骨髓来源的肥大细胞（BMMC）表面FcεRI表达调控及其生物学活性的研究
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摘要
实验目的
肥大细胞主要存在于粘膜和结缔组织中,借助于其表面表达的IgE的高亲合力受体FcεRI与抗变应原的特异性抗体IgE的交联,形成IgE/FcsRI复合物,进而引发Ⅰ型变态反应。肥大细胞胞浆中存在大量颗粒,其内储存组胺等生物介质,活化的肥大细胞也可新合成细胞因子如TNF-α、IL-6和IL-13等生物介质。正是借助于这些生物活性介质,肥大细胞在变态反应性疾病过程中发挥其生物学作用。因此,肥大细胞表面受体FcsRI表达以及其生物学功能调控机制的解析将为变态反应的防治奠定理论基础。
FcεRI由α-,β-和γ-链三部分构成,其中α-链是IgE特异性结合的亚基,p-和γ-链参与信号的转导。我们的前期试验结果显示转录因子Elf-1在体外可以结合到FcεRIα-链启动子的顺式元件周围的核苷酸序列。Elf-1是Ets转录因子家族成员之一,通过结合细胞核内的DNA序列,调节各种基因表达的诱导。Elf-1最初是从人类T细胞库克隆出来的一细胞型特异性转录因子,主要增强HIV-2启动子的转录。然而之后的研究发现,Elf-1在各种造血干细胞和免疫相关细胞中也可以呈现高表达,并且调控多种基因的表达,如T细胞中IL-2、GM-CSF、IL-5、IL-2RA和CD4；B细胞中免疫球蛋白重链、blk、lyn和CD1D1；巨核细胞IL-3和肥大细胞的干细胞白血病基因(SCL)。最近报道显示在人类嗜碱性粒细胞系KU812中,Elf-1还可抑制FcεRIγ-链的表达。而在FcεRIα-链启动子上具有结合位点的Elf-1是否能够影响小鼠肥大细胞FcεRI的基因表达,目前尚不可知。
除转录因子外,基因表达调控还与染色体上组蛋白乙酰化的状态有关。这种状态受控于两种关键的调节因子,即组蛋白乙酰化酶(HAT)和组蛋白去乙酰化酶(HDAC)。曲古菌素A(TSA),是一种强效的HDAC抑制剂(HDACi),属于羟肟酸类。研究显示TSA作为一种有效的抗癌制剂,能够抑制肿瘤细胞的生长,或者诱导肿瘤细胞的分化和凋亡。另外,TSA和另一种HDACi, SAHA,还可通过调节各种基因的表达而参与了免疫性疾病,如,系统性红斑狼疮(SLE)、移植物抗宿主病、类风湿性关节炎等。尤其在变态反应的小鼠模型中,TSA还显示了其潜在治疗效应。然而,TSA是否能够影响变态反应的重要效应细胞——肥大细胞表面FcεRI的表达及其功能,目前尚未见相关报道。
为此,本研究拟利用EIf-1 siRNA转染技术和体外TSA刺激等方式,探讨转录因子Elf-1和组蛋白去乙酰化酶抑制剂TSA对小鼠骨髓来源的肥大细胞(BMMC)表面FcεRI基因表达及其生物学功能的调控作用机制。进而,为肥大细胞相关的变态反应的研究提供新的实验依据。
实验方法
一、小鼠BMMC的制备
BALB/c小鼠,雌性,6-8周龄。钝性分离小鼠双侧股骨,用1ml注射器吸取少量RPMI-1640培养液冲洗骨髓内细胞至无菌平皿中。收集液体至10ml离心管中,1000rpm,4℃离心5min,弃上清。用10m1BMMC培养液重悬细胞,移至10cm无菌培养皿,37℃培养。常规换液,培养4周待用。
二、Elf-1 siRNA和Elf-1表达质粒体外转染
按照Mouse Macrophage Nucleofector试剂盒说明操作,选择Y-001程序,分别将20μM Elf-1 siRNA、Control siRNA和FITC-标记的寡核苷酸经电转至小鼠1×106BMMC中,37℃分别培养24h(待试剂盒转染效率、Elf-1 mRNA表达和FcεRIα、p、γ-链mRNA表达检测)和48h(待Elf-1蛋白表达检测)。同法将1μgpGV-B2-aNN0.6或pGL3-Basic分别与25ng pRL-CMV(内参)和20μM Elf-1 siRNA/Control siRNA共同电转染至1×106BMMC中；另将5μg pGV-B2-aNN0.6或pGL3-Basic分别与25ng pRL-CMV(内参)和10μgpCR3.1-Elf-1或对照pCR3.1经Bio-Rad Gene PulserⅡ电转染至BMMC中,37℃孵育24h后,收集BMMC(待FcεRI a-链启动子荧光素酶活性的分析)。
三、Elf-1表达鉴定
收集24h Elf-1及Control siRNA核转染的BMMC,按照RNeasy(?) Micro试剂盒的说明,提取细胞总RNA,利用High Capacity cDNA Reverse Transcription试剂盒反转录合成cDNA第一链,利用TaqMan Universal PCR Master Mix和7500Real-time PCR仪进行Real-time PCR,分析靶基因Elf-1 mRNA表达。Elf-1引物为(Mm00468217_ml)。另收集48h Elf-1及Control siRNA转染的BMMC,裂解细胞后,经7.5%SDS聚丙烯酰胺凝胶电泳,电转膜1.5h后,用anti-Elf-1 Ab和anti-actinAb作为第一抗体4℃过夜孵育。洗膜后用Alexa Fluor 680 goat anti-rabbit IgG和IRDye 800 goat anti-mouse IgG分别作为抗Elf-1和actin的第二抗体进行孵育1h,洗膜后用Odyssey infrared imaging system检测Elf-1蛋白的表达。
四、BMMC FcεRIα-链启动子活性检测
收集转染后的BMMC,按照Dualluciferase assay试剂盒说明,经Micro LumatPlus分析FcεRIα-链启动子荧光素酶活性。
五、FcεRIa-、β-和γ-链mRNA表达水平的检测
收集Elf-1 siRNA/Control siRNA转染后的BMMC。利用RNeasy Micro Kit提取总RNA,利用High Capacity cDNA Reverse Transcription Kit合成cDNA第一链,利用TaqMan Universal PCR Master Mix和7500 Real-Time PCR System with TaqMan Gene Expression,进行Realtime PCR,分析靶基因α-链(Mm00438867_ml)、p-链(Mm00442780_m1)和γ-链(Mm00438869_g1)mRNA表达水平。
六、PU.1结合FcεRIα-链启动子能力检测
收集Elf-1 siRN A/Control siRNA转染后的BMMC,经超声破碎细胞,沉淀染色质DNA。再分别与Anti-PU.1 goat Ab和goat IgG4℃孵育1h后,经7500Realtime PCR System分析PU.1结合FcεRIα-链启动子的能力。所用引物为小鼠FcεRIα-链启动子(-82／+1)：正义链-82／-56(5'-GGCATAGCTGATGAGTTAACCAGATAC-3'),反义链+1／-22(5'-TATGGCTTCGAAAATAGGCTTGA-3')和TaqMan probe-51/-33 (5'-FAM-CAGAAGACATTTCCTTCTC-MGB-3')。
七、TSA体外刺激BMMC
调制正常小鼠BMMC浓度至1×106／ml,经0,2,10,或50 nM TSA 37℃孵育24h(待BMMC FcεRI表达和功能检测)或48h(待BMMC凋亡检测)。
八、BMMC表面FcεRI表达检测
收集Elf-1 siRNA/对照siRNA转染的BMMC或TSA体外刺激的BMMC,经2.42G阻断细胞表面Fc受体后,再与PE-anti FcεRIα-链抗体4℃避光孵育1h,PBS洗细胞2遍,用FACSCalibur流式细胞仪分析细胞表面FcεRI表达。
九、BMMC凋亡检测
收集TSA体外刺激48h的BMMC,经5μg/ml propidium iodide (PI)和Annexin V-FITC(BD Biosciences)室温染色15min后,FACSCalibur流式细胞仪分析BMMC的凋亡情况。
十、肥大细胞脱颗粒能力检测
收集不同浓度TSA体外刺激的BMMC,经1μg／ml IgE 4℃致敏1h,重悬于Tyrode's缓冲液中,1×106／ml,再经1μg/ml anti-IgE37℃刺激45min后,收集上清液。加入p-nitrophenyl-N-acetyl-β-D-glucopyranoside作为底物37℃显色90min后,经酶标仪测定OD405值(ODsample)。IgE致敏细胞经2% Triton处理的培养上清液的OD值作为细胞颗粒的最大释放量(ODtotal)。IgE致敏细胞经Typrode's缓冲液孵育的上清液的OD值为ODbase。β-氨基己糖苷酶的释放量(%)=(ODsample-ODbase) /(ODtotal-ODbase)×100%。
十一、BMMC分泌合成IL-6检测
TSA体外刺激的BMMC,经1μg/ml IgE/anti-IgE体外刺激1h后,提取细胞总RNA,反转录成cDNA,经Realtime PCR检测IL-6mRNA水平；经体外刺激3h或6h后,收集培养上清,按照IL-6ELISA kit说明,检测上清中IL-6分泌水平。
十二、BMMC IL-6启动子上组蛋白乙酰化水平检测
TSA体外刺激的BMMC,经1μg/ml IgE/anti-IgE体外刺激30min后,超声破碎细胞,沉淀染色质DNA。再与Anti-acetyl-histone H3 rabbit IgG、anti-acetyl-histone H4 rabbit antiserum或rabbit IgG4℃孵育1h后,经7500Realtime PCR System(Applied Biosystems)分析乙酰化组蛋白H3和H4结合IL-6启动子的能力。所用引物为小鼠IL-6启动子(-84／-9)：正义链IL-6-84F(5'-CCCATGAGTCTCAAAATTAGAGAGTTG-3'),反义链IL-6-9R (5'-CAGAGCAGAATGAGCTACAGACATC-3')和TaqMan probe IL-6-56P (5'-CTCCTAATAAATATGAGACTGGG-3')。
实验结果
一、siRNA干扰技术阻断小鼠BMMC Elf-1的表达
1、小鼠巨噬细胞核转染试剂盒转染效果的鉴定
将Nucleofector转染仪的转染程序设定为Y-001,将FITC-标记的对照寡核苷酸转染至1×106BMMC中,37℃培养24h后,BMMC经光学显微镜和荧光显微镜下观察,结果表明此试剂盒对BMMC的转染率大约可达到70%。
2、转染后BMMC的Elf-1的表达
将Elf-1 siRNA和Control siRNA分别转染至1×106BMMC中,37℃培养24h后,Elf-1 mRNA的表达经Real-time PCR检测,结果显示Elf-1 siRNA转染的BMMC的Elf-1 mRNA表达量不足Control siRNA转染的BMMC的Elf-1mRNA表达量的1／5。转染48h后,经Western blot检测,Elf-1 siRNA转染的BMMC的Elf-1蛋白表达显著低于Control siRNA转染的BMMC的Elf-1蛋白表达,由此表明Elf-1 siRNA特异性地抑制BMMC Elf-1的转录和蛋白表达。
二、Elf-1对小鼠BMMC FcεRI基因表达调控的研究
1、Elf-1对小鼠BMMC FcεRIα-链启动子活性的影响
将携带受控于FcεRI a-链启动子(-605／+29)的荧光素酶的报告质粒pGV-B2-aNN0.6或对照质粒pGL3-Basic分别与Elf-1 siRNA/Control siRNA转染至BMMC之后,检测小鼠BMMC FcεRIα-链启动子活性。结果显示,Elf-1siRNA/pGL3-Basic共转染和Control siRNA/pGL3-Basic共转染的BMMC FcεRIα-链启动子活性均较低,而Elf-1 siRNA/pGV-B2-aNN0.6共转染的BMMC FcεRIα-链启动子活性却显著高于Control siRNA/pGV-B2-aNN0.6共转染组,这表明Elf-1的表达下降能够增强BMMC FcεRI a-链启动子的转录活性。相一致的是,Elf-1的过表达显著降低BMMC FcεRI a-链启动子的转录活性。由此提示,Elf-1对BMMCFcεRI a-链启动子活性具有抑制作用。
2、Elf-1对小鼠BMMC FcεRIα、β、γ-链mRNA表达的影响
Elf-1 siRNA/Control siRNA转染至BMMC,24h之后,Real-time PCR检测小鼠BMMC FcεRIα、β、γ-链mRNA的表达。结果显示,Elf-1 siRNA转染的BMMC FcεRI a-链mRNA表达水平显著高于Control siRNA转染的BMMC,而FcεRIβ和γ-链mRNA的表达水平两组未见显著性差异。由此提示,Elf-1能够抑制BMMCFcεRIα-链的转录水平。
3、Elf-1对转录因子PU.1向小鼠BMMC FcεRI a-链启动子募集的影响
Elf-1 siRNA/Control siRNA转染至BMMC,24h之后收集BMMC。经CHIPassay结果显示, Elf-1 siRNA转染的BMMC中与PU.1结合的FcεRI a-链启动子的量显著高于Control siRNA转染的BMMC,由此提示,Elf-1抑制了另一转录因子PU.1向BMMC FcεRIα-链启动子的募集。
4、Elf-1对小鼠BMMC表面FcεRI表达的影响
Elf-1 siRNA/Control siRNA转染至BMMC,48h之后收集BMMC。经FACS检测,结果显示,Elf-1 siRNA转染的BMMC表达FcεRI的量与Control siRNA转染的BMMC相似。由此提示,Elf-1未影响BMMC表面FcεRI的表达。
三、TSA对小鼠BMMC活化的影响
1、TSA对小鼠BMMC脱颗粒的影响
小鼠BMMC分别经0、2、10或50 nM TSA 37℃孵育24h后,经1μg／mlIgE/anti-IgE体外刺激,并检测细胞释放β-氨基己糖苷酶的量。结果显示,未经TSA孵育的BMMC的β-氨基己糖苷酶的释放可达到50.44+1.63%,TSA孵育后,BMMCβ-氨基己糖苷酶的释放以剂量依赖方式被抑制。
2、TSA对小鼠BMMC产生IL-6的影响
小鼠BMMC分别经0、2、10或50 nM TSA 37℃孵育24h后,经1μg／mlIgE/anti-IgE体外刺激1h,收集BMMC, Real-time PCR检测IL-6 mRNA的产生。结果显示,TSA以剂量依赖方式抑制小鼠FcεRI介导活化的BMMC IL-6 mRNA的合成。经1μg/ml IgE/anti-IgE体外刺激6h,收集BMMC培养上清,经ELISA方法检测IL-6的分泌情况,结果同样显示TSA以剂量依赖方式抑制小鼠FcsRI介导活化的BMMC IL-6的分泌。
3、TSA对小鼠BMMC FcεRI表达的影响
小鼠BMMC分别经0、2、10或50 nM TSA 37℃孵育24h后,经FACS检测细胞表面FcεRI的表达。结果显示,与未经TSA孵育的BMMC相比,50nM TSA孵育后的BMMC FcεRI表达降低,而2nM和10nM TSA孵育后的BMMC FcεRI表达没有明显变化。
4、TSA对小鼠BMMC凋亡的影响
小鼠BMMC分别经0、2、10或50 nM TSA 37℃孵育48h后,经FACS检测细胞的凋亡情况。结果显示,与未经TSA孵育的BMMC相比,50nM TSA孵育后Annexin V-FITC+/PI细胞即凋亡细胞明显增加,而2nM和10nM TSA孵育后凋亡细胞数未见明显变化。
5、TSA对小鼠BMMC IL-6启动子组蛋白乙酰化的影响
小鼠BMMC分别经0、2、10或50 nM TSA 37℃孵育24h后,经1μg／mlIgE/anti-IgE体外刺激0.5h,收集BMMC,沉淀DNA, CHIP assay检测结果显示,与未经TSA孵育的BMMC相比,10nM和50nM TSA均能增强小鼠FcεRI介导活化的BMMC IL-6启动子组蛋白H3和H4乙酰化的水平。
结论
1、小鼠巨噬细胞核转染试剂盒(Amaxa)同样适用于小鼠BMMC的核酸转染。
2、Elf-1 siRNA可特异性降低小鼠BMMC的Elf-1表达。
3、Elf-1通过抑制PU.1与α-链启动子之间的结合,抑制小鼠BMMC FcεRI a-链启动子的活性和转录。
4、Elf-1可抑制小鼠BMMC FcεRIa-链mRNA的表达,但对β-和γ-链的转录未见有意义的影响。
5、Elf-1单独作用尚不能影响小鼠BMMC FcεRI的表面表达。
6、TSA以剂量依赖方式抑制FcεRI-介导的BMMC活化。
7、50nM TSA通过诱导细胞凋亡降低小鼠BMMC FcεRI的表达,抑制FcεRI-介导的BMMC脱颗粒和IL-6合成分泌。
8、TSA增强小鼠FcεRI介导活化的BMMC IL-6启动子组蛋白乙酰化的水平。
Objective
