Multiple Genes and Diverse Hierarchical Pathways Affect Human Pigmentation

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Chapter 3

MULTIPLE GENES AND DIVERSE HIERARCHICAL PATHWAYS AFFECT HUMAN PIGMENTATION

C. Ganesh*1, Anita Damodaran1, Martin R. Green2, Sheila Rocha3, Nicole Pauloski3 and Shilpa Vora1 1Unilever R&D Bangalore, India 2Unilever R&D Colworth, UK 3Unilever R&D Trumbull, US

ABSTRACT

One of the most easily visible and recognized human physical attributes is skin color which is largely determined by the amount and type of melanin in skin and the influence of haemoglobin. Human genetic adaptation, multiple geographic origins and intermixing during migration of human population has resulted in a naturally wide palette of skin color. Exposure to sunlight (which varies in intensity across geographical locations), immune reactions, hormonal changes and aging alter skin appearance and pigmentation through multiple mechanisms. Many genes are involved in the control of the type and amount of melanin synthesized in melanocytes and its subsequent transfer to and distribution within keratinocytes. We report our observations on the differential changes in melanin content in human melanocytes, on modulating various

* Correspondence: [email protected]; [email protected]. 90 C. Ganesh, Anita Damodaran, Martin R. Green et al.

pigmentation pathways. Further, IL-1 was shown to be an upstream regulator of many of these pigmentation pathways. Multiple SNPs have been mapped in the genes linked to human pigmentation including MC1R, TYR, SLC45A2, KIT, EDNRB and SLC24A5. Our ground breaking studies demonstrated that variation of the non-synonymous SNP rs1426654 in SLC24A5 encoding the NCKX5 protein amino acid change A111T, accounted for over 30% of the variance in the constitutive skin color of South Asians. Diverse hyper- and hypopigmentation disorders have been well documented as local spots, vitiligo, melasma and mosaic pigmentation. Our investigations of such disorders have highlighted the role of genes involved in pigmentation, cell adhesion and communication, immune processes and lipid metabolism. Leveraging scientific advances in functional genomics has led to increased awareness of the intricate regulation of human pigmentation and altered modulation in pigmentary disorders. As understanding of pigmentary processes improves, our investigations will also translate discoveries into more effective safe cosmetic and pharmaceutical interventions for skin pigmentation benefits.

INTRODUCTION

By virtue of its immediate striking visibility, pigmentation has captured the attention of both scientists and laymen alike. There occurs naturally, wide variations in pigmentation and many pigmentary diseases and disorders have been well described [Goldsmith et al., 2012]. These have triggered intense discussion, hypotheses, commentaries and rigorous scientific investigations across centuries. It is evident that such complex phenomena are an outcome of complex genetics and environmental pressures. Multitudes of pigmentary genes have been identified, cloned and characterized [Lamoreux et al., Eds. 2010]. Complementary biochemical and cell biological investigations have uncovered fundamental processes governing pigmentation and its control. Excellent texts [Levine and Maibach, 2003; Nordlund et al., Eds., 2006; Quevedo WC and Holstein TJ, 2006; Borovansy and Riley, 2011] and other references [Yamaguchi and Hearing, 2009; Kondo and Hearing, 2011] abound in such areas of scientific investigations. The most striking example of phenotypic change due to underlying mutations in pigmentation genes, is seen in human albinism. Oculocutaneous albinism type 1 caused by mutations in the Tyrosinase gene, is one example of albinism. It is amongst the best understood [Oetting et al., 2003] and malfunction of tyrosinase compromises the quality of life (e.g. sunburn, vision problems etc.) due to little or no production of melanin. The fundamental Multiple Genes and Diverse Hierarchical Pathways … 91 importance of tyrosinase and related proteins to pigmentation have been well reviewed [Hearing, 2011] and it is understood that tyrosinase is regulated at multiple levels of transcription, post translation and enzymatic activity [Schallreuter et al., 2007; Ebanks et al., 2009]. Melanogenic proteins are the culmination points of diverse cellular signaling pathways in the melanocytes [Imokawa, 2004; Lin and Fisher, 2007; Schiaffino, 2010] which are regulated by intricate diverse interactions between melanocytes, keratinocytes and fibroblasts [Hirobe, 2005; Kondo and Hearing, 2011].

