1、細胞信號轉(zhuǎn)導(dǎo)與心血管疾病細胞信號轉(zhuǎn)導(dǎo)與心血管疾病病理生理學(xué)系病理生理學(xué)系李躍華細胞信號轉(zhuǎn)導(dǎo)系統(tǒng)概述細胞信號轉(zhuǎn)導(dǎo)系統(tǒng)概述TLRs介導(dǎo)的細胞信號轉(zhuǎn)導(dǎo)介導(dǎo)的細胞信號轉(zhuǎn)導(dǎo)TLRsTLRs信號通路與心血管疾病信號通路與心血管疾病 細胞信號轉(zhuǎn)導(dǎo)系統(tǒng)(signal transduction system)由受體或其他可接受信號的分子、細胞內(nèi)的信號轉(zhuǎn)導(dǎo)通路及其作用的終端所組成。 意義 對認識細胞在生命活動各方面的表現(xiàn)和調(diào)控方式，理解生命活動的本質(zhì)，在分子水平認識各種疾病的發(fā)病機制等都有重大意義。第一節(jié)第一節(jié) 細胞信號轉(zhuǎn)導(dǎo)系統(tǒng)概述細胞信號轉(zhuǎn)導(dǎo)系統(tǒng)概述Biological effectsEnd effectorR

2、eceptor Signal transductionPathwaySignal細胞通訊細胞通訊 細胞通訊細胞通訊（cell communication）：指一指一個細胞發(fā)出的信息通過介質(zhì)傳遞到另一個細胞發(fā)出的信息通過介質(zhì)傳遞到另一個細胞產(chǎn)生相應(yīng)反應(yīng)的過程。個細胞產(chǎn)生相應(yīng)反應(yīng)的過程。 細胞通訊主要有三種方式：細胞通訊主要有三種方式：細胞間隙連接細胞間隙連接(gap junction)：連接蛋白，直接通訊，可共享小分子物質(zhì)（1500D）膜表面分子接觸通訊膜表面分子接觸通訊(Contact-dependent signaling) ：蛋白、糖蛋白、糖脂，直接通訊化學(xué)信號通訊化學(xué)信號通訊(chemi

3、cal signaling )：以化學(xué)信號為介質(zhì)，間接通訊細胞間隙連接膜表面分子接觸通訊化學(xué)通訊 信號轉(zhuǎn)導(dǎo)信號轉(zhuǎn)導(dǎo)（signal transduction）： 指外界信號（如光、電、指外界信號（如光、電、化學(xué)分子）與細胞細胞表面化學(xué)分子）與細胞細胞表面受體作用，通過影響細胞內(nèi)受體作用，通過影響細胞內(nèi)信使的水平變化，進而引起信使的水平變化，進而引起細胞應(yīng)答反應(yīng)的一系列過程。細胞應(yīng)答反應(yīng)的一系列過程。信號轉(zhuǎn)導(dǎo)信號轉(zhuǎn)導(dǎo)細胞信號傳導(dǎo)的基本過程和機制細胞信號傳導(dǎo)的基本過程和機制 受體：受體： 識別外源信息分子( 配體 ligand); 信號轉(zhuǎn)換。膜受體介導(dǎo)的跨膜信號轉(zhuǎn)導(dǎo)膜受體介導(dǎo)的跨膜信號轉(zhuǎn)導(dǎo)核受體介導(dǎo)

4、的信號轉(zhuǎn)導(dǎo)核受體介導(dǎo)的信號轉(zhuǎn)導(dǎo) 信號的接受和轉(zhuǎn)導(dǎo)信號的接受和轉(zhuǎn)導(dǎo)細胞信號分子：細胞信號分子： 生物細胞所接受的信號既可以是物理信號（光、熱、電流、機械刺激），也可以是化學(xué)信號，但是在有機體間和細胞間的通訊中最廣泛的信號是化學(xué)信號。Biological effectsEnd effectorReceptor Signal transductionPathwaySignal信號分子信號分子 化學(xué)信號化學(xué)信號根據(jù)其溶解性可分為脂溶性脂溶性和水溶性水溶性兩類。 脂溶性信號分子脂溶性信號分子，主要是甾體類激素和甲狀腺素，它們分子小、疏水性強，可直接進入細胞，與細胞質(zhì)或細胞核中受體結(jié)合形成激素-受體復(fù)合物

