Immune regulation of 25-hydroxyvitamin D-1alpha-hydroxylase in human monocytic THP1 cells: mechanisms of interferon-gamma-mediated induction.
BACKGROUND
25-Hydroxyvitamin D can be activated to 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] by the rate-limiting enzyme 1alpha-hydroxylase in cells of the immune system under control of immune stimuli, such as interferon-gamma (IFNgamma). In pathological situations, such as sarcoidosis, this can lead to systemic excess of 1,25(OH)(2)D(3) and hypercalcemia.


OBJECTIVE
The aim of this study was to elucidate the intracellular pathways used by the immune system to tightly regulate 1,25(OH)(2)D(3) production in monocytes and macrophages.


METHODS
Human monocytic THP1-cells were differentiated and activated by IFNgamma and a secondary stimulus, such as lipopolysaccharide or phorbol myristate acetate. 1alpha-Hydroxylase mRNA levels were quantified by real-time RT-PCR. The involvement of different signaling pathways in the regulation of this enzyme was investigated using specific pharmacological inhibitors, whereas phosphorylation of signal transducer and activator of transcription 1alpha and CCAAT/enhancer binding protein beta was investigated by Western blotting.


RESULTS
In undifferentiated monocytic THP1 cells, IFNgamma needs to be combined with a second stimulus, such as lipopolysaccharide, to induce 1alpha-hydroxylase. In contrast, in phorbol myristate acetate-differentiated THP1 macrophages, IFNgamma alone induces 1alpha-hydroxylase and to much higher levels. Many different signaling pathways need to be activated concurrently to allow immune-mediated 1alpha-hydroxylase up-regulation. We show involvement of the Janus kinase-signal transducer and activator of transcription, MAPK, and nuclear factor-kappaB pathways, with a crucial role for the transcription factor CCAAT/enhancer binding protein beta. Furthermore, histone remodeling involving histone deacetylases and histone acetylase p300 is required.


CONCLUSIONS
The present findings indicate that IFNgamma-mediated 1,25(OH)(2)D(3) production, as observed in granulomatous diseases such as sarcoidosis, will take place only under conditions where the necessary other signaling pathways are also activated.
Overbergh
Lut
L
LEGENDO, UZ-Gasthuisberg, Onderwijs en Navorsing, Herestraat 49, B-3000 Leuven, Belgium.
Stoffels
Katinka
K
Waer
Mark
M
Verstuyf
Annemieke
A
Bouillon
Roger
R
Mathieu
Chantal
C
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000235:genetics / Q000276:immunology*; D000873:Anthracenes / Q000494:pharmacology; D015153:Blotting, Western; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics / Q000276:immunology; D002454:Cell Differentiation / Q000276:immunology; D002460:Cell Line; D004790:Enzyme Induction; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007371:Interferon-gamma / Q000276:immunology*; D020928:Mitogen-Activated Protein Kinases / Q000276:immunology; D009000:Monocytes / Q000166:cytology / Q000201:enzymology / Q000276:immunology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D012313:RNA / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050794:STAT1 Transcription Factor / Q000235:genetics / Q000276:immunology*; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000276:immunology
D000873:Anthracenes; D022782:CCAAT-Enhancer-Binding Protein-beta; D005419:Flavonoids; D007093:Imidazoles; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D050794:STAT1 Transcription Factor; C432165:pyrazolanthrone; D012313:RNA; D007371:Interferon-gamma; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D020928:Mitogen-Activated Protein Kinases; D013755:Tetradecanoylphorbol Acetate; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one