A basic way to tan Darker-skinned individuals have more melanin in their skin and a lower risk for skin cancer than fairer-skinned individuals. The production of melanin occurs in organelles called melanosomes and is regulated by melanosome pH. Zhou et al. found that cAMP generated by soluble adenylyl cyclase (sAC) resulted in decreases in melanosome pH and in the activity of tyrosinase, the rate-limiting enzyme in melanin synthesis. sAC deficiency or inhibitors increased melanosome pH and pigmentation in mice. These results define a mechanism of rapidly regulating melanin synthesis that could be exploited to reduce skin cancer risk for fair-skinned individuals.

Abstract The production of melanin increases skin pigmentation and reduces the risk of skin cancer. Melanin production depends on the pH of melanosomes, which are more acidic in lighter-skinned than in darker-skinned people. We showed that inhibition of soluble adenylyl cyclase (sAC) controlled pigmentation by increasing the pH of melanosomes both in cells and in vivo. Distinct from the canonical melanocortin 1 receptor (MC1R)–dependent cAMP pathway that controls pigmentation by altering gene expression, we found that inhibition of sAC increased pigmentation by increasing the activity of tyrosinase, the rate-limiting enzyme in melanin synthesis, which is more active at basic pH. We demonstrated that the effect of sAC activity on pH and melanin production in human melanocytes depended on the skin color of the donor. Last, we identified sAC inhibitors as a new class of drugs that increase melanosome pH and pigmentation in vivo, suggesting that pharmacologic inhibition of this pathway may affect skin cancer risk or pigmentation conditions.

INTRODUCTION Human pigmentation has psychosocial implications and affects skin cancer risk (1–5). Differences in pigmentation of the skin, hair, and eyes are the result of variation in the amount and type of melanin produced (5, 6). Melanin is produced in a specialized organelle called the melanosome (7–10). Canonical mechanisms that control melanin production involve changes in the expression of genes encoding synthetic enzymes such as tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and tyrosinase-related protein 2 (TYRP2) (11). Genetic analysis of human populations with differences in skin, hair, and eye pigmentation has identified polymorphisms in genes encoding melanosome channels, and functional analysis confirms that these channels influence pigmentation by altering melanosome pH (12–14). Although the discovery of these polymorphisms has identified proteins important for the maintenance of the melanosome pH set point, it remains unclear whether these proteins are dynamically regulated. The melanosome pH set point is not fixed: During early organelle development, the organelle is relatively acidic, progressively becoming more alkaline with maturity (15, 16). Mechanisms that control this active process are poorly understood. Tyrosinase is the rate-limiting enzyme in melanin synthesis, and its activity substantially affects human pigmentation. Signaling cascades, such as those mediated by activation of the melanocortin 1 receptor (MC1R) by melanocyte-stimulating hormone (MSH), can stimulate transcription factors, such as microphthalmia-associated transcription factor (MITF), to control pigmentation by altering the expression of TYR. Because tyrosinase is very pH sensitive and its activity increases as pH is elevated (15, 17), it has been therefore proposed that tyrosinase activity can be differentially regulated without altering its expression (18). However, signaling mechanisms that dynamically regulate melanosome pH to control tyrosinase activity and drugs capable of safely regulating pigmentation by altering melanosome pH have not been described. Human pigmentation is a reflection of the melanin content in the skin, hair, and eyes. There are two types of melanin: eumelanin and pheomelanin (6). Eumelanin is dark brown to black in color and is effective at blocking ultraviolet radiation (6). Pheomelanin is yellow to red in color and, because of its pro-oxidant chemistry, is carcinogenic (6, 19). Thus, the relative amount of these two types of melanin contributes to skin cancer risk (4). The visual difference in the skin or hair color of most people correlates with the presence of eumelanin in the tissue (20). The exception are redheads, who make a large amount of pheomelanin due to polymorphisms present in the MC1R gene. The pheomelanin content in people with wild-type MC1R is variable and is not clearly linked to a genetic polymorphism (21). Melanosome pH has been reported to be more