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哺乳动物生物钟昼夜节律同步机制的研究进展

哺乳动物生物钟昼夜节律同步机制的研究进展

谢朝晖王建林①

(兰州大学生命科学学院兰州730000)

E-mail xiezhh03@http://www.wendangku.net/doc/3da9540cf12d2af90242e65f.html

摘要昼夜生物钟机制主要包括钟振荡器、信号输入、信号输出和钟的整合,哺乳动物生物钟同步中枢位于下丘脑视交叉上核(SCN),与外周器官的下游振荡器协同调节昼夜节律。钟振荡器分子机制的研究框架建立在由钟基因转录和翻译调节组成的反馈环路之上;在核内,mCRY-mPER-CKIε/CKIδ和与之相关的CLOCK和BMAL1络合,构成“生物钟载体”。若干钟基因产物作为正向或负向调节子影响钟的振荡;翻译后事件对控时机制有重要作用。光信号通过视束(RHT)的光信号传导通路到达SCN,通过SCN的节律整合作用进而影响或调节下游振荡器的节律性。

关键词 生物钟; 昼夜节律;哺乳动物

中图分类号 Q41 文献标识码 A

几乎所有的生物体内的生理活动和外在行为都表现出其自身节律[1,2],这种节律由大脑控制,依靠从外界接受光信号,通过整个躯体的各种不同组织特有的节律协同产生作用。周期性节律是身体内部周期同步的外部表现,如睡眠/觉醒周期。节律同步机制是一个错综复杂的分子机制,研究哺乳动物周期性同步包括以基因、分子、生物化学的内在因素和精确的行为观察。哺乳动物通过位于下丘脑前部的视交叉上核(SCN)内的控时机制把周期性节律系统整合起来[3,4]。近来对“时钟结构”的研究方面取得了很大的发展,尤其在从视网膜到SCN的视觉通路的分子机制的研究、SCN控时机制对节律通路的整合和生理、行为上等的研究。

1.上下游振荡器的分布

哺乳动物周期性同步系统由多个上下游振荡器组成,生物钟支配的振荡器包括许多单极神经元[5],大约近20,000个神经元是SCN“生物钟细胞”,分子在SCN生物钟细胞发挥其独特的作用[6,7]。让人感兴趣的是,周期振荡器分散在整个身体内, SCN生物钟细胞内基因已经发现在大脑的其它脑区、外周器官(肾脏、心脏、睾丸、骨骼肌等)甚至在离体培养细胞内节律性地表达[7~9]。SCN振荡器在没有神经信号输入的条件下,可以持续呈24h周期节律表达几天[10]。SCN在其调节的生理系统有广泛的分布,每一处都有多个振荡器存在,通过SCN振荡器周期同步协调作用,依次在生理和行为上调节局部的节律,对整个身体时

作者简介: 谢朝晖(1969-), 男, 博士研究生.

①通讯联系人 jlwang@http://www.wendangku.net/doc/3da9540cf12d2af90242e65f.html

相控制赋予精确性和稳定性[11]。

对肝脏的培养细胞带基因研究表明,SCN 生物钟同步机制和下游的振荡器的分子合成非常相似[12],但对如何抑制下游振荡器的生物钟功能的真正机制还不清楚。研究仅仅停留在SCN 基因/蛋白质的特有表达的存在方面还是不够的,也许在蛋白质水平和/或蛋白质动力学水平的研究方面会有新的启示。

2.反馈还路的转录

果蝇(D osophila melanogaster )的周期时钟机制对研究哺乳动物时钟机制有很大的借

鉴意义,果蝇大多数生物钟同源基因在哺乳动物已经克隆r r r l r r r [13],小鼠和果蝇反馈环路的转录的核心机制相似,仍有几个根本成分的不同导致其功能的不同,因而使哺乳动物生物钟基因转录的复杂性增大。人们已经发现小鼠的基本生物钟基因和生物钟调节基因 [1,14,15]。

