Advertisement

Protein Profiling of the Human Epidermis from the Elderly Reveals Up-regulation of a Signature of Interferon-γ-induced Polypeptides That Includes Manganese-superoxide Dismutase and the p85β Subunit of Phosphatidylinositol 3-Kinase*

  • Pavel Gromov
    Correspondence
    To whom correspondence may be addressed: Inst. of Cancer Biology, The Danish Cancer Society, Strandboulevarden 49, DK-2100, Copenhagen. Fax: 45-35-25-77-21;
    Affiliations
    Department of Medical Biochemistry and Danish Centre for Molecular Gerontology, The University of Aarhus, Ole Worms Allé, build. 170, DK-8000 Aarhus C, Denmark

    Institute of Cancer Biology, The Danish Cancer Society, Strandboulevarden 49, DK-2100, Copenhagen, Denmark
    Search for articles by this author
  • Gunhild Lange Skovgaard
    Affiliations
    Department of Dermatology, Bispebjerg Hospital, The University of Copenhagen, Bispebjerg Bakke 23, DK-2400 Copenhagen NV, Denmark and Laboratory of Molecular Gerontology and Dermatology, Danish Centre for Molecular Gerontology, The University of Aarhus, CF Mollers Allé, build. 130, DK-8000 Aarhus C, Denmark
    Search for articles by this author
  • Hildur Palsdottir
    Footnotes
    Affiliations
    Department of Medical Biochemistry and Danish Centre for Molecular Gerontology, The University of Aarhus, Ole Worms Allé, build. 170, DK-8000 Aarhus C, Denmark
    Search for articles by this author
  • Irina Gromova
    Affiliations
    Department of Medical Biochemistry and Danish Centre for Molecular Gerontology, The University of Aarhus, Ole Worms Allé, build. 170, DK-8000 Aarhus C, Denmark

    Institute of Cancer Biology, The Danish Cancer Society, Strandboulevarden 49, DK-2100, Copenhagen, Denmark
    Search for articles by this author
  • Morten Østergaard
    Affiliations
    Department of Medical Biochemistry and Danish Centre for Molecular Gerontology, The University of Aarhus, Ole Worms Allé, build. 170, DK-8000 Aarhus C, Denmark
    Search for articles by this author
  • Julio E. Celis
    Correspondence
    To whom correspondence may be addressed: Inst. of Cancer Biology, The Danish Cancer Society, Strandboulevarden 49, DK-2100, Copenhagen. Fax: 45-35-25-73-76;
    Affiliations
    Department of Medical Biochemistry and Danish Centre for Molecular Gerontology, The University of Aarhus, Ole Worms Allé, build. 170, DK-8000 Aarhus C, Denmark

    Institute of Cancer Biology, The Danish Cancer Society, Strandboulevarden 49, DK-2100, Copenhagen, Denmark
    Search for articles by this author
  • Author Footnotes
    ** Present address: Max-Planck Institute of Biophysics, Heinrich-Hoffmann Strasse 7, 60528 Frankfurt am Main, Germany.
    * This study was supported by grants from the Danish Centre for Molecular Gerontology and the Danish Cancer Society.
Open AccessPublished:January 17, 2003DOI:https://doi.org/10.1074/mcp.M200051-MCP200
      Aging of the human skin is a complex process that consists of chronological and extrinsic aging, the latter caused mainly by exposure to ultraviolet radiation (photoaging). Here we present studies in which we have used proteomic profiling technologies and two-dimensional (2D) PAGE database resources to identify proteins whose expression is deregulated in the epidermis of the elderly. Fresh punch biopsies from the forearm of 20 pairs of young and old donors (21–30 and 75–92 years old, respectively) were dissected to yield an epidermal fraction that consisted mainly of differentiated cells. One- to two-mm3 epidermal pieces were labeled with [35S]methionine for 18 h, lysed, and subjected to 2D PAGE (isoelectric focusing and non-equilibrium pH gradient electrophoresis) and phosphorimage autoradiography. Proteins were identified by matching the gels with the master 2D gel image of human keratinocytes (proteomics.cancer.dk). In selected cases 2D PAGE immunoblotting and/or mass spectrometry confirmed the identity. Quantitative analysis of 172 well focused and abundant polypeptides showed that the level of most proteins (148) remains unaffected by the aging process. Twenty-two proteins were consistently deregulated by a factor of 1.5 or more across the 20 sample pairs. Among these we identified a group of six polypeptides (Mx-A, manganese-superoxide dismutase, tryptophanyl-tRNA synthetase, the p85β subunit of phosphatidylinositol 3-kinase, and proteasomal proteins PA28-α and SSP 0107) that is induced by interferon-γ in primary human keratinocytes and that represents a specific protein signature for the effect of this cytokine. Changes in the expression of the eukaryotic initiation factor 5A, NM23 H2, cyclophilin A, HSP60, annexin I, and plasminogen activator inhibitor 2 were also observed. Two proteins exhibited irregular behavior from individual to individual. Besides arguing for a role of interferon-γ in the aging process, the biological activities associated with the deregulated proteins support the contention that aging is linked with increased oxidative stress that could lead to apoptosis in vivo.
      Aging of the human skin is a complex process that comprises two components, chronological aging (replicative senescence) that is largely determined genetically and extrinsic aging, which is triggered by environmental factors, mainly exposure to UV radiation (photoaging) (Refs.
      • Jenkins G.
      Molecular mechanisms of skin ageing.
      and
      • Wlaschek M.
      • Tantcheva-Poor I.
      • Naderi L.
      • Ma W.
      • Schneider L.A.
      • Razi-Wolf Z.
      • Schuller J.
      • Scharffetter-Kochanek K.
      Solar UV irradiation and dermal photoaging.
      and references therein). UV radiation generates reactive oxygen species (ROS),
      The abbreviations used are: ROS, reactive oxygen species; 2D, two-dimensional; IEF, isoelectric focusing; NEPHGE, non-equilibrium pH gradient electrophoresis; IFN, interferon; TNF, tumor necrosis factor; SOD, superoxide dismutase; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; MS, mass spectrometry; eIF, eukaryotic initiation factor; PAI-2, plasminogen activator inhibitor 2; WRS, tryptophanyl-tRNA synthetase; HSP, heat shock protein; SSP, sample spot protein.
      1The abbreviations used are: ROS, reactive oxygen species; 2D, two-dimensional; IEF, isoelectric focusing; NEPHGE, non-equilibrium pH gradient electrophoresis; IFN, interferon; TNF, tumor necrosis factor; SOD, superoxide dismutase; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; MS, mass spectrometry; eIF, eukaryotic initiation factor; PAI-2, plasminogen activator inhibitor 2; WRS, tryptophanyl-tRNA synthetase; HSP, heat shock protein; SSP, sample spot protein.
      which in the dermal compartment lead to the accumulation of disorganized elastic fibers and microfibrillar components as well as loss of interstitial collagen, the main component of the dermal connective tissue (Refs.
      • Wlaschek M.
      • Tantcheva-Poor I.
      • Naderi L.
      • Ma W.
      • Schneider L.A.
      • Razi-Wolf Z.
      • Schuller J.
      • Scharffetter-Kochanek K.
      Solar UV irradiation and dermal photoaging.
      ,
      • Glogau R.G.
      Physiologic and structural changes associated with aging skin.
      ,
      • Gilchrest B.A.
      A review of skin aging and its medical therapy.
      ,
      • Pascuali-Ronchetti I.
      • Baccarani-Contri M.
      Elastic fibers during development and aging.
      and references therein).
      Currently there is mounting evidence indicating that the aging process of cells and organs is associated with increased oxidative stress (
      • Sohal R.S.
      Role of oxidative stress and protein oxidation in the aging process.
      ) as well as alterations in apoptosis (Ref.
      • Higami Y.
      • Schimokawa I.
      Apoptosis in the aging process.
      and references therein), a homeostatic mechanism that is exacerbated by increased production of ROS (
      • Curtin J.F.
      • Donovan M.
      • Cotter T.G.
      Regulation and measurement of oxidative stress in apoptosis.
      ,
      • Chandra J.
      • Samali A.
      • Orrenius S.
      Triggering and modulation of apoptosis by oxidative stress.
      ,
      • Clutton S.
      The importance of oxidative stress in apoptosis.
      ). Increased production of ROS, decline of the autoxidant cellular defenses to cope with oxidative stress, and the accumulation of mitochondrial DNA mutations and oxidized proteins are among the events that may play an important role in the aging process (Refs.
      • Sohal R.S.
      Role of oxidative stress and protein oxidation in the aging process.
      ,
      • Higami Y.
      • Schimokawa I.
      Apoptosis in the aging process.
      ,
      • Lenaz G.
      • Bovina C.
      • D’Aurelio M.
      • Fato R.
      • Formiggini G.
      • Genova M.L.
      • Giuliano G.
      • Pich M.M.
      • Paolucci U.
      • Castelli G.P.
      • Ventura B.
      Role of mitochondria in oxidative stress and aging.
      , and
      • Martindale J.L.
      • Holbrook N.J.
      Cellular response to oxidative stress: signaling for suicide and survival.
      and references therein). Identification of the molecular components that underlie these events is an area of priority in aging research today. While some of the components and pathways may play a common role in the aging process of various organs, others may be specific as tissues are differentiated to exert a defined function and are exposed to different environmental conditions.
      Gene expression profiling techniques such as two-dimensional (2D) PAGE and DNA microarrays have been used to reveal genes and proteins that may be associated with senescence and longevity, particularly in cultured fibroblasts (
      • Celis J.E.
      • Bravo R.
      Synthesis of the nuclear protein cyclin in growing, senescent and morphologically transformed human skin fibroblasts.
      ,
      • Toda T.
      • Satoh M.
      • Sugimoto M.
      • Goto M.
      • Furuichi Y.
      • Kimura N.
      A comparative analysis of the proteins between the fibroblasts from Werner’s syndrome patients and age-matched normal individuals using two-dimensional gel electrophoresis.
      ,
      • Toda T.
      • Kaji K.
      • Kimura N.
      TMIG-2DPAGE: a new concept of two-dimensional gel protein database for research on aging.
      ,
      • Dierick J.F.
      • Pascal T.
      • Chainiaux F.
      • Eliaers F.
      • Remacle J.
      • Larsen P.M.
      • Roepstorff P.
      • Toussaint O.
      Transcriptome and proteome analysis in human senescent fibroblasts and fibroblasts undergoing premature senescence induced by repeated sublethal stresses.
      ,
      • Kondo T.
      • Sakaguchi M.
      • Namba M.
      Two-dimensional gel electrophoretic studies on the cellular aging: accumulation of α-2-macroglobulin in human fibroblasts with aging.
      ,
      • Benvenuti S.
      • Cramer R.
      • Quinn C.C.
      • Bruce J.
      • Zvelebil M.
      • Corless S.
      • Bond J.
      • Yang A.
      • Hockfield S.
      • Burlingame A.L.
      • Waterfield M.D.
      • Jat P.S.
      Differential proteome analysis or replicative senescence in rat embryo fibroblasts.
      ). Similar studies at the tissue and organ level, however, have proven difficult due to the heterogeneous nature of the specimens. To date, tissue profiling of the aging process has been confined mainly to DNA microarray analysis of mouse and human muscle (
      • Lee C.-K.
      • Klopp R.G.
      • Weindruch R.
      • Prolla T.A.
      Gene expression profile of aging and its retardation by caloric restriction.
      ,
      • Welle S.
      • Bhatt K.
      • Thornton C.A.
      High-abundance mRNAs in human muscle: comparison between young and old.
      ,
      • Weindruch R.
      • Kayo T.
      • Lee C.K.
      • Prolla T.A.
      Gene expression profiling of aging using DNA microarrays.
      ) and mouse brain (
      • Lee C.K.
      • Weindruch R.
      • Prolla T.A.
      Gene-expression profile of the ageing brain in mice.
      ,
      • Jiang C.H.
      • Tsien J.Z.
      • Schultz P.G.
      • Hu Y.
      The effects of aging on gene expression in the hypothalamus and cortex of mice.
      ) and liver (
      • Han E.
      • Hilsenbeck S.G.
      • Richardson A.
      • Nelson J.F.
      cDNA expression arrays reveal incomplete reversal of age-related changes in gene expression by caloric restriction.
      ,
      • Dozmorov I.
      • Bartke A.
      • Miller R.A.
      Array-based expression analysis of mouse liver genes: effect of age and of the longevity mutant Prop1df.
      ). In general, these studies have shown that the expression of only a small fraction of the genes analyzed changed during the aging process. A comparison between the effect of senescence in the muscle of mice and men showed that of 70 homologous genes studied by oligonucleotide microarrays only 17 showed similar age-related changes (
      • Welle S.
      • Brooks A.
      • Thornton C.A.
      Senescence-related changes in gene expression in muscle: similarities and differences between mice and men.
      ). Interestingly there was no evidence indicating that human muscle from old individuals exhibited deregulated expression of stress response genes as observed in the old murine muscle indicating important differences among species (
      • Welle S.
      • Brooks A.
      • Thornton C.A.
      Senescence-related changes in gene expression in muscle: similarities and differences between mice and men.
      ).
      Our laboratory has for many years applied proteomic technologies to the study of human epidermal biopsies in health and disease, and we have gathered a substantial amount of information on keratinocyte proteins under various physiological conditions (Refs.
      • Celis J.E.
      • Madsen P.
      • Rasmussen H.H.
      • Leffers H.
      • Honore B.
      • Gesser B.
      • Dejgaard K.
      • Olsen E.
      • Magnusson N.
      • Kiil J.
      • Celis A.
      • Lauridsen J.B.
      • Basse B.
      • Ratz G.P.
      • Andersen A.H.
      • Walbum E.
      • Brandstrup B.
      • Pedersen P.S.
      • Brandt N.J.
      • Puype M.
      • Van Damme J.
      • Vandekerckhove J.
      A comprehensive two-dimensional gel protein database of noncultured unfractionated normal human epidermal keratinocytes: towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.
      ,
      • Celis J.E.
      • Rasmussen H.H.
      • Gromov P.
      • Olsen E.
      • Madsen P.
      • Leffers H.
      • Honore B.
      • Dejgaard K.
      • Vorum H.
      • Kristensen B.
      • Østergaard M.
      • Hauns⊘ A.
      • Jensen N.A.
      • Celis A.
      • Basse B.
      • Lauridsen J.B.
      • Ratz G.P.
      • Andersen A.H.
      • Walbum E.
      • Kjærgaard I.
      • Andersen I.
      • Puype M.
      • Van Damme J.
      • Vandekerckhove J.
      The human keratinocyte two-dimensional gel protein database (update 1995): mapping components of signal transduction pathways.
      ,
      • Celis J.E.
      • Østergaard M.
      • Jensen N.A.
      • Gromova I.I.
      • Rasmussen H.H.
      • Gromov P.
      Human and mouse proteomic databases: a novel resources in the proteome universe.
      ; proteomics.cancer.dk). Here we present a quantitative analysis of the proteome expression profiles of fresh human epidermal biopsies obtained from the forearm of young and old donors. Besides arguing for a role of IFN-γ in the aging process, the biological activities associated with the deregulated proteins support the contention that aging is linked with increased oxidative stress that could lead to apoptosis in vivo (Refs.
      • Sohal R.S.
      Role of oxidative stress and protein oxidation in the aging process.
      and
      • Higami Y.
      • Schimokawa I.
      Apoptosis in the aging process.
      and references therein).

