1 Department of Dermatology, University of Cincinnati College of Medicine, PO
Box 670592, Cincinnati, Ohio 45267-0592, USA
2 Fujita Health University School of Health Sciences, Toyoake, Aichi 470-1192,
Japan
3 Department of Dermatology, Nara Medical University, Kashihara, Nara 634-8522,
Japan
4 Department of Molecular Genetics, Biochemistry and Microbiology, University of
Cincinnati College of Medicine, PO Box 670524, Cincinnati, Ohio 45267-0524,
USA
5 POLA Laboratories, 560 Kashio-cho, Totsuka-ku, Yokohama 244-0812, Japan
6 Laboratory of Cell Biology, National Cancer Institute, National Institutes of
Health, 9000 Rockville Pike, Bethesda, MD 20892, USA
* Author for correspondence (e-mail: abdelmza{at}email.uc.edu )
Accepted 6 March 2002
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Summary |
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Key words: Melanocortin 1 receptor, Human melanocytes, MC1R variants
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Introduction |
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Eumelanin synthesis in melanocytes is stimulated by activation of the
-melanocyte stimulating hormone (
-MSH) receptor, termed the
melanocortin 1 receptor (MC1R) (Geschwind
et al., 1972
; Tamate and
Takeuchi, 1984
; Hunt et al.,
1995
). The human MC1R is more polymorphic than several
other pigment genes, including tyrosinase, suggesting its importance
in determining constitutive pigmentation in humans
(Sturm et al., 1998
;
Rees, 2000
). Furthermore,
mutations in the gene for proopiomelanocortin, the precursor for melanocortins
and other bioactive peptides, result in red hair color in addition to
metabolic abnormalities, including adrenal insufficiency and obesity
(Krude et al., 1998
). This
underscores the significance of melanocortins in regulating eumelanin
synthesis in humans.
The MC1R is a G-protein-coupled receptor with seven transmembrane domains
(Chhajlani and Wikberg, 1992;
Mountjoy et al., 1992
).
Binding of
-MSH to this receptor activates adenylate cyclase and
increases cAMP formation (Suzuki et al.,
1996
). More than 30 allelic variants of the human MC1R
have been identified mainly in northern European populations and in Australia
(Valverde et al., 1995
;
Box et al., 1997
;
Smith et al., 1998
;
Rees, 2000
). However, the
consequences of these variants on the physiological function of the MC1R
remain poorly understood. Among the variants so far reported, Arg142His,
Arg151Cys, Arg 160Trp and Asp294His are the mutations mostly associated with
the red hair phenotype and reduced tanning ability in several populations
(Box et al., 1997
;
Smith et al., 1998
;
Healy et al., 2000
). This
supports the notion that
-MSH and its receptor significantly affect the
response of melanocytes to UVR (Pawelek et
al., 1992
; Im et al.,
1998
). The above four MC1R variants are common in
melanoma patients, and increase the risk of melanoma by more than twofold
(Palmer et al., 2000
).
Expression of those variants in heterologous cell cultures reduced the
functional coupling of the MC1R to adenylate cyclase
(Frändberg et al., 1998
;
Schiöth et al., 1999
). As
yet, no studies have shown how allelic variants of MC1R would affect
the function of the receptor in human melanocytes, a main physiological target
for melanotropins in the skin. Therefore, we sought to analyze the biological
consequences of MC1R mutations by investigating the responses of
genetically different epidermal melanocyte cultures to
-MSH and
UVR.
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Materials and Methods |
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Determination of tyrosinase activity and proliferation
To determine the effects of -MSH on melanocyte proliferation and
tyrosinase activity, melanocytes were plated onto 60 mm dishes at a density of
2.5x105 cells. 72 hours later, and every other day thereafter
for a total of 6 days, the growth medium was changed and melanocytes were
treated with 0 (control), 0.1, 1 or 10 nM
-MSH, or with 1 µM
forskolin (n=3 dishes per group) (both were obtained from Sigma
Chemical Company, St Louis, Missouri). The tyrosine hydroxylase activity of
tyrosinase was determined as described previously
(Pomerantz, 1969
;
Abdel-Malek et al., 1992
). The
cell number was determined using a Coulter Counter. Each experiment was
repeated at least twice for each melanocyte culture. Statistical analysis was
carried out using the Student's t-test to compare the effects of
different concentrations of
-MSH on each individual melanocyte culture.
Two-way ANOVA was also used to compare the responses to different
concentrations of
-MSH of melanocyte cultures that expressed wild-type
MC1R and their MC1R mutant counterparts, which had reduced response to
-MSH.
