Skip to main content

Advertisement

SpringerLink
Log in

Antioxidant proteins and reactive oxygen species are decreased in a murine epidermal side population with stem cell-like characteristics

  • Original Paper
  • Published:
Histochemistry and Cell Biology Aims and scope Submit manuscript

Abstract

Reactive oxygen species (ROS) and antioxidants are essential to maintain a redox balance within tissues and cells. Intracellular ROS regulate key cellular functions such as proliferation, differentiation and apoptosis through cellular signaling, and response to injury. The redox environment is particularly important for stem/progenitor cells, as their self-renewal and differentiation has been shown to be redox sensitive. However, not much is known about ROS and antioxidant protein function in freshly isolated keratinocytes, notably the different keratinocyte subpopulations. Immunostaining of neonatal cutaneous sections revealed that antioxidant enzymes [catalase, SOD2, gluthatione peroxidase-1 (GPx)] and ROS are localized predominantly to the epidermis. We isolated keratinocyte subpopulations and found lower levels of SOD2, catalase and GPx, as well as decreased SOD and catalase activity in an epidermal side population with stem cell-like characteristics (EpSPs) compared to more differentiated (Non-SP) keratinocytes. EpSPs also exhibited less mitochondrial area, fewer peroxisomes and produced lower levels of ROS than Non-SPs. Finally, EpSPs were more resistant to UV radiation than their progeny. Together, our data indicate ROS and antioxidant levels are decreased in stem-like EpSPs.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Baillet A, Chanteperdrix V, Trocme C, Casez P, Garrel C, Besson G (2010) The role of oxidative stress in amyotrophic lateral sclerosis and Parkinson’s disease. Neurochem Res 35(10):1530–1537

    Article  PubMed  CAS  Google Scholar 

  • Barrandon Y, Green H (1985) Cell size as a determinant of the clone-forming ability of human keratinocytes. Proc Natl Acad Sci USA 82:5390–5394

    Article  PubMed  CAS  Google Scholar 

  • Beers RF Jr, Sizer IW (1952) A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 195(1):133–140

    PubMed  CAS  Google Scholar 

  • Bickenbach JR (1981) Identification and behavior of label retaining cells in oral mucosa and skin. J Dent Res 60C:1611–1620

    Google Scholar 

  • Cotsarelis G, Sun T-T, Lavker RM (1990) Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 61:1329–1337

    Article  PubMed  CAS  Google Scholar 

  • Das DK, George A, Liu XK, Rao PS (1989) Detection of hydroxyl radical in the mitochondria of ischemic-reperfused myocardium by trapping with salicylate. Biochem Biophys Res Commun 165(3):1004–1009

    Article  PubMed  CAS  Google Scholar 

  • Droge W (2002) Free radicals in the physiological control of cell function. Physiol Rev 82(1):47–95

    PubMed  CAS  Google Scholar 

  • Du J, Daniels DH, Asbury C, Venkataraman S, Liu J, Spitz DR, Oberley LW, Cullen JJ (2006) Mitochondrial production of reactive oxygen species mediate dicumarol-induced cytotoxicity in cancer cells. J Biol Chem 281(49):37416–37426

    Article  PubMed  CAS  Google Scholar 

  • Dunnwald M, Tomanek-Chalkey A, Alexandrunas D, Fishbaugh J, Bickenbach JR (2001) Isolating a pure population of epidermal stem cells for use in tissue engineering. Exp Dermatol 10(1):45–54

    Article  PubMed  CAS  Google Scholar 

  • Dunnwald M, Chinnathambi S, Alexandrunas D, Bickenbach JR (2003) Mouse epidermal stem cells proceed through the cell cycle. J Cell Physiol 195:194–201

    Article  PubMed  CAS  Google Scholar 

  • Giacco F, Brownlee M (2010) Oxidative stress and diabetic complications. Circ Res 107(9):1058–1070

    Article  PubMed  CAS  Google Scholar 

  • Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC (1996) Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 183:1797–1806

    Article  PubMed  CAS  Google Scholar 

  • Harman D (1956) Aging: a theory based on free radical and radiation chemistry. J Gerontol 11(3):298–300

    PubMed  CAS  Google Scholar 

  • He T, Peterson TE, Holmuhamedov EL, Terzic A, Caplice NM, Oberley LW, Katusic ZS (2004a) Human endothelial progenitor cells tolerate oxidative stress due to intrinsically high expression of manganese superoxide dismutase. Arterioscler Thromb Vasc Biol 24:2021–2027

