Mechanisms regulating melanogenesis (2024)

Chart 1

List of abbreviations

Stages of the melanogenesis process

Melanocytes originate in neural crest melanoblasts that migrate todifferent destinations, including the basal layer of the epidermis and hairfollicles, after closure of the neural tube.^3,11,12 Their migration, proliferation, and differentiation into melanin-producingcells depend on mediators produced by cells of the dorsal neural tube, ectoderm andkeratinocytes, such as the family of glycoproteins WNT, endothelin 3 (EDN3), and stemcell factor (SCF), which binds the c-Kit receptor tyrosine kinase in melanocytes and melanoblasts.^3,12 Bone morphogenic proteins antagonize these events, and their expression isreduced in melanocyte migration. Piebaldism, a genodermatosis withdepigmented macules, is caused by mutations in the c-kit and SCF genes (Chart 2).^3,10,12

Melanin synthesis occurs in melanosomes, lysosome-related organelles(LROs) whose defects are responsible for Chediak-Higashi Syndrome andHermansky-Pudlak Syndrome, diseases with cutaneous hypopigmentationand systemic manifestations (Chart 2).^6,10,11,13

The key proteins involved in skin pigmentation, such as the components of thefibrillar matrix that binds to melanin (glycoprotein Pmel17) and melanogenic enzymes,are located in melanosomes. In these organelles, the structural matrix is arranged,the tyrosinase enzyme is acquired, and melanin is synthesized along four maturation stages.^2,3,6,13 The acquisition of melanogenic enzymes is regulated by a membraneassociatedtransporter protein (MATP), and mutations of the respective gene determineOculocutaneous Albinism type 4.^3,6,10When melanin synthesis is completed, melanosomes move bi-directionally fromthe perinuclear area towards melanocyte dendrites, in a movement controlled bymicrotubule proteins (kinesin, dynein). This transport ends with melanosomes bindingactin filaments through a complex formed by myosin Va, Rab27a, and melanophilin (mlph).² Mutations in the corresponding genes determine various forms ofGriscelli Syndrome (Chart 2).^6,10 An increase in intramelanosomal pH from 5 to 6.8, which depends on the protonpump p-protein in the melanosomal membrane, is needed for full maturation of melanosomes.¹⁴ On the one hand, the importance of this stage is supported byOculocutaneous Albinism type 2, a disease which is caused by loss offunctional p-protein and, on the other hand, it is supported by lower response torepigmentation treatment options, which is observed in Vitiligo patientswho are also treated with proton pump inhibitors (Chart 2).^6,12,14

In the epidermis, each melanocyte interacts through dendrites with 30 to 40keratinocytes, allowing transfer of mature melanosomes to the cytoplasm ofkeratinocytes positioned strategically over nuclei.^1,11 This transfer is not fully understood, and different mechanisms such asexocytosis, citophagocytosis, fusion of plasma membranes, and transfer by membranevesicles are described.²

Phenotypic diversity of pigmentation and types of melanin

Phenotypic diversity of pigmentation is not due to a variation in melanocyte number,which is relatively constant in different ethnic groups, but to the size and numberof melanosomes, the amount and type of melanin, and melanin transfer and distributionin keratinocytes.^1,3,11 The melanosomes of dark-skinned individuals are larger, more numerous, andelongated, resulting in delayed degradation in keratinocytes and consequently inincreased visible pigmentation.^3,11,13 These differences in melanosomes are present at birth and are not determinedby extrinsic factors such as UVR.³

There are two types of melanin: eumelanin brown-black or dark insolublepolymer -and pheomelanin - red-yellow soluble polymer formed by theconjugation of cysteine or glutathione (Figure1).^11,13,15 Eumelanin is the major type in individuals with dark skin and hair and is moreefficient in photoprotection. Pheomelanin is predominantly found in individuals withred hair and skin phototypes I and II, in whom skin tumors are more common.^5,11