acidic in lighter-skinned people than in darker-skinned people; therefore, melanosome pH is important for human pigmentation (1, 2, 18). Nonphysiological disruption of vacuolar-type H+-ATPase (V-ATPase) activity after treatment with bafilomycin increases melanosome pH and can increase the ratio of eumelanin to pheomelanin (6, 15). However, signaling mechanisms that control melanin synthesis by dynamically regulating melanosome pH have not been described. Cyclic adenosine monophosphate (cAMP) regulates pigmentation by altering genes important for melanin synthesis (7). Signaling through this second messenger occurs locally in spatially restricted microdomains distributed throughout cells (22–24). cAMP signaling microdomains function independently: The cAMP produced in one microdomain within a cell has independent (and sometimes opposing) effects from cAMP produced in a distinct microdomain. In addition to being defined by their unique effects, cAMP signaling microdomains are also defined by the distinct mechanisms used to control the levels of the second messenger. cAMP is produced by adenylyl cyclases (ACs) and catabolized by phosphodiesterases (PDEs), and the activities of ACs and/or PDEs can regulate cAMP signaling in a microdomain. In mammalian cells, there are two distinct subfamilies of ACs (23). The canonical cAMP cascade is initiated by heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors, leading to G protein–dependent activation of the transmembrane AC (tmAC) located at the plasma membrane (23). There are nine tmAC genes (ADCY1–9) in mammalian cells, and the encoded proteins can signal at distinct locations within the plasma membrane or on recycling endosomes (24). The noncanonical cAMP cascade is initiated by soluble AC (sAC; which is encoded by ADCY10), which is not G protein stimulated but is instead stimulated by bicarbonate and Ca2+ ions (25, 26) and adenosine triphosphate (ATP) (27). Unlike tmACs, sAC is not linked to the plasma membrane and is present at multiple intracellular locations (28). sAC- and tmAC-defined cAMP cascades mediate both similar and disparate effects within a single cell type (27, 29). In melanocytes, tmAC-generated cAMP is responsible for MC1R-induced changes in gene expression. We explored whether other cAMP signaling cascades contribute to pigmentation in melanocytes. sAC regulates pH across the plasma membrane in the kidney and epididymis (30, 31) and the pH set point of lysosomes (32), which share many developmental and regulatory steps with melanosomes. Therefore, we hypothesized that sAC may also control melanosome pH and affect melanin production.

DISCUSSION Human skin and hair pigmentation has psychosocial and cancer risk importance; thus, understanding how pigmentation is controlled has numerous clinical implications. Much of our current understanding of human pigmentation is based on the characterization of polymorphisms in genes important for pigmentation such as MC1R (20, 56) and those encoding melanosome channels (12, 14, 67). Investigation of the melanocyte proteins containing polymorphisms has helped explain certain disorders of human pigmentation and the red hair phenotype; however, less well understood are the signaling pathways that directly alter melanosome biology to regulate pigment synthesis. Here, we demonstrated that the sAC-dependent cAMP cascade controlled pigmentation by regulating melanosome pH. Melanosome channels and proton pumps are important for establishing the melanosome pH set point. Biologically similar organelles, such as lysosomes, also dynamically regulate their pH (68). Mechanisms that dynamically regulate the pH of melanosomes are poorly understood. We revealed that melanosome pH rapidly changed in response to alterations in sAC-dependent cAMP signaling. Thus, melanosome pH regulation is not limited to a progressive pH increase as the melanosome matures (15, 16). Furthermore, because sAC modulates lysosomal pH in other cell types (32), sAC signaling cascades may represent a general mechanism of organelle pH control. Melanosome pH critically affects the activity of the rate-limiting, melanin-synthesizing enzyme tyrosinase (15, 17, 20, 64). In contrast to the mechanisms of tyrosinase regulation by changes in its expression over days, signaling pathways that acutely control tyrosinase activity by directly regulating melanosome pH are not well understood. Our data demonstrated that sAC-dependent cAMP signaling could rapidly (within hours) regulate melanosome pH and tyrosinase activity in cells and revealed a new cAMP-dependent mechanism of tyrosinase regulation that is independent of gene expression (Fig. 6E). The