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Torello LottiDepartment of Dermatologic Science
University of Florence, Italy
The International School of Vitiligo & Pigmentary Disorders
Barcelona, 2-5 November 2011
Biology of melanocyte
• A typical melanocyte is 7 μm in lenght
.
From: http://www.beautymagonline.com/pages/epidermal_turnover.htm
Melanocyte cytologyDIMENSION AND NUMBER
• Melanocytes comprise from 5% to
10% of the cells in the basal layer of
epidermis.
• There are typically between 1000 and
2000 melanocytes per mm2 of skin.
• Although almost everyone has the same amount of melanocytes, the
amount and size of the melanosomes and melanin particles produced
can differ immensely in humans, resulting in the different races of the
world.
From: http://news.softpedia.com/news/12-of-the-DNA-Differs-Amongst-Human-Races-and-Populations-40872.shtml
Melanocyte cytology
• After delivery, dendrites
are maintained by the
mitosis of the existing
ones..
• Dendritic prolongations
take contact with nearby
keratinocytes, and serve
as melanosomes carriers.
• Melanocytes have a round,
slightly pigmented cell body
with numerous dendrites
stemming from it.
From:http://www.chups.jussieu.fr/polys/histo/TPhis/imagespoly2&Melanocyte.jpg.11.html
DENDRITES
Melanocyte cytology
• Abundant rough endoplasmic
reticulum (RER).
• Well-developed Golgi
apparatus.
• Melanocytes are not attached
to neighboring cells by
desmosomes.
• The lack mechanisms of cell
union, typical of pavement
epithelial cells, gives them the
characteristic disaggregation
appearance.
Melanocyte from black human skin. From: http://bioeducate.ascb.org/images/FawcettTheCellPDFs/FawcettTheCellChapter11.pdf.
ORGANELLES
Melanocyte cytology
• Early stage melanosomes are characterized
morphologically by intralumenal fibrils upon
which melanins are deposited in later stages.
• The integral membrane protein Pmel17 is a
component of the fibrils, can nucleate fibril
formation in the absence of other pigment cell-
specific proteins, and forms amyloid-like fibrils
in vitro.
MELANOSOMES
• Melanosomes are ellipsoid lysosome-related organelles (LROs), around
0,2-0,7 μm long and limited by a membrane, filled with melanin and
proteins.
• Giant melanosomes have been in found in nevi: they can reach 5 μm
dimension.
• Melanosome transport requires the
microtubule and actin cytoskeleton. Melanosomes (green) marked with TRP-1.
Confocal photograph
showing melanosome (yellow), actin (red) and microtubules (violet)
Melanosome in motion:
filamentous actin (red), microtubules (blue) and melansomes (green)
Melanocytes distribution
Melanocytes of the epidermis
Melanocytes migration from the neural crest
Melanocytes of the hair follicle
Melanocytes of the ear
Melanocytes of the brain
Melanocytes of the eye
Melanocytes of the heart
Melanocytes of the adipose tissue
Melanocytes precursors, knew as melanoblasts, are formed in
the the neural crest: in the 11th week of fetal life, they migrate
to various sites, where they proliferate and then differentiate
into mature melanocyte.
- Melanocytes migration -
Melanocytes reside in:
skin (epidermis and hair follicle)
inner ear
eye (choroid and retina)
brain and
leptomeninges
- Melanocytes migration - Section of a mouse embryo at
11.5 days of gestation:
melanoblasts and melanocytes (in
blue) are migrating down from the
neural crest derived cells along the
spinal column and brain. Melanocytes are usually identified by
their expression of specific proteins
(e.g.,tyrosinase (TYR), TYRP1, DCT,
Pmel17/gp100, MART-1 and/or MITF).
Melanoblasts are more difficult to
identify since they don’t produce melanin,
therefore don’t usually express those
markers; only occasionally DCT and/or KIT
are detectable.
In human epidermic, melanocyte rside on the basement
membrane, at the epidermal–dermal junction, and they
form a close association with keratinocytes via their
dendrites
- Melanocyte of the epidermis -
Each
melanocyte
makes contact
with
around 30-40
keratinocytes.
