Altered cell surface expression of human MC1R variant receptor alleles associated with red hair and skin cancer risk

The human melanocortin-1 receptor gene (MC1R) encodes a G-protein coupled receptor that is primarily expressed on melanocytes, where it plays a key role in pigmentation regulation. Variant alleles are associated with red hair colour and fair skin, known as the RHC phenotype, as well as skin cancer risk. The R151C, R160W and D294H alleles, designated 'R', are strongly associated with the RHC phenotype and have been proposed to result in loss of function receptors due to impaired G-protein coupling. We recently provided evidence that the R151C and R160W variants can efficiently couple to G-proteins in response to alpha-melanocyte stimulating hormone. The possibility that altered cellular localization of the R151C and R160W variant receptors could underlie their association with RHC was therefore considered. Using immunofluorescence and ligand binding studies, we found that melanocytic cells exogenously or endogenously expressing MC1R show strong surface localization of the wild-type and D294H alleles but markedly reduced cell surface expression of the R151C and R160W receptors. In additional exogenous expression studies, the R variant D84E and the rare I155T variant, also demonstrated a significant reduction in plasma membrane receptor numbers. The V60L, V92M and R163Q weakly associated RHC alleles, designated 'r', were expressed with normal or intermediate cell surface receptor levels. These results indicate that reduced receptor coupling activity may not be the only contributing factor to the genetic association between the MC1R variants and the RHC phenotype, with MC1R polymorphisms now linked to a change in receptor localization.

Activation of the cAMP pathway by variant human MC1R alleles expressed in HEK and in melanoma cells

alpha-Melanocyte-stimulating hormone (alpha-MSH) activates the melanocortin-1 receptor (MC1R) on melanocytes to promote a switch from red/yellow pheomelanin synthesis to darker eumelanins via positive coupling to adenylate cyclase. The human MC1R locus is highly polymorphic with the specific variants associated with red hair and fair skin (RHC phenotype) postulated to be loss-of-function receptors. We have examined the ability of MC1R variants to activate the cAMP pathway in stably transfected REK293 cells. The RHC associated variants, Arg151Cys, Arg160Trp and Asp294His, demonstrated agonist-mediated increases in cAMP and phosphorylation of cAMP-responsive element-binding protein (CREB). Whereas the Asp294His variant showed severely impaired functional responses, the Arg151Cys and Arg160Trp variants retained considerable signaling capacity. Melanoma cells homozygous for either the Arg151Cys variant or consensus sequence both elicited CREB phosphorylation in response to alpha-MSH in the presence of IBMX. The common RHC alleles, Arg151Cys, Arg160Trp and Asp294His, are neither complete loss-of-function receptors nor are they functionally equivalent. (c) 2005 Elsevier Inc. All rights reserved.

A polymorphism in the agouti signalling protein (ASIP) is associated with decreased levels of mRNA

To date, a role for agouti signalling protein (ASIP) in human pigmentation has not been well characterized. It is known that agouti plays a pivotal role in the pigment switch from the dark eumelanin to the light pheomelanin in the mouse. However, because humans do not have an agouti banded hair pattern, its role in human pigmentation has been questioned. We previously identified a single polymorphism in the 3'-untranslated region (UTR) of ASIP that was found at a higher frequency in African-Americans compared with other population groups. To compare allele frequencies between European-Australians and indigenous Australians, the g.8818A -> G polymorphism was genotyped. Significant differences were seen in allele frequencies between these groups (P < 0.0001) with carriage of the G allele highest in Australian Aborigines. In the Caucasian sample set a strong association was observed between the G allele and dark hair colour (P = 0.004) (odds ratio 4.6; 95% CI 1.4-15.27). The functional consequences of this polymorphism are not known but it was postulated that it might result in message instability and premature degradation of the transcript. To test this hypothesis, ASIP mRNA levels were quantified in melanocytes carrying the variant and non-variant alleles. Using quantitative real-time polymerase chain reaction the mean ASIP mRNA ratio of the AA genotype to the AG genotype was 12 (P < 0.05). This study suggests that the 3'-UTR polymorphism results in decreased levels of ASIP and therefore less pheomelanin production.

