1、Title Page長庚大學生物醫學研究所長庚大學生物醫學研究所生理暨藥理組博士論文生理暨藥理組博士論文探討探討LPS誘發氣管平滑肌細胞調控黏附分子與誘發氣管平滑肌細胞調控黏附分子與cytosolic phospholipase A2 表現的機轉表現的機轉Mechanisms underlying lipopolysaccharide-induced expression of adhesion molecules and cytosolic phospholipase A2 in tracheal smooth muscle cells 研究生：林維寧研究生：林維寧(Wei-Ning Lin
2、)指導教授：楊春茂指導教授：楊春茂 博士博士(Chuen-Mao Yang,Ph.D)中華民國中華民國 九十七九十七 年年 七七 月月誌謝誌謝不要爲了明天憂慮，因為明天自有明天的憂慮，一天的難處一天當就夠了。不要爲了明天憂慮，因為明天自有明天的憂慮，一天的難處一天當就夠了。馬太福音馬太福音6:346:34從大二進實驗室到完成博士學位的現在，把握今天一直是我的目標，不用爲了明天擔憂，只要肯做就會有收穫，在這裡先要特別感謝我的指導老師楊春茂教授，另外，我也要感謝國防醫學院藥理研究所顏茂雄教授、台灣大學藥理研究所符文美教授、本校周美智副教授與黃聰龍副教授逾百忙之中對此論文內容的審閱與指正，並提供寶貴
3、的意見使我的論文更趨於完善，對未來的研究方向有更多的想法。我也要謝謝實驗室裡由古至今一起打拼的同志兄弟們：最後要感謝我的家人們:將我照顧的非常好的爸爸、媽媽、親媽、姨丈，還有陪伴長大的哥哥跟中閎，都是我非常穩固的精神後盾，因為有你們讓我一路走來無憂無慮放心自在，謝謝你們，我會永遠愛你們的。謹誌於 長庚大學 戊子年 夏AbbreviationsAP-1:Activating protein-1 BCECF:27-bis(carboxyethyl)-5(6)-carboxyfluorescein Pharmacological InhibitorsNameTargetGenisteinTyrosi
4、ne kinaseD609PC-PLCU73122PI-PLCStaurosporineNon-selective for PKCsGF109203XNon-selective for PKCsRo31-8220Non-selective for PKCsG-6976Ca2+-dependent PKCsPMAPKCs activatorBAPTAIntracellular Ca2+chelatorPD98059MEK1/2U0126MEK1/2SB202190P38 MAPKSP600125JNKPP1c-SrcAG1478EGFRWortmanninPI3-KLY294002PI3-KSH
5、-5AktCurcuminp300TanshinoneAP-1HelenalinNF-BAACOCF3Analog of arachidonic acid 中文摘要中文摘要(Abstract in Chinese)-1 呼吸道的發炎反應已被證實為呼吸系統疾病，包括：氣喘、慢性阻塞性肺疾病等的一個特殊表徵。在正常的生理環境或是病理狀態下，氣管平滑肌細胞也已被證實為神經傳導物質、發炎反應調控因數或外來物質作用的一種目標細胞(end-effect cells)。除此之外，氣管平滑肌細胞本身也是細胞激素、趨化激素、生長因數以及細胞外基質分泌的重要來源，在細胞病理狀態下的調節及結構重組中扮演重要的角色。
6、當呼吸道發炎時，氣管會表現cytosolic phospholipase A2(cPLA2)及vascular cell adhesion molecule-1(VCAM-1)等發炎性蛋白質進而調控整個發炎反應過程。再者，細胞激素及趨化激素已被證實可以調控cPLA2及VCAM-1的表現，因而調節呼吸道疾病的病程。研究調查指出，過敏原、毒害環境、病毒或細菌感染可以造成白血球細胞及淋巴細胞浸潤的現象進而導致細胞傷害或是支氣管的發炎反應。Lipopolysaccharide(LPS)已被發現存在於塵璊之中，是調控發炎反應的重要物質之。LPS本身是格蘭氏陰性菌細胞壁外膜的主要成分，是格蘭氏陰性細菌感染
7、引發宿主免疫反應的主要調控分子。雖然LPS已被證實可以刺激內皮細胞經由mitogen-activated protein kinases(MAPKs)、phosphoinositide 3-kinases(PI3-K)/AKT及其他訊息傳遞因數調控cPLA2及VCAM-1表現，然而對於LPS刺激氣管平滑肌細胞表現cPLA2及VCAM-1的分子機轉仍然並未被證實。因此，本篇論文主要是探討LPS刺激氣管平滑肌細胞表現cPLA2及VCAM-1等發炎蛋白的相關分子機制。我們將會建立細胞內訊號分子以及細胞核內共同活化因數(co-activators)間的相對關係模式。我們的假設為LPS經由增加cPLA2
8、及VCAM-1的表現量而影響氣管平滑肌細胞與白血球細胞之間的相互作用，進而促使呼吸道發炎反應的產生。為了說明這些問題，我們將一一探討細胞內的相關訊息傳遞因數，如：protein kinase Cs（PKCs）、MAPKs、Src、proline-rich tyrosine kinase(PyK2)、epidermal growth factor(EGFR)、PI3-K/Akt、nuclear factor-B(NF-B)、activator protein-1(AP-1)和histone acetyltransferase(HAT)等，是否參與在LPS刺激氣管平滑肌細胞引發cPLA2及VCAM
