Melanogenesis

Melanogenesis- formation and factors involved.

1. MELANOGENESIS

2. The skin has epidermal units - responsible for melanin production and distribution - melanogenesis Melanin is the primary determinant of skin, hair, and eye color . critical role in photoprotection d/t ability to absorb ultraviolet radiation (UVR) The Fitzpatrick system - mc distinguish different skin pigmentation phenotypes. It characterizes six phototypes (I-VI) by grading erythema and acquired pigmentation after exposure to UVR

3. Skin colour results from : 1. Concentration and admixture of the types of melanins in melanocyte 2. Carotenoid pigments 3. Haemoglobin (in both the oxygenated and reduced state).

4. Two types of melanin pigmentation occurs - constitutive skin colour , which is amount of melanin pigmentation genetically determined in absence of sun exposure and other influences. facultative (inducible) skin colour or ‘tan’, results from sun exposure. Increased pigmentation - due to endocrine, paracrine and autocrine factors

5. The major events are: • the origin and function of the melanocyte • the formation and function of the melanosome • Melanin biosynthesis and its regulations

6. The melanocyte is a neural crest-derived cell. During embryogenesis , Melanoblasts originate in neural crest , migrate first to dermis & then to basal lamina of epidermis Immunocytochemical marker studies with melanocyte ‐specific antibody - melanoblasts appear in epidermis by 7 weeks gestation They first appear in the head and neck region at ~10 weeks of gestation.

7. By the end of gestation, active dermal melanocytes have “disappeared”, except in three primary anatomic locations – - the head and neck, - the dorsal aspects of the distal extremities, - and the presacral area.

8. Distribution of melanocytes N umber shows little variation between different races or sexes. Capacity of melanocytes - synthesize melanin, in basal state and after stimulation by sunlight varies. Melanocytes in dark skin - great capacity to synthesize melanin and to transfer it to surrounding keratinocytes . (in contrast with fair skin) ↓ in number of melanocytes occur - ageing, ↓ se in melanocyte density of about 6–8% per decade.

9. Melanocytes - most numerous in epidermis, hair follicles and the eye Ear Melanins found in Striae vascularis of Inner ear. Eye Melanocytes - present in Iris stroma in front of Iris, Iris pigment epithelium on the back of Iris, Retinal pigment epithelium. Adrenal glands seen in Medula and Zona reticularis . Others Melanin is also found in heart, liver, muscles and intestine

10. Melanoblast migration and differentiation Melanoblasts - predominantly found in basal layer of epidermis & hair follicles , identified by expression of melanocyte -specific markers tyrosinase (TYR), tyrosinase -related protein 1 (TYRP1), tyrosinase -related protein-2 (TYRP2), Premelanosome protein 17 (Pmel17/gp1000), melan -A or melanoma antigen recognized by T cells 1 (MART-1) and microphthalmia -associated transcription factor (MITF)

11. Initial segregation of melanocyte lineage - the Wnt /β‐ catenin pathway . The transcription factor mi is specific for melanocytes differentiation. Mi encodes transcription factor MITF , regulates several melanocyte specific genes mutations in mi - Waardenburg syndrome type 2A,AD characterized by deafness and patchy abnormal pigmentation Pax3 - prime cells for differentiation , whereas Wnt signalling allows cells to proceed along this route

12. V ariation - density of epidermal melanocytes /mm2 when different regions of the body are analyzed , e.g. the density of melanocytes is greater in genital region (~1500/mm2) than with back (~900/mm2) There are smaller differences between individuals when same anatomic site is examined.

13. The major determinant of skin color is activity of the melanocytes , i.e. quantity and quality of pigment production , not density of melanocytes . Several factors play a role in melanocyte activity: - specific characteristics of individual melanosomes - baseline (constitutive) and stimulated (facultative) levels - activity of the enzymes in the melanin biosynthetic pathway.

