• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of fasebjClick here to go to the full-featured web site.Click here to learn how to submit an article to The FASEB Journal.Never miss an issue. Click here to subscribe to The FASEB Journal s full content.Click here for the latest news from The FASEB Journal.Find out what s being published every day. Click here to subscribe to RSS feeds.Click here to visit The FASEB Journal's full-featured site.
FASEB J. Oct 2010; 24(10): 3850–3860.
PMCID: PMC3229421

Melanocortin 1 receptor genotype: an important determinant of the damage response of melanocytes to ultraviolet radiation

Abstract

The melanocortin 1 receptor gene is a main determinant of human pigmentation, and a melanoma susceptibility gene, because its variants that are strongly associated with red hair color increase melanoma risk. To test experimentally the association between melanocortin 1 receptor genotype and melanoma susceptibility, we compared the responses of primary human melanocyte cultures naturally expressing different melanocortin 1 receptor variants to α-melanocortin and ultraviolet radiation. We found that expression of 2 red hair variants abolished the response to α-melanocortin and its photoprotective effects, evidenced by lack of functional coupling of the receptor, and absence of reduction in ultraviolet radiation-induced hydrogen peroxide generation or enhancement of repair of DNA photoproducts, respectively. These variants had different heterozygous effects on receptor function. Microarray data confirmed the observed differences in responses of melanocytes with functional vs. nonfunctional receptor to α-melanocortin and ultraviolet radiation, and identified DNA repair and antioxidant genes that are modulated by α-melanocortin. Our findings highlight the molecular mechanisms by which the melanocortin 1 receptor genotype controls genomic stability of and the mutagenic effect of ultraviolet radiation on human melanocytes.—Kadekaro, A. L., Leachman, S., Kavanagh, R. J., Swope, V., Cassidy, P., Supp, D., Sartor, M., Schwemberger, S., Babcock, G., Wakamatsu, K., Ito, S., Koshoffer, A., Boissy, R. E., Manga, P., Sturm, R. A., Abdel-Malek, Z. A. Melanocortin 1 receptor genotype: an important determinant of the damage response of melanocytes to ultraviolet radiation.

Keywords: DNA repair, melanoma susceptibility, MC1R variants, cAMP pathway

Epidermal melanocytes are the precursors for melanoma, the most fatal form of skin cancer. The incidence of melanoma continues to increase worldwide, especially in young adults (15). The resistance of melanoma tumors to conventional chemotherapy and radiation and lack of a cure for metastatic disease have made it necessary to identify genetic markers for melanoma that should lead to more accurate risk assessment and thus prevention.

The melanocortin 1 receptor (MC1R) has emerged as a melanoma susceptibility gene. The MC1R is a highly polymorphic gene and a major contributor to the diversity of human pigmentation (68) Some MC1R variants, particularly R151C, R160W, and D294H, are strongly associated with red hair color in humans (hence the name RHC alleles) and increased melanoma risk (710). An inverse correlation exists between the incidence of sporadic melanoma, on one hand, and constitutive pigmentation and tanning ability, on the other (2). Cutaneous pigmentation is determined by the relative rate of synthesis of the brown/black eumelanin and the red/yellow pheomelanin by epidermal melanocytes (11, 12). Eumelanin is the major contributor to skin pigmentation and photoprotection because of its efficiency in blocking ultraviolet rays (UV) and scavenging reactive oxygen species (13).

The principle physiological regulators of eumelanin synthesis in human melanocytes (HMs) are α-melanocortin (α-MSH) and adrenocorticotropic hormone, the physiological agonists of the MC1R (1416). The MC1R is a Gs-protein-coupled receptor with 7 transmembrane domains that is expressed on the cell surface of HMs (17, 18). Binding of the human MC1R by its ligands activates the cAMP signaling pathway, a major regulator of HM proliferation and pigmentation (19). We have made the seminal observation that in addition to their important role as the physiological regulators of pigmentation, melanocortins also modulate the UV response by enabling HMs to overcome the UV-induced growth arrest and apoptosis and reducing DNA damage (2022). We found that α-MSH enhances repair of UV-induced DNA photoproducts and reduces the induction of oxidative DNA damage (21, 22). These effects were dependent on activation of the MC1R and preceded the increase in melanin content in response to α-MSH treatment. Thus activation of MC1R is expected to reduce the genotoxic effects of UV exposure, independently of increased melanin content.

Evidence for the effect of MC1R genotype on melanoma risk came from the observation that RHC alleles increase the penetrance of mutations in cyclin-dependent kinase inhibitor 2A (2325) and that expression of an MC1R variant increases the chance for the somatic V600E BRAF-activating mutation (26). Expression of 2 RHC alleles results in loss of function of MC1R (27). However, the interaction of these variants with other alleles and their impact on the response of HMs to UV, a major environmental etiological factor for melanoma, are not understood. Unraveling the molecular mechanisms by which MC1R alleles affect genomic stability of melanocytes and the risk for melanoma will lead to improved preventive strategies and effective treatments for melanoma, one of the most challenging human cancers.

In this study we used primary HM cultures that naturally express different MC1R genotypes to determine systematically the impact of RHC alleles and other common MC1R variants on receptor function and response to UV. Our data demonstrate that loss of function of MC1R due to expression of 2 MC1R RHC variants, or an RHC allele and V60L variant, result in an aberrant UV response, and that the RHC alleles are not equivalent in their impact on receptor function. Our results provide compelling evidence that functional MC1R is required for optimal photoprotection and maintenance of genomic stability and elucidate mechanisms by which MC1R variants determine predisposition to melanoma.

