The Mechanism And Inhibitory Effect Of Recombinant Rat CC16 Protein On LPS-Induced Inflammatory Mediators Expression In Rat Tracheal Epithelial Cells

Clara cell protein（CC16） is a 16-k Da homodimeric protein secreted predominantly by non-ciliated airway epithelial（Clara） cells, and is regarded as an anti-inflammatory factor native to the lung and plays an important role in mediating immuno-inflammatory reaction. Levels of Clara cell secretory protein are dramatically reduced in either serum or airway lining fluid of patients with chronic lung diseases such as COPD, which is thought to be the contributing factor to the lung inflammations. The main inflammatory mediators involving in the pathogenesis of COPD include MMP-9, IL-6, IL-8 and TNF-alpha, and NF- kappa B signaling is a key pathway in the regulation of inflammatory reaction. Exogenous administration of the anti-inflammatory factor CC16, may have the potential inhibition of the production of inflammatory mediators and probably produce a therapeutic effect on inflammatory pulmonary diseases. In this study, gene engineering technology was conducted to clone,prokaryotic express and purify the rat CC16 protein which was proved to own the bioactivity. Then we evaluated the effect of recombinant CC16 on the expression of inflammatory factors in lipopolysaccharide（LPS）-stimulated rat tracheal epithelial cells and its underlying molecular mechanisms. The study paves the way for recombinant CC16 protein as an anti-inflammatory factor to treat the chronic lung inflammatoy disease such as COPD.Part 1 Gene-cloning,prokaryotic expression and purification of rat clara cell secretory proteinObjective: To clone,prokaryotic express and purify the rat clara cell secretory protein（CC16）which paves the way to study the function of CC16 protein and its intervention effect on chronic inflammatory airway diseases Methods:Total RNA was extracted from the lung tissue of Wistar rat. The open reading frame of CC16 gene was amplified with a pair of specific primers which was designed according to the coding sequence of CC16 gene（Gen Bank accession No: NM₀13051）,the product of RT-PCR was digested with double restrict enzyme and ligated into a p ET-30a（+） vector. The recombinant p ET-30a（+）-r CC16 plasmid was transferred into E. coli DH5α and the positive clones were selected through the colony-PCR and confirmed by the double restrict enzyme digestion and sequencing. The successful p ET-30a（+）-r CC16 construct was transformed into E. coli Rosetta（DE3） and induced with IPTG to express. The products of expression were analyzed through SDS-PAGE followed by coomassie blue staining and the recombinant r CC16 was purified using Ni2+-NTA affinity chromatograp Hy. A Western blotting assay with His primary antibody and goat anti-CC16 antibody was used to confirm the expression of r CC16.HPLC was performed to test the purity of the recombinant CC16 protein.Results: The product of RT-PCR was 290 bp. The recombinant plasmid p ET-30a（+）-r CC16 was confirmed successfully by colony-PCR, double restrict enzyme digestion and sequencing. A recombinant protein with a rough molecular weight of 16 KDa was analyzed by SDS-PAGE followed by coomassie blue staining. Meanwhile the r CC16 was detected efficiently by Western blotting analysis with the His primary antibody and with the goat anti-CC16 primary antibody respectively.Conclusion: The recombinant p ET30a（+）-r CC16 is successfully established and the purified dissoluble rat CC16 protein is obtained.The purity of r CC16 is more than 95%.Part 2 Recombinant Rat CC16 Protein Inhibits LPS-Induced Inflammatory mediators expression via NF-κB pathway in Rat Tracheal Epithelial CellsSection 1 Effect of rat recombinant CC16 protein on the expression of LPS induced IL-8,IL-6,TNF-α and MMP-9Objective: To explore the inhibitory effect of r CC16 on LPS induced expression of the inflammatory mediators.Methods:（1）The acid-base titration was applied to test the activity of p Hosp Holipase A2（PLA2） in the reaction system with or without the r CC16.（2） RTE cells were cultured with r CC16 at 5, 10, 20 μg/ml or PBS controls for 1, 3, 5, or 7 days.Cell Counting Kit-8 and Trypan Blue exclusion methods were used for cell proliferation and viability assays.（3）RTE cells were incubated with r CC16 at 0.5, 1.0, or 2.0 μg/ml in serum free media for 2 h prior to LPS（0.1 μg/ml） application and maintained for further 24 h. Cell culture supernatants or cells were then harvested. Elisa and RT-q PCR were used to determine the levels of protein and m RNA of MMP-9,IL-6,IL-8 and TNF-α. The enzyme activities of MMP-9 in the cell culture supernatants were determined with gelatin zymograp Hy assay.Results:（1） r CC16 inhibited PLA2 activity in a concentration-dependent manner. Significant PLA2 inhibition was observed at 0.5μg/ml r CC16 and reached higher level at 1.0 μg/ml and 2.0 μg/ml r CC16.