This constitutes
the
epidermal-
melanin
unit.
Epidermal
Melanin
Unit 36 K
KLM Unit 53 K
- Melanocyte of the epidermis -
- Melanocyte of the epidermis -
Immunohistochemical
analysis : melanocytes are
identified by using the marker
D5, which stains the nuclear
transcription factor.
Haematoxylin and eosin stain
of human skin: normal
melanocytes have smaller nuclei
and inconspicuous cytoplasm
compared with the surrounding
keratinocytes.
Longevity and resistance to apoptosis make melanocytes
vulnerable to
mutations that arise over the years, particularly due to sun
exposure, and
might culminate in melanoma formation in high-risk
individuals.
.
Immune suppression
INITIATION PROMOTION
Eicosanoid production
DNA DAMAGE
PRE CANCERDisplastic nevi
Reactive O2 species
CANCERGene mutation
Common nevi
MELANOMA
- Melanocyte of the epidermis -
Multipotent epidermal
stem cells exist in the
bulge region, at the
bottom of the
permanent portion of
the follicle
- Melanocytes in the hair follicle -
Differentiated
melanocyte reside in
the hair bulb
- Melanocyte of the eye -
• Uveal melanocytes (UM) are
differentiated melanocytes that
originate from the neural crest.
• Retinal pigment epithelium (RPE) is a
distinct type of melanocyte-like, that
develop in situ from the optic cup of
the brain, and is specifically present
only as a single layer of cells lying
behind the retina
• UM and RPE are located in the middle
(choroid) and inner (retinal) eye layers.
RPE
UM
• There are 2 distinct type of melanocyte in the
eye
- Melanocyte of the eye -
• Melanin has the ability to bind organic
amines and metal ions: this results in
the
accumulation of these substances in
the eye.
• As an antioxidant, melanin protects
eye cells
from chemical stress by binding ROS.
• RPE plays a critical role in the
metabolism of
retinoids, and in the active
phagocytosis and
turnover of the rod outer segments of
the
retina.
Hu D-N, Savage HE. Uveal Melanocytes, Ocular Pigment Epithelium, and Müller Cells in Culture: In Vitro Toxicology Int J Toxicology 2002;21(6):465-472.
Bok D. The retinal pigment epithelium: A versatile partner in vision. J Cell Sci Suppl 1993;17:189–195.
• UM contain both eu- and pheomelanin, whereas RPE contains mainly
eumelanin.
RPE
UM
- Melanocyte of the ear - COCHLEA
• Melanocytes are present as intermediate cells in the stria vascularis of
the cochlea.
• Strial intermediate cells are required for the generation of endolymph-
mediated
action potentials that are necessary for normal hearing.
• Hearing impairment can be associated with inherited pigmentary
disorders, e.g.
Waardenburg syndrome, and it has have shown that the extent of induce
temporary hearing loss is inversely related to skin pigment type.
• Melanocyte are likely to contribute to the hearing process.
Takeda K, Takahashi NH, Shibahara S. Neuroendocrine functions of melanocytes: Beyond the skindeep melanin maker. Tohoku J Exp Med 2007;211:201–221.
INNER EAR
Melanin granules produced by melanocytes in the inner ear play
important roles in
balance.
• Melanocytes have been identified in the brain and leptomeninges, and
may have
many neuroendocrine functions. In particular:
- melanocyte are likely to contribute to sleeping regulation
- a melanocyte-derived factor might be involved in controlling the
central
chemosensor that generates the respiratory rhythm.
• Melanocyte of neural system produce neuromelanin, which has a
protective
function by binding/removal of ROS and metals that are highly toxic to
neurons.
- Melanocyte of the brain -
Takeda K, Takahashi NH, Shibahara S. Neuroendocrine functions of melanocytes: Beyond the skindeep melanin maker. Tohoku J Exp Med 2007;211:201–221.
Zecca L, Bellei C, Costi P, et al. New melanic pigments in the human brain that accumulate in aging and block environmental toxic metals. Proc Natl Acad Sci U S A 2008;105:17567–17572.
