Yamamoto Joji, Ikeda Yukio, Iguchi Haruhisa, Fujino Takahiro, Tanaka Toshiya, Asaba Hiroshi, Iwasaki Satoshi, Ioka Ryoichi X, Kaneko Izumi W, Magoori Kenta, Takahashi Sadao, Mori Toshiyuki, Sakaue Hiroshi, Kodama Tatsuhiko, Yanagisawa Masashi, Yamamoto Tokuo T, Ito Sadayoshi, Sakai Juro
Division of Nephrology, Endocrinology, and Vascular Medicine, Department of Medicine, the Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan.
J Biol Chem. 2004 Apr 23;279(17):16954-62. doi: 10.1074/jbc.M312079200. Epub 2004 Feb 10.
Acetyl-CoA synthetase 2 (AceCS2) produces acetyl-CoA for oxidation through the citric acid cycle in the mitochondrial matrix. AceCS2 is highly expressed in the skeletal muscle and is robustly induced by fasting. Quantification of AceCS2 transcripts both in C2C12 and human myotubes indicated that fasting-induced AceCS2 gene expression appears to be independent on insulin action. Characterization of 5'-flanking region of the mouse AceCS2 gene demonstrates that Krüppel-like factor 15 (KLF15) plays a key role in the trans-activation of the AceCS2 gene. Deletion and mutation analyses of AceCS2 promoter region revealed that the most proximal KLF site is a curtail site for the trans-activation of the AceCS2 gene by KLF15. Using Sp-null Drosophila SL2 cells, we showed that the combination of KLF15 and Sp1 resulted in a synergistic activation of the AceCS2 promoter. Mutation analyses of three GC-boxes in the AceCS2 promoter indicated that the GC-box, located 8 bases downstream of the most proximal KLF15 site, is the most important GC-box in the synergistic trans-activation of the AceCS2 gene by KLF15 and Sp1. GST pull-down assays showed that KLF15 interacts with Sp1 in vitro. Quantification of various KLF transcripts revealed that 48 h fasting robustly induced the KLF15 transcripts in the skeletal muscle. Together with the trans-activation of the AceCS2 promoter, it is suggested that fasting-induced AceCS2 expression is largely contributed by KLF15. Furthermore, KLF15 overexpression induced the levels of AceCS2 transcripts both in myoblasts and in myotubes, indicating that AceCS2 gene expression in vivo is indeed induced by KLF15.
乙酰辅酶A合成酶2（AceCS2）通过线粒体基质中的柠檬酸循环产生用于氧化的乙酰辅酶A。AceCS2在骨骼肌中高度表达，并在禁食时强烈诱导。对C2C12和人肌管中AceCS2转录本的定量分析表明，禁食诱导的AceCS2基因表达似乎独立于胰岛素作用。对小鼠AceCS2基因5'-侧翼区域的表征表明，Krüppel样因子15（KLF15）在AceCS2基因的反式激活中起关键作用。AceCS2启动子区域的缺失和突变分析表明，最靠近近端的KLF位点是KLF15对AceCS2基因进行反式激活的关键位点。使用Sp基因缺失的果蝇SL2细胞，我们发现KLF15和Sp1的组合导致AceCS2启动子的协同激活。对AceCS2启动子中三个GC盒的突变分析表明，位于最靠近近端KLF15位点下游8个碱基处的GC盒是KLF15和Sp1协同反式激活AceCS2基因中最重要的GC盒。谷胱甘肽S-转移酶下拉实验表明，KLF15在体外与Sp1相互作用。对各种KLF转录本的定量分析表明，禁食48小时可强烈诱导骨骼肌中KLF15转录本的表达。结合AceCS2启动子的反式激活，提示禁食诱导的AceCS2表达很大程度上由KLF15促成。此外，KLF15的过表达在成肌细胞和肌管中均诱导了AceCS2转录本的水平，表明体内AceCS2基因表达确实由KLF15诱导。