balance between eumelanin and pheomelanin levels in skin has important implications for melanoma/skin cancer risk and aging (19, 69, 70). pH can differentially influence the synthesis of these two types of melanin (15, 17, 20, 43, 64), although the mechanisms that acutely control eumelanin and pheomelanin synthesis by directly altering melanosome pH have not been described. Here, we showed that sAC control of melanosome pH influenced the eumelanin-to-pheomelanin ratio in melanocytes both in vitro and in vivo. These data suggested that sAC activity may influence skin cancer risk and aging. Notably, sAC expression and subcellular localization change as melanocytes transition from benign to malignant cells (34, 71). We and others have shown that antibodies directed against sAC are an effective adjunct for the diagnosis of pigmented lesions, especially lentiginous growth melanomas (34, 71, 72). In addition to controlling skin, hair, and eye color, melanin production in multiple tissue types (for example, skin, retina, inner ear, and brain) is important for regulating reactive oxygen species (ROS), protecting tissues from toxins, and ion chelation (73), and these signals have been suggested to influence melanin production. sAC is a metabolic and pH sensor and is stimulated by many intracellular signals, such as HCO 3 −/pH, ATP, calcium, and ROS (Fig. 6E) (26, 27, 74, 75). Therefore, the sAC signaling cascade may provide a mechanism through which melanocytes and other melanin-producing cells can sense and respond to specific stimuli by altering melanin production. Melanocytes derived from people with different skin and hair color have distinct melanosome pH set points (15, 17, 20, 64). Our data revealed that sAC regulation of melanosome pH, tyrosinase activity, and pigmentation differed depending on the skin color of the melanocyte donor. Therefore, investigation of how sAC-dependent signaling pathways are different in people with varied skin colors may uncover new mechanisms that control pigmentation in humans. Our data suggested that PKA is not required for sAC-dependent regulation of melanosome pH. Instead, our data supported a role for EPAC in the control of melanosome pH downstream of sAC. The sAC/EPAC pathway and the canonical tmAC/PKA pathway may be completely distinct or may cooperate to control pigmentation (Fig. 6E); therefore, further investigation of the interplay between these two pathways will be important. Mammalian cells have distinct cAMP microdomains that can have similar or disparate effects on cellular biology (22–24). MSH/MC1R-induced, tmAC-generated cAMP is essential for the expression of pigmentation genes important for melanin production (20). Given the selective expression of the tmAC-encoding ADCY2 gene in melanocytes (76), ADCY2 may be the source of MSH-dependent cAMP. In addition, ADCY6, which links calcium and cAMP signaling to melanogenesis, is another potential source of tmAC-dependent cAMP (77). sAC-generated cAMP controlled melanosome pH and did not increase TYR gene expression (Fig. 6E). We demonstrated here that melanin production was enhanced by increased TYR gene expression, resulting from an increase in cAMP generated by the MSH/tmAC pathway, and an alkalinized melanosome pH, resulting from a decrease in cAMP generated by sAC (Fig. 5D). Thus, our data support a model in which tmAC- and sAC-defined microdomains lead to diametrically opposite changes in cAMP that both induce eumelanin synthesis. Distinct cAMP signaling cascades in melanocytes may help explain why some people with dysfunctional MC1Rs, altered tmAC activity, or aberrant MSH secretion can maintain normal skin and hair color (20, 78–80). In addition, extracutaneous melanocytes and other melanosome-containing cells, such as retinal pigment epithelium and stria vascularis, lack obvious MC1R-dependent signaling cascades (81), and the canonical melanin regulatory pathways do not appear to play a role in these cells. However, melanin synthesis in extracutaneous melanosome-containing cells is affected by melanosome pH (82, 83). Furthermore, diseases such as oculocutaneous albinism type 2 (OCA2) are thought to occur when melanosomes cannot alkalinize during organelle development (15). Pharmacologic correction of melanosome pH has been proposed as a method to restore pigmentation in OCA2 melanocytes to normal levels and reduce skin cancer risk (84). We found that sAC inhibitors increased melanosome pH and epidermal pigmentation in mice. Therefore, drugs targeting the sAC signaling cascade may represent a new therapeutic strategy for treating diseases of melanin synthesis or reducing skin cancer risk.

