127 - Thioredoxin reductase 1 knockdown disrupts pigment synthesis in melanocytes

Abstract

Our labs have shown that levels of the antioxidant enzyme thioredoxin reductase 1 (TR1) is increased in human malignant melanomas relative to benign nevi (moles), and that TR1 knockdown affects mitochondrial metabolism in human melanomas. Here we report our examination of the role(s) of TR1 in normal melanocytes, the cells from which melanomas arise. Cellular processes affected by the TR1/thioredoxin (Trx) system in melanocytes are not well characterized. We created an immortalized human melanocyte cell line that stably expresses an siRNA directed against the TR1 transcript. As has been reported in other cell types, TR1 knockdown resulted minor effects on cell growth, likely due to a reciprocal upregulation of the synthesis of reduced glutathione. However we also found that TR1 knock out resulted in a significant decrease in melanin levels as well as reduced activity of tyrosinase, the rate limiting enzyme involved in melanin synthesis. Chemical inhibition of Trx using a small molecule inhibitor had a similar phenotype. Knockdown of TR1 caused a delay in upregulation of the melanocyte master transcriptional regulator, Mitf, after elevation of cAMP levels by treatment with forskolin, a common method of promoting melanin synthesis. We examined the effects of forskolin treatment on the canonical targets of Mitf regulation, but only Tyrp1 was affected, suggesting TR1/Txn affects another transcriptional regulator specific to Tyrp1. Given the role of Tyrp1 in stabilizing tyrosinase, this likely explains the low tyrosinase activity in TR1 knockout cells. Our preliminary data from a mouse model where TR1 is knocked out specifically in melanocytes shows a phenotype that is consistent with our cell culture data. These studies provide new insight into the redox signaling that controls melanogenesis, and may lead to novel targets for the diagnosis and treatment of human diseases involving melanocytes such as melanoma and vitiligo.