Genetic Abnormalities, Melanosomal Transfer, and Degradation inside Keratinocytes Affect Skin Pigmentation

Abstract

Skin pigmentation is a specific and complex mechanism that occurs as a result of the quantity and quality of melanin produced, as well as the size, number, composition, mode of transfer, distribution, and degradation of the melanosomes inside keratinocytes and the handling of the melanin product by the keratinocyte consumer. Melanocyte numbers typically remain relatively constant. Melanin synthesis, melanosome maturation, and melanoblast translocation are considered to be responsible for hereditary pigmentary disorders. Keratinocytes play a significant role in regulating the adhesion, proliferation, survival, and morphology of melanocytes. In the epidermis, each melanocyte is surrounded by 30–40 keratinocytes through dendrites and transfers mature melanosomes into the cytoplasm of keratinocytes, which are then digested. Melanocytes are believed to transfer melanosomes to neighboring keratinocytes via exocytosis-endocytosis, microvesicle shedding, phagocytosis, or the fusion of the plasma membrane, protecting skin cells against ultraviolet (UV) damage by creating a physical barrier (cap structure) over the nucleus. An understanding of the factors of melanocytes and keratinocytes that induce pigmentation and the transfer mechanism of melanosomes to keratinocytes and how genetic abnormalities in keratinocytes affect pigmentary skin disorders will help us to elucidate hereditary pigmentary disorders more transparently and provide a conceptual framework for the importance of keratinocytes in the case of pigmentary disorders.

1. Introduction

The skin is the outermost organ, covering the whole body. It helps with temperature regulation, immune defense, vitamin production, and sensation. However, skin is also associated with many potential problems, with over 3,000 possible disorders [1]. Furthermore, its color has social, spiritual, cosmetic, and medical issues associated with it. The color of human skin depends on the distribution of melanin, a pigment produced in melanosomes in the melanocyte cytoplasm via the tyrosinase reaction. Skin produces melanin within the melanocytes of the interfollicular epidermis through a multi-stage process called melanogenesis. The subsequent transfer, translocation, and degradation of melanin to, in, and by the recipient keratinocytes, respectively, causes pigmentation. The skin can protect itself from solar irradiation, thanks to this specific and complex pigmentation mechanism. The nature of pigmentation is determined by the quantity and quality (pheo/eumelanin ratio) of melanin and the size, number, composition, mode of transfer, distribution, and degradation of the melanosomes inside keratinocytes, as well as the handling of the melanin product by macrophages. On the other hand, melanocyte numbers tend to remain relatively constant. The dysregulation of the process of melanogenesis can cause several types of pigmentary defects, classified as either hypopigmentation, hyperpigmentation, or mixed hyper/hypopigmentation [2, 3, 4]. Hereditary pigmentary disorders occur mostly due to genetic deficiency in melanin, irregular melanin synthesis in melanocytes, abnormal melanosome maturation, and melanoblast translocation. Various physiological factors, including autocrine and paracrine hormones/cytokines, also modulate skin pigmentation.

Keratins are specific to epithelial cells and one of the cytoskeletal proteins that provide structural support to keratinocytes through intermediate filament networks. Approximately 21 different keratins have been reported to be associated with different hereditary disorders [5]. Keratinocytes are crucial in the organization of cell adhesion, as well as the proliferation, survival, and morphology of melanocytes. Keratins also play a pivotal role in the uptake of melanosomes into keratinocytes, organelle transport, and nuclear anchorage, indicative of their involvement in intracellular transportation [6]. Several studies have claimed that the keratin 5 head domain interacts with heat shock cognate 70 (Hsc70) and is involved in organelle transport [7, 8] and chaperone-mediated autophagy.

Many studies have been performed to elucidate the role of keratinocytes in pigmentation; however, the transfer mechanism of melanin to keratinocytes remains ambiguous. It has been postulated that melanocytes transfer melanosomes to neighboring keratinocytes via exocytosis-endocytosis, microvesicle shedding, or phagocytosis. In this chapter, the factors of melanocytes and keratinocytes that induce pigmentation and the potential mechanism of melanosomal transportation to the surrounding keratinocytes, and how genetic abnormalities in keratinocytes affect pigmentary skin disorders are reviewed to develop a basis for the role of keratinocytes in pigmentation.