9、-1表現的機轉當中。在本篇論文的第一個部份主要是探討LPS刺激氣管平滑肌細胞調控cPLA2表現的機轉。LPS以時間依賴性(time-dependent)的方式刺激cPLA2的表現以及PGE2的釋放，此種現象可以受到前處理genistein(tyrosine kinase抑制劑)、D609(phosphatidycholine-phospholipase C抑制劑)、U73122(phosphatidylinositol-phospholipase C抑制劑)、GF109203X和staurosporine(PKC抑制劑)、BAPTA/AT plus EDTA(鈣離子敖合劑)、PD98059(M
10、EK1/2抑制劑)、SB202190(p38抑制劑)、SP600125(c-Jun terminal kinase(JNK)抑制劑)、LY294002與wortmannin(PI3-K抑制劑)等抑制劑與p42或p38 dominant negative mutants transfection所抑制。LPS刺激誘發cPLA2表現和PGE2釋放等現象也會受到前處理helenalin(NF-B選擇性抑制劑)或是NF-B inducing kinase(NIK)、IB kinase(IKK)-和IKK-dominant negative mutants transfection等作用而減低。並且，L
11、PS刺激也可以直接促進IB-分解使得NF-B轉移到細胞核內。另外，LPS刺激cPLA2的磷酸化增加會受到PD98059、GF109203X和staurosporine等抑制劑前處理所抑制，顯示LPS可以經由p42/p44 MAPK和PKC調控PLA2的活性。再者，經由AACOCF3(選擇性cPLA2抑制劑)抑制LPS所誘發的cPLA2與COX-2表現和PGE2釋放等現象證明cPLA2在此發炎反應中所扮演的角色。經由上述這些結果確認LPS刺激氣管平滑肌細胞可以經由PLC、鈣離子、PKC、tyrosine kinase、MAPKs、PI3-K和NF-B等訊號分子調控cPLA2表現。中文摘要中文摘要
12、(Abstract in Chinese)-2在本篇論文的第二個部分主要是探討MAPKs和NF-B在LPS刺激VCAM-1表現過程中所扮演的角色。LPS以時間依賴性的方式刺激VCAM-1蛋白質及mRNA表現量增加，此項作用可被MEK1/2(U0126)、p38、JNK、NF-B等專一性抑制劑或是MEK、p42、p38等siRNA前處理的方式所抑制。經由前處理抑制劑U0126，SB202190或是MEK、p42、p38等siRNA transfection也會減弱LPS所誘發的p42/p44MAPK和p38磷酸化作用。另外，LPS刺激也會造成IB-分解，和NF-B轉移到細胞核內。此項作用能夠受到
13、helenalin，U0126，SB202190，SP600125等藥理性抑制劑前處理所抑制。再者，LPS刺激VCAM-1的表現量增加可以促進多型性核白血球(PMNs)黏附到人類氣管平滑肌細胞，而前處理抑制劑helenalin，U0126或SP600125則可以抑制此情形。因此，經由上述這些結果推測，在人類氣管平滑肌細胞中，LPS的刺激可以經由p42/p44MAPK，p38 和 JNK調控NF-B的活性增加，進而引發VCAM-1基因的大量表現。中文摘要中文摘要(Abstract in Chinese)-3 論文的第三部份主要是探討LPS是否能經由Src/EGFR/PI3-K/Akt/p300等
14、一連串的訊息傳遞途徑調控VCAM-1基因表現。LPS刺激VCAM-1基因大量的表現所強化的嗜中性白血球黏附作用可被抑制劑LY294002和Wortmannin所抑制。LPS造成Src、PYK2、EGFR及AKT的磷酸化作用以及VCAM-1基因的表現也可被Src(PP1)、EGFR(AG1478)、PI3-K、AKT(SH-5)、p300(curcumin)等藥理性抑制劑，或是Src，AKT，p300等siRNA 以及p110 shRNA transfection前處理方式所抑制。利用免疫螢光，免疫沉澱，和ChIP(Chromatin immunoprecipition)assay等實驗進步發現
15、，LPS的刺激可以造成AKT的活化作用並且轉移到細胞核內，並且與細胞核內蛋白p300以及VCAM-1 promoter region連結在一起，進而調控VCAM-1的 promoter 活性促使VCAM-1 mRNA表現量增加。經由以上的結果，我們發現，當LPS刺激人類氣管平滑肌細胞，可以經由transactivation pathway(Src/PYK2/EGFR)來調控AKT的磷酸化以及增強p300活性，進而導致VCAM-1的基因表現增加。中文摘要中文摘要(Abstract in Chinese)-4中文摘要中文摘要(Abstract in Chinese)-5在論文的最後一個部分，我們將
16、焦點集中於AP-1是否也參與在LPS刺激VCAM-1的表現過程當中。前處理AP-1的抑制劑(tanshinone)，不管是在人類氣管平滑肌細胞或是在ICR老鼠等活體外或體內的試驗當中，都可明顯減少LPS所誘發的VCAM-1基因表現以及白血球細胞的吸附聚集現象。LPS可以刺激c-Fos的表現量增加，並且受到前處理GF109203X，Ro-318220、G-6976(conventional PKCs抑制劑)、U0126和SB202190等抑制劑所抑制。另外給予GF109203X、Ro31-8220、G-6976、U0126、SB202190和SP600125等抑制劑，皆可以減弱LPS刺激氣管平滑
17、肌細胞所誘發的c-Jun表現量。LPS刺激之下c-Fos和c-Jun的表現量可以進而促使AP-1 promoter的活性增加。活化的AP-1會結合到VCAM-1 promoter region，導致VCAM-1 promoter 的活性增加，促使VCAM-1 mRNA以及蛋白質的表現，進而強化人類氣管平滑肌細胞與白血球之間的黏附作用。此現象也可以藉由前處理GF109203X、Ro31-8220或G-6976等抑制劑而受到抑制作用。這些實驗結果說明，在人類氣管平滑肌細胞中，LPS也能夠透過活化PKCs/MAPKs/AP-1等一連串的訊息傳遞路徑來調節VCAM-1的表現。中文摘要中文摘要(Abst