      EXPERIMENTAL PROCEDURES

      Skin Samples—

      Skin punch biopsies were obtained from Rigshospitalet, Copenhagen. Biopsies were taken from both forearms of normal Danish individuals of different ages (from 21 to 92 years old), placed on ice, and immediately transported to the Department of Medical Biochemistry, Aarhus University. The forearm was selected to minimize the effect of solar irradiation.

      In Vivo [35S]Methionine Tissue Labeling and 2D Gel Electrophoresis—

      Fresh skin biopsies were dissected with the aid of a scalpel to yield enriched epidermis. One- to two-mm3 epidermal pieces were labeled with [35S]methionine for 14 h in 0.1 ml of Dulbecco's modified Eagle's medium containing 1% dialyzed fetal calf serum and 100 μCi of radioactivity (Amersham Biosciences, catalog number SJ204). Following labeling, the tissues were dissolved in 0.3 ml of lysis solution (
      • O’Farrell P.Z.
      • Goodman H.M.
      • O’Farrell P.H.
      High resolution two-dimensional electrophoresis of basic as well as acidic proteins.
      ) and kept at −20 °C until use. Whole protein lysates were then subjected to both IEF and NEPHGE 2D PAGE as described previously (
      • Celis J.E.
      • Ratz G.
      • Basse B.
      • Lauridsen J.B.
      • Celis A.
      • Gromov P.
      ). Several gels were run from each sample. Proteins were visualized using autoradiography and/or phosphorimaging.

      Cytokine Treatment and [35S]Methionine Labeling of Cultured Primary Human Epidermal Keratinocytes—

      Primary cultures of normal human keratinocytes were prepared and grown as described previously (
      • Madsen P.
      • Rasmussen H.H.
      • Leffers H.
      • Honore B.
      • Celis J.E.
      Molecular cloning and expression of a novel keratinocyte protein (psoriasis-associated fatty acid-binding protein [PA-FABP]) that is highly up-regulated in psoriatic skin and that shares similarity to fatty acid-binding proteins.
      ). Cells were labeled for 14 h in Dulbecco's modified Eagle's medium lacking methionine and containing 1% dialyzed fetal calf serum, 500 μCi/ml [35S]methionine, and 50 units/ml recombinant cytokines: IFN-γ, IFN-α, or TNF-α. After labeling, the medium was aspirated, and the cells were resuspended in lysis solution for 2D PAGE (see above and Ref.
      • O’Farrell P.Z.
      • Goodman H.M.
      • O’Farrell P.H.
      High resolution two-dimensional electrophoresis of basic as well as acidic proteins.
      ). Samples were kept at −20 °C until use. Control cells were labeled under the same conditions except that no cytokine was added.

      Quantitation of 2D Protein Phosphorimages—

      Phosphorimage autoradiographs were obtained with the aid of the Molecular Imager from Bio-Rad and were quantitated using the Multi-Analyst 1.0.1 software (manually driven) from the same company. Only gels depicting well focused spots and limited amount of protein remaining at the origin were selected for quantitation. The levels of actin (IEF) and of annexin II, which migrated both in IEF and NEPHGE gels, were used as reference to normalize protein levels in both gel types (
      • Celis J.E.
      • Rasmussen H.H.
      • Olsen E.
      • Madsen P.
      • Leffers H.
      • Honore B.
      • Dejgaard K.
      • Gromov P.
      • Vorum H.
      • Vassilev A.
      • Baskin Y.
      • Liu X.
      • Celis A.
      • Basse B.
      • Lauridsen J.B.
      • Ratz G.P.
      • Andersen A.H.
      • Walbum E.
      • Kjærgaard I.
      • Andersen I.
      • Puype M.
      • Van Damme J.
      • Vandekerckhove J.
      The human keratinocyte two-dimensional gel protein database (update 1994): towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.
      ). The levels of selected proteins were quantitated in 20 pairs of young and old individuals. The average means and S.D. were determined. Groups in which the average means differed by a factor 1.5 or more were compared by using the heteroscedastic t test and evaluated for several proteins with the nonparametric Wilcoxon-Mann-Whitney test.

      Protein Identification—

      Proteins were identified by matching the gels with the master image of the human keratinocyte 2D PAGE database (Refs.
      • Celis J.E.
      • Madsen P.
      • Rasmussen H.H.
      • Leffers H.
      • Honore B.
      • Gesser B.
      • Dejgaard K.
      • Olsen E.
      • Magnusson N.
      • Kiil J.
      • Celis A.
      • Lauridsen J.B.
      • Basse B.
      • Ratz G.P.
      • Andersen A.H.
      • Walbum E.
      • Brandstrup B.
      • Pedersen P.S.
      • Brandt N.J.
      • Puype M.
      • Van Damme J.
      • Vandekerckhove J.
      A comprehensive two-dimensional gel protein database of noncultured unfractionated normal human epidermal keratinocytes: towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.
      ,
      • Celis J.E.
      • Rasmussen H.H.
      • Gromov P.
      • Olsen E.
      • Madsen P.
      • Leffers H.
      • Honore B.
      • Dejgaard K.
      • Vorum H.
      • Kristensen B.
      • Østergaard M.
      • Hauns⊘ A.
      • Jensen N.A.
      • Celis A.
      • Basse B.
      • Lauridsen J.B.
      • Ratz G.P.
      • Andersen A.H.
      • Walbum E.
      • Kjærgaard I.
      • Andersen I.
      • Puype M.
      • Van Damme J.
      • Vandekerckhove J.
      The human keratinocyte two-dimensional gel protein database (update 1995): mapping components of signal transduction pathways.
      ,
      • Celis J.E.
      • Østergaard M.
      • Jensen N.A.
      • Gromova I.I.
      • Rasmussen H.H.
      • Gromov P.
      Human and mouse proteomic databases: a novel resources in the proteome universe.
      ; proteomics.cancer.dk). One or a combination of procedures that included Edman degradation (
      • Rasmussen H.H.
      • van Damme J.
      • Puype M.
      • Gesser B.
      • Celis J.E.
      • Vandekerckhove J.
      Microsequences of 145 proteins recorded in the two-dimensional gel protein database of normal human epidermal keratinocytes.
      ), mass spectrometry (
      • Rasmussen H.H.
      • Mortz E.
      • Mann M.
      • Roepstorff P.
      • Celis J.E.
      Identification of transformation sensitive proteins recorded in human two-dimensional gel protein databases by mass spectrometric peptide mapping alone and in combination with microsequencing.
      ), and 2D PAGE Western immunoblotting (
      • Celis J.E.
      • Gromov P.
      High-resolution two-dimensional gel electrophoresis and protein identification using western blotting and ECL detection.
      ) has identified proteins in the database. Mass spectrometry and/or 2D PAGE Western immunoblotting confirmed the identity of selected proteins. In short, for mass spectrometry, protein spots were cut out from the dry gel with the aid of the corresponding x-ray film and were prepared as described previously (
      • Rasmussen H.H.
      • Mortz E.
      • Mann M.
      • Roepstorff P.
      • Celis J.E.
      Identification of transformation sensitive proteins recorded in human two-dimensional gel protein databases by mass spectrometric peptide mapping alone and in combination with microsequencing.
      ,
      • Jensen O.N.
      • Wilm M.
      • Shevchenko A.
      • Mann M.
      Sample preparation methods for mass spectrometric peptide mapping directly from 2-DE gels.
      ,
      • Celis J.E.
      • Kruhoffer M.
      • Gromova I.
      • Frederiksen C.
      • Ostergaard M.
      • Thykjaer T.
      • Gromov P.
      • Yu J.
      • Palsdottir H.
      • Magnusson N.
      • Orntoft T.F.
      Gene expression profiling: monitoring transcription and translation products using DNA microarrays and proteomics.
      ). All MALDI-MS measurements were performed using a Bruker Reflex III MALDI-TOF-MS (Bruker Daltonik GmbH). Prior to peptide mass fingerprint analysis, the instrument was calibrated using the known masses of a mixture of synthetic peptides spotted on the target disc closer to the sample. Peptide masses were searched using the Expasy algorithm (www.expasy.ch/tools/). 2D PAGE Western immunoblotting was performed as described previously (
      • Celis J.E.
      • Gromov P.
      High-resolution two-dimensional gel electrophoresis and protein identification using western blotting and ECL detection.
      ).

      Indirect Immunofluorescence—

      Eight-μm cryostat sections from frozen human skin were placed on round coverslips, washed three times with phosphate-buffered saline, and treated for 5 min with methanol at −20 °C (
      • Celis J.E.
      • Celis P.
      • Ostergaard M.
      • Basse B.
      • Lauridsen J.B.
      • Ratz G.
      • Rasmussen H.H.
      • Orntoft T.F.
      • Hein B.
      • Wolf H.
      • Celis A.
      Proteomics and immunohistochemistry define some of the steps involved in the squamous differentiation of the bladder transitional epithelium: a novel strategy for identifying metaplastic lesions.
      ). Coverslips were washed several times with phosphate-buffered saline, covered with 20 μl of the primary antibody, and incubated for 60 min at 37 °C in a humidified box. Following incubation, the coverslips were washed several times with phosphate-buffered saline, covered with 20 μl of rhodamine-conjugated secondary antibody (dilution, 1:50), and incubated for 60 min at 37 °C in a humidified box. Coverslips were washed extensively with phosphate-buffered saline, washed once with distilled water, and covered with DAKO mounting medium. Samples were observed using a Leica photomicroscope equipped with epifluorescence and phase contrast optics.

