Summary
Binding of melanocortin peptide agonists to the melanocortin‐1 receptor of melanocytes results in eumelanin production, whereas binding of the agouti signalling protein inverse agonist ...results in pheomelanin synthesis. Recently, a novel melanocortin‐1 receptor ligand was reported. A β‐defensin gene mutation was found to be responsible for black coat colour in domestic dogs. Notably, the human equivalent, β‐defensin 3, was found to bind with high affinity to the melanocortin‐1 receptor; however, the action of β‐defensin as an agonist or antagonist was unknown. Here, we use in vitro assays to show that β‐defensin 3 is able to act as a weak partial agonist for cAMP signalling in human embryonic kidney (HEK) cells expressing human melanocortin‐1 receptor. β‐defensin 3 is also able to activate MAPK signalling in HEK cells stably expressing either wild type or variant melanocortin‐1 receptors. We suggest that β‐defensin 3 may be a novel melanocortin‐1 receptor agonist involved in regulating melanocyte responses in humans.
A portion of approximately 2-20% of cutaneous melanoma (CM) are diagnosed as amelanotic/hypopigmented melanoma (AHM) and represent a challenge for early diagnosis.BACKGROUNDA portion of approximately ...2-20% of cutaneous melanoma (CM) are diagnosed as amelanotic/hypopigmented melanoma (AHM) and represent a challenge for early diagnosis.Since the degree to which somatic mutations and copy number aberrations (CNA) in genes associated with skin-lightening or albinism may contribute to the loss of tumour pigmentation in AHM samples has not yet been addressed, we have investigated loss of function mutations of key pigmentation genes in matched germline and AHM as well as pigmented melanoma (PM) tumour DNA samples.OBJECTIVESSince the degree to which somatic mutations and copy number aberrations (CNA) in genes associated with skin-lightening or albinism may contribute to the loss of tumour pigmentation in AHM samples has not yet been addressed, we have investigated loss of function mutations of key pigmentation genes in matched germline and AHM as well as pigmented melanoma (PM) tumour DNA samples.An analysis of clinical and histopathological characteristics together with whole exome sequencing data of 34 fresh frozen primary CM, graded according to the amount of pigmentation present was performed. Together with germline and somatic variant analysis, 30 samples were previously analysed for CNA changes. This study focussed on germline and somatic variants in the coding region of 16 genes known to be associated with albinism/hypopigmentation or variation in human pigmentation in all samples. Chromosomal regions encompassing these 16 genes were examined for DNA copy loss or gain.METHODSAn analysis of clinical and histopathological characteristics together with whole exome sequencing data of 34 fresh frozen primary CM, graded according to the amount of pigmentation present was performed. Together with germline and somatic variant analysis, 30 samples were previously analysed for CNA changes. This study focussed on germline and somatic variants in the coding region of 16 genes known to be associated with albinism/hypopigmentation or variation in human pigmentation in all samples. Chromosomal regions encompassing these 16 genes were examined for DNA copy loss or gain.The finding that red hair related MC1R and TYR R402Q loss of activity gene variant alleles and genotypes are associated with AHM was validated in this study. Germline AHM-related gene variants were enriched in 70% (n=7 of 10) of AHM patients vs 8.3% (n=2 of 24) of PM patients. This surprisingly high frequency of rare germline variants in AHM patients constitutes the "first hit" and confirms that AHM patients are more likely to be albinism allele carriers than patients with PM. Next, in CNA analysis of each tumour sample, 50% (n=4 of 8) AHM samples with a pigmentation gene variant had LOH in the region containing the corresponding gene, and 25% (=2 of 8) had loss-of-heterozygosity (LOH) in chromosomal regions of two AHM-related genes.RESULTSThe finding that red hair related MC1R and TYR R402Q loss of activity gene variant alleles and genotypes are associated with AHM was validated in this study. Germline AHM-related gene variants were enriched in 70% (n=7 of 10) of AHM patients vs 8.3% (n=2 of 24) of PM patients. This surprisingly high frequency of rare germline variants in AHM patients constitutes the "first hit" and confirms that AHM patients are more likely to be albinism allele carriers than patients with PM. Next, in CNA analysis of each tumour sample, 50% (n=4 of 8) AHM samples with a pigmentation gene variant had LOH in the region containing the corresponding gene, and 25% (=2 of 8) had loss-of-heterozygosity (LOH) in chromosomal regions of two AHM-related genes.This study proposes that the likely molecular mechanism for development of amelanogenesis in AHM is carriage of an albinism/hypopigmentation allele followed by LOH of the corresponding gene in the tumour.CONCLUSIONSThis study proposes that the likely molecular mechanism for development of amelanogenesis in AHM is carriage of an albinism/hypopigmentation allele followed by LOH of the corresponding gene in the tumour.
