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


  1. Top of page
  2. Abstract
  7. Acknowledgements
  8. References

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.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  8. References

APART FROM ITS classical role in bone and mineral homeostasis, the activated form of vitamin D3, 1,25(OH)2D3, also modulates the immune system.(1) The nuclear receptor for the molecule can be found in cells throughout the immune system (e.g., in activated T lymphocytes), whereas macrophages can produce 1,25(OH)2D3.(2) Production of 1,25(OH)2D3 by macrophages is regulated in a completely different manner than its production by kidney cells, the place where 1,25(OH)2D3 for calcium and bone homeostasis is produced. In the proximal tubule cells of the kidney, the main site of 1,25(OH)2D3 synthesis, calcium, PTH, and 1,25(OH)2D3 itself are the key factors for regulation of production.(3,4) In contrast, macrophages produce 1,25(OH)2D3 independently of calcium-homeostasis signals.(2,5) This is observed in patients with sarcoidosis and related diseases, where activated macrophages continue to produce 1,25(OH)2D3 in the presence of overt hypercalcemia and high levels of 1,25(OH)2D3.(6–9) We and others have shown that, in macrophages, 1α-hydroxylase is under the regulation of immune stimuli, suggesting a role for this system in controlling the immune response.(2,10)

During an immune response, monocytes are activated by the T cell cytokine interferon (IFN)γ, resulting in a cross-talk between macrophages and T lymphocytes. Activation of monocytes/macrophages by IFNγ is classically mediated through the janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway.(11) Binding of IFNγ to its receptor leads to phosphorylation and dimerization of STAT1α, which can bind to γ-activated sequence (GAS) sites and directly activate transcription of several genes, the so-called “primary response genes,” including interferon regulatory factor-1 (IRF1).(12,13) IRF1, in turn activates transcription of secondary responsive genes, through binding to interferon-stimulated response element (ISRE) sites. On the other hand, activation of monocytes/macrophages through CD14 and Toll-like receptors (TLRs) leads to activation of mitogen-activated protein kinases (MAPKs) and NF-κB and subsequent expression of genes involved in inflammation.(14–16)

The aim of this study was to analyze the intracellular pathways involved in immune regulation of 1α-hydroxylase expression in monocytes/macrophages. As stimuli, we chose IFNγ, a major cytokine released by activated T lymphocytes, and CD14/TLR4 modulation. Our results show that, in peripheral blood monocytes, 1α-hydroxylase is synergistically induced by IFNγ and a CD14/TLR4 activator, such as lipopolysaccharide (LPS). The data show that, besides the expected involvement of the IFNγ-dependent JAK-STAT pathway and the activation of NF-κB, an intricate cross-talk between the signaling pathways exists and is essential for the induction of 1α-hydroxylase. Phosphorylation of C/EBPβ by the p38 MAPK pathway and binding to the 1α-hydroxylase promoter is crucial for this synergistic upregulation of 1α-hydroxylase.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  8. References

Cell culture conditions

Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient from buffy coats obtained from healthy blood donors. Monocytes were obtained using positive selection on a MACS column after labeling PBMCs with CD14-MicroBeads or by negative selection with the Monocyte Isolation kit II following the supplier's protocol (Miltenyi Biotec, Amsterdam, The Netherlands). Typically >90% purity of monocytes was obtained by either purification method as determined by fluorescence-activated cell sorting (FACS) analysis. Human PBMCs (1 × 106 cells/ml) and isolated monocytes (0.33 × 106 cells/ml) were cultured in Roswell Park Memorial Institute (RPMI) 1640 with 10% FCS and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) and stimulated with 12.5 U/ml human recombinant IFNγ (Roche, Vilvoorde, Belgium), 100 ng/ml LPS (Sigma, Bornem, Belgium), 10−8 M 1,25(OH)2D3, or 10−8 M 25(OH)D3 as indicated. The MAPK inhibitors PD98059 (5 μM; Calbiochem, Darmstadt, Germany), SP600125 (1 μM; SanverTech, Heerhugowaard, The Netherlands), SB203580 or its negative control SB202474 (5 μM; Calbiochem), the NF-κB inhibitor sulfasalazine (0.25 mM; Calbiochem), or the JAK inhibitor AG490 (50 μM; Calbiochem) were added to the cell cultures at the indicated concentrations in dimethyl sulfoxide (DMSO) 30 minutes before addition of the other stimuli and further incubated as indicated. Concentrations of the inhibitors were not toxic, as evaluated by unchanged β-actin mRNA levels during the incubation, visual microscopy of the cell cultures, and concerning SB203580, the use of the noninhibiting structural analog SB202474.

For transfection experiments, the murine RAW264.7 cell line (ATCC, Rockville, MD, USA) was grown in RPMI 1640 medium, 10% FCS, and antibiotics. Cells were stimulated with LPS (100 μg/ml) and murine recombinant IFNγ (100 U/ml; Roche, Brussels, Belgium) as indicated.

RNA extraction, cDNA synthesis, andreal-time RT-PCR

Cells were harvested and pooled, centrifuged at 930g for 5 minutes, and resuspended in 200 μl PBS. Total RNA was extracted using the High pure RNA Isolation Kit (Roche). Total RNA (0.5 μg) was reverse transcribed using 100 U Superscript II RT (Life Technologies, Merelbeke, Belgium) at 42°C for 80 minutes in the presence of 5 μM oligodT16.

