Clinical dermatology X Review article

Ageing and photoageing of keratinocytes and melanocytes

M. Yaar and B. A. Gilchrest
Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts, USA

Summary An overview of keratinocyte and melanocyte function is provided. The processes of

cutaneous ageing and photoageing are defined, and age-associated modulations in
gene expression are described. The changes in keratinocytes and melanocytes that
occur with skin ageing and photoageing and the characteristics of chronologically aged
vs. photoaged skin are delineated. Mutations that are found in malignant and
premalignant tumors of epidermal origin are described.

environmental stimuli to maintain homeostasis and

Introduction
Keratinocytes make up more than 95% of the epidermis,
a self-renewing stratified and cornified epithelium
approximately 100 mm thick. During embryogenesis,
epidermal keratinocytes also invaginate into the dermis
to form epidermal appendages, including hair follicles
that then cycle throughout the lifespan. The principal
product of the epidermis is the stratum corneum or
barrier layer, composed of highly compressed layers of
corneocytes in a `brick and mortar' configuration that
retains water in the viable epidermis and virtually
prohibits entry of microbes and most chemicals into the
body. Keratinocytic stem cells, located in the basal layer
of the epidermis and the `bulge' region of the hair follicle
reviewed in Latkowski et al. 1999,1 give rise to transient
amplifying daughter cells that divide several times
before undergoing terminal differentiation and ascend-
ing through the epidermis to the stratum corneum over
a period of approximately 2 weeks.1 The cells then
undergo an apoptosis-like process, flatten, and progress
outwardly over an additional 2 weeks to the skin
surface, from which they are continuously shed. In
addition to producing a barrier layer, keratinocytes are
highly metabolically active, secreting cytokines and
growth factors (reviewed in Stingl et al. 1999)2 that
regulate proliferation and differentiation of other cells in
the skin. In this way, they respond to a wide variety of
Correspondence: Department of Dermatology J-508, Boston University
School of Medicine, 609 Albany Street, Boston, MA 02118-2394 USA.
Tel.: 1617 638-5538. Fax: 1617 638-3550. E-mail: bgilchre@bu.edu
minimize injury to the tissue.
Melanocytes comprise approximately 1±2% of epider-
mal cells and are the second most numerous epidermal
cell type. They are derived from the neural crest and
migrate into the epidermis late in the first trimester of
development (reviewed in Reedy et al. 1998)3 after
which they reside in the basal layer in contact with
neighbouring keratinocytes to form the epidermal
melanin unit (reviewed in Jimbow et al. 1998)4 It is
not clear where the presumptive melanocytic stem cells
reside or which markers they express. Grichnik et al.
propose that melanocytic stem cells reside in the
follicular infundibula,5 from which they can migrate
either into the epidermis or down to the hair bulb
during each anagen cycle. These cells express the
receptor for stem cell factor, c-kit, an important cytokine
for melanocyte survival during embryogenesis. In
contrast, Botchkareva et al.6 provide evidence that
putative melanocytic stem cells reside in the secondary
hair germ (bulge) and that they are c-kit-negative.
Regardless, after relinquishing stem cell status, normal
melanocytes appear to have quite limited proliferative
potential. Melanocytes are the body's sole source of
melanin and function throughout life to produce this
protective pigment and to transfer it to surrounding
keratinocytes, where it is generally distributed as a
supranuclear cap, imposed between the keratinocyte
nucleus and the external environment. The extent and
character of melanogenesis has strong genetic determi-
nants that are as yet poorly understood. Melanin
production is also strongly up-regulated following