Zucca FA, Giaveri G, Gallorini M, et al.The neuromelanin of human substantia nigra: Physiological and pathogenic aspects. Pigment Cell Res 2004;17:610–617.
- Melanocyte of the heart -
• Melanocytes have been identified in the valves and septa of the heart
and their
numbers appear to reflect that of the skin.
• Cardiac melanocytes depend on the same the signaling molecules that
are crucial
for cutaneous melanocytes development, thus they may originate from
the same
precursor population.
• The function of cardiac melanocyte is still obscure.
• It has been hypotesized that the dysfunction of melanocyte-like cells in
the atrium
and in pulmonary venis may contribute to atrial arrhythmias.
• Probably, cardiac melanocytes are not essential in a healthy and non-
stressful
environment.
Yajima I, Larue L. The location of heart melanocytes is specified and the level of pigmentation in the heart may correlate with coat color. Pigment Cell Melanoma Res 2008;21:471–476.:471-476.
Brito FC, Kos L. Timeline and distribution of melanocyte precursors in the mouse heart. Pigment Cell Melanoma Res 2008;21:464–470.
Levin MD, Lu MM, Petrenko NB, et al. Melanocyte-like cells in the heart and pulmonary veins contribute to atrial arrhythmia triggers. J Clin Invest 2009; 119(11):3420-36.
Shosuke Ito. Melanins seem to be everywhere in the body, but for what? Pig Cell & Mel Res 2009;22(1):12-13.
• It has been recently demonstrated that melanin biosynthesis takes
place in the
visceral adipose tissue of morbidly obese humans.
• With the progression of obesity and the increase of cellular fat
deposition,
adipocytes become more exposed to endogenous apoptotic signals,
especially ROS.
• The obese adipocytes ectopically may activate melanogenesis in order
to neutralize
excess ROS and reduce oxidative damage. In addition, adipocytic
melanin might
suppress the secretion of proinflammatory molecules.
• It has been observed that fasting glucose levels correlated well to total
outputs of
the melanogenic pathway in adipose tissues of obese patients.
- Melanocyte of the adipose tissue -
Randhawa M, Huff T, Valencia JC, et al. Evidence for the ectopic synthesis of melanin in human adipose tissue. FASEB J 2009; 23(3):835-843.
Summing up….
Locations and functions of melanocytes
HAIR FOLLICLE
Melanocyte stem cell
reservoir for skin.
Hair pigmentation,
Removal of toxic
byproducts
EAR
Inner ear: balance
Cochlea: hearing
EYE
Choroid: Constitutive eye
pigmentation, protection against UV
Retinal pigment epithelium:
vision, metabolism of rod outer
segments and retinoids
BRAIN
Neuroendocrine function and detoxification
HEART
unknown
ADIPOSE TISSUE
Anti-inflammation,
reduction/binding of ROS
EPIDERMIS
Constitutive skin pigmentation.
Responses to and protection against the
environment (primarily UV)
And also….
• In lymphangioleiomyomatosis (LAM), muscle
cells
revert towards their developmental origins
and
express some melanocyte markers, such as
tyrosinase,
Pmel17, gp100, etc..
• LAM cells can immunoreact with CD63, PNL2,
both
markers for melanocyte differentiation.
• Gp 100 expression suggests that a least some
LAM
cells feature partial melanocytic
differentiation.
…. Melanocyte in the lung ?
LAM cells must be regarded as a fully abnormal type with the
unique
characteristic of having dual smooth cell/melanocyte
differentiation.
This type of differentiation suggests a neural crest cell origin.Zhe X,Schuger L. Combined Smooth Muscle and Melanocytic Differentiation in Lymphangioleiomyomatosis. J Histochem Cytochem 2004;52(12):1537-1542.
Ferrans VJ, Yu ZX, Nelson WK, et al.Lymphangioleiomyomatosis (LAM). a review of clinical and morphological features. J Nippon Med Sch 2000;67:311–329.
