The choroid within the human eye contains a rich milieu of cells including melanocytes. Human choroidal melanocytes (HCMs) absorb light, regulate free radical production, and were recently shown to... Show moreThe choroid within the human eye contains a rich milieu of cells including melanocytes. Human choroidal melanocytes (HCMs) absorb light, regulate free radical production, and were recently shown to modulate inflammation. This study aimed to identify key genes and pathways involved in the inflammatory response of HCMs through the use of RNA-seq. Primary HCMs were cultured from donor choroids, RNA was extracted from control and lipopolysaccharide (LPS)-treated HCMs, and mRNA was sequenced. Functional annotation and pathway analysis were performed using gene ontology and gene set enrichment analyses. Representative RNA-seq results were verified with RT-qPCR and protein measurements. We detected 100 differentially expressed genes including an array of CCL and CXCL cytokines and mediators of cell-cell and cell-matrix adhesion, such as ICAM1, CLDN1, CCN3, ITGA1 and ITGA11. Functional annotation showed that these gene sets control inflammatory pathways, immune cell trafficking, cell-cell adhesion, interactions with the extracellular matrix and blood vessels, angiogenesis and epithelial-to-mesenchymal transitions. Our study provides insights into the transcriptional regulation of primary HCMs in response to inflammatory stimuli and identifies novel melanocyte-driven mechanisms potentially involved in choroidal homeostasis and inflammation. Show less
Summary Melanoma antigen recognized by T cells 1 (MART-1) is a melanoma-specific antigen, which has been thoroughly studied in the context of immunotherapy against malignant melanoma and which is... Show moreSummary Melanoma antigen recognized by T cells 1 (MART-1) is a melanoma-specific antigen, which has been thoroughly studied in the context of immunotherapy against malignant melanoma and which is found only in the pigment cell lineage. However, its exact function and involvement in pigmentation is not clearly understood. Melanoma antigen recognized by T cells 1 has been shown to interact with the melanosomal proteins Pmel17 and OA1. To understand the function of MART-1 in pigmentation, we developed a new knockout mouse model. Mice deficient in MART-1 are viable, but loss of MART-1 leads to a coat color phenotype, with a reduction in total melanin content of the skin and hair. Lack of MART-1 did not affect localization of melanocytespecific proteins nor maturation of Pmel17. Melanosomes of hair follicle melanocytes in MART-1 knockout mice displayed morphological abnormalities, which were exclusive to stage III and IV melanosomes. In conclusion, our results suggest that MART-1 is a pigmentation gene that is required for melanosome biogenesis and⁄ or maintenance. Show less
Boorn, J.G. van den; Melief, C.J.; Luiten, R.M. 2011
G-protein-coupled receptors (GPCRs) have been implicated in the tumorigenesis and metastasis of human cancers and are considered amongst the most desirable targets for drug development. Utilizing a... Show moreG-protein-coupled receptors (GPCRs) have been implicated in the tumorigenesis and metastasis of human cancers and are considered amongst the most desirable targets for drug development. Utilizing a robust quantitative PCR array, we quantified expression of 94 human GPCRs, including 75 orphan GPCRs and 19 chemokine receptors, and 36 chemokine ligands, in 40 melanoma metastases from different individuals and benign nevi. Inter-metastatic site comparison revealed that orphan GPR174 and CCL28 are statistically significantly overexpressed in subcutaneous metastases, while P2RY5 is overexpressed in brain metastases. Comparison between metastases (all three metastatic sites) and benign nevi revealed that 16 genes, including six orphan receptors (GPR18, GPR34, GPR119, GPR160, GPR183 and P2RY10) and chemokine receptors CCR5, CXCR4, and CXCR6, were statistically significantly differentially expressed. Subsequent functional experiments in yeast and melanoma cells indicate that GPR18, the most abundantly overexpressed orphan GPCR in all melanoma metastases, is constitutively active and inhibits apoptosis, indicating an important role for GPR18 in tumor cell survival. GPR18 and five other orphan GPCRs with yet unknown biological function may be considered potential novel anticancer targets in metastatic melanoma. Show less
