MAPK信號轉(zhuǎn)導(dǎo)通路對炎癥反應(yīng)的調(diào)控

1、絲裂原活化蛋白激酶(mitogen-activated proteink inase,MAPK是介導(dǎo)細(xì)胞反應(yīng)的重要信號系統(tǒng),普遍存在于多種生物,包括酵母和哺乳動物細(xì)胞。自從1991年Sturgill 及其同事在哺乳動物細(xì)胞鑒定出細(xì)胞外信號調(diào)節(jié)激酶(extracellular-signal re gulatedprotein kinase,E RK,MAPK 信號轉(zhuǎn)導(dǎo)通路的研究取得了迅猛發(fā)展1,2。除ERK 外,還發(fā)現(xiàn)和克隆了c-Jun 氨基未端激酶(c-Jun amino-terminal kinase,JNK/267綜述MAPK 信號轉(zhuǎn)導(dǎo)通路對炎癥反應(yīng)的調(diào)控姜勇 ,龔小衛(wèi)(第一軍醫(yī)大學(xué)病理生

2、理教研室暨全軍休克微循環(huán)重點(diǎn)實(shí)驗(yàn)室,廣州510515摘要:絲裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK是生物體內(nèi)重要的信號轉(zhuǎn)導(dǎo)系統(tǒng)之一,參與介導(dǎo)生長、發(fā)育、分裂、分化、死亡以及細(xì)胞間的功能同步等多種細(xì)胞過程。在哺乳動物細(xì)胞中已發(fā)現(xiàn)和克隆了ERK JNK/S APK 、p38/RK 、ERK5/B MK1四個MAPK 亞族。這些MAPK 能被多種炎性刺激所激活,并對炎癥的發(fā)生、發(fā)展起重要調(diào)控作用。研究感染和炎癥反應(yīng)過程中這些MAPK 被激活的機(jī)制及其生物學(xué)效應(yīng),探討MAPK 特異性抑制劑的藥理學(xué)作用及分子基礎(chǔ),對于感染的防治及炎癥反應(yīng)的控制有著廣

3、泛的應(yīng)用前景。關(guān)鍵詞:絲裂原活化蛋白激酶;信號轉(zhuǎn)導(dǎo);炎癥反應(yīng)學(xué)科分類號:Q288Regulation of inflammatory responses byMAPK signal transduction pathwaysJIANG Yong ,GONG Xiao-Wei(De parment o f Pathophysiology and Key Laboratoryr Shock and Microcirculation o f PLA ,First Military Medical University ,Guangzhou 510515Abstract:Mitogen-activat

4、ed protein kinase (MAPK,which is one of the important signal transductionsyste ms in organisms,is involved in many cellular processes,such as cell growth,development,division,differentia tion,death and coordination of cellular func tions,and etc.Four subfamilies of MAP kinases,i. e.ERK,JNK/SAPK,p38/

5、RK and E RK5/B MK1,have been identified and cloned in mammalia m cells.TheseMAP kinas es are activated by many proinfla mmatory stimuli and play an important role in the pathogenesis anddevelop ment of inflam mation.In this article recent advances in the study of the mechanisms underlyingactivation

6、of MAPKs in infection and inflammation and the molecular basis of specific inhibitors for MAPKs arerevie wed,in special reference with the perspective prevention and treatment of inflam mation by these kinases.Keywords:mitogen-activated protein kinase;signal transduction;inflammatory responses 生理學(xué)報,

7、Aug.2000,52(4:267271Acta Physiologica SinicaRecei ved 1999-11-01Accep ted 2000-03-30This stud y we s upp orted b y Nati onal Science Fu nd for Dis tin gui shed You ng Sch olars (No.39925014,Gran td of National Natural Science Fou ndationofChina (No.39830400,No.39800074No.39870332,Grant for Outandin

8、g Young Inves ti gator of PLA (No.98J003,an d Grant of Natural Science Fou n d ationof Gu an gd on g Provi nce (No.980207.Corres pondin g au thor.Tel:(02085148231;Fax:(02087705671;E-mail:yjiangpu 268生理學(xué)報Acta Physiol.Sin.52卷應(yīng)激激活蛋白激酶(stress-activated protein knase, SAPK、p38和ERK5/B MK1(big MAP kinase1等

