Premature Graying as a Consequence of Compromised Antioxidant Activity in Hair Bulb Melanocytes and Their Precursors

Abstract

Intricate coordinated mechanisms that govern the synchrony of hair growth and melanin synthesis remain largely unclear. These two events can be uncoupled in prematurely gray hair, probably due to oxidative insults that lead to the death of oxidative stress-sensitive melanocytes. In this study, we examined the gene expression profiles of middle (bulge) and lower (hair bulb) segments that had been micro-dissected from unpigmented and from normally pigmented hair follicles from the same donors using quantitative real-time RT-PCR (qPCR) arrays. We found a significant down-regulation of melanogenesis-related genes (TYR, TYRP1, MITF, PAX3, POMC) in unpigmented hair bulbs and of marker genes typical for melanocyte precursor cells (PAX3, SOX10, DCT) in unpigmented mid-segments compared with their pigmented analogues. qPCR, western blotting and spin trapping assays revealed that catalase protein expression and hydroxyl radical scavenging activities are strongly repressed in unpigmented hair follicles. These data provide the first clear evidence that compromised antioxidant activity in gray hair follicles simultaneously affects mature hair bulb melanocytes and their immature precursor cells in the bulge region.

Full text

Premature Graying as a Consequence of Compromised
Antioxidant Activity in Hair Bulb Melanocytes and Their
Precursors
Ying Shi, Long-Fei Luo, Xiao-Ming Liu, Qiong Zhou, Shi-Zheng Xu, Tie-Chi Lei*
Department of Dermatology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, China
Abstract
Intricate coordinated mechanisms that govern the synchrony of hair growth and melanin synthesis remain largely unclear.
These two events can be uncoupled in prematurely gray hair, probably due to oxidative insults that lead to the death of
oxidative stress-sensitive melanocytes. In this study, we examined the gene expression profiles of middle (bulge) and lower
(hair bulb) segments that had been micro-dissected from unpigmented and from normally pigmented hair follicles from the
same donors using quantitative real-time RT-PCR (qPCR) arrays. We found a significant down-regulation of melanogenesis-
related genes (TYR, TYRP1, MITF, PAX3, POMC) in unpigmented hair bulbs and of marker genes typical for melanocyte
precursor cells (PAX3, SOX10, DCT) in unpigmented mid-segments compared with their pigmented analogues. qPCR,
western blotting and spin trapping assays revealed that catalase protein expression and hydroxyl radical scavenging
activities are strongly repressed in unpigmented hair follicles. These data provide the first clear evidence that compromised
antioxidant activity in gray hair follicles simultaneously affects mature hair bulb melanocytes and their immature precursor
cells in the bulge region.
Citation: Shi Y, Luo L-F, Liu X-M, Zhou Q, Xu S-Z, et al. (2014) Premature Graying as a Consequence of Compromised Antioxidant Activity in Hair Bulb
Melanocytes and Their Precursors. PLoS ONE 9(4): e93589. doi:10.1371/ journal.pone.0093589
Editor: Andrzej T. Slominski, University of Tennessee, United States of America
Received December 11, 2013; Accepted March 4, 2014; Published April 2, 2014
Copyright: ß 2014 Shi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the National Natural Science Foundation of China (No. 8107138) and a CMA-L’Oreal China Hair Grant (No.
H2010040414). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: We have the following interests: This study was funded in part by a grant from L’Oreal. We also contacted with Dr. Zhen Yaxian, a director
of L’Oreal R&D CHINA (yzhen@rd.loreal.com) to make clear no existence of any competing interests on this manuscript. There are no patents, products in
development or marketed products to declare. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.
* E-mail: tchlei@whu.edu.cn
Introduction
A recent worldwide survey showed that 74% of people between
the ages of 45 and 65 have grey hair, and that occurs earliest in
people of Caucasian descent, followed by Asians and Africans [1].
