Pharmacologic manipulation of skin pigmentation

Abstract

Skin complexion is among the most recognizable phenotypes between individuals and is mainly determined by the amount and type of melanin pigment deposited in the epidermis. Persons with dark skin complexion have more of a brown/black pigment known as eumelanin in their epidermis whereas those with fair skin complexions have less. Epidermal eumelanin acts as a natural sunblock by preventing incoming UV photons from penetrating into the skin and therefore protects against UV mutagenesis. By understanding the signaling pathways and regulation of pigmentation, strategies can be developed to manipulate skin pigmentation to improve UV resistance and to diminish skin cancer risk.

Skin Complexion

Skin complexion is a phenotype largely determined by the activity of melanocytes which are neural crest-derived pigment-producing cells found in the skin and other select locations in the body. Melanocytes associated with hair follicles in the dermis contribute melanin to growing hair shafts to give hair its color, while those found in the stratum basale of the skin melanize the epidermis itself. This review will focus on interfollicular epidermal melanocytes and pigmentation of the skin. The amount and type of melanin produced by melanocytes determines skin complexion, which can be described by the “Fitzpatrick Scale” (Del Bino et al., 2015; Fitzpatrick, 1988). In this way, the skin tone and UV sensitivity of individuals with very light complexions can be compared with persons of darker complexions (Table 1). In general, the darker a person’s complexion, the more melanin their epidermis contains and the more protected she/he/they will be from acute UV injury such as sunburn as well as long-term UV-dependent pathologies such as cancers of keratinocytes and melanocytes (Del Bino & Bernerd, 2013; Del Bino, Duval, & Bernerd, 2018; Fajuyigbe & Young, 2016). Melanogenesis – the process of melanin biogenesis – is a complex and dynamic process that is regulated by inherited as well as environmental factors. Skin complexion is a polygenic trait controlled by many genes. While each individual has his/her/their own basal complexion, skin tone can be modified by environmental factors, most notably from the damage and inflammation from UV radiation that can activate cutaneous damage response signaling pathways to up-regulate melanin production (Cui et al., 2007). This physiological response, termed “adaptive pigmentation” or tanning, is an important safeguard against further injury to the skin by the deposition of more UV-blocking melanin as well as epidermal thickening which together protect the inner layers of the skin against UV damage (D’Orazio et al., 2006; Scott et al., 2012). In this way, skin pigmentation is an important determinant of health, and loss-of-function mutations or polymorphisms in genes that regulate melanin production are associated with higher risk of UV-associated skin pathologies including sunburn risk, photoaging and many types of skin cancer (Brenner & Hearing, 2008). Organized as “epidermal melanin units”, melanocytes in the stratum basale contact many keratinocytes via dendritic processes (Fitzpatrick & Breathnach, 1963; Nordlund, 2007). This organizational structure facilitates transfer and uptake of melanin-bearing melanosomes to keratinocytes through phagocytic mechanisms (Cardinali et al., 2008; Singh et al., 2010). Indeed, while melanocytes are the sole melanin-producing cell of the skin, keratinocytes ultimately contain the bulk of the melanin found in the skin. In skin cells, melanin is deposited in a directional manner, positioned to block incoming UV photons and to protect the nuclei of keratinocytes. Importantly, melanocyte density is relatively constant across all skin complexions and it is due to differences in melanin distribution and production rather than melanocyte number that lead to varying complexions (Tadokoro et al., 2003; Tadokoro et al., 2005).

Table 1.

The Fitzpatrick Scale can be used to semi-objectively quantify skin pigmentation and UV risk.

Fitzpatrick Type	Skin complexion	Epidermal melanin content	Risk for UV pathologies
I	Very pale	Very low	Very high
II	Pale	Low	High
III	Light-to-medium	Modest	Moderate
IV	Medium-to-olive	Moderate	Somewhat
V	Dark	High	Modest
VI	Very dark	Abundant	Minimal

