Human Epidermal Keratinocytes Accumulate Superoxide Due to Low Activity of Mn- SOD, Leading to Mitochondrial Functional Impairment Hue-Tran Hornig-Do,

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Human Epidermal Keratinocytes Accumulate Superoxide Due to Low Activity of Mn- SOD, Leading to Mitochondrial Functional Impairment Hue-Tran Hornig-Do, Jürgen-Christoph von Kleist-Retzow, Katrin Lanz, Claudia Wickenhauser, Alexei P. Kudin, Wolfram S. Kunz, Rudolf J. Wiesner, Matthias Schauen Journal of Investigative Dermatology Volume 127, Issue 5, Pages 1084-1093 (May 2007) DOI: 10.1038/sj.jid.5700666 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Oxygen consumption and activity of mitochondrial enzymes. (a) Keratinocytes (black) and fibroblasts (white) were introduced into a polarographic chamber to monitor their oxygen consumption. After recording of intact cell respiration (CO), cells were permeabilized with digitonin, and the oxidation rates for pyruvate in the presence of malate (MPox), for malate in the presence of glutamate (MGox), for succinate in the presence of carbonyl cyanide m-chlorophenylhydrazone (Sox) and for glycerol-3-phosphate (GPox) as substrates were determined. Oxygen consumption per mg of cellular protein is shown. (b) RC complex activities in keratinocytes (black) and fibroblasts (white). The activities of complex II (SQDR), complexes II+III (SCCR), complex III (QCCR) and complex IV (COX) were determined as described in Materials and Methods. Activity per mg of cellular protein is shown. Data are the mean±SD obtained from keratinocytes and fibroblasts from three donors. NSP>0.05, *P<0.05, **P<0.01. Journal of Investigative Dermatology 2007 127, 1084-1093DOI: (10.1038/sj.jid.5700666) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Mitochondrial proteins in whole-cell lysates of keratinocytes and fibroblasts. The expression of the 39kDa subunit of complex I (C I), core-2 subunit of complex III (C III), subunit I of complex IV (C IV), cytochrome c and the mitochondrial transcription factor TFAM (TFAM) in fibroblasts from three donors was compared to that in keratinocytes from three donors using Western blot analysis. Equal protein loading was confirmed by reprobing for β-actin. Journal of Investigative Dermatology 2007 127, 1084-1093DOI: (10.1038/sj.jid.5700666) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Cellular levels of ROS determined by flow cytometric analysis in fibroblasts and keratinocytes. (a) Hydroethidine oxidation, (b) CM-H2DCFDA oxidation. Results represent relative fluorescence intensity (means±SD) obtained with keratinocytes (black) and fibroblasts (white) from three different donors in three independent experiments. At least 10,000 cells were analyzed in each experiment. *P<0.05, NS=P>0.05 Journal of Investigative Dermatology 2007 127, 1084-1093DOI: (10.1038/sj.jid.5700666) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Glutathione redox state in keratinocytes (black) and fibroblasts (white) from three different donors. (a) GSH, (b) GSSG, and (c) GSH/GSSG. Results are presented as the mean±SD of three separate experiments. *P<0.05, **P<0.01, ***P<0.001. Journal of Investigative Dermatology 2007 127, 1084-1093DOI: (10.1038/sj.jid.5700666) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Lipid peroxidation, mitochondrial membrane potential, and mitochondrial mass in keratinocytes and fibroblasts. (a) Lipid peroxidation (thiobarbituric acid-reactive substances) in keratinocytes and fibroblasts. Results are expressed as the mean±SD obtained in keratinocytes (black) and fibroblasts (white) from five donors. *P<0.05. (b) Mitochondrial membrane potential in keratinocytes (black, lined) and in fibroblasts (gray, filled) determined by DiOC6(3) staining. The figure shows a representative result of four independent experiments comparing fibroblasts and keratinocytes from four donors. (c) Mitochondrial mass in keratinocytes (black, lined) and in fibroblasts (gray, filled) determined by NAO-staining. The figure shows a representative result of three independent experiments comparing keratinocytes and fibroblasts from three donors. Typically a minimum of 10,000 cells per sample were measured in each experiment. Journal of Investigative Dermatology 2007 127, 1084-1093DOI: (10.1038/sj.jid.5700666) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Activity of total SOD, Mn-SOD, and Cu,Zn-SOD in keratinocytes and fibroblasts. All values are the mean±SD obtained with keratinocytes (black) from three donors and with fibroblasts (white) from four donors. nsP>0.05, *P<0.05. Journal of Investigative Dermatology 2007 127, 1084-1093DOI: (10.1038/sj.jid.5700666) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Activity of H2O2 degrading enzymes in keratinocytes and fibroblasts. (a) Activity of catalase. (b) Activity of GPX-1. Data are the mean±SD obtained with keratinocytes (black) and fibroblasts (white) from three different donors. NSP>0.05, **P<0.01. Journal of Investigative Dermatology 2007 127, 1084-1093DOI: (10.1038/sj.jid.5700666) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Western blot analysis of Mn-SOD, Cu,Zn-SOD, and GPX-1 expression in keratinocytes and fibroblasts from three donors. Equal protein loading was confirmed by reprobing for β-actin. Journal of Investigative Dermatology 2007 127, 1084-1093DOI: (10.1038/sj.jid.5700666) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 Histomorphology of formalin-fixed, paraffin-embedded normal human skin sections. Photographs of immunohistochemical stainings with Mn-SOD, GPX-1, COX I, cytochrome c, and TFAM antibodies are presented (one out of three comparable cases is shown). Arrows point to strongly stained keratinocytes, whereas arrowheads point to strongly stained fibroblasts. Bar=50μm. Journal of Investigative Dermatology 2007 127, 1084-1093DOI: (10.1038/sj.jid.5700666) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions