奶牛乳腺上皮细胞中SCAP和SREBP1蛋白调控SCD基因表达的作用研究
|
摘要
旨在研究奶牛乳腺上皮细胞中固醇调节元件结合蛋白裂解激活蛋白(SCAP)对固醇调节元件结合蛋白(SREBP1)调控的硬脂酰辅酶A去饱和酶基因(SCD)表达的影响。培养奶牛乳腺上皮细胞,在细胞中转染奶牛SCD基因启动子载体,同时转染SREBP1和SCAP真核表达载体作为处理,采用双荧光素酶报告基因系统检测转染SREBP1和SCAP对SCD基因启动子活性的影响;采用免疫荧光技术观察SREBP1在细胞核的表达,采用荧光定量PCR检测SCD基因mRNA的表达。结果表明,与转染pcDNA3.1载体的对照组相比,转染SCAP对SCD启动子活性无显著影响;转染SREBP1和共转染SCAP/SREBP1极显著增加SCD基因启动子活性值(P<0.01),并且SCAP的转染剂量与SCD的启动子活性值之间具有极显著的量效关系(P<0.01);在乳腺上皮细胞中转染SCAP后,能够增强SREBP1在细胞核的表达;细胞转染SREBP1和共转染SCAP/SREBP1后,SCD基因mRNA的表达分别显著上调1.23倍和1.54倍(P<0.05)。本研究表明,奶牛SCAP可以增加SREBP1蛋白在细胞核中的表达,促进对SCD基因的转录激活作用。
The aim of this study was to investigate the effect of sterol regulatory element-binding protein cleavage activating protein(SCAP)on the expression of stearoyl-CoA desaturase(SCD)gene regulated by steroid regulatory element binding protein(SREBP1)in dairy mammary epithelial cells.The dairy mammary epithelial cells were cultured and transfected with SCD gene promoter vectors,then SREBP1 and SCAP eukaryotic expression vectors were transfected into cells as treatment factor.The SCDpromoter activity was detected by dual luciferase reporter gene system.The expression of SREBP1 in nucleus was observed by immunofluorescence.qRT-PCR was used to measure the mRNA expression of SCD gene.The results showed that,compared with pcDNA3.1 control group,transfection of SCAP had no significant effect on SCD promoter activity.Transfection of SREBP1 and co-transfection of SCAP/SREBP1 significantly increased the SCD promoter activity(P<0.01).There was a significant dose-response relationship between thetransfection dose of SCAP and the promoter activity of SCD(P<0.01).The expression of SREBP1 in nucleus could be enhanced by SCAP transfection in mammary epithelial cells.The mRNA expression of SCD was up-regulated by 1.23-fold and 1.54-fold(P <0.05)after transfected SREBP1 or co-transfected SCAP/SREBP1,respectively.In conclusion,SCAP can increase the expression of SREBP1 protein in nucleus which enhance the transcriptional activation of SCD gene.
The aim of this study was to investigate the effect of sterol regulatory element-binding protein cleavage activating protein(SCAP)on the expression of stearoyl-CoA desaturase(SCD)gene regulated by steroid regulatory element binding protein(SREBP1)in dairy mammary epithelial cells.The dairy mammary epithelial cells were cultured and transfected with SCD gene promoter vectors,then SREBP1 and SCAP eukaryotic expression vectors were transfected into cells as treatment factor.The SCDpromoter activity was detected by dual luciferase reporter gene system.The expression of SREBP1 in nucleus was observed by immunofluorescence.qRT-PCR was used to measure the mRNA expression of SCD gene.The results showed that,compared with pcDNA3.1 control group,transfection of SCAP had no significant effect on SCD promoter activity.Transfection of SREBP1 and co-transfection of SCAP/SREBP1 significantly increased the SCD promoter activity(P<0.01).There was a significant dose-response relationship between thetransfection dose of SCAP and the promoter activity of SCD(P<0.01).The expression of SREBP1 in nucleus could be enhanced by SCAP transfection in mammary epithelial cells.The mRNA expression of SCD was up-regulated by 1.23-fold and 1.54-fold(P <0.05)after transfected SREBP1 or co-transfected SCAP/SREBP1,respectively.In conclusion,SCAP can increase the expression of SREBP1 protein in nucleus which enhance the transcriptional activation of SCD gene.
引文
[1]SHAO W,ESPENSHADE P J.Expanding roles for SREBP in metabolism[J].Cell Metab,2012,16(4):414-419.