2. Skin pigmentation is regulated by several factors of melanocytes

2.1 Melanosome biogenesis and melanogenesis process

Melanocytes are unique neural crest-derived cells that synthesize and store melanin pigments in melanosomes, which are specific membrane-bound organelles that share several features with lysosomes. In particular, they contain acid-dependent hydrolases and lysosomal-associated membrane proteins [9]. Melanosomes are members of the cell-specific organelle family, termed lysosome-related organelles (LROs), which also comprise lytic granules observed in cytotoxic T lymphocytes and natural killer cells, MHC class II compartments (MIICs) observed in antigen-presenting cells, basophil granules, platelet-dense granules, azurophil granules observed in neutrophils, and Weibel-Palade bodies observed in endothelial cells [10].

Most pigment-specific proteins are localized in melanosomes [11] and are divided into three distinct groups—structural fibrillar proteins required for melanosome structure and binding of melanin, enzymatic components required for melanin synthesis, and proteins required for melanosome transport and distribution [12, 13].

Melanosomes can be morphologically classified into four distinct stages (I–IV) based on melanosomal maturation and three important structural proteins that form melanosomes—melanosomal matrix protein (PMEL17/Silv/GP100), melanoma antigen recognized by T cell-1 (MART-1), and glycoprotein nonmetastatic melanoma protein b (GPNMB/DC-HIL/osteoactivin) (Figure 1). Intraluminal fibrils begin to form in amorphous spherical stage I melanosomes and develop a meshwork characteristic of stage II melanosomes, both of which are considered to be pre-melanosomes and do not contain melanin. MART-1 has been previously observed in earlier melanosomal stages [14]. In stage II, melanin synthesis begins within the fibrillar and is deposited uniformly on the internal fibrils that evolve into stage III. Melanin is deposited on the PMEL17 fibrils in stage III. MART-1 plays a significant role in the maturation of PMEL17 [14]. In the last stage (IV) of maturation, copious amounts of melanin fill the melanosomes and form a masked internal structure and dark color. GPNMB, a melanosome-specific and proteolytically released protein, is superabundant in late melanosomes [15] and is crucial for the formation of melanosomes in a microphthalmia transcription factor (MITF)-independent fashion [16]. Recent studies have shown that early melanosomes are derived from the endoplasmic reticulum (ER), coated vesicles, lysosomes, and endosomes [17, 18, 19].

Figure 1.

Factors of melanocyte that regulate skin pigmentation. The figure illustrates components that are involved in melanosomal transportation, enzymatic activates, motor activates, and transcriptional regulation.

The enzymatic components of melanosomes help melanosomes reach their ultimate stage of melanosomal maturation. Melanosomal enzymatic components, including TYR, tyrosinase-related protein-1 (TYRP1), and dopachrome tautomerase/tyrosinase-related protein-2 (DCT/TYRP2), play major roles in melanin synthesis (Figure 1). TYRP, a critical copper-dependent enzyme, catalyzes the conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), the rate-limiting step in melanin synthesis. Copper oxidization deactivates this enzyme but can be activated by electron donors, such as L-DOPA, ascorbic acid, superoxide anion, and nitric oxide (NO) [3, 20, 21]. Protein kinase C-β (PKC-β) phosphorylates two serine residues of the cytoplasmic domain and activates tyrosinase [22]. Mutations that inactivate this enzyme result in the most severe form of oculocutaneous albinism (OCAIa). TYRP1 and TYRP2 are also found in the membrane of melanosomes, and it is assumed that TYRP1 is involved in the activation and stabilization of tyrosinase, melanosome synthesis, increasing the eumelanin/pheomelanin ratio, and working against oxidative stress due to its peroxidase effect [3, 20].

