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The FASEB Journal • Research Communication

Human cathelicidin antimicrobial peptide (CAMP) gene

is a direct target of the vitamin D receptor and is
strongly up-regulated in myeloid cells by 1,25-
dihydroxyvitamin D3
Adrian F. Gombart,*,1 Niels Borregaard,† and H. Phillip Koeffler*
*Department of Medicine, Division of Hematology/Oncology, Cedars-Sinai Medical Center, David
Geffen School of Medicine at UCLA, Los Angeles, California, USA; and †The Granulocyte Research
Laboratory, Department of Hematology, Rigshospitalet, University of Copenhagen, Copenhagen,

ABSTRACT The innate immune system of mammals ocomial infections. In 2000, nearly 660,000 cases of
provides a rapid response to repel assaults from nu- sepsis developed in the United States. This resulted in
merous infectious agents including bacteria, viruses, an in-hospital mortality rate of nearly 18% (2). Among
fungi, and parasites. A major component of this system survivors of sepsis, an increased risk of death and
is a diverse combination of cationic antimicrobial pep- decreased quality of life occurred after discharge from
tides that include the ␣- and ␤-defensins and cathelici- the hospital (3, 4).
dins. In this study, we show that 1,25-dihydroxyvitamin This impending crisis has spurred the search for new
D3 and three of its analogs induced expression of the therapeutic agents to combat antibiotic resistance. One
human cathelicidin antimicrobial peptide (CAMP) potential solution lies within a system all animals are
gene. This induction was observed in acute myeloid “born with,” the innate immune system responsible for
leukemia (AML), immortalized keratinocyte, and colon keeping us healthy (5). It provides animals the capacity
cancer cell lines, as well as normal human bone marrow to repel assaults quickly from numerous infectious
(BM) -derived macrophages and fresh BM cells from agents including bacteria, viruses, fungi, and parasites
two normal individuals and one AML patient. The (6 –11). Diverse combinations of cationic antimicrobial
induction occurred via a consensus vitamin D response peptides (AMPs) including ␣- and ␤-defensins and
element (VDRE) in the CAMP promoter that was bound cathelicidins comprise a major component of this de-
by the vitamin D receptor (VDR). Induction of CAMP fense in mammals. Because bacteria have difficulty
in murine cells was not observed and expression of developing resistance against AMPs and are quickly
CAMP mRNA in murine VDR-deficient bone marrow killed by them, this class of antimicrobial agents is
was similar to wild-type levels. Comparison of mamma- being commercially developed as a source of peptide
lian genomes revealed evolutionary conservation of the antibiotics (1, 12, 13). The majority of the pharmaceu-
VDRE in a short interspersed nuclear element or SINE tical effort has concentrated on the development of
in the CAMP promoter of primates that was absent in topically applied agents (13). The expense and diffi-
the mouse, rat, and canine genomes. Our findings culty of preparing large amounts of peptide and the
reveal a novel activity of 1,25-dihydroxyvitamin D3 and uncertainty in systemic use of these peptides have
the VDR in regulation of primate innate immunity.— slowed their development beyond topical treatments.
Gombart, A. F., Borregaard, N., Koeffler, H. P. Human One AMP that shows promise is the human catheli-
cathelicidin antimicrobial peptide (CAMP) gene is a cidin antimicrobial peptide (CAMP), also known as
direct target of the vitamin D receptor and is strongly hCAP18/LL-37/FALL-39. It is the only known human
up-regulated in myeloid cells by 1,25-dihydroxyvitamin cathelicidin. The cathelicidins are a family of proteins
D3. FASEB J. 19, 1067–1077 (2005) consisting of a C-terminal cationic AMP domain that is
activated by cleavage from the N-terminal cathelin
Key Words: sepsis 䡠 wound healing 䡠 monocytes 䡠 VDRE portion of the propeptide. The majority of the CAMP
propeptide is stored in secondary or specific granules
of neutrophils from which it can be released at sites of
A major concern for public health in both developed microbial infection (14). In addition to neutrophils,
and developing countries is the alarming increase of
antibiotic resistance in bacteria (1). Drug-resistant bac- 1
Correspondence: Division of Hematology/Oncology, Ce-
teria such as Pseudomonas aeruginosa and Staphylococcus dars-Sinai Medical Center, Davis Bldg. 5019, 8700 Beverly Blvd.,
aureus pose serious problems for immunocompromised Los Angeles, CA 90048, USA. E-mail:
persons and are major sources of life-threatening nos- doi: 10.1096/fj.04-3284com

