Choi Hojung, Park Sung-Soo, Kim Seung-Jin, Kim Eungseok
Deptartment of Biological Sciences College of Natural Sciences, Chonnam National University, 77 Yong-bong-ro, Buk-Gu, Gwangju, 61186 Republic of Korea.
College of Pharmacy and Gachon Institute of Pharmaceutical Science, Gachon University, 191 Hambakmoe-ro, Yeonsu-gu, Incheon, 21936 Republic of Korea.
Cell Biosci. 2020 Oct 15;10:119. doi: 10.1186/s13578-020-00482-4. eCollection 2020.
TR4, an orphan nuclear receptor plays a key role in glucose and lipid metabolism by regulating the expression of genes involved in energy metabolism. We previously reported that overexpression of TR4 in 3T3-L1 adipocytes promotes lipid accumulation in part by facilitating fatty acid uptake and synthesis, indicating that TR4 tightly regulates lipid homeostasis during adipogenesis. Here, we report that β-catenin suppresses TR4 transcriptional activity and that this inhibition is achieved through induction of Slug gene, a well-known transcription repressor in a variety of cells.
To generate the stable cell line, 3T3-L1 cells were transfected with plasmids then cultured in presence of geneticin and/or blasticidin for 2 weeks. The lipid accumulation was measured by Oil Red O. The TR4-Slug and TR4-β-catenin interactions were checked by GST pull-down and mammalian two-hybrid assay. The TR4 transcriptional activities on various promoters were measured by luciferase activity. To check the binding affinity of TR4, we performed the gel shift and chromatin immunoprecipitation (ChIP) assay. Gene expression was detected by RT-qPCR at the mRNA level and western blotting at the protein level.
Stable overexpression of Slug gene in 3T3-L1 preadipocytes strongly inhibited differentiation of 3T3-L1 preadipocytes. Using GST pull-down, gel shift and ChIP assays, we found that Slug abolished the formation of TR4 homodimers through direct interaction with TR4 and reduced the binding affinity of TR4 for its response elements located in TR4 target gene promoters such as fatty acid transport protein 1 and pyruvate carboxylase. Consistently, Slug inhibited TR4 target gene expression and was accompanied by repression of TR4-induced lipid accumulation in 3T3-L1 adipocytes.
Our results demonstrated that Slug inhibits 3T3-L1 adipogenesis through suppression of TR4 transcriptional activity.
TR4是一种孤儿核受体，通过调节参与能量代谢的基因表达，在葡萄糖和脂质代谢中发挥关键作用。我们之前报道过，在3T3-L1脂肪细胞中过表达TR4会部分通过促进脂肪酸摄取和合成来促进脂质积累，这表明TR4在脂肪生成过程中严格调节脂质稳态。在此，我们报道β-连环蛋白抑制TR4转录活性，且这种抑制是通过诱导Slug基因实现的，Slug基因是多种细胞中一种著名的转录抑制因子。
为构建稳定细胞系，将质粒转染至3T3-L1细胞，然后在含遗传霉素和/或杀稻瘟菌素的条件下培养2周。通过油红O法测定脂质积累。通过谷胱甘肽S-转移酶（GST）下拉实验和哺乳动物双杂交实验检测TR4与Slug以及TR4与β-连环蛋白的相互作用。通过荧光素酶活性测定TR4在各种启动子上的转录活性。为检测TR4的结合亲和力，我们进行了凝胶迁移和染色质免疫沉淀（ChIP）实验。通过逆转录定量聚合酶链反应（RT-qPCR）在mRNA水平和蛋白质印迹法在蛋白质水平检测基因表达。
在3T3-L1前脂肪细胞中稳定过表达Slug基因强烈抑制3T3-L1前脂肪细胞的分化。通过GST下拉实验、凝胶迁移实验和ChIP实验，我们发现Slug通过与TR4直接相互作用消除TR4同源二聚体的形成，并降低TR4对其位于TR4靶基因启动子（如脂肪酸转运蛋白1和丙酮酸羧化酶）上的反应元件的结合亲和力。一致地，Slug抑制TR4靶基因表达，并伴随着抑制TR4诱导的3T3-L1脂肪细胞中的脂质积累。
我们的结果表明，Slug通过抑制TR4转录活性来抑制3T3-L1脂肪生成。
