1,25-Dihydroxyvitamin D3 Inhibits Nuclear Factor Kappa B Activation by Stabilizing Inhibitor IκBα via mRNA Stability and Reduced Phosphorylation in Passively Sensitized Human Airway Smooth Muscle Cells
Department of Pulmonary and Critical Care Medicine, Fuzhou General Hospital of Nanjing Military Command, Dongfang Hospital, Xiamen University, Fuzhou, China
Correspondence to: Guoxiang Lai, Department of Pulmonary and Critical Care Medicine, Fuzhou General Hospital of Nanjing Military Command, Dongfang Hospital, Xiamen University, 156 Xi'erhuan North Road, Fuzhou, Fujian, 350025, China. E-mail: firstname.lastname@example.org
Excessive activation of nuclear transcription factor-κB (NF-κB) is involved in human airway smooth muscle cells (HASMCs) activities in asthma. We investigated the effects of 1,25 – dihydroxyvitamin D3 [1,25 – (OH) 2D3] on the NF- κB signaling pathway in passively sensitized HASMCs and the molecular mechanisms involved. HASMCs were treated with either healthy controls’ serum, asthma patients’ serum or pretreated with 1,25 – (OH) 2D3 prior to treatment with asthmatics’ serum. At 1 h after serum treatment: electrophoretic mobility shift assay (EMSA) was used to detect NF-κB DNA binding activity; immunocytochemical staining was used to observe the nuclear translocation of NF-κB p65; Western blots were used for NF-κB p65, IκBα, and phospho-IκBα protein levels and the nuclear translocation of NF-κB p65; real-time quantitative PCR was used for NF-κB p65 and IκBα mRNA expressions; and actinomycin D treatment was used to determine IκBα mRNA stability. Our major findings were: (1) 1,25 – (OH) 2D3 significantly reduced asthma serum passively sensitized HASMCs NF-κB DNA binding activity and inhibited the nuclear translocation of NF-κB p65; (2) 1,25 – (OH) 2D3 increased the stability of IκBα mRNA with reduced IκBα phosphorylation in asthma serum passively sensitized HASMCs and significantly increased IκBα expression in these HASMCs. Inhibiting NF-κB signalling with 1,25 – dihydroxyvitamin D3 may be a therapeutic approach for controlling HASMC-related remodelling in asthma.
1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is the most important active metabolite of vitamin D that exerts its biological effects via binding to the vitamin D receptor (VDR). VDR's have been detected in multiple tissues, including pancreas, breast, spleen, skin and lung. 1,25-(OH)2D3 has extensive biological activities, including the regulation of calcium and phosphorus metabolism .
Recent studies have revealed that the VDR gene is an asthma susceptibility gene and that VDR gene polymorphisms are involved in asthma occurrence. This indicates that 1,25-(OH)2D3 can influence asthma occurrence via its binding to the VDR . Other studies have found that 1,25-(OH)2D3 significantly improved airway inflammation and regulated immune responses in asthma patients [3-5], which suggested that 1,25-(OH)2D3 could be used for treating asthma. Vitamin D and its active products have been shown to be important adjunctive drugs in treating steroid resistant (SR) asthma . 1,25-(OH)2D3 can also reduce asthma severity and improve steroid treatment responses in asthma patients .
Previous studies primarily focused on the effects of 1,25-(OH)2D3 on the immune responses and chronic airway inflammation in asthma patients, while possible effects of 1,25-(OH)2D3 on airway remodelling have not been extensively studied. Airway smooth muscle cells (ASMCs) are important cells involved in airway constriction and play critical roles in airway remodelling. Alterations in ASMCs, such as hyperplasia and phenotypic changes, are basic features of airway remodelling . In a previous study, we passively sensitized human ASMCs (HASMCs) with asthma patients’ serum to mimic the growth and microenvironment of HASMCs in the asthmatic state. We showed that 1,25-(OH)2D3 could inhibit HASMCs’ hyperplasia under and suppress MMP-9 expression in these cells . This was the first report that 1,25-(OH)2D3 had a regulatory role for HASMCs in asthma–associated airway remodelling at the cellular level.