0892-6638/05/0019-1067 © FASEB 1067

various white blood cell populations express hCAP18. cells isolated from either two normal or one acute myeloid
These include natural killer cells, ␥␦T cells, B cells, leukemia patient were cultured in RPMI1640 containing 10%
monocytes (15), and mast cells (16). CAMP/hCAP18 is FCS for short-term experiments. Bone marrow (BM) -derived
secreted into the blood and significant levels are found macrophages (M␾) were obtained by culturing normal hu-
man bone marrow (NHBM) cells in RPMI1640 containing
in the plasma (17). 10% FCS, 200 ng/mL GM-CSF, and 5% WeHi-3B conditioned
CAMP is synthesized and secreted in significant medium (source of IL-3) for 14 days. The bone marrow
amounts by those tissues that are exposed to environ- samples were obtained from patients after informed consent
mental microbes. This includes the squamous epithelia was given. Approval for the collection of these samples was
of the mouth, tongue, esophagus, lungs, intestine, obtained from the Cedars-Sinai Medical Center Institutional
cervix, and vagina (18, 19). In addition, it is produced Review Board. The immortalized keratinocyte cell line HaCat
by salivary and sweat glands (20, 20), epididymis, testis (a kind gift from Dr. Norbert Fusenig, Heidelberg, Germany)
(21), and mammary glands (22–24). Expression in and colon cancer cell line HT29 were cultured in DMEM
these tissues results in secretion of the polypeptide in containing 10% FCS. All media were supplemented with
antibiotics (100 units penicillin/streptomycin; Invitrogen).
wounds (25), sweat (26), airway surface fluids (19),
Cells were treated with various concentrations and durations
seminal plasma (27), and milk (22, 23). CAMP/ of 1,25(OH)2D3 , a vitamin D3 analog, or vehicle (ethanol).
hCAP18 possesses several important activities including The 1,25(OH)2D3 and compound I (1,25R,26-(OH)3-
bactericidal, anti-sepsis, chemoattraction, and promo- 22-ene-D3) were synthesized and generously provided by
tion of angiogenesis and wound healing. The possibility Dr. Milan Uskokovic at Hoffmann-LaRoche, Inc. (Nutley,
of extrinsically manipulating endogenous expression of NJ, USA). Analogs KH1060 (20-epi-22oxa-24a,26a,27a-tri-
CAMP for systemic and localized therapeutic benefit is homo-1,25(OH)2D3) and EB1089 (1,25-dihydroxy-22,24-
very attractive. diene, 24,26,27-trihomo) were synthesized by Leo Pharma-
Since their discovery more than a decade ago, the ceutical Products (Ballerup, Denmark) and generously pro-
vided by Dr. Lise Binderup. U937 cells were treated for 24 h
majority of expression studies have been focused on the
with vehicle (ethanol), LPS (1 ␮g/mL), 12-O-
detection of cathelicidins in various tissues; however, tetradecanoylphorbol 13-acetate (TPA, 10 ng/mL), TNF-␣ (1
the transcriptional mechanisms that regulate cathelici- ng/mL), INF-␣ (10 ng/mL), IFN␥ (50 ng/mL), IL-2 (2.5
din gene expression have not been adequately eluci- ng/mL), IL-6 (10 ng/mL), GM-CSF (1 ng/mL), G-CSF (60
dated. Understanding the signaling pathways and the ng/mL), estradiol (1⫻10⫺8 M), dihydrotestosterone (DHT,
downstream transcription factors that regulate CAMP 1⫻10⫺8 M), or all-trans retinoic acid (ATRA, 5⫻10⫺7 M).
gene expression in a tissue-specific manner is crucial Cyclohexamide (Sigma, St. Louis, MO, USA) was used at 20
for designing approaches for therapeutic manipulation ␮g/mL and the absence of protein synthesis was determined
of endogenous gene expression. Because AMPs serve a by measuring 35S-methionine incorporation. Cyclohexam-
role in host defense and may act as mediators of other inde was added 30 min before the vehicle or 1,25(OH)2D3.
Actinomycin D (Sigma) was used at 10 ␮g/mL and added at
biological processes, their expression is tightly regu- the same time as vehicle or 1,25(OH)2D3.
lated. Murine 32Dcl3 cells (a generous gift from Alan Friedman,
The experimental focus of this study was to identify Johns-Hopkins, Baltimore, MD, USA) were cultured in IMDM
extracellular signals and the downstream transcription (Invitrogen) supplemented with 10% FCS and 10% Wehi3B-
factors that activate transcription of the CAMP gene, conditioned medium. Cells were treated with 1,25-dihy-
with the ultimate goal of extrinsically manipulating its droxyvitamin D3 or ethanol for 0, 24, and 48 h and total RNA
endogenous expression for systemic and localized ther- was harvested. The 1,25(OH)2D3 and compound I (both 0.05
apeutic benefit. We provide evidence that the CAMP ␮g/mouse) were administered to beige/nude/x-linked
gene is a direct target of the transcription factor (bnx) nu/nu nude mice every 2 days for 6 wk. The bone
marrow cells were flushed from the femurs and total RNA was
vitamin D receptor (VDR) that mediates the strong
isolated. Bone marrow cells were flushed from femurs of
up-regulation of CAMP in response to treatment of VDR-deficient mice or wild-type littermates (28). Red blood
cells with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3 or cells were lysed and cells were plated in IMDM supplemented
vitamin D3] and its analogs. Induction of the endoge- with 10% FCS. Cells were treated with 1,25(OH)2D3 or
nous CAMP by these relatively safe (FDA approved) ethanol for 24 h and total RNA was harvested. BM-derived
compounds may provide important novel therapeutic macrophages were obtained from VDR-deficient and wild-
uses from promotion of wound healing to protection type murine femurs as described previously (29). Cells were
against bacteremia and sepsis after surgery, chemother- treated with ethanol or 1,25(OH)2D3 for 0, 24, and 48 h and
apy, or severe burns. total RNA was harvested.
U937 cells were electroporated using a BTX T820 (Gen-
etronics Biomedical, Ltd., San Diego, CA, USA). The settings
were low voltage, 200 V, 10 ms, 1 pulse in 250 ␮L of cells at
MATERIALS AND METHODS 2 ⫻ 107 cells/mL in a 4 mm cuvette. A total of 20 ␮g plasmid
was used per transfection. After transfection, cells were
treated with 1,25(OH)2D3 or vehicle at the concentration and
Tissue culture and reporter assays times indicated in the figure legends. Cell lysates were pre-
pared and luciferase activities determined using the dual
The human myeloid leukemia cell lines U937, NB4, HL60, luciferase assay system as described by the manufacturer
and ML1 were cultured in RPMI1640 (Invitrogen, Carlsbad, (Promega, Madison, WI, USA). Transfection efficiency
CA, USA) containing 10% fetal calf serum (FCS; Omega was normalized to the renilla luciferase expression vector
Scientific, Inc., Tarzana, CA, USA). Human bone marrow phTKRL (Promega).

