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. 1997 Feb 15;99(4):635–642. doi: 10.1172/JCI119206

Nitric oxide produced by ultraviolet-irradiated keratinocytes stimulates melanogenesis.

C Roméro-Graillet 1, E Aberdam 1, M Clément 1, J P Ortonne 1, R Ballotti 1
PMCID: PMC507845  PMID: 9045865

Abstract

Ultraviolet (UV) radiation is the main physiological stimulus for human skin pigmentation. Within the epidermal-melanin unit, melanocytes synthesize and transfer melanin to the surrounding keratinocytes. Keratinocytes produce paracrine factors that affect melanocyte proliferation, dendricity, and melanin synthesis. In this report, we show that normal human keratinocytes secrete nitric oxide (NO) in response to UVA and UVB radiation, and we demonstrate that the constitutive isoform of keratinocyte NO synthase is involved in this process. Next, we investigate the melanogenic effect of NO produced by keratinocytes in response to UV radiation using melanocyte and keratinocyte cocultures. Conditioned media from UV-exposed keratinocytes stimulate tyrosinase activity of melanocytes. This effect is reversed by NO scavengers, suggesting an important role for NO in UV-induced melanogenesis. Moreover, melanocytes respond to NO-donors by decreased growth, enhanced dendricity, and melanogenesis. The rise in melanogenesis induced by NO-generating compounds is associated with an increased amount of both tyrosinase and tyrosinase-related protein 1. These observations suggest that NO plays an important role in the paracrine mediation of UV-induced melanogenesis.

