Research ArticleSkin Biology

Mammalian pigmentation is regulated by a distinct cAMP-dependent mechanism that controls melanosome pH

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Science Signaling  06 Nov 2018:
Vol. 11, Issue 555, eaau7987
DOI: 10.1126/scisignal.aau7987
  • Fig. 1 sAC regulates melanosome pH in mouse and human melanocytes.

    (A) Confocal microscopic images of DAMP (green) and TYRP1 (red) immunofluorescence in sACFF [floxed/floxed (FF), top] and sACKO [knockout (KO), bottom] mouse melanocytes. (B) Frequency distribution (left) and median (right) DAMP fluorescence intensity at TYRP1+ (as detected by the TA99 antibody) melanosomes in sACFF (FF, black squares or bar) and sACKO (KO, red circles or bar) melanocytes. arb. units, arbitrary units. (C) Confocal microscopic images of DAMP (green) and HMB45 (red) immunofluorescence in sACFF (FF, top) and sACKO (KO, bottom) mouse melanocytes. (D) Frequency distribution (left) and median (right) DAMP fluorescence intensity at HMB45+ melanosomes (left) in sACFF (FF, black squares or bar) and sACKO (KO, red circles or bar) melanocytes. (E) Confocal microscopic images of DAMP (green) and HMB45 (red) immunofluorescence in sACFF cells treated with vehicle control (FF, top) or 30 μM KH7 (FF + KH7, bottom). (F) Frequency distribution (left) and median (right) DAMP fluorescence intensity at HMB45+ melanosomes (left) in sACFF cells treated with vehicle control (FF, black squares or bar) or with 30 μM KH7 (FF + KH7, blue triangles or bar) or LRE1 (FF + LRE1, gray Xs or bar). (G to I) Frequency distribution (left) and median (right) DAMP fluorescence intensity at HMB45+ melanosomes in human melanocytes derived from patients with lighter skin tone treated with vehicle control [C38 (G), C226 (H), C532 (I); black squares or bar] or with 30 μM KH7 [C38 + KH7 (G), C226 + KH7 (H), C532 + KH7 (I); blue triangles or bar]. (B, D, and F to I) Data are from the experiments performed on cells on four distinct coverslips per condition, where at least 15 cells and at least 1000 melanosomes were analyzed per coverslip. (B, D, F, and G to I) Mann-Whitney U test, ****P < 0.001. Scale bars, 10 μm.

  • Fig. 2 cAMP rescues the increase in melanosome pH after loss of sAC activity in mouse and human melanocytes.

    (A) Frequency distribution (left) and median (right) of DAMP fluorescence intensity at HMB45+ melanosomes in sACFF (FF, black squares or bar), sACKO (KO, red circles or bars), and cAMP-treated sACKO (KO + cAMP, green triangles or bars) mouse melanocytes. (B) Frequency distribution (left) and median (right) of DAMP fluorescence intensity at HMB45+ melanosomes in sACFF (FF, black squares or bar), KH7-treated sACFF (FF + KH7, blue triangles or bar), and KH7 + cAMP–treated sACFF (FF + KH7 + cAMP, green diamonds or bar) mouse melanocytes. (C) Frequency distribution (left) and median (right) of DAMP fluorescence at HMB45+ melanosomes in “light” human melanocytes (C38) after treatment with vehicle control (black squares or bar), KH7 (blue triangles or bar), or KH7 + cAMP (green diamonds or bar). (A to C) Data are from the experiments performed on cells on four distinct coverslips per condition, where at least 15 cells and at least 1000 melanosomes were analyzed per coverslip. “+KH7” consisted of incubation with 30 μM KH7 for 4 hours. “+KH7+cAMP” treatment consisted of incubation with 30 μM KH7 and 500 μM nonselective cAMP analog [Sp-8-CPT-cAMPs] for 4 hours. One-way analysis of variance (ANOVA) with Tukey post hoc analysis, ***P < 0.005, ****P < 0.001.

  • Fig. 3 Regulation of melanosome pH by EPAC.