Subsequently, Damera et al.  found that 1,25-(OH)2D3 inhibited PDGF-induced HASMCs proliferation via suppressing the phosphorylation of checkpoint kinase 1 (Chk1) and Rb protein, which confirmed a suppressive effect of 1,25-(OH)2D3 on HASMC proliferation. However, the suppressive effect of 1,25-(OH)2D3 on Chk1 and Rb protein phosphorylation does not account for the inhibition of MMP-9 expression. Thus, 1,25-(OH)2D3 might regulate HASMCs proliferation and MMP-9 expression via other signalling pathways.
Nuclear factor kappa B (NF-κB) is a transcription factor that has multiple regulatory roles. Numerous studies have confirmed that NF-κB is over-activated in asthma patients [11, 12]. During the pathogenesis of asthma, NF-κB may act at different levels of airway remodelling, such as regulating ASMCs proliferation and their expressions of inflammatory cytokines . Our previous work also demonstrated that the NF-κB signalling pathway was involved in regulating ASMCs by PAF . Interestingly, in a study to identify genes regulated by 1,25-(OH)2D3 it was found that 1,25-(OH)2D3 stimulation upregulated genes in bronchial smooth muscle cells involved with autorine, contractility and remodelling processes . However, whether the NF-κB signalling pathway involved in passively sensitized HASMCs can be regulated by 1,25-(OH)2D3 to control airway remodelling remains uncertain.
Thus, in the present study, we investigated the mechanisms underlying 1,25-(OH)2D3 effects on regulating NF-κB activity in passively sensitized HASMCs. Our findings provide evidence that NF-κB is a key player in regulating airway remodelling in asthma and may provide a theoretical basis for the clinical treatment of asthma with 1,25-(OH)2D3.
Materials and methods
The following cell line and reagents were used: HASMCs (Sciencell, Carlsbad, CA, USA); trypsin, actinomycin D (Sigma, St. Louis, MO, USA); DMEM (Gibco, Life Technologies, Grand Island, NY, USA); fetal bovine serum (FBS; Hangzhou Sijiqing Biotech Co., Ltd, Hangzhou, Zhejiang, China); SABC kit (Wuhan Boster Biotech Co., Ltd, Wuhan, Huibei, China); anti-NF-κB p65 antibody, anti-IκBα antibody and anti-p-IκBα antibody, ECL™ Advanced Western Blotting Detection Kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA); γ-32P ATP (Beijing Yahui Medical Engineering Company, Beijing, China); NE-PER™ kit (Pierce, Thermo Fisher Scientific Inc, Rockford, IL, USA); Gel Shift Assay System kit (Promega, San Luis Obispo, CA, USA) and FastStart SYBR Green I (Roche, Basel, Switzerland).
Serum samples and cell culture
Serum from asthma patients
We recruited 15 patients who had been admitted to our hospital because of acute asthma attacks from 2009 to 2010. They included 6 men and 9 women whose ages ranged from 20 to 45 years. Their clinical characteristics included: (1) asthma diagnosed based on guidelines for the prevention and treatment of bronchial asthma; (2) history of allergy, but no history of other respiratory diseases; and (3) not treated with glucocorticoids during the previous week. Peripheral venous blood was collected (100 ml per patient) and centrifuged at 250 g for 10 min. The supernatant (serum) was obtained under aseptic conditions and inactivated at 56 °C for 30 min. Serum aliquots were stored at −20 °C for later use.
Serum from controls
We recruited eight subjects without asthma who underwent routine physical examinations from 2009 to 2010. They included five men and three women whose ages ranged from 25 to 40 years. Their clinical characteristics included: (1) no history of allergy, asthma or other respiratory diseases and (2) not receiving pharmacotherapy. Serum samples were prepared as described above.
This study was approved by the Institutional Review Board of our hospital and informed consent was obtained from asthma patients and control subjects prior to their enrolment in this study.