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Ageing and photoageing of keratinocytes and melanocytes
epidermal injury, classically exposure to ultraviolet (UV)
light, but also thermal burns, harsh chemicals, mechan-
ical trauma, or other stimuli. This melanogenesis results
in part directly from DNA damage (reviewed in Gilchrest
and Eller 1999)7 and in part indirectly from the release
of hormones and inflammatory mediators by surround-
ing keratinocytes (reviewed in Hadley and Levine
1993).8 In hair, melanocytes transfer pigment to
differentiating keratinocytes that ultimately form the
hair shaft. They thus determine hair colour by the
amount of pigment transferred, as well as by the ratio of
eumelanin (black) to pheomelanin (red±yellow;
reviewed in Ito 1993).9
Ageing and photoageing have profound effects on
keratinocytes and melanocytes that can be appreciated
both in vivo and in vitro, as detailed below.
What is ageing?
Ageing is a basic biologic process characteristic of all
living organisms. The rate of ageing differs strikingly
among species and moderately among members of the
same species, but inevitably leads to reductions in
maximal function and reserve capacity in all organ
systems. From early to late adulthood, these reductions
are on the order of 50% and render the individual
progressively more susceptible to injury and disease.
Ultimately, the functional losses are incompatible with
life.
Ageing is widely acknowledged to be the consequence
of both a genetic program and cumulative environ-
mental wear and tear. A thorough discussion of the
multiple processes hypothesized to affect ageing is
beyond the scope of this article. However, central to
most theories of ageing is the balance between DNA
damage and repair capacity, with species lifespan
generally proportional to metabolic rate (reviewed in
Balin and Allen 1989)10 but also strongly influenced by
cellular ability to sustain and repair DNA damage
(reviewed in Jazwinski 1996).11 Cumulative oxidative
DNA damage due to cellular metabolism is presumed to
be the major contributor.11 As well, in lower organisms,
so-called longevity genes have uniformly been found to
encode proteins involved in the prevention or repair of
the effects of environmental stresses such as tempera-
ture, UV irradiation and oxidative damage.12,13 In a
recent study using the technique of gene arrays to
investigate modulations in gene expression between
young and old adult mice, of more than 6000 genes
surveyed, only 113 displayed more than a twofold
change with ageing.14 Interestingly, the expression of
several genes encoding proteins that participate in stress
584
X M. Yaar and B. A. Gilchrest
responses, including those induced by oxidative damage,
were increased in old animals. Also, genes encoding
proteins that are known to be up-regulated in response
to injury were increased, suggesting that incurred
damage to cellular components is not fully repaired
over the lifespan and/or that repair mechanisms
deteriorate with ageing, as has been shown recently
for human lymphocytes and dermal fibroblasts.15±17
Not surprisingly, with ageing there was a decrease in the
expression of genes encoding proteins that are involved
in energy metabolism and biosynthetic activity. How-
ever, the study did not determine the temporal order of
these gene modulations. Interestingly, most alterations
in gene expression were fully or partially abrogated in
caloric-restricted animals, known to age more slowly
than controls fed ad libitum, suggesting that caloric
restriction decreases the level of cumulative damage to
cellular components, perhaps by decreasing metabolic
oxidative damage. Moreover, in humans a major
progeroid syndrome, the recessively inherited Werner's
disease, has recently been shown to result from
mutation in a DNA helicase involved in DNA repair
and replication.18 Also, fibroblasts derived from patients
with progeria, a rare inherited condition with features of
premature and accelerated ageing, show gene modula-
tions similar to those observed in fibroblasts derived
from old adults. In particular, the expression of genes
encoding proteins that participate in cell cycle progres-
sion is decreased, including genes required for proper
assembly of the cytoskeleton during cell division,
suggesting that with ageing mitotic errors may lead to
defects in chromosome structure and consequently
affect their function.19
What is photoageing?
Cumulative DNA damage during ageing is expected to
result from the low but finite rate at which errors in
DNA replication occur spontaneously or result from
incompletely repaired DNA damage. The rate at which
exogenous environmental damage accumulates in tissue
might be expected to be greatest at interfaces between
the internal milieu and the environment e.g. the skin.
This is indeed the case, particularly in areas that are
habitually sun-exposed.
Photoageing is the term given to the superposition of
chronic sun damage on the intrinsic ageing process
(reviewed in Kligman and Kligman 1999).20 The rate of
photoageing varies even more strikingly among indivi-
duals than does the rate of intrinsic ageing, due in part
to variable sun exposure habits but also to widely
differing individual ability to block sun damage and/or
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 Ageing and photoageing of keratinocytes and melanocytes
Figure 1 Permanent hyperpigmentation of photoaged skin. This
54-year-old woman remained darkly tanned throughout the year
in areas exposed by her sunbathing attire during the summer.
to repair damage to irradiated tissue. In general,
photoageing affects fair-skinned persons most severely.20
A skin phototype I or II individual, particularly one of
Celtic ancestry, raised in a sunny climate, often has
permanent sharp demarcations at the collar line or
bathing suit line (Fig. 1) and is additionally at high risk
for skin cancer (Fig. 2).
Characteristics of aged skin
Keratinocyte-related changes
Intrinsically aged (sun protected) skin manifests
changes due to losses in keratinocyte proliferative
capacity (reviewed in Yaar and Gilchrest 1999),21
