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Prolonged treatment of fair-skinned mice with topicalforskolin causes persistent tanning and UV protectionMalinda L Spry1,2, Jillian C Vanover1,3, Timothy Scott1,2, Osama Abona-Ama1,Kazumasa Wakamatsu5, Shosuke Ito5 and John A D’Orazio1,4

1 Markey Cancer Center 2 Graduate Center for Toxicology 3 Department of Molecular and BiomedicalPharmacology and 4 Department of Pediatrics, University of Kentucky College of Medicine, Lexington, KY, USA5 Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake Aichi, Japan

CORRESPONDENCE John D'Orazio, e-mail: [email protected].

KEYWORDS Melanocyte ⁄ pigmentation ⁄ forskolin ⁄UV radiation ⁄ erythema ⁄ mouse model

PUBLICATION DATA Received 29 October 2008,revised and accepted for publication 25 November2008

doi: 10.1111/j.1755-148X.2008.00536.x

Summary

We previously reported that topical application of forskolin to the skin of fair-skinned MC1R-defective mice

with epidermal melanocytes resulted in accumulation of eumelanin in the epidermis and was highly protec-

tive against UV-mediated cutaneous injury. In this report, we describe the long-term effects of chronic topical

forskolin treatment in this animal model. Forskolin-induced eumelanin production persisted through 3 months

of daily applications, and forskolin-induced eumelanin remained protective against UV damage as assessed

by minimal erythematous dose (MED). No obvious toxic changes were noted in the skin or overall health of

animals exposed to prolonged forskolin therapy. Body weights were maintained throughout the course of

topical forskolin application. Topical application of forskolin was associated with an increase in the number

of melanocytes in the epidermis and thickening of the epidermis due, at least in part, to an accumulation of

nucleated keratinocytes. Together, these data suggest in this animal model, short-term topical regular

application of forskolin promotes eumelanin induction and that over time, topical forskolin treatment is

associated with persistent melanization, epidermal cell accumulation, and skin thickening.

Introduction

We previously reported the development of the fair-

skinned C57BL ⁄ 6 murine model of humanized skin

(D’Orazio et al., 2006). In this model, mice are charac-

terized by the presence of interfollicular melanocytes

localized to the stratum basale of the epidermis, and by

epidermal accumulation of the red ⁄ blonde pigment

pheomelanin due to defective signaling of the melano-

cortin 1 receptor (MC1R) pathway in melanocytes. Thus,

unlike normal murine skin, wherein melanocytes in the

skin are found almost exclusively in dermal hair follicles

where they impart pigment to the hairs of the coat, but

not to the actual skin itself, melanocytes in the K14-

stem cell factor (SCF) transgenic animal are recruited

and maintained in the interfollicular epidermis based on

constitutive expression of SCF by basal keratinocytes

(Kunisada et al., 1998). In this anatomic location

keratinocytes and melanocytes interact intimately, with

melanocytes transferring to keratinocytes the melanin

that accumulates to give skin its ‘color’ and protects the

animal against UV radiation (D’Orazio et al., 2006).

Significance

This manuscript shows prolonged topical administration of a pharmacologic agent capable of overcom-

ing a defective melanocytic cell surface receptor to promote sunless tanning is well-tolerated in an animal

model of the fair-skinned human. As eumelanin accumulation promotes lasting UV resistance, pharmaco-

logic manipulation of melanin synthesis may represent a novel approach for protecting against the acute

effects of solar radiation, as well as skin cancer prevention.

ª 2009 The Authors, Journal Compilation ª 2009 Blackwell Munksgaard 219

Pigment Cell Melanoma Res. 22; 219–229 ORIGINAL ARTICLE

Because this model was originally developed on the

wild type pigment background of the C57BL ⁄ 6 strain

with intact melanin biosynthetic pathways, original K14-

SCF C57Bl ⁄ 6 wild type mice were characterized by jet

black skin caused by abundant deposition of the dark

brown ⁄ black pigment eumelanin in the epidermis (Kunis-

ada et al., 1998). When this mouse was crossed with

the C57BL ⁄ 6 extension animal whose pheomelanotic

pigmentation results from a loss of function mutation in

the MC1R (Robbins et al., 1993), fair-skinned animals

were obtained whose epidermis, like the fair-skinned

human, had abundant levels of the red ⁄ blonde pigment

pheomelanin instead of eumelanin. The MC1R is a

transmembrane Gs-coupled protein that, when bound to

its cognate ligand a-melanocyte stimulating hormone

(a-MSH), triggers activation of adenylyl cyclase and

subsequent production of the second messenger cAMP

(Abdel-Malek et al., 1995, 2008; Cone, 2006). Raising

cytoplasmic cAMP levels in melanocytes, either via

a-MSH signaling or pharmacologically by direct activa-

tion of adenylyl cyclase, initiates a pro-differentiation

pathway that involves activation ⁄ induction of the CREB

and Mitf transcription factors, and up-regulation of a

variety of enzymes and effectors of pigmentation, which

results in production of the UV-protective pigmentary

bio-aggregate known as eumelanin (Bertolotto et al.,

1998; Price et al., 1998).

