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

4. •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).

5. •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

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

7. •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.

8. •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.

10. Distribution of melanocytes
•Number 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.

11. •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

12. 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)

13. • 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

15. •Variation - 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.

16. •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.

17. SURVIVAL
OF
NEURAL CREST DERIVED CELLS

18. 1. 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 .
.

19. •Germline mutations in KIT- that decrease ability of KIT receptor to be
activated by KIT ligand - Piebaldism
•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

21. 2. 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.

23. FORMATION
AND
FUNCTION OF
THE MELANOSOME

24. •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.

26. •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 .

27. Melanocyte
dendrite
formation
requires
actin
polymerizati
on,
controlled by
(GTP)‐bindin
g proteins
Rac &
Rho(regulato
ry associated
proteins)
transfer of
melanosom
e along
dendrites
occurs on
microtubul
es, process
driven by
dynein and
kinesin.
Dynein
binds
microtubul
es & ATP -
produces
forces
which
move
dynein and
melanoso
me
complex
along the
microtubul
e
Both dynein
& kinesin
regulates
direction of
melanosom
e
movmnt
along the
microtubule
s
Once
melanosomes
arrive at
cortical
regions of
melanocyte, 3
individual
proteins work
together in the
final stages of
melanosome
trafficking.

28. •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

30. 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
.

31. •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

32. •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.

33. STAGES OF MELANOSOMES

34. 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

35. 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.

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

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

41. EPIDERMAL- MELANIN UNIT

42. Approx,
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.

43. MELANIN BIOSYNTHESIS

44. •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

47. 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

48. ENZYMES OF MELANOGENESIS

49. •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 Cu2+ ions
bind

50. •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.

51. •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.
.

52. •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

53. •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

54. •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

55. Core Molecular Pathways
Influencing Melanin
Production

56. 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

57. •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.

58. •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

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

61. REGULATION OF MELANOGENESIS

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

63. 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

64. 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.

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

66. 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.

67. •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

68. II. 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.

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

70. III. 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 .

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

72. •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

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

74. 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.

75. • 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.

76. •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

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

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

80. •
•Melanin 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.

81. •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

82. 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

83. 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

84. 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