DOI:10.1111/j.1600-0625.2009.00997.

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Original Article

Topical treatment with the vitamin D analogue

calcipotriol enhances the upregulation of the
antimicrobial protein hCAP18/LL-37 during wounding
in human skin in vivo
Johan D. Heilborn1, Gunther Weber1,2, Alvar Gronberg1,3, Christine Dieterich1,3 and Mona Stahle1
1
Dermatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden;
2
GICC, UMR, CNRS 6239, Universite Francois Rabelais, Tours, France;
3
Lipopeptide AB, Stockholm, Sweden
Correspondence: Mona Stahle, Dermatology Unit, Department of Medicine, Karolinska Institutet, SE-171 76 Stockholm, Sweden,
Tel.: +46 8 517 733 48, Fax: +46 8 517 778 51, e-mail: mona.stahle@ki.se

Accepted for publication 3 September 2009

Abstract: Cathelicidin antimicrobial protein, hCAP18, is the sole
cathelin protein in human. Its C-terminal peptide, which is
released enzymatically from the holoprotein, has broad
antimicrobial activity but also has effects on eukaryotic cells.
hCAP18 is present in leukocytes and is produced at epithelial
interfaces as part of the innate immune system. In normal intact
skin, there is low constitutive expression of hCAP18, which is
rapidly upregulated upon injury. Accumulating evidence indicates
that hCAP18 LL-37 may serve a key role in protecting the
integrity of the epithelium and also actively promote re-
epithelialization and tissue repair. Molecular mechanisms
responsible for controlling hCAP18 gene expression in vivo are
only partly understood. Vitamin D3 and its analogue calcipotriol
were recently found to directly induce transcription of the
hCAP18 gene via functional vitamin D responsive elements in the
hCAP18 gene promoter. Skin is the major source for vitamin D3
in human, where its production is dependent on ultraviolet B
(UVB) radiation. We have shown that exposure to UVB, sufficient
to produce vitamin D3, upregulates hCAP18 in human skin in
vivo. In the present study, we demonstrate that the upregulation
of hCAP18 LL-37 following acute skin injury is further enhanced,
at both hCAP18 mRNA and protein levels, after topical treatment
with the vitamin D3 analogue calcipotriol. In chronic ulcers,
calcipotriol treatment upregulated hCAP18 mRNA, whereas no
consistent upregulation of hCAP18 protein was detected. Our
results further support the role of vitamin D3 as a key physiologic
regulator of hCAP18 LL-37 in human skin.
Key words: cathelicidin LL-37 skin vitamin D wound
healing

Please cite this paper as: Topical treatment with the vitamin D analogue calcipotriol enhances the upregulation of the antimicrobial protein hCAP18 LL-37
during wounding in human skin in vivo. Experimental Dermatology 2010; 19: 332338.

receptor-like 1 (FPRL1) (7,8). In addition, LL-37 was

Introduction
The cathelicidin gene family comprises a group of mamma-
lian antimicrobial peptides serving as a first line of defense
in the innate immune system (1,2). These proteins are syn-
thesized as precursors composed of a conserved N-terminal
cathelin-like domain, and a variable C-terminus released by
enzymatic processing that exerts broad antimicrobial activ-
ity (1,35). In human, hCAP18 is the only cathelicidin
protein and its C-terminal peptide LL-37 has lately been
shown to be multifunctional (2,6). The antimicrobial effect
of LL-37 is achieved by direct binding to and interaction
with the microbial membrane whereas the more recently
discovered effects on eukaryotic cells appear receptor-medi-
ated (1,2,6). LL-37 is chemotactic for leukocytes and mast
cells, mediated through binding to the formyl peptide
recently shown to upregulate the expression of FPRL1 in
HaCaT cells (9). Previous data also suggest that LL-37 can
act by transactivation of the epidermal growth factor recep-
tor (EGFR) which leads to the activation of MAPK signal-
ling in airway epithelium (10). A similar mechanism of
action was recently described in keratinocytes where it was
found that heparin binding epidermal growth factor is a
mediator for LL-37 EGFR transactivation (11)
hCAP18 LL-37 is produced in leukocytes and is widely
expressed in squamous epithelia of the skin and the air-
ways, in the gastrointestinal and genital tracts (1216) as
well as in exocrine and apocrine glands and their secretions
(1721). In normal intact skin there is low constitutive
expression of hCAP18 but upon inflammation there is
strong upregulation (15,22).