Mast cells, which mainly exist in the mucous and connective tissue, are activated upon stimulation by binding IgE to the high affinity surface receptor, FcεRI, thereby leading to allergic disease. In brief, cross-linking of FcεRI with IgE/allergen complex causes immediate reaction including degranulation and the de novo synthesis of proinflammatory cytokies such as TNF-α\ IL-6 and IL-13. Therefore, mast cells play key roles in the allergic diseases by means of these bioactivators. The elucidation of regulation on the surface expression of FcεRI and the bioactivities of mast cells will be helpful to the prevention and treatment of allergic diseases.
FcεRI is composed of three subunits:the IgE-bingding a-chain, and signal-transducingβ-and y-chain. In our previous studies, Elf-1 was found to recognize the nucleotide sequence around the cis-element of the FcεRI a-chain promoter in vitro. Elf-1 is a transcription factor belonging to the Ets family, which possesses a highly conserved DNA-binding region termed the Ets domain. Elf-1 cDNA was identified from human T cells as a cell-type specific transcription factor that enhanced the HIV-2 promoter. Subsequently, Elf-1 was shown to be involved in the expression of various immunorelated genes, including interleukin (IL)-2, GM-CSF, IL-5, IL-2RA, and CD4 in T cells; immunoglobulin heavy chain, blk, lyn, and CD1D1 in B cells; IL-3 in megakaryocytes; and stem cell leukemia gene in mast cells. Recently Elf-1 has been reported to inhibit the gene expression of FcεRI y-chain in human basophilic cell line KU812. However, it is remain unknown whether Elf-1, which binds to the promoter of FcεRI a-chain, can influence the the gene expression of FcεRI in mouse mast cells.
In addition to transcription factors, the expression of genes is associated with the status of histone acetylation in the chromatin, which is controlled by two key regulators, histone acetyltransferase (HAT), and histone deacetylase (HDAC). Trichostatin A (TSA), one of the most potent HDAC inhibitors (HDACi), belongs to the hydroxamic acids. Increasing evidence suggests that TSA is a potential anticancer agent as it was found to inhibit the growth of and to induce differentiation and apoptosis of tumor cells. TSA and another HDACi, suberoylanilide hydroxamic acid (SAHA), have also been shown to be effective in immune disease rodent models, including SLE, graft-versus-host-disease, rheumatoid arthritis, and experimental autoimmune encephalomyelitis. In addition, there is a report of therapeutic usage of TSA on allergic mouse model. However, the effects of TSA on the surface expression of FcεRI and related functions of mast cell, a key element in immune responses, have been rarely examined.
In this study, we use mouse bone marrow-derived mast cells (BMMC) transfected with Elf-1 siRNA or stimulated by TSA in vitro in order to investigate the role of Elf-1 and TSA on the gene expression of FcεRI on BMMC and its related bioactivities. It would provide new therapeutic approach for mast cell-mediated allergic diseases.
Methods
1、Preparation of mouse BMMC
Mouse BMMC were obtained by culturing femoral bone marrow cells from 6-8-week-old female BALB/c mice in BMMC culture medium (RPMI-1640 medium supplemented with 10% heat-inactivated FBS,100μM 2-mercaptoethanol,10 mM sodium pyruvate,10μM MEM nonessential amino acid olution,100 U/ml penicillin, 100μg/ml streptomycin and 10% pokeweed-mitogen-stimulated spleen-conditioned medium). In brief, both femurs were separated, and bone marrow cells were collected by rinse and centrifugation at 1000rpm for 5min at 4℃. The cells were cultured with BMMC culture medium for 4 weeks.
2、Transfection with Elf-1 siRNA and Elf-1 expression plasmid in vitro
Five microliters of 20μM double-stranded Elf-1 siRNA, control siRNA and FITC-labeled control oligonucleotide was introduced into 1×106 of BMMC for 24h (for detection of transfection rate, gene expression of Elf-1, FcεRIα-,β-, and y-chain) and 48h (for detection of Elf-1 protein expression) with Nucleofector II set at program Y-001 using a Mouse Macrophage Nucleofector Kit according to the manufacturer's instructions. One microgram of pGV-B2-aNN0.6 (reporter plasmid), or pGL3-Basic (promoter less control) was cotransfected with 25ng pRL-CMV (internal control for normalization of transfection efficiency) and siRNA into BMMC by Nucleofector II as described above. For overexpression of Elf-1, 10μg of pCR-Elf-1 or pCR3.1 control plasmid was introduced into BMMC with 5μg of reporter plasmid and 25ng of pRL-CMV by electroporation using Bio-Rad Gene PulserⅡ. The transfected cells were cultured for 24h at 37℃(for detection of the activity of the promoter of FcεRI a-chain).
3、The detection of Elf-1 expression
After 24 h of culture after electroporation, mRNA levels of Elf-1 were analyzed by real-time polymerase chain reaction (PCR). In brief, total RNA from cells was extracted using an RNeasy Micro Kit, and first-strand cDNA was synthesized from 1μg of total RNA using the High Capacity cDNA Reverse Transcription Kit.Quantitative PCR was performed using TaqMan Universal PCR Master Mix and a 7500 Real-Time PCR System with TaqMan Gene Expression. Assays of the mouse target genes, including Elf-1 (Mm00468217_ml) and the endogenous control glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Rodent GAPDH Control Reagents VIC Probe). Expression levels of Elf-1 were expressed relative to those of GAPDH by calculating cycle threshold (Ct) values in amplification plots with 7500 SDS software. In addition, After 48h of culture after electroporation,1×106 BMMC were collected, and whole cell lysates were subjected to electrophoresis on a 7.5% SDS polyacrylamide gel. Anti-Elf-1 antibody and anti-actin Ab were used as primary Abs. Alexa Fluor 680 goat antirabbit IgG and IR Dye 800 goat anti-mouse IgG (Molecular Probes) were used as secondary Abs against Elf-1 and actin, respectively. Infrared fluorescence on membranes was detected using the Odyssey infrared imaging system. The amount of protein was quantified by calculating band intensity using the Odyssey software.
4、The detection of promoter activities of FcεRI a-chain in BMMC
After 24h of cultivation, BMMC were harvested and treated with a Dual-luciferase assay kit for the measurement of luciferase activity. The luminescence was measured with Micro Lumat Plus.
5、The detection of mRNA expression of FcεRIα-,β-and y-chain
After 24 h of culture after electroporation, mRNA levels of FcεRIα-,β-and y-chain were analyzed by real-time polymerase chain reaction (PCR). In brief, total RNA from cells was extracted using an RNeasy Micro Kit, and first-strand cDNA was synthesized from 1μg of total RNA using the High Capacity cDNA Reverse Transcription Kit. Quantitative PCR was performed using TaqMan Universal PCR Master Mix and a 7500 Real-Time PCR System with TaqMan Gene Expression. Assays of the mouse target genes, including FcεRI a-chain (Mm00438867_ml), the FcεRI p-chain (Mm00442780_ml), the FcεRIγ-chain (Mm00438869_gl), and the endogenous control glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Rodent GAPDH Control Reagents VIC Probe). Expression levels of FcεRIα-,β-, and y-chain were expressed relative to those of GAPDH by calculating cycle threshold (Ct) values in amplification plots with 7500 SDS software.
