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Keywords:

  • vitamin D;
  • monocytes;
  • cytokines;
  • transcription factors;
  • molecular pathways

Abstract

Monocytes express 1α-hydroxylase, the enzyme responsible for final hydroxylation of vitamin D3, in response to IFNγ and CD14/TLR4 activation. Cross-talk between the JAK-STAT, the NF-κB, and the p38 MAPK pathways is necessary, and direct binding of C/EBPβ to its recognition sites in the promoter of the 1α-hydroxylase gene is a prerequisite.

Introduction: The activated form of vitamin D3, 1,25(OH)2D3, known for its action in bone and mineral homeostasis, has important immunomodulatory effects. 1,25(OH)2D3 modulates the immune system through specific nuclear receptors, whereas macrophages produce 1,25(OH)2D3. In monocytes, the expression of 1α-hydroxylase, the enzyme responsible for final hydroxylation of vitamin D3, is regulated by immune stimuli. The aim of this study was to elucidate the intracellular pathways through which interferon (IFN)γ and Toll-like receptor (TLR) modulation regulate expression of 1α-hydroxylase in monocytes/macrophages.

Materials and Methods: Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) and stimulated with IFNγ (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 μM), NF-κB inhibitor sulfasalazine (0.25 mM), p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (5 μM). 1α-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 IFNγ (100 U/ml) and LPS (100 μg/ml), and promoter activity was studied. Binding of signal transducer and activator of transcription (STAT)1α, NF-κB, and C/EBPβ to their respective binding sites in the promoter was analyzed by gel shift assays.

Results: 1α-hydroxylase mRNA expression in monocytes is synergistically induced by IFNγ and CD14/TLR4 ligation and paralleled by 1,25(OH)2D3 production. This induction requires the JAK-STAT, NF-κB, and p38 MAPK pathways. Each of them is essential, because blocking individual pathways is sufficient to block 1α-hydroxylase expression (JAK inhibitor, 60% inhibition, p < 0.01; NF-κB inhibitor, 70% inhibition, p < 0.05; p38 MAPK inhibitor, 95% inhibition, p < 0.005). In addition, we show the involvement of the p38 MAPK pathway in phosphorylation of C/EBPβ. Direct binding of C/EBPβ to its recognition sites in the 1α-hydroxylase promoter is necessary to enable its immune-stimulated upregulation.

Conclusion: IFNγ and CD14/TLR4 binding regulate expression of 1α-hydroxylase in monocytes in a synergistic way. Combined activation of the JAK-STAT, p38 MAPK, and NF-κB pathways is necessary, with C/EBPβ most probably being the essential transcription factor controlling immune-mediated transcription.