Melanocyte functions
Sinthesis of melanins (melanogenesis)
Reserve stem cell
Nervous system development
Tanning
Autocrine/paracrine function
o Melanocytes are cells specialized in the synthesis of the
pigment melanin
Melanogenesis
o The name 'melanin' comes from the ancient Greek melanos, meaning
'dark',
and the term was probably first applied by the Swedish chemist
Berzelius in 1840 o There are three types of melanin:
• EUMELANIN
• PHEOMELANIN
• NEUROMELANIN
o In addition to carotenoids and haemoglobin, melanin is the
main
contributor to pigmentation of the skin, hair and eyes .
Eumelanin, the best-know form of melanin, exists in two forms:
o Black melanin : produces black colours when it is present in large
quantities, and
grey colours when it is rarer. It colours hair black, dark brown and grey.
o Brown eumelanin : produces brown hair colours when it is present in
abundance,
but smaller anmounts produce lighter brown or blond hair colours.
EUMELANIN
o Pheomelanin produces reddish colours of hair, eye and
skin.
PHEOMELANIN
o Pheomelanin is more
abundant in
the skin of women than men
and
their skin is slightly redder.
Eumelanin is a
nitrogenous and
insoluble pigment.
Pheomelanin is a sulfur-
containing
and alkali-soluble pigment.
EUMELANIN VS PHEOMELANIN
Pheomelanin and eumelanin differ not only in colour
but also in the size, shape and packaging of their granules.
• Individual melanocyte can
synthesize
both eu- and pheomelanin.
• The synthesis of one the two
pigment is determined by a
balance
of some variables, including:
- pigment enzyme expression
- availability of tyrosine and
sulphydryl-containing
reducing
agents in the cell.
EUMELANIN VS PHEOMELANIN
• The ratio of eu-/pheomelanin ranges from 1.31 to exclusively eumelanic.
• Pheomelanin synthesis is characterized by the presence of spheroid
melanosomes
whereas eumelanin synthesis is ascribed to ellipsoid melanosomes.Nakagawa H, Imokawa G. Characterization of melanogenesis in normal human epidermal melanocytes by chemical and ultrastructural analysis.
Pigment Cell Res.1996;9(4):175-8.
Both melanins derive from a common tyrosinase-dependent pathway with
the same precursor, tyrosine. The obligatory step is hydroxylation of
tyrosine to dopaquinone, (DQ) from which L-DOPA can also be derived.
From DQ, the two pathways diverge.
EUMELANIN VS PHEOMELANIN
Pheomelanin is derived from conjugation by thiol-containing cysteine or
glutathione. It is more photolabile and can produce, among its by-products, hydrogen
peroxide, superoxide and hydroxyl radicals..
Land, E. J. & Riley, P. A. Spontaneous redox reactions of dopaquinone and the balance between the eumelanic and phaeomelanic pathways. Pigment Cell Res 2000; 13, 273–277..
NEUROMELANIN o Neuromelanin is the dark pigment that produces a black colour in
certain parts of
the brain. o Neuromelanin is primarily localized in dopaminergic neurons of the
substantia
nigra and in the locus coerulus, but virtually all brain tissue contain
significant
amounts of neuromelanin. With age, it accumulates in the substantia
nigra.
o Neuromelanin consists of a large, complex, eumelanin- covered
pheomelanin
core which may also contain aliphatics and peptides.
Neurocuteneous melanosis.
Boyd Smith A, Rushing E J, Smirniotopoulos JG. Pigmented Lesions of the Central Nervous System: Radiologic-Pathologic Correlation. Radiographics 2009;29:1503-1524.
Melanocyte functions
Sinthesis of melanins (melanogenesis)
Tanning
• Tanning results from a complex signalling pathway
involving
keratinocytes and melanocytes.
• It represents the physiological response of the
epidermis to UV
radiation.• Tanning process can be divided in 4 phases:
1. Cytonkines production (KERATINOCYTE
MELANOCYTE)
2. Gene transcription (MELANOCYTE)
3. Pigment sinthesis (MELANOCYTE)
4. Melanin transfer (MELANOCYTE KERATINOCYTE)
Tanning
Miller AJ, Tsao H. New Insights into Pigmentary Pathways and Skin Cancer. The British Journal of Dermatology 2010;162(1):22-28.
