Calcitriol decreases expression of importin α3 and attenuates RelA translocation in human bronchial smooth muscle cells

Abstract

Purpose: A potent immunomodulatory role of Vitamin D in both innate and adaptive immunity has recently been appreciated. In allergic asthma, activation of NF-кB induces transcription of various cytokines and chemokines involved in allergic airway inflammation. The nuclear import of activated NF-кB p50/RelA subunit is dependent on importin α3 (KPNA4) and importin α4 (KPNA3). In this study, we examined the role of importin α3 in immunomodulatory effect of calcitriol in human bronchial smooth muscle cells (HBSMCs).

Methods: Cultured HBSMCs were stimulated with calcitriol in the presence and absence of cytokines, TNF-α, IL-1β, and IL-10. The mRNA transcripts of importin α3 and α4 were analyzed using qPCR while protein expression of importin α3, α4 and nuclear RelA was analyzed by immunoblotting.

Results: Calcitriol significantly decreased mRNA and protein expression of importin α3 as well as nuclear protein expression of NF-кB p65 (RelA). The decreased activation of RelA by calcitriol was confirmed by decreased release of RelA-inducible molecules, including IL-5, IL-6 and IL-8, by HBSMCs upon calcitriol treatment. Calcitriol attenuated the effect of TNF-α and IL-1β to upregulate mRNA and protein expression of importin α3. IL-10 significantly decreased the TNF-α induced expression of importin α3 and this effect was further potentiated by calcitriol.

Conclusions: These data suggest that under inflammatory conditions, calcitriol decreases the expression of importin α3 resulting in decreased nuclear import of activated RelA. This could be a novel mechanism by which calcitriol could exert its immunomodulatory effects to reduce allergic airway inflammation and thus may alleviate the symptoms in allergic asthma.

Figure 1. Effect of Calcitriol treatment on mRNA and protein expression of VDR (A, B), in HBSMCs

Cultured HBSMCs were serum starved for 24 hr followed by treatment with different doses of calcitriol (0.1- 100 nM) for 24 hr. The mRNA and protein were isolated from cell lysates and subjected to qPCR and immunoblotting, respectively. Data is shown as mean ± SEM from three individual samples in each experiment; ***p<0.001, **p<0.01.

Figure 2. Effect of Calcitriol treatment on mRNA and protein expression of importin α3 (A, B) and importin α4 (C, D) in HBSMCs

Cultured HBSMCs were serum starved for 24 hr followed by treatment with different doses of calcitriol (0.1- 100 nM) for 24 hr. The mRNA and protein were isolated from cell lysates and subjected to qPCR and immunoblotting, respectively. Data is shown as mean ± SEM from three individual samples in each experiment; ***p<0.001, **p<0.01.

Figure 3. Effect of VDR siRNA on the expression of VDR (A) and the effect of calcitriol treatment on the protein expression of importin α3 (B) in VDR knockdown HBSMCs

Cultured HBSMCs were transfected with VDR siRNA or scrambled siRNA and were stimulated with calcitriol (100 nM) for 24 hr. Protein was isolated from cell lysates and subjected to immunoblotting. Data is shown as mean ± SEM from three individual samples in each experiment; ***p<0.001.

Figure 4. Effect of TNF-α on the expression of activated-RelA in the nuclear fraction of HBSMCs in a dose and time dependent manner(A,B), Effect of importin α3 siRNA on the expression of importin α3 (C) & Effect of calcitriol on nuclear protein expression of in importin α3 knockdown HBSMCs on stimulation with TNF- α

HBSMCs were treated with different doses of TNF-α (1-100ng/ml) for 20 min (A) and TNF-α (10ng/ml) at 0-60 min (B). The nuclear protein was extracted and analyzed for RelA by immunoblotting. HBSMCs were treated with calcitriol (100nM) for 24hr followed by TNF-α (10ng/ml) for 15min. Cultured HBSMCs were transfected with importin α3 siRNA or scrambled siRNA and were stimulated with calcitriol (100 nM) and TNF-α (10ng/ml) for 24 hr. The nuclear protein (D) was extracted for RelA by Immunoblotting. Lamin B was used as a housekeeping gene for nuclear protein. Data is shown as mean ± SEM from three individual samples in each experiment; **p<0.01, ***p<0.001.

