渗透压对人气道上皮细胞黏蛋白5AC分泌的调控研究
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摘要
目的:
气道黏液高分泌是慢性气道炎症性疾病包括慢性阻塞性肺疾病、支气管哮喘和肺囊性纤维化等疾病的一个重要病理特征。人支气管上皮细胞黏液增多是炎症级联反应的结果,水、无机盐离子和黏蛋白(mucin, MUC)等大分子构成气道黏液层,黏液潴留可导致病变气道阻塞加重,并为气道定植细菌提供良好的培养基,同时可释放多种促分泌因子加剧黏液潴留过程[1,2,3],从而加快疾病的进程并使病情恶化。理论上黏液层物理和化学性质的任何改变都会影响黏液性状和分泌量,已知MUC5AC是气道主要分泌型MUC,是超负荷生成病理性黏液中的主要成分,作为炎症气道黏蛋白的主要特征性表型,决定着高分泌黏液的病理特征[4,5]。目前对MUC5AC基因表达和分泌的相关分子机制已广泛开展,已知体内多种炎症介质和细胞外因素如白介素(interleukin, IL)-4、IL-13、IL-9、IL-6、IL-1β、肿瘤坏死因子(tumor necrosis factor, TNF-α)、中性粒细胞弹力酶(neutrophil elastase, NE) [11]、表皮生长因子受体(epidermal growth factor receptor, EGFR)配体[12]、空气污染物[13]、细菌产物[14]、类花生酸类、自由基[15]和氧化应激[16]等刺激均可以导致MUC5AC的异常,但是对临床上常见的高渗盐水可诱导排痰的分子机制却并不清楚,渗透压改变的条件下对MUC5AC分泌的效应尚无报道。既往研究发现气道上皮细胞黏液出胞过程中黏液分泌颗粒复合物形成必需热休克蛋白(heat shock proteins, HSP)70参与[17], HSP70可在多种刺激下诱导表达以保护气道细胞,并与蛋白激酶C(protein kinase C, PKC)有关[18],渗透压反应增强子结合蛋白(osmotic response element binding protein, OREBP/ nuclear factor of activated T cells, NFAT5/ toncity-responsive binding-protein, TonEBP)是哺乳动物细胞内唯一已知渗透压相关转录因子,可调控HSP70等的表达,保护细胞免受高渗应激[19]。本研究选用体外培养人支气道上皮细胞株HBE16的方法,以常见的高渗盐水作为干预因素,探讨渗透压刺激对MUC5AC分泌的效应,以及HSP70、PKC和OREBP在黏液分泌中的机制,研究高渗下OREBP对HSP70的调控作用及其对MUC5AC分泌的影响,从而深入阐明高渗在气道慢性炎症性疾病黏液分泌中的可能机制,并为能从基因和分子水平解释有效的临床防治措施提供实验依据。
方法:
细胞培养和高渗处理:体外培养正常人气道上皮HBE16细胞株,加入10%高渗氯化钠配成不同浓度的高渗培养基,渗透压计检测渗透压。高渗培养前用无激素和无血清培养基培养。四甲基偶氮唑盐光吸收法(methyl thiazolyl tetrazolium, MTT法)检测细胞活性。PKC各亚型抑制剂和OREBP siRNA做为干扰因素处理高渗培养HBE16细胞。
蛋白表达和转录水平的检测:采用酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA)检测培养上清中MUC5AC蛋白含量作为评价黏液分泌量的指标;Western Blot检测HSP70-2和OREBP蛋白表达,RT-PCR方法半定量检测HSP70-2转录水平。细胞免疫化学和激光共聚焦技术观察细胞在高渗下HSP70-2和OREBP蛋白表达的变化。
HSP70-2基因上游不同长度启动子序列荧光素酶(luciferase, Luc)报告基因质粒载体的设计和构建:从HBE16细胞中抽提人基因组DNA序列为模板,根据HSP70-2基因启动子上游调控区的基因序列及质粒表达载体pGL3-Basic的结构特点及实验需要设计扩增引物,PCR扩增HSP70-2基因启动子上游5’端不同片断,经酶切和DNA测序鉴定证实HSP70-2基因启动子上游序列系列截短的荧光素酶报告基因表达质粒构建成功。
含人HSP70-2基因启动子渗透压反应增强子(osmotic response element, ORE)位点定点突变序列荧光素酶报告基因质粒载体的设计和构建:基因启动子软件分析提示HSP70-2基因启动子上游存在两个潜在的ORE,ORE1位于转录起始点-1975位,ORE2位于-93位。ORE的共有序列为:TGGAANNNYNY (Y为T或C,N为任一碱基)。设计并导入定点突变引物使HSP70-2启动子上游的两个ORE位点分别单独失活。含人HSP70-2基因上游ORE位点定点突变序列荧光素酶报告基因质粒经酶切和DNA测序鉴定证实构建成功。
细胞转染和荧光素酶活性测定:报告基因质粒和OREBP siRNA通过脂质体2000共转染HBE16细胞,连续培养24h和48h后加高渗处理,12小时孵育后收集细胞,采用双荧光素酶报告基因检测系统计算报告基因的相对表达水平。
结果:
1、高渗能有效刺激HBE16细胞培养上清MUC5AC蛋白分泌量增加,与对照组相比较差异显著,有统计学意义,且结果显示在所选实验浓度范围内(500和600mOsm/kgH2O) MUC5AC随培养时间和培养浓度的改变对高渗盐水刺激有时间和剂量依赖关系(p < 0.05)。当渗透浓度增加到1000mOsm/kgH2O时HBE16细胞存活率降低,与对照组相比,差异有显著性(p < 0.05)。
2、高渗培养后,OREBP和HSP70-2蛋白在细胞内的荧光表达显著增加,HSP70-2蛋白和转录水平较对照组显著升高,并随着培养时间的延长而逐渐增加(p < 0.05),同时OREBP蛋白表达增加,且随培养时间延长而逐渐增加(p < 0.01)。
3、用PKCμ抑制剂G?6976处理高渗培养下HBE16细胞,培养上清MUC5AC蛋白含量、HSP70-2蛋白和转录水平较高渗组显著降低(p < 0.01),但仍高于对照组(p < 0.05);而PKCα抑制剂Safingol,PKCβ抑制剂LY333531和PKCδ抑制剂Rottlerin处理细胞后,上述指标水平无显著改变。
4、siRNA抑制OREBP表达后,高渗刺激下的OREBP、HSP70-2和上清MUC5AC蛋白相对含量显著减少(p均<0.05)。
5、成功构建系列截短的HSP70-2启动子荧光素酶报告基因质粒,真核细胞转染技术与OREBP siRNA共转染导入HBE16细胞,观察高渗刺激下的细胞,结果显示转染分别含2、1、1个ORE位点的pGL3-HSP70-2(-2181/+165)-Luc、pGL3-HSP70-2(-1394/+165)-Luc和pGL3-HSP70-2(-353/+165)-Luc细胞后在高渗刺激下的荧光素酶比活性无明显改变(p>0.05);而转染HSP70-2启动子不含ORE位点的pGL3-HSP70-2(-66/+165)-Luc细胞在高渗刺激下的荧光素酶比活性与高渗组的细胞相比显著降低(p<0.01)。
6、构建突变-1975位点的ORE1和位于-93位点的ORE2的HSP70-2启动子报告基因载体,与OREBP siRNA共转染细胞后检测,突变ORE1后细胞在高渗刺激下的荧光素酶表达与高渗组相比无显著改变(p>0.05);突变ORE2后高渗刺激下的荧光素酶比活性与高渗组比较显著降低有统计学意义(p<0.01)。
结论:
1、高渗刺激可诱导体外培养的HBE16细胞MUC5AC分泌,在所选实验浓度范围内呈现时间和浓度依赖关系,但同时高渗对HBE16细胞活力的影响也逐步加大,随渗透时间延长和渗透浓度加大细胞存活率下降。
2、高渗培养后,OREBP和HSP70-2蛋白在细胞内的荧光表达显著增加,HSP70-2蛋白和转录水平,OREBP蛋白水平均显著增加,提示HSP70-2和OREBP参与高渗致HBE16细胞MUC5AC分泌过程。用PKCμ抑制剂处理高渗培养下细胞,HSP70-2蛋白、转录水平和MUC5AC分泌较高渗组显著减少,但仍高于对照组;而用PKCα抑制剂、PKCβ抑制剂和PKCδ抑制剂处理后HSP70-2蛋白、转录水平和MUC5AC分泌均无改变,考虑高渗致黏液分泌可能是部分通过PKCμ-HSP70-2依赖的信号通路。siRNA抑制OREBP表达后,高渗刺激下的OREBP、HSP70-2和上清MUC5AC蛋白相对含量显著减少,进一步证实OREBP和HSP70-2参与高渗下黏液分泌的过程。