2.2 Melanosome trafficking

Melanosomes move from the perinuclear area to the periphery of melanocytes, and melanocytes transfer packaged melanin into adjacent keratinocytes because of the function of microtubules, actin filaments, and myosin, resulting in skin pigmentation [13]. Early melanosomes originate in the perinuclear area and move toward the periphery of melanocytes (i.e., dendrites) by kinesin and dynein mediate microtubule-dependent intracellular transportation systems (Figure 1). During this period, they eventually mature and turn into late (pigmented) melanosomes [23, 24]. Kinesins ensure the centrifugal movement of melanosomes, and melanosomal cargo is transferred from microtubules to F-actin in dendrites. RAB27A, melanophilin (MLPH), and myosin-Va (MYO5A) induce complexes to connect melanosomes to F-actin-based motors. Mutations in any of these genes induce a noticeable accumulation of pigments in the perinuclear region of mutant melanocytes due to the disruption of their transport to the dendrites [25], resulting in various forms of Griscelli syndrome (types II, III, and I, respectively) in humans [26]. This is manifested by mouse Melan-a cells by RAB27A linking to synaptotagmin-like 2 (SYTL2), prompting SYTL2 to dock melanosomes at the plasma membrane, suggesting that SYTL2 plays a role as a regulator of melanosome exocytosis [27, 28, 29].

2.3 Melanogenic regulation in melanocytes

The most vital transcription factor that regulates melanocyte function is MITF, which controls the expression of the melanogenesis enzymes TYR, TYRP1, and TYRP2 (Figure 1) [30]. Mutations in MITF result in Waardenburg syndrome type 2 (WS2) [31]. The MITF promoter is regulated by various other transcription factors, including a paired box protein 3 (PAX3), sex-determining region Y-box 9 and 10 (SOX9 and SOX10), lymphoid enhancer-binding factor 1 (LEF-1), and cyclic adenosine monophosphate (cAMP) responsive element-binding protein (CREB), which is phosphorylated by signals via the melanocortin-1 receptor (MC1R) [13]. The roles of polymorphisms in MC1R have been thoroughly investigated in response to UV radiation and/or in controlling constitutive skin pigmentation among racial/ethnic groups [32]. Several physiological factors from fibroblasts, keratinocytes, and other sources also regulate the expression levels and functions of MITF [33].

3. Skin pigmentation is regulated by several factors of keratinocytes

Keratinocytes derived from dark skin, such as microphthalmia-associated transcription factors and tyrosinase, significantly stimulate the expression of melanocyte-specific proteins. It has been suggested that keratinocytes regulate skin pigmentation, at least in part, regardless of whether the melanocytes are derived from light or dark skin [34, 35].

3.1 Keratinocyte-derived factors regulate melanocytes

Currently, it has been postulated that Foxn1/Whn/Hfh11, a transcription factor expressed by keratinocytes, is a regulator of keratinocyte growth and differentiation, and is also involved in melanocyte recruitment and induction of pigmentation in the skin through basic fibroblast growth factor (bFGF) production [36]. In summary, Foxn1 recruits melanocytes to their desired position and induces melanosome transfer, acting as an activator of the pigment-recipient phenotype. Keratinocyte-derived factors that act as activators of melanocytes also include stem cell factor (SCF), hepatocyte growth factor (HGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), nerve growth factor (NGF), α-melanocyte-stimulating hormone (α-MSH), adrenocorticotropic hormone (ACTH), endorphin, endothelin-1 (ET-1), prostaglandin (PG)E2/PGF2a, and leukemia inhibitory factor (LIF) (Figure 2) [37]. However, whether any of these factors are regulated by Foxn1 is unclear.

Figure 2.

Factors of Keratinocyte that regulate skin pigmentation. Foxn1 and p53 are directly involved in the up-regulation of pigmentation through bFGF and POMC derivatives, receptively. The figure shows all the relevant factors that enhance melanocyte growth and function.

3.2 Melanosome transfer

Synapses between melanocytes and keratinocytes are believed to exist, such as in the neural system, wherein melanosome transfer occurs through these synapses via some unknown mechanisms. Protease (proteinase)-activated receptor-2 (PAR-2), a G-protein coupled receptor, which is expressed on keratinocytes, seems to be closely involved in melanosome transfer. PAR-2 has a crucial role in mediating the phagocytosis of melanosomes in a Rho-dependent manner and in determining skin color phenotype [38]. Recently, it has been shown that the keratinocyte growth factor receptor (KGFR) plays a role similar to PAR-2 [39]. Keratinocyte phagocytosis of latex beads is enhanced by KGFR activation, and the addition of KGF to co-cultures induces the transfer of tyrosinase-positive granules. Phagocytosis via KGFR is dependent on the PAR-2-Rho pathway, as well as on Rac and Cdc42 activation [39]. Apart from phagocytosis, PAR-2 stimulates melanocyte dendricity and contributes to skin pigmentation. Stimulated keratinocytes release prostaglandins, PGE2, and PGF2a, which bind to the surface of melanocytes, thereby inducing dendrite formation [40]. Several physiological factors regulate the expression of PAR-2 (38), such as dickkopf 1 (DKK1), an inhibitor of the Wnt/b-catenin pathway [41].

