奶牛SCAP基因的克隆,表达定位及对FASN基因启动子的转录调控
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摘要
固醇调节元件结合蛋白裂解激活蛋白(srebpcieavage activating protein,SCAP)是细胞脂肪合成酶的表达调控因子。本研究旨在肝脏L02细胞中研究SCAP对FASN基因启动子的转录调控作用,有助于进一步明确SCAP/SREBP1对于靶基因的转录调控机制。本研究以荷斯坦奶牛组织c DNA为模板,克隆SCAP基因的编码序列,构建pc DNA3.1-SCAP表达载体,将SCAP表达载体转染L02肝脏细胞,采用荧光定量PCR检测SCAP基因m RNA在24 h、48 h、72 h的表达情况;采用免疫荧光的方法对SCAP标记,激光共聚焦观察SCAP蛋白的亚细胞定位;构建FASN基因启动子载体,转染至肝脏细胞,并分别转染pc DNA3.1-SREBP1和pc DNA3.1-SCAP作为处理,荧光素酶报告基因系统分析对FASN启动子活性的影响。结果发现克隆得到SCAP的PCR产物为3 836 bp的片段,通过与pc DNA3.1载体重组后获得pc DNA3.1-SCAP表达载体,经酶切和测序鉴定正确;将pc DNA3.1-SCAP载体转染肝脏细胞培养24 h、48 h、72 h后,Real-time PCR检测发现与对照组相比,48 h时细胞中SCAP基因m RNA的表达倍数增加了21倍(p<0.001);激光共聚焦观察发现,DAPI染色的细胞核呈蓝色,免疫荧光标记的SCAP呈红色,二者融合后发现SCAP定位于细胞质;启动子活性检测发现,与对照组相比,SREBP1质粒处理的细胞FASN启动子活性增加了4.1倍(p<0.001),而SCAP和SREBP1共同处理细胞FASN启动子活性极显著增加了7.4倍(p<0.000 1)。试验克隆构建奶牛SCAP基因表达载体,亚细胞定位SCAP蛋白主要位于肝脏细胞质中,表明SCAP可以通过结合SREBP1促进FASN基因启动子的转录。
SREBP cleavage activating protein(SCAP) is a regulator of fat synthesis. The objective of this study is to investigate the transcriptional regulation of FASN gene in L02 cells by SCAP, and to be beneficial to further understanding the transcriptional regulatory mechanism of SCAP/SREBP1. The coding sequence(CDS) of SCAP gene was cloned with the c DNA of Holstein tissues as the template. The pc DNA3.1-SCAP expression vector which was constructed was transfected tohepatocyte cells. The different expression of m RNA in SCAP gene was detected with Real-time PCR in 24 h, 48 h and 72 h. SCAP was marked by the immunofluorescence method, and the subcellular localization of protein SCAP was observed with laser con-focal microscopy. FASN gene promoter was constructed and transfected to pc DNA3.1-SREBP1 and pc DNA3.1-SCAP respectively, the effect on FASN promoter activity was analyzed with luciferase reporter gene system. The results showed that the PCR product of SCAP was cloned into 3 836 bp fragment and pc DNA3.1-SCAP expression vector was obtained by recombinant pc DNA3.1 vector, which was verified by enzyme digestion and sequencing. After vector pc DNA3.1-SCAP being transfected to hepatocyte cells for 24 h, 48 h and 72 h, compared with the control group of the empty transfected vector, the expression of m RNA in gene SCAP increased by 21 times(p<0.001) in the real-time PCR detection with48 h. As shown in the observation of the laser con-focal microscopy, the cell nucleus dyed by DAPI was blue, and SCAP with immunofluorescent labeling was red. After the two fusions, SCAP was found in cytoplasm. The results of the detection of the promoter activity showed that compared with the control group, the activity of FASN promoter of SREBP1 plasmid was increased by 4.1 times(p<0.01), SCAP and SREBP1 cotreatment of cell FASN promoter activity significantly increased by 7.4 times(p<0.001). The SCAP gene expression vector was constructed by cloning.The subcellular localization of SCAP protein was mainly in the cytoplasm of the liver, which indicated that SCAP could promote the transcription of FASN gene promoter by combining with SREBP1.