Sequencing of the MC1R gene
PCR amplification, sequencing, and restriction fragment length polymorphism
(RFLP) analysis of the MC1R gene were carried out as follows. Total
RNA was purified from cultured human melanocytes using an RNA Easy Kit
(Qiagen, Valencia, CA). The entire coding region of the MC1R was amplified
using reverse transcriptase and nested PCR amplification. 3 µg of total RNA
was amplified in a PCR reaction containing standard concentrations of reverse
transcriptase, Taq DNA polymerase, MgCl2, RNAse inhibitor, dNTPs,
buffer and primers, as described previously
(Koppula et al., 1997) in a
total volume of 50 µl. The complementary strand was synthesized at 43°C
for 1 hour, and the MC1R was amplified for 35 cycles (1 minute at
94°C, 1 minute at 60°C, and 2 minutes at 72°C) in an automated
thermal cycler (Gene Amp PCR System 9600, Perkin Elmer, Boston, MA). 1-5 µl
of the first reaction was amplified in two separate reactions using two sets
of M13-adjoined nested primers, first half N-terminal primer+M13up
(5'-TGTAAAACGACGGCCAGTCCTGGCAGCACCATGAACTAAGC-3'); first half
C-terminal primer+M13rp
(5'-CAGGAAACAGCTATGACCTGGTCGTAGTAGGCGATGAAGAGC-3'); second half
N-terminal primer+M13up
(5'-TGTAAAACGACGGCCAGTCGCTACATCTCCATCTTCTACGCAC3'); and second
half C-terminal primer+M13rp (5'-CAGGAAACAGCTATGACCCTCTGCCCACACTTAAAGC
3'). The standard concentrations of PCR reagents were added to the first
reaction and amplified for 25 cycles (30 seconds at 94°C, 1 minute at
62°C and 1 minute at 72°C). The final reaction yielded a 640 (first
half) and a 560 (second half) nucleotide product flanked by the M13 primers. 5
µl of the PCR reaction was electrophoresed on a 2% agarose gel. The
remainder was purified in a Centri-Spin Column (Princeton Separations,
Adelphia, NJ). The PCR products were sequenced by dye primer chemistry.
Briefly, M13 forward and reverse primers were labeled with four fluorescent
dyes in four separate base-specific tubes. The products were electrophoresed
and read by an automated sequencing machine (Perkin Elmer/Applied Biosystems
models 373A or 377). We confirmed some mutations by restriction fragment
length polymorphism analysis for variants at codons 151 (HhaI), 160
(SstII) and 294 (TaqI). The PCR products were digested using
standard conditions and run on 1-3% agarose gels.
Determination of cAMP levels
The dose-dependent effect of -MSH on cAMP formation in human
melanocytes was determined using a cyclic AMP radioimmunoassay kit (Dupont-New
England Nuclear, Boston, MA), as recommended by the manufacturer and as
described previously (Suzuki et al.,
1996
). Duplicate samples were assayed from each culture well
(triplicate wells/group) after the appropriate dilution (1:10 for groups
treated with 10 nM
-MSH, and 1:5 for the remaining groups). Each
culture was tested twice in two separate experiments. The data were analyzed
by two-way ANOVA and Student's t-test, as described above.
Analysis of eumelanin and pheomelanin content and total melanin
content
Melanocytes were lyophilized, and eumelanin and pheomelanin contents were
analyzed using a microassay developed previously
(Ito and Fujita, 1985).
Duplicate samples of melanocytes deprived of bovine pituitary extract
(approximately 1x106 melanocytes/sample) were oxidized by
permanganate to pyrrole 2,3,5-tricarboxylic acid (PTCA) and analyzed by HPLC
with UV detection to determine eumelanin content. Identical duplicate samples
were hydrolyzed with hydriodic acid to aminohydroxyphenylalanine (AHP), and
analyzed by HPLC with electrochemical detection to determine pheomelanin
content. Variations of PTCA and AHP values were approximately 10% or less when
determined on separate occasions in this study. The amount of eumelanin can be
obtained by multiplying the amount of PTCA by a conversion factor of 160,
while the amount of pheomelanin can be obtained by multiplying the amount of
AHP by a conversion factor of 10 (Ozeki et
al., 1996
). Statistical significance of differences was assessed
with the Mann-Whitney test. Differences were considered to be significant when
P values were less than 0.05.