1068 Vol. 19 July 2005 The FASEB Journal GOMBART ET AL.

Recombinant plasmids complexes were cross-linked in 1% formaldehyde for 10 min.
The reaction was terminated with the addition of glycine to
Primers 5⬘-CCGACGCGTCATACTGAGTCTCACTCTGTT- 0.125 M final concentration. The cells were washed in ice-cold
ACC-3⬘ and 5⬘-CCGCTCGAGGGTCCCCATGTCTGCCTC-3⬘ PBS containing PMSF (10 ␮g/mL), resuspended in 1 mL of
were used to amplify the human CAMP promoter (nucleo- SDS-lysis buffer containing protease inhibitors, and incubated
tides – 693 to ⫹14) from human genomic DNA (30). This on ice for 10 min. The lysates were sonicated 3⫻, 10 s at 30%
fragment was subcloned into the firefly luciferase reporter output to shear the DNA. The sonicated lysate was pelleted at
plasmid pXP2 (31) and called pXP2-CAMP-Luc. Subse- 13K rpm for 10 min at 4°C. Supernatant (0.2 mL) was mixed
quently deletion mutants pXP2-CAMP(⌬SmaI)-Luc and with 1.8 mL of dilution buffer and precleared with protein
pXP2-CAMP(⌬HindIII)-Luc were generated by restriction A-agarose for 1 h on ice. Antibody (2 ␮g) against VDR (mixed
enzyme digestion, fill-in, and religation of the purified linear SC-1008 [1 ␮g] and SC-1009 [1 ␮g], Santa Cruz Biotechnol-
plasmid. Constructs were verified by nucleotide sequencing. ogy, Santa Cruz, CA, USA), C/EBPε (2 ␮L) (34), preimmune
serum, or no antibody was added and the samples incubated
overnight at 4°C. A slurry of ssDNA/protein A agarose was
Analysis of RNA and protein expression added and the mixture was incubated with rocking overnight
at 4°C. The agarose/antibody/protein/DNA complex was
Total RNA was prepared using Trizol Reagent (Invitrogen), pelleted and washed in low salt (1⫻), high salt (1⫻), LiCl
electrophoresed through a formaldehyde-containing 1% aga- (1⫻), and TE (2⫻). The complex was removed from the
rose gel, and transferred to a positively charged nylon mem- protein A-agarose in elution buffer (2⫻500 ␮L); cross-links
brane (Hybond N⫹) for Northern analysis (Amersham Phar- were reversed in 100 mM NaCl at 65°C for 4 h, proteinase K
macia Biotech, Piscataway, NJ, USA). The blots were treated, phenol/chloroform extracted, and ethanol precipi-
sequentially probed with 32P-labeled DNA probes (Strip- tated. The promoter fragment was detected by PCR using
EZTM, Ambion, Inc., Austin, TX, USA) specific for the CAMP, primers against the CAMP promoter (forward, 5⬘-ACCGTGC-
CDllb, and ␤-actin mRNAs. CCTGCCTCATTC-3⬘ and reverse, 5⬘-TGGTCCCCATGTCT-
For quantitative real-time PCR (QRT-PCR), total RNA was GCCTC-3⬘). The 430 bp fragment was cloned and sequenced
prepared, treated with DnaseI (Invitrogen), and cDNAs were to verify that the CAMP promoter was amplified. QRT-PCR
synthesized by reverse transcription using Superscript II re- was performed using SYBR Green (Molecular Probes, Eu-
verse transcriptase as described by the manufacturer (Invitro- gene, OR, USA) essentially as described (32).
gen). The cDNAs were then analyzed by QRT-PCR using a
fluorescent probe (Applied Biosystems, Foster City, CA, USA)
against CAMP (5⬘-6fam-ACCCCAGGCCCACGATGGAT-tamra-
3⬘) or 18S (32) at a final concentration of 200 nM per reaction. RESULTS
Primers against CAMP (forward, 5⬘-GCTAACCTCTACCGC-
CTCCT-3⬘ and reverse, 5⬘-GGTCACTGTCCCCATACACC-3⬘) or Induction of CAMP gene expression by 1,25(OH)2D3
18S (32) were used at 600 nM per reaction. PCR was performed
using HotMasterTM Taq polymerase (Eppendorf AG, Hamburg, In an initial screen to identify extracellular signals that
Germany) on an iCycler PCR machine equipped with an optical might induce CAMP gene expression, we treated the
module (Bio-Rad Laboratories, Hercules, CA, USA). The proto-
col was 95°C, 1 min followed by 45 cycles of 95°C, 15 s and 60°C, myeloid leukemia cell line U937 with various inflamma-
1 min, during which time data were collected. Standard curves tory factors (LPS, TPA, TNF-␣, INF-␣, and INF-␥),
were generated by PCR using serial dilutions of known quanti- cytokines and growth factors (IL-2, IL-6, GM-CSF and
ties of CAMP or 18S cDNA and were included on each plate to G-CSF) and seco-steroid hormones (DHT, estradiol,
quantify the ng of CAMP or ng of 18S cDNA in each sample. ATRA and 1,25(OH)2D3) (Fig. 1A and data not shown).
PCR was performed in triplicate for each sample. As determined by QRT-PCR and Northern blot analy-
Primers against murine CAMP/CRAMP (forward, 5⬘-
ses, only 1,25(OH)2D3 induced CAMP expression sig-
GAAGGCACATTGC-3⬘) were used at 200 nM per reaction. nificantly (Fig. 1A, B). The induction was also observed
PCR was performed using SYBR green (Molecular Probes, in HL60 (Fig. 1B, C) and NB4 (Fig. 1C). Induction of
Eugene, OR, USA) as described previously (32). The protocol the CAMP gene occurred by day 1 in the U937 and
was 95°C, 1 min followed by 45 cycles of 95°C, 15 s; 60°C, 30 s; HL60 cell lines, but was stronger in the U937 cell line
and 65°C, 1 min, during which time data were collected. The (Fig. 1B). A time course of from 1 to 24 h in U937
relative fold change between samples was determined using indicated that CAMP induction began between 1 and
data normalized for 18S expression. Samples were analyzed in
3 h after addition of 1,25(OH)2D3 and prior to induc-
Western blot and immunfluorescent microscopy analyses tion of the differentiation marker CDllb at 12 h (Fig.
were performed essentially as described previously (33). The 1B). CAMP induction continued throughout the 5 days
total cell lysates were electrophoresed through 20% polyacryl- of treatment and was dose responsive (Fig. 1B, C). Each
amide-SDS gels. The hCAP18 antibody was used at 2.0 ␮g/mL of the chemically synthesized 1,25(OH)2D3 analogs—
for Western blot analysis and 4.0 ␮g/mL for immunofluores- KH1060, EB1089, and compound I—strongly induced
cent (IF) microscopy (14). The anti-GAPDH monoclonal CAMP gene expression (Fig. 1D). Levels of induction
antibody was used at a 1:10,000 dilution (Research Diagnos-
tics, Inc., Flanders, NJ, USA). were similar to those observed for 1,25(OH)2D3. No
induction was observed with ATRA (5⫻10⫺7 M, 1–5
Chromatin immunoprecipitation (ChIP) assays days) in U937, HL60, and NB4 (Fig. 1A and data not
shown). This is consistent with the inability of human
The ChIP assays were performed essentially as described by myeloid leukemia cell lines to express significant levels
the manufacturer (Upstate, Inc., Chalottesville, VA, USA). of mRNAs for secondary granule genes even when
Briefly, ⬃1 ⫻ 107 cells were incubated with vehicle or induced to undergo granulocytic differentiation by
1,25-dihydroxyvitamin D3 (1⫻10⫺7 M for 4 h). Protein/DNA ATRA (35).


