α‐MSH tripeptide analogs activate the melanocortin 1 receptor and reduce UV‐induced DNA damage in human melanocytes

One skin cancer prevention strategy that we are developing is based on synthesizing and testing melanocortin analogs that reduce and repair DNA damage resulting from exposure to solar ultraviolet (UV) radiation, in addition to stimulating pigmentation. Previously, we reported the effects of tetrapeptide analogs of α‐melanocortin (α‐MSH) that were more potent and stable than the physiological α‐MSH, and mimicked its photoprotective effects against UV‐induced DNA damage in human melanocytes. Here, we report on a panel of tripeptide analogs consisting of a modified α‐MSH core His⁶‐d ‐Phe⁷‐Arg⁸, which contained different N‐capping groups, C‐terminal modifications, or arginine mimics. The most potent tripeptides in activating cAMP formation and tyrosinase of human melanocytes were three analogs with C‐terminal modifications. The most effective C‐terminal tripeptide mimicked α‐MSH in reducing hydrogen peroxide generation and enhancing nucleotide excision repair following UV irradiation. The effects of these three analogs required functional MC1R, as they were absent in human melanocytes that expressed non‐functional receptor. These results demonstrate activation of the MC1R by tripeptide melanocortin analogs. Designing small analogs for topical delivery should prove practical and efficacious for skin cancer prevention.     

Endothelin‐1 and α‐melanocortin have redundant effects on global genome repair in UV‐irradiated human melanocytes despite distinct signaling pathways

Human melanocyte homeostasis is sustained by paracrine factors that reduce the genotoxic effects of ultraviolet radiation (UV), the major etiological factor for melanoma. The keratinocyte‐derived endothelin‐1 (End‐1) and α‐melanocyte‐stimulating hormone (α‐MSH) regulate human melanocyte function, proliferation and survival, and enhance repair of UV‐induced DNA photoproducts by binding to the G_q‐ and G_i‐protein‐coupled endothelin B receptor (EDNRB), and the G_s‐protein‐coupled melanocortin 1 receptor (MC1R), respectively. We hereby report that End‐1 and α‐MSH regulate common effectors of the DNA damage response to UV, despite distinct signaling pathways. Both factors activate the two DNA damage sensors ataxia telangiectasia and Rad3‐related and ataxia telangiectasia mutated, enhance DNA damage recognition by reducing soluble nuclear and chromatin‐bound DNA damage binding protein 2, and increase total and chromatin‐bound xeroderma pigmentosum (XP) C. Additionally, α‐MSH and End‐1 increase total levels and chromatin localization of the damage verification protein XPA, and the levels of γH2AX, which facilitates recruitment of DNA repair proteins to DNA lesions. Activation of EDNRB compensates for MC1R loss of function, thereby reducing the risk of malignant transformation of these vulnerable melanocytes. Therefore, MC1R and EDNRB signaling pathways represent redundant mechanisms that inhibit the genotoxic effects of UV and melanomagenesis.     

Melanin content and MC1R function independently affect UVR‐induced DNA damage in cultured human melanocytes

Malignant transformation of melanocytes leads to melanoma, the most fatal form of skin cancer. Ultraviolet radiation (UVR)‐induced DNA photoproducts play an important role in melanomagenesis. Cutaneous melanin content represents a major photoprotective mechanism against UVR‐induced DNA damage, and generally correlates inversely with the risk of skin cancer, including melanoma. Melanoma risk is also determined by susceptibility genes, one of which is the melanocortin 1 receptor (MC1R) gene. Certain MC1R alleles are strongly associated with melanoma. We hereby present experimental evidence for the role of two melanoma risk factors, constitutive pigmentation, as assessed by total melanin, eumelanin and pheomelanin contents, and MC1R genotype and function, in determining the induction and repair of DNA photoproducts in cultured human melanocytes after irradiation with increasing doses of UVR. We found that total melanin and eumelanin contents (MC and EC) correlated inversely with the extent of UVR‐induced growth arrest, apoptosis and induction of cyclobutane pyrimidine dimers (CPD), but not with hydrogen peroxide release in melanocytes expressing functional MC1R. In comparison, melanocytes with loss‐of‐function MC1R, regardless of their MC or EC, sustained more UVR‐induced apoptosis and CPD, and exhibited reduced CPD repair. Therefore, MC, mainly EC, and MC1R function are independent determinants of UVR‐induced DNA damage in melanocytes.     

