甲状腺素对气道黏蛋白5AC基因表达的影响及其调控机制
摘要
目的:
观察甲状腺素对气道上皮细胞黏蛋白(mucin,MUC)5AC表达的影响及其调控机制。
方法:
1、MUC5AC在A549细胞中的表达:体外培养A549细胞,以不同浓度的甲状腺素刺激A549细胞24h后,用ELISA法检测上清液中MUC5AC蛋白含量,用RT-PCR法检测细胞中MUC5ACmRNA水平。
2、设计、构建MUC5AC基因5'上游序列的表达质粒:根据MUC5AC基因5'调控区的DNA序列、表达载体pGL3质粒的结构特点及实验需要设计PCR扩增引物,以人基因组总DNA为模板扩增MUC5AC基因5'非编码区片段,双酶切后插入pGL3质粒构成重组质粒。重组质粒经酶切和DNA测序鉴定证实构建成功后,用脂质体将表达质粒和内参照质粒共转染A549细胞,培养24h后,加入甲状腺素(浓度为6.5ng/ml)处理,继续孵育24h收集细胞,采用双荧光素酶报告基因检测系统测定报告基因的表达水平。
结果:
1、用ELISA法检测A549细胞培养上清液中MUC5AC蛋白的表达显示,甲状腺素可成浓度、时间依赖性地使A549细胞MUC5AC蛋白表达水平下降,与对照组比较差异有统计学意义。用RT-PCR法检测MUC5ACmRNA表达显示,甲状腺素处理组A549细胞MUC5ACmRNA相对表达量明显低于对照组。
2、成功构建了4种含有MUC5AC基因启动子序列的表达质粒,分别导入A549细胞后予甲状腺素刺激,观察到甲状腺素可抑制重组质粒pGL3-MUC5AC-1330/+48、pGL3-MUC5AC-1250/+48荧光素酶的表达,而不能抑制重组质粒pGL3-MUC5AC-1200/+48、pGL3-MUC5AC-1020/+48荧光素酶的表达。
结论:
1、甲状腺素可抑制A549细胞黏蛋白MUC5AC基因转录及蛋白合成分泌,甲状腺素在基因转录水平抑制MUC5AC表达。
2、成功地构建了四种含人MUC5AC基因启动子系列截短的荧光素酶报告基因表达质粒,为研究甲状腺素及其它因素对MUC5AC基因表达的调控奠定了基础。
3、甲状腺素可抑制含有MUC5AC启动子5'-1330/+48、-1250/+48序列的报告基因质粒表达荧光素酶,而不能抑制含有该启动子5'-1200/+48、-1020/+48序列的报告基因质粒表达荧光素酶,表明甲状腺素调控MUC5AC基因表达依赖于启动子中-1250bp和-1200bp之间的序列。
Objective:
To explore the effects of thyroid hormone on mucin(MUC)5AC expression in the airway epithelial cells and its mechanism.
Methods:
Expression of MUC5AC in A549 cells:The well cultured airway epithelial cells of the line A549 were exposed to serial dilutions of thyroid hormone for 24 hours,the levels of MUC5AC protein secreted into the supernatants were determined by ELISA.The expression of MUC5AC in A549 cells was detected with RT-PCR on transcript levels.
Design and construction of luciferase reporter gene plasmids containing different length of human MUC5AC gene promoter:Using the restriction enzymes,PCR and gene recombination techniques,four luciferase reporter gene plasmids containing different length of human MUC5AC gene promoter were constructed.The plasmids and intracontrol plasmid pRL-TK were co-transfected into the human pulmonary A549 cells with eukaryotic gene transfected techniques,and the relative luciferase activities were detected in the transfected A549 cells.
Results:
Expression of MUC5AC in A549 cells:The expression of MUC5AC protein was decreased significantly in thyroid hormone treated groups compared with the without thyroid hormone treated.The expression of MUC5AC protein in different dose were lower than that of the control group.Thyroid hormone decreased MUC5AC mRNA levels in A549 cells.
Construction of luciferase reporter gene plasmids containing different length of human MUC5AC gene promoter:
Restriction enzyme digestion and DNA sequencing were performed to confirm the successful construction of four luciferase reporter gene plasmids containing different sequences of human MUC5AC promoter. Thyroid hormone could decrease the expression of luciferase reporter gene plasmids containing -1330bp and -1250bp version of MUC5AC promoter in transfected A549 cells,but could not decrease the the expression of luciferase reporter gene plasmids containing -1200bp and -1020bp version of MUC5AC promoter.
Conclusions:
1.Thyroid hormone can decrease the level of MUC5AC protein and mRNA respectively in the airway epithelial cells A549 by ELISA and RT-PCR.
2.We constructed a series of luciferase reporter gene plasmids containing different length of human MUC5AC gene promoter by gene recombination and PCR.The constructed luciferase reporter gene plasmids include pGL3-MUC5AC-1330/+48,pGL3-MUC5AC-1250/+48,pGL3-MUC5AC-1200/+48, pGL3-MUC5AC-1020/+48.
3.After above luciferase reporter plasmids and intracontrol plasmids pRL-TK were co-transfected into A549 cells,it is observed that thyroid hormone could decrease the expression of luciferase reporter gene plasmids containing -1330bp and -1250bp of MUC5AC promoter in the transfected A549 cells,whereas thyroid hormone could not decrease the expression of luciferase reporter gene plasmids containing -1200bp and -1020bp of MUC5AC promoter.These results suggest that there are thyroid hormone-responsive region between -1250bp and -1200bp of promoter.