[2]MOON Y A,LIANG G S,XIE X F,et al.The Scap/SREBP pathway is essential for developing diabetic fatty liver and carbohydrate-induced hypertriglyceridemia in animals[J].Cell Metab,2012,15(2):240-246.
[3]GOLDSTEIN J L,DEBOSE-BOYD R A,BROWN M S.Protein sensors for membrane sterols[J].Cell,2006,124(1):35-46.
[4]SHIMANO H.SREBPs:physiology and pathophysiology of the SREBP family[J].FEBS J,2009,276(3):616-621.
[5]SHAO W,ESPENSHADE P J.Sterol regulatory element-binding protein(SREBP)cleavage regulates Golgi-to-endoplasmic reticulum recycling of SREBP cleavage-activating protein(SCAP)[J].J Biol Chem,2014,289(11):7547-7557.
[6]MCFARLANE M R,CANTORIA M J,LINDEN A G,et al.Scap is required for sterol synthesis and crypt growth in intestinal mucosa[J].J Lipid Res,2015,56(8):1560-1571.
[7]SHIMIZU-ALBERGINE M,VAN YSERLOO B,GOLKOWSKI M G,et al.SCAP/SREBP pathway is required for the full steroidogenic response to cyclic AMP[J].Proc Natl Acad Sci U S A,2016,113(38):E5685-E5693.
[8]RU P,GUO D L.microRNA-29 mediates a novel negative feedback loop to regulate SCAP/SREBP-1and lipid metabolism[J].RNA Dis,2017,4(1):e1525.
[9]赵艳坤,邵伟,王立文,等.与脐带间充质干细胞共培养对乳腺上皮细胞乳脂合成及关键基因表达的影响[J].畜牧兽医学报,2017,48(5):846-853.ZHAO Y K,SHAO W,WANG L W,et al.Effect of Co-culturing with umbilical cord mesenchymal stem cells on milk fat synthesis and expression of key genes in bovine mammary gland epithelial cells[J].Acta Veterinaria et Zootechnica Sinica,2017,48(5):846-853.(in Chinese)
[10]王皓宇,秦彤,郝海生,等.胰岛素对体外培养奶牛乳腺上皮细胞乳蛋白、乳脂肪合成相关基因mRNA表达的影响[J].畜牧兽医学报,2013,44(5):710-718.WANG H Y,QIN T,HAO H S,et al.Effects of insulin on mRNA expression of genes related to milk protein and fat synthesis in bovine mammary epithelial cells cultured in vitro[J].Acta Veterinaria et Zootechnica Sinica,2013,44(5):710-718.(in Chinese)
[11]韩立强,王月影,王林枫,等.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[J].中国农业科学,2016,49(24):4797-4805.HAN L Q,WANG Y Y,WANG L F,et al,Expression and localization of bovine SREBP1 protein and regulation of the transcription of SCD1promoter in bovine mammary epithelial cell[J].Scientia Agricultura Sinica,2016,49(24):4797-4805.(in Chinese)
[12]韩立强,曹菁菁,付彤,等.奶牛硬脂酰辅酶A去饱和酶基因(SCD)启动子的克隆及活性分析[J].农业生物技术学报,2013,21(4):435-440.HAN L Q,CAO J J,FU T,et al.Cloning and activity analysis of promoter of bovine stearoyl-CoA desaturase gene(SCD)[J].Journal of Agricultural Biotechnology,2013,21(4):435-440.(in Chinese)
[13]NOHTURFFT A,DEBOSE-BOYD R A,SCHEEK S,et al.Sterols regulate cycling of SREBP cleavageactivating protein(SCAP)between endoplasmic reticulum and Golgi[J].Proc Natl Acad Sci U S A,1999,96(20):11235-11240.
[14]SAKAI J,NOHTURFFT A,CHENG D,et al.Identification of complexes between the COOH-terminal domains of sterol regulatory element-binding proteins(SREBPs)and SREBP cleavage-activating protein[J].J Biol Chem,1997,272(32):20213-20221.
[15]ZULKIFLI R M,PARR T,SALTER A M,et al.Regulation of ovine and porcine stearoyl coenzyme A desaturase gene promoters by fatty acids and sterols[J].J Anim Sci,2010,88(8):2565-2575.
[16]王苗,罗军,许会芬,等.山羊INSIG1基因超表达对乳腺上皮细胞中脂质合成的影响[J].畜牧兽医学报,2016,47(9):1806-1816.WANG M,LUO J,XU H F,et al.Effect of INSIG1 overexpression on the lipid synthesis in goat mammary gland epithelial cells[J].Acta Veterinaria et Zootechnica Sinica,2016,47(9):1806-1816.(in Chinese)
[17]XU D Q,WANG Z,ZHANG Y X,et al.PAQR3modulates cholesterol homeostasis by anchoring Scap/SREBP complex to the Golgi apparatus[J].Nat Commun,2015,6:8100.
