The Effect Of Vitamin D₃ Up-regulated Protein 1 In Bronchial Epithelium On Asthma And The Research Of Its Molecular Mechanisms

The research is supported by National Natural Science Foundation of China (No.30971328) and Specialized Research Fund for the Doctoral Program of Higher Education (No.20094433110011).BACKGROUNDBronchial asthma is one kind of chronic inflammatory disease of airway. Patients of asthmatic symptoms suffered from repeated gasp, dyspnoea, chest distress and cough because of airway hyper-responsiveness and obstruction. In the past twenty years the mobility of asthma and other allergy diseases in developed countries increased. Report from the world health organization show "the harm and the economic burden caused by asthma to human being were more than those by tuberculosis and AIDS".Scientists have performed plenty of penetrating researches on the mechanisms of asthmatic airway inflammation and remodeling for many years. There is some distinctive change in asthma such as the enhancement of Th2 type immunological reaction, thickening of basal membrane under the epithelium and hyperplasia of smooth muscles. However, the cause and the mechanisms of asthma are unclear. Inhaling corticosteroids, the first important therapy measure of asthma recommended by the asthma guide, can control the asthmatic symptoms effectively but cannot inhibit the potential development of disease.Bronchial epithelium is the first barrier of the airway. The airway epithelium not only defenses the environment with mucosal fluid and cilia but also secretes many mediators, cytokines and growth factors, which actively effect on the pathogenesis of respiratory diseases. The injury and shedding of airway epithelium is one of the important pathologic characters. Reduced barrier function might increase susceptibility to sensitization and lower the threshold of antigen exposures required to drive local antigen-dependent inflammation, which could initiate the following pathophysiologic courses. Bronchial epithelium is central to the pathogenesis of asthmatic airway inflammation and remodeling, and correlate with the pathophysiologic change such as airway obstruction and hyperresponsiveness.Airway epithelial cell (AEC), the primary composition of the airway epithelium, plays the central role in maintaining of epithelial structure and function. Epithelial cells secrete several key cytokines that drive Th2 chemotaxis after allergen challenge. Then, with the activation of the epithelial mesenchymal trophic unit (EMTU) the asthmatic airway epithelium is a major source of cytokines and chemokines that are strongly implicated in maintaining asthmatic inflammation including IL-5, RANTES, eotaxin etc. Otherwise, activated and repairing epithelial cells generate a range of growth factors such as TGFβ,VEGFs and so on, the majority of which are capable of interacting with fibroblasts to either cause or maintain the airway remodeling. So, different approaches to treatment can be devised focused more on protecting vulnerable airways against environmental injury rather than focusing on suppressing airway inflammation or manipulating the immune response.Thymic stromal lymphopoietin (TSLP) produced by epithelial cells might be a master switch for allergic inflammation. It is a novel IL-7-like cytokine. Human TSLP is produced primarily by epithelial cells in the lungs, gut, and skin but not by hematopoietic cells.The primary cell which TSLP effect on is DC. TSLP-activated DCs cannot induce the production of Thl-polarizing cytokines but help to create a Th2-permissive microenvironment. TSLP induces human mDCs to express the TNF superfamily protein OX40L. Signals triggered by OX40L induced the generation of inflammatory Th2 cells. At the same time, the expression of TSLP is induced in airway epithelial cells by stimulation with Th2 cytokines. The recruitment of Th2 cytokine producing cells may amplify Th2 inflammation via the induction of TSLP in the asthmatic airway.The expression of TSLP in asthmatic patients’airway epithelium and subepithelium were higher than healthy groups, and the level of TSLP correlated with the degree of airway obstruction, the amount of Th2 cytokines and the severity of disease. These results demonstrate that a high level of TSLP is associated with asthma in humans. A key molecular mechanism that links mediators and TSLP production is the NF-kB signaling pathway, which is activated by many proinflammatory cytokines. With the increase of our knowledge about the functions of