Biomolecules & Therapeutics

The Korean Society of Applied Pharmacology (한국응용약물학회)
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α-MSH Induces Intracellular ROS Generation in Melanoma Cells through NADPH Oxidase-1/4 Activation

  • Kyuri Kim (College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University) ;
  • Jihyun Yoon (College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University) ;
  • Hye Yeon Kim (College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University) ;
  • Kyung-Min Lim (College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University)
  • Received : 2025.12.12
  • Accepted : 2026.02.12
  • Published : 2026.05.01
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Abstract

α-Melanocyte-stimulating hormone (α-MSH) is a key physiological inducer of melanogenesis and melanocyte activation. Reactive oxygen species (ROS) function as secondary messengers in intracellular signaling pathways that regulate α-MSH-mediated melanogenesis. However, the mechanism by which α-MSH induces ROS generation in melanocytes remains incompletely understood. Here, we investigated the sources and regulatory mechanisms of α-MSH-induced ROS in murine B16F10 and human MNT-1 melanoma cells. Confocal microscopy revealed that α-MSH predominantly stimulated cytosolic rather than mitochondrial ROS generation. Western blot analysis showed that α-MSH upregulated the expression of NADPH oxidases NOX1 and NOX4 at early time points, key enzymes involved in cytosolic ROS production. Pharmacological inhibition of NOX1/4 significantly reduced α-MSH-induced ROS levels and melanin content, accompanied by decreased phosphorylation of MITF, a melanogenic transcription factor. These findings indicate that α-MSH is a key physiological inducer of melanogenesis and melanocyte-related signaling in melanoma cells. Our study highlights NOX4, together with NOX1, as potential targets for anti-pigmentation strategies.

Keywords

Acknowledgement

This study was supported by Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (RS-2024-00344565) and National Research Foundation of Korea (RS-2023-00273356).