Total melanin content was determined in 0.5-1x106
melanocytes. Cells were harvested, centrifuged, washed twice with PBS, counted
and centrifuged. The cell pellets were solubilized in 0.2 M NaOH
(1x106 cells/ml) for 1 hour, and melanin content was
determined spectrophotometrically by reading the absorbance at 475 nm. Melanin
content was calculated using a standard curve generated from the absorbance of
known concentrations of synthetic melanin, as described previously
(Barker et al., 1995).
Response of melanocytes to UVBR
The response of melanocytes to UVBR was determined by plating the cells in
complete growth medium at a density of 1x105cells/60 mm dish.
72 hours later, melanocytes were irradiated once with 21 mJ/cm2
UVBR as described previously (Barker et
al., 1995). The UV source is a bank of six FS20 sun lamps
(Westinghouse) with 75% emission in the UVB and 25% emission in the UVA range.
The peak emission of the lamps is at 313 nm. Percent cell death was determined
on days 2 and 4 after UV irradiation by calculating the number of dead
melanocytes that detached and incorporated Trypan blue dye and the number of
viable cells that remained attached to the culture dish and excluded Trypan
blue, as described before (Barker et al.,
1995
). The responses to UVR of the cultures with functional MC1R
were compared with those of the cultures with reduced response to
-MSH
using one-way ANOVA.
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Results |
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The sequencing data presented in Table
1 revealed that NHM 753-c was homozygous for Arg160Trp, NHM 830-c
was heterozygous for Arg160Trp and Asp294His, and NHM 849-b was heterozygous
for Arg151Cys and Asp294His substitutions in MC1R. All three cultures failed
to respond to -MSH with a significant dose-dependent increase in
tyrosinase activity (Fig. 1B).
NHM 755-c was homozygous for Val92Met substitution, and homozygous for a
silent mutation, Thr314Thr, in the MC1R. Four other cultures were heterozygous
for Arg151Cys (NHM 731-c), Val60Leu (NHM 777-c), Arg163Gln (NHM 790-c) and
Phe196Leu (NHM 780-b) substitutions. Two cultures were heterozygous for
Val92Met substitution (NHM 754-b and 765-c). NHM 754-b and 780-b were
heterozygous for a silent mutation, Thr314Thr and Thr177Thr, respectively.
Only three cultures, NHM 729-b, 747-c and 751-b carried the wild-type
MC1R genotype.
|
|
Analysis of eumelanin to pheomelanin content showed that NHM-b cultures
consistently expressed higher eumelanin to pheomelanin ratios than NHM-c
cultures. The mean (±s.d.) content of eumelanin of NHM-b cultures
(25.5±8.02; n=4) was more than tenfold higher
(P0.01) than that of NHM-c cultures (1.47±0.72;
n=7). Also, the mean (±s.d.) content of pheomelanin of NHM-b
cultures (3.37±0.77) was significantly higher (P
0.01) than
that of NHM-c (1.48±0.86). As a result, the mean (±s.d.)
eumelanin to pheomelanin ratio of NHM-b (8.15±4.17) was sevenfold
higher (P
0.01) than that of NHM-c (1.12±0.41).
Response of melanocytes with known MC1R genotype to
-MSH
Melanocyte cultures homozygous for the consensus MC1R or
heterozygous for one variant of the MC1R (represented by the data for
NHM 765-c) responded to -MSH with dose-dependent increases in cAMP
levels, tyrosinase activity and proliferation
(Fig. 1). Variations in the
magnitude of the response to
-MSH among cultures may be attributed to
differential expression of other genes involved in the regulation of
pigmentation. NHM 753-c, homozygous for Arg160Trp, 830-c, heterozygous for
Arg160Trp and Asp294His, and 849-b, heterozygous for Arg151Cys and Asp294His
substitutions in MC1R were unresponsive, or 100-times less responsive to
-MSH than melanocytes wild-type or heterozygous for MC1R
variants (Fig. 1). Comparing
the responses to
-MSH of NHM 753-c, 830-c and 849-b to that of NHM
765-c demonstrated significant differences. NHM 753-c, 830-c and 849-b showed
no significant change in cAMP levels after treatment with
-MSH, as
determined using Student's t-test.
(Fig. 1A). In contrast, NHM
765-c, heterozygous for Val92Met substitution and with a low eumelanin to
pheomelanin ratio, responded to 0.1 or 10 nM
-MSH with significant
increases in cAMP levels above control (66% and 4.5-fold, respectively;
P
0.001, as determined by Student's t-test). NHM 755-c
homozygous for the Val92Met substitution responded to 0.1 nM and 10 nM
-MSH with a 4-fold and 11-fold increase in cAMP formation
(Fig. 1A).