SREBP cleavage activating protein(SCAP) is a regulator of fat synthesis. The objective of this study is to investigate the transcriptional regulation of FASN gene in L02 cells by SCAP, and to be beneficial to further understanding the transcriptional regulatory mechanism of SCAP/SREBP1. The coding sequence(CDS) of SCAP gene was cloned with the c DNA of Holstein tissues as the template. The pc DNA3.1-SCAP expression vector which was constructed was transfected tohepatocyte cells. The different expression of m RNA in SCAP gene was detected with Real-time PCR in 24 h, 48 h and 72 h. SCAP was marked by the immunofluorescence method, and the subcellular localization of protein SCAP was observed with laser con-focal microscopy. FASN gene promoter was constructed and transfected to pc DNA3.1-SREBP1 and pc DNA3.1-SCAP respectively, the effect on FASN promoter activity was analyzed with luciferase reporter gene system. The results showed that the PCR product of SCAP was cloned into 3 836 bp fragment and pc DNA3.1-SCAP expression vector was obtained by recombinant pc DNA3.1 vector, which was verified by enzyme digestion and sequencing. After vector pc DNA3.1-SCAP being transfected to hepatocyte cells for 24 h, 48 h and 72 h, compared with the control group of the empty transfected vector, the expression of m RNA in gene SCAP increased by 21 times(p<0.001) in the real-time PCR detection with48 h. As shown in the observation of the laser con-focal microscopy, the cell nucleus dyed by DAPI was blue, and SCAP with immunofluorescent labeling was red. After the two fusions, SCAP was found in cytoplasm. The results of the detection of the promoter activity showed that compared with the control group, the activity of FASN promoter of SREBP1 plasmid was increased by 4.1 times(p<0.01), SCAP and SREBP1 cotreatment of cell FASN promoter activity significantly increased by 7.4 times(p<0.001). The SCAP gene expression vector was constructed by cloning.The subcellular localization of SCAP protein was mainly in the cytoplasm of the liver, which indicated that SCAP could promote the transcription of FASN gene promoter by combining with SREBP1.
引文
Asano L.,Watanabe M.,Ryoden Y.,Usuda K.,Yamaguchi T.,Khambu B.,Takashima M.,Sato S.I.,Sakai J.,Nagasawa K.,and Uesugi M.,2017,Vitamin D metabolite,25-Hydroxyvitamin D,regulates lipid metabolism by inducing degradation of SREBP/SCAP,Cell Chemical Biology,24(2):207-217
Cheng C.M.,Ru P.,Geng F.,Liu J.F.,Yoo J.Y.,Wu X.N.,Cheng X.,Euthine V.,Hu P.,Guo J.Y.,Lefai E.,Kaur B.,Nohturfft A.,Ma J.J.,Chakravarti A.,and Guo D.L.,2015,Glucose-Mediated N-glycosylation of SCAP is essential for SREBP-1 activation and tumor growth,Cancer Cell,28(5):569-581
Deng Q.,Li X.,Fu S.,Yin L.,Zhang Y.,Wang T.,Wang J.,Liu L.,Yuan X.,Sun G.,Wang Z.,Liu G.,and Li X.,2014,SREBP-1c gene silencing can decrease lipid deposits in bovine hepatocytes cultured in vitro,Cell Physiology and Biochemistry,33(5):1568-1578