3、成功构建系列截短的HSP70-2上游启动子荧光素酶报告基因质粒和含两个ORE位点突变的HSP70-2启动子荧光素酶报告基因质粒,为今后深入开展高渗对黏液分泌的调控机制奠定基础。
4、高渗刺激可分别诱导HSP70-2启动子含2、1、1个ORE位点的pGL3-HSP70-2(-2181/+165)-Luc、pGL3-HSP70-2(-1394/+165)-Luc和pGL3-HSP70-2(-353/+165)-Luc序列的报告基因质粒仍然表达荧光素酶,而不含ORE位点的pGL3-HSP70-2(-66/+165)-Luc序列的报告基因质粒对高渗刺激无反应,不表达荧光素酶,说明在HSP70-2启动子上游-66到-353序列之间存在ORE关键位点。
5、高渗刺激可诱导HSP70-2启动子-1975位点的ORE1发生定点突变序列的报告基因质粒表达荧光素酶,而含有-93位点的ORE2发生定点突变序列的报告基因质粒对高渗刺激无明显反映,提示高渗盐水诱导OREBP通过与HSP70-2启动子上-93位点的ORE结合而激活HSP70-2转录并促进HBE16细胞MUC5AC高分泌。
Objective:
Airway mucus hypersecretion is a main pathological feature of many chronic airway inflammatory diseases such as chronic obstructive pulmonary disease, bronchus asthma, lung cystic fibrosis and etc. The viscous mucin rich in human bronchial epithelial cells is a consequence of an inflammatory cascade reaction. Airway mucus layer consists of some large molecules which are water, inorganic salt ions, mucus protein (mucin, MUC) and etc. The excessive mucus leads to severe airway obstruction and provides a good medium for airway bacterial colonization. In addition, many excreted factors are crowded to aggravate the process of mucus obstruction and diseases [1, 2, 3]. Theoretically, it does not avoid that any change with physical and chemical properties of mucus layer would affect mucous traits and secretions. MUC5AC glycoprotein is the major respiratory mucins which present in secretion and is a main ingredient in generatin pathological mucus under overload [4, 5]. The molecular mechanism of the expression and the secretion of MUC5AC are under widely studied in recent years. It is well known that many mediators of inflammation and extracellular factors would lead disorder of MUC5AC. During the course of airway inflammatory disease, MUC5AC has been shown to be stimulated by a wide variety of stimuli, including proinflammatory cytokines such as interleukin (IL)-4[6], IL-13[7], IL-9[8], IL-6[9], IL-1βand tumor necrosis factor (TNF)-α[10], neutrophil elastase (NE)[11], epidermal growth factor receptor (EGFR) ligands[12], air pollutants[13], bacterial products[14], eicosanoids, and free radicals[15]. MUC5AC gene expression is also known to be regulated by oxidative stress [16]. However, the concrete molecular mechanisms of a common therapies that mucus overproduction was induced by hypertonic saline solution in clinic have not been clear at present. There still no report about hypertonicity on the mechanisms of MUC5AC secretion and regulation. Previous studies show that heat shock proteins 70 (HSP70) which are key related chaperones in the exocytosis process of mucin secretory granules formation by airway epithelial cells are expressed to protect airway cells induced by multiple stimuli[17] and are related with protein kinase C (PKC)[18]. Osmotic response element binding protein (OREBP/ nuclear factor of activated T cells, NFAT5/ toncity-responsive binding-protein, TonEBP) which is an important transcription factor in mammalian cells controlling the cellular response to osmotic stress regulate the express of HSP70 and protect cells in the stress of hypertonicy [19]. Our study was to observe the effect of hypertonic saline solution on MUC5AC protein
secretion in normal human bronchial epithelial cells cultured in vitro, and to disscuss the detailed molecular mechanism of HSP70, PKC and OREBP in the regulation of MUC5AC secretion, and to study the possible mechanism of OREBP in the transcriptional regulation of HSP70 and in MUC5AC secretion. This study tried to show the possible mechanism of mucin secretion in chronic airway inflammation disease with hypertonicy and to provide experimental experience for researchers to search effective clinical prevention and cure of mucus hypersecretion in a genic and molecular level.
