丙型肝炎病毒非结构蛋白NS4B诱导细胞非折叠蛋白反应

用RT-PCR和免疫印迹的方法检测稳定表达NS4B的HeLa细胞中的XBP1;通过RT-PCR的方法在表达NS4B的HeLa和Huh-7细胞中检测ATF6,Grp78和caspase-12的转录,并且通过报告基因的方法分析XBP1和Grp78启动子活性.实验结果表明在表达NS4B的HeLa细胞中检测到XBP1的两种形式(剪接和未剪接),此外,在细胞中ATF6、Grp78的转录水平和XBP1、Grp78启动子的荧光素酶活性较没有表达NS4B的HeLa和Huh-7细胞中的量有所增加;通过染色质免疫沉淀实验(ChIP)分析,这些增加可能是由于XBP1结合到了这些基因的启动子上引起的.总之,实验结果可提示HCV NS4B通过ATF6或XBP1途径引起内质网压力,导致UPR反应.NS4B可能在HCV的致病性中起着重要的作用,特别是在慢性肝炎,甚至肝细胞癌中.     

鼻病毒非结构蛋白2B 诱导细胞发生内质网应激

为探讨鼻病毒非结构蛋白2B诱导内质网应激和细胞凋亡的机制,本研究构建了鼻病毒非结构蛋白2B的真核表达载体p2B‐GFP ,通过转染BHK‐21细胞检测相关标志蛋白的变化情况。结果显示,非结构蛋白2B定位表达于BHK‐21细胞内质网,诱导内质网应激标志蛋白Grp78、CHOP的表达增加,并使活化转录因子6（ATF6）的转录活性增加,还诱导BHK‐21细胞发生核浓缩而凋亡,使凋亡标志蛋白PARP发生降解而减少。结果提示,鼻病毒非结构蛋白2B可诱导细胞发生内质网应激,并经该途径诱导细胞凋亡。     

人类不同类型细胞中X-盒结合蛋白1转录活性研究

人类X-盒结合蛋白1(X-box binding protein1,XBP1)作为一种重要的转录因子,在细胞中涉及了广泛的信号调控过程。为进一步研究XBP1的生物学功能,首先利用生物信息学技术确定XBP1基因的启动子区域和2个缺失突变体的基因序列,聚合酶链反应扩增XBP1启动子和2个缺失突变体的基因序列,分别克隆至真核报告载体pCAT3-Basic中,构建3个报告载体p1-XBP1p、p2-XBP1p和p3-XBP1p,确定启动子活性最强的序列,并以该序列分别转染正常人肝细胞L02、人肝母细胞瘤细胞HepG2、人肝癌细胞SMMC-7721、人类红白血病细胞K562、人皮肤成纤维细胞HSF和人贮脂细胞Lipocyte Ito Cell 6种不同类型的细胞后(FuGENE 6 transfection reagents),CAT-ELISA方法检测氯霉素乙酰转移酶(CAT)在不同细胞系中的表达活性。每一组CAT结果反应了XBP1启动子转录活性的大小,其中p3-XBP1p在HepG2细胞中活性最强,是pCAT3-Basic的12.4倍,其次是K562和SMMC-7721,分别是10.9倍和10.0倍;在L02细胞中CAT酶活性低于上述3种异常细胞,在HSF和Ito细胞中CAT酶活性低或没有活性。运用适时荧光PCR方法和免疫印迹分别从mRNA水平和蛋白水平检测了XBP1在不同细胞中的表达情况,结果均显示XBP1在HepG2、K562和SMMC-7721细胞中的转录和表达强于L02、HSF和Ito细胞,在HSF细胞和L02细胞中转录和表达较低,在Ito细胞中几乎检测不到XBP1的表达,与CAT-ELISA检测结果一致。因此,XBP1启动子的转录活性在不同细胞中是具有差异的,而XBP1启动子转录活性的大小直接调控下游基因XBP1的表达,导致不同细胞中XBP1的表达丰度也不相同,XBP1启动子的转录活性和表达与细胞类型、细胞周期和组织特异性密切相关。本研究发现XBP1启动子的ATG上游-227bp～66bp区域与XBP1的转录活性密切相关,属于XBP1启动子的核心区域;进一步比较XBP1基因核心启动子区在不同细胞中转录活性的差别和XBP1基因表达丰度的差异,为揭示真核细胞中XBP1的转录调控机制奠定基础。     

