Archives of Dermatological Research

, Volume 301, Issue 4, pp 301–306

Copper–GHK increases integrin expression and p63 positivity by keratinocytes

  • Youn-A Kang
  • Hye-Ryung Choi
  • Jung-Im Na
  • Chang-Hun Huh
  • Min-Ji Kim
  • Sang-Woong Youn
  • Kyu-Han Kim
  • Kyoung-Chan Park
Original Paper

Abstract

Glycyl-l-histidyl-l-lysyl (GHK) possesses a high affinity for copper(II) ions, with which it spontaneously forms a complex (copper–GHK). It is well known that copper–GHK plays a physiological role in the process of wound healing and tissue repair by stimulating collagen synthesis in fibroblasts. This study was conducted to investigate the effects of copper–GHK on keratinocytes. Proliferative effects were analyzed and hematoxylin and eosin staining and immunohistochemistry were conducted to evaluate the effects of copper–GHK in skin equivalent (SE) models. In addition, western blotting was performed. In monolayer cultured keratinocytes, copper–GHK increased the proliferation of keratinocytes. When the SE models were evaluated, basal cells became cuboidal when copper–GHK was added. Immunohistochemical analysis revealed that copper–GHK increased proliferating cell nuclear antigen (PCNA) and p63 positivity. Furthermore, the expression of integrin α6 and β1 increased in SE models, and these results were confirmed by Western blotting. The results of this study indicate that treatment with copper–GHK may increase the proliferative potential of basal keratinocytes by modulating the expression of integrins, p63 and PCNA. In addition, increased levels of p63, a putative stem cell marker of the skin, suggests that copper–GHK promotes the survival of basal stem cells in the skin.

