Reactive oxygen species facilitate adipocyte differentiation by accelerating mitotic clonal expansion

Abstract

Growth-arrested 3T3-L1 preadipocytes rapidly express CCAAT/enhancer-binding protein-beta (C/EBPbeta) upon hormonal induction of differentiation. However, the DNA binding activity of C/EBPbeta is not activated until the cells synchronously reenter S phase during the mitotic clonal expansion (MCE) phase of differentiation. In this period, C/EBPbeta is sequentially phosphorylated by MAPK and glycogen synthase kinase-3beta, inducing C/EBPbeta DNA binding activity and transcription of its target genes. Because the DNA binding activity of C/EBPbeta is further enhanced by oxidation in vitro, we investigated how redox state affects C/EBPbeta DNA binding and MCE during adipogenesis. When 3T3-L1 cells were treated with H(2)O(2) and hormonal stimuli, differentiation was accelerated with increased expression of peroxisome proliferator-activated receptor gamma. Interestingly, cell cycle progression (S to G(2)/M phase) was markedly enhanced by H(2)O(2), whereas antioxidants caused an S phase arrest during the MCE. H(2)O(2) treatment resulted in the early appearance of a punctate pattern observed by immunofluorescent staining of C/EBPbeta, which is a hallmark for C/EBPbeta binding to regulatory elements, whereas a short antioxidant treatment rapidly dispersed the centromeric localization of C/EBPbeta. Consistently, reactive oxygen species production was increased during 3T3-L1 differentiation. Our results indicate that redox-induced C/EBPbeta DNA binding activity, along with the dual phosphorylation of C/EBPbeta, is required for the MCE and terminal differentiation of adipocytes.

FIGURE 1.

The effects of ROS on differentiation of 3T3-L1 preadipocytes. Two-day postconfluent 3T3-L1 preadipocytes were induced to differentiate with the standard hormonal regimen (IBMX, dexamethasone, and insulin) in the presence or absence of H₂O₂ (100 μm), genistein (50 μm), resveratrol (50 μm) (A), or N-acetyl cysteine (10 mm) (B). After 2 days, all groups were fed with FBS, DMEM, and insulin for another 2 days and then with FBS + DMEM. On day 8, cells were fixed, and cytoplasmic triglyceride was stained with oil red O. In addition, whole cell extracts from day 1, 2, and 3 adipocytes were prepared and subjected to immunoblotting with antibody against PPARγ. C, cells were differentiated under the standard regimen (IBMX, dexamethasone, and insulin; MDI) or an incomplete regimen, lacking insulin (MD). D, cell lysates prepared from differentiating preadipocytes were subjected to SDS-PAGE and immunoblotted with antibody against C/EBPβ. LAP and LIP, 38-kDa liver-activating protein and 18-kDa liver-inhibiting protein, respectively. E, 3T3-L1 cells were treated with C/EBPβ siRNA at about 70% confluence using Lipofectamine RNAi/MAX reagent. After 24 h, medium was replaced with fresh DMEM containing 10% calf serum for 24 h before the induction of differentiation. Knockdown of C/EBPβ was verified by immunoblotting, and cells were differentiated with or without H₂O₂ (2-day treatment) and subjected to oil red O staining at day 8. F, 3T3-F442A cells were differentiated in the presence or absence of H₂O₂ (10 μm), genistein (50 μm), epigallocatechin 3-gallate (EGCG; 50 μm), or N-acetyl cysteine (10 mm).

FIGURE 2.

FACS analysis during MCE of adipocyte differentiation. Cells were induced to differentiate in the presence or absence of H₂O₂, genistein (50 μm), or resveratrol (50 μm), and at the times indicated, cells were trypsinized and fixed. Changes in DNA content were analyzed by FACS using a FACSCalibur flow cytometry system, and data were analyzed using CellQuest software. FACS analysis was performed during 0–72 h of differentiation induction (A) and after 24 h of induction (B). C, cells were differentiated with the standard protocol for 16 h, after which resveratrol (50 μm) was added to the medium. FACS analysis was done at 24 h after induction of differentiation.

FIGURE 3.

Effects of ROS on cell number and expression of cyclin A during MCE. A, on day 2 of differentiation, cells were trypsinized and subjected to cell counting. Cells were treated with H₂O₂ (100 μm), resveratrol (50 μm), or genistein (50 μm) for 48 or 32 h, as indicated. B, cell lysates were prepared at the times indicated and subjected to SDS-PAGE and immunoblotting (IB) to measure the expression of cyclin A.

FIGURE 4.

Effects of ROS on intracellular localization of C/EBPβ during MCE. 3T3-L1 preadipocytes were induced to differentiate with the standard protocol in the presence or absence of H₂O₂ (100 μm), resveratrol (50 μm), or N-acetyl cysteine (10 mm), as indicated. Cells were fixed and subjected to immunofluorescence analysis with antibody against C/EBPβ and 4′,6-diamidino-2-phenylindole. Fluorescence images were obtained by confocal microscopy. Resveratrol (A) and N-acetyl cysteine (B) prevented the characteristic punctate pattern at 16 h of induction and also rapidly dispersed the centromeric localization after short term treatment (14–18 h of induction). C, H₂O₂ caused the early appearance of a punctate pattern in immunofluorescent stainings.

FIGURE 5.

DNA-binding of C/EBPβ protein, in vivo and in vitro. A, chromatin immunoprecipitation (IP) analysis using the PPARγ promoter. At the indicated times after induction of differentiation with or without H₂O₂ (100 μm) or resveratrol (50 μm), cells were cross-linked with formaldehyde, the DNA was fragmented, and the chromatin-associated DNA was immunoprecipitated with IgG or antibodies against C/EBPβ. PCR amplification of the DNA fragments was conducted with specific primers corresponding to the PPARγ₂ promoter sequence. Relative -fold changes of binding were calculated using a densitometer. B, EMSA of recombinant C/EBPβ protein treated by H₂O₂. Recombinant protein doubly phosphorylated by MAPK and GSK3β was treated with the indicated concentrations of H₂O₂ and/or 5 mm DTT and subjected to nonreducing SDS-PAGE, without DTT, and immunoblotting (IB) with anti-C/EBPβ antibody (top). The same reaction using 1 mm H₂O₂ was used for EMSA, except the reaction was terminated with 20 mm iodoacetamide at the indicated times, and 5 ng of protein was used for the binding reaction with the C/EBP site (bottom). DTT was added as marked (0–5 mm).

FIGURE 6.

ROS production during MCE in adipogenesis. ROS were detected with the peroxide-sensitive fluorophore DCF-DA. At the times indicated, cells were incubated with DCF-DA (10 μm) for 1 h and analyzed by the FACSCalibur flow cytometry system; data are presented as relative intracellular ROS levels. A, ROS levels during MCE in the presence or absence of H₂O₂ (100 μm), genistein (50 μm), or resveratrol (50 μm); B, 3T3-L1 preadipocytes were treated with IBMX (M), dexamethasone (D), or insulin (I), respectively; ROS production was measured, as described above.

FIGURE 7.

Resveratrol effects are independent of SIRT1 pathway. A, 3T3-L1 preadipocytes were subjected to RNA interference against siSIRT1. A ∼50% reduction of SIRT1 expression was confirmed by Western blotting, and FACS showed that SIRT1 knockdown did not affect the S phase accumulation of cells driven by resveratrol. B, ROS measurement during MCE from cells treated with aminoimidazole carboxamide ribonucleotide (AICAR) (1 mm) or sirtinol (20 μm).