1alpha,25-dihydroxy-vitamin D3 stimulation of bronchial smooth muscle cells induces autocrine, contractility, and remodeling processes

Abstract

Genetic variants in the vitamin D receptor (VDR) gene were recently associated with asthma. The biological mechanisms explaining this association are unknown but are likely to involve many cell types given the pleiotropic effect of its ligand, 1alpha,25-dihydroxy-vitamin D3 [1alpha,25(OH)2D3]. Considering the prominent role of bronchial smooth muscle cells (BSMCs) in the pathogenesis of asthma, experiments were conducted to explore the gene regulatory effects of 1alpha,25(OH)2D3 in these cells. Using RT-PCR and Western blot, we showed that VDR is present both at the mRNA transcript and protein levels in human BSMCs. The functionality of the receptor was then demonstrated by showing a >200-fold change in the expression of the 24-hydroxylase (CYP24A1) gene following 1alpha,25(OH)2D3 stimulation. Microarray experiments were then performed to identify differentially regulated genes and pathways in BMSCs treated or not with 1alpha,25(OH)2D3. A total of 729 probe sets on the U133 plus 2.0 Affymetrix GeneChip showed fold-change differences above the 1.5 threshold using the Robust Multichip Average intensities. This corresponds to 231 unique genes that were upregulated and 215 unique genes that were down-regulated following 1alpha,25(OH)2D3 stimulation. A high similarity between microarray and real-time PCR results was observed for 13 random genes, with a concordance correlation coefficient of 0.91. Real-time PCR was also performed to confirm the regulation of asthma candidate genes. To identify the biological relevance of this regulation, biological pathways analyses were performed. The most significant network of upregulated genes included genes involved in morphogenesis, cell growth, and survival as well as genes encoding structural proteins, which are potentially involved in airway remodeling.