6、The detection of PU.1 binding to FcεRIα-chain promoter
BMMC transfected with siRNA were collected and lysis by ultrasonication. Chromatin DNA was precipitated and chromatin immunoprecipitation (ChIP) assay was performed. Anti-PU.1 goat IgG Ab and goat IgG were used. Quantitative PCR was performed using TaqMan Universal PCR Master Mix and a 7500 Real-Time PCR System. The following primers and TaqMan probe sequences were used for this analysis:for the promoter region of the a-chain gene,-82/+1, forward primer-82/-56 (5'-GGCATAGCTGATGAGTTAACCAGATAC-3'), reverse primer+1/-22 (5'-TATGGCTTCGAAAATAGGCTTGA-3'), and TaqMan probe-51/-33 (5'-FAMCAGAAGACATTTCCTTCTC-MGB-3').
7、BMMC were stimulated with TSA in vitro
BMMC from BALB/c mouse was adjusted to 1×106/ml and stimulated with TSA at the final concentration of 0,2,10 and 50nM at 37℃for 24h (for detection of expression of FcεRI and functions of BMMC) and 48h (for detection of apoptosis of BMMC), respectively.
8、The detection of the expression of FcεRI on BMMC
BMMC transfected with siRNA or stimulated with TSA were collected and then incubated with PE-labeled anti-FcεRI a-chain Ab for 1 h at 4℃after blocking Fc receptors on the cell surface with 2.4G2. Cells were washed with PBS twice. Then the expression of FcεRI on BMMC was analyzed using a FACSCalibur flow cytometer.
9、The detection of the apoptosis of BMMC
BMMC stimulated with TSA were collected. For analysis of apoptotic cells, BMMC were stained with 5μg/ml propidium iodide (PI) and Annexin V-FITC for 15min at room temperature. Cells were analyzed using a FACSCalibur flow cytometer.
10、β-hexosaminidase release assay
After incubation of BMMC with the indicated concentration of TSA for 24 h at 37℃in the complete culture medium, the cells were sensitized with 1μg/ml mouse IgE for 1 h at 4℃and then resuspended in Tyrode's buffer at a concentration of 1 x 106 cells/ml, and stimulated with 1μg/ml anti-mouse IgE for 45 min at 37℃. The cell supernatants were collected, andβ-hexosaminidase activity in the supernatants and cell lysates was quantified by spectrophotometric analysis of hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucopyranoside. The percentage ofβ-hexosaminidase release was calculated using the following formula:percent release (%)=(OD of the stimulated supernatant-OD of the unstimulated supernatant)/(OD of the total cell lysate-OD of the unstimulated supernatant)×100%.
11、Measurement of IL-6 production from BMMC
After incubation with TSA for 24h at 37℃, BMMC at concentration of 1 x 106/ml were stimulated with IgE/anti-IgE cross-linking for 1h at 37℃. The total RNA was collected and reversed transcripted into cDNA. The expression of IL-6 mRNA was analyzed by Real-time PCR. TSA incubated BMMC were stimulated with IgE/anti-IgE cross-linking for 6h at 37℃and the supernatant were collected. The concentration of IL-6 in culture supernatant was determined by an ELISA kit according to the manufacturer's instructions.
12、The detection of histone acetylation of IL-6 promoter in BMMC
BMMC incubated with TSA were collected and then stimulated with IgE/anti-IgE cross-linking for 30min at 37℃. Cells were lysis by ultrasonication. Chromatin DNA was precipitated and chromatin immunoprecipitation (ChIP) assay was performed. Anti-acetyl-histone H3 rabbit IgG, anti-acetyl-histone H4 rabbit antiserum and rabbit IgG were used. Quantitative PCR was performed using TaqMan Universal PCR Master Mix and a 7500 Real-Time PCR System. The following primers and TaqMan probe sequences were used for this analysis:for the mouse IL-6 promoter (-84/-9), forward primer IL-6-84F 5'-CCCATGAGTCTCAAAATTAGAGAGTTG-3'), reverse primer IL-6-9R (5'-CAGAGCAGAATGAGCTACAGACATC-3'), and TaqMan probe IL-6-56P (5'-CTCCTAATAAATATGAGACTGGG-3').
Results
1、Elf-1 expression on mouse BMMC knockdown by siRNA transfection
(1) The detection of transfection efficiency of Mouse Macrophage Nucleofector Kit
The FITC-labeled control oligonucleotides were introduced into BMMC with Nucleofector II set at program Y-001 using a Mouse Macrophage Nucleofector Kit. Transfection efficiency was confirmed to be approximately 70% by fluorescence microscopy and phase microscopy.
(2) The expression of Elf-1 in siRNA transfected BMMC
BMMC were transfected with Elf-1 siRNA and Control siRNA respectively.24h later, expression levels of Elf-1 mRNA were determined by real-time PCR. The amount of Elf-1 transcripts in Elf-1 siRNA-treated BMMC was less than one fifth of that in control BMMC.48h later, expression levels of Elf-1 protein were determined by Western blot. The band intensity of Elf-1 protein was markedly reduced by Elf-1 siRNA as compared with control siRNA-treated BMMC. These results indicate that Elf-1 siRNA generated in the present study successfully down regulated Elf-1 expression in BMMC.
2、The effects of Elf-1 on the gene expression of FcεRI in mouse BMMC
(1) The effects of Elf-1 on the promoter activity of FcεRI a-chain in mouse BMMC
To evaluate the effects of Elf-1 on FcεRI a-chain promoter activity, a reporter plasmid, pGV-B2-αNN0.6, carrying the luciferase gene under the control of the FcεRI a-chain promoter was introduced into BMMC in addition to the above-mentioned siRNA. When Elf-1 siRNA was co-introduced into BMMC with the a-chain reporter plasmid, luciferase activity derived from BMMC was significantly increased in comparison with that of control siRNA transfectants, whereas luciferase activity driven by the promoterless control plasmid, pGL3-Basic, was not markedly affected by Elf-1 siRNA. In addition, overexpression of Elf-1 by co-introduction of the Elf-1 expression plasmid decreased FcεRI a-chain-driven luciferase activity in BMMC in Elf-1 binding sequence-dependent manner. These results suggest that Elf-1 exhibits suppressive effect on the FcεRI a-chain promoter in BMMC.
(2) The effects of Elf-1 on FcεRIα-,β-, and y-chain transcription in mouse BMMC
After transfection with siRNA for 24h, the mRNA expression of FcεRIα-,β-, andγ-chain were analyzed by Real-time PCR. The amount of a-chain mRNA in Elf-1 siRNA-treated BMMC was significantly higher than that of control siRNA-treated BMMC. In contrast, significant differences were not observed inβ-andγ-chain between BMMC transfected with Elf-1 or control siRNA. Based on these results, Elf-1 leads to specific inhibition of FcεRI a-chain transcription.
(3) The effects of Elf-1 on recruitment of PU.1 toward FcεRI a-chain promoter in mouse BMMC
After transfection with siRNA for 24h, BMMC were performed to CHIP assay. Compared with that in control siRNA transfected BMMC, the amount of a-chain promoter region bound with PU.1 significantly increased in Elf-1 siRNA-treated BMMC, suggesting that Elf-1 specifically inhibits recruitment of transactivator PU.1 toward the a-chain promoter in mouse BMMC.
(4) The effects of Elf-1 on the surface expression of FcεRI on mouse BMMC
After transfection with siRNA for 48h, FACS was performed to detect the surface expression of FcεRI on mouse BMMC. Similar expression of FcεRI were shown on Elf-1 siRNA and control siRNA transfected BMMC, indicating that Elf-1 has little effect on the surface expression of FcεRI.
3、The effect of TSA on the activation of mouse BMMC
(1) The effects of TSA on the degranulation of mouse BMMC
Mouse BMMC were treated with TSA at the concentration of 0,2,10 and 50nM at 37℃for 24h before sensitization with IgE and then stimulated with anti-IgE Ab. The release ofβ-hexosaminidase was determined. The result indicated that TSA treatment significantly suppressedβ-hexosaminidase release from BMMC activated by IgE/FcεRI cross-linking in a dose-dependent manner, whereas the degranulation ability reached approximately 50% in the absence of TSA.
(2) The effects of TSA on the production of IL-6 in mouse BMMC
Mouse BMMC were treated with TSA at the concentration of 0,2,10 and 50nM at 37℃for 24h before sensitization with IgE and then stimulated with anti-IgE Ab. TSA treatment significantly suppressed the expression of IL-6 mRNA in BMMC determined by Real-time PCR and secretion of IL-6 in the supernatant of BMMC determined by ELISA.
(3) The effects of TSA on the surface expression of FcεRI on mouse BMMC
Mouse BMMC were treated with TSA at the concentration of 0,2,10 and 50nM at 37℃for 24h and then performed to FACS to detect the expression of FcεRI. In comparison to the TSA unstimulated BMMC, only 50nM TSA treatment led to the reduced expression of FcεRI, whereas,2 and 10nM TSA treatment did not.
(4) The effects of TSA on the apoptosis of mouse BMMC
Mouse BMMC were treated with TSA at the concentration of 0,2,10 and 50nM at 37℃for 48h and then performed to FACS to detect the apoptosis. In comparison to the TSA unstimulated BMMC, only 50nM TSA treatment led to the increased number of apoptotic cells, whereas,2 and 1 OnM TSA treatment did not.
(5) The effects of TSA on the histone acetylation of IL-6 promoter of mouse BMMC
Mouse BMMC were treated with TSA at the concentration of 0,2,10 and 50nM at 37℃for 24h before sensitization with IgE and then stimulated with anti-IgE Ab for 30min, and then performed to CHIP assay to detect the histone acetlylation of IL-6 promoter. In comparison to the TSA unstimulated BMMC,10 and 50nM TSA treatment led to the increased amount of acetylated H3 and H4 toward to mouse BMMC IL-6 promoter.
Conclusions
1、Mouse Macrophage Nucleofecter Kit (Amaxa) is suitable for mouse BMMC transfection.
2、Elf-1 siRNA can specifically knockdown the expression of Elf-1 in BMMC.
3、Elf-1 suppressed the mouse BMMC FcεRI a-chain promoter activity and transcription by inhibition of recruitment of PU.1 towards to a-chain promoter.
4、Elf-1 inhibited the mRNA expression of FcεRI a-chain, but not that ofβ-and y-chain.
5、Elf-1 alone had little effect on the surface expression of FcεRI on mouse BMMC.
6、TSA suppressed FcεRI-mediated activation of mouse BMMC in a dose-dependent mode.
7、50nM TSA treatment led to FcεRI-mediated degranulation and IL-6 production in BMMC supposedly by induction of apoptosis of BMMC and subsequently reduced expression of FcεRI on mouse BMMC.
8、TSA induced the accumulation of acetylated histones on the mouse IL-6 promoter in BMMC activated by IgE/FcεRI cross-linking.