ability to properly terminally differentiate in order to
form the stratum corneum,21 and ability to elaborate
cytokines and other cell±cell signals appropriately in
response to environmental stimuli (reviewed in Yaar
Figure 2 Concurrence of intrinsic ageing, photoageing, and skin
cancer. The squamous cell carcinoma on the dorsum of the hand is
surrounded by coarsely wrinkled elastotic skin.
q 2001 Blackwell Science Ltd X Clinical and Experimental Dermatology, 26, 583±591
X M. Yaar and B. A. Gilchrest
1995).22 Presumably, these decrements contribute,
respectively, to slow healing of minor injuries and
weaker surgical scars; as well as to a tendency to
nonhealing ulcers, the characteristically `dry', rough
and often pruritic skin of the elderly; and the decreased
inflammatory response of elderly skin to UV irradiation
or infection. Subtle changes can also be appreciated in
epidermal keratinocyte-derived skin appendages, in
which decreased cell proliferation results in thinner
and more slowly growing hair and nails and decreased
secretory rates for holocrine glands.21
Clinically, intrinsically aged skin is thin and pale. A
flattened dermal±epidermal junction can be appreciated
histologically, presumably reflecting a reduction in the
more proliferative keratinocyte population normally
located in the rete ridges. Functionally, the time required
to heal an epidermal wound, for example to reepithe-
lialize a de-roofed suction blister, increases approxi-
mately 50% between young adulthood and late
adulthood (reviewed in Eaglstein 1989).23 In addition,
increased ease of separation between the dermis and
epidermis can be documented as a reduced time for
creation of experimental blisters or as ease of blistering
or epidermal tears following minor trauma to elderly
skin. The stratum corneum appears relatively normal
and baseline stratum corneum barrier function is
minimally, if at all, reduced. However, as expected,
more striking age-associated differences are observed
after injury. Of probable direct clinical relevance, the
barrier to water loss is abrogated far more readily in
old adult than in young adult skin, and the time
required to reconstitute an effective stratum corneum
following tape stripping increases markedly, from
approximately 3 days in young adults to more than
6 days on average in elderly adults.24 Biochemical and
ultrastructural studies reveal that the decreased rate
of barrier regeneration is due at least in part to
decreased lipid synthetic capacity with ageing.24 A
significant influence of female hormones on stratum
corneum sphingolipid composition is also reported,25
with presumed consequent impact on postmenopausal
skin.
Melanocyte-related changes
Changes in the pigmentary system due to ageing alone
are minimal in skin, although the density of melano-
cytes (cells per unit area of skin surface) decrease
progressively during adulthood by approximately 10%
per decade.26 Loss of melanogenic capacity presumably
contributes to the pallor of old skin, which is also
attributable in part to decreased vascularity.21 Loss of
585
Ageing and photoageing of keratinocytes and melanocytes X M. Yaar and B. A. Gilchrest
Figure 3 Seborrhoeic keratoses as a biomarker of intrinsic skin
ageing. These benign neoplasms begin to appear in most
individuals during the third or fourth decade and become
increasingly numerous with age. Many elderly persons, such as
this 70-year-old man, have numerous, prominent keratoses over
the face, trunk, and extremities.
melanocytes with age has a more dramatic effect on
hair, with approximately half the population having
substantial `greying' or, more correctly, total depigmen-
tation of many scalp hairs by 50 years of age.27 White
hair is the result of decreased tyrosinase activity in hair
bulb melanocytes, reduced and less efficient melanoso-
mal transfer and faulty melanocyte migration and/or
proliferation from a presumptive storage area to an area
close to the dermal papilla.28 Absence of melanocytes
from the follicular epithelium of anagen hairs could also
reflect loss of proliferative capacity for stem cells or their
progeny. Apparently, proliferating melanocytes, presum-
ably of melanocytic stem cells, the progeny, are strongly
dependent on stem cell factor signaling6 and must
divide extensively during the anagen phase of each hair
586
cycle to repopulate the invaginating follicle and thus
contribute melanin pigment to the developing hair
shaft.
Seborrhoeic keratoses
Seborrhoeic keratoses are curious age-associated skin
lesions in which both keratinocytes and melanocytes
participate prominently (Fig. 3). These benign neo-
plasms, the majority of which are monoclonal in
origin,29 are highly variable in size and colour. They
first begin to appear in the third to fifth decade and
become increasingly numerous in most individuals
throughout life, independent of sun exposure (reviewed
in Ho 1999).30 Indeed, these lesions have been
suggested to be the best biomarker of intrinsic ageing
in the skin. Presumably they represent a focal subtle loss
of homeostasis, with resulting over-proliferation of
keratinocytes and melanocytes, although the pathogen-
esis is not known. Recently, keratinocytes with basaloid
morphology in seborrhoeic keratoses were reported to
express high levels of endothelin-1 (ET-1), associated
with increased tyrosinase expression in the melano-
cytes, compared with control perilesional skin,31 sug-
gesting that ET-1-induced melanogenesis,32
dendricity, and melanocyte proliferation34 may play
33
a role in the evolution of this neoplasm.
Characteristics of photoaged skin
Age-associated clinical and histological changes in
photoaged skin are generally more dramatic and also
more variable than those in sun-protected skin, being
strongly influenced by the individual's complexion and
history of sun exposure.
The epidermis may be hyperplastic or severely
atrophic, compared with sun-protected sites of the
same individual, and at least some degree of cytologic
atypia and disorderly maturation of keratinocytes is
common (reviewed in Kligman and Kligman 1999).20
More pronounced nuclear atypia and disorderly
