ROS and calcium signaling are critical determinant of skin pigmentation

Abstract

Pigmentation is a protective phenomenon that shields skin cells from UV-induced DNA damage. Perturbations in pigmentation pathways predispose to skin cancers and lead to pigmentary disorders. These ailments impart psychological trauma and severely affect the patients’ quality of life. Emerging literature suggests that reactive oxygen species (ROS) and calcium (Ca²⁺) signaling modules regulate physiological pigmentation. Further, pigmentary disorders are associated with dysregulated ROS homeostasis and changes in Ca²⁺ dynamics. Here, we systemically review the literature that demonstrates key role of ROS and Ca²⁺ signaling in pigmentation and pigmentary disorders. Further, we discuss recent studies, which have revealed that organelle-specific Ca²⁺ transport mechanisms are critical determinant of pigmentation. Importantly, we deliberate upon the possibility of clinical management of pigmentary disorders by therapeutically targeting ROS generation and cellular Ca²⁺ handling toolkit. Finally, we highlight the key outstanding questions in the field that demand critical and timely attention. Although an important role of ROS and Ca²⁺ signaling in regulating skin pigmentation has emerged, the underlying molecular mechanisms remain poorly understood. In future, it would be vital to investigate in detail the signaling cascades that connect perturbed ROS homeostasis and Ca²⁺ signaling to human pigmentary disorders.

1. Introduction

Skin is the largest organ of the human body that plays multifaceted roles in photoprotection, thermoregulation, sensory perception and inflammatory response against invading pathogens [1,2]. Skin pigmentation is a complex physiological phenomenon that protects from the UV-induced DNA damage, thus reducing the risk of skin cancers and various pigmentary disorders [3]. The diverse phenotypes of human skin are attributed to the melanin pigment synthesized by specialized dendritic cells called melanocytes. Melanocytes originate from the neuroectoderm but migrate to the epidermal and follicular region during embryonic development [4]. Upon migration, these specialized cells synthesize two types of melanin pigments: brownish-black eumelanin and reddish-yellow pheomelanin. The ratio of these two pigments is the key determinant of human skin color [5]. Eventually, melanin is transferred to keratinocytes thereby giving pigmentation phenotype. Melanin acts as a natural sunscreen by absorbing the harmful UV radiation and thus blocking it from penetrating into the skin. Melanin also serves as a powerful antioxidant and effectively neutralizes free radicals [6].

Emerging literature highlights the critical role of reactive oxygen species (ROS) and calcium (Ca²⁺) signaling modules in regulating pigmentation. In this review article, we will briefly discuss the physiological pigmentation process starting from melanogenesis within melanocytes to eventual transfer of mature melanosomes to keratinocytes. Then we will deliberate on literature implicating ROS and Ca²⁺ signaling pathways as key determinants of pigmentation. Further, we will debate on the possibilities of targeting ROS and Ca²⁺ signaling cascades for the clinical management of the pigmentary disorders. Importantly, we will review the key outstanding questions in the field that demand further investigation.

2. Physiological pigmentation

2.1. Melanogenesis

The two melanin (eumelanin and pheomelanin) types have a very stark difference in their biochemical structures. Pheomelanin is comprised of sulfur-containing benzothiazine and benzothiazole units, whereas eumelanin is a heterogenous polymer made up of 5,6-dihydroxyindole (DHI) and/or 5,6-dihydroxyindole-2-carboxylic acid (DHICA) [6]. Although the two pigmentary types are derived from the same precursor-dopaquinone, the biosynthesis of pheomelanin and eumelanin further diverges in subsequent steps. Pheomelanin formation does not require any further catalytic actions but only cysteine to generate the benzothiazine and benzothiazole units. Eumelanin synthesis, on the other hand, requires two additional tyrosine-related proteins, TRP1 and TRP2 (Dopachrome tautomerase [DCT]) to further produce 5,6-dihydroxyindole (DHI) and/or 5,6-dihydroxyindole-2-car-boxylic acid (DHICA) units [7]. The functional properties of the eumelanin and pheomelanin differ. The beneficial effects of melanin are primarily due to eumelanin which absorbs 50–75 % of UVR and scavenges the free radicals generated by UVR, thus blocking UVR penetration into the skin [6]. Pheomelanin, on the other hand, is photodegradable and contributes to the deleterious effects of UVR by depleting the scavengers of ROS and thus leading to the accumulation of free radicals and mutations in the melanocytes and eventually inducing tumorigenesis [3,6].

2.2. Tyrosine uptake and conversion

The process of melanogenesis begins with the acquisition of a non-essential amino acid, tyrosine, by the melanocytes. Solute carrier protein SLC7A5 or l-type amino acid transporter 1 (LAT-1) serves as a transporter of tyrosine into the melanocytes [8]. Melanocytes can also produce their own tyrosine using phenylalanine hydroxylase (PAH), which catalyzes the hydroxylation of phenylalanine’s aromatic side chain to produce tyrosine [9]. Once the tyrosine is acquired by the melanocyte, it is converted to dopaquinone (DQ), which serves as a substrate for melanin synthesis. This DQ conversion is a rate-limiting step of melanin synthesis and is catalyzed by a copper-containing enzyme, tyrosinase, which is synthesized exclusively in melanocytes [10]. Following synthesis, there are post-translational modifications in the tyrosinase enzyme, which help in its maturation and trafficking to Golgi. Subsequent processing of tyrosinase in the Golgi is essential for its localization to the melanosomes [11]. After processing, the tyrosinase is transported to melanosomes where it oxidizes the tyrosine to DQ. The catalytic activity of tyrosinase is attributed to the two copper ions, surrounded by the three histidine residues in its active site [10]. This copper addition is first introduced in the Golgi by ATP7A (copper--transporting ATPase1) but since this addition is transient, it is again introduced in the melanosomes [11].

2.3. Melanosome maturation and assembly

Melanosomes are unique lysosome-related organelles that are specialized to synthesize melanin within the melanocytes. Melanosomes undergo a series of four morphologically different stages to mature and synthesize melanin. Stage I melanosomes resemble early endosomal compartments and begin the assembly of proteinaceous fibrils within the intraluminal vesicles (ILVs). These fibrillar structures are composed of melanocyte-specific pre-melanosomal protein, PMEL17 (or Gp100) and are responsible for the elliptical shape of melanosomes. These structures serve as a scaffold for melanin synthesis. This assembly is completed by the end of stage II. Stage III involves the translocation of melanogenic enzymes - TYR, TRP1 and TRP2 along with the ion transporters, from the trans-Golgi network to the melanosomal membrane. This stage marks the initiation of melanogenesis and the deposition of synthesized melanin on the fibrillar matrix. By the end of stage IV, melanosomes can be seen as densely covered with melanin granules thus remarking a fully matured melanosome [11–13].