观察甲状腺素对气道上皮细胞黏蛋白(mucin,MUC)5AC表达的影响及其调控机制。
方法:
1、MUC5AC在A549细胞中的表达:体外培养A549细胞,以不同浓度的甲状腺素刺激A549细胞24h后,用ELISA法检测上清液中MUC5AC蛋白含量,用RT-PCR法检测细胞中MUC5ACmRNA水平。
2、设计、构建MUC5AC基因5'上游序列的表达质粒:根据MUC5AC基因5'调控区的DNA序列、表达载体pGL3质粒的结构特点及实验需要设计PCR扩增引物,以人基因组总DNA为模板扩增MUC5AC基因5'非编码区片段,双酶切后插入pGL3质粒构成重组质粒。重组质粒经酶切和DNA测序鉴定证实构建成功后,用脂质体将表达质粒和内参照质粒共转染A549细胞,培养24h后,加入甲状腺素(浓度为6.5ng/ml)处理,继续孵育24h收集细胞,采用双荧光素酶报告基因检测系统测定报告基因的表达水平。
结果:
1、用ELISA法检测A549细胞培养上清液中MUC5AC蛋白的表达显示,甲状腺素可成浓度、时间依赖性地使A549细胞MUC5AC蛋白表达水平下降,与对照组比较差异有统计学意义。用RT-PCR法检测MUC5ACmRNA表达显示,甲状腺素处理组A549细胞MUC5ACmRNA相对表达量明显低于对照组。
2、成功构建了4种含有MUC5AC基因启动子序列的表达质粒,分别导入A549细胞后予甲状腺素刺激,观察到甲状腺素可抑制重组质粒pGL3-MUC5AC-1330/+48、pGL3-MUC5AC-1250/+48荧光素酶的表达,而不能抑制重组质粒pGL3-MUC5AC-1200/+48、pGL3-MUC5AC-1020/+48荧光素酶的表达。
结论:
1、甲状腺素可抑制A549细胞黏蛋白MUC5AC基因转录及蛋白合成分泌,甲状腺素在基因转录水平抑制MUC5AC表达。
2、成功地构建了四种含人MUC5AC基因启动子系列截短的荧光素酶报告基因表达质粒,为研究甲状腺素及其它因素对MUC5AC基因表达的调控奠定了基础。
3、甲状腺素可抑制含有MUC5AC启动子5'-1330/+48、-1250/+48序列的报告基因质粒表达荧光素酶,而不能抑制含有该启动子5'-1200/+48、-1020/+48序列的报告基因质粒表达荧光素酶,表明甲状腺素调控MUC5AC基因表达依赖于启动子中-1250bp和-1200bp之间的序列。
Objective:
To explore the effects of thyroid hormone on mucin(MUC)5AC expression in the airway epithelial cells and its mechanism.
Methods:
Expression of MUC5AC in A549 cells:The well cultured airway epithelial cells of the line A549 were exposed to serial dilutions of thyroid hormone for 24 hours,the levels of MUC5AC protein secreted into the supernatants were determined by ELISA.The expression of MUC5AC in A549 cells was detected with RT-PCR on transcript levels.
Design and construction of luciferase reporter gene plasmids containing different length of human MUC5AC gene promoter:Using the restriction enzymes,PCR and gene recombination techniques,four luciferase reporter gene plasmids containing different length of human MUC5AC gene promoter were constructed.The plasmids and intracontrol plasmid pRL-TK were co-transfected into the human pulmonary A549 cells with eukaryotic gene transfected techniques,and the relative luciferase activities were detected in the transfected A549 cells.
Results:
Expression of MUC5AC in A549 cells:The expression of MUC5AC protein was decreased significantly in thyroid hormone treated groups compared with the without thyroid hormone treated.The expression of MUC5AC protein in different dose were lower than that of the control group.Thyroid hormone decreased MUC5AC mRNA levels in A549 cells.
Construction of luciferase reporter gene plasmids containing different length of human MUC5AC gene promoter:
Restriction enzyme digestion and DNA sequencing were performed to confirm the successful construction of four luciferase reporter gene plasmids containing different sequences of human MUC5AC promoter. Thyroid hormone could decrease the expression of luciferase reporter gene plasmids containing -1330bp and -1250bp version of MUC5AC promoter in transfected A549 cells,but could not decrease the the expression of luciferase reporter gene plasmids containing -1200bp and -1020bp version of MUC5AC promoter.
Conclusions:
1.Thyroid hormone can decrease the level of MUC5AC protein and mRNA respectively in the airway epithelial cells A549 by ELISA and RT-PCR.
2.We constructed a series of luciferase reporter gene plasmids containing different length of human MUC5AC gene promoter by gene recombination and PCR.The constructed luciferase reporter gene plasmids include pGL3-MUC5AC-1330/+48,pGL3-MUC5AC-1250/+48,pGL3-MUC5AC-1200/+48, pGL3-MUC5AC-1020/+48.
3.After above luciferase reporter plasmids and intracontrol plasmids pRL-TK were co-transfected into A549 cells,it is observed that thyroid hormone could decrease the expression of luciferase reporter gene plasmids containing -1330bp and -1250bp of MUC5AC promoter in the transfected A549 cells,whereas thyroid hormone could not decrease the expression of luciferase reporter gene plasmids containing -1200bp and -1020bp of MUC5AC promoter.These results suggest that there are thyroid hormone-responsive region between -1250bp and -1200bp of promoter.
引文
[1] Corfield AP and Shukla AK. Mucins: vital components of the mucosal defensive barrier[J]. Genomic/Proteomic Technol 2004, 3: 20-22
[2] Roqers DF. The role of airway secretions in COPD: pathophysiology, epidemiology and pharmacotherapeutic options[J]. COPD, 2005,2(3): 341-353
[3] Rose MC, Nickola TJ, and Voynow JA. Airway mucus obstruction: mucin glycoproteins, MUC gene regulation and goblet cell hyperplasia[J]. Respir Cell Mol Biol 2001, 25: 533-537
[4] Wedzicha JA, Donaldson GC. Exacerbations of chronic obstructive pulmonary disease[J]. Respir Care. 2003 , 48(12): 1204-13;
[5] Hauber HP, Foley SC, Hamid Q. Mucin overproduction in chronic inflammatory lung disease[J].Can Respir J,2006,13(6):327-335.
