TitlePage長(zhǎng)庚大學(xué)生物醫(yī)學(xué)研究所生理暨藥課件

TitlePage長(zhǎng)庚大學(xué)生物醫(yī)學(xué)研究所生理暨藥理組博士論文探討LPS誘發(fā)氣管平滑肌細(xì)胞調(diào)控黏附分子與cytosolic

phospholipaseA2

表現(xiàn)的機(jī)轉(zhuǎn)Mechanismsunderlyinglipopolysaccharide-inducedexpressionofadhesionmoleculesandcytosolic

phospholipaseA2intrachealsmoothmusclecells

研究生：林維寧

(Wei-NingLin)

指導(dǎo)教授：楊春茂

博士

(Chuen-MaoYang,Ph.D)中華民國(guó)九十七年七月誌謝『不要爲(wèi)了明天憂慮，因?yàn)槊魈熳杂忻魈斓膽n慮，一天的難處一天當(dāng)就夠了?！获R太福音6:34從大二進(jìn)實(shí)驗(yàn)室到完成博士學(xué)位的現(xiàn)在，「把握今天」一直是我的目標(biāo)，不用爲(wèi)了明天擔(dān)憂，只要肯做就會(huì)有收穫，……在這裡先要特別感謝我的指導(dǎo)老師楊春茂教授，………另外，我也要感謝國(guó)防醫(yī)學(xué)院藥理研究所顏茂雄教授、臺(tái)灣大學(xué)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(duì)此論文內(nèi)容的審閱與指正，並提供寶貴的意見(jiàn)使我的論文更趨於完善，對(duì)未來(lái)的研究方向有更多的想法。我也要謝謝實(shí)驗(yàn)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(zhǎng)大的哥哥跟中閎，都是我非常穩(wěn)固的精神後盾，因?yàn)橛心銈冏屛乙宦纷邅?lái)無(wú)憂無(wú)慮放心自在，謝謝你們，我會(huì)永遠(yuǎn)愛(ài)你們的。謹(jǐn)誌於長(zhǎng)庚大學(xué)戊子年夏AbbreviationsAP-1:Activatingprotein-1BCECF:2’7’-bis(carboxyethyl)-5(6)-carboxyfluorescein……

PharmacologicalInhibitorsNameTargetGenisteinTyrosinekinaseD609PC-PLCU73122PI-PLCStaurosporineNon-selectiveforPKCsGF109203XNon-selectiveforPKCsRo31-8220Non-selectiveforPKCsG?-6976Ca2+-dependentPKCsPMAPKCsactivatorBAPTAIntracellularCa2+

chelatorPD98059MEK1/2U0126MEK1/2SB202190P38MAPKSP600125JNKPP1c-SrcAG1478EGFRWortmanninPI3-KLY294002PI3-KSH-5AktCurcuminp300TanshinoneAP-1HelenalinNF-BAACOCF3Analogofarachidonicacid中文摘要

(AbstractinChinese)-1呼吸道的發(fā)炎反應(yīng)已被證實(shí)為呼吸系統(tǒng)疾病，包括：氣喘、慢性阻塞性肺疾病等的一個(gè)特殊表徵。在正常的生理環(huán)境或是病理狀態(tài)下，氣管平滑肌細(xì)胞也已被證實(shí)為神經(jīng)傳導(dǎo)物質(zhì)、發(fā)炎反應(yīng)調(diào)控因數(shù)或外來(lái)物質(zhì)作用的一種目標(biāo)細(xì)胞(end-effectcells)。除此之外，氣管平滑肌細(xì)胞本身也是細(xì)胞激素、趨化激素、生長(zhǎng)因數(shù)以及細(xì)胞外基質(zhì)分泌的重要來(lái)源，在細(xì)胞病理狀態(tài)下的調(diào)節(jié)及結(jié)構(gòu)重組中扮演重要的角色。當(dāng)呼吸道發(fā)炎時(shí)，氣管會(huì)表現(xiàn)cytosolic