肥大细胞主要存在于粘膜和结缔组织中,借助于其表面表达的IgE的高亲合力受体FcεRI与抗变应原的特异性抗体IgE的交联,形成IgE/FcsRI复合物,进而引发Ⅰ型变态反应。肥大细胞胞浆中存在大量颗粒,其内储存组胺等生物介质,活化的肥大细胞也可新合成细胞因子如TNF-α、IL-6和IL-13等生物介质。正是借助于这些生物活性介质,肥大细胞在变态反应性疾病过程中发挥其生物学作用。因此,肥大细胞表面受体FcsRI表达以及其生物学功能调控机制的解析将为变态反应的防治奠定理论基础。
FcεRI由α-,β-和γ-链三部分构成,其中α-链是IgE特异性结合的亚基,p-和γ-链参与信号的转导。我们的前期试验结果显示转录因子Elf-1在体外可以结合到FcεRIα-链启动子的顺式元件周围的核苷酸序列。Elf-1是Ets转录因子家族成员之一,通过结合细胞核内的DNA序列,调节各种基因表达的诱导。Elf-1最初是从人类T细胞库克隆出来的一细胞型特异性转录因子,主要增强HIV-2启动子的转录。然而之后的研究发现,Elf-1在各种造血干细胞和免疫相关细胞中也可以呈现高表达,并且调控多种基因的表达,如T细胞中IL-2、GM-CSF、IL-5、IL-2RA和CD4；B细胞中免疫球蛋白重链、blk、lyn和CD1D1；巨核细胞IL-3和肥大细胞的干细胞白血病基因(SCL)。最近报道显示在人类嗜碱性粒细胞系KU812中,Elf-1还可抑制FcεRIγ-链的表达。而在FcεRIα-链启动子上具有结合位点的Elf-1是否能够影响小鼠肥大细胞FcεRI的基因表达,目前尚不可知。
除转录因子外,基因表达调控还与染色体上组蛋白乙酰化的状态有关。这种状态受控于两种关键的调节因子,即组蛋白乙酰化酶(HAT)和组蛋白去乙酰化酶(HDAC)。曲古菌素A(TSA),是一种强效的HDAC抑制剂(HDACi),属于羟肟酸类。研究显示TSA作为一种有效的抗癌制剂,能够抑制肿瘤细胞的生长,或者诱导肿瘤细胞的分化和凋亡。另外,TSA和另一种HDACi, SAHA,还可通过调节各种基因的表达而参与了免疫性疾病,如,系统性红斑狼疮(SLE)、移植物抗宿主病、类风湿性关节炎等。尤其在变态反应的小鼠模型中,TSA还显示了其潜在治疗效应。然而,TSA是否能够影响变态反应的重要效应细胞——肥大细胞表面FcεRI的表达及其功能,目前尚未见相关报道。
为此,本研究拟利用EIf-1 siRNA转染技术和体外TSA刺激等方式,探讨转录因子Elf-1和组蛋白去乙酰化酶抑制剂TSA对小鼠骨髓来源的肥大细胞(BMMC)表面FcεRI基因表达及其生物学功能的调控作用机制。进而,为肥大细胞相关的变态反应的研究提供新的实验依据。
实验方法
一、小鼠BMMC的制备
BALB/c小鼠,雌性,6-8周龄。钝性分离小鼠双侧股骨,用1ml注射器吸取少量RPMI-1640培养液冲洗骨髓内细胞至无菌平皿中。收集液体至10ml离心管中,1000rpm,4℃离心5min,弃上清。用10m1BMMC培养液重悬细胞,移至10cm无菌培养皿,37℃培养。常规换液,培养4周待用。
二、Elf-1 siRNA和Elf-1表达质粒体外转染
按照Mouse Macrophage Nucleofector试剂盒说明操作,选择Y-001程序,分别将20μM Elf-1 siRNA、Control siRNA和FITC-标记的寡核苷酸经电转至小鼠1×106BMMC中,37℃分别培养24h(待试剂盒转染效率、Elf-1 mRNA表达和FcεRIα、p、γ-链mRNA表达检测)和48h(待Elf-1蛋白表达检测)。同法将1μgpGV-B2-aNN0.6或pGL3-Basic分别与25ng pRL-CMV(内参)和20μM Elf-1 siRNA/Control siRNA共同电转染至1×106BMMC中；另将5μg pGV-B2-aNN0.6或pGL3-Basic分别与25ng pRL-CMV(内参)和10μgpCR3.1-Elf-1或对照pCR3.1经Bio-Rad Gene PulserⅡ电转染至BMMC中,37℃孵育24h后,收集BMMC(待FcεRI a-链启动子荧光素酶活性的分析)。
三、Elf-1表达鉴定
收集24h Elf-1及Control siRNA核转染的BMMC,按照RNeasy(?) Micro试剂盒的说明,提取细胞总RNA,利用High Capacity cDNA Reverse Transcription试剂盒反转录合成cDNA第一链,利用TaqMan Universal PCR Master Mix和7500Real-time PCR仪进行Real-time PCR,分析靶基因Elf-1 mRNA表达。Elf-1引物为(Mm00468217_ml)。另收集48h Elf-1及Control siRNA转染的BMMC,裂解细胞后,经7.5%SDS聚丙烯酰胺凝胶电泳,电转膜1.5h后,用anti-Elf-1 Ab和anti-actinAb作为第一抗体4℃过夜孵育。洗膜后用Alexa Fluor 680 goat anti-rabbit IgG和IRDye 800 goat anti-mouse IgG分别作为抗Elf-1和actin的第二抗体进行孵育1h,洗膜后用Odyssey infrared imaging system检测Elf-1蛋白的表达。
四、BMMC FcεRIα-链启动子活性检测
收集转染后的BMMC,按照Dualluciferase assay试剂盒说明,经Micro LumatPlus分析FcεRIα-链启动子荧光素酶活性。
五、FcεRIa-、β-和γ-链mRNA表达水平的检测
收集Elf-1 siRNA/Control siRNA转染后的BMMC。利用RNeasy Micro Kit提取总RNA,利用High Capacity cDNA Reverse Transcription Kit合成cDNA第一链,利用TaqMan Universal PCR Master Mix和7500 Real-Time PCR System with TaqMan Gene Expression,进行Realtime PCR,分析靶基因α-链(Mm00438867_ml)、p-链(Mm00442780_m1)和γ-链(Mm00438869_g1)mRNA表达水平。
六、PU.1结合FcεRIα-链启动子能力检测
收集Elf-1 siRN A/Control siRNA转染后的BMMC,经超声破碎细胞,沉淀染色质DNA。再分别与Anti-PU.1 goat Ab和goat IgG4℃孵育1h后,经7500Realtime PCR System分析PU.1结合FcεRIα-链启动子的能力。所用引物为小鼠FcεRIα-链启动子(-82／+1)：正义链-82／-56(5'-GGCATAGCTGATGAGTTAACCAGATAC-3'),反义链+1／-22(5'-TATGGCTTCGAAAATAGGCTTGA-3')和TaqMan probe-51/-33 (5'-FAM-CAGAAGACATTTCCTTCTC-MGB-3')。
七、TSA体外刺激BMMC
调制正常小鼠BMMC浓度至1×106／ml,经0,2,10,或50 nM TSA 37℃孵育24h(待BMMC FcεRI表达和功能检测)或48h(待BMMC凋亡检测)。
八、BMMC表面FcεRI表达检测
收集Elf-1 siRNA/对照siRNA转染的BMMC或TSA体外刺激的BMMC,经2.42G阻断细胞表面Fc受体后,再与PE-anti FcεRIα-链抗体4℃避光孵育1h,PBS洗细胞2遍,用FACSCalibur流式细胞仪分析细胞表面FcεRI表达。
九、BMMC凋亡检测
收集TSA体外刺激48h的BMMC,经5μg/ml propidium iodide (PI)和Annexin V-FITC(BD Biosciences)室温染色15min后,FACSCalibur流式细胞仪分析BMMC的凋亡情况。
十、肥大细胞脱颗粒能力检测
收集不同浓度TSA体外刺激的BMMC,经1μg／ml IgE 4℃致敏1h,重悬于Tyrode's缓冲液中,1×106／ml,再经1μg/ml anti-IgE37℃刺激45min后,收集上清液。加入p-nitrophenyl-N-acetyl-β-D-glucopyranoside作为底物37℃显色90min后,经酶标仪测定OD405值(ODsample)。IgE致敏细胞经2% Triton处理的培养上清液的OD值作为细胞颗粒的最大释放量(ODtotal)。IgE致敏细胞经Typrode's缓冲液孵育的上清液的OD值为ODbase。β-氨基己糖苷酶的释放量(%)=(ODsample-ODbase) /(ODtotal-ODbase)×100%。
十一、BMMC分泌合成IL-6检测
TSA体外刺激的BMMC,经1μg/ml IgE/anti-IgE体外刺激1h后,提取细胞总RNA,反转录成cDNA,经Realtime PCR检测IL-6mRNA水平；经体外刺激3h或6h后,收集培养上清,按照IL-6ELISA kit说明,检测上清中IL-6分泌水平。
十二、BMMC IL-6启动子上组蛋白乙酰化水平检测
TSA体外刺激的BMMC,经1μg/ml IgE/anti-IgE体外刺激30min后,超声破碎细胞,沉淀染色质DNA。再与Anti-acetyl-histone H3 rabbit IgG、anti-acetyl-histone H4 rabbit antiserum或rabbit IgG4℃孵育1h后,经7500Realtime PCR System(Applied Biosystems)分析乙酰化组蛋白H3和H4结合IL-6启动子的能力。所用引物为小鼠IL-6启动子(-84／-9)：正义链IL-6-84F(5'-CCCATGAGTCTCAAAATTAGAGAGTTG-3'),反义链IL-6-9R (5'-CAGAGCAGAATGAGCTACAGACATC-3')和TaqMan probe IL-6-56P (5'-CTCCTAATAAATATGAGACTGGG-3')。
实验结果
一、siRNA干扰技术阻断小鼠BMMC Elf-1的表达
1、小鼠巨噬细胞核转染试剂盒转染效果的鉴定
将Nucleofector转染仪的转染程序设定为Y-001,将FITC-标记的对照寡核苷酸转染至1×106BMMC中,37℃培养24h后,BMMC经光学显微镜和荧光显微镜下观察,结果表明此试剂盒对BMMC的转染率大约可达到70%。
2、转染后BMMC的Elf-1的表达
将Elf-1 siRNA和Control siRNA分别转染至1×106BMMC中,37℃培养24h后,Elf-1 mRNA的表达经Real-time PCR检测,结果显示Elf-1 siRNA转染的BMMC的Elf-1 mRNA表达量不足Control siRNA转染的BMMC的Elf-1mRNA表达量的1／5。转染48h后,经Western blot检测,Elf-1 siRNA转染的BMMC的Elf-1蛋白表达显著低于Control siRNA转染的BMMC的Elf-1蛋白表达,由此表明Elf-1 siRNA特异性地抑制BMMC Elf-1的转录和蛋白表达。
二、Elf-1对小鼠BMMC FcεRI基因表达调控的研究
1、Elf-1对小鼠BMMC FcεRIα-链启动子活性的影响
将携带受控于FcεRI a-链启动子(-605／+29)的荧光素酶的报告质粒pGV-B2-aNN0.6或对照质粒pGL3-Basic分别与Elf-1 siRNA/Control siRNA转染至BMMC之后,检测小鼠BMMC FcεRIα-链启动子活性。结果显示,Elf-1siRNA/pGL3-Basic共转染和Control siRNA/pGL3-Basic共转染的BMMC FcεRIα-链启动子活性均较低,而Elf-1 siRNA/pGV-B2-aNN0.6共转染的BMMC FcεRIα-链启动子活性却显著高于Control siRNA/pGV-B2-aNN0.6共转染组,这表明Elf-1的表达下降能够增强BMMC FcεRI a-链启动子的转录活性。相一致的是,Elf-1的过表达显著降低BMMC FcεRI a-链启动子的转录活性。由此提示,Elf-1对BMMCFcεRI a-链启动子活性具有抑制作用。
2、Elf-1对小鼠BMMC FcεRIα、β、γ-链mRNA表达的影响
Elf-1 siRNA/Control siRNA转染至BMMC,24h之后,Real-time PCR检测小鼠BMMC FcεRIα、β、γ-链mRNA的表达。结果显示,Elf-1 siRNA转染的BMMC FcεRI a-链mRNA表达水平显著高于Control siRNA转染的BMMC,而FcεRIβ和γ-链mRNA的表达水平两组未见显著性差异。由此提示,Elf-1能够抑制BMMCFcεRIα-链的转录水平。
3、Elf-1对转录因子PU.1向小鼠BMMC FcεRI a-链启动子募集的影响
Elf-1 siRNA/Control siRNA转染至BMMC,24h之后收集BMMC。经CHIPassay结果显示, Elf-1 siRNA转染的BMMC中与PU.1结合的FcεRI a-链启动子的量显著高于Control siRNA转染的BMMC,由此提示,Elf-1抑制了另一转录因子PU.1向BMMC FcεRIα-链启动子的募集。
4、Elf-1对小鼠BMMC表面FcεRI表达的影响
Elf-1 siRNA/Control siRNA转染至BMMC,48h之后收集BMMC。经FACS检测,结果显示,Elf-1 siRNA转染的BMMC表达FcεRI的量与Control siRNA转染的BMMC相似。由此提示,Elf-1未影响BMMC表面FcεRI的表达。
三、TSA对小鼠BMMC活化的影响
1、TSA对小鼠BMMC脱颗粒的影响
小鼠BMMC分别经0、2、10或50 nM TSA 37℃孵育24h后,经1μg／mlIgE/anti-IgE体外刺激,并检测细胞释放β-氨基己糖苷酶的量。结果显示,未经TSA孵育的BMMC的β-氨基己糖苷酶的释放可达到50.44+1.63%,TSA孵育后,BMMCβ-氨基己糖苷酶的释放以剂量依赖方式被抑制。
2、TSA对小鼠BMMC产生IL-6的影响
小鼠BMMC分别经0、2、10或50 nM TSA 37℃孵育24h后,经1μg／mlIgE/anti-IgE体外刺激1h,收集BMMC, Real-time PCR检测IL-6 mRNA的产生。结果显示,TSA以剂量依赖方式抑制小鼠FcεRI介导活化的BMMC IL-6 mRNA的合成。经1μg/ml IgE/anti-IgE体外刺激6h,收集BMMC培养上清,经ELISA方法检测IL-6的分泌情况,结果同样显示TSA以剂量依赖方式抑制小鼠FcsRI介导活化的BMMC IL-6的分泌。
3、TSA对小鼠BMMC FcεRI表达的影响
小鼠BMMC分别经0、2、10或50 nM TSA 37℃孵育24h后,经FACS检测细胞表面FcεRI的表达。结果显示,与未经TSA孵育的BMMC相比,50nM TSA孵育后的BMMC FcεRI表达降低,而2nM和10nM TSA孵育后的BMMC FcεRI表达没有明显变化。
4、TSA对小鼠BMMC凋亡的影响
小鼠BMMC分别经0、2、10或50 nM TSA 37℃孵育48h后,经FACS检测细胞的凋亡情况。结果显示,与未经TSA孵育的BMMC相比,50nM TSA孵育后Annexin V-FITC+/PI细胞即凋亡细胞明显增加,而2nM和10nM TSA孵育后凋亡细胞数未见明显变化。
5、TSA对小鼠BMMC IL-6启动子组蛋白乙酰化的影响
小鼠BMMC分别经0、2、10或50 nM TSA 37℃孵育24h后,经1μg／mlIgE/anti-IgE体外刺激0.5h,收集BMMC,沉淀DNA, CHIP assay检测结果显示,与未经TSA孵育的BMMC相比,10nM和50nM TSA均能增强小鼠FcεRI介导活化的BMMC IL-6启动子组蛋白H3和H4乙酰化的水平。
结论
1、小鼠巨噬细胞核转染试剂盒(Amaxa)同样适用于小鼠BMMC的核酸转染。
2、Elf-1 siRNA可特异性降低小鼠BMMC的Elf-1表达。
3、Elf-1通过抑制PU.1与α-链启动子之间的结合,抑制小鼠BMMC FcεRI a-链启动子的活性和转录。
4、Elf-1可抑制小鼠BMMC FcεRIa-链mRNA的表达,但对β-和γ-链的转录未见有意义的影响。
5、Elf-1单独作用尚不能影响小鼠BMMC FcεRI的表面表达。
6、TSA以剂量依赖方式抑制FcεRI-介导的BMMC活化。
7、50nM TSA通过诱导细胞凋亡降低小鼠BMMC FcεRI的表达,抑制FcεRI-介导的BMMC脱颗粒和IL-6合成分泌。
8、TSA增强小鼠FcεRI介导活化的BMMC IL-6启动子组蛋白乙酰化的水平。
Objective
Mast cells, which mainly exist in the mucous and connective tissue, are activated upon stimulation by binding IgE to the high affinity surface receptor, FcεRI, thereby leading to allergic disease. In brief, cross-linking of FcεRI with IgE/allergen complex causes immediate reaction including degranulation and the de novo synthesis of proinflammatory cytokies such as TNF-α\ IL-6 and IL-13. Therefore, mast cells play key roles in the allergic diseases by means of these bioactivators. The elucidation of regulation on the surface expression of FcεRI and the bioactivities of mast cells will be helpful to the prevention and treatment of allergic diseases.