Figure 1. Induction of CAMP
mRNA expression by 1,25-
[OH]2D3. A) U937 cells were
treated with vehicle (–, etha-
nol) or the indicated com-
pounds for 24 h as described
in Materials and Methods.
Expression levels of CAMP
were determined by QRT-
PCR. Standard curves with
known amounts of CAMP or
18S cDNA were included to
measure the starting quantity
of CAMP (ng) and 18S (ng)
cDNA in each sample. Graphs
depict the ratio of CAMP/18S
in each sample (⫾sd). PCR
was performed in triplicate
for each sample. B) U937 or
HL60 cells were treated with
vehicle (0) or 1 ⫻ 10 –7 M
1,25[OH]2D3 for 1, 3, or 5
days (upper panel) or for 1,
3, 6, 12, or 24 h (lower
panel). Total RNA was ana-
lyzed by Northern blot using
probes against CAMP, CDllb,
or ␤-actin. C) U937, HL60, and NB4 cells were treated with either vehicle or decreasing molar concentrations of 1,25[OH]2D3
for 24 h. Total RNA was subjected to Northern blot analysis as described for panel B. D) U937 cells were treated with vehicle
(0), 1,25[OH]2D3 (VitD3), or one of its analogs at 1 ⫻ 10 –7 M for 12 or 24 h. Total RNA was subjected to Northern blot analysis
with probes against CAMP or ␤-actin. E) U937 cells were treated with vehicle (0) or 1 ⫻ 10 –7 M 1,25[OH]2D3 for 1, 2, 4, or 6 h
in the absence (–ActD) or presence (⫹ActD) of actinomycin D (10 ␮g/mL). Expression levels of CAMP were determined by
QRT-PCR and normalized to 18S. F) U937 cells were treated with vehicle (0) or 1,25[OH]2D3 for 0, 6, and 9 h in the absence
(–) or presence (⫹) of cyclohexamide (CHX, 20 ␮g/mL). Total RNA was subjected to Northern blot analysis as described in
panel D. G) U937 cells were treated with vehicle (0) or 1 ⫻ 10 –7 M 1,25[OH]2D3 for 12 or 24 h. The cDNAs from total RNA
were analyzed by RT-PCR using primers against myeloperoxidase (MPO), ␣-defensin (HNP-3), matrix metalloprotease 8
(MMP8), lactoferrin (LTF), CAMP, ␤-actin, and 18S. Amplification for all genes was 35 cycles except CAMP (30 cycles), ␤-actin
(25 cycles), and 18S (10 cycles). A negative control (c, ddH2O) and a positive control (normal bone marrow RNA, BM) were

