Tuberc Respir Dis.  2012 Nov;73(5):258-265. 10.4046/trd.2012.73.5.258.

Inhibition of Vitamin D Receptor Translocation by Cigarette Smoking Extracts

• Uh ST
• Koo SM
• Kim YK
• Kim KU
• Park SW
• Jang AS
• Kim DJ
• Kim YH
• Park CS

• KMID: 2050678
• DOI: http://doi.org/10.4046/trd.2012.73.5.258

Abstract

BACKGROUND
Vitamin D can translocate a vitamin D receptor (VDR) from the nucleus to the cell membranes. The meaning of this translocation is not elucidated in terms of a role in pathogenesis of chronic obstructive pulmonary disease (COPD) till now. VDR deficient mice are prone to develop emphysema, suggesting that abnormal function of VDR might influence a generation of COPD. The blood levels of vitamin D have known to be well correlated with that of lung function in patients with COPD, and smoking is the most important risk factor in development of COPD. This study was performed to investigate whether cigarette smoke extracts (CSE) can inhibit the translocation of VDR and whether mitogen activated protein kinases (MAPKs) are involved in this inhibition.
METHODS
Human alveolar basal epithelial cell line (A549) was used in this study. 1,25-(OH2)D3 and/or MAPKs inhibitors and antioxidants were pre-incubated before stimulation with 10% CSE, and then nucleus and microsomal proteins were extracted for a Western blot of VDR.
RESULTS
Five minutes treatment of 1,25-(OH2)D3 induced translocation of VDR from nucleus to microsomes by a dose-dependent manner. CSE inhibited 1,25-(OH2)D3-induced translocation of VDR in both concentrations of 10% and 20%. All MAPKs inhibitors did not suppress the inhibitory effects of CSE on the 1,25-(OH2)D3-induced translocation of VDR. Quercetin suppressed the inhibitory effects of CSE on the 1,25-(OH2)D3-induced translocation of VDR, but not in n-acetylcysteine.
CONCLUSION
CSE has an ability to inhibit vitamin D-induced VDR translocation, but MAPKs are not involved in this inhibition.

Keyword

Vitamin D; Receptors, Calcitriol; Extracelular Signal-Regulated MAP Kinases; Antioxidants; Pulmonary Disease, Chronic Obstructive

MeSH Terms

Animals
Antioxidants
Blotting, Western
Cell Membrane
Emphysema
Epithelial Cells
Humans
Lung
Mice
Microsomes
Mitogen-Activated Protein Kinases
Proteins
Pulmonary Disease, Chronic Obstructive
Quercetin
Receptors, Calcitriol
Risk Factors
Smoke
Smoking
Tobacco Products
Vitamin D
Vitamins
Antioxidants
Mitogen-Activated Protein Kinases
Proteins
Quercetin
Receptors, Calcitriol
Smoke
Vitamin D
Vitamins

Figure

• 

  Figure 1 Effects of 1,25-(OH)2D3 on the subcellular distribution of vitamin D receptor (VDR) in A549 cells. Proteins from microsomal and nuclear fractions from A549 cells treated for 5 min with 1 nM and 10 nM 1,25-(OH)2D3 were subjected to Western blot for anti-VDR antibody. Microsomal fractions of VDR are more increased by treatment with 1,25-(OH)2D3, compared to the control. Results of densitometric analysis are shown in the lower histogram.

• 

  Figure 2 Effects of cigarette smoke extracts (CSE) on the subcellular distribution of vitamin D receptor (VDR) in A549 cells. Proteins from microsomal and nuclear fractions from A549 cells treated for 10 min with 10% and 20% of CSE after pre-incubation of 5 min with 10 nM 1,25-(OH)2D3 were subjected to Western blot for anti-VDR antibody. Both 10% and 20% of CSE inhibit the 1,25-(OH)2D3-induced translocation of VDR. Results of densitometric analysis are shown in the lower histogram.

• 

  Figure 3 Effects of cigarette smoking extracts (CSE) on the activation of extraceullar signal-regulated kinases (ERK) in A549 cells. Proteins from A549 cells treated for 30 min with 1%, 5%, and 10% of CSE were subjected to Western blot for anti-p-ERK and anti-ERK antibody. ERK are activated with CSE treatment by dose-dependent pattern. Results of densitometric analysis are shown in the lower histogram.

• 

  Figure 4 Effects of mitogen activated protein kinases (MAPKs) on the 1,25-(OH)2D3-induced translocation of vitamin D receptor (VDR) in the presence of cigarette smoking extracts (CSE) in the A549 cells. Proteins from microsomal fractions from the A549 cells treated for 1 hr of MAPKs inhibitors before 10% of CSE treatment were subjected to Western blot for anti-VDR antibody. All MAPKs inhibitors cannot reverse the inhibitory effects of CSE on the 1,25-(OH)2D3-induced translocation of VDR. Results of densitometric analysis are shown in the lower histogram.

• 

  Figure 5 Effects of anti-oxidants on inhibitory effects of cigarette smoking extracts (CSE) on the1,25-(OH)2D3-induced translocation of vitamin D receptor (VDR) in the A549 cells. Proteins from microsomal fractions from the A549 cells treated for 1 hr of n-acetylcystein (NAC) (A) and quercetin (B) before 10% of CSE treatment were subjected to Western blot for anti-VDR antibody. Quercetin reversed the inhibitory effects of CSE on the 1,25-(OH)2D3-induced translocation of VDR, but NAC did not. Results of densitometric analysis are shown in the lower histogram.

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