Real-time PCR was performed for human β-actin and 1α-hydroxylase using the MyiQ system (Bio-Rad, Nazareth, Belgium) as described previously.(17,18) Primers were as follows −1α-hydroxylase: forward primer, 5′-CCCAGATCCTAACACATTTTGAGG-3′; reverse primer, 5′-AAAGGGTGATGATGACAGTCTCTTTC-3′; probe, 5′-FAM- ACCCAAGACCCGGACTGTCCTGGTTAMRA-3′ (Eurogentec, Liège, Belgium; GenBank no. NM_000785; amplicon length, 153 bp); β-actin: forward primer, 5′-ACCCCAAGGCCAACCG-3′; reverse primer, 5′-ACAGCCTGGATAGCAACGTACA-3′; and minor groove binding probe, 5′-TGACCCAGATCATGTTT-3′ (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands; GenBank no. NM_001101; amplicon length, 85 bp).

Measurement of 1α-hydroxylase enzymatic activity

Monocytes isolated by CD14 positive selection were incubated in RPMI 1640 with 10% FCS (20 × 106 cells in 6 ml), with or without 12.5 U/ml huIFNγ. After 24 h, cells were counted, and medium was removed and replaced by serum-free RPMI 1640 containing 0.1% fatty acid-free BSA (Sigma, tested for absence of bovine vitamin D binding protein) and incubated for 0.5 or 1.5 h with 58.894 dpm3H-25OHD3. Cells and media were pooled, sonicated, and stirred with an equal amount of acetonitrile. After centrifugation, supernatant was diluted with one volume of H2O before applying on an Oasis HLB 3-ml column (Waters, Milford, MA, USA). Samples were purified by washing with DMSO/H2O (70:30), methanol/H2O (60:40), chloroform/n-heptane (10:90), and 2-propanol/ethylacetate/n-heptane (1:1:98), eluted with 3 ml methanol/ethylacetate/n-heptane (1:35:64), vacuum-evaporated, and reconstituted in 100 μl high-performance liquid chromatography (HPLC) solvent. Eighty-three microliters of sample was separated on a calibrated Zorbax SIL column (5 μm; 4.6 × 250 mm) with n-heptane/2-propanol (91.5:8.5) at a flow rate of 2 ml/min {retention time for 1,25(OH)2D3 was 15 minutes}. Putative 1,25(OH)2D3 fractions were collected according calibration with a 1,25(OH)2D3 standard, evaporated, and counted on a LKB Wallace β counter (Perkin Elmer, Wellesley, MA, USA). Values are expressed as disintegrations per minute.

Immunoblotting analysis

Cells were cultured as described above and lysed in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% sodium dodecyl sulphate, 1% Igepal CA-630, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, Halt protease inhibitor cocktail (Pierce), phophatase inhibitor cocktail I (1/100), and phophatase inhibitor cocktail II (1/100) (Sigma). Protein concentrations were determined using the BCA Protein Assay Reagent Kit (Perbio Science, Aalst, Belgium). One hundred micrograms of cell lysate was denatured using NuPage Sample Buffer (4×) and NuPage 10× Reducing Agent and heated for 10 minutes at 70°C before loading on a 4–9% NuPage Bis-Tris gel. Proteins were transferred to an Invitrolon polyvinylidene difluoride (PVDF) membrane (Invitrogen, Merelbeke, Belgium). Protein electrophoresis and electroblotting were performed following the instructions of the manufacturer. Transfer efficiency was tested by transient Ponceau S staining. Previously blocked membranes were incubated (90 minutes at room temperature) with one of the following antibodies: anti-STAT1α (0.5 μg/ml), anti-phospho-STAT1α (S727; 0.5 μg/ml), anti-phospho-STAT1α (Y701; 1/1000; all from USBiological, Swampscott, MA, USA), anti-C/EBPβ (1/1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or anti-phospho-C/EBPβ Thr188 (1/1000; Cell Signaling Technology, Beverly, MA, USA). Bound antibody was detected using anti-rabbit horseradish peroxidase antibody conjugate (Dako, Leuven, Belgium) and Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer Life Sciences, Boston, MA, USA).

Construction of reporter plasmids

The full-length human 1α-hydroxylase promoter(19) was amplified by PCR using the following set of primers: FW1 5′-CTATGTTGCTCAAGCTTGTCTCAACCTCCT-3′ and RV1 5′-CGTCCGCTGGGCGCCCGAGTTAAGCTTGGG-3′(corresponding to −1600 to +101 bp). The PCR reaction was performed using the following conditions: 10 ng DNA, 1.25 U PfuII turbo polymerase (Stratagene, La Jolla, CA, USA), and incubation for 2 minutes at 95°C, followed by 30 cycles of 40 s at 95°C, 30 s at 63°C, and 2 minutes at 72°C, and concluded by 7 minutes at 72°C. A HindIII site was included in each primer for subsequent cloning into the pGL3 basic luciferase reporter plasmid (Promega, Leiden, the Netherlands).