MATERIALS AND METHODS

Culturing of human melanocytes

Primary cultures of HMs were established as described previously (15) from discarded neonatal foreskins or adult skin from patients undergoing elective plastic surgery procedures. The Institutional Review Board at the University of Cincinnati has deemed the protocol for obtaining these skin samples exempt from approval. Melanocyte cultures were also established from skin biopsies obtained from patients of the Melanoma Clinic at the Huntsman Cancer Institute after informed consent. For all experiments, HMs were maintained in medium devoid of bovine pituitary extract (BPE) to allow for response to exogenous α-MSH, as described previously (15).

Analysis of eumelanin and pheomelanin contents of cultured HMs

Melanocytes (2–4×106 cells) were harvested, pelleted, lyophilized, and used for quantitation of eumelanin and pheomelanin, respectively, as described previously (27).

Sequencing of the MC1R gene

The entire coding region of the MC1R was amplified using reverse transcriptase and nested PCR amplification, followed by sequencing as described previously (27).

Determination of cyclic AMP (cAMP) levels and tyrosinase activity

Cyclic AMP levels were determined using 125I-labeled radioimmunoassay kit (Perkin Elmer, Waltham, MA, USA), as described previously (18, 27), in HMs that were treated with 0 (control), 1, or 10 nM α-MSH, or 1 μM forskolin for 45 min in the presence of 0.1 mM isobutyl methylxanthine. Tyrosinase activity was measured after treatment of melanocytes with 1 or 10 nM α-MSH or 1 μM forskolin for 6 d, as described previously (15, 18, 27).

Irradiation of HMs with UV

Melanocytes were irradiated with a bank of FS 20 lamps, with 75% emission in the UVB, and 25% emission in the UVA spectra, with peak emission at 313 nm wavelength. Any emission of UVC rays from the UV source (FS 20 lamps) was blocked by a Kodacel filter (Eastman Kodak, Rochester, NY, USA). Prior to irradiation, the culture medium was replaced by PBS, which was removed after UV exposure, and fresh medium was added.

Measurement of UV-induced generation of hydrogen peroxide

Melanocytes were irradiated with 105 mJ/cm2 UV and treated immediately while in PBS with 0, 1 nM α-MSH or 1 μM forskolin. Generation of hydrogen peroxide was measured at 45 min after UV exposure, as described previously (21).

Quantitation of cyclobutane pyrimidine dimers (CPDs)

CPDs were detected using flow cytometry analysis. Melanocytes were pretreated with 0 (control), 10 nM α-MSH, or 1 μM forskolin for 4 d prior to, and 2 d after, exposure to 105 mJ/cm2 UV, harvested 48 h post-irradiation, and fixed with 70% ethanol; the DNA was denatured with 1N HCl, then renatured with sodium tetraborate. Melanocytes were incubated overnight at 4°C with the anti-CPD antibody (diluted 1:500; TDM-2 clone), then with goat anti-mouse IgG Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA) for 1 h, and finally resuspended in PBS containing RNase A and propidium iodide. Melanocytes were analyzed on a Coulter EPICS XL flow cytometer (Beckman Coulter, Miami, FL, USA) using a 488-nm argon ion laser.

Measurement of UV-induced apoptosis by Annexin Va staining

Melanocytes were maintained for the duration of the experiment in medium lacking BPE, and phorbol 12-myristate 13-acetate (PMA; TPA), to determine the survival effect of α-MSH in the absence of the antiapoptotic effect of TPA. Melanocytes were treated with 0, 1 nM α-MSH or 1 μM forskolin for 4 d prior to, and 24 h after, exposure to a dose of 105 mJ/cm2 UV, then stained for Annexin Va, as described previously (21).

Transfection of HMs expressing loss-of-function MC1R with the wild-type MC1R

Melanocytes naturally expressing nonfunctional MC1R (2n; R160W/D294H) were transfected with pcDNA3 plasmid containing the wild-type MC1R gene (pMC1R-WT) and pcDNA3 vector only, using the electroporation method from Amaxa Transfection System (Lonza Walkersville, Walkerville, MD, USA), then selected for stable transfection in medium containing geneticin.

Ultrastructural visualization of DOPA-stained HMs

Parental HMs expressing loss-of-function MC1R and the same HM strain stably transfected with wild-type MC1R were plated in 4-well chamber slides and treated for 4 d with 0 (control) or 1 nM α-MSH. Cells were processed for electron microscopy as described previously (28). Microtome sections were analyzed in a Jeol JEM-1230 transmission electron microscope (Jeol Ltd., Akishima, Japan), and photomicrographs (×15,000) of the perinuclear area and dendrites were taken for each experimental group. The number (n=200) and stage of the melanosomes in the perinuclear area and dendrites were determined in the control and α-MSH-treated cells from parental and transfected HM.