FcεRI is composed of three subunits:the IgE-bingding a-chain, and signal-transducingβ-and y-chain. In our previous studies, Elf-1 was found to recognize the nucleotide sequence around the cis-element of the FcεRI a-chain promoter in vitro. Elf-1 is a transcription factor belonging to the Ets family, which possesses a highly conserved DNA-binding region termed the Ets domain. Elf-1 cDNA was identified from human T cells as a cell-type specific transcription factor that enhanced the HIV-2 promoter. Subsequently, Elf-1 was shown to be involved in the expression of various immunorelated genes, including interleukin (IL)-2, GM-CSF, IL-5, IL-2RA, and CD4 in T cells; immunoglobulin heavy chain, blk, lyn, and CD1D1 in B cells; IL-3 in megakaryocytes; and stem cell leukemia gene in mast cells. Recently Elf-1 has been reported to inhibit the gene expression of FcεRI y-chain in human basophilic cell line KU812. However, it is remain unknown whether Elf-1, which binds to the promoter of FcεRI a-chain, can influence the the gene expression of FcεRI in mouse mast cells.
In addition to transcription factors, the expression of genes is associated with the status of histone acetylation in the chromatin, which is controlled by two key regulators, histone acetyltransferase (HAT), and histone deacetylase (HDAC). Trichostatin A (TSA), one of the most potent HDAC inhibitors (HDACi), belongs to the hydroxamic acids. Increasing evidence suggests that TSA is a potential anticancer agent as it was found to inhibit the growth of and to induce differentiation and apoptosis of tumor cells. TSA and another HDACi, suberoylanilide hydroxamic acid (SAHA), have also been shown to be effective in immune disease rodent models, including SLE, graft-versus-host-disease, rheumatoid arthritis, and experimental autoimmune encephalomyelitis. In addition, there is a report of therapeutic usage of TSA on allergic mouse model. However, the effects of TSA on the surface expression of FcεRI and related functions of mast cell, a key element in immune responses, have been rarely examined.
In this study, we use mouse bone marrow-derived mast cells (BMMC) transfected with Elf-1 siRNA or stimulated by TSA in vitro in order to investigate the role of Elf-1 and TSA on the gene expression of FcεRI on BMMC and its related bioactivities. It would provide new therapeutic approach for mast cell-mediated allergic diseases.
Methods
1、Preparation of mouse BMMC
Mouse BMMC were obtained by culturing femoral bone marrow cells from 6-8-week-old female BALB/c mice in BMMC culture medium (RPMI-1640 medium supplemented with 10% heat-inactivated FBS,100μM 2-mercaptoethanol,10 mM sodium pyruvate,10μM MEM nonessential amino acid olution,100 U/ml penicillin, 100μg/ml streptomycin and 10% pokeweed-mitogen-stimulated spleen-conditioned medium). In brief, both femurs were separated, and bone marrow cells were collected by rinse and centrifugation at 1000rpm for 5min at 4℃. The cells were cultured with BMMC culture medium for 4 weeks.
2、Transfection with Elf-1 siRNA and Elf-1 expression plasmid in vitro
Five microliters of 20μM double-stranded Elf-1 siRNA, control siRNA and FITC-labeled control oligonucleotide was introduced into 1×106 of BMMC for 24h (for detection of transfection rate, gene expression of Elf-1, FcεRIα-,β-, and y-chain) and 48h (for detection of Elf-1 protein expression) with Nucleofector II set at program Y-001 using a Mouse Macrophage Nucleofector Kit according to the manufacturer's instructions. One microgram of pGV-B2-aNN0.6 (reporter plasmid), or pGL3-Basic (promoter less control) was cotransfected with 25ng pRL-CMV (internal control for normalization of transfection efficiency) and siRNA into BMMC by Nucleofector II as described above. For overexpression of Elf-1, 10μg of pCR-Elf-1 or pCR3.1 control plasmid was introduced into BMMC with 5μg of reporter plasmid and 25ng of pRL-CMV by electroporation using Bio-Rad Gene PulserⅡ. The transfected cells were cultured for 24h at 37℃(for detection of the activity of the promoter of FcεRI a-chain).
3、The detection of Elf-1 expression
After 24 h of culture after electroporation, mRNA levels of Elf-1 were analyzed by real-time polymerase chain reaction (PCR). In brief, total RNA from cells was extracted using an RNeasy Micro Kit, and first-strand cDNA was synthesized from 1μg of total RNA using the High Capacity cDNA Reverse Transcription Kit.Quantitative PCR was performed using TaqMan Universal PCR Master Mix and a 7500 Real-Time PCR System with TaqMan Gene Expression. Assays of the mouse target genes, including Elf-1 (Mm00468217_ml) and the endogenous control glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Rodent GAPDH Control Reagents VIC Probe). Expression levels of Elf-1 were expressed relative to those of GAPDH by calculating cycle threshold (Ct) values in amplification plots with 7500 SDS software. In addition, After 48h of culture after electroporation,1×106 BMMC were collected, and whole cell lysates were subjected to electrophoresis on a 7.5% SDS polyacrylamide gel. Anti-Elf-1 antibody and anti-actin Ab were used as primary Abs. Alexa Fluor 680 goat antirabbit IgG and IR Dye 800 goat anti-mouse IgG (Molecular Probes) were used as secondary Abs against Elf-1 and actin, respectively. Infrared fluorescence on membranes was detected using the Odyssey infrared imaging system. The amount of protein was quantified by calculating band intensity using the Odyssey software.
4、The detection of promoter activities of FcεRI a-chain in BMMC
After 24h of cultivation, BMMC were harvested and treated with a Dual-luciferase assay kit for the measurement of luciferase activity. The luminescence was measured with Micro Lumat Plus.
5、The detection of mRNA expression of FcεRIα-,β-and y-chain
After 24 h of culture after electroporation, mRNA levels of FcεRIα-,β-and y-chain were analyzed by real-time polymerase chain reaction (PCR). In brief, total RNA from cells was extracted using an RNeasy Micro Kit, and first-strand cDNA was synthesized from 1μg of total RNA using the High Capacity cDNA Reverse Transcription Kit. Quantitative PCR was performed using TaqMan Universal PCR Master Mix and a 7500 Real-Time PCR System with TaqMan Gene Expression. Assays of the mouse target genes, including FcεRI a-chain (Mm00438867_ml), the FcεRI p-chain (Mm00442780_ml), the FcεRIγ-chain (Mm00438869_gl), and the endogenous control glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Rodent GAPDH Control Reagents VIC Probe). Expression levels of FcεRIα-,β-, and y-chain were expressed relative to those of GAPDH by calculating cycle threshold (Ct) values in amplification plots with 7500 SDS software.
6、The detection of PU.1 binding to FcεRIα-chain promoter
BMMC transfected with siRNA were collected and lysis by ultrasonication. Chromatin DNA was precipitated and chromatin immunoprecipitation (ChIP) assay was performed. Anti-PU.1 goat IgG Ab and goat IgG were used. Quantitative PCR was performed using TaqMan Universal PCR Master Mix and a 7500 Real-Time PCR System. The following primers and TaqMan probe sequences were used for this analysis:for the promoter region of the a-chain gene,-82/+1, forward primer-82/-56 (5'-GGCATAGCTGATGAGTTAACCAGATAC-3'), reverse primer+1/-22 (5'-TATGGCTTCGAAAATAGGCTTGA-3'), and TaqMan probe-51/-33 (5'-FAMCAGAAGACATTTCCTTCTC-MGB-3').
7、BMMC were stimulated with TSA in vitro
BMMC from BALB/c mouse was adjusted to 1×106/ml and stimulated with TSA at the final concentration of 0,2,10 and 50nM at 37℃for 24h (for detection of expression of FcεRI and functions of BMMC) and 48h (for detection of apoptosis of BMMC), respectively.
