Author response: Sex steroids regulate skin pigmentation through nonclassical membrane-bound receptors (2016)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The association between pregnancy and altered cutaneous pigmentation has been documented for over two millennia, suggesting that sex hormones play a role in regulating epidermal melanocyte (MC) homeostasis. Here we show that physiologic estrogen (17β-estradiol) and progesterone reciprocally regulate melanin synthesis. This is intriguing given that we also show that normal primary human MCs lack classical estrogen or progesterone receptors (ER or PR). Utilizing both genetic and pharmacologic approaches, we establish that sex steroid effects on human pigment synthesis are mediated by the membrane-bound, steroid hormone receptors G protein-coupled estrogen receptor (GPER), and progestin and adipoQ receptor 7 (PAQR7). Activity of these receptors was activated or inhibited by synthetic estrogen or progesterone analogs that do not bind to ER or PR. As safe and effective treatment options for skin pigmentation disorders are limited, these specific GPER and PAQR7 ligands may represent a novel class of therapeutics. https://doi.org/10.7554/eLife.15104.001 eLife digest Factors controlling pigment production in skin are complex and poorly understood. Cells called melanocytes produce a pigment called melanin, which makes the skin darker. It has been known for a long time that skin color often changes during pregnancy, which suggests that sex hormones may be involved. However, the specific hormones and signaling mechanisms responsible for the changes have remained largely undefined. Estrogen and progesterone are two of the main female sex hormones. Natale et al. now show that estrogen increases pigment production in human melanocytes, and progesterone decreases it. For hormones to signal to cells, they must bind to and activate particular receptor proteins. Further investigation by Natale et al. revealed that estrogen and progesterone regulate pigment production by binding to receptors that belong to a family called G protein-coupled receptors. These receptors can signal rapidly once activated by sex hormones, and may serve as therapeutic targets for treating pigmentation disorders. Skin diseases that cause inflammation often also cause changes in skin color. Natale et al. noticed several other G protein-coupled receptors that are likely to control pigmentation through similar mechanisms. Future analyses of the roles that these other receptors perform in melanocytes may therefore reveal how inflammation-based pigmentation changes occur. https://doi.org/10.7554/eLife.15104.002 Introduction Cutaneous pigmentary changes have been long recognized as common side effects of pregnancy. The British physician Daniel Turner, in his 1714 De Morbis Cutaneis (Turner, 1726), references Hippocrates (460–370 B.C.E.), "There is a spot on the face…more peculiar, according to our great master Hippoc., to Big Belly'd women, and recon'd as one of the Signs of Conception." Modern physicians recognize this common pregnancy-associated hyperpigmentation as melasma (Sheth and Pandya, 2011; Nicolaidou and Katsambas, 2014). Hippocrates also thought that the pigment was predictive of the sex of the fetus: Quae utero gerentes, maculum in facie veluti ex solis adustione habent, eae faemellas plerumque gestant. Translated to English: pregnant women who have a mark on the face as though stained by the sun, quite often give birth to girls. While Turner noted this association with fetal sex to be 'fallible', Hippocrates was remarkably astute in linking the pigment increases to the tanning response to DNA-damaging solar ultraviolet (UV) radiation. While early physicians attributed the pigment changes to "Retention of the menstrual Flux" (Turner and Cutaneis, 1726), the molecular mechanisms through which pregnancy-associated hormonal changes modulate skin color have remained elusive for over 2,000 years. Melanocytes in the basal epidermis control skin pigmentation through synthesis of melanin, a complex process thought to be primarily regulated by alpha-melanocyte stimulating hormone (αMSH) (Figure 1—figure supplement 1A and B). The αMSH peptide is secreted centrally by the anterior pituitary gland, and locally by keratinocytes in response to UV damage (Cui et al., 2007). αMSH binding to the melanocortin receptor 1 (MC1R), a G protein-coupled receptor (GPCR), activates adenylate cyclase, and increases cAMP. This