332 2009 John Wiley & Sons A/S, Experimental Dermatology, 19, 332338

Injury to the skin with a broken barrier sets in motion a
series of tightly coordinated and dynamic events with the
purpose to swiftly re-establish skin integrity. In accordance
with a prominent role in the first line of defense,
hCAP18 LL-37 is rapidly upregulated in wound edge epi-
thelium as a physiological response to injury (23,24). LL-37
has lately been suggested to be functionally involved in the
healing process. This was demonstrated by the ability of
LL-37 specific antibodies to inhibit the re-epithelialization
in wounded skin equivalents (24). In addition, LL-37 has
been shown to induce closure of epithelial wounds and
stimulate epithelial cell proliferation and migration (11,25)
and recent data have demonstrated that LL-37 gene transfer
enhances wound healing in vivo in diabetic mice (9).
The molecular mechanisms regulating hCAP18 gene
expression are insufficiently understood. Although insulin-
like growth factor (IGF-1) is reported to induce
hCAP18 LL-37 expression in human keratinocytes (26), a
host of other growth factors and cytokines have been inves-
tigated without demonstrating effect on hCAP18 LL-37
expression (16,27,28). We and others have demonstrated
that vitamin D3 directly upregulates the transcription of
hCAP18 in various cell types including keratinocytes via
functional vitamin D responsive elements (VDRE) in the
hCAP18 gene promoter (2931).
In the present study, we demonstrate that the physiologi-
cal upregulation of hCAP18 LL-37 occurring at acute skin
injury is further enhanced by topical treatment with calci-
potriol in vivo, peaking at 24 h at the RNA and protein
levels. These results underline the role of vitamin D3 as a
key regulator of hCAP18 LL-37 in skin. During recent
years is has been shown that keratinocytes contain the
enzymes to fully hydroxylate and produce active 1,25(OH)
2D3 (3234) and it has further been shown that the enzyme
responsible for converting inactive 25(OH)D3 to active
1,25(OH)2D3, CYP27B1, is upregulated in response to acute
skin injury (35). We here demonstrate that this upregula-
tion was even further enhanced by treatment with calcipot-
riol during acute wounding. In chronic ulcers, calcipotriol
treatment consistently increased hCAP18mRNA expression
levels, but not protein levels, nor was there upregulation of
CYP27B1 in these ulcers.
Methods
Tissue samples
Acute wounds
For the studies of topical treatment with calcipotriol during
normal wound healing in vivo, wounds were made with a
3-mm biopsy punch in the lower abdominal region of
healthy volunteers (n = 10). The investigation was non-
randomized, single-blinded including five females and five
2009 John Wiley & Sons A/S, Experimental Dermatology, 19, 332338
Vitamin D potentiates LL-37 induced during skin injury
males, age 2230 years. Only fair skinned, young and
healthy individuals were included and the tissue was
obtained from a sunprotected area in all individuals. Each
proband received two to three wounds in the left and the
right inguinal region, respectively. Topical treatment with
calcipotriol was applied on one side and control treatment
on the contralateral side. To each of the wounds on one
side, 25 lg calcipotriol in 0.5 g ointment (Daivonex; LEO
Pharma, Malmo, Sweden) was applied to a total test area
of 2 cm 2.25 cm. The control wounds in the opposite
inguinal region were treated with vaseline (ACO, Stock-
holm, Sweden). All wounds were covered with inert dress-
ings (Melolin, Smith and Nephew, Hull, UK; Mefix;
Molnlycke AB, Gothenburg, Sweden; Tegaderm; 3M Health
Care, St Paul, MN, USA). After 12 h the dressings were
changed and the treatment was repeated. In the first four
individuals the bandage was removed at 24 h and the
wounds were excised with a 6-mm biopsy punch and snap-
frozen. The following five individuals were treated with cal-
cipotriol and control for totally 24 h as described above.