Han L.Q.,Wang Y.Y.,Wang L.F.,Zhu H.S.,Zhong K.,Chu B.B.,and Yang G.Y.,2016,Expression and localization of bovine SREBP1 protein and regulation of the transcription of SCD1promoter in bovine mammary epithelial cell,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),49(24):4797-4805(韩立强,王月影,王林枫,朱河水,钟凯,褚贝贝,杨国宇,2016,奶牛SREBP1蛋白在乳腺上皮细胞的表达定位及对SCD1基因启动子的转录调控,中国农业科学,49(24):4797-4805)
Impheng H.,Pongcharoen S.,Richert L.,Pekthong D.,and Srisawang P.,2014,The selective target of capsaicin on FASNexpression and de novo fatty acid synthesis mediated through ROS generation triggers apoptosis in Hep G2 cells,PLo SOne,9(9):e107842
Li J.,Luo J.,Xu H.F.,Wang M.,Zhu J.J.,Shi H.B.,Haile A.B.,Wang H.,and Sun Y.T.,2015a,Fatty acid synthase promoter:characterization,and transcriptional regulation by sterol regulatory element binding protein-1 in goat mammary epithelial cells,Gene,561(1):157-164
Li N.,Zhao F.,Wei C.J.,Liang M.Y.,Zhang N.,Wang C.M.,Li Q.Z.,and Gao X.J.,2014,Function of SREBP1 in the milk fat synthesis of dairy cow mammary epithelial cells,International Journal of Molecular Sciences,15(9):16998-17013
Li X.W.,Huang W.K.,Gu J.M.,Du X.L.,Lei L.,Yuan X.,Sun G.Q.,Wang Z.,Li X.B.,and Liu G.W.,2015b,SREBP-1c overactivates ROS-mediated hepatic NF-κB inflammatory pathway in dairy cows with fatty liver,Cell Signalling,27(10):2099-2109
Magaa M.M.,and Osborne T.F.,1996,Two tandem binding sites for sterol regulatory element binding proteins are required for sterol regulation of fatty-acid synthase promoter,The Journal of Biological Chemistry,271(51):32689-32694
Makadia S.S.,Blaha M.,Keenan T.,Ndumele C.,Jones S.,DeFilippis A.,Martin S.,Kohli P.,Conceicao R.,Carvalho J.,Nasir K.,Blumenthal R.,and Santos R.D.,2013,Relation of hepatic steatosis to atherogenic dyslipidemia,The American Journal of Cardiology,112(10):1599-1604
Matsuda M.,Korn B.S.,Hammer R.E.,Moon Y.A.,Komuro R.,Horton J.D.,Goldstein J.L.,Brown M.S.,and Shimomura I.,2001,SREBP cleavage-activating protein(SCAP)is required for increased lipid synthesis in liver induced by cholesterol deprivation and insulin elevation,Genes&Development,15(10):1206-1216
Moon Y.A.,Lianq G.S.,Xie X.F.,Frank-Kamenetsky M.,Fitzgerald K.,Koteliansky V.,Brown M.S.,Goldstein J.L.,and Horton J.D.,2012,The Scap/SREBP pathway is essential for developing diabetic fatty liver and carbohydrate-induced hypertriglyceridemia in animals,Cell Metabolism,15(2):240-246
Moon Y.A.,2017,The SCAP/SREBP pathway:a mediator of hepatic steatosis,Endocrinology and Metabolism,32(1):6-10
Nigro D.,Menotti F.,Cento A.S.,Serpe L.,Chiazza F.,Dal Bello F.,Romaniello F.,Medana C.,Collino M.,Aragno M.,and Mastrocola R.,2017,Chronic administration of saturated fats and fructose differently affect SREBP activity resulting in different modulation of Nrf2 and Nirp3 inflammasome pathways in mice liver,The Journal of Nutritional Biochemistry,42:160-171
Nohturfft A.,Brown M.S.,and Goldstein J.L.,1998,Topology of SREBP cleavage-activating protein,a polytopic membrane protein with a sterol-sensing domain,The Journal of Biological Chemistry,273(27):17243-17250