8、The detection of the expression of FcεRI on BMMC
BMMC transfected with siRNA or stimulated with TSA were collected and then incubated with PE-labeled anti-FcεRI a-chain Ab for 1 h at 4℃after blocking Fc receptors on the cell surface with 2.4G2. Cells were washed with PBS twice. Then the expression of FcεRI on BMMC was analyzed using a FACSCalibur flow cytometer.
9、The detection of the apoptosis of BMMC
BMMC stimulated with TSA were collected. For analysis of apoptotic cells, BMMC were stained with 5μg/ml propidium iodide (PI) and Annexin V-FITC for 15min at room temperature. Cells were analyzed using a FACSCalibur flow cytometer.
10、β-hexosaminidase release assay
After incubation of BMMC with the indicated concentration of TSA for 24 h at 37℃in the complete culture medium, the cells were sensitized with 1μg/ml mouse IgE for 1 h at 4℃and then resuspended in Tyrode's buffer at a concentration of 1 x 106 cells/ml, and stimulated with 1μg/ml anti-mouse IgE for 45 min at 37℃. The cell supernatants were collected, andβ-hexosaminidase activity in the supernatants and cell lysates was quantified by spectrophotometric analysis of hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucopyranoside. The percentage ofβ-hexosaminidase release was calculated using the following formula:percent release (%)=(OD of the stimulated supernatant-OD of the unstimulated supernatant)/(OD of the total cell lysate-OD of the unstimulated supernatant)×100%.
11、Measurement of IL-6 production from BMMC
After incubation with TSA for 24h at 37℃, BMMC at concentration of 1 x 106/ml were stimulated with IgE/anti-IgE cross-linking for 1h at 37℃. The total RNA was collected and reversed transcripted into cDNA. The expression of IL-6 mRNA was analyzed by Real-time PCR. TSA incubated BMMC were stimulated with IgE/anti-IgE cross-linking for 6h at 37℃and the supernatant were collected. The concentration of IL-6 in culture supernatant was determined by an ELISA kit according to the manufacturer's instructions.
12、The detection of histone acetylation of IL-6 promoter in BMMC
BMMC incubated with TSA were collected and then stimulated with IgE/anti-IgE cross-linking for 30min at 37℃. Cells were lysis by ultrasonication. Chromatin DNA was precipitated and chromatin immunoprecipitation (ChIP) assay was performed. Anti-acetyl-histone H3 rabbit IgG, anti-acetyl-histone H4 rabbit antiserum and rabbit IgG were used. Quantitative PCR was performed using TaqMan Universal PCR Master Mix and a 7500 Real-Time PCR System. The following primers and TaqMan probe sequences were used for this analysis:for the mouse IL-6 promoter (-84/-9), forward primer IL-6-84F 5'-CCCATGAGTCTCAAAATTAGAGAGTTG-3'), reverse primer IL-6-9R (5'-CAGAGCAGAATGAGCTACAGACATC-3'), and TaqMan probe IL-6-56P (5'-CTCCTAATAAATATGAGACTGGG-3').
Results
1、Elf-1 expression on mouse BMMC knockdown by siRNA transfection
(1) The detection of transfection efficiency of Mouse Macrophage Nucleofector Kit
The FITC-labeled control oligonucleotides were introduced into BMMC with Nucleofector II set at program Y-001 using a Mouse Macrophage Nucleofector Kit. Transfection efficiency was confirmed to be approximately 70% by fluorescence microscopy and phase microscopy.
(2) The expression of Elf-1 in siRNA transfected BMMC
BMMC were transfected with Elf-1 siRNA and Control siRNA respectively.24h later, expression levels of Elf-1 mRNA were determined by real-time PCR. The amount of Elf-1 transcripts in Elf-1 siRNA-treated BMMC was less than one fifth of that in control BMMC.48h later, expression levels of Elf-1 protein were determined by Western blot. The band intensity of Elf-1 protein was markedly reduced by Elf-1 siRNA as compared with control siRNA-treated BMMC. These results indicate that Elf-1 siRNA generated in the present study successfully down regulated Elf-1 expression in BMMC.
2、The effects of Elf-1 on the gene expression of FcεRI in mouse BMMC
(1) The effects of Elf-1 on the promoter activity of FcεRI a-chain in mouse BMMC
To evaluate the effects of Elf-1 on FcεRI a-chain promoter activity, a reporter plasmid, pGV-B2-αNN0.6, carrying the luciferase gene under the control of the FcεRI a-chain promoter was introduced into BMMC in addition to the above-mentioned siRNA. When Elf-1 siRNA was co-introduced into BMMC with the a-chain reporter plasmid, luciferase activity derived from BMMC was significantly increased in comparison with that of control siRNA transfectants, whereas luciferase activity driven by the promoterless control plasmid, pGL3-Basic, was not markedly affected by Elf-1 siRNA. In addition, overexpression of Elf-1 by co-introduction of the Elf-1 expression plasmid decreased FcεRI a-chain-driven luciferase activity in BMMC in Elf-1 binding sequence-dependent manner. These results suggest that Elf-1 exhibits suppressive effect on the FcεRI a-chain promoter in BMMC.
(2) The effects of Elf-1 on FcεRIα-,β-, and y-chain transcription in mouse BMMC
After transfection with siRNA for 24h, the mRNA expression of FcεRIα-,β-, andγ-chain were analyzed by Real-time PCR. The amount of a-chain mRNA in Elf-1 siRNA-treated BMMC was significantly higher than that of control siRNA-treated BMMC. In contrast, significant differences were not observed inβ-andγ-chain between BMMC transfected with Elf-1 or control siRNA. Based on these results, Elf-1 leads to specific inhibition of FcεRI a-chain transcription.
(3) The effects of Elf-1 on recruitment of PU.1 toward FcεRI a-chain promoter in mouse BMMC
After transfection with siRNA for 24h, BMMC were performed to CHIP assay. Compared with that in control siRNA transfected BMMC, the amount of a-chain promoter region bound with PU.1 significantly increased in Elf-1 siRNA-treated BMMC, suggesting that Elf-1 specifically inhibits recruitment of transactivator PU.1 toward the a-chain promoter in mouse BMMC.
(4) The effects of Elf-1 on the surface expression of FcεRI on mouse BMMC
After transfection with siRNA for 48h, FACS was performed to detect the surface expression of FcεRI on mouse BMMC. Similar expression of FcεRI were shown on Elf-1 siRNA and control siRNA transfected BMMC, indicating that Elf-1 has little effect on the surface expression of FcεRI.
3、The effect of TSA on the activation of mouse BMMC
(1) The effects of TSA on the degranulation of mouse BMMC
Mouse BMMC were treated with TSA at the concentration of 0,2,10 and 50nM at 37℃for 24h before sensitization with IgE and then stimulated with anti-IgE Ab. The release ofβ-hexosaminidase was determined. The result indicated that TSA treatment significantly suppressedβ-hexosaminidase release from BMMC activated by IgE/FcεRI cross-linking in a dose-dependent manner, whereas the degranulation ability reached approximately 50% in the absence of TSA.
(2) The effects of TSA on the production of IL-6 in mouse BMMC
Mouse BMMC were treated with TSA at the concentration of 0,2,10 and 50nM at 37℃for 24h before sensitization with IgE and then stimulated with anti-IgE Ab. TSA treatment significantly suppressed the expression of IL-6 mRNA in BMMC determined by Real-time PCR and secretion of IL-6 in the supernatant of BMMC determined by ELISA.
(3) The effects of TSA on the surface expression of FcεRI on mouse BMMC
Mouse BMMC were treated with TSA at the concentration of 0,2,10 and 50nM at 37℃for 24h and then performed to FACS to detect the expression of FcεRI. In comparison to the TSA unstimulated BMMC, only 50nM TSA treatment led to the reduced expression of FcεRI, whereas,2 and 10nM TSA treatment did not.
(4) The effects of TSA on the apoptosis of mouse BMMC
Mouse BMMC were treated with TSA at the concentration of 0,2,10 and 50nM at 37℃for 48h and then performed to FACS to detect the apoptosis. In comparison to the TSA unstimulated BMMC, only 50nM TSA treatment led to the increased number of apoptotic cells, whereas,2 and 1 OnM TSA treatment did not.
(5) The effects of TSA on the histone acetylation of IL-6 promoter of mouse BMMC
Mouse BMMC were treated with TSA at the concentration of 0,2,10 and 50nM at 37℃for 24h before sensitization with IgE and then stimulated with anti-IgE Ab for 30min, and then performed to CHIP assay to detect the histone acetlylation of IL-6 promoter. In comparison to the TSA unstimulated BMMC,10 and 50nM TSA treatment led to the increased amount of acetylated H3 and H4 toward to mouse BMMC IL-6 promoter.
Conclusions
1、Mouse Macrophage Nucleofecter Kit (Amaxa) is suitable for mouse BMMC transfection.
2、Elf-1 siRNA can specifically knockdown the expression of Elf-1 in BMMC.
3、Elf-1 suppressed the mouse BMMC FcεRI a-chain promoter activity and transcription by inhibition of recruitment of PU.1 towards to a-chain promoter.
4、Elf-1 inhibited the mRNA expression of FcεRI a-chain, but not that ofβ-and y-chain.
5、Elf-1 alone had little effect on the surface expression of FcεRI on mouse BMMC.
6、TSA suppressed FcεRI-mediated activation of mouse BMMC in a dose-dependent mode.
7、50nM TSA treatment led to FcεRI-mediated degranulation and IL-6 production in BMMC supposedly by induction of apoptosis of BMMC and subsequently reduced expression of FcεRI on mouse BMMC.
8、TSA induced the accumulation of acetylated histones on the mouse IL-6 promoter in BMMC activated by IgE/FcεRI cross-linking.
引文
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26 Hasegawa M, Nishiyama C, Nishiyama M, et al. Regulation of the human FcepsilonRI alpha-chain distal promoter. J Immunol.2003;170:3732-3738.
27 Nishiyama C, Hasegawa M, Nishiyama M,et al. Cloning of full length genomic DNA encoding human FcepsilonRI alpha-chain and its transcriptional regulation. Biochem Biophys Res Commun.2001; 284:1056-1064.
28 Maeda K, Nishiyama C, Tokura T, et al. Regulation of cell type-specific mouse FcepsilonRI beta-chain gene expression by GATA-1 via four GATA motifs in the promoter. J Immunol. 2003; 170:334-340.
29 Maeda K, Nishiyama C, Tokura T, et al. FOG-1 represses GATA-1-dependent FcepsilonRI beta-chain transcription:transcriptional mechanism of mast-cell-specific gene expression in mice. Blood.2006; 108:262-269.
30 Gray SG, Teh BT. Histone acetylation/deacetylation and cancer:an "open" and "shut"case? Curr Mol Med.2001; 1:401-429.
31 Wolffe AP, Guschin D. Review:chromatin structural features and targets that regulate transcription. J Struct Biol.2000; 129:102-122.
32 32 Taddei A, Roche D, Bickmore WA, et al. The effects of histone deacetylase inhibitors on heterochromatin:implications for anticancer therapy? EMBO Rep.2005; 6:520-524.
33 33 Inokoshi J, Katagiri M, Arima S, et al. Neuronal differentiation of neuro 2a cells by inhibitors of cell cycle progression, trichostatin A and butyrolactone 1. Biochem Biophys Res Commun.1999; 256:372-376.
34 34 Dalgard CL, Van Quill KR, and O' Brien JM. Evaluation of the In vitro and In vivo AntitumorActivity of Histone Deacetylase Inhibitors for the Therapy of Retinoblastoma. Clin Cancer Res.2008; 14:3113-3123.
35 Mishra N, Brown DR, Olorenshaw IM, et al. Trichostatin A reverses skewed expression of CD154, interleukin-10, and interferon-gamma gene and protein expression in lupus T cells. Proc Natl Acad Sci USA.2001; 98:2628-2633.
36 eddy P, Maeda Y, Hotary K, et al. Histone deacetylase inhibitor suberoylanilide hydroxamic acid reduces acute graft-versus-host disease and preserves graft-versus-leukemia effect. Proc Natl Acad Sci USA.2004; 101:3921-3926.