Additionally, NHM 753-c, 830-c and 849-b showed no change, while NHM 765-c
showed significant increases, in tyrosinase activity in response to 0.1 or 1
nM -MSH (about 90% or 130% increase, respectively; P<0.0001
using Student's t-test) (Fig.
1B). NHM 753-c, 830-c and 849-b showed no significant stimulation,
while NHM 765-c showed a 160% increase in tyrosinase activity after treatment
with 10 nM
-MSH. NHM 753-c, 830-c and 849-b responded to 1 µM
forskolin, an activator of adenylate cyclase, with remarkable increases in
proliferation and tyrosinase activity. This indicated that their adenylate
cyclase is inducible and that their inability to respond to
-MSH is due
to a defect that lies upstream of adenylate cyclase
(Fig. 1B,C). NHM 755-c
responded dose-dependently to 1 or 10 nM
-MSH, with an 80 or 110%
increase in tyrosinase activity, respectively (P<0.001, as
determined by Student's t-test).
As expected, the cultures wild-type for MC1R responded with a
dose-dependent increase in proliferation beginning at a dose of 0.1 nM
(Abdel-Malek et al., 1995;
Suzuki et al., 1996
). NHM
753-c and 849-b demonstrated a significant increase (38% above control) in
cell number only following treatment with 10 nM
-MSH
(P<0.01); NHM 830-c showed no stimulation of proliferation at any
of the concentrations of
-MSH that were used
(Fig. 1C). In comparison, NHM
765-c demonstrated a 30 (P<0.1), 126 or 229%
(P<0.0001) increase, and NHM 755-c, homozygous for Val92Met
substitution, demonstrated a 25, 70 or 96% increase in cell number above
control (P<0.0001) in response to 0.1, 1 or 10 nM
-MSH,
respectively. Further statistical analysis using two-way ANOVA showed that the
effects of 1 and 10 nM
-MSH on cAMP levels, tyrosinase activity and
proliferation of NHM 753-c, 830-c and 849-b were significantly different than
the effects on NHM 747-c, 729-b and 751-b with wild-type MC1R
(P
0.01).
MC1R genotype and the responses of melanocytes to UVR
We assessed the survival of melanocyte cultures following a single exposure
to a dose of 21 mJ/cm2 UVBR. This treatment resulted in a 28, 31
and 34% cell death of NHM 753-c, 830-c and 849-b, respectively, compared with
only 6% cell death of NHM 765-c and 755-c, at 2 days after irradiation
(Fig. 2). NHM 753-c, 830-c and
849-b encountered a 17, 22 and 27% cell death, respectively, on day 4 after
UVB exposure, compared with 8 and 5% cell death in NHM 765-c and 755-c,
respectively (Fig. 2). The
differences in the extent of cell death between the cultures that were
unresponsive to -MSH and those with functional MC1R were statistically
significant, as determined by one-way ANOVA, which took into account the
responsiveness to
-MSH (P<0.0001). The responses of the
latter two cultures is comparable with that of many other cultures with
functional MC1R that we have tested. We did not detect significant
differences in the amounts of UVB-induced cyclobutane pyrimidine dimers or
pyrimidine 6,4-pyrimidone photoproducts in NHM 753-c with loss-of-function
MC1R and NHM 765-c with functional MC1R that have comparable
melanin contents (data not shown). Melanocyte cultures respond to
UVB-irradiation with a linear dose-dependent increase in the generation of
extracellular hydrogen peroxide. We did not detect significant differences in
the amounts of extracellular hydrogen peroxide generation in NHM 830-c with a
loss-of-function MC1R and a NHM-c with a functional MC1R and
comparable melanin content (data not shown).
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Discussion |
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The current study is the first to elucidate the impact of various allelic
variants of the human MC1R on the response of human melanocytes to
-MSH and UVR. Genetic sequencing of the MC1R in 13 different
melanocyte cultures revealed extensive polymorphism, with 31% of the cultures
homozygous for a MC1R allelic variant or compound heterozygous for
two different allelic variants, and 54% of the cultures heterozygous for one
MC1R variant (Table
1). Unexpectedly, NHM 849-b, with a relatively high eumelanin to
pheomelanin ratio (4.09), was heterozygous for Arg151Cys and Asp294His
substitutions in MC1R (Table
1), confirming that MC1R variants are necessary but not
sufficient for the red hair phenotype
(Sturm et al., 1998
).
All 13 melanocyte cultures were compared for their ability to respond to
-MSH with dose-dependent stimulation of cAMP formation, tyrosinase
activity and proliferation (Fig.