The remaining test areas were cleaned with saline solution,
covered with inert dressing and subsequently excised 48 h
postwounding. The initial 3-mm punch biopsies, employed
to create the wounds, were saved as control material of
intact skin.
Chronic ulcers
For the studies of topical treatment with calcipotriol of
non-healing ulcers, patients (n = 14) with chronic
(>6 months duration) leg ulcers because of venous insuffi-
ciency were recruited at the Department of Dermatology,
Karolinska Hospital, Stockholm. Individuals with a history
of diabetes mellitus, arterial insufficiency or chronic inflam-
matory disease were excluded. Patients with signs of
eczema in the ulcer margin, clinical signs of infection or
undergoing systemic or topical antibiotic treatment at the
time of biopsy were also excluded. The investigation was
non-randomized, single-blinded including eight females
and six males, age 5592 years. Bacterial culture from the
leg ulcers showed colonizing bacteria (e.g. Staphylococcus
aureus, mixed gram negative flora, Proteus mirabilis, Entero-
coccus species). All patients included were, prior to treat-
ment with calcipotriol, treated with inert local dressings
and standard compression bandaging. Topical treatment
with calcipotriol was applied to a total test area of
2 cm 2.25 cm on one side of the ulcer margin including
50% of the wound bed and 50% of the epithelium. The
control treatment was applied onto the contralateral side at
the wound margin of the same ulcer. To each ulcer, 25 lg
calcipotriol in 0.5 g ointment (Daivonex) was applied. The
control area on the contra lateral side was treated with vas-
eline as described above. The bandage was removed after
2448 h following treatment, punch-biopsies (4-mm) were
333
 Heilborn et al.
obtained from the ulcer margin of each treated site, includ-
ing 50% of the epithelialized area, and were snap-frozen.
All participants gave their written informed consent.
The study was approved by the Regional Committee of
Ethics and was conducted according to the Declaration of
Helsinki Principles.
Real-Time PCR
For hCAP18, frozen biopsies from acute wounds (n = 9)
and chronic ulcers (n = 9) were cut in 50 lm sections and
homogenized. RNA was extracted with the Qiagen RNeasy
kit (Operon Biotechnologies, Cologne, Germany) and
reverse transcribed with a first strand synthesis kit (Amer-
sham Biosciences, Norwalk, CT, USA). RNA was quantified
by Real-Time PCR on an ABI Prism 7700 (Applied Biosys-
tems Pleasanton, CA, USA) using 5 ng of cDNA according
to standard protocols. The samples were evaluated in tripli-
cates. Sequences were 5-GTCACCAGAGGATTGTGACTT-
CAA-3 and 5-TTGAGGGTCACTGTCCCCATA-3 for the Twenty-four hours after wounding, hCAP18 mRNA expres-
primers, and 6-FAM-5-CCGCTTCACCAGCCCGTCCTT-
3-BHQ1 for the fluorigenic probe. The results were nor-
malized by quantification of 18S-RNA (Assay on Demand,
Applied Biosystems). For analysis of CYP27B1 expression
frozen biopsies from acute wounds (n = 4) and chronic
ulcers (n = 4) were investigated. cDNA was made using
200 ng total RNA and 20 ng cDNA was amplified using
TaqMan Universal PCR master mix, no AmpErase UNG
and TaqMan predesigned gene expression assays (18S and
CYPB27B1) according to the manufacturers instruction.
Each reaction was performed in duplicate.
Fold change expression was evaluated relative to the
appropriate control samples using the comparative thresh-
old Ct method (36). Results are presented as the average
fold change together with the range of fold change.
Protein extraction
Frozen biopsies from acute wounds (n = 4) and chronic
ulcers (n = 4) were cut in 50 lm sections. Protein was
extracted by a buffer of 60% acetonitrile containing 1% tri-
fluoroacetic acid (24) or for the chronic ulcers, vortexed
2 20 s in RIPA buffer (Pierce; Thermo Fisher Scientific,
Rockford, IL, USA) with Halt protease cocktail (Pierce)
followed by cold centrifugation at 14 000 g for 15 min.