We previously reported that eumelanotic K14-SCF

C57BL ⁄ 6 transgenic animals were relatively protected

from UV-mediated skin damage and carcinogenesis

compared to their pheomelanotic counterparts. Further-

more, we showed topical treatment of fair-skinned

Mc1r-defective animals with forskolin, a direct activator

of adenylyl cyclase (Seamon and Daly, 1981), rescued

epidermal eumelanin production and rendered animals

resistant to UV injury (D’Orazio et al., 2006). In this

report, we extend our prior studies to demonstrate

eumelanin induction was robust, sustained, and UV-pro-

tective throughout 3 months of daily topical forskolin

treatment, and establish no overt toxicity occurred in

the animals treated with topical forskolin over this time

frame. Furthermore, whereas topically administered

forskolin did not cause accumulation of melanocytes in

the skin in short-term experiments (despite near maxi-

mal darkening of the skin), longer-term application of

forskolin was associated with epidermal thickening and

enhanced melanocyte numbers in the skin, raising the

possibility that cAMP manipulation may promote mela-

nocyte proliferation or interfere with interfollicular mel-

anocytic senescence and ⁄ or apoptosis.

Results

Chronic topical forskolin treatment causes

sustained melanogenesis in K14-SCF Mc1re/e mice

Daily topical application of forskolin to pheomelanotic

K14-SCF MC1R defective fair-skinned mice (Mc1re ⁄ e)

resulted in robust induction of eumelanin over the

course of 3 weeks and protection from UV injury

(D’Orazio et al., 2006). To investigate the effects of pro-

longed forskolin treatment, K14-SCF Mc1re ⁄ e animals

were treated topically (once-a-day, 5 days a week) for

three consecutive months with either forskolin

(80 lmoles per application) or vehicle control (70% etha-

nol, 30% propylene glycol), and cutaneous responses

were determined. We found that forskolin caused pro-

gressive darkening of the skin over the first 5–6 weeks

of treatment (Figure 1A). After this point, the skin failed

to darken further, suggesting that peak pharmacologi-

cally-induced melanin accumulation occurred after 5–

6 weeks of once-daily drug exposure. Forskolin failed to

cause skin darkening in genetically matched MC1R-

defective animals that lacked the K14-SCF transgene,

presumably because of the lack of interfollicular SCF-

induced melanocytes in the epidermis. The degree of

pigmentation caused by forskolin in K14-SCF animals

was dose-dependent. For example, twice daily applica-

tions of the drug accelerated the rate of melanin accumu-

lation, such that maximal levels of skin darkening were

obtained after only 5 days of treatment (Figure 1B). Con-

versely, pigmentation was less robust with less frequent

administration and ⁄ or if smaller doses of forskolin were

applied to the skin (data not shown). We chose a once

daily course of administration during chronic experiments

as it mimicked the dosing schedule used in prior studies.

With this schedule, forskolin-induced dark pigmentation

was sustained for the duration of topical treatment

through at least 3 months (Figure 1A, C).

To verify the skin darkening observed after 3 months

of daily topical forskolin administration was due to mela-

nin deposition rather than to non-pigment effect(s), such

as drug-induced tissue oxidation or dye effect, skin biop-

sies from treated animals were evaluated. Using the

Fontana–Masson method of staining (Zappi and Lom-

bardo, 1984), we found intense melanin deposition

throughout the epidermis in the forskolin, but not in

vehicle-treated, skin of K14-SCF transgenic animals after

3 months of topical treatment (Figure 2A). In contrast,

forskolin-treated non-transgenic animals exhibited little

epidermal melanin accumulation (Figure 2A), correlating

with their lack of observed skin darkening (Figure 1A).

We directly measured melanin levels in the depilated

skin of animals treated for 3 months with daily forskolin

or vehicle control (Figure 2B). Chronically administered

topical forskolin promoted robust accumulation of

eumelanin in the skin of fair-skinned MC1R-defective

K14-SCF animals. In fact, topically administered forskolin

seemed to essentially reverse melanin expression

from predominantly pheomelanin to primarily eumelanin

in the epidermis. Specifically, eumelanin:pheomelanin

ratios in the skin of forskolin- versus control-treated

K14-SCF Mc1re ⁄ e animals measured 4.0 ± 0.8 versus

0.4 ± 0.1, respectively. Taken together, these results

confirm daily topical treatment with topical forskolin led

Spry et al.

220 ª 2009 The Authors, Journal Compilation ª 2009 Blackwell Munksgaard

to progressive accumulation (over several weeks) of epi-

dermal eumelanin, which was maintained over a period

of months as long as daily treatments were continued.