      RESULTS

      To monitor the structural disorganization and fragmentation of the elastic fiber meshwork, which is a hallmark of skin aging (
      • Jenkins G.
      Molecular mechanisms of skin ageing.
      ,
      • Wlaschek M.
      • Tantcheva-Poor I.
      • Naderi L.
      • Ma W.
      • Schneider L.A.
      • Razi-Wolf Z.
      • Schuller J.
      • Scharffetter-Kochanek K.
      Solar UV irradiation and dermal photoaging.
      ), we performed immunohistochemistry of skin biopsies using a monoclonal antibody prepared in our laboratory (monoclonal antibody b9) that specifically decorates these fibers (
      • Palsdottir H.
      ). As shown in Fig. 1, the forearm skin from old individuals displayed disorganization of the elastic fiber meshwork as well as loss of anchoring of the fibers oriented perpendicularly to the dermal/epidermal junction (Fig. 1b). In young and middle-aged individuals, the latter formed arcades that projected toward the junction (Fig. 1a). Immunostaining of similar skin sections with a collagen IV antibody (DAKO AS), which decorates the basement membrane, revealed flattening of the dermal/epidermal junction, a feature that is characteristic of aging skin (Fig. 1, compare d with c (young)).
      Figure thumbnail gr1
      Fig. 1Immunofluorescence staining of frozen skin sections with monoclonal antibody b9 (a and b) and anti-collagen IV (c and d).a and c, young individuals; b and d, old individuals. Only cross-sections of the dermal-epidermal junction are shown (×200). The age-associated derangement of the elastic fiber network is clearly visible (compare white arrows in a and b). The progressive loss of the undulatory shape of this junction with age is shown by white arrowheads (compare e and d). MAb, monoclonal antibody; E, epidermis; D, dermis.
      Representative 2D gel phosphorimages of human epidermal proteins resolved in IEF and NEPHGE gels are shown in Fig. 2. Samples showed little contamination with connective tissue as judged by the low levels of expression of vimentin, a protein that is expressed by dermal fibroblasts (
      • Celis J.E.
      • Celis P.
      • Palsdottir H.
      • Østergaard M.
      • Gromov P.
      • Primdahl H.
      • Orntoft T.F.
      • Wolf H.
      • Celis A.
      • Gromova I.
      Proteomic strategies to reveal tumor heterogeneity among urothelial papillomas.
      ). A total of 172 well focused and abundant proteins were selected for quantitation (Table I). These are indicated with their name and/or SSP number in Fig. 2, A and B, and are listed in Table I together with their apparent Mr and pI values. Proteins were identified by matching the gels with the master image of the human keratinocyte 2D PAGE database (Fig. 3; proteomics.cancer.dk) (
      • Celis J.E.
      • Madsen P.
      • Rasmussen H.H.
      • Leffers H.
      • Honore B.
      • Gesser B.
      • Dejgaard K.
      • Olsen E.
      • Magnusson N.
      • Kiil J.
      • Celis A.
      • Lauridsen J.B.
      • Basse B.
      • Ratz G.P.
      • Andersen A.H.
      • Walbum E.
      • Brandstrup B.
      • Pedersen P.S.
      • Brandt N.J.
      • Puype M.
      • Van Damme J.
      • Vandekerckhove J.
      A comprehensive two-dimensional gel protein database of noncultured unfractionated normal human epidermal keratinocytes: towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.
      ,
      • Celis J.E.
      • Rasmussen H.H.
      • Gromov P.
      • Olsen E.
      • Madsen P.
      • Leffers H.
      • Honore B.
      • Dejgaard K.
      • Vorum H.
      • Kristensen B.
      • Østergaard M.
      • Hauns⊘ A.
      • Jensen N.A.
      • Celis A.
      • Basse B.
      • Lauridsen J.B.
      • Ratz G.P.
      • Andersen A.H.
      • Walbum E.
      • Kjærgaard I.
      • Andersen I.
      • Puype M.
      • Van Damme J.
      • Vandekerckhove J.
      The human keratinocyte two-dimensional gel protein database (update 1995): mapping components of signal transduction pathways.
      ,
      • Celis J.E.
      • Østergaard M.
      • Jensen N.A.
      • Gromova I.I.
      • Rasmussen H.H.
      • Gromov P.
      Human and mouse proteomic databases: a novel resources in the proteome universe.
      ). In selected cases, mass spectrometry and 2D PAGE Western immunoblotting further confirmed their identity. Known proteins listed in Table I are categorized according to the following functional groups: (i) energy metabolism; (ii) protein synthesis, folding, and degradation; (iii) cytoskeleton; (iv) RNA metabolism; (v) calcium-modulated metabolism; (vi) cell proliferation and differentiation; and (vii) others.
      Figure thumbnail gr2
      Fig. 22D gel (IEF and NEPHGE) phosphorimages of [35S]methionine-labeled proteins from human skin epidermis obtained from young (A, 24-year-old) and old (B, 89-year-old) individuals. Quantitated protein spots are indicated with their corresponding SSP numbers in the keratinocyte database (proteomics.cancer.dk). Deregulated proteins are highlighted with red (arrows and SSP numbers). Highly variable proteins are indicated with blue. The identity and relative levels of the proteins are given in . Protein levels were normalized with respect to the level of actin (SSP 6310) and annexin II (SSP 0210, IEF; and SSP 4205, NEPHGE).
      Table ILevels of [35S]methionine-labeled proteins in normal human skin biopsies obtained from young (21–30-year-old) and old (75–92-year-old) individuals
      Protein
      Proteins were identified by matching the gels with the master image of the human keratinocyte 2D PAGE database (proteomics.cancer.dk). In selected cases the identity was verified by mass spectrometry and/or 2D PAGE immunoblotting.
      Mr × 10−3
      Proteins in each functional category are listed in order of increasing Mr.
      pISSP
      SSP number from the human keratinocyte 2D PAGE database (proteomics.cancer.dk). I, IEF gel; N, NEPHGE gel.
      Spot volume (×102)
      Protein spots were quantitated as described under “Experimental Procedures.” The column shows the average means and S.D. Proteins changing by a factor of 1.5 or more are highlighted in bold.
      Ratio old/young
      p values were determined for groups in which the average means changed by a factor of 1.5 or more.
      YoungOld
      Energy metabolism
       1. Hydrogen-transporting ATP synthetase13.94.19010 (I)6.2 ± 1.57.9 ± 1.71.2
       2. Triose-phosphate isomerase28.37.35106 (N)15 ± 6.313 ± 4.00.9
       3. Phosphoglyceromutase28.67.01107 (I)12 ± 3.910 ± 3.40.8
       4. Phosphoglyceromutase30.47.25203 (N)24 ± 9.721 ± 7.70.9
       5. Aldose reductase37.56.53327 (I)5.6 ± 0.94.7 ± 0.70.8
       6. Glyceraldehyde-3-phosphate dehydrogenase38.48.91206 (N)167 ± 57166 ± 411.0
       7. Isocytrate dehydrogenase41.27.41320 (I)4.4 ± 0.95.2 ± 0.71.2
       8. Fructose 1,6-bisphosphate aldolase43.48.81302 (N)37 ± 1132 ± 7.50.8
       9. Phosphoglycerate kinase43.68.23308 (N)54 ± 2466 ± 301.2
       10. Citrate synthetase44.58.13302 (N)12 ± 3.015 ± 2.81.1
       11. α-Enolase46.87.41325 (I)72 ± 2182 ± 171.1
       12. α-Enolase48.77.45406 (N)152 ± 52168 ± 341.1
       13. Aldehyde dehydrogenase (mitochondrial)51.96.33303 (I)4.1 ± 1.25.3 ± 1.11.2
       14. Vacuolar ATPase68.95.36515 (I)7.8 ± 3.37.1 ± 3.00.9
      Protein synthesis, folding, and degradation
       15. Ubiquitin9.67.80013 (I)33 ± 1342 ± 111.3
       16. Cystatin11.25.36011 (I)5.5 ± 2.05.7 ± 1.51.0
       17. Ribosomal P-protein14.44.09005 (I)5.2 ± 1.16.0 ± 1.41.1
      18. eIF-5A16.04.98016 (I)6.9 ± 4.916 ± 4.02.3 (p < 0.001)
       19. eIF-5A, variant16.04.68010 (I)5.3 ÷ 21
      The division sign indicates values that were highly variable.
      1.6 ÷ 11
      The division sign indicates values that were highly variable.
      Variable, not determined
      20. Cyclophilin A17.38.32003 (N)26 ± 8.941 ± 111.6 (p < 0.001)
      21. Cyclophilin A, variant17.38.03004 (N)16 ± 6.326 ± 7.81.6 (p < 0.001)
       22. Cyclophilin B19.310.30117 (N)3.7 ± 2.05.1 ± 2.51.4
      23. Proteasomal protein28.97.70107 (I)2.6 ± 0.44.0 ± 0.81.5 (p < 0.001)
       24. Proteosome ζ28.14.49136 (I)4.0 ± 1.24.8 ± 1.41.2
       25. HSP2828.36.04110 (I)4.3 ± 1.93.2 ± 1.00.7
       26. HSP28 variant30.05.56111 (I)3.4 ± 0.83.3 ± 1.21.0
      27. PA28-α30.45.85111 (I)3.2 ± 0.75.6 ± 1.51.7 (p < 0.001)
       28. Proteasomal protein30.55.06226 (I)2.7 ± 0.43.2 ± 1.11.1
       29. Cathepsin D32.65.75215 (I)2.4 ± 0.43.1 ± 0.61.2
       30. eIF-2α36.75.27215 (I)4.1 ± 0.55.4 ± 0.61.3
       31. eIF-339.36.43212 (I)4.2 ± 1.84.9 ± 1.41.1
       32. SCC A139.56.62209 (I)3.4 ± 1.63.1 ± 1.90.9
       33. SCC A2, variant405.94333 (I)3.0 ± 1.13.0 ± 1.81.0
       34. SCC A240.15.914205 (I)12 ± 8.312 ± 8.21.0
       35. Ribosomal protein 1-β47.04.68302 (I)4.6 ± 1.16.4 ± 1.81.4
       36. CCT-β49.36.23301 (I)12.3 ± 3.017 ± 9.51.4
       37. Protein disulfide isomerase β-subunit56.24.68412 (I)5.6 ± 2.06.3 ± 2.01.1
       38. Protein disulfide isomerase ER-60 precursor56.55.75410 (I)7.3 ± 1.77.7 ± 2.11.1
      39. Tryptophanyl-tRNA synthetase56.95.93524 (I)3.0 ± 1.26.7 ± 2.52.2 (p < 0.001)
      40. HSP6057.65.26403 (I)30 ± 1148 ± 121.5 (p < 0.001)
       41. CCT-ζ58.26.52416 (I)14 ± 3.613 ± 5.70.9
       42. CCT-θ58.55.55403 (I)13 ± 3.618 ± 5.21.4
       43. Ribophorin II59.65.74523 (I)5.8 ± 1.27.7 ± 2.01.3
       44. CCT-α59.75.94406 (I)17 ± 1417 ± 4.81.0
       45. CCT-γ61.46.33408 (I)8.1 ± 2.111 ± 3.31.3
       46. HSX7065.05.46511 (I)78 ± 2589 ± 291.1
       47. HSP7065.65.36504 (I)124 ± 48104 ± 410.9
       48. grp75685.56513 (I)29 ± 1337 ± 131.3
       49. Endoplasmin99.84.68603 (I)27 ± 8.923 ± 110.9
       50. HSP110115.25.27717 (I)36 ± 1433 ± 110.9
      Cytoskeleton
       51. Myosin, light chain17.54.49019 (I)6.4 ± 1.36.4 ± 1.41.0
       52. Coffilin18.18.23008 (N)4.1 ± 1.63.7 ± 1.60.9
       53. Myosin, light chain18.24.49020 (I)5.1 ± 0.95.7 ± 1.11.1
       54. Rho A23.96.23102 (I)14 ± 5.212 ± 3.00.9
       55. Rho GDI GDP dissociation inhibitor, variant28.14.88120 (I)4.7 ± 0.84.8 ± 0.91.0
       56. Rho GDI GDP dissociation inhibitor29.34.78118 (I)2.6 ± 0.63.6 ± 0.91.4
       57. F-actin capping protein31.85.65106 (I)3.1 ± 0.53.3 ± 0.51.1
       58. p34 Arc32.87.60226 (I)4.2 ± 1.14.6 ± 0.91.1
       59. Tropomyosin36.64.439213 (I)59 ± 1644 ± 160.7
       60. gCap3941.05.94331 (I)6.3 ± 1.26.7 ± 1.31.1
       61. Elastase inhibitor41.86.04314 (I)17 ± 3.019 ± 9.01.1
       62. Actin435.26310 (I)Reference protein1.0
       63. Keratin 1747.44.68419 (I)4.3 ± 1.05.8 ± 1.71.3
       64. Keratin 17, variant47.54.78314 (I)3.7 ± 1.03.7 ± 1.31.0
       65. Keratin 1747.74.98312 (I)164 ± 45158 ± 471.0
       66. Keratin 1547.84.59307 (I)2.5 ± 0.83.6 ± 1.11.4
       67. Fascin, actin-binding protein50.07.11314 (I)7.0 ± 2.48.1 ± 2.11.2
       68. Arp 352.15.75313 (I)8.3 ± 1.57.9 ± 1.90.9
       69. Keratin 653.37.51418 (I)12 ± 3.116 ± 5.01.3
       70. Lamin C55.46.91414 (I)4.5 ± 1.55.0 ± 1.71.1
       71. β-Tubulin57.54.88411 (I)208 ± 38233 ± 541.1
       72. α-Tubulin59.65.07416 (I)72 ± 2296 ± 311.3
       73. Paxillin65.46.13525 (I)4.3 ± 0.95.7 ± 1.61.3
       74. Lamin B65.65.07510 (I)6.7 ± 2.08.3 ± 2.31.2
       75. Cytovillin (ezrin)77.96.33504 (I)46 ± 2139 ± 150.9
       76. Gelsolin94.65.75608 (I)3.1 ± 1.34.3 ± 1.61.4
       77. Cas (p130)126.95.26850 (I)12 ± 5.317 ± 101.4
      RNA metabolism
       78. hnRNP-related20.28.03104 (N)11 ± 4.114 ± 6.61.3
       79. hnRNP-A135.0100205 (N)8.5 ± 3.29.3 ± 3.21.1
       80. hnRNP B138.39.51306 (N)8.8 ± 3.48.8 ± 2.71.0
       81. Poly (rC)-binding protein (PCBP-1)40.87.01216 (I)15 ± 3.517 ± 3.31.1
       82. hnRNP H52.85.94410 (I)23 ± 7.723 ± 6.51.0
       83. hnRNP-related58.48.81502 (N)31 ± 1024 ± 8.10.8
       84. hnRNP K D′65.65.07512 (I)9.0 ± 2.69.9 ± 3.51.1
      Calcium-modulated metabolism
       85. MRP 8, S100A89.07.41003 (I)9.0 ± 5.29.3 ± 4.01.0
       86. CaN19, S100A29.04.29027 (I)99 ± 2686 ± 260.9
       87. Calcyclin, S100A69.85.07014 (I)2.7 ± 1.13.6 ± 1.21.3
       88. Calgizzarin, S100A1110.16.04006 (I)57 ± 1962 ± 141.1
       89. Psoriasin, S100A711.06.23002 (I)4.0 ÷ 477.0 ÷ 35
      The division sign indicates values that were highly variable.
      Variable, not determined
       90. MRP 14, S100A9 variant11.65.46010 (I)29 ± 9.636 ± 161.2
       91. MRP 14, S100A912.55.56017 (I)14 ± 3.914 ± 6.51.0
       92. Annexin III32.45.55204 (I)3.8 ± 0.74.3 ± 0.91.1
       93. Annexin V33.34.88213 (I)11 ± 2.912 ± 3.01.0
       94. Annexin IV34.15.85217 (I)2.4 ± 0.33.1 ± 0.71.3
       95. Annexin VIII35.75.65213 (I)5.5 ± 1.46.9 ± 1.91.3
       96. Annexin II, variant36.07.50202 (I)3.0 ± 0.72.9 ± 1.01.0
      97. Annexin I36.26.62216 (I)68 ± 2441 ± 140.6 (p < 0.001)
       98. Annexin II36.47.80210 (I)62 ± 1662 ± 211.0
       99. Annexin II36.47.94205 (N)Reference protein1.0
       100. Calreticulin, precursor60.74.169401 (I)4.8 ± 1.96.5 ± 1.81.3
