Skip to main content

Advertisement

SpringerLink
Log in

Extracellular glutathione promotes migration of hydrogen peroxide-stressed cultured chick embryonic skin cells

  • Published:
In Vitro Cellular & Developmental Biology - Animal Aims and scope Submit manuscript

Abstract

The ability of glutathione to affect melanocyte survival has fostered its use in a variety of applications related to epithelial cells. Our study focused on fibroblast migration and the effects of oxidative stress. We used scratch assays to measure cell migration: fibroblasts were harvested from embryonic chicks, grown to confluence in a monolayer, and the layer was scratched to initiate migration. Migration rates were measured over 8 h using photomicrographs, and vinculin expression as an indicator focal adhesion formation was measured using immunofluorescence. Addition of 200 μM glutathione to the culture media in which the cells grew resulted in a significantly increased rate of scratch closure. When the scratch assays were performed in the presence of 100 μM H2O2 (to simulate oxidative stress), the cells ceased to migrate. Addition of 200 μM glutathione to the H2O2-treated scratched layers resulted in a restoration of the scratch closure capabilities. At the subcellular level, addition of extracellular glutathione resulted in a redistribution of vinculin into fewer but larger aggregates. In cells at the edge of scratched monolayers that were treated with H2O2, vinculin particles were distributed throughout the cell in smaller aggregates; addition of glutathione resulted in vinculin aggregates that were larger and closer to the edges of the cell, indicating that these cells were more migratory. Our results suggest that glutathione promotes fibroblast migration, possibly via a mechanism that promotes the formation of focal adhesions.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1.
Figure 2.
Figure 3.
Figure 4.

Similar content being viewed by others

References

  • Alena F.; Dixon W.; Thomas P.; Jimbow K. Glutathione plays a key role in the depigmenting and melanocytotoxic action of N-acetyl-4-S-cyteaminylphenol in black and yellow hair follicles. J. Invest. Dermatol. 104: 792–797; 1995.

    Article  PubMed  CAS  Google Scholar 

  • Bradley R. A.; Couchman J. R.; Rees D. A. Comparison of the cell cytoskeleton in migratory and stationary chick fibroblasts. J. Musc. Res. Cell. Motil. 1: 5–14; 1980.

    Article  Google Scholar 

  • Burridge K.; Fath K.; Kelly T.; Nuckolls G.; Turner C. Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu. Rev. Cell. Biol. 4: 487–525; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Buschmann H. J.; Schollmeyer E. Applications of cyclodextrins in cosmetic products: a review. J. Cosmet. Sci. 53: 185–191; 2002.

    PubMed  CAS  Google Scholar 

  • Chen Q. M.; Tu V. C.; Catania J.; Burton M.; Toussaint O.; Dilley T. Involvement of Rb family proteins, focal adhesion proteins and protein synthesis in senescent morphogenesis induced by hydrogen peroxide. J. Cell. Sci. 113: 4087–4098; 2000.

    PubMed  CAS  Google Scholar 

  • Chow C. K.; Tappel A. L. An enzymatic protective mechanism against lipid peroxidation damage to lungs of ozone-exposed rats. Lipids 7: 518–524; 1972.

    Article  PubMed  CAS  Google Scholar 

  • Colston J. T.; Rosa S. D.; Freeman G. L. Impact of brief oxidant stress on primary adult cardiac fibroblasts. Biochem. Biophys. Res. Comm. 316: 258–262; 2004.

    Article  CAS  Google Scholar 

  • Dröge W.; Breitkreutz R. Glutathione and immune function. Proc. Nutr. Soc. 59: 595–600; 2000.

    Article  PubMed  Google Scholar 

  • Dunleavy J. R.; Couchman J. R. Controlled induction of focal adhesion disassembly and migration in primary fibroblasts. J. Cell. Sci. 105: 489–500; 1993.

    Google Scholar 

  • Engelmann J.; Janke V.; Volk J.; Leyhausen G.; Von Neuhoff N.; Schlegelberger B.; Geurtsen W. Effects of BisGMA on glutathione metabolism and apoptosis inhuman gingival fibroblasts in vitro. Biomaterials 25: 4573–4580; 2004.

    Article  PubMed  CAS  Google Scholar 

  • Gotti R.; Andrisano B.; Cavrini V.; Bongini A. Determination of glutathione in pharmaceuticals and cosmetics by HPLC with UV and fluorescence detection. Chromatographia 39: 23–28; 1994.

    Article  CAS  Google Scholar 

  • Gregersen N.; Bross P. Protein misfolding and cellular stress: an overview. Method Mol. Biol. 648: 3–23; 2010.

