Functional analysis of pig myostatin gene promoter with some adipogenesis- and myogenesis-related factors
Functional analysis of pig myostatin gene promoter with some adipogenesis- and myogenesis-related factors
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DOI：
10.1007/s11010-011-1181-y
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发表时间：
2012-04-01
影响因子：
4.3
通讯作者：
Jiang, Siwen
中科院分区：
文献类型：
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作者：
Deng, Bing;Wen, Jianghui;Jiang, Siwen
肌生长抑制素（Myostatin，MSTs）主要表达于肌肉中，在猪的肌肉和脂肪发育中起重要作用。然而，关于猪的监管信息很少。为了阐明猪Mcl 2基因是否受肌肉和脂肪相关因子的调控，本研究扩增了猪Mcl 2基因启动子，将其克隆到pGL 3-basic载体中，并转染细胞，通过双荧光素酶报告基因分析了猪Mcl 2基因启动子与肌肉和脂肪相关因子的转录活性。5 '端缺失表达结果显示，在-1519 ~-1236 bp之间存在负调控区，在-1236 ~-568 bp之间存在正调控区。最长的片段（1.7 kb）与肌肉相关的转录因子肌原性分化1（MyoD）共转染，导致启动子转录活性上调。用地塞米松、胰岛素和异丁基-1-甲基黄嘌呤（IBMX）等成脂剂（DIM）处理该片段。结果表明，IBMX能调控MMP 3启动子的转录活性，而DIM不能调控MMP 3启动子的转录活性。将DIM诱导的CCAAT/增强子结合蛋白（C/EBP）α和C/EBP β分别与1.7kb片段共转染，可下调MMP 3启动子的转录活性。用罗格列酮处理该片段，其诱导过氧化物酶体增殖物激活受体γ（PPAR γ）的表达，导致启动子转录活性上调。共转染实验证实了这一结果。综上所述，我们发现猪MMP 3可被IBMX、MyoD和PPAR γ上调，但被C/EBP α和C/EBP β下调。
Myostatin (MSTN) is primarily expressed in muscle and plays an important role in muscle and fat development in pigs. However, there is little information about the regulation of pig MSTN. In order to elucidate whether pig MSTN could be regulated by muscle- and fat-related factors, the porcine MSTN promoter was amplified and cloned into pGL3-basic vector, and transfected into cells to analyze the transcriptional activity of promoter with muscle- and fat-related factors through Dual-luciferase reporter assays. 5'-deletion expression showed that there was a negative-regulatory region located between nucleotides -1519 and -1236 bp, and there were some positive-regulatory regions located between -1236 and -568 bp. The longest fragment (1.7 kb) was cotransfected with muscle-related transcription factor myogenic differentiation 1 (MyoD), resulting in promoter transcriptional activity upregulation. The fragment was treated by the adipogenic agents (DIM) including dexamethasone, insulin, and isobutyl-1-methylxanthine (IBMX). We found that MSTN promoter transcriptional activity can be regulated by IBMX, but not by DIM. CCAAT/enhancer binding protein (C/EBP) alpha and C/EBP beta, two proteins which are induced by DIM during adipogenesis were cotransfected with the 1.7-kb fragment, respectively, resulting in promoter transcriptional activity downregulation. Treating the fragment with rosiglitazone which induce the expression of peroxisome proliferator-activated receptor gamma (PPAR gamma), resulting in promoter transcriptional activity upregulation. Cotransfection experiments confirmed this result. Taken together, we showed that porcine MSTN could be upregulated by IBMX, MyoD, and PPAR gamma but downregulated by C/EBP alpha and C/EBP beta.