    (A) Frequency distribution (left) and median (right) of DAMP fluorescence intensity at HMB45+ melanosomes in sACFF (FF, black squares or bars), sACKO (KO, red circles or bars), and ESI-09–treated sACFF (FF + ESI-09, purple diamonds or bars) mouse melanocytes. (B and C) Frequency distribution (left) and median (right) of DAMP fluorescence intensity at HMB45+ melanosomes in “light” human melanocytes after treatment with vehicle control [dimethyl sulfoxide (DMSO), C226 (B), C537 (C); black squares or bars], KH7 [C226 + KH7 (B), C537 + KH7 (C); blue triangles or bars], or ESI-09 [C226 + ESI-09 (B), C537 + ESI-09 (C); purple Xs or bars]. (D) Frequency distribution (left) and median (right) of DAMP fluorescence intensity at HMB45+ melanosomes in sACFF (FF, black squares and bars) and sACKO melanocytes in the absence (KO, red circles or bars) or presence of the EPAC-selective cAMP analog (KO + cAMPEPAC, pink Xs or bars). (E) Frequency distribution (left) and median (right) of DAMP fluorescence intensity at HMB45+ melanosomes of sACFF mouse melanocytes after a 4-hour treatment with vehicle (DMSO; FF, black squares or bars), KH7 alone (FF + KH7, blue triangles or bars), or KH7 + the EPAC-selective cAMP analog (FF + KH7 + cAMPEPAC, pink Xs or bars). (F) Frequency distribution (left) or median (right) of DAMP fluorescence intensity at HMB45 melanosomes in “light” human melanocytes after a 4-hour treatment with vehicle control (C226, black squares or bars), KH7 alone (C226 + KH7, blue triangles or bars), or KH7 + the EPAC-selective cAMP analog (C226 + KH7 + cAMPEPAC, pink Xs or bars). (A to F) Data are from the experiments performed on cells on four distinct coverslips per condition, where at least 15 cells and at least 1000 melanosomes were analyzed per coverslip. KH7 treatment consisted of incubation with 30 μM KH7 for 4 hours. ESI-09 treatment consisted of incubation with 10 μM ESI-09 for 4 hours. cAMP treatment consisted of incubation with 500 μM EPAC-selective cAMP analog (8-pHPT-2′-O-Me-cAMP) for 4 hours. Mann-Whitney U test, **P < 0.01, ****P <0.001. Scale bars, 10 μm.

  • Fig. 4 sAC regulates tyrosinase activity in cells independently of gene expression.

    (A) Tyrosinase activity of sACFF (FF) and sACKO (KO) mouse melanocytes as measured by production of 3H2O from 3H-tyrosine per cell over 4 hours (n = 3 independent experiments; each point is the average of duplicate determinations). DPM, disintegrations per minute. (B) Tyrosinase activity of “light” human melanocytes (C226, C38, and C611) treated with KH7 (30 μM), LRE1 (50 μM), or vehicle (DMSO) for 8 hours as measured by 3H2O production per cell (n = 3 distinct human cell lines; each point is the average of duplicate determinations). (C) Western blot for tyrosinase and MITF in sACFF (FF) and sACKO (KO) mouse melanocytes (left). Average quantitation of total tyrosinase immunoreactivity in sACFF (FF) and sACKO (KO) mouse melanocytes was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH; right) (n = 3 independent experiments; each experiment is the average of duplicate determinations). (D and E) Electron microscopy (EM) evaluation of melanosome morphology in sACFF (FF) and sACKO (KO) mouse melanocytes (D) and quantitation of stage IV melanosomes (organelles circled in red) per field expressed as percentage of total melanosomes (E) in sACFF (FF, black bars) and sACKO (KO, red bars) mouse melanocytes (n = 3 experiments, with 15 cells examined per experiment). (A and E) Student’s t test, (B) one-way ANOVA with Tukey post hoc analysis; *P < 0.05. Scale bars, 1 μm.

  • Fig. 5 sAC regulates melanin synthesis in mouse and human melanocytes.