Prolonged, but not short-term, topical

administration of forskolin promotes interfollicular

melanocyte retention

It is known that stimulation of melanocytes by MSH

through the MC1R pathway involves cAMP as a second

messenger, hence we reasoned forskolin treatment

should be expected to mimic functional MC1R signaling

in melanocytes. MSH-mediated activation of melano-

cytes via MC1R has been implicated in both differentia-

tion (e.g., melanogenesis) and proliferation (Burchill

et al., 1993; Busca and Ballotti, 2000; Hunt et al., 1994;

Kitano, 1976; Price et al., 1998), raising the possibility

that topical forskolin treatment might increase melano-

cyte numbers in the skin, in addition to promoting mela-

nin synthesis. We tested the hypothesis that repeated

topical forskolin application would lead to accumulation

of increased melanocytes in the epidermis. Cohorts of

K14-SCF Mc1re ⁄ e animals were treated with either vehi-

cle control or daily application of topical forskolin to the

dorsal skin. Importantly, animals used in this study

harbored the dopachrome tautomerase-b-galactosidase

(DCT-LacZ) transgene (Mackenzie et al., 1997) to facili-

tate the identification and enumeration of melanocytes

in the skin. We compared the numbers of b-galactosi-

dase-stained cells in the epidermis in skin biopsies

taken throughout the 3 months of daily treatments of

control- or forskolin-treated K14-SCF DCT-LacZ Mc1re ⁄ e

animals (Figure 3). The data suggest that there was

indeed a progressive difference in melanocyte numbers

between control- and forskolin-treated animals

over time. Specifically, we noted a gradual decline in

epidermal melanocyte number in control-treated

animals, whereas melanocyte numbers were maintained

in forskolin-treated counterparts (Figure 3A,B).

A

B C

Figure 1. Prolonged daily topical administration of forskolin results in persistent melanization of the skin of Mc1re ⁄ e K14-SCF mice. C57BL ⁄ 6Mc1re ⁄ e fair-skinned mice were treated topically on the dorsal skin of the back with either vehicle control (70% ethanol, 30% propylene

glycol) or with forskolin (80 micromoles) once daily 5 days per week for the indicated amount of time. (A) The color of treated, depillated skin

was measured by reflective colorimetry at the time indicated. Data are reported as reflectometry units ± SEM on the L* (black–white) CIE

color axis (Wagner et al., 2002). Non-transgenic Mc1re ⁄ e animals (that lacked interfollicular melanocytes) were included as a control for

non-specific drug effects. Note that only K14-SCF transgenic animals (which have epidermal melanocytes) treated with forskolin displayed

significant and persistent forskolin-induced skin darkening. To provide a reference for maximally and minimally pigmented skin among K14-

SCF mice, the white and black triangles on the y-axis depict mean skin reflectometry values for adult K14-SCF untreated albino (amelanotic;

75.48 ± 1.14) and wild type (eumelanotic; 27.09 ± 1.27) animals respectively. Differences in reflectometry units between forskolin-treated

K14-SCF animals were statistically significant (P < 0.005) at each time point examined except for day 0. (B) Kinetics of forskolin-induced

melanization are accelerated with more frequent pharmacologic dosing. Shown are the reflectometry results of treated dorsal depillated skin

of K14-SCF Mc1re ⁄ e mice treated twice-daily with the topical treatment for five consecutive days (***P < 0.005). Note that the amount of

skin darkening induced by forskolin administered twice daily in only 5 days approached that obtained after 2–3 weeks of once daily

application. (C) Side-by-side comparison photograph of control- and forskolin-treated K14-SCF Mc1re ⁄ e mice (those used in the experiment

whose results are shown in panel ‘A’) after 3 months of topical treatments. Please note that the scarred areas on the posterior dorsal skin of

the animals represent healing skin biopsy wounds.

Prolonged topical forskolin effects in fair mice


Chronic topical forskolin treatment is well-tolerated

As forskolin is a potent activator of adenylyl cyclase

with the potential to be absorbed systemically when

topically administered, we evaluated the treated animals

for signs of toxicity. Throughout a 3-month course of

topical treatments, we observed no apparent adverse

behavioral changes or general toxic reactions, such as

skin worrying, pruritis, ocular abnormalities, loose fecal

pellets, tremulousness, or other neurologic or gait

impairments, in the animals treated daily with either

vehicle (ethanol ⁄ propylene glycol) or with forskolin

(80 lmoles per dose). Because normal growth is fre-

quently affected by ongoing exposure to a noxious

agent, we reasoned that if topically applied forskolin

were toxic to the animals in the schedule and dose

provided, it should cause failure to thrive over time in

exposed animals. To investigate this, we compared the

body weights of animals treated with forskolin versus

vehicle control. Chronic exposure to daily topical forsko-

lin was not associated with weight loss despite

profound skin darkening over time (Figure 4). In fact,

forskolin-treated animals seemed to gain marginally

more weight after 3 months of daily topical therapy than

age- and gender-matched vehicle-treated counterparts

(Figure 4B). On examination of abdominal organs, we

found the liver size seemed to be greater in forskolin-

treated animals relative to their vehicle-treated counter-

parts (Figure 4E). Livers from both groups of animals

were biopsied and evaluated histologically, however, no

significant differences in hepatocellular size, density or

architecture were appreciated (Figure 4F). Of note,

there was no significant difference in spleen weights

between cohorts (data not shown). Collectively, these

data suggest prolonged topical treatment with forskolin

was well tolerated, and was not associated with overt

toxicity, as judged directly by behavioral observation or

by growth and development. Although we found

evidence that forskolin applied topically to the dorsal

skin had systemic effects in the fair-skinned mice, nota-

bly darkening of the skin of the ears, tail, legs and feet

where drug had not been applied, the only distinct glo-

bal effect of topical forskolin was mild hepatomegaly of

unclear etiology, and of apparently no untoward clinical

significance.

