Immune regulation of 25-hydroxyvitamin-D3-1alpha-hydroxylase in human monocytes.
Monocytes express 1alpha-hydroxylase, the enzyme responsible for final hydroxylation of vitamin D3, in response to IFNgamma and CD14/TLR4 activation. Cross-talk between the JAK-STAT, the NF-kappaB, and the p38 MAPK pathways is necessary, and direct binding of C/EBPbeta to its recognition sites in the promoter of the 1alpha-hydroxylase gene is a prerequisite.


BACKGROUND
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 1alpha-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)gamma and Toll-like receptor (TLR) modulation regulate expression of 1alpha-hydroxylase in monocytes/macrophages.


METHODS
Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) and stimulated with IFNgamma (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 microM), NF-kappaB inhibitor sulfasalazine (0.25 mM), p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (5 microM). 1alpha-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 IFNgamma (100 U/ml) and LPS (100 microg/ml), and promoter activity was studied. Binding of signal transducer and activator of transcription (STAT)1alpha, NF-kappaB, and C/EBPbeta to their respective binding sites in the promoter was analyzed by gel shift assays.


RESULTS
1alpha-hydroxylase mRNA expression in monocytes is synergistically induced by IFNgamma and CD14/TLR4 ligation and paralleled by 1,25(OH)2D3 production. This induction requires the JAK-STAT, NF-kappaB, and p38 MAPK pathways. Each of them is essential, because blocking individual pathways is sufficient to block 1alpha-hydroxylase expression (JAK inhibitor, 60% inhibition, p < 0.01; NF-kappaB 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/EBPbeta. Direct binding of C/EBPbeta to its recognition sites in the 1alpha-hydroxylase promoter is necessary to enable its immune-stimulated upregulation.


CONCLUSIONS
IFNgamma and CD14/TLR4 binding regulate expression of 1alpha-hydroxylase in monocytes in a synergistic way. Combined activation of the JAK-STAT, p38 MAPK, and NF-kappaB pathways is necessary, with C/EBPbeta most probably being the essential transcription factor controlling immune-mediated transcription.
Stoffels
Katinka
K
Laboratory for Experimental Medicine and Endocrinology (LEGENDO), University Hospital Gasthuisberg, Catholic University of Leuven, Leuven, Belgium.
Overbergh
Lut
L
Giulietti
Annapaula
A
Verlinden
Lieve
L
Bouillon
Roger
R
Mathieu
Chantal
C
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000096:biosynthesis* / Q000276:immunology; D050071:Bone Density Conservation Agents / Q000276:immunology / Q000494:pharmacology*; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000276:immunology / Q000378:metabolism; D002112:Calcifediol / Q000276:immunology / Q000494:pharmacology*; D002478:Cells, Cultured; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects* / Q000276:immunology; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology / Q000494:pharmacology; D018950:Lipopolysaccharide Receptors / Q000276:immunology / Q000378:metabolism; D009000:Monocytes / Q000166:cytology / Q000201:enzymology* / Q000276:immunology; D016328:NF-kappa B / Q000276:immunology / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects* / Q000276:immunology; D051197:Toll-Like Receptor 4 / Q000276:immunology / Q000378:metabolism
D050071:Bone Density Conservation Agents; D022782:CCAAT-Enhancer-Binding Protein-beta; D018950:Lipopolysaccharide Receptors; D016328:NF-kappa B; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D007371:Interferon-gamma; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D011505:Protein-Tyrosine Kinases; D002112:Calcifediol