phospholipaseA2(cPLA2)及vascularcelladhesionmolecule-1(VCAM-1)等發(fā)炎性蛋白質(zhì)進(jìn)而調(diào)控整個(gè)發(fā)炎反應(yīng)過(guò)程。再者，細(xì)胞激素及趨化激素已被證實(shí)可以調(diào)控cPLA2及VCAM-1的表現(xiàn)，因而調(diào)節(jié)呼吸道疾病的病程。研究調(diào)查指出，過(guò)敏原、毒害環(huán)境、病毒或細(xì)菌感染可以造成白血球細(xì)胞及淋巴細(xì)胞浸潤(rùn)的現(xiàn)象進(jìn)而導(dǎo)致細(xì)胞傷害或是支氣管的發(fā)炎反應(yīng)。Lipopolysaccharide(LPS)已被發(fā)現(xiàn)存在於塵璊之中，是調(diào)控發(fā)炎反應(yīng)的重要物質(zhì)之ㄧ。LPS本身是格蘭氏陰性菌細(xì)胞壁外膜的主要成分，是格蘭氏陰性細(xì)菌感染引發(fā)宿主免疫反應(yīng)的主要調(diào)控分子。雖然LPS已被證實(shí)可以刺激內(nèi)皮細(xì)胞經(jīng)由mitogen-activatedproteinkinases(MAPKs)、phosphoinositide3-kinases(PI3-K)/AKT及其他訊息傳遞因數(shù)調(diào)控cPLA2及VCAM-1表現(xiàn)，然而對(duì)於LPS刺激氣管平滑肌細(xì)胞表現(xiàn)cPLA2及VCAM-1的分子機(jī)轉(zhuǎn)仍然並未被證實(shí)。因此，本篇論文主要是探討LPS刺激氣管平滑肌細(xì)胞表現(xiàn)cPLA2及VCAM-1等發(fā)炎蛋白的相關(guān)分子機(jī)制。我們將會(huì)建立細(xì)胞內(nèi)訊號(hào)分子以及細(xì)胞核內(nèi)共同活化因數(shù)(co-activators)間的相對(duì)關(guān)係模式。我們的假設(shè)為L(zhǎng)PS經(jīng)由增加cPLA2及VCAM-1的表現(xiàn)量而影響氣管平滑肌細(xì)胞與白血球細(xì)胞之間的相互作用，進(jìn)而促使呼吸道發(fā)炎反應(yīng)的產(chǎn)生。為了說(shuō)明這些問(wèn)題，我們將一一探討細(xì)胞內(nèi)的相關(guān)訊息傳遞因數(shù)，如：proteinkinaseCs（PKCs）、MAPKs、Src、proline-richtyrosinekinase(PyK2)、epidermalgrowthfactor(EGFR)、PI3-K/Akt、

nuclearfactor-κB(NF-κB)、activatorprotein-1(AP-1)和histone

acetyltransferase(HAT)等，是否參與在LPS刺激氣管平滑肌細(xì)胞引發(fā)cPLA2及VCAM-1表現(xiàn)的機(jī)轉(zhuǎn)當(dāng)中。在本篇論文的第一個(gè)部份主要是探討LPS刺激氣管平滑肌細(xì)胞調(diào)控cPLA2表現(xiàn)的機(jī)轉(zhuǎn)。LPS以時(shí)間依賴性(time-dependent)的方式刺激cPLA2的表現(xiàn)以及PGE2的釋放，此種現(xiàn)象可以受到前處理genistein(tyrosinekinase抑制劑)、D609(phosphatidycholine-phospholipaseC抑制劑)、U73122(phosphatidylinositol-phospholipaseC抑制劑)、GF109203X和staurosporine(PKC抑制劑)、BAPTA/ATplusEDTA(鈣離子敖合劑)、PD98059(MEK1/2抑制劑)、SB202190(p38抑制劑)、SP600125(c-Junterminalkinase(JNK)抑制劑)、LY294002與wortmannin(PI3-K抑制劑)等抑制劑與p42或p38dominantnegativemutantstransfection所抑制。LPS刺激誘發(fā)cPLA2表現(xiàn)和PGE2釋放等現(xiàn)象也會(huì)受到前處理helenalin(NF-κB選擇性抑制劑)或是NF-Binducingkinase(NIK)、