Hair is considered to grey prematurely only if it occurs before the
age of 20 years in Whites, before 25 years in Asians and before 30
years in Africans [2]. Prematurely graying hair (also termed
canities) imposes a psychosocial burden on sufferers since it is often
regarded as a visible sign of rapidly progressing old age, ill health
and bodily decline [2–4]. In spite of the fact that the onset of hair
graying is genetically controlled and inheritable, there is very little
known about the mechanism(s) by which functional melanocytes
are lost from anagen graying hair follicles [1], [2]. Emerging
evidence shows that reactive oxygen species (ROS) accumulate in
human gray/white scalp hair follicles up to millimolar concentra-
tions, which likely causes oxidative damage to hair follicle
melanocytes [5], [6].
Mature melanocytes are densely distributed in hair bulbs to
sustain active melanogenesis that is strictly coupled to the anagen
stage of the hair cycle [7–11]. Thus far, the precise mechanism(s)
governing the synchrony of hair growth and melanin synthesis has
remained largely unclear. Isolation and short-term co-culture of
primary keratinocytes, melanocytes and dermal papilla fibroblasts
derived from human scalp skin tissues are common strategies to
dissect the regulation of anagen-coupled melanogenesis [12–14].
Unfortunately, in vitro co-culture studies with established cell lines
or primary cell cultures could have led to artificial outcomes and
some inaccuracies in earlier studies since hair follicles are
composed of several types of cells that span the range of
differentiation states, for which it is considered a dynamic
miniorgan [15]. Graying hair offers a unique opportunity to study
the uncoupling of melanin production with growth of the hair
shaft [8]. Although deficient antioxidant activity was reported in
human graying hair follicles [5], [6], it remains to be determined
whether an impaired antioxidant defense in gray hair follicles
simultaneously affects mature hair bulb melanocytes and their
immature precursor cells in the bulge region, which would have a
critical implication for restoring pigmentation to the affected gray
hair.
In this study, we micro-dissected hair bulbs and mid-segments
(corresponding to the bulge region) from unpigmented and from
pigmented hair follicles isolated from the same human donors.
The expression levels of genes encoding characteristic markers for
mature melanocytes, melanocyte stem cells and keratinocyte stem
cells in the hair bulbs and mid-segments were analyzed using
quantitative real-time PCR (qRT-PCR) arrays and the anti-
oxidative properties of these segmented hair follicle tissues was
investigated in parallel using a range of techniques [16]. The
results demonstrate that both mature hair bulb melanocytes and
immature melanocyte precursor cells in the bulge region of gray
hair follicles are depleted, at least to some extent, and those effects
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Table 1. Clinical and demographic characteristics of patients in this study.
Case No. Sex (F/M) Age Diagnosis* Surgical procedure Biopsy site
1 F 20 Canities+ Scar Scar removal Occipitalia
2 M 22 Canities+ AA Hair transplantation Occipitalia
3 M 25 Canities+ AA Hair transplantation Occipitalia
4 M 23 Canities+ AA Hair transplantation Occipitalia
5 M 21 Canities+ AA Hair transplantation Occipitalia
6 M 20 Canities+ AA Hair transplantation Occipitalia
7 M 24 Canities+ AA Hair transplantation Occipitalia
8 M 25 Canities Volunteer Occipitalia
9 M 23 Canities Volunteer Tempus
*AA: Androgenic alopecia.
doi:10.1371/journal.pone.0093589.t001
Figure 1. Hair bulbs and mid-segments of hair follicles were micro-dissected from human scalp skin tissues. (A) Pigmented (left) and
unpigmented (right) whole anagenic hair follicles; (B) Each hair follicle was further excised using an ophthalmic scalpel under a stereoscope to harvest
hair bulbs and mid-segments, as indicated by the arrows (left). Schematic view (right) showing the histological structures corresponding to hair bulbs
and mid-segments of hair follicles. Scale bar = 1 mm.
doi:10.1371/journal.pone.0093589.g001
In Vivo Evidence by Tissue Microdissection
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that could be ascribed to reduced levels of catalase protein and
activity.