Four different deletion clones were constructed by restriction digestion, starting from the full length promoter, using NheI (−1301 bp), SmaI (−1124 bp), SacI (−999 bp), and PvuII (−410 bp), and religation into pGL3. Two shorter deletion constructs were amplified by PCR and subsequently cloned into pGL3, using the following primer sets: FW2 5′-CCGCTCGAGACCACTCAGGAGGAGGGATTGGCTGAGG-3′ and RV1 (length −91 to +101 bp) and FW3 5′-CCGCTCGAGGAGCTTGGAGAGGGGGCGTCATCACCT-3′ and RV1 (length −65 to +101 bp). Promoter mutants were created by site-directed mutagenesis (Stratagene, La Jolla, CA, USA) of the GAS (5′-ATTCCAGAA-3′ at −1318/−1310 bp), the CCAAT 1 (5′-ATTGGCT-3′at 74/−68 bp), and the CCAAT 2 sites (5′-TCATTGC-3′ at −171/−165 bp), using the following primer sets: GAS-FW, 5′-CACATTGACCTTCAATACGCGTACTTCAGAGCTAGCTGACTGGCACAGAGCC-3′; GAS-RV, 5′-GGCTCTGTGCCAGTCAGCTAGCTCTGAAGTACGCGTATTGAAGGTCAATGTG-3′; CCAAT 1-FW, 5′-CACTCAGGAGGAGGGATATCCTGAGGAGCTTGGAGAGG-3′; CCAAT 1-RV, 5′-CCTCTCCAAGCTCCTCAGGATATCCCTCCTCCTGAGTG-3′; CCAAT 2-FW, 5′-CTCCCTCCCATGAGGGTGATATCAACATGAGACCCAAGGG-3′; CCAAT 2-RV, 5′-CCCTTGGGTCTCATGTTGATATCACCCTCATGGGAGGGAG-3′; NF-κB 2-FW, 5′-GCTGGGCTCACTGGTAGAAGTGGATATCTAAGAGACTGACTAGTGTAGCTTGG-3′; NF-κB 2-RV, 5′-CCAAGCTACACTAGTCAGTCTCTTAGATATCCACTTCTACCAGTGAGCCCAGC-3′; NF-κB 1-FW, 5′-CCTAGACAAAGGAGTACTACCAGGAGGAAAATCTTAGGCCCTCCCTCCC-3′; NF-κB 1-RV, 5′-GGGAGGGAGGGCCTAAGATTTTCCTCCTGGTAGTACTCCTTTGTCTAGG-3′; respectively. The mutated nucleotides are in bold letters.

Transfection and luciferase assays

RAW cells (1 × 106) were transfected with 2 μg pGL-1α-hydroxylase full-length promoter, deletion, or mutated constructs using Transfast reagent (Promega). Twenty-four hours after transfection, the cells were incubated for 24 h with LPS (100 μg/ml) + IFNγ (100 U/ml), after which the cells were harvested in 300 μl lysis buffer (Roche). Total protein content was determined using the BCA Protein Assay Reagent Kit (Perbio Science). Twenty-five microliters of extracted sample was subjected to measurement of luciferase activity in duplicate (Luminometer, Thermo Labsystems, Brussels, Belgium).

Electrophoretic mobility shift assay

Nuclear extracts were prepared from 40 × 106 PBMCs, incubated with or without 12.5 U/ml IFNγ + 100 ng/ml LPS for 30 minutes (at 4 × 106 cells/ml), using a modification of the method of Andrew and Faller.(20) The following oligonucleotides were used: GAS, 5′-ATTGACCTTCAATTCCAGAACTTCAGAGCTAGCTGA-3′; CCAAT 1, 5′-CTCAGGAGGAGGGATTGGCTGAGGAGCTTGGAGAGG-3′; CCAAT 2, 5′-CTCCCATGAGGGTCATTGCAACATGAGACCCAAGGG-3′; NF-κB 1 site, 5′-CTCCTAGACAAAGGCACTCTCCCAGGAGGAAAATCT-3′; NF-κB 2 site, 5′-ACTGGTAGAAGTGGGAATATCAGAGACTGACTAGTG-3′.

Oligonucleotides were labeled with α{32P}dCTP and 50,000 cpm of the probe were incubated with 15 μg of nuclear extracts at 32°C for 30 minutes. Binding reactions were performed in a total volume of 25 μl containing 10 mM HEPES, 2.5 mM MgCl2, 0.05 mM EDTA, 10% glycerol, 1 μg poly (dI-dC), 0.05% Triton X-100, and 1 mM dithiothreitol (DTT). The protein/DNA complexes were fractionated on a 4% polyacrylamide gel. Gels were transferred to Whatman filter paper, covered with plastic wrap, dried for 2 h, and exposed to Hyperfilm MP (−70°C; Amersham-Pharmacia). For Stat1α, NF-κB, and C/EBPβ supershift assays, nuclear extracts were incubated with 2 μl anti-Stat1α (US Biological), 2–4 μl anti-NF-κB p65 (SC-109X; Santa Cruz Biotechnology), or 2–4 μl anti-C/EBPβ (SC-150X; Santa Cruz Biotechnology), respectively.

