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The Journal of Immunology
Vitamin D Inhibits Monocyte/Macrophage Proinflammatory
Cytokine Production by Targeting MAPK Phosphatase-1
Yong Zhang,* Donald Y. M. Leung,*
,†
Brittany N. Richers,* Yusen Liu,
‡,x
Linda K. Remigio,* David W. Riches,* and Elena Goleva*
It is estimated that 1 billion people around the world are vitamin D deficient. Vitamin D deficiency has been linked to various
inflammatory diseases. However, the mechanism by which vitamin D reduces inflammation remains poorly understood. In this
study, we investigated the inhibitory effects of physiologic levels of vitamin D on LPS-stimulated inflammatory response in human
blood monocytes and explored potential mechanisms of vitamin D action. We observed that two forms of the vitamin D, 1,25
(OH)
2
D
3
, and 25(OH)D
3
, dose dependently inhibited LPS-induced p38 phosphorylation at physiologic concentrations, IL-6 and
TNF-aproduction by human monocytes. Upon vitamin D treatment, the expression of MAPK phosphatase-1 (MKP-1) was
significantly upregulated in human monocytes and murine bone marrow-derived macrophages (BMM). Increased binding of
the vitamin D receptor and increased histone H4 acetylation at the identified vitamin D response element of the murine and human
MKP-1 promoters were demonstrated. Moreover, in BMM from MKP1
2/2
mice, the inhibition of LPS-induced p38 phosphor-
ylation by vitamin D was completely abolished. Vitamin D inhibition of LPS-induced IL-6 and TNF-aproduction by BMM from
MKP-1
2/2
mice was significantly reduced as compared with wild-type mice. In conclusion, this study identified the upregulation of
MKP-1 by vitamin D as a novel pathway by which vitamin D inhibits LPS-induced p38 activation and cytokine production in
monocytes/macrophages. The Journal of Immunology, 2012, 188: 2127–2135.
Vitamin D is well known for its role in calcium homeostasis
and maintenance of bone metabolism (1). However, re-
cent evidence suggests that vitamin D plays important
roles in both innate and adaptive immunity (2). Vitamin D levels
are routinely tested by assessing the concentration of the major
circulating form of the vitamin D, 25(OH)D
3
, in serum; this form
of vitamin D has a half life of 15 d, whereas the active form of
vitamin D, 1,25(OH)
2
D
3
, has a short half life of ∼15 h (3–5).
1,25(OH)
2
D
3
acts as a ligand for the vitamin D receptor (VDR),
a member of the nuclear receptors superfamily (6). VDR forms
a heterodimer with a retinoid X receptor and regulates gene ex-
pression by binding to the vitamin D response element (VDRE).
VDRE had been shown to be predominantly located in introns
and intergenic intervals (7). VDRE is characterized by direct re-
peats of two hexameric core-binding motifs (preferentially being
AGTTCA) spaced by three nucleotides (8, 9). The binding of
VDR to VDRE recruits coactivators and enzymes with histone
acetylation activity, causing the structural changes in chromatin,
therefore, facilitating gene transcription (10).
LPS, a component of the Gram-negative bacterial cell wall,
induces cytokine production by monocytes/macrophages. LPS had
been implicated in sepsis caused by Gram-negative bacteria and
induces intense inflammatory and procoagulant responses, which
can be lethal (11). LPS is recognized by cell surface TLR4,
which initiates intracellular signal transduction cascades (12). The
MAPKs activated by LPS [ERK, JNK, and p38 (12)] are critical
regulators of proinflammatory cytokine production, including
TNF-aand IL-6 (13, 14). Although these proinflammatory cyto-
kines enhance host defense, excessive production leads to unre-
solved inflammation (15). Therefore, feedback control of MAPK
activation is necessary. MAPK phosphatases (MKP) inactivate
MAPKs by dephosphorylating conserved threonine and tyrosine
residues of the activated MAPK (16). MKP-1 is known to pref-
erentially inactivate p38 and JNK, leading to subsequent inhibition
of proinflammatory cytokines production (17, 18). In the current
study, we examined mechanisms of the vitamin D-mediated sup-
pression of LPS-activated monocytes/macrophages. We found that
vitamin D inhibits LPS-induced cytokine production by upregu-
lating MKP-1, thereby attenuating p38 activation.
Materials and Methods
Materials
1,25(OH)
2
D
3
, 25(OH)D
3
, and monoclonal anti–b-actin Ab were purchased
from Sigma-Aldrich (St. Louis, MO). HyQTase was purchased from
HyClone Laboratories (Logan, UT). TrypLE Express was purchased from
Invitrogen (Carlsbad, CA). Phosphorylated (p)-p38 and p38 Abs were
purchased from Cell Signaling Technology (Danvers, MA). Anti-mouse
and anti-rabbit HRP-labeled IgG were purchased from Amersham Bio-
sciences (Piscataway, NJ). Rabbit polyclonal Ab to VDR, rabbit polyclonal
Ab to MKP-1, Radioimmunoprecipitation Assay Lysis Buffer, and protein
A/G PLUS-agarose beads were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Rabbit polyclonal Ab to histone H4 and acetylated
histone H4 as well as the Magna Chromatin Immunoprecipitation (ChIP)
A/G ChIP Kit were purchased from Millipore (Temecula, CA). Chemilu-
*Department of Pediatrics, National Jewish Health, Denver, CO 80206;
†
De-
partment of Pediatrics, University of Colorado Denver, Aurora, CO 80045; and
‡
Center for Perinatal Research, Research Institute at Nationwide Children’s Hospital,
Columbus, OH 43205;
x
Department of Pediatrics, Ohio State University College of
Medicine, Columbus, OH 43205
Received for publication August 19, 2011. Accepted for publication December 28,
2011.