Identification and functional characterization of natural human melanocortin 1 receptor mutant alleles in Pakistani population

Melanocortin 1 receptor (MC1R), a Gs protein‐coupled receptor of the melanocyte's plasma membrane, is a major determinant of skin pigmentation and phototype. Upon activation by α‐melanocyte stimulating hormone, MC1R triggers the cAMP cascade to stimulate eumelanogenesis. We used whole‐exome sequencing to identify causative alleles in Pakistani families with skin and hair hypopigmentation. Six MC1R mutations segregated with the phenotype in seven families, including a p.Val174del in‐frame deletion and a p.Tyr298* nonsense mutation, that were analyzed for function in heterologous HEK293 cells. p.Tyr298* MC1R showed no agonist‐induced signaling to the cAMP or ERK pathways, nor detectable agonist binding. Conversely, signaling was comparable for p.Val174del and wild‐type in HEK cells overexpressing the proteins, but binding analysis suggested impaired cell surface expression. Flow cytometry and confocal imaging studies revealed reduced plasma membrane expression of p.Val174del and p.Tyr298*. Therefore, p.Tyr298* was a total loss‐of‐function (LOF) allele, while p.Val174del displayed a partial LOF attribute.     

α‐MSH activates immediate defense responses to UV‐induced oxidative stress in human melanocytes

Exposure of cultured human melanocytes to ultraviolet radiation (UV) results in DNA damage. In melanoma, UV‐signature mutations resulting from unrepaired photoproducts are rare, suggesting the possible involvement of oxidative DNA damage in melanocyte malignant transformation. Here we present data demonstrating immediate dose‐dependent generation of hydrogen peroxide in UV‐irradiated melanocytes, which correlated directly with a decrease in catalase activity. Pretreatment of melanocytes with α‐melanocortin (α‐MSH) reduced the UV‐induced generation of 7,8‐dihydro‐8‐oxyguanine (8‐oxodG), a major form of oxidative DNA damage. Pretreatment with α‐MSH also increased the protein levels of catalase and ferritin. The effect of α‐MSH on 8‐oxodG induction was mediated by activation of the melanocortin 1 receptor (MC1R), as it was absent in melanocytes expressing loss‐of‐function MC1R, and blocked by concomitant treatment with an analog of agouti signaling protein (ASIP), ASIP‐YY. This study provides unequivocal evidence for induction of oxidative DNA damage by UV in human melanocytes and reduction of this damage by α‐MSH. Our data unravel some mechanisms by which α‐MSH protects melanocytes from oxidative DNA damage, which partially explain the strong association of loss‐of‐function MC1R with melanoma.     

N-glycosylation of the human melanocortin 1 receptor: occupancy of glycosylation sequons and functional role

The melanocortin 1 receptor (MC1R), a major determinant of skin pigmentation and phototype, mediates the actions of α-melanocyte-stimulating hormone on melanocytes and is critical for melanocyte proliferation and differentiation. MC1R has two putative N-glycosylation targets, Asn15 and Asn29. It has been shown that MC1R is a glycoprotein with an unusual sensitivity to endoglycosidase H digestion. However, the occupancy and functional importance of each specific glycosylation sequon remains unknown. We demonstrate that MC1R is N-glycosylated at Asn15 and Asn29, with structurally and functionally different glycan chains. N-glycosylation is not necessary for high affinity agonist binding or functional coupling but has a strong effect on the availability of MC1R molecules on the plasma membrane, most likely by a combination of improved forward trafficking and decreased internalization. Finally, we found that MC1R variants exhibit different degrees of glycosylation which do not show a simple correlation with their functional status or intracellular trafficking.     

Cutaneous pharmacologic cAMP induction induces melanization of the skin and improves recovery from ultraviolet injury in melanocortin 1 receptor‐intact or heterozygous skin