    Article  CAS  Google Scholar 

  • Gurtner G. C.; Werner S.; Barrandon Y.; Longaker M. T. Wound repair and regeneration. Nature 453: 314–324; 2008.

    Article  PubMed  CAS  Google Scholar 

  • He Y.; Huang J.; Ramirez D. C.; Chignell C. F. Role of reduced glutathione efflux in apoptosis of immortalized human keratinocytes induced by UVA. J. Biol. Chem. 278: 8058–8064; 2003.

    Article  PubMed  CAS  Google Scholar 

  • Holmgren A. Glutathione-dependent synthesis of deoxyribonucleotides. Characterization of the enzymatic mechanism of Escherichia coli glutaredoxin. J. Biol. Chem. 254: 3672–3678; 1979.

    PubMed  CAS  Google Scholar 

  • Kalebic T.; Kinter A.; Poli G.; Anderson M. E.; Meister A.; Fauci A. S. Suppression of human immunodeficiency virus expression in chronically infected monocytic cells by glutathione, glutathione ester, and N-acetylcysteine. Proc. Natl. Acad. Sci. U.S.A. 88: 986–990; 1991.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Kim W.-S.; Park B.-S.; Kim H.-K.; Park J.-S.; Kim K.-J.; Choi J.-S.; Chung S.-J.; Kim D.-D.; Sung J.-H. Evidence supporting antioxidant action of adipose-derived stem cells: protection of human dermal fibroblasts from oxidative stress. J. Dermatol. Sci. 49: 133–142; 2008.

    Article  PubMed  CAS  Google Scholar 

  • Kopal C.; Deveci M.; Öztürk S.; Sengezer M. Effects of topical glutathione treatment in rat ischemic wound model. Ann. Plastic Surg. 58: 449–455; 2007.

    Article  CAS  Google Scholar 

  • Lai G.; Ozols R. F.; Young R. C.; Hamilton T. C. Effect of glutathione on DNA repair in cisplatin-resistant human ovarian cancer cell lines. J. Natl. Cancer. Inst. 81: 535–539; 1989.

    Article  PubMed  CAS  Google Scholar 

  • Liang C.-C.; Park A. Y.; Guan J.-L. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat. Protoc. 2: 329–333; 2007.

    Article  PubMed  CAS  Google Scholar 

  • Loo A. E. K.; Ho R.; Halliwell B. Mechanism of hydrogen peroxide-induced keratinocyte migration in a scratch-wound model. Free Rad. Biol. Med. 51: 884–892; 2011.

    Article  PubMed  CAS  Google Scholar 

  • Marionnet C.; Pierrard C.; Lejeune F.; Sok J.; Thomas M.; Bernerd F. Different oxidative stress response in keratinocytes and fibroblasts of reconstructed skin exposed to non extreme daily-ultraviolet radiation. PLoS ONE 5: e12059; 2010.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Martin P.; Parkhurst S. M. Parallels between tissue repair and embryo morphogenesis. Development 131: 3021–3034; 2004.

    Article  PubMed  CAS  Google Scholar 

  • Matsubayashi Y.; Ebisuya M.; Honjoh S.; Nishida E. ERK activation propagates in epithelial sheets and regulates their migration during wound healing. Curr. Biol. 14: 731–735; 2004.

    Article  PubMed  CAS  Google Scholar 

  • Mocali A.; Caldini C. R.; Chevanne M.; Paoletti F. Induction, effects, and quantification of sublethal oxidative stress by hydrogen peroxide on cultured human fibroblasts. Exp. Cell. Res. 216: 388–395; 1995.

    Article  PubMed  CAS  Google Scholar 

  • Nevin K. G.; Rajamohan T. Effect of topical application of virgin coconut oil on skin components and antioxidant status during dermal wound healing in young rats. Skin Pharmacol. Physiol. 23: 290–297; 2010.

    Article  PubMed  CAS  Google Scholar 

  • Niggli H. J.; Applegate L. A. Glutathione response after UVA irradiation in mitotic and postmitotic human skin fibroblasts and keratinocytes. Photochem. Photobiol. 65: 680–684; 1997.

    Article  PubMed  CAS  Google Scholar 

  • Nobes C. D.; Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81: 53–62; 1995.

    Article  PubMed  CAS  Google Scholar 

  • O’Toole E. A.; Goel M.; Woodley D. T. Hydrogen peroxide inhibits human keratinocyte migration. Dermatol. Surg 22: 525–529; 1996.

    PubMed  Google Scholar 

  • Orkin R. W.; Toole B. P. Isolation and characterization of hyaluronidase from cultures of chick embryo skin- and muscle-derived fibroblasts. J. Biol. Chem. 255: 1036–1042; 1980.

