关键蛋白酶激活因子Apaf-2/CytC在细胞凋亡中的作用

描述了Apaf-2/CytC在细胞凋亡中的作用,主要阐述了细胞凋亡相关因子Apaf-2/CytC的发现,Apaf-2与Apaf-1及Apaf-3在细胞凋亡中的作用及相互关系,CytC介导细胞凋亡的方式。探讨了线粒体CytC的泄漏机制,对MPTP假说、专一性通道假说和线粒体膨胀假说进行了阐述,并对细胞凋亡信号转导途径,如CytC途径、Fas/FasL途径、蛋白激酶途径、AIF凋亡途径及这些信号转导途径间的对话进行了分析,最后分析了研究CytC与细胞凋亡分子机制的意义并提出细胞凋亡分子机制中一些未完全研究清楚的问题。     

Fas/FasL系统与类风湿关节炎发病机制的研究进展

类风湿关节炎是机体对自身滑膜发生免疫反应的疾病,其主要病理特征是滑膜增生和多种炎症细胞的浸润。滑膜增生的有关机制仍不清楚,目前认为可能是滑膜细胞和炎症浸润细胞数量增加及凋亡相对减少,即细胞凋亡程度不及增殖程度所致。Fas/FasL系统是细胞凋亡的重要途径之一,通过影响滑膜细胞的Fas/FasL表达可以诱导其凋亡。本文对Fas/FasL的性质、结构、功能、Fas/FasL与RA发病机制及RA治疗的关系进行综述。     

胎盘发生过程中的细胞凋亡

细胞凋亡是一种正常的生理现象,在胚泡着床过程中,伴随有大量细胞凋亡。研究表明,胎盘发生过程中的细胞凋亡,对调控子宫内膜基质细胞的蜕膜化和滋养层细胞的浸润以形成胎盘具有重要意义;另外,由Fas/FasL系统介导的细胞凋亡可能与母体对胎儿的免疫耐受性有关。本文主要评述细胞凋亡的一般通路以及胎盘发生过程中细胞凋亡的调控。     

Fas介导细胞凋亡及相关免疫调节作用

Fas/CD95作为肿瘤坏死因子(TNF)/神经生长因子(NGF)分子受体超家族成员,在细胞凋亡及体内免疫调节方面发挥重要的作用。细胞在接收到经Fas传递的凋亡信号后,主要经胞浆caspase和线粒体两种途径引发细胞程序性死亡。Fas介导T/B淋巴细胞的凋亡,在这些细胞的早期分化发育和维持机体免疫平衡中起主要作用。CTL和NK等杀伤细胞也通过Fas-FasL介导的细胞凋亡而发挥对靶细胞的杀伤效应。因此,Fas-FasL系统在肿瘤、自身免疫性疾病、艾滋病等疾病过程中发挥重要的作用。     

人脑星形细胞瘤中p53，Fas／FasL的表达及其与凋亡关系的研究

目的：探讨细胞凋亡在星形细胞瘤中的作用及其与p53、Fas和Fas配体（Fas ligand,FasL）的关系。方法：对43例星形细胞瘤的标本分别进行HE染色,TUNEL及免疫组化分别标记p53,Fas和FasL。结果：高级别肿瘤和低级别肿瘤间的凋亡无显著差异（P〉0．05）。高级别星形细胞瘤的p53,Fas和FasL的表达均显著高于低级别肿瘤（P均〈0．05）。结论：突变型p53可作为评价星形细胞瘤生物学行为的参考指标。与低级别星形细胞瘤相比,高级别肿瘤中的细胞凋亡受到了抑制,且Fas与FasL的过表达对细胞凋亡可产生明显影响。     

人脑星形细胞瘤中p53，Fas/FasL 的表达及其与凋亡关系的研究

目的:探讨细胞凋亡在星形细胞瘤中的作用及其与p53、Fas和Fas配体(Fas ligand,FasL)的关系。方法:对43例星形细胞瘤的标本分别进行HE染色,TUNEL及免疫组化分别标记p53,Fas和FasL。结果:高级别肿瘤和低级别肿瘤间的凋亡无显著差异(P>0.05)。高级别星形细胞瘤的p53,Fas和FasL的表达均显著高于低级别肿瘤(P均<0.05)。结论:突变型p53可作为评价星形细胞瘤生物学行为的参考指标。与低级别星形细胞瘤相比,高级别肿瘤中的细胞凋亡受到了抑制,且Fas与FasL的过表达对细胞凋亡可产生明显影响。     