Ru P.,Hu P.,Geng F.,Geng F.,Mo X.K.,Cheng C.M.,Yoo J.Y.,Cheng X.,Wu X.N.,Guo J.Y.,Nakano I.,Lefai E.,Kaur B.,Chakravarti A.,and Guo D.L.,2016,Feedback loop regulation of SCAP/SREBP-1 by mi R-29 Modulates EGFRSignaling-Driven glioblastoma growth,Cell Reports,16(6):1527-1535
Salek L.,Lutucuta S.,Ballantyne C.M.,Gotto A.M.,and Marian A.,2002,Effects of SREBF-1a and SCAP polymorphisms on plasma levelsoflipids,severity,progression and regression of coronary atherosclerosis and response to therapy with fluvastatin,Journal of Molecular Medicine,80(11):737-744
Sawano T.,Shimizu T.,Yamada T.,Nanashima N.,Miura T.,Morohashi S.,Kudo D.,Hui F.M.,Kijima H.,Hakamada K.,and Tsuchida S.,2015,Fatty acid synthase-positive hepatocytes and subsequent steatosis in rat livers by irinotecan,Oncology Reports,33(5):2151-2160
Shao W.,and Esoenshade P.J.,2014,Sterol regulatory elementbinding protein(SREBP)cleavage regulates Golgi-to-endoplasmic reticulum recycling of SREBP cleavage-Activating protein(SCAP),Journal of Biological Chemistry,289(11):7547-7557
Shao W.,Machamer C.E.,and Espenshade P.J.,2016,Fatostatin blocks ER exit of SCAP but inhibits cell growth in a SC-AP-independent manner,Journal of Lipid Research,57(8):1564-1573
Shen L.,Cui A.F.,Xue Y.,Cui Y.,Dong X.Y.,Gao Y.,Yang H.,Fang F.,and Chang Y.S.,2014,Hepatic differentiated embryo-chondrocyte-expressed gene 1(Dec1)inhibits sterol regulatory element-binding protein-1c(Srebp-1c)expression and alleviates fatty liver phenotype,Journal of Biological Chemistry,289(34):23332-23342
Shimizu-Albergine M.,Van Yserloo B.,Golkowski M.G.,Ong S.E.,Beavo J.A.,and Bornfeldt K.E.,2016,SCAP/SREBP pathway is required for the full steroidogenic response to cyclic AMP,Proc.Natl.Acad.Sci.USA,113(38):E5685-E5693
Sun L.P.,Seemann J.,Goldstein J.L.,and Brown M.S.,2007,Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi:Insig renders sorting signal in Scap inaccessible to COPII proteins,Proc.Natl.Acad.Sci.USA,104(16):6519-6526
Wang D.,and Sul H.S.,1997,Upstream stimulatory factor binding to the E-box at-65 is required for insulin regulation of the fatty acid synthase promoter,Journal of Biological Chemistry,272(42):26367-26374
Xu D.Q.,Wang Z.,Zhang Y.X.,Jiang W.,Pan Y.,Song B.L.,and Chen Y.,2015,PAQR3 modulates cholesterol homeostasis by anchoring Scap/SREBP complex to the Golgi apparatus,Nature Communications,6:8100
Yabe D.,Xia Z.P.,Adams C.M.,and Rawson R.B.,2002,Three mutations in sterol-sensing domain of SCAP block interaction with insig and render SREBP cleavage insensitive to sterols,Proc.Natl.Acad.Sci.,99(26):16672-16677
Yang L.H.,and Chen D.F.,2007,Expression and role of SCAP in models of cultured steatosis hepatocytes,Chongqing Yixue(Chongqing Medical Journal),36(8):683-685(杨林辉,陈东风,2007,SCAP在培养肝细胞脂肪变性模型中的表达及意义,重庆医学,36(8):683-685)
Cheng C.M.,Ru P.,Geng F.,Liu J.F.,Yoo J.Y.,Wu X.N.,Cheng X.,Euthine V.,Hu P.,Guo J.Y.,Lefai E.,Kaur B.,Nohturfft A.,Ma J.J.,Chakravarti A.,and Guo D.L.,2015,Glucose-Mediated N-glycosylation of SCAP is essential for SREBP-1 activation and tumor growth,Cancer Cell,28(5):569-581