IBkinase(IKK)-和IKK-βdominantnegativemutantstransfection等作用而減低。並且，LPS刺激也可以直接促進(jìn)IκB-α分解使得NF-κB轉(zhuǎn)移到細(xì)胞核內(nèi)。另外，LPS刺激cPLA2的磷酸化增加會(huì)受到PD98059、GF109203X和staurosporine等抑制劑前處理所抑制，顯示LPS可以經(jīng)由p42/p44MAPK和PKC調(diào)控PLA2的活性。再者，經(jīng)由AACOCF3(選擇性cPLA2抑制劑)抑制LPS所誘發(fā)的cPLA2與COX-2表現(xiàn)和PGE2釋放等現(xiàn)象證明cPLA2在此發(fā)炎反應(yīng)中所扮演的角色。經(jīng)由上述這些結(jié)果確認(rèn)LPS刺激氣管平滑肌細(xì)胞可以經(jīng)由PLC、鈣離子、PKC、tyrosinekinase、MAPKs、PI3-K和NF-κB等訊號(hào)分子調(diào)控cPLA2表現(xiàn)。中文摘要

(AbstractinChinese)-2在本篇論文的第二個(gè)部分主要是探討MAPKs和NF-B在LPS刺激VCAM-1表現(xiàn)過(guò)程中所扮演的角色。LPS以時(shí)間依賴性的方式刺激VCAM-1蛋白質(zhì)及mRNA表現(xiàn)量增加，此項(xiàng)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(jīng)由前處理抑制劑U0126，SB202190或是MEK、p42、p38等siRNA

transfection也會(huì)減弱LPS所誘發(fā)的p42/p44MAPK和p38磷酸化作用。另外，LPS刺激也會(huì)造成IB-分解，和NF-B轉(zhuǎn)移到細(xì)胞核內(nèi)。此項(xiàng)作用能夠受到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(xiàn)量增加可以促進(jìn)多型性核白血球(PMNs)黏附到人類氣管平滑肌細(xì)胞，而前處理抑制劑helenalin，U0126或SP600125則可以抑制此情形。因此，經(jīng)由上述這些結(jié)果推測(cè)，在人類氣管平滑肌細(xì)胞中，LPS的刺激可以經(jīng)由p42/p44MAPK，p38和

JNK調(diào)控NF-B的活性增加，進(jìn)而引發(fā)VCAM-1基因的大量表現(xiàn)。中文摘要

(AbstractinChinese)-3

論文的第三部份主要是探討LPS是否能經(jīng)由Src/EGFR/PI3-K/Akt/p300等一連串的訊息傳遞途徑調(diào)控VCAM-1基因表現(xiàn)。LPS刺激VCAM-1基因大量的表現(xiàn)所強(qiáng)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(xiàn)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p110shRNA

transfection前處理方式所抑制。利用免疫螢光，免疫沉澱，和ChIP(Chromatinimmunoprecipition)assay等實(shí)驗(yàn)進(jìn)ㄧ步發(fā)現(xiàn)，LPS的刺激可以造成AKT的活化作用並且轉(zhuǎn)移到細(xì)胞核內(nèi)，並且與細(xì)胞核內(nèi)蛋白p300以及VCAM-1promoterregion連結(jié)在一起，進(jìn)而調(diào)控VCAM-1的

promoter活性促使VCAM-1mRNA表現(xiàn)量增加。經(jīng)由以上的結(jié)果，我們發(fā)現(xiàn)，當(dāng)LPS刺激人類氣管平滑肌細(xì)胞，可以經(jīng)由transactivationpathway(Src/PYK2/EGFR)來(lái)調(diào)控AKT的磷酸化以及增強(qiáng)p300活性，進(jìn)而導(dǎo)致VCAM-1的基因表現(xiàn)增加。中文摘要