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Figure 1

Synthesis of the two types of melanin and representation of the functions ofthe major enzymes involved

Enzymes of melanogenesis

Tyrosinase is a glycoprotein located in the melanossomal membrane, withan internal, a transmembrane, and a cytoplasmic domain. It is a copperdependentenzyme that catalyzes the conversion of Ltyrosine into L-DOPA, the rate-limitingstage in melanin synthesis (Figure 1).^2,14,15 Mutations that inactivate this enzyme are responsible for the most severe formof Albinism, that is, Oculocutaneous Albinism type 1 (Chart 2).⁶The cytoplasmic domain participates in the transport of the enzyme from thenucleus to melanosomes. The internal domain contains the catalytic region(approximately 90% of the protein) with histidine residues, where the copper ions bind.² Mutations in the copper carrier (ATP7A) result in Menkes Disease (Chart 2).⁶If copper is oxidized, the enzyme is inactivated and can be activated byelectron donors such as L-DOPA, ascorbic acid, superoxide anion, and possibly nitricoxide (NO).^14,15 Due to the fact that this enzyme can use superoxide anion as a substrate formelanogenesis, it may protect melanocytes from reactive oxygen species (ROS).^11,16 The phosphorylation of two serine residues from the cytoplasmic domain byprotein kinase C-β (PKC-β) is also important for tyrosinase activation.¹⁷

Tyrosine hydroxylase isoform I (THI) is present in the melanosomalmembrane adjacent to tyrosinase and catalyzes the conversion of L-tyrosine intoL-DOPA, promoting the activation of tyrosinase.

In cytosol, phenylalanine hydroxylase (PAH), cofactor 6BH4(6-tetrahydrobiopterin)-dependent, catalyzes the conversion of L-phenylalanine toL-tyrosine, the tyrosinase substrate, thus also promoting its activation.^14,15 Schallreuter et al., underlining the central role of tyrosinase, consider thatthese three enzymes are required for the beginning of melanogenesis.¹⁴

Two proteins similar to tyrosinase (40% hom*ologous amino acids),tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2),are also present in the membrane of melanosomes. Although its precise role isnot yet clarified, it is possible that TRP-1 has a role in the activation andstabilization of tyrosinase, melanosome synthesis, increased eumelanin/pheomelaninratio and a role against oxidative stress due to its peroxidase effect (Figure 1).^2,15 The results found by Jimbow et al. suggest that the premature death ofmelanocytes in Vitiligo is related to an increased sensitivity tooxidative stress caused by changes in TRP-1.¹⁸ Mutations in TRP-1, present in Oculocutaneous Albinism type 3,result in skin and hair hypopigmentation (Chart 2).⁶TRP-2 acts as a dopacrome tautomerase and, similarly to tyrosinase, requires ametal ion for its activity, zinc instead of copper (Figure 1).^2,14,15Figure 1 shows the synthesis of the two typesof melanin and the functions of the major enzymes involved.

Melanocortin 1 receptor (MC1-R)

Melanocortin receptors belong to the family of G-protein receptors. MC1-Rpredominates in melanocytes and its agonists include melanocyte stimulating hormone(α-MSH) and adrenocorticotropic hormone (ACTH), both cleavage products ofproopiomelanocortin (POMC). POMC is cleaved by carboxypeptidase-1 in ACTH andβ-lipotrophin and by carboxypeptidase-2 β in endorphin and ACTH. ACTHis fragmented in ACTH 1-17 and α-MSH. ACTH and α-MSH share thetetrapeptide His-Phe-Arg-Trp, which is essential for melanotropic activity. Thispeptide is the main intrinsic regulator of pigmentation, but its pituitary productionis insufficient to stimulate melanogenesis; thus, keratinocytes and melanocytes arethe main responsible for its production on the skin.^{1,5,11,13,15,16,19}Addison's Disease with high levels of ACTH, ACTH-producing tumors(Nelson Syndrome), and cases of prolonged administration of this hormoneare associated with hyperpigmentation, particularly in sun-exposed areas.^1,2,15