MAJOR PATHWAYS IN PIGMENTATION

The origins of signaling events in skin can be traced as responses to triggers such as UV radiation, hormones, growth factors and inflammation [Slominski et al., 2004; Yamaguchi and Hearing, 2009]. Classical studies have examined signaling systems such as MSH:MC1R [Abdel-Malek et al., 1995; Abdel-Malek et al., 1999; Suzuki et al., 1996; Millington, 2006; Eves and Haycock, 2010; Dessinioti et al., 2011], SCF:cKIT [Giebel and Spritz, 1991; Spritz et al., 1993; Lennartsson and Rönnstrand, 2012], WNT:FZD [Dorsky et al., 2000; Yamaguchi et al., 2008; Yamaguchi et al., 2009] and EDN:ENDR [Imokawa et al., 1992; Imokawa et al., 1995; Imokawa et al., 1996; Imokawa et al., 1997]. The cognate receptors are predominantly of the GPCR variety. In addition, literature describes the effect of mutations in pigmentation genes on melanin production [Lamoreux et al., 2010]. Some mutations result in unique changes in pigmentation, with albinism as the most severe phenotype. For long, these signaling pathways and their significance in the control of melanogenesis have been investigated, usually one pathway at a time. In tune with the paradigm shift in biology, integrated systems biology approaches now allow investigations on the extensive cross talk and relationship between pigmentation pathways [Imokawa et al., 2000; Bellei et al., 2011; Herraiz et al., 2011]. Physiological pathways operate not as isolated individual entities, but as coordinated interactive units. We chose the human primary melanocyte as the model system to discern contributions from multiple signaling pathways to melanin content. Either signaling pathways agonists and antagonists or siRNA (receptor gene specific transient knockdown) intervention was used, followed by quantitative photometric assessments of resultant changes in melanin content (the physiologically relevant end point). (figure 1 and table 1). While MSH increased melanin content and cKit phosphorylation inhibitor (ISCK03) led to 92 C. Ganesh, Anita Damodaran, Martin R. Green et al. substantial decrease in melanin content, other treatments resulted in no change (fig. 1). Experiments using agonists and antagonists are challenging to a certain extent, as typical cell culture media are replete complex mixtures, including critical growth factors. Necessary inclusion of such material in the media could result in an artificially blunted response, saturation of or even no effects in regard to an added test material.

Figure 1. Effect of select receptor specific agonist or antagonist on cellular melanin content in human primary melanocytes. Cells in culture were treated with the indicated amounts of either the agonist or antagonist, for three days. Total cellular melanin was estimated using the regular A405nm method and normalized with respect to control (reference 100%), after accounting for changes in cell count (neutral red assay). Only MSH (MC1R agonist) and ISCK03 (cKIT RTK activity inhibitor) altered melanin content. Results were from at least triplicate measurements.

A complementary approach is to alter cognate receptor levels by altering their gene expression (siRNA). Table 1 depicts the observed differential levels of reduction in melanin, upon reducing of the expression of melanocyte receptors. Interestingly, we observed that the knockdown of type A endothelin receptor was far more effective in reducing melanin content, than type B receptor. A complex pattern of results was observed that was dependent on siRNA duplex and time point. Our observations overall indicate that diverse signaling pathways contribute to melanin contentand that EDNRA and FZD agonist/antagonists do not exhibit the effect of mRNA knock down experiments.

Multiple Genes and Diverse Hierarchical Pathways … 93

Table 1. Effect of siRNA against select major melanogenic receptors on cellular melanin content in human primary melanocytes. The maximal extent of reduction in melanin content is shown. It should be noted that the maximal reduction in different cases occurred at different time points and with different concentrations of siRNA oligos (purchased from Invitrogen). Mirus reagent was used to transfect the siRNA oligos into melanocytes and each case was standardized with respect to oligo concentrations and time. % reduction in gene expression were measured by qRTPCR and melanin content was estimated by A405nm method

CYTOKINE REGULATION OF MELANOGENESIS

Epidermal keratinocytes are known to release many growth factors involved in inflammation and melanogenesis [Takashima & Bergstresser, 1996; Ansel et al., 1990]. It is well documented that external stimuli such as UV and allergic contact dermatitis can induce keratinocytes to secrete mitogens and pro-melanogenic factors including ET-1 [Imokawa, 1992], NGF [Tron et al., 1990], NO [Romero-Graillet et al., 1997], -MSH [Rousseau et al., 2007] and lipid mediators such as prostaglandins [Tomita et al., 1992; Scott et al., 2005], which increase proliferation, melanin synthesis or dendricity by melanocytes [reviewed in Imokawa, 2004; Hirobe, 2005; Yamaguchi and Hearing, 2009]. Melanocytes express specific receptors for growth factors and the ligand receptor interactions then signal for various melanocyte functions. 94 C. Ganesh, Anita Damodaran, Martin R. Green et al.

