Name: pharmacologic induction of epidermal melanin and protection against sunburn in a humanized mouse model
Uploaded: 2013-09-07T16:00:00
Description: Watch this Scientific Journal Video about Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model at JoVE.com

The authors wish to thank Malinda Spry for technical assistance. We also acknowledge current and past funding sources: the National Cancer Institute (R01 CA131075, R01 CA131075-02S1), the Wendy Will Case Cancer Research Fund, the Markey Cancer Foundation, the Children's Miracle Network and the Jennifer and David Dickens Melanoma Research Foundation.

1. Preparation of Forskolin for Topical Administration from a Crude Root Extract of the Plectranthus barbatus (Cohleus forskohlii) Plant
<ol>
	<li>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.</li>
	<li>Weigh 2...

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...

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 by inherited factors that influence cutaneous response to UV radiation. Skin pigmentation is one of the most important determinants of UV sensitivity, risk of sunburn and cancer risk. Melanoma occurs roughly twenty times more frequently in light-skinned persons compared to dark-skinned individuals 11-13.
	Melanin, a pigment produced by melanocytes in the epidermis, is the main determinant of skin complexion. Melanin comes in two major varieties: (1) eumelanin, a dark brown/black pigment effective at absorbing the energy of UV radiation, and (2) pheomelanin, a reddish/blonde pigment less effective at preventing penetration of UV photons into the skin. Skin color, UV sensitivity and melanoma risk are largely determined by epidermal eumelanin content 14-15. The more eumelanin in the epidermis, the less UV photons can penetrate into the skin. Because of low innate levels of eumelanin, fair-skinned individuals are much more prone to acute and chronic effects of UV radiation 16-18.	
	Skin pigmentation, melanoma risk and the ability to "tan" after UV exposure all correlate with the signaling ability of the melanocortin 1 receptor (Mc1r), a Gs-coupled seven transmembrane surface receptor on melanocytes 19-22. When Mc1r binds its cognate high-affinity ligand, &alpha;-melanocyte stimulating hormone (&alpha;-MSH), there is activation of adenylyl cyclase and production of the second messenger cAMP 23. The normal physiologic response of the skin after UV exposure includes epidermal production of &alpha;-MSH by keratinocytes 24-29. We and others hypothesize that keratinocyte-derived &alpha;-MSH binds to Mc1r on epidermal melanocytes, initiating downstream production of the cAMP second messenger through activation of adenylyl cyclase 30. cAMP levels control many aspects of melanocyte differentiation, including survival pathways, DNA repair and pigment synthesis. Mc1r signaling and cAMP clearly induce pigment enzyme levels and eumelanin production. When Mc1r signaling is intact and melanocytic cAMP levels are robust, eumelanin is produced and the skin darkens. However, if Mc1r signaling is defective and cytoplasmic cAMP levels remain low, pheomelanin is produced instead 1. Eumelanin synthesis can be stimulated pharmacologically by agents that raise cAMP levels 1,14,31-35.	
	Since the Mc1r protein is a major regulator of melanoma risk in humans 36-46, we are interested in mechanisms by which Mc1r protects melanocytes against UV-induced carcinogenesis. As a foundation for our studies, we generated a transgenic Mc1r-variant murine model on a pure C57BL/6 genetic background 1. In this model, stem cell factor (Scf) is constitutively expressed in the basal epidermis and epidermal interfollicular melanocytes are retained in the skin throughout life 47, in contrast to the non-transgenic mice in which melanocytes localize to the dermis in hair follicles. With the K14-Scf transgene incorporated, the epidermis becomes pigmented with the particular melanin pigments characteristic of the pigment strain of the animal 1. K14-Scf mice on the C57BL/6 genetic background with wild type Mc1r signaling have jet-black skin characterized by very high levels of eumelanin pigment. Not surprisingly, these animals are highly UV-resistant. In contrast, genetically matched K14-Scf C57BL/6 animals that harbor a mutant inactive Mc1r have almost no eumelanin in the epidermis. Instead, these "extension" animals (Mc1re/e) have a fair skin complexion caused by deposition of pheomelanin pigment (Figure 1A) and are much more UV-sensitive 48-49.	
	Pharmacologic compounds with chemical properties that allow penetration into the skin have been shown to potently induce eumelanin in the extension (Mc1re/e) K14-Scf animal model by directly manipulating cAMP levels in epidermal melanocytes in the skin. Melanin upregulation in this model has been reported by adenylyl cyclase activation 1 as well as phosphodiesterase 4 inhibition 35. In this article, we demonstrate the preparation and topical application of forskolin in extension (Mc1re/e) K14-Scf animals which model the fair-skinned UV-sensitive human. We show that twice daily application of the drug promotes accelerated melanization, that skin darkening is due to epidermal deposition of melanin pigment and that induced epidermal melanin protects against UV-induced sunburn through measurement of "minimal erythematous dose" (MED) 48.

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

pharmacologic induction of epidermal melanin and protection against sunburn in a humanized mouse model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-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.

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 (Mc1re/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 &mu;M per dose) for 5 days (Figure 2B). Effects of topical treatments on epidermal pigmentation were ...

Watch this Scientific Journal Video about Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model at JoVE.com

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

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.

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

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