secondary messenger activates a cascade of downstream transcriptional events leading to expression of genes required for melanin synthesis (Rodríguez and Setaluri, 2014). Exogenous broadly-acting adenylate cyclase activators such as plant-derived forskolin, also stimulate melanin production (D'Orazio et al., 2006), but the degree to which other endogenous molecules, other than αMSH regulate melanin synthesis in tissue is unclear. However, the observation that melasma frequently occurs in non-pregnant women using oral contraceptive pills, which contain only steroid hormone analogs (Sheth and Pandya, 2011; Resnik, 1967a), suggests that humans may maintain αMSH-independent pigment control mechanisms. Identifying these pathways, and strategies to specifically access them pharmacologically to modulate skin pigmentation, may have substantial therapeutic utility. Results Estrogen and progesterone reciprocally regulate melanin synthesis To examine whether sex steroids influence melanin synthesis, we treated primary human melanocytes with estrogen (17β-estradiol). This resulted in a dose-dependent melanin increase (Figure 1—figure supplement 1C). After 4 days of exposure to 25 nM estrogen, a medically-relevant concentration observed during pregnancy (Abbassi-Ghanavati et al., 2009), melanin was markedly increased (208% +/- 27%) in three individual isolates of primary human melanocytes (Figure 1A). The magnitude of this change was similar to that observed with αMSH (Figure 1—figure supplement 1D), and is consistent with prior in vitro studies implicating estrogen in melanin synthesis (McLeod et al., 1994; Ranson et al., 1988). Hormonal oral contraceptives, most of which incorporate ethinyl estradiol, are associated with melasma (Resnik, 1967b). Ethinyl estradiol also increased melanin to levels similar to those observed with native estrogen. To examine the effects of estrogen on melanocyte homeostasis in the context of intact human epidermis, architecturally-faithful three-dimensional organotypic skin was established utilizing normal primary epidermal keratinocytes and melanocytes in native human stroma (Ridky et al., 2010; Monteleon et al., 2015; Duperret et al., 2014; McNeal et al., 2015). After one week, estrogen-treated skin displayed a threefold increase in melanin content (Figure 1B), without changes in melanocyte number or density (Figure 1C). Figure 1 with 2 supplements see all Download asset Open asset Estrogen and progesterone reciprocally regulate melanin synthesis. (A) Melanin content of primary human melanocytes treated with estrogen (E2), compared to vehicle-treated controls. (B) Fontana-Masson (melanin) staining of organotypic skin treated with vehicle or estrogen. Relative melanin content is quantified below. (C) MITF immunohistochemistry of organotypic skin treated with vehicle or estrogen. Melanocyte population density is quantified below. (D) Melanin content of primary human melanocytes treated with progesterone (P4), compared to vehicle. (E), Fontana-Masson (melanin) staining of organotypic skin tissues treated with progesterone or vehicle. Relative melanin content is quantified below. (F) MITF immunohistochemistry of organotypic skin tissues treated with vehicle or progesterone. Melanocyte population density is quantified below. n=3 biologic replicates for each experiment. Error bars denote +/- s.d., *p<0.05, scale bar = 50 μm. https://doi.org/10.7554/eLife.15104.003 Estrogen effects in other tissue types are often counter-balanced by progesterone (Ismail et al., 2015), which also increases during pregnancy. To determine whether this reciprocal relationship is active in melanocytes, we treated cells with physiologic levels of progesterone, which resulted in a dose-dependent decrease in melanin production (Figure 1—figure supplement 1F). At 500 nM, a concentration observed in third trimester pregnancy (Abbassi-Ghanavati et al., 2009), progesterone decreased melanin production by half (58% +/- 11.4%), both in culture (Figure 1D) and in skin tissue (Figure 1E), without altering melanocyte cell number (Figure 1F). Most of our primary melanocytes were derived from newborn male foreskin. To determine whether female cells also responded similarly, we treated female iPS-derived melanocytes with estrogen and progesterone and noted responses similar to those observed with the male cells (Figure 1—figure supplement 1G). To determine whether melanocytes isolated from body sites other than foreskin also responded similarly to