UV initiates several signaling cascades which are also common to various growth factor and cytokine mediated pathways. However, how UV initiates these signals is still unclear [Gilchrest et al., 1996: Rosette and Karin 1996; Fischer et al., 2002]. Various intermediates speculated in UV responses are reactive oxygen species, ROS [Gross et al., 1999], cytokines like IL-1 [Griswold et al., 1991] or lipid mediators [Tomita et al., 1992; Scott et al., 2005]. In skin keratinocytes, IL-1 exists as preformed protein complex, which can be released immediately in response to a noxious stimulus [Brink et al., 2000; Ashida et al., 2001; Luo et al., 2004; de Jongh et al., 2007] and plays an important role in the pathophysiology of skin inflammation and wound healing [Kominine et al., 2000; Murphy et al., 2000; Freedberg et al., 2001]. Also, it has been reported [Okazaki et al., 2005] that the amount of IL-1 secreted by keratinocytes from aged volunteers was higher than the young volunteers, which suggests a role of Il-1 in age related changes in skin. Studies have revealed that both release [Murphy et al., 1989] and synthesis [Griswold et al., 1991; Lew et al., 1995] of IL-1is maximum 1 hr. post UV exposure in epidermis. Meanwhile, others studies report extended release and synthesis of IL-1 for 3-72 hrs. [Imokawa, 1995; Luo et al., 2004] in skin epidermis. Since IL-1 is the only factor which is preformed and stored in keratinocytes, we hypothesised IL-1 to be one of the intermediate as well as a common initiator, through which UV effects both inflammatory and melanogenic changes in epidermis. Our studies demonstrated that keratinocytes do indeed constitutively produce large amounts of IL-1(fig. 2). UV irradiation further increased the production and secretion of IL-1α in a short period (4hrs.) without any change in cell viability, suggesting a specific mechanism for release of preformed IL-1 by keratinocytes in response to UV. The IL-1 gene family comprises of IL-1α & β and the IL-1Ra receptor antagonist. IL-1α & β are formed as a 31 kDa precursor protein [Dinarello, 2002]. This leaderless peptide gets processed into active 17 kDa forms by the action of proteases, specifically Caspase-1 [Faustin and Reed, 2007; Martinon and Tschopp, 2007]. A set of proteins forming the ‘inflammasomes’, a multi- protein innate immune complex, have been implicated in the maturation and secretion of IL-1in various immune cells [Martinon and Tschopp, 2007]. It has been demonstrated [Feldmeyer et al., 2008] that UVB mediated enhancement of cytoplasmic Ca+2 is required for the activation of caspase 1 by the inflammasomes, for maturation of IL-1 in keratinocytes and implicated the process in UV induced sunburn. Recently, new mechanisms involving Multiple Genes and Diverse Hierarchical Pathways … 95 cAMP and NO activating NLRP3 inflammasome in the processing of IL-1 has been suggested [Leavy, 2013]. However, it is unclear if the process is same for IL-1maturationin keratinocytes, though it has been demonstrated that both proIL-1 and processed IL-1 are functionally active [Werman et al., 2004]. UV induced IL-1 has been demonstrated in various studies in skin [Murphy et al., 1989; Griswold et al., 1991; Lew et al., 1995] and keratinocytes [Kupper et al., 1987 and fig. 2]. However, unlike an earlier report [Swope et al., 1994], we did not detect IL-1 or  in melanocytes (fig. 3). The receptors for IL-1 were expressed by both keratinocytes and melanocytes, suggesting that IL-1 produced by keratinocytes can regulate functions of both keratinocytes and melanocytes in autocrine and paracrine manners respectively. Thus the keratinocyte appears to be the major and most important source of active preformed IL-1 in skin [Murphy et al., 2000].

