Regulation Of Hypertonicity On Respiratory Mucin MUC5AC Secretion In Human Airway Epithelial Cells

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.