1). Binding of
-MSH to the MC1R stimulates cAMP formation,
a major pathway for stimulation of melanogenesis, particularly eumelanin
synthesis, and proliferation in human melanocytes
(Abdel-Malek et al., 1992
;
Suzuki et al., 1996
;
Sakai et al., 1997
). Our
finding that increases in tyrosinase activity did not correlate perfectly with
increases in cAMP following treatment of responsive melanocyte cultures to
-MSH suggest that the MC1R activates other pathways, such as protein
kinase Cß (Park et al.,
1996
). As expected, melanocyte cultures homozygous for wild-type
MC1R demonstrated a typical dose-dependent response to
-MSH
beginning at a dose of 0.1 nM (Fig.
1) (Abdel-Malek et al.,
1995
; Suzuki et al.,
1996
). All cultures heterozygous for a MC1R variant had
dose-dependent responses to
-MSH similar to those of cultures
expressing the wild-type gene, showing that a single wild-type functional copy
of MC1R is sufficient for receptor function. In contrast, NHM 753-c,
homozygous for Arg160Trp, 830-c and 849-b, heterozygous for Arg160Trp and
Asp294His and for Arg151Cys and Asp294His, respectively, had a drastically
reduced response to
-MSH (Fig.
1). The inability of NHM 753-c, 830-c and 849-b to respond to
-MSH with a dose-dependent stimulation of cAMP formation is not due to
lack of expression of the MC1R gene, as all three cultures expressed
MC1R mRNA as determined by northern blot analysis (data not shown),
nor to a defect in adenylate cyclase since they were responsive to forskolin
(Fig. 1B,C). The high eumelanin
to pheomelanin ratio of 849-b is not the result of a constitutively active
MC1R that could not be stimulated by
-MSH, as in the sombre mouse
(Robbins et al., 1993
;
Abdel-Malek et al., 2001
).
Basal cAMP level in 849-b was comparable with the levels in 747-c and 751-c,
which expressed the consensus MC1R, as described in the legend for
Fig. 1A. Our findings are
corroborated by previous reports and demonstrate that Arg151Cys, Arg160Trp,
and Asp294His substitutions in the MC1R significantly diminish the functional
coupling of the receptor as shown by poor stimulation of intracellular cAMP
production in response to
-MSH (Fig.
1A) (Frändberg et al.,
1998
; Schiöth et al.,
1999
). In transfected cells, those allelic variants did not
significantly reduce the binding affinity of
-MSH for MC1R since
Arg151Cys and Arg160Trp lie within the second intracellular loop of the MC1R,
a region unlikely to be involved in receptor binding
(Prusis et al., 1997
).
One report (Xu et al.,
1996) suggested that the Val92Met substitution reduces the binding
affinity of MC1R for
-MSH, while another found no alteration in the
functional coupling of the receptor to adenylate cyclase
(Koppula et al., 1997
). This
variant was identified in Chinese individuals, and therefore is not associated
with a red hair phenotype (Box et al.,
1997
). Here, we showed that NHM 755-c, homozygous for Val92Met
substitution, responds dose-dependently to
-MSH with stimulation of
cAMP formation, tyrosinase activity and proliferation, suggesting that this
polymorphism does not represent a loss-of-function in the MC1R
(Fig. 1A-C).
A role for the melanocortins and MC1R in the response of melanocytes to UVR
and in determining the tanning ability of individuals has been proposed
(Pawelek et al., 1992;
Im et al., 1998
;
Healy et al., 2000
). The
observed exaggerated sensitivity of melanocyte cultures with loss-of-function
MC1R to UVR emphasizes the significance of the MC1R in the cutaneous
response to UVR (Fig. 2).
Comparison of the UV responses of NHM 765-c and 755-c with functional MC1R to
the UV responses of NHM 753-c and 830-c with loss-of-function MC1R revealed a
striking difference in the extent of cell death. Despite similar melanin
contents in all four melanocyte cultures, NHM 753-c and 830-c demonstrated a
greater extent of cell death than NHM 765-c and 755-c. Additionally, the
observation that NHM 849-b, with a high eumelanin to pheomelanin ratio and
loss-of-function MC1R, is more sensitive to UVR-induced cytotoxicity than NHM
765-c or 755-c suggests that regardless of constitutive pigmentation,
inability of melanocytes to respond to
-MSH reduces their defense
mechanisms against UVR genotoxicity. The mechanism for the increased
susceptibility of these melanocytes to UVR is now being investigated. Our
preliminary results showed that the amounts of cyclobutane pyrimidine dimers
and pyrimidine(6-4)pyrimidone photoproducts, the major types of DNA damage
induced by UVBR (Brash, 1988
),
were not increased in NHM 753-c. However, the rate of removal of these
photoproducts is yet to be determined. Our results suggest that the extensive
cell death observed in NHM 753-c and, by extension, in NHM 830-c and 849-b,
possibly result from impaired DNA repair and/or oxidative DNA damage. Our
preliminary data show a linear dose-dependent increase in the generation of
hydrogen peroxide by UVB-irradiated melanocytes, suggesting an induction of a
prooxidant state. Recently, it was reported that
-MSH protects from
oxidative stress that may result from exposure of cells to UVR
(Haycock et al., 2000
).