内耳免疫反应诱导Fas和FasL表达与凋亡的关系

目的研究内耳免疫反应过程中是否存在细胞凋亡,以及细胞凋亡是否与Fas和FasL信号转导有关.方法选用雌性白色豚鼠16只,随机分为实验组和对照组各8只,以钥孔虫戚血蓝蛋白(keyhole limpet hemocyanin,KLH)全身免疫后,实验组以相同抗原进行内耳免疫,对照组内耳注射等量的磷酸盐缓冲生理盐水(phosphate buffered saline,PBS),在内耳免疫5d后处死动物,取内耳免疫侧耳蜗做石蜡切片.通过脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术(terminal-deoxynucleotidyl transferase mediated nick end labeling,TUNEL)检测内耳凋亡细胞,免疫组化检测内耳Fas和FasL的表达.结果实验组豚鼠内耳Corti器毛细胞,血管纹的缘细胞和螺旋神经节细胞存在TUNEL染色阳性细胞,而对照组动物切片仅在支持细胞、血管纹和螺旋神经节细胞中发现极少数TUNEL染色阳性细胞.免疫组化染色实验组Corti器、螺旋神经节细胞、血管纹和螺旋韧带Fas和FasL蛋白表达阳性,而对照组只有螺旋神经节细胞和血管纹有较弱的Fas蛋白表达,FasL蛋白表达阴性.结论内耳免疫反应可诱导细胞凋亡的发生,Fas-FasL途径是参与此过程重要的信号转导途径之一.     

汉滩病毒诱导小鼠骨髓瘤SP2/0细胞凋亡的初步研究

为研究汉滩病毒对肿瘤细胞的诱导凋亡作用,以一定量病毒悬液感染体外培养的SP2/0细胞,接种后定时间将细胞消化甩片行Gimsa染色观察凋亡细胞核的变化,制细胞悬液以流式细胞仪测细胞周期,并用免疫组化的方法检测凋亡分子Fas和FasL的表达.结果示经病毒诱导后细胞出现生长特性及形态学变化,Giemsa染色观察到典型凋亡细胞;流式细胞仪显示有凋亡峰出现;免疫组化检测出感染后SP2/0细胞中Fas和FasL表达明显升高.该结果表明汉滩病毒可诱导体外培养SP2/0细胞凋亡,其发生可能与凋亡分子Fas和FasL有关.     

FasL-Fas:免疫豁免还是炎症?

FasL,又称死亡配体,与其受体Fas分子（Fas）结合,能诱导Fas阳性细胞的凋亡。免疫豁免区组织中组成性表达的FasL被认为是介导免疫豁免的关键分子,一些肿瘤细胞也表达FasL,被认为是用来逃脱免疫攻击的手段。然而,最近大量设想利用FasL来诱导异体移植的耐受却得出了相反的结果,FasL引起严重的炎症反应使得移植排斥更为迅速。这使得人们对于FasL在介导免疫豁免中的作用提出了异议。本就最近几年FasL的研究进展,对这些非淋巴细胞表达的FasL在机体维持免疫豁免及诱发炎症中的关系作一讨论。     

牙龈卟啉单胞菌诱导T淋巴细胞活化、凋亡的体外研究

目的检测牙龈卟啉单胞菌对人外周血中T淋巴细胞活化及凋亡的作用,并检测Fas/FasL在牙龈卟啉单胞菌(Porphyrom onas gingivalis,Pg)诱导的T淋巴细胞凋亡中的表达。方法选取10例全身及牙周组织健康受试者,分离外周血中T淋巴细胞,在有/无Pg情况下培养0~96 h,用荧光探针(Annexin V-FITC、PI、CD69)及特殊的单克隆抗体(Fas、FasL)进行标记,并进行流式细胞仪检测。结果 CD69+淋巴细胞+Pg组Annexin V+/PI-细胞百分数在各个时间点上都明显高于T淋巴细胞+Pg组(P0.01)。Fas和FasL的表达量明显上调。用抗Fas单克隆抗体阻滞Fas-FasL相互作用导致T细胞凋亡的明显减少,百分比为(20.56±2.43)%,未加抗体的为(50.41±2.68)%。但残余的细胞凋亡活动与阴性对照相比仍高。结论 Pg能够诱导人外周血中T淋巴细胞活化,并且能够通过活化促进其凋亡,Pg诱导T淋巴细胞凋亡主要通过Fas-FasL途径,并具有时间依赖性。     

Germ cell apoptosis in the testes of Sprague Dawley rats following testosterone withdrawal by ethane 1,2-dimethanesulfonate administration: relationship to Fas?

