Melanocortin 1 Receptor as regulator of protective responses against oxidative stress and UVR-induced DNA damage

Abstract

The melanocortin 1 receptor (MC1R) gene expressed in melanocytes encodes a G protein-coupled receptor (GPCR) activated by MSH. It is a well-established melanoma susceptibility gene that regulates the amount and type of melanin pigments formed within epidermal melanocytes. In response to ultraviolet radiation (UVR), the main external etiologic factor for melanoma, WT MC1R protects against melanomagenesis by a combination of pigmentation-dependent and independent mechanisms. The pigmentation-dependent component of MC1R protective action is accounted for by a switch from basal pheomelanogenesis to eumelanogenesis. Eumelanin is a photoprotective pigment owing to its absorption properties in the UVR spectrum and its free radical scavenging properties. Conversely, pheomelanin is a photosensitizer promoting reactive oxygen species (ROS) production upon exposure to UVR, and can reduce the intracellular antioxidant pool even in the absence of UVR. Concerning non-pigmentary actions, WT MC1R signaling activates antioxidant enzymes and DNA repair pathways. These processes have been thought to rely on the cAMP-pathway and are triggered by WT MC1R to maintain genomic stability. Accordingly, it is widely accepted that the efficient UVR response of melanocytes is strongly dependent on MC1R activity. Therefore, the role of intergenic splice isoforms and of skin cancer-associated (R-type) variants of poorly defined functional properties in the regulation of this response required further investigation. Human MC1R has an inefficient poly(A) site allowing intergenic splicing with its downstream neighbour TUBB3. Intergenic splicing produces 2 MC1R isoforms, Iso1 and Iso2, bearing the complete seven transmembrane helices from MC1R fused to TUBB3-derived C-terminal extensions, in-frame for Iso1 and out-of-frame for Iso2. Exposure to UVR might promote an isoform switch from canonical MC1R (MC1R-001) to the MC1R-TUBB3 chimeras, which might lead to novel phenotypes required for tanning. We expressed the Flag epitope-tagged intergenic isoforms in heterologous HEK293T cells and HMCs, for functional characterization. Iso1 was expressed with the expected size. Iso2 yielded a doublet of Mr significantly lower than predicted, and impaired intracellular stability. Iso1 and Iso2 bound radiolabeled agonist with the same affinity as MC1R-001, but their plasma membrane expression was strongly reduced. Decreased surface expression mostly resulted from aberrant forward trafficking, rather than high rates of endocytosis. Functional coupling of both isoforms to cAMP was lower than WT, but ERK activation upon binding of MSH was not impaired, suggesting imbalanced signaling from the splice variants. Heterodimerization of differentially labelled MC1R-001 with the splicing isoforms analyzed by co-immunoprecipitation was efficient and caused decreased surface expression of binding sites. Thus, UVR-induced MC1R isoforms might contribute to fine-tune the tanning response by modulating MC1R-001 availability and functional parameters. Accordingly, these isoforms show a similar behavior to many natural MC1R variant alleles associated with the RHC phenotype and with increased skin cancer risk. However, the possible role of R variants in protecting against UVR-induced DNA damage by pigment-independent mechanisms remained to be determined. MC1R variants associated with increased melanoma risk promote production of photosensitizing pheomelanins as opposed to photoprotective eumelanins. WT MC1R activates DNA repair and antioxidant defenses in a cAMP-dependent fashion. Since melanoma-associated MC1R variants are hypomorphic in cAMP signaling, these non-pigmentary actions are thought to be defective in MC1R-variant human melanoma cells and epidermal melanocytes, consistent with a higher mutation load in MC1R-variant melanomas. We compared induction of antioxidant enzymes and DNA damage responses in melanocytic cells of defined MC1R genotype. We also analyzed the enzymatic machinery and signaling pathway(s) responsible for these protective responses downstream of MC1R. Increased expression of CAT and superoxide dismutase (SOD1) genes following MC1R activation was cAMP-dependent and required a WT MC1R genotype. Conversely, pretreatment of melanocytic cells with an MC1R agonist before an oxidative challenge with Luperox decreased i) accumulation of 8-oxo-7,8-dihydroguanine (8-oxodG), a major product of oxidative DNA damage, ii) phosphorylation of histone H2AX, a marker of DNA double strand breaks and iii) accumulation of DNA breaks as estimated with comet assays. The repair of oxidative lesions was accounted for by induction of two key BER enzymes OGG and APE-1/Ref1 downstream of MC1R. These responses were comparable in cells WT for MC1R or harboring hypomorphic MC1R variants without detectable cAMP signaling. In MC1R-variant melanocytic cells, the DNA-protective responses were mediated by AKT via NOX stimulation. Conversely, in MC1R-WT melanocytic cells, high cAMP production downstream of MC1R blocked AKT activation, likely by blocking NOX-dependent increases in intracellular ROS, and was responsible for inducing BER enzymes and DNA repair. Accordingly, MC1R activation could promote repair of oxidative DNA damage by a cAMP-dependent pathway downstream of WT receptor, or via AKT in cells of variant MC1R genotype. Moreover, in MC1R-WT cells cAMP would efficiently induce antioxidant enzymes, mainly catalase, and the BER enzymes OGG and APE-1/Ref1 whereas variant MC1R would augment efficiently the expression of the two key BER enzymes but should have no effect on catalase levels or activity.