Crystallization and preliminary x-ray diffraction analysis of the unliganded human growth hormone receptor

The crystal structure of the extracellular domain of growth hormone receptor complexed to its ligand, growth hormone, has been known since 1992. However, no information exists for the unliganded form of the receptor. The human growth hormone receptor's extracellular ligand-binding domain, encompassing amino-acid residues 1 - 238, has been expressed in Escherichia coli, purified by anion ion-exchange chromatography and crystallized in its unliganded state by the hanging-drop vapour-diffusion method in 100 mM HEPES pH 7.0 containing 27.5%(w/v) PEG 5000 monomethyl ether and 200 mM ammonium sulfate as the co-precipitants. The crystals belong to the othorhombic space group C222(1), have unit-cell parameters a = 99.7, b = 112.2, c = 93.2 Angstrom and diffract to 2.5 Angstrom resolution using synchrotron radiation. The crystal structure will shed light on the nature of any conformation changes that occur upon ligand binding and will provide information to develop potential low-molecular-weight agonists/antagonists to treat clinical diseases in which the growth hormone receptor is implicated.

Diversity of pigmentation in cultured human melanocytes is due to differences in the type as well as quantity of melanin

Cultured human melanocytes differ tremendously in visual pigmentation, and recapitulate the pigmentary phenotype of the donor's skin. This diversity arises from variation in type as well as quantity of melanin produced. Here, we measured contents of eumelanin (EM) and pheomelanin (PM) in 60 primary human melanocyte cultures (51 neonatal and nine adults), and correlated some of these values with the respective activity and protein levels of tyrosinase, and the melanocortin-1 receptor (MC1R) genotype. Melanocytes were classified into four phenotypes (L, L+, D, D+) as depicted by visual pigmentation using light microscopy, and by the pigmentary phenotype of the donor's skin. There were large differences in total melanin (TM) and EM, which increased progressively for L, L+, D and D+ melanocytes. TM content, the sum of EM and PM, showed a good correlation with TM measured spectrophotometrically, and with the activity and protein levels of tyrosinase. Log EM/PM ratio did not correlate with MC1R genotype. We conclude that: (i) EM consistently correlates with the visual phenotype; (ii) lighter melanocytes tend to be more pheomelanic in composition than darker melanocytes; (iii) in adult melanocyte cultures, EM correlates with the ethnic background of the donors (African-American > Indian > Caucasian); and (iv) MC1R loss-of-function mutations do not necessarily alter the phenotype of cultured melanocytes.

Quantitative analysis of MC1R gene expression in human skin cell cultures

To address the issue of melanocortin-1 receptor (MC1R) expression in non-melanocytic cells, we have quantitatively evaluated the relative expression levels of both MC1R mRNA and protein in a subset of different cell types. Using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) at high cycle numbers, we detected MC1R mRNA in all cell types examined, including human embryonic kidney-293 (HEK 293) cells, a cell type widely used as a negative control in melanocortin expression studies. Quantitative real-time PCR revealed the highest levels of MC1R transcripts were in melanocytic cells, whereas the keratinocyte and fibroblast cell cultures examined had only a low level of expression, similar to that of HEK 293 cells. Antibody mediated detection of MC1R protein in membrane extracts demonstrated exogenous receptor in MC1R transfected cell lines, as well as endogenous MC1R in melanoma cells. However, radioligand binding procedures were required to detect MC1R protein of normal human melanocytes and no surface expression of MC1R was detected in any of the non-melanocytic cells examined. This was consistent with their low level of mRNA, and suggests that, if present, the levels of surface receptor are significantly lower than that in melanocytes. The capacity of such limited levels of MC1R protein to influence non-melanocytic skin cell biology would likely be severely compromised. Indeed, the MC1R agonist [NIe(4), D-Phe(7)] alpha-melanocyte stimulating hormone (NDP-MSH) was unable to elevate intracellular cyclic adenosine monophosphate (cAMP) levels in the keratinocyte and fibroblast cells examined, whereas a robust increase was elicited in melanocytes. Although there are a variety of cell types with detectable MC1R mRNA, the expression of physiologically significant levels of the receptor may be more restricted than the current literature indicates, and within epidermal tissue may be limited to the melanocyte