[6] Danahay H, Jackson AD. Epithelial mucus-hypersecretion and respiratory disease [J].Curr Drug Targets Inflamm Allergy, 2005,4(6):651 -664
[7] Jeffery PK, Li D. Airway mucosa: secretory cells, mucus and mucin genes[J].Eur Respir. 1997 , 10(7): 1655-62
[8] Davies JR, Herrmann A, Russell W, et al. Respiratory tract mucins: structure and expression patterns[J]. Novartis Found Symp. 2002, 248:76-88;
[9] Williams OW, Sharafkhaneh A, Kim V, et al. Airway mucus: From production to secretion[J]. Respir Cell Mol Biol.2006 ,34(5):527-36.
[10] Reid C, Gould S, and Harris A. Developmental expression of mucin genes in the human respiratory tract[J]. Respir Cell Mol Biol 1997, 17: 592-598
[11]Rose MC and Gendler SJ. Airway mucin genes and gene products. In: Airway Mucus: Basic Mechanisms and Clinical Perspectives[J]. Basel: Birkhauser, 1997, 41-66
[12]Leikauf GD, Borchers MT, Prows DR, et al. Mucin apoprotein expression in COPD[J]. Chest 2002, 121: 166S-182S
[13] Williams SJ, Wreschner DH, Tran M, et al. Muc13, a novel human cell surface mucin expressed by epithelial and hemopoietic cells[J]. Biol Chem 276: 2001, 18327-18336
[14]Pallesen LT, Berglund L, Rasmussen LK,et al. Isolation and characterization of MUC15, a novel cell membrane-associated mucin[J]. Biochem 2002, 269: 2755,
[15]Chen Y,Zhao YH,Kalaslavadi TB,et al.Genome-wide search and identification of a novel gel-forming mucin MUC19/MucI9 in glandular tissues[J].Respir Cell Mol Biol 2004,30:155-165
[16]Higuchi T,Orita T,Nakanishi S,et al.Molecular cloning,genomic structure,and expression analysis of MUC20,a novel mucin protein,up-regulated in injured kidney[J].Biol Chem.2004,279(3):1968-79
[17]Alcorta DA,Li H,Yamamoto T,et al.Molecular cloning,genomic structure,and expression analysis of MUC20,a novel mucin protein,up-regulated in injured kidney[J].Biol Chem 2004,279:1968-1979
[18]Rose MC,Voynow JA.Respiratory tract mucin genes and mucin glycoproteins in health and disease[J].Physiol Rev.2006,86(1):245-78.Review
[19]Rose MC,Nickola T J,and Voynow JA.Airway mucus obstruction:mucin glycoproteins,MUC gene regulation and goblet cell hyperplasia[J].Respir Cell Mol Biol2001,25:533-537
[20]Alimam MZ,Piazza FM,Selby D,et al.Muc-5/5ac mRNA and protein expression is a marker of goblet cell metaplasia in murine airways[J].Respir Cell Mol Biol 2000,22:253-260
[21]Gerber V,Robinson NE,Venta R J,et al.Mucin genes in horse airways:MUC5AC,but not MUC2,may play a role in recurrent airway obstruction[J].Equine Vet J 2003,35:252-257
[22]黄奕江,冯夏,郑万川.慢性阻塞性肺病患者免疫功能的研究[J].临床内科杂志,1997,14:30-31
[23]Karadag F,Ozcan H,Karul AB,et,al.Correlates of non-thyroidal illness syndrome in Alacrimaobstructive pulmonary disease[J].Respir Med.2007,101(7):1439-1446
[24]Okutan O,Kartaloglu Z,Onde ME,et al.Pulmonary function tests and thyroid hormone concentrations in patients with chronic obstructive pulmonary disease[J].Med Princ Pract.2004,13(3):126-128
[25]Mancini A,Corbo GM,Gaballo A,et al.Relationships between plasma CoQ10levels and thyroid hormones in chronic obstructive pulmonary disease [J].Biofactors,2005,25(1-4):201-204
[26]Moon SK,Yoo JH,Kim HN,et al.Effects ofretinoic acid,triiodothyronine and hydrocortisone on mucin and lysozyme expression in cultured human middle ear epithelial cells[J].Acta Otolaryngol,2000,120(8):944-949
[27]Yoon JH,Gray T,Guzman K,et al.Regulation of the secretory phenotype of human airway epithelium by retinoic acid,triiodothyronine,and extracellular matrix.[J]Respir Cell Mol Biol.1997,16(6):724-31.
[28]周向东,兰箭,童瑾.慢性阻塞性肺疾病气道黏蛋白表型的研究[J].中华结核和呼吸杂志,2002,25(7):437
[29]Vovnow JA.What dose mucin have to do with lung disease[J]? Paediatr Respir Rev,2002,3(2):98-103
[30]Kirkham S,Sheehan JK,Knight D,et al.Heterogeneity of airways mucus:variations in the amounts and glycoforms of the major oligomeric mucins MUC5AC and MUC5B[J].Biochem J.2002,361:537-546
[31]Shapiro DL,Nardone LL,Rooney SA.Phospholipid biosynthesis and secretion by a cell line(A549)which resembles type Ⅱ aleveolar epithelial cells[J].Biochim Biophys Acta.1978,530(2):197-207.
[32]Croce MV,Colussi AG,Price MR,et al.Identification and characterization of different subpopulations in a human lung adenocarcinoma cell line(A549)[J].Pathol Oncol Res.1999,5(3):197-204.
[33]Kode A,Yang SR,Rahman I.Differential effects of cigarette smoke on oxidative stress and proinflammatory cytokine release in primary human airway epithelial cells and in a variety of transformed alveolar epithelial cells[J].Respir Res.2006,7:132.
[34]Li S,Bobek LA.Functional analysis of human MUC7 mucin gene 5'-flanking region in lung epithelial cells[J].Am J Respir Cell Mol Biol.2006,35(5):593-601.
[35]Moon Y,Lee M,Yang H.Involvement of early growth response gene 1 in the modulation of microsomal prostaglandin E synthase 1 by epigallocatechin gallate in A549 human pulmonary epithelial cells[J].Biochem Pharmacol.2007,73(1):125-35
[36]梁荩忠,李秀均.内分泌病诊疗手册.人民卫生出版社出版.2002,129.