9、MAPK亞族13。MAPK信號轉(zhuǎn)導(dǎo)通路采用高度保守的三級激酶級聯(lián)傳遞信號;細(xì)胞外刺激通過某些環(huán)節(jié)使MKKK(MAP kinase kinase kinase激活,轉(zhuǎn)而激活MKK(MAP kinase kinase;然后通過對蘇氨酸(threonine,T和酪氨酸(tyrossine,Y雙位點(diǎn)磷酸化激活MAPK13。MAPK不同成員具有明顯的序列同源性,都是通過 T-X-Y 雙位點(diǎn)磷酸化達(dá)到最大激活46。細(xì)胞外刺激作用于細(xì)胞,通過多級激酶級聯(lián)使MAPK激活,激活的MAPK可通過磷酸化轉(zhuǎn)錄因子、細(xì)胞骨架相關(guān)蛋白、酶類等多種底物來調(diào)節(jié)多種細(xì)胞生理過程69。MAPK在介導(dǎo)炎癥反應(yīng)和細(xì)胞因子生成中起著重

10、要的作用,近年來我們在這方面開展了一些工作,本文結(jié)合我們的一些發(fā)現(xiàn)和國內(nèi)、外的研究進(jìn)展,就MAPK信號系統(tǒng)在炎癥過程中的激活、與炎癥因子生成的調(diào)控關(guān)系以及MAPK 通路抑制劑在炎癥控制中的應(yīng)用等問題重點(diǎn)展開討論。1MAPK在炎癥條件下的激活1.1ERK通路ERK亞族至少包括兩個亞型: ERK1和E RK2。ERK通路首先被證實(shí)在EGF (epithelia growth factor、NGF(neuronal gro wth factor和PDGF(platelet-derived growth factor等生長因子刺激引起的細(xì)胞反應(yīng)中起重要調(diào)控作用1,2。然而,研究發(fā)現(xiàn)ERK通路還參與應(yīng)激

11、刺激、細(xì)菌產(chǎn)物、炎癥介質(zhì)等引起的細(xì)胞反應(yīng),表明ERK通路的激活與炎癥反應(yīng)也有著密切關(guān)系1,2,10。Dziarski等報道,在MW264.7巨噬細(xì)胞中,可溶性葡萄球菌肽聚糖(solublpeptidoglycan,sPGN強(qiáng)烈激活ERK1和ERK2,中度激活JNK,僅輕微地激活p38;這與脂多糖(lipopolysaccharide,LPS的作用不同,LPS能強(qiáng)烈地激活所有這些MAPKs10。ERK通路在介導(dǎo)炎癥反應(yīng)中的確切作用尚有待進(jìn)一步研究。1.2JNK通路除了被生長因子激活外,JNK通路還能被LPS、腫瘤壞死因子 (TNF- 、白細(xì)胞介素1(IL-1、紫外線、射線、熱休克、細(xì)胞外高滲及D

12、NA變性劑等激活1,2。目前JNK亞族已克隆了3個基因:JNK1、JNK2和JNK3,共包含至少10種剪切亞型1,2。生長因子刺激激活JNK依賴于Ha-Ras,而TNF- 和IL-1等致炎細(xì)胞因子則通過Ha-Ras 非依賴性方式激活JNK。JNK被激活后,轉(zhuǎn)而磷酸化轉(zhuǎn)錄因子c-Jun的氨基末端的特定位點(diǎn)。c-Jun是序列特異性轉(zhuǎn)錄激活因子AP-1(activating protein1的成分之一1,2,5。磷酸化的c-Jun通過誘導(dǎo)同源或異源二聚體形成,與AP-1位點(diǎn)的順式作用元件結(jié)合而啟動某些效應(yīng)基因的轉(zhuǎn)錄5,11。1.3p38通路p38最初被認(rèn)為是一種能被LPS激活的激酶12。Lee等人在

13、篩選抑制炎癥因子生成的化合物(cytokine-suppressive anti-infla mmatory drugs,CSAID時,發(fā)現(xiàn)一族吡啶-異咪噠唑化合物能夠明顯抑制IL-1和TNF- 的生成13。該類化合物能夠與細(xì)胞內(nèi)兩個非常相似的MAPK同源體結(jié)合,他們稱之為CSBP1/2(CSAID-binding protein1/ 2。CSAID通過與CSBP結(jié)合并抑制其激酶活性而顯著減少細(xì)胞因子的生成。測序證實(shí)C SBP1/2與小鼠p38MAPK為同一基因13。除了LPS之外,生血細(xì)胞因子,如促紅細(xì)胞生成素(erythropoietin,EPO和白細(xì)胞介素3(I L-3、致炎細(xì)胞因子、細(xì)