Statistical analysis

Mean mRNA values from different experimental conditions or different time-points were compared by one-way ANOVA. When ANOVA was significant, the Student's t-test was performed. Differences were considered significant at p < 0.05. Data are presented as means ± SE.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  8. References

IFNγ and CD14/TLR4 ligation induce 1α-hydroxylase in human monocytes

Induction of 1α-hydroxylase mRNA was measured in PBMCs. Incubation of these cells for 48 h with IFNγ alone did not result in induction of 1α-hydroxylase transcription. Incubation of cells with LPS alone resulted in an 18-fold increase of 1α-hydroxylase levels (p < 0.001). Simultaneous addition of LPS and IFNγ resulted in a major increase in 1α-hydroxylase mRNA levels (36-fold over baseline levels; p < 0.001; Fig. 1A).

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Figure FIG. 1.. (A) Expression of 1α-hydroxylase mRNA in PBMCs. Total PBMCs were cultured for 48 h without stimulation, with IFNγ (12.5 U/ml), with LPS (100 ng/ml), or with a combination of LPS (100 ng/ml) and IFNγ (12.5 U/ml). 1α-hydroxylase mRNA levels were quantified by real-time RT-PCR and normalized to β-actin levels. Each bar is the mean of five experiments ± SE. ****p < 0.001 vs. medium control. (B) Expression of 1α-hydroxylase mRNA in purified monocytes. Monocytes were cultured for 48 h without stimulation, with CD14-binding, with IFNγ (12.5 U/ml), with LPS (100 ng/ml), or with a combination of stimuli. 1α-hydroxylase mRNA levels were quantified by real-time RT-PCR and normalized to β-actin levels. Each bar is the mean of five experiments ± SE. ****p < 0.001 vs. medium control, §§§§p < 0.001 LPS + IFNγ vs. IFNγ, #p < 0.05 LPS + IFNγ + CD14 vs. LPS + CD14 or IFNγ + CD14, &p < 0.05 LPS + IFNγ + CD14 vs. LPS + IFNγ, ££p < 0.01 IFNγ vs. IFNγ + CD14. Absolute values of ratio of copies 1α-hydroxylase to copies β-actin (±SE) from a representative experiment, consisting of three samples for each condition, were 1.45 ± 0.11 × 104 for medium controls of monocytes isolated by negative selection and 1.52 ± 0.02 × 104 for medium controls of monocytes isolated by CD14-binding.20

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When monocytes were isolated by negative selection and thus without direct CD14 binding, incubation for 48 h with IFNγ alone resulted in an induction of 1α-hydroxylase levels (5-fold over baseline levels; p < 0.001) and an 8-fold increase (p < 0.001) when incubated with LPS alone (Fig. 1B). Simultaneous addition of LPS and IFNγ resulted in an 11-fold induction of 1α-hydroxylase levels (p < 0.001 versus baseline levels).

When monocytes were isolated by CD14 binding, a greater induction of 1α-hydroxylase was observed after stimulation with IFNγ (8.5-fold; p < 0.001), LPS (12-fold; p < 0.001), or a combination of both stimuli (24.5-fold; p < 0.001). No differences were observed in absolute 1α-hydroxylase mRNA levels between nontreated monocytes isolated by CD14 binding and nontreated monocytes isolated by negative selection.

A time-course analysis for the mRNA induction of 1α-hydroxylase in CD14-isolated monocytes was performed, stimulating the cells with IFNγ for 0–48 h (Fig. 2A). A gradual increase of 1α-hydroxylase expression was observed between 3 and 12 h of incubation, followed by a strong induction that reached maximal levels after 24 h of incubation (8.5-fold; p < 0.001).

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Figure FIG. 2.. (A) Effects of IFNγ (12.5 U/ml; •) on 1α-hydroxylase mRNA expression in CD14-activated monocytes. Cells were cultured for different time periods (0-3, 6–12, 24–48 h), and 1α-hydroxylase mRNA levels were quantified by real-time RT-PCR and normalized to β-actin levels. Each point is the mean of samples obtained from 13 individuals ± SE. p < 0.001 vs. 0 h at all time-points. (B) Effect of IFNγ (12.5 U/ml) on 1α-hydroxylase activity. Cells were incubated for 24 h, and {3H}-25OHD3 was included for the final 0.5 or 1.5 h, as indicated on the y axis. Data are shown as the ratio of production of {3H}-1,25(OH)2D3 in dpm (1α-hydroxylase activity) in stimulated cells over nonstimulated cells and corrected for cell number in each condition. (C) Effect of 1,25(OH)2D3 and 25(OH)D3 on IFNγ-stimulated upregulation in CD14-activated monocytes. Cells were cultured for 48 h with 12.5 U/ml IFNγ with or without 10−8 M 1,25(OH)2D3 or 10−8 M 25(OH)D3, and 1α-hydroxylase mRNA levels were quantified by real-time RT-PCR and normalized to β-actin levels. Each point is the mean of samples obtained from 10 individuals ± SE. *p < 0.005 vs. 0 h.20

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Furthermore, we studied the enzymatic activity of 1α-hydroxylase in CD14-isolated monocytes. A low basal enzymatic activity was measured in noninduced monocytes, whereas a clear induction of enzymatic activity, measured by the conversion of {3H}-25(OH)D3 into {3H}-1,25(OH)2D3, was measured on incubation with IFNγ (Fig. 2B).