    Article  PubMed  CAS  Google Scholar 

  • He ZP, Tan WQ, Tang YF, Zhang HJ, Feng MF (2004b) Activation, isolation, identification and in vitro proliferation of oval cells from adult rat livers. Cell Prolif 37(2):177–187

    Article  PubMed  CAS  Google Scholar 

  • Ho YS, Xiong Y, Ma W, Spector A, Ho DS (2004) Mice lacking catalase develop normally but show differential sensitivity to oxidant tissue injury. J Biol Chem 279(31):32804–32812

    Article  PubMed  CAS  Google Scholar 

  • Hornig-Do HT, von Kleist-Retzow JC, Lanz K, Wickenhauser C, Kudin AP, Kunz WS, Wiesner RJ, Schauen M (2007) Human epidermal keratinocytes accumulate superoxide due to low activity of Mn-SOD, leading to mitochondrial functional impairment. J Invest Dermatol 127(5):1084–1093

    Article  PubMed  CAS  Google Scholar 

  • Ingram DA, Krier TR, Mead LE, McGuire C, Prater DN, Bhavsar J, Saadatzadeh MR, Bijangi-Vishehsaraei K, Li F, Yoder MC, Haneline LS (2007) Clonogenic endothelial progenitor cells are sensitive to oxidative stress. Stem Cells 25(2):297–304

    Article  PubMed  CAS  Google Scholar 

  • Ito K, Hirao A, Arai F, Takubo K, Matsuoka S, Miyamoto K, Ohmura M, Naka K, Hosokawa K, Ikeda Y, Suda T (2006) Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells. Nature Med 12(4):446–451

    Article  PubMed  CAS  Google Scholar 

  • Ivanova NB, Dimos JT, Schaniel C, Hackney J, Moore KA, Lemischka IR (2002) A stem cell molecular signature. Science 298:601–604

    Article  PubMed  CAS  Google Scholar 

  • Jaks V, Barker N, Kasper M, van Es JH, Snippert HJ, Clevers H, Toftgard R (2008) Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat Genet 40(11):1291–1299

    Article  PubMed  CAS  Google Scholar 

  • Jiao C, Bronner SJ, Mercer KLN, Sheriff DD, Schatteman GC, Dunnwald M (2004) Epidermal cells accelerate the restoration of the blood flow in diabetic ischemic limbs. J Cell Sci 117:1055–1063

    Article  PubMed  CAS  Google Scholar 

  • Juntilla MM, Patil VD, Calamito M, Joshi RP, Birnbaum MJ, Koretzky GA (2010) AKT1 and AKT2 maintain hematopoietic stem cell function by regulating reactive oxygen species. Blood 115(20):4030–4038

    PubMed  CAS  Google Scholar 

  • Larouche D, Tong X, Fradette J, Coulombe PA, Germain L (2008) Vibrissa hair bulge houses two populations of skin epithelial stem cells distinct by their keratin profile. Faseb J 22(5):1404–1415

    Article  PubMed  CAS  Google Scholar 

  • Li TS, Marban E (2010) Physiological levels of reactive oxygen species are required to maintain genomic stability in stem cells. Stem Cells 28(7):1178–1185

    PubMed  CAS  Google Scholar 

  • Li L, Xie T (2005) Stem cell niche: structure and function. Annu Rev Cell Dev Biol 21:605–631

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Liu Y, Lyle S, Yang Z, Cotsarelis G (2003) Keratin 15 promoter targets putative epithelial stem cells in the hair follicle bulge. J Invest Dermatol 121:963–968

    Google Scholar 

  • Madhavan L, Ourednik V, Ourednik J (2006) Increased “vigilance” of antioxidant mechanisms in neural stem cells potentiates their capability to resist oxidative stress. Stem Cells 24(9):2110–2119

    Article  PubMed  CAS  Google Scholar 

  • Menon SG, Sarsour EH, Spitz DR, Higashikubo R, Sturm M, Zhang H, Goswami PC (2003) Redox regulation of the G1 to S phase transition in the mouse embryo fibroblast cell cycle. Cancer Res 63(9):2109–2117

    PubMed  CAS  Google Scholar 

  • Michel M, Török N, Godbout M-J, Lussier M, Gaudreau P, Royal A, Germain L (1996) Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage. J Cell Sci 109:1017–1028

    PubMed  CAS  Google Scholar 

  • Michel M, L’Heureux N, Pouliot R, Xu W, Auger FA, Germain L (1999) Characterization of a new tissue-engineered human skin equivalent with hair. In Vitro Cell Dev Biol 35:318–326