Figure 2. Constitutive and UV induced release of IL-1 α by keratinocytes. Confluent HaCaT (immortal transformed human keratinocyte cell line) and primary human keratinocytes (1o K)were irradiated (100 mJ/cm2 UVA and 20mJ/cm2 UVB). Culture supernatant media was collected 4 hrs. post UV exposure and analyzed by ELISA for IL-1α. HaCaT cells were a kind gift from Dr. Norbert Fusenig.

Earlier studies have demonstrated that IL-1α can induce the production of ET-1 [Imokawa et al., 1995], POMC/MSH/ACTH [Funasaka et al., 1998, Scholzen et al., 2000], SCF [Da Silva et al., 2003] and COX2 [Kessler- Beckker et al., 2004]. ET-1, SCF and POMC are melanogenic mediators produced by keratinocytes upon UV exposure. However, the mechanism or 96 C. Ganesh, Anita Damodaran, Martin R. Green et al. signaling events involved in their synthesis is unclear. In our study, expression of these molecules was induced by IL-1 (fig. 4) to a higher level than by UV during early time points. This suggests that the direct effect of IL-1 is faster than that of UV as the latter needs a preceding build up of IL-1. Further, induction by UV was inhibited by anti-IL-1 antibody confirming that UV induction of these melanogenic molecules is under the regulation of IL-1.

b Figure 3. Endogenous levels of IL-1α mRNA and IL-1 mRNA in melanocytes and keratinocytes. Total RNA was extracted from confluent cultures of HaCaT cells, primary human melanocytes and keratinocytes. Real time PCR was carried out to determine the endogenous level of IL-1(A) and IL-1 (B) mRNA. Multiple Genes and Diverse Hierarchical Pathways … 97

Figure 4. Induction of COX-2, SCF, POMC and ET1 in keratinocytes on IL-1 treatment. Confluent primary keratinocytes were treated with 10ng of IL-1 and 2g of IL-1antibody for 1 hr. and UV (100 mJ/cm2 UVA and 20mJ/cm2 UVB). Total RNA was isolated and semi-quantitative PCR was performed using specific primers for COX-2, SCF, POMC, ET1 and GAPDH (internal control). PCR products were then separated through 2% agarose gel and visualized by ethidium bromide staining. Photocap software was used to digitize the data and converted to fold changes. Lanes 1-4 are: Control, 10ng IL-1treatment, UV followed by 2g of IL-1 Antibody and UV treatment.

IL-1 regulation of melanogenesis has been reported in organ cultured guinea pig skin [Maeda et al., 1996] and in vitro in mouse melanoblasts [Hirobe and Ootaka, 2007], while it has been demonstrated that IL1 treatment reduced tyrosinase activity in human melanocytes [Swope et al 1991; Abdel- Malek et al., 1993]. In our study, we observed that UV and IL-1 exposed keratinocytes induced increase in melanin content (fig. 5) and tyrosinase activity (data not shown), which could be abolished by anti-IL-1 antibody in co-cultures. Primary human keratinocytes were treated with IL-1 for 6hrs, supernatant collected and primary melanocytes were treated with those supernatant for 4hrs. Tyrosinase expression in melanocytes was quantified by 98 C. Ganesh, Anita Damodaran, Martin R. Green et al. qRTPCR (GAPDH reference gene). We observed that tyrosinase expression increased to ~3, 9 and 7 fold upon treatment of melanocytes with spent media from control keratinocytes or IL1 treated keratinocytes or IL1 treated keratinocytes respectively (reference 1-fold in untreated melanocytes). By contrast, direct addition of IL-1/ to melanocyte culture did not affect either melanin synthesis or tyrosinase activity or expression.

Figure 5. Melanin content estimation in co-cultures where keratinocytes were treated with IL-1 prior to melanocyte addition. Primary keratinocytes were treated with IL- 1α, UV and UV followed by addition of anti-IL-1α antibody (ab). After 24hrs., melanocytes were added to the keratinocytes culture and incubated for further 72hrs, prior to the estimation of melanin content (change by IL1 treatment was significant over control at p<0.01).