14、菌成分、紫外線照射、細(xì)胞外高滲、H2O2、熱休克等刺激都能激活這條通路3,6。由于其他MAPK亞族存在不同亞型,我們對p38是否存在其他亞型進(jìn)行了研究,相繼發(fā)現(xiàn)了3個新的p38亞型,即p38 ,p38 ,p38 7,8, 14。4種p38亞型在炎癥條件下的激活具有各自不同的特性。譬如,MKK3和MKK6能激活p38和p38 ,但膠內(nèi)激酶分析顯示,p38 還能被某些未知的MKK所激活8.另外,Hale報道,在LPS刺激的巨噬細(xì)胞內(nèi),p38 被強(qiáng)烈激活,而p38 的激活程度較低;I L-1則能激活內(nèi)皮細(xì)胞中的p38 和p38 15。這提示,在炎癥反應(yīng)中,p38 可能起主要作用。我們對細(xì)胞內(nèi)p38的

15、定位及其對刺激的反應(yīng)進(jìn)行了研究,發(fā)現(xiàn)在RAW264.7細(xì)胞、心肌細(xì)胞、內(nèi)皮細(xì)胞等在靜息狀態(tài)下,主要散在分布于胞漿內(nèi), LIS、UV、高溫刺激后,p38被激活并移位入核16, 17。這提示轉(zhuǎn)錄因子可能是p38MAPK的重要作用目標(biāo)。目前已發(fā)現(xiàn)p38能激活轉(zhuǎn)錄因子AW F2、C HOP10、MEF2C(myocyteenhancer factor2C等3,18。通過對轉(zhuǎn)錄因子磷酸化來調(diào)節(jié)參與細(xì)胞反應(yīng)的基因的轉(zhuǎn)錄表達(dá)是MAPK的重要細(xì)胞功能。除了轉(zhuǎn)錄因子之外,p38還能激活細(xì)胞內(nèi)的一些蛋白激酶,如MAPKAPK2/3(MAPKactivated protein kinase2/3和PRAK(p38r

16、egulated/activated protein kinase3,9。這些絲氨酸/蘇氨酸家族的成員被磷酸化激活后,轉(zhuǎn)而激活低分子量熱休克蛋白(small heat-shock proteinHSP27,從而介導(dǎo)細(xì)胞骨架重構(gòu),參與細(xì)胞應(yīng)激反應(yīng)9。p38亞族的不同激酶可轉(zhuǎn)移到特定的細(xì)胞內(nèi)部位,作用到細(xì)胞內(nèi)相應(yīng)的目標(biāo),從而發(fā)揮不同的調(diào)節(jié)功能3,6。1.4E RK5通路至今只發(fā)現(xiàn)一個ERK5/B MK1亞型能被TNF- 、H2O2、細(xì)胞外高滲等刺激激活,說明該通路可能也參與某些條件下的炎癥反應(yīng)調(diào)19,20。最近,我們對p38MAPK和B MK1在TNF- 誘導(dǎo)c-jun表達(dá)中的調(diào)控作用進(jìn)行了研究,

17、發(fā)現(xiàn)p38和B MK1在TNF- 誘導(dǎo)c-jun轉(zhuǎn)錄的調(diào)控中具有協(xié)同作用。p38和B MK1可分別上調(diào)MEF2和MEF2D的轉(zhuǎn)錄活性。TNF- 通過激活p38和B MK1,使MEF2A和MEF2D磷酸化,從而誘導(dǎo)ME T2A/MEF2D異源二聚體形成,這在TNF- 誘導(dǎo)的c-jun基因轉(zhuǎn)錄表達(dá)調(diào)控中起著重要的作用20。2MA PK與炎癥因子生成的調(diào)控關(guān)系LPS介導(dǎo)的細(xì)胞炎癥反應(yīng)是一個典型的病原體與機(jī)體相互作用的過程3,6。LPS刺激細(xì)胞能夠激活ERK、JNK和p38,然后作用于各自的底物,影響多種轉(zhuǎn)錄因子的活性,從而調(diào)節(jié)包括TNF、IL-1、IL-6、I L-8等多種細(xì)胞因子在內(nèi)的基因的表達(dá)。