Germ cell apoptosis, which occurs normally during spermatogenesis, increases after testosterone withdrawal from the testis. The molecular mechanism by which this occurs remains uncertain. The Fas system has been implicated as a possible key regulator of apoptosis in various cells: binding of Fas ligand (FasL), a type II transmembrane protein, to Fas, a type I transmembrane receptor protein, triggers apoptosis in cells expressing Fas. Recently, Fas has been localized to germ cells, and FasL to Sertoli cells, within the rat testis. We hypothesized that Fas protein content would rise in response to reduced levels of testosterone as part of a suicide pathway that would result in germ cell apoptosis. To test this hypothesis, ethane 1,2-dimethanesulfonate (EDS), a Leydig cell toxicant, was used to kill Leydig cells and thus reduce intratesticular testosterone levels in Sprague Dawley rats. Apoptosis was examined in situ and biochemically, and Fas protein content in the testis was monitored by Western blot analysis. We show that EDS injection results in the following sequence of events: apoptotic death of Leydig cells by a mechanism that does not involve Fas; reduced testosterone; increased testicular Fas content; and germ cell apoptosis. These results suggest that Fas may play a role in the apoptotic death of germ cells that results from reduced intratesticular testosterone levels, and that testosterone may play a role in germ cell survival via its suppression of Fas.     

TNF-alpha and IFN-gamma regulate expression and function of the Fas system in the seminiferous epithelium

Sertoli cells have long been considered to be involved in the regulation of the immune response in the testis. More recently, the Fas system has been implicated in the maintenance of the immune privilege in the testis as well as in the regulation of germ cell apoptosis. However, the control of Fas and Fas ligand (FasL) expression in the testis remains unknown. In the present study, we demonstrate that cultured mouse Sertoli cells constitutively express a low level of membrane-bound Fas protein, but not a soluble form of Fas. Sertoli cells stimulated with TNF-alpha and IFN-gamma markedly increase the expression of both soluble and membrane-bound Fas in a dose-dependent manner. The up-regulated membrane-bound Fas protein is functionally active because it induces a significant level of Sertoli cell death in the presence of Neuro-2a FasL+ effector cells. Interestingly, the soluble form of Fas, which is induced by the same cytokines but has an antiapoptotic effect, is also functional. In fact, conditioned media from TNF-alpha-stimulated Sertoli cell cultures inhibit Neuro-2a FasL+-induced cell death. Taken together, our data suggest a possible regulatory role of TNF-alpha and IFN-gamma on Fas-mediated apoptosis in the testis through disruption of the balance between different forms of Fas.     

Palmitoylation of human FasL modulates its cell death-inducing function

Fas ligand (FasL) is a transmembrane protein that regulates cell death in Fas-bearing cells. FasL-mediated cell death is essential for immune system homeostasis and the elimination of viral or transformed cells. Because of its potent cytotoxic activity, FasL expression at the cell surface is tightly regulated, for example, via processing by ADAM10 and SPPL2a generating soluble FasL and the intracellular fragments APL (ADAM10-processed FasL form) and SPA (SPPL2a-processed APL). In this study, we report that FasL processing by ADAM10 counteracts Fas-mediated cell death and is strictly regulated by membrane localization, interactions and modifications of FasL. According to our observations, FasL processing occurs preferentially within cholesterol and sphingolipid-rich nanodomains (rafts) where efficient Fas–FasL contact occurs, Fas receptor and FasL interaction is also required for efficient FasL processing, and FasL palmitoylation, which occurs within its transmembrane domain, is critical for efficient FasL-mediated killing and FasL processing.     

