Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Summary

Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.

Abstract

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a G_s-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection ¹. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Introduction

The incidence of melanoma, the most deadly form of skin cancer, has increased dramatically over the last several decades in United States, particularly among fair-skinned individuals. Strong molecular and epidemiologic evidence implicates UV radiation as a major causative environmental factor ^2-5. Increased UV exposure in the form of sun exposure and tanning bed use is likely to be responsible for much of increases in melanoma incidence ^6-7. Melanoma risk seems particularly linked with sunburns ⁸, especially those early in life ^9-10. Risk of sunburn is linked not only to dose and intensity of UV exposure, but also b....

Protocol

1. Preparation of Forskolin for Topical Administration from a Crude Root Extract of the Plectranthus barbatus (Cohleus forskohlii) Plant

1. Protocols for murine experiments followed the guidelines for ethical conduct in the care and use of animals and were approved by the Institutional Animal Care and Use Committee at the University of Kentucky (Protocol # 00768M2004). The root extract is made up at 40% weight/volume in a standard dermatologic base of 70% ethanol, 30% propylene glycol.
2. Weigh 200 g of crude forskolin root extract and transfer it to a beaker. To make 500 ml of a 40% (w/v) solution, resuspend 200 g of crude forskolin root extr....

Results

C57BL/6 mice were generated on eumelanotic, pheomelanotic or amelanotic backgrounds incorporating the K14-Scf transgene as described (Figure 1A). Cohorts of fair-skinned extension (Mc1r^e/e, Tyr^+/+) mice were treated topically with twice daily doses of either vehicle (70% ethanol, 30% propylene glycol) or 40% crude Coleus forskohlii root extract (80 μM per dose) for 5 days (Figure 2B). Effects of topical treatments on epidermal pigmentation were det.......

Discussion

Using an animal model of the fair-skinned human, we find that topical application of a forskolin-rich crude root extract robustly darkens the epidermis by stimulating melanin production in the skin. Epidermal melanization is dependent on the expression of stem cell factor in the basal epidermis, as occurs in human skin but not in genetically-unmodified mouse skin. The dorsal skin of genetically-unmodified mice lacks sufficient numbers of interfollicular melanocytes to impart pigment to the skin. Only in the setting of co.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
Coleus Forskoli extract 20%	Buckton Scott USA Inc.	n/a	Princeton, NJ
Isothesia, Isoflurane , USP	Butler Schein	NCD 11695-6776-1	Dublin, OH, USA
Xylazine	Anased Injection	LA04612	Shenandoah, Iowa, USA
Ketamine HCl, USP	Putney	NDC 26637-411-01	St. Joseph, MO, USA
Ethanol	Decon Labs.	2705
Propylene glycol	Adesco	05751L	Solon, OH, USA
Depilatory cream, Nair	Church Dwight	JF-11 4381322	Priceton, NJ
EQUIPMENT
Germicidal Hg Lamp UV-B	Westinghouse	F15T8UV-B
Radiometer photometer	International light	1LT400A	Peabody, MA,USA
Chromameter	Konica Minolta	CR-400	Ramsey, NJ, USA
Data Processor for Chromameter CR-400	Konica Monilta	DR-400	Ramsey, NJ, USA