Our study also demonstrated an autocrine effect of IL-1 on keratinocytes resulting in production of more IL-1 (data not shown) as well as other melanogenic gene products, suggesting a self sustained keratinocyte initiated IL-1 signal cascade. It is known that IL-1R levels on the keratinocyte surface increase 9-20 fold on differentiation, although IL-1 protein level does not change much on cell differentiation [Blanton et al., 1989]. This suggests that differentiated keratinocytes, on external stress, release IL-1 and can be activated in an autocrine manner, to produce keratinocyte and melanocyte growth and melanogenic factors. This serves to propagate the UV signal via IL-1. We have thus identified and confirmed the role of IL-1α in regulating melanogenesis by activation of keratinocytes to release melanogenic factors. Further studies are required to identify the molecular mechanism involved in IL-1α induction of melanogenic factors.

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GENES INFLUENCING NATURAL VARIATION IN HUMAN SKIN COLOUR

Human, visible skin colour variation is largely due to amount and type of melanin in skin [Alaluf et al., 2002a]. Darker skin colours are dominated by darker eumelanin while the lighter yellow/orange pheomelanin contributes to fairer skin types. Blood and the organisation of blood-vessels can provide a reddish hue while carotenoids contribute to skin yellowness [Alaluf et al., 2002b], and other structures in paler skin such as ‘blue’ veins are caused by differential light reflection and absorption. Over the past 30,000y skin colour variation has been thought to be due to human adaptation to variations in sun light intensity particularly UVB, as mankind moved north and occupied a wide range of geographical locations [Beleza et al., 2012]. The ancestral colour of human skin is considered to be black with lightening suggested to be driven by need for UVB catalysed vitamin D synthesis and/or protection of folate in skin. However recently the ‘vitamin D’ theory has been challenged by the hypothesis that pigmentation is essential to prevent dehydration and sun burn, by protecting the skin epidermal water barrier from the destructive power of sun light. Universally, females within populations appear to be lighter in skin colour, an observation variously ascribed to sexual selection or the need by females for higher levels of vitamin D for successful reproduction. Many gene products are required for and influence the amount and nature of melanin production in skin. For example the color-genes web site http:// www.espcr.org/micemut/ on Dec 2012 listed 378 loci linked to pigmentation of which 171 were cloned genes having human homologues. A siRNA based functional genomics study identified at least 92 genes affecting the degree of melanin production by human melanocytes (MNT-1 cells). Interestingly despite the number of genes required for pigment production, human albinism is restricted to inactivating mutations in just 4 genes (TYR, OCA2, TRP1 and SLC45A2) the protein products of all of which are specifically located in functional form in the melanosome. Melanosomes are lysosome-related organelles [Raposo et al., 2007] and melanin production has parallels with autophagy [Ganesan et al., 2008]. One might speculate that other inactivating mutations that might give rise to albinism in humans are selected against because of essential, life sustaining function(s) in other cellular pathways.

Table 2. Genes linked to normal (constitutive) variation of skin-colour in human populations

Table 2. (Continued)

102 C. Ganesh, Anita Damodaran, Martin R. Green et al.

Several pigmentation gene variants have shown a remarkable degree of selection in human populations with some gene variants moving to fixation very recently, that is within the last 11 to19,000y [Beleza et al., 2012]. Such genetic adaptation of skin colour genes is almost certainly driven by the human survival advantages noted above. Other sets of genes may also have been subject to recent selection for example the worldwide variation in human mitochondrial DNA responding to climate [Balloux et al., 2009]. A striking example of variant selection linked to skin colour is provided by SLC24A5 which codes for the potassium-dependent sodium-calcium exchanger family member 5 (NCKX5). In a genome wide association study of skin pigmentation in a South Asian population, SLC24A5 was found to account for >30% of the variance in dichotomously defined light and dark skin colour [Stokowski et al., 2007]. Data from the HapMap project [International HapMap Consortium, 2005] shows that alternate alleles of the non-synonymous (ns) SNP rs1426654 in the SLC24A5 gene are present almost mutually exclusively in African and European populations. A different inactivating mutation in the zebrafish homologue is responsible for the lighter golden pigment phenotype [Lamason et al., 2005]. Recently a predicted inactivating 4-nucleotide insertion into SLC24A5 has been described in a person showing extreme cutaneous hyperpigmentation (Mondal et al., 2012). The nsSNP (rs1426654) changes the coding amino acid at position 111 in NCKX5 from alanine to threonine (pA111T) and results in a greatly reduced exchange function of the protein expressed in ‘High Five’ insect cells [Ginger et al., 2007]. NCKX5 is primarily located in the trans-Golgi network and the mechanism by which altered ion exchange activity regulates pigmentation remains unconfirmed, though Lamason et al., [2005] have proposed a role in melanosomal calcium uptake and a location for NCKX5 in the melanosomal membrane. Surprisingly NCKX5 knockdown perturbs sterol and particularly cholesterol metabolism in melanocytes [Wilson et al., 2012] a lipid which has been shown to regulate melanogenesis [Hall et al., 2004; Jin et al., 2008; Schallreuter et al., 2009]. Apart from SLC24A5 other pigment genes showing a high degree of selection in human populations are ASIP, KITLG, MC1R, OCA2, SLC45A2, TYR, TYRP1 [Norton et al., 2007; Sturm et al., 2009; Edwards et al., 2010; Donnelly et al., 2012; see also table 2]. Given the complex genetic nature of many continuous human traits, remarkably few genes to date have a confirmed role in natural, constitutive variation in human skin colour. Genes are listed in table 2 as confirmed if there is at least two independent pieces of evidence from human skin colour Multiple Genes and Diverse Hierarchical Pathways … 103 studies, or candidates if the link is inferred, of borderline significance and/or supported by only one reference. The table also lists pigmentation informative SNPs, the function of the SNP if known, and the human populations affective by the alternate allele. Accordingly there are so far perhaps 8 confirmed (ASIP, IRF4, KITG, MC1R, OCA2, SLC24A5, SLC45A2, TYR) and 9 candidate genes that have had variants selected through a process of human adaptation to new environments.