Protein concentrations were measured using Protein Assay
Kit (Bio-Rad Laboratories, Hercules, CA, USA) based on
the Bradford method (37) or using (chronic ulcers) BCA
Protein Assay (Pierce).
Western blot analysis
For the detection of hCAP18 protein, the extracts were sep-
arated on a 16.5% Tris-Tricine gel (38). The total protein
amount in each sample was corrected to 5 lg. Affinity
purified anti-LL-37 antiserum (24) was used at 1:1000 dilu-
334
tion. After electroblotting onto PVDF membranes (BioRad,
Hercules, CA, USA), and sequential incubation with pri-
mary antibodies and horse-radish-peroxidase conjugated
IgG anti-rabbit (SantaCruz Biotechnology, Santa Cruz, CA,
USA), signals from enhanced chemiluminiscence (Amer-
sham Biosciences, Piscataway, NJ, USA) were captured with
a CCD camera (LAS 1000, Fujifilm). To confirm that equal
amounts of protein were blotted, the membranes were
stained with 0.1% Amidoblack 10B (Sigma) solution in
methanol acetic acid H20 at 45 10 45 (v v). Blots for
chronic ulcers were also probed with a rabbit monoclonal
antibody against b-actin (Cell Signaling Technology, Inc.,
Danvers, MA, USA).
Results
Calcipotriol enhanced the upregulation hCAP18
mRNA in acute wounds
sion increased significantly in all wounds treated with calci-
potriol compared with control wounds (Fig. 1). In the five
individuals investigated at 0, 24 and 48 h, the most promi-
nent hCAP18 mRNA expression was found at 24 h post-
wounding (Fig. 2). By 48 h, hCAP18 mRNA had declined
to levels similar to the control wounds. One individual
demonstrated lower hCAP18 mRNA levels in the calcipotri-
ol treated wound 48 h postwounding compared with the
control. In all five individuals there was a slight increase in
hCAP18 mRNA of the control wounds treated with vase-
line compared with normal intact skin (data not shown).
Figure 1. Topical calcipotriol treatment enhanced the upregulation of
hCAP18 mRNA in acute wounds. Real-Time RT-PCR expression analysis
of nine probands (no 19) at 24 h. RNA was extracted from excision
biopsies of acute wounds locally treated with calcipotriol or vaseline
(control) for 24 h. The stimulation of hCAP18 gene expression after
treatment is shown in arbitrary units and standardized to 18S mRNA
expression. For each individual, values are presented relative to the
expression of hCAP18 mRNA of the respective control wound, which is
set as 1 (not shown). In all probands there is a pronounced
upregulation of the hCAP18 mRNA expression after topical calcipotriol
treatment.
2009 John Wiley & Sons A/S, Experimental Dermatology, 19, 332338

Figure 2. Enhanced upregulation of hCAP18 mRNA following topical
calcipotriol treatment peaks at 24 h in acute wounds. Real-Time RT-PCR
expression analysis of five probands (no 59) at 0, 24 and 48 h
postwounding. RNA was extracted from excision biopsies of acute
wounds locally treated with (D) vitamin D (calcipotriol) or (C) control
(vaseline) for 24 h. The stimulation of hCAP18 gene expression after
treatment is shown in arbitrary units and normalized to18S RNA
expression. The most prominent hCAP18 mRNA expression is
demonstrated at 24 h postwounding and rapidly declining by 48 h.
Calcipotriol treatment enhanced the upregulation
of hCAP18 protein and the LL-37 peptide in acute
wounds
Three individuals were investigated by immunoblotting
demonstrating stronger immunoreactivity for hCAP18 in
wounds treated with calcipotriol compared with control
wounds (Fig. 3). Overall the strongest bands for hCAP18
Figure 3. Topical calcipotriol treatment enhances the upregulation of
hCAP18 LL37 protein in acute wounds. Western blot analysis of one
representative proband (no 6) at 24 and 48 h. Protein was extracted
from excision biopsies of acute wounds locally treated with calcipotriol
or control (vaseline) for 24 h. Overall, stronger immunoreactive bands,
corresponding to the holoprotein and to the processed LL-37 peptide,
are found for the calcipotriol treated wounds collected at 24 and 48 h
compared with the bands of the control wounds.
