Mouse Anti-Human Microphthalmia Transcription Factor (MiTF) monoclonal antibody, clone JID733

It has been reported that 5-hydroxytryptamine (5-HT) is related to melanogenesis in mice and melanoma cells. However, the underlying mechanisms of 5-HT in regulating pigmentation remains unknown. In this study, we aim to clarify the regulatory mechanism of 5-HT in the pigmentation of zebrafish embryos and B16F10 cells. Our results show that 5-HT induces the pigmentation of zebrafish embryos in a dosage-dependent manner at concentrations of 0.01-1 mM. Whole mount in situ hybridizations and qRT-PCR in zebrafish embryos indicate that the expression of neural crest cells marker genesox10is not changed in embryos treated with 5-HT compared to control group. The expression ofmitfa, the marker gene of melanoblasts, is increased in the presence of 5-HT. Furthermore, 5-HT increased the expression of regeneration associated genes, namelykita,mitfa, anddct,after ablation of the melanogenic cells in zebrafish embryos. The experiments in B16F10 cells show that 5-HT promotes melanin synthesis by up-regulating the expression of key proteins MITF, TYR, TRP-1, and TRP-2. Especially, the small molecule inhibitor of PKA signaling, but not AKT and MAPK signaling, attenuates the up-regulation of MITF and TYR resulted from 5-HT induction in B16F10 cells. These results will help us to further understand the regulatory network of vertebrate pigmentation.

The MC1R/cAMP/MITF pathway is a key determinant for growth, differentiation, and survival of melanocytes and melanoma. MITF-M is the melanocyte-specific isoform of Microphthalmia-associated Transcription Factor (MITF) in human melanoma. Here we use two melanocyte cell lines to show that forced expression of hemagglutinin (HA) -tagged MITF-M through lentiviral transduction represents an oncogenic insult leading to consistent cell transformation of the immortalized melanocyte cell line Hermes 4C, being a melanocortin-1 receptor (MC1R) compound heterozygote, while not causing transformation of the MC1R wild type cell line Hermes 3C. The transformed HA-tagged MITF-M transduced Hermes 4C cells form colonies in soft agar and tumors in mice. Further, Hermes 4C cells display increased MITF chromatin binding, and transcriptional reprogramming consistent with an invasive melanoma phenotype. Mechanistically, forced expression of MITF-M drives the upregulation of the AXL tyrosine receptor kinase (AXL), with concomitant downregulation of phosphatase and tensin homolog (PTEN), leading to increased activation of the PI3K/AKT pathway. Treatment with AXL inhibitors reduces growth of the transformed cells by reverting AKT activation. In conclusion, we present a model system of melanoma development, driven by MITF-M in the context of MC1R loss of function, and independent of UV exposure. This model provides a basis for further studies of critical changes in the melanocyte transformation process.