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. Author manuscript; available in PMC: 2010 Jan 1.
Published in final edited form as: J Invest Dermatol. 2008 Jun 26;129(1):162–166. doi: 10.1038/jid.2008.182

Forskolin protects keratinocytes from ultraviolet (UV) B-induced apoptosis and increases DNA repair independent of its effects on melanogenesis

Thierry Passeron 1, Takeshi Namiki 1, Hélène Passeron 1, Elodie Le Pape 1, Vincent J Hearing 1
PMCID: PMC2654621  NIHMSID: NIHMS95137  PMID: 18580960

Abstract

Melanin pigments provide efficient protection against ultraviolet (UV) B radiation but DNA repair also plays a key role in eliminating UV-induced damage and preventing the development of skin cancers. In this study, we demonstrate that forskolin, an agent that increases intracellular levels of cAMP, protects keratinocytes from UVB-induced apoptosis independently from the amount of melanin in the skin. Forskolin enhances the removal of the two major types of UVB-induced DNA damage, cyclobutane pyrimidine dimers and 6,4-photoproducts, by facilitating DNA repair. These findings suggest new preventive approaches with topical formulations of forskolin or other bioactive agents that could be applied to the skin before sun exposure to increase its ability to repair DNA damage.

Introduction

The relationship between ultraviolet (UV) B exposure and skin cancers has been clearly demonstrated (Cleaver and Crowley, 2002). Melanin pigments provide efficient protection against UVB radiation and their levels within the skin are inversely correlated with the DNA damage induced by UV exposure (Tadokoro et al., 2003; Yamaguchi et al., 2006). However, melanin content is not the only factor that plays a role in protecting the skin against UV radiation. The increased risk of UV-induced skin cancers in patients suffering from defects in DNA repair, such as occurs in xeroderma pigmentosum, clearly illustrates the critical role of DNA repair mechanisms (Yarosh et al., 2005a). It has been recently demonstrated that inducing pigmentation with forskolin (FSK), a chemical agent that increases intracellular levels of cAMP, provides effective protection against UVB-induced DNA damage and skin cancer development in mice deficient for a DNA repair enzyme (D’Orazio et al., 2006). However, controversial effects of FSK have been reported. FSK promotes the differentiation of melanocytes and stimulates pigmentation (Busca and Ballotti, 2000) but it has also been shown to increase the metastatic potential of melanoma cells and other cancer cells (Ormerod and Hart, 1989; Hill et al., 1990; DeVos et al., 1990; Wu et al., 2003). The action of FSK on keratinocytes is less studied but it has been shown to decrease the invasiveness of transformed murine keratinocytes (Santibanez et al., 2003). Interestingly, the repair of DNA can also be modulated by chemical compounds and in that context, calcineurin inhibitors have been recently shown to decrease DNA repair in keratinocytes after UVB exposure (Yarosh et al., 2005b). IL-12 and IL-18 were also shown to reduce UV-induced apoptosis by inducing DNA repair (Schwarz et al., 2002; 2006). A recent study has shown the critical role played by p53 in UV responses in the skin, which is effected by its stimulation of the POMC/MC1R cascade (Cui et al., 2007). In this study, we investigated the ability of FSK to enhance DNA repair in keratinocytes and we show its protective role against UVB-induced DNA damage beyond its previously known role in stimulating melanogenesis.

Results and Discussion

We first pretreated normal human keratinocytes with or without FSK for 30 min before irradiating them with 21 mJ/cm2 UVB. After subsequent solubilization of the cells, their constituent proteins were characterized by immunoblotting using an antibody to cleaved PARP to study the UV-induced apoptosis. Cleaved PARP was highly expressed 6 h after the UVB exposure but its expression was significantly reduced in cells pretreated with FSK (Figure 1A). We then repeated that experiment in a reconstructed skin model containing both melanocytes and keratinocytes. Skin tissues were fixed 6 h, 24 h and 48 h after UVB exposure at 21 mJ/cm2. The numbers of sunburn (apoptotic) cells were then counted and were significantly (p<0.05) reduced at all time points in skin pretreated with FSK (Figure 1B). The protective effect of FSK against UVB-induced apoptosis was confirmed by TUNEL assay (Figure 1C). Interestingly, since FSK was added only 30 min before the UVB irradiation, the melanin content was similar between skins treated or untreated with FSK, showing that the protection against apoptosis was not due to an increase in melanin levels within the 2 day time frame of the experiment (Figure 2A).

Figure 1. FSK protects keratinocytes from UVB-induced apoptosis.

