缺氧预处理的抗炎机制研究
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摘要
第一部分HPC通过调节细胞内cAMP水平发挥抗炎效应
缺氧预处理(Hypoxia Preconditioning, HPC)是近年对缺氧病理生理机制研究比较集中的热点之一。目前,缺氧预处理被认为是一个复杂的,涉及多因素、多机制的保护过程。
最近有研究提示环磷酸腺苷(cyclic adenosine monophosphate, cAMP)与缺氧预处理时细胞内信号转导密切相关。cAMP是细胞内普遍存在的第二信使,参与调节细胞的许多活动。细胞内的cAMP处于一个动态平衡,胞外刺激可以通过细胞膜上G蛋白耦联受体(G protein coupled recptors, GPCRs)如肾上腺素受体、腺苷受体(A2A及A2B受体)等激活腺苷酸环化酶(adenylyl cyclase, AC),催化5’-AMP环化形成cAMP;一旦cAMP达到一定的水平,负反馈机制便自动启动,主要通过两条途径:一是通过β-arrestin使GPCRs失活,减少cAMP的生成,二是通过磷酸二酯酶(PDEs)的作用将其降解为5’-AMP。
研究目的
本研究利用所在实验室已建立的缺氧预处理动物及细胞模型,探讨:1.缺氧预处理对细胞内cAMP水平的调节机制;2.cAMP信号通路参与缺氧预处理抗炎作用的机制。
实验方法和材料
一、细胞缺氧预处理模型:
细胞置于低氧密闭容器(2%O2)中缺氧45分钟,取出于正常氧环境(21%O2)中复氧20分钟,此为一个循环,重复3个循环。单纯缺氧组一直置于低氧密闭容器(2%O2)中,正常对照组则一直置于正常氧环境(21%O2)中。
二、动物缺氧预处理模型:
C57BL/6小鼠或CD73基因缺陷小鼠随机分成3组,分别是正常对照组、单纯缺氧组和缺氧预处理组。缺氧预处理组小鼠被放置于可调节氧浓度的湿化密闭容器内,调节氧浓度到8%,维持10分钟,然后氧浓度转换为21%持续10min,如此循环3次。然后取出,置于常氧环境中2小时;单纯缺氧组小鼠也被放置同种容器内,调节氧浓度到8%,维持1小时,取出,置于常氧环境中2小时;正常对照组小鼠在正常氧环境中(氧浓度21%),不作任何处理。随后各组的小鼠均被置于容器内,调节氧浓度到5%,维持10分钟,令其死亡,手术取出肺组织,立即投入液氮快速冷冻,-80℃冰箱保存备用。
三、缺氧预处理对cAMP水平及效应的调节:
1、ELISA法检测细胞内cAMP水平
2、Western blot方法检测β-arrestin、PDE4B、p38MAPK等蛋白表达水平的变化
①细胞总蛋白提取法:常规总蛋白裂解液提取
②细胞蛋白分步提取法(两步法):第一步提取胞浆/胞膜蛋白;第二步提取脂质体/核蛋白
3、双荧光素酶报告分析(Dual luciferase reporter assay)检测NF-кB活性:将构建有luciferase reporter的NFкB质粒转染入Hela细胞,在缺氧预处理后,继续缺氧24小时,然后检测Luciferase强度,即NFкB活性。
4、RT-PCR检测PDE4B、白介素-6(IL-6)的mRNA水平
5、免疫荧光染色检测NFкB的核移位情况
研究结果
一、缺氧预处理可以明显提高Calu-3细胞和T84细胞内cAMP水平:ELISA检测显示,与单纯缺氧组和正常氧环境组比,缺氧预处理组cAMP水平明显提高。
二、缺氧预处理不影响细胞内β-arrestin蛋白总量,但可使位于脂质体上的“有效”β-arrestin减少,阻止GPCRs的失活,从而使cAMP生成增多。
三、缺氧预处理可降低PDE4B的转录及表达水平,减少cAMP的降解。
四、缺氧预处理能抑制cAMP-PKA-CREB通路下游的p38MAPK的磷酸化。
五、缺氧预处理能抑制NF-кB的核移位。
六、缺氧预处理能抑制NF-кB的转录活性。
七、缺氧预处理能阻止IL-6 mRNA水平的升高。
结论
一、缺氧预处理可提高细胞内cAMP水平:缺氧预处理一方面可以通过细胞外刺激信号如腺苷酸等的增强,激活腺苷酸环化酶;另一方面可通过对β-arrestin和PDE4B的调节,抑制负反馈途径,共同促进细胞内cAMP的提升。
二、缺氧预处理对cAMP的提升,可通过cAMP-PKA-CREB-p38MAPK通路,抑制NF-кB介导的炎症反应。
第二部分IκBα的SUMO化修饰参与HPC对NF-κB炎症通路的抑制
SUMO化修饰是蛋白质表达后修饰的一种方式,是指小泛素相关修饰物(small ubiquitin-ralated modifier,SUMO)共价结合于靶蛋白的赖氨酸残基上。这个过程类似但又不同于泛素化,与泛素介导蛋白质的降解不同,SUMO化修饰的蛋白质更加稳定。SUMO参与了广泛的细胞内代谢途径,在蛋白-蛋白之间的相互作用、信号转导、核质运输、转录调控等方面均发挥着重要的作用。
缺氧预处理是目前对缺氧病理生理机制研究的热点之一,主要指在进行缺氧处理以前,对研究对象给予几次时间较短,程度较轻的“低氧-复氧-低氧-复氧”预适应处理。缺氧预处理是一个复杂的,涉及多因素、多机制的保护过程,其中,对NF-кB介导的炎症反应的抑制是一个比较重要的环节。
有研究发现SUMO化修饰可阻止IκBα泛素化,从而抑制IκBα的降解,稳定NF-κB,减轻炎症反应。我们推测,IκBα的SUMO化修饰可能参与缺氧预处理对NF-κB炎症通路的抑制。
研究目的
本研究利用所在实验室已建立的缺氧及缺氧预处理动物及细胞模型,探讨IκBα的SUMO化修饰是否参与缺氧预处理对NF-κB炎症通路的抑制。
实验方法
一、细胞缺氧预处理模型:(方法同第一章)
二、动物缺氧预处理模型:(方法同第一章)
三、在Hela细胞模型中,利用SAE-1过表达上调SUMO-1水平,然后进行缺氧预处理实验,与正常对照组对比研究:
1、Western blot方法检测IκBα、SUMO-1-IκBα、pIκBα等蛋白表达的变化。
2、双荧光素酶报告法检测NF-кB活性。
3、RT-PCR检测IL-6 mRNA水平。
四、在Hela细胞模型中,利用shRNA下调SUMO-1水平,然后进行缺氧预处理实验,与正常组对比研究:
1、双荧光素酶报告法分析检测NF-кB活性。
2、RT-PCR检测IL-6 mRNA水平
研究结果
小鼠模型中,缺氧预处理可以上调肺组织内SUMO-1修饰的IκBα水平。
Hela细胞体外实验中,缺氧可下调上调细胞内SUMO-1修饰的IκBα水平,且呈时间依赖性;反之,缺氧预处理可以上调细胞内SUMO-1修饰的IκBα水平。SAE-1过表达可以阻止缺氧时IκBα的磷酸化及降解。SAE-1过表达可以阻止缺氧时NF-кB由胞浆到胞核的移位。SAE-1过表达可以阻止缺氧时NF-кB转录活性的升高。SAE-1过表达可以阻止缺氧时IL-6 mRNA水平的升高。SUMO-1 shRNA可减弱缺氧预处理对NF-кB核移位的抑制。SUMO-1 shRNA可减弱缺氧预处理对NF-кB转录活性的抑SUMO-1 shRNA可减弱缺氧预处理对IL-6 mRNA水平的抑制。
结论
一、缺氧预处理可以上调SUMO-1修饰的IκBα水平。
二、SAE-1过表达可以阻止缺氧时IκBα的磷酸化及降解,进而抑制NF-кB炎症通路,起到类似于缺氧预处理的保护作用。
三、SUMO-1的沉默则可减弱缺氧预处理对NF-кB炎症通路的抑制,即削弱缺氧预处理的保护作用。简而言之,IκBα的SUMO化修饰在缺氧预处理对细胞内NF-кB炎症通路的抑制过程中起重要作用。
第三部分IκBα的SUMO化修饰受腺苷相关通路的调节
腺苷(Adenosine,Ado)是核苷(Nucleotide)的一种,除参与能量代谢外,还在细胞信号转导途径中的扮演重要角色:腺苷可与细胞膜上的受体结合,通过G蛋白,参与调节多种细胞内活动,包括对炎症反应的调节。
研究发现,腺苷与缺氧预处理(Hypoxia Preconditioning, HPC)关系密切。缺氧预处理是目前对缺氧病理生理机制研究的热点之一,主要指在进行缺氧处理以前,对研究对象给予几次时间较短,程度较轻的“低氧-复氧-低氧-复氧”预适应处理。缺氧预处理是一个复杂的,涉及多因素、多机制的保护过程,其中,对NF-кB介导的炎症反应的抑制是一个比较重要的环节。
缺氧预处理可以通过腺苷介导的Cullin-1 deneddylation,阻止IκBα的降解,稳定NF-κB,从而阻断其下游炎症反应。SUMO化修饰也可以阻止IκBα泛素化,从而抑制IκBα的降解,稳定NF-κB,减轻炎症反应。从细胞信号转导的角度来看,处于上游的腺苷对下游的NF-κB炎症通路的调节应该是多途径的。我们推测,IκBα的SUMO化修饰可能也是其中重要的一条,换言之,IκBα的SUMO化修饰可能也是腺苷介导的抗炎通路之一。
研究目的
本研究利用所在实验室已建立的缺氧及缺氧预处理动物及细胞模型,探讨IκBα的SUMO化修饰与腺苷通路的关系。
实验方法
一、细胞缺氧预处理模型:(方法同第一章)
二、动物缺氧预处理模型:(方法同第一章)
三、在Hela细胞模型中,利用腺苷拟似剂NECA及阻断剂8-PT对腺苷通路进行激活/阻断实验,然后与正常对照组进行以下对比研究:
1、正常氧环境下,激活/阻断腺苷通路后,SUMO-1-IκBα结合蛋白水平的变化(Western blot方法检测)。
2、缺氧环境下,激活/阻断腺苷通路后,SUMO-1-IκBα结合蛋白水平的变化(Western blot方法检测)。
四、过表达A2AR和A2BR,然后与野生型及阴性对照组比较SUMO-1-IκBα结合蛋白水平的变化。
五、动物实验中阻断腺苷通路:敲除小鼠CD73基因(内源性腺苷生成中的关键基因),然后进行缺氧预处理实验,对比研究,SUMO-1- IκBα结合蛋白水平在阻断前、后的变化。
六、在Hela细胞模型中,利用shRNA下调SUMO-1水平,然后进行缺氧预处理实验,双荧光素酶报告法分析检测NF-кB活性,对比研究SUMO-1水平变化对腺苷通路介导的抗炎作用的影响。
研究结果
在正常氧环境中,添加外源性腺苷可以上调细胞内SUMO-1- IκBα结合蛋白水平,且呈剂量依赖性。
在缺氧环境中,添加外源性腺苷仍然可以上调细胞内SUMO-1-IκBα结合蛋白水平,且呈剂量依赖性。
在正常氧和缺氧环境中,外源性腺苷上调细胞内SUMO-1- IκBα结合蛋白水平的作用均可被腺苷受体阻断剂8-PT抑制。
过表达A2AR和A2BR,可以上调细胞内SUMO-1-IκBα结合蛋白水平。
小鼠CD73基因敲除后(CD73-/-),SUMO-1-IκBα结合蛋白水平明显降低。
SUMO-1的沉默可以使外源性腺苷失去对NF-кB活性的抑制作用。
结论
通过以上研究,我们发现:IκBα的SUMO化修饰受到腺苷相关通路的调节。缺氧预处理可以通过腺苷相关通路,促进IκBα的SUMO化修饰,稳定NF-кB,并因此抑制缺氧时细胞内NF-кB介导的炎症反应。
Cyclic adenosine monophosphate(cAMP)-mediated pathway is one of the most important signal pathways. It was found might play a roll in the anti-inflammatory modification by hypoxia precondiaitoning (HPC), which is kown for its protection for cells in the following hypoixa procedure. During HPC, extra cellular adenosine (ADO) increases due to the chang of oxygen concentration, this may enhance the simulation to adenylyl cyclase (AC), which will lead to an increase of cAMP in turn. On the other hand, it was recently found that cAMP inhibits the activation of p38 mitogen-activated protein kinase (p38MAPK) via cAMP response element-binding protein (CREB)-induced dynein light chain (DLC). Taken together, we hypothesize that cAMP may be increased by HPC treatment, and inhibits the activation of p38MAPK, therefore, attenuates the NF-κB mediated inflammation. However, the mechnism relates cAMP to HPC is still unknown. In general, cAMP is produeced from 5’-AMP when AC is stimulated by some G-protein coupled receptors (GPCRs), such as ADO receptor 2A, 2B, and degrades to 5’-AMP when Phosphodiesterase (PDEs) is activated. To confirm our hypothesize, we studied the intracellular cAMP level after the treatment of HPC vesus hypoxia and normoxia control, and further tried to explore the mechnism responsible to this change.