37 hung YL, Lee MY, Wang AJ, et al. A therapeutic strategy uses histone deacetylase inhibitors to modulate the expression of genes involved in the pathogenesis of rheumatoid arthritis. Mol Ther.2003; 8:707-717.
38 Choi JH, Oh SW, Kang MS, et al. Trichostatin A attenuates airway inflammation in mouse asthma model. Clin Exp Allergy.2005; 35:89-96.
39 39 Yee SB, Kim MS, Baek SJ, et al. Trichostatin A induces apoptosis of p815 mastocytoma cells in histone acetylation-and mitochondria-dependent fashion. Int J Oncol.2004; 25: 1431-1436.
40 40 Assem el-SK, Peh KH, Wan BY, et al. Effects of a selection of histone deacetylase inhibitors on mast cell activation and airway and colonic smooth muscle contraction. Int Immunopharmacol.2008; 8:1793-1801.
41 41 Mishra N, Reilly CM, Brown DR,et al. Histone deacetylase inhibitors modulate renal disease in the MRL-lpr/lpr mouse. J Clin Invest.2003; 111:539-552.
42 42 Luhrs H, Gerke T, Muller JG, et al. Butyrate inhibits NF-kappaB activation in lamina propria macrophages of patients with ulcerative colitis. Scand J Gastroenterol.2002; 37:458-466.
43 43 Inan MS, Rasoulpour RJ, Yin L, et al. The luminal short-chain fatty acid butyrate modulates NF-kappaB activity in a human colonic epithelial cell line. Gastroenterology.2000; 118:724-734.
1 Matsuda A, Okayama Y, Ebihara N, et al. Monomeric IgE sensitization increased beta-chain expression as well as mature alpha-chain expression in mast cells. J Immunol Methods.2008; 336:229-234.
2 Masuda A, Yoshida M, Shiomi H, et al. Role of Fc Receptors as a therapeutic targetlnflamm Allergy Drug Targets.2009; 8:80-86.
3 Miyahara S, Miyahara N, Takeda K, et al. Physiologic assessment of allergic rhinitis in mice: role of the high-affinity IgE receptor (FcepsilonRI). J Allergy Clin Immunol.2005; 116:1020-1027.
4 Andrasfalvy M, Peterfy H, Toth G, et al. The beta subunit of the type I Fcepsilon receptor is a target for peptides inhibiting IgE-mediated secretory response of mast cells. J Immunol.2005; 175:2801-2086.
5 Hanashiro K, Sunagawa M, Nakasone T, et al. Inhibition of IgE-mediated phosphorylation of FcepsilonRIgamma protein by antiallergic drugs in rat basophilic leukemia (RBL-2H3) cells:a novel action of antiallergic drugs. Int Immunopharmacol.2007; 7:994-1002.
6 Nishiyama C, Hasegawa M, Nishiyama M, et al. Regulation of human FcεRI α-chain gene expression by multiple transcription factors. J Immunol.2002; 168:4546-4552.
7 Nishiyama C, Takahashi K, Nishiyama M, et al. Splice isoforms of transcription factor Elf-1 affecting its regulatory function in transcription:molecular cloning of rat Elf-1. Biosci Biotechnol Biochem.2000; 64:2601-2607.
8 Nerlov C, Querfurth E, Kulessa H, et al. GATA-1 interacts with the myeloid PU.1 transcription factor and represses PU.1-dependent transcription. Blood.2000; 95:2543-2551.
9 Walsh JC, DeKoter RP, Lee HJ, et al. Cooperative and antagonistic interplay between PU.1 and GATA-2 in the specification of myeloid cell fates. Immunity.2002; 17:665-676.
10 Geng Y, Laslo P, Barton K, et al. Transcriptional regulation of CD ID by Ets family transcription factors. J Immunol.2005; 175:1022-1029.
11 Juang YT, Sumibcay L, Tolnay M, et al. Elf-1 binds to GGAA elements on the FcRgamma promoter and repress its expression. J Immunol.2007; 179:4884-4889.
12 Luo D, Dai Y, Duffy LB, et al. Inhibition of message for FcepsilonRI alpha chain blocks mast cell IL-4 production induced by co-culture with Mycoplasma pneumoniae. Microb Pathog. 2008; 44:286-292.
13 Bischoff SC, Sellge G, Lorentz A, et al. IL-4 enhances proliferation and mediator release in mature human mast cells. Proc Natl Acad Sci. USA.1999; 96:8080-8085.
14 Gosset P, Lamblin-Degros C, Tillie-Leblond I, et al. Modulation of high-affinity IgE receptor expression in blood monocytes:opposite effect of IL-4 and glucocorticoids. J Allergy Clin Immunol.2001; 107:114-122.
15 Ryan JJ, DeSimone S, Klish G, et al. IL-4 inhibits mouse mast cell FcεRI expression through a STAT6-dependent mechanism. J Immunol.1998; 161:6915-6923.
16 Nishiyama C, Hasegawa M, Nishiyama M, et al. Cloning of full-length genomic DNA encoding human FcεRI α-chain and its transcriptional regulation. Biochem Biophys Res Commun.2001; 284:1056-1064.
17 Hasegawa M, Nishiyama C, Nishiyama M, et al. Regulation of the human FcεRI a-chain distal promoter. J Immunol.2003; 170:3732-3738.
18 Smith SJ, Ying S, Meng Q, et al. Blood eosinophils from atopic donors express messenger RNA for the α, β, and y subunits of the high-affinity IgE receptor (FcεRI) and intracellular, but not cell surface, a subunit protein. J Allergy Clin Immunol.2000; 105:309-317.
19 Donnadieu E, Jouvin MH, and Kinet JP. A second amplifier function for the allergy-associated FcεRIβ subunit. Immunity.2000; 12:515-523.
20 Matsuda A, Okayama Y, Ebihara N, et al. Hyperexpression of the high-affinity IgE receptor-{beta} chain in chronic allergic keratoconjunctivitis. Invest Ophthalmol Vis Sci.2009; 50:2871-2877.
21 Maeda K, Nishiyama C, Tokura T, et al. Regulation of cell type-specific mouse FcεRI β-chain gene expression by GATA-1 via four GATA motifs in the promoter. J Immunol.2003; 170: 334-340.
22 Akizawa Y, Nishiyama C, Hasegawa M, et al. Regulation of human FcεRI β chain gene expression by Oct-1. Int Immunol.2003; 15:549-556.
23 Takahashi K, Nishiyama C, Hasegawa M, et al. Regulation of the human high affinity IgE receptor β-chain gene expression via an intronic element. J Immunol.2003; 171:2478-2484.
24 Nishiyama C, Ito T, Nishiyama M, et al. GATA-1 is required for expression of FcεRI on mast cells:analysis of mast cells derived from GATA-1 knockdown mouse bone marrow. Int Immunol.2005; 17:847-856.
25 Maeda K, Nishiyama C, Tokura T, et al. FOG-1 represses GATA-1-dependent FcepsilonRI beta-chain transcription:transcriptional mechanism of mast-cell-specific gene expression in mice. Blood.2006; 108):262-269.
26 Takahashi K, Matsumoto C, Ra C. FHL3 negatively regulates human high-affinity IgE receptor β-chain gene expression by acting as a transcriptional co-repressor of MZF-1. Biochem J.2005; 385:191-200.
27 Laprise C, Boulet LP, Morissette J, et al. Evidence for association and linkage between atopy, airway hyper-responsiveness, and the beta subunit Glu237Gly variant of the high-affinity receptor for immunoglobulin E in the French-Canadian population. Immunogenetics.2000; 51: 695-702.
28 Nagata H, Mutoh H, Kumahara K, et al. Association between nasal allergy and a coding variant of the FcεRIβ gene Glu237Gly in a Japanese population. Hum Genet.2001; 109: 262-266.
29 Takabayashi A, Ihara K, Sasaki Y, et al. Childhood atopic asthma:positive association with a polymorphism of IL-4 receptor a gene but not with that of IL-4 promoter or Fcε receptor 1β gene. Exp Clin Immunogenet.2000; 17:63-70.
30 Furumoto Y, Hiraoka S, Kawamoto K, et al. Polymorphisms in FcεR1β chain do not affect IgE-mediated mast cell activation. Biochem Biophys Res Commun.2000; 273:765-771.
31 Donnadieu E, Cookson WO, Jouvin MH, et al. Allergy-associated polymorphisms of the FcεR1β subunit do not impact its two amplification functions. J Immunol.2000; 165:3917-3922.
32 Nishiyama C, Akizawa Y, Nishiyama M, et al. Polymorphisms in the FcεRIβ promoter region affecting transcription activity:a possible promoter-dependent mechanism for association between FcεR1β and atopy. J Immunol.2004; 173:6458-6464.
33 Kim YK, Park HW, Yang JS, et al. Association and functional relevance of E237G, a polymorphism of the high-affinity immunoglobulin E-receptor beta chain gene, to airway hyper-responsiveness. Clin Exp Allergy.2007; 37:592-598.
34 Asai K, Kitaura J, Kawakami Y, et al. Regulation of mast cell survival by IgE. Immunity. 2001; 14:791-800.
35 Kalesnikoff J, Huber M, Lam V, et al. Monomeric IgE stimulates signaling pathways in mast cells and leads to cytokine production and cell survival. Immunity.2001;14:801-811.
36 Cookson WO, Ubhi B, Lawrence R, et al. Genetic linkage of childhood atopic dermatitis to psoriasis susceptibility loci. Nat Genet.2001; 27:372-373.
37 Hasegawa M, Nishiyama C, Nishiyama M, et al. A novel-66T/C polymorphism in FcεRI α-chain promoter affecting the transcription activity:possible relationship to allergic diseases. J Immunol.2003; 171:1927-1933.
38 Kanada S, Nakano N, Potaczek DP, et al. Two different transcription factors discriminate the-315C>T polymorphism of the Fc epsilon RI alpha gene:binding of Spl to-315C and of a high mobility group-related molecule to-315T. J Immunol.2008; 180:8204-8210.
39 Anderson KL, Perkin H, Surh CD, et al. Transcription factor PU.1 is necessary for development of thymic and myeloid progenitor-derived dendritic cells. J Immunol.2000; 164: 1855-1861.
40 Guerriero A, Langmuir PB, Spain LM, et al. PU.1 is required for myeloid-derived but not lymphoid-derived dendritic cells. Blood.2000; 95(3):879-885.
41 DeKoter RP. Singh H. Regulation of B lymphocyte and macrophage development by graded expression of PU.1. Science.2000; 288:1439-1441.
42 Dahl R, Walsh JC, Lancki D, et al. Regulation of macrophages and neutrophil cell fates by the PU.1:C/EBPa ratio and granulocyte colony-stimulating factor. Nat Immunol.2003; 4: 1029-1036.
43 Nishiyama C, Nishiyama M, Ito T, et al. Overproduction of PU.1 in mast cell progenitors:its effect on monocyte-and mast cell-specific gene expression. Biochem Biophys Res Commun. 2004;313:516-521.
44 Ito T, Nishiyama C, Nishiyama M, et al. Mast cells acquire monocyte-specific gene expression and monocyte-like morphology by overproduction of PU.1. J Immunol.2005; 174: 376-383.
45 Nishiyama C, Nishiyama M, Ito T, et al. Functional analysis of PU.1 domains in monocyte-specific gene regulation. FEBS Lett.2004; 561:63-68.
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13 Juang YT, Sumibcay L, Tolnay M, et al. Elf-1 binds to GGAA elements on the FcRgamma promoter and repress its expression. J Immunol.2007; 179:4884-4889.
14 Dombrowicz D, Flamand V, Brigman KK, et al. Abolition of anaphylaxis by targeted disruption of the high affinity immunoglobulin E receptor a chain gene. Cell.1993;
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24 Nishiyama C, Hasegawa M, Nishiyama M, et al. Regulation of human FcεRI α-chain gene expression by multiple transcription factors. J Immunol.2002;168:4546-4552.
25 Nishiyama C, Yokota T, Okumura K, et al. The transcription factors Elf-1 and GATA-1 bind to cell-specific enhancer elements of human high-affinity IgE receptor a-chain gene. J Immunol.1999; 163:623-630.