The induction of CAMP was blocked by actinomycin secondary [MMP8 (matrix metalloproteinase 8) and
D, indicating it occurred at the level of transcription LTF (lactoferrin)] granule genes for induction. We did
(Fig. 1E). The data suggested that the human CAMP not observe induction of these genes after 24 h of
gene was a direct transcriptional target of the VDR. The treatment, whereas CAMP was significantly up-regu-
steroid hormone receptor family members are gener- lated (Fig. 1G). The data demonstrate that the
ally present in the cytosol or bound to the DNA in an 1,25(OH)2D3 induction of neutrophil granule genes is
inactive state and require activation by binding ligand restricted primarily to CAMP.
(36). Upon binding to ligand, they immediately trans-
locate to the nucleus and bind vitamin D response
elements (VDREs) in target genes and induce gene Induction of CAMP is independent of monocytic
expression. The model predicts that ongoing protein differentiation
synthesis is not required for this process to occur. To
test this, we treated U937 cells with 1,25(OH)2D3 in the Vitamin D3 promotes macrophage-like differentiation
presence or absence of cyclohexamide (CHX) to block of U937 and HL-60 (37). To determine whether differ-
protein synthesis. Induction of CAMP gene expression entiation was responsible for the induction of the
occurred in the absence of ongoing protein synthesis CAMP gene in AML cell lines, we treated HL-60 and
(presence of CHX) (Fig. 1F ). CHX did not induce U937 cells with 1,25(OH)2D3 or TPA. Both compounds
CAMP gene expression (data not shown). These data promoted macrophage-like differentiation of these
further support the hypothesis that the CAMP gene is a cells as demonstrated by the induction of the differen-
direct target of the VDR and not activated by secondary tiation marker CDllb, but induction of CAMP mRNA
events such as the synthesis of other transcription was observed only with 1,25(OH)2D3 , not TPA (Fig.
factors that are induced by VDR. 2A). The data suggested that induction of differentia-
To determine the specificity of CAMP induction by tion was not sufficient for CAMP expression. Further-
1,25(OH)2D3 , we tested other neutrophil primary more, 1,25(OH)2D3 induced expression of CAMP was
[MPO (myeloperoxidase) and HNP3 (␣-defensin)] and observed in the AML cell line NB4, which does not

1070 Vol. 19 July 2005 The FASEB Journal GOMBART ET AL.

Figure 2. CAMP mRNA induction occurs in
the absence of monocytic differentiation in
patient samples and in nonmyeloid cells.
A) The myeloid leukemia cell lines HL60
and U937 were treated with vehicle (0),
1,25[OH]2D3 (1⫻10 –7 M), or TPA (5 ng/
mL) for 1, 3, or 5 days. Total RNA was
extracted and analyzed by Northern blot. B)
The HL60 sub-lines HL60R (pan-resistant)
and HL60⌬404 (ATRA-resistant) were treated
with vehicle (–) or 1,25[OH]2D3 (⫹, 1⫻10 –7
M) for 24 h. Northern blot analysis was per-
formed. C) Bone marrow cells from normal
human patients (NHBM, upper panel) were
cultured in RPMI1640 ⫹ 10% FCS with vehi-
cle (0) or 1,25[OH]2D3 for 72 or 120 h.
BM-derived M␾ were treated for 24 h with
vehicle (0) or an increasing concentration of
1,25[OH]2D3. AML BM cells were treated for
24 h with vehicle (0) or 1,25[OH]2D3 (lower
panel). Duration of treatment and the concen-
trations of 1,25[OH]2D3 are indicated along
the bottom of the graphs. Total RNA was
prepared and cDNAs were analyzed using
QRT-PCR. The fold change within each set is
indicated inside the bar. As a positive control
for induction, U937 cells were treated with
vehicle (0) or 1,25[OH]2D3 for 12 and 24 h.
D) 1,25[OH]2D3 induces CAMP expression in keratinocyte (HaCat) and colon cancer (Ht-29) cell lines. Cells were treated with
vehicle (–) or 1,25[OH]2D3 (⫹, 1⫻10 –7 M) for 24 h. Total RNA was prepared; cDNAs were synthesized and analyzed by QRT-PCR.

undergo macrophage differentiation when treated with level of CAMP mRNA in normal and diseased human
1,25(OH)2D3 (Fig. 1C). Finally, to demonstrate that BM cells and that the induction is not a cell line
CAMP induction occurs in the absence of differentia- phenomenon.
tion, we treated sub-lines derived from HL-60, which The induction of CAMP by 1,25(OH)2D3 was not
are unable to differentiate in response to vitamin D3 limited to myeloid cells. We observed induction of
(HL60R) or ATRA (HL60⌬404) with 1,25(OH)2D3 and CAMP mRNA in the keratinocyte cell line HaCat and
found that CAMP was induced in the absence or the colon cancer cell line HT-29 by QRT-PCR (Fig. 2D).
presence of differentiation (Fig. 2B, HL60R vs. Induction was not as robust as that observed in the
HL60⌬404, respectively). myeloid cells.
To determine whether the induction of CAMP
Induction of the CAMP gene occurs in bone marrow mRNA expression resulted in an increase of CAMP
cells from normal humans and a patient suffering (hCAP18) protein expression, Western blot and immu-
from acute myeloid leukemia nofluorescent microscopy analyses were performed on
U937 cells treated with 1,25(OH)2D3 (Fig. 3A, B). At
18 h and 36 h post-treatment, increased levels of
To determine whether CAMP induction by vitamin D3 hCAP18 were observed compared with untreated cells
occurs in hematopoietic cells other than leukemia cell (Fig. 3A, B). An ELISA performed on the medium from
lines, we treated total bone marrow (BM) cells and U937 cells treated for 24 h with ethanol or
BM-derived macrophages (BM M␾) from two normal 1,25(OH)2D3 showed that CAMP was secreted into the
individuals and BM cells from one AML patient with medium (Fig. 3C).
1,25(OH)2D3 in vitro. Total RNA was harvested, cDNAs
synthesized, and the quantity of CAMP mRNA expres-
sion was determined by QRT-PCR using a Taqman Identification of a functional VDRE in the human
probe assay (Fig. 2C). As demonstrated previously, a CAMP promoter
strong induction of CAMP was observed for U937
treated with 1,25(OH)2D3 (Fig. 2C, upper panel). Sim- We hypothesized the existence of a VDRE in the CAMP
ilarly strong induction of CAMP was observed in two promoter to explain the strong induction of CAMP
normal human bone marrow cell samples and in BM mRNA expression by exposure to vitamin D3. A search
M␾ (Fig. 2C, upper panel). The AML cells had a high of the upstream region revealed a classical DR3-type
baseline level of CAMP expression, which was induced VDRE (38) at – 615 bp from the transcriptional start site
further in a dose-responsive manner by 6- and 11-fold (Fig. 4A) (30). PCR was used to amplify the human
(Fig. 2C, lower panel). These data demonstrate that CAMP promoter from nucleotides – 693 to ⫹14 (30).
1,25(OH)2D3 can markedly enhance the expression This fragment was subcloned into the firefly luciferase