Methods
Murine preconditioning model.
C57BL/6 (Charles River Laboratories) male mice of 8–10 weeks of age were used in this study. The Animal Care and Use Committee of Brigham and Women’s Hospital approved all procedures. Whole-body preconditioning was performed in a manner similar to that previously described. Briefly, mice were placed in humidified environmental chambers and subjected to Fi O2 8% for 10 minutes followed by Fi O2 21% for 10 minutes for 3 cycles. The O2 concentrations in the chambers were continuously measured by an O2 analyzer. Following preconditioning, mice were subjected to FiO2 21% for 120 minutes. Preconditioned and nonpreconditioned animals were subsequently placed in the chambers and subjected to FiO2 5% for 10 minutes. Animals were then removed from the chamber and sacrificed, and pulmonary tissue was collected. For each experiment, 3 animals were treated in triplicate.
Cell culture, and in vitro preconditioning.
HeLa cells (ATCC) were maintained in DMEM plus 10% FBS, Calu-3 cells (ATCC) in MEM plus 10%FBS, T84(ATCC) cells in a mixture of Ham’s F12 and DMEM (1:1), plus 10% FBS, HMEC cells in MCDB131 plus 10% FBS at 37_C in a humidified incubator with 5% CO2 in room air. Cellular preconditioning was performed on cells following a modified in vivo protocol optimized for cells. Hela or Calu-3 cells were placed in a hypoxia chamber (Coy Laboratory Products Inc.) in pre-equilibrated hypoxic media at 2% O2 for 45 minutes, then re-oxygenated in normoxic conditions (21% O2) for 20 minutes. When cells were returned to hypoxia, media was once again replaced with fresh hypoxic medium in order to minimize the effects of oxygen present. This protocol was followed for 3 cycles.
ELISA assay.
Calu-3 or T84 cells were plated in BD culture dishes (10 cm in diameter) and allowed to grow to approximately 70-80% confluence, treated in hypoxia chamber following the in vitro preconditioning protocol, then stimulated with 10uM adenosin for 30 minutes or not, cyclic AMP concentration of cell lysates were measured immediately using cAMP assay kit (R&D Systems, Inc. #KGE002) following steps instructed by the manufacturer: cells were lysed by cell lysis buffer, applied to the 96-well plates coated with goat anti-mouse antibody in triplicate, incubated at room temperature with primary antibody and cAMP conjugate on a orbital shaker at a speed of 500rpm for 3 hours, washed 3 times with wash buffer, optical density were then measured sequentially at 450nm and 570nm using a spectrophotometer (Bio-Rad). All reagents and the coated plates were provided in the kit.
Transfections and NF-κB reporter assays.
Transfection of Hela cells was carried out using Fugene 6 transfection reagent, with pNF-κB-Luc plasmid(pNRE; Clontech) at a concentration of 1.6μg per well along with 0.08μg of Renilla for 24 hours and then either left at normoxia or subjected to a protocol of preconditioning. Following treatments, cells were rinsed in PBS, lysed in passive lysis buffer (Promega) for 15 minutes, and spun down, and 20μl of lysates was assayed using the Dual-Glo Luciferase assay system (Promega) with the use of a luminometer (Turner BioSystems).
Transcriptional analysis.
cDNA was collected Using“Clonetech sprint powerscript”after extraction of total RNA. Real-time PCR (Taqman, Apllied Biosystems) was employed to examine PDE4B in Calu-3 cells and IL-6 expression in HeLa cells following the instructions from the manufacturer. PDE4B primers:forward 5’-ATGGGCAGATTTGGTACAGC-3’; reverse 5’–TAGCC GTCCAGAAATGGTTT-3’. Ready-to-use IL-6 primer was ordered from Applied Biosystems. Samples were controlled forβ-actin using the following primers: forward, 5'-GGTGGCTTTTAATGGCAAG-3'; reverse, 5'-ACTGGAACGGTGAAGGTGACAG-3'
Western blot.
Total protein was isolated from cells using cell lysis buffer (Cell signaling Technology, #9803), or extracted from lungs of mice with RIPA buffer (Cell signaling Technology, #9806), both containing protease inhibitor cocktail (Roche Diagnostics, #04693124001) and PMSF 1mM. Protein concentrations were measured by DC protein assay (Bio-Rad). An equal amount of protein was boiled in SDS loading buffer (Bio-Rad), then resolved on 8% polyacrylamide denaturing gels and transferred to nitrocellulose (Bio-Rad). After transfer, the membranes were stained with ponceau S stain in order to verify equal loading. Antibodies used for Western blotting included rabbit anti–PDE4B (1:1000; Abcam), rabbit anti-p38 and P-p38 (1:1000; Cell signaling Technology), rabbit anti-actin (1:2,000; Cell signaling Technology). Blots were washed, and species-matched peroxidase-conjugated secondary antibody was added. Labeled bands from washed blots were detected by enhanced chemiluminescence (Thermol Scientific).
Immunofluorescence study.
For p65 nuclear translocation studies, HeLa cells were plated on 4-well glass chamber slides (Nalgene Nunc International) and allowed to grow to approximately 50-70% confluence, treated in hypoxia chamber following in vitro preconditioning protocol. Cells were fixed in 1% paraformaldehyde/PBS at 4℃for 10 minutes and permeabilized with prechilled 0.2% Triton X-100/PBS/2% BSA. Cells were incubated with rabbit anti-p65 (1:200; Rockland Immunochemicals) in 1% normal goat serum in PBS for 1 hour followed by anti-rabbit Oregon Green 488 (1:100, Molecular Probes; Invitrogen) in the same buffer for 30 minutes. Cell images were captured on a fluorescence microscope.
Results
Intracellular cyclic AMP level was obviously lifted by HPC treatment
Initially, Calu-3 cells and T84 cells were treated in nomoxia, hypoxia and HPC respectively, right after the treatment, cyclic AMP was extracted using the lysis buffer provieded in the kit, then measured through ELISA assay (see Metholds for protocol). An increase of cyclic AMP was found in cells subjected to HPC, compared with hypoxia and normoxia groups (2-fold increase compared with normoxia control; P < 0.05), indicating cyclic AMP may play a roll in the anti-inflammatory modification caused by HPC treatment.
Recruitment ofβ-arrestin to lipid raft was blocked by HPC treatment
To define the mechnism accont for cyclic AMP increase, we first studied the recruitment ofβ-arrestin to lipid raft, which is reponsive to the desensitization of GPCRs, including A2AR and A2BR. Protein was extracted from HeLa cells had been treated in nomoxia, hypoxia or HPC, in two steps: first step, with lysis buffer containing triton x-100 to get cytoplasmic and most membrane proteins; second with lysis buffer containing OCG to extract lipid raft and nuclei proteins. Result of western blot assay revealed an sharp decrease ofβ-arrestin protein level in the lipid raft extraction of cells subjected to HPC, compared with nomoxia and hypoxia control groups, indicating the recruitment ofβ-arrestin to lipid raft was blocked by HPC. As an result, this may lead to an decrase of the desensitization of A2AR and A2BR, make the recptors“keeping open”, in turn, cells keep produce more cyclic AMP.
HPC treated cells has a higher efficency in producing cAMP when stimulated with ADO
To test if HPC affects the ability of cells to produce cAMP, after the HPC/Hypoxia/ Normoxia treatment, we stimulated cells with ADO for 30minutes, measured cAMP immediately. A higher increase of cAMP in HPC group after ADO stimulation was noticed, compared with other two groups, however, in hypoxia group, cAMP was not lifted, actualy, even lower than that of non-stimulated cells, confirming the hypothesis from the result ofβ-arrestin.
HPC treatment transciptionaly down-regulates PDE4B both in vitro and in vivo
Total protein of Calu-3、Hela、T84 and HMEC cells treated in either nomoxia, hypoxia or HPC were extracted, then, PDE4B, a well-known mediator of cyclic AMP degradation, was measured using western blot respectively. Consistent to the increase of cyclic AMP level, PDE4B was found to be much lower in HPC group compared with normoxia and hypoxia treated cells. Western blot assay of total protein of lung tissue from mice subjected to normoxia, hypoxia or HPC shows similar result with in vitro experiment. Further more, we measured the mRNA level of PDE4B in Calu-3 through RT-PCR, result shows a similar change with PDE4B protein, indicating the change is transcriptional. These data give us the information that the degradation of cyclic AMP was inhibited during HPC treatment.
HPC treatment inhibits p38 phosphoralytion both in vitro and in vivo
Sequentially, to confirm if HPC treatment can inhibit p38 phosphoralytion, which is thought to be a down-stream activity to cyclic AMP while up-stream to NF-κB translocation, we meseaured both p38 and phospho-p38 in cell whole lysates at the same time using western blot. In all 4 cell lines, phospho-p38 dropped dramatically to a lower level in HPC group compared with normoxia and hypoxia, wihle no difference of p38 was found, suggesting an inhibition of p38 avtivation caused by HPC treatment.
HPC treatment inhibits NF-κB activity
To define the functional attributes of NF-κB inhibition by HPC, we used an NF-κB luciferase reporter. HeLa cells were transfected pNRE-Luc vector followed by exposure to normoxia, hypoxia (2% O2, 24 hours), or HPC followed by hypoxia (see HPC protocol in Methods). These studies revealed that cells subjected to HPC displayed a significant attenuation of NF-κB activation compared with those exposed to hypoxia alone.
More over, we still visualized the inhibition of NF-κB by showing the translocated NF-κB in nucleis through immunoflurecence stain. Consistent to the luciferase data, Hela cells subjected to HPC has a much less NF-κB translocation rate than hypoxia control, while close to the low level of normoxia group.
HPC treatment inhibits IL-6 activity
To get more evidence of the inhibition of NF-κB inflammatory, we further meseaured the mRNA level of IL-6, which is a well-established reporter gene for NF-κB activation. As shown by the result, hypoxia was a strong stimulus for induction of IL-6 mRNA in Hela cells. Parallel examination in HeLa cells subjected to HPC revealed a loss of hypoxia-induced IL-6, thus confirming our findings from the NF-κB experiments.
Conclusions
In conclusion, HPC up-regulates intracellar cyclic AMP by both decreasing the desensitization of AC stimulators through inhibition on the cruitment ofβ-arrestin to lipid raft and blocking the degradation of cAMP through the transcriptional down-regulation PDE4B. Secondly, the lift of cyclic AMP plays a key roll in the anti-inflammatory activity through the cAMP- PKA-CREB-p38MAPK pathway during HPC treatment.
A SUMO-1 modified IκBαform has been described to actively participate in the regulation of NFκB metabolism. After proteosomal degradation of IκBα, an auto-regulatory loop consisting of transcriptional activation of IκBαgene and SUMO-1 modification of newly synthesized IκBαexists. The SUMOylated IκBαform is resistant to signal induced degradation consequently halting NFκB activation. We describe a physiologic environment by which rapid induction and deactivation of NFκB results in significant accumulation of SUMO-1 modified IκBα, with subsequent decreased NFκB mediated gene expression. Initially we demonstrated that the relative protein levels of IκBα/SUMO were significantly increased after hypoxia preconditioning (HPC). To confirm that in fact IκBα/SUMO control NFκB activation in the presence of hypoxia, assays targeting shRNA and over-expression of SUMO-1 were designed. With the use of a NF-κB-luciferase reporter plasmid, cells were co-transfected with shRNA plasmid and the full-length cDNA for SUMO-1. A significant increase in NF-κB activation was noted during hypoxia in shRNA-transfected cells, compared to wild type cells. Conversely, over-expression of SUMO-1 resulted in a significant reduction in NF-κB activation despite hypoxia. As proof of principle for the physiologic significance of SUMO-1 contribution to NF-κB activation, p65 subunit in nucleis and IL-6 mRNA levels of wild type compared to shRNA and over-expressing cells were also studied. In summary, we present an endogenous mechanism by which cells acquire anti-inflammatory properties by recruiting a non-degradable form of IκBα, a major control point for NF-κB activation.