      Cell proliferation and differentiation
       101. PA-FABP12.46.33007 (I)18 ± 1225 ± 9.41.4
      102. NM23 H2 (nucleoside diphosphate kinase)18.08.71009 (N)10 ± 4.116 ± 4.41.6 (p < 0.001)
       103. Translationally controlled human tumor protein24.24.68114 (I)49 ± 1560 ± 141.2
       104. 14-3-3-σ, stratifin304.49126 (I)713 ± 139705 ± 1411.0
       105. 14-3-3-β30.24.58102 (I)56 ± 1455 ± 111.0
       106. 14-3-3-related30.84.29106 (I)2.7 ± 0.52.9 ± 0.61.1
       107. 14-3-3-η31.14.49125 (I)17 ± 3.418 ± 7.41.1
       108. Purine nucleoside phosphorylase31.36.52108 (I)2.1 ± 0.71.9 ± 0.40.8
       109. Receptor for activated C-kinase32.77.80205 (I)9.6 ± 2.312 ± 1.71.3
       110. Nucleophosmin37.84.68207 (I)7.7 ± 1.89.1 ± 2.51.2
       111. G-protein, β238.15.56208 (I)5.9 ± 0.67.0 ± 1.71.2
       112. Maspin38.75.85220 (I)50 ± 1050 ± 131.0
       113. Maspin, variant38.75.65207 (I)3.3 ± 0.33.4 ± 0.41.0
       114. G-protein, β139.15.56214 (I)6.9 ± 1.77.8 ± 1.31.1
       115. MEK 2, variant47.56.13428 (I)6.7 ± 1.98.5 ± 1.81.3
       116. MEK 247.56.23313 (I)7.3 ± 1.48.9 ± 1.61.2
       117. NCK48.46.33306 (I)8.5 ± 2.611 ± 2.91.3
       118. Signal-transducing adaptor67.34.59507 (I)2.3 ± 0.82.1 ± 0.61.1
      119. PI3K, p85β isoform90.86.32634 (I)2.8 ± 1.35.9 ± 2.12.1 (p < 0.001)
      Others
       120. Thioredoxin11.44.608006 (I)13 ± 4.012 ± 4.00.9
       121. Phosphoneuroprotein18.94.29104 (I)2.8 ± 0.53.0 ± 1.01.1
      122. Mn-superoxide dismutase21.77.70105 (I)7.0 ± 2.417 ± 122.4 (p = 0.0013)
       123. NKCEF-A24.98.32108 (N)21 ± 6.415 ± 4.40.8
       124. GST-π, variant25.45.36125 (I)11 ± 4.011 ± 3.01.0
       125. GST-π25.45.45101 (I)6.4 ± 0.96.6 ± 1.41.0
       126. CCL P protein31.95.07107 (I)2.1 ± 0.52.4 ± 0.31.1
       127. Adenosine deaminase41.65.55305 (I)10.3 ± 4.77.9 ± 3.10.8
      128. PAI-241.75.56314 (I)232 ± 56123 ± 380.5 (p < 0.001)
      129. PAI-2, variant41.75.46316 (I)46 ± 1324 ± 6.00.5 (p < 0.001)
      130. Mx-A protein77.65.65507 (I)2.2 ± 1.315 ± 8.76.8 (p < 0.001)
      Unknown
       131. Unknown9.26.60204 (I)7.6 ± 2.88.8 ± 3.11.2
      132. Unknown157.51015 (I)9.9 ± 7.120 ± 8.12.0 (p < 0.001)
       133. Unknown20.07.35111 (N)5.5 ± 3.17.4 ± 5.41.3
       134. Unknown26.06.51131 (I)14 ± 3.613 ± 5.70.9
       135. Unknown31.17.51112 (I)3.0 ± 0.44.1 ± 1.91.4
       136. Unknown328.32204 (N)14 ± 4.316 ± 4.91.1
       137. Unknown32.85.07205 (I)2.4 ± 0.32.8 ± 0.31.1
       138. Unknown33.98.13205 (N)22 ± 5.624 ± 6.01.1
       139. Unknown34.26.14210 (I)5.4 ± 1.05.1 ± 0.71.0
       140. Unknown35.27.31223 (I)4.8 ± 1.46.2 ± 1.61.2
       141. Unknown35.65.94203 (I)3.6 ± 0.84.0 ± 1.11.1
       142. Unknown35.86.72215 (I)5.1 ± 0.94.9 ± 0.71.0
      143. Unknown36.25.84201 (I)1.4 ± 0.52.1 ± 0.51.5 (p < 0.001)
       144. Unknown36.65.74218 (I)2.3 ± 0.32.6 ± 0.41.1
       145. Unknown36.75.36203 (I)3.7 ± 0.33.5 ± 0.70.9
       146. Unknown37.25.07210 (I)3.6 ± 1.14.4 ± 1.61.3
       147. Unknown37.77.70319 (I)6.2 ± 3.14.9 ± 1.30.8
       148. Unknown39.96.52210 (I)9.5 ± 3.410 ± 3.61.0
       149. Unknown40.18.22304 (N)24 ± 1621 ± 5.70.9
       150. Unknown41.46.14316 (I)4.9 ± 1.16.2 ± 2.01.3
      151. Unknown41.94.58323 (I)2.0 ± 0.63.8 ± 1.01.9 (p < 0.001)
       152. Unknown45.37.25304 (N)28 ± 8.132 ± 9.91.1
       153. Unknown46.47.64303 (N)8.3 ± 1.810 ± 2.01.2
       154. Unknown476.81303 (I)8.9 ± 4.29.6 ± 2.31.1
      155. Unknown47.19.02411 (N)8.8 ± 3.015 ± 7.11.7 (p = 0.0035)
       156. Unknown47.87.51324 (I)3.3 ± 0.63.3 ± 0.91.0
      157. Unknown47.95.55307 (I)46 ± 1516 ± 7.80.3 (p < 0.001)
      158. Unknown47.95.46322 (I)155 ± 6348 ± 430.3 (p < 0.001)
       159. Unknown48.66.91307 (I)5.6 ± 1.25.3 ± 1.50.9
       160. Unknown48.65.94301 (I)4.9 ± 1.25.5 ± 1.01.1
       161. Unknown49.27.41430 (I)2.6 ± 0.43.4 ± 1.01.3
       162. Unknown51.38.12404 (N)13 ± 3.014 ± 5.01.1
       163. Unknown54.85.65405 (I)4.6 ± 1.05.8 ± 1.31.3
       164. Unknown55.36.33410 (I)4.9 ± 1.04.4 ± 1.30.9
      165. Unknown55.65.15428 (I)3.3 ± 1.66.0 ± 2.51.8 (p < 0.001)
       166. Unknown63.46.14414 (I)17 ± 3.720 ± 5.01.2
       167. Unknown63.68.42505 (N)15 ± 6.011 ± 6.00.7
      168. Unknown67.24.68510 (I)5.0 ± 1.67.7 ± 2.81.5 (p < 0.001)
       169. Unknown67.54.78509 (I)3.5 ± 1.22.8 ± 0.60.8
       170. Unknown72.36.04509 (I)13 ± 7.013 ± 121.0
       171. Unknown72.66.62507 (I)4.6 ± 1.76.0 ± 2.11.3
       172. Unknown96.64.68608 (I)7.9 ± 3.17.9 ± 3.20.9
      a Proteins were identified by matching the gels with the master image of the human keratinocyte 2D PAGE database (proteomics.cancer.dk). In selected cases the identity was verified by mass spectrometry and/or 2D PAGE immunoblotting.
      b Proteins in each functional category are listed in order of increasing Mr.
      c SSP number from the human keratinocyte 2D PAGE database (proteomics.cancer.dk). I, IEF gel; N, NEPHGE gel.
      d Protein spots were quantitated as described under “Experimental Procedures.” The column shows the average means and S.D. Proteins changing by a factor of 1.5 or more are highlighted in bold.
      e p values were determined for groups in which the average means changed by a factor of 1.5 or more.
      f The division sign indicates values that were highly variable.
      Figure thumbnail gr3
      Fig. 3Master synthetic images of human epidermal keratinocyte proteins separated by IEF (A) and NEPHGE (B) as depicted on the World Wide Web at proteomics.cancer.dk. Proteins flagged with a red cross correspond to known proteins. By clicking on any given spot at the Web site it is possible to open a file that contains protein information as well as links to other World Wide Web sites.
      Steady state levels of [35S]methionine incorporation were estimated as an average mean value for each protein spot in all 20 sample pairs. Only gels that exhibited a limited amount of protein remaining at the origin of the first dimension gel and that were devoid of major streaking were selected for quantitation. The levels of actin (IEF) and annexin II, which migrated in both IEF and NEPHGE gels, were used to normalize the amount of labeled protein that entered the gels. The -fold change column in Table I shows the ratios of [35S]methionine incorporation calculated as an arithmetic mean value of the spot volumes for each sample pair. As depicted in Table I, the great majority of the 172 proteins (148 proteins), which represented essentially the most abundant components of the epidermal proteome, showed no significant alterations in their levels in the old individuals. Two proteins exhibited irregular behavior from individual to individual and are indicated in blue in Fig. 2 and are listed in Table I. The identity of the proteins was determined by comparison with the master image in the keratinocyte 2D PAGE database.
      Twenty-two proteins were consistently deregulated by a factor of 1.5 or more across the 20 samples and are indicated in Fig. 2 (in red) and Table I. Statistical evaluation of the data showed that in most cases the p values were less than 0.001 (α = 0.05) suggesting that the observed changes are statistically significant. This was also the case for manganese-superoxide dismutase (Mn-SOD) and the unknown protein SSP 2411, which yielded p values of 0.0013 and 0.0035, respectively.
      The 22 proteins included Mx-A (↑6.8), Mn-SOD (↑2.4), tryptophanyl-tRNA synthetase (↑2.2), the p85β subunit of phosphatidylinositol 3-kinase (PI3K, ↑2.1), the proteasome regulator PA28-α (↑1.7), the eukaryotic initiation factor 5A (eIF-5A, ↑2.3), NM23 H2, (↑1.6), cyclophilin A and its variant (↑1.6), proteasomal protein SSP 0107 (↑1.5), HSP60 (↑1.5), annexin I (↓1.7), and plasminogen activator inhibitor 2 (PAI-2) and its variant (↓2.0) as well as seven acidic and one basic protein of still unknown identity (SSPs: 1015, ↑2.0; 5307, ↓2.8; 6322, ↓3.2; 4201, ↑1.5; 8323, ↑1.9; 2411, ↑1.7; 5428, ↑1.8; and 8510, ↑1.5). Close-up areas of 2D gels from young and old epidermis illustrating some of the changes are shown in Fig. 4. The identity of some of these proteins was confirmed by immunohistochemistry (p85β subunit of PI3K, tryptophanyl-tRNA synthetase, and Mx-A; Fig. 5) and mass spectrometry (PA28-α, Mn-SOD, and eIF-5A; Fig. 6).
      Figure thumbnail gr4
      Fig. 4Age-associated alterations in the levels of proteins that are up-regulated by a factor of 2 or more in the epidermis of elderly individuals. Only fractions of the 2D gel phosphorimages are shown in each case. A and B, p85β subunit of PI3K and Mx-A protein; C and D, tryptophanyl-tRNA synthetase; E and F, Mn-SOD; G and H, eIF-5A and eIF-5A variant.
      Figure thumbnail gr5
      Fig. 5Enhanced chemiluminescence 2D PAGE Western blotting analysis of proteins from normal human skin epidermis.A, p85β subunit of PI3K; B, tryptophanyl-tRNA synthetase; C, Mx-A protein. The antibodies were kindly provided by I. Gout and M. Waterfield, J. Justesen, and J. Pavlovic, respectively.
      Figure thumbnail gr6
      Fig. 6Identification of PA28-α, Mn-SOD, and eIF-5A by MALDI-TOF-MS.A, number of identified peptides and sequence coverage from the analysis of the three protein digest mixtures. B–D, spectra and search results. The left panels show the MALDI-TOF-MS spectra. The internal calibration was performed using picks generated by trypsin autodigestion (805.417, 1153.574, and 2163.057). The right panels represent the identification probability plot from the search of the mass spectrometry protein sequence database (MSDB) with the MASCOT software.
      Careful inspection of the data recorded in the keratinocyte 2D PAGE database (
      • Celis J.E.
      • Madsen P.
      • Rasmussen H.H.
      • Leffers H.
      • Honore B.
      • Gesser B.
      • Dejgaard K.
      • Olsen E.
      • Magnusson N.
      • Kiil J.
      • Celis A.
      • Lauridsen J.B.
      • Basse B.
      • Ratz G.P.
      • Andersen A.H.
      • Walbum E.
      • Brandstrup B.
      • Pedersen P.S.
      • Brandt N.J.
      • Puype M.
      • Van Damme J.
      • Vandekerckhove J.
      A comprehensive two-dimensional gel protein database of noncultured unfractionated normal human epidermal keratinocytes: towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.
      ,
      • Celis J.E.
      • Rasmussen H.H.
      • Gromov P.
      • Olsen E.
      • Madsen P.
      • Leffers H.
      • Honore B.
      • Dejgaard K.
      • Vorum H.
      • Kristensen B.
      • Østergaard M.
      • Hauns⊘ A.
      • Jensen N.A.
      • Celis A.
      • Basse B.
      • Lauridsen J.B.
      • Ratz G.P.
      • Andersen A.H.
      • Walbum E.
      • Kjærgaard I.
      • Andersen I.
      • Puype M.
      • Van Damme J.
      • Vandekerckhove J.
      The human keratinocyte two-dimensional gel protein database (update 1995): mapping components of signal transduction pathways.
      ,
      • Celis J.E.
      • Østergaard M.
      • Jensen N.A.
      • Gromova I.I.
      • Rasmussen H.H.
      • Gromov P.
      Human and mouse proteomic databases: a novel resources in the proteome universe.
      ) revealed that six of the deregulated proteins, namely Mx-A, Mn-SOD, tryptophanyl-tRNA synthetase, the p85β subunit of PI3K, and the proteasomal proteins PA28-α and SSP 0107, correspond to a protein signature that is induced by treatment of cultured human keratinocytes with IFN-γ (
      • Honore B.
      • Leffers H.
      • Madsen P.
      • Celis J.E.
      Interferon-γ up-regulates a unique set of proteins in human keratinocytes. Molecular cloning and expression of the cDNA encoding the RGD-sequence-containing protein IGUP I-5111.
      ). To corroborate this observation we treated normal primary human keratinocytes with IFN-γ as described under “Experimental Procedures,” and the results are presented in Fig. 7. Clearly all six proteins were up-regulated in the IFN-γ-treated cells (Fig. 7B) confirming previous studies. Induction of these proteins as a group represents a specific signature for IFN-γ as deregulation of the polypeptide set has not been observed in keratinocytes treated with other cytokines (IFN-α (Fig. 7C) and -β; interleukins 1α, 1β, 2, 3, 6, 7, and 8; and TNF-α (Fig. 7D)) or growth factors (transforming growth factor-β and fibroblast growth factor) (proteomics.cancer.dk) (
      • Rasmussen H.H.
      • Celis J.E.
      Evidence for an altered protein kinase C (PKC) signaling pathway in psoriasis.
      ). IFN-α (Fig. 7C) and -β up-regulated Mx-A, while interleukins 1α and -β and TNF-α (Fig. 7D) up-regulated Mn-SOD.
      Figure thumbnail gr7
      Fig. 72D PAGE protein profiles of primary human keratinocytes labeled with [35S]methionine in the absence (A) and presence of IFN-γ (B), IFN-α (C), and TNF-α (D). Only fractions of the 2D gel phosphorimages are shown. The IFN-γ-induced polypeptide signature is indicated with arrows in B.