Gene expression profiling and data analysis

Total RNA was isolated from HMs treated with 1 nM α-MSH as described for experiments to measure CPD, 8 and 24 h after UV exposure, using RNeasy columns following the manufacturer's protocol (Qiagen, Valencia, CA, USA). After quality checks were performed with the Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), RNA was used to generate the cDNA that was hybridized with in-house (Genomics and Microarray Core of the University of Cincinnati) glass slide arrays (OmniGrid, multiaxis microarrayer). The slides were prepared using the DNA source from the Research Genetics Human UNIGENE Library (31,742 clones) and scanned, and differential gene expression levels were determined by the calculated ratio of Cy-3 to Cy-5 emission, using the Axon GenePixPro 3.0 software (Axon Instruments, Foster City, CA). For data analysis, only genes with an expected false positive less than one and more than 2-fold change in expression were considered. A complete list of genes included in the microarray analysis can be found online (http://microarray.uc.edu). The arrays were normalized and tested for differential expression using the methods described in Borchers et al. (29).

Detection of MC1R cell surface expression

Melanocytes were treated with 0 (control) or 10 nM α-MSH and/or irradiated with 105 mJ/cm2 UV. At 14 h after α-MSH treatment or 24 h after UV exposure, the culture dishes were placed at 4°C for 2 h, and HMs were harvested with cold 5 mM EDTA. The cells were incubated with 4 μg/ml rabbit anti-MC1R (H-60; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at 4°C, then incubated with 20 μM goat anti-rabbit Quantum Dot 655 for 1 h (Invitrogen). The cells were postfixed in 1% paraformaldehyde and analyzed using a BD LSRII flow cytometer (Becton-Dickinson, San Jose, CA, USA). Ten thousand events were collected for each sample.

Western blot analysis

Melanocytes were treated as described for the microarray experiments and cell extracts were obtained 24 or 48 h after UV irradiation. Western blotting was carried out to validate some of the microarray data, using the following antibodies for Mitf (a gift from David Fisher, Harvard University, Cambridge, MA, USA), Cdk2 (M2), PCNA (F-2), (Santa Cruz Biotechnology), Peroxiredoxin 1 (Prx1; AbCam, Cambridge, MA, USA), Bcl2 (Ab-1) (EMD, San Diego, CA, USA), and DDB1 (Zymed Laboratories, San Francisco, CA, USA). As control for loading, antibodies against histone H1 (AE-4) and actin (C-11) were used (Santa Cruz Biotechnology).

RESULTS

MC1R genotype and activation of MC1R

A total of 21 different primary HM cultures, 14 derived from neonatal foreskins (n) and 7 from individual adult (a) skin samples, were established as described previously (15) and utilized for the experiments described below. MC1R genotypes, as well as eumelanin and pheomelanin contents, were determined (Table 1), as described previously (27). Responsiveness to α-MSH was first determined by measuring the increase in cAMP levels and tyrosinase activity. Strains highly responsive to α-MSH were those homozygous for the wild-type MC1R (+/+; 17n-19n, 20a, and 21a), or heterozygous for the pseudoalleles (p/+) V92M (14n and 15a) or R163Q (16n), or the mild allele V60L (r/+; 13n) (Fig. 1). These strains exhibited >70% increase in cAMP levels and ≥2-fold increase in tyrosinase activity above control in response to 10 nM α-MSH. In contrast, HM strains that were unresponsive to α-MSH were those homozygous or compound heterozygous for 2 RHC alleles (R/R; 1n, 2n, 3a, 4n, 5n), or compound heterozygous for the RHC allele R160W and V60L (R/r; 6n and 7a).

Table 1.
MC1R genotype, eumelanin and pheomelanin content, and response to α-MSH in various assays of HM strains
Figure 1.
Effects of α-MSH on cAMP generation (A) and tyrosinase activity (B) of HM strains with different MC1R genotypes. Data points represent the mean ± se percentage of control of triplicate determinations. Data presented are representative ...

Results from HMs heterozygous for RHC alleles (R/+) were variable. Strain 12a (R151C/+) was highly responsive to α-MSH in both assays, whereas 10n (R160W/+) and 11n (D294H/+) demonstrated a relatively low (~50% increase above control), yet statistically significant, stimulation of cAMP and no or modest increase in tyrosinase activity, respectively, following α-MSH treatment (Fig. 1). Statistical analysis revealed that 12a had a significantly higher response than 10n to 10 or 1nM α-MSH, and in turn 10n had a significantly higher response than 11n to 1 nM α-MSH in the cAMP assay, as determined by one-way ANOVA. As for activation of tyrosinase activity, 12a had a statistically higher than 11n in response to 1 or 10 nM α-MSH, as determined by Student's t test. Expression of R163Q with R160W in 8n did not contribute further to the disruptive effect of the latter RHC allele (10n) on MC1R activation by α-MSH, because there was no statistical difference in the response of the 2 strains to 1 or 10 nM α-MSH in the cAMP assay. Melanocytes with this genotype exhibited a modest increase in cAMP (50% above control) and marked (2.5-fold) stimulation of tyrosinase activity in response to 10 nM α-MSH. As in 12a, which is heterozygous for R151C, 9a, compound heterozygous for R151C and V92M, responded dramatically to 10 nM α-MSH with 4-fold increase in cAMP formation, and 2.5-fold stimulation of tyrosinase activity. Depending on the availability of HMs, forskolin, a direct activator of adenylate cyclase, was used as a positive control and found to increase cAMP levels and tyrosinase activity, regardless of MC1R genotype.

MC1R genotype and response of HMs to UV

We have reported that α-MSH inhibits the UV-induced generation of hydrogen peroxide by HMs within minutes (21, 22). Melanocyte strains 7a (V60L/R160W), and 2n, 3a, 4n, and 5n, which express 2 RHC alleles, with the exception of 1n, which was homozygous for R160W, failed to respond to α-MSH with significant inhibition of hydrogen peroxide generation, as measured 45 min after exposure to 105 mJ/cm2 UV (Fig. 2A). In contrast, all remaining strains showed a marked decrease (25–65%) in hydrogen peroxide generation after α-MSH treatment.

