mouse FBI-1 homologue LRF at different time points during differentiation of 3T3-L1 preadipocytes. Data were normalized by GAPDH expression.A, representative image of a 2% agarose gel stained by ethidium bromide.B, densitometry ofn = 3 independent experiments (mean ± S.E.). The effect of single components of the differentiation mixture (C) and of rosiglitazone (D) on FBI-1 expression during 3T3-L1 adipogenesis is shown. Results of the semiquantitative RT-PCR are shown on a 2% agarose gel with ethidium bromide staining. Data were normalized by GAPDH expression. *,p < 0.05.FCS, fetal calf serum;Ins, insulin;Dex, dexamethasone;IBMX, 1-methyl-3-isobutylxanthine;MDI, 1-methyl-3-isobutylxanthine + dexamethasone + insulin (full differentiation mixture);rel., relative.
Fig. 4
Fig. 4. Effect of FBI-1 on terminal differentiation
A, generation of FBI-1-overexpressing 3T3-L1 preadipocytes. Semiquantitative RT-PCR for FBI-1 expression in preconfluent 3T3-L1 preadipocytes (2% agarose gel, ethidium bromide staining) is shown.B, expression of molecular markers of differentiation on RNA level. Control cells and FBI-1-overexpressing 3T3-L1 preadipocytes were differentiated, and RNA was isolated before and 2 days after induction. Subsequently expression of the two molecular markers of adipogenesis aP2 and PPARγ2 was analyzed by real time PCR (Taqman). Results are shown as -fold difference compared with control cells of five independent experiments for PPARγ2 and three independent experiments for aP2.Gray columns, control cells;black columns, FBI-1 cells (mean ± S.E.).C, expression of molecular markers of differentiation on the protein level. Control cells and FBI-1-overexpressing 3T3-L1 preadipocytes were differentiated and lysed at the given time points, and whole cell proteins were subjected to Western blotting.D, oil red O staining. Control cells and FBI-1-overexpressing 3T3-L1 preadipocytes were differentiated and stained for lipids 6 days after induction of differentiation. *,p < 0.05.EV, empty vector control;rel., relative;C-EBPα, CCAAT/enhancer-binding proteinα.
Fig. 5
Fig. 5. Effect of FBI-1 on mitotic clonal expansion
A, thymidine incorporation assay. Control cells and FBI-1-overexpressing 3T3-L1 preadipocytes were differentiated into mature adipocytes. At the given time points, cells were incubated in medium containing [3H]thymidine for 60 min before cells were lysed, and activity was measured by scintillation counting. —, control cells; ---, FBI-1 cells (n = 4 experiments using two independently generated FBI-1-overexpressing cell lines, mean ± S.E.).B, expression of proteins involved in regulation of mitotic clonal expansion. Differentiation of control cells and FBI-1-overexpressing cells was induced, and total cellular protein was extracted at the given time points followed by Western blotting. *,p < 0.05.
Fig. 6
Fig. 6. Effect of FBI-1 on transcriptional activity
HepG2 cells were transiently transfected with 0.1μg of a promoter-reporter gene construct carrying the firefly luciferase under control of the mouse cyclin A promoter and the given amounts of pcDNA3.1-FBI-1. Transfection efficiency was normalized by co-transfection of a plasmid carrying theRenilla luciferase under control of the CMV promoter. Cells were lysed 48 h after transfection using FuGENE (n = 4 independent experiments, mean ± S.E.). **,p < 0.01.
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