Methods:
Murine model.
(See part one)
Cell culture, in vitro hypoxia and chemical treatments
HeLa cells (ATCC) were maintained in DMEM plus 10% FBS, at 37℃in a humidified incubator with 5% CO2 in room air. Cellular preconditioning was performed on cells following a modified in vivo protocol optimized for cells (See part one)
Transfections and NF-κB reporter assays.
Transfection of Hela cells was carried out using Fugene 6 transfection reagent (Roche Diagnostics) as directed by the manufacturer. Plasmids used in transfections were SAE-1(SUMO activating enzyme-1, SAE-1) plasmid, shRNA for SUMO-1 and control plasmids (Origen). On the second day of the transfection, HeLa cells in 6-well plates were transfected for the second time with pNF-κB-Luc (pNRE; Clontech) at a concentration of 1.6μg per well along with 0.08μg of Renilla for 24 hours and then either left at normoxia or subjected to a protocol of hypoxia preconditioning. Following treatments: cells were rinsed in PBS, lysed in passive lysis buffer (Promega) for 15 minutes, and spun down, and 20μl of lysates was assayed using the Dual-Glo Luciferase assay system (Promega) with the use of a luminometer (Turner BioSystems).
Transcriptional analysis.
cDNA was collected Using“Clonetech sprint powerscript”after extraction of total RNA. Real-time PCR (Taq-man) was employed to examine IL-6 expression levels in HeLa cells. IL-6 Primer was ready-to-use, ordered from Applied Biosystems. Samples were controlled forβ-actin using the following primers: sense , 5'-GGTGGCTTTTAGGATGGCAA G-3'; antisense , 5'-ACTGGAACGGTGA AGGTGACAG-3'; 162 bp).
Western blot assay
(See part one for method)
Antibodies used for Western blotting included rabbit anti-SUMO-1 (1:1000), rabbit anti–IκBα(1:1000), rabbit anti–Phospho-IκBα(1:1000), all from Cell Signaling Technology, Inc. Blots were washed, and species-matched peroxidase-conjugated secondary antibody was added. Labeled bands from washed blots were detected by enhanced chemiluminescence (Thermol Scientific).
Immunoprecipitation.
Total protein was isolated from HeLa cells and measured as described above. After pre-cleared with 60ul of Immobilized Protein A/G (Thermol Scientific) for 2 hours, rabbit anti-SUMO-1 antibody or normal rabbit serum (Sigma-Aldrich) were added into the protein, gently shaked for overnight. 60ul of Immobilized Protein A/G were added into each tube, incubated for 2 hours, centrifuged at 7500rpm for 4 minutes, complex-bound gel were then collected and washed with cell lysis buffer for 3 times. Finally, 100ul of 2% SDS loading buffer was added into each tube, boiled at 95℃for 5 minutes, centrifuged at 14000rpm for 30 seconds, loaded to 12% gel and followed western blot steps mentioned above.
Results
HPC-induced SUMO-1 modification prevents phosphorylation of IκBαduring hypoxia
SUMO-1 modification prevents phosphorylation and, in turn degradation of IκBα, therefore helps to stabilize NF-κB p65/p50 subunits. This has been demonstrated in inflammatory model induced by TNF-α. However, does it work in other models, for example, hypoxia-induced inflammation? In our study, Hela cells were subjected to normoxia, hypoxia or HPC treatment for 3 cycles, at the end of each cycle; proteins were extracted for western blot assay. Result shows a decrease of IκBαalong with the dropping of SUMO-1-conjugated IκBαin hypoxia, while in HPC, when SUMO-1-conjugated IκBαgoes up, IκBαstops to decrease as well. Furthermore, HeLa cells transfected with SAE-1 plasmid or control vector were subjected to hypoxia treatment for 3-24 hours, western assay shows: IκBαand SUMO-1-conjugated IκBαdecreased dramatically in wild type cells while is stable in SUMO-1 over-expressed cells; phospho-IκBαincreased in wild type cells while kept low in SUMO-1 over-expressed cells; Iκκβ, no significant change in both groups. From above experiments, we can draw the conclusion that SUMO-1 modification prevents phosphorylation of IκBαduring hypoxia.
HPC-induced SUMO-1 modification of IκBαinhibits NF-κB mediated inflammatory during hypoxia
To confirm if the SUMO-1 modification of IκBαinhibits NF-κB mediated inflammatory induced by hypoxia, we first used the luciferise reporter assay to measure NF-κB activity and its downstream changes related to inflammatory. HeLa cells were transfected with either SAE-1 plasmid or SUMO-1 shRNA, subjected to normoxia; hypoxia or HPC treatment followed by hypoxia, and then measured NF-κB activity by luciferise reporter assay. As we can see, cells transfected with SUMO-1 plasmid showed a significant (P < 0.05, SAE-1 versus control vector) decrease in NF-κB activity, meanwhile, in HPC group, those had been transfected with shRNA for SUMO-1 showed an increase of NF-κB activity (P < 0.05, SUMO-1 shRNA versus control vector).
Moreover, we still analyzed p65 protein, which reflects the translocation rate of NF-κB into nuclei, in the nuclear lysates extracted from Hela cells in parallel experiments. Data shows a similar pattern with luciferase assay: in SUMO-1 over-expressing cells, nuclear p65 was decreased despite of hypoxia treatment; while in shRNA group, HPC failed to inhibit the increase of nuclear p65.
Finally, IL-6, which is the downstream target of NF-κB in the inflammatory chain, was meseaured at transcriptional level by using Real Time-PCR. The result is consistent to the change of NF-κB activity.
Taken together, these changes indicate NF-κB mediated inflammation after hypoxia treatment can be inhibited by HPC induced SUMO-1 modification of IκBα.
Conclusions
In summary, we present an endogenous mechanism by which cells acquire anti-inflammatory properties by recruiting a non-degradable form of IκBα, a major control point for NF-κB activation
Adenosine (ADO) signaling has been found to play an key roll in hypoxia preconditioning (HPC), which enhancs cell anti-infalammatory ability. In our previous work, SUMO-1 modified IκBαform has been demonstrated to actively participate in the inhibition of NFκB activation. Using models of HPC, we have found a cyclic dependent increase in extra cellular Adenosine and theorize that the increase in cytoplasmic pool of IκBα/SUMO is mediated by Adenosine signaling, just like the adenosine–mediated cullin-1 deneddylation. Initially, We stimulated the ADO signaling pathway both in normoxia and hypoxia with the treatment of general Adenosine receptor agonist NECA, found an increase of IκBα/SUMO protein by western blot assay. In turn, when the stimulation was dampened by the general ADO receptor antagonist---8-PT, the increase was blocked, confirming the effect of ADO on the up-regulation of IκBα/SUMO. Further, functional evaluation of the effect was done by using luciferase assay on NFκB activity after genetically knocked SUMO-1 down by shRNA and stimualated with NECA. Result shows a loss of inhibition on NFκB activity after NECA stimulation when SUMO-1 had been knocked down, indicating SUMO-1 is crucial in the ADO pathway. More over, in cells over-expressing A2AR or A2BR have a higher level of IκBα/SUMO. Finally, in vivo experiments were conducted with cd73-knocked out (cd73-/-) mice, which were lack of ability to generate endogenous ADO. Mice were subjected to normoxia/hypoxia/HPC, and then total proteins of mice lung were analyzed by western blot. A signifcantly drop of IκBα/SUMO was found in cd73-/- mice compared with wild type mice. It is more important that, for cd73-/- mice, there was no increase of IκBα/SUMO in HPC group over normoxia and hypoxia groups, indicating the block the endogenous ADO pathway caused the failure for HPC to increas IκBα/SUMO, in another word, ADO is critical in the regulation of IκBα/SUMO.
Methods
Murine model.
CD73-/- mouse model was generated as described in our previous work. C57BL/6 (Charles River Laboratories) male mice of 8–10 weeks of age were used as control in this study. The Animal Care and Use Committee of Brigham and Women’s Hospital approved all procedures. Whole-body preconditioning was performed in a manner similar to that previously described. (See part one)
Cell culture, in vitro hypoxia and chemical treatments
HeLa cells (ATCC) were maintained in DMEM plus 10% FBS, at 37℃in a humidified incubator with 5% CO2 in room air. Cellular preconditioning was performed on cells following a modified in vivo protocol optimized for cells (See part one). Cells were treated with NECA in DMSO at a rang from 100nM to 100μM or pretreatment with 8-phenyltheophylline (8-PT) in ddH2O at concentration of 100nM. Treatment time is indicated in results.
Transfections and NF-κB reporter assays.
Transfection of Hela cells was carried out using Fugene 6 transfection reagent (Roche Diagnostics) as directed by the manufacturer. Plasmids used in transfections SUMO-1 shRNA or control plasmids (Origen). On the second day of the transfection, HeLa cells were transfected for the second time with pNF-κB-Luc (See part one) for 24 hours and then treated with NECA for 30 minutes. Following treatments: cells were rinsed in PBS, lysed in passive lysis buffer (Promega) for 15 minutes, and spun down, and 20μl of lysates was assayed using the Dual-Glo Luciferase assay system (Promega) with the use of a luminometer (Turner BioSystems).
Western blot assay
(See part two)
Results
Adenosine regulates SUMO-1 modification of IκBαin vitro
To define the functional attributes of SUMO-1 modification of IκB by Ado signaling, NECA stimulation on hela cells was conducted to get direct evidence of Ado’s up-regulation on SUMO-1 modification of IκB . Data showed a dose-dependent increase of IκBα/SUMO after hela cells were treated with NECA for about 30 minutes in normoxia, expectedly, this increase can be blocked by pre-treatment with 8-PT, a general inhibitor for Ado receptors. Not only in normal condition, but also in hypoxia, a similar pattern of the change of IκBα-SUMO after cells was treated with NECA or 8-PT/NECA was noticed. More over, when transfected with A2AR or A2BR, a higher of IκBα-SUMO was found compared with wild type cells, confirming above results that ADO pathway was involved in regulation of SUMO-1 modification of IκB.
SUMO-1 modification of IκBαis mediated by Ado in mouse models
To make clear if SUMO-1 modification of IκBαis mediated by Ado, we used a CD73 (-/-) mouse model which lacks the ability to generate Ado endogenously in our study. Wild type C57BL/6 mice were used as control. Both types of mice were subjected to normoxia, hypoxia or HPC treatment, total protein of mice lung was analyzed by western blot. SUMO-1-conjugated IκB in both groups shows a similar pattern with cell model. Down-regulated in hypoxia, while up-regulated in HPC treated mice. What is more important, in CD73 (-/-) mice, SUMO-1-conjugated IκB level is much less than that in wild type mice, indicating Ado is crucial in SUMO-1 modification of IκBα. Conclusions
In summary, we demonstrated that ADO signaling mediateds SUMO-1 modificaion of IκBα, which in turn induces anti-inflammatory properties by recruiting a non-degradable form of IκBα, a major control point for NFκB activation.