丙型肝炎病毒NS2蛋白在Huh-7细胞中抑制NF—κB活性

将丙型肝炎病毒（HCV)非结构蛋白NS2基因的全长序列插入到真核表达载体pCDNA3．1(-)CMV启动子下游,构建成重组质粒pCNS2。用脂质体Lipo Vec^TM转染Huh-7细胞,转染细胞内可检出NS2的mRNA和蛋白质,表明构建的pCNS2可在Huh-7细胞内成功表达;把不同剂量的pCNS2质粒DNA与报告质粒pNF-κ B-Luc共转染Huh-7细胞,48h后检测荧光素酶活性,结果显示与pCNS2共转染的细胞中pNF-κB-Luc表达出的荧光素酶的活性比对照细胞降低了约2—4倍,并呈明显的剂量相关性。表明HCV NS2对NF-κ B激活转录活性有明显的抑制作用。这可能与HCV慢性持续性感染的致病性有一定的相关性。     

IRE1α上调人骨肿瘤细胞XBP1启动子转录活性的研究

目的研究内质网跨膜蛋白肌醇酶1α（inositol-requiring enzyme 1α,IRE1α）对其下游转录因子XBP1启动子转录活性的影响。方法在成功构建人IREla基因全长重组质粒的基础上,设计合成并构建靶向IRE1α基因的siRNA干扰载体（pS1;pS2）;采用巢式PCR扩增XBP1启动子,插入到pGL3．Basic载体,构建携带XBP1启动子的报告基因重组质粒pGL3-XBP1。分别将siIRE1α干扰质粒与pGL3-XBPl报告基因重组质粒共转染人SW1353细胞和Saos-2细胞中,48h后采用双荧光报告系统检测IRE1α对XBP1启动子转录活性的调控作用。结果酶切及测序结果证实siRNA1干扰质粒和XBPl启动子真核表达载体均构建成功,双荧光报告系统检测显示SW1353细胞和Saos-2细胞中,直接载体pcDNA3．15（-）IRE1α与pGL3-XBP1共转实验组的荧光素酶活性明显高于其他实验组（P〈0．5）,并随着IRE1α转染剂量的增加逐渐达到饱和;而silRE1α与pGL3-XBP1共转实验组的荧光素酶活性明显降低。免疫印迹结果显示,过表达IRE1α明显上调剪接型XBPlS蛋白的表达。结论人恶性骨肿瘤细胞中,过表达IRE1α明显上调XBP1启动子的转录与表达,并能有效促进XBPIU剪切生成XBPIU;通过siRNA方法抑制IRE1α后,XBP1启动子的转录与表达受到抑制。本实验为进一步研究骨肿瘤细胞中IRE1α调控XBP1启动子转录与剪接的分子机制奠定基础,为临床骨肿瘤的诊断与治疗提供一定的实验依据。     

丙型肝炎病毒NS2蛋白在Huh-7细胞中抑制NF-κB活性

将丙型肝炎病毒(HCV)非结构蛋白NS2基因的全长序列插入到真核表达载体pCDNA3.1(-)CMV启动子下游,构建成重组质粒pCNS2.用脂质体LipoVecTM转染Huh-7细胞,转染细胞内可检出NS2的mRNA和蛋白质,表明构建的pCNS2可在Huh-7细胞内成功表达;把不同剂量的pCNS2质粒DNA与报告质粒pNF-κ B-Luc共转染Huh-7细胞,48h后检测荧光素酶活性,结果显示与pCNS2共转染的细胞中pNF-κ B-Luc表达出的荧光素酶的活性比对照细胞降低了约2～4倍,并呈明显的剂量相关性.表明HCV NS2对NF-κ B激活转录活性有明显的抑制作用.这可能与HCV慢性持续性感染的致病性有一定的相关性.     

靶向XBP1S的RNA干扰质粒对细胞增殖的影响

目的 构建靶向人XBP1S的siRNA真核表达载体（pSUPER-XBP1S）并观察其对人HeLa细胞和HepG2细胞增殖能力的影响.方法 设计并合成针对XBP1S基因的siRNA,退火成互补双链后克隆至真核表达载体pSUPER构建重组质粒,并将其转染入HeLa细胞和HepG2细胞中.采用RT-PCR检测转染前后XBP1S在HeLa细胞和HepG2细胞中的转录,Western印迹检测转染前后XBP1S蛋白的表达;MTT法、细胞计数检测重组质粒对HeLa细胞和HepG2细胞增殖能力的影响.结果 重组质粒能有效地抑制HeLa细胞和HepG2细胞中XBP1S基因的转录和表达;转染HeLa细胞和HepG2细胞后,细胞增殖抑制率及细胞增殖数与对照组比较,差异有统计学意义（P〈0.05）.结论 成功构建了靶向人XBP1S的siRNA表达载体pSUPER-XBP1S,并且有效的抑制了HeLa细胞和HepG2细胞中XBP1S的转录和表达,有效抑制了细胞的增殖能力.     