Deng Q.,Li X.,Fu S.,Yin L.,Zhang Y.,Wang T.,Wang J.,Liu L.,Yuan X.,Sun G.,Wang Z.,Liu G.,and Li X.,2014,SREBP-1c gene silencing can decrease lipid deposits in bovine hepatocytes cultured in vitro,Cell Physiology and Biochemistry,33(5):1568-1578
Han L.Q.,Wang Y.Y.,Wang L.F.,Zhu H.S.,Zhong K.,Chu B.B.,and Yang G.Y.,2016,Expression and localization of bovine SREBP1 protein and regulation of the transcription of SCD1promoter in bovine mammary epithelial cell,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),49(24):4797-4805(韩立强,王月影,王林枫,朱河水,钟凯,褚贝贝,杨国宇,2016,奶牛SREBP1蛋白在乳腺上皮细胞的表达定位及对SCD1基因启动子的转录调控,中国农业科学,49(24):4797-4805)
Impheng H.,Pongcharoen S.,Richert L.,Pekthong D.,and Srisawang P.,2014,The selective target of capsaicin on FASNexpression and de novo fatty acid synthesis mediated through ROS generation triggers apoptosis in Hep G2 cells,PLo SOne,9(9):e107842
Li J.,Luo J.,Xu H.F.,Wang M.,Zhu J.J.,Shi H.B.,Haile A.B.,Wang H.,and Sun Y.T.,2015a,Fatty acid synthase promoter:characterization,and transcriptional regulation by sterol regulatory element binding protein-1 in goat mammary epithelial cells,Gene,561(1):157-164
Li N.,Zhao F.,Wei C.J.,Liang M.Y.,Zhang N.,Wang C.M.,Li Q.Z.,and Gao X.J.,2014,Function of SREBP1 in the milk fat synthesis of dairy cow mammary epithelial cells,International Journal of Molecular Sciences,15(9):16998-17013
Li X.W.,Huang W.K.,Gu J.M.,Du X.L.,Lei L.,Yuan X.,Sun G.Q.,Wang Z.,Li X.B.,and Liu G.W.,2015b,SREBP-1c overactivates ROS-mediated hepatic NF-κB inflammatory pathway in dairy cows with fatty liver,Cell Signalling,27(10):2099-2109
Magaa M.M.,and Osborne T.F.,1996,Two tandem binding sites for sterol regulatory element binding proteins are required for sterol regulation of fatty-acid synthase promoter,The Journal of Biological Chemistry,271(51):32689-32694
Makadia S.S.,Blaha M.,Keenan T.,Ndumele C.,Jones S.,DeFilippis A.,Martin S.,Kohli P.,Conceicao R.,Carvalho J.,Nasir K.,Blumenthal R.,and Santos R.D.,2013,Relation of hepatic steatosis to atherogenic dyslipidemia,The American Journal of Cardiology,112(10):1599-1604
Matsuda M.,Korn B.S.,Hammer R.E.,Moon Y.A.,Komuro R.,Horton J.D.,Goldstein J.L.,Brown M.S.,and Shimomura I.,2001,SREBP cleavage-activating protein(SCAP)is required for increased lipid synthesis in liver induced by cholesterol deprivation and insulin elevation,Genes&Development,15(10):1206-1216
Moon Y.A.,Lianq G.S.,Xie X.F.,Frank-Kamenetsky M.,Fitzgerald K.,Koteliansky V.,Brown M.S.,Goldstein J.L.,and Horton J.D.,2012,The Scap/SREBP pathway is essential for developing diabetic fatty liver and carbohydrate-induced hypertriglyceridemia in animals,Cell Metabolism,15(2):240-246
Moon Y.A.,2017,The SCAP/SREBP pathway:a mediator of hepatic steatosis,Endocrinology and Metabolism,32(1):6-10
Nigro D.,Menotti F.,Cento A.S.,Serpe L.,Chiazza F.,Dal Bello F.,Romaniello F.,Medana C.,Collino M.,Aragno M.,and Mastrocola R.,2017,Chronic administration of saturated fats and fructose differently affect SREBP activity resulting in different modulation of Nrf2 and Nirp3 inflammasome pathways in mice liver,The Journal of Nutritional Biochemistry,42:160-171