Homozygous loss of function of the melanocortin 1 receptor (MC1R) is associated with a pheomelanotic pigment phenotype and increased melanoma risk. MC1R heterozygosity is less well studied, although individuals inheriting one loss‐of‐function MC1R allele are also melanoma‐prone. Using the K14‐Scf C57BL/6J animal model whose skin is characterized by lifelong retention of interfollicular epidermal melanocytes like that of the human, we studied pigmentary, UV responses, and DNA repair capacity in the skin of variant Mc1r background. Topical application of forskolin, a skin‐permeable pharmacologic activator of cAMP induction to mimic native Mc1r signaling, increased epidermal eumelanin levels, increased the capacity of Mc1r‐heterozygous skin to resist UV‐mediated inflammation, and enhanced the skin's ability to clear UV photolesions from DNA. Interestingly, topical cAMP induction also promoted melanin accumulation, UV resistance, and accelerated clearance in Mc1r fully intact skin. Together, our findings suggest that heterozygous Mc1r loss is associated with an intermediately melanized and DNA repair‐proficient epidermal phenotype and that topical cAMP induction enhances UV resistance in Mc1r‐heterozygous or Mc1r‐wild‐type individuals by increasing eumelanin deposition and by improving nucleotide excision repair.     

p53 suppression overwhelms DNA polymerase eta deficiency in determining the cellular UV DNA damage response

Xeroderma pigmentosum variant (XP-V) cells lack the damage-specific DNA polymerase eta and have normal excision repair but show defective DNA replication after UV irradiation. Previous studies using cells transformed with SV40 or HPV16 (E6/E7) suggested that the S-phase response to UV damage is altered in XP-V cells with non-functional p53. To investigate the role of p53 directly we targeted p53 in normal and XP-V fibroblasts using short hairpin RNA. The shRNA reduced expression of p53, and the downstream cell cycle effector p21, in control and UV irradiated cells. Cells accumulated in late S phase after UV, but after down-regulation of p53 they accumulated earlier in S. Cells in which p53 was inhibited showed ongoing genomic instability at the replication fork. Cells exhibited high levels of UV induced S-phase gammaH2Ax phosphorylation representative of exposed single strand regions of DNA and foci of Mre11/Rad50/Nbs1 representative of double strand breaks. Cells also showed increased variability of genomic copy numbers after long-term inhibition of p53. Inhibition of p53 expression dominated the DNA damage response. Comparison with earlier results indicates that in virally transformed cells cellular targets other than p53 play important roles in the UV DNA damage response.     

The effect of MC1R variants and sunscreen on the response of human melanocytes in vivo to ultraviolet radiation and implications for melanoma

We conducted a clinical trial to compare the molecular and cellular responses of human melanocytes and keratinocytes in vivo to solar‐simulated ultraviolet radiation (SSUVR) in 57 Caucasian participants grouped according to MC1R genotype. We found that, on average, the density of epidermal melanocytes 14 days after exposure to 2 minimal erythemal dose (MED) SSUVR was twofold higher than baseline (unirradiated) skin. However, the change in epidermal melanocyte counts among people carrying germline MC1R variants (97% increase) was significantly less than those with wild‐type MC1R (164% increase; P = 0.01). We also found that sunscreen applied to the skin before exposure to 2 MED SSUVR completely blocked the effects of DNA damage, p53 induction, and cellular proliferation in both melanocytes and keratinocytes.     

Variation of the melanocortin 1 receptor gene in the macaques

Melanocortin 1 receptor (MC1R), a G-coupled seven-transmembrane receptor protein, plays a key role in the regulation of melanin synthesis in mammals. Sequence variation of the MC1R gene (MC1R) has been associated with pigmentation phenotypes in humans and in several animal species. The macaques (genus Macaca) are known to show a marked inter-specific variation in coat color although the causative genetic variation remains unclear. We investigated nucleotide sequences of the MC1R in 67 individuals of 18 macaque species with different coat color phenotypes including black and agouti. Twenty-eight amino acid replacements were identified in the macaques, but none of these amino acid replacements could explain the black coat color of Macaca silenus and the Sulawesi macaque species. Our molecular evolutionary analysis has revealed that nonsynonymous substitution/synonymous substitution (dN/dS) ratio of the MC1R has not been uniform in the macaque groups and, moreover, their coat color and dN/dS ratio were not related. These results suggest that the MC1R is unlikely to be responsible for the coat color variation of the macaques and functions of MC1R other than pigmentation might be associated with the different selective pressures on the MC1R in macaques.     