Interactive effects of MC1R and OCA2 on melanoma risk phenotypes

The relationships between MC1R gene variants and red hair, skin reflectance, degree of freckling and nevus count were investigated in 2331 adolescent twins, their sibs and parents in 645 twin families. Penetrance of each MC1R variant allele was consistent with an allelic model where effects were multiplicative for red hair but additive for skin reflectance. Of nine MC1R variant alleles assayed, four common alleles were strongly associated with red hair and fair skin (Asp84Glu, Arg151Cys, Arg160Trp and Asp294His), with a further three alleles having low penetrance (Val60Leu, Val92Met and Arg163Gln). These variants were separately combined for the purposes of this analysis and designated as strong 'R' (OR=63.3; 95% CI 31.9-139.6) and weak 'r ' (OR=5.1; 95% CI 2.5-11.3) red hair alleles. Red-haired individuals are predominantly seen in the R/R and R/r groups with 67.1 and 10.8%, respectively. To assess the interaction of the brown eye color gene OCA2 on the phenotypic effects of variant MC1R alleles we included eye color as a covariate, and also genotyped two OCA2 SNPs (Arg305Trp and Arg419Gln), which were confirmed as modifying eye color. MC1R genotype effects on constitutive skin color, freckling and mole count were modified by eye color, but not genotype for these two OCA2 SNPs. This is probably due to the association of these OCA2 SNPs with brown/green not blue eye color. Amongst individuals with a R/R genotype (but not R/r), those who also had brown eyes had a mole count twice that of those with blue eyes. This suggests that other OCA2 polymorphisms influence mole count and remain to be described.

T1/ST2 - an IL-1 receptor-like modulator of immune responses

Th2-associated factors such as IL-4 are involved in both the development of Th2 responses (via modulating Th2 cell differentiation) and in the effector phase of Th2 responses (via modulating macrophage activation). The IL-1 receptor-like protein ST2 (T1, Fit-1, or DER4) is expressed as a membrane-bound (ST2L) or secreted form (sST2), and has been clearly implicated as a regulator of both the development and effector phases of Th2-type responses. Here we analyze the mechanisms and therapeutic implications of the unique ability of ST2 to promote development and function of type 2 helper T cells through a positive feedback loop, as well as to act as a negative feedback modulator of macrophage pro-inflammatory function. (C) 2004 Elsevier Ltd. All rights reserved.

The colony-stimulating factor 1 receptor is expressed on dendritic cells during differentiation and regulates their expansion

The lineage of dendritic cells (DC), and in particular their relationship to monocytes and macrophages, remains obscure. Furthermore, the requirement for the macrophage growth factor CSF-1 during DC homeostasis is unclear. Using a transgenic mouse in which the promoter for the CSF-1R (c-fms) directs the expression of enhanced GFP in cells of the myeloid lineage, we determined that although the c-fms promoter is inactive in DC precursors, it is up-regulated in all DC subsets during differentiation. Furthermore, plasmacytoid DC and all CD11c(high) DC subsets are reduced by 50-70% in CSF-1-deficient osteopetrotic mice, confirming that CSF-1 signaling is required for the optimal differentiation of DC in vivo. These data provide additional evidence that the majority of tissue DC is of myeloid origin during steady state and supports a close relationship between DC and macrophage biology in vivo.