Figure 2.
Effects of α-MSH on the UV-induced hydrogen peroxide generation, repair of CPD, and apoptosis of HM strains with different MC1R genotypes. A) Effects of 1 nM α-MSH or 1 μM forskolin on UV-induced generation of hydrogen peroxide ...

In preliminary experiments, we compared induction of CPD immediately after UV irradiation in untreated vs. α-MSH-treated HMs and found that α-MSH did not affect initial CPD formation (data not shown). However, treatment with α-MSH enhanced the repair of CPD in HMs, as measured 48 h after UV exposure, except in those expressing 2 RHC alleles (1n–5n) or compound heterozygous for V60L and R160W (7a) (Fig. 2B). The latter strains maintained high levels of CPD with no evidence of significant removal. These results were validated using Southwestern blot analysis of 2 HM strains, 1n (R160W/R160W) and 4n (R151C/D294W) (data not shown).

We examined the effect of α-MSH on UV-induced apoptosis (Fig. 2C). With the exception of HM strains expressing 2 RHC alleles (1n, 2n, 3a, 4n, 5n), 6n and 7a (V60L/R160W), 14n (V92M/+) which was the most resistant to UV-induced apoptosis, and the 2 strains 9a (R151C/V92M), and 10n (R160W/+), the remaining HM strains responded to 1 nM α-MSH with reduction of the apoptotic effect of 105 mJ/cm2 UV. The latter 2 HM strains (9a and 10n) were responsive to the antiapoptotic effect of forskolin but not α-MSH, despite significant reduction in hydrogen peroxide generation and enhanced CPD repair in response to α-MSH treatment, suggesting inefficient activation of survival pathways by the dose of 1 nM α-MSH (Fig. 2A, B).

Transfection with wild-type MC1R restores MC1R function and normalizes the UV response of HMs naturally expressing 2 RHC alleles

To establish the causality between lack of response to α-MSH and the aberrant response to UV, we stably transfected the HM strain 2n (R160W/D294H) with wild-type MC1R. Electron microscopic examination of HMs transfected with the wild-type MC1R and incubated with L-DOPA, the substrate for tyrosinase, showed that unlike the parental strain, they responded to α-MSH with stimulation of melanogenesis (Fig. 3A), evidenced by enhanced maturation of melanosomes (an increase in the number of mature stage III and IV melanosomes) in the perinuclear area and dendrites, due to increased deposition of melanin. In HMs transfected with the wild-type MC1R, α-MSH reduced UV (105 mJ/cm2)-induced hydrogen peroxide production, enhanced repair of CPD, and inhibited apoptosis (Fig. 3B–D).

Figure 3.
Transfection of HMs with loss-of-function MC1R with the wild-type MC1R restores responsiveness to α-MSH and normalizes the response to UV. HM strain 2n (R160W/D294H) was stably transfected with empty vector or wild-type MC1R. A) Electron microscopic ...

α-MSH- and UV-altered gene expression

We compared global gene expression in 2 HM strains, 13n (V60L/+), highly responsive to α-MSH (HM-F), and 2n (R160W/D294H) with loss-of-function MC1R (HM-LOF) (Fig. 4A), as determined by cAMP and tyrosinase activity assays (Table 1 and Fig. 1). The groups that were directly compared against each other are shown in Fig. 4A. The changes in gene expression resulting from α-MSH-treatment were observed exclusively in 13n, HM-F (compare lanes 1 and 2 that were almost black, indicating no alteration in gene expression, and lanes 3 and 4 of the α-MSH treated groups in the heat map; Fig. 4C). In HM-F, we identified changes in expression of genes associated with functional categories such as cell adhesion, transcription factors, intracellular signaling, protein trafficking, and biosynthesis in response to α-MSH (Fig. 4B). Global gene expression in HM-F (13n) showed that a total of 3458 genes were significantly altered in expression, with 668 genes affected only by α-MSH, 454 genes affected only by UV, and 2336 genes influenced by both α-MSH and UV.

Figure 4.
Microarray experimental design, heat map, and data analysis. Microarray experiments were carried out on 13n that expresses functional MC1R (HM-F) and 2n that expresses loss-of-function MC1R (HM-LOF). A) Schematic representation of microarray experimental ...

Because our aim was to investigate the photoprotective mechanisms of α-MSH, we focused our analysis of the microarray data on gene categories that were melanocyte-specific or involved in control of melanogenesis, oxidative stress, DNA repair, cell cycle, and apoptosis. Data analysis of genes belonging to these functional categories revealed that α-MSH had the opposite effect of UV on the expression of 34.6% of all UV and α-MSH-responsive genes. Of those, 78% were up-regulated by α-MSH (Fig. 4D). In contrast, in HM-LOF, comparison of the group treated with α-MSH and irradiated with UV to that only irradiated with UV revealed no differences in gene expression between the 2 groups, indicating lack of modulation of the UV response in HM-LOF by α-MSH. Similarly, comparison of HM-LOF treated with α-MSH and irradiated with UV to the same HMs treated with α-MSH showed that the genes that were altered were those only affected with UV, further confirming lack of response of these HMs to α-MSH. Important conclusions derived from the microarray data were that 1) α-MSH mostly induced, while UV mainly suppressed, gene expression; 2) α-MSH and UV had opposing effects on many of the analyzed genes, and α-MSH modulated the effects of UV on these genes (Supplemental Data); and 3) loss of function of MC1R compromised the UV response of HMs, due to absence of the effects of α-MSH on important genes that affect the DNA damage response.