This work was supported in part by National Institutes of Health Grants AI070140,
HL68628, HL37260, AI57798, and AI68956.
The content is solely the responsibility of the authors and does not necessarily
represent the official views of the National Institute of Allergy and Infectious Dis-
eases, National Heart, Lung, and Blood Institute or the National Institutes of Health.
Address correspondence and reprint requests to Dr. Elena Goleva, National Jewish
Health, 1400 Jackson Street, Room K1016A, Denver, CO 80206. E-mail address:
golevae@njhealth.org
Abbreviations used in this article: BMM, bone marrow-derived macrophage; ChIP,
chromatin immunoprecipitation; MKP, MAPK phosphatase; p, phosphorylated; VDR,
vitamin D receptor; VDRE, vitamin D response element.
Copyright Ó2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102412
minescent reagents were purchased from Perkin-Elmer Life Sciences
(Waltham, MA). All the reagents and conjugated Abs against p-p38, p-
ERK1/2, p-JNK, and IL-6 in flow cytometry analysis were purchased from
BD Biosciences (San Diego, CA), whereas the TLR4 Ab was purchased
from eBioscience (San Diego, CA).
Study subjects
Blood samples were collected from normal healthy adults. Approval was
received from the National Jewish Health Institutional Review Board
(Denver, CO) for the study.
Mice
C57BL/6 3129 mice were purchased from The Jackson Laboratory (Bar
Harbor, ME). MKP-1
2/2
mice were provided by Bristol–Myers Squibb
(19). Six- to 8-wk-old males were used in the experiments. All experiments
using these animals were approved by the Institutional Animal Care and
Use Committee at National Jewish Health. This institution has an animal
welfare assurance number (A3026-1) on file with the Office of Protection
and Research Risks.
Cell culture and treatment
PBMC were isolated from heparinized, venous blood of healthy donors by
Ficoll-Hypaque density gradient centrifugation as described elsewhere.
PBMC were cultured in hormone-free medium (phenol-red free RPMI 1640
medium containing 5% charcoal-stripped FCS, 50 mg/ml streptomycin,
and 50 U/ml penicillin) with a range of doses of 1,25(OH)
2
D
3
or 25(OH)
D
3
for 24 h at 37˚C with 5% CO
2
. Equal volume of ethanol was used as
vehicle control. Following pretreatment with vitamin D, the cells were
stimulated with 10 ng/ml LPS, and the effects of vitamin D on LPS
responses in CD14
+
monocytes were examined.
Bone marrow cells from wild-type (C57BL/6 3129) and MKP
2/2
mice
were isolated as previously described (20, 21) and cultured in DMEM
containing 10% FBS, 10% L929 cell-conditioned medium (as a source of
CSF-1), 50 mg/ml streptomycin, and 50 U/ml penicillin at 37˚C with 10%
CO
2
for 5 d to produce bone marrow-derived macrophages (BMM).
Flow cytometry analysis
To analyze the effects of vitamin D on p38 activation by LPS, PBMC were
preincubated in hormone-free medium containing 1,25(OH)
2
D
3
or 25(OH)
D
3
for 24 h, followed by stimulation with 10 ng/ml LPS for 10 min. Cells
were then fixed with 2% formaldehyde at 37˚C for 10 min. Adherent cells
were collected by using HyQTase and TrypLE consecutively and com-
bined with suspension cells. Cells were permeabilized in 500 ml13perm/
wash buffer I (BD Pharmingen) at 4˚C for 30 min, incubated in 100 ml13
perm/wash buffer I containing FITC-conjugated anti-CD14 and allophy-
cocyanin-conjugated anti–p-p38 Abs at 4˚C for 1 h, and washed with 13
perm/wash buffer I. The samples were then analyzed by flow cytometry
(BD FACSCalibur Flow Cytometer; BD Biosciences, Franklin Lakes, NJ)
and CellQuest Pro software. The flow data were displayed as a percentage
of CD14
+
cells that express p-p38 (the gate for p-p38
+
cells was set on the
basis of isotype control binding).
To examine theeffects of vitaminD on IL-6 production byLPS-activated
monocytes, PBMC were cultured in hormone-free medium containing 1,25
(OH)
2
D
3
or 25(OH)D
3
for 24 h, followed by 10 ng/ml LPS stimulation for
6 h with GolgiPlug added for the last 4 h of the incubation. Cells were
collected as above and stained with allophycocyanin-conjugated anti-
CD14 Ab in staining buffer at 4˚C for 30 min. The cells were per-
meabilized in 500 ml13perm/wash buffer at 4˚C for 30 min, incubated in
100 ml13perm/wash buffer containing FITC-conjugated anti–IL-6 Ab at
4˚C for 30 min, and washed with 13perm/wash buffer before testing on
FACSCalibur as above.