18、而TNF- 、I L-1、花生四烯酸(arachdonic acid,AA產(chǎn)物、內(nèi)皮素等多種炎癥介質(zhì)能激活不同MAPK,通過促進(jìn)或抑制基因的轉(zhuǎn)錄來調(diào)控其它炎癥介質(zhì)的生成。2.1TMF- TNF- 是導(dǎo)致內(nèi)毒素休克的關(guān)鍵介質(zhì)17。LPS刺激單核/巨噬細(xì)胞可引起TNF- 合成和釋放,后者再誘導(dǎo)其它細(xì)胞因子和血小板激活因子(platelet-activated factor,PAF等的釋放。極低濃度的PAF就可刺激中性粒細(xì)胞和血管內(nèi)皮細(xì)胞釋放大量的TNF- 和IL-1,而TNF- 、IL-1又可刺激巨噬細(xì)胞和血管內(nèi)皮細(xì)胞產(chǎn)生和釋放P AF。這些細(xì)胞因子協(xié)同作用,導(dǎo)致炎癥反應(yīng)失控,甚至引起致死性休克。

19、我們發(fā)現(xiàn)LPS可激活RAW264.3細(xì)胞p38,使其移位入核;p38激活入核后參與了LPS誘導(dǎo)的TNF- 基因轉(zhuǎn)錄表達(dá)的調(diào)控16,17。E RK、JNK和p38通路都能被TNF- 強(qiáng)烈激活,通過對轉(zhuǎn)錄因子的作用而影響TNF- 的產(chǎn)生和生物學(xué)效應(yīng)20。2.2IL-1IL-1也是參與炎癥反應(yīng)的重要介質(zhì)。它在炎癥中的主要作用包括刺激其它炎癥介質(zhì),如TNF- 、IL-6、I L-8、P M和PGs等的釋放、激活T和B淋巴細(xì)胞。增加粘附分子的表達(dá)、刺激急性期蛋白(可能是內(nèi)生性致熱原的主要成分的生成和釋放等。Guan等最近報道,IL-1 能通過激活p38而上調(diào)PGE2和下調(diào)NO的合成21。2.3AA代謝產(chǎn)

20、物AA代謝產(chǎn)物前列腺素(PGs和白三烯類(LTs在促進(jìn)炎癥的過程中起重要作用。ERK1/2激活可誘導(dǎo)環(huán)加氧酶2(cyclooxygenase2, C OX2生成,限制氧化應(yīng)激對心肌細(xì)胞的損傷22。C OX是PG合成的限速酶,經(jīng)典抗炎藥水楊酸類通過抑制其活性而減少PG的產(chǎn)生,抑制炎癥反應(yīng)。p38通路和JNK通路激活后能上調(diào)IL-1 誘導(dǎo)的C OX 表達(dá),使PGE2合成增加,利用p38抑制劑SC68376能完全抑制這種作用23。2.4粘附分子炎癥反應(yīng)所生成的炎癥介質(zhì)可通過激活MAPK通路增加細(xì)胞表面多種粘附分子的表達(dá),促進(jìn)細(xì)胞間粘附和炎癥的進(jìn)展。內(nèi)皮細(xì)胞表達(dá)E-選擇素(E-selectin對于炎癥

21、反應(yīng)中白細(xì)胞的聚集至關(guān)重要。JNK和p38通過增加E-選擇素的啟動子轉(zhuǎn)錄活性而促進(jìn)E-選擇素的大量表達(dá)24。2.5其它體液因子還有一些體液因子在炎癥中的作用也是通過MAPK介導(dǎo)的。Bruder等報道,腺病毒感染通過激活ERK通路使細(xì)胞產(chǎn)生趨化因子IL-825.3MAPK亞族間的協(xié)同作用在多細(xì)胞生物,尤其是哺乳動物細(xì)胞,要準(zhǔn)確完成某種細(xì)胞功能,往往需要多條信號通道協(xié)同作用3。MPAK通路的協(xié)同作用可發(fā)生在信號轉(zhuǎn)導(dǎo)級聯(lián)的各個水平,也可采用不同的協(xié)同作用方式和/或機(jī)制1,3,5。p38MAPK對炎性分子的表達(dá)有明顯影響,在感染引起的細(xì)胞反應(yīng)中具有重要的調(diào)控作用。我們用酵母雙雜交技術(shù)(two-hybr

22、id system首次報道了p38轉(zhuǎn)導(dǎo)通路和心肌細(xì)胞增強(qiáng)因子(myocyte-enhancer factor2,MEF2轉(zhuǎn)錄因子家族的成員ME T2C之間的功能聯(lián)系18。在單核細(xì)胞受到LPS的刺激時,p38和JNK發(fā)生雙位點(diǎn)磷酸化而被同時激活。p38的激活使ME T2C磷酸化;使c-jun轉(zhuǎn)錄增加,從而增加c-Jun蛋白表達(dá);而另外一條與應(yīng)激有關(guān)的MAPK通路?JNK通路的激活使c-Jun磷酸化。磷酸化消耗的c-jun可由ME F2C活性增加引起的c-Jun蛋白表達(dá)補(bǔ)充。在多種細(xì)胞因子的啟動子區(qū)都存在AP1位點(diǎn),磷酸化的c-Jun可以和AP1位點(diǎn)結(jié)合,從而使炎癥相關(guān)基因表達(dá)增加18。通過激活一