Metabotropic glutamate receptor 6 signaling enhances TRPM1 calcium channel function and increases melanin content in human melanocytes

Mutations in TRPM1, a calcium channel expressed in retinal bipolar cells and epidermal melanocytes, cause complete congenital stationary night blindness with no discernible skin phenotype. In the retina, TRPM1 activity is negatively coupled to metabotropic glutamate receptor 6 (mGluR6) signaling through Gα_o and TRPM1 mutations result in the loss of responsiveness of TRPM1 to mGluR6 signaling. Here, we show that human melanocytes express mGluR6, and treatment of melanocytes with L‐AP4, a type III mGluR‐selective agonist, enhances Ca²⁺ uptake. Knockdown of TRPM1 or mGluR6 by shRNA abolished L‐AP4‐induced Ca²⁺ influx and TRPM1 currents, showing that TRPM1 activity in melanocytes is positively coupled to mGluR6 signaling. Gα_o protein is absent in melanocytes. However, forced expression of Gα_o restored negative coupling of TRPM1 to mGluR6 signaling, but treatment with pertussis toxin, an inhibitor of G_i/G_o proteins, did not affect basal or mGluR6‐induced Ca²⁺ uptake. Additionally, chronic stimulation of mGluR6 altered melanocyte morphology and increased melanin content. These data suggest differences in coupling of TRPM1 function to mGluR6 signaling explain different cellular responses to glutamate in the retina and the skin.     

DNA damage response in microcephaly development of MCPH1 mouse model

MCPH1 encodes BRCT-containing protein MCPH1/Microcephalin/BRIT1, mutations of which in humans cause autosomal recessive disorder primary microcephaly type 1 (MCPH1), characterized by a congenital reduction of brain size particularly in the cerebral cortex. We have shown previously that a deletion of Mcph1 in mice results in microcephaly because of a premature switch from symmetric to asymmetric division of the neuroprogenitors, which is regulated by MCPH1's function in the centrosome. Because MCPH1 has been implicated in ATM and ATR-mediated DNA damage response (DDR) and defective DDR is often associated with neurodevelopmental diseases, we wonder whether the DDR-related function of MCPH1 prevents microcephaly. Here, we show that a deletion of Mcph1 results in a specific reduction of the cerebral cortex at birth, which is persistent through life. Due to an effect on premature neurogenic production, Mcph1-deficient progenitors give rise to a high level of early-born neurons that form deep layers (IV–VI), while generate less late-born neurons that form a thinner outer layer (II–III) of the cortex. However, neuronal migration seems to be unaffected by Mcph1 deletion. Ionizing radiation (IR) induces a massive apoptosis in the Mcph1-null neocortex and also embryonic lethality. Finally, Mcph1 deletion compromises homologous recombination repair and increases genomic instability. Altogether, our data suggest that MCPH1 ensures proper neuroprogenitor expansion and differentiation not only through its function in the centrosome, but also in the DDR.     

Biological weighting functions for DNA damage in sea urchin embryos exposed to ultraviolet radiation

Laboratory experiments examining the effects of ultraviolet radiation (UVR, 290-400 nm) on DNA damage were carried out using the embryos of three species of sea urchins from different habitats; Strongylocentrotus droebachiensis from the Gulf of Maine, Sterechinus neumayeri from the Antarctic, and Evechinus chloroticus from New Zealand. All three species exhibited significant amounts of accumulated DNA damage, measured as cyclobutane pyrimidine dimers (CPD) photoproducts, when exposed to UVR in the laboratory. Biological weighting functions (BWFs) revealed that S. neumayeri has significantly higher sensitivity to UVR-induced DNA damage across most of the UVR spectrum compared to the other two species, and all species were observed to have weightings in the ultraviolet-A (UVA, 320-400 nm) portion of the spectrum. The increased sensitivity to ultraviolet-B (290-320 nm) and UVA in S. neumayeri is correlated with the lowest concentration of UVR absorbing compounds observed in the embryos of the three species of urchin used in this study. Sea urchin embryos and larvae in the respective habitats of the species tested are known to occur within 5 m of the surface of the ocean where both UVB and UVA wavelengths occur. Solar irradiances of UVR at a depth of 5 m, weighted using the urchin DNA damage BWFs, show that E. chloroticus receives the greatest amount of biologically effective UVR despite having the lowest wavelength dependent weightings for DNA damage when compared to the other two species.     

mTORC1 pathway in DNA damage response

Living organisms have evolved various mechanisms to control their metabolism and response to various stresses, allowing them to survive and grow in different environments. In eukaryotes, the highly conserved mechanistic target of rapamycin (mTOR) signaling pathway integrates both intracellular and extracellular signals and serves as a central regulator of cellular metabolism, proliferation and survival. A growing body of evidence indicates that mTOR signaling is closely related to another cellular protection mechanism, the DNA damage response (DDR). Many factors important for the DDR are also involved in the mTOR pathway. In this review, we discuss how these two pathways communicate to ensure an efficient protection of the cell against metabolic and genotoxic stresses. We also describe how anticancer therapies benefit from simultaneous targeting of the DDR and mTOR pathways.     