Real-time PCR
Total RNA from human PBMC and murine BMM at the specified time point
was prepared using the RNeasy Mini kit (Qiagen, Valencia, CA). After
reverse transcription, 500 ng cDNA from each sample was analyzed by real-
time PCR using the dual-labeled fluorogenic probe method on an ABI Prism
7300 real-time PCR system (Applied Biosystems). The expression of hu-
man and murine MKP-1, b-actin, TNF-a, IL-6, and human MKP-5 mRNA
was determined. All primers were purchased from Applied Biosystems
(Foster City, CA).
Western blot analysis
Whole-cell extracts were prepared by incubating cells with radio-
immunoprecipitation assay lysis buffer with protease inhibitors on ice for
30 min, followed by centrifugation at 4˚C for 10 min and collection of
supernatants. Protein samples from vitamin D/LPS-treated PBMC and
murine bone marrow cultures were resolved on Invitrogen 4–12% Bis-tris
gel and transferred to polyvinylidene difluoride membranes. The mem-
branes were incubated in PBS containing specific Abs, 5% dry milk, and
0.1% Tween 20 at 4˚C overnight. Subsequently, membranes were washed
in PBS/0.1% Tween 20, incubated for 1 h at room temperature with HRP-
labeled secondary Abs, washed, incubated with chemiluminescent reagent,
and processed for immunodetection. Densitometry was used to evaluate
the intensity of the bands.
ELISA analysis
PBMC and murine BMM were treated with 1,25(OH)
2
D
3
for 24 h, followed
by 10 ng/ml LPS stimulation for 24 h. Supernatants were collected, human
IL-6 levels from PBMC were tested using the Human IL-6 ELISA ready-
set-go! Kit, and murine IL6 and TNF-alevels from BMM were tested
using the Mouse IL-6 ELISA ready-set-go! Kit and the Mouse TNF-a
ELISA ready-set-go! Kit, respectively. All the ELISA kits were purchased
from eBioscience (San Diego, CA).
ChIP assay
VDR binding to VDRE and histone H4 acetylation at the human MKP-1
promoter were assessed using the Magna ChIP A/G ChIP Kit following
the manufacturer’s instructions. VDR binding to VDRE and histone H4
acetylation at the murine MKP-1 promoter were assessed by ChIP assay as
previously described (22) with modifications. Briefly, 2 310
6
cells were
used in each precipitation. After sonication, chromatin solution was pre-
cleared with 50 ml protein A/G PLUS-agarose beads and 10 mg herring
DNA at 4˚C for 2 h and incubated with specific Ab or isotype control at
4˚C overnight, followed by precipitation with 50 ml protein A/G PLUS-
agarose beads at room temperature for 2 h. Precipitated chromatin com-
plexes were removed from the beads through incubation at 65˚C for 30 min
with elution buffer (50 mM Tris [pH 8], 1 mM EDTA, and 1% SDS). A
total of 200 ml eluate were mixed with 10 ml 5 M NaCl and 0.5 ml RNase
A (DNase-free; 10 mg/ml) and incubated at 65˚C overnight. Precipitated
DNA was quantified by real-time PCR using SYBR green (Applied Bio-
systems). Primers used to detect the potential VDRE in the human MKP-1
promoter were as follows: 59-AGCTGGGATTCTAATCCAGGCAGT-39
(E4.7 forward) and 59-CTTTGGAAGGTGGAGTTCCTCTCA-39(E4.7 re-
verse). Primers used to detect the potential VDRE in the murine MKP-1
promoter were as follows: 59-ACCCTTGCTCTCTCCCAAGTTCAT-39
(E33 forward), 59-AGAGTGTACCCAACACGACCCAAT-39(E33 reverse),
59-ACGAGGGCAGATGCCTTTAATCCT-39(E0.9 forward), and 59-TGCT-
GTGTAGCTCTGGCTAGTCTT-39(E0.9 reverse).
Statistical analyses
Results were expressed as the mean 6SEM. Statistical analysis was
conducted using GraphPad Prism, version 5 (GraphPad Software, La Jolla,
CA). The data were analyzed by the paired Student ttest, pairing by ex-
periment. Before testing, paired difference distributions were examined for
outliers, which can indicate violation to the normality assumption of the t
test. No outliers were apparent. Tests were performed between specific
treatments and LPS treatments or between vitamin D treatment and con-
trol. Unpaired ttest was used for comparison of responses between cells
from wild-type and MKP-1
2/2
mice. Differences were considered sig-
nificant at p,0.05. A minimum of three independent experiments was
conducted to allow for statistical comparisons.
Results
Pretreatment of human PBMC with vitamin D inhibits
LPS-induced p38 phosphorylation in human monocytes
In this study, we examined the role of vitamin D in the regulation of
LPS responses. Human PBMC were pretreated with vitamin D for
24 h, followed by stimulation with 10 ng/ml LPS. Both forms of the
vitamin D, an active form, 1,25(OH)
2
D
3
, and 25(OH)D
3
, were
tested. Monocytes/macrophages have previously been shown to be
able to locally convert 25(OH)D
3
into an active form (23). Vita-
min D levels are normally measured by serum 25(OH)D
3
levels,
because this form of vitamin D is more stable, whereas the active
form of vitamin D has a short half life (5). In these experiments,
we assessed the anti-inflammatory effects of 25(OH)D
3
doses that
are related to vitamin D deficiency (15 ng/ml) and vitamin D
2128 VITAMIN D INHIBITS LPS ACTION
sufficiency (30 ng/ml, lower normal range; 50 and 70 ng/ml, upper
normal range for the serum vitamin D levels) (1, 23, 24).
