Grants and Contracts Details
A. SPECIFIC AIMS Fairness of skin correlates with diminished epidermal expression of eumelanin, the brown/black pigment responsible for dark complexion2 . Instead, there is preferential expression of pheomelanin, a sulfated blonde/red melanin species that is soluble and has poor UV-blocking abilities. Persons with high levels of pheomelanin and low levels of eumelanin in the skin have little basal protection from UV radiation, and therefore suffer the highest incidence of UV-mediated skin damage and skin cancer. To delineate the relationship between UV exposure and pigmentation and develop novel pigment-based UV protective strategies, we have developed a mouse model of "humanized skin" with inducible pigmentation based on topical administration of a natural root extract from the Coleus forskohlii plant. Like in humans, the fair-skinned phenotype in our animal model is caused by defective signaling in melanocytes by the melanocortin-1 receptor (MC1R), a protein that mediates adenylate cyclase activation and cAMP generation when bound by its natural ligand, melanocyte stimulating hormone (MSH)4. Similar to fair-skinned humans who tend to burn rather than tan after sunlight exposure, our fair-skinned animals demonstrate heightened UV sensitivity and failure to tan adaptively. Using this novel mouse model, we study melanin induction by forskolin, a labdane diterpenoid that directly activates adenylate cyclase and promotes intracellular accumulation of cAMP. By topically applying root extract from the C. forskohlii plant (the natural source of forskolin) to the skin, we can induce the production of UV-protective eumelanin by chemically circumventing the defective cAMP signaling associated with the mutant MC1R. Interestingly, we have observed that other root extract compounds, including the alkaloid piperine, enhance pro-pigmentation effects relative to pure forskolin. Using our convenient animal model of human skin of different melanin composition, we will directly modulate cAMP signaling and melanin levels to clarify the protective effects of C. forskohlii extract against UV-mediated skin damage. Specifically, we hypothesize that up-regulation of cutaneous pigmentation by a natural extract from the C. forskohlii plant will impart protection from UV-mediated DNA damage. To address our hypothesis, we propose three specific aims: Specific Aim 1. Define the protective effects of forskolin-mediated pigmentation in our murine model. To discern whether enhanced pigmentation imparts protection at the DNA level, we will study the effects of topical administration of either C. forskohlii extract or pure forskolin on UV-induced DNA damage and repair. Toward this end, we will i) characterize UV-induced DNA damage and repair in animals of varying skin pigmentation, and ii) quantify the melanin content and type for each murine line to compare DNA damage and repair with the melanin constitution. We will utilize appropriate statistical analyses to correlate DNA damage and repair with melanin type/content and forskolin source. As we have already determined that piperine (a root extract component) enhances forskolin-mediated pigmentation (see section C, Preliminary Data), we will include, in tandem, DNA damage and repair studies exploring the effects of purified forskolin in combination with piperine. Specific Aim 2. Characterize skin permeation of forskolin extract in our murine model system. To delineate differential permeation of forskolin vs. C. forskohlii root extract, we will compare pure vs. crude forskolin permeation in mouse skin, using statistical analyses to also explore any correlation between skin penetration and DNA damage and/or repair (as studied in Aim 1). Similar to Aim 1, we will perform a parallel permeation study exploring the effects of purified forskolin in combination with piperine. Specific Aim 3. Define the effects of forskolin, extract and pure, and piperine on cultured melanocytes and keratinocytes. Although our genetic system suggests that melanin induction by C. forskohlii extract likely occurs via a direct effect of the forskolin on melanocytes (through cAMP activation), it is feasible that keratinocyte-mediated signals playa role in C. forskohlii-induced pigmentation. This possibility is supported by the unique and intimate interaction that occurs between the melanocytes and keratinocytes in the basal layer of the epidermis (in humans and in our animal model). In this aim, we will expose cultured melanocytes and keratinocytes (individually and in co-culture) to forskolin extract, purified forskolin, purified forskolin with piperine, and piperine alone to determine their effects on cell activation, proliferation, calcium flux, and pigment synthesis. Aim 3 studies will set the foundation for future experiments that will address the molecular and cellular events driving eumelanin up-regulation via forskolin extract and facilitate the identification of other bioactive components in the forskolin extract.
|Effective start/end date||7/1/08 → 6/30/10|
- National Cancer Institute: $329,626.00
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