Methods:
Cell culture and hypertonicy incubation: Normal human bronchial epithelial cells, HBE16 cells were cultured in vitro. Hypertonic medium with different concentration were added by 10 percent hypertonic saline salution and the osmotic pressure were detected by osmometer. Cells were starved in serum- and hormone-free medium before cells were cultured with hypertonicity. Cells activity was assessed by methyl thiazolyl tetrazolium (MTT) method. Inhibitors of PKC isoforms and OREBP siRNA were served to interference HBE16 cells with hypertonicy.
Level of proteins expression and transcription: MUC5AC protein content in supernatant as an index to evaluate mucus secretary volume was analyzed by ELISA. The expression of HSP70-2 and OREBP were determined by Western blot analysis. RT-PCR was used to detect the transcriptional level of HSP70-2. The changes of OREBP and HSP70-2 protein in cells with hypertonicity were observed by immunofluorescence and confocal laser technology.
Design and construction of luciferase reporter gene plasmids containing different length of human HSP70-2 gene promoter: DNA template was extracted from human genomic DNA in HBE16 cells. PCR amplification primers were designed according to gene sequence of upstream promoter region of HSP70-2 gene and construction features of plasmid expression vector pGL3-Basic. 5 'end different fragments of HSP70-2 upstream were amplified and observed by sequence analysis. Different HSP70-2 promoter sequences length luciferase reporter gene plasmid vector constructed successfully were confirmed by restriction enzymes digestion and DNA sequences alignment.
Design and construction of luciferase reporter gene plasmids containing osmotic response element (ORE) directed mutagenesis site of human HSP70-2 gene promoter: Promoter sequence analysis showed that there were two potential ORE sites in the promoter of HSP70-2. ORE1 was located in -1975 bp of promoter and ORE2 was in -93 bp of promoter. Consensus sequence of ORE was:GGAANNNYNY (Y is T or C, N is any base). Two point mutations were introduced at the ORE1 and ORE2 sites of HSP70-2 promoter separately. Site directed mutagenesis HSP70-2 promoter sequences length luciferase reporter gene plasmid vector constructed successfully were confirmed by restriction enzymes digestion and DNA sequences alignment.
Cell transfection and luciferase activity assay: Reporter gene plasmid and OREBP siRNA were co-transfected using lipofectinamine 2000 in HBE16 cells. Cells were continuously cultured. 24 h and 48 h later, cells were induced by hypertonic saline solution. Cells were collected after 12 h. Dual-luciferase reporter assay system was used to calculate relative expression level of reporter genes.
Results:
1. The secretion of MUC5AC protein in supernatant were elevated significantly in hypertonicity stimulating group (500 and 600mOsm/kgH2O) compared with that in control group. Hypertonic saline solution could increase the level of secretion MUC5AC protein in a concentration and a time dependent manner (p < 0.05). While HBE16 cells activity decrease significantly in hypertonic conditions (1000mOsm/kgH2O) compared with that in control group (p < 0.05).
2. The flourescence expression of OREBP and HSP70-2 intracellular were increased significantly after cells were cultured with hypertonic saline solution. Hypertonicity increased the level of HSP70-2 protein and mRNA in a time dependent manner (p < 0.05). The expression of OREBP protein was significantly increased in hypertonic group compared with that in control group and in a time dependent manner (p < 0.01).