The binding of LPS to TLR4 on monocytes triggers immediate
activation of MAPK, which together with activation of the ca-
nonical IKK pathway regulate NF-kB activation to induce pro-
inflammatory cytokine production (25). We investigated whether
the pretreatment of cells with vitamin D affects the activation of
MAPK. The phosphorylation of three subfamilies of MAPK—
ERK, JNK, and p38—was examined by flow cytometry. Human
PBMC were pretreated with vitamin D for 24 h, followed by 10
min of treatment with 10 ng/ml LPS. After short-term stimulation
with LPS, phosphorylation of p38 MAPK in CD14
+
cells was
induced. Monocytes cultured in media for 24 h had high level of
p-JNK, which was not changed after LPS stimulation for 10 min.
No ERK phosphorylation was observed 10 min after LPS treat-
ment in these cells. As expected, LPS treatment did not activate
CD3
+
T cells.
As shown by flow cytometry (Fig. 1A), 6.5 61.2% CD14
+
cells expressed p-p38 MAPK (p-p38) prior to LPS treatment, and
23.6 62.5% CD14
+
cells expressed p-p38 after 10 min of LPS
treatment (n=4,p,0.05). After LPS stimulation, a significant
increase in the percentage of CD14
+
cells that express p-p38 was
observed; there was no change in p-p38 mean fluorescence in-
tensity (Fig. 1C). It was found that 15 ng/ml 25(OH)D
3
, a con-
centration corresponding to the insufficient serum vitamin D levels
in humans (24), did not suppress LPS-induced p38 phosphoryla-
FIGURE 1. Vitamin D inhibits LPS-induced p38
phosphorylation in human monocytes. PBMC were cul-
tured in hormone-free medium containing 25(OH)D
3
(A)
or 1,25(OH)
2
D
3
(B) for 24 h, followed by stimulation with
10 ng/ml LPS for 10 min. (Aand B) As shown by flow
cytometry, vitamin D pretreatment inhibits LPS-induced
p38 phosphorylation in CD14
+
cells (n= 4). (C) Repre-
sentative flow cytometry data on the effects of LPS and
vitamin D/LPS on p38 activation in human monocytes is
shown. (D) Pretreatment with vitamin D significantly
inhibits p-p38 expression by the cells as shown by Western
blot. Whole-cell extracts from LPS or vitamin D/LPS-
treated adherent PBMC fraction were prepared and blotted
against p-p38 and total p38. (E) Fold changes in the
densitometry of p-p38 normalized to total p38 MAPK
expression are provided. Values represent mean 6SEM
(n= 4 experiments).
The Journal of Immunology 2129
tion (Fig. 1A), whereas significant inhibition of LPS-induced p38
phosphorylation was achieved with $30 ng/ml 25(OH)D
3
(Fig.
1A). Maximum inhibition was achieved with 50 ng/ml 25(OH)D
3
(a mean inhibition of 78% [n=4;p,0.01]). Similarly, a dose-
dependent inhibition of LPS-induced p38 activation was observed
in human monocytes when the cells were pretreated with active
vitamin D (Fig. 1B, 1C). The maximum inhibitory effect was
achieved when the cells were preincubated with 0.4 ng/ml (1 nM)
1,25(OH)
2
D
3
(mean inhibition of 70% [n=4;p,0.01]). Inhi-
bition of LPS-induced p38 phosphorylation by vitamin D was
confirmed by Western blot analysis, because 10 nM 1,25(OH)
2
D
3
significantly suppressed LPS-induced p38 phosphorylation by
75% in adherent fraction of the PBMC (n=4;p,0.01) (Fig.
1D, 1E). As confirmed by flow cytometry, this adherent fraction
of PBMC consisted of 85–90% CD14
+
monocytes. Both flow
cytometry and Western blot evaluations demonstrated that in vi-
tamin D-pretreated cells, LPS-induced p-38 phosphorylation was
inhibited to the level of p-p38 phosphorylation observed in cells
cultured with media alone.
Pretreatment with vitamin D inhibits LPS-induced IL-6 and
TNF-aproduction by human monocytes
Upon LPS stimulation, monocytes produce proinflammatory
cytokines, such as TNF-aand IL-6 (26). Persistent inflammatory
FIGURE 2. Vitamin D inhibits LPS-induced cytokine production in human monocytes. PBMC were cultured in hormone-free medium containing 25
(OH)D
3
(A,C) or 1,25(OH)
2
D
3
(B,D,E) for 24 h, followed by 24 h of treatment with 10 ng/ml LPS. IL-6 (A,B) and TNF-a(C,D) mRNA levels in the total
PBMC were detected by real-time PCR after 24 h of stimulation with LPS (n= 4). IL-6 protein levels (E) in the culture supernatants following LPS
stimulation were detected by ELISA (n= 4). (F) IL-6 expression in CD14
+
cells was detected in human monocytes by flow cytometry after 24 h of
pretreatment with 10 nM 1,25(OH)
2
D
3
, followed by 6 h of stimulation with LPS. The percentage of CD14
+
cells expressing IL-6 was calculated. Values
represent mean 6SEM (n= 4 experiments). (G) Representative flow cytometry data on the effects of LPS and vitamin D/LPS on IL-6 production by human
monocytes are shown.