14. SURVIVAL OF NEURAL CREST DERIVED CELLS

15. Depends upon interactions between cell surface specific receptors and extracellular ligands . - Eg , KIT ligand ( steel factor or stem cell growth factor) binds to the transmembrane KIT receptor on melanocytes and melanoblasts Melanoblasts require expression of KIT receptor to maintain normal chemotactic migration directed by production of KIT ligand . .

16. G ermline mutations in KIT- that decrease ability of KIT receptor to be activated by KIT ligand - P iebaldism In developing embryo, melanoblasts express endothelin receptor type B (EDNRB) -stimulated to migrate by endothelin-3 (ET3 [EDN3]) Mutations in one or both copies of EDN3 or EDNRB can result in Waardenburg syndrome

17. Transcription factors group of proteins - essential role during embryogenesis. They bind DNA and influence the activity of other genes, able to regulate the complex interplay of genes required for embryonic development.

18. FORMATION AND FUNCTION OF THE MELANOSOME

19. Within cytoplasm of melanocytes - unique organelle - melanosome - melanin pigments are synthesized, deposited & transported. The melanosome - closely related to lysosome . Both organelles provide protection for the cell – lysosomes protect against pro-enzymes (proteases ) & melanosomes protect against melanin precursors (e.g. phenols, quinones ) that can oxidize lipid membranes.

20. The melanosomes transported in melanocytes from cell centre → periphery. Melanosome transport depends upon effective dendrite formation by melanocytes . UV radiation and melanocyte ‐stimulating hormone – stimulate this process .

21. Myosin Va , ‘ capture’ of melanosomes at actin ‐rich tip of the dendrite. Another protein, Rab27A , associates with membrane of melanocytes and forms complex with myosin Va and a third protein, melanophylin . The ability of melanophylin to bind actin - final part of transport process prior to melanosome transfer . The importance of interactions between these 3 proteins – is illustrated by the three different forms of Griscelli syndrome

22. Melanosome transfer to keratinocytes Both UV radiation and hormone(MSH ) stimulate transfer, while niacinamide suppress it . the exact mechanism unclear . One possibility is exocytosis of melanosomes from the tips of dendrites with subsequent keratinocyte uptake by endocytosis .

23. In vivo high resolution time‐lapse digital images identified long dynamic filopedia arising from melanocyte dendrite tips packed with melanosomes The filopedia attach and detach from the keratinocyte membrane: melanocytes observed travelling both directions within the filopedia

24. other hypotheses – - melanosome ‐laden protrusions from dendritic tips of melanocyte breach cell membrane of keratinocyte & protrusions engulfed by the keratinocyte . A final theory suggests - formation of membrane vesicles containing melanosome globules released from melanocytes & fusion of these with keratinocyte cell membrane or phagocytosis .

25. STAGES OF MELANOSOMES

26. Stage 1 Melanosomes - spherical vacuoles lacking (TYR) activity and no internal structural components. No melanin is present yet. They contain the melanosomal protein Pmel17 within the organelle. The presence of Pmel17 gives rise to structurally important intraluminal fibrils that characterise stage II melanosomes

27. Stage 2 Melanosomes - ellipsoidal, 0.5 micro-mm diameter. the presence Pmel17, determine transformation of stage I to elongated, fibrillar organelles → stage II melanosomes . They contain tyrosinase , TYRP1. They exhibit minimal deposition of melanin. Melanin is deposited within cross-linked longitudinal filaments.

28. Stage 3 Melanosomes - ellipsoidal. Melanin deposition increases by enzymatic activity The pigment is uniformly deposited on the internal fibrils

29. Stage 4 Melanosomes are ellipsoidal. Melanosome is fully developed and is filled with electron-opaque organelles. Melanin production is through polymerization.

30. EPIDERMAL- MELANIN UNIT

31. A pprox , every tenth cell in basal layer is a melanocyte . Melanosomes are transferred from the dendrites of the melanocyte into neighboring keratinocytes of the epidermis, hair matrices and mucous membranes; no transfer occurs in the pigment epithelium of the retina. The epidermal melanin unit refers to the association of a melanocyte with ~30–40 surrounding keratinocytes to which it transfers melanosomes .