Nohturfft A.,Brown M.S.,and Goldstein J.L.,1998,Topology of SREBP cleavage-activating protein,a polytopic membrane protein with a sterol-sensing domain,The Journal of Biological Chemistry,273(27):17243-17250
Ru P.,Hu P.,Geng F.,Geng F.,Mo X.K.,Cheng C.M.,Yoo J.Y.,Cheng X.,Wu X.N.,Guo J.Y.,Nakano I.,Lefai E.,Kaur B.,Chakravarti A.,and Guo D.L.,2016,Feedback loop regulation of SCAP/SREBP-1 by mi R-29 Modulates EGFRSignaling-Driven glioblastoma growth,Cell Reports,16(6):1527-1535
Salek L.,Lutucuta S.,Ballantyne C.M.,Gotto A.M.,and Marian A.,2002,Effects of SREBF-1a and SCAP polymorphisms on plasma levelsoflipids,severity,progression and regression of coronary atherosclerosis and response to therapy with fluvastatin,Journal of Molecular Medicine,80(11):737-744
Sawano T.,Shimizu T.,Yamada T.,Nanashima N.,Miura T.,Morohashi S.,Kudo D.,Hui F.M.,Kijima H.,Hakamada K.,and Tsuchida S.,2015,Fatty acid synthase-positive hepatocytes and subsequent steatosis in rat livers by irinotecan,Oncology Reports,33(5):2151-2160
Shao W.,and Esoenshade P.J.,2014,Sterol regulatory elementbinding protein(SREBP)cleavage regulates Golgi-to-endoplasmic reticulum recycling of SREBP cleavage-Activating protein(SCAP),Journal of Biological Chemistry,289(11):7547-7557
Shao W.,Machamer C.E.,and Espenshade P.J.,2016,Fatostatin blocks ER exit of SCAP but inhibits cell growth in a SC-AP-independent manner,Journal of Lipid Research,57(8):1564-1573
Shen L.,Cui A.F.,Xue Y.,Cui Y.,Dong X.Y.,Gao Y.,Yang H.,Fang F.,and Chang Y.S.,2014,Hepatic differentiated embryo-chondrocyte-expressed gene 1(Dec1)inhibits sterol regulatory element-binding protein-1c(Srebp-1c)expression and alleviates fatty liver phenotype,Journal of Biological Chemistry,289(34):23332-23342
Shimizu-Albergine M.,Van Yserloo B.,Golkowski M.G.,Ong S.E.,Beavo J.A.,and Bornfeldt K.E.,2016,SCAP/SREBP pathway is required for the full steroidogenic response to cyclic AMP,Proc.Natl.Acad.Sci.USA,113(38):E5685-E5693
Sun L.P.,Seemann J.,Goldstein J.L.,and Brown M.S.,2007,Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi:Insig renders sorting signal in Scap inaccessible to COPII proteins,Proc.Natl.Acad.Sci.USA,104(16):6519-6526
Wang D.,and Sul H.S.,1997,Upstream stimulatory factor binding to the E-box at-65 is required for insulin regulation of the fatty acid synthase promoter,Journal of Biological Chemistry,272(42):26367-26374
Xu D.Q.,Wang Z.,Zhang Y.X.,Jiang W.,Pan Y.,Song B.L.,and Chen Y.,2015,PAQR3 modulates cholesterol homeostasis by anchoring Scap/SREBP complex to the Golgi apparatus,Nature Communications,6:8100
Yabe D.,Xia Z.P.,Adams C.M.,and Rawson R.B.,2002,Three mutations in sterol-sensing domain of SCAP block interaction with insig and render SREBP cleavage insensitive to sterols,Proc.Natl.Acad.Sci.,99(26):16672-16677
Yang L.H.,and Chen D.F.,2007,Expression and role of SCAP in models of cultured steatosis hepatocytes,Chongqing Yixue(Chongqing Medical Journal),36(8):683-685(杨林辉,陈东风,2007,SCAP在培养肝细胞脂肪变性模型中的表达及意义,重庆医学,36(8):683-685)