Forskolin treatment promotes skin thickening

As we studied biopsies of treated skin from K14-SCF

Mc1re ⁄ e animals treated with prolonged topical therapy,

we noted the epidermis was thicker in animals treated

with daily forskolin than in the vehicle-treated counter-

parts (Figure 5A). Careful quantization of epidermal

thickness (from stratum corneum to the epidermal–

dermal junction below the stratum basale) revealed a

divergence in thickness between treatment groups

beginning early in the course of daily topical treatments

and continuing throughout 3 months (Figure 5B). To dis-

tinguish whether forskolin caused epidermal thickening

through an increase in epidermal cell numbers, skin sec-

tions from control- versus forskolin-treated animals were

stained with 4¢,6-diamidino-2-phenylindole (DAPI), which

binds strongly to DNA and facilitates identification of

nuclei under fluorescent light. Application of forskolin to

the skin clearly caused an increase in the numbers of

cells in the epidermis (Figure 5C, D), suggesting drug-

induced thickening of the epidermis is due, at least in

part, to accumulation of nucleated keratinocytes in the

epidermis. Furthermore, forskolin-induced thickening of

the epidermis seemed to be a direct effect of the drug

on keratinocytes independent of either melanocyte

involvement or pigment deposition, as comparable fors-

kolin-induced epidermal thickening was noted among

the K14-SCF negative animals, which lack epidermal

melanocytes and do not respond to topical forskolin-

mediated eumelaninization (Figure 2A). We conclude

topically applied forskolin induces epidermal keratinocyte

accumulation, which may involve induction of prolifera-

tion, cellular hypertrophy and ⁄ or interference with termi-

nal differentiation (enucleation).

A

B

Figure 2. Prolonged forskolin treatment causes induction of

eumelanin in the epidermis. K14-SCF Mc1re ⁄ e animals were

treated for 3 months with either forskolin or vehicle control as

described. (A) Dorsal skin biopsies from treated areas were stained

for melanin using the Fontana–Masson procedure wherein melanin

pigments appear black in section. Representative images at 400·magnification are shown. (B) Levels of pheomelanin (white bars)

and eumelanin (black bars) were quantified by HPLC in treated

depillated skin of K14-SCF Mc1re ⁄ e animals treated for 3 months

with either forskolin or vehicle control as described. Data represent

the mean of five animals per condition ± SEM and analysis of

variance is shown (***P < 0.005).

Spry et al.


Eumelanin induction by chronically applied forskolin

is UV-protective

Lastly, we were interested in whether forskolin-induced

eumelanin accumulation after chronic application was

protective against UV injury. Cohorts of K14-SCF

Mc1re ⁄ e animals were treated topically with either fors-

kolin or with vehicle control for 3 months, and were

evaluated for UV sensitivity by determining the minimal

erythematous dose (MED). MED, defined as the mini-

mal amount of UV radiation required to induce either

erythema or edema in the exposed skin, is a conven-

tional dermatologic measure of UV sensitivity that quan-

tifies UV-induced global inflammation in the skin (Noda

et al., 1993). As in prior experiments, we found robust

melanin-dependent darkening of the skin in forskolin-

treated, but not vehicle-treated, animals (Figure 6A).

Animals with forskolin-induced melanin induction in the

skin were significantly more resistant to cutaneous UV

damage, as manifested by a significantly higher MED

(Figure 6A, B) that was similar to that of shorter term

forskolin applications (Figure 6C). Thus, we conclude

that in this fair-skinned animal model with epidermal

melanocytes, forskolin-induced eumelanin up-regulation

was highly protective against UV-mediated skin damage

regardless of duration of pharmacologic stimulation. As

a whole, these data extend our prior studies of pharma-

cologic rescue of melanin in a fair-skinned mouse model

by showing (i) persistence of melanin up-regulation and

UV protection with ongoing forskolin administration, and

(ii) low overt toxicity of the topically administered drug

over time.

Discussion

In an earlier report, we showed that application of topi-

cal forskolin could rescue production of UV-protective

melanin pigment in fair-skinned mice otherwise incapa-

ble of making eumelanin due to loss of function of the

MC1R (D’Orazio et al., 2006). Mechanistically, forskolin-

induced eumelanization is thought to occur by direct

activation of adenylyl cyclase in melanocytes and

up-regulation of melanocyte cAMP levels, thereby effec-

tively ‘rescuing’ the dysfunctional MC1R that is respon-

sible for the fair-skinned pheomelanotic phenotype in

our model. For this report, we performed further experi-

ments to determine the effects of prolonged forskolin

treatment on these animals. In summary, our results

indicate chronic forskolin treatment leads to robust and

sustained eumelanin production without overt toxicity to

the animals, further supporting the theoretical use of

pharmacologic melanin up-regulation as a potential novel

UV-protective strategy in fair-skinned individuals.