The process of melanosome transfer to keratinocytes is not completely understood at this time, although various assumptions have been proposed, including exocytosis, cytophagocytosis, fusion, and membrane vesicle transport [26].

4. Proposed mechanism of melanosomal transfer to keratinocytes

4.1 Exo/endocytosis

Regulated exocytosis is a multistage process in which the membranes of cytoplasmic organelles fuse with the plasma membrane in response to stimulation through which secretions are released from the vesicle to the cell exterior. Several types of regulated secretory exocytosis exist, including the exocytosis of synaptic vesicles and dense-core vesicles in the presynaptic compartment of neuronal cells, exocytosis of these vesicles (all over the plasma membrane) by neuroendocrine and endocrine cells, exocytosis of secretory granules at the apical plasma membrane of exocrine cells, and exocytosis by hematopoietic cells of various types of secretory organelles (Figure 3) [42].

Figure 3.

Exocytosis as a mode of melanosomal transfer to the keratinocyte. Melanosomal membrane fuses with the plasma membrane, thus release melanin. This released melanin is taken up by keratinocytes either endocytosis or phagocytosis.

According to the exocytosis theory, the melanosomal membrane fuses with the melanocyte plasma membrane, resulting in extracellular melanin, and the melanin that is released is phagocytosed by a neighboring keratinocyte. As a result, melanin is transferred from melanocytes to keratinocytes (Figure 3).

This hypothesis is based on observations of human skin and hair follicles using an electron microscope, depicting ranased is phagocytosed by a neighboring keratinocyte. As a result, melanin is trannin is tranratinocyte. As a resultclathrin-coated pits [43, 44].

Melanocytes release melanin in the extracellular space in vitro after being stimulated either with α-melanocyte-stimulating hormone or with the soluble domain of the β-amyloid precursor protein [45, 46]. The process is referred to as exocytosis because the released melanin is not surrounded by a membrane, as observed by electron microscopy, and melanocytes are known to express SNAREs and Rab GTPases molecules, which are involved in the regulation of exocytosis.

SNAREs contain three conserved families of membrane-associated proteins (synaptobrevin/VAMP, syntaxin, and SNAP25 families) that play a significant role in the later stages of membrane fusion. Several SNAREs have been identified in melanosome-enriched fractions, including SNAP23, SNAP25, VAMP2, syntaxin 4, and syntaxin 6 [47, 48]. SNAP25 and syntaxin on the plasma membrane bind VAMP to the vesicle membrane. Some immunoprecipitation experiments have reported a degree of interaction between VAMP2 and SNAP23, but not with syntaxin 4, to achieve fusion.

Rab GTPases are another family of proteins that act in the tethering and docking of membranes before fusion. RAB27A contributes to the regulated exocytosis of various types of organelles [27]. Synaptotagmin-like protein2-a (Slp2-a) is a concurrent RAB27A effector that has been found in melanocytes [27]. It links RAB27A with phosphatidylserine and facilitates the attachment of melanosomes to the plasma membrane, which is an essential step in exocytosis. Interestingly, Slp2-a is a putative regulator of exocytosis at the neuronal synapse and is structurally homologous to synaptotagmin. Coupled exo/endocytosis of melanin transfer from melanocytes to keratinocytes in the core of the melanosome, termed melanocore [49] and the small GTPase Rab11b mediates the final steps of melanocore exocytosis prior to the transfer to keratinocytes [49]. One study found that the depletion of Rab11b, but not RAB27A, causes a decrease in keratinocyte-induced melanin exocytosis [49]. The exocyst is an evolutionarily conserved protein complex comprising eight subunits, including Sec8, Sec15, and Exo70 [50]. This complex plays an essential role in various processes, including cell migration, vesicle tethering, membrane trafficking, ciliogenesis, autophagy, and cytokinesis. It is postulated that the exocyst is essential for melanocyte exocytosis and keratinocyte transfer [51]. Rab3, consisting of Rab3a-d, is also involved in exocytosis in several cell types [52, 53]. Rab3a is expressed in melanocytes, and its expression is downregulated by UV irradiation [47, 54].