3. Inhibitor of PKCμ(G?6976) was served to interference HBE16 cells with hypertonicity. The content of MUC5AC secretion in supernatant, the level of HSP70-2 protein and mRNA were significantly decreased (p < 0.01) and were still higher than that in control group (p < 0.05). However, treatment with Safingol (PKCαinhibitor), LY333531 (PKCβinhibitor) and rottlerin (PKCδinhibitor) exerted no effects on hypertonicity-induced the content of HSP70-2, HSP70-2mRNA and MUC5AC secretion proteins.
4. After OREBP was knocked down by RNAi, the level of OREBP, HSP70-2 and MUC5AC in supernatant were significantly decreased compared with control group (all p <0.05).
5. Luciferase reporter gene plasmids containing different length of human HSP70-2 gene promoter were successfully constructed. After luciferase reporter plasmid containing different length of human HSP70-2 gene promoter and OREBP siRNA were co-transfected into HBE16 cells by eukaryocyte transfection technology, it is observed that hypertonicity could also increase the expression of luciferase reporter gene plasmid containing pGL3-HSP70-2(-2181/+165)-Luc (including 2 ORE sites), pGL3-HSP70-2(-1394/+165)-Luc (including 1 ORE site) and pGL3-HSP70-2(-353/+165)-Luc (including 1 ORE site) of HSP70-2 promoter in the transfected HBE16 cells with hyptertonicity. Relative luciferase unit (RLU) of cells with hypertonicity had no obviously alteration (p > 0.05). Whereas deletion derivative pGL3-HSP70-2(-66/+165)-Luc (including no ORE site) of HBE16 promoter prevented hypertonicity-induced increase in the expression of luciferase reporter gene plasmid which was significantly decreased RLU than that in hypertonic group (p < 0.01).
6. Luciferase reporter gene plasmids containing ORE1 (-1975bp) and ORE2 (-93bp) directed mutagenesis site of human HSP70-2 gene promoter were successfully constructed separately. After luciferase reporter plasmid containing ORE1 and ORE2 directed mutagenesis site of human HSP70-2 gene promoter and OREBP siRNA co-transfected into HBE16 cells by eukaryocyte transfection technology, it is observed that hypertonicity could increase the expression of luciferase reporter gene plasmid containing directed mutagenesis ORE1 site of HSP70-2 promoter in the transfected HBE16 cells with hyptertonicity (p > 0.05). The expression of luciferase reporter gene plasmid containing directed mutagenesis ORE2 site of HSP70-2 promoter in the transfected HBE16 cells with hyptertonicity had significantly decreased compared with that in hypertonic group (p < 0.01).
Conclusions:
1. Hypertonicity could increase the level of MUC5AC secretion in a concentration and a time dependent manner within experimental density, while the cells activity and motility rate decreases with the increase of hypertonic density and the duration of incubation.
2. The flourescence expression of OREBP and HSP70-2 intracellular were increased significantly after cells were cultured with hypertonicity. The expression of HSP70-2 protein and transcriptional level, OREBP protein were significantly increased after cells induced by hypertonicity. It suggests that HSP70-2 and OREBP are required in the process of MUC5AC secretion in HBE16 cells with hypertonicity. The level of HSP70-2 protein, transcription and the MUC5AC was decreased after cells with hypertonicity were handled with inhibitor of PKCμcompared with that in hypertonic group, while these levels were still higher than that in control group. The level of HSP70-2 protein, transcription and the MUC5AC secretion had no change after cells were interfered with inhibitor of PKCα/β/δ. The results show that hypertonic stress induces MUC5AC hypersecretion partly through PKCμ-HSP70-2 dependent signaling pathways. The expression of OREBP, HSP70-2 and MUC5AC secretion in supernatant were significantly decreased after OREBP were knocked down by OREBP RNAi. The results further confirm that OREBP and HSP70-2 may participate in the process of mucin hypersecretion with hypertonicity.
3. Luciferase reporter gene plasmids containing different length and ORE directed mutagenesis site of human HSP70-2 gene promoter were successfully constructed separately. It provides an experimental basis for advanced develop the regulation mechanism of mucin hypersecretion in hypertonicity in the future days.
4. Hypertonicity could increase the expression of luciferase reporter gene plasmid containing pGL3-HSP70-2(-2181/+165)-Luc (including 2 ORE sites), pGL3-HSP70-2(-1394/+165)-Luc (including 1 ORE site) and pGL3-HSP70-2(-353/+165)-Luc (including 1 ORE site) of HSP70-2 promoter in the transfected HBE16 cells with hyptertonicity. While deletion derivative pGL3-HSP70-2(-66/+165)-Luc (including no ORE site) of HBE16 promoter prevented hypertonicity-induced RLU increase in the luciferase reporter gene plasmid. Luciferase assay indicated that an important ORE site in HSP70-2 promoter was in the region from -66bp to -353 bp.
5. hypertonicity could increase the expression of luciferase RLU in reporter gene plasmid containing directed mutagenesis ORE1 site (-1975 bp) of HSP70-2 promoter in the transfected HBE16 cells with hyptertonicity, while the inactivation of the ORE2 site at -93 bp using site-directed mutagenesisi led to complete loss of HSP70-2 promoter activity. It suggests that One ORE site at -93 bp in the HSP70-2 promoter was found to be essential roles to induce HSP70-2 transcription and MUC5AC hypersecretion in human bronchial epithelial cells in response to hypertonicity.