2009 John Wiley & Sons A/S, Experimental Dermatology, 19, 332338
Vitamin D potentiates LL-37 induced during skin injury
were detected at 24 h, but by 48 h the difference between
wounds treated with calcipotriol and control wounds was
even more pronounced (Fig. 3). In addition, in all three
individuals, stronger immunoreactive bands, corresponding
to LL-37, were present in wounds treated with calcipotriol
compared with control wounds. In normal intact skin,
hCAP18 protein was barely detectable, with no immuno-
reactivity for LL-37 (data not shown).
Calcipotriol treatment enhanced the upregulation
of hCAP18 mRNA in chronic non-healing ulcers
Twenty-four hours after topical treatment with calcipotriol
the hCAP18 mRNA expression increased significantly in all
non-healing leg ulcers compared with the control-treated
area of the same ulcer (Fig. 4). In all nine individuals there
was minor increase in hCAP18 mRNA of the control
wounds treated with vaseline compared with normal intact
skin. In addition, four individuals were investigated by
immunoblotting, but no consistent upregulation on the pro-
tein level was detected after calcipotriol treatment (Fig. 5).
CYP27B1mRNA was upregulated during acute
wounding
In accordance with published data, CYP27B1 mRNA was
upregulated 24 h following wounding in normal skin
(Fig. 6a). Individual data are presented (n = 4) demon-
strating increased expression of CYP27B1 mRNA in three
of four individuals.
Calcipotriol treatment further enhanced the
upregulation of CYP27B1 mRNA in acute wounds,
but not in chronic ulcers
In the acute wounds the expression of CYP27B1 mRNA
was further enhanced by treatment with calcipotriol in three
Figure 4. Topical calcipotriol treatment enhances the upregulation of
hCAP18 mRNA in chronic ulcers. Real-Time RT-PCR expression analysis of
nine probands (no 19) at 24 h. RNA was extracted from biopsies of
chronic non-healing ulcers locally treated with calcipotriol or vaseline
(control) for 24 h. The stimulation of hCAP18 gene expression after
treatment is shown in arbitrary units and normalized to 18S RNA
expression. For each individual, values are presented relative to the
expression of hCAP18 mRNA of the respective control wound, which is set
as 1 (not shown). Prominent hCAP18 mRNA expression was found in all of
the investigated non-healing leg ulcers treated with topical calcipotriol.
335
 Heilborn et al.
(a)
(b)
Figure 5. Effect of topical calcipotriol treatment on hCAP18 LL37
protein in chronic ulcers. Western blot analysis of four probands (no
1114) at 48 h. Protein was extracted from excision biopsies of chronic
ulcers locally treated with calcipotriol or control (vaseline) for 48 hours.
(a) hCAP18 and b-actin protein bands, (b) hCAP18 expression shown in
arbitrary units after normalization to b-actin expression.
out of four investigated individuals (Fig. 6b). In the chronic
ulcers however, there was no upregulation of CYP27B1
mRNA after calcipotriol treatment (Fig. 6c). These data
support the notion that the production of active vitamin
D3 may be vital for efficient wound healing to occur.
Discussion
In the present study, we demonstrate that topical treatment
with calcipotriol enhances the upregulation of hCAP18
LL-37 occurring during human skin wounding in vivo.
Accumulating evidence supports a role for hCAP18 LL-37
in tissue repair and wound healing. In addition to its
increased and dynamic expression in wound edge epithe-
lium upon injury, we have previously shown that LL-37
specific antiserum inhibited re-epithelialization in an
ex vivo wound healing model (24). These data suggest that
hCAP LL-37 is a key component in the repertoire of
wound edge keratinocytes during normal repair. Moreover,
we and others have demonstrated that exogenous treatment
with LL-37 peptide or endogenous upregulation of hCAP18
increase proliferation and migration of epithelial cells
(9,11,24,25). In this context it is notable that hCAP18 pro-
tein is virtually absent in the wound edge epithelium of
chronic ulcers (24). In further support of a prominent role
for LL-37 in wound repair, are data indicating that LL-37
stimulates angiogenesis and neovascularization (39).