Figure 1

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(a) Human epidermal keratinocytes were pretreated for 30 min with FSK (20 μM) or with DMSO alone (control) after which the medium was removed and new medium (without DMSO or FSK) was added during the exposure to UVB (21 mJ/cm2). After the UVB exposure, 20 μM FSK or DMSO as appropriate was added back to the medium and proteins were extracted 6 h later and immunoblotted with the cleaved PARP antibody. (b) Hematoxylin and eosin staining of reconstructed skins exposed to UVB (21 mJ/cm2) and treated for 30 min before exposure with or without 20 μM FSK. Skin specimens were fixed 6, 24 and 48 h after UV exposure and sunburn cells (arrowheads point to some examples) were counted. The mean number (± SD, n=3) of sunburn cells per cm is shown below each panel and the statistical significance from the untreated control is indicated. (c) TUNEL assays of reconstructed skins exposed to 21 mJ/cm2 UVB and treated 30 min before exposure with or without 20 μM FSK. Skin specimens were fixed 5 min, 6 h, 24 h and 48 h after the UV exposure. Apoptotic cells are stained green (arrowheads point to some examples) and nuclei are stained blue with DAPI. The mean number (± SD, n=3) of TUNEL-positive cells per cm is shown below each panel and the statistical significance from the untreated control is indicated (NS = not significant). All images in each panel are at the same magnification; bars = 100 μm.

Figure 2. FSK promotes the removal of UVB-induced CPD and 64PP but is independent of pigmentation.

Figure 2

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(a)Fontana-Masson staining of reconstructed skins exposed to 21 mJ/cm2 UVB and treated 30 min before UV exposure with or without 20 μM FSK. Skin specimens were fixed 6, 24 and 48 h after the UVB exposure. The mean (± SD, n=2) melanin content (in arbitrary units) is shown below each panel and the statistical significance from the untreated control is indicated (NS – not significant). (b) Immunostaining of reconstructed skins exposed to 21 mJ/cm2 UVB and treated for 30 min before exposure with or without 20 μM FSK. Skin specimens were fixed 5 min, 6 h, 24 h and 48 h after the UVB exposure. CPD (left) and 64PP (right) are stained red, nuclei in all images are stained blue with DAPI. The mean density (± SD, n=3) of CPD or 64PP damage per nucleus is shown below each panel and the statistical significance from the untreated control is indicated (NS = not significant). All images in each panel are at the same magnification; bars = 100 μm.

Thus, we hypothesized that the difference in protection against apoptosis elicited by FSK was not due to the amount of the initial DNA damage but was due to its more efficient removal. To test that hypothesis, we stained the reconstructed skins with antibodies to the two major types of DNA damage resulting from UVB, cyclobutane pyrimidine dimers (CPD) and 6,4-photoproducts (64PP). FSK-treated and untreated control skins showed similar levels of CPD and 64PP 5 min after UVB exposure (Figure 2B). However, after 6, 24 and 48 h, the amounts of CPD and 64PP damage were significantly (p<0.05) reduced in skins pretreated with FSK compared to untreated skins, which shows that FSK is able to enhance the removal of UVB-induced DNA damage. 64PP has been reported to be removed more rapidly than CPD, but not all 64PP are repaired in a few hours. In a previous study (Yamaguchi et al., 2006), we showed that removal of 64PP (and CPD) depends on the phototype of the skin. In dark skin, almost all 64PP staining was removed within 1 day. In contrast, in fair skin a significant amount of 64PP staining was still observed after 1 day and some even remained after 7 days. In this study, we used reconstructed skin from Asian donors which had small amounts of pigmentation at the moment of UV exposure. This could explain the relatively high level of 64PP still observed at 24 and at 48 h. Interestingly, in the reconstructed skins treated with FSK, almost all 64PP were removed at 48 h while significant amounts of 64PP were still observed in the untreated controls. Moreover, we also observed a quicker removal of 64PP than of CPD, particularly in FSK-treated skin. Previous studies have shown that FSK or α-MSH decreases the UV–induced apoptosis of melanocytes (Bohm et al., 2005; 2005; Hauser et al., 2006). We hypothesize that FSK promotes DNA repair via its well-known effect on increasing cAMP levels, perhaps by modulating DNA repair mechanisms, for example by increasing XPC or other DNA repair enzymes. Some studies linking the α-MSH/MC1R/ pathway (and thus cAMP signaling) to enhanced DNA repair have been reported (Bohm et al., 2005; Hauser et al., 2006; Abdel-Malek et al., 2008; Smith et al., 2008).

In sum, these results show that FSK is able to protect keratinocytes from UVB-induced apoptosis and increases DNA repair independent of its effects on melanogenesis. Although the induction of new pigment synthesis requires several days, the action of FSK on DNA repair appears to be efficient when applied only a few minutes before UVB exposure. Although the best time point to use FSK remains to be determined, the speed of action and efficiency of FSK to enhance DNA repair suggests new preventive approaches with topical formulations of FSK or other agonist agents that could be applied to the skin before sun exposure to enhance the DNA repair processes of skin cells.