      DISCUSSION

      To our knowledge the studies reported here represent the first attempt to apply quantitative proteomic technologies in combination with 2D PAGE database resources (proteomics.cancer.dk) for the analysis of aging of the human epidermis using fresh tissue biopsies. Clearly our studies have been restricted to the analysis of major proteome alterations that take place in the differentiated compartment of the epidermis as basal cells comprise only a small fraction of the tissue. In general, the results indicate that aging of the epidermis is accompanied by changes in the relative levels of a few abundant proteins that are expressed throughout the life span of keratinocytes rather than by the appearance or disappearance of polypeptides. These results are in line with recent studies by Benvenuti and colleagues (
      • Benvenuti S.
      • Cramer R.
      • Quinn C.C.
      • Bruce J.
      • Zvelebil M.
      • Corless S.
      • Bond J.
      • Yang A.
      • Hockfield S.
      • Burlingame A.L.
      • Waterfield M.D.
      • Jat P.S.
      Differential proteome analysis or replicative senescence in rat embryo fibroblasts.
      ) of serially passaged rat embryo fibroblasts by 2D PAGE, which showed that 49 proteins were altered by a factor of 2-fold or more in the senescence cells. The majority of these proteins, with roles in the cytoskeleton, heat shock, trafficking, differentiation, protein synthesis, modification, and turnover, has not been previously associated with senescence and represent important targets for future studies. Our data are also in agreement with DNA microarray studies of mouse and human tissues, which have shown that expression of only a small fraction of the genes studied changed during the aging process (
      • Lee C.-K.
      • Klopp R.G.
      • Weindruch R.
      • Prolla T.A.
      Gene expression profile of aging and its retardation by caloric restriction.
      ,
      • Welle S.
      • Bhatt K.
      • Thornton C.A.
      High-abundance mRNAs in human muscle: comparison between young and old.
      ,
      • Weindruch R.
      • Kayo T.
      • Lee C.K.
      • Prolla T.A.
      Gene expression profiling of aging using DNA microarrays.
      ,
      • Lee C.K.
      • Weindruch R.
      • Prolla T.A.
      Gene-expression profile of the ageing brain in mice.
      ,
      • Jiang C.H.
      • Tsien J.Z.
      • Schultz P.G.
      • Hu Y.
      The effects of aging on gene expression in the hypothalamus and cortex of mice.
      ,
      • Han E.
      • Hilsenbeck S.G.
      • Richardson A.
      • Nelson J.F.
      cDNA expression arrays reveal incomplete reversal of age-related changes in gene expression by caloric restriction.
      ,
      • Dozmorov I.
      • Bartke A.
      • Miller R.A.
      Array-based expression analysis of mouse liver genes: effect of age and of the longevity mutant Prop1df.
      ,
      • Welle S.
      • Brooks A.
      • Thornton C.A.
      Senescence-related changes in gene expression in muscle: similarities and differences between mice and men.
      ).

      Signature of IFN-γ-induced Proteins in Aging Epidermis

      Among the proteins up-regulated in the epidermis of the elderly we identified a group of six polypeptides (Mn-SOD, p85β subunit of PI3K, proteasomal proteins PA28-α and SSP 0107, Mx-A, and tryptophanyl-tRNA synthetase) that we have previously shown to be induced by IFN-γ in primary human keratinocytes (Ref.
      • Honore B.
      • Leffers H.
      • Madsen P.
      • Celis J.E.
      Interferon-γ up-regulates a unique set of proteins in human keratinocytes. Molecular cloning and expression of the cDNA encoding the RGD-sequence-containing protein IGUP I-5111.
      ; proteomics.cancer.dk). These proteins are not induced as a group by other cytokines or growth factors (
      • Rasmussen H.H.
      • Celis J.E.
      Evidence for an altered protein kinase C (PKC) signaling pathway in psoriasis.
      ) and represent a specific protein signature for the effect of this cytokine on human keratinocytes (proteomics.cancer.dk). The putative role of these proteins in the aging process is discussed below taking into consideration the whole skin.