Figure 4. Effect of TNF-α on the expression of activated-RelA in the nuclear fraction of HBSMCs in a dose and time dependent manner(A,B), Effect of importin α3 siRNA on the expression of importin α3 (C) & Effect of calcitriol on nuclear protein expression of in importin α3 knockdown HBSMCs on stimulation with TNF- α

HBSMCs were treated with different doses of TNF-α (1-100ng/ml) for 20 min (A) and TNF-α (10ng/ml) at 0-60 min (B). The nuclear protein was extracted and analyzed for RelA by immunoblotting. HBSMCs were treated with calcitriol (100nM) for 24hr followed by TNF-α (10ng/ml) for 15min. Cultured HBSMCs were transfected with importin α3 siRNA or scrambled siRNA and were stimulated with calcitriol (100 nM) and TNF-α (10ng/ml) for 24 hr. The nuclear protein (D) was extracted for RelA by Immunoblotting. Lamin B was used as a housekeeping gene for nuclear protein. Data is shown as mean ± SEM from three individual samples in each experiment; **p<0.01, ***p<0.001.

Figure 5. Effect of calcitriol on TNF-α Induced RelA (A) and importin α3 (B) Transactivation

HBSMCs were seeded in 96-well plate for 24 hr, followed by transfection with 100 ng NF-κB-responsive firefly luciferase reporter (A) and importin α3 luciferase reporter (B) using FuGENE-HD transfection reagent. Thirty hours after transfection, cells were treated with calcitriol (100 nM) for 20 hr followed by treatment with TNF-α (10 ng/ml) for an additional 4 hr. Luciferase activities were assessed using Dual-Glo® Luciferase Assay System. Data is shown as mean ± SEM from three individual samples in each experiment; **p<0.01, ***p<0.001.

Figure 6. Effect of calcitriol ± TNF-α/IL-1β on mRNA transcript and protein expression of importin α3 in HBSMCs

Cultured HBSMCs were serum starved for 24 hr followed by treatment with TNF-α (10ng/ml)(A,B)/IL-1β (10ng/ml)(C,D) ± calcitriol (100 nM) for 24 hr. The mRNA and protein was isolated from whole cell lysates and subjected to qPCR and immunoblotting respectively. Data is shown as mean ± SEM from three individual samples in each experiment *p <0.05, **p<0.01, ***p <0.001.

Figure 7. Effect of IL-10±TNF-α ±calcitriol on mRNA transcript (A) and protein expression (B) of importin α3 in HBSMCs

Cultured HBSMCs were serum starved for 24 hr followed by treatment with IL-10 (10ng/ml) ± TNF-α (10 ng/ml) ± calcitriol (100 nM) for 24 hr. mRNA and protein was isolated from whole cell lysates and subjected to qPCR (A) and immunoblotting (B), respectively. Data is shown as mean ± SEM from three individual samples in each experiment *p <0.05, **p<0.01,***p <0.001.

Figure 8. Effect of calcitriol ± Dexamethasone on TNF-α induced IL-5, IL-6 and IL-8 secretion

Cultured HBSMCs were serum starved for 24 hours followed by treatment with TNF-α (10 ng/ml) ± dexamethasone ± calcitriol for 24h. The secretion of cytokines- IL-5 (A), IL-6 (B) and IL-8 (C) secretion in the culture medium by HBSMCs were determined by ELISA. Data is shown as mean ± SEM from three individual samples in each experiment *p <0.05, **p<0.01, ***p <0.001.