(AbstractinChinese)-4中文摘要

(AbstractinChinese)-5在論文的最後一個(gè)部分，我們將焦點(diǎn)集中於AP-1是否也參與在LPS刺激VCAM-1的表現(xiàn)過(guò)程當(dāng)中。前處理AP-1的抑制劑(tanshinone)，不管是在人類氣管平滑肌細(xì)胞或是在ICR老鼠等活體外或體內(nèi)的試驗(yàn)當(dāng)中，都可明顯減少LPS所誘發(fā)的VCAM-1基因表現(xiàn)以及白血球細(xì)胞的吸附聚集現(xiàn)象。LPS可以刺激c-Fos的表現(xiàn)量增加，並且受到前處理GF109203X，Ro-318220、G?-6976(conventionalPKCs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刺激氣管平滑肌細(xì)胞所誘發(fā)的c-Jun表現(xiàn)量。LPS刺激之下c-Fos和c-Jun的表現(xiàn)量可以進(jìn)而促使AP-1promoter的活性增加?；罨腁P-1會(huì)結(jié)合到VCAM-1promoterregion，導(dǎo)致VCAM-1promoter的活性增加，促使VCAM-1mRNA以及蛋白質(zhì)的表現(xiàn)，進(jìn)而強(qiáng)化人類氣管平滑肌細(xì)胞與白血球之間的黏附作用。此現(xiàn)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(shí)驗(yàn)結(jié)果說(shuō)明，在人類氣管平滑肌細(xì)胞中，LPS也能夠透過(guò)活化PKCs/MAPKs/AP-1等一連串的訊息傳遞路徑來(lái)調(diào)節(jié)VCAM-1的表現(xiàn)。中文摘要

(AbstractinChinese)-6

本篇論文深入探討LPS在人類氣管平滑肌細(xì)胞中的作用機(jī)制，證實(shí)我們於研究開(kāi)始時(shí)所設(shè)立的假設(shè)，發(fā)現(xiàn)LPS能夠經(jīng)由增加氣管平滑肌細(xì)胞與白血球細(xì)胞之間的交互作用，進(jìn)而促使發(fā)炎反應(yīng)的發(fā)生，而參與整個(gè)呼吸道疾病病程的發(fā)展。希望能藉由增加對(duì)於cPLA2和VCAM-1等發(fā)炎基因調(diào)控相關(guān)訊息傳遞機(jī)轉(zhuǎn)的瞭解，發(fā)展出更適當(dāng)?shù)目拱l(fā)炎的新療程。英文摘要

(AbstractinEnglish)Airwayinflammationhasbeenprovenassignificantfeaturesofairwaydiseasesincludingasthmaandchronicobstructivepulmonarydisease(COPD).Trachealsmoothmusclecells(TSMCs)functionasend-effectorcellsinvolvinginresponsetostimulationofvariousneurotransmitters,proinflammatorymediators,andexogenoussubstancesreleasedunderhomeostaticorpathologicconditions.Inaddition,TSMCsarethesourceofproinflammatorycytokines,chemokines,growthfactors,andECM,whichfurtherplayasignificantroleinregulatingcellularpathologyandstructureremodeling.Duringtheairwayinflammation,cytosolic

phospholipaseA2(cPLA2).…….TableofContents(1)Page(頁(yè)數(shù))指導(dǎo)教授推薦書…………………i口試委員會(huì)審定書………………ii博士論文著作授權(quán)書……………iiiAcknowledgements(誌謝)…………ivAbbreviations(縮寫表)…………….vPharmacologicalinhibitors(藥理性抑制劑)……...viiAbstractinChinese(中文摘要)…………………...viiiAbstractinEnglish(英文摘要)……………………xiiTableofContents(目錄)…………...xviCHAPTERIINTRODUCTION……………1SECTION1.Backgroundandsignificance……2SECTION2.Specificaims………………………18SECTION3.Figuresandtables………………19CHAPTERIIMATERIALSANDMETHODS……………57SECTION1.Materials…………...58SECTION2.Methods…………60TableofContents(2)CHAPTERIII(Results)…………...69Inductionofcytosolic