Melanin

Melanin, a bioaggregate of molecules ultimately derived from the amino acid tyrosine, is not just one compound but a mixture of various pigment species (d’Ischia et al., 2013; Prota, 2000; Vincensi et al., 1998). Two main melanin forms exist in humans – the brown/black pigment eumelanin which functions as an effective absorber of UV radiation, and the lighter brown/red pigment pheomelanin which is less able to block incoming UV photons into the skin and is more chemically reactive (Nasti & Timares, 2015). The amount and ratio of eumelanin to pheomelanin deposits in the epidermis ultimately determine an individual’s skin complexion. Tyrosine is the common synthetic precursor for both eumelanin and pheomelanin, but it is the incorporation of sulfur-containing cysteine residues that yields the red-yellow coloration of pheomelanin (Fig. 1) (d’Ischia et al., 2015). Synthesis of melanin in the skin occurs exclusively in melanocytes which produce and store melanin within membrane bound organelles termed melanosomes that contain melanogenic enzymes and provide the appropriate conditions for melanin biosynthesis (Jimbow et al., 1997). Arising as trans-Golgi endosomes, melanosomes undergo a multi-stage maturation process and accumulate in the intracellular periphery via dynein and kinesin motors to mobilize along microtubules and actin filaments (Jimbow, Hua, et al., 2000; Jimbow, Park, et al., 2000). Melanosomes commit themselves to the production of either eumelanin or pheomelanin but melanocytes possess populations of both eumelanogenic and pheomelanogenic melanosomes (Liu et al., 2004). Aspects of melanogenesis including tyrosinase activity and the ratio of eumelanin to pheomelanin are dictated in part by the internal pH of melanosomes, indicating the importance of endosomal ion transporters, protons pumps, and soluble adenylyl cyclase to pigmentation (D. Zhou et al., 2018).

Figure 1. Overview of Melanogenesis.

Melanin, a bioaggregate of pigmented compounds, is found in two predominant forms, both of which derive from the amino acid tyrosine. Eumelanin is a dark brown/black species that is highly UV protective whereas pheomelanin, as a result of incorporation of a sulfur molecule donated by cysteine, is a reddish/blonde pigment that is less able to block UV photons. Tyrosinase is the rate-limiting enzyme that catalyzes the first two steps in the synthesis of both melanin types, and pigment-diluting phenotypes such as albinism result when tyrosinase or other melanogenic enzymes are defective.

Regulation of Skin Pigmentation

The genetic basis for basal skin pigmentation in humans is complex and involves a variety of genes affecting melanocytic functions including migration, development, melanin synthesis, and melanosome structure (Table 2) (Baxter, Watkins-Chow, Pavan, & Loftus, 2019; Pavan & Sturm, 2019). In general, defects in melanocyte-specific genes result in hypopigmentation, perhaps best exemplified by tyrosinase and oculocutaneous albinism type 1 (OCA-1). Tyrosinase is the rate-limiting enzyme that catalyzes the first two steps of melanin synthesis (Fig. 1), and mutations in tyrosinase can result in OCA-1, a disorder of dense hypopigmentation in which affected individuals lack the photoprotective effects provided by melanin and thus are highly UV-sensitive (Oetting, 2000). In fact, there are multiple types of OCA, each one caused by distinct mutations in gene products needed for melanin biosynthesis. The varying severity of albinism phenotypes caused by mutations in different genes illustrates the polygenic nature of skin complexion. Moreover, just as mutations exist that lead to the underproduction of pigment, there are those that cause its overproduction including an activating mutation of the Guanine Nucleotide Binding Protein (Gnaq) linked to development of port-wine stains and blue nevi (Shirley et al., 2013; Van Raamsdonk, Barsh, Wakamatsu, & Ito, 2009). While many genes encode for enzymes directly involved in the synthesis of melanin, others impact melanosome structure and development. As melanosomes mature, ion transporters and proton pumps regulate their pH to promote melanogenesis. Promelanosome protein PMEL (encoded by Pmel17) serves as a structural protein in immature melanosomes and as a scaffold upon which melanin is deposited in mature melanosomes (Berson, Harper, Tenza, Raposo, & Marks, 2001).

Table 2.

Partial list of genes that regulate skin pigmentation.