缺氧预处理(Hypoxia Preconditioning, HPC)是近年对缺氧病理生理机制研究比较集中的热点之一。目前,缺氧预处理被认为是一个复杂的,涉及多因素、多机制的保护过程。
最近有研究提示环磷酸腺苷(cyclic adenosine monophosphate, cAMP)与缺氧预处理时细胞内信号转导密切相关。cAMP是细胞内普遍存在的第二信使,参与调节细胞的许多活动。细胞内的cAMP处于一个动态平衡,胞外刺激可以通过细胞膜上G蛋白耦联受体(G protein coupled recptors, GPCRs)如肾上腺素受体、腺苷受体(A2A及A2B受体)等激活腺苷酸环化酶(adenylyl cyclase, AC),催化5’-AMP环化形成cAMP;一旦cAMP达到一定的水平,负反馈机制便自动启动,主要通过两条途径:一是通过β-arrestin使GPCRs失活,减少cAMP的生成,二是通过磷酸二酯酶(PDEs)的作用将其降解为5’-AMP。
研究目的
本研究利用所在实验室已建立的缺氧预处理动物及细胞模型,探讨:1.缺氧预处理对细胞内cAMP水平的调节机制;2.cAMP信号通路参与缺氧预处理抗炎作用的机制。
实验方法和材料
一、细胞缺氧预处理模型:
细胞置于低氧密闭容器(2%O2)中缺氧45分钟,取出于正常氧环境(21%O2)中复氧20分钟,此为一个循环,重复3个循环。单纯缺氧组一直置于低氧密闭容器(2%O2)中,正常对照组则一直置于正常氧环境(21%O2)中。
二、动物缺氧预处理模型:
C57BL/6小鼠或CD73基因缺陷小鼠随机分成3组,分别是正常对照组、单纯缺氧组和缺氧预处理组。缺氧预处理组小鼠被放置于可调节氧浓度的湿化密闭容器内,调节氧浓度到8%,维持10分钟,然后氧浓度转换为21%持续10min,如此循环3次。然后取出,置于常氧环境中2小时;单纯缺氧组小鼠也被放置同种容器内,调节氧浓度到8%,维持1小时,取出,置于常氧环境中2小时;正常对照组小鼠在正常氧环境中(氧浓度21%),不作任何处理。随后各组的小鼠均被置于容器内,调节氧浓度到5%,维持10分钟,令其死亡,手术取出肺组织,立即投入液氮快速冷冻,-80℃冰箱保存备用。
三、缺氧预处理对cAMP水平及效应的调节:
1、ELISA法检测细胞内cAMP水平
2、Western blot方法检测β-arrestin、PDE4B、p38MAPK等蛋白表达水平的变化
①细胞总蛋白提取法:常规总蛋白裂解液提取
②细胞蛋白分步提取法(两步法):第一步提取胞浆/胞膜蛋白;第二步提取脂质体/核蛋白
3、双荧光素酶报告分析(Dual luciferase reporter assay)检测NF-кB活性:将构建有luciferase reporter的NFкB质粒转染入Hela细胞,在缺氧预处理后,继续缺氧24小时,然后检测Luciferase强度,即NFкB活性。
4、RT-PCR检测PDE4B、白介素-6(IL-6)的mRNA水平
5、免疫荧光染色检测NFкB的核移位情况
研究结果
一、缺氧预处理可以明显提高Calu-3细胞和T84细胞内cAMP水平:ELISA检测显示,与单纯缺氧组和正常氧环境组比,缺氧预处理组cAMP水平明显提高。
二、缺氧预处理不影响细胞内β-arrestin蛋白总量,但可使位于脂质体上的“有效”β-arrestin减少,阻止GPCRs的失活,从而使cAMP生成增多。
三、缺氧预处理可降低PDE4B的转录及表达水平,减少cAMP的降解。
四、缺氧预处理能抑制cAMP-PKA-CREB通路下游的p38MAPK的磷酸化。
五、缺氧预处理能抑制NF-кB的核移位。
六、缺氧预处理能抑制NF-кB的转录活性。
七、缺氧预处理能阻止IL-6 mRNA水平的升高。
结论
一、缺氧预处理可提高细胞内cAMP水平:缺氧预处理一方面可以通过细胞外刺激信号如腺苷酸等的增强,激活腺苷酸环化酶;另一方面可通过对β-arrestin和PDE4B的调节,抑制负反馈途径,共同促进细胞内cAMP的提升。
二、缺氧预处理对cAMP的提升,可通过cAMP-PKA-CREB-p38MAPK通路,抑制NF-кB介导的炎症反应。
第二部分IκBα的SUMO化修饰参与HPC对NF-κB炎症通路的抑制
SUMO化修饰是蛋白质表达后修饰的一种方式,是指小泛素相关修饰物(small ubiquitin-ralated modifier,SUMO)共价结合于靶蛋白的赖氨酸残基上。这个过程类似但又不同于泛素化,与泛素介导蛋白质的降解不同,SUMO化修饰的蛋白质更加稳定。SUMO参与了广泛的细胞内代谢途径,在蛋白-蛋白之间的相互作用、信号转导、核质运输、转录调控等方面均发挥着重要的作用。
缺氧预处理是目前对缺氧病理生理机制研究的热点之一,主要指在进行缺氧处理以前,对研究对象给予几次时间较短,程度较轻的“低氧-复氧-低氧-复氧”预适应处理。缺氧预处理是一个复杂的,涉及多因素、多机制的保护过程,其中,对NF-кB介导的炎症反应的抑制是一个比较重要的环节。
有研究发现SUMO化修饰可阻止IκBα泛素化,从而抑制IκBα的降解,稳定NF-κB,减轻炎症反应。我们推测,IκBα的SUMO化修饰可能参与缺氧预处理对NF-κB炎症通路的抑制。
研究目的
本研究利用所在实验室已建立的缺氧及缺氧预处理动物及细胞模型,探讨IκBα的SUMO化修饰是否参与缺氧预处理对NF-κB炎症通路的抑制。
实验方法
一、细胞缺氧预处理模型:(方法同第一章)
二、动物缺氧预处理模型:(方法同第一章)
三、在Hela细胞模型中,利用SAE-1过表达上调SUMO-1水平,然后进行缺氧预处理实验,与正常对照组对比研究:
1、Western blot方法检测IκBα、SUMO-1-IκBα、pIκBα等蛋白表达的变化。
2、双荧光素酶报告法检测NF-кB活性。
3、RT-PCR检测IL-6 mRNA水平。
四、在Hela细胞模型中,利用shRNA下调SUMO-1水平,然后进行缺氧预处理实验,与正常组对比研究:
1、双荧光素酶报告法分析检测NF-кB活性。
2、RT-PCR检测IL-6 mRNA水平
研究结果
小鼠模型中,缺氧预处理可以上调肺组织内SUMO-1修饰的IκBα水平。
Hela细胞体外实验中,缺氧可下调上调细胞内SUMO-1修饰的IκBα水平,且呈时间依赖性;反之,缺氧预处理可以上调细胞内SUMO-1修饰的IκBα水平。SAE-1过表达可以阻止缺氧时IκBα的磷酸化及降解。SAE-1过表达可以阻止缺氧时NF-кB由胞浆到胞核的移位。SAE-1过表达可以阻止缺氧时NF-кB转录活性的升高。SAE-1过表达可以阻止缺氧时IL-6 mRNA水平的升高。SUMO-1 shRNA可减弱缺氧预处理对NF-кB核移位的抑制。SUMO-1 shRNA可减弱缺氧预处理对NF-кB转录活性的抑SUMO-1 shRNA可减弱缺氧预处理对IL-6 mRNA水平的抑制。
结论
一、缺氧预处理可以上调SUMO-1修饰的IκBα水平。
二、SAE-1过表达可以阻止缺氧时IκBα的磷酸化及降解,进而抑制NF-кB炎症通路,起到类似于缺氧预处理的保护作用。
三、SUMO-1的沉默则可减弱缺氧预处理对NF-кB炎症通路的抑制,即削弱缺氧预处理的保护作用。简而言之,IκBα的SUMO化修饰在缺氧预处理对细胞内NF-кB炎症通路的抑制过程中起重要作用。
第三部分IκBα的SUMO化修饰受腺苷相关通路的调节
腺苷(Adenosine,Ado)是核苷(Nucleotide)的一种,除参与能量代谢外,还在细胞信号转导途径中的扮演重要角色:腺苷可与细胞膜上的受体结合,通过G蛋白,参与调节多种细胞内活动,包括对炎症反应的调节。
研究发现,腺苷与缺氧预处理(Hypoxia Preconditioning, HPC)关系密切。缺氧预处理是目前对缺氧病理生理机制研究的热点之一,主要指在进行缺氧处理以前,对研究对象给予几次时间较短,程度较轻的“低氧-复氧-低氧-复氧”预适应处理。缺氧预处理是一个复杂的,涉及多因素、多机制的保护过程,其中,对NF-кB介导的炎症反应的抑制是一个比较重要的环节。
缺氧预处理可以通过腺苷介导的Cullin-1 deneddylation,阻止IκBα的降解,稳定NF-κB,从而阻断其下游炎症反应。SUMO化修饰也可以阻止IκBα泛素化,从而抑制IκBα的降解,稳定NF-κB,减轻炎症反应。从细胞信号转导的角度来看,处于上游的腺苷对下游的NF-κB炎症通路的调节应该是多途径的。我们推测,IκBα的SUMO化修饰可能也是其中重要的一条,换言之,IκBα的SUMO化修饰可能也是腺苷介导的抗炎通路之一。
研究目的
本研究利用所在实验室已建立的缺氧及缺氧预处理动物及细胞模型,探讨IκBα的SUMO化修饰与腺苷通路的关系。
实验方法
一、细胞缺氧预处理模型:(方法同第一章)
二、动物缺氧预处理模型:(方法同第一章)
三、在Hela细胞模型中,利用腺苷拟似剂NECA及阻断剂8-PT对腺苷通路进行激活/阻断实验,然后与正常对照组进行以下对比研究:
1、正常氧环境下,激活/阻断腺苷通路后,SUMO-1-IκBα结合蛋白水平的变化(Western blot方法检测)。
2、缺氧环境下,激活/阻断腺苷通路后,SUMO-1-IκBα结合蛋白水平的变化(Western blot方法检测)。
四、过表达A2AR和A2BR,然后与野生型及阴性对照组比较SUMO-1-IκBα结合蛋白水平的变化。
五、动物实验中阻断腺苷通路:敲除小鼠CD73基因(内源性腺苷生成中的关键基因),然后进行缺氧预处理实验,对比研究,SUMO-1- IκBα结合蛋白水平在阻断前、后的变化。
六、在Hela细胞模型中,利用shRNA下调SUMO-1水平,然后进行缺氧预处理实验,双荧光素酶报告法分析检测NF-кB活性,对比研究SUMO-1水平变化对腺苷通路介导的抗炎作用的影响。
研究结果
在正常氧环境中,添加外源性腺苷可以上调细胞内SUMO-1- IκBα结合蛋白水平,且呈剂量依赖性。
在缺氧环境中,添加外源性腺苷仍然可以上调细胞内SUMO-1-IκBα结合蛋白水平,且呈剂量依赖性。
在正常氧和缺氧环境中,外源性腺苷上调细胞内SUMO-1- IκBα结合蛋白水平的作用均可被腺苷受体阻断剂8-PT抑制。
过表达A2AR和A2BR,可以上调细胞内SUMO-1-IκBα结合蛋白水平。
小鼠CD73基因敲除后(CD73-/-),SUMO-1-IκBα结合蛋白水平明显降低。
SUMO-1的沉默可以使外源性腺苷失去对NF-кB活性的抑制作用。
结论
通过以上研究,我们发现:IκBα的SUMO化修饰受到腺苷相关通路的调节。缺氧预处理可以通过腺苷相关通路,促进IκBα的SUMO化修饰,稳定NF-кB,并因此抑制缺氧时细胞内NF-кB介导的炎症反应。
Cyclic adenosine monophosphate(cAMP)-mediated pathway is one of the most important signal pathways. It was found might play a roll in the anti-inflammatory modification by hypoxia precondiaitoning (HPC), which is kown for its protection for cells in the following hypoixa procedure. During HPC, extra cellular adenosine (ADO) increases due to the chang of oxygen concentration, this may enhance the simulation to adenylyl cyclase (AC), which will lead to an increase of cAMP in turn. On the other hand, it was recently found that cAMP inhibits the activation of p38 mitogen-activated protein kinase (p38MAPK) via cAMP response element-binding protein (CREB)-induced dynein light chain (DLC). Taken together, we hypothesize that cAMP may be increased by HPC treatment, and inhibits the activation of p38MAPK, therefore, attenuates the NF-κB mediated inflammation. However, the mechnism relates cAMP to HPC is still unknown. In general, cAMP is produeced from 5’-AMP when AC is stimulated by some G-protein coupled receptors (GPCRs), such as ADO receptor 2A, 2B, and degrades to 5’-AMP when Phosphodiesterase (PDEs) is activated. To confirm our hypothesize, we studied the intracellular cAMP level after the treatment of HPC vesus hypoxia and normoxia control, and further tried to explore the mechnism responsible to this change.
Methods
Murine preconditioning model.
C57BL/6 (Charles River Laboratories) male mice of 8–10 weeks of age were used in this study. The Animal Care and Use Committee of Brigham and Women’s Hospital approved all procedures. Whole-body preconditioning was performed in a manner similar to that previously described. Briefly, mice were placed in humidified environmental chambers and subjected to Fi O2 8% for 10 minutes followed by Fi O2 21% for 10 minutes for 3 cycles. The O2 concentrations in the chambers were continuously measured by an O2 analyzer. Following preconditioning, mice were subjected to FiO2 21% for 120 minutes. Preconditioned and nonpreconditioned animals were subsequently placed in the chambers and subjected to FiO2 5% for 10 minutes. Animals were then removed from the chamber and sacrificed, and pulmonary tissue was collected. For each experiment, 3 animals were treated in triplicate.
Cell culture, and in vitro preconditioning.