Validation of microarray data

To validate the microarray data, we selected genes representative of the functional categories of interest. The microarray result showed that MC1R expression was suppressed 7-fold by UV, and induced 7-fold by α-MSH. We confirmed these results by demonstrating with immunostaining increased cell surface expression of MC1R in HMs following α-MSH treatment, and its reduction following exposure to UV (Fig. 5A). By Western blotting, we verified that the transcription factor Mitf, the master regulator of melanogenesis and melanocyte survival (30), was induced by α-MSH and repressed by UV (Fig. 5B). Peroxiredoxin1 (Prx1), which reduces endogenous peroxides (31, 32), was one of the most α-MSH up-regulated oxidative stress regulatory genes that was suppressed by UV, according to the microarray data. Western blotting showed increased protein levels of Prx1 in response to α-MSH, and decreased levels following UV exposure (Fig. 5B). The 2 DNA repair genes, damaged DNA binding protein 1 (DDB1), which promotes global genomic repair in the nucleotide excision repair pathway and regulates DNA replication (33), and proliferating cell nuclear antigen (PCNA), which plays important roles in DNA replication and repair as well as DNA methylation, chromatin assembly, and remodeling (34, 35), were found to be increased by α-MSH, and the latter to be repressed by UV. Western blot analysis of DDB1 and PCNA revealed that their levels were increased by α-MSH and reduced by UV, and that α-MSH partially reversed the inhibitory effects of UV (Fig. 5B). We validated by Western blotting the expression of cyclin-dependent kinase 2 (CDK2), which binds to cyclin E, to allow entry of cells into S phase, or to cyclin A, to enhance progression to G2 phase (36) and found CDK2 protein level to be increased by α-MSH and diminished by UV, in agreement with the microarray data (Fig. 5B). The antiapoptotic Bcl2 is critical for melanocyte survival (37); it was reduced at the protein level by UV and increased by α-MSH, which confirmed the microarray results and our previous findings (21).

Figure 5.
Validation of altered expression of selected genes belonging to the melanocyte-specific, oxidative stress, DNA damage, cell cycle, and apoptosis functional categories. A) Altered cell surface expression of MC1R in HM-F, as determined by immunostaining ...

DISCUSSION

In the present study, our goal was to elucidate the impact of RHC MC1R alleles expressed as heterozygous, homozygous, or compound heterozygous with other commonly expressed alleles, in comparison to pseudoalleles (V92M and R163Q) and wild-type allele, on MC1R function and the UV response of HMs. The MC1R is a melanoma susceptibility gene (7, 8, 10, 2325); however, the molecular mechanisms by which MC1R protects HMs from malignant transformation to melanoma are not understood. By comparing a panel of HM strains that naturally express different MC1R genotypes (Table 1), we found that those expressing 2 RHC alleles (1n-5n) or compound heterozygous for V60L and R160W (6n, 7a) were refractory to α-MSH (Table 1 and Fig. 1) (27). Expression of these genotypes was associated with persistence of CPD and, with the exception of R160W/R160W (1n), sustained hydrogen peroxide generation in UV-irradiated HM, in the presence of α-MSH (Fig. 2A, B). These results are consistent with earlier genetic studies that RHC alleles reduce MC1R function by different extents (38) and suggest that MC1R is linked to signaling pathways other than cAMP, such as inositol phospholipids and protein kinase C (39, 40).

We have examined the possible dosage effect of various MC1R variants suggested by Palmer et al. (7) (Table 1 and Figs. 1 and and2).2). Melanocyte strain 10n (R160W/+) and 11n (D294H/+) had markedly reduced ability to respond to α-MSH with increased cAMP levels and tyrosinase activity (Fig. 1), yet were efficient in reducing UV-induced hydrogen peroxide production and apoptosis, and in repairing CPD when treated with α-MSH (Fig. 2). In contrast, HMs heterozygous for the RHC allele, R151C (12a), or for V60L (13n), V92M (14n, 15a), or R163Q (16n), showed no evidence for reduced MC1R activity. We conclude that RHC alleles differ in their heterozygous effects, and that signaling pathways other than cAMP possibly mediate the effects of α-MSH and/or that mild activation of cAMP is sufficient to activate antioxidant and nucleotide excision repair pathways. Others have reported that expression of any MC1R variant allele with an RHC allele further increases the risk for melanoma (8). In the HM strains 8n and 9a (R/p), we found no evidence for further disruption of MC1R by coexpression of a pseudoallele R163Q or V92M with RHC allele. These novel results define the interactions of these allelic variants based on multiple biochemical assays that determine MC1R function and its role in the UV response of HMs.

To confirm that lack of response of HMs expressing 2 RHC alleles to α-MSH and UV is due to their MC1R genotype, we stably transfected HM strain 2n (R160W/D294H) with the wild-type MC1R and found that this restored the response to α-MSH and reduced the burden of UV-induced oxidative stress and DNA damage (Fig. 3). Interestingly, in the absence of α-MSH, the extent of UV-induced apoptosis of HMs transfected with wild-type MC1R was markedly less than that of the LOF parental HMs, possibly due to increased constitutive activity of the receptor encoded for by the wild-type MC1R.