[37]Werner,S.C.,Ingbar,S.H.,Braverman,L.E.,et al.Werner & Ingbar's the Thyroid:a Fundamental and Clinical Text,2004,9th Ed.
[38]Harvey,C.B.,and Williams,G.R.Mechanism of thyroid hormone action.Thyroid,2002(12),441-446
[39]Moon SK,Yoo JH,Kim HN,et al.Effects of retinoic acid,triiodothyronine and hydrocortisone on mucin and lysozyme expression in cultured human middle ear epithelial cells[J]. Acta Otolaryngol. 2000 , 20(8):944-9.
[40]Koo JS, Jetten AM, Belloni P, et al.Role of retinoid receptors in the regulation of mucin gene expression by retinoic acid in human tracheobronchial epithelial cells[J]. Biochem .1999, 338 ( Pt 2):351-7
[1]Ayoubi TA,Van De Ven WJ.Regulation of gene expression by alternative promoters.FASEB[J].1996,10(4):453-60
[2]Smale ST.Transcription initiation from TATA-Iess promoters within eukaryotic protein-coding genes.Biochim Biophys Acta[J].1997,1351(1-2):73-88
[3]Parker LM,Fierro-Monti I,Reichman TW,et al.Double-stranded RNA-binding proteins and the control of protein synthesis and cell growth[J].Cold Spring Harb Syrup Quant Biol.2001,66:485-97
[4]Meijer HA,Thomas AA.Control of eukaryotic protein synthesis by upstream open reading frames in the 5'ountranslated region of an mRNA[J].Biochem,2002,367(Pt 1):1-11
[5]Hovenberg HW,Davies JR,Carlstedt I.Different mucins are produced by the surface epithelium and the submucosa in human trachea:identification of MUC5AC as a major mucin from the goblet cells[J].Biochem.,1996,318:319-24.
[6]Alimam MZ,Piazza FM,Selby D,et al.Muc-5/5ac mRNA and protein expression is a marker of goblet cell metaplasia in murine airways[J].Am J Respir Cell Mol Biol 2000,22:253-260.
[7]Davies JR,Herrmann A,Russell W,ET AL.Respiratory tract mucins:structure and expression[J]patterns.Novartis Found Symp.2002,248:76-88
[8]冯作化主编,医学分子生物学.北京:人民卫生出版社,2005,53-120
[9]Bjorklund S,Almouzni G,Davidson I,et al.Global transcription regulators of eukaryotes[J].Cell,1999,96(6):759-767
[10]Cosma MP,Tanaka T,Nasmyth K.Ordered recruitment of transcription and chromatin remodeling factors to a cell cycle-and developmentally promoter regulated promoter[J].cell,! 999,97(3):299-311
[11]Boeger H,Bushnell DA,Davis R,et al.Structural basis ofeukaryotic gene transcription[J].FEBS Lett,2005,579(4):899-903[12][Novina CD,Roy AL.Core promoters and transcriptional control[J].Trends Genet,1996,12(9):351-355
[13]Schumacher MA,Lau AO,Johnson PJ.Structural basis of core promoter recognition in a primitive eukaryote[J].Cell,2003,115(4):413-424
[14]Maim,J.Thyroid hormone ligands and metabolic diseases[J].Curr.Pharm.Des. 2004,10,3525-3532
[15]Harvey, C. B., and Williams, G. R. Mechanism of thyroid hormone action[J]. Thyroid 2002,12, 441 -446
[16]Puzianowska-Kuznicka M, Nauman A, Madej A, et al. Expression of thuroid hormone receptors is disturbed in human renalclear cell carcinoma Cancer[J]. Lett, 2000, 155(1): 145-152
[17]Carr FE, Wong NC. Characteristics of a negative thyroid hormone response element[J]. Biol. Chem.1994, 269:4175-4179
[18]Hollenberg AN, Monden T, Flynn TR,et al.The human thyrotropin- releasing hormone gene is regulated by thyroid hormone through two distinct classes of negative thyroid hormone response elements[J]. Mol Endocrinol, 1995,9: 540-550,
[19] Waters KM, Miller CW, Ntambi JM. Localization of a negative thyroid hormone-response region in hepatic stearoyl-CoA desaturase gene 1 [J]. Biochem Biophys Res Commun., 1997: 838-43
[20]Lopez G, Schaufele F, Webb P, et al.Positive and negative modulation of Jun action by thyroid hormone receptor at a unique AP1 site[J]. Mol Cell Biol. 1993 ,3(5): 3042-3049
[1]arin M,Liu Z,Zandi E.AP-I function and regulation[J].Curr Opin Cell Biol,1997,9(2):2404246.
[2]Fujioka S,Niu J,Schmidt C,el al.NF-κB and AP-1 connection:mechanism of NF-κB-dependent regulation of AP-1 activity[J].Mol Cell Biol,2004,24(17): 7806-7819.
[3]Barila D,Mangano R,Gonfloni S,el al.A nuclear tyrosine phosphorylation circuit:c-Jun as an activator and substrate of c-Abl and JNK[J].EMBO J,2000,19(2):273-281.
[4]Claret F X,Hibi M,Dhut S,el al.A new group of conserved coactivators that increase the specificity of AP-I transcription factors[J].Nature,1996,383(6599):453-457.
[5]Nead MA,Baglia LA,Antinore M J,el al.Rb binds c-Jun and activates transcription [J].EMBOJ,I998,17(8):2342-2352.
[6]Caramori G,Ito K,Adcock IM.Transcription factors in asthma and COPD[J].IDrugs,2004,7(8):764-770.
[7]Domagala-Kulawik J,Maskey-Warzechowska M,Kraszewska I,el al.The cellular composition and macrophage phenotype in induced sputum in smokers and ex-smokers with COPD[J].Chest 2003,123(4):1054-1059.
[8]Zhang Q,Adiseshaiah P,Reddy SP.Matrix metalloproteinase/epidermal growth factor receptor/mitogen-activated protein kinase signaling regulate fra-1 induction by cigarette smoke in lung epithelial cells[J].Respir Cell Mol Biol,2005,32(1):72-78.