气道黏液高分泌是慢性气道炎症性疾病包括慢性阻塞性肺疾病、支气管哮喘和肺囊性纤维化等疾病的一个重要病理特征。人支气管上皮细胞黏液增多是炎症级联反应的结果,水、无机盐离子和黏蛋白(mucin, MUC)等大分子构成气道黏液层,黏液潴留可导致病变气道阻塞加重,并为气道定植细菌提供良好的培养基,同时可释放多种促分泌因子加剧黏液潴留过程[1,2,3],从而加快疾病的进程并使病情恶化。理论上黏液层物理和化学性质的任何改变都会影响黏液性状和分泌量,已知MUC5AC是气道主要分泌型MUC,是超负荷生成病理性黏液中的主要成分,作为炎症气道黏蛋白的主要特征性表型,决定着高分泌黏液的病理特征[4,5]。目前对MUC5AC基因表达和分泌的相关分子机制已广泛开展,已知体内多种炎症介质和细胞外因素如白介素(interleukin, IL)-4、IL-13、IL-9、IL-6、IL-1β、肿瘤坏死因子(tumor necrosis factor, TNF-α)、中性粒细胞弹力酶(neutrophil elastase, NE) [11]、表皮生长因子受体(epidermal growth factor receptor, EGFR)配体[12]、空气污染物[13]、细菌产物[14]、类花生酸类、自由基[15]和氧化应激[16]等刺激均可以导致MUC5AC的异常,但是对临床上常见的高渗盐水可诱导排痰的分子机制却并不清楚,渗透压改变的条件下对MUC5AC分泌的效应尚无报道。既往研究发现气道上皮细胞黏液出胞过程中黏液分泌颗粒复合物形成必需热休克蛋白(heat shock proteins, HSP)70参与[17], HSP70可在多种刺激下诱导表达以保护气道细胞,并与蛋白激酶C(protein kinase C, PKC)有关[18],渗透压反应增强子结合蛋白(osmotic response element binding protein, OREBP/ nuclear factor of activated T cells, NFAT5/ toncity-responsive binding-protein, TonEBP)是哺乳动物细胞内唯一已知渗透压相关转录因子,可调控HSP70等的表达,保护细胞免受高渗应激[19]。本研究选用体外培养人支气道上皮细胞株HBE16的方法,以常见的高渗盐水作为干预因素,探讨渗透压刺激对MUC5AC分泌的效应,以及HSP70、PKC和OREBP在黏液分泌中的机制,研究高渗下OREBP对HSP70的调控作用及其对MUC5AC分泌的影响,从而深入阐明高渗在气道慢性炎症性疾病黏液分泌中的可能机制,并为能从基因和分子水平解释有效的临床防治措施提供实验依据。
方法:
细胞培养和高渗处理:体外培养正常人气道上皮HBE16细胞株,加入10%高渗氯化钠配成不同浓度的高渗培养基,渗透压计检测渗透压。高渗培养前用无激素和无血清培养基培养。四甲基偶氮唑盐光吸收法(methyl thiazolyl tetrazolium, MTT法)检测细胞活性。PKC各亚型抑制剂和OREBP siRNA做为干扰因素处理高渗培养HBE16细胞。
蛋白表达和转录水平的检测:采用酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA)检测培养上清中MUC5AC蛋白含量作为评价黏液分泌量的指标;Western Blot检测HSP70-2和OREBP蛋白表达,RT-PCR方法半定量检测HSP70-2转录水平。细胞免疫化学和激光共聚焦技术观察细胞在高渗下HSP70-2和OREBP蛋白表达的变化。
HSP70-2基因上游不同长度启动子序列荧光素酶(luciferase, Luc)报告基因质粒载体的设计和构建:从HBE16细胞中抽提人基因组DNA序列为模板,根据HSP70-2基因启动子上游调控区的基因序列及质粒表达载体pGL3-Basic的结构特点及实验需要设计扩增引物,PCR扩增HSP70-2基因启动子上游5’端不同片断,经酶切和DNA测序鉴定证实HSP70-2基因启动子上游序列系列截短的荧光素酶报告基因表达质粒构建成功。
含人HSP70-2基因启动子渗透压反应增强子(osmotic response element, ORE)位点定点突变序列荧光素酶报告基因质粒载体的设计和构建:基因启动子软件分析提示HSP70-2基因启动子上游存在两个潜在的ORE,ORE1位于转录起始点-1975位,ORE2位于-93位。ORE的共有序列为:TGGAANNNYNY (Y为T或C,N为任一碱基)。设计并导入定点突变引物使HSP70-2启动子上游的两个ORE位点分别单独失活。含人HSP70-2基因上游ORE位点定点突变序列荧光素酶报告基因质粒经酶切和DNA测序鉴定证实构建成功。
细胞转染和荧光素酶活性测定:报告基因质粒和OREBP siRNA通过脂质体2000共转染HBE16细胞,连续培养24h和48h后加高渗处理,12小时孵育后收集细胞,采用双荧光素酶报告基因检测系统计算报告基因的相对表达水平。
结果:
1、高渗能有效刺激HBE16细胞培养上清MUC5AC蛋白分泌量增加,与对照组相比较差异显著,有统计学意义,且结果显示在所选实验浓度范围内(500和600mOsm/kgH2O) MUC5AC随培养时间和培养浓度的改变对高渗盐水刺激有时间和剂量依赖关系(p < 0.05)。当渗透浓度增加到1000mOsm/kgH2O时HBE16细胞存活率降低,与对照组相比,差异有显著性(p < 0.05)。
2、高渗培养后,OREBP和HSP70-2蛋白在细胞内的荧光表达显著增加,HSP70-2蛋白和转录水平较对照组显著升高,并随着培养时间的延长而逐渐增加(p < 0.05),同时OREBP蛋白表达增加,且随培养时间延长而逐渐增加(p < 0.01)。
3、用PKCμ抑制剂G?6976处理高渗培养下HBE16细胞,培养上清MUC5AC蛋白含量、HSP70-2蛋白和转录水平较高渗组显著降低(p < 0.01),但仍高于对照组(p < 0.05);而PKCα抑制剂Safingol,PKCβ抑制剂LY333531和PKCδ抑制剂Rottlerin处理细胞后,上述指标水平无显著改变。
4、siRNA抑制OREBP表达后,高渗刺激下的OREBP、HSP70-2和上清MUC5AC蛋白相对含量显著减少(p均<0.05)。
5、成功构建系列截短的HSP70-2启动子荧光素酶报告基因质粒,真核细胞转染技术与OREBP siRNA共转染导入HBE16细胞,观察高渗刺激下的细胞,结果显示转染分别含2、1、1个ORE位点的pGL3-HSP70-2(-2181/+165)-Luc、pGL3-HSP70-2(-1394/+165)-Luc和pGL3-HSP70-2(-353/+165)-Luc细胞后在高渗刺激下的荧光素酶比活性无明显改变(p>0.05);而转染HSP70-2启动子不含ORE位点的pGL3-HSP70-2(-66/+165)-Luc细胞在高渗刺激下的荧光素酶比活性与高渗组的细胞相比显著降低(p<0.01)。
6、构建突变-1975位点的ORE1和位于-93位点的ORE2的HSP70-2启动子报告基因载体,与OREBP siRNA共转染细胞后检测,突变ORE1后细胞在高渗刺激下的荧光素酶表达与高渗组相比无显著改变(p>0.05);突变ORE2后高渗刺激下的荧光素酶比活性与高渗组比较显著降低有统计学意义(p<0.01)。
结论:
1、高渗刺激可诱导体外培养的HBE16细胞MUC5AC分泌,在所选实验浓度范围内呈现时间和浓度依赖关系,但同时高渗对HBE16细胞活力的影响也逐步加大,随渗透时间延长和渗透浓度加大细胞存活率下降。
2、高渗培养后,OREBP和HSP70-2蛋白在细胞内的荧光表达显著增加,HSP70-2蛋白和转录水平,OREBP蛋白水平均显著增加,提示HSP70-2和OREBP参与高渗致HBE16细胞MUC5AC分泌过程。用PKCμ抑制剂处理高渗培养下细胞,HSP70-2蛋白、转录水平和MUC5AC分泌较高渗组显著减少,但仍高于对照组;而用PKCα抑制剂、PKCβ抑制剂和PKCδ抑制剂处理后HSP70-2蛋白、转录水平和MUC5AC分泌均无改变,考虑高渗致黏液分泌可能是部分通过PKCμ-HSP70-2依赖的信号通路。siRNA抑制OREBP表达后,高渗刺激下的OREBP、HSP70-2和上清MUC5AC蛋白相对含量显著减少,进一步证实OREBP和HSP70-2参与高渗下黏液分泌的过程。
3、成功构建系列截短的HSP70-2上游启动子荧光素酶报告基因质粒和含两个ORE位点突变的HSP70-2启动子荧光素酶报告基因质粒,为今后深入开展高渗对黏液分泌的调控机制奠定基础。
