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Renalase Inhibition Induces Melanogenesis Via MAPK and PI3K/AKT Signaling Pathways
*Lindsay Hollander1,2,3, *Xiaojia Guo1,3, *Robert Safirstein1,3, *Gary Desir1,3, Charles Cha1,3
1Yale University, New Haven, CT;2University of Connecticut, Farmington, CT;3VACHS, West Haven, CT
Objective: Renalase (RNLS) is a secreted flavoprotein we have previously demonstrated to promote the growth and progression of melanoma via signaling through the PI3K/AKT and MAPK pathways. The same pathways are vital to the melanocyte’s ability to regulate melanogenesis. Activation of ERK1/2 phosphorylates microphthalmia transcription factor (MITF) at serine 73, which leads to MITF ubiquitination and degradation. p38 activation leads to MITF activation and increased tyrosinase (Tyr) expression. PI3K/AKT inhibition leads to GSK3β activation and MITF phosphorylation at serine 289, which facilitates its binding to the M-box of the Tyr promoter. We tested the hypothesis that RNLS could downregulate melanogenesis by examining the effect of RNLS inhibition on melanin production. Design: Lentiviral particles were used to transduce and stably knockdown RNLS expression in B16-F10 melanoma cells. Intra- and extracellular melanin contents were compared via 475nm absorbance measurements following dissolution in 1N NaOH. Cell lysate mRNA and protein were evaluated with qRT-PCR and western blots to illustrate the involved pathways. Results: Inhibition of RNLS signaling significantly enhanced the melanogenesis of the melanoma cells. Intracellular melanin content increased 2.2-fold (p<0.001), and extracellular melanin content increased 2.0-fold (p<0.001) when RNLS expression was blocked. MITF gene expression was increased 2.6-fold (p=0.003) with the knockdown of RNLS. Western blot analysis demonstrated decreased ERK1/2 and PI3K/AKT activation and increased p38 activation, as well as increased expression of Tyr, tyrosinase-related protein 1 (TRP1), and tyrosinase-related protein 2 (TRP2) in the absence of RNLS. Conclusions: These results further validate that RNLS acts through the PI3K/AKT and MAPK signaling pathways in melanoma. Inhibition of RNLS signaling through these pathways leads to increased expression of MITF, Tyr, TRP1, and TRP2 and downstream increased cellular melanogenesis.
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