[9]Shibata Y,Berclaz PY,Chroneos ZC,el al.GM-CSF regulates alveolar macrophage differentiation and innate immunity in the lung through PU.1[J].Immunity.2001,15(4):557-567.
[10]Laan M.Bozinovski S.Anderson GR Cigarette smoke inhibits lipopolysaccharideinduced production of inflammatory cytokines by suppressing the activation of activator protein-1 in bronchial epithelial cells[J].Immunol,2004,173(6):4164-4170.
[11]Chen CC,Chow MP,Huang WC,et al.Flavonoids inhibit tumor necrosis factor-α-induced up-regulation of intercellular adhesion molecule-1(ICAM-1)in respiratory epithelial cells through activator protein-1 and nuclear factor-κB:structureactivity relationships[J].Mol Pharmacol.2004,66(3):683-693.
[12]Woo CH. Lim JH. Kim JH. Lipopolysaccharide induces matrix metalloproteinase- 9 expression via a mitochondrial reactive oxygen species-p38 kinase-activator protein-1 pathway in Raw 264.7 cells [J]. Immunol, 2004, 173(11):6973-6980.
[13]Desmet C, Gosset P, Henry E,et al. Treatment of experimental asthma by decoy-mediated local inhibition of activator protein-1[J].. Respir Crit Care Med. 2005, 172(6):671-678.
[14]Funkhouser AW, Kang JA, Tan A,et al. Rhinovirus 16 3C protease induces interleukin -8 and granulocyte-macrophage colony- stimulating factor expression in human bronchial epithelial cells[J].. Pediatr Res. 2004,55(1):13-18.
[15]Spahn JD, Leung DY, Szefler SJ. New insights into the pathogenesis and management of steroid-resistant asthma [J] . Asthma. 1997;34(3): 177-194.
[16]Shukla A, Flanders T, Lounsbury KM, et al. The γ-glutamylcysteine synthetase and glutathione regulate asbestos-induced expression of activator protein-1 family members and activity [J]. Cancer Res, 2004, 64(21): 7780-7786.
[17] Andrew AS, Klei LR, Barchowsky A. AP-1-dependent induction of plasminogen activator inhibitor-1 by nickel does not require reactive oxygen [J]. Physiol Lung Cell Mol Physiol,2001,281(3):L616-L623
[1]Takeyama K,Dabbagh K,Lee H Met al.Epidermal growth factor system regulates mucin production in airways[J].Proc Natl Acad Sci U S A.1999,96(6):3081-6.
[2]Perrais M,Pigny P,Copin MC et al.Induction of MUC2 and MUC5AC mucins by factors of the epidermal growth factor(EGF)family is mediated by EGF receptor/Ras/Raf/extracellular signal-regulated kinase cascade and Spl[J].Biol Chem.2002,277(35):32258-67
[3]Shao MX,Nakanaqa T,Nadel JA.Cigarette smoke induces MUC5AC mucin overproduction via tumor necrosis factor-alpha-converting enzyme in human airway epithelial(NCI-H292)cells[J].Physiol Lung Cell Mol Physiol.2004,287(2):420-7
[4]Shao MX,Ueki IF,and Nadel JA.Tumor necrosis factor alpha-converting enzyme mediates MUC5AC mucin expression in cultured human airway epithelial cells[J].Proc Natl Acad Sci.2003,100(20):11618-11623.
[5]O'Donnell RA,Richter A,Ward J et al.Expression of ErbB receptors and mucins in the airways of long term current smokers[J].Thorax,2004,59(12):1032-1040
[6]Kohri K, Ueki IF, Shim JJ et al. Pseudomonas aeruginosa induces MUC5AC production via epidermal growth factor receptor[J]. Eur Respir 2002; 20(5):1263-1270
[7]Burgel PR, Lazarus SC, Tarn DC et al. Human Eosinophils Induce Mucin Production in Airway Epithelial Cells Via Epidermal Growth Factor Receptor Activation[J]. Immunol, 2001, 167: 5948-5954
[8]Jeffrey W. Tyner, Edy Y. Kim, Kyotaro Ide et al. Blocking airway mucous cell metaplasia by inhibiting EGFR antiapoptosis and IL-13 transdifferentiation signals[J]. Clin Invest. 2006 , 116(2): 309-321
[9]Takeyama K, Dabbagh K, Jeong Shim J et al. Oxidative stress causes mucin synthesis via transactivation of epidermal growth factor receptor: role of neutrophils[J]. Immunol. 2000 , 164(3): 1546-52
[10]Deshmukh HS, Case LM, Wesselkamper SC et al. Metalloproteinases Mediate Mucin 5AC Expression by Epidermal Growth Factor Receptor Activation[J]. Respir Crit Care Med. 2005 , 171(4):305-314
[11]Casalino-Matsuda SM, Monzon ME, Forteza RM . Epidermal growth factor receptor activation by epidermal growth factor mediates oxidant-induced goblet cell metaplasia in human airway epithelium[J]. Respir Cell Mol Biol. 2006, 34(5):581-591
[12]Hewson CA, Edbrooke MR, Johnston SL.PMA induces the MUC5AC respiratory mucin in human bronchial epithelial cells, via PKC, EGF/TGF-alpha, Ras/Raf, MEK, ERK and Sp1-dependent mechanisms[J]. Mol Biol. 2004 , 344(3):683-695
[13]Song JS, Cho KS, Yoon HK. Neutrophil elastase causes MUC5AC mucin synthesis via EGF receptor, ERK and NF-kB pathways in A549 cells[J]. Intern Med. 2005 , 20(4):275-283.
[2] Roqers DF. The role of airway secretions in COPD: pathophysiology, epidemiology and pharmacotherapeutic options[J]. COPD, 2005,2(3): 341-353
[3] Rose MC, Nickola TJ, and Voynow JA. Airway mucus obstruction: mucin glycoproteins, MUC gene regulation and goblet cell hyperplasia[J]. Respir Cell Mol Biol 2001, 25: 533-537
[4] Wedzicha JA, Donaldson GC. Exacerbations of chronic obstructive pulmonary disease[J]. Respir Care. 2003 , 48(12): 1204-13;
[5] Hauber HP, Foley SC, Hamid Q. Mucin overproduction in chronic inflammatory lung disease[J].Can Respir J,2006,13(6):327-335.
