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Calcitriol Decreases Expression of Importin α3 and Attenuates RelA Translocation in Human Bronchial Smooth Muscle Cells

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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.

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Acknowledgments

This work was supported by NIH grants R01HL085680 and R01AI75315 to DKA.

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Authors and Affiliations

  1. Department of Biomedical Sciences and Center for Clinical & Translational Science, Creighton University School of Medicine, CRISS II Room 510, 2500 California Plaza, Omaha, NE, 68178, USA

    Tanupriya Agrawal, Gaurav K. Gupta & Devendra K. Agrawal

  2. Internal Medicine, Creighton University School of Medicine, CRISS II Room 510, 2500 California Plaza, Omaha, NE, 68178, USA

    Devendra K. Agrawal

  3. Medical Microbiology and Immunology, Creighton University School of Medicine, CRISS II Room 510, 2500 California Plaza, Omaha, NE, 68178, USA

    Devendra K. Agrawal

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  1. Tanupriya Agrawal
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  2. Gaurav K. Gupta
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Correspondence to Devendra K. Agrawal.

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Figure S1

Effect of Calcitriol treatment on mRNA and protein expression of CYP24A1 (A, B) and CYP27B1 (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. (TIFF 220 kb)

Figure S2

Effect of Calcitriol treatment on mRNA expression of VDR in HBSMCs: Cultured HBSMCs were serum starved for 24 hr followed by treatment with calcitriol (100 nM) for at times 0-36hr. The mRNA were isolated from cell lysates and subjected to qPCR. Data is shown as mean ± SEM from three individual samples; ***p<0.001 (TIFF 125 kb)

Figure S3

Effect of Calcitriol treatment on mRNA expression of importin α3 in HBSMCs: Cultured HBSMCs were serum starved for 24 hr followed by treatment with calcitriol (100 nM) for at times 0-36hr. The mRNA was isolated from cell lysates and subjected to qPCR. Data is shown as mean ± SEM from three individual samples; *p <0.05, **p<0.01, ***p <0.001. (TIFF 123 kb)

Figure S4

Effect of Calcitriol treatment on mRNA(A)and protein(B) expression of importin α4in 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 (TIFF 122 kb)

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Agrawal, T., Gupta, G.K. & Agrawal, D.K. Calcitriol Decreases Expression of Importin α3 and Attenuates RelA Translocation in Human Bronchial Smooth Muscle Cells. J Clin Immunol 32, 1093–1103 (2012). https://doi.org/10.1007/s10875-012-9696-x

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