Agouti signal protein regulation in human melanoma cells.

Abstract

Production of the pigment eumelanin is controlled by alpha-melanocyte stimulating hormone (alpha-MSH) stimulation of melanocortin 1 receptor (Mc1r), whereas production of pheomelanin results from agouti antagonism of alpha-MSH signalling through Mc1r. The role of agouti in mouse pigmentation has been extensively investigated but a role for agouti signalling protein (ASIP) in human pigmentation has not been determined. To determine whether ASIP regulates known melanogenic genes in humans, ASIP was over-expressed in a human melanoma cell line. Levels of mRNA and protein were measured in genes known to be up or down-regulated by agouti in the mouse, namely microphthalmia (Mitf), tyrosinase (Tyr), tyrosinase-related protein 1 (Tyrp1), dopachrome tautomerase (Dct), Mc1r, silver, initiation transcription factor 2 (Itf2) and mini chromosome maintenance protein 6 (Mcm6). These melanogenic genes were not found to be significantly up or down-regulated by ASIP at the transcriptional level in human melanoma cells. However, ASIP down-regulation of tyrp1 was observed at the translational level. To identify novel genes that may be regulated by ASIP in melanoma cells, microarrays were used to determine differences in gene expression between the control and ASIP transfected melanoma cells. The expression level of human RNAs were determined by microarray analysis using a 19,200 cDNA and a 19,200 oligonucleotide array representing 13,000 and 18,864 individual genes, respectively. Genes observed to be modulated by ASIP were confirmed by quantitative real-time polymerase chain reaction. Results identify five genes, namely PPARbeta, eIF-4B, RRM2, MINOR and EVI2B that are down-regulated by ASIP, indicating a likely role for ASIP in human melanogenesis.