Physiology and Pathophysiology of

Melanocytes

Torello Lotti, MD Professor of Dermatology and Venereology

Florence, Italy

Melanocytes are pigment-producing cells that originate from the dorsal portion of the closing neural tube in vertebrate embryos.

MELANOCYTES

MELANOCYTES AND PIGMENTATION PHYSIOLOGY

Pluripotent neural crest cell

Melanoblast migration and differentiation into melanocytes is influenced by a number of signaling molecules produced by neighboring cells that interact with their their specific cell surface receptors.

Wnt

Endothelin-3 (ET-3)

Stem Cell Factor (SCF), c-Kit-ligand

Bone morphogenetic factor (BMPs)

Hepatocyte growth factor (HGF)

Wnt Family 16 different secreted glycoproteins; Directs the maturation of pluripotent neural crest cell into melanoblasts

Wnt Frizzled receptor

Induction and

accumulation of

Β- catenin

Transcription of

Microphthalmia-assocciated

transcription factor (Mitf)

Induce the transcription of 3 key

enzyme in melanin synthesis:

- Tyrosinase;

- TRP-1;

- TRP-2.

MITF is central to Melanocyte viability and function

Waardenburg syndrome type 2A - different colored irises; - white forelock; - congenital cochlear deafness.

normal MITF activity is

completely lost in these

animals

usually MITF activity

is only partially lost

Endothelins Family

ET-1, ET-2, ET-3 EdnrA, EdnrB (receptor)

ET-3 + EdnrB: - required for survival, prolifeartion and migration of melanoblasts; - also affect the development of other neural crest cells. - exracutaneous symptomatology in type IV Waardenburg syndrome and in Hirschsrung syndrome.

Stem Cell Factor

SCF: expressed by keratinocytes

c-Kit (its receptor): expressed on melanoblasts

Drive melanoblasts to their final destination

Mutations of c-Kit or SCF:

melanoblast unable to migrate to the skin and/or survive there

PIEBALDISM

MELANOBLAST

Inner ear Cochlea

Choroid Ciliary body Iris

Leptomeninges

Skin and Hair Follicle

CUTANEOUS MELANOCYTES

Melanocyte density/mm2: 550-1200 (highest concentration in genitalia and face)

Epidermal melanin unit: one melanocyte surrounded by several keratynocites

Melanocytes syntesize melanine, stored in cyosolic organelles (melanosomes) transferred to keratinocytes through dendritic process.

Keratinocytes signals regulate epidermal melanocyte survival, dendricity, melanogenesis…

MELANIZATION

The synthesis and distribution of melanin in the epidermis (pigmentation) involves several step:

Transcription of proteins required to melanogenesis

Melanosome biogenesis

Sorting of melanogenic proteins into the melanosomes

Transport of melanosomes to the tips of melanocyte dendritic cells

Transfer of melanosomes to keratinocytes

Disruption in any of these events results in Hypopigmentation

Melanosome Biogenesis

Unique menbrane bound organelle (modified version of lysosomes?) in which melanin biosynthesis take place.

Eumelanosomes Pheomelanosomes

Eumelanin Pheomelanin

Melanosome Biogenesis

MELANIN BIOSYNTHESIS

Two types of melanin

Eumelanin Dark, brown/black

Pheomelanin Light, red/yellow

• Melanin provide protection against UV (280-400 nm)- induced DNA damage; • UV absorbed is converted into heat (less toxic form of energy).

Melanin and its intermediates can be harmful to melanocytes: ROS generation: DNA damage: melanoma

MELANOGENIC PROTEINS

Tyrosinase: Chromosome 11

OCULOCUTANEOUS ALBINISM TYPE I

Mutations (missense, nonsense frameshift, deletion):

• Synthesized in Endoplasmic reticulus • Glycosilation in Golgi apparatus • Packaged in endosomes • Fuse in melanosome stage II

Enzymes and proteins involved in melanosomal maturation

Tyrosinase-Related Proteins (TRP)

TRP-1: chromosome 9

- Same Tyrosinase maturation pathway - Tyrosinase activation/stabilization ? - Melanosome biogenesis ?