2130 VITAMIN D INHIBITS LPS ACTION
responses can damage host tissues (27, 28). To examine whether
changes in LPS-induced p38 activation in the presence of vitamin
D influenced cytokine production, human PBMC were pre-
incubated as above with either 1,25(OH)
2
D
3
or 25(OH)D
3
for 24
h, followed by stimulation with 10 ng/ml LPS for 24 h. LPS
treatment significantly induced IL-6 mRNA production by the
cells (p,0.05). When the cells were preincubated with $30 ng/
ml 25(OH)D
3
, a significant inhibition of LPS-induced IL-6 mRNA
expression was observed (p,0.01) (Fig. 2A). No inhibition of
LPS-induced IL-6 expression was observed when the cells were
cultured with 15 ng/ml 25(OH)D
3
(Fig. 2A). All doses of the
active form of vitamin D significantly inhibited LPS-induced IL-6
mRNA expression (Fig. 2B). The degree of suppression of IL-6
mRNA by 30 ng/ml (70 nM) 25(OH)D
3
was comparable to the
inhibition achieved with 0.04 ng/ml (0.1 nM) of the active form of
vitamin D (Fig. 2A, 2B). Similar vitamin D effects were observed
in LPS-induced TNF-amRNA expression (Fig. 2C, 2D).
Upon stimulation with LPS, the amounts of IL-6 protein in
culture supernatants increased from a basal level of 330 696 to
1385 6277 pg/ml (p,0.05). When the cells were pretreated
with 10 nM 1,25(OH)
2
D
3
for 24 h, LPS-induced IL-6 production
by the cells was significantly inhibited by a mean of 77% (n=4;
p,0.01) (Fig. 2E). The inhibition of IL-6 production was more
efficient with 10 nM 1,25(OH)
2
D
3
as compared with 1 nM 1,25
(OH)
2
D
3
(p,0.05) (Fig. 2E). These data were confirmed by flow
cytometry as the amount of IL-6–producing monocytes was sig-
nificantly increased after the stimulation with LPS from basal
0.8 60.4 to 29.7 64.9% (n=4;p,0.01) (Fig. 2F, 2G). How-
ever, when the cells were pretreated with 10 nM 1,25(OH)
2
D
3
for
24 h, the amount of IL-6–producing CD14
+
cells induced by LPS
was significantly inhibited by a mean of 67% (n=4;p,0.05)
(Fig. 2F, 2G).
Vitamin D pretreatment induces MKP-1 expression
MKP-1 plays a critical role in switching off p38 signaling and
cytokine production in monocytes/macrophages after the inflam-
matory stimuli (18). Because vitamin D pretreatment signifi-
cantly inhibited LPS-induced p38 phosphorylation, we examined
whether this process was mediated via MKP-1 or other phos-
phatases. Pretreatment of human PBMC with 10 nM 1,25(OH)
2
D
3
for 24 h resulted in significant increases in MKP-1 mRNA (2.5 6
0.1-fold; p,0.05) (Fig. 3A) and protein (3.1 60.4-fold; p,
0.05) (Fig. 3C, 3D) expressions by the adherent PBMC fraction,
which consisted mainly of monocytes. The expression of MKP-5,
another phosphatase that has been reported to dephosphorylate
p38, was not changed in adherent PBMC fraction after 24 h of
culture with 1,25(OH)
2
D
3
(Fig. 3B).
To examine the mechanism of MKP-1 upregulation by vitamin
D, we used murine BMM cultures. Similarly to human monocytes,
when murine BMM from wild-type mice (C57BL/6 3129) were
preincubated with 10 nM 1,25(OH)
2
D
3
, a significant increase in
MKP-1 mRNA expression was observed. The maximum increase
in MKP-1 mRNA expression was found after 6 h of treatment with
vitamin D (2.6 60.2-fold increase, n=3;p,0.01) (Fig. 3E).
We examined the presence of potential VDRE sites in the human
and murine MKP-1 promoter. A VDRE (labeled E4.7) sequence
AGTTCAAATCATTCA was located at 24708 to 24694 from the
transcriptional start site of human MKP-1 gene. A VDRE (labeled
E33) sequence AGTTCATGGCCTTCA was located at 233410 to
233396 from the transcriptional start site of murine MKP-1 gene.
Underlined nucleotides directly interact with VDR. Several ad-
ditional half VDRE sites were also found at the murine MKP-1
promoter, with the closest one with a sequence AGTTCA (la-
beled E0.9) located at 2930 to 2925 from the transcriptional start
FIGURE 3. Vitamin D induces MKP-1 by human and mouse mono-
cytes/macrophages. Human PBMC were cultured in hormone-free medium
containing 10 nM 1,25(OH)
2
D
3
or vehicle control for 24 h. An adherent
PBMC fraction was collected. (Aand B) Human MKP-1 and MKP-5
mRNA levels were tested by real-time PCR and normalized to b-actin
mRNA levels (n= 6 experiments). (C) Human MKP-1 protein levels were
tested by Western blot. (D) Fold changes in the densitometry of human
MKP-1 to b-actin expression from Western blot are provided (n=4
experiments). (E) Murine BMM cells were cultured in DMEM for 18 h and
then treated with 10 nM 1,25(OH)
2
D
3
or vehicle control for 6 h. mRNA
levels of murine MKP-1 were tested and normalized to b-actin mRNA (n=
3 experiments). All values represent mean 6SEM.