MATERIALS AND METHODS Antibody reagents for Western blot analysis Tyrosinase antibody (used at 1:1000) was a gift from R. Halaban, Yale University School of Medicine (85). Tyrosinase antibody (T311) was purchased from Santa Cruz Biotechnology (1:200; catalog no. sc-20035, lot no. J0616). MITF antibody was purchased from Abcam (1:500; catalog no. ab13703, lot no. GR27425-10). MITF (D5G7V; 1:1000; catalog no. 12590S, lot no. 1), phospho-(Ser/Thr) PKA substrate (1:1000; catalog no. 9621, lot no. 14), and GAPDH (14C10; 1:1000; catalog no. 2118S, lot no. 8) antibodies were purchased from Cell Signaling Technology. Antibodies directed against (R21) (3.2 mg/ml, 1:2000) were used as previously described (28). sACKO mouse melanocyte generation and culture All experiments involving mice were approved by the Weill Cornell Medical College Institutional Animal Care and Use Committee and were performed in accordance with institutional guidelines. Immortalized mouse melanocytes were derived from 1- to 3-day-old Adcy10fl/fl agouti newborn mice as previously described (41). Briefly, newborn mice were euthanized, and the skin was removed from the back, placed in a petri dish epidermis side up, and incubated in 2.5 ml of dispase in Eagle’s minimal essential medium without calcium and magnesium overnight at 4°C. On the next day, the dermis was discarded, and the epidermis was incubated in trypsin solution until the cells dissociated. Cells were washed to remove the trypsin solution and then cultured in mouse melanocyte medium [Ham’s F12 plus glutamine, penicillin-streptomycin, horse serum (7%), fetal bovine serum (FBS; 7%), dibutyryl cAMP (dbcAMP; 500 μM), Na 3 VO 4 (1 μM)]. Once the immortalized line was established, the medium was changed to normal mouse melanocytes culture media [Opti-MEM medium supplemented with 10% FBS, 7% horse serum, 1% penicillin-streptomycin, 400 μM dbcAMP, 0.3 nM cholera toxin (CT), and 1.6 μM 12-O-tetradecanoylphorbol-13-acetate (TPA)]. To generate Adcy10−/− melanocytes (sACKO), parental Adcy10fl/fl cells were infected with either Ad5-CMV-GFP or Ad5-CMV-CREGFP (Vector BioLabs, Malvern, PA, USA) at 200 multiplicity of infection. Forty-eight hours after infection, cells were fluorescence-activated cell sorted for GFP fluorescence, and only cells that were in the upper 25% of fluorescence were collected and cultured. Independent pairs of Adcy10fl/fl (sACFF) and Adcy10−/− (sACKO) cells were generated. Genetic deletion of sAC was confirmed by PCR and functional assay. All experiments using mouse melanocytes were performed between passages 15 and 28. Before the experiments, melanocytes were cultured in “cAMP starvation media” without dbcAMP and without CT for 48 to 96 hours. The cell growth rate of each cell line under different media conditions was measured using the CyQUANT assay (Thermo Fisher Scientific) at the time point indicated. Human melanocyte culture Primary human melanocytes derived from neonatal foreskins were obtained from the Biospecimen Core of the Yale Specialized Programs of Research Excellence (SPORE) in Skin Cancer (New Haven, CT, USA) and grown in Opti-MEM medium supplemented with 5% FBS, 1% penicillin-streptomycin, fibroblast growth factor-2 (10 ng/ml), heparin (1 ng/ml), 0.1 μM dbcAMP, and 0.1 mM 3-isobutyl-1-methylxanthine (IBMX). Designations of “light,” “medium,” or “dark” are based on the skin color of the donor as per the Biospecimen Core. Before experiments, melanocytes were cultured in cAMP starvation media without dbcAMP and without IBMX for 24 hours. Immunocytochemistry Cells were cultured on sterile glass coverslips in 24-mm wells at 5.0 × 104 cells per coverslip in cAMP starvation media for 48 hours. Cells were then fixed with 3% (w/v) paraformaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100 in Buffer A (125 mM