Cell groups and culture
HASMCs were maintained in high glucose DMEM medium containing 10% FBS at 37 °C in a 5% CO2 atmosphere. HASMCs at passage 4-8 were used in these experiments. Using a random number table, cells were randomly divided into three groups: (1) control group: cells were grown in serum-free DMEM for 48 h for synchronization and then treated with 10% control serum; (2) asthma group: cells were grown in serum-free DMEM for 48 h for synchronization and then treated with 10% serum from asthma patients; (3) Vitamin D (VD) group: cells were grown in serum-free DMEM containing 10−7m of 1,25-(OH)2D3 for 48 h for synchronization and then treated with 10% serum from asthma patients.
NF-κB activity determinations by EMSA
At 1 h after serum treatment, cells were harvested and nuclear proteins were extracted with a NE-PER™ kit. NF-κB oligonucleotide probes (5′-AGT TGA GGG GAC TTT CCC AGG C-3′;3′-TCA ACT CCC CTG A AA GGG TCC G-5′) were labelled according to the Gel Shift Assay System kit instructions. DNA binding was also determined. A standard was included in each gel, which was prepared with nuclear proteins from HeLa cells and γ-32P labelled SP1 probes. After non-denaturing polyacrylamide gel electrophoresis, the gel was dried, followed by autoradiography for 48 h at −70 °C. A Promega Gel Drying Kit (Promega Corp., Madison, WI, USA) was used for an image analysis system. Image J software was used for gel analysis. The optical density (OD) of samples was normalized to that of the standard used for NF-κB activity.
NF-κB p65, IκBα and p-IκBα protein expression by Western blot assay
At 1 h after serum treatment, cells from the different groups were harvested. Total proteins, plasma proteins and nuclear proteins were extracted. Total proteins were subjected to 10% SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was treated separately with an NF-κB p65 antibody, an IκBα antibody and a p-IκBα antibody (1:1000). Visualization used an ECL kit. The bands were scanned and their OD's were determined with the gel image analysis system. The OD of a target protein was normalized to that of β-actin used as an internal reference. Experiments were repeated three times. Using the same procedures, NF-κB p65 protein expression was determined in nuclear and plasma proteins.
Immunohistochemistry for nuclear translocation of NF-κB p65 in HASMCs
At 1 h after serum treatment, cells from the different groups were harvested and fixed in formaldehyde. The SABC method was used for immunohistochemsitry using an SABC kit. Goat anti-human NF-κB p65 antibody (1:1000) was the primary antibody. PBS was used in place of the primary antibody for a negative control. Cells that were positive for NF-κB p65 had yellow-brown granules in the cytoplasm and/or nucleus. Under a light microscope, 10 fields were randomly selected at high magnification. Cells that were positive for NF-κB p65 in the nucleus were counted, after which the positive rate (PR) was determined. For this experiment, two people independently determined the positive counts after viewing at least 200 cells per field.
Real-time fluorescence quantitative RT-PCR for NF-κB p65 and IκBα mRNA expression in HASMCs
Total RNA was extracted with Trizol reagent and reverse transcribed into cDNA. Then, 2 μl of cDNA was used as a template for PCR amplification. The primers for NF-κB p65 were: 5′-GCACTTACGGATTCTGGTGG-3′ (forward), 5′-CTCAAACGCTGGTG TTAGGC-3′ (reverse); the expected size was 426 bp. The primers for IκBα were: 5′-CGG GTTCCTGCACTTGGCCATC-3′ (forward), 5′-GTCCGGCCATTACAGGGCTC-3′ (reverse); the expected size was 410 bp. The primers for GAPDH were: 5′-GGTATCGTGGAAGGACTCATGAC-3′ (forward), 5′-ATGCCAGTGAGCTTCCCGTTCAGC-3′ (reverse); the expected size was 188 bp. PCR conditions were: NF-κB p65: 30 cycles of 94 °C for 30 s, 57°C for 30 s and 72 °C for 1 min; IκBα: 30 cycles of 95 °C for 30 s, 57 °C for 30 s and 72 °C for 40 s; GAPDH: 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 2 min. The mRNA expression of a target gene was normalized to that of GAPDH used as an internal reference. Experiments were carried out in triplicate.