The Journal of Immunology 2131
site (the scheme of the potential VDR binding sites in the human
and murine MKP-1 promoter is shown in Fig. 4A). VDR binding
and histone H4 acetylation at the E4.7, E33, and E0.9 sites of
the human and murine MKP-1 promoters were tested by ChIP
assays. We found that treatment of adherent PBMC with 10 nM
1,25(OH)
2
D
3
for 24 h enhanced VDR binding and histone H4
acetylation by 2.4 60.5-fold (p,0.05) and 3.0 60.8-fold ( p,
0.05), respectively, at the E4.7 site of the human MKP-1 promoter
(Fig. 4B, 4C). We found that treatment of murine BMM with 10
nM 1,25(OH)
2
D
3
for 6 h enhanced VDR binding and histone H4
acetylation by 3.7 60.4-fold (p,0.05) and 6.26 60.04-fold
(p,0.05), respectively, at the E33 site of the murine MKP-1
promoter (Fig. 4D, 4E). No increase in VDR binding and only
a slight yet significant (p,0.01) increase in histone H4 acety-
lation were observed at the E0.9 site (Fig. 4D, 4E). These data
suggest that vitamin D-bound VDR interacts with the VDRE
upstream of MKP-1 gene, thus, potentially influencing MKP-1
transcription.
Vitamin D inhibits LPS-induced p38 phosphorylation in
macrophages derived from the bone marrow of the wild-type
but not the MKP-1
2/2
mice
Next, we tested the ability of vitamin D to inhibit LPS-induced p38
phosphorylation in macrophages derived from the bone marrow of
the wild-type and MKP-1
2/2
mice (29). Ten minutes of treatment
with LPS resulted in a significant increase in p38 phosphorylation
by 4.8 60.2-fold (n=3;p,0.05) and 8.2 60.2-fold (n=3;p,
0.01) in wild-type and MKP-1
2/2
macrophages, respectively (Fig.
5A, 5B). As shown by Western blot analysis, pretreatment with 10
nM 1,25(OH)
2
D
3
significantly inhibited LPS-induced p38 MAPK
activation by a mean of 93% (n=3;p,0.05) in BMM from wild-
type mice (Fig. 5B), but this inhibition was abolished in BMM
from MKP1
2/2
mice (Fig. 5B).
Inhibition of LPS-induced production of IL-6 and TNF-ain
bone marrow macrophages from wild-type mice by vitamin D
was significantly compromised in MKP-1
2/2
mice
To examine whether inhibition of LPS-induced p38 activation by
vitamin D in mouse macrophages influenced cytokine produc-
tion, macrophages derived from the bone marrow of wild-type
and MKP-1
2/2
mice were preincubated with either 10 nM 1,25
(OH)
2
D
3
or 75 nM (30 ng/ml) 25(OH)D
3
for 24 h, followed by
stimulation with 10 ng/ml LPS for 24 h. Significantly higher levels
of IL-6 and TNF-awere observed in culture supernatants of the
LPS-treated BMM from MKP-1
2/2
mice as compared with wild-
type mice (p,0.01 and p,0.05, respectively). The amounts of
LPS-induced IL-6 protein in culture supernatants were inhibited
67 and 61% by 1,25(OH)
2
D
3
and 25(OH)D
3
, respectively, in wild-
type macrophages. LPS-induced IL-6 production was inhibited
only to 18 and 33%, respectively, in MKP-1
2/2
macrophages (Fig.
6A). Similarly, we observed that 1,25(OH)
2
D
3
and 25(OH)D
3
inhibited LPS-induced TNF-aprotein in culture supernatants by
48 and 57%, respectively, in wild-type macrophages, whereas the
inhibition of LPS-induced production of these cytokines was only
16 and 29%, respectively, in MKP-1
2/2
macrophages (Fig. 6B).
Discussion
In this study, we examined the effects of vitamin D at physio-
logic concentrations on LPS-stimulated inflammatory responses in
monocytes/macrophages. We found that both 1,25(OH)
2
D
3
, an ac-
tive form of vitamin D, and 25(OH)D
3
, which requires conver-
sion by monocytes into an active form (23), dose dependently
inhibited p38 phosphorylation and cytokines, IL-6 and TNF-a,
and production in LPS-stimulated human monocytes. Upon vita-
min D treatment, the expression of MKP-1 phosphatase mRNA
and protein was significantly upregulated in both human mono-
cytes and murine BMM. We demonstrated that vitamin D treat-
FIGURE 4. Vitamin D regulates MKP-1 expression. (A) Schematic
representation of the potential VDRE in human and murine MKP-1 pro-
moter. (Band C) The recruitment of VDR to E4.7 of human MKP-1
promoter and histone H4 acetylation at this site as determined by ChIP
assay after 24 h of treatment of human adherent PBMC with vitamin D. (D
and E) The recruitment of VDR to E33 and E0.9 VDRE sites of the murine
MKP-1 promoter and histone H4 acetylation at these sites as determined
by ChIP assay after 6 h of treatment of murine BMM with vitamin D. The
quantity of anti-VDR Ab-precipitated DNA was normalized to Input DNA,
and anti-acetylated histone H4 Ab-precipitated DNA was normalized to
anti-histone H4 Ab precipitated DNA. Values represent mean 6SEM (n=
3 experiments). TSS, transcriptional start site.
