Physiology and Pathophysiology of Melanocytes

Torello Lotti, MD
Professor of Dermatology and Venereology Florence, Italy

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

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.

Bone morphogenetic factor (BMPs) Wnt Hepatocyte growth factor (HGF) Endothelin-3 (ET-3) Stem Cell Factor (SCF), c-Kit-ligand

Wnt Family
16 different secreted glycoproteins;

Directs the maturation of pluripotent neural crest cell into melanoblasts

Wnt

Frizzled receptor

Induction and accumulation of

- catenin

Induce the transcription of 3 key enzyme in melanin synthesis: Transcription of - Tyrosinase; - TRP-1; - TRP-2.

Microphthalmia-assocciated transcription factor (Mitf)

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 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.

EdnrA, EdnrB (receptor)

Stem Cell Factor

SCF: expressed by keratinocytes
Drive melanoblasts to their final destination

c-Kit (its receptor): expressed on melanoblasts

Mutations of c-Kit or SCF: melanoblast unable to migrate to the skin and/or survive there

PIEBALDISM

Skin and Hair Follicle

Inner ear Cochlea

Leptomeninges

MELANOBLAST

Choroid

Ciliary body

Iris

CUTANEOUS MELANOCYTES
Melanocyte density/mm2: 550-1200 (highest concentration in genitalia and face) Melanocytes syntesize melanine, stored in cyosolic organelles (melanosomes) transferred to keratinocytes through dendritic process.

Keratinocytes signals regulate epidermal melanocyte survival, dendricity, melanogenesis

Epidermal melanin unit: one melanocyte surrounded by several keratynocites

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 Eumelanin

Pheomelanosomes Pheomelanin

Melanosome Biogenesis

MELANIN BIOSYNTHESIS
Two types of melanin

Dark, brown/black

Eumelanin

Light, red/yellow

Pheomelanin

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
Enzymes and proteins involved in melanosomal maturation

Tyrosinase: Chromosome 11
Synthesized in Endoplasmic reticulus Glycosilation in Golgi apparatus Packaged in endosomes Fuse in melanosome stage II Mutations (missense, nonsense frameshift, deletion):

OCULOCUTANEOUS ALBINISM TYPE I

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 cAMPelevating agents such MSH.

Microphtalmia-Associated Transcription Factor (Mitf)

Mitf upregulate the expression of anti-apoptotic protein BCl2

Melanocyte survival +
Cdk2

Mitf

-/+ p21

(Melanocyte proliferation)

G1 to S phase

Role in melanocyte proliferation ?

Melanocortin Receptor (MCR)

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

-MSH MC1R
cAMP

ACTH Eumelanin synthesis

transcription

Mitf

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

Propiomelancortin (POMC) encodes -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
crucial for the initiation of melanogenesis

(PAH), tyrosine hydroxylase isoform I (THI) and tyrosinase, are

6BH4 in turn acts as the essential electron donor for PAH to produce Ltyrosine from Lphenylalanine and for THI to convert l-tyrosine to L-DOPA. 6BH4 is an allosteric inhibitor of tyrosinase.
Schallreuter KU et al. Regulation of melanogenesis controversies and new concepts. Experimental Dermatology 2008; 17: 395404.

MELANIN BIOSYNTHESIS

Dessinioti C et al.A review of genetic disorders of hypopigmentation: Lessons learned from the biology of melanocytes. Experimental Dermatology 2009; 18: 741749.

MELANOCYTE DENDRITES
Branching protoplasmatic process that interact with keratinocytes. Actin is a major structural component of dendrites; Several keratinocytesderived factors (ET-1, NGF, PGE2, -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 Transfer by membrane vesicles

(keratinocytes phagocytose the tip of a melanocyte dendrite)

Cytophagocitosis

Fusion of plasma menbranes

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. 2.

3. 4.

Loss or reduction of melanocytes; Reduced melanine production from melanocytes (altered tyrosinase activity, altered structure/activity of rough endoplasmic reticulum, lack of specific melanocyte receptors); Decreased melanine transfer from melanocytes to keratinocytes; Primary disorder of keratinocytes.

Hypomelanoses
Normal Albinism
Functional defect in melanine synthesis

Vitiligo
Localized loss / inactivation of melanocytes

Development and Migration

Disorders of Melanocyte

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: 741749.

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: 741749.

VITILIGO ETIOPATHOGENESIS
GENETIC PREDISPOSITION Autoimmune Susceptibility Locus (AIS1) NEURAL HYPOTHESIS AUTOIMMUNE HYPOTHESIS

MELANOCYTE DESTRUCTION

AUTOCYTOTOXIC/ RADICALIC HYPOTHESIS

ECLECTIC HYPOTHESIS MELANOCYTORRAGY SYNERGISTIC THEORY

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: 90100. 2003

Metabolic Pathogenesis
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

Vitiligo: whats 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: whats new in 2011

Melanocytes are not completely absent in the depigmented epidermis

Normal Skin

Perilesional Skin

Lesional Skin

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

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

Whats 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.

Whats 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;54:157167

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;54:157167

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;127:26127.

mRNA for TNF- 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;151:9951003. 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;22:335338

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;54:157167; 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;22:335338.
Prignano F, Pescitelli L, Ricceri F, Lotti T. The importance of genetical link in immuno-mediated dermatoses: psoriasis and vitiligo. Int J Dermatol 2008;47:10601062;
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