A

B

Figure 3. Topical application of forskolin promotes melanocyte retention in the skin. K14-SCF DCT-LacZ Mc1re ⁄ e mice were treated with

topically administered vehicle or forskolin for the indicated amount of time as described. Treated skin was biopsied and stained for

b-galactosidase-expressing cells to quantify cells of melanocytic origin which stain blue by virtue of an active dopachrome tautomerase (DCT)

promoter and b-galactosidase expression. Representative images are shown for each treatment group; magnification 400·. (B) Quantification

of melanocyte numbers in the skin of K14-SCF DCT-LacZ Mc1re ⁄ e mice treated topically with either vehicle control (white bars) or forskolin

(black bars) for the indicated amount of time. Cells were considered to be of melanocytic lineage if they stained blue. Data are reported as

mean number of b-galactosidase-positive cells per high powered field ± SEM; analysis of variance is shown (ns, not significant; *P < 0.05;

***P < 0.005).



Over 3 months of daily forskolin treatments in K14-

SCF Mc1re ⁄ e animals, we found melanization was

sustained as long as topical forskolin treatments were

continued. Fontana–Masson staining of tissue sections,

as well as direct HPLC-based quantification of the two

melanin types, confirmed forskolin-induced skin darken-

ing was due to accumulation of eumelanin. Eumelanin

induction was not observed in vehicle-treated animals,

ruling out non-specific effects due to the ethanol–propyl-

ene glycol vehicle diluent. Similarly, fair-skinned MC1R-

defective animals lacking the K14-SCF transgene did not

manifest forskolin-induced skin darkening, suggesting

there is a requirement for interfollicular epidermal mela-

nocytes to be present in order for skin darkening to

occur. However, as the anatomic presence of epidermal

melanocytes (and therefore epidermal melanin) in this

animal model is SCF-dependent, the data presented in

this report do not exclude the possibility that SCF-cKit

signaling contributes to melanogenesis. However, SCF

alone does not alter the type of melanin produced by

the melanocytes in our model, as evidenced by the fact

the presence of the K14-SCF transgene does not rescue

A B

C D

E F

Figure 4. Effect of prolonged application of forskolin on body weight and liver size. Age- and gender-matched (female) K14-SCF Mc1re ⁄ e

mice were treated for 3 months with topically applied forskolin or vehicle control. Dashed lines represent data from vehicle control-treated

animals whereas solid lines represent data from forskolin-treated animals. (A) Weights of individual animals at the beginning

(time = 0 months) and at the end of the experiment (time = 3 months). (B) Mean body weight ± SEM of cohorts of animals described

individually in ‘A’; analysis of variance between groups showed no significant difference in body weights on day 0 or at 3 months (P > 0.05).

(C, D) Skin color measurement of individual animals (C) and groups of animals (D) treated and labeled as described above; analysis of variance

between groups showed no significant difference in skin color on day 0 but statistical difference (P < 0.005) at 3 months. (E) Mean liver

size ± SEM described as percent of body weight of groups of K14-SCF Mc1re ⁄ e mice treated for 3 months with either vehicle control (white

bar) or forskolin (black bar); analysis of variance is shown (P = 0.02). (F) Representative images of hematoxylin & eosin stained sections of

K14-SCF Mc1re ⁄ e mice treated with either vehicle or with forskolin as described; magnification 400·. Note the lack of overt pathology in

either section.

Spry et al.


(independently of forskolin) defective eumelanin produc-

tion in Mc1re ⁄ e animals.

We monitored forskolin-treated animals for signs of

toxicity throughout the three consecutive months of

daily topical drug application, but found no overt worri-

some signs or symptoms of toxicity, including lethargy,

interference with feeding, loose stool, hunching, gait or

neurologic imbalances, or coat changes. We reasoned

weight changes would be an appropriate gauge for glo-

bal toxicity in our animal model, and predicted failure to

thrive might be an appropriate measure of chronic toxic-

ity from the drug (Ellis et al., 1984; Pavan et al., 2003;

Preece et al., 1989). We found both control- and forsko-

lin-treated groups exhibited weight gain over time, as

would be expected in maturing young adult animals. In

fact, rather than failing to thrive, forskolin-treated ani-

mals seemed to exhibit a slightly increased weight at

the end of a 3-month course of topical therapy than

their vehicle-treated counterparts. Although mild hepa-

tomegaly among forskolin-treated animals was noted,

histologic examination of livers failed to identify a patho-

logic cause for liver enlargement (including fatty liver).

Nonetheless, it is clear that some amount of topically

administered forskolin gets absorbed and acts systemi-

cally, as robust melanization of the ears, tail, legs, and

feet were noted in animals that were treated only on

the dorsal skin.

Although forskolin treatment over 3 months caused a

robust increase in melanin production without apparent

inflammation, we did note thickening of the epidermis

in animals exposed to daily forskolin treatments.