Mutations: OCULOCUTANEOUS ALBINISM TYPE III

TRP-2: chromosome 13

Microphtalmia-Associated Transcription Factor (Mitf)

• Master gene for melanocyte survival; • Key factor regulating transcription of melanogenic proteins: Tyrosinase, TRP-1, TRP-2. • 9 isoforms: Mitf-M (specific for melanocytes), -A, -B, -C, -D, -E, -H, -J and –Mc.

Mitf activity is induced by binding of SCF to c-Kit receptor and by cAMP-elevating agents such α-MSH.

Microphtalmia-Associated Transcription Factor (Mitf)

Mitf upregulate the expression of anti-apoptotic protein BCl2

Melanocyte survival

Mitf

G1 to S phase (Melanocyte proliferation)

Cdk2

+

+

p21

-/+

-

Role in melanocyte proliferation ?

Melanocortin Receptor (MCR)

Family of five related receptors (MC1-5R). MC1R: melanocytes

Polimorphisms within the MC1R gene are largely responsible for the different skin/hair color among different ethnic group.

α-MSH ACTH

MC1R

cAMP Mitf transcription

Eumelanin synthesis

Propiomelancortin (POMC) encodes α-MSH and other hormones

Both pituitary gland anf epidermal keratinocytes are able to synthesized POMC

UV light activates p53 in keratinocytes,

p53 induces expression of POMC in keratinocytes

UV light activates a cascade that results in elevated melanin synthesis and transport

The “THREE ENZYME THEORY” and the crucial role of 6BH4

3 enzymes, phenylalanine hydroxylase activity (PAH), tyrosine hydroxylase isoform I (THI) and tyrosinase, are crucial for the initiation of melanogenesis

Schallreuter KU et al. Regulation of melanogenesis – controversies and new concepts. Experimental Dermatology 2008;

17: 395–404.

6BH4 in turn acts as the essential electron donor for PAH to produce L-tyrosine from L-phenylalanine and for THI to convert l-tyrosine to L-DOPA. 6BH4 is an allosteric inhibitor of tyrosinase.

MELANIN BIOSYNTHESIS

Dessinioti C et al.A review of genetic disorders of hypopigmentation: Lessons learned from the biology of melanocytes.

Experimental Dermatology 2009; 18: 741–749.

MELANOCYTE DENDRITES

• Branching protoplasmatic process that interact with keratinocytes.

• Actin is a major structural component of dendrites;

• Several keratinocytes-derived factors (ET-1, NGF, PGE2, β-endorphin) play a role in melanocyte dendricity; • Integrins also play a role in dendrite formation.

MELANOSOME TRANSPORT (in Melanocyte)

Microtubules (arranged parallel to the long axis of the dendrite)

Microtubule-associated motor proteins: Kinesins (centrifugal movement) and Dyneins (Ccentripetal movement)

Other partecipants:

Rab27a Myosin-Va melanophilin

+

Mutated in Griscelli syndrome

MELANOSOME TRANSPORT (to Keratinocytes)

Several potential ways involved

Exocytosis

Cytophagocitosis (keratinocytes

phagocytose the tip of a melanocyte dendrite)

Fusion of plasma menbranes

Transfer by membrane vesicles

REGULATION OF MELANOCYTE FUNCTION

Imokawa G. Autocrine and paracrine regulation of melanocytes in human skin and in pigmentary disorders. Pigment Cell Res 2004

Hypomelanoses: Why ?

1. Loss or reduction of melanocytes; 2. Reduced melanine production from

melanocytes (altered tyrosinase activity, altered structure/activity of rough endoplasmic reticulum, lack of specific melanocyte receptors…);

3. Decreased melanine transfer from melanocytes to keratinocytes;

4. Primary disorder of keratinocytes.

Hypomelanoses

Normal

Albinism

Vitiligo

Functional defect in melanine synthesis

Localized loss / inactivation of melanocytes

Disorders of Melanin Synthesis

Dessinioti C et al.A review of genetic disorders of hypopigmentation: Lessons learned from the biology of melanocytes.

Experimental Dermatology 2009; 18: 741–749.

Disorders of Melanocyte Development and Migration

Disorders of Melanosome Formation and Transfer to Keratinocytes

Dessinioti C et al.A review of genetic disorders of hypopigmentation: Lessons learned from the biology of melanocytes.

Experimental Dermatology 2009; 18: 741–749.