sex hormones, we treated melanocytes from adult facial skin with estrogen and progesterone and again observed responses that were similar to those observed with the foreskin melanocytes (Figure 1—figure supplement 1H). Consistent with other groups who have noted that steroid hormones have variable effects on melanocyte proliferation in culture (Im et al., 2002), we observed modest changes in proliferation when isolated primary cells were treated with estrogen or progesterone in vitro. Estrogen treated melanocytes tended to proliferate slightly slower, while progesterone treated cells tended to proliferate slightly faster (Figure 1—figure supplement 2A–B). The effects varied with the basal level of melanin production. Melanocytes from dark skin were more sensitive to progesterone than estrogen, while melanocytes from light skin were more sensitive to estrogen. These proliferation changes are likely an in vitro artifact, as adult interfollicular epidermal melanocytes are relatively nonproliferative in vivo, and we did not note any changes in melanocyte numbers in sex steroid-treated 3-D organotypic tissues. Consistent with this lack of melanocyte proliferation in interfollicular epidermis, another group thoroughly examined 280 tissue sections from normal human skin from 18 donors, and identified only 2 proliferative interfollicular melanocytes (Jimbow et al., 1975). Primary human melanocytes do not express nuclear estrogen or progesterone receptors (ER/PR), and respond to sex steroids via altered cAMP signaling To determine the mechanisms mediating estrogen and progesterone pigment effects, we examined components of the canonical pigment production pathway, and observed a cAMP increase upon estrogen treatment (Figure 2A), suggesting that estrogen accesses the canonical pigment production pathway downstream of MC1R. Consistent with this, pCREB and MITF proteins were similarly induced (Figure 2B). In contrast, progesterone reciprocally decreased melanin, cAMP, pCREB and MITF (Figures 2C–D). Figure 2 with 2 supplements see all Download asset Open asset Estrogen and progesterone access the classical melanin production pathway through nonclassical receptors. (A) cAMP ELISA from estrogen-treated melanocytes (B) Western blot demonstrating changes in classical melanin pathway regulators after a 16 hr estrogen treatment. (C) cAMP ELISA from progesterone-treated melanocytes. (D) Western blot demonstrating changes in classical melanin pathway regulators after a 16 hr progesterone treatment. (E) Melanin assay from melanocytes treated with estrogen and progesterone simultaneously. (F) Western blot for estrogen and progesterone receptors in MCF7 cells and melanocytes. (G) Melanin content of melanocytes transduced with control shRNA or shRNA targeting GPER. Cells were treated with either vehicle or estrogen. (H) Melanin assay performed on melanocytes transduced with control shRNA or shRNA targeting PAQR7. Cells were treated with either vehicle or progesterone. n=3 biologic replicates for each experiment. Error bars denote +/- s.d., *p<0.05. https://doi.org/10.7554/eLife.15104.006 Figure 2—source data 1 List of GPCR transcripts expressed in primary human melanocytes. https://doi.org/10.7554/eLife.15104.007 Download elife-15104-fig2-data1-v2.xlsx These data indicate that estrogen, progesterone, and αMSH converge on adenylate cyclase to reciprocally modulate melanin synthesis, and suggest that individual steroid effects may counter-balance each other. Consistent with this, the estrogen effects were significantly attenuated in the presence of progesterone (Figure 2E). This likely helps explain why pregnancy-associated hyperpigmentation is characteristically limited to specific areas where melanocyte or UV radiation exposure is highest including the face, genital, and areolar regions (Szabo, 1954; Staricco and Pinkus, 1957). It is also possible that in the complex hormonal milieu of pregnancy, additional factors beyond the sex steroid activated pathways described here also contribute to skin color modulation. As steroid hormones are not predicted to signal through MC1R, whose natural ligand is the peptide αMSH, we sought to identify the specific receptors mediating the estrogen and progesterone pigment effects. We did not detect classical estrogen (ER) or progesterone (PR) receptors in melanocytes using qRT-PCR, although transcripts were observed in breast cells (Figure 2—figure supplement 1A). Previous RNAseq studies in human melanocytes, conducted