32. MELANIN BIOSYNTHESIS

33. The “starting material” for production of melanin, both the brown–black eumelanin and the yellow–red pheomelanin , is amino acid tyrosine . The key regulatory enzyme in the pathway is Tyrosinase , which controls the initial biochemical reactions in this pathway (RATE LIMITING STEP) Melanin from tyrosine through a series of enzymatic and spontaneous chemical reactions is termed the - Raper –Mason pathway

34. MAIN TYPES OF EPIDERMAL MELANIN PIGMENTS. Eumelanins • Brown or black nitrogenous pigments, insoluble in all solvents, arise by oxidative polymerization of 5,6‐dihydroxyindoles from tyrosine Phaeomelanins • Alkali‐soluble pigments, yellow to reddish brown; arise by oxidative polymerization of cysteinyl‐dopa Trichochromes • A variety of sulphur‐containing phaeomelanic pigments with a well‐defined structure, characterized by a bi(1,4‐benzothiazine) chromophore

35. ENZYMES OF MELANOGENESIS

36. Tyrosinase - glycoprotein - melanosomal membrane, with an internal transmembrane & a cytoplasmic domain . copper dependent enzyme , rate-limiting stage in melanin synthesis Mutations inactivating enzyme -severe form of Albinism - OCA type 1 . cytoplasmic domain → transport of enzyme from nucleus to melanosomes . Internal domain contains catalytic region ( 90% of the protein) with histidine residues, where the Cu 2+ ions bind

37. Enzyme use superoxide anion as substrate for melanogenesis , protect melanocytes from ROS The phosphorylation of two serine residues from the cytoplasmic domain by protein kinase C-β (PKC-β) - important for tyrosinase activation.

38. In OCA1A - mutations in both copies of the tyrosinase gene lead to complete loss of enzyme activity, no melanin is found in the hair, skin, or eyes In OCA1B , there is decreased enzyme activity, pheomelanin is produced, especially in the hair as the patient ages. The activity of tyrosinase - enhanced by DOPA and is stabilized by tyrosinase -related protein 1 (TYRP1). Competitive inhibitors of tyrosinase activity – hydroquinone( melasma ) and L-phenylalanine. .

39. In phenylketonuria (PKU), ↑ L-phenylalanine(deficiency enzyme L-phenylalanine hydroxylase ) The characteristic blonde hair of PKU undergo darkening when pt is on a low-phenylalanine diet. Tyrosinase is a copper-requiring enzyme In patients with Menkes disease , a transmembrane Cu2+-transporting ATPase (delivers copper to melanosomes ) is dysfunctional, the kinky hair is hypopigmented

40. Two proteins similar to tyrosinase , tyrosinase -related protein-1 (TRP-1) and tyrosinase -related protein-2 (TRP-2), - membrane of melanosomes . TRP-1 - role in activation & stabilization of tyrosinase , melanosome synthesis, oxidative stress due to its peroxidase effect

41. The premature death of melanocytes in Vitiligo - increased sensitivity to oxidative stress caused by changes in TRP-1. Mutations in TRP-1, present in OCA type 3, skin and hair hypopigmentation TRP-2 acts similarly to tyrosinase , requires a metal ion for its activity, zinc instead of copper

42. Core Molecular Pathways Influencing Melanin Production

43. Melanocortin 1 receptor (MC1-R) Melanocortin receptors belong to the family of G-protein receptors includes MC1R to MC5R Eumelanin synthesis stimulated via MC1R agonists - ᾳ -MSH & ACTH while pheomelanin synthesis via ASP ᾳ-MSH cleaved from → pro- opiomelanocortin (POMC) produced by pituitary gland & keratinocytes UVR stimulates POMC gene expression, UVR-triggered oxidative stress leads to POMC peptide production this signaling pathway - involved in physiological adaptations of skin to environmental factors such as UV exposure

44. The Agouti signaling protein , is only known antagonist of MC1-R, competing with α-MSH – stimulating pheomelanogenesis . MC1-R activation by POMC peptides stimulates the accumulation of eumelanin instead of pheomelanin .