Gene	Function	Mutant/polymorphism Phenotype
Melanocortin 1 Receptor (MC1R)	Generation of cAMP signal	Inability to tan, red hair, increased risk of melanoma
Agouti-Signaling Protein (ASIP)	MC1R Antagonist	Eumelanin synthesis favored over pheomelanin synthesis
Tyrosinase (TYR)	Melanin Synthesis (first and rate-limiting step)	Oculocutaneous Albinism Type 1
Solute Carrier Family 24 Member 5 (SLC24A5)	Endosomal ion transport	Light Skin, Oculocutaneous Albinism Type 6
Microphthalmia-associated transcription factor (MITF)	Melanoblast survival and development	Waardenburg Syndrome Type 2
Tyrosine-related protein-1 (TRP1)	Melanin Synthesis, Tyrosinase Stability	Oculocutaneous Albinism Type 3, Melanoma
Dopachrome tautomerase (TRP2)	Melanin Synthesis	Mild Oculocutaneous Albinism
KIT Ligand (KITLG)	Melanoblast survival and development	Piebaldism, Hyper/Hypopigmentation
Pre-Melanosome Protein (PMEL17)	Structural development of melanosomes	Unknown
P Protein (OCA2)	Melanosome development, Melanogenesis	Oculocutaneous Albinism Type 2
Paired-box gene 3 (PAX3)	Melanocyte survival and development	Waardenburg Syndrome Types 1/3, Melanoma
Guanine Nucleotide Binding Protein (GNAQ)	Encodes GPCR alpha subunit	Sturge-Weber Syndrome, Port-Wine Stains, Blue Nevi

A central determinant of the capacity of melanocytes to produce melanin is the melanocortin 1 receptor (MC1R), a 317 amino acid G protein-coupled receptor (GPCR) associated with the plasma membrane of melanocytes. Cloned in 1992 (Mountjoy, Robbins, Mortrud, & Cone, 1992), the MC1R is among the most important genetic determinants of pigmentation and skin cancer risk in the general population because it regulates eumelanin-pheomelanin balance and also how melanocytes respond to UV exposure. In its functional wild type state, the MC1R interacts with either of two high-affinity agonistic ligands – adrenocorticotropic hormone (ACTH) or alpha-melanocyte stimulating hormone (α-MSH). Each activates transmembrane adenylyl cyclase (TMAC) through their binding to MC1R to induce cAMP second messenger production and downstream signaling pathway activation. In melanocytes, cAMP signaling pathway activates key transcriptional networks that induce melanocyte differentiation. Microphthalmia (Mitf) is one key cAMP-activated protein (Price et al., 1998). It is a master transcription factor in melanocytes that promotes differentiation and pigment induction by binding to specific DNA sequences termed “E boxes” in key pigmentation and differentiation gene regulatory regions and stimulating transcription of tyrosinase and other pigment enzyme genes to upregulate eumelanin production (Bertolotto et al., 1998; Bertolotto, Bille, Ortonne, & Ballotti, 1996; Levy, Khaled, & Fisher, 2006).

The human MC1R gene is highly polymorphic with loss of function mutations likely selected among populations that migrated away from UV-rich equatorial regions in order to diminish epidermal eumelanin levels to maximize UV-mediated vitamin D production in more temperate UV-poor climates (Harding et al., 2000). These polymorphisms correlate with a fair-skinned phenotype and impaired adaptive pigmentation (tanning) response (Valverde, Healy, Jackson, Rees, & Thody, 1995). Indeed, Cui and co-workers reported that MC1R is central to the skin’s response to UV damage, via a p53-dependent upregulation of pro-opiomelanocortin (POMC) in keratinocytes that produce MSH and ACTH to stimulate eumelanin production by epidermal melanocytes in a paracrine manner (Cui et al., 2007). MC1R signaling is also pertinent to DNA damage and repair responses in melanocytes, with abundant evidence that cAMP signaling enhances the ability of melanocytes to resist UV photodamage (Bautista et al., 2020; Im et al., 1998; Kadekaro et al., 2012; Kadekaro et al., 2005; Song et al., 2009; Swope et al., 2012). MC1R loss-of-function polymorphisms are associated with increased risk of malignancy (Valverde et al., 1996), therefore there is great interest in attenuating cancer risk by cAMP-enhancing strategies.