2.4. Melanin biosynthesis

Once inside the melanosomes, l-tyrosine is oxidized by TYR to l-dihydroxyphenylalanine (L-DOPA) and further to l-dopaquinone (DQ). Dopaquinone serves as a common precursor of eumelanin and pheomelanin. The formation of either of them depends on the availability of cysteine. When cysteine availability is low, the production of brownish-black eumelanin is favored. In the eumelanin synthesis, DQ is spontaneously cyclized and oxidized to form DOPAchrome (DC). DC formation proceeds to either DHI or DHICA. The presence of DCT/TYRP2 leads to the synthesis of DHICA whereas the absence of DCT/TRP2 leads to spontaneous decarboxylation of DC to form DHI. Further, both DHI and DHICA oxidize and polymerize to produce the eumelanin. At higher concentrations of cysteine, there is a predominant synthesis of reddish-yellow pheomelanin. Following the import of cystine into the melanocytes via SLCA11 transporter (or xCT) and subsequent reduction to cysteine. MFDS12 transports the cysteine into the melanosomes where it is incorporated into the DQ to form 5-S-cysteine DOPA (5SCD) and 2-S-cysteine DOPA (2SCD). These cysteinyl-DOPA isomers are further oxidized to form cysteinyl DQ. Cysteinyl DQ undergoes cyclization and rearrangement to form benzothiazine and benzothiazole intermediates, which are the monomer subunits of pheomelanin [11,14].

2.5. Transfer to keratinocytes

Once the melanosomes have completely matured within the melanocytes, they are transferred to the neighboring keratinocytes. A ratio of 1:36 (approximately) melanocytes to keratinocytes usually comprises an epidermal melanin unit [15]. The molecular mechanisms involved in the melanin transfer have been a controversial topic since the very first observation of melanin transfer to keratinocytes [16]. Several models have been proposed to explain this transfer process: here, we have discussed four main hypotheses. The first model of cyto-phagocytosis suggests that when the melanocyte extends its dendrites towards the keratinocyte, the keratinocyte pinches off the dendritic tip of the melanocyte and phagocytose it. Once internalized, this newly formed phagosome fuses with the lysosome which leads to the degradation of internal membrane of phagosome and thus dispersion of melanin in the cytoplasm of keratinocytes [15,17]. Second one is the tunneling nanotube model, which proposes that there is a direct fusion of melanocytes with the keratinocytes. This connects both cell types to facilitate the transfer of intact melanin pigments from the melanocyte to the keratinocyte [15,18]. A third model, the shedding-vesicle model, proposes that melanosomes are packaged into small vesicles which are released into the surrounding environment where they are engulfed by the keratinocytes [18,19]. Another model, the exocytosis-endocytosis model postulates that upon stimulation by keratinocytes, melanosomes fuse with the melanocyte membrane, which leads to the release of melanosomes into the extracellular milieu and subsequent uptake by the surrounding keratinocytes [18,20]. This interaction between melanocyte and keratinocyte triggers a Ca²⁺ signaling cascade which drives the melanin transfer and internalization by the keratinocyte machinery [21]. The melanin is further processed, leading to its polarization in the supranuclear region. The polarized melanin forms a supranuclear cap that protects the genetic material inside the keratinocyte nucleus from degradative effects of UV exposure. This localization of melanin is carried out by a dynein-dynactin motor complex. However, the precise molecular mechanism behind this polarization is still unknown [14,15].

2.6. Regulation of melanogenesis

In humans, pigmentation is a tightly regulated process involving more than 150 genes and several signaling cascades that regulate the key melanogenic players both transcriptionally and post-transcriptionally [22]. Moreover, there are other factors such as UV exposure and hormonal fluctuations, that can modulate skin pigmentation. Environmental factor such as UV exposure is a potent regulator of melanogenesis process. It induces DNA damage in the keratinocytes present in the epidermal layer of the skin. This leads to p53 activation which further causes stimulates the pro-opiomelanocortin (POMC) gene. POMC is post-translationally processed to produce α-melanocyte stimulating hormone (α-MSH). Upon secretion, α-MSH binds to its receptor melanocortin 1 receptor (MC1R) present on the melanocytes. This binding triggers cAMP production and therefore activation of PI3K/AKT pathway. Upon phosphorylation by protein kinase A (PKA), CREB mediates microphthalmia-associated transcription factor (MITF) activation [23]. MITF is one of the crucial players of the pigmentation process. It regulates cellular differentiation of melanocytes as well as the transcription of three major pigmentation enzymes, namely, DCT, TYRP1 and TYR and some melanosomal structural proteins like PMEL17 and MART-1 [24].

The production of cAMP by α-MSH binding on the melanocytes involves a crosstalk between ER Ca²⁺ and cAMP. Our group has shown that α-MSH treatment results in IP₃ generation, followed by release of Ca²⁺ from ER stores and this activates the ER Ca²⁺ sensor, Stromal Interaction Molecule 1 (STIM1). Upon activation, STIM1 oligomerizes and moves towards ER-plasma membrane junctions to potentiate adenylyl cyclase 6 (ADCY6). The ADCY6 activation further leads to the cAMP generation and subsequently MITF-mediated transcription of melanogenic genes. Thus, STIM1 establishes a positive feedback loop connecting the Ca²⁺and cAMP signaling pathways [25,26]. It is important to highlight that in addition to α-MSH, UV exposure also triggers keratinocytes to release endothelin-1 (ET-1), which then binds to the ET-B receptors on the neighboring melanocytes, triggering IP₃ formation and ER Ca²⁺ stores depletion through ER membrane localized-IP₃Rs [27]. This IP₃-mediated Ca²⁺ store depletion further activates STIM1/-STIM2 and consequently increases intracellular Ca²⁺ via the activation of Orai1 channels. This in turn enhances the melanogenesis. Thus, both cAMP and IP₃ production via STIM-Orai axis ensures enhanced mela-nogenesis upon UV exposure [25–27].

Since MITF is a central player of the melanogenesis process, its expression and activity are kept under a constant check by several transcription factors such as cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB), paired box family of transcription factor 3 (PAX3), sex-determining region box 9 and 10 (SOX9 and SOX10) and Wnt/β-catenin pathway effector lymphoid enhancer-binding factor 1 (LEF1). Post-transcriptional levels of MITF are regulated by phosphorylation through several kinases such as MAPK, GSK3β, p38 and ribosomal S6 kinase (RSK) [22]. MC1R is highly polymorphic and is responsible for the pigmentation differences among various human populations as well as differences in the responses to UV exposures. MC1R variants in individuals with red hair and light skin are associated with reduced function, eumelanin production and thus increased risk of skin cancer [28]. In addition to the MC1R receptor, there are other melanocyte receptors which are associated with cAMP production and CREB-mediated MITF transcription. Some of them are estrogen receptors, muscarinic receptors and catecholamines [29]. It has been reported that higher estrogen levels during pregnancy lead to hyperpigmentation [29]. In addition to these regulators, Ca²⁺ and reactive oxygen species (ROS) are important regulators of melanogenesis production.

Fig. 1: Schematic illustration of physiological pigmentation process.

Fig. 1. Schematic illustration of physiological pigmentation process.