HBV X基因的表达及在真核细胞中对内质网压力的作用

运用PCR技术获得HBx基因,分别克隆到原核表达载体pET-his和真核表达载体pcDNA3.1(-)上。重组质粒pET-his-HBx转化大肠杆菌BL21(DE3)后,IPTG诱导表达,利用Ni柱纯化后的蛋白免疫家兔,获得特异性的抗-HBx兔抗血清。重组质粒pcDNA3.1(-)-HBx分别转染HepG2和Hep3B细胞系后,经RT-PCR和Westernblot检测,证明HBx可以在这两种细胞系中表达。通过报告基因的表达研究了HBx对XBP1和GRP78启动子的激活活性,结果表明瞬时转染HBx的细胞系中,XBP1和GRP78启动子介导的荧光素酶活性比相应的对照细胞增加了3～7倍。通过RT-PCR分析证明,转染了HBx的细胞中XBP1mRNA发生了剪切。因此,可以初步推断HBx在HepG2和Hep3B细胞中的表达可以引起内质网压力反应,为进一步阐明HBx表达对内质网的影响和肝脏病原发生机制奠定了基础。     

HBV X基因的表达及在真核细胞中对内质网压力的作用

运用PCR技术获得HBx基因,分别克隆到原核表达载体pET-his和真核表达载体pcDNA3.1(-)上.重组质粒pET-his-HBx转化大肠杆菌BL21(DE3)后, IPTG诱导表达,利用Ni柱纯化后的蛋白免疫家兔,获得特异性的抗-HBx兔抗血清.重组质粒pcDNA3.1(-)-HBx分别转染HepG2和Hep3B细胞系后,经RT-PCR和Western blot检测,证明HBx可以在这两种细胞系中表达.通过报告基因的表达研究了HBx对XBP1和GRP78启动子的激活活性,结果表明瞬时转染HBx的细胞系中,XBP1和GRP78启动子介导的荧光素酶活性比相应的对照细胞增加了3～7倍.通过RT-PCR分析证明,转染了HBx的细胞中XBP1 mRNA发生了剪切.因此,可以初步推断HBx在HepG2和Hep3B细胞中的表达可以引起内质网压力反应,为进一步阐明HBx表达对内质网的影响和肝脏病原发生机制奠定了基础.     

慢性乙型肝炎病毒感染病毒复制与肝细胞内质网应激反应相关性的初步研究

目的:研究体内、体外乙型肝炎病毒(hepatitis B virus,HBV)复制水平与肝细胞内质网应激(endoplasmic reticulum stress,ER stress)反应基因表达的相关性。方法:选择7例慢性HBV感染者作体内研究,用液氮冷冻保存肝脏穿刺活检组织,检测肝功能并提取m RNA;体外研究分三组,转染组(HepG2.2.15)为HBV基因转染人肝癌细胞株,对照组(HepG2)为人肝癌细胞株,干预组(3TC)为50μg/m L拉米夫定作用于HBV基因转染人肝癌细胞。分别培养2天、4天、6天后收集细胞提取m RNA。HBV DNA载量的测定用实时定量聚合酶链反应(PCR);活化转录因子4 (ATF4)、转录因子C/EBP同源蛋白(CHOP)、活化转录因子6(ATF6)、X盒结合蛋白1(XBP1)及葡萄糖调节蛋白78(GRP78)的m RNA表达,用SYBR Green荧光定量逆转录聚合酶链反应(SYBR Green RT-PCR)检测,以β-actin为内参照;GRP78的表达用蛋白免疫印迹法(WB)检测。结果:体内研究结果表明:XBP1、GRP78、CHOP及ATF6基因表达水平与病毒载量、炎症程度及肝组织纤维化程度无显著相关性(P0.05);ATF6与GRP78基因表达水平和HBV DNA水平呈显著正相关(P0.05)。体外研究结果表明:干预组细胞培养2天至6天HBV DNA水平下降,CHOP、GRP78、ATF6及XBP1基因表达水平在三组之间差异无统计学意义(P0.05);WB结果表明,转染组细胞GRP78的表达培养4天与6天相比差异无统计学意义(P0.05),干预组培养6天后与转染组比较差异无统计学意义(P0.05)。结论:研究结果提示体内HBV复制水平与肝细胞ER stress反应显著相关,而肝脏炎症程度及肝脏纤维化程度等与ER stress反应无明显相关性;体外三组细胞之间ER stress基因的表达无显著差异(P0.05)。     