This report is significant since it is the first to document the effects of
four known MC1R variants, namely Arg151Cys, Arg160Trp, Asp294His and
Val92Met, on the responses of human melanocytes to -MSH and UVR. Our
results suggest that the MC1R genotype is a useful marker that is
more reliable than melanin content for predicting the sensitivity of
individuals to sun exposure and their susceptibility to skin cancer.
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Acknowledgments |
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References |
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Abdel-Malek, Z., Swope, V. B., Pallas, J., Krug, K. and Nordlund, J. J. (1992). Mitogenic, melanogenic and cAMP responses of cultured neonatal human melanocytes to commonly used mitogens. J. Cell. Physiol. 150,416 -425.[Medline]
Abdel-Malek, Z., Swope, V., Collins, C., Boissy, R., Zhao, H.
and Nordlund, J. (1993). Contribution of melanogenic proteins
to the heterogeneous pigmentation of human melanocytes. J. Cell
Sci. 106,1323
-1331.
Abdel-Malek, Z., Swope, V. B., Suzuki, I., Akcali, C., Harriger, M. D., Boyce, S. T., Urabe, K. and Hearing, V. J. (1995). Mitogenic and melanogenic stimulation of normal human melanocytes by melanotropic peptides. Proc. Natl. Acad. Sci. USA 92,1789 -1793.[Abstract]
Abdel-Malek, Z. A., Scott, M. C., Furumura, M., Lamoreux, M. L.,
Ollmann, M., Barsh, G. S. and Hearing, V. J. (2001). The
melanocortin 1 receptor is the principal mediator of the effects of agouti
signaling protein on mammalian melanocytes. J. Cell
Sci. 114,1019
-1024.
Barker, D., Dixon, K., Medrano, E. E., Smalara, D., Im, S., Mitchell, D., Babcock, G. and Abdel-Malek, Z. A. (1995). Comparison of the responses of human melanocytes with different melanin contents to ultraviolet B irradiation. Cancer Res. 55,4041 -4046.[Abstract]
Bastiaens, M. T., ter Huurne, J. A. C., Kielich, C., Gruis, N. A., Westendorp, R. G. J., Vermeer, B. J. and Bouwes Bavinck, J. N. (2001). Melanocortin-1 receptor gene variants determine the risk of non-melanoma skin cancer independent of fair skin type and red hair. Am. J. Hum. Genet. 68,884 -894.[Medline]
Box, N. F., Wyeth, J. R., O'Gorman, L. E., Martin, N. G. and
Sturm, R. A. (1997). Characterization of melanocyte
stimulating hormone receptor variant alleles in twins with red hair.
Hum. Mol. Genet. 6,1891
-1897.
Box, N. F., Duffy, D. L., Irving, R. E., Russell, A., Chen, W.,
Griffiths, L. R., Parsons, P. G., Green, A. C. and Sturm, R. A.
(2001). Melanocortin-1 receptor genotype is a risk factor for
basal and squamous cell carcinoma. J. Invest.
Dermatol. 116,224
-229.
Brash, D. E. (1988). UV mutagenic photoproducts in Escherichia coli and human cells: A molecular genetics perspective on human skin cancer. Photochem. Photobiol. 48, 59-66.[Medline]
Chhajlani, V. and Wikberg, J. E. S. (1992). Molecular cloning and expression of the human melanocyte stimulating hormone receptor cDNA. FEBS Lett. 309,417 -420.[Medline]
Fitzpatrick, T. B., Sober, A. J., Pearson, B. J. and Lew, R. (1976). Cutaneous carcinogenic effects of sunlight in humans. In Research in Photobiology (ed. A. Castellani), pp.485 -490. New York: Plenum Press.
