Ruan ChongMei, Li Xiu, Hu JunJie, Zhang Yong, Zhao XingXu
College of Veterinary Medicine, Gansu Agriculture University, Lanzhou, 730070 China.
College of Animal Science and Technology, Anhui Agriculture University, Hefei, 230036 China.
Cell Mol Biol Lett. 2017 Jan 17;22:2. doi: 10.1186/s11658-016-0032-y. eCollection 2017.
PU box-binding protein (PU.1) is a master gene of hematopoietic lineage and an important specific transcription factor in osteoclast lineage. There is proof of its expression in adipose tissue, and it is known to significantly and negatively affect adipogenesis. However, it is unclear whether there are any other molecules involved in this process.
We wished to explore the effect of PU.1's co-activator microphthalmia-associated transcription factor (MITF) on the adipogenic differentiation of ovine primary preadipocytes. The expression vectors pcDNA-MITF and pcDNA-PU.1, and MITF siRNA and PU.1 siRNA were transfected or co-transfected into ovine tail primary preadipocytes. Real-time PCR and western blot analysis were applied to investigate the expression levels of PU.1 and MITF. The morphologic changes in the cells were observed under a microscope at a magnification of × 200 after staining with Oil Red O. The triglyceride (TG) content in cells was also determined after transfection.
MITF and its co-activator PU.1 synergistically exhibited an opposite expression pattern to that of CCAAT-enhancer-binding protein-β (C/EBPβ) during adipogenic differentiation of ovine primary preadipocytes. Before induction of differentiation, overexpression of MITF or PU.1 inhibited the expression of C/EBPβ and adipogenesis in the cells; and knockdown of MITF or PU.1 promoted the expression of C/EBPβ and adipogenesis in the cells. The inhibitory or promotive effect was enhanced when MITF and PU.1 were co-overexpressed or co-silenced. However, when MITF and/or PU.1 were overexpressed after day 2 of differentiation, no changes in adipogenesis of the cells were observed.
MITF and its co-activator PU.1 inhibited adipogenesis of ovine primary preadipocytes by restraining C/EBPβ.
PU盒结合蛋白（PU.1）是造血谱系的主控基因，也是破骨细胞谱系中一种重要的特异性转录因子。有证据表明其在脂肪组织中表达，并且已知它会对脂肪生成产生显著的负面影响。然而，尚不清楚在此过程中是否有其他分子参与。
我们希望探究PU.1的共激活因子小眼相关转录因子（MITF）对绵羊原代前体脂肪细胞成脂分化的影响。将表达载体pcDNA-MITF和pcDNA-PU.1，以及MITF siRNA和PU.1 siRNA转染或共转染到绵羊尾部原代前体脂肪细胞中。应用实时PCR和蛋白质印迹分析来研究PU.1和MITF的表达水平。用油红O染色后，在200倍显微镜下观察细胞的形态变化。转染后还测定了细胞中的甘油三酯（TG）含量。
在绵羊原代前体脂肪细胞成脂分化过程中，MITF及其共激活因子PU.1协同呈现出与CCAAT增强子结合蛋白β（C/EBPβ）相反的表达模式。在诱导分化前，MITF或PU.1的过表达抑制了细胞中C/EBPβ的表达和成脂作用；而MITF或PU.1的敲低则促进了细胞中C/EBPβ的表达和成脂作用。当MITF和PU.1共过表达或共沉默时，抑制或促进作用增强。然而，在分化第2天后过表达MITF和/或PU.1时，未观察到细胞成脂作用的变化。
MITF及其共激活因子PU.1通过抑制C/EBPβ来抑制绵羊原代前体脂肪细胞的成脂作用。