IκBα mRNA stability in HASMCs after actinomycin D treatment
At 1 h after serum treatment, cells were treated with 10 mg/l actinomycin D to stop gene transcription. At 0, 1, 2, 3, 4, 5 and 6 h after actinomycin D treatment, real-time PCR was used to determine IκBα mRNA expression. The changes in IκBα mRNA expression over time after actinomycin D treatment were used to determine the half-life of IκBα mRNA.
Results are given as means and standard errors of the mean (SEs). For NF-κBDNA binding activity, a one-sample t-test was used to compare the mean ratio of the results for asthma serum-treated cells and the mean ratio of the VD group cells each compared to control serum-treated cells. A two-sample t-test was used to compare the asthma serum-treated cells and the VD group cells. Results for protein expressions, positive rates of nuclear NF-κB p65 and mRNA expressions among control, asthma and VD groups were compared by one-way anova followed by Bonferroni pair-wise comparisons. All statistical assessments were two-sided and significance was set at P <0.05. An adjusted significance level of P =0.0167 (0.05/3) was used for pair-wise comparisons. Statistical analyses used pasw statistics software version 18.0 (SPSS Inc, Chicago, IL, USA).
1,25-(OH)2D3 reduces NF-κB DNA binding in HASMCs passively sensitized with asthmatics’ sera
HASMCs were passively sensitized with serum from either healthy controls (control group) or asthma patients (asthma group). In addition, HASMCs were first treated with 1,25-(OH)2D3 for 48 h, and then passively sensitized with serum from asthma patients (Vitamin D; VD group). After 1 h of serum treatment, NF-κB DNA binding activity in HASMCs was determined by EMSA.
Figure 1a shows representative experimental results for: (1) non-specific competition; (2) specific competition; (3) control group; (4) asthma group and (5) VD group. A summary of the results for the control, asthma and VD groups is shown in Fig. 1b. The mean ratio of the asthma group to the control group was 7.8 ± 0.5 and the mean ratio of the VD group to the control group was 5.2 ± 0.3. By one-sample t-test, these ratios were significantly different (P < 0.0167). Thus, 1,25-(OH)2D3 inhibited NF-κB DNA binding activity in HASMCs that were passively sensitized with asthmatics’ sera.
Effects of 1,25-(OH)2D3 on NF-κB p65, IκBα and phospho-IκBα protein expressions in HASMCs passively sensitized with asthmatics’ sera
At 1 h after serum treatment, protein extracts were prepared from HASMCs for the three experimental groups, including total protein and nuclear and cytoplasmic proteins. Western blots were used for each of these fractions to determine the expressions of nuclear and cytoplasmic NF-κB p65, total NF-κB p65, total IκBα and total phospho-IκBα proteins relative to that of β-actin (used as an internal control). Figure 2a shows a representative Western blot.
Figure 2b summarizes the results of these proteins’ expressions in the total protein extracts for the control, asthma and VD groups. Relative to cells treated with control serum, after treatment with asthma serum, NF-κB p65 and p-IκBα protein levels were increased, whereas the IκBα protein level was decreased in HASMCs. However, relative to cells that were passively sensitized with asthmatics’ sera and without treatment, 1,25-(OH)2D3 inhibited the protein expressions of NF-κB p65 and phospho-IκBα and increased the protein expression of IκBα in HASMCs.