The results of the above biochemical assays were consistent with microarrray data, which showed that in HM-F, but not in HM-LOF, α-MSH altered gene expression (Fig. 4B), and antagonized the effects of UV on many genes, such as those regulating melanogenesis, oxidative stress, DNA repair, cell cycle, and apoptosis (Fig. 4D). Generally, α-MSH up-regulated, while UV down-regulated, global gene expression (Fig. 4C, D). The suppressive effects of UV on gene expression was also observed by others (4143) and is thought to be due to UV-induced DNA damage that inhibits transcription by stalling RNA polymerases (42). Because of the stochastic nature of UV-induced DNA damage, the inhibition of a particular gene is proportional to its size (43). We propose that by increasing CPD repair, α-MSH reduces the number of these offending lesions and alleviates the repressive effect of UV on gene transcription.

The effect of α-MSH on the induction and repair of UV-induced DNA damage was independent of increased eumelanin synthesis, because eumelanin content was not changed by any of our experimental protocols (data not shown). In addition to pigmentation, inefficient DNA repair is a risk factor for melanoma (44, 45). Our results underscore the significance of functional MC1R in reducing the genotoxic effects of UV by enhancing the repair of DNA photoproducts and reducing the extent of oxidative damage, in addition to its classic role as regulator of melanogenesis. Reduced DNA photoproducts and increased expression of DNA repair genes by melanocortins were reported by others (46); our recent findings revealed reduction of UV-induced oxidative DNA damage by α-MSH (22). Elucidating the impact of different MC1R genotypes on the responses of HMs to α-MSH and UV sheds light on the molecular mechanisms by which MC1R functions as a melanoma susceptibility gene, and explains the differential susceptibility to melanoma.

Supplementary Material

Supplemental Data:

Acknowledgments

The authors thank Silva Terzieva for technical assistance, Toshio Mori (Nara Medical University, Kashihara, Japan) for donating TDM-2 antibody, Jose Carlos Garcia Borron (University of Murcia, Murcia, Spain) for his gift of the pMC1R-WT plasmid, and David Fisher (Harvard University, Cambridge, MA, USA) for providing the Mitf antibody.

This work was supported in part by U.S. National Institutes of Health grants R01ES009110 (Z.A.M.), R011ES017561 (Z.A.M. and S.L.), and P30 ES06096 (Z.A.M. and D.S.); the University of Cincinnati Cancer Center Pilot Project (Z.A.M.); Procter and Gamble Co. (Cincinnati, OH, USA) (Z.A.M.), a Dermatology Foundation research grant (S.L.); Ohio Cancer Research Associates (A.L.K.); and the Japan Society for the Promotion of Sciences, KAKENHI grant 20591357 (K.W. and S.I.).