[6] Danahay H, Jackson AD. Epithelial mucus-hypersecretion and respiratory disease [J].Curr Drug Targets Inflamm Allergy, 2005,4(6):651 -664
[7] Jeffery PK, Li D. Airway mucosa: secretory cells, mucus and mucin genes[J].Eur Respir. 1997 , 10(7): 1655-62
[8] Davies JR, Herrmann A, Russell W, et al. Respiratory tract mucins: structure and expression patterns[J]. Novartis Found Symp. 2002, 248:76-88;
[9] Williams OW, Sharafkhaneh A, Kim V, et al. Airway mucus: From production to secretion[J]. Respir Cell Mol Biol.2006 ,34(5):527-36.
[10] Reid C, Gould S, and Harris A. Developmental expression of mucin genes in the human respiratory tract[J]. Respir Cell Mol Biol 1997, 17: 592-598
[11]Rose MC and Gendler SJ. Airway mucin genes and gene products. In: Airway Mucus: Basic Mechanisms and Clinical Perspectives[J]. Basel: Birkhauser, 1997, 41-66
[12]Leikauf GD, Borchers MT, Prows DR, et al. Mucin apoprotein expression in COPD[J]. Chest 2002, 121: 166S-182S
[13] Williams SJ, Wreschner DH, Tran M, et al. Muc13, a novel human cell surface mucin expressed by epithelial and hemopoietic cells[J]. Biol Chem 276: 2001, 18327-18336
[14]Pallesen LT, Berglund L, Rasmussen LK,et al. Isolation and characterization of MUC15, a novel cell membrane-associated mucin[J]. Biochem 2002, 269: 2755,
[15]Chen Y,Zhao YH,Kalaslavadi TB,et al.Genome-wide search and identification of a novel gel-forming mucin MUC19/MucI9 in glandular tissues[J].Respir Cell Mol Biol 2004,30:155-165
[16]Higuchi T,Orita T,Nakanishi S,et al.Molecular cloning,genomic structure,and expression analysis of MUC20,a novel mucin protein,up-regulated in injured kidney[J].Biol Chem.2004,279(3):1968-79
[17]Alcorta DA,Li H,Yamamoto T,et al.Molecular cloning,genomic structure,and expression analysis of MUC20,a novel mucin protein,up-regulated in injured kidney[J].Biol Chem 2004,279:1968-1979
[18]Rose MC,Voynow JA.Respiratory tract mucin genes and mucin glycoproteins in health and disease[J].Physiol Rev.2006,86(1):245-78.Review
[19]Rose MC,Nickola T J,and Voynow JA.Airway mucus obstruction:mucin glycoproteins,MUC gene regulation and goblet cell hyperplasia[J].Respir Cell Mol Biol2001,25:533-537
[20]Alimam MZ,Piazza FM,Selby D,et al.Muc-5/5ac mRNA and protein expression is a marker of goblet cell metaplasia in murine airways[J].Respir Cell Mol Biol 2000,22:253-260
[21]Gerber V,Robinson NE,Venta R J,et al.Mucin genes in horse airways:MUC5AC,but not MUC2,may play a role in recurrent airway obstruction[J].Equine Vet J 2003,35:252-257
[22]黄奕江,冯夏,郑万川.慢性阻塞性肺病患者免疫功能的研究[J].临床内科杂志,1997,14:30-31
[23]Karadag F,Ozcan H,Karul AB,et,al.Correlates of non-thyroidal illness syndrome in Alacrimaobstructive pulmonary disease[J].Respir Med.2007,101(7):1439-1446
[24]Okutan O,Kartaloglu Z,Onde ME,et al.Pulmonary function tests and thyroid hormone concentrations in patients with chronic obstructive pulmonary disease[J].Med Princ Pract.2004,13(3):126-128
[25]Mancini A,Corbo GM,Gaballo A,et al.Relationships between plasma CoQ10levels and thyroid hormones in chronic obstructive pulmonary disease [J].Biofactors,2005,25(1-4):201-204
[26]Moon SK,Yoo JH,Kim HN,et al.Effects ofretinoic acid,triiodothyronine and hydrocortisone on mucin and lysozyme expression in cultured human middle ear epithelial cells[J].Acta Otolaryngol,2000,120(8):944-949
[27]Yoon JH,Gray T,Guzman K,et al.Regulation of the secretory phenotype of human airway epithelium by retinoic acid,triiodothyronine,and extracellular matrix.[J]Respir Cell Mol Biol.1997,16(6):724-31.
[28]周向东,兰箭,童瑾.慢性阻塞性肺疾病气道黏蛋白表型的研究[J].中华结核和呼吸杂志,2002,25(7):437
[29]Vovnow JA.What dose mucin have to do with lung disease[J]? Paediatr Respir Rev,2002,3(2):98-103
[30]Kirkham S,Sheehan JK,Knight D,et al.Heterogeneity of airways mucus:variations in the amounts and glycoforms of the major oligomeric mucins MUC5AC and MUC5B[J].Biochem J.2002,361:537-546
[31]Shapiro DL,Nardone LL,Rooney SA.Phospholipid biosynthesis and secretion by a cell line(A549)which resembles type Ⅱ aleveolar epithelial cells[J].Biochim Biophys Acta.1978,530(2):197-207.
[32]Croce MV,Colussi AG,Price MR,et al.Identification and characterization of different subpopulations in a human lung adenocarcinoma cell line(A549)[J].Pathol Oncol Res.1999,5(3):197-204.
[33]Kode A,Yang SR,Rahman I.Differential effects of cigarette smoke on oxidative stress and proinflammatory cytokine release in primary human airway epithelial cells and in a variety of transformed alveolar epithelial cells[J].Respir Res.2006,7:132.
[34]Li S,Bobek LA.Functional analysis of human MUC7 mucin gene 5'-flanking region in lung epithelial cells[J].Am J Respir Cell Mol Biol.2006,35(5):593-601.
