Original Contribution
Nuclear factor erythroid-derived factor 2-related factor 2 regulates transcription of CCAAT/enhancer-binding protein β during adipogenesis

https://doi.org/10.1016/j.freeradbiomed.2011.10.453Get rights and content

Abstract

Nuclear factor erythroid-derived factor 2-related factor 2 (Nrf2) is a cap-n-collar basic leucine zipper transcription factor that is involved in the cellular adaptive response to oxidative stress. Our previous study reported that targeted disruption of the Nrf2 gene in mice decreases adipose tissue mass and protects against obesity induced by a high-fat diet. Deficiency of Nrf2 in preadipocytes and mouse embryonic fibroblasts led to impaired adipogenesis. Consistent with these findings, the current study found that lack of Nrf2 in primary cultured mouse preadipocytes and 3T3-L1 cells hampered adipogenic differentiation induced by hormonal cocktails. Stable knockdown of Nrf2 in 3T3-L1 cells blocked the enhanced adipogenesis caused by deficiency of kelch-like ECH-associated protein 1 (Keap1), a Cul3-adapter protein that allows for Nrf2 to be ubiquinated and degraded by the 26S protesome complex. In addition, increased production of reactive oxygen species (ROS) and activation of Nrf2 occurred at the very early stage upon adipogenic hormonal challenge in 3T3-L1 cells, followed by an immediate induction of CCAAT/enhancer-binding protein β (C/EBPβ). Knockdown of Nrf2 led to reduced expression of C/EBPβ induced by adipogenic hormonal cocktails, chemical Nrf2 activators or Keap1 silencing. Cebpβ promoter-driven reporter assays and chromatin immunoprecipitation suggested that Nrf2 associates with a consensus antioxidant response element (ARE) binding site in the promoter of the Cebpβ gene during adipogenesis and upregulates its expression. These findings demonstrate a novel role of Nrf2 beyond xenobiotic detoxification and antioxidant response, and suggest that Nrf2 is one of the transcription factors that control the early events of adipogenesis by regulating expression of Cebpβ.

Graphical abstract

Highlights

► Lack of Nrf2 in preadipocytes hampers adipogenic differentiation. ► Knockdown of Nrf2 blocks the enhanced adipogenesis caused by Keap1 silencing. ► Knockdown of Nrf2 leads to reduced expression of C/EBPβ. ► Nrf2 binds to an ARE in the promoter of Cebpβ gene during adipogenesis. ► Nrf2 is one of the nuclear factors that control the early events of adipogenesis.

Introduction

Adipogenesis (differentiation of adipogenic precursor cells, so called preadipocytes, into adipocytes) is regulated by a complex network of transcription factors that coordinate expression of hundreds of proteins responsible for establishing the mature fat-cell phenotype [1], [2], [3], [4]. While it was firmly established that peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) are the terminal factors responsible for adipogenesis, the signaling cascade underlying the differentiation process, especially the early events, are only partially resolved.

C/EBPs, including C/EBPα, C/EBPβ, C/EBPγ, C/EBPδ, C/EBPε and C/EBPζ (also known as CHOP10), belong to basic region/leucine zipper (bZIP) transcription factors and are expressed in adipocytes [2]. C/EBPβ and C/EBPδ are transiently expressed and function at an early stage of differentiation by sensing adipogenic stimuli and initiating expression of PPARγ and C/EBPα [5], [6]. C/EBPα and PPARγ, forming a positive feedback loop by activating each other's expression, play roles at a later stage by inducing and maintaining expression of adipocyte-specific genes [7], [8]. Although the forced expression of C/EBPα in fibroblasts can trigger adipogenic differentiation, C/EBPα is incapable of inducing adipogenesis in the absence of PPARγ [9]. In contrast, PPARγ can induce adipogenic differentiation in C/EBPα-null cells indicating that PPARγ is sufficient in effecting adipogenesis [10]. C/EBPβ is thought to initiate mitotic clonal expansion of preadipocytes and to later coordinate the transcription network by turning on C/EBPα and PPARγ [11]. The induction of C/EBPβ occurs rapidly (within 2–4 h) on stimulation of differentiation [12], whereas the acquisition of DNA binding activity by C/EBPβ requires various posttranslational modifications, including phosphorylation, acetylation, disulfide bond formation and dimerization [11], [13], [14], [15]. Apart from cAMP response element-binding protein (CREB) [16], early growth response 2 (EGR2, also known as KROX20) [17] and Kruppel-like factor 4 (KLF4) [18], relatively few transcription factors have been identified that bind to the C/EBPβ promoter and positively regulate its transcription during adipogenesis. Other C/EBP isoforms, including C/EBPγ and CHOP10, seem to suppress adipogenesis [19].