18、ract in Chinese)-6 本篇論文深入探討LPS在人類氣管平滑肌細胞中的作用機制，證實我們於研究開始時所設立的假設，發現LPS能夠經由增加氣管平滑肌細胞與白血球細胞之間的交互作用，進而促使發炎反應的發生，而參與整個呼吸道疾病病程的發展。希望能藉由增加對於cPLA2和VCAM-1等發炎基因調控相關訊息傳遞機轉的瞭解，發展出更適當的抗發炎的新療程。英文摘要英文摘要(Abstract in English)Airway inflammation has been proven as significant features of airway diseases including ast
19、hma and chronic obstructive pulmonary disease(COPD).Tracheal smooth muscle cells(TSMCs)function as end-effector cells involving in response to stimulation of various neurotransmitters,proinflammatory mediators,and exogenous substances released under homeostatic or pathologic conditions.In addition,T
20、SMCs are the source of proinflammatory cytokines,chemokines,growth factors,and ECM,which further play a significant role in regulating cellular pathology and structure remodeling.During the airway inflammation,cytosolic phospholipase A2(cPLA2).Table of Contents(1)Page(頁數)指導教授推薦書指導教授推薦書.i口試委員會審定書口試委員
21、會審定書.ii博士論文著作授權書博士論文著作授權書.iiiAcknowledgements(誌謝誌謝)ivAbbreviations(縮寫表縮寫表).vPharmacological inhibitors(藥理性抑制劑藥理性抑制劑).viiAbstract in Chinese(中文摘要中文摘要).viiiAbstract in English(英文摘要英文摘要)xiiTable of Contents(目錄目錄).xviCHAPTER I INTRODUCTION.1SECTION 1.Background and significance.2SECTION 2.Specific aims1
22、8SECTION 3.Figures and tables.19CHAPTER II MATERIALS AND METHODS.57SECTION 1.Materials.58SECTION 2.Methods.60Table of Contents(2)CHAPTER III(Results).69Induction of cytosolic phospholipase A2 by lipopolysaccharide in canine tracheal smooth muscle cells:Involvement of MAPKs and NF-B pathwaysSECTION 1
23、:Background70SECTION 2:Results.73SECTION 3:Discussion.79SECTION 4:Figures and legends83CHAPTER IV(Results).100Involvement of MAPKs and NF-B in LPS-induced VCAM-1 expression in human tracheal smooth muscle cellsCHAPTER V(Results).127Lipopolysaccharide induces VCAM-1 expression and neutrophil adhesion
24、 to human tracheal smooth muscle cells:Involvement of Src/EGFR/PI3-K/Akt pathwayCHAPTER VI(Results).157Lipopolysaccharide up-regulates VCAM-1 expression in human tracheal smooth muscle cells via PKC/MAPKs/AP-1 activationCHAPTER VII CONCLUSION AND PERSPECTIVES.188SECTION 1:Conclusion.189SECTION 2:Per
25、spectives.192SECTION 3:Schemes193PUBLICATIONS.194REFERENCES.196SECTION 1.Background and significanceRoles of proinflammatory protein expression in airway inflammatory diseasesI.1-1 The importance of airway smooth muscle cells in airway inflammatory diseasess I.1-2 Role of lipopolysaccharide in airwa