HeLa cells (ATCC) were maintained in DMEM plus 10% FBS, Calu-3 cells (ATCC) in MEM plus 10%FBS, T84(ATCC) cells in a mixture of Ham’s F12 and DMEM (1:1), plus 10% FBS, HMEC cells in MCDB131 plus 10% FBS at 37_C in a humidified incubator with 5% CO2 in room air. Cellular preconditioning was performed on cells following a modified in vivo protocol optimized for cells. Hela or Calu-3 cells were placed in a hypoxia chamber (Coy Laboratory Products Inc.) in pre-equilibrated hypoxic media at 2% O2 for 45 minutes, then re-oxygenated in normoxic conditions (21% O2) for 20 minutes. When cells were returned to hypoxia, media was once again replaced with fresh hypoxic medium in order to minimize the effects of oxygen present. This protocol was followed for 3 cycles.
ELISA assay.
Calu-3 or T84 cells were plated in BD culture dishes (10 cm in diameter) and allowed to grow to approximately 70-80% confluence, treated in hypoxia chamber following the in vitro preconditioning protocol, then stimulated with 10uM adenosin for 30 minutes or not, cyclic AMP concentration of cell lysates were measured immediately using cAMP assay kit (R&D Systems, Inc. #KGE002) following steps instructed by the manufacturer: cells were lysed by cell lysis buffer, applied to the 96-well plates coated with goat anti-mouse antibody in triplicate, incubated at room temperature with primary antibody and cAMP conjugate on a orbital shaker at a speed of 500rpm for 3 hours, washed 3 times with wash buffer, optical density were then measured sequentially at 450nm and 570nm using a spectrophotometer (Bio-Rad). All reagents and the coated plates were provided in the kit.
Transfections and NF-κB reporter assays.
Transfection of Hela cells was carried out using Fugene 6 transfection reagent, with pNF-κB-Luc plasmid(pNRE; Clontech) at a concentration of 1.6μg per well along with 0.08μg of Renilla for 24 hours and then either left at normoxia or subjected to a protocol of preconditioning. Following treatments, cells were rinsed in PBS, lysed in passive lysis buffer (Promega) for 15 minutes, and spun down, and 20μl of lysates was assayed using the Dual-Glo Luciferase assay system (Promega) with the use of a luminometer (Turner BioSystems).
Transcriptional analysis.
cDNA was collected Using“Clonetech sprint powerscript”after extraction of total RNA. Real-time PCR (Taqman, Apllied Biosystems) was employed to examine PDE4B in Calu-3 cells and IL-6 expression in HeLa cells following the instructions from the manufacturer. PDE4B primers:forward 5’-ATGGGCAGATTTGGTACAGC-3’; reverse 5’–TAGCC GTCCAGAAATGGTTT-3’. Ready-to-use IL-6 primer was ordered from Applied Biosystems. Samples were controlled forβ-actin using the following primers: forward, 5'-GGTGGCTTTTAATGGCAAG-3'; reverse, 5'-ACTGGAACGGTGAAGGTGACAG-3'
Western blot.
Total protein was isolated from cells using cell lysis buffer (Cell signaling Technology, #9803), or extracted from lungs of mice with RIPA buffer (Cell signaling Technology, #9806), both containing protease inhibitor cocktail (Roche Diagnostics, #04693124001) and PMSF 1mM. Protein concentrations were measured by DC protein assay (Bio-Rad). An equal amount of protein was boiled in SDS loading buffer (Bio-Rad), then resolved on 8% polyacrylamide denaturing gels and transferred to nitrocellulose (Bio-Rad). After transfer, the membranes were stained with ponceau S stain in order to verify equal loading. Antibodies used for Western blotting included rabbit anti–PDE4B (1:1000; Abcam), rabbit anti-p38 and P-p38 (1:1000; Cell signaling Technology), rabbit anti-actin (1:2,000; Cell signaling Technology). Blots were washed, and species-matched peroxidase-conjugated secondary antibody was added. Labeled bands from washed blots were detected by enhanced chemiluminescence (Thermol Scientific).
Immunofluorescence study.
For p65 nuclear translocation studies, HeLa cells were plated on 4-well glass chamber slides (Nalgene Nunc International) and allowed to grow to approximately 50-70% confluence, treated in hypoxia chamber following in vitro preconditioning protocol. Cells were fixed in 1% paraformaldehyde/PBS at 4℃for 10 minutes and permeabilized with prechilled 0.2% Triton X-100/PBS/2% BSA. Cells were incubated with rabbit anti-p65 (1:200; Rockland Immunochemicals) in 1% normal goat serum in PBS for 1 hour followed by anti-rabbit Oregon Green 488 (1:100, Molecular Probes; Invitrogen) in the same buffer for 30 minutes. Cell images were captured on a fluorescence microscope.
Results
Intracellular cyclic AMP level was obviously lifted by HPC treatment
Initially, Calu-3 cells and T84 cells were treated in nomoxia, hypoxia and HPC respectively, right after the treatment, cyclic AMP was extracted using the lysis buffer provieded in the kit, then measured through ELISA assay (see Metholds for protocol). An increase of cyclic AMP was found in cells subjected to HPC, compared with hypoxia and normoxia groups (2-fold increase compared with normoxia control; P < 0.05), indicating cyclic AMP may play a roll in the anti-inflammatory modification caused by HPC treatment.
Recruitment ofβ-arrestin to lipid raft was blocked by HPC treatment
To define the mechnism accont for cyclic AMP increase, we first studied the recruitment ofβ-arrestin to lipid raft, which is reponsive to the desensitization of GPCRs, including A2AR and A2BR. Protein was extracted from HeLa cells had been treated in nomoxia, hypoxia or HPC, in two steps: first step, with lysis buffer containing triton x-100 to get cytoplasmic and most membrane proteins; second with lysis buffer containing OCG to extract lipid raft and nuclei proteins. Result of western blot assay revealed an sharp decrease ofβ-arrestin protein level in the lipid raft extraction of cells subjected to HPC, compared with nomoxia and hypoxia control groups, indicating the recruitment ofβ-arrestin to lipid raft was blocked by HPC. As an result, this may lead to an decrase of the desensitization of A2AR and A2BR, make the recptors“keeping open”, in turn, cells keep produce more cyclic AMP.
HPC treated cells has a higher efficency in producing cAMP when stimulated with ADO
To test if HPC affects the ability of cells to produce cAMP, after the HPC/Hypoxia/ Normoxia treatment, we stimulated cells with ADO for 30minutes, measured cAMP immediately. A higher increase of cAMP in HPC group after ADO stimulation was noticed, compared with other two groups, however, in hypoxia group, cAMP was not lifted, actualy, even lower than that of non-stimulated cells, confirming the hypothesis from the result ofβ-arrestin.
HPC treatment transciptionaly down-regulates PDE4B both in vitro and in vivo
Total protein of Calu-3、Hela、T84 and HMEC cells treated in either nomoxia, hypoxia or HPC were extracted, then, PDE4B, a well-known mediator of cyclic AMP degradation, was measured using western blot respectively. Consistent to the increase of cyclic AMP level, PDE4B was found to be much lower in HPC group compared with normoxia and hypoxia treated cells. Western blot assay of total protein of lung tissue from mice subjected to normoxia, hypoxia or HPC shows similar result with in vitro experiment. Further more, we measured the mRNA level of PDE4B in Calu-3 through RT-PCR, result shows a similar change with PDE4B protein, indicating the change is transcriptional. These data give us the information that the degradation of cyclic AMP was inhibited during HPC treatment.
HPC treatment inhibits p38 phosphoralytion both in vitro and in vivo
Sequentially, to confirm if HPC treatment can inhibit p38 phosphoralytion, which is thought to be a down-stream activity to cyclic AMP while up-stream to NF-κB translocation, we meseaured both p38 and phospho-p38 in cell whole lysates at the same time using western blot. In all 4 cell lines, phospho-p38 dropped dramatically to a lower level in HPC group compared with normoxia and hypoxia, wihle no difference of p38 was found, suggesting an inhibition of p38 avtivation caused by HPC treatment.
HPC treatment inhibits NF-κB activity
To define the functional attributes of NF-κB inhibition by HPC, we used an NF-κB luciferase reporter. HeLa cells were transfected pNRE-Luc vector followed by exposure to normoxia, hypoxia (2% O2, 24 hours), or HPC followed by hypoxia (see HPC protocol in Methods). These studies revealed that cells subjected to HPC displayed a significant attenuation of NF-κB activation compared with those exposed to hypoxia alone.
More over, we still visualized the inhibition of NF-κB by showing the translocated NF-κB in nucleis through immunoflurecence stain. Consistent to the luciferase data, Hela cells subjected to HPC has a much less NF-κB translocation rate than hypoxia control, while close to the low level of normoxia group.
HPC treatment inhibits IL-6 activity
To get more evidence of the inhibition of NF-κB inflammatory, we further meseaured the mRNA level of IL-6, which is a well-established reporter gene for NF-κB activation. As shown by the result, hypoxia was a strong stimulus for induction of IL-6 mRNA in Hela cells. Parallel examination in HeLa cells subjected to HPC revealed a loss of hypoxia-induced IL-6, thus confirming our findings from the NF-κB experiments.
Conclusions
In conclusion, HPC up-regulates intracellar cyclic AMP by both decreasing the desensitization of AC stimulators through inhibition on the cruitment ofβ-arrestin to lipid raft and blocking the degradation of cAMP through the transcriptional down-regulation PDE4B. Secondly, the lift of cyclic AMP plays a key roll in the anti-inflammatory activity through the cAMP- PKA-CREB-p38MAPK pathway during HPC treatment.
A SUMO-1 modified IκBαform has been described to actively participate in the regulation of NFκB metabolism. After proteosomal degradation of IκBα, an auto-regulatory loop consisting of transcriptional activation of IκBαgene and SUMO-1 modification of newly synthesized IκBαexists. The SUMOylated IκBαform is resistant to signal induced degradation consequently halting NFκB activation. We describe a physiologic environment by which rapid induction and deactivation of NFκB results in significant accumulation of SUMO-1 modified IκBα, with subsequent decreased NFκB mediated gene expression. Initially we demonstrated that the relative protein levels of IκBα/SUMO were significantly increased after hypoxia preconditioning (HPC). To confirm that in fact IκBα/SUMO control NFκB activation in the presence of hypoxia, assays targeting shRNA and over-expression of SUMO-1 were designed. With the use of a NF-κB-luciferase reporter plasmid, cells were co-transfected with shRNA plasmid and the full-length cDNA for SUMO-1. A significant increase in NF-κB activation was noted during hypoxia in shRNA-transfected cells, compared to wild type cells. Conversely, over-expression of SUMO-1 resulted in a significant reduction in NF-κB activation despite hypoxia. As proof of principle for the physiologic significance of SUMO-1 contribution to NF-κB activation, p65 subunit in nucleis and IL-6 mRNA levels of wild type compared to shRNA and over-expressing cells were also studied. In summary, we present an endogenous mechanism by which cells acquire anti-inflammatory properties by recruiting a non-degradable form of IκBα, a major control point for NF-κB activation.
Methods:
Murine model.
(See part one)
Cell culture, in vitro hypoxia and chemical treatments
HeLa cells (ATCC) were maintained in DMEM plus 10% FBS, at 37℃in a humidified incubator with 5% CO2 in room air. Cellular preconditioning was performed on cells following a modified in vivo protocol optimized for cells (See part one)
Transfections and NF-κB reporter assays.
Transfection of Hela cells was carried out using Fugene 6 transfection reagent (Roche Diagnostics) as directed by the manufacturer. Plasmids used in transfections were SAE-1(SUMO activating enzyme-1, SAE-1) plasmid, shRNA for SUMO-1 and control plasmids (Origen). On the second day of the transfection, HeLa cells in 6-well plates were transfected for the second time with pNF-κB-Luc (pNRE; Clontech) at a concentration of 1.6μg per well along with 0.08μg of Renilla for 24 hours and then either left at normoxia or subjected to a protocol of hypoxia preconditioning. Following treatments: cells were rinsed in PBS, lysed in passive lysis buffer (Promega) for 15 minutes, and spun down, and 20μl of lysates was assayed using the Dual-Glo Luciferase assay system (Promega) with the use of a luminometer (Turner BioSystems).
Transcriptional analysis.
cDNA was collected Using“Clonetech sprint powerscript”after extraction of total RNA. Real-time PCR (Taq-man) was employed to examine IL-6 expression levels in HeLa cells. IL-6 Primer was ready-to-use, ordered from Applied Biosystems. Samples were controlled forβ-actin using the following primers: sense , 5'-GGTGGCTTTTAGGATGGCAA G-3'; antisense , 5'-ACTGGAACGGTGA AGGTGACAG-3'; 162 bp).