Because epidermal thickening was seen in both K14-

SCF, as well as non-transgenic animals, we conclude

forskolin-induced epidermal thickening is independent of

melanin production, and likely represents a direct effect

of the drug on epidermal keratinocytes. It is possible

forskolin-induced skin thickening contributes significantly

to protection from UV-induced skin injury and that the

contribution of melanogenesis to UV protection may be

over-estimated in our animal model. However, similar

BA

DC

Figure 5. Topically-applied forskolin promotes epidermal thickening and accumulation of epidermal cells over time. K14-SCF Mc1re ⁄ e mice

were treated for 3 months with topically applied forskolin or vehicle control as indicated and skin biopsies were collected. (A) Fontana–

Masson melanin staining was performed and representative images from either control-treated or forskolin-treated mice are shown; note the

marked difference in epidermal thickness (along with melanin deposition) between the two. (B) Epidermal thickness of treated skin was

quantified and results are shown as average thickness (microns) ± SEM of the epidermis of control-treated (white bars) or forskolin-treated

(black bars) animals. (C) Skin biopsies of control- versus forskolin-treated animals were stained with DAPI to stain nuclei and representative

sections are shown. (D) Average numbers of nucleated cells in the epidermis (as determined by DAPI staining) ± SEM were quantified in

animals exposed to prolonged topical vehicle control (white bars) or forskolin (black bars). Please note the standard errors of the mean are

very small due to the numbers of sections counted per sample (29–61 each); analysis of variance is shown (***P < 0.005).



MED’s were noted among forskolin-exposed K14-SCF

Mc1re ⁄ e animals treated for only 2 weeks (such that pig-

mentation but not skin thickening was stimulated) as

animals treated for a full 3 months (Figure 6B,C), sug-

gesting that melanization plays more of a role in forsko-

lin-induced UV protection than does forskolin-mediated

epidermal thickening. Nonetheless, future UV protection

experiments will include non-transgenic ⁄ forskolin-

treated animals to formally test the contribution of skin

thickening to UV protection, as non-transgenic animals

demonstrate forskolin-induced skin thickening but not

melanization (Figure 2A).

We investigated topical forskolin’s effect on melano-

cyte numbers in the skin by quantifying LacZ-expressing

cells in the skin of forskolin- or control-treated K14-SCF

Mc1re ⁄ e animals that also harbored the DCT-LacZ

transgene. We found animals treated with daily topical

forskolin exhibited higher numbers of epidermal melano-

cytes than control-treated counterparts, and that forsko-

lin seemed to interfere with age-related decline in

epidermal melanocyte number. We have not yet deter-

mined if the molecular mechanism of forskolin-induced

melanocyte retention involves increased proliferation of

melanocytes, recruitment of melanocytes from the fol-

licular stem cell niche, and ⁄ or prolongation of melano-

cytic survival in the skin. As no significant changes in

melanocyte numbers were noted between forskolin and

control-treated animals during the first few weeks of

treatment, despite near maximal darkening of the skin

by forskolin in this time frame, we can exclude the pos-

sibility that forskolin-induced eumelanization is due to

increased melanocyte density. What effect this forsko-

lin-induced increase in the melanocyte population

imparts on melanomagenesis is unclear, thus we antici-

pate further investigation of this effect in the context of

our model system.

Our hope is that a pharmacologically suitable agent

capable of manipulating melanin production in the skin

may ultimately prove safe for human use as a novel UV

protective strategy. In this manner, eumelanin accumu-

lation would be expected to protect against UV-induced

skin damage and skin cancers by blocking UV photons

from interacting with melanocytes and keratinocytes in

the deep layers of the epidermis, and ⁄ or by functioning

as a scavenger of UV-induced free radicals (Rozanowska

et al., 1999). In addition, cutaneous cAMP manipulation

might promote pharmacologic repair of UV damage in

the skin, as clearance of UV-induced DNA lesions in

melanocytes was found to be enhanced by a-MSH or

its analogues (Abdel-Malek et al., 2006; Bohm et al.,

2005; Hauser et al., 2006) and more recently, topical

forskolin was found to accelerate disappearance of UV-

induced cyclobutane dimers in keratinocytes in a human

epidermal skin model (Passeron et al., 2008). Thus topi-

cal forskolin or other mediators of cAMP accumulation

might protect against UV damage in the skin through

several mechanisms. If observations from our animal

A

B

C

Figure 6. Melanization induced by forskolin treatment protects

against UV-mediated inflammation. K14-SCF Mc1re ⁄ e mice were

treated once daily for 3 months with topically applied forskolin or

vehicle control. As with other cohorts of mice, forskolin, but not

vehicle, promoted darkening of the skin (skin reflectometry CIE L*

values of 61.7 ± 2.0 for vehicle-treated animals versus 29.5 ± 2.8

for forskolin treated animals respectively). UV-occlusive tape was

applied to the dorsal surface with cut-out circular apertures through

which various doses of UV-B (as labeled) were administered. MED

was determined 24 h after exposure to various doses of UV, and

was quantified as the minimal dose of UV per animal that caused

either erythema or edema of the entire circle of exposed skin for

the given dose. (A) Representative images of UV-exposed control-

or forskolin-treated animals 24 h post-irradiation (with doses in

kJ ⁄ m2 indicated). (B) Average MED ± SEM of animals treated for

3 months with either vehicle or forskolin; analysis of variance is

shown (***P < 0.005). Note that a higher MED value indicates a

higher dose of UV needed to cause inflammation and correlates

with UV resistance. (C) Average MED ± SEM of animals treated

once daily for only 2 weeks with daily vehicle or forskolin); analysis

of variance is shown (***P < 0.005). Skin reflectometry

measurements and skin thickness were similar among animals

treated for 2 weeks as for those treated for 3 months.