4、高渗刺激可分别诱导HSP70-2启动子含2、1、1个ORE位点的pGL3-HSP70-2(-2181/+165)-Luc、pGL3-HSP70-2(-1394/+165)-Luc和pGL3-HSP70-2(-353/+165)-Luc序列的报告基因质粒仍然表达荧光素酶,而不含ORE位点的pGL3-HSP70-2(-66/+165)-Luc序列的报告基因质粒对高渗刺激无反应,不表达荧光素酶,说明在HSP70-2启动子上游-66到-353序列之间存在ORE关键位点。
5、高渗刺激可诱导HSP70-2启动子-1975位点的ORE1发生定点突变序列的报告基因质粒表达荧光素酶,而含有-93位点的ORE2发生定点突变序列的报告基因质粒对高渗刺激无明显反映,提示高渗盐水诱导OREBP通过与HSP70-2启动子上-93位点的ORE结合而激活HSP70-2转录并促进HBE16细胞MUC5AC高分泌。
Objective:
Airway mucus hypersecretion is a main pathological feature of many chronic airway inflammatory diseases such as chronic obstructive pulmonary disease, bronchus asthma, lung cystic fibrosis and etc. The viscous mucin rich in human bronchial epithelial cells is a consequence of an inflammatory cascade reaction. Airway mucus layer consists of some large molecules which are water, inorganic salt ions, mucus protein (mucin, MUC) and etc. The excessive mucus leads to severe airway obstruction and provides a good medium for airway bacterial colonization. In addition, many excreted factors are crowded to aggravate the process of mucus obstruction and diseases [1, 2, 3]. Theoretically, it does not avoid that any change with physical and chemical properties of mucus layer would affect mucous traits and secretions. MUC5AC glycoprotein is the major respiratory mucins which present in secretion and is a main ingredient in generatin pathological mucus under overload [4, 5]. The molecular mechanism of the expression and the secretion of MUC5AC are under widely studied in recent years. It is well known that many mediators of inflammation and extracellular factors would lead disorder of MUC5AC. During the course of airway inflammatory disease, MUC5AC has been shown to be stimulated by a wide variety of stimuli, including proinflammatory cytokines such as interleukin (IL)-4[6], IL-13[7], IL-9[8], IL-6[9], IL-1βand tumor necrosis factor (TNF)-α[10], neutrophil elastase (NE)[11], epidermal growth factor receptor (EGFR) ligands[12], air pollutants[13], bacterial products[14], eicosanoids, and free radicals[15]. MUC5AC gene expression is also known to be regulated by oxidative stress [16]. However, the concrete molecular mechanisms of a common therapies that mucus overproduction was induced by hypertonic saline solution in clinic have not been clear at present. There still no report about hypertonicity on the mechanisms of MUC5AC secretion and regulation. Previous studies show that heat shock proteins 70 (HSP70) which are key related chaperones in the exocytosis process of mucin secretory granules formation by airway epithelial cells are expressed to protect airway cells induced by multiple stimuli[17] and are related with protein kinase C (PKC)[18]. Osmotic response element binding protein (OREBP/ nuclear factor of activated T cells, NFAT5/ toncity-responsive binding-protein, TonEBP) which is an important transcription factor in mammalian cells controlling the cellular response to osmotic stress regulate the express of HSP70 and protect cells in the stress of hypertonicy [19]. Our study was to observe the effect of hypertonic saline solution on MUC5AC protein
secretion in normal human bronchial epithelial cells cultured in vitro, and to disscuss the detailed molecular mechanism of HSP70, PKC and OREBP in the regulation of MUC5AC secretion, and to study the possible mechanism of OREBP in the transcriptional regulation of HSP70 and in MUC5AC secretion. This study tried to show the possible mechanism of mucin secretion in chronic airway inflammation disease with hypertonicy and to provide experimental experience for researchers to search effective clinical prevention and cure of mucus hypersecretion in a genic and molecular level.