Finally, we studied the effect of 1,25(OH)2D3 or 25(OH)D3 on the IFNγ-induced 1α-hydroxylase upregulation in CD14-isolated monocytes. Immune-stimulated 1α-hydroxylase mRNA expression was not influenced by these vitamin D3 derivatives (Fig. 2C).

Intracellular signaling through the JAK-STATand NF-κB pathway contributes to 1α-hydroxylase induction

To confirm the role of the JAK-STAT pathway, commonly known to be activated by IFNγ, in the evaluated upregulation of 1α-hydroxylase in response to IFNγ stimulation in monocytes, cells were treated with the JAK inhibitor AG490 (50 μM). As expected, the effect of IFNγ in the upregulation was partially abolished, and the 1α-hydroxylase level was 60% inhibited (p < 0.01; Fig. 3A).

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Figure FIG. 3.. (A) Inhibitory effects of the JAK inhibitor AG490 and the NF-κB inhibitor sulfasalazine (SFZ) on the IFNγ-induced 1α-hydroxylase mRNA expression in CD14-activated monocytes. Cells were pretreated for 30 minutes with the inhibitor, followed by stimulation with 12.5 U/ml IFNγ in the presence of the inhibitor for 48 h. 1α-hydroxylase mRNA levels were quantified by real-time RT-PCR and normalized to β-actin levels. *p < 0.05 and **p < 0.01 compared with the positive control. One of three independently performed experiments is shown. Each bar is the mean of three samples ± SE. (B) Inhibitory effects of the MEK inhibitor PD98059, p38 MAPK inhibitor SB203580, and JNK inhibitor SP600125 on the IFNγ-induced 1α-hydroxylase mRNA expression in CD14-activated monocytes. Cells were pretreated for 30 minutes with each inhibitor, followed by stimulation with 12.5 U/ml IFNγ in the presence of the inhibitor during 48 h. 1α-hydroxylase mRNA levels were quantified by real-time RT-PCR and normalized to β-actin levels. ***p < 0.005 compared with the positive control. One of three independently performed experiments is shown. Each bar is the mean of three samples ± SE.20

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The possible involvement of the NF-κB pathway, known to be activated by CD14/TLR4-signaling, in 1α-hydroxylase induction, was studied by incubating CD14-isolated monocytes with IFNγ for 48 h in the presence of the NF-κB inhibitor sulfasalazine (0.25 mM). 1α-hydroxylase mRNA upregulation was blocked (70% inhibition compared with control; p < 0.05; Fig. 3A), confirming a role for the NF-κB pathway in the observed induction.

Cross-talk between IFNγ- and CD14/TLR4-stimulated signaling cascades

The possible involvement of different MAPK pathways in the synergistic induction of 1α-hydroxylase was studied by determining the effects of PD98059 (MEK inhibitor), SB203580 (p38 MAPK inhibitor), and SP600125 (JNK inhibitor) on 1α-hydroxylase levels. CD14-isolated (and thus activated) monocytes were pretreated for 30 minutes with each inhibitor, followed by stimulation with IFNγ for 48 h in the presence of the respective inhibitor. Addition of SB203580 resulted in an almost complete (95%) inhibition of IFNγ-induced 1α-hydroxylase mRNA expression (p < 0.005), whereas addition of PD98059 or SP600125 to the cultures had no statistically relevant inhibitory effect on 1α-hydroxylase induction (Fig. 3B). Incubation with the vehicle DMSO or with the negative control inhibitor SB202474 did not inhibit 1α-hydroxylase expression, excluding a nonspecific or toxic effect of the p38 MAPK inhibitor or DMSO (data not shown). These results suggest an important role for the p38 MAPK pathway in the induction of 1α-hydroxylase.

Phosphorylation of STAT1α and C/EBPβ, both important transcription factors in IFNγ signaling, was studied by Western blotting (Figs. 4A and 4B). Phosphorylation of STAT1αY701 required the presence of IFNγ, whereas phosphorylation of STAT1αS727 and C/EBPβ was already evident after CD14 activation and further increased by IFNγ. Interestingly, in the presence of the p38 MAPK inhibitor SB203580, a slight reduction of the phosphorylation of STAT1αS727 and a strong inhibitory effect on the phosphorylation of C/EBPβ was observed. This shows a cross-talk between the p38 MAPK pathway and the IFNγ-activated pathway. In contrast, C/EBPβ phosphorylation was not influenced by the JAK inhibitor AG490 or the NF-κB inhibitor sulfasalazine (Fig. 4B).

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Figure FIG. 4.. Western blot analysis of phosphorylation of STAT1α and C/EBPβ in monocytes. Cells were incubated without stimulation or with IFNγ (12.5 U/ml) for 45 minutes as indicated. (A)SB203580, (B) AG490, or sulfasalazine (SFZ) were added 30 minutes before stimulation. Cells were harvested and analyzed by Western blot. Equal gel loading was confirmed by Ponceau S staining. Data shown are representative for three independent experiments.20

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Necessity of C/EBPβ binding sites in the 1α-hydroxylase promoter