[35]Moon Y,Lee M,Yang H.Involvement of early growth response gene 1 in the modulation of microsomal prostaglandin E synthase 1 by epigallocatechin gallate in A549 human pulmonary epithelial cells[J].Biochem Pharmacol.2007,73(1):125-35
[36]梁荩忠,李秀均.内分泌病诊疗手册.人民卫生出版社出版.2002,129.
[37]Werner,S.C.,Ingbar,S.H.,Braverman,L.E.,et al.Werner & Ingbar's the Thyroid:a Fundamental and Clinical Text,2004,9th Ed.
[38]Harvey,C.B.,and Williams,G.R.Mechanism of thyroid hormone action.Thyroid,2002(12),441-446
[39]Moon SK,Yoo JH,Kim HN,et al.Effects of retinoic acid,triiodothyronine and hydrocortisone on mucin and lysozyme expression in cultured human middle ear epithelial cells[J]. Acta Otolaryngol. 2000 , 20(8):944-9.
[40]Koo JS, Jetten AM, Belloni P, et al.Role of retinoid receptors in the regulation of mucin gene expression by retinoic acid in human tracheobronchial epithelial cells[J]. Biochem .1999, 338 ( Pt 2):351-7
[1]Ayoubi TA,Van De Ven WJ.Regulation of gene expression by alternative promoters.FASEB[J].1996,10(4):453-60
[2]Smale ST.Transcription initiation from TATA-Iess promoters within eukaryotic protein-coding genes.Biochim Biophys Acta[J].1997,1351(1-2):73-88
[3]Parker LM,Fierro-Monti I,Reichman TW,et al.Double-stranded RNA-binding proteins and the control of protein synthesis and cell growth[J].Cold Spring Harb Syrup Quant Biol.2001,66:485-97
[4]Meijer HA,Thomas AA.Control of eukaryotic protein synthesis by upstream open reading frames in the 5'ountranslated region of an mRNA[J].Biochem,2002,367(Pt 1):1-11
[5]Hovenberg HW,Davies JR,Carlstedt I.Different mucins are produced by the surface epithelium and the submucosa in human trachea:identification of MUC5AC as a major mucin from the goblet cells[J].Biochem.,1996,318:319-24.
[6]Alimam MZ,Piazza FM,Selby D,et al.Muc-5/5ac mRNA and protein expression is a marker of goblet cell metaplasia in murine airways[J].Am J Respir Cell Mol Biol 2000,22:253-260.
[7]Davies JR,Herrmann A,Russell W,ET AL.Respiratory tract mucins:structure and expression[J]patterns.Novartis Found Symp.2002,248:76-88
[8]冯作化主编,医学分子生物学.北京:人民卫生出版社,2005,53-120
[9]Bjorklund S,Almouzni G,Davidson I,et al.Global transcription regulators of eukaryotes[J].Cell,1999,96(6):759-767
[10]Cosma MP,Tanaka T,Nasmyth K.Ordered recruitment of transcription and chromatin remodeling factors to a cell cycle-and developmentally promoter regulated promoter[J].cell,! 999,97(3):299-311
[11]Boeger H,Bushnell DA,Davis R,et al.Structural basis ofeukaryotic gene transcription[J].FEBS Lett,2005,579(4):899-903[12][Novina CD,Roy AL.Core promoters and transcriptional control[J].Trends Genet,1996,12(9):351-355
[13]Schumacher MA,Lau AO,Johnson PJ.Structural basis of core promoter recognition in a primitive eukaryote[J].Cell,2003,115(4):413-424
[14]Maim,J.Thyroid hormone ligands and metabolic diseases[J].Curr.Pharm.Des. 2004,10,3525-3532
[15]Harvey, C. B., and Williams, G. R. Mechanism of thyroid hormone action[J]. Thyroid 2002,12, 441 -446
[16]Puzianowska-Kuznicka M, Nauman A, Madej A, et al. Expression of thuroid hormone receptors is disturbed in human renalclear cell carcinoma Cancer[J]. Lett, 2000, 155(1): 145-152
[17]Carr FE, Wong NC. Characteristics of a negative thyroid hormone response element[J]. Biol. Chem.1994, 269:4175-4179
[18]Hollenberg AN, Monden T, Flynn TR,et al.The human thyrotropin- releasing hormone gene is regulated by thyroid hormone through two distinct classes of negative thyroid hormone response elements[J]. Mol Endocrinol, 1995,9: 540-550,
[19] Waters KM, Miller CW, Ntambi JM. Localization of a negative thyroid hormone-response region in hepatic stearoyl-CoA desaturase gene 1 [J]. Biochem Biophys Res Commun., 1997: 838-43
[20]Lopez G, Schaufele F, Webb P, et al.Positive and negative modulation of Jun action by thyroid hormone receptor at a unique AP1 site[J]. Mol Cell Biol. 1993 ,3(5): 3042-3049
[1]arin M,Liu Z,Zandi E.AP-I function and regulation[J].Curr Opin Cell Biol,1997,9(2):2404246.
[2]Fujioka S,Niu J,Schmidt C,el al.NF-κB and AP-1 connection:mechanism of NF-κB-dependent regulation of AP-1 activity[J].Mol Cell Biol,2004,24(17): 7806-7819.
[3]Barila D,Mangano R,Gonfloni S,el al.A nuclear tyrosine phosphorylation circuit:c-Jun as an activator and substrate of c-Abl and JNK[J].EMBO J,2000,19(2):273-281.
[4]Claret F X,Hibi M,Dhut S,el al.A new group of conserved coactivators that increase the specificity of AP-I transcription factors[J].Nature,1996,383(6599):453-457.
[5]Nead MA,Baglia LA,Antinore M J,el al.Rb binds c-Jun and activates transcription [J].EMBOJ,I998,17(8):2342-2352.
[6]Caramori G,Ito K,Adcock IM.Transcription factors in asthma and COPD[J].IDrugs,2004,7(8):764-770.