phospholipaseA2bylipopolysaccharideincaninetrachealsmoothmusclecells:InvolvementofMAPKsandNF-κBpathwaysSECTION1:Background……………70SECTION2:Results………………73SECTION3:Discussion……………..79SECTION4:Figuresandlegends……………………83CHAPTERIV(Results)…….……………………..100InvolvementofMAPKsandNF-κBinLPS-inducedVCAM-1expressioninhumantrachealsmoothmusclecellsCHAPTERV(Results)..…..……………………...127LipopolysaccharideinducesVCAM-1expressionandneutrophiladhesiontohumantrachealsmoothmusclecells:InvolvementofSrc/EGFR/PI3-K/AktpathwayCHAPTERVI(Results)…………..157Lipopolysaccharideup-regulatesVCAM-1expressioninhumantrachealsmoothmusclecellsviaPKC/MAPKs/AP-1activationCHAPTERVIICONCLUSIONANDPERSPECTIVES188SECTION1:Conclusion...189SECTION2:Perspectives...192SECTION3:Schemes………………193PUBLICATIONS…………………..194REFERENCES...196SECTION1.BackgroundandsignificanceRolesofproinflammatoryproteinexpressioninairwayinflammatorydiseasesI.1-1Theimportanceofairwaysmoothmusclecellsinairwayinflammatorydiseasess

I.1-2RoleoflipopolysaccharideinairwayinflammatorydiseasessI.1-3Roleofcytosolic

phospholipaseA2inairwayinflammatorydiseasesI.1-4RoleofadhesionmoleculesinairwayinflammatorydiseasesCHAPTERIINTRODUCTIONI.1-6RolesofMAPKpathwaysinairwayinflammatorydiseasesI.1-7RoleofPI3-kinase/AktpathwaysinairwayinflammatorydiseaseI.1-8RoleofCalciuminairwayinflammatorydiseasesI.1-9RoleofPKCinairwayinflammatorydiseasesI.1-10RoleofRTKsinairwayinflammatorydiseaseI.1-11RoleofNF-BandAP-1inairwayinflammatorydiseasesI.1-12Roleofp300inairwayinflammatorydiseasesTheintracellularsignalingsofToll-likereceptors(TLRs)SECTION2.SpecificaimsTheoverallobjectiveofthisworkistoestablishthemechanismsofLPSactionontheTSMCsthatregulatestheexpressionofVCAM-1thusresultsinenhancementofcelladhesionandinflammation.Ourhypothesisofthisproposalisthatup-regulationofcPLA2andVCAM-1inducedbyLPSmaycontributetoleukocyte/TSMCsinteractionandexaggerateinflammatoryresponsesinairways.Toevaluatethehypothesis,thespecificaimsaddressedare:1.ToinvestigatetheproteinandmRNAexpressionofcPLA2andVCAM-1inducedbyLPSin

TSMCs.2.TodifferentiatethepathwaysofMAPKsandNF-BactivationinvolvedinexpressionofcPLA2andVCAM-1inducedbyLPSinTSMCs.3.ToinvestigatewhetheralternativepathwaysrelatedtoLPS-inducedcPLA2andVCAM-1expressioninTSMCs:RTKtransactivation,SrcandPI3K/Akt.4.TodetermineifCa2+orPKCisozymesactivationrelatedtoLPScorrelatedtocPLA2andVCAM-1expressioninTSMCs.5.ToconstructacPLA2andVCAM-1promoterconstructanddetecttheactivityofcPLA2andVCAM-1promoterstimulatedbyLPS.6.Toestablishtherelationshipofp300andNF-B,AP-1relatedtotheexpressionofcPLA2andVCAM-1inTSMCs.7.TodeterminethefunctionalactivityofcPLA2andVCAM-1expressedbyLPSinTSMCs.TheseresultswillprovidenewinsightsintothemechanismsofLPSaction,supportingthehypothesisthatLPSmaycontributetoleukocyte/TSMCsinteractionandpromoteinflammatoryresponsesinvolvedinthedevelopmentofairwaydiseases.IncreasedunderstandingofsignaltransductionmechanismsunderlyingcPLA2andVCAM-1generegulationwillcreateopportunitiesforthedevelopmentofanti-inflammationtherapeuticstrategies.

