Journal of Steroid Biochemistry & Molecular Biology 103 (2007) 451–456

In vivo relevance for photoprotection by the

vitamin D rapid response pathway
K.M. Dixon a , S.S. Deo a , A.W. Norman b , J.E. Bishop b , G.M. Halliday c ,
V.E. Reeve d , R.S. Mason a,∗
a Department of Physiology and Bosch Institute, University of Sydney, NSW 2006, Australia
b Department of Biochemistry, University of California, Riverside, CA 92521, USA
c Department of Medicine (Dermatology), University of Sydney, NSW 2006, Australia
d Department of Veterinary Science, University of Sydney, NSW 2006, Australia

Abstract

Vitamin D is produced by exposure of 7-dehydrocholesterol in the skin to UV irradiation (UVR) and further converted in the skin to
the biologically active metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ) and other compounds. UVR also results in DNA damage
producing cyclobutane pyrimidine dimers (CPD). We previously reported that 1,25(OH)2 D3 at picomolar concentrations, protects human skin
cells from UVR-induced apoptosis, and decreases CPD in surviving cells. 1,25(OH)2 D3 has been shown to generate biological responses
via two pathways—the classical steroid receptor/genomic pathway or a rapid, non-genomic pathway mediated by a putative membrane
receptor. Whether the rapid response pathway is physiologically relevant is unclear. A cis-locked, rapid-acting agonist 1,25(OH)2 lumisterol3
(JN), entirely mimicked the actions of 1,25(OH)2 D3 to reduce fibroblast and keratinocyte loss and CPD damage after UVR. The effects
of 1,25(OH)2 D3 were abolished by a rapid-acting antagonist, but not by a genomic antagonist. Skh:hr1 mice exposed to three times the
minimal erythemal dose of solar-simulated UVR and treated topically with 1,25(OH)2 D3 or JN immediately after UVR showed reduction
in UVR-induced UVR-induced sunburn cells (p < 0.01 and <0.05, respectively), CPD (p < 0.01 for both) and immunosuppression (p < 0.001
for both) compared with vehicle-treated mice. These results show for the first time an in vivo biological response mediated by a rapid-acting
analog of the vitamin D system. The data support the hypothesis that 1,25(OH)2 D3 exerts its photoprotective effects via the rapid pathway
and raise the possibility that other D compounds produced in skin may contribute to the photoprotective effects.
© 2006 Elsevier Ltd. All rights reserved.

Keywords: Vitamin D; 1,25-Dihydroxyvitamin D3 ; Vitamin D analogs; Cyclobutane pyrimidine dimers (CPD); Sunburn cells; Ultraviolet radiation; Immuno-
suppression; Skh:hr1 hairless mice

1. Introduction in the liver, followed by the kidney, forming the biologically

Exposure to ultraviolet radiation (UVR) from sunlight can
result in a variety of responses in the skin including muta-
genic DNA damage such as cyclobutane pyrimidine dimers
(CPD) [1] and immunosuppression [2], both of which play
a role in skin carcinogenesis. UVR is also the catalyst for
the formation of vitamin D3 in skin. Upon the interaction of
UVR with 7-dehydrocholesterol in skin, pre-vitamin D3 is
formed, which thermally isomerizes to vitamin D3 [3]. Vita-
min D3 undergoes two sequential hydroxylation reactions
∗ Corresponding author.
E-mail address: rebeccam@physiol.usyd.edu.au (R.S. Mason).
active metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ).
There is evidence that the entire pathway to 1,25(OH)2 D3 is
present in skin [4].
Previous studies in this laboratory have shown that
1,25(OH)2 D3 not only reduces the level of UVR-induced cell
death in human skin cells, but also reduces CPD in surviving
cells in a dose-dependent manner when added to skin cell
cultures prior to and/or after UVR [5–7].
The various biological responses produced by 1,25(OH)2
D3 appear to be mediated by two major pathways. The
well-studied genomic pathway depends on the interaction
between 1,25(OH)2 D3 and the nuclear vitamin D recep-
tor (VDR) followed by interaction of this steroid receptor