[18]JENSEN K K,TADIN-STRAPPS M,WANG S P,et al.Dose-dependent effects of siRNA-mediated inhibition of SCAP on PCSK9,LDLR,and plasma lipids in mouse and rhesus monkey[J].J Lipid Res,2016,57(12):2150-2162.
[19]KUWABARA P E,LABOUESSE M.The sterolsensing domain:multiple families,a unique role?[J].Trends Genet,2002,18(4):193-201.
[20]MOON Y A.The SCAP/SREBP pathway:a mediator of hepatic steatosis[J].Endocrinol Metab(Seoul),2017,32(1):6-10.
[21]CHENG C M,RU P,GENG F,et al.Glucose-mediated N-glycosylation of SCAP is essential for SREBP-1activation and tumor growth[J].Cancer Cell,2015,28(5):569-581.
[22]BIONAZ M,LOOR J J.Gene networks driving bovine milk fat synthesis during the lactation cycle[J].BMC Genomics,2008,9:366.
[23]RUDOLPH M C,MONKS J,BURNS V,et al.Sterol regulatory element binding protein and dietary lipid regulation of fatty acid synthesis in the mammary epithelium[J].Am J Physiol Endocrinol Metab,2010,299(6):E918-E927.
[24]MA L,CORL B A.Transcriptional regulation of lipid synthesis in bovine mammary epithelial cells by sterol regulatory element binding protein-1[J].J Dairy Sci,2012,95(7):3743-3755.
[25]XU H F,LUO J,ZHAO W S,et al.Overexpression of SREBP1(sterol regulatory element binding protein1)promotes de novo fatty acid synthesis and triacylglycerol accumulation in goat mammary epithelial cells[J].J Dairy Sci,2016,99(1):783-795.
[26]GONG X,LI J X,SHAO W,et al.Structure of the WD40domain of SCAP from fission yeast reveals the molecular basis for SREBP recognition[J].Cell Res,2015,25(4):401-411.
[2]MOON Y A,LIANG G S,XIE X F,et al.The Scap/SREBP pathway is essential for developing diabetic fatty liver and carbohydrate-induced hypertriglyceridemia in animals[J].Cell Metab,2012,15(2):240-246.
[3]GOLDSTEIN J L,DEBOSE-BOYD R A,BROWN M S.Protein sensors for membrane sterols[J].Cell,2006,124(1):35-46.
[4]SHIMANO H.SREBPs:physiology and pathophysiology of the SREBP family[J].FEBS J,2009,276(3):616-621.
[5]SHAO W,ESPENSHADE P J.Sterol regulatory element-binding protein(SREBP)cleavage regulates Golgi-to-endoplasmic reticulum recycling of SREBP cleavage-activating protein(SCAP)[J].J Biol Chem,2014,289(11):7547-7557.
[6]MCFARLANE M R,CANTORIA M J,LINDEN A G,et al.Scap is required for sterol synthesis and crypt growth in intestinal mucosa[J].J Lipid Res,2015,56(8):1560-1571.
[7]SHIMIZU-ALBERGINE M,VAN YSERLOO B,GOLKOWSKI M G,et al.SCAP/SREBP pathway is required for the full steroidogenic response to cyclic AMP[J].Proc Natl Acad Sci U S A,2016,113(38):E5685-E5693.
[8]RU P,GUO D L.microRNA-29 mediates a novel negative feedback loop to regulate SCAP/SREBP-1and lipid metabolism[J].RNA Dis,2017,4(1):e1525.