小鼠细胞内生物钟是正负反馈环路相互作用的结果,此环路推动生物钟RNA 和蛋白质

水平周期性节律表达(Table 1),bHLH (basic helix-loop-helix,bHL )-PAS(Period-Arnt-Single-minded ,PAS)-转录因子、CLOCK 和BMAL1(亦叫MOP3)是生物钟机制中基本成分[16~19]。CLOCK-BMAL1异二聚体结合E-box 增强子和高选择性的核甘酸序列CACGTG 结合激活节律的转录。负反馈参与负调节子参与的CLOCK-BMAL1的节律抑制,特别是CLOCK-BMAL1异二聚体激活三种period 基因 (小鼠mPer1~mPe 3)和两种隐染色质(mC y1和mCry2)的节律性转录。mPER 和mCRY 蛋白转移回核内,在核内mPER 和mCRY 作为负调节子直接作用CLOCK 和/或BMAL1抑制转录,关闭负反馈环路[20~23]。 Bmal1参与正反馈环路转录的节律的调节,Bma 1的RNA 水平高峰相与mPer 和mC y 相关,呈12h 高峰相[23,24], 当CLOCK-BMAL1异二聚体激活 mPer 和mC y 转录,也同时激活核内受体Rev-E b α基因的转录,REV-ERBα蛋白反过来通过Rev-Erb/ROR反应抑制Bmal1的转录[25,26],结果Bmal1RNA水平下降,同时mPer 和mCry 水平升高。当mCRY蛋白进入核内抑制mPer 和mCry 的转录(通过激活CLOCK-BMAL1),同时又抑制Rev-Erbα(通过不抑制或激活

Bmal1的转录)[25,27].当Rev-Erbα表达受抑时,mPER2可能提供Bmal1转录的正反馈驱动[23]。

因此,正负反馈环路由ClOCK-BMAL1异二聚体协同调节,不同的环路动力来自不同蛋白质的协同调节。

负反馈环路输出的mPER和mCRY对于维护生物钟功能是必须的,在恒定条件下,当mPer 和mCry 或mCry基因敲除,动物会在行为上立刻出现无节律变化[21,28~32]。在恒定条件下生物钟单个基因发生突变时,mPer 基因家族成员有部分补偿作用。但在维持生物钟核心反馈环路中,mPER3不起关键作用,在mPer3基因突变的小鼠,其分子和行为节律不变,也不能与mPer1和mPer2协同性基因突变[28,33],mPER3可能具有输出信号的作用。

Protein Protein Protein Protein Protein Protein Protein Protein

Family Member

Gene(s) Behavioralphenotype

bHLH-PAS Clock clock Longperiod,then

arrhythmic

BMAL1(MOP3)

Mmal(Mop3) Arrhythmic

PER-PAS PER1 Per1 or Per1+per3

r Short period, then arrhythmic

PER2 Per1 or Per2+per3Short period, then arrhythmic

PER3 Per3 Short

period

Per1+Pe2 Arrhythmic Flavoproteins CRY1 Cry1 Shortperiod

CRY2 Cry2 Longperiod

Cry1+Cry2 Arrhythmic

Casein kinase CKIεCKIε Shortperiod

Orphan nuclear receptor REV-ERBαRev-ErbαShortperiod

表1 小鼠时钟机制的成分

根据Steven M & David R.Weaver, Nature 2002,418:935~941.

3.生物钟的翻译后控制

生物钟蛋白的周期模式对控时机制有重要作用。翻译后磷酸化、蛋白质的降解等控时机制可以调节mPer RNA和蛋白质之间的动态平衡。目前,研究者对翻译后进程控制已进行了很好的研究。

3.1生物钟蛋白质的磷酸化

在细胞质中,CLOCK,BMAL1和mPER2可被磷酸化, mPer/mCry转录负反馈环的时间与磷酸化程度有关。有趣的是,当CLOCK和BMAL1在核内处于最低水平时,转录才开始[34],提示转录因子的磷酸化状态对于CLOCK-BMAL1异二聚体的转录补偿很重要[35,36],在细胞核内,CLOCK和BMAL1磷酸化移位也很重要,复合体蛋白质抑制CLOCK-BMAL1调节的转录(Figure 1)。

Lowrey等2000年报道酪蛋白激酶CKIε (casein kinaseε,CKIε) 在叙利亚仓鼠体内是一个重要的调节子,因为叙利亚仓鼠CKIε基因缺乏相当于短周期 tau 基因的突变,CKIε能磷酸化 mPER1和 mPER2,仓鼠突变体蛋白激酶磷酸化水平很低[37]。另外,遗传紊乱可导致人的生物钟时相变短、睡眠时间延长,这与CKIε低效的磷酸化有关[38],值得注意的是,PER1 和PER2水平在同型tau突变仓鼠并没有变化。近来发现CKIε也能磷酸化CRY1,mCRY2和MBMAL1,这可能是在tau突变仓鼠,这些蛋白质被短暂磷酸化可以使生物钟表型改变[35]。