2132 VITAMIN D INHIBITS LPS ACTION
ment increased VDR binding to a putative VDRE in both human
and murine MKP-1 promoter and enhanced histone H4 acetylation
near this VDRE site. With macrophages derived from MKP-1
2/2
mice, we further demonstrated that vitamin D was no longer able
to suppress LPS-induced p38 activation, followed by the com-
promised ability to inhibit LPS-induced IL-6 and TNF-apro-
duction in the absence of MKP-1. Our current study therefore
identified the upregulation of MKP-1 by physiologic concen-
trations of vitamin D as a novel pathway by which vitamin D
inhibits LPS-induced p38 activation and cytokine production in
monocytes/macrophages.
In recent years, vitamin D deficiency in humans has received
significant attention (1, 24). According to the recent brief of the
National Center for the Health Statistics in 2001–2006, 32% of the
U.S. population had serum 25(OH)D
3
levels ,20 ng/ml; 8% of
the U.S. population had serum 25(OH)D
3
levels ,12 ng/ml (30).
Aside from its classical role as a modulator of calcium metabo-
lism and bone health, vitamin D has been shown to have potent
anti-inflammatory effects and consequently has been considered
for adjunctive therapy for numerous chronic diseases including
asthma, arthritis, and prostate cancer (4, 31, 32). A variety of pro-
and anti-inflammatory effects for the vitamin D had been reported
previously (33, 34). It has been shown that vitamin D can di-
rectly induce the production of the important antimicrobial pep-
tides, cathelicidin and human bdefensin 4, by human monocytes/
macrophages and epithelial cells (23, 35). Antiproliferative and
proapoptotic activity has been shown in vitamin D-treated tumor
cells because of the induction of cyclin-dependent kinase inhib-
itors p21
Waf/Cip1
and p27
Kip1
and inhibition of c-Myc and anti-
apoptotic Bcl-2 (32). Vitamin D had been demonstrated to sup-
press prostaglandin pathways in the tumor cell lines via inhibition
of cyclooxygenase-2 production and stimulation of 15-hydroxy-
prostaglandin production by the cells (32). Vitamin D has also
been shown to interfere with NF-kB activation and signaling by
increasing the expression of IkBain the cells, thus interfering
with the nuclear translocation of the activated NF-kB subunits
(36). It has also been reported that vitamin D can influence den-
dritic cells’ maturation and function (37). Several studies have
highlighted the capacity of vitamin D to modulate the population and
function of FOXP3
+
and IL-10–producing T regulatory cells (37).
The current study found that human monocytes are capable of
responding to treatment with two different forms of vitamin D: 1,25
(OH)
2
D
3
and 25(OH)D
3
. 25(OH)D
3
is converted into a function-
ally active form, 1,25(OH)
2
D
3
, by the enzyme 25-hydroxyvitamin
D
3
-1a-hydroxylase (CYP27b1), a process that primarily occurs in
FIGURE 5. Vitamin D inhibits LPS-induced p38 phos-
phorylation in BMM from wild-type but not MKP-1
2/2
mice. (A) BMMs from wild-type and MKP1
2/2
mice were
cultured in DMEM containing 10 nM 1,25(OH)
2
D
3
or
vehicle control for 24 h, followed by 10 ng/ml LPS stim-
ulation for 10 min. Whole-cell extracts were prepared and
blotted against p-p38 and total p38. (B) Fold changes in the
densitometry of p-p38 normalized to total p38 are pro-
vided. Values represent mean 6SEM (n= 3 experiments).
FIGURE 6. Inhibition of LPS-induced production of IL-6 and TNF-ain
BMM from wild-type mice by vitamin D was significantly compromised in
MKP-1
2/2
mice. BMM from wild-type and MKP1
2/2
mice were cultured
in DMEM containing 10 nM 1,25(OH)
2
D
3
or 75 nM (30 ng/ml) 25(OH)D
3
for 24 h, followed by 10 ng/ml LPS stimulation for 24 h. IL-6 (A) and
TNF-a(B) protein levels in the culture supernatants were detected by
ELISA. Values represent mean 6SEM (n= 3 experiments).
The Journal of Immunology 2133
kidneys (38, 39). However, it has been shown that monocytes,
macrophages, and dendritic cells also express CYP27b1 (35, 40).
Therefore, 1,25(OH)
2
D
3
can be produced locally and exert im-
munomodulatory effects (41). We demonstrated that 15 ng/ml 25
(OH)D
3
[a concentration amount considered in this study vitamin
D deficiency (24)] did not suppress LPS-induced IL-6 and TNF-a
production in human monocytes. We found that 25(OH)D
3
at $30
ng/ml (levels considered vitamin D sufficient in humans) signif-
icantly inhibited IL-6 and TNF-aproduction induced by LPS.
Furthermore, the degree of suppression of IL-6 and TNF-apro-
duction by 30 ng/ml 25(OH)D
3
was comparable to the effects
achieved by 0.04 ng/ml (0.1 nM) of the active form of the vitamin
D. These data suggest that human monocytes have the ability
to convert 25(OH)D
3
to an active form. Furthermore, our study
demonstrates that serum concentrations of 25(OH)D
3
does matter
for the optimal anti-inflammatory response of human monocytes,
because the amount of the available circulating 25(OH)D
3
influ-
ences local tissue concentrations of the active vitamin D (23,
35). These data support the idea that to achieve optimal anti-
inflammatory effects by vitamin D it is important to maintain
serum vitamin D levels .30 ng/ml in the physiologic range (23,
42).
Dual-specificity phosphatases are a heterogeneous group of
protein phosphatases that can dephosphorylate both phosphotyr-
osine and phosphothreonine residues within the same substrate.