sodium chloride, 10 mM sodium phosphate, 2 mM magnesium chloride) at −20°C for 5 min. Melanosomal labeling was performed using polyclonal antibodies against TRP1/TYRP1 [Abcam, 1:1000, catalog no. ab83774, lot no. GR272706-6; and Novus Biologicals (TA99), 1:200, catalog no. NBP2-32906, lot no. 7306-1P170816] and HMB45 [Melanoma Marker Antibody (HMB45), Santa Cruz Biotechnology, 1:200, catalog no. sc-59305, lot no. E1314]. Melanosome pH was measured using goat anti-dinitrophenol antibody (anti-DNP, Oxford Biomedical Research, 1:200, catalog no. D04, lot no. d4.111212) as described below. Fluorescence was detected after secondary staining with Alexa Fluor at the excitation/emission (Ex/Em) spectra indicated. We surveyed Alexa Fluor secondary antibodies over a range of Ex/Em spectra in melanocytes with a range of melanin contents to identify the best Ex/Em spectra. We found that Ex/Em spectra >500 nM were unaffected by melanin (fig. S3, L to N). Images were acquired using a Zeiss LSM 880 and analyzed using NIS-Elements AR 4.60 (Nikon) as described in the next section. General immunofluorescence quantitation Quantitative analyses were performed using the Object Count tool in Nikon AR 4.60. The entire cell was imaged to ensure that no specific region of the cell was favored; however, when analyzing each cell, an equivalent diameter (EqDiameter) was restricted to 1.85 to 15.00 pixels and circularity to 0.20 to 1.00 for optimal identification of individual puncta and exclusion of larger structures such as the Golgi apparatus. The lower intensity threshold limit of each fluorescence channel was defined as the intensity of the dimmest punctum returned using the 3 points circle threshold tool. The upper intensity threshold limit was set to the maximum value. This method assured that while the intensity of fluorescence changed between cell lines and conditions, all melanosome specific data were captured and subjected to analysis. DAMP synthesis, imaging, and quantification DAMP was synthesized and verified by nuclear magnetic resonance at our institutional Chemistry Core Facility and was prepared as a stock (1 mg/ml) in 80% ethanol. Cells were cultured on sterile glass coverslips in 24-mm wells at 5.0 × 104 cells per coverslip and treated or not for 4 hours with 30 μM KH7 or LRE1 in the presence or absence of the nonselective cAMP analog Sp-8-CPT-cAMPs (500 μM; Biolog), the EPAC-selective cAMP analog 8-pHPT-2′-O-Me-cAMP (500 μM; Biolog), the pan-EPAC inhibitor ESI-09 (10 μM; EMD Millipore), the NDP-MSH (100 nM; Sigma-Aldrich), the PKA inhibitor PKI (1 μM; Sigma-Aldrich) or H89 (10 μM; Sigma-Aldrich), or cycloheximide (10 μM; Sigma-Aldrich), as indicated. Cells were washed with fresh cAMP starvation media and incubated with 10 μM DAMP for 30 min, fixed with 3% (w/v) paraformaldehyde for 15 min at room temperature, and washed with 50 mM ammonium chloride. After permeabilization with 0.1% Triton X-100 in Buffer A at −20°C for 5 min, cells were labeled with goat anti-DNP antibody (anti-DNP, Oxford Biomedical Research, 1:200, catalog no. D04, lot no. d4.111212). Melanosomes were identified using a mouse monoclonal antibody against TRP1/TYRP1 [Abcam, 1:1000, catalog no. ab83774, lot no. GR272706-6; and Novus Biologicals (TA99), 1:200, catalog no. NBP2-32906, lot no. 7306-1P170816] or HMB45 [Melanoma Marker Antibody (HMB45), Santa Cruz Biotechnology, 1:200, catalog no. sc-59305, lot no. E1314]. For most of the images, fluorescence was detected after secondary staining with Alexa Fluor 546 donkey anti-goat immunoglobulin G (IgG) antibody (Invitrogen, 1:1000, catalog no. A11056, lot no. 1714714) and Alexa Fluor 647 donkey anti-mouse IgG antibody (Invitrogen, 1:1000, catalog no. A31571, lot no. 1839633). We found minimal effect of melanin on the Ex/Em spectra of these secondary