Pharmacologic strategies for eumelanin induction

NDP-MSH

[Nle4,D-Phe7]-α-MSH (NDP-MSH) is a well-known agonist of MC1R that is identical to native α-MSH with the exception of norleucine replacing the position-4 methionine and the position-7 phenylalanine replaced with its dextro-isomer that stabilizes the oligopeptide by making it more resistant to degradation (Hruby et al., 1980; Sawyer, Hruby, Darman, & Hadley, 1982). These structural modifications create an analog more resistant to proteolysis than native α-MSH. NDP-MSH significantly increases eumelanogenesis (production of eumelanin) in humans, one study citing a 41% increase in Caucasian subjects following subcutaneous injection (Barnetson et al., 2006). While NDP-MSH is a potent α-MSH analog, it is not specific for MC1R and exerts a systemic effect through its interactions with multiple melanocortin receptors. Selectivity and specificity of MSH analogues has been ongoing, with various reports describing amino acid substitutions (Y. Zhou et al., 2017). The search for NDP-MSH alternatives does not mean that it has been rendered obsolete as a melanogenic agent. Under the brand name SCENESSE® (afamelanotide) NDP-MSH recently was approved by the Food and Drug Administration for treatment of erythropoietic protoporphyria, a rare condition characterized by UV phototoxicity.

MSH peptides

Another strategy used to pharmacologically induce melanin production is to increase MC1R activity in melanocytes via MSH peptide agonists. Small peptides consisting of only three or four amino acids have been of particular interest. The His-Phe-Arg-Trp amino acid sequence is of importance in the construction of these peptide agonists because it is responsible for the melanogenic effect and is conserved among melanocortins (Holder, Bauzo, Xiang, & Haskell-Luevano, 2002). One study produced a tetrapeptide with a 1000-fold increase in potency as well as longer-lasting effect compared to α-MSH in human melanocytes (Abdel-Malek et al., 2006). Abdel-Malek and colleagues also investigated the efficacy of tripeptides by creating a peptide with 10-fold less potency than α-MSH, showing that deletion of Trp from the core sequence still creates a viable agonist (Abdel-Malek et al., 2009). While less potent, the advantage to these tripeptides is greater selectivity for MC1R and lower selectivity for other melanocortin receptors. This advantage was proven through a study in which the half-maximal effective concentration (EC₅₀) of numerous modified tripeptides for MC1R was calculated to be far lower than the EC₅₀ for MC3R, MC4R, and MC5R (Ruwe et al., 2009). While tetrapeptides are potent and tripeptides are selective, neither can exert any effect in MC1R-deficient models. Individuals with homozygous loss-of-function MC1R polymorphisms who tend to have red hair and poor tanning ability may not benefit from peptide-induced protection from melanoma, however this approach could benefit MC1R heterozygous persons who number in the millions and are at increased risk of UV-induced skin pathologies including melanoma. It is also worth noting that a tetrapeptide and tripeptide agonists are only two “lengths” of MC1R peptide agonists with demonstrated melanogenic activity; a host of oligopeptides with varying amino acid lengths have been found to manipulate melanogenesis and are worth further investigation (Boo, 2020). Abdel-Malek and co-workers recently published that MSH tetra- and tripeptide agonists highly selective for MC1R stimulated melanogenesis enhanced repair of UV damage and reduced apoptosis in human melanocytes (Koikov et al., 2021), therefore MSH peptide agonists are an active area in development.