26、y inflammatory diseasessI.1-3 Role of cytosolic phospholipase A2 in airway inflammatory diseasesI.1-4 Role of adhesion molecules in airway inflammatory diseasesCHAPTER I INTRODUCTIONI.1-6 Roles of MAPK pathways in airway inflammatory diseasesI.1-7 Role of PI3-kinase/Akt pathways in airway inflammato
27、ry diseaseI.1-8 Role of Calcium in airway inflammatory diseasesI.1-9 Role of PKC in airway inflammatory diseasesI.1-10 Role of RTKs in airway inflammatory diseaseI.1-11 Role of NF-B and AP-1 in airway inflammatory diseasesI.1-12 Role of p300 in airway inflammatory diseasesThe intracellular signaling
28、s of Toll-like receptors(TLRs)SECTION 2.Specific aimsThe overall objective of this work is to establish the mechanisms of LPS action on the TSMCs that regulates the expression of VCAM-1 thus results in enhancement of cell adhesion and inflammation.Our hypothesis of this proposal is that up-regulatio
29、n of cPLA2 and VCAM-1 induced by LPS may contribute to leukocyte/TSMCs interaction and exaggerate inflammatory responses in airways.To evaluate the hypothesis,the specific aims addressed are:1.To investigate the protein and mRNA expression of cPLA2 and VCAM-1 induced by LPS in TSMCs.2.To differentia
30、te the pathways of MAPKs and NF-B activation involved in expression of cPLA2 and VCAM-1 induced by LPS in TSMCs.3.To investigate whether alternative pathways related to LPS-induced cPLA2 and VCAM-1 expression in TSMCs:RTK transactivation,Src and PI3K/Akt.4.To determine if Ca2+or PKC isozymes activat
31、ion related to LPS correlated to cPLA2 and VCAM-1 expression in TSMCs.5.To construct a cPLA2 and VCAM-1 promoter construct and detect the activity of cPLA2 and VCAM-1 promoter stimulated by LPS.6.To establish the relationship of p300 and NF-B,AP-1 related to the expression of cPLA2 and VCAM-1 in TSM
32、Cs.7.To determine the functional activity of cPLA2 and VCAM-1 expressed by LPS in TSMCs.These results will provide new insights into the mechanisms of LPS action,supporting the hypothesis that LPS may contribute to leukocyte/TSMCs interaction and promote inflammatory responses involved in the develo
33、pment of airway diseases.Increased understanding of signal transduction mechanisms underlying cPLA2 and VCAM-1 gene regulation will create opportunities for the development of anti-inflammation therapeutic strategies.CHAPTER I INTRODUCTION CHAPTER II MATERIALS AND METHODSSECTION 1.MaterialsII.1-1 Ch