To further study which of the above-mentioned transcription factors actually bind to the promoter of 1α-hydroxylase, transfection experiments were performed. The human full-length promoter and several deletion constructs were cloned into the pGL3 plasmid and transfected in RAW cells. After transfection, cells were incubated for 24 h with IFNγ and LPS (a TLR4 stimulator). Activation of transcription by the promoter constructs was monitored by assaying luciferase activity. The luciferase activity induced by the full-length promoter was 2.9-fold upregulated after IFNγ + LPS stimulation compared with medium controls (p < 0.05; Fig. 5). Deletion of the GAS site, as in the construct generated by NheI digestion of the complete promoter, did not result in any decrease of the luciferase activity. Furthermore, gradual deletion of the first two NF-κB sites did not have an effect on the upregulation. When the last two NF-κB sites and the putative C/EBPβ site (CCAAT 2) were deleted, resulting in the −91 to +101 construct, a dramatic decrease of the luciferase activity could be observed (p < 0.005 versus full length). The −91 to +101 construct contains a consensus CCAAT box. Once this site was removed, resulting in the −65 to +101 construct, the luciferase activity further decreased 1.8-fold (p < 0.005 −91 to +101 construct versus −65 to +101-construct).

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Figure FIG. 5.. (A) Overview of the different human 1α-hydroxylase promoter-luciferase constructs, displaying transcription factor binding sites. (B) Transfection of RAW cells with human 1α-hydroxylase promoter-luciferase constructs. After transfection, cells were stimulated for 24 h with IFNγ (100 U/ml) and LPS (100 μg/ml). Fold induction in ratio of luciferase activity to protein content is shown for a representative experiment. All plasmids were transfected a minimum of three times in separate experiments. *p < 0.05 IFNγ + LPS vs. medium, $$$p < 0.005 full-length vs. −91 to +101, §§§p < 0.005 full-length vs. −65 to +101, ###p < 0.005 −91 to +101 vs. −65 to +101. (C) Overview of the different human 1α-hydroxylase promoter-luciferase mutations. X indicates a mutated transcription factor binding site. (D) Transfection of RAW cells with human 1α-hydroxylase promoter-luciferase mutated constructs. Fold induction in ratio of luciferase activity to protein content is shown for a representative experiment. All plasmids were transfected a minimum of three times in separate experiments. *p < 0.05 vs. nonmutated, ***p < 0.005 vs. nonmutated.20

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In addition, several mutated constructs were generated (Fig. 5C), in which the GAS site, two NF-kB sites (NF-kB 1 or NF-kB 2), the putative C/EBPβ site (CCAAT 2), or the consensus CCAAT box (CCAAT 1) were mutated. Mutation of the GAS site or the NF-κB sites did not result in any reduction of the promoter activity, whereas mutation of the CCAAT 2 or the CCAAT 1 site resulted in 66.7% (p < 0.05 versus nonmutated) and 99% inhibition (p < 0.005 versus nonmutated), respectively (Fig. 5D).

Next, we studied the actual binding of STAT1α, NF-κB, and C/EBPβ to the 1α-hydroxylase promoter by gel shift assays. We could not show enhanced binding of STAT1α or NF-κB to the GAS or NF-κB 1 and NF-κB 2 sites, respectively, in immune-stimulated cells when compared with nonstimulated cells (data not shown). In contrast, C/EBPβ did clearly bind to the CCAAT 1 and CCAAT 2 sites in immune-stimulated cells (Fig. 6). This binding was absent in control nonstimulated cells. Furthermore, addition of anti-C/EBPβ resulted in a supershift of the formed complex. These results, combined with the results from the transfections, confirm that C/EBPβ is most probably the primary transcription factor involved in immune-regulated transcription of 1α-hydroxylase.

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Figure FIG. 6.. C/EBPβ binding to the 1α-hydroxylase promoter. Nuclear extracts from PBMCs either untreated (lanes 1 and 5) or treated with IFNγ + LPS for 30 minutes (lanes 2 and 6), were incubated with the {32P}labeled oligonucleotides CCAAT 1 (lanes 1–4) or CCAAT 2 (lanes 5–8); 2 (lanes 3 and 7) or 4 μl (lanes 4 and 8) anti-C/EBPβ was added. White arrows indicate the complexes of interest; black arrows indicate supershifted complexes.20

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  1. Top of page
  2. Abstract
  7. Acknowledgements
  8. References

1,25(OH)2D3, the central hormone in calcium metabolism, is not only produced by kidney cells, but is also secreted by macrophages, and several reports have shown that this system is regulated by immune signals rather than by the classical signals of bone and mineral homeostasis.(2,10,21) In this study, we show that 1α-hydroxylase, the enzyme regulating 1,25(OH)2D3 production, is subject to a tight transcriptional regulation by immune stimuli in monocytes. Regulation of the 1α-hydroxylase in monocytes is clearly different from the regulation of this enzyme in kidney cells, with absence of downregulation by 1,25(OH)2D3.(2) Analysis of the human 1α-hydroxylase promoter revealed several potential binding sites for transcription factors whose activities can be modulated by inflammatory stimuli: binding sites for NF-κB, AP1, AP2 and Sp1, cAMP binding sites (CRE), a CCAAT box, and a GAS site are present.(3,19,22,23) The presence of this multitude of putative regulatory elements suggests a complex regulatory pathway. In this study, we investigated the intracellular mechanisms and pathways involved in 1α-hydroxylase upregulation in monocytes after activation by CD14/TLR4 binding and stimulation with the cytokine IFNγ.