23、條MAPK通路使某種轉(zhuǎn)錄因子表達(dá)增加,從而為另外一條通路MAPK磷酸化作用提供底物,以促進(jìn)炎癥反應(yīng)基因表達(dá),這是MAPK通路間協(xié)同作用的一種重要形式(圖1。4MAPK通路抑制劑在抑制炎癥反應(yīng)中的作用4.1MAPK通路抑制劑對p38特異性抑制劑的研究較多,包括SK&F86002、SB206718、SB220025、SB202190和SB203580等13,26。我們發(fā)現(xiàn)磷酸化環(huán)狀結(jié)構(gòu)是決定下游底物的關(guān)鍵結(jié)構(gòu),而與上游激酶選擇無明顯關(guān)系27。由于結(jié)構(gòu)的差異,不同的p38亞型對特異性抑制劑的反應(yīng)不同。例如,與其它三種亞型不同,p38 的活性不被SB202190所抑4期姜勇等:MAPK信號轉(zhuǎn)導(dǎo)

24、通路對炎癥反應(yīng)的調(diào)控269制8,14。p38特異性抑制劑并不阻斷上游激酶對p38的激活,而是作用于p38本身。根據(jù)蛋白質(zhì)一級結(jié)構(gòu)、分子構(gòu)型和X線晶體衍射結(jié)構(gòu)確定p38特異性抑制劑主要作用于ATP結(jié)合活性位點(diǎn)T106,且M109能增加這些特異性抑制劑與p38的親和力28。這些特異性抑制劑使p38失去了與ATP 結(jié)合的能力,從而使其失去激酶活性。作為E RK通路的特異性抑制劑,PD98059通過特異性地抑制其上游激酶ME K1/2,而阻斷E RK通路的激活。PD98059能抑制CD3/PMA或C D3/C D28共刺激T細(xì)胞時產(chǎn)生TNF- 、I L-3、GM-CSF、IFN- 、IL-6及IL-1

25、,但能使IL-4、IL-5及I L-3的生成增加29。目前有關(guān)JNK的特異性抑制劑尚未見報道。4.2MAPK通路抑制劑的抑炎效應(yīng)及臨床應(yīng)用前景利用MAPK通路抑制劑阻斷其作用,可抑制炎癥反應(yīng)的進(jìn)一步發(fā)展。Guan等報道,一種能選擇性抑制系膜細(xì)胞內(nèi)p38通路的藥物SC68376,能抑制腎小球系膜細(xì)胞內(nèi)I L-15對p38的激活,從而增加NO的合成并抑制前列腺素的釋放21。動物實(shí)驗(yàn)表明,SB203580能降低鼠內(nèi)毒素性休克模型的死亡率30;SB220025能抑制血管形成,對慢性增殖性炎癥有效。MAPK通路抑制劑能抑制多種炎癥因子的產(chǎn)生,阻斷炎癥反應(yīng),并且具有特異性好、作用強(qiáng)等特點(diǎn)。因此,這些藥物一

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43、armacologicalprofile of SB203580,a selective inhibi tor of cytokinesuppres sive binding protein/p38kinase in animal models ofarthritis, bone resorption,endotoxin shock and immunefunction.J Pha rmacol Exp Ther,1996,279(3:14531461.4期姜勇等:MAPK信號轉(zhuǎn)導(dǎo)通路對炎癥反應(yīng)的調(diào)控271280 生 理 學(xué) 報 Acta Physiol. Sin. 52 卷 因存在。近年來, 肝器官特異性早期反應(yīng)基因時有發(fā) 現(xiàn), 它們對解釋肝再生的器官特異性具有重要意義。 本研究利用RDA 技術(shù)構(gòu)建了 2/ 3 肝部切除后 1 h 再生 肝選擇性表達(dá)基因 EST 庫, 該庫約含 600 50 ( 3 104 個獨(dú)立克隆, 其中 95% 以上的克隆含有插入片 段, 長度約 200 700 bp 不等。選擇術(shù)后 1 h 是依據(jù)大 多數(shù)早期反應(yīng)基因的表達(dá)高峰均在術(shù)后 1