After UV exposure, Keratinocytes release two key hormones – α-MSH and Endothelin-1 (ET-1). α-MSH binds to MC1R, whereas ET-1 binds to ET-BR on the neighbouring melanocytes, triggering IP₃-mediated ER Ca²⁺ store depletion and STIM1 activation, which in turn, activate adenylyl cyclase (ADCY6) to generate cAMP. The resulting cAMP/PKA/CREB pathway stimulates MITF-driven melanogenesis. Melanin production occurs in specialized lysosome-related organelles – melanosomes which progress through a series of four morphologically distinct stages. The melanin production begins at stage III of melanosomes with the acquisition of tyrosine or its conversion from phenylalanine, thus leading to the production of either brownish-black eumelanin or reddish-yellow pheomelanin depending on the availability of cystine. The detailed biochemical pathway of melanin formation is shown in the figure. The process is completed by the end of stage IV and, thereby, the mature melanosomes are transferred to the surrounding keratinocytes.

3. Role of ROS in pigmentation

Reactive oxygen species (ROS) are a group of non-radical oxygen derivatives and chemically reactive oxygen radicals [30]. Inside the cell, the production of ROS occurs via both enzymatic and non-enzymatic reactions as a byproduct of normal cellular metabolism occurring in oxidative conditions, specifically in mitochondrial respiratory processes [31]. Mitochondria is the primary source of ROS in most mammalian cells and the most prominent ROS being superoxide (O²⁻) [32,33]. ROS are also produced in several cellular processes like cell growth, differentiation, and apoptosis [34]. The concentration of ROS in cells dictates its effect on cellular events. Low levels of ROS promote DNA mutations, moderate levels cause cell senescence, and high ROS levels induce apoptosis and necroptosis [35]. Since ROS are highly reactive molecules causing reduction reactions of nucleic acids, lipids, and proteins, maintaining ROS levels (redox homeostasis) is crucial for normal cellular physiology [36,37]. Cells have developed various antioxidant systems to detoxify ROS prior to its effect on cellular compartments [38] Nevertheless, if there is an imbalance in ROS production and the antioxidant system’s ability to eliminate ROS, cells undergo oxidative stress, leading to different pathological conditions [39]. These pathological conditions in skin result in pigmentary defects, melanoma, and other skin disorders [40].

3.1. Sources of oxidative stress in melanocytes

3.1.1. Exogenous sources

The skin, being the largest organ in the human body, serves as the primary barrier against the external environment. Due to this, skin cells, especially epidermal melanocytes, are susceptible to oxidative stress caused via exposure to various environmental factors like solar ultra-violet (UV) rays, environmental pollutants, and particulate matter [41]. The main triggers for ROS production in the skin are the UV rays as the action spectrum for ROS production primarily falls within the UVA/UVB range (320–420 nm) [42]. UV irradiation-induced ROS production starts with the absorption of photons by cellular photosensitive proteins like cytochromes, heme, porphyrin, and riboflavin. These excited proteins react with oxygen to form ROS like O²⁻, which is converted to H₂O₂ and OH. [43]. Moreover, Dumbaya et al. showed that physiological doses of UVA induce two sources of ROS which are spatiotemporally distinct, one which is upstream of G-protein activation and another in the mitochondria (ROS_mito) leading to melanin synthesis [44]. Besides UV rays, the visible light spectrum can generate ROS like singlet oxygen and hydroxyl radicals in the skin [45]. Infrared radiation has also been reported to initiate ROS production, especially in the mitochondria [45].

Pollutants from the environment, like particulate matter from combustion of fuel consists of polycyclic aromatic hydrocarbons (PAH). PAH are highly photoreactive and cause oxidative stress in collaboration with UV rays [46]. Fuel combustion also produces nitrogen oxides like NO and NO₂, which provoke oxidative stress on the skin’s surface, together with ROS produced by particulate matter [46]. Environmental pollutants and UV rays react to form ozone (O₃). Even if O₃ cannot penetrate the skin, the outermost skin layers produce lipid peroxidation products which induces oxidative stress. O₃ also lessens antioxidant levels in the skin, like vitamin C and E, and can react with sebum on the outermost layer of the epidermis, i.e., stratum corneum, producing reactive carbonyls and aldehydes [47]. Cutaneous microbiota present on the skin is majorly affected by exposure to UV and pollutants via UV-induced immunosuppression and inflammation, respectively [48]. Alterations in the cutaneous microbiota is reported in several dermatological conditions and skin aging [49]. As described above, the role of UV rays and pollutants in oxidative stress production in the skin is well known, but whether skin microbiota plays a role in oxidative stress remains to be explored. Associations between gut dysbiosis and various skin conditions like psoriasis, acne vulgaris, and atopic dermatitis were recently reported [50] Interestingly, a study suggested that the interrelation between gut dysbiosis and skin could be due to inflammatory responses in the circulation system [51]. Earlier studies have shown a positive correlation between the severity of these skin conditions and oxidative stress levels, implicating that ROS and elevated oxidative stress in the skin enhance dermal inflammation [52]. Overall, cutaneous microbiota could regulate oxidative stress in the skin, and microbiota dysbiosis via UV and environmental pollutants triggers skin conditions by ROS overproduction and inflammation.

3.1.2. Endogenous sources

Synthesis of melanin occurs via oxidation reactions resulting in ROS (like O2− and H2O2) generation, leading to increased ROS levels in melanocytes. Restraining melanin synthesis in melanosomes prevents oxidative damage to other cellular compartments [53]. Melanin synthesis involves a chain of oxidative reactions, which are as follows:Oxidation of l-tyrosine to l-3,4-dihydroxy-phenylalanine (L-DOPA) and then to dopaquinone. This reaction is catalyzed by tyrosinase (rate--limiting enzyme), which results in O²⁻ generation. Dopaquinone undergoes a redox reaction to form dopachrome, which is further converted to either 5,6-dihydroxyindole (5,6-DHI) or 5,6 dihydroxyindole-2-carboxylic acid (5,6-DHICA). 5,6-DHI is oxidized to indole quinone by tyrosinase, forming H₂O₂ as a byproduct, while 5, 6-DHICA is converted to its corresponding quinone. Finally, the reactive quinones are polymerized to form black/brown eumelanin. Dopaquinone is also converted to cysteinyl-DOPA and pheomelanin, which has a higher sulphur-to-quinone ratio than eumelanin [54].