哺乳动物生物钟昼夜节律同步机制的研究进展

Figure 1哺乳动物时钟机制模式图

根据Steven M & David R.Weaver, Nature 2002,418:935~941,有所修改。

有证据表明[30],CKIε是第二种哺乳动物生物钟重要的蛋白激酶,CKIδ(与 CKIε也有很高的同源性,在人氨基酸水平达76 %)可磷酸化mPER1,mPER2,mCRY1,mCRY2和BMAL1;有丝分裂原活化性蛋白激酶(mitogen-activated protein kinase,MAPK)亦能磷酸化BMAL1,MAPK的昼夜节律性激活/失活周期与钟的振荡密切相关[35,39]。近期对果蝇的研究表明,糖原合成激酶-3和相关激酶应该用于对哺乳动物生物钟的研究[40],虽然对CLOCK节律性磷酸化的反应并不确定,但生物钟短暂的磷酸化过程可能与生物钟的原动力有关,该过程中有许多酶包括磷酸酶的参与。

3.2入核的调节

mPER和mCRY蛋白的入核是生物钟机制循环进程的很重要的检验点。对体内肝脏振荡器研究表明,在细胞质中,mPER蛋白限制mPER-mCRY异二聚体的形成,反过来,mPER蛋白又为核内复合体积累所必需[34]。mPER蛋白核内的积累推动生物钟机制向前,当生物钟蛋白在适当的时间进入核内,参与负转录环路。可以解释当单个基因发生突变(mPer1,mPer2,mCry1或mCry2)时生物钟循环并不中断,但当双重敲除出mPer1和mPer2或mCry1和mCry2时循环停止。生物钟蛋白核内外平衡也与其细胞定位有关,可以提供一个正确的循环周期时相[41,42]。

mPER1和mPER2蛋白与CKIε和CKIδ在细胞质中结合成多聚复合体,在核内积累的PER 蛋白有mCRY和CKIε/CKIδ的结合位点,PER不仅是三聚体组装的桥连蛋白,而且是mCRY被酪蛋白激酶磷酸化反应所必需[35,39]。

值得注意的是,mCRY蛋白对mPER2磷酸化的稳定性很重要,对mPER2磷酸化提供蛋白载体,保护mPER2磷酸化。mCRY蛋白对mPER1磷酸化稳定性不是必需的,在生物钟机制中,mCRY 对mPER1和mPER2的不同作用机理目前尚未可知[34,43]。

3.3核内的生物钟载体

在核内,mCRY-mPER-CKIε/CKIδ和与之相关的CLOCK和BMAL1络合,构成“生物钟载体”,通过降解转录复合体的活性达到负相调节mPer,mCry和Erbα基因的转录的目的(Figure 2)。肝脏振荡器染色质免疫沉淀反应表明,CLOCK和BMAL1与mPer1 E-Box结合与周期循环无关,负调节子与DNA锚定CLOCK-BMAL1异二聚体节律性地结合,在转录过程钟产生节律[34]。振荡器在脑室组织的细胞核内激素受视黄酸受体α(RARα)和维生素A类受体α(RXRα)的活化可能对mCRY蛋白质负调节CLOCK-BMAL1异二聚体转录调节有整合的作用[44]。其结果说明有很多调节子参与调节复合体的形成,CLOCK和BMAL1与DNA牢固地结合与修饰CLOCK-BMAL1异二聚体与E-boxes的结合的氧化还原作用不同[45,46]。

在抑制相的终末,时钟载体通过过磷酸化将生物钟蛋白降解[39,42],CLOCK-BMAL1异二聚体与DNA持久结合可能反映一个新合成的“衰老(older)”转录复合体,其功能也是一个未知数。

哺乳动物生物钟昼夜节律同步机制的研究进展

Figure 2 转录复合体模式图

根据Steven M & David R.Weaver, Nature 2002,418:935~941.

4.光信号输入SCN

4.1“周期性”的光受体

过磷酸化可能是时钟机制把外界环境因素的刺激与生物体内周期性同步的重要过程。在哺乳类动物,光线刺激产生信号,通过视网膜到下丘脑的视束(RHT)的视觉通路将信息传递到SCN[47,48]。最近的研究表明,光敏黑色素视蛋白型的感光分子(photopigment melanopsin)在周期性光受体中有作用。