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Selected References

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  1. Aberdam E., Roméro C., Ortonne J. P. Repeated UVB irradiations do not have the same potential to promote stimulation of melanogenesis in cultured normal human melanocytes. J Cell Sci. 1993 Dec;106(Pt 4):1015–1022. doi: 10.1242/jcs.106.4.1015. [DOI] [PubMed] [Google Scholar]
  2. Baudouin J. E., Tachon P. Constitutive nitric oxide synthase is present in normal human keratinocytes. J Invest Dermatol. 1996 Mar;106(3):428–431. doi: 10.1111/1523-1747.ep12343523. [DOI] [PubMed] [Google Scholar]
  3. Beitner H. Immediate pigment-darkening reaction. Photodermatol. 1988 Apr;5(2):96–100. [PubMed] [Google Scholar]
  4. Bos J. D., Kapsenberg M. L. The skin immune system: progress in cutaneous biology. Immunol Today. 1993 Feb;14(2):75–78. doi: 10.1016/0167-5699(93)90062-P. [DOI] [PubMed] [Google Scholar]
  5. Bredt D. S., Snyder S. H. Nitric oxide: a physiologic messenger molecule. Annu Rev Biochem. 1994;63:175–195. doi: 10.1146/annurev.bi.63.070194.001135. [DOI] [PubMed] [Google Scholar]
  6. Brouet I., Ohshima H. Curcumin, an anti-tumour promoter and anti-inflammatory agent, inhibits induction of nitric oxide synthase in activated macrophages. Biochem Biophys Res Commun. 1995 Jan 17;206(2):533–540. doi: 10.1006/bbrc.1995.1076. [DOI] [PubMed] [Google Scholar]
  7. Bécherel P. A., Le Goff L., Ktorza S., Ouaaz F., Mencia-Huerta J. M., Dugas B., Debré P., Mossalayi M. D., Arock M. Interleukin-10 inhibits IgE-mediated nitric oxide synthase induction and cytokine synthesis in normal human keratinocytes. Eur J Immunol. 1995 Oct;25(10):2992–2995. doi: 10.1002/eji.1830251042. [DOI] [PubMed] [Google Scholar]
  8. Bécherel P. A., Mossalayi M. D., Ouaaz F., Le Goff L., Dugas B., Paul-Eugène N., Frances C., Chosidow O., Kilchherr E., Guillosson J. J. Involvement of cyclic AMP and nitric oxide in immunoglobulin E-dependent activation of Fc epsilon RII/CD23+ normal human keratinocytes. J Clin Invest. 1994 May;93(5):2275–2279. doi: 10.1172/JCI117227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. De Luca M., D'Anna F., Bondanza S., Franzi A. T., Cancedda R. Human epithelial cells induce human melanocyte growth in vitro but only skin keratinocytes regulate its proper differentiation in the absence of dermis. J Cell Biol. 1988 Nov;107(5):1919–1926. doi: 10.1083/jcb.107.5.1919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. DeLeo V., Scheide S., Meshulam J., Hanson D., Cardullo A. Ultraviolet radiation alters choline phospholipid metabolism in human keratinocytes. J Invest Dermatol. 1988 Oct;91(4):303–308. doi: 10.1111/1523-1747.ep12475636. [DOI] [PubMed] [Google Scholar]
  11. Deliconstantinos G., Villiotou V., Stravrides J. C. Release by ultraviolet B (u.v.B) radiation of nitric oxide (NO) from human keratinocytes: a potential role for nitric oxide in erythema production. Br J Pharmacol. 1995 Mar;114(6):1257–1265. doi: 10.1111/j.1476-5381.1995.tb13341.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Donatien P., Surlève-Bazeille J. E., Thody A. J., Taïeb A. Growth and differentiation of normal human melanocytes in a TPA-free, cholera toxin-free, low-serum medium and influence of keratinocytes. Arch Dermatol Res. 1993;285(7):385–392. doi: 10.1007/BF00372130. [DOI] [PubMed] [Google Scholar]
  13. Eisinger M., Marko O. Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin. Proc Natl Acad Sci U S A. 1982 Mar;79(6):2018–2022. doi: 10.1073/pnas.79.6.2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Eller M. S., Ostrom K., Gilchrest B. A. DNA damage enhances melanogenesis. Proc Natl Acad Sci U S A. 1996 Feb 6;93(3):1087–1092. doi: 10.1073/pnas.93.3.1087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Englaro W., Rezzonico R., Durand-Clément M., Lallemand D., Ortonne J. P., Ballotti R. Mitogen-activated protein kinase pathway and AP-1 are activated during cAMP-induced melanogenesis in B-16 melanoma cells. J Biol Chem. 1995 Oct 13;270(41):24315–24320. doi: 10.1074/jbc.270.41.24315. [DOI] [PubMed] [Google Scholar]
  16. Evans T., Carpenter A., Cohen J. Purification of a distinctive form of endotoxin-induced nitric oxide synthase from rat liver. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5361–5365. doi: 10.1073/pnas.89.12.5361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Friedmann P. S., Gilchrest B. A. Ultraviolet radiation directly induces pigment production by cultured human melanocytes. J Cell Physiol. 1987 Oct;133(1):88–94. doi: 10.1002/jcp.1041330111. [DOI] [PubMed] [Google Scholar]