1 2 3
• Ultraviolet (UV) radiation reaching
keratinocytes
leads to the production of a large number of
cytokines, including MSH(melanocyte
stimulationg factor).
1.CYTOKINES PRODUCTION
• In the other cases, p53 triggers cell cycle
arrest and
the expression of pro-piomelanocortin
(POMC),
which is the precursor for α-MSH.
• UV-induced photoproduct formation
triggering a
repair response mediated by the xeroderma
pigmentosum (XP) DNA repair enzymes. • In cases of sever damage, p53 may induce apoptosis.
2. GENE TRANSCRIPTION
• MSH secreted from keratinocytes binds to
melanocortin-1 receptor (MC1R) on nearby
melanocytes.
• MSH binding promote MC1R
transcription, the elevation of cAMP levels
and then the expression of MITF
(microphthalmia transcription factor), that
stimultes eumelanin production.
• MSH and Agouti Signalling Peptide (ASIP)
are both ligands which could bind to MC1R:
MSH is an agonist, so promote MC1R
transcription, while ASIP is an antagonist.
3. PIGMENT SYNTHESIS
• Melanin production occurs predominantly in a
lysosome-like structure known as the
melanosomes.• Melanin is packaged and delivered to
keratinocytes
by melanosomes. The formation, maturation
and
trafficking of melanosomes is crucial to
pigmentation,
Sinthesis passages of eu- and pheomelanin from tyrosine
Melanosomes are formed from two sources:
- In the Smooth Endoplasmic Reticulum (SER), pre-melanosomes are
produced by coalescence of vesicles, and appear to contain parallel
filamentous contents.
- In the Rough Endoplasmic Reticulum (RER) and Golgi complex,
tyrosinase and other enzymes are synthesised and packaged into
vesicles.
Melanosomes synthesis
These fuse with the
pre-melanosomes.
Localization of melanogenesis phases within the melanocyte
• Stage I to IV
melanosomes are formed
in cytosol and distributed
long the dendritic
prolongation .
• The structural proteins of
melanosomes and
tyrosinase and are
synthesized on ribosomes
associated with RER • In the Golgi, these are
packaged in small
vesicles which elongate
as the lamellar
framework of the
melanosome is
assembled.
Post-Golgi transport of melanosomes in melanocyte citosol
• On the keratinocyte
side, the protease-
activated receptor-2
(PAR2), a seven-
transmembrane
receptor on
keratinocytes, has a
central role in
melanosome transfer.
• Melanosomes are
tranferred to
keratinocytes trough
dendrites
4. MELANIN
TRANSFER
Once in
keratinocytes,
melanosomes are
distributed and
-in response to
UVR- positioned
strategically over
the ‘sun-exposed’
side of nuclei, to
form
cap-like
structures
resembling
umbrellas.
Melanocyte functions
Sinthesis of melanins (melanogenesis)
Autocrine/paracrine function
Tanning
Summary of the major participants in the paracrine/autocrine network
that regulates melanocyte functions and survival.
Factors of the paracrine/autocrine network that regulates
melanocyte functions, proliferation and survival, with their
cellular origin, role in pigmentation and effects on
melanocytes
Abdel-Malek ZA, Swope VB. Epidermal melanocytes: regulation of their survivial, proliferation and function in human skin. In: Melanoma
Development: Molecular Biology, Genetics and Clinical Application. A Bosserhoff ed. Springer-Verlag, Wien 2011. p.11.
Key molecules and signaling pathways implicated
in melanocyte-keratinocyte interactions
Role of ET-1 and melanocortins in the DNA Damage Response
Abdel-Malek ZA, Swope VB. Epidermal melanocytes: regulation of their survivial, proliferation and function in human skin. In: Melanoma
Development: Molecular Biology, Genetics and Clinical Application. A Bosserhoff ed. Springer-Verlag, Wien 2011. p.22.
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Tsatmali M, Ancas J, Thody JA. Melanocyte functions and its control by melanocortin peptides. J Histoc & Cytochemistry 2002; 50(2):125-133.
