biology of melanocyte - professor torello lotti, md - university g.marconi, rome, italy - and linda...

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



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


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.


• 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.


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.

http://radiographics.rsna.org/content/29/5/1503/F2.large.jpg



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.




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



Tanning


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.



Reserve stem cells


Tanning


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.


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).

…for your

attention. yo for your for

attention….

Thank

you…

www.torellolotti.it

professor@

torellolotti.it

http://www.torellolotti.it/

biology of melanocyte - professor torello lotti, md - university g.marconi, rome, italy - and linda...

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