Nuclear factor erythroid-derived factor 2-related factor 2 (Nrf2) belongs to the cap-n-collar subfamily of bZIP transcription factors [20], which also includes Nrf1 [21], Nrf3 [22] and the NF-E2 p45 subunit [23], as well as the more distantly related factor BTB and CNC homology 1 (BACH1) and BACH2 [24]. In response to oxidative/electrophilic stress Nrf2 heterodimerizes with small Maf proteins, as well as other bZIP proteins, bind to cis-acting element(s) termed antioxidant response elements (AREs) in the promoters of target genes [25], inducing transcriptional responses. ARE-dependent genes encode various enzymes, including detoxification enzymes such as glutathione S-transferases (GSTs), NAD(P)H quinone oxidoreductase 1 (NQO1), and heme oxygenase 1 (HO-1), as well as antioxidant enzymes such as catalase (CAT), sulfiredoxin (SRX), γ-glutamate cysteine ligase catalytic subunit (GCLC) and regulatory subunit (GCLM). Nrf2 therefore plays a central role in the cellular adaptive response to oxidative stress. Although Nrf2 is abundantly expressed in adipose tissue, its function in adipocyte biology has not been fully explored.

Our previous study [26] showed that mice deficient in Nrf2 display decreased fat mass in association with small adipocytes and are resistant to high-fat diet (HFD)-induced obesity. Mouse embryonic fibroblasts (MEFs) deficient in Nrf2 have impaired adipogenesis, and in 3T3-L1 and human subcutaneous preadipocytes, knockdown of Nrf2 expression inhibits adipogenic differentiation. Conversely, stable knockdown of kelch-like ECH-associated protein 1 (Keap1), which is a Cul3-adapter protein that allows for Nrf2 to be ubiquinated and degraded by the 26S protesome complex [27], leads to accelerated and enhanced adipogenesis in 3T3-L1 cells. In addition, we showed that the impaired adipogenesis induced by knockout or knockdown of Nrf2 is related at least in part to down-regulation of PPARγ expression. The current study explores the mechanisms further and reveals that Nrf2 is involved in transcriptional regulation of C/EBPβ expression during adipogenesis. These findings demonstrate a novel biologic role of Nrf2 beyond its participation in detoxification and antioxidant pathways, and suggest Nrf2 is one of the transcription factors that control the early events of adipogenesis by regulating expression of Cebpβ.

Section snippets

Reagents

Insulin solution (human, I9278), 3-isobutyl-1-methylxanthine (IBMX, I7018), dexamethasone (D1756), indomethacin (I7378), sulforaphane (SFN, S6317), tert-butylhydroquinone (tBHQ, #19986), sodium arsenite (iAs, #71287), Oil-red O (ORO, #75087), N-acetyl-L-cysteine (NAC, A7250), glutathione reduced ethyl ester (GSH-EE, G1404), diphenyleneiodonium (DPI, D2926), mesoxalonitrile 4-trifluoromethoxyphenylhydrazone (FCCP, C2920), diethyl maleate (DEM, W505005) and puromycin (P8833) were obtained from

Deficiency of Nrf2 impairs adipogenic differentiation in mouse preadipocytes

To determine the regulatory role of Nrf2 in adipogenesis, preadipocytes derived from white adipose tissues (WAT) of Nrf2-knockout (Nrf2−/−) and wildtype (Nrf2+/+) mice were isolated, cultured and differentiated in vitro. As shown in Fig. 2A, on the day 5 of hormonal cocktail DMIRI-induced adipogenesis Nrf2−/− preadipocytes showed substantially reduced levels of lipid accumulation, revealing lack of Nrf2 expression impairs adipogenic differentiation in preadipocytes. Consistent with the

Discussion

The current model for adipogenesis begins with increased expression of C/EBPβ presumably through transcriptional activation by CREB, KLF4 and KROX20 [16], [17], [18], [41]. C/EBPβ then induces expression of PPARγ and C/EBPα, which leads to the expression of factors associated with a mature adipocyte phenotype. Therefore, identifying the factors that regulate C/EBPβ expression as well as other factors that cooperate with other C/EBPs should provide additional insight into the mechanisms

Acknowledgments

The content is solely the responsibility of the authors. All authors have agreed to its content and there are no financial or other conflicts of interest. Y.H., P.X., C.G.W, K.Y., J.F., Q.Z., M.E.A. and J.P. are employees of The Hamner Institutes for Health Sciences. The Hamner is a 501(c)3 not-for-profit organization that has a diverse research portfolio that includes funding from the American Chemistry Council, a trade association that represents chemical manufacturers. This work was

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