Induction Of Thymic Stromal Lymphopoietin Expression In16-HBE Human Bronchial Epithelial Cells By25-hydroxyvitamin D3and1,25-dihydroxy-Vitamin D3

IntroductionBronchial asthma is a chronic inflammatory disease in airways, which are related to airway inflammatory cells, cell structure and cytokines. In1990, the collaborative group of pediatric asthma investigated children aged0to14from27provinces, showing a prevalence of asthma in children of0.09%-2.60%, with an average0.91percent. In2000, the prevalence was surveyed again and found it was increased to0.12%-3.34%, with an average of1.54%, comparing with the result10years ago, the average increase rate was64.84%. Many patients are suffered from asthma in different ways in their daily life. According to WHO, in the world the number of disability years is estimated to reach15million due to the asthma, which accounting for about1%of the total global health burden. Economic burden brought about by the asthma hospitalization and drug use in terms of direct medical costs, such as lost income and non-normal or death caused by indirect non-medical costs are quite large. Although the number of new treatments and methods have been confirmed in clinical treatments, but there are many problems need to be solved. On the one hand, a lot of treatment did not manage the root of asthma, some showed various reversal effects. On the other hand there are a number of different side effects of treatment, and some also need to continue to monitor the drug concentration of patients, which caused great inconvenience. Therefore, explore more effective means to treat is still urgent, discover and explore the way asthma occurred may be a nice choice.Emerging evidence indicates that vitamin D insufficiency is associated with several lung diseases, including asthma and chronic obstructive pulmonary disease (COPD), which may be associated with impaired pulmonary function, and the increased incidence of inflammation and infection. The exact underlying mechanisms are unknown; however, vitamin D appears to affect the function of inflammatory and structural cells, including lymphocytes, dendritic cells, monocytes and epithelial cells, la-hydroxylase is responsible for the final and rate-limiting step in the synthesis of active1,25-dihydroxyvitamin D3(1,25D3) from the circulating or storage form,25-hydroxyvitamin D3(25D3).In2008, Hansdottir et al reported that airway epithelial cells express la-hydroxylase and are able to convert vitamin D from an inactive form into an active one, and that vitamin D affects the expression of vitamin D-driven genes that play a major role in host defense. The expression of la-hydroxylase in airway epithelial cells suggests a broader role of vitamin D in respiratory diseases.Airway epithelial cells are not only physical barriers to invaders, but also produce cytokines, such as thymic stromal lymphopoietin (TSLP) in response to allergens. Thymic stromal lymphopoietin (TSLP) is a novel interleukin-7(IL-7) similar cytokines, which plays important roles in allergic inflammation and asthma as a pleiotropic cytokine in lungs. Recent research has focused on TSLP production mechanism, which helps us to clarify how TSLP bronchial asthma and development works. The overexpression of the TSLP gene in airway epithelial cells has been shown to lead to asthma and TSLP has been suggested to play an important role in the initiation and maintenance of the allergic immune response. TSLP expression has been shown tobe induced by1,25D3in SCC25human epithelial tumor cells (derived from a tongue squamous cell carcinoma). It has also been demonstrated that the topical application of1,25D3leads to the induction of TSLP expression in mouse epidermal keratinocytes. However, whether vitamin D affects the expression of TSLP in human bronchial epithelial (HBE) cells remains unresolved.Vitamin D3upregulated protein1(VDUP1) is an endogenous inhibitor of thioredoxin. VDUP1was originally identifi ed as a differentially expressed gene in1,25D3-treated HL-60leukemia cells. It is cytoplasmically located and has been shown to play a multifunctional role in a variety of cellular responses. Recently, VDUP1has been shown to play a role in vascular endothelial growth factor (VEGF)-and interleukin-1β-mediated signal transduction pathways. However, little is known about the expression of VDUP1in bronchial epithelial cells and its involvement in the activation of these cells.In this study, to determine whether vitamin D enhances the expression of TSLP in airway epithelial cells and whether VDUP1is involved in this process,16-HBE cells were used. This SV40large T antigen-transformed bronchial epithelial cell line is widely used for the investigation of the functional properties of bronchial epithelial cells. The results from this study