ERR促进Glucokinase表达的功能研究PGC-1β调节ALAS1及线粒体相关基因的启动子转录机制的初步探讨

【作者】 刘阳；

【导师】 方福德； 常永生；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 孤儿核受体ERRα和ERRγ均能被PPARγ的共激活因子PGC-1α所激活,PGC-1α是细胞能量代谢的关键调节子。葡萄糖激酶在调节肝脏中葡萄糖的利用发挥重要的作用,同时胰岛素可以促进其表达。我们的研究发现,葡萄糖激酶的启动子区有ERRα和ERRγ的结合位点,通过染色质免疫沉淀法进一步明确ERRγ可以结合在葡萄糖激酶的启动子区。同时发现在大鼠肝原代细胞中PGC-1α可以共激活ERR促进葡萄糖激酶基因的表达。这表明ERR在调节葡萄糖代谢方面发挥着重要的作用。当对葡萄糖激酶启动子区的ERR的结合元件进行突变,PGC-1α和ERR的共激活作用受到了抑制。对葡萄糖激酶的启动子区进行了一系列的截短实验,PGC-1α和ERR可以显著的共激活全长的葡萄糖激酶的启动子,但当截短至不含ERR的结合元件后,这种共激活作用受到了显著抑制。通过用合成的ERRα的抑制剂XCT790对ERRα进行抑制,明确了ERR在胰岛素促进葡萄糖激酶表达中起到了一定的作用。进一步的实验表明ERR和共激活子PGC-1α共同促进了葡萄糖激酶的mRNA,蛋白以及酶活性的表达。以上结果表明ERR能直接激活肝中葡萄糖激酶的表达并可以促进2型糖尿病葡萄糖的稳态的形成。PGC-1β是近年来发现的转录共激活子,它的组织分布和序列都和PGC-1α有高度的相似性。但是它的许多生物学功能还不清楚。和PGC-1α相似,PGC-1β在分化的肌管细胞中可以显著地激活线粒体的生物合成并且可以促进细胞呼吸。血红素是血红蛋白的重要组成成分,它的功能包括氧的运输和能量代谢。ALAS1是血红素合成的限速酶,它为呼吸链中的细胞色素提供血红素。通过5’端系列截短实验,我们发现PGC-1β可以通过ALAS1启动子的NRF-1结合位点促进其表达。ALAS1启动子有两个NRF1的识别位点,这两个位点对于PGC-1β促进ALAS1的转录起着重要的作用。当对其中一个结合位点进行突变,则大约60-70%的ALAS1的启动子活性受到抑制。当将两个结合位点都进行突变,则大约90%的ALAS1的启动子活性受到抑制。为了进一步明确NRF-1结合位点对于PGC-1β促进ALAS1的转录起着重要的作用,我们将NRF-1的显性负突变和PGC-1β共转染HepG2细胞,这时PGC-1β对ALAS1的促进作用明显受到了抑制。PGC-1β同样也可以通过结合和激活NRF-1,促进线粒体相关基因的转录。为了明确NRF-1对于表达内源性的PGC-1β的靶基因是必需的,我们通过RNA干扰的方法来降低内源性的NRF-1的表达,使其表达降低约70%,然后通过real-timePCR来检测由PGC-1β调节的线粒体相关基因的mRNA水平。实验结果表明,PGC-1β可以促进线粒体相关基因如ALAS1, Cytc, ATPase, Tfb2m, Tfam, COX7b, MRPL46的表达。当通过RNAi干扰NRF-1的表达后,PGC-1β对线粒体相关基因的促进作用明显受到了抑制。实验结果显示,PGC-1β在调节线粒体氧化代谢方面起着非常重要的作用。更多还原

【Abstract】 The orphan nuclear receptor estrogen-related receptors(ERR a and ERRγ) are activated by the transcriptional coactivator peroxisome proliferator-activated receptorγ(PPARγ) coactivator 1α(PGC-1α), a critical regulator of cellular energy metabolism. Glucokinase plays a key role in the regulation of glucose utilization in liver and its expression is strongly enhanced by insulin. We report here that the regulatory sequences of the GK gene harbor a functional ERRαand ERRγbinding site and the co-stimulating effects of ERRs and PGC-1αon GK gene expression in the rat primary hepatocytes, suggesting a novel role for ERRs in controlling glucose metabolism. The co-stimulating effect was inhibited when the GK promoter fragment was mutated at the ERRE. In contrast to the dramatic effects of ERRs and PGC-1αon full length GK, they had only a small effect on the truncated GK construct that lacked the ERRE. By using a synthetic inhibitor of ERRα, the inhibitory result revealed that ERRαplay a key role in insulin stimulating GK expression. Furthermore, ERRαand ERRγincreased endogenous GK mRNA, protein and GK activity. Our data demonstrate that ERRs can directly activate GK expression in liver and may contribute to improving glucose homeostasis in type 2 diabetes. PGC-1βis a recently identified transcriptional coactivator closely related to PGC-1αwhose biological activities are largely unknown. Like PGC-1α, PGC-1βstrongly activates mitochondrial biogenesis and cellular respiration in differentiated myotubes. Heme is an essential component of numerous hemoproteins with functions including oxygen transport, energy metabolism.5-aminolevulinate synthase (ALAS1) is the rate-limiting enzyme in heme biosynthesis which provide heme for cytochromes in the respiratory chain. By 5’promoter deletion assay, we found PGC-1βcould activate ALAS 1 promoter that depended on NRF 1 for full activity. The ALAS 1 promoter has two functional NRF-1 recognition sites that are essential for basal promoter activity.When either of the two NRF-1 elements was mutated, about 60-70% of promoter activity was lost; when both were mutated, full activity was diminished by approximately 90%. Furthermore, expression of a dominant negative NRF-1 inhibited the PGC-1βmediated increase in ALAS1 gene. PGC-1βcould also bind and co-activate NRF-1 and augment transcriptional activation of mitochondrial related gene. To determine whether NRF-1 is required for the expression of endogenous PGC-1βtargets, we used an adenovial RNAi vector (Ad-RNAi) toward NRF-1. Treatment of C2C12 myotubes with this Ad-RNAi reduces endogenous NRF-1 protein by approximately 70%. Ad-PGC-1βexpression in C2C12 myotubes stimulated mRNA abundance of several mitochondrial related gene such as ALAS-1, Cytc, ATPase, Tfb2m, Tfam, COX7b, MRPL46. The induction of all these genes in response to PGC-1β, however, is greatly impaired in the cells infected with Ad-RNAi compared to the control GFP. These results suggest that PGC-1βplay an important role in controlling mitochondrial oxidative energy metabolism.更多还原