[7]Domagala-Kulawik J,Maskey-Warzechowska M,Kraszewska I,el al.The cellular composition and macrophage phenotype in induced sputum in smokers and ex-smokers with COPD[J].Chest 2003,123(4):1054-1059.
[8]Zhang Q,Adiseshaiah P,Reddy SP.Matrix metalloproteinase/epidermal growth factor receptor/mitogen-activated protein kinase signaling regulate fra-1 induction by cigarette smoke in lung epithelial cells[J].Respir Cell Mol Biol,2005,32(1):72-78.
[9]Shibata Y,Berclaz PY,Chroneos ZC,el al.GM-CSF regulates alveolar macrophage differentiation and innate immunity in the lung through PU.1[J].Immunity.2001,15(4):557-567.
[10]Laan M.Bozinovski S.Anderson GR Cigarette smoke inhibits lipopolysaccharideinduced production of inflammatory cytokines by suppressing the activation of activator protein-1 in bronchial epithelial cells[J].Immunol,2004,173(6):4164-4170.
[11]Chen CC,Chow MP,Huang WC,et al.Flavonoids inhibit tumor necrosis factor-α-induced up-regulation of intercellular adhesion molecule-1(ICAM-1)in respiratory epithelial cells through activator protein-1 and nuclear factor-κB:structureactivity relationships[J].Mol Pharmacol.2004,66(3):683-693.
[12]Woo CH. Lim JH. Kim JH. Lipopolysaccharide induces matrix metalloproteinase- 9 expression via a mitochondrial reactive oxygen species-p38 kinase-activator protein-1 pathway in Raw 264.7 cells [J]. Immunol, 2004, 173(11):6973-6980.
[13]Desmet C, Gosset P, Henry E,et al. Treatment of experimental asthma by decoy-mediated local inhibition of activator protein-1[J].. Respir Crit Care Med. 2005, 172(6):671-678.
[14]Funkhouser AW, Kang JA, Tan A,et al. Rhinovirus 16 3C protease induces interleukin -8 and granulocyte-macrophage colony- stimulating factor expression in human bronchial epithelial cells[J].. Pediatr Res. 2004,55(1):13-18.
[15]Spahn JD, Leung DY, Szefler SJ. New insights into the pathogenesis and management of steroid-resistant asthma [J] . Asthma. 1997;34(3): 177-194.
[16]Shukla A, Flanders T, Lounsbury KM, et al. The γ-glutamylcysteine synthetase and glutathione regulate asbestos-induced expression of activator protein-1 family members and activity [J]. Cancer Res, 2004, 64(21): 7780-7786.
[17] Andrew AS, Klei LR, Barchowsky A. AP-1-dependent induction of plasminogen activator inhibitor-1 by nickel does not require reactive oxygen [J]. Physiol Lung Cell Mol Physiol,2001,281(3):L616-L623
[1]Takeyama K,Dabbagh K,Lee H Met al.Epidermal growth factor system regulates mucin production in airways[J].Proc Natl Acad Sci U S A.1999,96(6):3081-6.
[2]Perrais M,Pigny P,Copin MC et al.Induction of MUC2 and MUC5AC mucins by factors of the epidermal growth factor(EGF)family is mediated by EGF receptor/Ras/Raf/extracellular signal-regulated kinase cascade and Spl[J].Biol Chem.2002,277(35):32258-67
[3]Shao MX,Nakanaqa T,Nadel JA.Cigarette smoke induces MUC5AC mucin overproduction via tumor necrosis factor-alpha-converting enzyme in human airway epithelial(NCI-H292)cells[J].Physiol Lung Cell Mol Physiol.2004,287(2):420-7
[4]Shao MX,Ueki IF,and Nadel JA.Tumor necrosis factor alpha-converting enzyme mediates MUC5AC mucin expression in cultured human airway epithelial cells[J].Proc Natl Acad Sci.2003,100(20):11618-11623.
[5]O'Donnell RA,Richter A,Ward J et al.Expression of ErbB receptors and mucins in the airways of long term current smokers[J].Thorax,2004,59(12):1032-1040
[6]Kohri K, Ueki IF, Shim JJ et al. Pseudomonas aeruginosa induces MUC5AC production via epidermal growth factor receptor[J]. Eur Respir 2002; 20(5):1263-1270
[7]Burgel PR, Lazarus SC, Tarn DC et al. Human Eosinophils Induce Mucin Production in Airway Epithelial Cells Via Epidermal Growth Factor Receptor Activation[J]. Immunol, 2001, 167: 5948-5954
[8]Jeffrey W. Tyner, Edy Y. Kim, Kyotaro Ide et al. Blocking airway mucous cell metaplasia by inhibiting EGFR antiapoptosis and IL-13 transdifferentiation signals[J]. Clin Invest. 2006 , 116(2): 309-321
[9]Takeyama K, Dabbagh K, Jeong Shim J et al. Oxidative stress causes mucin synthesis via transactivation of epidermal growth factor receptor: role of neutrophils[J]. Immunol. 2000 , 164(3): 1546-52
[10]Deshmukh HS, Case LM, Wesselkamper SC et al. Metalloproteinases Mediate Mucin 5AC Expression by Epidermal Growth Factor Receptor Activation[J]. Respir Crit Care Med. 2005 , 171(4):305-314
[11]Casalino-Matsuda SM, Monzon ME, Forteza RM . Epidermal growth factor receptor activation by epidermal growth factor mediates oxidant-induced goblet cell metaplasia in human airway epithelium[J]. Respir Cell Mol Biol. 2006, 34(5):581-591
[12]Hewson CA, Edbrooke MR, Johnston SL.PMA induces the MUC5AC respiratory mucin in human bronchial epithelial cells, via PKC, EGF/TGF-alpha, Ras/Raf, MEK, ERK and Sp1-dependent mechanisms[J]. Mol Biol. 2004 , 344(3):683-695
[13]Song JS, Cho KS, Yoon HK. Neutrophil elastase causes MUC5AC mucin synthesis via EGF receptor, ERK and NF-kB pathways in A549 cells[J]. Intern Med. 2005 , 20(4):275-283.