Materials and Methods
1. Patient Recruitment and Isolation of Whole Anagen
Hair Follicles
This study was carried out on 9 Chinese patients under 25 years
of age who suffered from premature gray hair (Table 1). Written
informed consent was obtained from each participant before
enrollment. The Ethical Committee of the Renmin Hospital of
Wuhan University approved this study and supervised its
compliance with the Declaration of Helsinki Guidelines. Pigment-
ed and unpigmented hair follicles were individually extracted from
the scalp using a micromotor-driven skin punch device (Mecica-
mat S.A., Malakoff, France) [17], [18]. Each hair follicle was
further excised using an ophthalmic scalpel under a stereoscope to
harvest the hair bulbs and mid-segments (corresponding to the
bulge region) (Figure 1A), which were then used for subsequent
isolation of RNA and protein extraction.
2. Fenton Reaction and Hydroxyl Radical Measurement
using a Spin Trapping Assay
The effects of hair bulbs and mid-segments from pigmented and
from unpigmented hair follicles on hydroxyl radical (NOH)
generation in the Fenton reaction were studied using a spin
trapping method, according to our previous report [19]. FeSO
4
was dissolved in distilled water, while all other solutions were
dissolved in 0.1 M phosphate buffer (pH 7.4). The spin trap
compound 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was pur-
chased from Sigma Chemical Co. (St. Louis, MO. Catalog#
D5766). Each reaction was carried out in a total of 50 mlinan
Eppendorf tube containing 260 mM H
2
O
2
, 0.4 mM FeSO
4
,
400 mM DMPO and identical amounts of minced tissue samples
as noted. In the control, metal-free water was substituted for the
sample. The Fenton reaction was initiated by the addition of
H
2
O
2
, then 50 ml of the reaction mixture was placed in an ESR
quartz flat cell. Exactly 20 s after the addition of H
2
O
2
, the ESR
spectra of the DMPO-NOH spin adducts were recorded at room
temperature using a Bruker ER 200D-SRC ESR spectrometer
(Bruker Analytische Messtechnik GmbH, Rheinstetten, Germany)
operating at 9.53 GHz microwave frequency, 20 mW microwave
power, 100 kHz modulation frequency, and 0.05 mT modulation
amplitude. NOH scavenging activity was calculated using the
equation: NOH scavenging activity = [12(H/H
0
)]6100%, in
which H and H
0
represent relative peak height (amplitude) of
the second peak of the DMPO-NOH spin adduct with or without
sample, respectively.
3. RNA Extraction and qRT- PCR Arrays
Thirty to 50 hair bulbs and mid-segments of isolated hair
follicles were dissected from the same patients using a micro-
dissecting protocol [20] and were pooled in a RNase-free
Eppendorf tube containing 20 mL TRIzol reagent (Invitrogen,
Eugene, OR, USA). The tissues were then homogenized with a
micro-homogenizer (Kimble, Toledo, OH, USA). Total RNAs
Table 2. Primers used in RT-PCR assays.
Gene symbol Accession number Primer sequence
ACTB NM_001101.3 Forward: 59-AGCGAGCATCCCCCAAAGTT-39
Reverse: 59-GGGCACGAAGGCTCATCATT-39
CAT NM_001752.3 Forward: 59-GATGTGCATGCAGGACAATCAG-39
Reverse: 59-GCTTCTCAGCATTGTACTTGTCC-39
GPX1 NM_000581.2 Forward: 59-ACGATGTTGCCTGGAACTTT-39
Reverse: 59-TCGATGTCAATGGTCTGGAA-39
SOD1 NM_000454.4 Forward: 59-AGGGCATCATCAATTTCGAG-39
Reverse: 59-TGCCTCTCTTCATCCTTTGG-39