References

  1. Angelova, P. R., Dinkova-Kostova, A. T. and Abramov, A. Y. (2021) Assessment of ROS production in the mitochondria of live cells. In Reactive Oxygen Species: Methods and Protocols, pp. 33-42.
  2. Autier, P. and Doré, J.-F. (2020) Ultraviolet radiation and cutaneous melanoma: a historical perspective. Melanoma Res. 30, 113-125. https://doi.org/10.1097/CMR.0000000000000609
  3. Bento-Lopes, L., Cabaço, L. C., Charneca, J., Neto, M. V., Seabra, M. C. and Barral, D. C. (2023) Melanin's journey from melanocytes to keratinocytes: uncovering the molecular mechanisms of melanin transfer and processing. Int. J. Mol. Sci. 24, 11289.
  4. Bienert, G. P., Møller, A. L., Kristiansen, K. A., Schulz, A., Møller, I. M., Schjoerring, J. K. and Jahn, T. P. (2007) Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes. J. Biol. Chem. 282, 1183-1192. https://doi.org/10.1074/jbc.M603761200
  5. Bin, B. H., Bhin, J., Yang, S. H., Choi, D. H., Park, K., Shin, D. W., Lee, A. Y., Hwang, D., Cho, E. G. and Lee, T. R. (2014) Hyperosmotic stress reduces melanin production by altering melanosome formation. PLoS One 9, e105965. https://doi.org/10.1371/journal.pone.0105965
  6. Block, K. and Gorin, Y. (2012) Aiding and abetting roles of NOX oxidases in cellular transformation. Nat. Rev. Cancer 12, 627-637. https://doi.org/10.1038/nrc3339
  7. Brar, S. S., Kennedy, T. P., Sturrock, A. B., Huecksteadt, T. P., Quinn, M. T., Whorton, A. R. and Hoidal, J. R. (2002) An NAD(P)H oxidase regulates growth and transcription in melanoma cells. Am. J. Physiol. Cell Physiol. 282, C1212-C1224. https://doi.org/10.1152/ajpcell.00496.2001
  8. Cargnello, M. and Roux, P. P. (2011) Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases. Microbiol. Mol. Biol. Rev. 75, 50-83. https://doi.org/10.1128/MMBR.00031-10
  9. Castejón-Griñán, M., Cerdido, S., Sánchez-Beltrán, J., Lambertos, A., Abrisqueta, M., Herraiz, C., Jiménez-Cervantes, C. and GarcíaBorrón, J. C. (2024) Melanoma-associated melanocortin 1 receptor variants confer redox signaling-dependent protection against oxidative DNA damage. Redox Biol. 72, 103135. https://doi.org/10.1016/j.redox.2024.103135
  10. Costin, G.-E. and Hearing, V. J. (2007) Human skin pigmentation: melanocytes modulate skin color in response to stress. FASEB J. 21, 976-994. https://doi.org/10.1096/fsb2.v21.4
  11. d'Ischia, M., Wakamatsu, K., Cicoira, F., Di Mauro, E., Garcia-Borron, J. C., Commo, S., Galván, I., Ghanem, G., Kenzo, K. and Meredith, P. (2015) Melanins and melanogenesis: from pigment cells to human health and technological applications. Pigment Cell Melanoma Res. 28, 520-544. https://doi.org/10.1111/pcmr.2015.28.issue-5
  12. D'Mello, S. A., Finlay, G. J., Baguley, B. C. and Askarian-Amiri, M. E. (2016) Signaling pathways in melanogenesis. Int. J. Mol. Sci. 17, 1144.
  13. D'Orazio, J., Jarrett, S., Amaro-Ortiz, A. and Scott, T. (2013) UV radiation and the skin. Int. J. Mol. Sci. 14, 12222-12248. https://doi.org/10.3390/ijms140612222
  14. Daiber, A., Di Lisa, F., Oelze, M., Kröller-Schön, S., Steven, S., Schulz, E. and Münzel, T. (2017) Crosstalk of mitochondria with NADPH oxidase via reactive oxygen and nitrogen species signalling and its role for vascular function. Br. J. Pharmacol. 174, 1670-1689. https://doi.org/10.1111/bph.v174.12
  15. De Jager, T., Cockrell, A. and Du Plessis, S. (2017) Ultraviolet light induced generation of reactive oxygen species. In Ultraviolet Light in Human Health, Diseases and Environment, pp. 15-23.
  16. Dikalov, S. I., Dikalova, A. E., Bikineyeva, A. T., Schmidt, H. H., Harrison, D. G. and Griendling, K. K. (2008) Distinct roles of Nox1 and Nox4 in basal and angiotensin II-stimulated superoxide and hydrogen peroxide production. Free Radic. Biol. Med. 45, 1340-1351. https://doi.org/10.1016/j.freeradbiomed.2008.08.013