Western blot assay
(See part one for method)
Antibodies used for Western blotting included rabbit anti-SUMO-1 (1:1000), rabbit anti–IκBα(1:1000), rabbit anti–Phospho-IκBα(1:1000), all from Cell Signaling Technology, Inc. Blots were washed, and species-matched peroxidase-conjugated secondary antibody was added. Labeled bands from washed blots were detected by enhanced chemiluminescence (Thermol Scientific).
Immunoprecipitation.
Total protein was isolated from HeLa cells and measured as described above. After pre-cleared with 60ul of Immobilized Protein A/G (Thermol Scientific) for 2 hours, rabbit anti-SUMO-1 antibody or normal rabbit serum (Sigma-Aldrich) were added into the protein, gently shaked for overnight. 60ul of Immobilized Protein A/G were added into each tube, incubated for 2 hours, centrifuged at 7500rpm for 4 minutes, complex-bound gel were then collected and washed with cell lysis buffer for 3 times. Finally, 100ul of 2% SDS loading buffer was added into each tube, boiled at 95℃for 5 minutes, centrifuged at 14000rpm for 30 seconds, loaded to 12% gel and followed western blot steps mentioned above.
Results
HPC-induced SUMO-1 modification prevents phosphorylation of IκBαduring hypoxia
SUMO-1 modification prevents phosphorylation and, in turn degradation of IκBα, therefore helps to stabilize NF-κB p65/p50 subunits. This has been demonstrated in inflammatory model induced by TNF-α. However, does it work in other models, for example, hypoxia-induced inflammation? In our study, Hela cells were subjected to normoxia, hypoxia or HPC treatment for 3 cycles, at the end of each cycle; proteins were extracted for western blot assay. Result shows a decrease of IκBαalong with the dropping of SUMO-1-conjugated IκBαin hypoxia, while in HPC, when SUMO-1-conjugated IκBαgoes up, IκBαstops to decrease as well. Furthermore, HeLa cells transfected with SAE-1 plasmid or control vector were subjected to hypoxia treatment for 3-24 hours, western assay shows: IκBαand SUMO-1-conjugated IκBαdecreased dramatically in wild type cells while is stable in SUMO-1 over-expressed cells; phospho-IκBαincreased in wild type cells while kept low in SUMO-1 over-expressed cells; Iκκβ, no significant change in both groups. From above experiments, we can draw the conclusion that SUMO-1 modification prevents phosphorylation of IκBαduring hypoxia.
HPC-induced SUMO-1 modification of IκBαinhibits NF-κB mediated inflammatory during hypoxia
To confirm if the SUMO-1 modification of IκBαinhibits NF-κB mediated inflammatory induced by hypoxia, we first used the luciferise reporter assay to measure NF-κB activity and its downstream changes related to inflammatory. HeLa cells were transfected with either SAE-1 plasmid or SUMO-1 shRNA, subjected to normoxia; hypoxia or HPC treatment followed by hypoxia, and then measured NF-κB activity by luciferise reporter assay. As we can see, cells transfected with SUMO-1 plasmid showed a significant (P < 0.05, SAE-1 versus control vector) decrease in NF-κB activity, meanwhile, in HPC group, those had been transfected with shRNA for SUMO-1 showed an increase of NF-κB activity (P < 0.05, SUMO-1 shRNA versus control vector).
Moreover, we still analyzed p65 protein, which reflects the translocation rate of NF-κB into nuclei, in the nuclear lysates extracted from Hela cells in parallel experiments. Data shows a similar pattern with luciferase assay: in SUMO-1 over-expressing cells, nuclear p65 was decreased despite of hypoxia treatment; while in shRNA group, HPC failed to inhibit the increase of nuclear p65.
Finally, IL-6, which is the downstream target of NF-κB in the inflammatory chain, was meseaured at transcriptional level by using Real Time-PCR. The result is consistent to the change of NF-κB activity.
Taken together, these changes indicate NF-κB mediated inflammation after hypoxia treatment can be inhibited by HPC induced SUMO-1 modification of IκBα.
Conclusions
In summary, we present an endogenous mechanism by which cells acquire anti-inflammatory properties by recruiting a non-degradable form of IκBα, a major control point for NF-κB activation
Adenosine (ADO) signaling has been found to play an key roll in hypoxia preconditioning (HPC), which enhancs cell anti-infalammatory ability. In our previous work, SUMO-1 modified IκBαform has been demonstrated to actively participate in the inhibition of NFκB activation. Using models of HPC, we have found a cyclic dependent increase in extra cellular Adenosine and theorize that the increase in cytoplasmic pool of IκBα/SUMO is mediated by Adenosine signaling, just like the adenosine–mediated cullin-1 deneddylation. Initially, We stimulated the ADO signaling pathway both in normoxia and hypoxia with the treatment of general Adenosine receptor agonist NECA, found an increase of IκBα/SUMO protein by western blot assay. In turn, when the stimulation was dampened by the general ADO receptor antagonist---8-PT, the increase was blocked, confirming the effect of ADO on the up-regulation of IκBα/SUMO. Further, functional evaluation of the effect was done by using luciferase assay on NFκB activity after genetically knocked SUMO-1 down by shRNA and stimualated with NECA. Result shows a loss of inhibition on NFκB activity after NECA stimulation when SUMO-1 had been knocked down, indicating SUMO-1 is crucial in the ADO pathway. More over, in cells over-expressing A2AR or A2BR have a higher level of IκBα/SUMO. Finally, in vivo experiments were conducted with cd73-knocked out (cd73-/-) mice, which were lack of ability to generate endogenous ADO. Mice were subjected to normoxia/hypoxia/HPC, and then total proteins of mice lung were analyzed by western blot. A signifcantly drop of IκBα/SUMO was found in cd73-/- mice compared with wild type mice. It is more important that, for cd73-/- mice, there was no increase of IκBα/SUMO in HPC group over normoxia and hypoxia groups, indicating the block the endogenous ADO pathway caused the failure for HPC to increas IκBα/SUMO, in another word, ADO is critical in the regulation of IκBα/SUMO.
Methods
Murine model.
CD73-/- mouse model was generated as described in our previous work. C57BL/6 (Charles River Laboratories) male mice of 8–10 weeks of age were used as control in this study. The Animal Care and Use Committee of Brigham and Women’s Hospital approved all procedures. Whole-body preconditioning was performed in a manner similar to that previously described. (See part one)
Cell culture, in vitro hypoxia and chemical treatments
HeLa cells (ATCC) were maintained in DMEM plus 10% FBS, at 37℃in a humidified incubator with 5% CO2 in room air. Cellular preconditioning was performed on cells following a modified in vivo protocol optimized for cells (See part one). Cells were treated with NECA in DMSO at a rang from 100nM to 100μM or pretreatment with 8-phenyltheophylline (8-PT) in ddH2O at concentration of 100nM. Treatment time is indicated in results.
Transfections and NF-κB reporter assays.
Transfection of Hela cells was carried out using Fugene 6 transfection reagent (Roche Diagnostics) as directed by the manufacturer. Plasmids used in transfections SUMO-1 shRNA or control plasmids (Origen). On the second day of the transfection, HeLa cells were transfected for the second time with pNF-κB-Luc (See part one) for 24 hours and then treated with NECA for 30 minutes. Following treatments: cells were rinsed in PBS, lysed in passive lysis buffer (Promega) for 15 minutes, and spun down, and 20μl of lysates was assayed using the Dual-Glo Luciferase assay system (Promega) with the use of a luminometer (Turner BioSystems).
Western blot assay
(See part two)
Results
Adenosine regulates SUMO-1 modification of IκBαin vitro
To define the functional attributes of SUMO-1 modification of IκB by Ado signaling, NECA stimulation on hela cells was conducted to get direct evidence of Ado’s up-regulation on SUMO-1 modification of IκB . Data showed a dose-dependent increase of IκBα/SUMO after hela cells were treated with NECA for about 30 minutes in normoxia, expectedly, this increase can be blocked by pre-treatment with 8-PT, a general inhibitor for Ado receptors. Not only in normal condition, but also in hypoxia, a similar pattern of the change of IκBα-SUMO after cells was treated with NECA or 8-PT/NECA was noticed. More over, when transfected with A2AR or A2BR, a higher of IκBα-SUMO was found compared with wild type cells, confirming above results that ADO pathway was involved in regulation of SUMO-1 modification of IκB.
SUMO-1 modification of IκBαis mediated by Ado in mouse models
To make clear if SUMO-1 modification of IκBαis mediated by Ado, we used a CD73 (-/-) mouse model which lacks the ability to generate Ado endogenously in our study. Wild type C57BL/6 mice were used as control. Both types of mice were subjected to normoxia, hypoxia or HPC treatment, total protein of mice lung was analyzed by western blot. SUMO-1-conjugated IκB in both groups shows a similar pattern with cell model. Down-regulated in hypoxia, while up-regulated in HPC treated mice. What is more important, in CD73 (-/-) mice, SUMO-1-conjugated IκB level is much less than that in wild type mice, indicating Ado is crucial in SUMO-1 modification of IκBα. Conclusions
In summary, we demonstrated that ADO signaling mediateds SUMO-1 modificaion of IκBα, which in turn induces anti-inflammatory properties by recruiting a non-degradable form of IκBα, a major control point for NFκB activation.
引文
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[4] Hay R T. Protein modification by SUMO [J]. Trends in Biochem Sci, 2001, 26: 332 ~ 333.
[5] Müller S, Hoege C, Pyrowolakis G, et al. SUMO, ubiquitin's mysterious cousin[J]. Nat Rev Mol Cell Biol, 2001, 2: 202~213
[6] SuHL, LiStevenS-L. Molecular features of human ubiquitin like-SUMO genes and their encoded proteins[J]. Gene, 2002,296: 65～73
[7] Sen R ,Baltimore D. Inducibility of the immunuglobulin enhancer binding protein NF - κB by a posttranslational mechanism[J]. Cell ,1986 ,47 :921 - 928
[8] 尹会男,柴家科. Toll 样受体/ 核因子κB 信号通路与脓毒症的研究进展[J]. 中华实验外科杂志,2003 ,20 (11) :1051-1052
[9] Hayden MS, Ghosh S. Shared principles in NF-kappaB signaling [J]. Cell. 2008, 132(3): 344-362
[10] Brasier AR. The NF-kappaB regulatory network [J]. Cardiovasc Toxicol. 2006, 6(2): 111-130
[11] 林振和. 核因子-κB 信号转导途径的调节研究进展[J ] . 国外医学免疫学分册,2004 ,27 (4) :234 – 238
[12] Thompson J E , Phillips RJ , Erdjument - Bromage H , et al . Ikappa B - beta regulates t he persistent response in a biphasic activation of NF - kappa B [J]. Cell, 1995 , 80 (4) :573 - 582
[13] 王文珊. TNF-α信号转导通路的研究进展[J]. 福建医科大学学报, 2005 ,39 (1) :27 – 31
[14] Martin P Kracklauer, Christian Schmidt. At the crossroad of SUMO and NF-κB [J]. Molecular Cancer. 2003, 2:39-43
[15] Joana M.P. Desterro, Manuel S. Rodriguez, Ronald T. Hay. SUMO-1 modification of IκBα inhibits NF-κB activation [J]. Molecular Cell. 1998,2:233-239
[16] Shao G, Zhang R, Wang ZL, et al. Hypoxic Preconditioning Improves Spatial Cognitive Ability in Mice [J]. Neurosignals. 2007, 15(6):314-321
[17] Zhang SX, Miller J, Gozal D, et al. Whole body hypoxic preconditioning protects mice against acute hypoxia by improving lung function [J]. J Appl Physiol, 2004, 96 (1): 392-397.
[18] Cai Z, Semenza GL. Phosphatidylinositol-3-kinase signaling is required for erythro- poietin mediated acute p rotection against myocardial ischemia / reperfusion injury [J]. Circulation, 2004, 109 (17): 2050-2053.
[19] Wu LY, Ding AS, Zhao T, et al. Involvement of increased stability of mitochondrial membrane potential and overexpression of Bcl2 in enhanced anoxic tolerance induced by hypoxic preconditioning in cultured hypothalamic neurons [J]. Brain Res, 2004, 999 (2): 149-154.
[20] Joseph Khoury, Juan Ibla, Andrew s.Neish, et al. Antiinflammatory adaptation to hypoxia through adenosine-mediated cullin-1 deneddylation. J Clin Invest. 2007, 117(3): 703-711.
[1] Joseph Khoury, Juan Ibla, Andrew s.Neish, et al. Antiinflammatory adaptation to hypoxia through adenosine-mediated cullin-1 deneddylation[J]. J Clin Invest. 2007, 117(3): 703-711.