Zinc protects against ultraviolet A1-induced DNA damage and apoptosis in cultured human fibroblasts

Ultraviolet Al (UVA1) radiation generates reactive oxygen species and the oxidative stress is known as a mediator of DNA damage and of apoptosis. We exposed cultured human cutaneous fibroblasts to UVA1 radiation (wavelengths in the 340–450-nm range with emission peak at 365 nm) and, using the alkaline unwinding method, we showed an immediate significant increase of DNA strand breaks in exposed cells. Apoptosis was determined by detecting cytoplasmic nucleosomes (enzyme-linked immunosorbent assay method) at different time points in fibroblasts exposed to different irradiation doses. In our conditions, UVA1 radiation induced an early (8 h) and a delayed (18 h) apoptosis. Delayed apoptosis increased in a UVA dosedependent manner. Zinc is an important metal for DNA protection and has been shown to have inhibitory effects on apoptosis. The addition of zinc (6.5 mg/L) as zinc chloride to the culture medium significantly decreased immediate DNA strand breaks in human skin fibroblasts. Moreover, zinc chloride significantly decreased UVA1-induced early and delayed apoptosis. Thus, these data show for the first time in normal cutaneous cultured cells that UVA1 radiation induces apoptosis. This apoptosis is biphasic and appears higher 18 h after the stress. Zinc supplementation can prevent both immediate DNA strand breakage and early and delayed apoptosis, suggesting that this metal could be of interest for skin cell protection against UVA1 irradiation.     

Mutation in the melanocortin 1 receptor is associated with amber colour in the Norwegian Forest Cat

Amber (previously called X-Colour) is a yellow recessive coat colour observed in the Norwegian Forest Cat (NFC) population and apparently absent in other cat breeds. Until now, there has never been any scientific evidence of yellow recessive mutation ( e ) reported in the extension gene in Felidae. We sequenced the complete coding sequence region for the melanocortin 1 receptor in 12 amber, three carriers, two wild-type NFCs, one wild-type European Shorthair and two 'golden' Siberian cats and identified two single nucleotide polymorphisms (SNPs): a non-synonymous (FM180571: c.250G>A) and a synonymous (FM180571: c.840T>C) mutation. The c.250G>A SNP, further genotyped on 56 cats using PCR-RFLP, is associated with amber colour and only present in the amber cat lineages. It replaced an aspartic acid with a neutral polar asparagine in the second transmembrane helix (p.Asp84Asn), a position where e mutations have already been described. Three-dimensional models were built and showed electrostatic potential modification in the mutant receptor. With these results and together with those in the scientific literature, we can conclude that amber colour in NFCs is caused by a single MC1R allele called e , which has never been documented.     

Molecular response to climate change: temperature dependence of UV-induced DNA damage and repair in the freshwater crustacean Daphnia pulicaria

In temperate lakes, asynchronous cycles in surface water temperatures and incident ultraviolet (UV) radiation expose aquatic organisms to damaging UV radiation at different temperatures. The enzyme systems that repair UV‐induced DNA damage are temperature dependent, and thus potentially less effective at repairing DNA damage at lower temperatures. This hypothesis was tested by examining the levels of UV‐induced DNA damage in the freshwater crustacean Daphnia pulicaria in the presence and absence of longer‐wavelength photoreactivating radiation (PRR) that induces photoenzymatic repair (PER) of DNA damage. By exposing both live and dead (freeze‐killed) Daphnia as well as raw DNA to UV‐B in the presence and absence of PRR, we were able to estimate the relative importance and temperature dependence of PER (light repair), nucleotide excision repair (NER, dark repair), and photoprotection (PP). Total DNA damage increased with increasing temperature. However, the even greater increase in DNA repair rates at higher temperatures led net DNA damage (total DNA damage minus repair) to be greater at lower temperatures. Photoprotection accounted for a much greater proportion of the reduction in DNA damage than did repair. Experiments that looked at survival rates following UV exposure demonstrated that PER increased survival rates. The important implication is that aquatic organisms that depend heavily on DNA repair processes may be less able to survive high UV exposure in low temperature environments. Photoprotection may be more effective under the low temperature, high UV conditions such as are found in early spring or at high elevations.