5、，調(diào)控基因表達； 水溶性信號分子：水溶性信號分子：大部分激素、遞質(zhì)和細胞因子都是親水性的，他們不能穿過細胞膜，只能通過與靶細胞表面受體結(jié)合，經(jīng)信號轉(zhuǎn)導(dǎo)機制，在胞內(nèi)產(chǎn)生第二信使或激活蛋白激酶的活性，引起細胞效應(yīng)。信號分子信號分子 化學(xué)信號的作用方式： 內(nèi)分泌(endocrine)系統(tǒng)的化學(xué)信號：激素(hormone)，作用距離最遠（m），膜受體或胞內(nèi)受體 旁分泌(paracrine)系統(tǒng)的化學(xué)信號：細胞因子，作用于周圍細胞（m），膜受體。神經(jīng)元間的突觸信號發(fā)放也屬于旁分泌，神經(jīng)遞質(zhì)(neural transmitter)，如乙酰膽堿(acetyl-choline)，作用距離最短（nm），膜受體。

6、 自分泌(autocrine)：作用于自身，常見于病理條件，如腫瘤。 內(nèi)在分泌(intracrine):某些配體分子如激素等在合成以后并未分泌出細胞，而直接與其胞內(nèi)的受體結(jié)合啟動信號轉(zhuǎn)導(dǎo)，主要見于核受體家族?；瘜W(xué)信號的作用方式：（Transmembrane signal transduction mediated by membrane receptor） 1)離子通道受體（離子通道受體（Direct ligand-gated ion-channel type） 即有信號接收部位，又是離子通道，跨膜轉(zhuǎn)導(dǎo)無需中間步驟，反應(yīng)快速（ms），如Ach受體通道（N型Ach受體）。 Ionotropic

7、receptor - receptor + ion channel 2) G蛋白偶聯(lián)型受體（蛋白偶聯(lián)型受體（G protein coupled receptor (GPCR)） AC： AMP cAMP a subunit + GTP PLC： PIP2 DAG，IP3 3) 具有酶活性的受體（具有酶活性的受體（Protein kinase-linked type） 既具有受體功能，又具有酶活性，可將胞外信號直接轉(zhuǎn)化為胞內(nèi)效應(yīng)。 受體酪氨酸蛋白激酶(receptor tyrosine kinase，RTK) 絲蘇氨酸蛋白激酶(protein serinethreonine kinase，PST

8、K)型受體膜受體介導(dǎo)的跨膜信號轉(zhuǎn)導(dǎo)膜受體介導(dǎo)的跨膜信號轉(zhuǎn)導(dǎo)G protein coupled receptor (GPCR) Ligand: hormone, light, odorous molecules, neurotransmitter, neuropeptide, chemotactic factor, etc b a g GDP GTP AC-cAMP-PKA (cAMP dependent protein kinase) a PLC-b-DAG-PKC (Ca2+ , PL dependent protein kinase) PLC-g-IP3 -Ca2+ (Ca2+-CaM d

9、ependant protein kinase) GTP PI-3K-PIP2, PIP3-PDK-PKB Ion channel（ Signal transduction mediated by nuclear receptor ） Neuclear receptor (NR): Steroid hormone receptor (e.g. GR); Thyroxine receptor (TR); Retinoid acid receptor (RAR); 1,25 (OH)2D3 receptor (VDR); TF = transcriptional factor DNA-bindin

10、g area hormone-binding area N A/B C E C - HSP (GR) HER Gene (TR, RAR, VDR) N A/B C E C + TIFHER = hormone response element HSP = heat shock proteinTIF = transcriptional intermediary factor (co-activator, co-repressor) 核受體介導(dǎo)的信號轉(zhuǎn)導(dǎo)核受體介導(dǎo)的信號轉(zhuǎn)導(dǎo)細胞信號傳導(dǎo)的基本過程和機制細胞信號傳導(dǎo)的基本過程和機制（二）信號的抑制和終止（二）信號的抑制和終止(1) Signalin

11、g termination 1) dissociation of receptor with ligand 2) receptor internalization and degradation (down-regulation) 3) transfer of GP or small GP from active to inactive type 4) dephosphorization by PP or degradation of 2nd messenger(2) Signal inhibition and antagonist between signal pathway 1) inhi

12、bitory receptors and inhibitory transduction components 2) negative feedback regulation of signal pathway receptor phosphorylation leading to affinity to ligand receptor down-regulation inhibitory element (CIS) induced by signal pathway (JAK-STAT)1、啟始、啟始 配體與受體結(jié)合，啟動膜內(nèi)側(cè)級聯(lián)反應(yīng)，將信號傳給第二信使分子。2、轉(zhuǎn)導(dǎo)（放大和整合）、轉(zhuǎn)導(dǎo)（