    Article  CAS  Google Scholar 

  • Moldovan L, Moldovan NI (2004) Oxygen free radicals and redox biology of organelles. Histochem Cell Biol 122(4):395–412

    Article  PubMed  CAS  Google Scholar 

  • Monteiro HP, Stern A (1996) Redox modulation of tyrosine phosphorylation-dependent signal transduction pathways. Free Radic Biol Med 21(3):323–333

    Article  PubMed  CAS  Google Scholar 

  • Moreno S, Mugnaini E, Ceru MP (1995) Immunocytochemical localization of catalase in the central nervous system of the rat. J Histochem Cytochem 43(12):1253–1267

    PubMed  CAS  Google Scholar 

  • Noble M, Mayer-Proschel M, Proschel C (2005) Redox regulation of precursor cell function: insights and paradoxes. Antioxid Redox Signal 7(11&12):1456–1467

    Article  PubMed  CAS  Google Scholar 

  • Oberley TD (2002) Oxidative damage and cancer. Am J Pathol 160(2):403–408

    Article  PubMed  CAS  Google Scholar 

  • Oberley CC, Gourronc F, Hakimi S, Riordan M, Bronner S, Jiao C, Dunnwald M (2008) Murine epidermal side population possesses unique angiogenic properties. Exp Cell Res 314(4):720–728

    Article  PubMed  CAS  Google Scholar 

  • Oberley-Deegan RE, Rebits BW, Weaver MR, Tollefson AK, Bai X, McGibney M, Ovrutsky AR, Chan ED, Crapo JD (2010) An oxidative environment promotes growth of Mycobacterium abscessus. Free Radic Biol Med 49:1666–1673

    Article  PubMed  CAS  Google Scholar 

  • Owusu-Ansah E, Banerjee U (2009) Reactive oxygen species prime Drosophila haematopoietic progenitors for differentiation. Nature 461(7263):537–541

    Article  PubMed  CAS  Google Scholar 

  • Pellegrini G, Dellambra E, Golisano O, Martinelli E, Fantozzi I, Bondanza S, Ponzin D, McKeon F, De Luca M (2001) P63 identifies keratinocyte stem cells. Proc Natl Acad Sci USA 98(6):3156–3161

    Article  PubMed  CAS  Google Scholar 

  • Pervaiz S, Taneja R, Ghaffari S (2009) Oxidative stress regulation of stem and progenitor cells. Antioxid Redox Signal 11(11):2777–2789

    Article  PubMed  CAS  Google Scholar 

  • Potten CS, Morris RJ (1988) Epithelial stem cells in vivo. J Cell Sci 10(suppl):45–62

    CAS  Google Scholar 

  • Redvers RP, Li A, Kaur P (2006) Side population in adult murine epidermis exhibits phenotypic and functional characteristics of keratinocyte stem cells. Proc Natl Acad Sci USA 103(35):13168–13173

    Article  PubMed  CAS  Google Scholar 

  • Rezvani HR, Mazurier F, Cario-Andre M, Pain C, Ged C, Taieb A, de Verneuil H (2006) Protective effects of catalase overexpression on UVB-induced apoptosis in normal human keratinocytes. J Biol Chem 281(26):17999–18007

    Article  PubMed  CAS  Google Scholar 

  • Rezvani HR, Cario-Andre M, Pain C, Ged C, de Verneuil H, Taieb A (2007) Protection of normal human reconstructed epidermis from UV by catalase overexpression. Cancer Gene Ther 14(2):174–186

    Article  PubMed  CAS  Google Scholar 

  • Rezvani HR, Ged C, Bouadjar B, de Verneuil H, Taieb A (2008) Catalase overexpression reduces UVB-induced apoptosis in a human xeroderma pigmentosum reconstructed epidermis. Cancer Gene Ther 15(4):241–251

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Sarsour EH, Agarwal M, Pandita TK, Oberley LW, Goswami PC (2005) Manganese superoxide dismutase protects the proliferative capacity of confluent normal human fibroblasts. J Biol Chem 280:18033–18041

    Article  PubMed  CAS  Google Scholar 

  • Schafer M, Dutsch S, auf dem Keller U, Navid F, Schwarz A, Johnson DA, Johnson JA, Werner S (2010a) Nrf2 establishes a glutathione-mediated gradient of UVB cytoprotection in the epidermis. Genes Dev 24(10):1045–1058

    Article  PubMed  CAS  Google Scholar 

  • Schafer M, Dutsch S, auf dem Keller U, Werner S (2010b) Nrf2: a central regulator of UV protection in the epidermis. Cell Cycle 9(15):2917–2918