1,25-(OH)2D3 inhibits NF-κBp65 nuclear translocation in HASMCs passively sensitized with asthmatics’ sera
At 1 h after serum treatment, the percentage changes of the ratios of cytoplasmic NF-κBp65 and nuclear NF-κBp65 proteins versus total NF-κBp65 protein are shown in Figure 3 (n = 5 for each group). The nuclear NF-κBp65/total NF-κBp65 ratios were: 31.6 ± 1.1% in the control group; 78.3 ± 0.9% in the asthma group and 60.0 ± 0.9% in the VD group. The cytoplasmic NF-κBp65/total NF-κBp65 ratios were: 68.4 ± 1.1% in the control group; 21.7 ± 0.9% in the asthma group and 40.0 ± 1.3% in the VD group. Thus, for HASMCs that had been passively sensitized with asthmatics’ sera, 1,25-(OH)2D3 reduced the protein expression of NF-κB p65 in the nuclear fraction rather than in the cytoplasmic fraction. We next focused on NF-κBp65 protein nuclear translocation in HASMCs. For each group of cells and using a light microscope, 10 fields were randomly selected at high magnification to identify NF-κB p65 positive staining in the nuclei and cytoplasm of HASMCs, with results given as means ± SEs (n = 5 for each group). A quantitative summary of these results is given in Fig. 4. Compared to treatment with control serum, treatment with asthmatics’ serum resulted in increased NF-κB p65 protein in the nuclei of HASMCs. However, 1,25-(OH) 2D3 reduced the nuclear protein expression of NF-κB p65 in HASMCs that had been passively sensitized with asthmatics’ sera.
Effects of 1,25-(OH)2D3 on NF-κB p65 and IκBα mRNA expressions in HASMCs passively sensitized with asthmatics’ sera
We next used real-time fluorescence quantitative RT-PCR to determine the relative NF-κBp65 and IκBα mRNA expressions. mRNA expressions were normalized to that of GADPH mRNA (used as a housekeeping gene). Figure 5 shows these results at 1 h after serum treatment. Similar to the trend for protein expression (Fig. 2a), treatment with asthmatics’ serum resulted in increased NF-κB p65 mRNA and reduced IκBα mRNA compared to HASMCs treated with control serum. However, 1,25-(OH) 2D3 inhibited NF-κB p65 mRNA expression and enhanced IκBα mRNA expression in HASMCs that had been passively sensitized with asthmatics’ sera.
Effects of 1,25-(OH)2D3 on IκBα mRNA stability in HASMCs passively sensitized with asthmatics’ sera
At 1 h after serum treatment, cells were treated with actinomycin D (10 mg/L) to stop gene transcription. At 0, 1, 2, 3, 4, 5 and 6 h after actinomycin D treatment, real-time PCR was used to determine IκBα mRNA expression (relative to GADPH mRNA). These results are shown in Fig. 6. The half-life of IκBα mRNA in cells treated with control serum was relatively long (>300 min), while it was appreciably shorter in cells treated with asthmatics’ sera (≈190 min). Pretreating cells with 1,25-(OH)2D3 significantly increased the half-life to ≈260 min. Thus, 1,25-(OH)2D3 increased the stability of IκBα mRNA in HASMCs that had been passively sensitized with asthmatics’ sera.
Alterations in HASMCs are the structural basis of airway remodelling in asthma . As an in vitro model, passive stimulation of HASMCs with asthmatic patients’ sera has provided important insights into the complex phenomenology of airway remodelling. Passive stimulation promotes changes in airway smooth muscle tone, growth and cytokine production  and induces these cells’ production of extracellular matrix proteins . Although asthmatic patients’ sera undoubtedly contains numerous factors (e.g., growth factors, inflammatory mediators, and others), it has been shown to be useful for unravelling the details of these processes at the molecular level, such as protein kinase C (PKC) involvement in passively sensitized HASMCs proliferation .
As a key factor that regulates HASMCs proliferation and their expressions of inflammatory cytokines, NF-κB is intimately involved in the dysfunction of HASMCs under the asthmatic state . Thus, a change in NF-κB activity in ASMCs is a surrogate for airway remodelling in asthma.
In the present study, NF-κB p65 expressions among nuclear, cytoplasmic and total proteins were compared. These results demonstrated by EMSA that 1,25-(OH)2D3 could effectively inhibit asthma-serum induced nuclear translocation of NF-κB in HASMCs and markedly inhibited the DNA binding activity of NF-κB in passively sensitized HASMCs. These findings confirmed that 1,25-(OH)2D3 could inhibit NF-κB activation in passively sensitized HASMCs.
IκB is a major inhibitor of NF-κB. To date, five IκB family members have been identified: IκBα, IκBβ, IκBγ, IκBδ and IκBε, among which IκBα is most closely associated with NF-κB activation. The phosphorylation and degradation of IκBα is required for NF-κB activation. Thus, changes in IκBα concentrations may indirectly reflect the extent of NF-κB activation .