      Mn-SOD—

      Mn-SOD is a mitochondrial enzyme that disposes of the superoxide anion (O⨪2) derived from the reduction of molecular oxygen to hydrogen peroxide (H2O2) and thus is essential for maintaining the normal function of this organelle after oxidative stress (
      • Macmillan-Crow L.A.
      • Cruthirds D.L.
      Invited review: manganese superoxide dismutase in disease.
      ,
      • Wallace D.C.
      Animal models for mitochondrial disease.
      ). Homologous deletion of sod2 (−/−) causes neonatal lethality in mice highlighting the deleterious effect that severe oxidative stress has on organisms (
      • Li Y.
      • Huang T.T.
      • Carlson E.J.
      • Melov S.
      • Ursell P.C.
      • Olson J.L.
      • Noble L.J.
      • Yoshimura M.P.
      • Berger C.
      • Chan P.H.
      • Wallace D.C.
      • Epstein C.J.
      Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase.
      ). Decreased levels of Mn-SOD have been associated with increased apoptosis as hepatocytes from old sod2 (+/−) mice show more than twice the number of apoptotic cells as compared with matched-age controls (
      • Kokoszka J.E.
      • Coskun P.
      • Esposito L.A.
      • Wallace D.C.
      Increased mitochondrial oxidative stress in the Sod2 (+/−) mouse results in the age-related decline of mitochondrial function culminating in increased apoptosis.
      ). The augmentation in apoptotic cells may be due to an increased sensitivity of the opening of the mitochondria transition pore, which is a key event in the intrinsic pathway of apoptosis activated by oxidative stress (
      • Halestrap A.P.
      • McStay G.P.
      • Clarke S.J.
      The permeability transition pore complex: another view.
      ). The up-regulation of Mn-SOD in the epidermis from the elderly is most likely triggered by increased ROS (
      • Poswig A.
      • Wenk J.
      • Brenneisen P.
      • Wlaschek M.
      • Hommel C.
      • Quel G.
      • Faisst K.
      • Dissemond J.
      • Briviba K.
      • Krieg T.
      • Scharffetter-Kochanek K.
      Adaptive antioxidant response of manganese-superoxide dismutase following repetitive UV irradiation.
      ,
      • Warner B.B.
      • Stuart L.
      • Gebb S.
      • Wispe J.R.
      Redox regulation of manganese superoxide dismutase.
      ) derived from a combination of intrinsic changes related to longevity as well as environmental factors, primarily exposure to UV irradiation (photoaging) (Fig. 8). We hypothesized that the Mn-SOD levels are sustained by the action of IFN-γ produced by T-cells that undergo activation and selective homing to the skin (Fig. 8). Indeed several groups (
      • Hirokawa K.
      Age-related changes of signal transduction in T cells.
      ,
      • Sakata-Kaneko S.
      • Wakatsuki Y.
      • Matsunaga Y.
      • Usui T.
      • Kita T.
      Altered Th1/Th2 commitment in human CD4+ T cells with ageing.
      ,
      • Bandres E.
      • Merino J.
      • Vazquez B.
      • Inoges S.
      • Moreno C.
      • Subira M.L.
      • Sanchez-Ibarrola A.
      The increase of IFN-γ production through aging correlates with the expanded CD8(+high)CD28(−)CD57(+) subpopulation.
      ,
      • Eylar E.H.
      • Lefranc C.E.
      • Yamamura Y.
      • Baez I.
      • Colon-Martinez S.L.
      • Rodriguez N.
      • Breithaupt T.B.
      HIV infection and aging: enhanced interferon- and tumor necrosis factor-α production by the CD8+ CD28− T subset.
      ,
      • McNerlan S.E.
      • Rea I.M.
      • Alexander H.D.
      A whole blood method for measurement of intracellular TNF-α, IFN-γ and IL-2 expression in stimulated CD3+ lymphocytes: differences between young and elderly subjects.
      ) have reported increased production of IFN-γ in the elderly by subsets of T-cells. In particular, studies of Bandres and colleagues (
      • Bandres E.
      • Merino J.
      • Vazquez B.
      • Inoges S.
      • Moreno C.
      • Subira M.L.
      • Sanchez-Ibarrola A.
      The increase of IFN-γ production through aging correlates with the expanded CD8(+high)CD28(−)CD57(+) subpopulation.
      ) have shown that the increase of IFN-γ through aging in healthy individuals correlates with an expanded CD8 (+high) CD28 (−) CD57 (+) subpopulation of T-cells. The exact role of this T-cell subpopulation is at present unknown, although it is believed that it may play a regulatory role as a Tc1 response in aging individuals.
      Figure thumbnail gr8
      Fig. 8Some age-associated changes in human skin.

      PI3K—

      PI3K is an important regulatory component of pathways that control key cellular functions (Ref.
      • Cantley L.C.
      The phosphoinositide 3-kinase pathway.
      and references therein), and there is a growing body of evidence suggesting that it is involved in the control of cell aging (
      • Weng L.
      • Brown J.
      • Eng C.
      PTEN induces apoptosis and cell cycle arrest through phosphoinositol-3-kinase/Akt-dependent and -independent pathways.
      ). Studies of human skin have shown that the activation of the PI3K/Akt survival pathway by UV radiation is initiated via ROS (Fig. 8) and is sustained by feedback activation of p38 induced by cytokines released in response to UV radiation (
      • Zhang Q.S.
      • Maddock D.A.
      • Chen J.P.
      • Heo S.
      • Chiu C.
      • Lai D.
      • Souza K.
      • Mehta S.
      • Wan Y.S.
      Cytokine-induced p38 activation feedback regulates the prolonged activation of AKT cell survival pathway initiated by reactive oxygen species in response to UV irradiation in human keratinocytes.
      ). PI3K activates Akt/PKB, a serine/threonine kinase that promotes cell survival and inhibits apoptosis by phosphorylating BAD, a proapoptotic member of the BCL-2 family (
      • del Peso L.
      • Gonzalez-Garcia M.
      • Page C.
      • Herrera R.
      • Nunez G.
      Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt.
      ,
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery.
      ). The up-regulation of the p85β subunit of PI3K observed in the epidermis of the elderly is likely triggered by ROS (
      • Zhang Q.S.
      • Maddock D.A.
      • Chen J.P.
      • Heo S.
      • Chiu C.
      • Lai D.
      • Souza K.
      • Mehta S.
      • Wan Y.S.
      Cytokine-induced p38 activation feedback regulates the prolonged activation of AKT cell survival pathway initiated by reactive oxygen species in response to UV irradiation in human keratinocytes.
      ) and sustained by the effect of IFN-γ as may be the case for Mn-SOD (Fig. 8). We were unable to detect the levels of the p110 subunit of PI3K, most likely due to the fact that this protein may not focus in the first dimension.

      PA28-α—

      One of the highlights of age-related changes brought about by ROS is the accumulation of oxidized proteins that can lead to cellular deterioration and loss of function (
      • Lee H.C.
      • Wei Y.H.
      Mitochondrial alterations, cellular response to oxidative stress and defective degradation of proteins in aging.
      ,
      • Squier T.C.
      Oxidative stress and protein aggregation during biological aging.
      ). The proteasome, a multienzymatic proteolytic complex, is the major proteolytic system responsible for the removal of oxidized cytosolic proteins (
      • Grune T.
      Oxidative stress, aging and the proteasomal system.
      ,
      • Mehlhase J.
      • Grune T.
      Proteolytic response to oxidative stress in mammalian cells.
      ). In animal cells there are several distinct molecular forms of proteasomes that contribute to different functions. Exposure of cells to IFN-γ leads to a gradual replacement of the standard proteasome by the immunoproteasome, which is considered more efficient at producing antigenic peptides for presentation to CD8 (+) T-cells (
      • Khan S.
      • van den Broek M.
      • Schwarz K.
      • de Giuli R.
      • Diener P.A.
      • Groettrup M.
      Immunoproteasomes largely replace constitutive proteasomes during an antiviral and antibacterial immune response in the liver.
      ,
      • Rivett A.J.
      • Bose S.
      • Brooks P.
      • Broadfoot K.I.
      Regulation of proteasome complexes by γ-interferon and phosphorylation.
      ). Immunoproteasomes contain LMP2, LMP7, and MECL1 as well as the proteasome activator PA28 (11S REG), which is composed of two homologous subunits termed PA28-α and -β (
      • Griffin T.A.
      • Nandi D.
      • Cruz M.
      • Fehling H.J.
      • Kaer L.V.
      • Monaco J.J.
      • Colbert R.A.
      Immunoproteasome assembly: cooperative incorporation of interferon γ (IFN-γ)-inducible subunits.
      ). Recently it was shown that treatment of COS-7 cells with IFN-γ, which increases immunoproteasomes and PA28 complexes, protected these cells from apoptosis (
      • Brophy V.A.
      • Tavare J.M.
      • Rivett A.J.
      Treatment of COS-7 cells with proteasome inhibitors or γ-interferon reduces the increase in caspase 3 activity associated with staurosporine-induced apoptosis.
      ). The increase in PA28-α and proteasomal protein 0107 observed in the epidermis of the elderly is most likely associated with enhanced oxidative damage to proteins produced by ROS. It should be stressed, however, that not all proteasomal proteins analyzed showed deregulation suggesting that the phenomena may be restricted to only some components in line with recent studies of Bulteau and colleagues (
      • Bulteau A.-L.
      • Petropoulos I.
      • Friguet B.
      Age-related alterations of proteasome structure and function in aging epidermis.
      ).

      Mx-A—

      Mx-A is a member of the dynamin family that is induced by IFN-α, -β, and -γ and that exhibits antiviral activity against pathogenic RNA viruses (
      • Haller O.
      • Frese M.
      • Kochs G.
      MX proteins: mediators of innate resistance to RNA viruses.
      ,
      • Brod S.A.
      • Nelson L.
      • Jin R.
      • Wolinsky J.S.
      Ingested interferon-α induces Mx mRNA.
      ). Elevated levels of Mx-A and IFN-α have been reported in reactive microglia and white matter microglia, respectively, as well as in the brain of Alzheimer's patients (
      • Yamada T.
      • Horisberger M.A.
      • Kawaguchi N.
      • Moroo I.
      • Toyoda T.
      Immunohistochemistry using antibodies to α-interferon and its induced protein, MxA, in Alzheimer’s and Parkinson’s disease brain tissues.
      ) suggesting a role in aging. In addition, it has been shown that Mx-A may suppress multiplication of influenza virus by affecting various cellular pathways, apoptosis included (
      • Mibayashi M.
      • Nakad K.
      • Nagata K.
      Promoted cell death of cells expressing human MxA by influenza virus infection.
      ). Also it has been reported that overexpression of Mx-A in Hep38 cells enhances the sensitivity to mitomycin C and induces apoptosis (
      • Li Y.
      • Youssoufian H.
      MxA overexpression reveals a common genetic link in four Fanconi anemia complementation groups.
      ). The levels of Mx-A increase in response to viral infection and uptake of IFN-α/β (
      • Chieux V.
      • Hober D.
      • Harvey J.
      • Lion G.
      • Lucidarme D.
      • Forzy G.
      • Duhamel M.
      • Cousin J.
      • Ducoulombier H.
      • Wattre P.
      The MxA protein levels in whole blood lysates of patients with various viral infections.
      ,
      • Fernandez M.
      • Quiroga J.A.
      • Martin J.
      • Herrero M.
      • Pardo M.
      • Horisberger M.A.
      • Carreno V.
      In vivo and in vitro induction of MxA protein in peripheral blood mononuclear cells from patients chronically infected with hepatitis C virus.
      ) but returns to normal shortly after treatment. It seems unlikely, therefore, that the high increase in Mx-A observed in aging skin may be the result of viral infections as young individuals are also susceptible to similar infections. However, this possibility cannot be excluded completely since it has been observed that in stable IFN-β-treated multiple sclerosis patients the Mx-A protein levels in blood leukocytes increased as compared with untreated patients (
      • Kracke A.
      • von Wussow P.
      • Al-Masri A.N.
      • Dalley G.
      • Windhagen A.
      • Heidenreich F.
      Mx proteins in blood leukocytes for monitoring interferon β-1b therapy in patients with MS.
      ).