Spry et al.


model prove extendable to humans, there seems to be

a temporal window for forskolin-mediated melanization

before skin thickening and melanocyte accumulation

occur in response to the drug. This temporal difference

between the onset of pigment synthesis and other cuta-

neous effects suggests that if topical cAMP rescue-

mediated eumelanization were extended to clinical use

in humans, it might be prudent to use such agents on a

short-term, temporary basis instead of by prolonged

usage.

In conclusion, we report prolonged exposure to topi-

cal forskolin (i) was effective at inducing sustained

eumelanin production in the skin of MC1R-defective

mice, (ii) caused negligible systemic toxicity, with mild

hepatomegaly being noted, (iii) effected profound UV

protection, as measured by the MED, (iv) triggered

keratinocyte accumulation in the epidermis, leading to

skin thickening, and (v) led to enhanced number of

melanocytes in the skin. At present, topical forskolin-

induced eumelanin induction remains a proof-of-concept

demonstration that fair-skinned individuals may be able

to produce eumelanin when melanocytes are appropri-

ately signaled. Nonetheless, caution must be taken

against using similar agents in humans given the poten-

tial for both systemic absorption and unknown local

consequences in the skin.

Methods

AnimalsC57BL ⁄ 6JJ mice harboring the extension mutation in the Mc1r

gene (Mountjoy et al., 1992; Robbins et al., 1993) were purchased

from the Jackson Laboratory (Bar Harbor, ME, USA) and were

crossed with K14-SCF transgenic animals also on the C57BL ⁄ 6JJJ

background (Kunisada et al., 1998) originally donated by Dr. Takahi-

ro Kunisada, Gifu University, Gifu City, Japan. The DCT-LacZ trans-

gene originally reported on a mixed CBA · C57BL ⁄ 6JJ background

(Mackenzie et al., 1997) was introduced into the system after back-

crossing onto the C57BL ⁄ 6JJ background (10 generations), and

then by crossing with K14-SCF Mc1re ⁄ e animals also on the

C57BL ⁄ 6 background. Presence of the K14-SCF and DCT-LacZ

transgenes were assessed by phenotype (in the case of K14-SCF

because of obvious skin color characteristics) and ⁄ or by PCR ampli-

fication of DNA obtained by tail snip of a fragment specific to the

K14-SCF and DCT-LacZ transgenes (Kunisada et al., 1998; Macken-

zie et al., 1997). Amelanotic K14-SCF animals and eumelanotic

C57BL ⁄ 6JJ animals were developed by crosses with tyrosinase-

deficient (Le Fur et al., 1996) or Mc1rE ⁄ E animals respectively. All

experiments were carried out in accordance with institutionally-

approved animal protocols.

Forskolin preparationForskolin preparation was prepared as a crude extract of Coleus

forskohlii root preparation as described (D’Orazio et al., 2006).

Briefly, C. forskohlii root extract (20% w ⁄ w forskolin) was

purchased from Phytotech Extracts Pvt. Ltd (Waterloo, ON,

Canada), distributed through Buckton Scott USA, Inc. (Princeton,

NJ, USA) and used as a working source of forskolin. This root

extract was prepared as a 40% weight:volume solution in a stan-

dard dermatologic vehicle of 70% ethanol, 30% propylene glycol

(Sigma-Aldrich Chemical Corporation, St. Louis, MO, USA). Fors-

kolin extract was prepared by mixing the dry root powder with

vehicle for 1 h at room temperature on a stir plate with constant

agitation. Next, the solution was centrifuged (10 min, room tem-

perature, 2000 ·g), after which the supernatant was decanted

and filtered through a 0.45-micron cellulose acetate filter to

remove particulate matter. The C. forskohlii extract was stored at

room temperature. Independent assay of the amount of forskolin

in the 40% (w ⁄ v) working solution revealed a concentration of

approximately 200 mM.

Topical treatmentsC57BL ⁄ 6JJ Mc1re ⁄ e K14-SCF gender-matched animals between 4

and 8 weeks of age were used for these experiments unless other-

wise noted. Dorsal hairs were initially trimmed using electric animal

shears with a 0.25-mm head (Fisher, Pittsburgh, PA, USA) and

were re-trimmed as needed (usually once weekly) to keep hairs at

a minimal length throughout the course. Preparations of topical

agents were applied to the sheared skin with a micropipette, first

by dripping the solution onto the skin using the end of the pipette

tip and then smoothing it out over the dorsal skin with the side of

a pipette tip so that a similar amount of solution would be applied

to the entire dorsal skin. Solvent (vehicle) control consisted of the

same volume of 70% ethanol ⁄ 30% propylene glycol applied to

the skin of age-matched congenic cohorts. Unless otherwise

indicated, animals were treated once daily on their dorsal surface

with 400 ll of topical agent for 5 days a week; for forskolin-treated

animals, this equated to roughly 80 lmoles of drug applied daily to

the skin.