视网膜光受体是由视杆细胞、视锥细胞和感光色素组成,但视杆细胞、视锥细胞对几种光反应并不是必需的。光诱导SCN生物钟时相转换,没有视杆、视锥细胞的器官,如松果

体、垂体的周期性反应调节[48]。通过对光敏黑色素视蛋白分子离析,推测视网膜内存在调节这些反应的光敏黑色素视蛋白分子。

感光色素视蛋白基因在视网膜节细胞表达定位免疫反应提示:黑色素视蛋白是一种周期性光受体,是视网膜节细胞内的特有物质[49~51]。垂体腺苷酸环化酶激活肽PACAP (adenylate cyclase-activiting peptide, PACAP)亦是黑色素视蛋白,在形态上有很大的树状分枝,直接投射到SCN和内侧膝状体[50,52,53],更重要的是黑素视蛋白免疫反应阳性的节细胞可能直接投射到SCN参与光敏反应,尽管这些反应不排除有其它黑色素视蛋白阳性细胞的光敏反应,提示:黑色素视蛋白调节无视杆细胞和视锥细胞的光信号的传导。是否黑色素视蛋白完全是周期性光受体尚无定论,黑色素视蛋白可能是传统光受体系统通路的补充(Figure 3)

哺乳动物生物钟昼夜节律同步机制的研究进展

Figure 3 从视网膜到SCN的视觉通路激活SCN神经元时钟机制示意图。

根据Steven M & David R.Weaver, Nature 2002,418:935~941.

4.2视网膜输入的传导

视网膜节细胞通过RHT的神经递质谷氨酸盐和PACAP等接受和处理光信号到达SCN神经元,神经递质在有光信号和无光信号输入的情况下同样可以直接到达SCN,向后延伸至内侧膝状体和中脑,光信号输入SCN通路中神经递质GABA(γ-aminobutyric acid,GABA)、神经肽Y和5-羟色氨有重要作用[54,55]。

视网膜节细胞是怎样激活分布在SCN神经元分子生物钟的呢?光信号不管在夜晚开始或结束,都能使mPer1基因迅速表达[56]。但mPer2在夜晚的初期表达,在夜晚持续一段时间,就检测不到mPer2的表达[8]。说明,mPer2是黑夜初期的调节子(相延迟),mPer1是相后调节子[57]。对小鼠的一项研究表明,mPER1和mPER2的降解也与上述情况一致,说明mPer1基因对光诱导时相转换不是必需的[32]。

在夜间给予光信号数小时,SCN内mPER1和mCRY1免疫反应阳性增强[58],且发现当不进行光照且mCry RNA水平低的情况下,mCRY1仍伴随mPER1含量增加,表明有丰富的mCRY进入核内趋同mPER的增长水平。因此,PER依靠CRY在核内积累可能重新设置周期时相,这是同步机制的关键,也是控时机制的核心[34,42]。

染色质的变化和CREB(cyclic AMP responsive element-binding protein ,CREB)和CRE (cAMP-responsive element,CRE)结 合,可能调节光引导的 mPer1和mPer2基因表达[59,60],值得注意的是,光诱导是E-box调节在很大程度上是独立的,如光诱导Per表达在clock/clock突变小鼠十分有效[60,61]。

5.从SCN到下游振荡器

SCN周期性信号输出到其它脑区依赖Na+的动作电位[54],至少两种离子参与此生物种的控制,一种是Ca2+ ,另一种是K+。两种离子对于维持膜电位所必需[5,62],但这两种离子如何在生物钟机制中发挥作用,Ca2+通道和K+通道的亚单位能够节律性地调节转录和翻译水平,离子通道也能在翻译后进程中起调节作用,如磷酸化和/或调节子蛋白间的反应,这种调节子蛋白能被包含E-box增强子的控时基因CCDs(clock-controllerd genes, CCDs)控制,所以可以直接被CLOCK-BMAL1异二聚体(因此CCDs可以认为是第一指令)控制,或在下游被CLOCK-BMAL1异二聚体间接控制。

从SCN传出神经信号分子包括神经递质和分泌因子,其中一种分泌因子来自SCN在啮齿类动物的移植研究,提示源于SCN的抑制和激活因子之间的转换驱动运转节律(休息/活动,睡眠/觉醒周期),最近确定两种神经肽转移因子(TGF-α)和蛋白激酶-2(PK2)作为周期性输出因子。TGF-α是一种膜电位抑制物质,进入第三脑室抑制膜电位运转活动,生长因子在SCN和视网膜表达从而抑制在室旁核的受体活动,室旁核是SCN传出神经的中继站,生长因子明显影响室旁核发挥其功能,

因此生长因子可以调节光诱导的 周期性控制。TGF-α增强夜间活动的周期性控

制[63]。

PK2是一种多肽,其RNA水平在小鼠SCN高通量、节律性地表达,大多直接控制活动的周期性[64],PK2是SCN中的一种第一指令CCG,在RNA水平由作用于E-box增强子的CLOCK和BMAL1调节,PK2受体在SCN投射的许多靶点表达,也在SCN表达,值得注意的是PK2却不在室旁核SPZ(subparaventricular zone, SPZ)表达。当夜间PK2水平降低时,诱导PK2浸液在向脑室内侧移动[64]。在白天,当小鼠处于静止状态,被诱导含量水平降低的PK2和一种内生肽抑制这种转移。生物钟控制的PK2在白天表达形式为周期性生物钟和行为输出之间提供了关键线索(包括睡眠)。