  18. Förstermann U., Closs E. I., Pollock J. S., Nakane M., Schwarz P., Gath I., Kleinert H. Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions. Hypertension. 1994 Jun;23(6 Pt 2):1121–1131. doi: 10.1161/01.hyp.23.6.1121. [DOI] [PubMed] [Google Scholar]
  19. Förstermann U., Schmidt H. H., Pollock J. S., Sheng H., Mitchell J. A., Warner T. D., Nakane M., Murad F. Isoforms of nitric oxide synthase. Characterization and purification from different cell types. Biochem Pharmacol. 1991 Oct 24;42(10):1849–1857. doi: 10.1016/0006-2952(91)90581-o. [DOI] [PubMed] [Google Scholar]
  20. Gilchrest B. A., Park H. Y., Eller M. S., Yaar M. Mechanisms of ultraviolet light-induced pigmentation. Photochem Photobiol. 1996 Jan;63(1):1–10. doi: 10.1111/j.1751-1097.1996.tb02988.x. [DOI] [PubMed] [Google Scholar]
  21. Halaban R., Langdon R., Birchall N., Cuono C., Baird A., Scott G., Moellmann G., McGuire J. Basic fibroblast growth factor from human keratinocytes is a natural mitogen for melanocytes. J Cell Biol. 1988 Oct;107(4):1611–1619. doi: 10.1083/jcb.107.4.1611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hearing V. J., Ekel T. M. Mammalian tyrosinase. A comparison of tyrosine hydroxylation and melanin formation. Biochem J. 1976 Sep 1;157(3):549–557. doi: 10.1042/bj1570549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Heck D. E., Laskin D. L., Gardner C. R., Laskin J. D. Epidermal growth factor suppresses nitric oxide and hydrogen peroxide production by keratinocytes. Potential role for nitric oxide in the regulation of wound healing. J Biol Chem. 1992 Oct 25;267(30):21277–21280. [PubMed] [Google Scholar]
  24. Imokawa G., Yada Y., Kimura M., Morisaki N. Granulocyte/macrophage colony-stimulating factor is an intrinsic keratinocyte-derived growth factor for human melanocytes in UVA-induced melanosis. Biochem J. 1996 Jan 15;313(Pt 2):625–631. doi: 10.1042/bj3130625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Imokawa G., Yada Y., Miyagishi M. Endothelins secreted from human keratinocytes are intrinsic mitogens for human melanocytes. J Biol Chem. 1992 Dec 5;267(34):24675–24680. [PubMed] [Google Scholar]
  26. Jiménez M., Tsukamoto K., Hearing V. J. Tyrosinases from two different loci are expressed by normal and by transformed melanocytes. J Biol Chem. 1991 Jan 15;266(2):1147–1156. [PubMed] [Google Scholar]
  27. Joshi M., Strandhoy J., White W. L. Nitric oxide synthase activity is up-regulated in melanoma cell lines: a potential mechanism for metastases formation. Melanoma Res. 1996 Apr;6(2):121–126. doi: 10.1097/00008390-199604000-00006. [DOI] [PubMed] [Google Scholar]
  28. Knowles R. G., Moncada S. Nitric oxide synthases in mammals. Biochem J. 1994 Mar 1;298(Pt 2):249–258. doi: 10.1042/bj2980249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kondo S., Kono T., Sauder D. N., McKenzie R. C. IL-8 gene expression and production in human keratinocytes and their modulation by UVB. J Invest Dermatol. 1993 Nov;101(5):690–694. doi: 10.1111/1523-1747.ep12371677. [DOI] [PubMed] [Google Scholar]
  30. Kupper T. S., Chua A. O., Flood P., McGuire J., Gubler U. Interleukin 1 gene expression in cultured human keratinocytes is augmented by ultraviolet irradiation. J Clin Invest. 1987 Aug;80(2):430–436. doi: 10.1172/JCI113090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kupper T. S., Min K., Sehgal P., Mizutani H., Birchall N., Ray A., May L. Production of IL-6 by keratinocytes. Implications for epidermal inflammation and immunity. Ann N Y Acad Sci. 1989;557:454–465. [PubMed] [Google Scholar]
  32. Lacour J. P., Gordon P. R., Eller M., Bhawan J., Gilchrest B. A. Cytoskeletal events underlying dendrite formation by cultured pigment cells. J Cell Physiol. 1992 May;151(2):287–299. doi: 10.1002/jcp.1041510210. [DOI] [PubMed] [Google Scholar]
  33. Libow L. F., Scheide S., DeLeo V. A. Ultraviolet radiation acts as an independent mitogen for normal human melanocytes in culture. Pigment Cell Res. 1988;1(6):397–401. doi: 10.1111/j.1600-0749.1988.tb00142.x. [DOI] [PubMed] [Google Scholar]
  34. Lotan R., Lotan D. Stimulation of melanogenesis in a human melanoma cell line by retinoids. Cancer Res. 1980 Sep;40(9):3345–3350. [PubMed] [Google Scholar]
  35. Moncada S., Palmer R. M., Higgs E. A. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev. 1991 Jun;43(2):109–142. [PubMed] [Google Scholar]