Figure 6. Genes whose variants are associated with natural variations in human skin colour. Both the well documented genes and candidate genes have been depicted. It is interesting to note that although most genes are expressed in melanocytes, they participate in diverse stages of melanogenesis.

As hundreds of genes are required for pigmentation but so few are linked to natural human skin colour variation (table 2 and fig. 6), it is instructive to ask why this may be. Although further genes influencing constitutive human skin colour variation may remain to be discovered it is highly likely that all the major skin colour ‘effect’ genes such as SLC24A5, have been discovered in genome wide association investigations. It is possible that over the last 30,000y the stochastic gene variant selection process may not have sampled all the available possibilities leaving safe intervention targets still to be discovered. Alternatively and more likely is that very few genes essential for 104 C. Ganesh, Anita Damodaran, Martin R. Green et al. pigment production can have their function altered to reduce pigmentation in a safe and effective manner without adversely affecting other important cellular processes in the human body. As noted above, melanosomes are lysosomal- related organelles [Raposo et al., 2007] and pigment synthesis shares processes with autophagy [Ganesan et al., 2008] and both processes that might therefore be adversely affected by changes in pigment genes. Brinkman et al., [2006] argued a parallel case by suggesting that the study of the human phenome [the set of all human phenotypes] and associated underpinning genetic variation is a good place to start in order to discover safe and effective drug targets. Hence it is probable that many gene variants have been ‘tested’ by survival pressures as human adapted to new environments, but very few of those genes variants have been ‘selected’ having safe function and provided sufficient advantage to become established in the human population.