Endoplasmic reticulum stress response promotes cytotoxic phenotype of CD8αβ+ intraepithelial lymphocytes in a mouse model for Crohn's disease-like ileitis

Endoplasmic reticulum (ER) unfolded protein responses (UPR) are implicated in the pathogenesis of inflammatory bowel disease. Cytotoxic CD8αβ(+) intraepithelial lymphocytes (IEL) contribute to the development of Crohn's disease-like ileitis in TNF(ΔARE/+) mice. In this study, we characterized the role of ER-UPR mechanisms in contributing to the disease-associated phenotype of cytotoxic IEL under conditions of chronic inflammation. Inflamed TNF(ΔARE/+) mice exhibited increased expression of Grp78, ATF6, ATF4, and spliced XBP1 in CD8αβ(+) IEL but not in CD8αα(+) IEL or in lamina propria lymphocytes. Chromatin immunoprecipitation analysis in CD8αβ(+) T cells showed selective recruitment of ER-UPR transducers to the granzyme B gene promoter. Heterozygous Grp78(-/+) mice exhibited an attenuated granzyme B-dependent cytotoxicity of CD8αβ(+) T cells against intestinal epithelial cells, suggesting a critical activity of this ER-associated chaperone in maintaining a cytotoxic T cell phenotype. Granzyme B-deficient CD8αβ(+) T cells showed a defect in IL-2-mediated proliferation in Grp78(-/+) mice. Adoptively transferred Grp78(-/+) CD8αβ(+) T cells had a decreased frequency of accumulation in the intestine of RAG2(-/-) recipient mice. The tissue pathology in TNF(ΔARE/+) × Grp78(-/+) mice was similar to TNF(ΔARE/+) mice, even though the cytotoxic effector functions of CD8αβ(+) T cells were significantly reduced. In conclusion, ER stress-associated UPR mechanisms promote the development and maintenance of the pathogenic cytotoxic CD8αβ(+) IEL phenotype in the mouse model of Crohn's disease-like ileitis.     

Decreased ER-associated degradation of alpha-TCR induced by Grp78 depletion with the SubAB cytotoxin

HeLa cells stably expressing the α chain of T-cell receptor (αTCR), a model substrate of ER-associated degradation (ERAD), were used to analyze the effects of BiP/Grp78 depletion by the SubAB cytotoxin. SubAB induced XBP1 splicing, followed by JNK phosphorylation, eIF2α phosphorylation, upregulation of ATF3/4 and partial ATF6 cleavage. Other markers of ER stress, including elements of ERAD pathway, as well as markers of cytoplasmic stress, were not induced. SubAB treatment decreased absolute levels of αTCR, which was caused by inhibition of protein synthesis. At the same time, the half-life of αTCR was extended almost fourfold from 70 min to 210 min, suggesting that BiP normally facilitates ERAD. Depletion of p97/VCP partially rescued SubAB-induced depletion of αTCR, confirming the role of VCP in ERAD of αTCR. It therefore appears that ERAD of αTCR is driven by at least two different ATP-ase systems located at two sides of the ER membrane, BiP located on the lumenal side, while p97/VCP on the cytoplasmic side. While SubAB altered cell morphology by inducing cytoplasm vacuolization and accumulation of lipid droplets, caspase activation was partial and subsided after prolonged incubation. Expression of CHOP/GADD153 occurred only after prolonged incubation and was not associated with apoptosis.     

Differences in Unfolded Protein Response Pathway Activation in the Lenses of Three Types of Cataracts

Purpose

To investigate the activation of three unfolded protein response (UPR) pathways in the lenses of age-related, high myopia-related and congenital cataracts.

Methods and Materials

Lens specimens were collected from patients during small incision cataract surgery. Lenses from young cadaver eyes were collected as normal controls. Real-time PCR and Western blotting were performed to detect the expression of GRP78, p-eIF2α, spliced XBP1, ATF6, ATF4 and p-IRE1α in the lenses of normal human subjects and patients with age-related, myopia-related or congenital cataracts.

Results

In the lenses of the age-related and high myopia-related cataract groups, the protein levels of ATF6, p-eIF2α and p-IRE1α and the gene expression levels of spliced XBP1, GRP78, ATF6 and ATF4 were greatly increased. Additionally, in the congenital cataract group, the protein levels of p-eIF2α and p-IRE1α and the gene expression levels of spliced XBP1, GRP78 and ATF4 were greatly increased. However, the protein and gene expression levels of ATF6 were not up-regulated in the congenital cataract group compared with the normal control group.