  36. Nakane M., Schmidt H. H., Pollock J. S., Förstermann U., Murad F. Cloned human brain nitric oxide synthase is highly expressed in skeletal muscle. FEBS Lett. 1993 Jan 25;316(2):175–180. doi: 10.1016/0014-5793(93)81210-q. [DOI] [PubMed] [Google Scholar]
  37. Nathan C., Xie Q. W. Nitric oxide synthases: roles, tolls, and controls. Cell. 1994 Sep 23;78(6):915–918. doi: 10.1016/0092-8674(94)90266-6. [DOI] [PubMed] [Google Scholar]
  38. O'Keefe E. J., Chiu M. L. Stimulation of thymidine incorporation in keratinocytes by insulin, epidermal growth factor, and placental extract: comparison with cell number to assess growth. J Invest Dermatol. 1988 Jan;90(1):2–7. doi: 10.1111/1523-1747.ep12462409. [DOI] [PubMed] [Google Scholar]
  39. Oikawa A., Nakayasu M., Nohara M., Tchen T. T. Fate of L-(3,5- 3H) tyrosine in cell-free extracts and tissue cultures of melanoma cells: a new assay method for tyrosinase in living cells. Arch Biochem Biophys. 1972 Feb;148(2):548–557. doi: 10.1016/0003-9861(72)90173-7. [DOI] [PubMed] [Google Scholar]
  40. Peunova N., Enikolopov G. Amplification of calcium-induced gene transcription by nitric oxide in neuronal cells. Nature. 1993 Jul 29;364(6436):450–453. doi: 10.1038/364450a0. [DOI] [PubMed] [Google Scholar]
  41. Pomerantz S. H. L-tyrosine-3,5-3H assay for tyrosinase development in skin of newborn hamsters. Science. 1969 May 16;164(3881):838–839. doi: 10.1126/science.164.3881.838. [DOI] [PubMed] [Google Scholar]
  42. Rheinwald J. G., Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell. 1975 Nov;6(3):331–343. doi: 10.1016/s0092-8674(75)80001-8. [DOI] [PubMed] [Google Scholar]
  43. Roméro C., Aberdam E., Larnier C., Ortonne J. P. Retinoic acid as modulator of UVB-induced melanocyte differentiation. Involvement of the melanogenic enzymes expression. J Cell Sci. 1994 Apr;107(Pt 4):1095–1103. doi: 10.1242/jcs.107.4.1095. [DOI] [PubMed] [Google Scholar]
  44. Rosen C. F., Seki Y., Farinelli W., Stern R. S., Fitzpatrick T. B., Pathak M. A., Gange R. W. A comparison of the melanocyte response to narrow band UVA and UVB exposure in vivo. J Invest Dermatol. 1987 Jun;88(6):774–779. doi: 10.1111/1523-1747.ep12470474. [DOI] [PubMed] [Google Scholar]
  45. Schallreuter K. U., Wood J. M., Pittelkow M. R., Gütlich M., Lemke K. R., Rödl W., Swanson N. N., Hitzemann K., Ziegler I. Regulation of melanin biosynthesis in the human epidermis by tetrahydrobiopterin. Science. 1994 Mar 11;263(5152):1444–1446. doi: 10.1126/science.8128228. [DOI] [PubMed] [Google Scholar]
  46. Schauer E., Trautinger F., Köck A., Schwarz A., Bhardwaj R., Simon M., Ansel J. C., Schwarz T., Luger T. A. Proopiomelanocortin-derived peptides are synthesized and released by human keratinocytes. J Clin Invest. 1994 May;93(5):2258–2262. doi: 10.1172/JCI117224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schmidt H. H., Walter U. NO at work. Cell. 1994 Sep 23;78(6):919–925. doi: 10.1016/0092-8674(94)90267-4. [DOI] [PubMed] [Google Scholar]
  48. Wang R., Ghahary A., Shen Y. J., Scott P. G., Tredget E. E. Human dermal fibroblasts produce nitric oxide and express both constitutive and inducible nitric oxide synthase isoforms. J Invest Dermatol. 1996 Mar;106(3):419–427. doi: 10.1111/1523-1747.ep12343428. [DOI] [PubMed] [Google Scholar]
  49. Warren J. B. Nitric oxide and human skin blood flow responses to acetylcholine and ultraviolet light. FASEB J. 1994 Feb;8(2):247–251. doi: 10.1096/fasebj.8.2.7509761. [DOI] [PubMed] [Google Scholar]
  50. Wittbjer A., Odh G., Rosengren E., Rorsman H. A sensitive tyrosinase method for human skin. Acta Derm Venereol. 1991;71(5):399–402. [PubMed] [Google Scholar]
  51. Wood J. M., Schallreuter-Wood K. U., Lindsey N. J., Callaghan S., Gardner M. L. A specific tetrahydrobiopterin binding domain on tyrosinase controls melanogenesis. Biochem Biophys Res Commun. 1995 Jan 17;206(2):480–485. doi: 10.1006/bbrc.1995.1068. [DOI] [PubMed] [Google Scholar]
  52. Yaar M., Gilchrest B. A. Human melanocyte growth and differentiation: a decade of new data. J Invest Dermatol. 1991 Oct;97(4):611–617. doi: 10.1111/1523-1747.ep12482985. [DOI] [PubMed] [Google Scholar]
  53. Yada Y., Higuchi K., Imokawa G. Effects of endothelins on signal transduction and proliferation in human melanocytes. J Biol Chem. 1991 Sep 25;266(27):18352–18357. [PubMed] [Google Scholar]
  54. Yohn J. J., Morelli J. G., Walchak S. J., Rundell K. B., Norris D. A., Zamora M. R. Cultured human keratinocytes synthesize and secrete endothelin-1. J Invest Dermatol. 1993 Jan;100(1):23–26. doi: 10.1111/1523-1747.ep12349932. [DOI] [PubMed] [Google Scholar]

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