Summary
A
SNP
within intron4 of the interferon regulatory factor4 (
IRF
4
) gene, rs12203592*C/T, has been independently associated with pigmentation and age‐specific effects on naevus count in ...European‐derived populations. We have characterized the cis‐regulatory activity of this intronic region and using human foreskin‐derived melanoblast strains, we have explored the correlation between
IRF
4
rs12203592 homozygous C/C and T/T genotypes with
TYR
enzyme activity, supporting its association with pigmentation traits. Further, higher
IRF
4 protein levels directed by the rs12203592*C allele were associated with increased basal proliferation but decreased cell viability following
UVR
, an etiological factor in melanoma development. Since
UVR
, and accompanying
IFN
γ‐mediated inflammatory response, is associated with melanomagenesis, we evaluated its effects in the context of
IRF
4
status. Manipulation of
IRF
4 levels followed by
IFN
γ treatment revealed a subset of chemokines and immuno‐evasive molecules that are sensitive to
IRF
4 expression level and genotype including
CTLA
4
and
PD
‐L1
.
Peroxisome proliferator-activated receptor-gamma (PPARγ) is a member of the nuclear hormone receptor (NHR) superfamily of ligand-activated transcriptional regulators. Accumulating evidence suggests ...that PPARγ agonists such as the thiazolidinediones (TZDs) may prove to be useful anti-cancer agents exhibiting anti-proliferative and/or pro-apoptotic affects in a range of cancer cell types including melanoma, however, the mechanisms underlying this effect remain unclear. We have demonstrated the anti-proliferative effects of full and partial PPARγ modulators in human melanoma cell lines. Ablation of PPARγ expression in the MM96L melanoma cell line by siRNA mediated mechanisms attenuates the anti-proliferative effect of these agents suggesting this effect is directly mediated by PPARγ. The mechanisms underlying the anti-proliferative effects of PPARγ in melanoma cells involve the regulation of expression of a number of critical cell cycle genes and β-catenin. Moreover, our data indicate that PPARγ modulates Wnt/β-catenin mediated signalling in melanoma cells in an agonist dependent manner.
Expression of the beta(3) integrin subunit in melanoma in situ has been found to correlate with tumor thickness, the ability to invade and metastasize, and poor prognosis. Transition from the radial ...growth phase (RGP) to the vertical growth phase (VGP) is a critical step in melanoma progression and survival and is distinguished by the expression of beta(3) integrin. The molecular pathways that operate in melanoma cells associated with invasion and metastasis were examined by ectopic induction of the beta(3) integrin subunit in RGP SBcl2 and WM1552C melanoma cells, which converts these cells to a VGP phenotype. We used cDNA representational difference analysis subtractive hybridization between beta(3)-positive and -negative melanoma cells to assess gene expression profile changes accompanying RGP to VGP transition. Fourteen fragments from known genes including osteonectin (also known as SPARC and BM-40) were identified after three rounds of representational difference analysis. Induction of osteonectin was confirmed by Northern and Western blot analysis and immunohistochemistry and correlated in organotypic cultures with the beta(3)-induced progression from RGP to VGP melanoma. Expression of osteonectin was also associated with reduced adhesion to vitronectin, but not to fibronectin. Osteonectin expression was not blocked when melanoma cells were cultured with anti-alpha(v)beta(3) LM609 mAb, mitogen-activated protein kinase, or protein kinase C inhibitors, indicating that other signaling pathway(s) operate through alpha(v)beta(3) integrin during conversion from RGP to VGP.