SCN起步器控制大脑外周组织的下游振荡器,从SCN到外周器官的神经输出由自主神经完成,由神经和激素信号控制外周振荡器[65]。另外小鼠肾上腺皮质激素也能作用于外周振荡器,也是SCN控制下游振荡器在外周组织的间接通路[66]。SCN对下游振荡器(如肝脏)通过调节休息/活动周期实现时相的同步[67,68]。因此,在啮齿类,人工干扰可以使外周振荡器完全脱离SCN的控制,环路诱导信号、肾上腺皮质激素等同样影响外周振荡器[69]。神经的控制、激素信号、行为变化可以改变周期同步,也能影响Per1和Per2在外周组织的表达。

下游振荡器被SCN协调起来后,其相关分子转导机制使局部振荡器的节律同步化,一种通路是通过局部第一指令CCGs发生[70,71]。新的证据表明,DBP(D-element binding protein,DBP)也是一种CCGs,在肝的新陈代谢进程中调节关键酶的节律性转录[72]。DBP与以亮氨酸为基础的转录因子E4BP4同时作用,E4BP4的节律相与DBP的节律相相反(DBP可能负相调节Per基因的转录,E4BP4可能正相调节Per基因的转录)[73,74],不同程度地影响如cis-acting等成分,随之驱动应答基因的节律性[75]。E4BP4的节律性很可能由REV-ERBα受体的调节,与BMAL1的节律性生物钟的核心调节机制相似。

DNA重组技术和基因组测序证明哺乳动物组织有大量的CCGs[76],对于人类和啮齿动物基因组研究表明:在稳态RNA水平,有2%~10%的周期振荡器的基因表达在特有组织(少于5%在组织间重复表达)。在大多数情况下,节律由器官的功能决定。然而,实际仅有小部分节律基因直接由CLOCK-BMAL异二聚体控制转录[77~79],生物钟蛋白第一指令CCGs(如PK2和DBP),似乎是周期信息的关键调节子,大多数直接通道通过核心振荡器的转换调节下游的振荡器。

6.问题及展望

目前可以把克隆生物钟基因和其蛋白质生物钟机制模板组装,而且这些模板可以继续参与反应(修正、调节正负反馈环路),说明大多数哺乳动物生物钟机制业已清楚,如小鼠节律丧失与VPAC2受体的分解有关,细胞内信号对细胞内周期振荡器有重要的驱动作用等。尽管如此,组织水平上单个振荡子内部反应机制仍是未来研究的一个难点。

近来,DNA基因重组提示,直接的核心反馈环路节律基因表达的情况很少,关键是如何控制外周器官节律基因的表达。利用染色质免疫沉淀反应结合DNA重组分析,提示第一指令CCGs直接参与SCN和外周振荡器中CLOCK-BMAL1异二聚体的控制,以及CCGs的网络级联反应与之相联系的生物钟机制的表达节律。在生物钟周期研究中,局部的控制很重要,如在生理条件下器官和系统的水平上节律基因表达和病理条件下节律性丧失的控制的研究有重要意义。

致谢

本研究得到了兰州大学生命科学学院邵宝平、丁艳平、李东波和刘重兵博士及兰州大学草地农业科技学院杨思忠老师的修改建议,在此深表诚挚的谢意。

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Advances in Study on Timing Mechanism of Circadian Clock in Mammalians

XIE Zhao-Hui and WANG Jian-Lin

School of Life Science Lanzhou University, Lanzhou 730000

Abstract Circadian rhythms mechanism is mainly composed with oscillators, input signal, output signal and circadian timing. The focal point of this system is

master clock located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus in the mammalian, which orchestrates the circadian rhythms with oscillators of peripheral organs. The fundamental molecular framework of circadian clock is composed of a transcription/translation-based autoregulatory feedback loop. In the nucleus, mCRY-mPER-CKIε/CKIδ complexes with CLOCK and BMAL1 form “timesome”. Some products of the clock genes serve as positive or negative regulators influencing the clock oscillation. It is important that the circadian time-keeping mechanism is involved in several posttranslational events. In mammals, light is the most potent entraining signal, with the retinohypothalamic tract (RHT) being the principal retinal pathway through which entraining information reaches the SCN in which it can effect and mediate to slave oscillators to local rhythms.

Key words Biology clock; Circadian rhythm; mammal