Topical forskolin

One of the most effective pharmacologic approaches to upregulating epidermal eumelanin levels was shown to be by topically applying forskolin, a direct activator of adenylyl cyclase, to the skin. For these studies, a mouse model of the fair-skinned MC1R-defective human was developed by studying “extension” mice with defective Mc1r activiy. Extension mice display a phenotype switch from eumelanin to pheomelanin on the C57BL6/J background due to a genetic mutation in Mc1r that truncates the protein to result in its loss of function. Because interfollicular epidermal melanocytes are not a feature of adult mouse skin except somewhat on the tail and ears, the extension (Mc1r-defective) animals were crossed with K14-Scf transgenic mice developed by Takahiro Kunisada’s group (Kunisada et al., 1998). In this model, stem cell factor (c-Kit ligand) is constitutively expressed by basal keratinocytes in the epidermis, and as a result, melanocytes are chronically retained in the epidermis and epidermal pigmentation can occur. In the Mc1r wild type C57BL6/J background, the epidermis of K14-Scf mice is jet black and full of eumelanin, whereas in the Mc1r-defective extension background K14-Scf mice have fair skin pigmentation that is associated with preferential expression of pheomelanin, similar to lightly complexioned MC1R-defective persons (D’Orazio et al., 2006). Forskolin, a hydrophobic small molecule naturally derived from the roots of the Coleus forsksohlii plant, was applied to the skin of extension K14-Scf mice, and as a result of cutaneous cAMP signaling, the skin darkened because of a marked upregulation in eumelanogenesis. Analysis of the skin confirmed increased melanin deposition by Fontana-Masson staining and by chemical melanin analysis and animals were more resistant to UV damage, thus proving that fair skin complexion and the UV sensitivity caused by defective Mc1r signaling could be pharmacologically rescued by restoring cutaneous cAMP signaling (D’Orazio et al., 2006). This proof-of-concept study clearly showed the ability of cAMP inducing agents to rescue eumelanotic complexion in fair-skinned individuals, however forskolin is not generally regarded as a useful therapeutic agent because of its lack of specificity.

SIK Inhibitors

Salt-inducible kinases (SIK) are widely expressed and serve in a variety of physiological roles including gut inflammation, bone resorption and formation, and hepatic gluconeogenesis. SIKs counter-act a GPCR/cAMP/PKA axis that induces MITF-dependent melanogenesis, thus leading to hypopigmentation (Wein, Foretz, Fisher, Xavier, & Kronenberg, 2018). Upon discovery that downregulation of SIK2 in mice could induce eumelanogenesis in mice (Horike et al., 2010), a number of SIK inhibitors were developed for utilization as pharmacological enhancers of pigmentation. Treatment of human melanocytes with SIK inhibitor compound HG 9–91–01 increased MITF mRNA expression in a dose-dependent manner, and these observations were extended to in vivo studies, demonstrating a noticeable darkening of skin in Mc1r-deficient mice compared to control after 7 days of topical SIK inhibitor application (Mujahid et al., 2017). To address the poor cutaneous penetration of HG 9–91–01, Mujahid and colleagues created two “second generation” SIK inhibitors, YKL 06–061 and YKL 06–062, which had a darkening effect on human breast tissue extracts. Several SIK inhibitors have been developed but few have been evaluated for efficacy in manipulating skin pigmentation (Mujahid et al., 2017). There are three isoforms of SIK (SIK1/2/3), and the inhibitors described so far affect all of them. Several SIK inhibitors, such as compounds ARN 3236 and pterosin B, have SIK-specific inhibitory effect, however more work needs to be done to evaluate their efficacy as melanogenesis-inducing agents. Furthermore, several pharmacologicals such as dasatinib and bosutinib exert a non-specific inhibitory effect on SIKs through their interactions with various kinases (Sundberg et al., 2014). These compounds represent a potential route of investigation in the discovery of new pigmenting pharmacologicals.

Phosphodiesterase inhibition

Rolipram, a selective phosphodiesterase-4 inhibitor developed as a potential antidepressant drug in the early 1990s, inhibits phosphodiesterase (PDE) activity. PDEs terminate cAMP activity via enzymatic cleavage and inactivation (Huang & Mancini, 2006). Given that cAMP can cause melanin production via a MC1R-cAMP-PKA-MITF axis, inhibitors of PDEs are of obvious translational interest. Dozens of PDEs are expressed in human tissue but the PDE4D isoform is of particular interest to melanin synthesis given that knockout of PDE4D yields darker fur coloration in mice (Jin, Richard, Kuo, D’Ercole, & Conti, 1999). In an important study, Khaled, Levy & Fisher (Khaled, Levy, & Fisher, 2010) provided in vitro evidence that MITF regulates expression of PDE4D in human melanocytes, suggesting that a “homeostatic circuit” is formed in which a downstream product of cAMP activity inhibits MITF. Additionally, MITF expression was prolonged in melanocytes treated with rolipram. As a proof of principle, the group reported in vivo data clearly showing darker pigmentation and upregulation of melanin synthesis using the same K14-Scf extension mouse model that previously validated topical forskolin-mediated epidermal melanization. Interestingly, co-administration with forskolin led to even greater induction of skin pigmentation, suggesting a synergistic effect. While these initial results are promising, they must be viewed as proof-of-concept since past preclinical and clinical trials demonstrated that rolipram administration was associated with several concerning side effects including plasma osmolality imbalances and nausea/vomiting (Larson, Pino, Geiger, & Simeone, 1996). Given that these trials observed oral and intravenous administration of rolipram rather than topical application, further research is warranted to gauge the value of rolipram as a translational product.