Methods:
Cell culture and hypertonicy incubation: Normal human bronchial epithelial cells, HBE16 cells were cultured in vitro. Hypertonic medium with different concentration were added by 10 percent hypertonic saline salution and the osmotic pressure were detected by osmometer. Cells were starved in serum- and hormone-free medium before cells were cultured with hypertonicity. Cells activity was assessed by methyl thiazolyl tetrazolium (MTT) method. Inhibitors of PKC isoforms and OREBP siRNA were served to interference HBE16 cells with hypertonicy.
Level of proteins expression and transcription: MUC5AC protein content in supernatant as an index to evaluate mucus secretary volume was analyzed by ELISA. The expression of HSP70-2 and OREBP were determined by Western blot analysis. RT-PCR was used to detect the transcriptional level of HSP70-2. The changes of OREBP and HSP70-2 protein in cells with hypertonicity were observed by immunofluorescence and confocal laser technology.
Design and construction of luciferase reporter gene plasmids containing different length of human HSP70-2 gene promoter: DNA template was extracted from human genomic DNA in HBE16 cells. PCR amplification primers were designed according to gene sequence of upstream promoter region of HSP70-2 gene and construction features of plasmid expression vector pGL3-Basic. 5 'end different fragments of HSP70-2 upstream were amplified and observed by sequence analysis. Different HSP70-2 promoter sequences length luciferase reporter gene plasmid vector constructed successfully were confirmed by restriction enzymes digestion and DNA sequences alignment.
Design and construction of luciferase reporter gene plasmids containing osmotic response element (ORE) directed mutagenesis site of human HSP70-2 gene promoter: Promoter sequence analysis showed that there were two potential ORE sites in the promoter of HSP70-2. ORE1 was located in -1975 bp of promoter and ORE2 was in -93 bp of promoter. Consensus sequence of ORE was:GGAANNNYNY (Y is T or C, N is any base). Two point mutations were introduced at the ORE1 and ORE2 sites of HSP70-2 promoter separately. Site directed mutagenesis HSP70-2 promoter sequences length luciferase reporter gene plasmid vector constructed successfully were confirmed by restriction enzymes digestion and DNA sequences alignment.
Cell transfection and luciferase activity assay: Reporter gene plasmid and OREBP siRNA were co-transfected using lipofectinamine 2000 in HBE16 cells. Cells were continuously cultured. 24 h and 48 h later, cells were induced by hypertonic saline solution. Cells were collected after 12 h. Dual-luciferase reporter assay system was used to calculate relative expression level of reporter genes.
Results:
1. The secretion of MUC5AC protein in supernatant were elevated significantly in hypertonicity stimulating group (500 and 600mOsm/kgH2O) compared with that in control group. Hypertonic saline solution could increase the level of secretion MUC5AC protein in a concentration and a time dependent manner (p < 0.05). While HBE16 cells activity decrease significantly in hypertonic conditions (1000mOsm/kgH2O) compared with that in control group (p < 0.05).
2. The flourescence expression of OREBP and HSP70-2 intracellular were increased significantly after cells were cultured with hypertonic saline solution. Hypertonicity increased the level of HSP70-2 protein and mRNA in a time dependent manner (p < 0.05). The expression of OREBP protein was significantly increased in hypertonic group compared with that in control group and in a time dependent manner (p < 0.01).