Further support for the exocytosis hypothesis is based on findings regarding melanin in the keratinocyte cytoplasm, which are present in the same way it exogenously administered melanin by melanocytes [45, 55, 56, 57, 58]. The distribution pattern of melanin granules seems to be dependent on size. The phagocytosis of small or large latex beads also results in aggregates and singly dispersed beads, respectively. Melanin granules aggregate after being ingested as single granules, indicative of the final stage in the lifecycle of melanin in keratinocytes.

Finally, melanocytes are closely related to both neuronal and hematopoietic cells because of their neural crest origin and secretory lysosome family belonging, respectively [59]. Both synaptic vesicles and secretory lysosomes help regulate exocytosis upon stimulation, which suggests that melanin transfer occurs via similar mechanisms.

4.2 Cytophagocytosis

Phagocytosis is an essential process that maintains cellular homeostasis and is defined as the cellular engulfment of particles with a diameter of more than 0.5 mm. In the epidermis, the phagocytosis of melanosomes into keratinocytes is vital to protect their DNA against damage from ultraviolet B (UVB) radiation and is essential for triggering host defenses against invading pathogens, as well as in the elimination of damaged, senescent, and apoptotic cells in mammals. Phagocytes can be classified into two types—professional, such as macrophages, dendritic cells, and granulocytes and non-professional (or amateur), such as keratinocytes. Amateur phagocytes are slower, less mobile, and have a limited range of particles that they can take up [60]. Their phagocytic nature has been shown both in vitro [55] and in vivo [56].

The phagocytosis of a viable cell or an intact part of a viable cell is known as cytophagocytosis. The cytophagocytosis hypothesis of melanin transfer denotes the phagocytosis of intact melanocytic dendrite cells, known as the “dendrite tip.” First, the melanocytes extend their dendrites towards the surrounding keratinocytes to make contact. The keratinocytes respond with extensive membrane ruffling and engulf the dendrite tip using villus-like cytoplasmic projections. Next, the dendrite tip is squeezed and pinched off, thus forming a cytoplasmic poach filled with melanosomes. Then, a phagolysosome is formed by the fusion of lysosomes, and the degradation of the melanocyte membranes and cytoplasmic constituents occurs. Meanwhile, phagolysosomes are transported to the supranuclear region. Finally, the phagolysosome disintegrates into smaller vesicles containing a single melanin granule or aggregates of melanin granules, and are then dispersed over the cytoplasm (Figure 4) [61].

Figure 4.

Cytophagocytosis as a mode of melanosomal transfer to the keratinocyte. In this process, melanocytic dendrite is pinched off and phagocytosed, and resulting in the phagolysosome. Melanin granules are dispersed throughout the cytoplasm from this phagolysosome.

The hypothesis of the cytophagocytosis of melanosomes by keratinocytes is supported by evidence obtained using electron microscopy [43, 62, 63, 64]. Additionally, measuring the internalization of latex microsphere beads by keratinocyte phagocytosis has been shown to be activated by keratinocyte growth factor, which acts only on the recipient keratinocytes [39, 45]. The activation or inhibition of PAR-2 expressed by keratinocytes, but not by melanocytes [65], regulates melanosome transfer via keratinocyte phagocytosis [39, 66]. Light and electron microscopy showed that exogenously added melanosomes are taken up by normal human keratinocytes in a time-dependent manner, reflecting a possible melanosome transfer process in which melanosomes released into the extracellular space are phagocytosed by keratinocytes [67].

4.3 Fusion of plasma membranes as a mode of transfer

The melanocyte plasma membrane fuses with the keratinocyte plasma membrane, thus creating a pore or channel that connects the cytoplasm of both cells and through which melanosomes are transported (Figure 5).

Figure 5.

Fusion as a mode of melanosomal transfer to the keratinocyte. Melanosomes are passage through a channel. The plasma membrane of the melanocyte fuses with the plasma membrane of Keratinocyte to form this channel.