Frändberg, P.-A., Doufexis, M., Kapas, S. and Chhajlani, V. (1998). Human pigmentation phenotype: a point mutation generates nonfunctional MSH receptor. Biochem. Biophys. Res. Commun. 245,490 -492.[Medline]
Geschwind, I. I., Huseby, R. A. and Nishioka, R. (1972). The effect of melanocyte-stimulating hormone on coat color in the mouse. Rec. Prog. Hormone Res. 28, 91-130.[Medline]
Harding, R. M., Healy, E., Ray, A. J., Ellis, N. S., Flanagan, N., Todd, C., Dixon, C., Sajantila, A., Jackson, I. J., Birch-Machin, M. A. and Rees, J. L. (2000). Evidence for variable selective pressures at MC1R. Am. J. Hum. Genet. 66,1351 -1361.[Medline]
Haycock, J. W., Rowe, S. J., Cartledge, S., Wyatt, A., Ghanem,
G., Morandini, R., Rennie, I. G. and MacNeil, S. (2000).
-Melanocyte-stimulating hormone reduces impact of proinflammatory
cytokine and peroxide-generated oxidative stress on keratinocyte and melanoma
cell lines. J. Biol. Chem.
275,15629
-15636.
Healy, E., Flannagan, N., Ray, A., Todd, C., Jackson, I. J., Matthews, J. N., Birch-Machin, M. A. and Rees, J. L. (2000). Melanocortin-1 receptor gene and sun sensitivity in individuals without red hair. Lancet 355,1072 -1073.[Medline]
Hunt, G., Kyne, S., Wakamatsu, K., Ito, S. and Thody, A. J.
(1995). Nle4DPhe7
-Melanocyte-stimulating hormone increases the eumelanin: Phaeomelanin
ratio in cultured human melanocytes. J. Invest.
Dermatol. 104,83
-85.[Abstract]
Im, S., Moro, O., Peng, F., Medrano, E. E., Cornelius, J.,
Babcock, G., Nordlund, J. and Abdel-Malek, Z. (1998).
Activation of the cAMP pathway by -melanotropin mediates the response
of human melanocytes to UVB light. Cancer Res.
58, 47-54.[Abstract]
Ito, S. and Fujita, K. (1985). Microanalysis of eumelanin and pheomelanin in hair and melanomas by chemical degradation and liquid chromatography. Anal. Biochem. 144,527 -536.[Medline]
Kaidbey, K. H., Poh Agin, P., Sayre, R. M. and Kligman, A. M. (1979). Photoprotection by melanin a comparison of black and Caucasian skin. J. Am. Acad. Dermatol. 1, 249-260.[Medline]
Kennedy, C., ter Huurne, J., Berkhout, M., Gruis, N., Bastiaens,
M., Bergman, W., Willemze, R. and 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.
Kobayashi, N., Nakagawa, A., Muramatsu, T., Yamashina, Y., Shirai, T., Hashimoto, M. W., Ishigaki, Y., Ohnishi, T. and Mori, T. (1998). Supranuclear melanin caps reduce ultraviolet induced DNA photoproducts in human epidermis. J. Invest. Dermatol. 110,806 -810.[Abstract]
Koppula, S. V., Robbins, L. S., Lu, D., Baack, E., White, C. R., Jr, Swanson, N. A. and Cone, R. D. (1997). Identification of common polymorphisms in the coding sequence of the human MSH receptor (MC1R) with possible biological effects. Hum. Mutat. 9, 30-36.[Medline]
Krude, H., Biebermann, H., Luck, W., Horn, R., Brabant, G. and Grüters, A. (1998). Severe early-onset obesity, adrenal insufficiency and red hair pigmentation caused by POMC mutations in humans. Nat. Genet. 19,155 -157.[Medline]
Menon, A., Persad, A., Ranadine, N. S. and Haberman, H. F. (1983). Effects of ultraviolet-visible radiation in the presence of melanin isolated from human black or red hair upon Ehrlich ascites carcinoma cells. Cancer Res. 43,3165 -3169.[Abstract]
Mountjoy, K. G., Robbins, L. S., Mortrud, M. T. and Cone, R. D. (1992). The cloning of a family of genes that encode the melanocortin receptors. Science 257,1248 -1251.[Medline]
Ozeki, H., Ito, S., Wakamatsu, K. and Thody, A. J. (1996). Spectrophotometric characterization of eumelanin and pheomelanin in hair. Pigment Cell Res. 9, 265-270.[Medline]
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. and 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.[Medline]
Park, H.-Y., Russakovsky, V., Ao, Y., Fernandez, E. and
Gilchrest, B. A. (1996). -Melanocyte stimulating
hormone-induced pigmentation is blocked by depletion of protein kinase C.