Skin color measurementSkin reflective colorimetry measurements were assessed with a

CR-400 Colorimeter (Minolta Corporation, Japan) calibrated against

the white standard background provided by the manufacturer

before use. Degree of melanization (darkness) was quantified as

the colorimetric measurement on the *L axis (white–black axis) of

the CIE standard color axis (Wagner et al., 2002). Photographs of

animals treated as described were taken using a Canon EOS 20D

camera using a white dissecting board (Richard-Allan Scientific,

Kalamazoo, MI, USA) as a background.

Melanin staining, melanocyte quantification,

determination of epidermal thickness, and

enumeration of epidermal nucleated cellsAnimals were either killed by CO2 narcosis or anesthetized with

isoflurane anesthesia prior to skin sampling. Approximately 1 cm2

skin biopsies were obtained from sheared skin of animals

exposed to either forskolin or vehicle control at the indicated

times using institutionally approved protocols. Samples were

fixed in 10% buffered formalin (Sigma) and were paraffin embed-

ded and sectioned (6 microns) by the University of Kentucky his-

topathology core laboratory. Skin biopsies were stained for

melanin using the Fontana–Masson Staining Kit (American Mas-

ter*Tech Scientific, Inc., Lodi, CA, USA) (Zappi and Lombardo,

1984), which stains melanin black. Fontana–Masson stained sec-

tions were used for measurements of epidermal thickness, with

values reported for the perpendicular length of the epidermis

from the outside of the skin (edge of the stratum corneum) to

the bottom of the stratum basale. For epidermal thickness mea-

surements, only those fields were chosen that lacked obvious or

potential sectioning artifacts that might confound the data. For

enumeration of epidermal nucleated cells, paraffin sections

of treated skin were stained with DAPI (Sigma) as described,

and numbers of nuclei in the epidermis were quantified.



For LacZ-mediated identification of melanocytes in DCT-LacZ trans-

genic animals, frozen sections (6 microns) were prepared, b-galac-

tosidase staining and counterstaining with nuclear fast red were

performed as described (Franco et al., 2001; Mackenzie et al.,

1997), and numbers of blue-stained cells were quantified. Micro-

scopic evaluation of skin biopsies was performed using an Olym-

pus BX51 microscope (Center Valley, PA, USA), and images were

captured using the QCapture Pro program (QImaging Software,

Surrey, BC, Canada).

Melanin quantificationEumelanin and pheomelanin were quantitatively analyzed by HPLC

based on the formation of pyrrole-2,3,5-tricarboxylic acid (PTCA) by

permanganate oxidation of eumelanin and 4-amino-3-hydroxy-

phenylalanine (4-AHP) by hydriodic acid reductive hydrolysis of

pheomelanin, respectively. PTCA determination was performed by

a method modified from a previous report (Wakamatsu et al.,

2003). The eumelanin and pheomelanin content were calculated by

multiplying those of PTCA and 4-AHP by factors of 25 and 9,

respectively (Wakamatsu and Ito, 2002).

UV exposure and MED testingMc1re ⁄ e K14-SCF animals were treated topically as described with

either forskolin or vehicle control for the indicated amount of time.

They were then depillated by trimming hairs with surgical shears

as described above in conjunction with topical depilatory cream

(NairTM, Princeton, NJ, USA) used as directed 1 day prior to irradia-

tion. Mice were then sedated with ketamine ⁄ xylazine according to

standard veterinary dosing (Xu et al., 2007), and UV-occlusive tape

with holes punched in it was applied to the dorsal skin in order to

facilitate multiple UVB dosing on the same animal. Mice were

exposed to UV irradiation in a custom-made lucite chamber (Plastic

Design Corporation, Chelmsford, MA, USA) outfitted with a double

bank of UVB lamps (UV Products, Upland, CA, USA). UV emittance

was measured with the use of a UV photometer (UV Products)

equipped with UVB measuring head; the spectral output of the

lamps was determined to be roughly 75% UV-B and 25% UV-A.

Edema and ⁄ or erythema of the UV-exposed areas was scored

visually 24 h after irradiation, and the MED was calculated as the

minimal dose of radiation needed to cause erythema and ⁄ or edema

of the entire circle of exposed skin.

Statistical analysisStatistical comparisons between cohorts of control- versus

forskolin-treated animals were evaluated by a Tukey’s post-test.

Differences were considered statistically significant if the P value

was <0.05.

Acknowledgements

We wish to thank Cynthia Long (University of KY histopathology

core) for technical help, Dr. Michael Jay and Ronan O’Carra (Univer-

sity of KY College of Pharmacy) for assistance with tissue lyophil-

ization and Drs. Adria Hartmann and Eun Lee (University of KY

Department of Pathology) for interpretation of liver histology. We

also thank Dr. David Fisher (Harvard Medical School) for general

support and helpful suggestions. Funding sources include the

Wendy Will Case Cancer Research Fund, the National Cancer Insti-

tute (R03 CA125782-01A1; R21 CA127052-01A1), the Kentucky

Tobacco Research and Development Council, the Markey Cancer

Foundation and the Jennifer and David Dickens Melanoma

Research Foundation. MLS was supported by the Graduate Center

for Toxicology’s Department of Health and Human Service Public

Health Services Grant T32 ES-07266-17.

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