1. Physiology and Pathophysiology of Melanocytes Torello Lotti, MDProfessor of Dermatology and Venereology Florence, Italy
2. MELANOCYTESMelanocytes are pigment-producing cellsthat originate from the dorsal portion ofthe closing neural tube in vertebrateembryos.
3. MELANOCYTES ANDPIGMENTATION PHYSIOLOGY
4. Pluripotent neural crest cell
5. Melanoblast migration and differentiation intomelanocytes is influenced by a number of signalingmolecules produced by neighboring cells that interact withtheir their specific cell surface receptors.Wnt Bone morphogenetic factor (BMPs)Endothelin-3 (ET-3) Hepatocyte growth factor (HGF)Stem Cell Factor (SCF), c-Kit-ligand
6. Wnt Family16 different secreted glycoproteins;Directs the maturation of pluripotent neural crest cellinto melanoblasts Induction and Wnt Frizzled receptor accumulation of Β- cateninInduce the transcription of 3 keyenzyme in melanin synthesis: Transcription of- Tyrosinase; Microphthalmia-assocciated- TRP-1; transcription factor (Mitf)- TRP-2.
7. MITF is central to Melanocyte viability and function Waardenburg syndrome type 2A - different colored irises; - white forelock; - congenital cochlear deafness. usually MITF activitynormal MITF activity is is only partially lostcompletely lost in theseanimals
8. Endothelins FamilyET-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.
9. Stem Cell FactorSCF: expressed by keratinocytes Drive melanoblasts to their final destination c-Kit (its receptor): expressed on melanoblastsMutations of c-Kit or SCF:melanoblast unable to migrate to the skin and/or survive there PIEBALDISM
10. Skin and Hair FollicleInner ear Cochlea Leptomeninges MELANOBLAST Choroid Ciliary body Iris
11. CUTANEOUS MELANOCYTESMelanocyte density/mm2:550-1200 (highest concentration in genitalia and face)Melanocytes syntesize melanine,stored in cyosolic organelles(melanosomes) transferred tokeratinocytes through dendriticprocess.Keratinocytes signals regulate epidermal melanocytesurvival, dendricity, melanogenesis…Epidermal melanin unit: one melanocytesurrounded by several keratynocites
12. MELANIZATIONThe 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 keratinocytesDisruption in any of these events results in Hypopigmentation
13. Melanosome BiogenesisUnique menbrane bound organelle (modified version oflysosomes?) in which melanin biosynthesis take place. Eumelanosomes Pheomelanosomes Eumelanin Pheomelanin
15. MELANIN BIOSYNTHESIS Two types of melanin Eumelanin PheomelaninDark, brown/black 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 tomelanocytes:ROS generation: DNA damage: melanoma
16. MELANOGENIC PROTEINSEnzymes and proteins involved in melanosomal maturationTyrosinase: 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
18. 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.
19. Microphtalmia-Associated Transcription Factor (Mitf)Mitf upregulate the expression of anti-apoptotic protein BCl2 Melanocyte survival Mitf + -/+ Cdk2 - p21 + G1 to S phase Role in melanocyte (Melanocyte proliferation) proliferation ?
20. Melanocortin Receptor (MCR)Family of five related receptors (MC1-5R).MC1R: melanocytes α-MSH ACTH MC1R Mitf Eumelanin cAMP transcription synthesisPolimorphisms within the MC1Rgene are largely responsible forthe different skin/hair coloramong different ethnic group.
21. Propiomelancortin (POMC) encodes α-MSH and other hormonesBoth pituitary gland anf epidermal keratinocytes are able to synthesized POMC
22. UV light activates p53 in keratinocytes,p53 induces expression of POMC in keratinocytes
23. UV light activates a cascade that resultsin elevated melanin synthesis and transport
24. 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 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.Schallreuter KU et al. Regulation of melanogenesis – controversies and new concepts. Experimental Dermatology 2008;17: 395–404.
25. MELANIN BIOSYNTHESISDessinioti C et al.A review of genetic disorders of hypopigmentation: Lessons learned from the biology of melanocytes.Experimental Dermatology 2009; 18: 741–749.
26. MELANOCYTE DENDRITES• Branching protoplasmatic process that interact withkeratinocytes.• Actin is a major structural component of dendrites;• Several keratinocytes-derived factors (ET-1,NGF, PGE2, β-endorphin)play a role in melanocytedendricity;• Integrins also play arole in dendriteformation.