34、emicals and reagentsII.1-2 AntibodiesII.1-3 si-RNAs,shRNAs and plasmidsSECTION 2.MethodsII.2-1 Isolation of HTSMCsII.2-2 Animal treatmentII.2-3 Isolation of CTSMCsII.2-4 Preparation of cell extracts and Western blotting analysisII.2-5 Total RNA extraction and RT-PCR analysisII.2-6 Isolation of bronc
35、hoalveolar lavage(BAL)II.2-7 Promoter luciferase activity assayII.2-8 Transient transfection with small interference RNA,short hairpin RNA and dominent negative plasmidII.2-9 Cell Fraction isolationII.2-10 Immunofluorescence stainingII.2-11 Coimmunoprecipitation AssayII.2-12 Chromatin immunoprecipit
36、ation assayII.2-13 Neutrophil adhesion assayII.2-14 Measurement of PGE2 levelsII.2-15 Analysis of data SECTION 1.MaterialsII.1-1.Chemicals and ReagentsII.1-2.AntibodiesII.1-3.siRNAs,shRNAs and plasmidsCHAPTER II MATERIALS AND METHODSSECTION 2.MethodsII.2-1.Isolation of HTSMCsII.2-2.Animal treatmentI
37、I.2-3.Isolation of CTSMCsII.2-4.Preparation of Cell Extracts and Western Blotting AnalysisII.2-5.Total RNA Extraction and RT-PCR AnalysisII.2-6.Isolation of bronchoalveolar lavage(BAL)II.2-7.Promoter Luciferase Activity AssayII.2-8.Transient Transfection with Small Interference RNA,Short Hairpin RNA
38、 and Dominent Negative PlasmidII.2-9.Cell Fraction IoslationII.2-10.Immunofluorescence StainingII.2-11.Coimmunoprecipitation AssayII.2-12.Chromatin Immunoprecipitation AssayII.2-13.Neutrophil adhesion assayII.2-14.Measurement of PGE2 levelsII.2-13.Analysis of DataCHAPTER II MATERIALS AND METHODSIndu
39、ction of cytosolic phospholipase A2 by lipopolysaccharide in canine tracheal smooth muscle cells:Involvement of MAPKs and NF-B pathwaysSECTION 1:BackgroundSECTION 2:ResultsSECTION 3:DiscussionSECTION 4:Figures and legendsThis part of thesis had been published on Cellular Signalling,2006,18:1201-1211
40、CHAPTER III(Results)Involvement of MAPKs and NF-B in LPS-induced VCAM-1 expression in human tracheal smooth muscle cellsSECTION 1:BackgroundSECTION 2:ResultsSECTION 3:DiscussionSECTION 4:Figures and legendsThis part of thesis had been published on Cellular Signalling,2007,19:1258-1267CHAPTER IV(Resu
41、lts)Lipopolysaccharide induces VCAM-1 expression and neutrophil adhesion to human tracheal smooth muscle cells:Involvement of Src/EGFR/PI3-K/Akt pathwaySECTION 1:BackgroundSECTION 2:ResultsSECTION 3:DiscussionSECTION 4:Figures and legendsThis part of thesis had been published on Toxicology and Appli
