Abstract
Adipose tissue stores energy and plays an important role in energy homeostasis. CCAAT/enhancer-binding protein β (C/EBPβ) is an important early transcription factor for 3T3-L1 preadipocyte differentiation, facilitating mitotic clonal expansion (MCE) and transactivating C/EBPα and peroxisome proliferator-activated receptor-γ (PPARγ) to promote adipogenesis. C/EBPβ is induced early, but the expression of antimitotic C/EBPα and PPARγ is not induced until ∼48 h. The delayed expression of C/EBPα and PPARγ is thought to ensure MCE progression, but the molecular mechanism for this delay remains elusive. Here, we show that the zinc-finger transcription factor Krüppel-like factor 10 (KLF10) is induced after adipogenic induction and that its expression positively correlates with that of C/EBPβ but inversely correlates with expression of C/EBPα and PPARγ. C/EBPβ bound to the KLF10 promoter and transactivated its expression during MCE. KLF10 overexpression in 3T3-L1 preadipocyte repressed adipogenesis and decreased C/EBPα and PPARγ expression, whereas siRNA-mediated down-regulation of KLF10 enhanced adipogenesis and increased C/EBPα and PPARγ expression. Luciferase assays revealed an inhibitory effect of KLF10 on C/EBPα promoter activity. Using promoter deletion and mutation analysis, we identified a KLF10-binding site within the proximal promoter region of C/EBPα. Furthermore, KLF10 interacted with and recruited histone deacetylase 1 (HDAC1) to the C/EBPα promoter, decreasing acetylated histone H4 on the C/EBPα promoter and inactivating C/EBPα transcription. Because C/EBPα can transactivate PPARγ, our results suggest a mechanism by which expression of C/EBPα and PPARγ is delayed via KLF10 expression and shed light on the negative feedback loop for C/EBPβ-regulated adipogenesis in 3T3-L1 preadipocyte.
Highlights
Adipose tissue stores energy and plays an important role in energy homeostasis
Because CCAAT/enhancer-binding proteins (C/EBP)␣ can transactivate PPAR␥, our results suggest a mechanism by which expression of C/EBP␣ and PPAR␥ is delayed via Krüppel-like factor 10 (KLF10) expression and shed light on the negative feedback loop for C/EBP-regulated adipogenesis in 3T3-L1 preadipocyte
Overexpression of C/EBP enhanced (Fig. 2, E and F), while knockdown of C/EBP impaired (Fig. 2, G and H), the expression of KLF10 at 24 h postinduction. These results demonstrate that KLF10 is a bona fide target of C/EBP during the mitotic clonal expansion (MCE) of 3T3-L1 preadipocyte differentiation
Summary
C/EBP, CCAAT/enhancer-binding protein ; C/EBP␣, CCAAT enhancer-binding protein ␣; HDAC1, histone deacetylase 1; KLF10, Krüppel-like factor 10; MCE, mitotic clonal expansion; PPAR␥, peroxisome proliferator–activated receptor ␥; qPCR, real-time quantitative PCR; TSA, trichostatin A. KLF4 is expressed in the early stage of the differentiation and promotes adipogenesis by inducing C/EBP expression [15]. KLF5, which is induced by C/EBP/␦, activates PPAR␥ expression and promotes adipocyte differentiation [16]. According to the database online, KLF10 is widely expressed in many tissues and organs, especially in bone marrow, lung, and skin. Our laboratory has identified a series of potential target genes of C/EBP by using ChIP-on-chip analysis at the early stage of 3T3-L1 preadipocyte differentiation (20 h after induction) [27]. KLF10 may be involved in the self-restricted pathway of C/EBP, which could help to maintain the adipogenesis at an appropriate level
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