Figure 3. Induction of CAMP protein hCAP18
in U937 treated with vitamin D3. Cells were
treated with either vehicle (–) or 1,25[OH]2D3
(⫹, 1⫻10 –7 M) for 18 and 36 h. A) Cytospins of
cells treated for 36 h were prepared and IF for
hCAP18 was performed. Photographs were
taken at 200⫻ magnification. Examples of
strongly positive cells are indicated by black
arrows B) Total cell lysates were analyzed by
Western blot for hCAP18 expression. The posi-
tion of hCAP18 is indicated by arrow at the
right (upper panel). The positions of the mo-
lecular weight markers are indicated at the left.
Subsequent probing of the same blot for
GAPDH demonstrated equivalent loading of
protein in each lane (lower panel). (C) Levels
of hCAP18 in the medium of U937 cells treated
with vehicle or 1,25[OH]2D3 were determined

reporter plasmid pXP2 and called pXP2-CAMP-Luc that specifically amplifies the CAMP promoter (Fig.
(Fig. 4A). Subsequently, deletion mutants pXP2- 4A). The chromatin was sheared to an average size of
CAMP(⌬SmaI)-Luc and pXP2-CAMP(⌬HindIII)-Luc ⬃1 kb and immunoprecipitated with antibodies (Ab)
were generated by restriction enzyme digestion using specific for the VDR and C/EBPε proteins. C/EBPε
the SmaI and HindIII sites, respectively (Fig. 4A). activates CAMP gene expression (39) and was included
The CAMP promoter constructs were transfected as a positive control. For negative controls chromatin
into U937 cells that were subsequently treated with was immunoprecipitated with protein A-Sepharose (No
vehicle or 1,25(OH)2D3. After 18 h treatment, cell Ab) or preimmune serum (Pre). The samples were
lysates were prepared and dual luciferase assays were amplified by conventional PCR and visualized by
performed. In the absence of 1,25(OH)2D3 , lucif- ethidium bromide staining (Fig. 4C, upper panel) or
erase activity for all reporter constructs, including QRT-PCR (Fig. 4C, lower panel). Extremely low back-
the empty parental vector, was similarly low (Fig. 4B). ground levels were detected in the negative controls
This is consistent with the very low levels of endoge- (Fig. 4C, No Ab or Pre). A significant level of the
nous CAMP mRNA expression in untreated U937. promoter was immunoprecipitated by anti-VDR Ab
Upon treatment, the full-length promoter construct (22-fold above background) without 1,25(OH)2D3
pXP2-CAMP-Luc was consistently activated 2- to 2.5-fold treatment, and this increased by ⬎ 2-fold (48-fold above
(Fig. 4B). The deletion mutants pXP2-CAMP(⌬SmaI)- background) with treatment (Fig. 4C, lower panel).
Luc and pXP2-CAMP(⌬HindIII)-Luc were not activated. Binding of C/EBPε to the promoter was similar under
pXP2-CAMP(⌬SmaI)-Luc still possesses the VDRE; both conditions (76- and 89-fold), demonstrating that
however, the SmaI site used for the generation of the vitamin D3 treatment is not increasing the amount of
construct is immediately adjacent to the VDRE (Fig. C/EBPε binding to the promoter (Fig. 4C). These
4A), suggesting that a single or several nucleotides results indicated that VDR is binding to the CAMP
located 5⬘ to the VDRE is required for the response. promoter in both a ligand-dependent and -indepen-
These data demonstrate that this VDRE is required for dent manner, consistent with current models of steroid-
activation of the CAMP promoter by vitamin D3. hormone gene regulation.

VDR binds to the CAMP promoter in cells Induction of CAMP by vitamin D3 is not
evolutionarily conserved
To determine whether VDR complexes were actually
binding to the CAMP promoter, we performed ChIP To elucidate further the role of the VDR in regulating
assays on chromatin prepared from U937 cells treated CAMP gene expression, we examined the expression of
with vehicle or 1,25(OH)2D3 for 4 h (Fig. 4C). Because the murine CAMP/CRAMP gene in RNA from un-
the VDRE is located in a repetitive DNA element or treated bone marrow cells from a VDR-deficient mouse
short interspersed nuclear element (SINE), it was dif- and its wild-type littermate (Fig. 5A, left panel). Bone
ficult to design primers for PCR that specifically ampli- marrow RNAs from C/EBPε-deficient and wild-type
fied that region of the CAMP promoter (Fig. 4A, mice were included as controls (Fig. 5A, left panel). As
shaded boxes). Therefore, we designed primers to the expected, the C/EBPε-deficient bone marrow lacked
nonrepetitive region near the transcriptional start site expression of CRAMP (40). In contrast, CRAMP was