0960-0760/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jsbmb.2006.11.016
452 K.M. Dixon et al. / Journal of Steroid Biochemistry & Molecular Biology 103 (2007) 451–456
Fig. 1. Structures of 1,25(OH)2 -lumisterol3 (JN) and 1,25(OH)2 D3 in cis- and trans-form.
complex with vitamin D response elements present in the
promoter regions of the target genes, resulting in up- or down-
regulation of transcription [8]. There is now considerable
evidence to suggest that 1,25(OH)2 D3 can also utilize other
signal transduction mechanisms in order to generate rapid,
non-genomic responses. A rapid-acting, non-genomic path-
way for 1,25(OH)2 D3 action has been proposed to generate a
variety of cell specific responses within seconds to minutes.
This involves the hormone binding to a membrane receptor,
the identity of which is still not clear [9,10].
In the current study, vitamin D analogs were used
to differentiate between the vitamin D pathways. The
steroid hormone, 1,25(OH)2 D3 , because it is conforma-
tionally flexible, is able to generate biological responses
either by the classical genomic pathway or by the
membrane receptor-initiated rapid response path. It has
been shown that analogs permanently locked in the cis-
configuration, as in 1␣,25(OH)2 lumisterol3 (JN), can activate
only the rapid response pathway [11] (Fig. 1). The com-
pound 1␤,25(OH)2 D3 (HL) acts only as a rapid response
antagonist, whilst (23S)-25-dehydro-1␣-hydroxyvitamin D3 -
26,23-lactone (TEI-9647) acts as a genomic antagonist. No
specific agonist of the genomic pathway has been reported.
In our preliminary studies we showed that the photoprotec-
tive effects of 1,25(OH)2 D3 in human skin cells were likely
to be acting via the rapid pathway [6], and showed that the
photoprotective effects of 1,25(OH)2 D3 were present in vivo.
We now provide further data to support these proposals, and
show for the first time, an in vivo photoprotective effect of a
low-calcemic analog that can only activate the rapid pathway.
2. Methods
Fibroblasts and keratinocytes from human neonatal fore-
skins were cultured in 96-well plates on 5 mm glass coverslips
coated with poly-l-lysine as described in [12]. Cells were
treated with 1,25(OH)2 D3 or analogs 24 h prior to irradiation
and media containing treatments were added immediately
after UVR. Treatments were replaced with Martinez buffer
solution containing d-glucose for the duration of irradiation.
The sham-irradiated plate was subjected to similar conditions
but covered in aluminium foil for the duration of irradiation.
The UVR source for in vitro studies consisted of one
UVA and one UVB fluorescent lamp, with irradiance of
203 mJ/cm2 UVB and 1168 mJ/cm2 UVA (Phillips, Amster-
dam, Holland), filtered through 0.5 mm cellulose tri-acetate
(Eastman Chemical Products, Kingsport, TN) to eliminate
wavelengths below 290 nm [6,7].
Cell loss after UVR was measured as previously described
[7]. Cyclobutane pyrimidine dimers (CPD) were detected
by immunohistochemistry and image analysis as described
previously [7].
Female Skh:hr1 (albino, inbred) hairless mice were used
for in vivo studies. Housing and irradiation conditions were as
previously described [7]. Mice were subjected to three times
the minimal erythemal dose (MED) of UVR (3.98 kJ/m2
UVB and 63.8 kJ/m2 UVA).
Treatments were diluted in a base lotion containing
ethanol, propylene glycol and water at a final solvent ratio
of 2:1:1. Mice were treated with vehicle, 1,25(OH)2 D3 , or
the low calcemic, rapid-acting analog JN. All treatments
were applied dorsally, once, immediately after irradiation
only. Skin samples were taken from UV-irradiated back skin
24 h post-UVR, fixed in Histochoice (Amresco) and paraffin-
embedded for sunburn cell analysis by routine haematoxylin
and eosin staining. Sunburn cells were identified under a light
microscope as cells with pyknotic nuclei surrounded by an
eosinophilic cytoplasm, and were counted along the length
of each skin section. Detection of CPD was by immunohis-
tochemical analysis as previously described [7].
The contact hypersensitivity response was performed as
previously described [7]. Briefly, mice were sensitized 1 week
after irradiation with 100 ␮L of 2% oxazolone (Sigma, St.
Louis, MO, USA) (w/v) in absolute alcohol applied to non-
irradiated abdominal skin. Sensitization was repeated on the
subsequent day. The sensitized mice were challenged 2 weeks
after irradiation. Ear thickness measurements were recorded
before challenge and at 20 h after challenge.
Results are based on quadruplicates of each treatment from
experiments performed at least twice with similar results.
Comparisons between treatment groups were made by one-
 K.M. Dixon et al. / Journal of Steroid Biochemistry & Molecular Biology 103 (2007) 451–456 453