Apart from melanogenesis, contribution to the ROS pool is made by mitochondria, NADPH oxidases (NOX), cytochrome p450, endoplasmic reticulum, and peroxidases. In mitochondria, the electron transport chain majorly produces O²⁻specifically, the NADPH-ubiquinone oxidoreductase complex (complex I) [32]. Further, glycerol-3-phosphate dehydrogenase, pyruvate dehydrogenase, 2-oxo-glutarate, and flavoprotein Q oxidoreductase also contribute to mtROS levels [55,56]. NOX is an important ROS source, producing O²⁻or H₂O₂ when NADPH binds to the dehydrogenase domain, transferring electrons to the FAD cofactor and sequentially to the final electron acceptor, oxygen [57,58]. We have demonstrated that knockdown of MFN-2 (Mitofusin-2), involved in mediating mitochondria and melanosome crosstalk, leads to an increase in mitochondrial ROS levels and thereby increase in melanogenesis. Further, quenching ROS by antioxidant N-acetyl cysteine (NAC) rescues MFN-2 knockdown mediated melanogenic increase [59]. Cytochrome p450 is a monooxygenase enzyme involved in metabolizing drugs and dietary chemicals that produce ROS via monooxygenation reactions. The endoplasmic reticulum also participates in ROS production via ER oxirreductin-1 (ERO-1), as optimal ROS levels are critical for disulfide bond formation during protein folding [60]. Along with this, Ca²⁺ release by ER initiates the production of ROS by hyperactivating NADPH oxidase in the mitochondria [61].

3.1.3. Antioxidant defenses in melanocytes

Melanocytes maintain ROS levels through complex antioxidant systems, paracrine regulation, and other signaling networks to prevent cellular oxidative cytotoxicity. Antioxidant systems comprise enzymatic antioxidants like superoxide dismutase, glutathione peroxidase, thioredoxin, and catalase as well as non-enzymatic antioxidants like ascorbic acid (Vitamin C), tocopherol (Vitamin E), carotenoids, coenzyme Q10, ubiquinol, uric acid, sulfhydryl, and glutathione [62–64]. In the process of melanogenesis, tyrosinase-related protein 2 (TRP2), which synthesizes 5,6-DHI, increases glutathione levels to decrease oxidative damage [65].

The normal functioning of melanocytes is sustained mainly by paracrine signals consisting of cytokines and growth factors from surrounding keratinocytes and dermal fibroblasts [66]. One of the cytokines, endothelin-1, is a potent melanogenic factor that reduces H₂O₂ and promotes the repair of UV-induced DNA damage [67]. α-MSH (α-Melanocyte stimulating hormone), which is synthesized in keratinocytes, rapidly reduces H₂O₂ by increasing the expression and activity of catalase enzyme and reduces H₂O₂-induced DNA damage [68]. α-MSH binds to and activates the melanocortin 1 receptor (MC1R) which upregulates expression of antioxidant genes like ferritin, peroxiredoxin-1 and heme oxygenase-1 [69]. α-MSH also activates transcription factors like Nrf-2 and induces y-gluta-mylcysteine-synthetase and glutathione S-transferase expression [53, 70]

3.4.1. Crosstalk between oxidative stress signaling pathways and melanogenesis

3.1.4.1. ROS/RNS associated signaling

H₂O₂ can directly activate melanogenic proteins like MITF, tyrosinase, and epidermal phenyl hydroxylase (PAH), which converts l-phenylalanine to l-tyrosine [71]. Hence, H₂O₂ promotes melanogenesis by increasing l-tyrosine levels, which is the initial substrate in melanin synthesis.

Nitrogen oxide radical (NO) plays a critical role in UV-induced hyperpigmentation. A study showed that exogenous NO or cGMP analog mimics the effect of UV-stimulated melanogenesis while cGMP kinase inhibitors inhibit melanogenesis [72]. The same group later demonstrated that UV-irradiated keratinocytes release nitric oxide (NO), which promotes melanogenesis in co-cultured melanocytes [44]. NO also enhances MC1R expression and stimulates melanin synthesis via α-MSH pathway [45]. In vivo studies on guinea pigs have shown that applying l-NAME (a NOS antagonist) topically reduces pigmentation levels following UVB exposure [46]. At the molecular level, NO is linked to guanylate cyclase and cGMP-dependent protein kinase (PKG), which further activates tyrosinase [43], though the precise mechanism of NO-induced pigmentation is still unclear.

Apart from melanogenesis, ROS/RNS can also influence the melanin oxidation/polymerization process and thereby pigmentation. UV exposure leads to an immediate tanning response and persistent pigment-darkening reaction, in which the pre-existing melanin is oxidized and polymerized [73]. Importantly, ROS is involved in these darkening reactions. Studies have also reported that the oxidation of melanin-related metabolites under oxidative stress leads to hyperpigmentation [74]. NO reacts with DHICA in the presence of oxygen, leading to the deposition of melanin-like pigments [75].

3.1.4.2. Nrf-2-ARE pathway

Physiologically, the Nrf-2 (nuclear erythroid 2-related factor 2) - ARE (antioxidant response element) pathway plays a crucial role in antioxidant defenses by initiating gene expression of cellular antioxidants and protecting mitochondria [76,77]. Nrf-2 is activated by MC1R and mtROS [78]. Interestingly, Nrf-2 activates the PI3K/Akt signaling pathway, thereby inducing phosphorylation-mediated degradation of MITF and inhibition of melanogenesis [79]. Moreover, treatment with Nrf-2 agonists like tert-butylhydroquinone, sulforaphane, and curcumin induced depigmentation in black guinea pigs [80]. Nonetheless, the Nrf-2-Akt axis remains to be explored in detail, and whether intracellular ROS plays a role in regulating Akt signaling remains to be examined.

3.1.4.3. Wnt signaling pathway

Wnt signaling and oxidative stress reciprocate to each other. High ROS levels dissociate nucleoredoxin (NXN) and disheveled (DVL) interactions, causing DVL to associate with frizzled (FZD). This interaction induces AXIN2 (Axis inhibitor protein 2) gene expression and nuclear translocation of β-catenin, activating Wnt signaling [81]. Kim et.al showed that knockdown of WIF-1 (Wnt inhibitory factor-1) reduced phosphorylation of glycogen synthase kinase-3b (GSK-3b), β-catenin and NFATc2 (nuclear factor of activated T cells cytoplasmic-2) and increased expression of microphthalmia-associated transcription factor (MITF) [82]. Expression of Cadherin-11 in keratinocytes and fibroblasts enhances melanogenesis through canonical Wnt and Akt pathways in cocultured melanocytes [83]. In hyperpigmentation disorders, Wnt expression and the β-catenin pathway are highly activated, promoting melanin synthesis. Hence, inhibitors of Wnt signaling like cardamonin (degrades intracellular β-catenin) and ICG-001 are being considered as potential treatment options for hyperpigmentation disorders [84,85]. In summary, high levels of ROS can activate Wnt signaling, further leading to increased melanin production. But the molecular mechanism of Wnt signaling activation by ROS is still poorly understood. Likewise, whether dysregulated Wnt signaling module in turn contributes to oxidative stress remains to be investigated.