demonstrate that TSLP expression can be manipulated by both inactive and active vitamin D via the VDUP1pathway, therefore suggesting a novel mechanism by which vitamin D regulates immune function in the lungs.Materials and methodsCell culture, treatment and transfection.16-HBE bronchial epithelial cells were cultured in MEM growth medium supplemented with10%fetal calf serum and maintained at37℃in a humidifi ed incubator in the presence of5%CO2. Cells were cultured for at least2days prior to stimulation with25D3and1,25D3for6h with or without pre-treatment with1,000nM itraconazole for2h.The sequence of the VDUP1-targeting small interfering RNA (siRNA) was as follows:siRNA1sense,5’-GUCAGAGG CAAUCAUAUUATT-3’and antisense,5’-UAAUAUGAUUG CCUCUGACTG-31; siRNA2sense,5’-CUGUGAAGGUGA UGAUAUUTT-3’and antisense,5’-AAUCUCAUCACCUUC ACAGTT-3’; siRNA3sense,5’-GAAACAAAUAUGAGUACAATT-3’andantisense,5’-UUGUACUCAUAUUUGUUU CCA-3’;and negative control (NO)sense, 5’-UUCUCCGAAC GUGUCACGUTT-3’and antisense,5’-ACGUGACACGUU CGGAGAATT-3’. The16-HBE cells were seeded at a density of1.2x105cells/well in12-well culture plates and cultured for2days. Cells that were40-60%confluent, were transfected with5nM VDUP1-specific siRNA or control siRNA using the HiPerFect transfection reagent. The viability of the cells was determined by MTT assay. The transfected cells were cultured for an additional48h and then stimulated with500nM25D3and50nM1,25D3for6h.Quantitative determination of1,25D3.1,25D3was quantifi ed using an enzyme immunoassay kit for1,25D3.RNA isolation and real-time polymerase chain reaction (PCR). Gene expression was analyzed by real-time RT-PCR using SYBR(?) Premix Ex TaqTM and the Mx3005p real-time qPCR system. Total RNA was extracted with RNAisoPLUS following the manufacturer’s instructions and cDNA was synthesized from500ng of RNA using the PrimeScriptTM RT kit. The qRT-PCR reaction mixture contained1X SYBR Premix Ex Taq,200nM forward and reverse primers and2μl cDNA in a fi nal volume of25μ1. The primers used were:human TSLP (forward,5’-GCCC AGGCTATTCGGAAAC-31and reverse,5’-GAAGCGACG CCACAATCC-31) VDUP1(forward,5’-ACTCGTGTCAAAG CCGTTAGGA-3’and reverse,5’-AGCTCAAAGCCGAACTT GTACTCA-31) and β-actin (forward,5’-GTGGACATCCGC AAAGAC-3’and reverse,5’-GAAAGGGTGTAACGC AACT-3’). The cycle threshold (Ct) values were used to calculate the relative expression levels of the messenger RNA (mRNA). The expression levels of all genes were normalized to the expression of a reference gene (β-actin)Western blot analysis. The cells were washed3times with phosphate-buffered saline (PBS) and disrupted in ice-cold lysis buffer (20mM Tris,20mM P-glycerophosphate,150mM NaCl,3mM EDTA,3mM EGTA,1mM Na3VO4,0.5%Nonidet P-40and1mM dithiothreitol). The protein concentration of the lysates was determined using the Bradford protein assay was separated on a10%SDS-polyacrylamide gel and transferred onto a PVDF membrane. The membrane was blocked with5%skim milk in Tris-buffered saline with Tween-20(TBST)(50mM Tris-HCl, pH7.5,150mM NaCl and0.05%Tween-20) for1h at room temperature and then incubated overnight with rabbit anti-human VDUP1polyclonal antibody1:600at4℃. The membrane was washed with TBST (3x15-min washes) and incubated in anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase1:5,000for1h at room temperature. Protein signal was detected using the SuperECL system and detected by radiography. The immunoreactive bands were visualized using a Kodak2000M camera. An anti-GAPDH goat polyclonal antibody was used to confirm equal loading.TSLP enzyme-linked immunosorbent assay (ELISA). TSLP in culture supernatants were detected by ELISA and the data were normalized to the total protein amounts in the samples, as determined by Bradford assay. The minimal detectable level of TSLP was31.25pg/ml.Statistical analysis. Data are presented as the means±SEM. Differences among multiple groups were assessed for statistical signifi cance by one-way analysis of variance; Bonferroni’s method was used for multiple comparisons. When2groups were being compared we used a one-or two-tailed Student’s t-test depending on the hypothesis in question. A P-value<0.05was considered to indicate a statistically significant difference.Results1.25D3induces TSLP expression in16-HBE cells.To determine whether inactive25D3affects TSLP expression in airway epithelial cells, we first investigated the effects of25D3on the viability of16-HBE cells. We found no loss in cell viability when the16-HBE cells were stimulated with various concentrations of25D3(50-1,000nM). Based on our concentration-response curve, TSLP mRNA levels significantly increased at concentrations of100nM and peaked at concentrations500nM25D3. Therefore, we used the concentration of500nM in the subsequent experiments. The time-response results revealed that TSLP