TYR NM_000372 Forward: 59-CAGCTTTCAGGCAGAGGTTC-39
Reverse: 59-GCTTCATGGGCAAAATCAAT-39
DCT NM_001129889 Forward: 59-AGTGATTCGGCAGAACATCC-39
Reverse: 59-AGTTCCAGTAGGGCAAAGCA-39
TYRP1 NM_000550.2 Forward: 59-GCAGAATGAGTGCTCCTAAACTCC-39
Reverse: 59-CCTGATGATGAGCCACAGCG-39
MITF-M NM_198178.2 Forward: 59-TTATAGTACCTTCTCTTTGCC-39
Reverse: 59-GCTTGCTGTATGTGGTACTTG-39
MC1R NM_002386.3 Forward: 59-GCAGCAGCTGGACAATGTCA-39
Reverse: 59-GCCCCAGCAGAGGAAGAAAA-39
KRT15 NM_002275.3 Forward: 59-GAGAACTCACTGGCCGAGAC-39
Reverse: 59-CTGAAGAGGCTTCCCTGATG-39
KRT19 NM_002276.4 Forward: 59-TTTGAGACGGAACAGGCTCT-39
Reverse: 59-AATCCACCTCCACACTGACC-39
TGFB1
NM_000660.4 Forward: 59-GCCCTGGACACCAACTATTGCT-39
Reverse: 59-AGGCTCCAAATGTAGGGGCAGG-39
doi:10.1371/journal.pone.0093589.t002
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were extracted from tissues derived from pigmented and from
unpigmented hair follicles using TRIzol reagent according to the
instructions of the manufacturer and were quantified by measuring
absorbance (A value) at 260 nm. A total of 1 mg RNA for each
sample was used for reverse transcription using an RT-PCR Kit
(Catalog#CTB101; CT Biosciences, China) on an ABI 9700
Thermocycler (ABI, Foster City, CA). PCR arrays were performed
with customized PCR containing pre-dispensed primers (CT
Biosciences, China) on a LightCycler 480 (Roche Diagnostics,
Mannheim, Germany) using SYBR MasterMix (Cata-
log#CTB101; CT Biosciences, China). Each PCR assay con-
tained 10 ng of synthesized cDNA. The thermocycler parameters
were performed with an initial denaturation at 95uC for 5 min
followed by 45 cycles of denaturation at 95uC for 10 sec,
annealing at 60uC for 10 sec and extension at 72uC for 10 sec.
Relative changes in gene expression were calculated using
theDDCt (threshold cycle) method. Housekeeping genes such as
B2M, ACTB, GAPDH, RPL27, HPRT1 and OAZ1 were used to
normalize the amounts of RNA. Fold change values were
calculated using the formula of 2
–DDCt
. The amplification products
of 3 panels of genes associated with melanogenic enzymes, anti-
oxidative enzymes and specific marker genes for stem cells or
precursors of melanocytes and keratinocytes (Table 2) were also
confirmed by visualization of ethidium bromide-stained DNA after
agarose gel electrophoresis [20].
4. Western Blot Analysis
The tissue samples were washed in PBS and lysed in extraction
buffer containing 1% Nonidet P-40, 0.01% SDS and a protease
inhibitor cocktail (Roche, Indianapolis, IN, USA). Protein contents
were determined with a BCA assay kit (Pierce, Rockford, IL, USA)
and equal amounts of each protein extract (10 mg per lane) were
resolved using 10% SDS polyacrylamide gel electrophoresis (SDS-
PAGE). Following transblotting to Immobilon-P membranes
(Millipore, Bedford, MA, USA) and blocking with 5% nonfat
milk in saline buffer, the membranes were incubated with anti-
catalase antibody (Abcam, Cambridge, MA, USA) at a 1:1000
dilution, washed with PBS-T, and then were incubated with
horseradish peroxidase-conjugated anti-rabbit IgG (Amersham,
Piscataway, NJ, USA) at a dilution of 1:10,000. Immunoreactive
Table 3. Fold changes of gene expression in unpigmented hair follicles compared with pigmented hair follicles (qRT-PCR array
analysis was performed on one subject).