Depalmitoylation Inhibitors

Palmitoylation of GPCRs has been reported to impact their signaling (Qanbar & Bouvier, 2003). Palmitoylation of MC1R was recently studied by Chen and co-workers who identified residue C315 as the major palmitoylation site of MC1R. C315S mutant human melanocytes failed to produce cAMP following α-MSH application and UVB exposure unlike controls, suggesting that palmitoylation affects MC1R-mediated melanogenesis (Chen et al., 2017). Using human melanocytes carrying an R151C mutation that models the red-headed, poor-tanning phenotype, Chen and co-workers found that application of Palm-B, a deacylating enzyme inhibitor, decreased MC1R depalmitoylation. In C57BL/6 mice carrying the R151C Mc1r mutation, administration of Palm-B both increased palmitoylation and enhanced photodamage repair after UVB exposure compared to Mc1r-r151c mice that did not receive Palm-B. A second depalmitoylation inhibitor, ML349, was shown to reduce melanomagenesis in a mouse model, indicating that inhibition of MC1R depalmitoylation was photoprotective (Chen et al., 2017). In these studies, Palm-B and ML349 were injected intraperitoneally when mice were used. While effective, topical applications might prove more translationally practical (Garcia-Borron & Jimenez-Cervantes, 2018), thus further work is indicated for their development.

Translational potential of pharmacologic melanin induction

The ability to increase the amount of melanin in the skin has clear implications for UV health and skin cancer prevention. If a safe and effective means to induce epidermal eumelanin were developed, this approach could potentially be used by individuals seeking to darken their skin (look tanner) by increasing epidermal eumelanin levels. Currently, use of tanning beds and purposeful recreational sun exposure in order to darken the skin is widespread and a clear risk factor for melanoma as well as non-melanomatous skin cancer (Rodriguez-Acevedo, Green, Sinclair, van Deventer, & Gordon, 2020; Schulman & Fisher, 2009). As reviewed above, UV-mediated adaptive pigmentation is based on damage to skin cells by UV photons, including the production of oxidative free radicals and DNA photodamage, which promote photoaging, inflammation and long-term malignancy risk. Therefore there is no “safe tan” that can come from UV exposure. Pharmacologic pigment induction would theoretically achieve the same result – more epidermal eumelanin deposition – without the long-term inherent risks of UV. Moreover, having more epidermal eumelanin would help protect the skin resist UV penetration, so melanized individuals would be more protected from the immediate and long-term toxicities of UV exposure. Indeed, epidermal melanin offers many advantages over commercially-available sunscreens that have to be reapplied at regular intervals, have had mixed results with respect to cancer prevention (Mulliken, Russak, & Rigel, 2012; Wehner, 2018; Weinstock, 1999), can leach out into the environment (Langford & Thomas, 2008; Raffa, Pergolizzi, Taylor, Kitzen, & Group, 2019) and can be absorbed systemically raising the possibility of adverse health consequences (Abbasi, 2020; Matta et al., 2020). Epidermal eumelanin induction represents an innate mechanism of UV resistance by functioning as a built-in sunblock.

Despite remarkable progress made in the fight against cutaneous malignancies – notably those based on targeted inhibition of oncogenic driver signaling pathways and on immune checkpoint blockade – skin cancer incidence is still rising and new preventive approaches are needed. By 2030 the cost of treatment for U.S. patients with melanoma is expected to rise to $1.6 billion (Guy, Berkowitz, Holman, & Hartman, 2015). This does not even take into account the terrible morbidity and mortality associated with skin malignancies and UV pathology. The pharmacological induction of innate pigment represents an excellent candidate approach for reducing risk of melanoma and other UV-driven non-melanomatous skin cancers.