MKP-1 is the best-studied member of this family and is charac-
terized for its role in the regulation of MAPK signaling cascades
(43, 44). In this study, we demonstrated that vitamin D can up-
regulate MKP-1 expression by monocytes/macrophages and use
this pathway for the inhibition of the LPS-induced p38 phos-
phorylation. In this study, we focused on the early stages of
monocyte activation by LPS to assess the contribution of vitamin
D-regulated phosphatases on LPS-induced MAPK activation. We
did not extend this assessment to 30 min post-LPS stimulation of
avoid the effects of LPS-induced MKP-1 (18). Another member
of the dual specificity phosphatase family, MKP-5, was recently
shown to be induced by vitamin D treatment in normal prostate
epithelial cells, resulting in inhibition of p38 activation in these
cells (45). Vitamin D did not alter MKP-5 expression in human
monocytes in our study.
Recently, it has been reported that vitamin D treatment down-
regulates TLR2 and TLR4 expression by human monocytes and
upregulates CD14 expression by these cells (46). The authors
concluded that downregulation of TLR expression can substan-
tially reduce LPS and lipoteichoic acid-mediated MAPK activa-
tion. MKP-1 expression under these conditions was not studied.
Importantly, in this study, 100 nM active vitamin D was used. In
our study, 10 nM active vitamin D was the highest dose used, and
we did not observe changes in TLR4 protein expression by human
monocytes in response to 24 h of pretreatment with the 10 nM
active form of vitamin D (data not shown).
Vitamin D has been reported to regulate a variety of human
and mouse genes through VDR-mediated VDRE binding (47,
48). Although VDRE have been well studied in genes such as
CYP24A1 (48, 49), no VDRE has been discovered in MKP-1 gene
to date. In this study, we identified a putative VDRE site ∼4.7 kb
upstream of transcriptional start site in the human MKP-1 pro-
moter and a putative VDRE site ∼33 kb upstream of transcrip-
tional start site in the distal murine MKP-1 promoter. We con-
firmed VDR binding to these two sites using ChIP assays. The far
upstream location of the putative VDRE in murine MKP-1 pro-
moter is not surprising because genome-wide mapping of VDRE
have indicated that VDREs are predominantly located in introns
and intergenic intervals (7). Following the binding of VDR to
VDRE, coactivators, corepressors, or mediator proteins may be
recruited to the site depending on the effects of vitamin D on
a specific gene transcription. Because vitamin D treatment sig-
nificantly induces MKP-1 mRNA expression, it is likely that VDR
binding to the MKP-1 promoter induces recruitment of the
coactivators and initiates histone acetylation, leading to transcrip-
tional activation (10). Our ChIP assays confirmed significantly
enhanced histone H4 acetylation near the putative VDRE sites
(E4.7 and E33) in humans and in the distal murine MKP-1 pro-
moter, respectively, after vitamin D treatment.
Selective inhibitors for MKP-1 are currently unavailable, and
inhibition of MKP-1 expression is difficult to accomplish via small
interfering RNA in primary monocytes/macrophages. For these
reasons, we explored the role of MKP-1 in vitamin D-mediated
anti-inflammatory effects in monocytes by using BMM from
MKP-1
2/2
mice and wild-type mice. Our data suggest that vita-
min D regulation of MKP-1 is one of the essential pathways that
inhibits LPS-induced cytokine production by monocytes/macro-
phages. We demonstrated that vitamin D-mediated suppression of
the LPS-induced p38 phosphorylation was abolished, and the in-
hibition of LPS-induced production of IL-6 and TNF-awas sig-
nificantly reduced in macrophages derived from MKP-1
2/2
mice.
The fact that MKP-1 knockout did not completely abolish the
vitamin D inhibition effect on LPS-induced cytokine production
indicates that vitamin D engages additional pathways aside from
MKP-1 to regulate LPS-induced proinflammatory cytokine pro-
duction. This may include but not be limited to transrepression of
NF-kB–mediated responses by VDR as reported previously (33,
36, 46). It was recently reported that LPS controls MKP-1 in
activated monocytes via upregulation of the microRNA 101 (50).
We did not find changes in microRNA 101 expression in vitamin
D-pretreated human monocytes (E. Goleva, Y. Zhang, C. Hall, and
D.Y.M. Leung, unpublished observations).
In summary, our study provides several novel discoveries: first,
physiologic levels of vitamin D can modulate inflammatory ac-
tivities, because 30–50 ng/ml 25(OH)D
3
is sufficient to inhibit
LPS-induced p38 activation and cytokine production in human
monocytes (Figs. 1, 2). Second, the study identified the upregu-
lation of MKP-1 by vitamin D as a novel mechanism by which
vitamin D inhibits LPS-induced p38 activation and cytokine
production in monocytes/macrophages. Finally, a putative VDR
binding site was identified in the distal murine MKP-1 promoter
and human MKP-1 promoter. Our current studies suggest that
patients with chronic inflammatory diseases that are vitamin D
deficient (,20 ng/ml) may benefit from oral supplementation of
vitamin D to get their serum vitamin D level .30 ng/ml.
Acknowledgments
We thank Maureen Sandoval and Shih-Yun Lyman for help in preparation
of this manuscript. We thank Bristol–Myers Squibb, which developed
MKP-1
2/2
mice.
Disclosures
The authors have no financial conflicts of interest.
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The Journal of Immunology 2135