26 Hasegawa M, Nishiyama C, Nishiyama M, et al. Regulation of the human FcepsilonRI alpha-chain distal promoter. J Immunol.2003;170:3732-3738.
27 Nishiyama C, Hasegawa M, Nishiyama M,et al. Cloning of full length genomic DNA encoding human FcepsilonRI alpha-chain and its transcriptional regulation. Biochem Biophys Res Commun.2001; 284:1056-1064.
28 Maeda K, Nishiyama C, Tokura T, et al. Regulation of cell type-specific mouse FcepsilonRI beta-chain gene expression by GATA-1 via four GATA motifs in the promoter. J Immunol. 2003; 170:334-340.
29 Maeda K, Nishiyama C, Tokura T, et al. FOG-1 represses GATA-1-dependent FcepsilonRI beta-chain transcription:transcriptional mechanism of mast-cell-specific gene expression in mice. Blood.2006; 108:262-269.
30 Gray SG, Teh BT. Histone acetylation/deacetylation and cancer:an "open" and "shut"case? Curr Mol Med.2001; 1:401-429.
31 Wolffe AP, Guschin D. Review:chromatin structural features and targets that regulate transcription. J Struct Biol.2000; 129:102-122.
32 32 Taddei A, Roche D, Bickmore WA, et al. The effects of histone deacetylase inhibitors on heterochromatin:implications for anticancer therapy? EMBO Rep.2005; 6:520-524.
33 33 Inokoshi J, Katagiri M, Arima S, et al. Neuronal differentiation of neuro 2a cells by inhibitors of cell cycle progression, trichostatin A and butyrolactone 1. Biochem Biophys Res Commun.1999; 256:372-376.
34 34 Dalgard CL, Van Quill KR, and O' Brien JM. Evaluation of the In vitro and In vivo AntitumorActivity of Histone Deacetylase Inhibitors for the Therapy of Retinoblastoma. Clin Cancer Res.2008; 14:3113-3123.
35 Mishra N, Brown DR, Olorenshaw IM, et al. Trichostatin A reverses skewed expression of CD154, interleukin-10, and interferon-gamma gene and protein expression in lupus T cells. Proc Natl Acad Sci USA.2001; 98:2628-2633.
36 eddy P, Maeda Y, Hotary K, et al. Histone deacetylase inhibitor suberoylanilide hydroxamic acid reduces acute graft-versus-host disease and preserves graft-versus-leukemia effect. Proc Natl Acad Sci USA.2004; 101:3921-3926.
37 hung YL, Lee MY, Wang AJ, et al. A therapeutic strategy uses histone deacetylase inhibitors to modulate the expression of genes involved in the pathogenesis of rheumatoid arthritis. Mol Ther.2003; 8:707-717.
38 Choi JH, Oh SW, Kang MS, et al. Trichostatin A attenuates airway inflammation in mouse asthma model. Clin Exp Allergy.2005; 35:89-96.
39 39 Yee SB, Kim MS, Baek SJ, et al. Trichostatin A induces apoptosis of p815 mastocytoma cells in histone acetylation-and mitochondria-dependent fashion. Int J Oncol.2004; 25: 1431-1436.
40 40 Assem el-SK, Peh KH, Wan BY, et al. Effects of a selection of histone deacetylase inhibitors on mast cell activation and airway and colonic smooth muscle contraction. Int Immunopharmacol.2008; 8:1793-1801.
41 41 Mishra N, Reilly CM, Brown DR,et al. Histone deacetylase inhibitors modulate renal disease in the MRL-lpr/lpr mouse. J Clin Invest.2003; 111:539-552.
42 42 Luhrs H, Gerke T, Muller JG, et al. Butyrate inhibits NF-kappaB activation in lamina propria macrophages of patients with ulcerative colitis. Scand J Gastroenterol.2002; 37:458-466.
43 43 Inan MS, Rasoulpour RJ, Yin L, et al. The luminal short-chain fatty acid butyrate modulates NF-kappaB activity in a human colonic epithelial cell line. Gastroenterology.2000; 118:724-734.
1 Matsuda A, Okayama Y, Ebihara N, et al. Monomeric IgE sensitization increased beta-chain expression as well as mature alpha-chain expression in mast cells. J Immunol Methods.2008; 336:229-234.
2 Masuda A, Yoshida M, Shiomi H, et al. Role of Fc Receptors as a therapeutic targetlnflamm Allergy Drug Targets.2009; 8:80-86.
3 Miyahara S, Miyahara N, Takeda K, et al. Physiologic assessment of allergic rhinitis in mice: role of the high-affinity IgE receptor (FcepsilonRI). J Allergy Clin Immunol.2005; 116:1020-1027.
4 Andrasfalvy M, Peterfy H, Toth G, et al. The beta subunit of the type I Fcepsilon receptor is a target for peptides inhibiting IgE-mediated secretory response of mast cells. J Immunol.2005; 175:2801-2086.
5 Hanashiro K, Sunagawa M, Nakasone T, et al. Inhibition of IgE-mediated phosphorylation of FcepsilonRIgamma protein by antiallergic drugs in rat basophilic leukemia (RBL-2H3) cells:a novel action of antiallergic drugs. Int Immunopharmacol.2007; 7:994-1002.
6 Nishiyama C, Hasegawa M, Nishiyama M, et al. Regulation of human FcεRI α-chain gene expression by multiple transcription factors. J Immunol.2002; 168:4546-4552.
7 Nishiyama C, Takahashi K, Nishiyama M, et al. Splice isoforms of transcription factor Elf-1 affecting its regulatory function in transcription:molecular cloning of rat Elf-1. Biosci Biotechnol Biochem.2000; 64:2601-2607.
8 Nerlov C, Querfurth E, Kulessa H, et al. GATA-1 interacts with the myeloid PU.1 transcription factor and represses PU.1-dependent transcription. Blood.2000; 95:2543-2551.
9 Walsh JC, DeKoter RP, Lee HJ, et al. Cooperative and antagonistic interplay between PU.1 and GATA-2 in the specification of myeloid cell fates. Immunity.2002; 17:665-676.
10 Geng Y, Laslo P, Barton K, et al. Transcriptional regulation of CD ID by Ets family transcription factors. J Immunol.2005; 175:1022-1029.
11 Juang YT, Sumibcay L, Tolnay M, et al. Elf-1 binds to GGAA elements on the FcRgamma promoter and repress its expression. J Immunol.2007; 179:4884-4889.
12 Luo D, Dai Y, Duffy LB, et al. Inhibition of message for FcepsilonRI alpha chain blocks mast cell IL-4 production induced by co-culture with Mycoplasma pneumoniae. Microb Pathog. 2008; 44:286-292.
13 Bischoff SC, Sellge G, Lorentz A, et al. IL-4 enhances proliferation and mediator release in mature human mast cells. Proc Natl Acad Sci. USA.1999; 96:8080-8085.
14 Gosset P, Lamblin-Degros C, Tillie-Leblond I, et al. Modulation of high-affinity IgE receptor expression in blood monocytes:opposite effect of IL-4 and glucocorticoids. J Allergy Clin Immunol.2001; 107:114-122.
15 Ryan JJ, DeSimone S, Klish G, et al. IL-4 inhibits mouse mast cell FcεRI expression through a STAT6-dependent mechanism. J Immunol.1998; 161:6915-6923.
16 Nishiyama C, Hasegawa M, Nishiyama M, et al. Cloning of full-length genomic DNA encoding human FcεRI α-chain and its transcriptional regulation. Biochem Biophys Res Commun.2001; 284:1056-1064.
17 Hasegawa M, Nishiyama C, Nishiyama M, et al. Regulation of the human FcεRI a-chain distal promoter. J Immunol.2003; 170:3732-3738.
18 Smith SJ, Ying S, Meng Q, et al. Blood eosinophils from atopic donors express messenger RNA for the α, β, and y subunits of the high-affinity IgE receptor (FcεRI) and intracellular, but not cell surface, a subunit protein. J Allergy Clin Immunol.2000; 105:309-317.
19 Donnadieu E, Jouvin MH, and Kinet JP. A second amplifier function for the allergy-associated FcεRIβ subunit. Immunity.2000; 12:515-523.
20 Matsuda A, Okayama Y, Ebihara N, et al. Hyperexpression of the high-affinity IgE receptor-{beta} chain in chronic allergic keratoconjunctivitis. Invest Ophthalmol Vis Sci.2009; 50:2871-2877.
21 Maeda K, Nishiyama C, Tokura T, et al. Regulation of cell type-specific mouse FcεRI β-chain gene expression by GATA-1 via four GATA motifs in the promoter. J Immunol.2003; 170: 334-340.
22 Akizawa Y, Nishiyama C, Hasegawa M, et al. Regulation of human FcεRI β chain gene expression by Oct-1. Int Immunol.2003; 15:549-556.
23 Takahashi K, Nishiyama C, Hasegawa M, et al. Regulation of the human high affinity IgE receptor β-chain gene expression via an intronic element. J Immunol.2003; 171:2478-2484.
24 Nishiyama C, Ito T, Nishiyama M, et al. GATA-1 is required for expression of FcεRI on mast cells:analysis of mast cells derived from GATA-1 knockdown mouse bone marrow. Int Immunol.2005; 17:847-856.
25 Maeda K, Nishiyama C, Tokura T, et al. FOG-1 represses GATA-1-dependent FcepsilonRI beta-chain transcription:transcriptional mechanism of mast-cell-specific gene expression in mice. Blood.2006; 108):262-269.
26 Takahashi K, Matsumoto C, Ra C. FHL3 negatively regulates human high-affinity IgE receptor β-chain gene expression by acting as a transcriptional co-repressor of MZF-1. Biochem J.2005; 385:191-200.
27 Laprise C, Boulet LP, Morissette J, et al. Evidence for association and linkage between atopy, airway hyper-responsiveness, and the beta subunit Glu237Gly variant of the high-affinity receptor for immunoglobulin E in the French-Canadian population. Immunogenetics.2000; 51: 695-702.
28 Nagata H, Mutoh H, Kumahara K, et al. Association between nasal allergy and a coding variant of the FcεRIβ gene Glu237Gly in a Japanese population. Hum Genet.2001; 109: 262-266.
29 Takabayashi A, Ihara K, Sasaki Y, et al. Childhood atopic asthma:positive association with a polymorphism of IL-4 receptor a gene but not with that of IL-4 promoter or Fcε receptor 1β gene. Exp Clin Immunogenet.2000; 17:63-70.
30 Furumoto Y, Hiraoka S, Kawamoto K, et al. Polymorphisms in FcεR1β chain do not affect IgE-mediated mast cell activation. Biochem Biophys Res Commun.2000; 273:765-771.
31 Donnadieu E, Cookson WO, Jouvin MH, et al. Allergy-associated polymorphisms of the FcεR1β subunit do not impact its two amplification functions. J Immunol.2000; 165:3917-3922.
32 Nishiyama C, Akizawa Y, Nishiyama M, et al. Polymorphisms in the FcεRIβ promoter region affecting transcription activity:a possible promoter-dependent mechanism for association between FcεR1β and atopy. J Immunol.2004; 173:6458-6464.
33 Kim YK, Park HW, Yang JS, et al. Association and functional relevance of E237G, a polymorphism of the high-affinity immunoglobulin E-receptor beta chain gene, to airway hyper-responsiveness. Clin Exp Allergy.2007; 37:592-598.
34 Asai K, Kitaura J, Kawakami Y, et al. Regulation of mast cell survival by IgE. Immunity. 2001; 14:791-800.
35 Kalesnikoff J, Huber M, Lam V, et al. Monomeric IgE stimulates signaling pathways in mast cells and leads to cytokine production and cell survival. Immunity.2001;14:801-811.
36 Cookson WO, Ubhi B, Lawrence R, et al. Genetic linkage of childhood atopic dermatitis to psoriasis susceptibility loci. Nat Genet.2001; 27:372-373.
37 Hasegawa M, Nishiyama C, Nishiyama M, et al. A novel-66T/C polymorphism in FcεRI α-chain promoter affecting the transcription activity:possible relationship to allergic diseases. J Immunol.2003; 171:1927-1933.
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