Experiments using monocytes isolated from human PBMCs showed that 1α-hydroxylase mRNA expression is synergistically inducible by combined IFNγ and CD14/TLR4 activation. This upregulation correlates with enzymatic 1α-hydroxylase activity and production of 1,25(OH)2D3 by monocytes. Moreover, confirming previous observations, we show that this induction is not abrogated by 1,25(OH)2D3, which is a negative regulator of 1α-hydroxylase in kidney cells.(2,24) The induction by IFNγ and CD14/TLR4 stimulation was completely blocked on addition of the p38 MAPK inhibitor SB203580 and partly blocked by the JAK inhibitor AG490 and the NF-κB inhibitor sulfasalazine, clearly indicating the involvement of these pathways in the upregulation of 1α-hydroxylase. To perform a more in-depth analysis of the intracellular signaling pathways involved in this regulation, promoter-luciferase assays were performed in the macrophage RAW cell line. In these cells, as in monocytes, the expression of 1α-hydroxylase was 3.3-fold induced by IFNγ and LPS, a TLR4 modulator (data not shown). Thus, simultaneous activation of IFNγ-dependent and -independent pathways is indispensable for the IFNγ-mediated induction to occur.

An essential transcription factor in interferon signaling is STAT1α. On binding of IFNγ to its receptor, JAKs are activated and phosphorylate STAT1α at Tyr701. In this study, we showed the involvement of the JAK-STAT pathway in the upregulation of 1α-hydroxylase, using the pharmacological JAK inhibitor AG490. Furthermore, we showed cross-talk between IFNγ-mediated signaling and the p38 MAPK pathway in phosphorylation of STAT1αS727, which was decreased by the p38 MAPK inhibitor SB203580. However, no important direct effect of STAT1α through the GAS site on the 1α-hydroxylase promoter could be detected. Indeed, IFNγ + LPS stimulation of cells transfected with the −1301 to +101 deletion construct, which does not contain the GAS site, did not result in lower luciferase activity compared with the full-length promoter. In addition, mutation of the GAS site did not result in a significant decrease of promoter activity, and gel shift assays failed to show STAT1α binding to the 1α-hydroxylase promoter. Therefore, the effect of IFNγ on the upregulation of 1α-hydroxylase is rather an indirect effect, possibly by upregulating other factors such as C/EBPβ.(25)

CD14/TLR4 binding is known to activate the NF-κB pathway.(15,26,27) In its inactive form, NF-κB is bound by members of the IκB family in the cytoplasm. The various stimuli that activate NF-κB cause phosphorylation of IκB, which is followed by its ubiquitination and subsequent degradation. This results in the exposure of the nuclear localization signals on NF-κB subunits and the subsequent translocation of the molecule to the nucleus. In the nucleus, NF-κB binds to a consensus sequence (5′-GGGACTTTCC-3′) of various genes and thus activates their transcription. Because inhibition of either the NF-κB pathway or the p38 MAPK pathway results in an inhibition of 1α-hydroxylase induction, activation of both pathways is required for 1α-hydroxylase induction. These data suggest a cross-talk between different TLR4-mediated pathways, thereby further increasing the complexity of the signaling network leading to 1α-hydroxylase upregulation.

Transfection experiments using the 1α-hydroxylase promoter and several deletion constructs of it might point to a role of NF-κB recognition sites, as luciferase activity is only dramatically decreased after all the NF-κB binding sites are deleted from the promoter. However, in addition to the last two NF-κB sites, a putative C/EBPβ binding site (CCAAT 2) was deleted when generating the −91/+101-bp deletion construct, which may contribute to the loss of promoter activity. To evaluate the relative contribution of the NF-κB 1, NF-κB 2, or CCAAT 2 sites, several mutation constructs were designed. Mutation of either of the two NF-κB sites did not influence luciferase activity. Furthermore, NF-κB binding to these sites could not be confirmed in gel shift assays. Therefore, as for STAT1α, NF-κB has only indirect effects on 1α-hydroxylase immune-mediated upregulation. Indeed, NF-kB-mediated activation of transcription has been described before to be independent of its binding site in the promoter, and rather, acts through cooperation with other transcription factors.(28–30)

The 1α-hydroxylase promoter contains a consensus CCAAT box, which is a recognition site for C/EBPβ. In addition, a second putative C/EBPβ-binding site is present.(3) C/EBPβ is a transcription factor involved in many immunological processes, which can become activated by several signals, including LPS and IFNγ itself.(31,32) Phosphorylation of several serine and threonine residues regulates the activity of C/EBPβ. Phosphorylation within the DNA-binding domain has been associated with decreased DNA binding, whereas phosphorylation in the transactivating domain has been linked with increased transcriptional activity. C/EBPβ also contains a regulatory domain. Phosphorylation in this domain reduces the transcriptional activity. Several kinases have been reported to phosphorylate C/EBPβ, including ERK1/2, protein kinase A (PKA), protein kinase C (PKC), and calcium/calmodulin-dependent protein kinase.(16,25,31,33–40) Esteban et al.(41) have recently shown an important involvement of C/EBPβ in the upregulation of 1α-hydroxylase in response to IFNγ in murine macrophages. In our setting, Thr235 of C/EBPβ, located in the transactivating domain, was phosphorylated by CD14 activation, which was further increased on addition of IFNγ. Furthermore, this phosphorylation of C/EBPβ was strongly affected by the p38 MAPK inhibitor, indicating the involvement of this kinase in its phosphorylation. Moreover, C/EBPβ mRNA levels increased during the immune stimulation (data not shown), which can again attribute to the increased C/EBPβ-mediated 1α-hydroxylase induction. The important role of C/EBPβ in the upregulation of 1α-hydroxylase transcription was further confirmed by transfection experiments. Thus, the −91 to +101-bp deletion construct, containing a consensus C/EBPβ binding site (CCAAT 1 in Fig. 5), still displays a 1.8-fold higher luciferase activity than the −65 to +101 construct. In addition, mutation of the consensus CCAAT 1 site completely abolishes promoter activity. Moreover, the putative C/EBPβ-binding site (CCAAT 2 in Fig. 5) plays a role in immune-stimulated upregulation of 1α-hydroxylase, because mutation of this site results in 66.7% inhibition of luciferase activity. Gel shift assays confirmed binding of C/EBPβ to both CCAAT sites, further emphasizing the direct role of this transcription factor.