In this study, real-time fluorescence quantitative PCR and Western blot were used to detect the mRNA and protein expressions of IκBα in HASMCs after 1,25-(OH)2D3 treatment. These results showed that 1,25-(OH)2D3 either significantly up-regulated IκBα expression in passively sensitized HASMCs or inhibited the degradation of IκBα. Additional experiments revealed that this effect could be attributed to an increase in IκBα mRNA stability. This indirectly confirms that 1,25-(OH)2D3 can exert a suppressive effect on NF-κB activation in HASMCs. These results are consistent with a study that showed that IκBα protein expression was reduced in cells in which the VDR had been deleted .
Because NF-κB is a key transcriptional factor that regulates the functions of ASMCs under the asthmatic state, we postulate that the suppressive effect of 1,25-(OH)2D3 on NF-κB activation in HASMCs plays an important role in regulating HASMC function. This is similar to the mechanism associated with the therapeutic effects of glucocorticoids on asthma. Traditionally, the roles of HASMCs in asthma were thought to be due only to their contractile properties. However, the emerging view is that these cells may adopt an immuno-effector role in chronic asthma by proliferating, secreting cytokines and expressing adhesion molecules . Thus, therapeutically targeting these cells should aid in ameliorating the pathological features of chronic asthma, including airway hyperresponsiveness, remodelling, and inflammation . As examples, vitamin D levels are associated with lung function and steroid responses in adult asthma  and can modulate T helper type 2-driven asthmatic responses . Our findings confirmed that 1,25-(OH)2D3 could regulate NF-κB activation in ASMCs, which suggests a new strategy for asthma therapy and possibly other diseases with smooth muscle cell hyperplasia, such as chronic obstructive pulmonary disease, atherosclerosis, post-angioplasty restenosis and others.
The mechanism(s) underlying the regulatory effects of 1,25-(OH)2D3 on NF-κB activation in passively sensitized HASMCs is not entirely certain. The biological effects of 1,25-(OH)2D3 appear to be closely related to the extent of VDR expression in target tissues. 1,25-(OH)2D3 can up-regulate VDR expression via increasing VDR stability and by other mechanisms . Of note, VDR is an important up-stream molecule of NF-κB activation. VDR not only directly up-regulates IκBα expression and increases IκBα stability , it also physiologically binds to the NF-κB p65 subunit. The VDR may directly interrupt the nuclear translocation of the p65 subunit and reduce the DNA binding activity of the p65 subunit, which would directly inhibit the activity of NF-κB p65 .
1,25-(OH)2D3 has been shown to regulate the NF-κB signalling pathway via binding to VDR and thereby specifically modulate the biological functions of embryonic fibroblasts, monocytes, endothelial cells and dendritic cells [26, 27]. Interestingly, in a clinical study of children with asthma, increased airway smooth muscle mass and worse asthma control and lung function were associated with reduced vitamin D levels . Of interest, our previous study also revealed that 1,25-(OH)2D3 could induce VDR expression and functional responses by HASMCs . Based on these previous findings and those in the present study, we postulate that the suppressive effect of 1,25-(OH)2D3 on NF-κB activation in HASMCs is possibly dependent on the VDR. We will study this mechanism in detail in future studies.
Our results show that 1,25-(OH)2D3 can up-regulate the expression of the inhibitory protein IκBα in passively sensitized HASMCs, which then suppresses asthma serum-induced NF-κB activation. Over-activation of NF-κB has been found to be involved in the dysfunction of HASMCs under the asthmatic state. Thus, the suppressive effect of 1,25-(OH)2D3 on the NF-κB signalling pathway is an important mechanism underlying its regulation of passively sensitized HASMCs. Our results not only provide a better understanding of the role of 1,25-(OH)2D3 in the pathogenesis of asthma at the molecular level, but also provide a theoretical basis for the clinical application of 1,25-(OH)2D3 in the treatment of asthma.
This study was supported by the National Natural Science Foundation of China (Grant No. 81100011). The authors have declared that no competing interests exist.