    (A) Cellular eumelanin content in sACFF (FF, black bar) and sACKO (KO, red bar) mouse melanocytes. Average of triplicate determinations per experiment (n = 3 experiments). Below are representative cellular pellets for each condition. (B and C) Cellular eumelanin level of “light” (B, n = 8) and “dark” (C, n = 5) human melanocytes treated with KH7 (30 μM, blue bars) or vehicle (DMSO, black and gray bars) for 48 hours expressed as fold over vehicle control. Triplicate determinations were performed for each cell line. n is number of distinct human primary cell lines. Representative cell pellets pictured below. (D) Cellular eumelanin content in sACFF (FF, black bars) and sACKO (KO, red bars) mouse melanocytes in the absence (−) or presence (+) of NDP-MSH (MSH, 10 nM) for 72 hours. Average of triplicate determinations per experiment (n = 3 experiments). Asterisks above the bars correspond to comparison of − to + MSH conditions in each genotype. Below are representative cellular pellets for each condition. (E) Cellular eumelanin level of “light” human melanocytes (n = 3) treated with ESI-09 (10 μM, purple bar) or vehicle (DMSO, black bar) for 48 hours expressed as fold over vehicle control. Triplicate determinations were performed for each line. n is number of distinct human primary cell lines. Representative cell pellets pictured below. (F) Cellular pheomelanin content in sACFF (FF, black bar) and sACKO (KO, red bar) mouse melanocytes. Average of triplicate determinations per experiment (n = 3 experiments). (G) Ratio of pheomelanin to eumelanin in sACFF (FF, black bar) and sACKO (KO, red bar) mouse melanocytes. Average of triplicate determinations per experiment (n = 3 experiments). (A to G) Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001.

  • Fig. 6 sAC regulates pigmentation in mice.

    (A) Representative photographs of C3H/HeJ Tyr::CRE-ERT2;sACfl/fl (wild type, sACfl/fl left pair) and C3H/HeJ Tyr::CRE-ERT2+;sAC−/− (sACKO, right pair) mice showing a darkening of hair after loss of sAC in melanocytes. (B) Average percentage of total AWL hairs with agouti banding in sACfl/fl (FF, black bar; n = 13 mice) and sAC−/− (KO, red bar; n = 7 mice). (C) Average ratio of pheomelanin to eumelanin content in hair from Tyr::CRE-ERT2;sACfl/fl (FF, black bar; n = 13 mice) and Tyr::CRE-ERT2+;sAC−/− (KO, red bar; n = 7 mice) mice. Student’s t test; *P < 0.05, **P < 0.01. (D) Representative photographs of mouse ears (top inset) and accompanying Fontana-Masson stain (bottom inset) after treatment with vehicle (DMSO) on both ears (left; n = 6 mice), vehicle on the left ear and LRE1 on the right ear (middle; n = 6 mice), or vehicle on the left ear and KH7 on the right ear (right; n = 6 mice) twice daily for 2 weeks. Arrowheads indicate positive Fontana-Masson staining of melanin content in epidermis. Topical treatments were performed with 20 μl of KH7 (42 mg/ml), LRE1 (28 mg/ml), or vehicle alone (DMSO). Scale bar, 50 μm. (E) Schematic of sAC- and tmAC-dependent cAMP signaling domains in melanocytes. MC1R (gray box at plasma membrane) binds to MSH (blue circle), leading to activation of tmACs (brown box at plasma membrane). tmAC-dependent cAMP activates PKA, leading to CREB (cAMP response element–binding protein)–dependent MITF expression and, ultimately, increased abundance of TYR (gray transmembrane melanosome protein, tyrosinase). sAC (blue oval), which responds to changes in HCO3, ATP, and Ca2+ (gray circles), stimulates EPAC, leading to altered melanosome pH, likely by regulating melanosome ion channels, and changes in the activity of pH-sensitive tyrosinase. pH regulation of tyrosinase differentially affects eumelanin and pheomelanin synthesis.

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.

  • This PDF file includes:

    • 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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