Gene symbol (full name) Gene ID
Fold change (W vs. B)
Hair bulbs Mid-segments
Melanogenesis-related genes
DCT (
dopachrome tautomerase
) 1638 Q 5.59 Q 14.12
TYR (
tyrosinase
) 7299 Q 45.95 Q 109.14
TYRP1 (
tyrosinase-related protein 1
) 7306 Q125.53 Q 54.95
MITF (
microphthalmia-associated transcription factor
) 4286 Q 52.42 Q 1.80
PAX3 (
paired box 3
) 5077 Q 21.74 Q 16.68
POMC (
proopiomelanocortin
) 5443 Q104.84 Q 2.99
ASIP (
agouti signal protein
) 434 Q 9.66 q 1.042
KIT (
proto-oncogene c-kit
) 3815 Q 30.74 q 16.68
SOX10 (
SRY-box containing gene 10
) 6663 Q125.53 Q 1.01
Antioxidant enzyme genes
CAT (
catalase
) 847 Q 44.08 Q 18.25
SOD1 (
superoxide dismutase 1, soluble
) 6647 q 4.88 q 1.80
SOD2 (
Mn superoxide dismu
tase) 6648 q 5.425 q 1.46
GPX1 (
glutathione peroxidase 1
) 2876 Q 1.88 q 1.35
MSRA (
methionine sulfoxide reductase A
) 4482 Q 11.33 Q 1.30
MSRB1 (
methionine sulfoxide reductase B1
) 51734 Q 2.50 Q 1.57
Putative marker gen es for stem cell and niche
KRT15 (
keratin 15
) 3866 Q 20.87 Q 9.18
KRT19
(keratin 19
) 3880 Q 25.17 Q 5.17
TNC (
tenascin C
) 3371 Q 10.94 Q 5.43
ITGB1 (
integrin beta 1
) 3688 q 5.49 q 1.80
CD200 (
CD200 molecule
) 4345 q 1.17 q 1.79
LGR5 (
leucine-rich repeat containing G protein-coupled
receptor 5
)
8549 Q 5.51 q 5.06
TGFB1
(transforming growth factor beta1
) 7040 Q18.05 Q 5.57
ITGA8 (
integrin alpha 8
) 8516 Q 17.53 q 1.02
NPNT (
nephronectin
) 255743 Q 12.5 Q 7.26
TP53 (
tumor P53
) 7157 q 3.41 q 50.91
*q: up-regulation; Q: down = regulation; W: unpigmented hair follicle (HF); B: pigmented HF.
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bands were detected by enhanced chemiluminescence using an
ECL kit (Amersham, Piscataway, NJ, USA). Immunoblotting of b-
actin served as a loading control.
5. Catalase Activity Analysis
Catalase activity was determined spectrophotometrically using a
commercial catalase analysis kit (Beyotime Biotechnology Co.,
Nanjing, China), as described previously [21]. Briefly, tissue
extracts were treated with excess H
2
O
2
to decompose catalase for
specific times as noted in the text, after which the remaining H
2
O
2
coupled with a substrate was treated with peroxidase to generate a
red product, N-4-antipyryl-3-chloro-5-sulfonate-p-benzoquinone
monoimine, which absorbed maximally at 520 nm. The H
2
O
2
consumption per min was converted to units of enzymatic activity
on the basis of a standard curve obtained testing scalar units of
bovine catalase. Units were corrected for the protein content of
each tissue extract.
Figure 2. Gene expression profiles of isolated hair follicles analyzed by semi-quantitative RT-PCR. (A) Total RNA was extracted from a
pool of 30–50 hair bulbs and mid-segments of hair follicle tissues. RT-PCR amplification was performed using primers specific for the molecular
signature genes of the mature hair bulb melanocytes, the immature precursor cells of melanocytes, and anti-oxidant enzymes etc. as indicated in the
gels and in Table 2. RT-PCR products were analyzed by electrophoresis on 1.0% agarose gels and images of PCR products are presented in reversed
black and white in which the DNA band is black. PCR product sizes for each set of primers are noted in parentheses and were determined by
comparison with a 100-bp DNA ladder (far left lane of each panel). (B) The intensity of each band was quantified using Image J densitometry software
(NIH, Bethesda, MD, USA). The relative expression level of each targeted gene was normalized to expression of the housekeeping gene b-actin and is
reported as relative expression from 3 independent experiments. *P,0.05.
doi:10.1371/journal.pone.0093589.g002
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6. Statistical Analyses
All data are expressed as means 6 standard deviation (SD).
Differences between two groups were determined using the two-
tailed Student t-test. P,0.05 is considered to be statistically
significant. All statistical analyses were performed using GraphPad
Prism (Ver. 5) (GraphPad Software, San Diego, CA, USA).