13、放大和整合） 多種信使分子通過不同通路激活一系列蛋白激酶傳導(dǎo)信息，實現(xiàn)信號的放大與整合。3、細胞反應(yīng)（效應(yīng)）、細胞反應(yīng)（效應(yīng)） 信號分子或轉(zhuǎn)錄因子進入細胞核，與基因相互作用，調(diào)節(jié)蛋白質(zhì)合成或細胞分泌、運動、形態(tài)變化和凋亡。4、中止、中止 通過負反饋途徑，活化抑制因子或滅活因子，終止信號的啟動作用。受體介導(dǎo)的信號傳導(dǎo)的基本過程受體介導(dǎo)的信號傳導(dǎo)的基本過程Cross-Talk細胞信號轉(zhuǎn)導(dǎo)大事記細胞信號轉(zhuǎn)導(dǎo)大事記 1955年，cAMP第二信使學(xué)說，獲1971年諾 貝爾生理和醫(yī)學(xué)獎 1963年，cGMP作為胞內(nèi)信使的發(fā)現(xiàn) 1978年，Ca2+第二信使學(xué)說 1983年，IP3和DG作為胞內(nèi)信使的發(fā)現(xiàn) 8

14、0年代，G蛋白的研究，獲1994年諾貝爾生理和醫(yī)學(xué)獎 80-90年代，酪氨酸蛋白激酶與信號轉(zhuǎn)導(dǎo)的研究 1997年，Janeway等第一次報道了人的Toll同源物TLR4 ，到目前為止至少已有11種人TLRs和13種小鼠TLRs被克隆Dr. Janeway. In drosophila, Toll is a receptor for dorso-ventral polarity during embryogenesis. Later, it plays a role in the immune response against fungal infection. In 1997, human h

15、omologues of Toll, designated as Toll-like receptors (TLRs) were discovered. (Janeway, et al. (1997) Nature, v388,394) At present, eleven members of TLRs have been actually identified in human.J Immunol,1998;539.第二節(jié)第二節(jié) TLRsTLRs介導(dǎo)的細胞信號轉(zhuǎn)導(dǎo)介導(dǎo)的細胞信號轉(zhuǎn)導(dǎo)Abbreviations:TLRs (Toll-like receptors) TIR (Toll/IL-1

16、 resistance) MyD88 (Myeloid Differentiation protein-88 ） MAL (MyD88-adapter-like; also known as TIRAP) TIRAP(TIR domain containing adaptor protein) IRAK (IL-1 receptor-associated kinase)TRAF6 (TNF receptor-associated factor 6) TAK-1(TGF-activated kinase-1) TAB2 (TAK1 binding protein 2)TRIF (TIR-doma

17、in-containing adapter protein inducing interferon- )TRAM (TRIF-related adapter molecule)TICAM-1(TIR domain containing adaptor molecular 1)Tollip (Toll-interacting protein) PAMP (pathogen associated molecular pattern) HSP (heat shock protein) IRF (interferon regulatory factor )TLRs structure include

18、a LRR and a TIR domainThe extracellular portion of the TLRs contain a leucine-rich repeat (LRR) motif. LRR domains are involved in pathogens recognition. TLRs is a type I transmembrane receptor.What the hToll will ectodomain look like ?cytoplasmic regions:TLRs like IL-1R share a conserved stretch of

19、 200 amino acids, the Toll/IL-1R (TIR) domain.Ligand specificities of mammalian TLRs TLR Signaling PathwaysCell membraneTLR3 TLR7 TLR8 TLR9H+H+H+H+H+H+H+H+H+H+H+H+TLR2/TLR1 TLR2/TLR6 TLR4MAL MyD88 TRIF TRAMNF- BTRIF MyD88NF- BInflammatory CytokinesInterferon PathwayEndosomeTLR3TRIF IRF3IRF7MAL MyD88

20、MALMyD88Cell membraneLBPsCD14TLR4LPSMD-2MD-2LPSp65p50p50NF-kBIRF-3PPPPp65p50p50NF-kBMyD88 Dependent and Independent Pathways IFN-b bNF- BNF- BTNFCOX2IL-18ChemokinesChemokines:Rantes, IP-10IFNb bMyD88 has a Death domain (DD) MyD88 is an adapter molecule. It recruits IRAK the Toll like receptor comple

21、xes following stimulation. The association between MyD88 and IRAK is mediated through a DD-DD (blue) interaction (DD mean death domain).TLR4LBPsCD14LPSMD-2MD-2LPSMALMyD88IRAK4IRAK2IRAK1TRAF6TAB1TAB2TAK1IKK-g gIKK-a aIKK-b bp65I Bp50NF- BTNFCOX2IL-18UBC13UBV1A(-)TOLLIPMEKK3MKK3MKK7JNKp38Cell membrane