3.1.4.4. DNA damage-associated signaling

UV exposure to skin cells causes structural DNA damage in both direct and indirect ways. UVB is directly absorbed by DNA, leading to ionization and the formation of cyclobutane pyrimidine dimers (CPDs), while UVA indirectly damages DNA through the production of reactive oxygen species (ROS), causing single-strand breaks and DNA-protein crosslinks [57]. Several studies have demonstrated that UV-induced DNA damage triggers melanogenesis following UV exposure [58]. Additionally, hyperpigmentation has been observed in disorders related to nucleotide excision repair, with affected individuals typically being sensitive to sunlight [59]. Treatment of melanocytes with DNA-damaging agents such as methyl methanesulfonate or 4-nitroquinoline 1-oxide (4-NQO) increased melanin production by 70 % and elevated tyrosinase mRNA levels [60]. Production of small single-strand DNA fragments like thymidine dinucleotides (pTpT) also leads to increase in melanin content and tyrosinase levels. Notably, topical application of pTpT on guinea pig skin induces visible tan [86]. Hence, DNA damage and melanogenesis are closely interlinked, but the exact mechanism of altered gene expression by DNA damage promoting melanin synthesis needs to be elucidated.

3.1.4.5. p53 nexus

The PI3K-Akt pathway, Wnt signaling module, and DNA repair are intertwined via p53. Hence, as expected, studies have shown an association between p53 and hyperpigmentation. p53 directly regulates melanogenesis by increasing tyrosinase and propiomelanocortin expression in human melanocytes [87]. Casein kinase 1α (CK1α) negatively regulates Wnt signaling, and knockout of CK1α in keratinocytes activates p53 and Wnt signaling. This results in increased eumelanin levels and the number of epidermal melanocytes. The double-knockout of CK1α and p53 did not show epidermal hyperpig-mentation, suggesting that hyperpigmentation is p53-dependent [88]. Expression of p53 and its phosphorylation is increased in epidermal cells of hyperpigmented spots along with the expression of p53 transcriptional targets and melanogenic cytokines [89]. However, there were no significant changes in the levels of p53 between facial melasma and surrounding normal cells [90]. Therefore, activation of p53 seems to be fine-tuned in different pigmentary disorders to augment melanin synthesis. However, the interplay of oxidative stress and p53 in pigmentation warrants further investigation.

3.1.4.6. Autophagy

Physiologically, autophagy plays a crucial role in maintaining redox homeostasis. ROS activates autophagy, which degrades oxidized macromolecules and organelles [91]. Autophagy also protects melanocytes from oxidative stress-induced apoptosis [92]. Activation of autophagy is facilitated by the Nrf-2 signaling pathway. Under oxidative stress, degradation of Nrf-2 is blocked, causing Nrf-2-ARE pathway activation and induction of autophagy. The major role of autophagy in pigmentation is the degradation of melanosomes, i. e., melanophagy [93]. It has been demonstrated that Caucasian skin has higher autophagic activity compared to African American skin. Similarly, melanin levels were significantly decreased by autophagy activators, while the melanin levels increased upon inhibition of autophagy [94]. Hence, induction of autophagy can potentially help in treating hyperpigmentary disorders. Indeed, autophagy inducer PTPD-12 increases autophagic flux and promotes melanosome degradation without altering MITF levels [95]. Additionally, exposure to 585 nm light-emitting diodes decreased melanin levels in human skin by inducing melanophagy, accumulation of autophagosome, and LC3-II in melanocytes [96]. In senile lentigo, hyperpigmented lesions showed lower autophagic flux and premature skin aging. While activating autophagy in ex vivo lesioned skin reduced melanin content [97]. Hence, autophagy could be a potential target for the treatment of pigmentary disorders. Please refer to Fig. 1 for the diagrammatic summary highlighting role of ROS in pigmentation.

Fig. 2: ROS mediated regulation of skin pigmentation.

Fig. 2. Schematic representation of the regulation of melanogenesis by ROS.

Various external and internal sources of ROS contribute to oxidative stress in melanocytes. Subsequently, ROS triggers melanogenesis by activating Nrf-2-ARE pathway, Wnt signaling and p53 axis. Conversely, ROS can decrease pigmentation by inducing autophagy. Enzymatic and non-enzymatic antioxidant systems are activated in melanocytes to keep ROS levels under control. Further, paracrine signals from surrounding keratinocytes like Endothelin-1 and α-MSH also help in preventing oxidative stress in melanocytes.

4. Melanogenesis regulation by Ca^2þsignaling pathways

Ca²⁺ is an essential secondary messenger that rheostats various physiological processes like cell proliferation, apoptosis, autophagy, exocytosis etc. [98]. Downstream of Ca²⁺signals, there are specific Ca²⁺ responder proteins, channels and transporters that work in synergy to form a versatile, adaptable and robust signaling framework. Perturbations in the Ca²⁺ homeostasis are linked with several cell anomalies (ER stress, redox imbalance, mitochondrial dysfunction, cell death to name a few) as well as disorders like cancer, diabetes, neurodegenerative diseases and cardiac abnormalities [99–102].

Beyond the diverse roles of Ca²⁺in physiology and pathophysiology, is its emerging role in the regulation of skin pigmentation biology or melanogenesis. Recent studies focusing on the regulation of the skin pigmentation process are delving into organellar contribution and the role of organellar Ca²⁺ signaling cascades towards the same. A vast multitude of Ca²⁺ handling proteins, channels and transporters localized to the plasma membrane, endoplasmic reticulum (ER), mitochondria, melanosomes, lysosomes have been shown to connect Ca²⁺ signaling to melanogenesis that have been described below.

4.1. Plasma membrane Ca²⁺channels

Human skin is constantly exposed to the carcinogenic ultraviolet radiations (UVR) from the sun. UVR comprises of 95 % long-wavelength UVR (320–400 nm), known as ultraviolet A (UVA) and 5 % of short-wavelength UVR (290–320 nm), called ultraviolet B (UVB) [1,103,104]. UVA and UVB mediated mechanisms of pigmentation induction are distinct, with UVB induced skin pigmentation pathways being characterized in more detail [104]. UVB typically induces thymidine breaks in DNA that stimulate the expression of key melanogenic regulators, resulting in delayed skin pigmentation over a course of few days [1,103]. UVA primarily induces oxidative damage and is responsible for immediate pigmentary response (within minutes) [103]. In order to understand the details of phototransduction in skin, Oancea lab irradiated primary human epidermal melanocytes (HEMs) with UVR. In these experiments, UVA elicited significantly higher Ca²⁺mobilization from the intracellular ER Ca²⁺stores than UVB [103]. The induction of Ca²⁺mobilization downstream of UVR was retinal dependent, which is the chromophore essential for light activation of an opsin GPCR. Inhibition of G protein or phospholipase C (PLC) abrogated the UVR induced Ca²⁺ mobilization, suggesting the role of PLC activation in this process. Expression of a photopigment-rhodopsin was validated in the HEMs and shown to mediate the UVR phototransduction process. UVR exposure induced significantly faster Ca²⁺and retinal-dependent melanin synthesis, thus contributing towards the mechanistic knowledge about immediate pigment darkening (IPD). Next, the significance of Transient Receptor Potential (TRP) channels, specifically subfamily A member 1 (TRPA1) in extraocular phototransduction was elucidated by the same group [104]. TRP channels are non-selective cation channels that serve as multimodal sensors of noxious chemical and physical stimuli such as light, heat and pressure [104,105]. Bellono et al. stimulated the human melanocytes with UVR doses equivalent to seconds/minutes of full sun exposure and observed induction of retinal-dependent currents [104]. Inhibition of TRP channels using ruthenium red (RR) blocked the UVR activated photocurrent. TRPA1 being expressed in melanocytes and possessing a current-voltage relationship comparable to the UVR photocurrent was further studied using TRPA1 agonists and antagonists. UVA induced current was shown to be mediated via TRPA1, after activation of GPCR and PLC signaling. Further, TRPA1 mediated the influx of the extracellular Ca²⁺ accompanied by UVR induced Ca²⁺ release from the ER stores. This signaling cascade elicited an increase in the early melanin synthesis, thus imparting protection from the UVR mediated DNA damage.