  17. Feichtinger, R. G., Sperl, W., Bauer, J. W. and Kofler, B. (2014) Mitochondrial dysfunction: a neglected component of skin diseases. Exp. Dermatol. 23, 607-614. https://doi.org/10.1111/exd.2014.23.issue-9
  18. Finkel, T. and Holbrook, N. J. (2000) Oxidants, oxidative stress and the biology of ageing. Nature 408, 239-247. https://doi.org/10.1038/35041687
  19. García-Borrón, J. C., Sánchez-Laorden, B. L. and Jiménez-Cervantes, C. (2005) Melanocortin-1 receptor structure and functional regulation. Pigment Cell Res. 18, 393-410. https://doi.org/10.1111/pcr.2005.18.issue-6
  20. Ghali, L., Wong, S.-T., Tidman, N., Quinn, A., Philpott, M. P. and Leigh, I. M. (2004) Epidermal and hair follicle progenitor cells express melanoma-associated chondroitin sulfate proteoglycan core protein. J. Invest. Dermatol. 122, 433-442. https://doi.org/10.1046/j.0022-202X.2004.22207.x
  21. Hearing, V. J. (2011) Determination of melanin synthetic pathways. J. Invest. Dermatol. 131, E8-E11. https://doi.org/10.1038/skinbio.2011.4
  22. Hemesath, T. J., Price, E. R., Takemoto, C., Badalian, T. and Fisher, D. E. (1998) MAP kinase links the transcription factor Microphthalmia to c-Kit signalling in melanocytes. Nature 391, 298-301. https://doi.org/10.1038/34681
  23. Hosoi, J., Abe, E., Suda, T. and Kuroki, T. (1985) Regulation of melanin synthesis of B16 mouse melanoma cells by 1α, 25-dihydroxyvitamin D3 and retinoic acid. Cancer Res. 45, 1474-1478.
  24. Jakubczyk, K., Dec, K., Kałduńska, J., Kawczuga, D., Kochman, J. and Janda, K. (2020) Reactive oxygen species-sources, functions, oxidative damage. Pol. Merk. Lek. 48, 124-127.
  25. Jeayeng, S., Wongkajornsilp, A., Slominski, A. T., Jirawatnotai, S., Sampattavanich, S. and Panich, U. (2017) Nrf2 in keratinocytes modulates UVB-induced DNA damage and apoptosis in melanocytes through MAPK signaling. Free Radic. Biol. Med. 108, 918-928. https://doi.org/10.1016/j.freeradbiomed.2017.05.009
  26. Kalyanaraman, B., Darley-Usmar, V., Davies, K. J., Dennery, P. A., Forman, H. J., Grisham, M. B., Mann, G. E., Moore, K., Roberts II, L. J. and Ischiropoulos, H. (2012) Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations. Free Radic. Biol. Med. 52, 1-6.
  27. Kawahara, T., Quinn, M. T. and Lambeth, J. D. (2007) Molecular evolution of the reactive oxygen-generating NADPH oxidase (Nox/Duox) family of enzymes. BMC Evol. Biol. 7, 1-21.
  28. Kim, E. S., Park, S. J., Goh, M. J., Na, Y. J., Jo, D. S., Jo, Y. K., Shin, J. H., Choi, E. S., Lee, H. K. and Kim, J. Y. (2014) Mitochondrial dynamics regulate melanogenesis through proteasomal degradation of MITF via ROS-ERK activation. Pigment Cell Melanoma Res. 27, 1051-1062. https://doi.org/10.1111/pcmr.2014.27.issue-6
  29. Kim, M. and Lim, K. M. (2024) DEHP (di(2-ethylhexyl)phthalate) stimulates skin pigmentation by perturbing cytoskeletal homeostasis. Toxicol. Res. 40, 487-497.
  30. Kim, S. Y. and Choi, Y. H. (2025) Honokiol induces paraptosis-like cell death through mitochondrial ROS-dependent endoplasmic reticulum stress in hepatocellular carcinoma Hep3B cells. Toxicol. Res. 41, 385-396. https://doi.org/10.1007/s43188-025-00291-2
  31. Lambeth, J. D. (2004) NOX enzymes and the biology of reactive oxygen. Nat. Rev. Immunol. 4, 181-189.  https://doi.org/10.1038/nri1312
  32. Leto, T. L. and Geiszt, M. (2006) Role of Nox family NADPH oxidases in host defense. Antioxid. Redox Signal. 8, 1549-1561. https://doi.org/10.1089/ars.2006.8.1549
  33. Liu-Smith, F., Dellinger, R. and Meyskens, F. L., Jr. (2014) Updates of reactive oxygen species in melanoma etiology and progression. Arch. Biochem. Biophys. 563, 51-55. https://doi.org/10.1016/j.abb.2014.04.007