45. Addison’s Disease with high levels of ACTH, ACTH-producing tumors ( Nelson Syndrome), are associated with hyperpigmentation , particularly in sun-exposed areas. MC1-R genetic polymorphisms -responsible for ethnic differences of constitutive pigmentation & for different responses to UVR exposure. In individuals with red hair and light skin - high incidence of MC1-R mutations, responsible for ↓ sed response to α-MSH, reduced pigmentation induced by UVR exposure

46. The SCF-KIT receptor tyrosine kinase pathway -- involved in melanocyte pigmentation & development via the activation of the MITF transcription factor

47. REGULATION OF MELANOGENESIS

48. Regulation of Enzyme Activity in Melanogenesis ᾳ - Melanocyte -Stimulating Hormone ( ᾳ -MSH) Microphthalmia -Associated Transcription Factor (MITF) Protein Kinase C Sox Family

49. 1. ᾳ - Melanocyte -Stimulating Hormone ( ᾳ -MSH) The activity of tyrosinase is stimulated by ᾳ -MSH through the cAMP pathway. ᾳ -MSH binds to MC1R (melanocortin-1 receptor) on cell surface and activates adenylate cyclase - an ↑ intracellular cAMP The expression of tyrosinase , TYRP1 and TYRP2 is induced by cAMP

50. 2.MITF only member of microphthalmia family of transcription factors - essential for melanocyte development contains multiple promoters. The M promoter is selectively used in melanocytes and targeted by transcriptional factors - PAX3 , SOX9, SOX10, and MITF itself MITF regulates transcription of TYR, TYRP1 and TYRP2 ; The regulation of multiple pigmentation and differentiation related-genes by MITF shows it as a central regulator of melanogenesis .

51. 3. protein kinase C (PKC)-dependent pathway also regulates melanogenesis . T hrough phosphorylation and activation of tyrosinase . The activity of tyrosinase - dependent on phosphorylation of serine residues in its cytoplasmic domain.

52. 4.SOX family – About 20 transcription factors containing domains that mediate sequence-specific DNA binding. 9 groups of SOX proteins known in mammals (SOXA, B1, B2 and C–H). SOXE includes SOX9 and 10 - essential developmental regulators of melanogenesis .

53. Melanocytes originate from neuroectodermal tissue under SOX proteins influence ( SOX8, 9 and 10 are expressed in the dorsal neural tube and the neural crest ) SOX10 controls transcription of MITF ;critical for melanogenesis In the absence of SOX10, MITF cannot induce tyrosinase . The most important role of SOX9 in melanoblast development - ability to induce the expression of SOX10

54. Melanocyte regulation by endocrine factors ↑ levels oestrogens - ↑ sed pigmentation ( face, areola, lower central abdomen and genitalia.) Melanocytes - oestrogen receptors and ↑ oestradiol stimulate enzymes involved in melanogenesis In Addison disease,diffuse brown hyperpigmentation results from melanocortins from pituitary.

55. In Cushing syndrome, hyperpigmentation is caused by an overproduction of ACTH from a corticotrophic adenoma or an ectopic non‐pituitary tumour.

56. Melanocyte regulation by paracrine and autocrine factors Human melanocytes synthesize IL‐1α and IL‐1β, an autocrine as well as paracrine regulatory role . Melanocytes respond to PGE2 with ↑ sed melanogenesis and dendrite formation . Prostaglandins, leukotrienes and thromboxanes are the main inducers of tyrosinase .

57. Basic fibroblast growth factor - first paracrine factor for melanocytes - identified. exerts effect by binding to a tyrosine kinase receptor expressed on melanocytes .