Fig. 2. Protective effects of 1,25(OH)2 D3 on UVR-induced skin cell loss (a) and CPD (b) reversed by rapid-acting antagonist HL but not by genomic antagonist
TEI-9647. Cells were treated for 24 h with vehicle, 1,25(OH)2 D3 , TEI-9647, HL, or a combination of 1,25(OH)2 D3 and either TEI-9647 or HL. Treatments
were replaced with Martinez solution for the duration of irradiation, and post-treatments added immediately after irradiation. Cell loss was determined 24 h
after UVR (a). Significantly different from vehicle: *** p < 0.001 and * p < 0.05. CPD were detected 3 h after UVR (b). Significantly different from vehicle:
** p < 0.01.

way analysis of variance (ANOVA) using the GraphPad Instat
statistical program (GraphPad Software Inc., San Diego, CA).
3. Results
The level of UVR-induced cell loss was reduced in
cells treated with either 1,25(OH)2 D3 or JN, compared with
vehicle-treated cells. In pooled data from two separate exper-
iments, mean cell loss in vehicle-treated skin fibroblasts was
37.1 ± 4.0%. This was significantly reduced to 15.4 ± 4.5%
in cells treated with 1,25(OH)2 D3 (p < 0.01). The rapid-
acting, low-calcemic analog JN completely mimicked the
effects of 1,25(OH)2 D3 , reducing cell loss to 15.2 ± 5.8%
(p < 0.01). Similar results were obtained in keratinocytes.
Furthermore, both 1,25(OH)2 D3 and JN significantly reduced
CPD in keratinocytes from 18.6 ± 6.0 to 3.7 ± 1.0 (p < 0.01)
and 5.3 ± 1.7 (p < 0.05), respectively.
As shown in Fig. 2a, the addition of the genomic path-
way antagonist, TEI-9647 to skin fibroblasts had no effect on
the photoprotective action of 1,25(OH)2 D3 . Mean cell loss
in vehicle-treated skin fibroblasts was 36.3 ± 3.0%. This was
reduced to 21.0 ± 2.2% in cells treated with 1,25(OH)2 D3
(p < 0.001). Mean loss in cells treated with TEI-9647 or HL
alone was not significantly different to that of vehicle-treated
cells. When cells were treated with a combination of TEI-
9647 and 1,25(OH)2 D3 , mean cell loss was 28.8 ± 1.7%,
significantly lower than in vehicle-treated cells (p < 0.05)
and not significantly different to mean loss in cells treated
with 1,25(OH)2 D3 alone. In contrast, the protective effect of
1,25(OH)2 D3 on cell loss was abolished in the presence of
HL (Fig. 2a).
The proportion of positive CPD staining in cells treated
with a combination of TEI-9647 and 1,25(OH)2 D3 did not
differ from that in cells treated with 1,25(OH)2 D3 alone
(Fig. 2b). In cells treated with the rapid response pathway
454 K.M. Dixon et al. / Journal of Steroid Biochemistry & Molecular Biology 103 (2007) 451–456

Fig. 3. Effects of rapid response pathway antagonist HL and genomic pathway antagonist TEI-9647 on UV-induced CPD in skin fibroblasts. Images are
representative of fields captured during image analysis for (A) UV-irradiated vehicle; (B) UV-irradiated 1␣,25(OH)2 D3 ; (C) UV-irradiated Tei-9647; (D)
UV-irradiated Tei-9647 + 1␣,25(OH)2 D3 ; (E) UV-irradiated HL; (F) UV-irradiated HL + 1␣,25(OH)2 D3 .