      Tryptophanyl-tRNA Synthetase—

      Tryptophanyl-tRNA synthetase (WRS) is an enzyme that catalyzes the ATP-dependent formation of tryptophanyl-tRNA. The IFN-γ induction of the gene encoding WRS results in the production of two mRNA species differing in size (
      • Wakasugi K.
      • Slike B.M.
      • Hood J.
      • Otani A.
      • Ewalt K.L.
      • Friedlander M.
      • Cheresh D.A.
      • Schimmel P.
      A human aminoacyl-tRNA synthetase as a regulator of angiogenesis.
      ). Under apoptotic conditions secretion of aminoacyl-tRNA synthetase may contribute to this process both by arresting translation and by producing cytokines derived from their cleavage (
      • Wakasugi K.
      • Schimmel P.
      Two distinct cytokines released from a human aminoacyl-tRNA synthetase.
      ). The WRS-encoding gene contains IFN-response regulatory elements (
      • Frolova L.Y.
      • Grigorieva A.Y.
      • Sudomoina M.A.
      • Kisselev L.L.
      The human gene encoding tryptophanyl-tRNA synthetase: interferon-response elements and exon-intron organization.
      ), and it has been recently suggested that the induction of WRS by IFN may play a role in safeguarding tryptophan incorporation for the IFN-enhanced synthesis of immunological molecules (
      • Xue H.
      • Wong J.T.
      Interferon induction of human tryptophanyl-tRNA synthetase safeguards the synthesis of tryptophan-rich immune-system proteins: a hypothesis.
      ). In addition, strong induction of the WRS gene during the delayed-type hypersensitivity reaction suggests its involvement in the immune response in vivo (
      • Yang D.
      • Nakada-Tsukui K.
      • Ohtani M.
      • Goto R.
      • Yoshimura T.
      • Kobayashi Y.
      • Watanabe N.
      Identification and cloning of genes associated with the guinea pig skin delayed-type hypersensitivity reaction.
      ).

      Other Deregulated Proteins

      Among the proteins up-regulated in the epidermis of the elderly that are not induced by IFN-γ, cyclophilin A, eIF-5A, HSP60, and annexin I merit some discussion as these proteins have been associated with a role in apoptosis.

      Cyclophilin A—

      Cyclophilin A possesses a peptidyl-prolyl cis-trans isomerase activity and belongs to a superfamily of immunosuppressant-binding proteins (
      • Ivery M.T.
      Immunophilins: switched on protein binding domains?.
      ,
      • Bukrinsky M.I.
      Cyclophilins: unexpected messengers in intercellular communications.
      ). Cyclophilin distributes in cell compartments where protein folding takes place, and complexes of cyclosporin A with cyclophilin have been shown to inhibit calcineurin, a serine/threonine phosphatase (
      • Ivery M.T.
      A proposed molecular model for the interaction of calcineurin with the cyclosporin A-cyclophilin A complex.
      ) that has been reported to play a role in apoptosis (
      • Nomura T.
      • Yamamoto H.
      • Mimata H.
      • Shitashige M.
      • Shibasaki F.
      • Miyamoto E.
      • Nomura Y.
      Enhancement by cyclosporin A of Taxol-induced apoptosis of human urinary bladder cancer cells.
      ). Recently it was shown that cyclophilin A participates in the activation of the caspase cascade in neuronal cells (
      • Capano M.
      • Virji S.
      • Crompton M.
      Cyclophilin-A is involved in excitotoxin-induced caspase activation in rat neuronal B50 cells.
      ) and has been identified as a secreted growth factor that mediates extracellular signal-regulated kinase (ERK1/2) activation and vascular smooth muscle cell growth by reactive oxygen species (
      • Jin G.
      • Melaragno M.G.
      • Liao D.F.
      • Yan C.
      • Haendeler J.
      • Suh Y.A.
      • Lambeth J.D.
      • Berk B.C.
      Cyclophilin A is a secreted growth factor induced by oxidative stress.
      ). Interestingly secreted cyclophilin B, a member of the cyclophilin family, has been shown to enhance chemotaxis as well as adhesion of memory CD4 (+) T-cells (
      • Allain F.
      • Vanpouille C.
      • Carpentier M.
      • Slomianny M.C.
      • Durieux S.
      • Spik G.
      Interaction with glycosaminoglycans is required for cyclophilin B to trigger integrin-mediated adhesion of peripheral blood T lymphocytes to extracellular matrix.
      ).

      eIF-5A—

      eIF-5A plays a key role in the initiation phase of the translation process, and it has been involved in cell proliferation, tumorigenesis, and apoptosis (
      • Caraglia M.
      • Marra M.
      • Giuberti G.
      • D’Alessandro A.M.
      • Budillon A.
      • del Prete S.
      • Lentini A.
      • Beninati S.
      • Abbruzzese A.
      The role of eukaryotic initiation factor 5A in the control of cell proliferation and apoptosis.
      ). eIF-5A is the only cellular protein that contains the basic amino acid hypusine (Nε-(4-amino-2-hydroxybutyl)lysine) (
      • Chen K.Y.
      • Liu A.Y.
      Biochemistry and function of hypusine formation on eukaryotic initiation factor 5A.
      ). Tome and coauthors (
      • Tome M.E.
      • Fiser S.M.
      • Payne C.M.
      • Gerner E.W.
      Excess putrescine accumulation inhibits the formation of modified eukaryotic initiation factor 5A (eIF-5A) and induces apoptosis.
      ) reported that inhibition of modified eIF-5A formation is one mechanism by which cells may be induced to undergo apoptosis. The non-modified (SSP 8016) and modified (SSP 8010) forms of eIF-5A can be easily distinguished in the 2D gels of young and old epidermis (Figs. 2 and 4), and it is clear that the non-modified form is more prominent in the epidermal cells of the elderly (Table I).

      HSP60—

      Cell exposed to adverse environmental conditions respond by synthesizing stress proteins that are believed to function in maturation oligomerization and/or repair of nascent or damaged proteins in specific cellular compartments (
      • Kregel K.C.
      Heat shock proteins: modifying factors in physiological stress responses and acquired thermotolerance.
      ). HSP60 is a mitochondrial chaperonin (
      • Ranford J.C.
      • Henderson B.
      Chaperonins in disease: mechanisms, models and treatments.
      ) that plays a role in oxidative stress defense (
      • Cabiscol E.
      • Belli G.
      • Tamarit J.
      • Echave P.
      • Herrero E.
      • Ros J.
      Mitochondrial Hsp60, resistance to oxidative stress and the labile iron pool are closely in Saccharomyces cerevisiae.
      ,
      • Mitsumoto A.
      • Takeuchi A.
      • Okawa K.
      • Nakagawa Y.
      A subset of newly synthesized polypeptides in mitochondria from human endothelial cells exposed to hydroperoxide stress.
      ) and that has been shown to interact with Bax and/or Bak to regulate apoptosis in cardiac myocytes (
      • Kirchhoff S.R.
      • Gupta S.
      • Knowlton A.A.
      Cytosolic heat shock protein 60, apoptosis, and myocardial injury.
      ). The striking up-regulation of HSP60 in the epidermis of the elderly must reflect mitochondrial oxidative stress as we did not detect deregulation of other chaperones such as HSP28 and HSP70.

      Annexin I—

      Annexins are a group of structurally related proteins that bind membrane phospholipids in a calcium-dependent fashion (Ref.
      • Gerke V.
      • Moss S.E.
      Annexins: from structure to function.
      and references therein). Annexin I plays a major role in cell proliferation, differentiation, and neutrophil migration (
      • Flower R.J.
      • Rothwell N.J.
      Lipocortin-1: cellular mechanisms and clinical relevance.
      ). Recently Solito and co-workers (
      • Solito E.
      • de Coupade C.
      • Canaider S.
      • Goulding N.J.
      • Perretti M.
      Transfection of annexin I in monocytic cells produces a high degree of spontaneous and stimulated apoptosis associated with caspase-3 activation.
      ) showed that overexpression of annexin I, also termed lipocortin I, in pre-monomyelocytic U937 cells results in cell death by promoting apoptosis. The down-regulation of annexin I in the epidermis of the elderly might be associated with the apoptotic effect of oxidative stress.
      At present we have no clear hint as to the putative role of both PAI-2 and NM23 H2 in the aging process. Expression of PAI-2 has been associated with invasive tumor growth and increased metastatic ability (
      • Andreasen P.A.
      • Egelund R.
      • Petersen H.H.
      The plasminogen activation system in tumor growth, invasion, and metastasis.
      ), while NM23 H2 has been involved in suppression of tumor metastasis (Refs.
      • Hartsough M.T.
      • Steeg P.S.
      Nm23/nucleoside diphosphate kinase in human cancers.
      and
      • Otero A.S.
      NM23/nucleoside diphosphate kinase and signal transduction.
      and references therein). Postel and co-authors (
      • Postel E.H.
      • Berberich S.J.
      • Flint S.J.
      • Ferrone C.A.
      Human c-myc transcription factor PuF identified as nm23-H2 nucleoside diphosphate kinase, a candidate suppressor of tumor metastasis.
      ) have shown that the product of the NM23 H2 gene corresponds to the c-Myc purine binding transcription factor PuF, suggesting that this protein may play a role in transcription activation of c-myc, a proto-oncogene playing pivotal roles in cell cycle progression, apoptosis, and differentiation (Refs.
      • Henriksson M.
      • Selivanova G.
      • Lindstrom M.
      • Wiman K.G.
      Inactivation of Myc-induced p53-dependent apoptosis in human tumors.
      and
      • Nasi S.
      • Ciarapica R.
      • Jucker R.
      • Rosati J.
      • Soucek L.
      Making decisions through Myc.
      and references therein).
      One protein, psoriasin, exhibited a high degree of variation both in young and old epidermis (Table I) most likely reflecting tissue heterogeneity generated by microdifferentiation of the epidermal stem cells. Indeed we have previously shown that this calcium-binding protein is synthesized primarily by stratified squamous epithelia (
      • Madsen P.
      • Rasmussen H.H.
      • Leffers H.
      • Honore B.
      • Dejgaard K.
      • Olsen E.
      • Kii J.
      • Walbum E.
      • Andersen A.H.
      • Basse B.
      • Lauridsen J.B.
      • Ratz G.P.
      • Celis A.
      • Vandekerckhove J.
      • Celis J.E.
      Molecular cloning, occurrence, and expression of a novel partially secreted protein “psoriasin” that is highly up-regulated in psoriatic skin.
      ) and is expressed in a mosaic-like fashion in differentiated squamous metaplasias (
      • Celis J.E.
      • Celis P.
      • Ostergaard M.
      • Basse B.
      • Lauridsen J.B.
      • Ratz G.
      • Rasmussen H.H.
      • Orntoft T.F.
      • Hein B.
      • Wolf H.
      • Celis A.
      Proteomics and immunohistochemistry define some of the steps involved in the squamous differentiation of the bladder transitional epithelium: a novel strategy for identifying metaplastic lesions.
      ).

      Concluding Remarks

      One of the most intriguing observations of the present study is the putative role for IFN-γ in the aging process. This cytokine is known to play a role in preventing infectious diseases and promoting a host response to tumors, but it has not been, to our knowledge, associated with aging of the epidermis (
      • Shtrichman R.
      • Samuel C.E.
      The role of γ interferon in antimicrobial immunity.
      ,
      • Ikeda H.
      • Old L.J.
      • Schreiber R.D.
      The roles of IFN γ in protection against tumor development and cancer immunoediting.
      ). Wei and colleagues (
      • Wei Y.P.
      • Kita M.
      • Shinmura K.
      • Yan X.Q.
      • Fukuyama R.
      • Fushiki S.
      • Imanishi J.
      Expression of IFN-γ in cerebrovascular endothelial cells from aged mice.
      ) have presented evidence indicating that IFN-γ may play a role in age-associated changes that take place in mouse brain since they have found increased expression of IFN-γ mRNA and protein during aging. Using immunofluorescence they traced the source of IFN-γ to the cerebrovascular endothelial cells. Although we have not identified the cellular source of IFN-γ in our study, all available information suggest that it is derived from a subpopulation of T-cells as discussed above.
      As mentioned at the start of the Introduction, our study suffers from some obvious limitations that are in part due to the fact that we chose to analyze the proteome profiles of fresh tissue biopsies obtained from young and old individuals rather that cultured cells derived from them. The size of the punch biopsies was relatively small, and as a result we were compelled to focus on the analysis of the more abundant proteins of the differentiated compartment of the epidermis. Analysis of the proliferative compartment may require immunoaffinity purification of basal cells using specific antibodies and/or laser microdissection techniques (
      • Bonner R.F.
      • Emmert-Buck M.
      • Cole K.
      • Pohida T.
      • Chuaqui R.
      • Goldstein S.
      • Liotta L.A.
      Laser capture microdissection: molecular analysis of tissue.
      ,
      • Bichsel V.E.
      • Petricion III, E.F.
      • Liotta L.A.
      The state of the art microdissection and its downstream applications.
      ) in combination with immunohistochemistry. In both cases, the analysis of less abundant proteins may require the use of fractionation techniques to enrich for particular sets of proteins.
      In conclusion, our studies have provided insight into the major protein changes that take place during epidermal aging in vivo. As a whole they support the contention that aging is associated with increased severe oxidative stress as well as with alterations in apoptosis signaling (Refs.
      • Sohal R.S.
      Role of oxidative stress and protein oxidation in the aging process.
      ,
      • Higami Y.
      • Schimokawa I.
      Apoptosis in the aging process.
      , and
      • Lenaz G.
      • Bovina C.
      • D’Aurelio M.
      • Fato R.
      • Formiggini G.
      • Genova M.L.
      • Giuliano G.
      • Pich M.M.
      • Paolucci U.
      • Castelli G.P.
      • Ventura B.
      Role of mitochondria in oxidative stress and aging.
      and references therein).