TRPM1, a member of the TRP cation channels family that localizes to the plasma membrane [106]. TRPM1 regulates cellular Ca²⁺homeostasis as its knockdown reduces the intracellular Ca²⁺ levels and its uptake [107]. TRPM1 knockdown also decreased the tyrosinase activity and cellular melanin content. Oancea et al. demonstrated a positive correlation between TRPM1 expression and pigmentation in the HEMs [106]. TRPM1 expression is also positively associated with pigmentation phenotype in the Appaloosa horse [108]. TRPM1 mRNA expression is strongly correlated with MITF and tyrosinase expression [109]. TRPM1 was identified as one of the p53 target genes in a genome wide analysis, which is supported by the fact that it harbors a putative p53-binding motif, 25 kb upstream of TRPM1 transcription start site [107]. Interestingly, UVB stimulation mediates an increase in p53 expression in the melanocytes followed by a reduction in the expression of TRPM1 and a concordant reduction in the intracellular Ca²⁺ levels [107].

Ca²⁺ release activated Ca²⁺ (CRAC) channels on the plasma membrane comprise of Orai(1/2/3) channels and their activating partners stromal interaction molecules 1 and 2 (STIM1 and STIM2) [110]. Keratinocytes secrete Endothelin-1 (ET-1) in response to UVR, which binds to and activates endothelin B (ET-B) receptors present on the melanocytes [110]. Activated ET-B receptors trigger the PLC mediated IP₃ and diacylglycerol (DAG) production. IP₃ binds to the IP₃ receptors (IP₃Rs) on the ER membrane and induce Ca²⁺ mobilization from the ER stores. This leads to activation of Store operated Ca²⁺ entry (SOCE). It is a cellular phenomenon that involves oligomerization of Ca²⁺ sensing ER membrane resident proteins STIMs upon ER Ca²⁺ store depletion [111]. This leads to the activation of plasma membrane store-operated Ca²⁺ (SOC) channels thus inducing Ca²⁺entry. Orai1 channels form the major Ca²⁺ influx channels and STIM2 is the more abundant isoform in primary human melanocytes [110]. Treatment with ET-1 stimulates the tyrosinase enzyme activity and melanin production that is abrogated upon Orai1 inhibition using 2-APB. UVR also stimulates keratinocytes and melanocytes to secrete corticotropin-releasing hormone that increases ACTH, αMSH and endorphin production locally. These molecules have also been implicated in elevating intracellular Ca²⁺ levels via IP₃ production. Taken together, the plasma membrane localized TRP channels (TRPM1, TRPA1) and Orai channels increase intracellular Ca²⁺ levels upon activation and contribute towards pigmentation in a context dependent manner.

4.2. Endoplasmic reticulum Ca²⁺ signaling

Increasing evidence supports that SOCE plays a critical role in regulating melanocyte physiology and pigmentation [25]. We showed that upon αMSH mediated ER Ca²⁺ stores depletion, STIM1 is recruited to ER-PM junctions leading to its interaction with adenylyl cyclase 6 (ADCY6) thereby activating ADCY6 [25]. Downstream, cAMP generation is stimulated leading to amplification of cAMP mediated melanogenesis. This, coupled with the well-studied αMSH-cAMP-MITF axis forms a positive feedback loop in the melanocytes. Ca²⁺ and cAMP are vital secondary messengers that are known to regulate each other, and in this scenario, also modulate melanogenesis cooperatively. While this study clearly demonstrated that STIM1 expression is positively correlated with pigmentation induction, our subsequent study delineated the molecular choreography behind the process [26]. The study utilized well established B16 LD pigmentation model, αMSH mediated pigmentation induction in B16 cells and primary human melanocytes. We demonstrated that the transcriptional regulation of STIM1 is augmented upon αMSH treatment. Further, αMSH stimulation enhanced SOCE as a result of increase in STIM1 expression while Orai(1/2/3) levels remain unchanged. It was demonstrated that MITF mediates the augmentation of STIM1 expression and SOCE as a result of αMSH stimulation. Bioin-formatics analysis revealed that the STIM1 promoter has four putative MITF binding sites, out of which two sites (−540 bp and −407 bp) contributed to STIM1 promoter activity as revealed by in vitro luciferase assays. The −407 bp MITF binding site appeared as the most crucial regulator of STIM1 transcription. The ER Ca²⁺contribution towards melanogenesis is highly relevant physiologically as evident via the bioinformatics based human tanning data, cellular models and in vivo zebrafish system [25,26].

4.3. Mitochondrial Ca²⁺channels

UVA phototransduction in HEMs involved the crosstalk of Ca²⁺ and ROS mediated pigment induction [44]. A portion of UVA induced ROS was retinal-independent and was not accompanied by a measurable Ca²⁺ response, whereas cells treated with retinal displayed significantly high Ca²⁺ and ROS response. Mitochondrial ROS was also elevated in response to the UVA stimulus. The authors demonstrated that G-protein activation is necessary for retinal-dependent ROS production using BIM, a Gα_q/11 inhibitor. BAPTA-AM treatment of the melanocytes to suppress the UVA stimulated intracellular Ca²⁺ levels also suppressed the ROS response. Mitochondrial ROS production was analyzed downstream of Ca²⁺ and treatment with mitochondrial antioxidant-mitoTEMPO reduced the total UVA-mediated ROS levels. Further, retinal-independent ROS response potentiates the UVA induced, retinal-dependent Ca²⁺ response. This also promotes mitochondrial Ca²⁺ uptake, mitochondrial ROS production and ultimately, melanin production in HEMs.

Further, our lab delved into the role of mitochondrial Ca²⁺ signaling in pigmentation. We revealed the significance of Mitochondrial Ca²⁺ Uniporter (MCU) mediated mitochondrial Ca²⁺ uptake in pigmentation biology. It is widely acknowledged that mitochondrial Ca²⁺ signaling controls processes like cell division, ROS metabolism, respiration, autophagy and so on [112]. For the first time, we revealed that mitochondrial Ca²⁺ uptake is a critical determinant of vertebrate pigmentation. Using a variety of cellular systems, zebrafish and transgenic mouse models we demonstrated that MCU mediated mitochondrial Ca²⁺ uptake positively regulates melanogenesis, whereas its dominant negative subunit MCUb negatively controls pigmentation. Downstream, NFAT2 transcription factor activation augments keratin expression (Keratin 5, 7 and 8) [112]. Keratins in turn potentiate melanogenesis by enhancing melanosome biogenesis and maturation. Importantly, inhibition of MCU with mitoxantrone (an FDA approved drug) reduces pigmentation.

Role of SLC24A5 or NCKX5 has been implicated in oculocutaneous albinism type 6 (OCA6). NCKX5 belongs to the K⁺-dependent Na⁺/Ca²⁺ exchanger family (NCKX) [113]. NCKX5 was recently shown to be localized on the mitochondria and the trans-Golgi network instead of melanosomes [113]. Pharmacological inhibition or the loss of NCKX5 inhibits melanogenesis via mitochondrial dysfunction and attenuation of Ca²⁺ transfer to the melanosomes. Compromised melanosomal Ca²⁺levels lead to reduced PMEL expression and therefore hypopigmentation. It appears that NCKX5 may mediate mitochondrial Ca²⁺ efflux. Its presence or function at the mitochondria-melanosome contact sites and exact mechanism of Ca²⁺ transfer remains to be elucidated.

Recently, the role of VDAC1, an outer mitochondrial membrane (OMM) protein has been highlighted in the pigmentation biology [114]. VDAC1 forms a multimeric channel on the OMM that contributes towards the bidirectional transfer of various ions including Ca²⁺, metabolites and small molecules. The literature suggests that VDAC1 depletion or chemical inhibition induces hyperpigmentation in various cellular models [114,115]. Wang et al. reported an increase in MITF transcriptional and translational levels upon VDAC1 knockdown and further examined the CREB and CRTC-1 axis, where nuclear levels of CRTC-1 were enhanced [114]. Depletion of VDAC1 increased the cytosolic Ca²⁺ levels, further activating calnexin (CaN) via calmodulin (CaM), leading to dephosphorylation and migration of CRTC1 into the nucleus. Inhibition of VDAC1 using VBIT-12 and NSC-15,364 induces hyperpig-mentation in a similar mechanistic manner [115]. These observations were also strengthened by utilizing zebrafish model system and human skin explants.

4.4. Lysosomal/Melanosomal Ca²⁺ channels

Various ions such as Ca²⁺, H⁺, Na⁺ and Cl⁻ are known regulators of melanogenesis [116]. Interplay of cellular ionic homeostasis and pH critically influence the process of pigmentation [116]. Organellar pH like melanosomal or lysosomal pH regulates key steps of melanogenesis such as the enzymatic activity. TPC2, a non-specific cation channel, found on lysosomes and melanosomes mediates Ca²⁺ and Na⁺ homeostasis that ultimately regulates melanogenesis via the control of melanosomal pH and size. TPC2 mediates Ca²⁺/Na⁺ efflux in lysosomes and melanosomes whereas CLC7/OCA2 mediates H⁺/Cl⁻ efflux. Collectively, they regulate V-ATPase activity to maintain lumen pH and ionic homeostasis. Melanosomal lumen pH is responsible for the regulation of tyrosinase activity, thereby regulating pigmentation. TPC2 has been shown to be a negative regulator of pigmentation and its gain of function mutations-M484 L and G734E found in the European population are linked with hypopigmentation. Recently, a TPC2 gain of function mutation R210C was shown to be associated with albinism in a Chinese patient [117]. The downstream mechanism is based on Ca²⁺ mediated pH regulation of the lysosomes. Since TPC2 is present on melanosomes as well, further studies are required to precisely delineate the contribution of lysosomal v/s melanosomal TPC2 in regulating pigmentation.

4.5. Intercellular Ca²⁺signaling

With well-established roots in the arenas of aging and inflammation, a scarcely explored angle towards melanogenesis is the impact of heat stress [118]. The Zeng group studied this phenomenon and obtained strong leads from high-throughput RNA sequencing that highlighted the activation of Hedgehog (Hh) signaling pathway in the keratinocytes during heat stress. The Hh pathway agonists stimulated melanogenesis through their downstream paracrine effects. Melanocyte-keratinocyte co-culture model systems were used to reveal that the conditioned medium obtained from heat-treated keratinocytes stimulated melanogenic pathways such as MAPK and Wnt/β-catenin pathway in melanocytes [118]. Further, the study highlighted the role of heat and pain sensitive channels-Transient receptor potential vanilloid (TRPV) in mediating the heat induced hyperpigmentation. Both the activation of TRPV3 and heat stress stimulate Ca²⁺ uptake in the HaCaT keratinocytes. TRPV3 inhibition abrogated the heat-induced Ca²⁺ influx into the cells. TRPV3 agonists and antagonists stimulated and inhibited the heat-induced pigmentation, respectively. Overall, this study throws light on the TRPV3/Ca²⁺/Hh signaling pathway in heat-induced pigmentation. Taken together, recent studies are highlighting the potential of managing pathophysiological pigmentary conditions by specifically targeting organellar Ca²⁺dynamics.

In an exciting study by Belote and Simon, ion beam scanning EM (FIB-SEM) technique was utilized to show that keratinocyte processes wrap around melanocyte dendrites [119]. This study revealed that paracrine factors secreted by keratinocytes-endothelin-1 (ET-1) and acetylcholine (ACh) mediate induction of local Ca²⁺ transients in the melanocyte dendrites. Conversely, the addition of the endothelin receptor B (ET_B) antagonist BQ788 and muscarinic Ach receptor antagonist atropine reduced the number of melanocytes producing Ca²⁺ transients and the frequency of these transients per cell respectively. This might further regulate melanogenesis locally within dendrites via the control of mRNA transcription or regulation of enzymatic activity.

Melanocytes transfer melanosomes to the keratinocytes via mechanisms that are scarcely understood. Pigment transfer does involve physical contact amongst the two cell types. Fontana-Masson staining in B16 or primary human melanocytes co-cultured with A431 cells or HaCaT keratinocytes respectively clearly demonstrated pigment transfer from the former cell type to the latter [21]. Mechanistically, induction of transient Ca²⁺ signals was observed upon melanocyte plasma membrane addition to the keratinocytes. The study shows store dependent increase in intracellular Ca²⁺ in the A431 keratinocytes was only specific to the B16 plasma membrane addition and not that of other cell types such as U87MG glioblastoma cells and primary rat brain astrocytes. Further, pigment transfer was abrogated upon chelation of intracellular Ca²⁺ using BAPTA-AM in the A431 keratinocytes. Therefore, emerging literature indicates that Ca²⁺ signaling could be one of the key modulators guiding the melanin transfer. Please refer to Fig. 2 for the diagrammatic summary highlighting role of Ca²⁺ signaling in pigmentation.

4.7. Ca²⁺signaling and pigmentary disorders

Ca²⁺ levels are distinctly and tightly regulated in the intracellular and extracellular milieu. Cytosolic Ca²⁺ levels are low in the cytosol (∼100 nM); in contrast to the sequestration of the ion in the Ca²⁺ stores like endoplasmic reticulum (ER) (∼200μ M), sarcoplasmic reticulum (SR), mitochondria (∼100 nM), lysosomes (∼500μ M) and melanosomes [120,121]. Ca²⁺gradient exists within the cells as well as within the distinct skin layers. Highest Ca²⁺ concentration is found in stratum granulosum and the lowest in the stratum corneum, essential to preserve skin barrier function [122]. Cellular Ca²⁺signaling has various pathophysiological implications. While we emphasize its importance in the physiological process of melanogenesis, imbalance in the Ca²⁺homeostasis can result in melanogenic and dermatological disorders like skin cancer, melanoma, Darier’s disease etc. [122–125]

Darier’s disease (DD) is an autosomal dominant skin disease occurring as a consequence of SERCA2 (Sarcoplasmic/Endoplasmic Reticulum Ca²⁺ATPase) mutations, which is an important Ca²⁺ influx channel on the ER membrane [125]. Briefly, DD manifests as skin blisters, warty papules, plaques with unpleasant odor, fragile skin, dyskeratosis and mottled pigmentation. SERCA2 haploinsufficiency observed during the classical DD phenotype results in the reduction of ER Ca²⁺stores in keratinocytes. Compensatory mechanisms to maintain Ca²⁺homeostasis in SERCA2-compromised keratinocytes such as the augmentation of TRPC1, PMCA and hSPCA1 expression and function have been reported by different groups [126–128]. Interestingly, changes in STIM1 subcellular localization was suggested as a biomarker for distinguishing DD from Hailey-Hailey (HH) disease in a small cohort of patients [129]. Further studies are required to validate this observation. The pleiotropic nature of the disease, age-specific manifestation, focal nature pose challenges in DD management, however, a multi-faceted approach including Ca²⁺channel modulators to maintain the ionic balance may be explored and exploited.

Fig. 3. Overview of pigmentation modulation by inter- and intra-cellular Ca²⁺ signaling.

Fig. 3. Schematic representation of the regulation of pigmentation by Ca²⁺ signaling.

Keratinocytes trigger melanogenesis via paracrine and Ca²⁺ induced signaling cascades within the melanocytes. UVR stimulation acts via plasma membrane opsin receptors, GPCRs and TRPA1 channels, that cumulatively increase cytosolic Ca²⁺ levels through PLC mediated cascade. In addition to the classical αMSH-cAMP-MITF axis, STIM1 interaction with ADCY6 forms a positive feedback loop and stimulates cAMP mediated melanogenesis. Critical regulation of mitochondrial matrix and cytosolic Ca²⁺ levels by mitochondrial inner and outer membrane channels, MCU and VDAC1, respectively, control melanogenic transcription activation. NCKX5 mediates Ca²⁺ transfer between mitochondria and melanosomes that regulates melanogenic protein synthesis. Melanosome localised Na⁺/Ca²⁺efflux channel TPC2 regulates lumen pH through V-ATPase. Hyper-acidification of melanosome lumen may inhibit melanin biosynthesis.

5. Concluding remarks and future perspective

Melanogenesis is a multimodal cellular process. As discussed above, signaling modules involving ROS and Ca²⁺ play a key role in pigmentation biology. Pathways like the Nrf-2-ARE pathway, Wnt signaling and the p53 axis modulate melanin biogenesis. While these pathways are associated with ROS generation, the precise mechanisms through which ROS levels regulate these pathways remain poorly appreciated.

Alterations in cellular Ca²⁺ homeostasis modulate several downstream metabolic cascades in the pigmentation process, both physiological and pathological. Numerous studies suggest that oxidants or cellular oxidative stress can modulate the cytosolic Ca²⁺ levels, via regulation of several Ca²⁺ handling proteins [130–132]. SERCA [132], IP₃ receptors (IP₃Rs) [133,134], SOCE pathway players like Orais [135], voltage-gated calcium channels (VGCCs) [136] and plasma membrane Ca²⁺ATPase have been demonstrated to be under regulation of oxidative stress. S-glutathionylation at cysteine 56 of STIM1 acts as a sensor for oxidative stress, leading to CRAC channel mediated constitutive Ca²⁺ entry independent of intracellular Ca²⁺ stores [137]. This also results in mitochondrial Ca²⁺ overload and bioenergetic changes in the cells. STIM2, which has 10 more additional cytosolic cysteines than STIM1 can also suppress SOCE in response to oxidative stress via Cys313 oxidation [138]. Extracellularly localized reactive cysteine 195 also imparts redox sensitivity to Orai1 channel, but not Orai3 [139]. SOCE can lead to influx of extracellular Na⁺in the cell, thus activating mitochondria localized Na⁺/Ca²⁺ exchanger (NCLX) [140]. This enhances the mitochondrial Na⁺uptake and efflux of matrix Ca²⁺, also activating a mitochondrial redox transient. Further, NCLX dependent mitochondrial redox exerts its control over SOCE via Orai1 Cys195 leading to SOCE and CRAC current alterations [140]. Thus, existing literature clearly defines the interplay between ROS and Ca²⁺ signaling in various cellular systems. However, role of ROS in regulating function of the melanocyte Ca²⁺ signaling toolkit and its subsequent effect on pigmentation is largely unknown.

Autophagy is a crucial player in deciding the levels of melanin in melanocytes by degradation of melanosomes, and dysregulation of melanosomal autophagy (melanophagy) is observed in pigmentary defects. Previous studies have demonstrated that autophagy induction reduces melanin content in ex vivo lesioned skin and in senile lentigo, the hyperpigmented lesions showed lower autophagic flux. Considering these factors, targeting autophagy could be a potential treatment option for hyperpigmentary disorders. Therefore, future studies in this direction are required.

Mitochondria play a crucial role in the overall maintenance of cellular Ca²⁺ metabolism. Mitochondrial damage, loss of mitochondrial membrane potential, disruption of the electron transport chain (ETC) can trigger or occur as a consequence of imbalances in Ca²⁺ signaling pathways. Another well documented outcome of these events is the generation of excess amounts of ROS and oxidative stress [141]. Still role of mitochondria in melanosome biology and pigmentation has only started to emerge.

Ca²⁺ and oxidative stress pathways act in unison, with ETC and ATP production being dependent on Ca²⁺sensitive dehydrogenases of the Kreb’s (TCA) cycle, mitochondrial membrane potential regulation, ATP synthase enzyme activity and overall stability of the mitochondrial respiration [141]. Mitochondrial Ca²⁺ overload can interfere with the bioenergetic supply, leading to ROS accumulation, mitochondrial membrane potential alterations and ultimately, cell death. Further, mitochondrial Ca²⁺ signaling pathways crosstalk to the ROS pathways via several molecular players such as p53. While some of the key ROS and Ca²⁺modulators have been shown to affect the melanogenesis and melanogenic disorders, the literature regarding the crosstalk of Ca²⁺ and ROS in pigmentation is limited, making this an intriguing area for further exploration.

Data availability

No data was used for the research described in the article.