[9]赵艳坤,邵伟,王立文,等.与脐带间充质干细胞共培养对乳腺上皮细胞乳脂合成及关键基因表达的影响[J].畜牧兽医学报,2017,48(5):846-853.ZHAO Y K,SHAO W,WANG L W,et al.Effect of Co-culturing with umbilical cord mesenchymal stem cells on milk fat synthesis and expression of key genes in bovine mammary gland epithelial cells[J].Acta Veterinaria et Zootechnica Sinica,2017,48(5):846-853.(in Chinese)
[10]王皓宇,秦彤,郝海生,等.胰岛素对体外培养奶牛乳腺上皮细胞乳蛋白、乳脂肪合成相关基因mRNA表达的影响[J].畜牧兽医学报,2013,44(5):710-718.WANG H Y,QIN T,HAO H S,et al.Effects of insulin on mRNA expression of genes related to milk protein and fat synthesis in bovine mammary epithelial cells cultured in vitro[J].Acta Veterinaria et Zootechnica Sinica,2013,44(5):710-718.(in Chinese)
[11]韩立强,王月影,王林枫,等.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[J].中国农业科学,2016,49(24):4797-4805.HAN L Q,WANG Y Y,WANG L F,et al,Expression and localization of bovine SREBP1 protein and regulation of the transcription of SCD1promoter in bovine mammary epithelial cell[J].Scientia Agricultura Sinica,2016,49(24):4797-4805.(in Chinese)
[12]韩立强,曹菁菁,付彤,等.奶牛硬脂酰辅酶A去饱和酶基因(SCD)启动子的克隆及活性分析[J].农业生物技术学报,2013,21(4):435-440.HAN L Q,CAO J J,FU T,et al.Cloning and activity analysis of promoter of bovine stearoyl-CoA desaturase gene(SCD)[J].Journal of Agricultural Biotechnology,2013,21(4):435-440.(in Chinese)
[13]NOHTURFFT A,DEBOSE-BOYD R A,SCHEEK S,et al.Sterols regulate cycling of SREBP cleavageactivating protein(SCAP)between endoplasmic reticulum and Golgi[J].Proc Natl Acad Sci U S A,1999,96(20):11235-11240.
[14]SAKAI J,NOHTURFFT A,CHENG D,et al.Identification of complexes between the COOH-terminal domains of sterol regulatory element-binding proteins(SREBPs)and SREBP cleavage-activating protein[J].J Biol Chem,1997,272(32):20213-20221.
[15]ZULKIFLI R M,PARR T,SALTER A M,et al.Regulation of ovine and porcine stearoyl coenzyme A desaturase gene promoters by fatty acids and sterols[J].J Anim Sci,2010,88(8):2565-2575.
[16]王苗,罗军,许会芬,等.山羊INSIG1基因超表达对乳腺上皮细胞中脂质合成的影响[J].畜牧兽医学报,2016,47(9):1806-1816.WANG M,LUO J,XU H F,et al.Effect of INSIG1 overexpression on the lipid synthesis in goat mammary gland epithelial cells[J].Acta Veterinaria et Zootechnica Sinica,2016,47(9):1806-1816.(in Chinese)
[17]XU D Q,WANG Z,ZHANG Y X,et al.PAQR3modulates cholesterol homeostasis by anchoring Scap/SREBP complex to the Golgi apparatus[J].Nat Commun,2015,6:8100.
[18]JENSEN K K,TADIN-STRAPPS M,WANG S P,et al.Dose-dependent effects of siRNA-mediated inhibition of SCAP on PCSK9,LDLR,and plasma lipids in mouse and rhesus monkey[J].J Lipid Res,2016,57(12):2150-2162.
[19]KUWABARA P E,LABOUESSE M.The sterolsensing domain:multiple families,a unique role?[J].Trends Genet,2002,18(4):193-201.
[20]MOON Y A.The SCAP/SREBP pathway:a mediator of hepatic steatosis[J].Endocrinol Metab(Seoul),2017,32(1):6-10.
[21]CHENG C M,RU P,GENG F,et al.Glucose-mediated N-glycosylation of SCAP is essential for SREBP-1activation and tumor growth[J].Cancer Cell,2015,28(5):569-581.
[22]BIONAZ M,LOOR J J.Gene networks driving bovine milk fat synthesis during the lactation cycle[J].BMC Genomics,2008,9:366.
[23]RUDOLPH M C,MONKS J,BURNS V,et al.Sterol regulatory element binding protein and dietary lipid regulation of fatty acid synthesis in the mammary epithelium[J].Am J Physiol Endocrinol Metab,2010,299(6):E918-E927.
[24]MA L,CORL B A.Transcriptional regulation of lipid synthesis in bovine mammary epithelial cells by sterol regulatory element binding protein-1[J].J Dairy Sci,2012,95(7):3743-3755.
[25]XU H F,LUO J,ZHAO W S,et al.Overexpression of SREBP1(sterol regulatory element binding protein1)promotes de novo fatty acid synthesis and triacylglycerol accumulation in goat mammary epithelial cells[J].J Dairy Sci,2016,99(1):783-795.
[26]GONG X,LI J X,SHAO W,et al.Structure of the WD40domain of SCAP from fission yeast reveals the molecular basis for SREBP recognition[J].Cell Res,2015,25(4):401-411.