Exp. Cell Res. 227,70
-79.[Medline]
Pathak, M. A. (1995). Functions of melanin and protection by melanin. In Melanin: Its Role in Human Photoprotection (ed. L. Zeise, M. R. Chedekel and T. B. Fitzpatrick), pp. 125-133. Overland Park: Valdenmar Publishing Company.
Pathak, M. A., Hori, Y., Szabó, G. and Fitzpatrick, T. B. (1971). The photobiology of melanin pigmentation in human skin. In Biology of Normal and Abnormal Melanocytes (ed. T. Kawamura, T. B. Fitzpatrick and M. Seiji), pp.149 -169. Baltimore: University Park Press.
Pathak, M. A., Jimbow, K. and Fitzpatrick, T. (1980). Photobiology of pigment cells. In Phenotypic Expression in Pigment Cells (ed. M. Seiji), pp.655 -670. Japan: University of Tokyo Press, Tokyo.
Pawelek, J. M., Chakraborty, A. K., Osber, M. P., Orlow, S. J., Min, K. K., Rosenzweig, K. E. and Bolognia, J. L. (1992). Molecular cascades in UV-induced melanogenesis: a central role for melanotropins? Pigment Cell Res. 5, 348-356.[Medline]
Pomerantz, S. H. (1969). L-tyrosine-3,5-3H assay for tyrosinase development in skin of newborn hamsters. Science 164,838 -839.[Medline]
Prusis, P., Schiöth, H. B., Muceniece, R., Herzyk, P., Asfar, M., Hubbard, R. and Wikberg, J. E. S. (1997). Modeling of the three dimensional structure of the human melanocortin 1 receptor, using an automated method and docking of a rigid cyclic melanocyte-stimulating hormone core peptide. J. Mol. Graph. Model. 15,307 -317.[Medline]
Rees, J. L. (2000). The melanocortin 1 receptor (MC1R): more than just red hair. Pigment Cell Res. 13,135 -140.[Medline]
Robbins, L. S., Nadeau, J. H., Johnson, K. R., Kelly, M. A., Roselli-Rehfuss, L., Baack, E., Mountjoy, K. G. and Cone, R. D. (1993). Pigmentation phenotypes of variant extension locus alleles result from point mutations that alter MSH receptor function. Cell 72,827 -834.[Medline]
Sakai, C., Ollmann, M., Kobayashi, T., Abdel-Malek, Z., Muller,
J., Vieira, W. D., Imokawa, G., Barsh, G. S. and Hearing, V. J.
(1997). Modulation of murine melanocyte function in
vitro by agouti signal protein. EMBO J.
16,3544
-3552.
Schiöth, H. B., Phillips, S. R., Rudzish, R., Birch-Machin, M. A., Wikberg, J. E. S. and Rees, J. L. (1999). Loss of function mutations of the human melanocortin 1 receptor are common and are associated with red hair. Biochem. Biophys. Res. Commun. 260,488 -491.[Medline]
Smith, R., Healy, E., Siddiqui, S., Flanagan, N., Steijlen, P. M., Rosdahl, I., Jacques, J. P., Rogers, S., Turner, R., Jackson, I. J. et al. (1998). Melanocortin 1 receptor variants in Irish population. J. Invest. Dermatol. 111,119 -122.[Abstract]
Sturm, R. A., Box, N. F. and Ramsay, M. (1998). Human pigmentation genetics: the difference is only skin deep. BioEssays 20,712 -721.[Medline]
Suzuki, I., Cone, R., Im, S., Nordlund, J. and Abdel-Malek, Z. (1996). Binding capacity and activation of the MC1 receptors by melanotropic hormones correlate directly with their mitogenic and melanogenic effects on human melanocytes. Endocrinology 137,1627 -1633.[Abstract]
Tamate, H. B. and Takeuchi, T. (1984). Action
of the e locus of mice in the response of phaeomelanic hair follicles
to -melanocyte-stimulating hormone in vitro.
Science 224,1241
-1242.[Medline]
Valverde, P., Healy, E., Jackson, I., Rees, J. L. and Thody, A. J. (1995). Variants of the melanocyte-stimulating hormone receptor gene are associated with red hair and fair skin in humans. Nat. Genet. 11,328 -330.[Medline]
Xu, X., Thörnwall, M., Lundin, L.-G. and Chhajlani, V. (1996). Val92Met variant of the melanocyte stimulating hormone receptor gene. Nat. Genet. 14, 384.[Medline]