maturation, so-called `loss of polarity' in the epidermis,
is perceived clinically as an actinic keratosis, a rough
scaly often erythematous patch (reviewed in Schwartz
and Stoll 1999).35 At the molecular level, this lesion is
characterized by mutation of the p53 tumour suppres-
sor protein36 that is presumably then nonfunctional but
is often overexpressed at the protein level.
Changes in melanocyte number and function are also
prominent in sun-exposed skin, beginning in childhood.
In fair skinned individuals, freckling begins within the
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 Ageing and photoageing of keratinocytes and melanocytes
first years of life and at a histological level consists of
enlarged overactive melanocytes with an overall slight
increase in melanocyte density.37 Depending on the
individual's complexion and total insolation, within a
few decades exposed skin becomes chronically hyper-
pigmented (`tanned' or `bronzed'), remaining darker
than the sun-protected skin indefinitely, even in the
absence of further sun exposure (Fig. 1), in part because
of increased melanocyte density and increased epider-
mal melanin and in part because of increased number of
dermal melanophages.38 The density of melanocytes in
such skin is approximately twice that in comparable
body sites not habitually sun exposed,26 presumably
reflecting melanocyte division in response to acute sun
exposures in the past. Increased melanin per cell is also
observed, however, in late passage melanocytes `aged' in
culture,39 suggesting that a differentiation-like phenom-
enon may also contribute to this hyperpigmentation.
Well circumscribed dark brown macules, termed solar
lentigines or less appropriately `age spots' or `liver spots',
also appear on habitually sun-exposed skin (Fig. 4a).
Histologically, they consist of an increase in both
number and activity of the melanocytes, as well as a
characteristic increased prominence of rete ridges with a
deeply convoluted dermal±epidermal junction,40 sug-
gesting that proliferative keratinocytes also contribute in
some way to the development of these pigmented
lesions.41 Interestingly, it was shown recently that ET-
1 levels are increased in keratinocytes in solar lentigines
and that the level of the ET-1 receptor is increased in the
melanocytes,42 in which tyrosinase expression is
enhanced. Particularly in fair skinned individuals,
habitually sun-exposed skin may also develop areas of
total depigmentation (Fig. 4b), from which melanocytes
are absent, presumably reflecting UV-mediated destruc-
tion of this cell population. Naevi or moles are local
proliferations of melanocytes at the dermal±epidermal
junction, often followed by invagination of the naevo-
cellular nests into the dermis. Although naevi can occur
on any body site and in individuals of any complexion,
they are statistically linked to fair complexion and sun
exposure43±46 and characteristically appear in sun-
exposed areas during childhood, following sufficient sun
exposure.43 Most acquired naevi are harmless, but a
subset of acquired naevi appear clinically and histolo-
gically atypical and have a higher risk of evolving into
malignant melanoma.44
Relationship to malignancy
Both keratinocyte-derived malignancies [basal cell
carcinomas (BCC) or squamous cell carcinomas (SCC)]
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X M. Yaar and B. A. Gilchrest
and melanocyte-derived malignancies (melanomas)
occur with exponentially increasing incidence during
adulthood,47,48 relating risk strongly to chronologic
age. In addition, both types of malignancies are strongly
associated with photoageing.
SCC and BCC occur overwhelmingly in habitually
sun-exposed skin of fair-skinned individuals (reviewed
in Leffell and Fitzgerald 1999;49 Schwartz and Stoll
1999).50 At least two-thirds of all melanomas are also
epidemiologically linked to sun exposure, although
these lesions tend to occur in intensely but intermit-
tently sun-exposed skin, rather than in areas with
maximal lifetime exposure.51 UV-induced mutations in
key cell cycle regulatory genes are widely believed to
be the principal cause of all three types of skin
malignancies. In the case of SCC, UV-induced muta-
tions in the p53 tumour suppressor gene are almost
universal and are believed to confer a critical growth
advantage to the mutated cells through impairing
apoptosis of cells that have suffered severe DNA
damage, thus allowing accumulation of multiple
mutations in single cells, sufficient to result in
malignancy.52,53 UV signature p53 mutations are also
very common in BCC, and are found in approximately
half of the tumours including those in xeroderma
pigmentosum (XP) patients.53,54 However, the patched
gene (PTCH), which encodes a transmembrane protein
that prevents cellular proliferation, is the gene that is
specifically mutated in BCC.55 Mutations of other genes
that participate in the PTCH signalling pathway are
present as well.56,57 These include mutations in the
growth promoting smoothened (SMO) gene that
encodes the transmembrane protein that complexes
with PTCH, and the growth promoting SHH gene that
encodes sonic hedgehog, a protein that binds and
inactivates PTCH.
The equivalent early mutation required for malignant
conversion of melanocytes has not been identified.
Germline p16 mutations are frequently found in familial
melanomas and in atypical melanocytic naevi in these
kindreds,58 but p16 UV signature mutations are
reported only occasionally in sporadic melanomas.59,60
Still, the observation that melanoma cell lines have a
high frequency of p16 mutations occurring at dipyr-
imidine sites and occasional CC!TT mutations of the
gene,61 suggests that this gene plays a role in UV-
induced sporadic melanoma as well. Moreover, in many
atypical (dysplastic) naevi there is loss of heterozygosity
on the short arm of chromosome 9 at a location
corresponding to the tumour suppressor gene p16/
CDKN2A.62 Other highly prevalent mutations in spora-
dic atypical melanocytic naevi, mutations expected to
587
Ageing and photoageing of keratinocytes and melanocytes X M. Yaar and B. A. Gilchrest