Significant role for Fas in the pathogenesis of autoimmune diabetes

Programmed cell death represents an important pathogenic mechanism in various autoimmune diseases. Type I diabetes mellitus (IDDM) is a T cell-dependent autoimmune disease resulting in selective destruction of the beta cells of the islets of Langerhans. beta cell apoptosis has been associated with IDDM onset in both animal models and newly diagnosed diabetic patients. Several apoptotic pathways have been implicated in beta cell destruction, including Fas, perforin, and TNF-alpha. Evidence for Fas-mediated lysis of beta cells in the pathogenesis of IDDM in nonobese diabetic (NOD) mice includes: 1) Fas-deficient NOD mice bearing the lpr mutation (NOD-lpr/lpr) fail to develop IDDM; 2) transgenic expression of Fas ligand (FasL) on beta cells in NOD mice may result in accelerated IDDM; and 3) irradiated NOD-lpr/lpr mice are resistant to adoptive transfer of diabetes by cells from NOD mice. However, the interpretation of these results is complicated by the abnormal immune phenotype of NOD-lpr/lpr mice. Here we present novel evidence for the role of Fas/FasL interactions in the progression of NOD diabetes using two newly derived mouse strains. We show that NOD mice heterozygous for the FasL mutation gld, which have reduced functional FasL expression on T cells but no lymphadenopathy, fail to develop IDDM. Further, we show that NOD-lpr/lpr mice bearing the scid mutation (NOD-lpr/lpr-scid/scid), which eliminates the enhanced FasL-mediated lytic activity induced by Fas deficiency, still have delayed onset and reduced incidence of IDDM after adoptive transfer of diabetogenic NOD spleen cells. These results provide evidence that Fas/FasL-mediated programmed cell death plays a significant role in the pathogenesis of autoimmune diabetes.     

IL-4 diminishes perforin-mediated and increases Fas ligand-mediated cytotoxicity In vivo

CTL have evolved two major mechanisms for target cell killing: one mediated by perforin/granzyme secretion and the other by Fas/Fas ligand (L) interaction. Although cytokines are integral to the development of naive CTL into cytolytic effectors, the role of cytokines on mechanisms of CTL killing is just emerging. In this study, we evaluate the effects of IL-4 in Fas(CD95)/FasL(CD95L)-mediated killing of Fas-overexpressing target cells. Recombinant vaccinia viruses (vv) were constructed to express respiratory syncytial virus M2 Ag alone (vvM2) or coexpress M2 and IL-4 (vvM2/IL-4). MHC-matched Fas-overexpressing target cells (L1210Fas+) were used to measure both perforin- and FasL-mediated killing pathways. In contrast to Fas-deficient (L1210Fas-) target cells, effectors from vvM2/IL-4-immunized mice were able to lyse L1210Fas+ target cells with similar magnitude as vvM2-infected mice. Addition of EGTA/Mg2+ revealed that effectors from vvM2/IL-4-infected mice primarily lyse targets by a Ca2+-independent Fas/FasL pathway. Analysis of FasL expression by flow cytometry showed that IL-4 increased cell surface FasL expression on CD4+ and CD8+ splenocytes, with peak expression on day 4 after infection. These data demonstrate that IL-4 increases FasL expression on T cells, resulting in a shift of the mechanism of CTL killing from a dominant perforin-mediated cytolytic pathway to a dominant FasL-mediated cytolytic pathway.     

The metalloproteinase matrilysin proteolytically generates active soluble Fas ligand and potentiates epithelial cell apoptosis

BACKGROUND: The Fas ligand/Fas receptor (FasL/Fas) system is an important mediator of apoptosis in the immune system where the juxtaposition of cells expressing the cell-surface ligand induces the apoptotic pathway in Fas-expressing lymphocytes. The FasL/Fas system has also been shown to be involved in apoptosis in epithelial tissues, including the involuting rodent prostate. FasL can be shed through the action of an hitherto unidentified metalloproteinase to yield soluble FasL (sFasL), although the biological activity of sFasL has been disputed. RESULTS: Here we report that the matrix metalloproteinase matrilysin can process recombinant and cell-associated FasL to sFasL, and that matrilysin-generated sFasL was effective at inducing apoptosis in a target epithelial cell population. In the involuting mouse prostate, FasL and matrilysin colocalized to the cell surface in a restricted population of epithelial cells. Mice deficient in matrilysin demonstrated a 67% reduction in the apoptotic index in the involuting prostate compared with wild-type animals, implicating matrilysin in this FasL-mediated process. CONCLUSIONS: The results show that a functional form of sFasL was generated by the action of the metalloproteinase matrilysin, and suggest that matrilysin cleavage of FasL is an important mediator of epithelial cell apoptosis.     