MC1-R genetic polymorphisms are responsible for ethnic differences of constitutivepigmentation and for different responses to UVR exposure.^2,5,11,16 In individuals with red hair and light skin there is a high incidence of MC1-Rmutations, which may be responsible for a decreased response to α-MSH,resulting in decreased eumelanogenesis and reduced pigmentation induced by UVR exposure.¹¹

The Agouti signaling protein, although poorly documented, is theonly known antagonist of MC1-R, competing with a-MSH and, therefore,stimulating pheomelanogenesis. MC1-R activation by POMC peptides stimulates theaccumulation of eumelanin instead of pheomelanin.

MC1-R agonists activate the adenylate cyclase enzyme, increasing intracellular cAMPand activating protein kinase A (PKA). PKA phosphorylates CREB (cAMP responseelement), which acts as a transcription factor in several genes, including themicrophthalmia-associated transcription factor (MITF). MITF in its phosphorylatedactive form regulates the expression of melanogenic enzymes, promoting eumelanogenis.^2,5,11,13,15 Its phosphorylation depends on kinases of mitogen-activated protein (MAP)whose activity is induced by the binding of keratinocyte-produced-SCF to the c-kitreceptor tyrosine kinase.^11,12 In addition to CREB, the expression of the MITF protein is regulated by othertranscription factors and mediators produced by keratinocytes and fibroblasts.^6,20 Moreover, the MITF protein regulates the expression of the Rab27a protein,which is important in melanosome transport, the melanosomal matrix protein Pmel17,and an anti-apoptotic protein (bcl-2) of melanocytes, which is often expressed in melanomas.^2,10,12,20 MITF gene mutations are responsible forWaardenburg Syndrome type 2, with cutaneous and iris hypopigmentation, andTietz Syndrome, with hypopigmentation and deafness (Chart 2).^1,10,20

Intrinsic regulation of skin pigmentation

Melanocytes produce POMC peptides, cytokines, NO, prostaglandins, and leukotrienes,which act via an autocrine or paracrine way on keratinocytes, and are involved inimmune and inflammatory responses. Keratinocytes also produce several factors inresponse to UVR exposure, with paracrine action on melanocytes, which may stimulateor inhibit melanogenesis (Chart 3).^2,6,11,12Our group investigated the role of the recently described cutaneousendocannabinoid system in melanogenesis and demonstrated that UVR also activatesendocannabinoid production by keratinocytes and that a paracrine cannabinoid receptortype 1-mediated endocannabinoid signaling negatively regulates melanin synthesis.²¹

Although POMC/MC1-R/cAMP is the main pathway, there are other melanocyte receptorsassociated with adenyl cyclase and cAMP production, such as muscarinic receptors andα and β estrogen receptors. The increase in estrogen levels duringpregnancy can cause hyperpigmentation (melasma, areolar hyperpigmentation andline nigricans).⁶ Catecholamines may be produced by keratinocytes from L-DOPA, the melaninprecursor, and can bind to α1 and β2 adrenergic receptors inmelanocytes, stimulating melanogenesis via the cAMP pathway and PKC-β.^15,22 This redundancy of cAMP production reveals the importance of this secondmessenger in melanogenesis. However, norepinephrine/α1 adrenergic receptor,ACTH 1-17/MC1-R can also activate the inositol trisphosphate/diacylglycerol (IP3/DAG)pathway, which promotes the release of calcium in the cytoplasm of melanocytes.^2,14,22 DAG is important for the activation of PKC-β, which phosphorylatestyrosinase, and can also be released from melanocytes through UVR action in the lipid membrane.^3,14,17Figure 2 illustrates some of the differentpathways, receptors, second messengers, and melanogenic enzymes involved inmelanogenesis.