AEC in asthma, we need to perform further researches on the regulation mechanism of TSLP in asthmatic AEC. It will provide the theoretical and experimental basement to future therapy for asthma.It is clear that environment and life style have effect on asthma. Ecologically, many of the patterns of vitamin D deficiency appear to parallel the patterns of the asthma epidemic. Although an intriguing suggestion, the link between vitamin D and the asthma epidemic is premature. In contrast with infectious pulmonary disease, where a clear association has been demonstrated, the data for asthma are less clear.Vitamin D3 up-regulated protein 1 (VDUP1) was originally identified in HL60 cells stimulated with 1,25-(OH)2D3. It is a multifunctional protein involved in maintaining cellular homeostasis in particular containing cellular proliferation, apoptosis and differentiation. Besides, VDUP1 is a major messenger in intracellular physiological processes triggered by various stress stimulation. The development of fetus’lungs in sheep needs VDUP1. VDUP1 correlates with complex diseases such as diabetes, tumor, and atopic dermatitis and so on.However, the effects of VDUP1 on apoptosis were reported to be dependent on cell types. VDUP1 was reported to bind the reduced thioredoxin (TRX) by yeast-two hybrid assay, hence it is thought to be a negative regulator of TRX. TRX is one of the cellular important antioxidants involved in oxidative stress. VDUP1 blocks the reducing activity of TRX and inhibits the interaction between TRX and other factors, such as ASK-1 and PAG. Recent research suggested that VDUP1 induces inflammation through chromatin modification in renal capillary endothelial cells under diabetic conditions.Our research focus on VDUP1, which has been identified to be related to eosinophils activation and pulmonary function in our previous studies. Up to now the effect of VDUP1 on asthma has been unclear. But TRX benefits to asthma. Exogenous TRX1 inhibits airway hyperresponsiveness and pulmonary inflammation accompanied by eosinophilia in mouse models of asthma. Moreover, TRX not only prevents the development of airway remodeling but also improves established airway remodeling in mouse asthma model.OBJECTIVETo further investigate effects and mechanism of VDUP1 on the pathogenesis of asthma, and to provide novel target for intervention of asthma.METHODS1. Animals’bronchus were obtained in asthmatic animal models. Then the expressions of VDUP1 mRNA were measured by real-time RT-PCR.2. Bronchial mucosa of asthmatic patients were obtained by bronchofibroscope. The expressions of VDUP1 mRNA in mucosa were measured by real-time RT-PCR.3. To determine whether the level of VDUP1 in bronchial epithelial cells in vitro is affected by some environmental stimulations involved in asthma.4. Real-time RT-PCR and ELISA were performed to determine the effect of overexpression of VDUP1 on the expression of TSLP in AEC5. To determine the effect of knockdown of VDUP1 on the expressing of TSLP by real-time RT-PCR in gene level and by ELISA in protein level respectively.CONTENT AND COURSE OF RESEARCH1. To measure the expression of VDUP1 in bronchus.1.1 The levels of VDUP1 in asthmatic animals’bronchus.Eighteen BALB/c mice were divided equally into a control, asthmatic and treated groups. The asthmatic mice were sensitized with ovalbumin. The treated mice were injected with dexamethasone via intraperitoneal. After the challenge, we detected the airway responsiveness with Penh. The bronehialalveolar lavage fluid(BALF) was collected for examining the cell number and the level of IL-4, and the bronchus tissue was taken to evaluate the airway inflammation. The expressions of VDUP1 mRNA were measured by real-time RT-PCR.Results:We succeeded to set up the asthmatic mode of mouse. These asthmatic mice appeared dyspneic. We found hyperresponsiveness, recruitment of inflammatory cells including eosinophils and lymphocytes and increasing IL-4 in the asthmatic mice’s BALF. We also observed the characteristic pathological changes in the asthmatic mice’s bronchus, including thickening of bronchial wall and infiltration of inflammatory cells. The levels of VDUP1 mRNA in asthmatic mice’s bronchus (2.74±0.99) were significantly higher than those in control (1.01±0.18, P=0.020) or those in treated groups (0.94±0.34, P=0.015).1.2 levels of VDUP1 in asthmatic animals’bronchus.We selected patients according to the diagnostic criteria of asthma. Asthmatic patients had a clear history of relevant symptoms, documented