Abstract
Distal-less homeobox 5 (Dlx5) is a negative regulator of adipogenesis. Dlx5 expression is decreased by adipogenic stimuli, but the mechanisms of Dlx5 downregulation by adipogenic stimuli have not yet been determined. Here, we tested the impact of cAMP/PKA (protein kinase A) signaling induced by 3-isobutyl-1 methyl xanthine (IBMX), forskolin, and 8-CPT-cAMP on the expression of Dlx5 in 3T3-L1 preadipocytes. Significant downregulation of Dlx5 mRNA expression and protein production levels were observed via cAMP/PKA-dependent signaling. Forced expression of cAMP-responsive element-binding protein (CREB) and CCAAT/enhancer-binding protein β (C/EBPβ) was sufficient for downregulation of Dlx5 expression and revealed that CREB functions upstream of C/EBPβ. In addition, C/EBPβ knockdown by siRNA rescued Dlx5 expression in IBMX-treated 3T3-L1 preadipocytes. Luciferase assays using a Dlx5-luc-2935 reporter construct demonstrated the requirement of the Dlx5 promoter region, ranging from −774 to −95 bp that contains two putative C/EBPβ binding elements (site-1: −517 to −510 bp and site-2: −164 to −157 bp), in the suppression of Dlx5 transcription. Consequently, chromatin immunoprecipitation analysis confirmed the importance of site-1, but not site-2, in C/EBPβ binding and transcriptional suppression of Dlx5. In conclusion, we elucidated the underling mechanism of Dlx5 downregulation in IBMX-induced adipogenesis. IBMX activated cAMP/PKA/CREB signaling and subsequently upregulated C/EBPβ, which binds to the Dlx5 promoter to suppress Dlx5 transcription.
Highlights
Obesity has become a rising issue related with various health problems in modern society [1]
Because our previous study demonstrated that an adipogenesis induction mixture suppresses Distal-less homeobox 5 (Dlx5) expression [4], we first examined the effect of each component of the induction mix on the expression of Dlx5. 3T3-L1 cells were incubated for 24 h in the presence of each adipogenic component (0.5 mM isobutyl-1 methyl xanthine (IBMX), 0.1 μM dexamethasone, 10 μg/mL insulin, or 50 μM indomethacin), and Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and Western blot analyses were performed to detect Dlx5 mRNA and protein expression, respectively
Because Dlx5 promoter activity was suppressed by CCAAT/enhancer-binding protein β (C/EBPβ) (Figure 4B,C), we examined whether the binding of RNA polymerase II (pol II) to the Dlx5 promoter region was affected by C/EBPβ
Summary
Obesity has become a rising issue related with various health problems in modern society [1]. Fat tissue is mainly composed of adipocytes that play an important role in energy metabolism; studying the molecular mechanisms of adipogenic differentiation and its regulatory factors might produce clues to the etiology of obesity and other metabolic diseases. Distal-less homeobox 5 (Dlx5) is a well-known transcription factor involved in bone development. Dlx upregulates osteogenesis by activating Runx, a master transcriptional regulator for osteoblast differentiation [2]. Bone morphogenetic protein 2 (Bmp2) induces Dlx expression, which in turn binds to the Runx promoter and enhances Runx expression. Dlx mediates Bmp2-induced expression of Sp7, another key transcription factor for osteoblastic differentiation [3]. Among the many constituents responsible for adipogenesis induction, insulin downregulates Dlx expression by inducing miR-124 [5]. The mechanism by which Dlx expression is inhibited has not yet been characterized
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