1072 Vol. 19 July 2005 The FASEB Journal GOMBART ET AL.

expressed in the VDR-deficient cells at a level compa-
rable to the wild-type littermate. Furthermore, intra-
peritoneal treatment of BNX mice with 1,25(OH)2D3
or vitamin D3 compound I over 6 wk did not signifi-
cantly alter CRAMP expression in bone marrow com-
pared with a vehicle-treated mouse (Fig. 5A, middle
panel). We did not observe induction of CRAMP in
murine cell lines 32Dcl3 (Fig. 5, right panel), NIH3T3
and Wehi3B (data not shown). Finally, CRAMP induc-
tion was not observed in C/EBPε-deficient or wild-type
bone marrow cells cultured in vitro with 1,25(OH)2D3
(Fig. 5B) or in BM M␾ from VDR-deficient or wild-type
mice (Fig. 5C). Indeed, an ⬃ 2-fold decrease was
observed by 24 h post-treatment (Fig. 5B, C) and 5-fold
by 48 h (Fig. 5C).
We compared genomes from human, chimpanzee,
rat, dog, and mouse to determine conservation of the
promoter region for each CAMP gene (Fig. 5D).
Though significant homology was observed, a gap was
identified at – 409 bp upstream from the start site of
transcription in the human promoter. This was a due to
a SINE conserved only in the human and chimpanzee
genomes and absent in the others (Fig. 5D). The VDRE
is located in this SINE. Thus, the mouse gene lacks a
VDRE. This is consistent with the observed absence of
CRAMP induction by vitamin D3.


In this study, we showed that 1,25-dihydroxyvitamin D3

and three of its analogs induced expression of the
antimicrobial peptide gene CAMP in AML, immortal-
ized keratinocyte, and colon cancer cell lines, as well as
bone marrow-derived macrophages and fresh bone
marrow cells from normal individuals and one AML
patient. The induction of antimicrobial protein genes
by vitamin D3 in myeloid cell lines was restricted
primarily to CAMP.
Figure 4. Identification of a functional VDRE in the human
Activation of the CAMP gene occurred via a consen-
CAMP promoter. A) Sequence of the human CAMP promoter
(– 693 to ⫹14) (30) as it was cloned into the firefly luciferase sus VDRE in the promoter that is bound by VDR. The
reporter vector pXP2. Restriction enzyme sites are indicated VDR is expressed in a wide range of tissues; potentially,
across the top of the sequence and transcription factor CAMP can be induced in all of these tissues. A recent
binding sites are indicated across the top and bottom. These report published during the preparation and submis-
include CCAATT displacement protein (CDP), STAT3, sion of this manuscript reported findings consistent
C/EBP, PU.1, and VDR. The sequences of the primers used with those reported here (41). They observed induc-
for chromatin IP are underlined (line with filled circles at
each end). Two additional constructs were generated by tion by 1,25(OH)2D3 in purified monocytes, neutro-
deleting from the 5⬘-end with Smal and with HindIII. The phils, and cell lines from lung as well as head and neck
shaded box indicates the position of a repetitive element squamous cell carcinomas (41). This study expands on
(SINE) in the promoter (schematic diagram). B) U937 cells
were transfected (twice in duplicate) with the CAMP pro-
moter-firefly luciferase reporter constructs and a renilla ex-
pression vector, phTKRL. Each transfection was treated with (upper panel) PCR (reverse image of ethidium bromide
vehicle (–) or 1,25[OH]2D3 (⫹, 1⫻10⫺7 M) for 18 h. Dual stained gel; 30 cycles) and QRT-PCR (lower panel). The
luciferase assays were performed and firefly luciferase activity relative amount of CAMP promoter gDNA was determined in
was normalized to renilla luciferase activity. The untreated each sample by SYBR Green QRT-PCR. Differences (fold
and treated conditions for each construct were compared. change) were normalized to the preimmune (Pre, average of
CAMP-Luc (pXP2-CAMP-Luc); ⌬SmaI [pXP2-CAMP(⌬SmaI)- untreated and treated) and indicated by the number within
Luc], and ⌬HindIII [pXP2-CAMP(⌬HindIII)-Luc]. C) ⬃ 1 ⫻ each bar. The positions of the DNA markers are indicated in
107 cells were incubated in the absence (–) or presence (⫹) base pairs (bp) at the left of the panel and the size of the
of 1,25[OH]2D3 at 1 ⫻ 10⫺7 M for 4 h. ChIP assays were expected promoter product is indicated at the right. Anti-ε,
performed and the promoter was detected by conventional rabbit anti-C/EBPε antiserum.


Figure 5. Vitamin D3 induction of CAMP is not
conserved in the murine system. A) Total RNA
from bone marrow cells flushed from the fe-
murs of C/EBPε, VDR wild-type (WT), or
knockout (KO) mice were analyzed for murine
CAMP (CRAMP) expression by Northern blot
(left panel). Total RNA of BM cells from BNX
mice treated for 6 wk with vehicle (–),
1,25[OH ]2D3 (D3), or vitamin D3 analog
compound I were examined for CRAMP ex-
pression (middle panel). The murine my-
eloid cell line 32Dcl3 was treated with vehicle
(0) or 1,25[OH ]2D3 at 1 ⫻ 10⫺7 M for 24 and
48 h. Total RNA was analyzed by Northern
blot for CRAMP expression (right panel).
␤-Actin levels were used to demonstrate even
loading of the samples. B) The BM cells from
C/EBPε WT or KO mice were cultured with
vehicle (–) or 1,25[OH ]2D3 for 24 h. Relative
levels of CRAMP were determined by QRT-
PCR. (C) BM M␾ from VDR WT or KO mice
were treated with vehicle (–) or 1,25[OH ]2D3
for 24 or 48 h. Relative levels for CRAMP were
determined by QRT-PCR. D) Screen shot
(Human May 2004 Assembly) of the human
CAMP genomic region (chr3: 48, 237, 952-48,
423, 990; UCSC Genome Browser; http:// (64, 65). Conservation of
the human genome {International Human
Genome Sequencing Consortium) compared
with the chimpanzee (Chimpanzee Genome
Sequencing Consortium), dog (The Broad
Institute and Agencourt Bioscience), rat (Rat Genome Sequencing Consortium) and mouse (Mouse Genome Sequencing
Consortium) genomes are depicted by histograms and the Alignment Net. The positions of SINEs and LINEs are indicated.
The location of the VDRE within the SINE is indicated by the arrow.