The fusion assumption of pigment transfer has been suggested in pigmented basal cell carcinoma and the skin of black guinea pig ears [68]. Recently, it has been suggested that filopodia extend from the dendrite tips and cell body of melanocytes and fuse with the neighboring keratinocyte membrane, allowing for the passage of melanosomes [69] towards the keratinocyte membrane. Although the melanosomal transfer was observed via these protrusions, proof of membrane fusion was not unambiguously obtained. The similar optical properties of melanocyte and keratinocyte membranes make it difficult to distinguish fusion via light microscopy. In contrast, thin projections were found directly connecting the melanocyte and keratinocyte cytoplasm in the electron micrographs of their co-cultures.

This mode of cell-cell communication transport could be considered as tunneling nanotubes [69], providing a network of various cultured cells and functioning as channels for organelle transport [70].

Filopodial fusion with neighboring cells forms a tubular structure composed of actin filaments, with a diameter of 50–200 nm, directly connecting the cytoplasm of cells. The tube allows for the unidirectional transport of organelles and plasma membrane molecules, as opposed to soluble cytoplasmic molecules.

Similarly, interconnecting channels have been observed between cytotoxic T lymphocytes or natural killer cells, antigen-presenting cells to B cells and their respective targets, allowing for membrane transfer [71, 72, 73]. Tunneling nanotubes are observed in several cell types, indicating that they can provide a general mode of intercellular communication. However, further research on the phenomena of intercellular communication is still needed.

4.4 Membrane vesicles as a mode of transfer

These pieces of the membrane have been reported to travel from cell to cell. Proteins and lipids destined for transfer are concentrated on the plasma membrane, resulting in the formation of an extracellular vesicle, which travels to distant cells (Figure 6).

Figure 6.

Membrane vesicles as a mode of melanosomal transfer to the keratinocyte. Melanosomal vesicles either fuse with the plasma membrane of keratinocytes or ingest through phagocytosis.

This mode of melanin transfer is usually not considered a mode of pigment transfer. However, two studies suggest that melanosome-containing membrane vesicles are phagocytosed by keratinocytes or fused with the keratinocyte plasma membrane as a model of melanin transfer. Flow cytometry analysis of a human melanoma cell line revealed that vesicles can be identified according to their size and fluorescent properties upon neoglycoprotein binding [74]. The addition of neoglycoproteins could partially inhibit vesicle adhesion to keratinocytes because of the participation of carbohydrates in this interaction. The vesicles are finally swallowed by keratinocytes, thereby delivering melanin.

Another study showed that melanin transfers melanophores to fibroblasts [75]. Double membrane-covered melanin is transferred to distinct groups of recipient cells, some of which are located at a distance from the melanophore, suggesting that melanophores release melanin by the shedding of vesicles, and are subsequently recognized by fibroblasts through specific interactions.

Microparticles or microvesicles shed by live cells are believed to be formed upon the induction of cell stress, including cell activation and apoptosis, indicating true vectors of information exchange between cells [76]. This ubiquitous mode of material transfer for the delivery of melanosomes should be considered as a potential model.

In short, it is difficult to draw conclusions because none of the hypotheses provide concrete evidence. Naturally, these mechanisms are not mutually exclusive. Within this context, phagocytosis seems to be a necessary step for all proposed mechanisms, except for the fusion of plasma membranes.

5. Translocation, distribution, and degradation of melanosomes by the keratinocyte

After being transferred into recipient keratinocytes, melanosomes are selectively and predominantly translocated to the apical pole of the keratinocyte. As a result, they protect the underlying nucleus from mutagenic damage by absorbing UV light. This trafficking is mediated by cytoskeletal elements and microtubule-associated motor proteins. Studies have reported that dynein co-localizes with phagocytosed melanosomal aggregates throughout the cytoplasm, predominantly at the microtubule-organizing center in keratinocytes [24].

The distribution of recipient melanosomes within the keratinocytes varies according to complexion coloration, as demonstrated over a quarter of a century ago [77, 78]. Melanosomes are maintained as individual organelles throughout the cytosol of keratinocytes in the dark. In light-skinned individuals, melanosomes are significantly smaller and aggregated into membrane-bound clusters of 4–8 organelles. Whether these distinct distribution patterns are determined by factors within the transferred melanosome or are innate to the recipient keratinocytes remains unclear.