（2） r CC16 at the concentration 10μg/ml and 20μg/ml but not 5μg/ml could supress the proliferation of RTE cells.No difference of cell viability was observed in cells treated with different concentrations of r CC16. These data indicated that the r CC16 we produced was biological active and displayed no cytotoxic effect at low concentrations（or lower）.（3） Increased expressions of MMP-9,IL-6,IL-8 and TNF-α were observed on both m RNA and protein levels in LPS-treated RTE cells, and these LPS-stimulated MMP-9,IL-6 and IL-8 expressions were inhibited by r CC16 in a dose-dependent manner.But for TNF-α, lower concentration of r CC16 showed the higher capacity of inhibition.Conclusion: The recombinant CC16 that we generated had the anti-inflammatory capacity. r CC16 could suppress the expression of LPS-induced MMP-9,IL-6,IL-8 and TNF-α on bothm RNA and protein levels in LPS-treated RTE cells.Section 2 Effect and molecular mechanism of rat recombinant CC16 protein on the NF-κB activation induced by LPS in RTE cellObjective: To elucidate the molecular mechanism of inhibitory effect of r CC16 on LPS induced the inflammatory mediators.Methods: RTE cells were incubated with r CC16 at 0.5, 1.0, or 2.0 μg/ml in serum free media for 2 h prior to LPS（0.1 μg/ml） application and maintained for further 1 h. The transcriptional activities of NF-κB were determined by the Dual-Luciferase Reporter Assay（Firefly and Renilla luciferases） and expressed as relative luciferase activities. Nuclear proteins were extracted from RTE cells, and Electrop Horesis mobility shift assay was used to assay the capacity of NF-κB-DNA binding.Western blotting was applied to test the level of NF-κB in cytoplasm and nuclear respectively. The subcellular distributions of NF-κB/p65 were assessed by immunofluorescence staining. IκBα, phospho-IκBα（Ser32）, p38 MAPK, phospho-p38 MAPK（Thr180/Tyr182） and phospho-p65（Ser276） were also indicated with western blotting.Results:（1） LPS caused an approximate 9-fold increase in relative luciferase activity, and r CC16 inhibited LPS-induced NF-κB transcriptional activity in a concentration-dependent manner, from 38.8% at 1.0 μg/ml to 62.6% reduction at 2.0 μg/ml.（2） LPS treatment augmented the DNA binding activity significantly, and r CC16 suppressed LPS-induced NF-κB-DNA binding at 1.0 and 2.0 μg/ml in a concentration-dependent manner.（3） An increased level of NF-κB/p65 in the nucleus was seen in LPS-treated cells, and this nuclear increase of NF-κB/p65 protein was suppressed by r CC16 in a concentration-dependent manner.（4） We found that LPS exposure resulted in increased p Hosp Horylation of IκBα, p38 MAPK and NF-κB/p65 and that pretreatment of cells with r CC16 suppressed this phosphorylation.Conclusion: r CC16 suppresses LPS-induced NF-κB activation by down-regulating both of its translocation- and phosphorylation-dependent pathways in RTE cells.（1）Inhibit IκBα phosphorylation and nucleus translocation of NF-κB.（2）Inhibit LPS-induced phosphorylation of p38 MAPK and p65 subunit of NF-κB at Ser276.Part 3 Study of r CC16 uptake by RTE cell through clathrin-mediated endocytosisObjective: To explore the role of clathrin mediated endocytosis in r CC16 uptaken by RTE cells and its anti-inflammatory effect.Methods:（1） RTE cells were cultured and grouped as control, r CC16 and r CC16+Chlorpromazine. Cells were treated with r CC16（2μg/ml） or Chlorpromazine（30μM） prior to r CC16. The subcellular locations of His-r CC16 were analysed via immunofluorescence staining.（2） RTE cells were exposed to 30 μM chlorpromazine or controls for 30 min, and then treated with r CC16 prior to LPS（0.1 μg/ml） application. The subcellular distributions of NF-κB/p65 were assessed by immunofluorescence staining and western blotting. Elisa was used to assay the MMP-9 in the the cell culture supernatants.Results:（1） r CC16 was uptaken and located in cytoplasm.RTE cells pre-incubated with chlorpromazine at 30μM for 30 min displayed strong resistance against endocytosis of r CC16（His-tagged） as shown by immunofluorescence staining with anti-His antibodies.（2） Pretreatment of RTE cells with chlorpromazine significantly blocked the inhibitory roles of r CC16 on LPS-induced NF-κB/p65 nuclear translocation. In accordance, chlorpromazine rescued the nuclear NF-κB/p65 distribution in presence of r CC16 and LPS.（3） High levels of MMP-9 production were visualized from chlorpromazine-pretreated RTE cells which were exposed to r CC16 and LPS, suggesting that chlorpromazine abolished the inhibitory effect of r CC16 on LPS-stimulated expression of MMP-9.Conclusion: Clathrin-mediated endocytosis of r CC16 is involved in its inhibitory role in LPS-induced MMP-9 production during inflammatory responses.