reversible airway obstruction either spontaneously or after administration of inhaledβ2 agonist, and/or methacholine provocation before biopsy. Each subject provided written, informed consent. Endobronchial biopsies were obtained from six asthmatics and five normal controls. The expressions of VDUP1 mRNA were measured by real-time RT-PCR.Results:The levels of VDUP1 mRNA in asthmatic patients bronchus (2.11±1.15) were higher than those in health persons (1.00±0.10), but there is no significant difference between two groups (P=0.063). The levels of VDUP1 mRNA in asthmatic patients’bronchus without treatment (2.49±1.62) were higher than that of asthmatic patients with treatment (1.72±0.48).2. The effect of various kinds of environmental factors on the expression of VDUP1 in AEC.After isolated culture 16HBE were stimulated with one of the following at the indicated concentration:200U/L Derp1,100μg/ml TDI-HSA,200ng/ml 25(OH)D3 for 6 hours. The expressions of VDUP1 mRNA were measured by real-time RT-PCR.Results:The levels of VDUP1 mRNA were significantly up-regulated with the treatments of Derp1 (1.69±0.59 vs.1,95% CI:1.07-2.31), TDI-HSA (1.56±0.32 vs.1, 95%CI:1.23-1.89), and 25(OH)D3 (2.04±0.65 vs.l,95%CI:1.36-2.71).3. To explore the mechanism of VDUP1 in AEC participating pathogenesis of asthma.3.1 The effect of VDUP1 on cell viability.VDUP1 were overexpressed by transient transfection of corresponding plasmids at 60-80% confluence of cells. On the other hand,16HBE were transfected with siRNA against VDUP1 or control RNA at 5nM using HiPerFect transfection reagent following the manufacturer’s instructions at 40-60% confluence. MTT assay was used to evaluate cell viability.Results:Cell viability of AECs with overexpression of VDUP1 was lower than those of empty vector (71.94±13.72% vs.100.02±9.20%, P=0.003). Cell viability of AECs with knockdown of VDUP1 was higher than those of control RNA (109.57±1.93% vs.99.98±4.30%, P=0.002).3.2 The effect of overexpression of VDUP1 on the level of TSLP in AEC.After overexpression, real-time RT-PCR and ELISA were used to determine whether overexpression of VDUP1 affected on the expression of TSLP in mRNA level and protein level respectively.Results:The levels of TSLP mRNA in AEC with overexpression of VDUP1 were higher than those of empty vector (2.13±0.84 vs.l,95%CI:1.25-3.02). Moreover, the protein levels of secreted TSLP in medium were higher too (156.86±77.68 pg/ml vs.60.10±18.45 pg/ml, P=0.048).3.3 The effect of various kinds of environmental factors on the expression of TSLP of AEC.16HBE were stimulated with one of the following at the indicated concentration: 200U/L Derp1, 100μg/ml TDI-HSA, and 200ng/ml 25(OH)D3 for 30 min,2 hours,6 hours,12 hours and 24 hours respectively. The expressions of TSLP mRNA were measured with real-time RT-PCR.Results:The levels of TSLP mRNA were significantly up-regulated by TDI-HSA for 24 hours (1.96±0.59 vs.1,95%CI:1.35～2.58) and 25(OH)D3 for 2 hours and 6 hours (respectively 2.38±0.65 vs.1,95%CI:1.70-3.06 and 1.95±0.55 vs.1, 95%CI:1.38-2.53), and not affected by Derpl in NHBE (P>0.05).3.4 The effect of knockdown of VDUP1 on the expression of TSLP in AEC. Transfected cells were further cultured for 48h and then stimulated with 100μg/ml TDI-HSA or 200ng/ml 25(OH)D3 for 6h. Real-time RT-PCR and ELISA were used to determine whether knockdown of VDUP1 affected on the expression of TSLP in mRNA level and protein level respectively.Results:With the treatment of TDI-HSA, the levels of TSLP mRNA in AECs with knockdown of VDUP1 were lower than those of control (1.19±0.14 vs. 1.91±0.50, P=0.029).With the treatment of vitamin D, the levels of TSLP mRNA in AECs with knockdown of VDUP1 were also lower than those of control (0.78±0.08 vs.1.74±0.23, P<0.001). Moreover, the protein levels of secreted TSLP showed consistent results (55.77±19.88 pg/ml vs.141.20±49.05 pg/ml, P=0.008).CONCLUSION1. VDUP1 in bronchial epithelium participates in the pathogenesis of asthma. Related therapies for asthma maybe affect the level of VDUP1 mRNA in airway epithelium.2. Sensibilisinogens including Derp1 and TDI-HSA induce the expression of VDUP1 in AEC in vitro.3. VDUP1 in AEC plays an important role in the pathogenesis of asthma by affecting the proliferation of cells and the expression of TSLP.4. One of the mechanisms of TDI-induced asthma is TDI-HSA induces the expression of TSLP of AEC by up-regulating the expression of VDUP1.5. Vitamin D induces the expression of TSLP of AEC by up-regulating the expression of VDUP1.