HYPERPIGMENTATION OF SKIN

Hyperpigmentation is a localised darkening of the skin. It is most common in people with darker skin tones of Asian, Mediterranean, or African descent. It is a major clinical problem that can affect the life of a person by altering his or her appearance. The visual impact of hyperpigmentation often causes considerable psychological distress [Brown et al., 2008]. In fact it has been ranked the top cosmetic dermatological concern for which people seek a dermatologist. Hyperpigmentation can result from many factors and can present itself in different forms throughout a person’s life. Chronic sun damage, inflammation due to acne vulgaris or other skin injuries and hormonal imbalances can all lead to some form of hyperpigmentation. It can be diffuse or local and is often associated with underlying medical conditions. However, ultimately it is strongly linked to an over production of melanin by the melanocytes. It can occur anywhere on body especially on sun exposed skin, but the areas of greatest esthetic concern are the face, hands and upper body. Hyperpigmentation has been associated with numerous diseases or conditions. It has been linked to adrenal hormonal imbalance in cases of Addison’s disease and Cushing’s disease. It is a symptom of sex hormone imbalance as in cases of melasma or chloasma and Linea nigra, associated with insulin resistance in cases of Acanthosis nigricans and linked to many other syndromes or disorders such as Nelson’s syndrome. Other forms of hyper pigmentation such as Leopard syndrome, freckles, and nevi have been linked to genomic mutations or SNPs. However, the forms of Multiple Genes and Diverse Hierarchical Pathways … 105 hyperpigmentation that impact even-skin tone in the majority of the population, are melasma, post-inflammatory hyperpigmentation (PIH) and solar lentigines. Melasma is a patchy brown, tan, or blue-gray discoloration that occurs mainly on the face. It is most common in women, however more recently there have been reports of melasma in men [Vachiramon et al., 2012]. Some reports link a genetic predisposition as a major factor in male melasma [Sarkar et al., 2010]. Although a high number of men with melasma have a family history of the condition, interestingly none of them reported melasma in their fathers [Vazquez et al., 2010]. Therefore, there is a high potential for an X-linked chromosome aberration responsible for the condition in men. In the female population it is most common among pregnant women with olive or darker skin tone such as Hispanic, Asian, and Middle Eastern individuals [Grimes 1995]. Despite a great deal of research in the area, the exact cause of melasma remains largely unknown. It has been reported to be triggered by several factors such as pregnancy, birth control pills, hormone [HRT and progesterone] etc. and predisposition due to family history and race. Sun exposure is also a key factor, especially in individuals with a genetic predisposition for developing melasma. Once present it is responsive to sun exposure and therefore darkens during summer months and fades slightly over the winter months. Post-inflammatory hyperpigmentation (PIH) can occur as a consequence of exposure to certain chemicals or inflammatory agents such as in acne or fungal infections. PIH has also been linked to shaving or plucking in the axilla [Evans 2012]. The sun has been implicated in exacerbating various forms of PIH. It is less characterized because it is a sensitive area to biopsy without significant risk of further worsening the condition. It is believed that the inflammatory cytokines released by the inflammatory cells that infiltrate the area in response to the skin challenge can trigger the melanocytes to produce more melanin. Chronic sun exposure is responsible for the development of another form of hyperpigmentation, solar lentigines. Solar Lentigines or age spots develop on sun-damaged skin of the face, the back of the hands, lateral forearms, the back and chest. They are characterized by a hyperpigmented basal layer, elongated rete ridges and increased numbers of melanocytes. Aside from the apparent activated state, the melanocytes appear otherwise normal with melanosomes present in all stages of maturation in the cytoplasm and in dendrites. Increased expressions of melanogenesis-specific genes and proteins such as POMC, TYR, TYRP-1, DCT, PMEL-17, and OCA2 have been 106 C. Ganesh, Anita Damodaran, Martin R. Green et al. confirmed. It has been proposed that there is a perturbation in keratinocyte differentiation. Genomic profiling studies have been completed to aid in characterizing solar lentigines. Insights generated in these studies confirm the up regulation of melanogenesis specific gene and inflammatory pathways. In order to expand on this work we have investigated the expression profile of microRNAs of solar Lentigines. Only a subset of the genes in the human genome has been confirmed to be active in any cell type and it is now known that around 98% of the human genome consists of non-protein coding regions (The ENCODE project consortium, 2012). However approximately 76% of human DNA is actively transcribed into functional primary RNA transcripts or non-coding RNAs (ncRNAs). Some of these ncRNAs are involved in post-transcriptional regulation, mainly small interfering RNAs (siRNAs) and microRNAs (miRNAs). MicroRNAs are small endogenous RNA molecules that play essential roles in regulation of gene expression and a wide range of cellular processes. They are believed to be coded for about 1-3% of the mammalian genome. It is estimated that one-third of the protein-coding mRNAs are regulated by miRNAs. Although their functions have not been completely elucidated it has been confirmed that many regulate mRNAs by targeting them for degradation or inhibiting their translation. MicroRNAs have recently been shown to play a pivotal a role in a variety of skin diseases. Several miRNAs have been implicated in wound healing and inflammatory skin conditions. In order to gain insight into the genomic profile of hyperpigmented lesions we explored the expression profile of miRNAs and mRNAs in solar lentigines compared and photo-exposed and [peri-lesion] and photo protected skin. Through the analysis of over 160 skin biopsy samples we have identified characteristic expression profiles in these hyperpigmented lesions that can guide our understanding of its etiology. The microRNA profile for age spots reveals that there is a gradual increased expression of specific microRNAs from photo-protected to peri-lesional skin to solar Lentigines (fig. 7). The profile of these microRNA reveal a significant change in microRNAs that regulate genes involved in lipid and fatty acid metabolism as well as inflammation. Further studies are on going to understand the role of microRNAs in the etiology of solar Lentigines.