336
(a) (b)
(c)
Figure 6. Upregulation of CYP27B1 expression after wounding and
effect of topical calcipotriol treatment on CYP27B1 mRNA. (a) Real-
Time RT-PCR expression analysis of four probands (no 57 and 10) at
24 h. CYP27B1 expression was upregulated 24 h after wounding. RNA
was extracted from excision biopsies of acute wounds. The stimulation
of CYP27B1 gene expression after treatment is presented relative to
normal intact skin (0 h) using the comparative threshold cycle (Ct)
method. (b) Real-Time RT-PCR expression analysis of four probands (no
5, 7, 8 and 10) at 24 h. Topical calcipotriol treatment enhanced the
upregulation of CYP27B1 mRNA in acute wounds. RNA was extracted
from excision biopsies of acute wounds. Fold change expression was
evaluated relative to the appropriate control samples (acute wound at
24 h). (c) Real-Time RT-PCR expression analysis of four probands (no 7
10) at 24 h. Effect of topical calcipotriol treatment on CYP27B1 mRNA
in chronic wounds. RNA was extracted from biopsies of four chronic
ulcers locally treated with calcipotriol or vaseline (control) for 24 h. In
the chronic ulcers there was no upregulation of CYP27B1 mRNA after
calcipotriol treatment. The changes in CYP27B1 expression in treated
samples are shown relative to their respective control samples.
Expression in control samples = 1. Each reaction was performed in
duplicate.
Lately, substantial efforts have been made to understand
the regulation of the hCAP18 gene, since such mechanisms
may be crucial for novel pharmaceutical approaches to
modulate the endogenous gene expression. Short chain
fatty acids were reported to induce hCAP18 expression in
colon epithelial cells (27) and insulin-like growth factor
(IGF-1) was shown to elicit expression of hCAP18 LL-37
in cultured human keratinocytes (26), which may indicate
a role for IGF-1 in regulating hCAP18 LL-37 during
wounding. Other growth factors involved in wound healing
such as TGF-a, TGF-b1 and bFGF have not shown any
effect (26). IL-1a treatment of cultured keratinocytes
induced hCAP18 gene expression, whereas there is no evi-
dence so far for direct regulation of hCAP18 LL-37 by
other proinflammatory cytokines (28,40).
Interestingly, vitamin D3 was reported to directly induce
antimicrobial gene expression and activity by binding of 1,
25(OH)2D3 to the vitamin D receptor, which subsequently
binds to VDRE located in the promoter region of hCAP18
gene (29). The induction of gene expression was demon-
2009 John Wiley & Sons A/S, Experimental Dermatology, 19, 332338

strated in human adult and neonatal primary keratinocytes
in addition to neutrophils, monocytes and various tumor
cell lines. Our group confirmed these data and in addition
delineated the effect exerted by various vitamin D
analogues such as 25(OH)D3 and calcipotriol as well as
analogues of vitamin D2 whereas the vitamin D precursor,
7-dehydroxycholesterol, was ineffective. Furthermore, we
demonstrated that this mechanism is functional in human
skin in vivo (30). The direct regulation of hCAP18 expres-
sion by vitamin D was again confirmed by a publication
where it was suggested that the mechanism is restricted to
primates since the functional VDRE is lacking in the mur-
ine, rat and canine cathelicidin gene promoters (31).