these observations by demonstrating that not only does it mediates some of those immunosuppressive proper-
1,25(OH)2D3 induce CAMP gene expression, but so do ties of vitamin D3 or acts to counter them needs
analogs of vitamin D3. We showed that induction occurs clarification.
in the cells of the bone marrow. Moreover, we discov- While these observations further expand the role of
ered that the induction of CAMP by vitamin D3 does vitamin D3 in immunomodulation in humans (44, 45),
not occur in mice. In fact, it appears that this mecha- they also indicate that the use of vitamin D3 and its
nism is conserved in primates (humans and chimpan- analogs provides a method to manipulate extrinsically
zees) and not in other mammals as suggested by the the expression of CAMP. This may provide additional
absence of the VDRE in the murine, rat, and canine avenues for using relatively safe compounds in the
CAMP promoters. The VDRE is present in a SINE treatment of human disease and injury. Enhancing the
element of the Alu-Sx subfamily. These elements can expression of CAMP expression could prove advanta-
retrotranspose from a progenitor element to other geous. Protective effects of CAMP overexpression in
locations in the genome during evolution. It would respiratory epithelia were observed in a cystic fibrosis
appear that this event occurred in a primate progeni- model (46). The systemic expression of CAMP/
tor. Whether this element is conserved in all primates hCAP18 in mice improved survival rates after intrave-
(other than humans and chimpanzees), as well as in nous injection of lipopolysaccharide (LPS) (47). LPS is
New World and Old World monkeys, has not been a component of the bacterial cell wall of gram-negative
determined. bacteria such as Escherichia coli or P. aeruginosa. Massive
The biological importance of this regulation is in- gram-negative bacterial infection can result in septic
triguing. Unfortunately, the murine model for VDR- shock due to the large amounts of LPS present in the
deficiency will not prove useful. Perhaps examination blood. Thus, hCAP18 may not only aid in clearance of
of vitamin D-resistant rickets patient samples for CAMP bacterial infection, but may protect against the sepsis.
expression may elucidate the importance of vitamin D3 This protection probably derives from the ability of
regulation of CAMP. It is interesting that these patients CAMP to bind to LPS and neutralize it (48 –51). The
suffer from frequent dental abscesses (42). Decreased hCAP18 peptide has been shown to inhibit LPS-in-
expression of CAMP/hCAP18 protein may contribute duced cellular responses such as release of TNF-␣,
to this as it does in Kostmann syndrome patients who tissue factor, and nitric oxide, protecting mice and pigs
lack hCAP18 (43). Finally, CAMP/hCAP18 has immu- from septic shock (48, 52). In vitro, hCAP18 inhibits
nomodulating properties ascribed to it. Whether or not macrophage activation by LPS and other bacterial

1074 Vol. 19 July 2005 The FASEB Journal GOMBART ET AL.

components (51). If endogenous hCAP18 levels can be LPS, or PMA (58, 63). Combining those cytokines or
increased by extrinsic manipulation, then the potential growth factors with vitamin D3 offers the possibility of
exists to treat conditions that are susceptible to the obtaining synergistic activation of the CAMP gene.
development of sepsis. Boosting CAMP/hCAP18 levels Such synergy was reported for LPS and vitamin D3 in
potentially could protect against this condition after neutrophils (41). Synergistic activation of the CAMP
surgery and speed wound healing. gene could prove useful in treating skin grafts for burn
Like VDR expression, CAMP expression is wide- patients or in boosting immunity to opportunistic in-
spread. It is important for barrier defenses in the skin. fections in chemotherapy patients.
Mice deficient in CAMP are much more susceptible to
skin infection than wild-type mice (53). CAMP expres- We thank Ook Kim, Dih-Yih Chen, and Jonathan Frank for
sion is up-regulated during cutaneous infection, injury, technical assistance and James O’Kelly for insightful discus-
sions and helpful suggestions. We are grateful to Drs. Lise
or inflammation (psoriasis) of the skin (54 –56). De- Binderup, Milan Uskokovic, Norbert Fusenig, and Alan Fried-
creased levels of hCAP18 in the skin of individuals with man for providing reagents. This work was supported by NIH
atopic dermatitis (AD) correlates with their increased grant CA26038-20, the Cindy and Allan Horn Foundation,
susceptibility to skin infection compared with those Parker Hughes Trust, the King Harbor Yacht Club Tom
with psoriasis (55). Vitamin D3 and its analogs have Collier Memorial Fund, and the Inger Fund. H.P.K. holds the
proven safe and effective in the treatment of psoriasis. Mark Goodson endowed chair for Cancer Research and is a
member of the Jonsson Cancer Center.
It remains to be determined whether CAMP induction
occurs with the topical application of vitamin D3. If so,
treatment of CAMP-deficient AD with vitamin D3 may
prove beneficial also.
Increasing CAMP expression by vitamin D3 treatment
may prove beneficial in other instances. CAMP is
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