Immune regulation of 25-hydroxyvitamin-D-3-1 alpha-hydroxylase in human monocytes

Monocytes express 1 alpha-hydroxylase, the enzyme responsible for final hydroxylation of vitamin D-3, in response to IFN gamma and CD14/TLR4 activation. Cross-talk between the JAK-STAT, the NF-kappa B, and the p38 MAPK pathways is necessary, and direct binding of C/EBP beta to its recognition sites in the promoter of the lot-hydroxylase gene is a prerequisite. Introduction: The activated form of vitamin D-3, 1,25(OH)(2)D-3, known for its action in bone and mineral homeostasis, has important immunomodulatory effects. 1,25(OH)(2)D-3 modulates the immune system through specific nuclear receptors, whereas macrophages produce 1,25(OH)(2)D-3. In monocytes, the expression of 1 alpha-hydroxylase, the enzyme responsible for final hydroxylation of vitamin D-3, is regulated by immune stimuli. The aim of this study was to elucidate the intracellular pathways through which interferon (IFN)gamma and Toll-like receptor (TLR) modulation regulate expression of 1 alpha-hydroxylase in monocytes/macrophages. Materials and Methods: Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) and stimulated with IFN gamma (12.5 U/ml) and/or lipopolysaccharide (LPS; 100 ng/ml) for 48 h. The following inhibitors were used: janus kinase (JAK) inhibitor AG490 (50 mu M), NF-kappa B inhibitor sulfasalazine (0.25 mM), p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (5 mu M). la-hydroxylase mRNA expression was monitored by qRT-PCR. Phosphorylation of transcription factors was studied by Western blotting. Transfection of mutated or deletion promoter constructs, cloned in the pGL3-luciferase reporter plasmid, were performed in the RAW264.7 cell line. Cells were stimulated with IFN gamma (100 U/ml) and LPS (100 mu g/ml), and promoter activity was studied. Binding of signal transducer and activator of transcription (STAT)1 alpha, NF-kappa B, and C/EBP beta to their respective binding sites in the promoter was analyzed by gel shift assays. Results: 1 alpha-hydroxylase mRNA expression in monocytes is synergistically induced by IFN gamma and CD14/TLR4 ligation and paralleled by 1,25(OH)(2)D-3 production. This induction requires the JAK-STAT, NF-kappa B, and p38 MAPK pathways. Each of them is essential, because blocking individual pathways is sufficient to block p < 0.01; NF-kappa B inhibitor, 70% inhibition, p < 0.05; 1 alpha-hydroxylase expression (JAK inhibitor, 60% inhibition, p38 MAPK inhibitor, 95% inhibition, p < 0.005). In addition, we show the involvement of the p38 MAPK pathway in phosphorylation of C/EBPP. Direct binding of C/EBPP to its recognition sites in the 1 alpha-hydroxylase promoter is necessary to enable its immune-stimulated upregulation. Conclusion: IFN gamma and CD14/TLR4 binding regulate expression of 1 alpha-hydroxylase in monocytes in a synergistic way. Combined activation of the JAK-STAT, p38 MAPK, and NF-kappa B pathways is necessary, with C/EBPP most probably being the essential transcription factor controlling immune-mediated transcription.