Multiple Genes and Diverse Hierarchical Pathways … 107

Figure 7. miRNA array data on solar lentigines. Statistical testing was performed on the array data assuming a linear model of condition as an ordinal variable [photo- protected < peri-lesion < lesion] and subject to test the effects of each condition relative to photo-protected. Probes were filtered to ensure FDR < 1% and monotonic increase or decrease across the three conditions. 49 miRNAs were considered significant. On the heat map, red indicates relatively higher expression and green indicates relatively lower expression.

Although several topical over the counter and prescription creams are marketed for ameliorating various forms of hyperpigmentation, to date the most efficacious treatments require in office visits to dermatologists. In the cases that involve hormonal imbalance, medical treatments for these conditions usually lead to some relief of the hyperpigmentation. For the aesthetic conditions that do not respond to those types of treatments the only options available today are chemical peels, cryosurgery or laser treatments. Therefore, in order to provide more efficacious, specific and less invasive treatment options for hyperpigmentation conditions, further characterization of each type of hyperpigmentation is needed.

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MOSAIC HYPOPIGMENTATION OF SKIN

Depigmentation of skin is also widespread in human population. Classical examples of hypopigmented skin conditions include the earlier discussed albinism as well as vitiligo [Gawkrodger et al., 2010; Guerra et al., 2010]. However, there exists other distinct hypopigmented conditions, which are being characterized. We have observed and described one such [Mollet et al., 2007] mosaic hypo pigmentary pattern, characterized by hypopigmented patches in the background of normal pigmentation in hands, legs and torso. This pattern is present from birth and persists through the life of the affected individuals, who otherwise lead normal healthy lives. The condition is very similar to nevus depigmentosus [Lee et al., 1999; Khandpur and Sumanth, 2005; Kim et al., 2006] but distinct from vitiligo, according to the opinion of expert dermatologists. A family tree (figure 8) analysis suggests possible genetic association, in a family native to the southern part of India.

Figure 8. Family tree of individuals from southern part of India, who display blotchy skin hypopigmentation patterns. Numbers indicate individuals whose skin samples were analyzed by histology as well as microarray. Multiple Genes and Diverse Hierarchical Pathways … 109

It was evident from Mason-Fontana stained sections of the normal and hypopigmented patches (data not shown) that the overall melanin content in the lighter patches was much reduced but not completely absent compared to adjacent normal skin. The regular basal layer in normal skin was seen enriched in melanin, while melanin was grainy in the lighter patches. RNA was isolated from such skin biopsies and analyzed by microarray. It was observed that ~200 genes were differentially regulated (comparable numbers up and down regulated). Broadly, those genes were under the categories of cell adhesion, phosphatidyl inositol signaling, lipids metabolism, immune function related, steroid metabolism etc. In terms of gene ontology, significant categories include signal transduction (GO:4871) and lipid/fatty acid metabolism (GO:6629/31). Genes with links to organelle biology/intracellular transport were also found to be differentially regulated. In contrast, although several melanogenic genes were represented by multiple spots in the microarray chip, their expression did not vary uniformly between samples. It is clear that substantial reduction in pigmentation/skin colour can be observed, even when the expression of critical core melanogenic genes are unaffected. As noted elsewhere pigmentary differences can arise from altered cellular signaling enhancing the possibility of intervening at different levels, to safely modulate skin colour.

CONCLUSION

Our work discussed in this chapter exemplifies the complex nature of signaling pathways related to pigmentation. Although multiple pathways are involved, there appears to be a graded contribution towards melanin content. Being a primary organ for the defence of the body, skin is unique by virtue of its intrinsically highly visible nature. Human adaptation to new geographic environments has generated a continuous palette of skin colours biologically enabled by highly regulated intricate signaling pathways. Deeper and wider understanding of cellular signaling events in pigmentation can open up new vista to safely modulate skin colour and tackle a wide range of problems associated with pigmentation. Both Pharma and Cosmetics domains can benefit by leveraging a wide variety of scientific approaches and modern genomic tools to address the fundamental biology of pigmentation.

110 C. Ganesh, Anita Damodaran, Martin R. Green et al.

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