Results
1. Gene Expression Profiles in Hair Bulbs and in Mid-
segments of Hair Follicles
We first analyzed the gene expression patterns typical for
mature melanocytes (TYR, TYRP1, MITF), melanocyte precursor
cells (DCT, KIT, PAX3), keratinocyte stem cells (KRT15,
KRT19) and antioxidant enzymes (CAT, SOD, GPX1) in the
hair bulbs and mid-segments of unpigmented (white) and of
pigmented (black) hair follicles using qRT-PCR arrays, as
summarized in Table 3. A panel of genes encoding melanogen-
esis-related genes (TYR, TYRP1, MITF, PAX3, POMC, KIT,
SOX10) in unpigmented hair bulbs was suppressed more than 20-
fold compared with pigmented hair bulbs. Putative marker genes
of melanocyte precursor cells (PAX3, SOX10, DCT) were
markedly decreased in unpigmented mid-segments compared
with pigmented mid-segments. These findings showed that
functional melanocytes in the hair bulbs and immature melanocyte
precursor cells in the bulge region were depleted in gray hair. The
expression level of genes encoding antioxidant enzymes (except
SOD2) was significantly decreased in unpigmented hair bulbs
compared with pigmented hair bulbs, especially catalase, which
was reduced 44-fold in unpigmented hair bulbs and 18-fold in
unpigmented mid-segments relative to the pigmented analogues.
To confirm the array data, we selected one or two genes from each
category and performed RT-PCR analyses. Similar molecular
changes in the gray hair follicles were found by semi-quantitative
RT-PCR, as shown in Figure 2. Interestingly, a significant down-
regulation of marker genes typical of keratinocyte stem cells
(KRT15 and KRT19) and up-regulation (.50-fold) of the P53
gene was discerned in unpigmented mid-segments, which might
indicate the existence of activated keratinocyte stem cells [22] and
alternative P53-mediated antioxidation [23].
2. Suppression of Catalase Protein and Catalytic Activity
in Hair Bulbs and in M id-segments of Hair Follicles
Based on the above observations, we further proposed that an
intrinsic deficiency of catalase protein might be a major cause of
the oxidative damage of melanocytes in gray hair. Since mRNA
expression levels are not always consistent with protein levels [24],
we verified catalase protein levels in the hair bulbs and mid-
segments of unpigmented hair follicles using western blotting and
determined its catalytic activity using a spectrophotometric assay.
Figures 3 and 4 show that levels of catalase protein expression and
catalytic activity were significantly decreased in hair bulbs and
mid-segments of unpigmented hair follicles compared with those
areas of pigmented hair follicles. These results reveal that
compromised catalase activity may contribute to the pathogenesis
of premature graying hair.
3. Reduced Scavenging Activities against Hydroxyl Free
Radicals in Hair Bulbs and in Mid-segments of Hair
Follicles
A high concentration of H
2
O
2
accumulates in graying hair
follicles, as described in published reports [5], [6], which might be
explained in part by the intrinsic deficiency of catalase in gray hair
since catalase is an important antioxidant enzyme that catalyzes
the conversion of H
2
O
2
to water and molecular oxygen [6]. We
Figure 3. Expression level of catalase protein in hair follicles
determined by western blotting. Equal amounts (15 mg per lane) of
each protein extract were resolved using 10% SDS-PAGE electropho-
resis. Protein loading variations were determined by immunoblotting
with an anti-b-actin antibody. Representative blots are shown (A). The
histogram (B) shows the densitometric quantification of data with
means 6 SD of 3 independent experiments, *P,0.05, compared to
pigmented hair follicles.
doi:10.1371/journal.pone.0093589.g003
Figure 4. Catalase enzyme activity in hair follicles estimated by
spectrophotometric assay. Catalase activity was determined spec-
trophotometrically using a commercial catalase analysis kit, as
described in the text. Activities (unit per mg tissue protein) are
expressed as means 6 SD of 3 independent experiments, *P,0.05,
compared to pigmented hair follicles.