CHAPTERIINTRODUCTION

CHAPTERIIMATERIALSANDMETHODSSECTION1.MaterialsII.1-1ChemicalsandreagentsII.1-2AntibodiesII.1-3si-RNAs,shRNAsandplasmidsSECTION2.MethodsII.2-1IsolationofHTSMCsII.2-2AnimaltreatmentII.2-3IsolationofCTSMCsII.2-4PreparationofcellextractsandWesternblottinganalysisII.2-5TotalRNAextractionandRT-PCRanalysisII.2-6Isolationofbronchoalveolar

lavage(BAL)II.2-7PromoterluciferaseactivityassayII.2-8TransienttransfectionwithsmallinterferenceRNA,shorthairpinRNAanddominentnegativeplasmidII.2-9CellFractionisolationII.2-10ImmunofluorescencestainingII.2-11CoimmunoprecipitationAssayII.2-12ChromatinimmunoprecipitationassayII.2-13NeutrophiladhesionassayII.2-14MeasurementofPGE2levelsII.2-15Analysisofdata

SECTION1.MaterialsII.1-1.ChemicalsandReagentsII.1-2.AntibodiesII.1-3.siRNAs,shRNAsandplasmidsCHAPTERIIMATERIALSANDMETHODSSECTION2.MethodsII.2-1.IsolationofHTSMCsII.2-2.AnimaltreatmentII.2-3.IsolationofCTSMCsII.2-4.PreparationofCellExtractsandWesternBlottingAnalysisII.2-5.TotalRNAExtractionandRT-PCRAnalysisII.2-6.Isolationofbronchoalveolar

lavage(BAL)II.2-7.PromoterLuciferaseActivityAssayII.2-8.TransientTransfectionwithSmallInterferenceRNA,ShortHairpinRNAandDominent

NegativePlasmidII.2-9.CellFractionIoslationII.2-10.ImmunofluorescenceStainingII.2-11.CoimmunoprecipitationAssayII.2-12.ChromatinImmunoprecipitationAssayII.2-13.NeutrophiladhesionassayII.2-14.MeasurementofPGE2levelsII.2-13.AnalysisofDataCHAPTERIIMATERIALSANDMETHODSInductionofcytosolic

phospholipaseA2bylipopolysaccharideincaninetrachealsmoothmusclecells:InvolvementofMAPKsandNF-BpathwaysSECTION1:BackgroundSECTION2:ResultsSECTION3:DiscussionSECTION4:FiguresandlegendsThispartofthesishadbeenpublishedonCellularSignalling,2006,18:1201-1211CHAPTERIII(Results)InvolvementofMAPKsandNF-BinLPS-inducedVCAM-1expressioninhumantrachealsmoothmusclecellsSECTION1:BackgroundSECTION2:ResultsSECTION3:DiscussionSECTION4:FiguresandlegendsThispartofthesishadbeenpublishedonCellularSignalling,2007,19:1258-1267CHAPTERIV(Results)LipopolysaccharideinducesVCAM-1expressionandneutrophiladhesiontohumantrachealsmoothmusclecells:InvolvementofSrc/EGFR/PI3-K/AktpathwaySECTION1:BackgroundSECTION2:ResultsSECTION3:DiscussionSECTION4:FiguresandlegendsThispartofthesishadbeenpublishedonToxicologyandAppliedPharmacology,2008,228:256-268CHAPTERV(Results)Lipopolysaccharideup-regulatesVCAM-1expressioninhumantrachealsmoothmusclecellsviaPKC/MAPKs/AP-1activationSECTION1:BackgroundSECTION2:ResultsSECTION3:FiguresandlegendsSECTION4:DiscussionCHAPTERVI(Results)

Fig.10.LPSstimulatesVCAM-1expressionthroughPKCs/MAPKs/AP-1pathway.SECTION1:ConclusionSECTION2:PerspectivesSECTION3:SchemesCONCLUSIONANDPERSPECTIVESCHAPTERVIISECTION3:Schemes

Wang,C.C.,C.W.Lee,W.N.Lin,C.C.Lin,S.F.Luo,J.S.Wang,andC.M.Yang.Involvementofp42/p44MAPK,p38MAPK,JNKandNF-Bininterleukin-1-inducedVCAM-1expressioninhumantrachealsmoothmusclecells.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,andL.D.Hsiao.Inductionofcytosolic

phospholipaseA2bylipopolysaccharideincaninetrachealsmoothmusclecells:involvementofMAPKsandNF-Bpathways.Cell.Signal.18:1201-1211,2006.3.Lee,C.W.,W.N.Lin,C.C.Lin,S.F.Luo,J.S.Wang,J.Pouyssegur,andC.M.Yang.TranscriptionalregulationofVCAM-1expressionbytumornecrosisfactor-inhumantrachealsmoothmuscle