58. Endothelins - important group of peptides that act upon melanocytes in a paracrine manner. ET1 acts synergistically with α‐MSH and basic fibroblast growth factor - stimulate melanocytes proliferation Furthermore, endothelins appear to have a role in protecting melanocytes : treatment of melanocytes with ET1 reduced UVR‐induced apoptosis and prolonged melanocyte survival

59. Melanocyte response to UV radiation UVR - most important extrinsic factor -regulation of melanogenesis . The main stimulus for induced or acquired pigmentation, known as “tanning”

60. Immediate Pigmentation , appears 5-10 minutes after exposure , disappears mins or days later, largely due to UVA, dependent on oxidation of pre-existing melanin & redistribution of melanosomes to epidermal upper layers. Delayed pigmentation, occurs 3-4 days after exposure to UVR, disappears within weeks, due to UVA & mainly UVB radiation, results from an ↑ sed epidermal melanin, particularly eumelanin , providing photoprotection .

61. UVR ↑ ses proliferation / recruitment of melanocytes , no. of dendrites, transfer of melanosomes to keratinocytes for DNA photoprotection . The expression of POMC peptides, MC1-R, and melanogenic enzymes ↑ ses in keratinocytes and melanocytes . DNA, directly absorbs UVR with formation of thymine dimers & pyrimidine derivatives, and defects in DNA repair increase the risk of skin cancer.

62. UVR - enhances (ROS) formation in keratinocytes and melanocytes → consequent DNA damage. An elderly individual, depending on constitutive pigmentation & cumulative UVR dose, may have hyperpigmented lesions (solar lentigines ) indicate photoaging . Explained - aged melanocytes possess enhanced functional activity after years of cumulative UVR exposure. However, with aging, there is ↓ se in number of functional melanocytes

63. Eumelanin acts as a natural sunscreen against photoaging and photocarcinogenesis , by reducing ROS and increasing repair of DNA damage.

64. Melanin Production in Hair Shaft Hair follicle pigmentary unit interactions - follicular melanocytes , keratinocytes & dermal papilla fibroblasts → production of hair shaft melanin.

65. M elanin in follicular melanocytes → transfer of melanin granules → cortical and medullary keratinocytes → pigmented hair shafts. Hair pigmentation active only during anagen stage (growth phase) of hair cycle. Melanogenesis is switched off in catagen stage and remains absent through telogen .

66. Epidermal and follicular melanins - independent units and the co-expression of white hair on highly pigmented skin - clear affirmation Melanocytes of hair follicle produce larger melanosomes than those in epidermis. Follicular-melanin units are larger, more dendritic , and have more extensive Golgi and rough endoplasmic reticulum

67. Defects in melanocyte lineage migration Inheritance Genes Clinical features Piebaldism AD KIT SLUG Well‐demarcated ventral midline hypopigmentary macules , white forelock Waardenburg syndrome WS1-4 AD PAX3 White forelock, hypopigmented patches, iris heterochromia , deafness, and mild facial dysmorphism (broad nose root) Hypopigmentation disorders

68. Albinism: defects in melanin synthesis OCA1A AR TYR Absent skin and hair pigmentation, no ability to tan (0CA1A). Partial albinism, hair darkens with age (OCA1B) OCA2 AR OCA2 Prevalent in black people, blond to red‐brown hair with age, ephelides AR OCA2 Prevalent in black people, blond to red‐brown hair with age OCA3 AR TYRP1 Rufus albinism in black people

69. Defects in lysosomal biogenesis and transport, including melanosomes Hermansky–Pudlak syndrome Chediak –Higashi syndrome Griscelli–Pruniéras syndrome Hyperpigmentation disorders Generalized/diffuse Familial progressive hyperpigmentation Linear Incontinentia pigmenti Linear and whorled naevoid Hypermelanosis Punctate/reticulate Dyskeratosis congenita Dowling– Degos disease