Keywords

Peptide Skin equivalents Integrin Keratinocytes Copper–GHK p63 Stem cells 

References

  1. 1.
    Bell E, Ivarsson B, Merrill C (1979) Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc Natl Acad Sci USA 76:1274–1278. doi:10.1073/pnas.76.3.1274 PubMedCrossRefGoogle Scholar
  2. 2.
    Canapp SO Jr, Farese JP, Schultz GS, Gowda S, Ishak AM, Swaim SF, Vangilder J, Lee-Ambrose L, Martin FG (2003) The effect of topical tripeptide–copper complex on healing of ischemic open wounds. Vet Surg 32:515–523. doi:10.1111/j.1532-950X.2003.00515.x PubMedCrossRefGoogle Scholar
  3. 3.
    Carroll DK, Carroll JS, Leong CO, Cheng F, Brown M, Mills AA, Brugge JS, Ellisen LW (2006) p63 regulates an adhesion programme and cell survival in epithelial cells. Nat Cell Biol 8:551–561. doi:10.1038/ncb1420 PubMedCrossRefGoogle Scholar
  4. 4.
    Grose R, Hutter C, Bloch W, Thorey I, Watt FM, Fassler R, Brakebusch C, Werner SA (2002) Crucial role of beta 1 integrins for keratinocyte migration in vitro and during cutaneous wound repair. Development 129:2303–2315PubMedGoogle Scholar
  5. 5.
    Ivanova IA, D’Souza SJ, Dagnino L (2005) Signalling in the epidermis: the E2F cell cycle regulatory pathway in epidermal morphogenesis, regeneration and transformation. Int J Biol Sci 1:87–95PubMedGoogle Scholar
  6. 6.
    Kamoun A, Landeau JM, Godeau G, Wallach J, Duchesnay A, Pellat B, Hornebeck W (1995) Growth stimulation of human skin fibroblasts by elastin-derived peptides. Cell Adhes Commun 3:273–281. doi:10.3109/15419069509081013 PubMedCrossRefGoogle Scholar
  7. 7.
    Kim DS, Cho HJ, Choi HR, Kwon SB, Park KC (2004) Isolation of human epidermal stem cells by adherence and the reconstruction of skin equivalents. Cell Mol Life Sci 61:2774–2781. doi:10.1007/s00018-004-4288-4 PubMedCrossRefGoogle Scholar
  8. 8.
    Kim SW, Lee IW, Cho HJ, Cho KH, Kim KH, Chung JH, Song PI, Park KC (2002) Fibroblasts and ascorbate regulate epidermalization in reconstructed human epidermis. J Dermatol Sci 30:215–223. doi:10.1016/S0923-1811(02)00108-1 CrossRefGoogle Scholar
  9. 9.
    Li A, Simmons PJ, Kaur P (1998) Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci USA 95:3902–3907. doi:10.1073/pnas.95.7.3902 PubMedCrossRefGoogle Scholar
  10. 10.
    Maquart FX, Bellon G, Chaqour B, Wegrowski J, Patt LM, Trachy RE, Monboisse JC, Chastang F, Birembaut P, Gillery P et al (1993) In vivo stimulation of connective tissue accumulation by the tripeptide-copper complex glycyl-l-histidyl-l-lysine-Cu2+ in rat experimental wounds. J Clin Invest 92:2368–2376. doi:10.1172/JCI116842 PubMedCrossRefGoogle Scholar
  11. 11.
    Maquart FX, Pickart L, Laurent M, Gillery P, Monboisse JC, Borel JP (1988) Stimulation of collagen synthesis in fibroblast cultures by the tripeptide–copper complex glycyl-l-histidyl-l-lysine-Cu2+. FEBS Lett 238:343–346. doi:10.1016/0014-5793(88)80509-X PubMedCrossRefGoogle Scholar
  12. 12.
    Park KC, Choi HR, Cho HJ, Kim SK, Kwon SB, Kim DS, Cho YJ, Youn SW (2004) Effects of ascorbic acid on keratinocyte and epidermalization of skin. Ann Dermatol 16:45–51Google Scholar
  13. 13.
    Pickart L (2008) The human tri-peptide GHK and tissue remodeling. J Biomater Sci Polym Ed 19:969–988. doi:10.1163/156856208784909435 PubMedCrossRefGoogle Scholar
  14. 14.
    Pickart L, Thaler MM (1973) Tripeptide in human serum which prolongs survival of normal liver cells and stimulates growth in neoplastic liver. Nat New Biol 243:85–87PubMedGoogle Scholar
  15. 15.
    Pollard JD, Quan S, Kang T, Koch RJ (2005) Effects of copper tripeptide on the growth and expression of growth factors by normal and irradiated fibroblasts. Arch Facial Plast Surg 7:27–31. doi:10.1001/archfaci.7.1.27 PubMedCrossRefGoogle Scholar
  16. 16.
    Rheinwald JG, Green H (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331–343. doi:10.1016/S0092-8674(75)80001-8 PubMedCrossRefGoogle Scholar
  17. 17.
    Rodius S, Indra G, Thibault C, Pfister V, Georges-Labouesse E (2007) Loss of alpha6 integrins in keratinocytes leads to an increase in TGFbeta and AP1 signaling and in expression of differentiation genes. J Cell Physiol 212:439–449. doi:10.1002/jcp.21040 PubMedCrossRefGoogle Scholar
  18. 18.
    Russell AJ, Fincher EF, Millman L, Smith R, Vela V, Waterman EA, Dey CN, Guide S, Weaver VM, Marinkovich MP (2003) Alpha 6 beta 4 integrin regulates keratinocyte chemotaxis through differential GTPase activation and antagonism of alpha 3 beta 1 integrin. J Cell Sci 116:3543–3556. doi:10.1242/jcs.00663 PubMedCrossRefGoogle Scholar
  19. 19.
    Simeon A, Monier F, Emonard H, Gillery P, Birembaut P, Hornebeck W, Maquart FX (1999) Expression and activation of matrix metalloproteinases in wounds: modulation by the tripeptide-copper complex glycyl-l-histidyl-l-lysine-Cu2+. J Invest Dermatol 112:957–964. doi:10.1046/j.1523-1747.1999.00606.x PubMedCrossRefGoogle Scholar
  20. 20.
    Simeon A, Wegrowski Y, Bontemps Y, Maquart FX (2000) Expression of glycosaminoglycans and small proteoglycans in wounds: modulation by the tripeptide-copper complex glycyl-l-histidyl-l-lysine-Cu(2+). J Invest Dermatol 115:962–968. doi:10.1046/j.1523-1747.2000.00166.x PubMedCrossRefGoogle Scholar
  21. 21.
    Sonnenberg A, Calafat J, Janssen H, Daams H, van der Raaij-Helmer LM, Falcioni R, Kennel SJ, Aplin JD, Baker J, Loizidou M et al (1991) Integrin alpha 6/beta 4 complex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion. J Cell Biol 113:907–917. doi:10.1083/jcb.113.4.907 PubMedCrossRefGoogle Scholar
  22. 22.
    Tenaud I, Sainte-Marie I, Jumbou O, Litoux P, Dreno B (1999) In vitro modulation of keratinocyte wound healing integrins by zinc, copper and manganese. Br J Dermatol 140:26–34. doi:10.1046/j.1365-2133.1999.02603.x PubMedCrossRefGoogle Scholar
  23. 23.
    Tiberio R, Marconi A, Fila C, Fumelli C, Pignatti M, Krajewski S, Giannetti A, Reed JC, Pincelli C (2002) Keratinocytes enriched for stem cells are protected from anoikis via an integrin signaling pathway in a Bcl-2 dependent manner. FEBS Lett 524:139–144. doi:10.1016/S0014-5793(02)03040-5 PubMedCrossRefGoogle Scholar
  24. 24.
    Wegrowski Y, Maquart FX, Borel JP (1992) Stimulation of sulfated glycosaminoglycan synthesis by the tripeptide–copper complex glycyl-l-histidyl-l-lysine-Cu2+. Life Sci 51:1049–1056. doi:10.1016/0024-3205(92)90504-I PubMedCrossRefGoogle Scholar
  25. 25.
    Yang A, Schweitzer R, Sun D, Kaghad M, Walker N, Bronson RT, Tabin C, Sharpe A, Caput D, Crum C, McKeon F (1999) p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398:714–718. doi:10.1038/19539 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Youn-A Kang
    • 1
  • Hye-Ryung Choi
    • 1
  • Jung-Im Na
    • 1
  • Chang-Hun Huh
    • 1
  • Min-Ji Kim
    • 1
  • Sang-Woong Youn
    • 1
  • Kyu-Han Kim
    • 1
  • Kyoung-Chan Park
    • 1
    • 2
  1. 1.Department of DermatologySeoul National University College of MedicineSeoulRepublic of Korea
  2. 2.Department of DermatologySeoul National University Bundang HospitalSeongnam-siRepublic of Korea

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