for unrelated experimental questions (Flockhart et al., 2012), also failed to detect ER or PR transcripts (Figure 2—figure supplement 1B). Consistent with this, ER or PR protein was not observed in MC via western blotting, although both receptors were readily apparent in breast cells (Figure 2F). Sex steroid signaling in melanocytes is dependent on the nonclassical membrane bound G-protein coupled sex hormone receptors GPER and PAQR7 Considering MC1R is a G protein-coupled receptor (GPCR), we hypothesized that alternative GPCRs mediate sex steroid pigment effects. To identify possible candidates, we analyzed whole transcriptome melanocyte RNAseq data. Of 412 known or predicted 7-pass human GPCRs (Alexander et al., 2013), 61 distinct GPCRs were expressed in MCs, including the membrane-bound, G protein-coupled estrogen receptor (Filardo et al., 2002) (GPER) (Figure 2—source data 1 and Figure 2—figure supplement 1B). Given that prior work in breast cancer cell lines and fish oocytes determined that estrogen binding to GPER modulates cAMP (Filardo et al., 2002; Thomas et al., 2005; Cabas et al., 2013; Majumder et al., 2015; Pang and Thomas, 2010), and that cAMP signaling stimulates melanin synthesis, we thought it possible that GPER may be the physiologically relevant human melanocyte estrogen receptor. The melanocyte RNAseq studies also demonstrated that an analogous, noncanonical G protein-coupled progesterone receptor, progestin and adipoQ receptor 7 (PAQR7) (Zhu et al., 2003; Tang et al., 2005), was also expressed (Figure 2—figure supplement 1B). We next used qRT-PCR to verify that GPER and PAQR7 are both expressed in primary human MC (Figure 2—figure supplement 1C). Notably, GPER and PAQR7 expression was markedly lower in other skin cells including keratinocytes and fibroblasts (Figure 2—figure supplement 1D). To establish the necessity of GPER and PAQR7 in mediating sex hormone effects in MCs, we first depleted GPER using either of two independent shRNA hairpins, which completely eliminated the melanocyte pigmentation response to estrogen (Figure 2G and Figure 2—figure supplement 1E–F). Analogous shRNA-mediated PAQR7 depletion ablated the pigmentary response to progesterone (Figure 2H and Figure 2—figure supplement 1F). To verify these results, we next used a complementary genetic approach based on CRISPR-Cas9 mediated gene disruption of GPER or PAQR7, which also completely blocked the pigmentary response to estrogen and progesterone, respectively (Figure 2—figure supplement 1G–H). Consistent with our model in which these receptors access melanin synthesis at the level of adenylate cyclase, PAQR7 was found to bind progesterone and regulate the final stages of sea trout oocyte meiosis through cAMP reduction (Zhu et al., 2003; Tokumoto et al., 2012). In that fish system, PAQR7 signals through G protein complexes containing the inhibitory G subunit (Gi), which represses adenylate cyclase. To examine whether this signaling mechanism is functional in melanocytes, we treated melanocytes with progesterone in the presence of pertussis toxin (PTX), an exotoxin that specifically inactivates Gi subunits via ADP-ribosylation. With PTX treatment alone, we observed a small increase in melanin production. This likely reflects inhibition of Gi released from basal PAQR7 activity, as well Gi subunits from other GPCRs that collectively contribute to the basal level of cAMP signaling observed in culture. Importantly, PTX blocked progesterone effects, establishing that progesterone signals through Gi subunits (Figure 2—figure supplement 2A). Specific activation of GPER or PAQR7 is sufficient to alter pigment production in human skin tissue To complement these genetic studies establishing that GPER and PAQR7 are the melanocyte sex steroid receptors, we utilized a pharmacologic approach employing synthetic steroid analogs with specific agonist or antagonist activity on ER, PR, GPER, or PAQR7. Tamoxifen, an ER antagonist, is associated with melasma in breast cancer patients (Kim and Yoon, 2009). The mechanistic basis for the pigment change was previously unknown. However, tamoxifen is a GPER agonist (Thomas et al., 2005; Li et al., 2010), and increased melanin to levels comparable to those observed with estrogen (Figure 3—figure supplement 1A). To determine whether GPER signaling was sufficient to increase melanin, we utilized the specific GPER agonist G-1 (Bologa et al., 2006), an estrogen analog developed for mechanistic studies in other systems that does not bind ER. G-1 drove a dose-dependent increase in melanin production through pCREB and MITF that was GPER dependent (Figure 3A–C and Figure 3—figure supplement 2A–D). Further establishing that GPER is the melanocyte estrogen receptor, G-1 and estrogen effects were blocked by either of two specific GPER antagonists, G-15 and G-36 (Figure 3—figure supplement 2E), which do not have inhibitory activity against ER (Dennis et al., 2009; 2011). To establish that PAQR7 signaling is sufficient to decrease melanin production, we used a specific PAQR7 agonist Org OD-02 (CH2P4), which does not bind PR (Kelder et al., 2010). CH2P4 caused a dose-dependent decrease in melanin production through pCREB and MITF that was PAQR7 dependent (Figure 3D–F and Figure 3—figure supplement 3A–D). Figure 3 with 3 supplements see all Download asset Open asset GPER and PAQR7 signaling is sufficient to alter melanin production in organotypic human tissue. (A) Organotypic skin treated with vehicle (left) or G-1 (right). (B) Fontana-Masson (melanin) staining of organotypic skin treated with vehicle or G-1. Quantification of melanin content is shown on the right. (C) MITF immunohistochemistry of organotypic skin treated with vehicle or G-1. Quantification of melanocyte population density is shown on the right. (D) Organotypic skin treated with vehicle (left) or CH2P4 (right). (E) Fontana-Masson (melanin) staining of organotypic skin treated with vehicle or CH2P4. Quantification of melanin content is shown on the right. (F) MITF immunohistochemistry of organotypic skin treated with vehicle or CH2P4. Quantification of melanocyte population density is shown on the right. n=3 biologic replicates for each experiment. Error bars denote +/- s.d., *p<0.05, scale bar = 50 μm. https://doi.org/10.7554/eLife.15104.010 Topical delivery of a GPER-specific synthetic estrogen analog in vivo increases epidermal melanin To demonstrate that GPER was sufficient to promote melanin production in vivo, we synthesized G-1 to 95% purity (Figure 4—figure supplement 1A–B), and formulated G-1 for topical application. We treated the right ears of mice daily for 3 weeks with vehicle or 2% (w/v) G-1 in DMSO, and observed a gradual increase pigmentation compared to vehicle-treated controls over 3 weeks (Figure 4A). Melanin content was increased 1.6-fold, a cosmetically significant change, and was consistent with the magnitude of change seen in vitro (Figure 4B–C). Clinically apparent skin darkening on mice increased over 2–3 weeks while pigment changes in culture were more rapid. It is likely that the synthetic GPCR ligands are metabolized and/or distributed differently in vivo then in in vitro culture, such that the effective local concentration of steroid in the in vivo setting is relatively transient. Optimization of an ideal topical formulation and dosing schedule will require additional study in the context of a human clinical trial. Figure 4 with 1 supplement see all Download asset Open asset Topical GPER agonists increase pigmentation in vivo. (A) Mouse ear skin treated for 3 weeks with vehicle only on the left ear, and 2% (w/v) G-1 on the right ear. (B) Melanin assay on whole ear tissue that was treated with either vehicle or 2% G-1 for 3 weeks. (C) Fontana-Masson (melanin) staining of tissue sections from ears treated with either vehicle or 2% G-1, quantification of staining on right. (D) Schematic model of estrogen and progesterone signaling in melanocytes. n=3 biologic replicates for each experiment. Error bars denote +/- s.d., *p<0.05, scale bar = 20 μm. https://doi.org/10.7554/eLife.15104.014 Discussion Safe and effective approaches for modulating skin melanocyte function for therapeutic benefit are lacking, largely because the factors normally regulating melanocyte homeostasis are complex and incompletely deciphered. Defining these mechanisms is important however, as myriad genetic and acquired conditions including common afflictions such as acne, eczema, vitiligo, ultraviolet (UV) radiation exposure, traumatic injury, and pregnancy are associated with alterations in skin pigmentation that can be extensive and long-lasting (James et al., 2011). Another population that could potentially benefit from modulating skin pigment are people with naturally light skin, especially those with red hair, who have a markedly decreased ability to synthesize UV-protective brown eumelanin as a result of inactivating mutations in MC1R (Valverde et al., 1995). This large