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Figure 2

Melanocyte role and representation of the different signaling pathwaysregulating melanogenesis: activation factors, receptors, second messengers, andmelanogenic enzymes

Extrinsic regulation of skin pigmentation by ultraviolet radiation (UVR)

UVR is the most important extrinsic factor in the regulation of melanogenesis. It isthe main stimulus for induced or acquired pigmentation, known as "tanning".^1,2,6,11 There are two types of induced pigmentation, which depends on genetic factorsand is more evident in individuals with dark skin and hair.^3,23Immediate pigmentation, which appears 5-10 minutes after exposure toUVR, disappears minutes or days later, is largely due to UVA, and is not dependent onincreased melanin synthesis, but on the oxidation of pre-existingmelanin and redistribution of melanosomes to the epidermal upper layers.Delayed pigmentation, which occurs 3-4 days after exposure to UVR,disappears within weeks, is due to UVA and mainly UVB radiation, and results from anincreased level of epidermal melanin, particularly eumelanin, providing photoprotection.^{3,12,13,16,23}

On the one hand, UVR increases proliferation and/or recruitment of melanocytes, thenumber of dendrites, and the transfer of melanosomes to a supranuclear location onthe keratinocytes for DNA photoprotection. On the other hand, the expression of POMCpeptides, MC1-R, and melanogenic enzymes increases in keratinocytes and melanocytes respectively.^11,13,16,23

DNA, the main cellular chromophore, directly absorbs UVR with the formation ofthymine dimers and other pyrimidine derivatives, and defects in DNA repair increasethe risk of skin cancer.^1-3,5 The key observation that the UVR spectra for producing a delayed tan and forinduction of thymine dimers following UVR were virtually identical and within the UVBrange suggested for the first time a cause-effect relation between DNA damage and melanogenesis.^24,25 In the last decade, it was recognized that the tumor-supressor protein p53 is atranscription factor that plays a pivotal role in the tanning response afterUVB-induced DNA damage.²⁶ Using an elegant mouse model in which UVR causes tanning, it was demonstratedthat α-MSH production is regulated in keratinocytes by p53 via a p53consensus sequence in the POMC gene promoter.²⁷ Furthermore, Eller et al. demonstrated in vitro that small fragments of DNAinduced pigmentation by increasing tyrosinase expression and activity and the levelsof the tumor suppressor protein p53.^1,28 Cui et al. found that activation of p53 in keratinocytes increases theexpression and release of POMC peptides.²⁷ Even in the absence of keratinocytes, there is a strong melanogenic responseto UVR mediated by p53 in human melanocytes and melanoma cells invitro, which can be explained by the fact that p53 regulates thetranscription of the hepatocyte nuclear factor 1α (HNF1 α), atyrosinase transcription factor, in melanocytes.^2,14,26

UVR also enhances reactive oxygen species (ROS) formation in keratinocytes andmelanocytes, with consequent DNA damage.^3,5

Regardless of the specific mechanisms, acquired pigmentation is part of the skinadaptive response, mediated by exposure to UVR and will provide skin protection infuture exposures.^2,26

It has been shown that plasma membrane lipids are also affected by UVR to releasemembrane-associated diacylglycerol (DAG), which activates PKC-β In turn,PKC-β activates tyrosinase, resulting in stimulation of melanogenesis.¹⁴

An elderly individual, depending on constitutive pigmentation and cumulative UVRdose, may have hyperpigmented lesions (solar lentigines) that indicate photoaging.This can be explained by the fact that aged melanocytes possess an enhancedfunctional activity after years of cumulative UVR exposure. However, with aging,there is also a decrease in the number of functional melanocytes.³ Eumelanin acts as a natural sunscreen against photoaging andphotocarcinogenesis, in part by reducing ROS and increasing repair of DNA damage.^5,13,15

Conflict of interest: None

Financial Support: None

* Work conducted at the Institute of Pharmacology and Therapeutics, School ofMedicine, University of Porto (IFT -FMUP) - Porto, Portugal.

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