22、I BProteasomePaz S., Nakhaei P,( 2005)TLR4LBPsCD14LPSMD-2MD-2TRIFTRAMTRAF6TBK1IKK LPSIKK-g gIKK-a aIKK-b bTLR4 MyD88-Independent Signalingp65I Bp50NF- BIFN-b bPPIRF-3PPPPCell membraneI BProteasomePaz S., Nakhaei P,( 2005)Late inductionTLR4IKK-g gIKK-a aIKK-b bp65I Bp50NF- BTNFCOX2IL-18MD-2MD-2MALMyD

23、88IRAK4IRAK1UBC13TRAF6UBV1A(-)Cell membraneST2SIGIRR(-)SOCS1Cytoplasmic molecules: IRAK-M : inducible by TLR stimulation; anti-inflammatory SOCS1(Supressor of cytokine signaling 1) : inhibits JAK-STAT signaling A20 : Creates negative feedback loop inhibiting NFkappaB and TNF-mediated programmed cell

24、 death. Tollip : inhibits TLR 2 and 4 signaling MyD88 forms: alternative splicing creates short molecules that interfere with signaling.Membrane bound molecules: SIGIRR (single immunoglobulin IL-1R-related molecule) ST2Negative Regulation of TLR SignalingIRAK-M(-)I BProteasome(-)A20MyD88s(-)Tollip第三

25、節(jié)第三節(jié) TLRsTLRs信號通路與心血管疾病信號通路與心血管疾病Inflammation Cause Heart AttackAtherosclerosis and inflammationHansson GK N Engl J Med 2005;352:1685-95Recently, a possible role for TLR4 signaling in arterial remodeling has been revealed in mouse models. TLRs have recently been associated with atherosclerosis.In mo

26、use models of hyperlipidemia, a potential role for the toll-like receptor pathway has been suggested in hypercholesterolemia-induced atherosclerosis.In addition, oxidized lipoproteins interact with toll-like receptors. Lipid-rich atherosclerotic plaques express TLR4, and that TLR4 expression in macr

27、ophages is up-regulated by oxidized but not native low-density lipoprotein (LDL). Human epidemiologic studies have linked TLR4 polymorphism with atherosclerosis.Role of TLR signaling in atherosclerosis MyD88 and TLR4 deficiency reduces the extent of aortic atherosclerosisAortas stained for lipid dep

28、osition with Oil red OLipid content, macrophage infiltration, and COX-2 expression in aortic sinus plaques is reduced in Apoe-/-MyD88-/-mice.Atherosclerotic plaques stained for lipid deposition with Oil red OMacrophage infiltration in aortic sinus plaquesThe sections were incubated withMOMA-2, a mac

29、rophage-specific antibody, or control IgG antibody.COX-2 expression in aortic sinus plaquesQuantitative analysis of COX-2 immunofluorescent staining in sclerotic plaquesSerum concentration of IL-12p40 and MCP-1 are reduced in Apoe-/-MyD88-/-mice.IL-12p40 and MCP-1 concentrations in the sera of mice

30、were measured by ELISAs. Increased inflammatory cytokine (TNF, IL-1 and IL-6) expression have been found in CHF and ischemic hearts. Reperfusion injury is related closely to inflammatory reactions such as activation of inflammatory cells and expression of cytotoxic cytokines. Molecules released from

31、 damaged blood vessels or extracellular matrix have been identified as endogenous activators of TLRs.Role of TLR Mediated Signaling in Cardiac Ischemia InjuryRole of TLR Mediated Signaling in Cardiac Ischemia InjuryRole of TLR Mediated Signaling in Cardiac Ischemia InjuryRole of TLR Mediated Signali

32、ng in Cardiac Ischemia InjuryReduced Myocardial Ischemia-Reperfusion Injury in Toll-Like Receptor 4Deficient MiceRole of TLR Mediated Signaling in Cardiac Ischemia Injury TLR4 deficient mice has significantly smaller myocardial infarctions compared with wild mice given similar areas at risk after MI

33、R. Fewer neutrophils infiltrated the myocardium of TLR4-deficient mice after MIR. Fewer lipid peroxides and less complement deposition. Serum levels of interleukin-12, interferon-, and endotoxin were not increased after MIR. In addition to its role in innate immune responses, TLR4 serves a proinflam

34、matory role in MIR injury.A20 Is Dynamically Regulated in the Heart and Inhibits the Hypertrophic ResponseStuart A. Cook, et al. Circulation 2003;108:664-668Editorial: Circulation 2003;108:638-640Is NF B an Attractive Therapeutic Target for Treating Cardiac Hypertrophy?Nicole H. Purcell, PhD; Jeffery D. Molkentin, PhD To investigate the importance of NF-B signaling in the heart as a potential mediator of hypertrophy and/or apoptosis through a manipulation of the NF-B inhibitory protein A20A20 Infection of cardio