[2] Gao ZG, Jacobson KA. Emerging adenosine receptor agonists [J]. Expert Opin Emerg Drugs. 2007, 12(3): 479-492
[3] Conlon BA, Ross JD, Law WR. Advances in understanding adenosine as a plurisystem modulator in sepsis and the systemic inflammatory response syndrome (SIRS) [J]. Front Biosci. 2005, 10: 2548- 2565
[4] Benarroch EE. Adenosine and its receptors: multiple modulatory functions and potential therapeutic targets for neurologic disease [J]. Neurology. 2008, 70(3): 231-236
[5] Auchampach JA. Adenosine receptors and angiogenesis [J]. Circ Res. 2007, 101(11): 1075-1077
[6] Bar-Yehuda S, Silverman MH, Kerns WD, Ochaion A, et al. The anti-inflammatory effect of A3 adenosine receptor agonists: a novel targeted therapy for rheumatoid arthritis [J].Expert Opin Investig Drugs. 2007, 16(10): 1601-1613.
[7] Sullivan GW. Adenosine A2A receptor agonists as anti-inflammatory agents [J]. Curr Opin Investig Drugs. 2003, 4(11): 1313-1319
[8] Michail V. Sitkovsky, Dmitriy Lukashev, Sergey Apasov, et al. Physiological control of immune response and inflammatory tissue damage by hypoxia-inducible factors and adenosine A2A receptors [J]. Annu. Rev. Immunol. 2004. 22: 657-682
[9] Kong T, Westerman KA, Faigle M, et al. HIF-dependent induction of adenosine A2B receptor in hypoxia [J]. FASEB J. 2006, 20(13): 2242-2250
[10] Thompson LF, Eltzschig HK, Ibla JC, Van De Wiele CJ, Resta R, Morote-Garcia JC, Colgan SPCrucial Role for Ecto-5-Nucleotidase (CD73) in Vascular Leakage during Hypoxia[J]. J Exp Med,2004 Dec 6;200(11):1395-405
[11] Eltzschig HK, Ibla JC, Furuta GT, et al. Coordinated adenine nucleotide phosphohydrolysis and nucleoside signaling in posthypoxic endothelium: role of ectonucleotidases and adenosine A2B receptors [J]. J Exp Med. 2003, 198(5): 783-796
[12] Olanrewaju HA, Qin W, Feoktistov I, et al. Adenosine A(2A) and A(2B) receptors in cultured human and porcine coronary artery endothelial cells [J]. Am J Physiol Heart Circ Physiol. 2000, 279(2): H650- 656.
[13] 柯荣湖,刘颖.腺苷受体在脑缺血中的研究进展[J].国际病理科学与临床杂志[J]. 2007, 27(5): 443-447
[14] Moro S, Spalluto G, Jacobson KA. Techniques: Recent developments in computer-aided engineering of GPCR ligands using the human adenosine A3 receptor as an example [J]. Trends Pharmacol Sci. 2005, 26(1): 44-51
[15] Alexander Choukèr, Manfred Thiel, Dmitriy Lukashev, et al. Critical Role of Hypoxia and A2A Adenosine Receptors in Liver Tissue-Protecting Physiological Anti-Inflammatory Pathway [J]. Mol Med. 2008, 14(3-4): 116–123
[16] 李晓波,殷莲华,周平. 5’2 核苷酸酶的研究进展[J].国外医学·生理、病理科学与临床分册[J]. 2004,24(2): 122-124
[17] Takedachi M, Qu D, Ebisuno Y, et al. CD73-Generated Adenosine Restricts Lymphocyte Migration into Draining Lymph Nodes [J]. J Immunol. 2008, 180(9): 6288-6296
[18] Eckle T, Füllbier L, Wehrmann M, et al. Identification of ectonucleotidases CD39 and CD73 in innate protection during acute lung injury [J]. J Immunol. 2007, 178(12): 8127-8137
[1] BOHREN KM, NADKARN IV , SONG J H, et al A M55V polymorphism in a novel SUMO gene ( SUMO24 ) differentially activates heat shock transcription factors and is associated with susceptibility to type I diabetes mellitus [J]. J Biol Chem, 2004, 279(26):27233～272381.
[2] Bayer P, Arndt A, Metzger S, et al. Structure determination of the smallubiquitin-related modifier SUMO-1[J]. J Mol Biol, 1998, 280: 275~286
[3] Su HL, LiStevenS-L. Molecular features of human ubiquitinlike SUMO genes and their encoded proteins[J]. Gene, 2002, 296:65~73
[4] Hay R T. Protein modification by SUMO [J]. Trends in Biochem Sci, 2001, 26: 332~333.
[5] Müller S, Hoege C, Pyrowolakis G, et al. SUMO, ubiquitin's mysterious cousin[J]. Nat Rev Mol Cell Biol, 2001, 2:202~213
[6] SuHL, LiStevenS-L. Molecular features of human ubiquitin like-SUMO genes and their encoded proteins[J]. Gene, 2002,296:65～73
[7] 叶晓峰,吴乔. 类泛素蛋白- SUMO[J]. 细胞生物学杂志,2004,26(1): 10～14.
[8] SONG J, DURR IN L K, W ILKINSON TA, et al Identification of a SUMO binding motif that recognizes SUMO modified proteins[J].Proc Natl Acad Sci USA, 2004,101(40):14373～143781
[9] DOHMEN R J. SUMO protein modification [J].Biochim Biophys Acta, 2004, 1695 (1-3):113～131.
[10] JOHNSON E S. Protein modification by SUMO[J]. Annu Rev Biochem, 2004, 73: 355～382.
[11] GRESKO E, MOLLER A, ROSCIC A, et al. Covalent modification of human homeodomain interacting protein kinase 2 by SUMO-1 at lysine 25 affects its stability [J]. Biochem Biophys Res Commun, 2005, 329 (4) : 1293～1299.
[12] GillG. Post translational modification by the small ubiquitin related modifier SUMO has big effects on transcription factor activity[J]. Curr Opin in Gene Devel, 2003, 13: 108~113.
[13] SCHW IENHORST I, JOHNSON E S, DOHMEN R J. SUMO conjugation anddeconjugation[J]. Mol Gen Genet, 2000, 263 (5):771～786
[14] Sen R ,Baltimore D. Inducibility of the immunuglobulin enhancer binding protein NF-κB by a posttranslational mechanism[J]. Cell, 1986, 47:921 – 928
[15] 尹会男,柴家科. Toll 样受体/ 核因子κB 信号通路与脓毒症的研究进展[J]. 中华实验外科杂志,2003,20(11) :1051-1052
[16] Hayden MS, Ghosh S. Shared principles in NF-kappaB signaling [J]. Cell. 2008, 132(3): 344-362
[17] Brasier AR. The NF-kappaB regulatory network [J]. Cardiovasc Toxicol. 2006, 6(2): 111-130
[18] 林振和. 核因子-κB 信号转导途径的调节研究进展[J].国外医学免疫学分册, 2004, 27(4): 234 – 238
[19] Thompson J E , Phillips RJ , Erdjument-Bromage H , et al. Ikappa B-beta regulates the persistent response in a biphasic activation of NF-kappa B [J]. Cell, 1995, 80 (4):573 - 582
[20] 王文珊. TNF-α信号转导通路的研究进展[J]. 福建医科大学学报,2005, 39(1): 27 – 31
[21] Perkins ND. Integrating cell-signalling pathways with NF-kappaB and IKK function [J]. Nat Rev Mol Cell Biol. 2007, 8(1): 49-62
[22] Ronald T. Hay, Laurent Vuilllard, Joana M P Desterro, et al. Control of NF-kappaB transcriptional activation by signal induced proteolysis of I kappa B alpha[J].The royal society , 1999,354:1601～1609.
[2] Zhang SX, Miller J, Gozal D, et al. Whole body hypoxic preconditioning protects mice against acute hypoxia by improving lung function [J]. J Appl Physiol, 2004, 96 (1): 392-397.
[3] Joseph Khoury, Juan Ibla, Andrew s.Neish, et al. Antiinflammatory adaptation to hypoxia through adenosine-mediated cullin-1 deneddylation. J Clin Invest. 2007, 117(3): 703-711.
[4] Wu LY, Ding AS, Zhao T, et al. Involvement of increased stability of mitochondrial membrane potential and overexpression of Bcl2 in enhanced anoxic tolerance induced by hypoxic preconditioning in cultured hypothalamic neurons [J]. Brain Res, 2004, 999 (2): 149-154.
[5] Cai Z, Semenza GL. Phosphatidylinositol-3-kinase signaling is required for erythro- poietin mediated acute p rotection against myocardial ischemia / reperfusion injury[J]. Circulation, 2004, 109 (17): 2050-2053.
[6] 王正朝、石放雄.PDE4 与 cAMP 信号区域化[J].科学通报. 2006,51(22): 2577-2586
[7] Houslay M D, Milligan G. Tailoring cAMP signalling responses through isoform multiplicity. Trends Biochem Sci, 1997, 22: 217-224
[8] Evellin S, Mongillo M, Terrin A, et al. Measuring dynamic changes in cAMP using fluorescence resonance energy transfer. Methods Mol Biol, 2004, 284: 259-270
[9] Courtney M. Lappas, Yuan-Ji Day, Melissa A. Marshall et al. Adenosine A2A receptor activation reduces hepatic ischemia reperfusion injury by inhibiting CD1- dependent NKT cell activation [J]. The Journal of Experiment Medicine 2006, 203(12): 2639-2648
[10] Ohta, A., M. Sitkovsky. Role of G-protein-coupled adenosine receptors in down regulation of inflammation and protection from tissue damage [J]. Nature. 2001, 414:916-920.
[11] Lukashev D, Ohta A, Sitkovsky M. Hypoxia-dependent anti-inflammatory pathways in protection of cancerous tissues [J]. Cancer Metastasis Rev. 2007, 26(2):273-279.
[12] Cronstein, B.N. Adenosine, an endogenous anti-inflammatory reagent [J]. J. Appl. Physiol. 1994, 76: 5–13.
[13] Alexander Choukèr, Manfred Thiel, Dmitriy Lukashev, et al. Critical Role of Hypoxia and A2A Adenosine Receptors in Liver Tissue-Protecting Physiological Anti-Inflammatory Pathway [J]. Mol Med. 2008, 14(3-4): 116–123
[14] Pfleger KD, Dalrymple MB, Dromey JR, et al. Monitoring interactions between G-protein-coupled receptors and beta-arrestins. Biochem Soc Trans. 2007, 35(Pt 4):764-766
[15] Klaasse EC, Ijzerman AP, de Grip WJ, Beukers MW. Internalization and desensitization of adenosine receptors [J]. Purinergic Signal. 2008, 4(1): 21-37.
[16] George S Balillie, Miles S Houslay. Arrestin times for compartmentalized cAMP signaling and phosphodiesterase-4 enzymes [J]. Current Opinion in Cell Biology. 2005, 17: 129-134
[17] Houslay MD, Baillie GS, Maurice DH. cAMP-Specific phosphodiesterase-4 enzymes in the cardiovascular system: a molecular toolbox for generating compartmentalized cAMP signaling[J]. Circ Res. 2007, 100(7): 950-966
[18] Omori K, Kotera J. Overview of PDEs and their regulation. Circ Res. 2007, 100(3): 309-327
[19] Makranz C, Cohen G, Reichert F, et al. cAMP cascade (PKA, Epac, adenylyl cyclase, Gi, and phosphodiesterases) regulates myelin phagocytosis mediated by complement receptor-3 and scavenger receptor-AI/II in microglia and macrophages[J]. Glia. 2006, 53(4): 441-448
[20] Gao ZG, Jacobson KA. Emerging adenosine receptor agonists [J]. Expert Opin Emerg Drugs. 2007, 12(3): 479-92
[21] 柯荣湖,刘颖.腺苷受体在脑缺血中的研究进展[J].国际病理科学与临床杂志. 2007, 27(5): 443-447
[22] Palmer TM, Trevethick. MA.Suppression of inflammatory and immune responses by the A (2A) adenosine receptor: an introduction [J]. Br J Pharmacol. 2008, 153 Suppl 1:S27-34
[23] Gurevich VV, Gurevich EV. The structural basis of arrestin-mediated regulation of G-protein- coupled receptors [J]. Pharmacol Ther. 2006, 110(3): 465-502
[24] Shenoy SK, Lefkowitz RJ. Seven-transmembrane receptor signaling throughbeta-arrestin [J].Sci STKE. 2005, (308):cm10
[25] DeFea KA. Stop that cell! Beta-arrestin-dependent chemotaxis: a tale of localized actin assembly and receptor desensitization [J]. Annu Rev Physiol. 2007, 69:535-560
[26] Ma L, Pei G. Beta-arrestin signaling and regulation of transcription [J]. J Cell Sci. 2007, 120(Pt 2):213-218
[27] Houslay M D, Schafer P, Zhang K Y. Keynote review: Phosphodiesterase-4 as a therapeutic target[J]. Drug Discov Today, 2005, 10(22): 1503-1519
[28] Conti M, Richter W, Mehats C, et al. Cyclic AMP-specific PDE4phosphodiesterases as critical components of cyclic AMP signaling [J]. J Biol Chem, 2003,278(8):5493-5496
[29] Houslay M D, Milligan G. Tailoring cAMP signalling responses through isoform multiplicity [J]. Trends Biochem Sci, 1997, 22: 217-224
[1] BOHREN KM, NADKARN IV , SONG J H, et al A M55V polymorphism in a novel SUMO gene ( SUMO24 ) differentially activates heat shock transcription factors and is associated with susceptibility to type I diabetes mellitus [ J ] J Biol Chem, 2004, 279(26) : 27233～272381.