REFERENCES

1. Purdue M. P., Freeman L. E., Anderson W. F., Tucker M. A. (2008) Recent trends in incidence of cutaneous melanoma among US Caucasian young adults. J. Invest. Dermatol. 128, 2905–2908 [PMC free article] [PubMed]
2. Geller A. C., Miller D. R., Annas G. D., Demierre M. F., Gilchrest B. A., Koh H. K. (2002) Melanoma incidence and mortality among US whites, 1969–1999. JAMA 288, 1719–1720 [PubMed]
3. De Vries E., Coebergh J. W. (2004) Cutaneous malignant melanoma in Europe. Eur. J. Cancer 40, 2355–2366 [PubMed]
4. Stang A., Pukkala E., Sankila R., Soderman B., Hakulinen T. (2006) Time trend analysis of the skin melanoma incidence of Finland from 1953 through 2003 including 16,414 cases. Int. J. Cancer 119, 380–384 [PubMed]
5. Linos E., Swetter S. M., Cockburn M. G., Colditz G. A., Clarke C. A. (2009) Increasing burden of melanoma in the United States. J. Invest. Dermatol. 129, 1666–1674 [PMC free article] [PubMed]
6. Garcia-Borron J. C., Sanchez-Laorden B. L., Jimenez-Cervantes C. (2005) Melanocortin-1 receptor structure and functional regulation. Pigment Cell Res. 18, 393–410 [PubMed]
7. Palmer J. S., Duffy D. L., Box N. F., Aitken J. F., O'Gorman L. E., Green A. C., Hayward N. K., Martin N. G., Sturm R. A. (2000) Melanocortin-1 receptor polymorphisms and risk of melanoma: is the association explained solely by pigmentation phenotype? Am. J. Hum. Genet. 66, 176–186 [PMC free article] [PubMed]
8. Kennedy C., ter Huurne J., Berkhout M., Gruis N., Bastiaens M., Bergman W., Willemze R., Bouwes Bavinck J. N. (2001) Melanocortin 1 receptor (MC1R) gene variants are associated with an increased risk for cutaneous melanoma which is largely independent of skin type and hair color. J. Invest. Dermatol. 117, 294–300 [PubMed]
9. Smith R., Healy E., Siddiqui S., Flanagan N., Steijlen P. M., Rosdahl I., Jacques J. P., Rogers S., Turner R., Jackson I. J., Birch-Machin M. A., Rees J. L. (1998) Melanocortin 1 receptor variants in Irish population. J. Invest. Dermatol. 111, 119–122 [PubMed]
10. Kanetsky P. A., Rebbeck T. R., Hummer A. J., Panossian S., Armstrong B. K., Kricker A., Marrett L. D., Millikan R. C., Gruber S. B., Culver H. A., Zanetti R., Gallagher R. P., Dwyer T., Busam K., From L., Mujumdar U., Wilcox H., Begg C. B., Berwick M. (2006) Population-based study of natural variation in the melanocortin-1 receptor gene and melanoma. Cancer Res. 66, 9330–9337 [PubMed]
11. Hunt G., Kyne S., Ito S., Wakamatsu K., Todd C., Thody A. J. (1995) Eumelanin and pheomelanin contents of human epidermis and cultured melanocytes. Pigment Cell Res. 8, 202–208 [PubMed]
12. Hennessy A., Oh C., Diffey B., Wakamatsu K., Ito S., Rees J. (2005) Eumelanin and pheomelanin concentrations in human epidermis before and after UVB irradiation. Pigment Cell Res. 18, 220–223 [PubMed]
13. Bustamante J., Bredeston L., Malanga G., Mordoh J. (1993) Role of melanin as a scavenger of active oxygen species. Pigment Cell Res. 6, 348–353 [PubMed]
14. Hunt G., Todd C., Cresswell J. E., Thody A. J. (1994) α-Melanocyte stimulating hormone and its analogue Nle4DPhe7 α-MSH affect morphology, tyrosinase activity and melanogenesis in cultured human melanocytes. J. Cell Sci. 107, 205–211 [PubMed]
15. Abdel-Malek Z., Swope V. B., Suzuki I., Akcali C., Harriger M. D., Boyce S. T., Urabe K., Hearing V. J. (1995) Mitogenic and melanogenic stimulation of normal human melanocytes by melanotropic peptides. Proc. Natl. Acad. Sci. U. S. A. 92, 1789–1793 [PMC free article] [PubMed]
16. Wakamatsu K., Graham A., Cook D., Thody A. J. (1997) Characterization of ACTH peptides in human skin and their activation of the melanocortin-1 receptor. Pigment Cell Res. 10, 288–297 [PubMed]
17. Mountjoy K. G., Robbins L. S., Mortrud M. T., Cone R. D. (1992) The cloning of a family of genes that encode the melanocortin receptors. Science 257, 1248–1251 [PubMed]
18. Suzuki I., Cone R., Im S., Nordlund J., Abdel-Malek Z. (1996) Binding of melanotropic hormones to the melanocortin receptor MC1R on human melanocytes stimulates proliferation and melanogenesis. Endocrinology 137, 1627–1633 [PubMed]
19. Abdel-Malek Z., Swope V., Collins C., Boissy R., Zhao H., Nordlund J. (1993) Contribution of melanogenic proteins to the heterogeneous pigmentation of human melanocytes. J. Cell Sci. 106, 1323–1331 [PubMed]
20. Im S., Moro O., Peng F., Medrano E. E., Cornelius J., Babcock G., Nordlund J., Abdel-Malek Z. (1998) Activation of the cyclic AMP pathway by α-melanotropin mediates the response of human melanocytes to ultraviolet B radiation. Cancer Res. 58, 47–54 [PubMed]
21. Kadekaro A. L., Kavanagh R., Kanto H., Terzieva S., Hauser J., Kobayashi N., Schwemberger S., Cornelius J., Babcock G., Shertzer H. G., Scott G., Abdel-Malek Z. A. (2005) α-Melanocortin and endothelin-1 activate anti-apoptotic pathways and reduce DNA damage in human melanocytes. Cancer Res. 65, 4292–4299 [PubMed]
22. Song X., Mosby N., Yang J., Xu A., Abdel-Malek Z., Kadekaro A. L. (2009) alpha-MSH activates immediate defense responses to UV-induced oxidative stress in human melanocytes. Pigment Cell Melanoma Res. 22, 809–818 [PubMed]
23. Van der Velden P. A., Sandkuijl L. A., Bergman W., Pavel S., van Mourik L., Frants R. R., Gruis N. A. (2001) Melanocortin-1 receptor variant R151C modifies melanoma risk in Dutch families with melanoma. Am. J. Hum. Genet. 69, 774–779 [PMC free article] [PubMed]