Murine male germ cell apoptosis induced by busulfan treatment correlates with loss of c-kit-expression in a Fas/FasL- and p53-independent manner

Male germ cell apoptosis has been extensively explored in rodents. In contrast, very little is known about the susceptibility of developing germ cells to apoptosis in response to busulfan treatment. Spontaneous apoptosis of germ cells is rarely observed in the adult mouse testis, but under the experimental conditions described here, busulfan-treated mice exhibited a marked increase in apoptosis and a decrease in testis weight. TdT-mediated dUTP-X nicked end labeling analysis indicates that at one week following busulfan treatment, apoptosis was confined mainly to spermatogonia, with lesser effects on spermatocytes. The percentage of apoptosis-positive tubules and the apoptotic cell index increased in a time-dependent manner. An immediate effect was observed in spermatogonia within one week of treatment, and in the following week, secondary effects were observed in spermatocytes. RT-PCR analysis showed that expression of the spermatogonia-specific markers c-kit and Stra 8 was reduced but that Gli I gene expression remained constant, which is indicative of primary apoptosis of differentiating type A spermatogonia. Three and four weeks after busulfan treatment, RAD51 and FasL expression decreased to nearly undetectable levels, indicating that meiotic spermatocytes and post-meiotic cells, respectively, were lost. The period of germ cell depletion did not coincide with increased p53 or Fas/FasL expression in the busulfan-treated testis, although p110Rb phosphorylation and PCNA expression were inhibited. These data suggest that increased depletion of male germ cells in the busulfan-treated mouse is mediated by loss of c-kit/SCF signaling but not by p53- or Fas/FasL-dependent mechanisms. Spermatogonial stem cells may be protected from cell death by modulating cell cycle signaling such that E2F-dependent protein expression, which is critical for G1 phase progression, is inhibited.     

Deciphering the pathways of germ cell apoptosis in the testis

A growing body of evidence demonstrates that germ cell death both spontaneous (during normal spermatogenesis) and that induced by suppression of hormonal support or increased scrotal temperature occurs via apoptosis. The mechanisms by which these proapoptotic stimuli activate germ cell apoptosis are not well understood. In order to provide some insight, here we report the key molecular components of the effector pathways leading to caspase activation and increased germ cells apoptosis triggered by mildly increased scrotal temperature. Short-term exposure (43 °C for 15 min) of the testis to mild heat results, within 6 h, in stage- and cell-specific activation of germ cell apoptosis in rats. Initiation of apoptosis was preceded by a redistribution of Bax from a cytoplasmic to paranuclear localization in heat-susceptible germ cells. Such relocation of Bax is further accompanied by sequestration of mitochondria and endoplasmic reticulum (ER) into paranuclear areas, cytosolic translocation of cytochrome c and is associated with activation of the initiator caspase 9 and the executioner caspases 3, 6, and 7, and cleavage of PARP. Furthermore, Bax is co-localized with ER in the susceptible germ cells as assessed by combined two-photon and confocal microscopy and Western blot analyses of fractionated testicular lysates. In additional studies, using gld and lpr^cg mice, which harbor loss-of-function mutations in Fas-ligand (FasL) and Fas, respectively, we demonstrated that heat-induced germ cell apoptosis is not blocked, thus providing further evidence that the Fas signaling system is dispensable for heat-induced germ cell apoptosis in the testis. Taken together, these results demonstrate that the mitochondria- and possibly also ER-dependent pathways are the key apoptotic pathways for heat induced germ cell death in the testis.     

Doxycycline induces Fas/Fas ligand-mediated apoptosis in Jurkat T lymphocytes.

Tetracyclines have been used in the treatment of chronic inflammatory diseases associated with local infiltration of inflammatory cells and matrix destruction as observed in rheumatoid arthritis and periodontal disease. Fas/Fas ligand (FasL)-mediated apoptosis plays an important role in maintaining T lymphocyte homeostasis and modulating immune response. The present study demonstrates that doxycycline inhibits Jurkat T lymphocyte proliferation and induces apoptosis. The phytohemagglutinin (PHA)-activated Jurkat cells are more susceptible to doxycycline-induced apoptosis. Furthermore, doxycycline-induced apoptosis is associated with increased Fas/FasL expression in Jurkat cells. The increase of apoptosis in Jurkat cells treated with doxycycline is consistent with the increase of FasL expression. These results suggest that doxycycline may downregulate the inflammatory process in certain diseases by eliminating activated T lymphocytes through Fas/FasL-mediated apoptosis.