antibodies. Images were acquired using a Zeiss LSM 880 and analyzed using NIS-Elements AR 4.60 (Nikon). Mean DAMP fluorescence intensity was measured for all DAMP+ puncta. Melanosomes were identified as HMB45+ or TYRP1+ puncta. Melanosome DAMP measurements were only recorded when colocalized with HMB45 or TYRP1, as indicated (fig. S3D). Frequency distributions were generated for each sample from the mean DAMP fluorescence intensity of all DAMP+ melanosomes. All analyses were performed on two replicate coverslips (n ≥ 15 cells per coverslip), which were then pooled for each condition (n ≥ 30 cells per condition) to ensure adequate power when generating frequency distributions and determining statistical significance. We found that to achieve the necessary power to detect a statistically significant pH change (P values; table S1), we needed to measure at least 15 cells, which allowed for the measurement of at least 1500 puncta and the detection of DAMP+ melanosomes at various stages. LysoSensor DND-160 imaging and quantification Cells were incubated with 1 μM LysoSensor DND-160 (Invitrogen, catalog no. L7545, lot no. 846175) for 5 min at 37°C. LysoSensor was excited at 405 nm, and its emission was detected at 417 to 483 nm (W1) and 490 to 530 nm (W2). The ratio of emissions (W1/W2) in LysoSensor-stained puncta was assigned to a pH value based on a calibration curve generated for each experiment using solutions containing 125 mM KCl, 25 mM NaCl, and 24 μM monensin and using varying concentrations of MES to adjust the pH to 4, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5. The fluorescence ratio was linear for pH 5.0 to 7.0. Mean W1 and W2 fluorescence intensities at each punctum were measured and used to calculate the W1/W2 ratio. The W1/W2 ratio for each LysoSensor+ puncta was compared to the pH standard curve to generate a pH value, as described above. Frequency distributions were generated for each sample from the predicted pH values of LysoSensor+ organelles. All analyses were performed on two replicate coverslips (n ≥ 10 cells per coverslip). Transmission EM As previously published (52), cell monolayers were fixed with a modified Karnovsky’s fix (86) and a secondary fixation in reduced osmium tetroxide (87). After dehydration, the monolayers were embedded in an epon analog resin. En face ultrathin sections (65 nm) were contrasted with lead citrate (88) and viewed on a JEM 1400 electron microscope (JEOL) operated at 100 kV. Digital images were captured on a Veleta 2K × 2K charge-coupled device camera (Olympus Soft Imaging Solutions). Tritium in vivo tyrosinase assay Tyrosinase activity of melanocytes in vivo was determined by measuring the amount of radioactive H 2 O produced from L-[Ring-3,5-3H]-Tyrosine, as previously described (15). Mouse melanocytes were incubated in six-well plates with cAMP starvation media containing L-[Ring-3,5-3H]-Tyrosine (5 μCi/ml; PerkinElmer) for 4 or 8 hours. Media (1.5 ml) from each well were removed and centrifuged at 1200 rpm in a microfuge (Eppendorf 5154 D) for 5 min. Supernatant (1 ml) was combined with 1 ml of 0.1 M citric acid containing 10% (w/v) activated charcoal to remove excess tyrosine and then centrifuged at 12,000 rpm for 5 min. 3H activity of the supernatant was determined using a scintillation counter. In human cells, tyrosinase activity with and without pharmacologic inhibition of sAC was performed by incubating cells in six-well plates with media containing L-[Ring-3,5-3H]-Tyrosine (5 μCi/ml) and 30 μM KH7, 30 μM LRE1, or DMSO (vehicle control) for 8 hours. Media (1.5 ml) from each well were put through the same process as above. In all experiments, media incubated in parallel wells containing no cells were used as a negative control for tyrosinase activity. Genetic deletion of melanocyte sAC in vivo