Interplay and association of several transcription factors and co-activators has been described for the promoter of various genes, such as iNOS and IL12p40.(31,42–45) In analogy, our results show that activation of different pathways and transcription factors is essential in the induction of 1α-hydroxylase by IFNγ and CD14/TLR4-signaling (Fig. 7). The JAK/STAT pathway becomes activated, leading to subsequent phosphorylation of STAT1α and transcription of primary responsive genes. In addition, the NF-κB pathway is induced, resulting in degradation of IκB and phosphorylation of NF-κB. Furthermore, activation of the p38 MAPK pathway results in additional phosphorylation of C/EBPβ in IFNγ and CD14/TLR4-stimulated monocytes. Specific inhibition of each of these signaling pathways blocks the 1α-hydroxylase upregulation, suggesting that each of them constitutes a prerequisite for the initiation of transcription. Moreover, we showed that cross-talk exists between IFNγ-mediated and CD14/TLR-mediated signaling. Indeed, both contribute to the phosphorylation of STAT1α and C/EBPβ, and blocking the p38 MAPK pathway results in inhibition of this phosphorylation. The precise way in which the involved transcription factors are interacting with each other, as well as the possible role of certain co-activators, needs further study.

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Figure FIG. 7.. Proposed model for 1α-hydroxylase induction by IFNγ and CD14/TLR4-binding in monocytes. As represented in this simplified model, different pathways are essential in the induction of 1α-hydroxylase: (1) induction of the JAK-STAT pathway and subsequent initiation of transcription of primary response genes, (2) induction of the NF-κB pathway, (3) induction of the p38 MAPK pathway, subsequent phosphorylation of C/EBPβ, and binding of phosphorylated C/EBPβ to the promoter. Moreover, at different levels, cross-talk between the pathways exists, leading to synergistic effects.20

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The observed synergistic action between the IFNγ- and CD14/TLR4-induced pathways in upregulation of the 1α-hydroxylase expression in monocytes again clearly indicates a totally different regulation in these immune cells than in kidney cells. Ebert et al.(46) have shown an NF-κB-mediated downregulation of 1α-hydroxylase in kidney cells.

Upregulation of 1α-hydroxylase expression in macrophages has a potential physiological role in the immune reactivity. In the early phase of inflammation, when only macrophages are involved, 1α-hydroxylase is absent or low. On recruitment of IFNγ-producing lymphocytes and NK cells, the activated macrophages receive the second and necessary signal for 1α-hydroxylase induction. Once 1,25(OH)2D3 is produced, it can perform its immunosuppressive action and shut down the ongoing immune reaction, thus preventing unrestricted immune responses. A defect in this negative feedback loop may possibly lead to autoimmunity, whereas administration of 1,25(OH)2D3 or its analogs can prevent this.(1) This has been shown in a model for type 1 diabetes, the NOD mouse, that carries a defect in 1α-hydroxylase upregulation, and where treatment with high doses of 1,25(OH)2D3 or analogs prevents the disease.(2,47)

In conclusion, 1α-hydroxylase transcription in monocytes/macrophages is subject to a complex immune regulation. We showed the involvement of the JAK-STAT, the p38 MAPK, and the NF-κB pathway in the synergistic induction of 1α-hydroxylase transcription by IFNγ and CD14/TLR4 signaling. Moreover, we showed that CD14/TLR4 binding plays a role in the phosphorylation of STAT1αS727 and C/EBPβ through activation of the p38 MAPK pathway. Because specific inhibition of each of these pathways almost completely blocks the 1α-hydroxylase induction, activation of each of them is required for the initiation of transcription. The presence of C/EBPβ recognition sites in the 1α-hydroxylase promoter is a prerequisite for immune-mediated transcription, with C/EBPβ binding to them and most probably interacting with other transcription factors or co-activators in a cooperative manner.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  8. References

The expert technical assistance of Dirk Valckx, Wim Cockx, and Ivo Jans is gratefully acknowledged. This work was supported by the Catholic University of Leuven (Geconcerteerde Onderzoeksacties 2004/10), a clinical research fellowship (FWO) of CM, a predoctoral fellowship (FWO) of KS, and a doctoral scholarship from the KUL (Interfaculty Council for Development Cooperation) of AG.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  8. References
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