Recent reports have underscored the vital importance of
vitamin D mediated upregulation of hCAP18 in the defense
against infectious diseases such as tuberculosis (41,42). In
human, skin is the major source of vitamin D3 where its
production is dependent on UVB acting on the precursor
7-DHC to form previtamin D3. This precursor is
subsequently transformed thermally to produce vitamin
D3, which is then further hydroxylated to produce
1,252(OH)D3. We have shown that vitamin D regulation of
hCAP18 expression seems to be a physiologic mechanism
since exposure to low doses of UVB upregulated hCAP18
expression in human skin whereas UVA was without effect
(43). These data underline the significance of vitamin D3 as
a major regulator of hCAP18 in innate immunity, which is
further underscored by data showing that vitamin D3
induces expression of the microbial pattern recognition
receptors, CD14 and TLR2 (35). Interestingly, it was
recently reported that a novel, structurally different vitamin
D3 analogue (ZK203278) exerts considerable immunomod-
ulatory activity at non-hypercalcaemic dosages (44).
In addition, the enzyme, CYP27B1, responsible for
hydroxylating inactive 25(OH)D3 to active 1,25(OH)2D3 is
highly upregulated upon injury, which even further empha-
sizes the role of vitamin D3 in controlling barrier defense
(35). In the present study, we confirm the upregulation of
CYP27B1 in association with acute wounding (Fig. 6a),
which was even further enhanced by calcipotriol treatment
(Fig 6b). In the chronic ulcers however, calcipotriol treat-
ment failed to convincingly induce CYP27B1 mRNA in the
four samples tested (Fig. 6c), supporting the notion that
cells within the chronic ulcer environment do not respond
adequately to physiologic stimuli (45,46).
The data presented herein show significant upregulation
of hCAP18 on both mRNA and protein levels, including
the processed LL-37 peptide following calcipotriol treat-
ment upon acute injury. The most prominent expression of
both mRNA and protein was recorded at 24 h postwoun-
ding, which is in agreement with our previous data, where
protein levels peaked at 1248 h (24). Even though we
aimed at minimizing heterogeneity among study subjects,
2009 John Wiley & Sons A/S, Experimental Dermatology, 19, 332338
Vitamin D potentiates LL-37 induced during skin injury
there was biological variation in response between individ-
uals (Figs 1 and 2). Upregulation at the protein level was
confirmed in three individuals (Fig. 3). However, in
chronic ulcers, we could not confirm upregulation of
hCAP18 at the protein level after calcipotriol treatment
despite pronounced induction at the mRNA level (Fig. 4).
These data are in accordance with our previous observation
in chronic ulcers, where a prominent signal for hCAP18
mRNA was detected in the epithelium by in situ hybridiza-
tion but no immunoreactivity for hCAP18 protein (24).
The mechanisms responsible for the lack of hCAP18 pro-
tein in non-healing ulcers are still unclear. Chronic ulcers
are characterized by colonization with a variety of bacteria
and one can speculate that they may interfere with expres-
sion and or may inactivate AMPs as has previously been
described (47).
In the present study, calcipotriol was applied only in the
early phase following injury, when the hCAP18 LL-37 lev-
els are at its peak (24). It has recently been reported that
in vivo delivery of an adenoviral vector containing the
human LL-37 improves wound healing by increasing
re-epithelialization and granulation tissue formation in
wounds of diabetic mice (9). In what way application of
vitamin D3 during later phases of the healing process or
repeated treatments will affect the levels of hCAP18 in the
wound remains to be investigated. Whether the upregula-
tion of hCAP18 expression will affect the time to heal
and or the quality of tissue repair are key questions that
also need to be addressed.
The biological consequences of these findings are poten-
tially intriguing and further studies are needed to fully eval-
uate the clinical effects of vitamin D3 treatment in tissue
repair. Vitamin D3 could prove useful in topical treatment
of various clinical conditions such as surgical wounds,
donor site of split skin grafts, burn wounds and conditions
found to have diminished expression levels of antimicrobial
proteins, e.g. chronic leg ulcers (24). In atopic dermatitis,
it would be attractive to upregulate the levels of
hCAP18 LL-37, but existing vitamin D3 analogues may be
too irritative for clinical use (48).
Acknowledgement
Financial support is acknowledged from the Medical Research Council,
Karolinska Institutet, the Swedish Cancer Society and the Welander-Finsen
Foundations. We thank Lisa Sundblad, Katarina Nystrom and Anna-Lena
Kastman for skilful technical assistance.
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