Effects of melanocortins (MSH and ACTH) stimulation on
melanocyte
in response to environmental stress
Corticotropin Releasing Hormon central role in the skin
response to stress
Slominski A, Wortsman J, Pisarchik A, et al. Cutaneous expression of corticotropin-releasing hormone (CRH), urocortin, and CRH receptors . The FASEB Journal. 2001;15:1678-1693
• Human melanocytes express FP receptor (the receptor for PGF2α) in
vitro and in vivo, and expression of this receptor was upregulated upon
UV exposure
Eicosanoids as Paracrine/Autocrine factors for Melanocytes
Starner RJ, McClelland L, Abdel-Malek Z, et al.PGE(2) is a UVR-inducible autocrine factor for human melanocytes that stimulates
tyrosinase activation. Exp Dermatol 2010;19:682–684
• Melanocytes respond to PGF2α with
stimulation of dendricity, and
melanogenesis that is evidenced by
increased activity and protein levels of
tyrosinase.• The leukotrienes LTC4 and D4 were
found to be potent mitogens for
cultured human melanocytes
Morelli JG, Yohn JJ, Lyons MB, et al. Leukotrienes C4 and D4 as potent mitogens for cultured human melanocytes. J Investig Dermatol 1989;93:719–722
Melanocyte functions
Sinthesis of melanins (melanogenesis)
Tanning
Autocrine/paracrine function
Reserve stem cells
Melanocyte of the hair follicle
• Melanocyte Stem Cell (MSCs) and hair follicle stem cells (SCs)
reside in a common niche within the permanent portion of the hair
follicle (HF), the bulge.
• Progressive loss of MSCs determine
hair
graying in both humans and mice.
• TGF-β produced in this specialized niche regulates the function of MSCs in the HF.
Nishimura EK, Suzuki M, Igras V, et al. Key roles for transforming grow factor-β in melanocyte stem cell mainteinance. Cell Stem Cell 2010; 6:130-140.
In catagen, all cells of the lower HF, including the mature melanocytes
and rapidly cycling transit-amplifying (TA) matrix cells, die by apoptosis,
except MSCs and SCs.
During anagen, MSCs and SCs proliferate, migrate downwards, and
give rise to TA matrix cells and TA melanocytes that differentiate to
produce the colored hair shaft.
Melanocyte of the hair follicle
Nishimura EK, Suzuki M, Igras V, et al. Key roles for transforming grow factor-β in melanocyte stem cell mainteinance. Cell Stem Cell 2010; 6:130-140.
Melanocyte functions
Sinthesis of melanins (melanogenesis)
Reserve stem cells
Nervous system development
Tanning
Autocrine/paracrine function
Melanocytes and melanin play critical
roles during embryonic development.
This can be seen in individuals with
oculocutaneous albinism type 1 (OCA1).
OCA1 results from the dysfunction of TYR
which leads to impaired pigmentation of
skin, hair and eyes but also to misrouting
of the optic nerves at the chiasm.
King, RA; Hearing, VJ.; Creel, DJ., et al. Albinism. In: Scriver, CR.; Beaudet, AL.; Sly, WS.;Valle, D., editors. The metabolic and molecular bases of inherited disease. New York: McGraw-Hill 2001. p. 5587-5627.
Le, Pape E.; Passeron, T.; Giubellino, A.; et al. Microarray analysis reveals the complex effects of MC1R signaling in melanocytes. Proc Natl Acad Sci U S A. 2009;10;106(6):1802-7.
Nervous system development
The antagonist agouti signaling
protein (ASP) was shown recently to
modulate the expression of genes
involved in morphogenesis, especially
in nervous system development.
Melanocytes are not simply melanin-producing cells, but have
several
functions
Besides the skin, melanocyte reside in multiple tissues of the
body
They are capable of secreting a wide range of signaling
molecules, within
the paracrine/autocrine network of the skin.
Melanocytes are likely to function as regulator cells in
mantaining
epidermal homeostasis.
Conclusions
Co-culture of human epidermal keratinocytes (red) and melanocytes (green), showing melanosome transfer into
keratinocytes with DNA counterstaining (blue).
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