27. MELANOSOME TRANSPORT (in Melanocyte)Microtubules(arranged parallel to the long axis +of the dendrite)Microtubule-associated motor proteins:Kinesins (centrifugal movement)andDyneins (Ccentripetal movement)Other partecipants:Rab27aMyosin-Va Mutated in Griscelli syndromemelanophilin
28. MELANOSOME TRANSPORT (to Keratinocytes) Several potential ways involvedExocytosis Transfer by Cytophagocitosis (keratinocytes membrane vesicles phagocytose the tip of a melanocyte dendrite) Fusion of plasma menbranes
29. REGULATION OF MELANOCYTE FUNCTIONImokawa G. Autocrine and paracrine regulation of melanocytes in human skin and inpigmentary disorders. Pigment Cell Res 2004
30. 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.
31. Hypomelanoses Normal Albinism Functional defect in melanine synthesis VitiligoLocalized loss / inactivation of melanocytes
32. Disorders of Melanocyte Disorders of MelaninDevelopment and Migration SynthesisDessinioti C et al.A review of genetic disorders of hypopigmentation: Lessons learned from the biology of melanocytes.Experimental Dermatology 2009; 18: 741–749.
33. Disorders of Melanosome Formation and Transfer to KeratinocytesDessinioti C et al.A review of genetic disorders of hypopigmentation: Lessons learned from the biology of melanocytes.Experimental Dermatology 2009; 18: 741–749.
34. VITILIGO ETIOPATHOGENESIS GENETIC PREDISPOSITION Autoimmune Susceptibility Locus (AIS1)NEURAL HYPOTHESIS AUTOIMMUNE HYPOTHESIS MELANOCYTE DESTRUCTIONAUTOCYTOTOXIC/RADICALIC HYPOTHESIS ECLECTIC HYPOTHESIS MELANOCYTORRAGY SYNERGISTIC THEORY
36. 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.
37. Autoimmune PathogenesisOngenae K et al. Evidence for an Autoimmune Pathogenesis of Vitiligo. Pigment Cell Res 16: 90–100. 2003
38. Metabolic Pathogenesis Altered antioxidant Increased activity and scavenger of superoxide mechanism dismutase High levels of epidermic 7-BH4 and H2O2Inhibition of enzyme function (phenylalanine-hydroxilase and tyrosinase) and abnormal expression of Tyrosinase Related Protein-1 (TRP-1). impaired melanine synthesis
39. Vitiligo: what’s new in 2011Melanocytes 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
40. Vitiligo: what’s new in 2011Melanocytes are not completely absentin 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
41. Vitiligo: what’s new in 2011Melanocytes are not completely absent in the depigmented epidermisComment: – 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
42. VITILIGO:NOT ONLY A MELANOCYTIC DISEASE?
43. Imokawa G. Autocrine and paracrine regulation of melanocytes in human skin and inImokawa G. Autocrine and paracrine regulation of melanocytes in human skin and inpigmentary disorders. Pigment Cell Res 2004pigmentary disorders. Pigment Cell Res 2004
44. 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.
45. 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
46. 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
47. 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
48. 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
49. 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
50. 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
51. OUR CONTRIBUTIONS
52. Berti S, Bellandi S, Bertelli A, Colucci R, Lotti T, Moretti S. Vitiligoin an Italian outpatient center: a clinical and serologic study of 204patients 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 andimmunophenotypical changes upon nb-UVB in vitiligo skin. ArchDermatol Res. 2010Arunachalam M, Sanzo M, Lotti T, Colucci R, Berti S, Moretti S.Common variable immunodeficiency in vitiligo. G Ital DermatolVenereol. 2010;145(6):783-8.Becatti M, Prignano F, Fiorillo C, Pescitelli L, Nassi P, Lotti T, TaddeiN. The involvement of Smac/DIABLO, p53, NF-kB, and MAPKpathways in apoptosis of keratinocytes from perilesional vitiligoskin: Protective effects of curcumin and capsaicin. Antioxid RedoxSignal. 2010, 1;13(9):1309-1321.
53. Prignano F, Pescitelli L, Becatti M, Di Gennaro P, Fiorillo C, TaddeiN, Lotti T. Ultrastructural and functional alterations of mitochondriain 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 keratinocyteapoptosis. 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 MassiD. Protease-activated receptor-2 downregulation is associated tovitiligo lesions. Pigment Cell Melanoma Res. 2009;22:335–338.Prignano F, Pescitelli L, Ricceri F, Lotti T. The importance of genetical linkin immuno-mediated dermatoses: psoriasis and vitiligo. Int J Dermatol2008;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