Conclusions

The UPR is activated via different pathways in the lenses of age-related, high myopia-related and congenital cataracts. UPR activation via distinct pathways might play important roles in cataractogenesis mechanisms in different types of cataracts.     

Unfolding protein response signaling is involved in development,maintenance, and regression of the corpus luteum during the bovine estrous cycle

The corpus luteum (CL) is a transient endocrine organ. Development, maintenance, and regression of CL are effectively controlled by dynamic changes in gene expression. However, it is unknown what types of gene are affected during the CL life span of the estrous cycle in bovine. Here, we determined whether unfolded protein response (UPR) signaling via eIF2α/ATF4/GADD34, p90ATF6/p50ATF6, and IRE1/XBP1, which is a cellular stress response associated with the endoplasmic reticulum (ER), is involved in the bovine CL life span. Our results indicated that expression of Grp78/Bip, the master UPR regulator, was increased during the maintenance stage and rapidly decreased at the regression stage. Additionally, UPR signaling pathways genes were found to be involved in luteal phase progression during the estrous cycle. Our findings suggested that Grp78/Bip, ATF6, and XBP1 act as ER chaperones for initiating CL development and maintaining the CL. In addition, we investigated whether ER stress-mediated apoptosis is occurred through three UPR signaling pathways in CL regression stage. Interestingly, pIRE1 and CHOP were found to be involved in both the adaptive response and ER stress-mediated apoptosis. During the CL regression stage, increased expression of pJNK and CHOP, two components of ER stress-mediated apoptotic cascades, occurred before increased level of cleaved caspase 3 were observed. The present investigation was performed to identify a functional link between UPR signaling and CL life span during the bovine estrous cycle. Taken together, results from this study demonstrated that UPR protein/gene expression levels were different at various stages of the bovine CL life span. Variations in the expression of these protein/genes may play important roles in luteal stage progression during the estrous cycle.     

Coupling cystic fibrosis to endoplasmic reticulum stress: Differential role of Grp78 and ATF6

Cystic fibrosis (CF) is the most common Caucasian autosomal recessive disease. It is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding the CFTR protein, which is a chloride (Cl(-)) channel. The most common mutation leads to a missing phenylalanine at position 508 (DeltaF508). The DeltaF508-CFTR protein is misfolded and retained in the endoplasmic reticulum and may trigger the unfolded protein response (UPR). Furthermore, CF is accompanied by inflammation and infection, which are also involved in the UPR. To date, the UPR transducer ATF6 and ER stress sensor Grp78 have been used as UPR markers. Therefore, our aim was to study the activation of ATF6 and Grp78 in transfected human epithelial cells expressing the DeltaF508-CFTR protein, and we showed that they are activated in these cells. We investigated the effect of exogenous UPR inducers thapsigargin (Tg) and tunicamycin (Tu) on Grp78 and ATF6 expression. Whereas the cells reacted to the UPR induction, we show a difference in the electrophoretic pattern of ATF6. The Grp78/ATF6 complex was previously described, but its stability during UPR is controversial. Using co-immunoprecipitation we show that it is stable in DeltaF508-CFTR-expressing cells and is maintained under UPR conditions. Finally, using siRNA, we show that decreased ATF6 expression induces increased cAMP-dependent halide flux through DeltaF508-CFTR due to its increased membrane localization. Therefore, our results suggest that UPR may be triggered in CF and that ATF6 may be a therapeutic target.     

Rat synapsin 1 promoter mediated transgene expression in testicular cell types

Previous reports described the rat synapsin 1 promoter as primarily neuron selective. However, ectopic expression of a transgene under the rat synapsin 1 promoter was also detected in testis from some transgenic mouse lines. Here we investigate which cells within the testis express a transgene consisting of the rat synapsin 1 promoter fused with luciferase. Synapsin 1-luciferase expression vectors were introduced into HeLa cells, into TM3 cells derived from mouse testicular Leydig cells, and into one-cell embryos to make transgenic mice. Indirect immunofluorescence suggests that nontransfected TM3 cells do not express endogenous synapsin 1. TM3 stable transfectants, however, expressed luciferase under the direction of the synapsin 1 promoter, in both promoter orientations. HeLa cells displayed only low levels of activity. Transgenic mice carrying the synapsin 1-luciferase construct displayed high levels of luciferase activity in the brain, spinal cord, and testis. Enriched populations of prepuberal types A and B spermatogonia and adult Leydig cells, pachytene spermatocytes, and round spermatids prepared from transgenic mice all displayed substantial luciferase activity. Thus, the rat synapsin 1 promoter can mediate reporter gene expression in neurons and testicular cell types.