Figure 4 Pigmentary changes in photoaged skin. (a) Lentigines. These hyperpigmented macules are extremely common in some exposed
areas of fair-skinned and darker-skinned individuals. This 68-year-old man has numerous lentigines over the face and neck, interspersed
with seborrhoeic keratoses and with other markers of photoageing. (b) Depigmented pseudoscars. The dorsal forearm is a common site of
these lesions, here interspersed with actinic purpura and other photoageing changes.

influence melanoma development, have not been
identified to date. However, cells isolated from sporadic
atypical naevi display deficient DNA-damage repair,63,64
suggesting that an a priori or acquired decreased
capacity to repair DNA damage may predispose to the
development of melanoma.
Particularly for SCC, a causative role for UV-induced
DNA damage has been established by the finding of p53
mutations at dipyrimidine sites CC!TT or C!T, the so-
called `UV signature' mutations.52,65 UVB induces
lesions between two adjacent pyrimidines on the same
DNA strand and the majority of these are removed by

588 q 2001 Blackwell Science Ltd X Clinical and Experimental Dermatology, 26, 583±591
 Ageing and photoageing of keratinocytes and melanocytes
nucleotide excision repair enzymes before the cell
divides. However, occasionally the cell undergoes DNA
replication and division before all dimers have been
removed, in which case, DNA polymerase, the enzyme
that synthesizes the new DNA strand, places an adenine
dinucleotide (AA) opposite each `unreadable' pyrimidine
dimer (the `A rule').66,67 As AA is the correct dinucleo-
tide to pair with thymidine dinucleotides (TT), no
mutations occur. However, CC or CT dimers would be
mistakenly replaced by thymidine dinucleotide (TT)
giving rise to CC!TT and C!T mutations, respectively.
Because no other mutagen affects dipyrimidine
bases68,69 mutations at these sites constitute statistical
proof of UV causality. p53 contains six mutations hot
spots, but because five out of these six are methylated
CG sequences68 and many mutagens preferentially bind
these sites, it is impossible to establish specific causality.
The striking increase with age in incidence of skin
malignancies, parallel to that for malignancies in
general, is attributable in part to straightforward
cumulative DNA damage over the lifespan (in skin due
primarily to UV irradiation), and in part to age-
associated decreases in DNA repair capacity,15±17
making mutations increasingly likely after DNA damage
as the individual ages. Changes in surrounding dermal
matrix, the transformed cells' local environment, and/or
diminished immunosurveillance with age may also
contribute.
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