  34. Liu, F., Garcia, A. M. G. and Meyskens, F. L., Jr. (2012a) NADPH oxidase 1 overexpression enhances invasion via matrix metalloproteinase-2 and epithelial–mesenchymal transition in melanoma cells. J. Invest. Dermatol. 132, 2033-2041. https://doi.org/10.1038/jid.2012.119
  35. Liu, G.-S., Peshavariya, H., Higuchi, M., Brewer, A. C., Chang, C. W., Chan, E. C. and Dusting, G. J. (2012b) Microphthalmia-associated transcription factor modulates expression of NADPH oxidase type 4: a negative regulator of melanogenesis. Free Radic. Biol. Med. 52, 1835-1843. https://doi.org/10.1016/j.freeradbiomed.2012.02.040
  36. Liu, G.-S., Wu, J.-C., Tsai, H.-E., Dusting, G. J., Chan, E. C., Wu, C.-S. and Tai, M.-H. (2014) Proopiomelanocortin gene delivery induces apoptosis in melanoma through NADPH oxidase 4-mediated ROS generation. Free Radic. Biol. Med. 70, 14-22. https://doi.org/10.1016/j.freeradbiomed.2013.12.024
  37. Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63. https://doi.org/10.1016/0022-1759(83)90303-4
  38. Nisimoto, Y., Jackson, H. M., Ogawa, H., Kawahara, T. and Lambeth, J. D. (2010) Constitutive NADPH-dependent electron transferase activity of the Nox4 dehydrogenase domain. Biochemistry 49, 2433-2442. https://doi.org/10.1021/bi9022285
  39. Oh, S.-Y. and Hyun, C.-G. (2022) Chrysoeriol enhances melanogenesis in B16F10 cells through the modulation of the MAPK, AKT, PKA, and Wnt/β-catenin signaling pathways. Nat. Prod. Commun. 17, 1934578X211069204. https://doi.org/10.1177/1934578X211069204
  40. Rudolf, J., Raad, H., Taieb, A. and Rezvani, H. R. (2018) NADPH oxidases and their roles in skin homeostasis and carcinogenesis. Antioxid. Redox Signal. 28, 1238-1261. https://doi.org/10.1089/ars.2017.7282
  41. Sander, C. S., Chang, H., Hamm, F., Elsner, P. and Thiele, J. J. (2004) Role of oxidative stress and the antioxidant network in cutaneous carcinogenesis. Int. J. Dermatol. 43, 326-335. https://doi.org/10.1111/ijd.2004.43.issue-5
  42. Schieber, M. and Chandel, N. S. (2014) ROS function in redox signaling and oxidative stress. Curr. Biol. 24, R453-R462. https://doi.org/10.1016/j.cub.2014.03.034
  43. Sreedhar, A., Aguilera-Aguirre, L. and Singh, K. K. (2020) Mitochondria in skin health, aging, and disease. Cell Death Dis. 11, 444. https://doi.org/10.1038/s41419-020-2649-z
  44. Sun, J. and Nan, G. (2016) The mitogen-activated protein kinase (MAPK) signaling pathway as a discovery target in stroke. J. Mol. Neurosci. 59, 90-98. https://doi.org/10.1007/s12031-016-0717-8
  45. Thody, A. J. (1999) α-MSH and the regulation of melanocyte function. Ann. N. Y. Acad. Sci. 885, 217-229. https://doi.org/10.1111/nyas.1999.885.issue-1
  46. Venza, I., Venza, M., Visalli, M., Lentini, G., Teti, D. and d' Alcontres, F. S. (2021) ROS as regulators of cellular processes in melanoma. Oxid. Med. Cell. Longev. 2021, 1208690.
  47. Vermot, A., Petit-Härtlein, I., Smith, S. M. and Fieschi, F. (2021) NADPH oxidases (NOX): an overview from discovery, molecular mechanisms to physiology and pathology. Antioxidants 10, 890. https://doi.org/10.3390/antiox10060890
  48. Yasumoto, K. i., Takeda, K., Saito, H., Watanabe, K., Takahashi, K. and Shibahara, S. (2002) Microphthalmia-associated transcription factor interacts with LEF-1, a mediator of Wnt signaling. EMBO J. 21, 2703-2714. https://doi.org/10.1093/emboj/21.11.2703
  49. Ye, Y., Wang, H., Chu, J. H., Chou, G. X. and Yu, Z. L. (2011) Activation of p38 MAPK pathway contributes to the melanogenic property of apigenin in B16 cells. Exp. Dermatol. 20, 755-757. https://doi.org/10.1111/exd.2011.20.issue-9