24. Box N. F., Duffy D. L., Chen W., Stark M., Martin N. G., Sturm R. A., Hayward N. K. (2001) MC1R genotype modifies risk of melanoma in families segregating CDKN2A mutations. Am. J. Hum. Genet. 69, 765–773 [PMC free article] [PubMed]
25. Goldstein A. M., Landi M. T., Tsang S., Fraser M. C., Munroe D. J., Tucker M. A. (2005) Association of MC1R variants and risk of melanoma in melanoma-prone families with CDKN2A mutations. Cancer Epidemiol. Biomarkers Prev. 14, 2208–2212 [PubMed]
26. Landi M. T., Bauer J., Pfeiffer R. M., Elder D. F., Hulley B., Minghetti P., Calista D., Kanetsky P. A., Pinkel D., Bastian B. C. (2006) MC1R germline variants confer risk for BRAF-mutant melanoma. Science 313, 521–522 [PubMed]
27. Scott M. C., Wakamatsu K., Ito S., Kadekaro A. L., Kobayashi N., Groden J., Kavanagh R., Takakuwa T., Virador V., Hearing V. J., Abdel-Malek Z. A. (2002) Human melanocortin 1 receptor variants, receptor function and melanocyte response to UV radiation. J. Cell Sci. 115, 2349–2355 [PubMed]
28. Wang R., Tang P., Wang P., Boissy R. E., Zheng H. (2006) Regulation of tyrosinase trafficking and processing by presenilins: partial loss of function by familial Alzheimer's disease mutation. Proc. Natl. Acad. Sci. U. S. A. 103, 353–358 [PMC free article] [PubMed]
29. Borchers M. T., Wesselkamper S. C., Eppert B. L., Motz G. T., Sartor M. A., Tomlinson C. R., Medvedovic M., Tichelaar J. W. (2008) Nonredundant functions of αβ and γδ T cells in acrolein-induced pulmonary pathology. Toxicol. Sci. 105, 188–199 [PMC free article] [PubMed]
30. McGill G. G., Horstmann M., Widlund H. R., Du J., Motyckova G., Nishimura E. K., Lin Y.-L., Ramaswamy S., Avery W., Ding H.-F., Jordan S. A., Jackson I. J., Korsmeyer S. J., Golub T. R., Fisher D. E. (2002) Bcl2 regulation by the melanocyte master regulator Mitf modulates lineage survival and melanoma cell viability. Cell 109, 707–718 [PubMed]
31. Wood Z. A., Schroder E., Robin Harris J., Poole L. B. (2003) Structure, mechanism and regulation of peroxiredoxins. Trends Biochem. Sci. 28, 32–40 [PubMed]
32. Bryk R., Griffin P., Nathan C. (2000) Peroxynitrite reductase activity of bacterial peroxiredoxins. Nature 407, 211–215 [PubMed]
33. Lovejoy C. A., Lock K., Yenamandra A., Cortez D. (2006) DDB1 maintains genome integrity through regulation of Cdt1. Mol. Cell. Biol. 26, 7977–7990 [PMC free article] [PubMed]
34. Maga G., Hubscher U. (2003) Proliferating cell nuclear antigen (PCNA): a dancer with many partners. J. Cell Sci. 116, 3051–3060 [PubMed]
35. Prosperi E. (2006) The fellowship of the rings: distinct pools of proliferating cell nuclear antigen trimer at work. FASEB J. 20, 833–837 [PubMed]
36. Kaldis P., Aleem E. (2005) Cell cycle sibling rivalry: Cdc2 vs. Cdk2. Cell. Cycle 4, 1491–1494 [PubMed]
37. Yamamura K., Kamada S., Ito S., Nakagawa K., Ichihashi M., Tsujimoto Y. (1996) Accelerated disappearance of melanocytes in bcl-2-deficient mice. Cancer Res. 56, 3546–3550 [PubMed]
38. Healy E., Jordan S. A., Budd P. S., Suffolk R., Rees J. L., Jackson I. J. (2001) Functional variation of MC1R alleles from red-haired individuals. Hum. Mol. Genet. 10, 2397–2402 [PubMed]
39. Konda Y., Gantz I., DelValle J., Shimoto Y., Miwa H., Yamada T. (1994) Interaction of dual intracellular signaling pathways activated by the melanocortin-3 receptor. J. Biol. Chem. 269, 13162–13166 [PubMed]
40. Park H.-Y., Russakovsky V., Ao Y., Fernandez E., Gilchrest B. A. (1996) Alpha-melanocyte stimulating hormone-induced pigmentation is blocked by depletion of protein kinase C. Exp. Cell. Res. 227, 70–79 [PubMed]
41. McKay B. C., Stubbert L. J., Fowler C. C., Smith J. M., Cardamore R. A., Spronck J. C. (2004) Regulation of ultraviolet light-induced gene expression by gene size. Proc. Natl. Acad. Sci. U. S. A. 101, 6582–6586 [PMC free article] [PubMed]
42. Tornaletti S. (2005) Transcription arrest at DNA damage sites. Mutat. Res. 577, 131–145 [PubMed]
43. April C. S., Barsh G. S. (2007) Distinct pigmentary and melanocortin 1 receptor-dependent components of cutaneous defense against ultraviolet radiation. PLoS Genet. 3, e9 [PMC free article] [PubMed]
44. Wei Q., Lee J. E., Gershenwald J. E., Ross M. I., Mansfield P. F., Strom S. S., Wang L. E., Guo Z., Qiao Y., Amos C. I., Spitz M. R., Duvic M. (2003) Repair of UV light-induced DNA damage and risk of cutaneous malignant melanoma. J. Natl. Cancer Inst. 95, 308–315 [PubMed]
45. Sarkar-Agrawal P., Vergilis I., Sharpless N. E., DePinho R. A., Runger T. M. (2004) Impaired processing of DNA photoproducts and ultraviolet hypermutability with loss of p16INK4a or p19ARF. J. Natl. Cancer Inst. 96, 1790–1793 [PubMed]
46. Bohm M., Wolff I., Scholsen T. E., Robinson S. J., Healy E., Luger T. A., Scharz T., Schwarz A. (2005) Alpha-melanocyte-stimulating hormone protects from ultraviolet radiation-induced apoptosis and DNA damage. J. Biol. Chem. 280, 5795–5802 [PubMed]
47. Smith A. G., Luk N., Newton R. A., Roberts D. W., Sturm R. A., Muscat G. E. (2008) Melanocortin-1 receptor signaling markedly induces the expression of the NR4A nuclear receptor subgroup in melanocytic cells. J. Biol. Chem. 283, 12564–12570 [PubMed]

Articles from The FASEB Journal are provided here courtesy of The Federation of American Societies for Experimental Biology
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...