mRNA and protein levels were significantly upregulated in the cells stimulated with500nM25D3for2to24h.2.25D3induces VDUP1expression in16-HBE cells. The mRNA and protein expression of VDUP1following treatment with25D3from0.5to24h were determined. The results indicated that VDUP1mRNA and protein expression was significantly upregulated in the16-HBE cells treated with500nM25D3for2to24h when compared with the untreated cells.3.VDUP1silencing by RNAi.To determine the biological function of VDUP1, its expression was silenced by RNA interference. Three RNA duplexes (siRNA1, siRNA2and siRNA3) directed against VDUP1were synthesized (see Materials and methods). Extracts prepared from the cells transfected with either the siRNA2or siRNA3duplex showed reduced VDUP1levels. This effect was more pronounced with the use of siRNA3, wherein VDUP1expression was<35%of the control. siRNAl and siRNA2reduced VDUP1expression to40-80%of the control. Therefore, we selected VDUP1siRNA3in the subsequent experiments.4. Silencing of VDUP1decreases25D3-induced TSLP production in16-HBE cells.To elucidate the mechanism behind the25D3-induced TSLP production, we investigated whether VDUP1is involved in this process. We used VDUP1siRNA3to suppress VDUP1expression in the16-HBE cells. The viability of the cells was>90%in the siRNA-transfected cells. We observed a significantly lower level of TSLP mRNA and protein expression in the VDUP1-silenced cells when compared with the control siRNA-treated cells following treatment with500nM25D3.5. Inhibition of la-hydroxylase blocks the conversion of25D3to1,25D3andattenuates the upregulation of TSLP expression in16-HBE cells.To determine the effect of1,25D3, the16-HBE cells were first treated with increasing concentrations of25D3, and the levels of1,25D3in the supernatants were measured after24h. Consistent with a previous study, our results indicated that the16-HBE cells converted25D3to1,25D3when exposed to25D3without other stimuli. To further link the enzymatic machinery expressed by16-HBE cells to the1, 25D3generation and induction of TSLP expression, we used a chemical inhibitor of la-hydroxylase, itraconazole. Pre-treatment of the16-HBE cells with itraconazole (1,000nM) significantly reduced their ability to convert25D3to1,25D3. We then examined the effects of itraconazole on the induction of TSLP by25D3. The results revealed that in the presence of itraconazole (1,000nM), there was a signifi cantly lower production of TSLP mRNA and protein by25D3. These results indicate that in the presence of itraconazole, less25D3is being converted to1,25D3, resulting in a reduced production of TSLP.6. VDUP1silencing decreases1,25D3-induced TSLP expression in16-HBE cells.To further investigate the effect of active vitamin D on TSLP expression in airway epithelial cells, we directly used1,25D3as the stimulator. We found no loss in cellviability when the16-HBE cells were stimulated with various concentrations of1,25D3(0.1to100nM). Based on the concentration-response curve, TSLP mRNA and protein levels significantly increased at0.1nM, and peaked at concentrations of50nM1,25D3. The levels of TSLP mRNA and protein expression significantly increased at2h and peaked at12h in the cells exposed to50nM1,25D3. To determine the role of VDUP1in this process, we examined the mRNA expression level of VDUP1at the same time following treatment with50nM1,25D3for6h and VDUP1was also manipulated via the siRNA-mediated silencing of VDUP1. The results revealed that VDUP1mRNA expression was significantly upregulated in the16-HBE cells treated with50nM1,25D3for6h when compared with the untreated cells. In comparison with the control siRNA-treated cells, following treatment with50nM1,25D3, we observed a significantly lower level of TSLP mRNA and protein in the cells in which VDUP1was silenced. These results suggest that the VDUP1pathway is involved in the1,25D3-induced TSLP expression in16-HBE cells.7. Inhibition of1α-hydroxylase exerts no effects on1,25D3-induced TSLP expression in16-HBE cells.To confirm the specificity of the TSLP production induced by1,25D3, we treated the cells with itraconazole, as well as1,25D3. We found that in the presence of itraconazole (1,000nM), a similar induction of TSLP by1,25D3was obsevered. This further supports our primary hypothesis that1αhydroxylase converts inactive vitamin D to active vitamin D and induces TSLP expression in16-HBE cells.Conclusion1. Both inactive25D3and active1,25D3induce TSLP expression in16-HBE cells;2. The inhibition of1α-hydroxylase by itraconazole partially suppresses the25D3-but not the1,25D3-induced TSLP expression in16-HBE cells.3.0.1nM1,25D3significantly increased the expression of TSLP in airway epithelial cells, not25D3;4. The effect of vitamin D on TSLP gene expression may be an indirect effect.