We have examined melanocortin‐1 receptor (MC1R) variant allele frequencies in the general population and in a collection of adolescent dizygotic and monozygotic twins to determine statistical ...associations of pigmentation phenotypes with increased skin cancer risk. This included hair and skin color, freckling, mole count and sun exposed skin reflectance. Nine variants were studied and designated as either strong R (OR = 63; 95% CI 32–140) or weak r (OR = 5; 95% CI 3–11) red hair alleles. Penetrance of each MC1R variant allele was consistent with an allelic model where effects were multiplicative for red hair but additive for skin reflectance. To assess the interaction of the brown eye color gene BEY2/OCA2 on the phenotypic effects of variant MC1R alleles we imputed OCA2 genotype in the twin collection. A modifying effect of OCA2 on MC1R variant alleles was seen on constitutive skin color, freckling and mole count. In order to study the individual effects of these variants on pigmentation phenotype we have established a series of human primary melanocyte strains genotyped for the MC1R receptor. These include strains which are MC1R wild‐type consensus, variant heterozygotes, and homozygotes for strong R alleles Arg151Cys and Arg160Trp. Ultrastructural analysis demonstrated that only consensus strains contained stage III and IV melanosomes in their terminal dendrites whereas Arg151Cys and Arg160Trp homozygous strains contained only immature stage I and II melanosomes. Such genetic association studies combined with the functional analysis of MC1R variant alleles in melanocytic cells should provide a link in understanding the association between pigmentary phototypes and skin cancer risk.
The complete sequence of the
MC1R locus has been assembled, the coding region of the gene is intronless and placed within a 12 kb region flanked by the
NULP1 and
TUBB4 genes. The immediate promoter ...region has an E-box site with homology to the M-box consensus known to bind the microphthalmia transcription factor (MITF); however, promoter deletion analysis and transactivation studies have failed to show activation through this element by MITF. Polymorphism within the coding region, immediate 5′ promoter region and a variable number tandem repeat (VNTR) minisatellite within the locus have been examined in a collection of Caucasian families and African individuals. Haplotype analysis shows linkage disequilibrium between the VNTR and
MC1R coding region red hair variant alleles which can be used to estimate the age of these missense changes. Assuming a mean VNTR mutation rate of 1% and a star phylogeny, we estimate the Arg151Cys variant arose 7500 years before the present day, suggesting these variants may have arisen in the Caucasian population more recently than previously thought.
Paralogous regions are duplicated segments of chromosomal DNA that have been acquired during the evolution of the genome. Subsequent divergent evolution of the genes within paralogous regions can ...lead to the formation of gene families. Here, we report the identification of a region on Chromosome (Chr) 6 at 6p21.3 that is paralogous with the Spinal Muscular Atrophy (SMA) gene region on Chr 5 at 5q13.1. Partial characterization of this region identified nine sequences all of which are highly homologous to DNA sequences of the SMA gene region at 5q13.1. These sequences include four beta-glucuronidase sequences, two retrotransposon sequences, a novel cDNA, a Sequence Tagged Site (STS), and one that is homologous to exon 9 of the Neuronal Apoptosis Inhibitor Protein (NAIP) gene. The 6p21.3 paralogous SMA region may contain genes that are related to those in the SMA region at 5q13.1; however, a direct association of this region with SMA is unlikely given that no linkage of SMA with Chr 6 has been reported.
Mass extinctions manifest in Earth's geologic record were turning points in biotic evolution. We present 40 Ar/ 39 Ar data that establish synchrony between the Cretaceous-Paleogene boundary and ...associated mass extinctions with the Chicxulub bolide impact to within 32,000 years. Perturbation of the atmospheric carbon cycle at the boundary likely lasted less than 5000 years, exhibiting a recovery time scale two to three orders of magnitude shorter than that of the major ocean basins. Low-diversity mammalian fauna in the western Williston Basin persisted for as little as 20,000 years after the impact. The Chicxulub impact likely triggered a state shift of ecosystems already under near-critical stress.
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