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also determined the non-specific hydroxyl radical scavenging
activities of graying hair follicles in a Fenton reaction system using
a spin trapping ESR assay. As shown in Figure 5, the hydroxyl
radical-scavenging activities of hair bulbs and mid-segments of
unpigmented hair follicles were significantly reduced compared
with those areas of pigmented hair follicles, and are more
prominent in the mid-segments (P,0.01). These results indicate
that the loss of antioxidative activities in graying hair bulbs and
bulge regions contributes to the abnormal accumulation of
hydroxyl free radicals.
Discussion
The regulation of anagen-coupled melanogenesis in human hair
follicles has been enigmatic. Recently, a plethora of genes which
play roles in either hair cycling growth or hair pigmentation has
been identified by constructing transgenic mice [25], [26]. Despite
the fact that incomplete melanocyte stem cell maintenance in the
bulge region causes a hair graying phenotype in mice [27], as far
as we know, characteristic premature hair graying has not been
reported in wild-type C57 mice [28]. It seems plausible that data
harvested from mouse models cannot fully explain the disappear-
ance of functional melanocytes seen in gray hair bulbs of human
anagen scalp hair follicles. We further propose that immature
melanocyte precursor cells in the bulge region may also be
destroyed along with hair bulb melanocytes, which has a critical
implication for restoring pigment to gray hair in the clinical setting
[29], [30]. Herein, we clearly provide qRT-PCR array results that
indicate that genes encoding melanogenesis-related genes (TYR,
TYRP1, MITF, PAX3, POMC, KIT, SOX10) in unpigmented
hair bulbs are reduced more than 20-fold compared with
pigmented hair bulbs. Meanwhile, marker genes of melanocyte
precursor cells (PAX3, SOX10, DCT) were markedly decreased in
unpigmented mid-segments compared with their pigmented
analogues. These results demonstrate that melanocyte precursor
cells in the bulge region are affected in graying hair follicles. More
recently, Ito et al. noticed that over-expression of Wnt protein in
mice potentiates hair neogenesis following wounding, but the new
hair was unpigmented. This raised the possibility that differently
coordinated activation of the Wnt pathway was required to
modulate repopulation of keratinocyte stem cells and melanocyte
stem cells in such regenerated follicles [31]. Our results reveal that
significant down-regulation of the KRT15 and KRT19 genes is
also detected in unpigmented mid-segments, which may represent
the activation of quiescent keratinocyte stem cells in the bulge
region [22], which in turn promotes the hair shaft growth of
unpigmented hair [32].
Another focus of this study was the analysis of the antioxidative
properties of isolated gray hair follicles. Those results show that
hair bulbs and mid-segments of pigmented hair follicles express
significant amounts of catalase protein and activity, whereas the
level of catalase expression is significantly suppressed in unpig-
mented hair follicles. Our ESR data also reinforce the conclusion
that compromised antioxidative activities in graying hair bulbs and
bulge regions contribute to the abnormal accumulation of
hydroxyl free radicals and the resulting oxidative destruction of
hair follicle melanocytes.
Taken together, our findings add new understanding to whether
immature melanocyte precursor cells in the bulge region of gray
hair follicles are destroyed along with mature hair bulb
melanocytes. They also reveal that an intrinsic deficiency of
catalase protein may contribute to the abnormal accumulation of
hydroxyl free radicals in gray hair follicles. In the future, it will be
interesting to induce the targeted differentiation of bulge neural
crest-derived stem cells into functional melanocytes in order to
restore pigment to gray hair [33].
Acknowledgments
The authors wish to thank Dr. Vincent J. Hearing of the National Institutes
of Health, Bethesda, MD, USA, for helpful discussions of this manuscript.
Author Contributions
Performed the experiments: YS LFL XML. Analyzed the data: QZ SZX
TCL. Wrote the paper: QZ SZX TCL. Designed the research study: QZ
SZX TCL.
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Hydroxyl radicals were generated by the Fenton reaction (DMPO:
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doi:10.1371/journal.pone.0093589.g005
In Vivo Evidence by Tissue Microdissection
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In Vivo Evidence by Tissue Microdissection
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