antagonist HL, there was no difference in the level of
CPD compared with vehicle-treated cells. When cells were
treated with a combination of HL and 1,25(OH)2 D3 , the
inhibitory effects of 1,25(OH)2 D3 on CPD were abolished
(Figs. 2b and 3).
Sunburn cells (apoptotic keratinocytes) in mouse skin
were reduced by both 1,25(OH)2 D3 and JN. In mice treated
with 1,25(OH)2 D3 at doses of 22.8 and 4.6 pmol/cm2 , the
numbers of sunburn cells per 245 linear micrometres of
skin were reduced from 0.5 ± 0.1 to 0.2 ± 0.1 (p < 0.01) and
0.4 ± 0.1 (p < 0.05), respectively. In mice treated with JN
at a dose of 22.8 pmol/cm2 , sunburn cells were reduced to
0.3 ± 0.0 (p < 0.05) (Fig. 4).
Topical 1,25(OH)2 D3 or JN at similar doses reduced CPD
measured 24 h post-UVR from 9 ± 3% to 3 ± 1% (p < 0.01)
and 4 ± 2% (p < 0.01), respectively.
1,25(OH)2 D3 as well as JN, significantly reduced lev-
els of UVR-induced immunosuppression in Skh:hr1 mice.
As shown in Fig. 5, the level of immunosuppression in
vehicle-treated mice at 20 h post-challenge was 23.4 ± 1.3%.
In mice treated with 1,25(OH)2 D3 at a dose of 22.8 pmol/cm2
this was reduced to 3.9 ± 1.5% (p < 0.001). JN at the same
dose (22.8 pmol/cm2 ) also significantly reduced immunosup-
pression at 20 h post-challenge to −3.0 ± 1.5% (p < 0.001)
(Fig. 5).
4. Discussion
We have previously provided preliminary evidence to sup-
port the proposal that the rapid pathway is involved in the
photoprotective effect of 1,25(OH)2 D3 [6,7]. In the current
 K.M. Dixon et al. / Journal of Steroid Biochemistry & Molecular Biology 103 (2007) 451–456
Fig. 4. Reduction in sunburn cells (apoptotic keratinocytes) by 1,25(OH)2 D3 in Skh:hr1 mouse skin. Mice were exposed to 3 MED solar-simulated UVR and
treated topically with vehicle or 1,25(OH)2 D3 immediately after exposure. Skin biopsies were taken 24 h after UVR. Results presented as mean number of
sunburn cells per 245 linear micrometres of skin section, n = 3. Significantly different from vehicle: ** p < 0.01 and * p < 0.05.
study, we show that the rapid-acting analog, JN, entirely mim-
ics the photoprotective effects of 1,25(OH)2 D3 in human skin
cells. Furthermore, both the reduction in UVR-induced cell
loss and in CPD damage by 1,25(OH)2 D3 were fully reversed
by the rapid response antagonist HL and unaffected by the
genomic antagonist TEI-9647.
We have shown for the first time an in vivo complex
biological response using an analog that is only capable
of acting via the rapid pathway. The cis-locked compound
JN, which has no transcriptional activity, entirely mimicked
the effects of 1,25(OH)2 D3 at equivalent doses by reducing
UVR-induced sunburn cells, DNA damage and immuno-
suppression in Skh:hr1 hairless mice. These results provide
strong and consistent evidence for a physiological role of the
rapid response pathway in photoprotection.
Fig. 5. Reduction in UVR-induced systemic immunosuppression by
1,25(OH)2 D3 and JN in Skh:hr1 mice. Mice were exposed to 3 MED solar-
simulated UVR and treated immediately after exposure once with vehicle,
1,25(OH)2 D3 or JN. Mice were sensitized on non-irradiated abdominal skin
7 and 8 d after UVR with 2% oxazolone. Mice were challenged on ears
7 d after sensitization and ear swelling recorded 20 h later. Results were
calculated as the difference between pre- and post-challenge ear thick-
ness measurements of non-irradiated mice as a proportion of the difference
between pre- and post-challenge ear thickness measurements of irradiated
mice for each treatment. Immunosuppression expressed as 100% minus this
value, ± S.E.M. *** p < 0.001 significantly different from vehicle. n = 5.
455
These functions are likely to be important, as there is
ample evidence that both production of CPD and immunosup-
pression are key elements in skin carcinogenesis [2]. Since
CPD are produced by the direct interaction of UVR with
DNA, and the protective effects of vitamin D compounds are
seen following application after irradiation, it is reasonable
to speculate that the CPD reduction is the result, at least in
part, of increased repair, probably in turn a result of increased
p53 expression and reduced nitric oxide products, previously
demonstrated in the presence of 1,25(OH)2 D3 [7]. Repair
of CPDs reduces skin cell apoptosis, and prevents UVR-
induced mutation but also immunosuppression [13], thus
providing a link between the three effects described in vivo.
The process of vitamin D3 production in skin following
exposure to UVR takes several hours. The conversion of vita-
min D3 to 1,25(OH)2 D3 takes place over several further hours
[14]. Because of this long time-course, it is unlikely that the
1,25(OH)2 D3 produced locally in skin protects from the UVR
exposure responsible for its production. It is more likely that
the locally produced vitamin D compounds protect from fur-
ther UVR exposure, as is the case for UVR-induced increases
in cornification and pigmentation [15,16]. Overirradiation
products have some structural similarities to the rapid-acting
agonists [3] and it is possible to speculate that these com-
pounds may also contribute to the photoprotective effect.
The data presented are consistent with the proposal that
locally produced 1,25(OH)2 D3 contributes to endogenous
photoprotection in skin. The data also provide evidence that
the rapid response pathway for vitamin D plays an important
physiological role in a whole animal.
Acknowledgements
This work was supported by the National Health and
Medical Research Council of Australia and by the Cancer
Council of New South Wales. K.M. Dixon was the recipient
456 K.M. Dixon et al. / Journal of Steroid Biochemistry & Molecular Biology 103 (2007) 451–456

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