A recent study showed that the distribution pattern of recipient melanosomes is dictated by the type of donor melanocyte. An in vitro skin reconstruction model was assessed using combinations of keratinocytes and melanocytes from different complexion colorations [79]. In contrast, the skin type from which the recipient keratinocyte was derived regulates the distribution pattern of transferred melanosomes regardless of their size, as illustrated in the melanocyte/keratinocyte co-culture experiment [34]. However, the experimental context of these approaches casts doubt on the results. Therefore, the mechanism underlying the regulation of the distribution of melanosomes in the skin remains to be fully elucidated.

Melanosomes undergo degradation by the time they differentiate into corneocytes. As observed in the interface between the stratum granulosum and the stratum corneum, few melanosome structures remain in corneocytes of very darkly pigmented skin 49, and no apparent melanosomes remain in the corneocytes of light skin. There is a need to identify the hydrolytic processes used by keratinocytes to degrade the dense melanosome/melanin. Hydrolytic enzymes have been found to be involved in melanosome degradation by keratinocytes [80].

6. Dermatological evidence of abnormal pigmentation due to abnormalities in keratinocytes

6.1 EBS with mottled pigmentation (EBS-MP)

Epidermolysis bullosa simplex (EBS) is an autosomal dominant inherited skin disease characterized by blistering. EBS with mottled pigmentation (EBS-MP) is a rare form of generalized EBS characterized by non-scarring blistering and small hyper- and hypopigmented spots that form a mottled to the reticulate pattern. Using electron microscopy, an increased number of melanosomes within basal keratinocytes, dermal macrophages, and Schwann cells has been reported in EBS-MP patients. EBS-MP mostly involves the distal extremities and progressive mottled hyperpigmentation. During the neonatal period, differentiating EBS-MP from other subtypes is challenging due to the fact that pigmentary changes usually start later in infancy or childhood [81]. In the literature, most cases of EBS-MP have been attributed to a heterozygous missense mutation of KRT5 that results in the substitution of proline 25 with leucine (p25L) in the nonhelical V1 domain of KRT5 [81]. A 25-year-old Japanese woman and her cousin and a Chinese family have been reported as rare cases of EBS-MP due to a 1649delG mutation in the KRT5 tail domain (V2) [82, 83]. Figure 7 shows a familial case of a 1-year-old girl and her father with EBS-MP, who developed mottled to reticulate pattern hyperpigmentation on the extremities, trunk, and face (Figure 7). Electron microscopy was used to evaluate a skin sample from the father (unpublished).

Figure 7.

Many brown macules scattered on arms, legs and face of a 1-year-old girl with EBS-MP.

6.2 Dowling-Degos disease (DDD)

DDD is characterized by progressive and disfiguring hyperpigmentation primarily in the flexural areas, which is a rare autosomal dominant genodermatosis with variable penetrance due to haploinsufficiency of the KRT5 gene [84, 85] on chromosome 12q [84]. KRT5/KRT14 is a crucial element of the basal keratinocyte cytoskeleton. KRT5 dysfunction alters organelle transportation and epidermal differentiation. DDD usually develops after puberty and is clinically distinguished by brownish hyperpigmented macules that alter a reticular pattern. These macules are mostly located in the skin flexures (sub-mammary, axillae, and groin), cervical region, trunk, and anterior surface of the thighs and upper arms. Pinpoint papules with keratin plugs that simulate comedones are also found in the palmar, axillary, cervical, perioral, and gluteal regions. Some studies have shown that DDD is associated with Hidradenitis suppurativa and can develop depressed perioral scars [84, 86, 87]. An interconnected hyperpigmented epidermal proliferation projection in the dermis denoted as the “antler-like” pattern, is characterized by a filiform pattern and is a key differentiating feature. In the case of DDD, hyperpigmented proliferation is derived from both the epidermis and the follicular wall. This feature makes DDD different from other keratinized disorders. At present, there have not been any successful medicinal therapeutic approaches reported in the literature.

6.3 Galli-Galli disease (GGD)

GGD is a rare autosomal dominant genodermatosis that is considered an acantholytic variant of DDD, characterized by hyperkeratotic papules and progressive reticular hyperpigmentation involving the neck, trunk, and proximal extremities. GGD is associated with autosomal dominant mutations in KRT5, POGLUT1, and POFUT1 genes, and is clinically characterized as red-to-brown hyperkeratotic papules that gradually develop into brown reticulate lentigo-like macules over the trunk, neck, flexor, and extensor surfaces of the extremities. Severe flares and more diffuse distribution of cutaneous participation may be observed in rare cases [88]. A successful medicinal therapeutic approach has yet to be reported in the literature.