[2] Bayer P, Arndt A, Metzger S, et al. Structure determination of the smallubiquitin-related modifier SUMO-1[J]. J Mol Biol, 1998, 280: 275~286
[3] SuHL, LiStevenS-L. Molecular features of human ubiquitinlike SUMO genes and their encoded proteins[J]. Gene, 2002, 296: 65~73
[4] Hay R T. Protein modification by SUMO [J]. Trends in Biochem Sci, 2001, 26: 332 ~ 333.
[5] Müller S, Hoege C, Pyrowolakis G, et al. SUMO, ubiquitin's mysterious cousin[J]. Nat Rev Mol Cell Biol, 2001, 2: 202~213
[6] SuHL, LiStevenS-L. Molecular features of human ubiquitin like-SUMO genes and their encoded proteins[J]. Gene, 2002,296: 65～73
[7] Sen R ,Baltimore D. Inducibility of the immunuglobulin enhancer binding protein NF - κB by a posttranslational mechanism[J]. Cell ,1986 ,47 :921 - 928
[8] 尹会男,柴家科. Toll 样受体/ 核因子κB 信号通路与脓毒症的研究进展[J]. 中华实验外科杂志,2003 ,20 (11) :1051-1052
[9] Hayden MS, Ghosh S. Shared principles in NF-kappaB signaling [J]. Cell. 2008, 132(3): 344-362
[10] Brasier AR. The NF-kappaB regulatory network [J]. Cardiovasc Toxicol. 2006, 6(2): 111-130
[11] 林振和. 核因子-κB 信号转导途径的调节研究进展[J ] . 国外医学免疫学分册,2004 ,27 (4) :234 – 238
[12] Thompson J E , Phillips RJ , Erdjument - Bromage H , et al . Ikappa B - beta regulates t he persistent response in a biphasic activation of NF - kappa B [J]. Cell, 1995 , 80 (4) :573 - 582
[13] 王文珊. TNF-α信号转导通路的研究进展[J]. 福建医科大学学报, 2005 ,39 (1) :27 – 31
[14] Martin P Kracklauer, Christian Schmidt. At the crossroad of SUMO and NF-κB [J]. Molecular Cancer. 2003, 2:39-43
[15] Joana M.P. Desterro, Manuel S. Rodriguez, Ronald T. Hay. SUMO-1 modification of IκBα inhibits NF-κB activation [J]. Molecular Cell. 1998,2:233-239
[16] Shao G, Zhang R, Wang ZL, et al. Hypoxic Preconditioning Improves Spatial Cognitive Ability in Mice [J]. Neurosignals. 2007, 15(6):314-321
[17] Zhang SX, Miller J, Gozal D, et al. Whole body hypoxic preconditioning protects mice against acute hypoxia by improving lung function [J]. J Appl Physiol, 2004, 96 (1): 392-397.
[18] Cai Z, Semenza GL. Phosphatidylinositol-3-kinase signaling is required for erythro- poietin mediated acute p rotection against myocardial ischemia / reperfusion injury [J]. Circulation, 2004, 109 (17): 2050-2053.
[19] Wu LY, Ding AS, Zhao T, et al. Involvement of increased stability of mitochondrial membrane potential and overexpression of Bcl2 in enhanced anoxic tolerance induced by hypoxic preconditioning in cultured hypothalamic neurons [J]. Brain Res, 2004, 999 (2): 149-154.
[20] Joseph Khoury, Juan Ibla, Andrew s.Neish, et al. Antiinflammatory adaptation to hypoxia through adenosine-mediated cullin-1 deneddylation. J Clin Invest. 2007, 117(3): 703-711.
[1] Joseph Khoury, Juan Ibla, Andrew s.Neish, et al. Antiinflammatory adaptation to hypoxia through adenosine-mediated cullin-1 deneddylation[J]. J Clin Invest. 2007, 117(3): 703-711.
[2] Gao ZG, Jacobson KA. Emerging adenosine receptor agonists [J]. Expert Opin Emerg Drugs. 2007, 12(3): 479-492
[3] Conlon BA, Ross JD, Law WR. Advances in understanding adenosine as a plurisystem modulator in sepsis and the systemic inflammatory response syndrome (SIRS) [J]. Front Biosci. 2005, 10: 2548- 2565
[4] Benarroch EE. Adenosine and its receptors: multiple modulatory functions and potential therapeutic targets for neurologic disease [J]. Neurology. 2008, 70(3): 231-236
[5] Auchampach JA. Adenosine receptors and angiogenesis [J]. Circ Res. 2007, 101(11): 1075-1077
[6] Bar-Yehuda S, Silverman MH, Kerns WD, Ochaion A, et al. The anti-inflammatory effect of A3 adenosine receptor agonists: a novel targeted therapy for rheumatoid arthritis [J].Expert Opin Investig Drugs. 2007, 16(10): 1601-1613.
[7] Sullivan GW. Adenosine A2A receptor agonists as anti-inflammatory agents [J]. Curr Opin Investig Drugs. 2003, 4(11): 1313-1319
[8] Michail V. Sitkovsky, Dmitriy Lukashev, Sergey Apasov, et al. Physiological control of immune response and inflammatory tissue damage by hypoxia-inducible factors and adenosine A2A receptors [J]. Annu. Rev. Immunol. 2004. 22: 657-682
[9] Kong T, Westerman KA, Faigle M, et al. HIF-dependent induction of adenosine A2B receptor in hypoxia [J]. FASEB J. 2006, 20(13): 2242-2250
[10] Thompson LF, Eltzschig HK, Ibla JC, Van De Wiele CJ, Resta R, Morote-Garcia JC, Colgan SPCrucial Role for Ecto-5-Nucleotidase (CD73) in Vascular Leakage during Hypoxia[J]. J Exp Med,2004 Dec 6;200(11):1395-405
[11] Eltzschig HK, Ibla JC, Furuta GT, et al. Coordinated adenine nucleotide phosphohydrolysis and nucleoside signaling in posthypoxic endothelium: role of ectonucleotidases and adenosine A2B receptors [J]. J Exp Med. 2003, 198(5): 783-796
[12] Olanrewaju HA, Qin W, Feoktistov I, et al. Adenosine A(2A) and A(2B) receptors in cultured human and porcine coronary artery endothelial cells [J]. Am J Physiol Heart Circ Physiol. 2000, 279(2): H650- 656.
[13] 柯荣湖,刘颖.腺苷受体在脑缺血中的研究进展[J].国际病理科学与临床杂志[J]. 2007, 27(5): 443-447
[14] Moro S, Spalluto G, Jacobson KA. Techniques: Recent developments in computer-aided engineering of GPCR ligands using the human adenosine A3 receptor as an example [J]. Trends Pharmacol Sci. 2005, 26(1): 44-51
[15] Alexander Choukèr, Manfred Thiel, Dmitriy Lukashev, et al. Critical Role of Hypoxia and A2A Adenosine Receptors in Liver Tissue-Protecting Physiological Anti-Inflammatory Pathway [J]. Mol Med. 2008, 14(3-4): 116–123
[16] 李晓波,殷莲华,周平. 5’2 核苷酸酶的研究进展[J].国外医学·生理、病理科学与临床分册[J]. 2004,24(2): 122-124
[17] Takedachi M, Qu D, Ebisuno Y, et al. CD73-Generated Adenosine Restricts Lymphocyte Migration into Draining Lymph Nodes [J]. J Immunol. 2008, 180(9): 6288-6296
[18] Eckle T, Füllbier L, Wehrmann M, et al. Identification of ectonucleotidases CD39 and CD73 in innate protection during acute lung injury [J]. J Immunol. 2007, 178(12): 8127-8137
[1] BOHREN KM, NADKARN IV , SONG J H, et al A M55V polymorphism in a novel SUMO gene ( SUMO24 ) differentially activates heat shock transcription factors and is associated with susceptibility to type I diabetes mellitus [J]. J Biol Chem, 2004, 279(26):27233～272381.
[2] Bayer P, Arndt A, Metzger S, et al. Structure determination of the smallubiquitin-related modifier SUMO-1[J]. J Mol Biol, 1998, 280: 275~286
[3] Su HL, LiStevenS-L. Molecular features of human ubiquitinlike SUMO genes and their encoded proteins[J]. Gene, 2002, 296:65~73
[4] Hay R T. Protein modification by SUMO [J]. Trends in Biochem Sci, 2001, 26: 332~333.
[5] Müller S, Hoege C, Pyrowolakis G, et al. SUMO, ubiquitin's mysterious cousin[J]. Nat Rev Mol Cell Biol, 2001, 2:202~213
[6] SuHL, LiStevenS-L. Molecular features of human ubiquitin like-SUMO genes and their encoded proteins[J]. Gene, 2002,296:65～73
[7] 叶晓峰,吴乔. 类泛素蛋白- SUMO[J]. 细胞生物学杂志,2004,26(1): 10～14.
[8] SONG J, DURR IN L K, W ILKINSON TA, et al Identification of a SUMO binding motif that recognizes SUMO modified proteins[J].Proc Natl Acad Sci USA, 2004,101(40):14373～143781
[9] DOHMEN R J. SUMO protein modification [J].Biochim Biophys Acta, 2004, 1695 (1-3):113～131.
[10] JOHNSON E S. Protein modification by SUMO[J]. Annu Rev Biochem, 2004, 73: 355～382.
[11] GRESKO E, MOLLER A, ROSCIC A, et al. Covalent modification of human homeodomain interacting protein kinase 2 by SUMO-1 at lysine 25 affects its stability [J]. Biochem Biophys Res Commun, 2005, 329 (4) : 1293～1299.
[12] GillG. Post translational modification by the small ubiquitin related modifier SUMO has big effects on transcription factor activity[J]. Curr Opin in Gene Devel, 2003, 13: 108~113.
[13] SCHW IENHORST I, JOHNSON E S, DOHMEN R J. SUMO conjugation anddeconjugation[J]. Mol Gen Genet, 2000, 263 (5):771～786
[14] Sen R ,Baltimore D. Inducibility of the immunuglobulin enhancer binding protein NF-κB by a posttranslational mechanism[J]. Cell, 1986, 47:921 – 928
[15] 尹会男,柴家科. Toll 样受体/ 核因子κB 信号通路与脓毒症的研究进展[J]. 中华实验外科杂志,2003,20(11) :1051-1052
[16] Hayden MS, Ghosh S. Shared principles in NF-kappaB signaling [J]. Cell. 2008, 132(3): 344-362
[17] Brasier AR. The NF-kappaB regulatory network [J]. Cardiovasc Toxicol. 2006, 6(2): 111-130
[18] 林振和. 核因子-κB 信号转导途径的调节研究进展[J].国外医学免疫学分册, 2004, 27(4): 234 – 238
[19] Thompson J E , Phillips RJ , Erdjument-Bromage H , et al. Ikappa B-beta regulates the persistent response in a biphasic activation of NF-kappa B [J]. Cell, 1995, 80 (4):573 - 582
[20] 王文珊. TNF-α信号转导通路的研究进展[J]. 福建医科大学学报,2005, 39(1): 27 – 31
[21] Perkins ND. Integrating cell-signalling pathways with NF-kappaB and IKK function [J]. Nat Rev Mol Cell Biol. 2007, 8(1): 49-62
[22] Ronald T. Hay, Laurent Vuilllard, Joana M P Desterro, et al. Control of NF-kappaB transcriptional activation by signal induced proteolysis of I kappa B alpha[J].The royal society , 1999,354:1601～1609.