Tyr::CRE-ERT2 (89) mice were mated with Adcy10/sACfl/fl mice (39, 40). These mice were then backcrossed to the C3H/HeJ agouti (brown hair color) strain to generate progeny with brown hair color containing both eumelanin and pheomelanin. Tyr::CRE-ERT2+/−;sACfl/fl mice were then mated with C3H/HeJ sACfl/fl or sACfl/+ mice lacking the Tyr::CRE-ERT2 cassette. The inducible knockout sAC allele was activated by painting the dorsum of all mice in the litter with 20 mM 4-hydroxytamoxifen on postnatal days 2, 3, and 4. Tamoxifen binds to the ERT2 protein fused to the Cre recombinase, thereby revealing a nuclear localization domain and allowing for Cre entry into the nucleus and gene recombination. Hair color during the first hair cycle was blindly evaluated using a (+) “light brown,” (++) “medium brown,” and (+++) “dark brown” scoring system. At 21 days, hair from the dorsum of each mouse was shaved for melanin quantitation (see below) or plucked for microscopic evaluation by a blinded observer, and then (after euthanasia), the epidermis was sampled for genotyping. As expected, roughly 25% of the mice were Cre+, and a subset had recombination of both sAC alleles (n = 7 mice). Although they had received tamoxifen, none of the Cre− mice showed sAC allele rearrangement (n = 13 mice). The percentage of hairs with and without an agouti band was measured in a blinded fashion under a stereo microscope. The treated skin was submitted to an animal pathologist who performed histological evaluation of the epidermis in a blinded fashion. Melanin analysis All melanin quantitation was performed in a blinded fashion. Cell samples (0.2 to 1.15 million) were ultrasonicated in 400 μl of Milli-Q H 2 O, or mouse hair was homogenized at a concentration of H 2 O (10 mg/ml), and 100 μl of water suspensions of samples was subjected to alkaline hydrogen peroxide oxidation (90) and hydroiodic acid hydrolysis (91). Eumelanin was analyzed as a specific degradation product pyrrole-2,3,5-tricarboxylic acid (PTCA) produced by the alkaline hydrogen peroxide oxidation, whereas pheomelanin was analyzed as the degradation product 4-amino-3-hydroxyphenylalanine (4-AHP) produced by the hydroiodic acid hydrolysis. Eumelanin and pheomelanin were calculated by multiplying the PTCA and 4-AHP contents by factors of 25 and 7, respectively (73). Pharmacologic inhibition of sAC in vivo Animal experiments were performed in accordance with the approved Institutional Animal Care and Use Committee protocol at Weill Cornell Medicine. Age- and gender-matched C3H/HeJ mice (female, 7 weeks old) were purchased from the Jackson laboratory. For the analysis of epidermal pigmentation, C3H/HeJ mouse ears were topically treated with 20 μl of KH7 (42 mg/ml) or LRE1 (28 mg/ml) on the right ear, and DMSO (vehicle control) was topically applied on the left ear twice a day for 2 weeks. In parallel, a different group of mice was treated with DMSO on both ears. Ear skin was monitored daily for irritation and changes in pigmentation. After the final treatment, mice were euthanized, and the treated skin was submitted to an animal pathologist for blinded histological evaluation of the epidermis and specific staining (Fontana-Masson). This experiment was performed twice with three mice per cohort (for a total of six mice). Statistical analysis All statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software). Proliferation rate was assessed by linear regression. LysoSensor calibration curves were generated using a third-order polynomial regression. Comparisons of median DAMP frequency distributions between conditions were analyzed using a Mann-Whitney U test. For all other data, comparison of means was performed using an unpaired, two-tailed t test (for two groups) or a one-way ANOVA with Tukey correction (for groups of three or more).