7. Potential mechanism of keratin mutation affects melanin distribution

It has been reported that the KRT5 head domain is noticeably more stable than the KRT14 head, and its distribution is altered following the depolymerization of microtubules. Several studies have reported that the KRT5 head domain interacts with heat shock cognate 70 (HSC70) and is involved in organelle transport [8, 89] and chaperone-mediated autophagy. In this context, the KRT5 mutation may affect melanosome degradation by modulating the interaction between KRT5 and HSC70, resulting in an abnormal accumulation of melanin in the keratinocytes (Figure 8).

Figure 8.

Speculated mechanism of how keratin mutation affects melanin distribution.

8. Conclusion

The regulation of pigmentation in melanogenesis in melanocytes has been thoroughly investigated. However, the mechanism by which melanosomes are transferred into keratinocytes and the interaction among different molecules during this transfer process have yet to be well characterized. Recently, keratinocytes have become an interesting subject for pigmentary disorders. Surprisingly, little is known about the mechanism by which human melanosomes are transferred to keratinocytes and the degradation of melanosomes inside keratinocytes, and how genetic abnormalities in keratinocytes, but not in melanocytes, cause pigmentary skin disorders. This chapter covers the most important aspects of melanocytes and keratinocytes that induce pigmentation and summarizes the mechanism underlying the transfer of melanosomes to keratinocytes and the degradation of melanosomes inside keratinocytes. How genetic abnormalities in keratinocytes affect pigmentary skin disorders are also discussed in an attempt to shed light on hereditary pigmentary disorders and provide a conceptual framework for the role of keratinocytes in pigmentary disorders.

Acknowledgments

I would like to express my gratitude to my primary supervisor, Professor Mayumi Komine, and my Chairman, Professor Mamitaro Ohtsuki, who guided me throughout this work. I would also like to express my deep appreciation to my family and colleagues who helped me finalize my book chapter.

Conflicts of interest

The author declares no conflict of interest.

Funding

This research did not receive any external funding.

Abbreviations

ER	Endoplasmic reticulum
PMEL17	Melanocytes lineage-specific antigen gp100
MART-1	Melanoma antigen recognized by T cell-1
GPNMB	Glycoprotein nonmetastatic melanoma protein b
TYR	Tyrosinase
TYRP1	Tyrosinase-related protein-1
TYRP2	Tyrosinase-related protein-2
BLOCs	Biogenesis of lysosome-related organelles complexes
AP-1	Activator protein 1
MLPH	Melanophilin
MYO5A	Myosin-Va
RAB27A	Ras-related protein Rab27A
MITF	Microphthalmia-associated transcription factor
MIICs	MHC class II compartments
Hsc70	Heat shock cognate 70
NO	Nitric oxide
CREB	cAMP response element-binding protein
MC1R	Melanocortin-1 receptor
PAX3	Paired box protein 3
SOX9, SOX10	Sex-determining region Y-box 9 and 10
LEF-1	Lymphoid enhancer-binding factor 1
WS2	Waardenburg syndrome type 2
Foxn1	Forkhead box protein N1
bFGF	Fibroblast growth factor
HGF	Hepatocyte growth factor
NGF	Nerve growth factor
SCF	Stem cell factor
LIF	Leukemia inhibitory factor
KGFR	Keratinocyte growth factor receptor
PAR-2	Protease (proteinase)-activated receptor-2
DKK1	Dickkopf 1
UV	Ultraviolet
NFR	Tumor necrosis factor receptor
EGFR	Epidermal growth factor receptor
PGE2	Prostaglandin E2
PGF2α	Prostaglandin F2α
GM-CSF	Granulocyte-macrophage colony stimulating factor
α-MSH	α-Melanocyte-stimulating hormone
ACTH	Adrenocorticotropic hormone
SNAP	Soluble NSF attachment proteins
VAMP2	Vesicle-associated membrane protein 2
cAMP	Cyclic adenosine monophosphate
KRT	Keratinocyte
POGLUT1	Protein O-Glucosyltransferase 1
POFUT1	Protein O-Fucosyltransferase 1