While genetic and biochemical investigations have led to the identification of genes that regulate pigment production, the mechanisms by which melanocytes couple UV irradiation to melanin production has not been identified. Genome wide siRNA screening was performed to identify human genes that regulate melanogenesis in melanocytes and melanoma cells. Validation studies determined that this novel, unbiased approach had a low false positive and off-target rate, and identified novel genes that regulate melanin secretion and melanin biosynthesis. Two validated gene targets identified in this analysis, aldehyde dehydrogenase 1 and aldehyde dehydrogenase 9, are cellular genes involved in metabolizing byproducts of lipid peroxidation reactions. Preliminary data indicates that Aldhl and AldhQ impact the expression of tyrosinase and MITF in normal melanocytes and melanoma cells. The overall goal of this proposal is to understand the mechanism by which Aldhl and Aldh9 regulate melanin biosynthesis.
Specific aim 1 will determine the mechanism by which Aldhl and AldhQ regulate tyrosinase and MITF protein levels.
Specific aim 2 couples siRNA based loss of function approaches with chemical approaches to quantitate melanin to determine if Aldh activity impacts pheomelanin production.
Specific aim 3 will couple loss of function approaches with standard plate assays to determine whether Aldh activity is required for UVA and UVB induced pigment production. Followup experiments will determine if Aldhl and Aldh9 maintain a permissive environment for melanin production by maintaining low intracellular levels of reactive oxygen species and lipid peroxidation products.
Specific aim 4 will use novel imaging modalities and shRNA loss of funciton technology to determine whether Aldhl and Aldh9 impact pigment production in a human skin equivalent model. Through these studies, we will determine the mechanism by which Aldhl and Aldh9 regulate melanin production and gain a more complete understanding of the molecular mediators of UV induced pigment production.
UV induced melanin production protects the skin from the carcinogenic effects of UV light. While core genes that regulate pigment production have been identified, little is understood about how melanocytes produce more melanin in response to UV. In this proposal, we characterize putative regulators (Aldhl and Aldh9) of UV induced tanning and determine whether these genes regulate tanning in human skin models.
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