    Article  PubMed  CAS  Google Scholar 

  • Smith J, Ladi E, Mayer-Proschel M, Noble M (2000) Redox state is a central modulator of the balance between self-renewal and differentiation in a dividing glial precursor cell. Proc Natl Acad Sci USA 97(18):10032–10037

    Article  PubMed  CAS  Google Scholar 

  • Stern MM, Bickenbach JR (2007) Epidermal stem cells are resistant to cellular aging. Aging Cell 6(4):439–452

    Article  PubMed  CAS  Google Scholar 

  • Tani H, Morris RJ, Kaur P (2000) Enrichment for murine keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci USA 97(20):10960–10965

    Article  PubMed  CAS  Google Scholar 

  • Trempus C, Morris RJ, Bortner CD, Cotsarelis G, Faircloth RS, Reece JM, Tennant RW (2003) Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34. J Invest Dermatol 120:501–511

    Article  PubMed  CAS  Google Scholar 

  • Trouba KJ, Hamadeh HK, Amin RP, Germolec DR (2002) Oxidative stress and its role in skin disease. Antioxid Redox Signal 4(4):665–673

    Article  PubMed  CAS  Google Scholar 

  • von Wangenheim KH, Peterson HP (1998) Control of cell proliferation by progress in differentiation: clues to mechanisms of aging, cancer causation and therapy. J Theor Biol 193(4):663–678

    Article  Google Scholar 

  • Weisiger RA, Fridovich I (1973) Mitochondrial superoxide simutase site of synthesis and intramitochondrial localization. J Biol Chem 248(13):4793–4796

    PubMed  CAS  Google Scholar 

  • Weydert CJ, Cullen JJ (2010) Measurement of superoxide dismutase, catalase and glutathione peroxidase in cultured cells and tissue. Nat Protoc 5(1):51–66

    Article  PubMed  CAS  Google Scholar 

  • Xie T, Spradling AC (2000) A niche maintaining germ line stem cells in the Drosophila ovary. Science 290(5490):328–330

    Article  PubMed  CAS  Google Scholar 

  • Zelko IN, Mariani TJ, Folz RJ (2002) Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. Free Radic Biol Med 33(3):337–349

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This manuscript is dedicated to Dr. Larry Oberley, who was the inspiration for these studies. The authors would like to acknowledge Dr. N. Akhins-Burns for helpful discussion. Also, the authors would like to thank Justin Fishbaugh, Gene Hess and George Rasmussen from Flow Cytometry Core Facility at the University of Iowa for technical assistance, and Jian Shao for electron microscopy expertise. This work was supported in part by the Bioscience Funding Program from the University of Iowa to M.D., NIH R03AR055313 and R01DK059965 to M.D., and NIH R01CA115438 and P01CA066081 to L.W.O. W.J.C was a fellow of the Iowa Bioscience Advantage program (R25 GM58939).

Author information

Authors and Affiliations

  1. Department of Pediatrics, 206 MRC, The University of Iowa, Iowa City, IA, 52242, USA

    Wanakee J. Carr, Christopher C. Oberley & Martine Dunnwald

  2. Department of Radiation Oncology, The University of Iowa, Iowa City, IA, 52242, USA

    Yuping Zhang & Larry W. Oberley

  3. Department of Free Radical Biology Program, The University of Iowa, Iowa City, IA, 52242, USA

    Yuping Zhang & Larry W. Oberley

  4. Interdisciplinary Program in Molecular and Cellular Biology, The University of Iowa, Iowa City, IA, 52242, USA

    Martine Dunnwald

  5. Interdisciplinary Program in Genetics, The University of Iowa, Iowa City, IA, 52242, USA

    Martine Dunnwald

  6. Department of Medicine, National Jewish Health, Denver, CO, 80206, USA

    Rebecca E. Oberley-Deegan

Authors
  1. Wanakee J. Carr
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rebecca E. Oberley-Deegan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yuping Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christopher C. Oberley
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Larry W. Oberley
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Martine Dunnwald
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Martine Dunnwald.

Additional information

L. W. Oberley: Deceased.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Carr, W.J., Oberley-Deegan, R.E., Zhang, Y. et al. Antioxidant proteins and reactive oxygen species are decreased in a murine epidermal side population with stem cell-like characteristics. Histochem Cell Biol 135, 293–304 (2011). https://doi.org/10.1007/s00418-011-0786-2

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00418-011-0786-2

Keywords

Search

Navigation