3. Inhibitor of PKCμ(G?6976) was served to interference HBE16 cells with hypertonicity. The content of MUC5AC secretion in supernatant, the level of HSP70-2 protein and mRNA were significantly decreased (p < 0.01) and were still higher than that in control group (p < 0.05). However, treatment with Safingol (PKCαinhibitor), LY333531 (PKCβinhibitor) and rottlerin (PKCδinhibitor) exerted no effects on hypertonicity-induced the content of HSP70-2, HSP70-2mRNA and MUC5AC secretion proteins.
4. After OREBP was knocked down by RNAi, the level of OREBP, HSP70-2 and MUC5AC in supernatant were significantly decreased compared with control group (all p <0.05).
5. Luciferase reporter gene plasmids containing different length of human HSP70-2 gene promoter were successfully constructed. After luciferase reporter plasmid containing different length of human HSP70-2 gene promoter and OREBP siRNA were co-transfected into HBE16 cells by eukaryocyte transfection technology, it is observed that hypertonicity could also increase the expression of luciferase reporter gene plasmid containing pGL3-HSP70-2(-2181/+165)-Luc (including 2 ORE sites), pGL3-HSP70-2(-1394/+165)-Luc (including 1 ORE site) and pGL3-HSP70-2(-353/+165)-Luc (including 1 ORE site) of HSP70-2 promoter in the transfected HBE16 cells with hyptertonicity. Relative luciferase unit (RLU) of cells with hypertonicity had no obviously alteration (p > 0.05). Whereas deletion derivative pGL3-HSP70-2(-66/+165)-Luc (including no ORE site) of HBE16 promoter prevented hypertonicity-induced increase in the expression of luciferase reporter gene plasmid which was significantly decreased RLU than that in hypertonic group (p < 0.01).
6. Luciferase reporter gene plasmids containing ORE1 (-1975bp) and ORE2 (-93bp) directed mutagenesis site of human HSP70-2 gene promoter were successfully constructed separately. After luciferase reporter plasmid containing ORE1 and ORE2 directed mutagenesis site of human HSP70-2 gene promoter and OREBP siRNA co-transfected into HBE16 cells by eukaryocyte transfection technology, it is observed that hypertonicity could increase the expression of luciferase reporter gene plasmid containing directed mutagenesis ORE1 site of HSP70-2 promoter in the transfected HBE16 cells with hyptertonicity (p > 0.05). The expression of luciferase reporter gene plasmid containing directed mutagenesis ORE2 site of HSP70-2 promoter in the transfected HBE16 cells with hyptertonicity had significantly decreased compared with that in hypertonic group (p < 0.01).
Conclusions:
1. Hypertonicity could increase the level of MUC5AC secretion in a concentration and a time dependent manner within experimental density, while the cells activity and motility rate decreases with the increase of hypertonic density and the duration of incubation.
2. The flourescence expression of OREBP and HSP70-2 intracellular were increased significantly after cells were cultured with hypertonicity. The expression of HSP70-2 protein and transcriptional level, OREBP protein were significantly increased after cells induced by hypertonicity. It suggests that HSP70-2 and OREBP are required in the process of MUC5AC secretion in HBE16 cells with hypertonicity. The level of HSP70-2 protein, transcription and the MUC5AC was decreased after cells with hypertonicity were handled with inhibitor of PKCμcompared with that in hypertonic group, while these levels were still higher than that in control group. The level of HSP70-2 protein, transcription and the MUC5AC secretion had no change after cells were interfered with inhibitor of PKCα/β/δ. The results show that hypertonic stress induces MUC5AC hypersecretion partly through PKCμ-HSP70-2 dependent signaling pathways. The expression of OREBP, HSP70-2 and MUC5AC secretion in supernatant were significantly decreased after OREBP were knocked down by OREBP RNAi. The results further confirm that OREBP and HSP70-2 may participate in the process of mucin hypersecretion with hypertonicity.
3. Luciferase reporter gene plasmids containing different length and ORE directed mutagenesis site of human HSP70-2 gene promoter were successfully constructed separately. It provides an experimental basis for advanced develop the regulation mechanism of mucin hypersecretion in hypertonicity in the future days.
4. Hypertonicity could increase the expression of luciferase reporter gene plasmid containing pGL3-HSP70-2(-2181/+165)-Luc (including 2 ORE sites), pGL3-HSP70-2(-1394/+165)-Luc (including 1 ORE site) and pGL3-HSP70-2(-353/+165)-Luc (including 1 ORE site) of HSP70-2 promoter in the transfected HBE16 cells with hyptertonicity. While deletion derivative pGL3-HSP70-2(-66/+165)-Luc (including no ORE site) of HBE16 promoter prevented hypertonicity-induced RLU increase in the luciferase reporter gene plasmid. Luciferase assay indicated that an important ORE site in HSP70-2 promoter was in the region from -66bp to -353 bp.
5. hypertonicity could increase the expression of luciferase RLU in reporter gene plasmid containing directed mutagenesis ORE1 site (-1975 bp) of HSP70-2 promoter in the transfected HBE16 cells with hyptertonicity, while the inactivation of the ORE2 site at -93 bp using site-directed mutagenesisi led to complete loss of HSP70-2 promoter activity. It suggests that One ORE site at -93 bp in the HSP70-2 promoter was found to be essential roles to induce HSP70-2 transcription and MUC5AC hypersecretion in human bronchial epithelial cells in response to hypertonicity.
引文
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