The estrogen receptor 1 G594A polymorphism is associated with migraine susceptibility in two independent case/control groups

Migraine is a painful and debilitating disorder with a significant genetic component. Steroid hormones, in particular estrogen, have long been considered to play a role in migraine, as variations in hormone levels are associated with migraine onset in many sufferers of the disorder. Steroid hormones mediate their activity via hormone receptors, which have a wide tissue distribution. Estrogen receptors have been localized to the brain in regions considered to be involved in migraine pathogenesis. Hence it is possible that genetic variation in the estrogen receptor gene may play a role in migraine susceptibility. This study thus examined the estrogen receptor 1 (ESRalpha) gene for a potential role in migraine pathogenesis and susceptibility. A population-based cohort of 224 migraine sufferers and 224 matched controls were genotyped for the G594A polymorphism located in exon 8 of the ESR1 gene. Statistical analysis indicated a significant difference between migraineurs and non-migraineurs in both the allele frequencies (P=0.003) and genotype distributions (P=0.008) in this sample. An independent follow-up study was then undertaken using this marker in an additional population-based cohort of 260 migraine sufferers and 260 matched controls. This resulted in a significant association between the two groups with regard to allele frequencies (P=8x10(-6)) and genotype distributions (P=4x10(-5)). Our findings support the hypothesis that genetic variation in hormone receptors, in particular the ESR1 gene, may play a role in migraine.

Patched 1 conditional null allele in mice

The patched gene (Ptc) is a member of the hedgehog signaling pathway which plays a central role in the development of many invertebrate and vertebrate tissues. In addition, Ptc and a number of other pathway members are mutated in some common human cancers. Patched is the receptor for the hedgehog ligand and in the mouse ablation of the Ptc gene leads to developmental defects and an embryonic lethal phenotype. Here we describe a conditional Ptc allele in mice which will have utility for the temporospatial ablation of Ptc function. genesis 36:158-161, 2003. (C) 2003 Wiley-Liss, Inc.

Expression of caveolin-1 enhances cholesterol efflux in hepatic cells

HepG2 cells were stably transfected with human caveolin-1 (HepG2/cav cells). Transfection resulted in expression of caveolin-1 mRNA, a high abundance of caveolin-1 protein, and the formation of caveolae on the plasma membrane. Cholesterol efflux from HepG2/cav cells was 280 and 45% higher than that from parent HepG2 cells when human plasma and human apoA-I, respectively, were used as acceptors. The difference in efflux was eliminated by treatment of cells with progesterone. There was no difference in cholesterol efflux to cyclodextrin. Cholesterol efflux from plasma membrane vesicles was similar for the two cell types. Transfection led to a 40% increase in the amount of plasma membrane cholesterol in cholesterol-rich domains ( caveolae and/or rafts) and a 67% increase in the rate of cholesterol trafficking from intracellular compartments to these domains. Cholesterol biosynthesis in HepG2/cav cells was increased by 2-fold, and cholesterol esterification was reduced by 50% compared with parent HepG2 cells. The proliferation rate of transfected cells was significantly lower than that of non-transfected cells. Transfection did not affect expression of ABCA1 or the abundance of ABCA1 protein, but decreased secretion of apoA-I. We conclude that overexpression of caveolin-1 in hepatic cells stimulates cholesterol efflux by enhancing transfer of cholesterol to cholesterol-rich domains in the plasma membrane.

Overexpression of sonic hedgehog suppresses embryonic hair follicle morphogenesis

The Sonic Hedgehog (Shh) signalling pathway plays a central role in the development of the skin and hair follicle and is a major determinant of skin tumorigenesis, most notably of basal cell carcinoma (BCC). Various mouse models involving either ablation or overexpression of key members of the Shh signalling pathway display a range of skin tumours. To further examine the role of Shh in skin development. we have overexpressed Shh in a subset of interfollicular basal cells from 12.5 dpc under the control of the human keratin 1 (HK1) promoter. The HK1-Shh transgenic mice display a range of skin anomalies, including highly pigmented inguinal lesions and regions of alopecia. The most striking hair follicle phenotype is a suppression in embryonic follicle development between 14.0 and 19.0 dpc, resulting in a complete absence of guard, awl, and auchene hair fibres. These data indicate that alternative signals are responsible for the development of different hair follicles and point to a major role of Shh signalling in the morphogenesis of guard, awl, and auchene hair fibres. Through a comparison with other mouse models, the characteristics of the HK1-Shh transgenic mice suggest that the precise timing and site of Shh expression are key in dictating the resultant skin and tumour phenotype. 2003 Elsevier Inc. All rights reserved.