VITILIGO ETIOPATHOGENESIS

NEURAL HYPOTHESIS AUTOIMMUNE HYPOTHESIS

AUTOCYTOTOXIC/ RADICALIC HYPOTHESIS

GENETIC PREDISPOSITION Autoimmune Susceptibility Locus (AIS1)

ECLECTIC HYPOTHESIS MELANOCYTORRAGY SYNERGISTIC THEORY

MELANOCYTE DESTRUCTION

Vitiligo etiopathogenesis

GENETIC PREDISPOSITION

Autoimmune Susceptibility Locus (AIS1)

AUTOIMMUNE

Umoral mechanism -Autoantibodies

Citotoxic mechanism – Cell mediated

METABOLIC

Hydrogen peroxide accumulation

Abnormal expression of Tyrosine-Related Protein -1

OTHERS

Viral hypothesis

Neuronal toxicity

Autoimmune Pathogenesis

Presence of “vitiligo antibodies” in patients;

Vitiligo is associated with several autoimmune disease (vitiligo is a syndrome, not a disease…): tyroiditis (up to 40%), diabetes type I (1-7%), autoimmune gastritis, autoimmune polyglandular syndromes, alopecia areata…;

Most effective therapies in inducing repigmentation have also immunosuppressive effects (i.e.corticosteroids, ultraviolet, cytotoxic drugs);

Immunotherapies for melanoma often cause vitiligo patches.

Autoimmune Pathogenesis

Ongenae K et al. Evidence for an Autoimmune Pathogenesis of Vitiligo. Pigment Cell Res 16: 90–100. 2003

Altered antioxidant and scavenger

mechanism

Increased activity of superoxide

dismutase

High levels of epidermic 7-BH4 and H2O2

Inhibition of enzyme function (phenylalanine-hydroxilase and tyrosinase) and abnormal expression of Tyrosinase

Related Protein-1 (TRP-1).

impaired melanine synthesis

Metabolic Pathogenesis

Vitiligo: what’s new in 2011

Melanocytes are completely absent in the depigmented epidermis

Nordlund JJ and Lerner AB – Arch Dermatol, 1982;118:5-8

Le Poole IC et Al. J Invest Dermatol, 1993;100:816-822

Vs.

Melanocytes are not completely absent in the depigmented epidermis

Bertosi KJ et Al. Eur J Dermatol 1998;8:95-97

Tobin DJ et Al. J Pathol 2000;191:407-416

Gottschalk GM, Kidson SH. Int J Dermatol. 2007;46(3):268-72

Vitiligo: what’s new in 2011

Melanocytes are not completely absent in the depigmented epidermis

Massi D. Histopathological and ultrastructural features of vitiligo. In: Lotti T & Hercogova J (Eds.) Vitiligo – Problems and solutions. Marcel Dekker Inc, New York 2004

Normal Skin Perilesional Skin Lesional Skin

Vitiligo: what’s new in 2011

Melanocytes are not completely absent in the depigmented epidermis

Comment: – A subpopulation of “resistant” epidermal melanocytes can persist independent of disease duration

– Repigmentation can always occur independent of disease duration and with non-perifollicular pattern

VITILIGO: NOT ONLY A MELANOCYTIC DISEASE?

Imokawa G. Autocrine and paracrine regulation of melanocytes in human skin and in pigmentary disorders. Pigment Cell Res 2004

Imokawa G. Autocrine and paracrine regulation of melanocytes in human skin and in pigmentary disorders. Pigment Cell Res 2004

What’s new in 2011: A focus on keratinocytes

Impaired scavenging mechanisms can lead to ROS increase and subsequent melanocyte and keratinocyte damaging;

Altered function of PAR-2 receptor can impair calcium homeostasis in keratinocytes and alter melanosome intake and processing.

What’s new in 2011: the focus on keratinocytes

The importance in mitochondria in keratinocytes from perilesional skin and the role of oxidative stress.

Prignano F, et al. Ultrastructural and functional alterations

of mitochondria in perilesional vitiligo skin. J Derm Sci

2009;54:157–167

Mitochondrial alterations in perilesional keratinocytes

Mitochondrial activity plays a crucial role in normal cell function

Mitochondrial alterations observed in perilesional keratinocytes appear to be very similar to those described in the same cell types during apoptosis

The mitochondrial damage is associated with an increase in ROS production and, hence, oxidative stress.