population is especially susceptible to photodamage, sunburns, and has an increased lifetime risk of keratinocyte and melanocyte-derived skin cancers (Han et al., 2006). There is currently no available therapeutic that promotes protective eumelanin pigment production. However, the specific activation of GPER alternatively activates cAMP signaling, bypassing MC1R, to stimulate melanin synthesis, and could therefore be especially useful in this sun-vulnerable population. Selective GPER activation in skin could potentially be a safe alternative to intentional UV radiation exposure (via natural sunlight or tanning beds) for individuals seeking what they perceive as an aesthetically desirable tan. The only method currently available to increase skin melanin is UV exposure. While effective at darkening skin, the requisite damage promotes and skin utilized approaches for skin melanin are also often and of or especially common in and but also in the and in a from the of or a which has been in because of possible association with cancer 2007). small sex steroid without these that modulate pigment production (Figure of GPER or PAQR7 could potentially have effects on cells other than epidermal melanocytes. While topical delivery of such likely effects in the of effects the However, we did not note any significant in the epidermis from our in vitro or in vivo skin tissues treated with the sex GPER and PAQR7 have been identified only relatively but are expressed in several and may mediate at of the estrogen and progesterone effects that be attributed to the classical nuclear hormone receptors. GPER has been identified in the and systems and 2011). In cells, GPER expression on cells has been shown to play a role in and PAQR7 is expressed in the and systems et al., and in et al., and in human cells et al., although the functional role of PAQR7 in those tissues relatively unclear. Given that the increased estrogen and progesterone associated with pregnancy does not result in skin cancer or significant in other we it likely that the specific GPER and PAQR7 agonists will be studies and of human skin treated in clinical will be The that PAQR7 through inhibitory Gi subunits is especially as it is the first of a melanocyte signaling cascade that represses melanin synthesis at the level of G-protein signaling, as to pigment control mechanisms that modulate the of the MC1R In the protein decreases pigment production via binding to MC1R and αMSH et al., than through an that normal primary melanocytes lack nuclear ER or PR a prior (Im et al., This group performed immunohistochemistry and to the that nuclear estrogen receptors are expressed in melanocytes, but in our the data in that work is not especially and is no in that work that nuclear hormone signaling changes in melanin synthesis. Another group demonstrated that melanocyte protein have the ability to bind estrogen, but that work did not identify the specific responsible for the binding activity et al., We do not the that in tissue including and melanocytes express nuclear as is no known signaling pathway linking nuclear sex hormone receptors to the melanin synthesis it is most likely that the effects of estrogen and progesterone on pigment production are mediated through the and Gi coupled GPCRs identified in our We have shown that signaling through GPCRs other than MC1R melanin production. While all the 7-pass proteins expressed in melanocytes, we noted expression of several additional receptors that may also influence melanin production. These et al., et al., and receptors, which in other are known to signal through and Gi subunits et al., 2005; et al., 2010). Future functional of these and other GPCRs in melanocytes may the mechanisms responsible for the pigmentation changes that frequently skin diseases associated with These studies may identify additional and useful receptors in melanocytes, and will an of how GPCR signaling is to regulate melanin production in human Materials and methods Melanocyte culture a Primary melanocytes were from human foreskin and as described previously described with as After in the epidermis was from the and treated with for Cells were and on MC with Melanocyte and and primary melanocytes were utilized for estrogen and GPER agonist effects, and primary melanocytes were utilized for progesterone and PAQR7 agonist effects in melanin production. iPS-derived human melanocytes were a from and αMSH were from G-1 G-15 and G-36 were from CH2P4 was from toxin was from