SUPPLEMENTARY MATERIALS www.sciencesignaling.org/cgi/content/full/11/555/eaau7987/DC1 Fig. S1. sAC expression in human melanocytes. Fig. S2. Establishment of Adcy10−/− (sACKO) mouse melanocytes. Fig. S3. Measurement of melanosome pH using DAMP. Fig. S4. sAC regulates organelle pH in melanocytes. Fig. S5. Modulation of sAC-dependent cAMP signaling does not affect melanosome marker fluorescence. Fig. S6. Effects of sAC inhibition on melanosome pH and tyrosinase abundance in human melanocytes. Fig. S7. Effects of distinct sources of cAMP on melanosome pH. Fig. S8. Inhibition of protein synthesis does not affect melanosome pH. Fig. S9. Regulation of melanosome pH in human and mouse melanocytes by distinct cAMP effector proteins. Fig. S10. Pharmacologic inhibition or genetic ablation of sAC increases melanization. Fig. S11. Inhibition of sAC signaling increases eumelanin production. Table S1. Mann-Whitney analysis of median cellular DAMP fluorescence. Table S2. Assessment of Tyr::CRE-ERT2;sACf/f mice.

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Acknowledgments: We thank R. Halaban and A. Bacchiocchi (Biospecimen Core of the Yale SPORE in Skin Cancer, Yale University School of Medicine) for assistance in the development of the mouse melanocyte cell lines. We also thank L. Cohen-Gould, S. Mukherjee, and the Optical Microscopy Core at Weill Cornell Medical College for technical assistance; M. Lunquist and A. Bolin at Weill Cornell Medical College for computational assistance; J. D. Warren and the Milstein Chemistry Core Facility at Weill Cornell Medical College for the synthesis of DAMP; C. Burd (Ohio State University), E. Piskounova (Weill Cornell Medical College), and members of the Zippin laboratory for the critical reading of the manuscript; and P. Christos (Weill Cornell Medical College) for the independent analysis of the statistics of the manuscript. The content of this study is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Funding: D. Z. was supported by a Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the National Institutes of Health under award number T32GM007739 to the Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program. C.N. was funded in part by Université de Franche Comté Sciences Médicales et Pharmaceutiques–Année-Recherche 2013, Société Française de Dermatologie–AO bourse de soutien pour la formation à la recherche en dermatologie, and Collège des Enseignants de Dermatologie En France–Bourse CEDEF d’aide à la mobilité. J.H.Z. was funded in part by a Melanoma Research Alliance Team Science Award, a Clinique Clinical Scholars Award, an American Skin Association Calder Research Scholar Award, and the NCI (K08 CA 160657-01). K.W. and S.I. were supported, in part, by the Japan Society for the Promotion of Science (JSPS) (grant nos. 26461705 and 15K09794). Author contributions: D.Z., K.O., and J.H.Z. designed the experiments. D.Z., K.O., A.W., M.F., M.R., O.W., A.S., K.W., and S.I. generated the figures. C.N. generated the melanocyte cell lines. K.W. and S.I. analyzed all melanin levels. D.Z., L.R.L., J.B., and J.H.Z wrote the manuscript, with all authors providing feedback. Competing interests: L.R.L., J.B., and J.H.Z. own equity interest in CEP Biotech, which has licensed commercialization of a panel of monoclonal antibodies directed against sAC. J.H.Z. is a paid consultant and on the medical advisory board of Hoth Therapeutics, is on the medical advisory board of SHADE Inc., and an inventor on an international patent application PCT/US2017/040428 on “Methods of modulating melanosome pH and melanin level in cells.” L.R.L., J.B., and J.H.Z. are inventors on a U.S. patent 8,859,213 on the use of antibodies directed against sAC for the diagnosis of melanocytic proliferations. Data and materials availability: All data needed to evaluate the conclusions are present in the paper or the Supplementary Materials.