Prignano F, et al. J Derm Sci 2009;54:157–167

Functional alterations in vitiligo skin

High levels of TNF-alpha and FasL in the depigmented epidermis (role in increasing apoptosis) – Kim NH, et al. J Invest Dermatol 2007;127:2612–7.

mRNA for TNF-α and IL-6, with an inhibitory effect on pigmentation, was increased in the epidermis from vitiligo biopsies.

This could contribute to keratinocyte apoptosis, which results in reduced release of melanogenic cytokines and in melanocyte disappearance. – Moretti S, et al. Histol Histopathol 2009:24:849-857

Functional alterations in vitiligo skin

Apoptotic keratinocytes may cause a decrease in SCF synthesis, which plays an important role in melanocyte survival and proliferation

Keratinocyte apoptosis induces a decrease in the synthesis of other melanocyte growth factors, such as bFGF, resulting in melanocyte disappearance. – Lee AY, et al. Br J Dermatol

2004;151:995–1003.

– Moretti S, et al. Histol Histopathol

2009:24:849-857

Functional alterations in vitiligo skin

Endothelin-1 (ET-1) mRNA seems to be significantly reduced in lesional as compared to perilesional epidermis

SCF and ET-1 may contribute to melanocyte survival – Moretti S, et al. Histol Histopathol 2009:24:849-857

Functional alterations in vitiligo skin

Protease-activated receptor (PARs) 2 is abundantly expressed by keratinocytes, and seems to contribute to the pigmentation process

PAR-2 impairment is seen in vitiligo, and may contribute to the epidermal pigment deficit through a reduced melanosome uptake in keratinocytes.

To date, a precise cause and effect relationship between these two conditions cannot be determined. – Moretti S, et al. Pigment Cell Melanoma Res 2009;22:335–338

OUR CONTRIBUTIONS

Berti S, Bellandi S, Bertelli A, Colucci R, Lotti T, Moretti S. Vitiligo

in an Italian outpatient center: a clinical and serologic study of 204

patients in Tuscany. Am J Clin Dermatol. 2011;12(1):43-9.

Prignano F, Ricceri F, Bianchi B, Guasti D, Bonciolini V, Lotti T,

Pimpinelli N. Dendritic cells: ultrastructural and

immunophenotypical changes upon nb-UVB in vitiligo skin. Arch

Dermatol Res. 2010

Arunachalam M, Sanzo M, Lotti T, Colucci R, Berti S, Moretti S.

Common variable immunodeficiency in vitiligo. G Ital Dermatol

Venereol. 2010;145(6):783-8.

Becatti M, Prignano F, Fiorillo C, Pescitelli L, Nassi P, Lotti T, Taddei

N. The involvement of Smac/DIABLO, p53, NF-kB, and MAPK

pathways in apoptosis of keratinocytes from perilesional vitiligo

skin: Protective effects of curcumin and capsaicin. Antioxid Redox

Signal. 2010, 1;13(9):1309-1321.

Prignano F, Pescitelli L, Becatti M, Di Gennaro P, Fiorillo C, Taddei

N, Lotti T. Ultrastructural and functional alterations of mitochondria

in perilesional vitiligo skin. J Derm Sci 2009;54:157–167;

Moretti S, Fabbri P, Baroni G, Berti S, Bani D, Berti E, Nassini R,

Lotti T and Massi D. Keratinocyte dysfunction in vitiligo epidermis:

cytokine microenvironment and correlation to keratinocyte

apoptosis. Histol Histopathol 2009;24:849-857;

Moretti S, Nassini R, Prignano F, Pacini A, Materazzi S, Naldini A,

Simoni A, Baroni G, Pellerito S, Filippi I, Lotti T, Geppetti P and Massi

D. Protease-activated receptor-2 downregulation is associated to

vitiligo lesions. Pigment Cell Melanoma Res. 2009;22:335–338.

Prignano F, Pescitelli L, Ricceri F, Lotti T. The importance of genetical link

in immuno-mediated dermatoses: psoriasis and vitiligo. Int J Dermatol

2008;47:1060–1062;

Prignano F, Betts CM, Lotti T. Vogt-Koyanagi-Harada disease and vitiligo:

where does the illness begin? J Electron Microsc (Tokyo). 2008

Thank you for your attention