Materials and Methods

Reagents, Cell Lines and Culture Conditions

Human epidermal keratinocytes (HEKa-APF) were grown at 37°C under 5% CO2 in EpiLife Medium supplemented with Human Keratinocyte Growth Supplement (HKGS) (Cascade Biologics, Portland, OR). FSK (20 μM) was purchased from Sigma-Aldrich (St-Louis, MO, USA). The UVB source used was a Phillips TL20W/12RS lamp (Philips, Eindhoven, Holland); the spectrum of the lamp is available at http://www.prismaecat.lighting.philips.com. A UVB detector (PMA2101, Solar Light Company Inc, Glenside, PA), coupled to a radiometer (PMA2100, Solar Light Company Inc) was used during each exposure to measure the dose applied.

Reconstructed Skin

The epidermal equivalent MelanoDerm® was obtained from MatTek Corp. (Ashland, MA, USA), using normal human keratinocytes and melanocytes obtained from Asian neonatal foreskin tissues. MelanoDerms were grown at the air/liquid interface of the maintenance medium MEL-NHM-113 (MatTek Corp) and culture medium was renewed every two days. Where noted, the epidermal samples were supplemented with 20 μM FSK 30 min before UVB exposure. FSK was dissolved in DMSO and the same concentration of DMSO was employed for mock-treated controls. Each experiment was performed in duplicate with similar results.

Immunoblotting

Cultures in 100 mm dishes were solubilized in M-PER® mammalian protein extraction reagent according to the instructions of the manufacturer (Pierce Biotechnology, Rockford, IL, USA) and Protease Inhibitor cocktail (Roche, Mannheim, Germany). Protein concentrations of extracts were measured using the BCA protein assay kit (Pierce). Cell extracts (15 μg) were separated on SDS polyacrylamide gels (Invitrogen, Carlsbad, CA). After electrophoresis, proteins were transferred electrophoretically from the gels to Invitrolon PVDF transfer membranes (Invitrogen). The PVDF membranes were incubated with antibodies to cleaved PARP (rabbit, 1:2000, Abcam) or GAPDH (rabbit 1:5000; Cell Signaling) overnight at 4°C. They were then incubated with horseradish peroxidase-linked anti-rabbit antibodies (at 1:1,0000, GE Healthcare, Buckinghamshire, UK) at room temperature for 1 h. Antigens were detected using the ECL-plus Western Blotting Detection System (GE Healthcare). Each experiment was performed at least in duplicate.

Histochemistry

Epidermal equivalents were fixed in paraformaldehyde, embedded in paraffin and sections were cut using standard techniques. Sections were deparaffinized in xylene and hydrated through a series of graded ethanols. Specimens were observed following haematoxylin and eosin staining, Fontana-Masson staining and immunohistochemistry. The expression of proteins of interest was detected by indirect immunofluorescence using the following as primary antibodies: CPD (TDM2) (mouse 1:20000, kind gift of Dr. Toshio Mori, Nara Medical University, Nara, Japan) or 64PP (mouse 1:1000, kind gift of Dr. Mori). Bound antibodies were visualized with appropriate secondary antibodies, Alexa Fluor® 488 goat anti-rabbit IgG (H+L), Alexa Fluor® 594 mouse anti-rabbit IgG (H+L), Alexa Fluor® 488 goat anti-mouse IgG (H+L) or Alexa Fluor® 594 goat anti-mouse IgG (H+L) (all from Molecular Probes, Eugene, OR, USA) at room temperature for 1 h at 1: 400 dilution with 10% goat serum. DAPI (Vector, Burlingame, CA; USA) was used as a counter-stain. The TUNEL assay was performed using the ApopTag Plus kit (Chemicon, Temecula, CA, USA) according to the recommendation of the manufacturer. Images were captured using a Leica DMR B/D MLD fluorescence microscope (Leica, Wetzlar, Germany) and a Dage-MTI 3CCD 3-chip color video camera (Dage-MTI, Michigan City, IN, USA) and were analyzed using Scion Image Software (Scion Corp., Frederick, MD, USA). This system allows one to eliminate background fluorescence and to quantitate fluorescence intensity from the original images; each value was recorded as integrated density over a given epidermal area. The formation of CPD or 64PP in epidermal nuclei was then expressed as the ratio of the intensity of red fluorescence (for DNA damage) over the intensity of blue fluorescence (for localization of nuclei) as described previously (Yamaguchi et al., 2006).Ten randomly selected areas of each specimen were photographed and quantitated for each condition. Melanin content was measured after Fontana-Masson staining and was also analyzed by Scion Image Software as described previously (Yamaguchi et al., 2008).

Statistical Analysis

Data are presented as means ± SD. Student’s t test was used to analyze differences. Values of p<0.05 are considered significant.

Acknowledgments

This research was supported by the Intramural Research Program of the NIH, National Cancer Institute. We would like to thank Dr. Ettore Appella, National Cancer Institute, for his useful comments and suggestions.

Abbreviations

64PP

6,4-photoproducts

CPD

cyclobutane pyrimidine dimers

UV

ultraviolet

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