42、ed Pharmacology,2008,228:256-268CHAPTER V(Results)Lipopolysaccharide up-regulates VCAM-1 expression in human tracheal smooth muscle cells via PKC/MAPKs/AP-1 activationSECTION 1:BackgroundSECTION 2:ResultsSECTION 3:Figures and legendsSECTION 4:DiscussionCHAPTER VI(Results)Fig.10.LPS stimulates VCAM-1
43、 expression through PKCs/MAPKs/AP-1 pathway.SECTION 1:ConclusionSECTION 2:PerspectivesSECTION 3:SchemesCONCLUSION AND PERSPECTIVESCHAPTER VIISECTION 3:Schemes 1.Wang,C.C.,C.W.Lee,W.N.Lin,C.C.Lin,S.F.Luo,J.S.Wang,and C.M.Yang.Involvement of p42/p44 MAPK,p38 MAPK,JNK and NF-B in interleukin-1-induced
44、VCAM-1 expression in human tracheal smooth muscle cells.Am.J.Physiol.288:L227-L237,2005.2.Luo,S.F.,W.N.Lin,C.M.Yang,C.W.Lee,C.H.Liao,Y.L.Leu,and L.D.Hsiao.Induction of cytosolic phospholipase A2 by lipopolysaccharide in canine tracheal smooth muscle cells:involvement of MAPKs and NF-B pathways.Cell.
45、Signal.18:1201-1211,2006.3.Lee,C.W.,W.N.Lin,C.C.Lin,S.F.Luo,J.S.Wang,J.Pouyssegur,and C.M.Yang.Transcriptional regulation of VCAM-1 expression by tumor necrosis factor-in human tracheal smooth muscle cells:Involvement of MAPKs,NF-B,p300,and histone acetylation.J.Cell.Physiol.207:174-186,2006.4.Liang
46、,K.C.,C.W.Lee,W.N.Lin,C.C.Lin,C.B.Wu,S.F.Luo,and C.M.Yang.Interleukin-1 induces MMP-9 expression via p42/p44 MAPK,p38 MAPK,JNK and nuclear factor-B signaling pathways in human tracheal smooth muscle cells.J.Cell Physiol.211:759-770,2007.5.Lee,C.W.,C.C.Lin,W.N.Lin,K.C.Liang,S.F.Luo,C.B.Wu,S.W.Wang,an
47、d C.M.Yang.TNF-induces MMP-9 expression via activation of Src/EGFR,PDGFR/PI3K/Akt cascade and promotion of NF-B/p300 binding in human tracheal smooth muscle cells.Am J Physiol Lung Cell Mol Physiol.292:L799-L812,2007.6.Lin,W.N.,C.W.Lee,C.J.Wang,S.F.Luo,J.S.Wang,and C.M.Yang.Involvement of p42/p44 MA
48、PK,p38 MAPK,JNK and NF-B in LPS-induced VCAM-1 expression in human tracheal smooth muscle cells.Cell Signal.19:1258-1267,2007.7.Lin,W.N.,S.F.Luo,C.B.Wu,Lin C.C.,and Yang C.M.Lipopolysaccharide induces VCAM-1 expression and neutrophil adhesion to human tracheal smooth muscle cells:Involvement of Src/
49、EGFR/PI3-K/Akt pathway.Toxicol.Appl.Pharmacol.228:256-268,2008.8.Luo,S.F.,C.C.Lin,H.C.Chen,W.N.Lin,I.T.Lee,C.W.Lee,L.D.Hsiao,and C.M.Yang.Involvement of MAPKs,NF-B and p300 co-activator in IL-1-induced cytosolic phospholipase A2 expression in canine tracheal smooth muscle cells.Toxicol.Appl.Pharmacol.In press,2008.9.Lin,W.N.,C.C.Lin,S.F.Luo,and C.M.Yang.Lipopolysaccharide up-regulates VCAM-1 expression in human tracheal smooth muscle cells via PKC/MAPKs/AP-1 activation.In preparation.PUBLICATIONS