    PubMed  CAS  Google Scholar 

  • Ridley A. Rho GTPases and cell migration. J. Cell. Sci. 114: 2713–2722; 2001.

    PubMed  CAS  Google Scholar 

  • Rifkin D. B.; Crowe R. M. Tumor promoters induce changes in the chick embryo fibroblast cytoskeleton. Cell 18: 361–368; 1979.

    Article  PubMed  CAS  Google Scholar 

  • Schäfer M.; Werner S. Oxidative stress and normal and impaired wound repair. Pharmacol. Res. 58: 165–171; 2008.

    Article  PubMed  CAS  Google Scholar 

  • Shen H.-M.; Liu Z. JNK signaling pathway is a key modulator in cell death mediated by reactive oxygen and nitrogen species. Free Radic. Biol. Med 40: 928–939; 2006.

    Article  PubMed  CAS  Google Scholar 

  • Shukla A.; Rasik A. M.; Patnaik G. K. Depletion of reduced glutathione, ascorbic acid, vitamin E and antioxidant defence enzymes in a healing cutaneous wound. Free Radic. Res. 26: 93–101; 1997.

    Article  PubMed  CAS  Google Scholar 

  • Steiling H.; Munz B.; Werner S.; Brauchle M. Different types of ROS-scavenging enzymes are expressed during cutaneous wound repair. Exp. Cell Res. 247: 484–494; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Takahashi M.; Nagai T.; Hamano S.; Kuwayama M.; Okamura N.; Okano A. Effect of thiol compounds on in vitro development and intracellular glutathione content of bovine embryos. Biol. Reprod. 49: 228–232; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Tyrrell R. M.; Pidoux M. Endogenous glutathione protects human skin fibroblasts against the cytotoxic action of UVB, UVA and near-visible radiations. Photochem. Photobiol. 44: 561–564; 1986.

    Article  PubMed  CAS  Google Scholar 

  • Veal E. A.; Day A. M.; Morgan B. A. Hydrogen peroxide sensing and signaling. Molec. Cell 26: 1–14; 2007.

    Article  PubMed  CAS  Google Scholar 

  • Vessey D. A.; Lee K. Inactivation of enzymes of the glutathione antioxidant system by treatment of cultured human keratinocytes with peroxides. J. Invest. Dermatol. 100: 829–833; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Vessey D. A.; Lee K.; Blacker K. L. Characterization of the oxidative stress initiated in cultured human keratinocytes by treatment with peroxides. J. Invest. Dermatol. 99: 859–863; 1992.

    Article  PubMed  CAS  Google Scholar 

  • Villarama C. D.; Maibach H. I. Glutathione as a depigmenting agent: an overview. Int. J. Cosmet. Sci. 27: 147–153; 2005.

    Article  PubMed  CAS  Google Scholar 

  • Wall I. B.; Moseley R.; Baird D. M.; Kipling D.; Giles P.; Laffafian I.; Price P. E.; Thomas D. W.; Stephens P. Fibroblast dysfunction is a key factor in the non-healing of chronic venous leg ulcers. J. Invest. Dermatol. 128: 2526–2540; 2008.

    Article  PubMed  CAS  Google Scholar 

  • Werner S.; Krieg T.; Smola H. Keratinocyte–fibroblast interactions in wound healing. J. Invest. Dermatol. 127: 998–1008; 2007.

    Article  PubMed  CAS  Google Scholar 

  • Yohn J. J.; Norris D. A.; Yrastorza D. G.; Bruno I. J.; Leff J. A.; Hake S. S.; Repine J. E. Disparate antioxidant enzyme activities in cultured human cutaneous fibroblasts, keratinocytes, and melanocytes. J. Invest. Dermatol. 97: 405–409; 1991.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Biology Department, Fordham University, 113 W. 60th Street, New York, NY, 10023, USA

    Mia DeNunzio

  2. Biology Department, University of Scranton, 800 Linden Street, Scranton, PA, 18510, USA

    George Gomez

  3. Biology Department, University of Scranton, LSC 395 204 Monroe Ave, Scranton, PA, 18510, USA

    George Gomez

Authors
  1. Mia DeNunzio
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. George Gomez
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to George Gomez.

Additional information

Editor: T. Okamoto

Rights and permissions

Reprints and permissions

About this article

Cite this article

DeNunzio, M., Gomez, G. Extracellular glutathione promotes migration of hydrogen peroxide-stressed cultured chick embryonic skin cells. In Vitro Cell.Dev.Biol.-Animal 50, 350–357 (2014). https://doi.org/10.1007/s11626-013-9696-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11626-013-9696-z

Keywords

Search

Navigation