      Acknowledgments

      We thank G. Ratz and P. Celis for expert technical assistance. We also thank Professor B. F. C Clark and Professor W. Bohr for critical reading of the manuscript and valuable comments and Dr. B. Thomsen for kind help with the statistical evaluation of the data.

      REFERENCES

        • Jenkins G.
        Molecular mechanisms of skin ageing.
        Mech. Ageing Dev. 2002; 123: 801-810
        • Wlaschek M.
        • Tantcheva-Poor I.
        • Naderi L.
        • Ma W.
        • Schneider L.A.
        • Razi-Wolf Z.
        • Schuller J.
        • Scharffetter-Kochanek K.
        Solar UV irradiation and dermal photoaging.
        J. Photochem. Photobiol. 2001; 63: 41-51
        • Glogau R.G.
        Physiologic and structural changes associated with aging skin.
        Dermatol. Clin. 1997; 15: 555-559
        • Gilchrest B.A.
        A review of skin aging and its medical therapy.
        Br. J. Dermatol. 1996; 135: 867-875
        • Pascuali-Ronchetti I.
        • Baccarani-Contri M.
        Elastic fibers during development and aging.
        Microsc. Res. Tech. 1997; 38: 428-435
        • Sohal R.S.
        Role of oxidative stress and protein oxidation in the aging process.
        Free Radic. Biol. Med. 2002; 33: 37-44
        • Higami Y.
        • Schimokawa I.
        Apoptosis in the aging process.
        Cell Tissue Res. 2000; 301: 125-132
        • Curtin J.F.
        • Donovan M.
        • Cotter T.G.
        Regulation and measurement of oxidative stress in apoptosis.
        J. Immunol. Methods. 2002; 265: 49-72
        • Chandra J.
        • Samali A.
        • Orrenius S.
        Triggering and modulation of apoptosis by oxidative stress.
        Free Radic. Biol. Med. 2000; 29: 323-333
        • Clutton S.
        The importance of oxidative stress in apoptosis.
        Br. Med. Bull. 1997; 53: 662-668
        • Lenaz G.
        • Bovina C.
        • D’Aurelio M.
        • Fato R.
        • Formiggini G.
        • Genova M.L.
        • Giuliano G.
        • Pich M.M.
        • Paolucci U.
        • Castelli G.P.
        • Ventura B.
        Role of mitochondria in oxidative stress and aging.
        Ann. N. Y. Acad. Sci. 2002; 959: 199-213
        • Martindale J.L.
        • Holbrook N.J.
        Cellular response to oxidative stress: signaling for suicide and survival.
        J. Cell. Physiol. 2002; 192: 1-15
        • Celis J.E.
        • Bravo R.
        Synthesis of the nuclear protein cyclin in growing, senescent and morphologically transformed human skin fibroblasts.
        FEBS Lett. 1984; 165: 21-25
        • Toda T.
        • Satoh M.
        • Sugimoto M.
        • Goto M.
        • Furuichi Y.
        • Kimura N.
        A comparative analysis of the proteins between the fibroblasts from Werner’s syndrome patients and age-matched normal individuals using two-dimensional gel electrophoresis.
        Mech. Ageing Dev. 1998; 100: 133-143
        • Toda T.
        • Kaji K.
        • Kimura N.
        TMIG-2DPAGE: a new concept of two-dimensional gel protein database for research on aging.
        Electrophoresis. 1998; 19: 344-348
        • Dierick J.F.
        • Pascal T.
        • Chainiaux F.
        • Eliaers F.
        • Remacle J.
        • Larsen P.M.
        • Roepstorff P.
        • Toussaint O.
        Transcriptome and proteome analysis in human senescent fibroblasts and fibroblasts undergoing premature senescence induced by repeated sublethal stresses.
        Ann. N. Y. Acad. Sci. 2000; 908: 302-305
        • Kondo T.
        • Sakaguchi M.
        • Namba M.
        Two-dimensional gel electrophoretic studies on the cellular aging: accumulation of α-2-macroglobulin in human fibroblasts with aging.
        Exp. Gerontol. 2001; 36: 487-495
        • Benvenuti S.
        • Cramer R.
        • Quinn C.C.
        • Bruce J.
        • Zvelebil M.
        • Corless S.
        • Bond J.
        • Yang A.
        • Hockfield S.
        • Burlingame A.L.
        • Waterfield M.D.
        • Jat P.S.
        Differential proteome analysis or replicative senescence in rat embryo fibroblasts.
        Mol. Cell Proteomics. 2002; 1: 280-292
        • Lee C.-K.
        • Klopp R.G.
        • Weindruch R.
        • Prolla T.A.
        Gene expression profile of aging and its retardation by caloric restriction.
        Science. 1999; 285: 1390-1393
        • Welle S.
        • Bhatt K.
        • Thornton C.A.
        High-abundance mRNAs in human muscle: comparison between young and old.
        J. Appl. Physiol. 2000; 89: 297-304
        • Weindruch R.
        • Kayo T.
        • Lee C.K.
        • Prolla T.A.
        Gene expression profiling of aging using DNA microarrays.
        Mech. Ageing Dev. 2002; 123: 177-193
        • Lee C.K.
        • Weindruch R.
        • Prolla T.A.
        Gene-expression profile of the ageing brain in mice.
        Nat. Genet. 2000; 25: 294-297
        • Jiang C.H.
        • Tsien J.Z.
        • Schultz P.G.
        • Hu Y.
        The effects of aging on gene expression in the hypothalamus and cortex of mice.
        Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1930-1934
        • Han E.
        • Hilsenbeck S.G.
        • Richardson A.
        • Nelson J.F.
        cDNA expression arrays reveal incomplete reversal of age-related changes in gene expression by caloric restriction.
        Mech. Ageing Dev. 2000; 115: 157-174
        • Dozmorov I.
        • Bartke A.
        • Miller R.A.
        Array-based expression analysis of mouse liver genes: effect of age and of the longevity mutant Prop1df.
        J. Gerontol. A Biol. Sci. Med. Sci. 2001; 56: B72-B80
        • Welle S.
        • Brooks A.
        • Thornton C.A.
        Senescence-related changes in gene expression in muscle: similarities and differences between mice and men.
        Physiol. Genomics. 2001; 5: 67-73
        • Celis J.E.
        • Madsen P.
        • Rasmussen H.H.
        • Leffers H.
        • Honore B.
        • Gesser B.
        • Dejgaard K.
        • Olsen E.
        • Magnusson N.
        • Kiil J.
        • Celis A.
        • Lauridsen J.B.
        • Basse B.
        • Ratz G.P.
        • Andersen A.H.
        • Walbum E.
        • Brandstrup B.
        • Pedersen P.S.
        • Brandt N.J.
        • Puype M.
        • Van Damme J.
        • Vandekerckhove J.
        A comprehensive two-dimensional gel protein database of noncultured unfractionated normal human epidermal keratinocytes: towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.
        Electrophoresis. 1991; 12: 802-872
        • Celis J.E.
        • Rasmussen H.H.
        • Gromov P.
        • Olsen E.
        • Madsen P.
        • Leffers H.
        • Honore B.
        • Dejgaard K.
        • Vorum H.
        • Kristensen B.
        • Østergaard M.
        • Hauns⊘ A.
        • Jensen N.A.
        • Celis A.
        • Basse B.
        • Lauridsen J.B.
        • Ratz G.P.
        • Andersen A.H.
        • Walbum E.
        • Kjærgaard I.
        • Andersen I.
        • Puype M.
        • Van Damme J.
        • Vandekerckhove J.
        The human keratinocyte two-dimensional gel protein database (update 1995): mapping components of signal transduction pathways.
        Electrophoresis. 1995; 16: 2177-2240
        • Celis J.E.
        • Østergaard M.
        • Jensen N.A.
        • Gromova I.I.
        • Rasmussen H.H.
        • Gromov P.
        Human and mouse proteomic databases: a novel resources in the proteome universe.
        FEBS Lett. 1998; 430: 64-72
        • O’Farrell P.Z.
        • Goodman H.M.
        • O’Farrell P.H.
        High resolution two-dimensional electrophoresis of basic as well as acidic proteins.
        Cell. 1977; 12: 1133-1141
        • Celis J.E.
        • Ratz G.
        • Basse B.
        • Lauridsen J.B.
        • Celis A.
        • Gromov P.
        Celis J.E. Carter N. Hunter T. Shotton D. Simons K. Small J.V. Cell Biology. A Laboratory Handbook. 4. Academic Press, New York1998: 375-385
        • Madsen P.
        • Rasmussen H.H.
        • Leffers H.
        • Honore B.
        • Celis J.E.
        Molecular cloning and expression of a novel keratinocyte protein (psoriasis-associated fatty acid-binding protein [PA-FABP]) that is highly up-regulated in psoriatic skin and that shares similarity to fatty acid-binding proteins.
        J. Investig. Dermatol. 1992; 99: 299-305
        • Celis J.E.
        • Rasmussen H.H.
        • Olsen E.
        • Madsen P.
        • Leffers H.
        • Honore B.
        • Dejgaard K.
        • Gromov P.
        • Vorum H.
        • Vassilev A.
        • Baskin Y.
        • Liu X.
        • Celis A.
        • Basse B.
        • Lauridsen J.B.
        • Ratz G.P.
        • Andersen A.H.
        • Walbum E.
        • Kjærgaard I.
        • Andersen I.
        • Puype M.
        • Van Damme J.
        • Vandekerckhove J.
        The human keratinocyte two-dimensional gel protein database (update 1994): towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.
        Electrophoresis. 1994; 15: 1349-1458
        • Rasmussen H.H.
        • van Damme J.
        • Puype M.
        • Gesser B.
        • Celis J.E.
        • Vandekerckhove J.
        Microsequences of 145 proteins recorded in the two-dimensional gel protein database of normal human epidermal keratinocytes.
        Electrophoresis. 1992; 13: 960-969
        • Rasmussen H.H.
        • Mortz E.
        • Mann M.
        • Roepstorff P.
        • Celis J.E.
        Identification of transformation sensitive proteins recorded in human two-dimensional gel protein databases by mass spectrometric peptide mapping alone and in combination with microsequencing.
        Electrophoresis. 1994; 15: 406-416
        • Celis J.E.
        • Gromov P.
        High-resolution two-dimensional gel electrophoresis and protein identification using western blotting and ECL detection.
        EXS (Basel). 2000; 88: 55-67
        • Jensen O.N.
        • Wilm M.
        • Shevchenko A.
        • Mann M.
        Sample preparation methods for mass spectrometric peptide mapping directly from 2-DE gels.
        Methods Mol. Biol. 1999; 112: 513-530
        • Celis J.E.
        • Kruhoffer M.
        • Gromova I.
        • Frederiksen C.
        • Ostergaard M.
        • Thykjaer T.
        • Gromov P.
        • Yu J.
        • Palsdottir H.
        • Magnusson N.
        • Orntoft T.F.
        Gene expression profiling: monitoring transcription and translation products using DNA microarrays and proteomics.
        FEBS Lett. 2000; 480: 2-16
        • Celis J.E.
        • Celis P.
        • Ostergaard M.
        • Basse B.
        • Lauridsen J.B.
        • Ratz G.
        • Rasmussen H.H.
        • Orntoft T.F.
        • Hein B.
        • Wolf H.
        • Celis A.
        Proteomics and immunohistochemistry define some of the steps involved in the squamous differentiation of the bladder transitional epithelium: a novel strategy for identifying metaplastic lesions.
        Cancer Res. 1999; 59: 3003-3009
        • Palsdottir H.
        Preparation and Characterization of Monoclonal Antibodies against Urines from Bladder Cancer Patients with Invasive Disease. Institute for Molecular and Structural Biology, Aarhus Universitet, Aarhus, Denmark2000 (M.Sc. thesis)
        • Celis J.E.
        • Celis P.
        • Palsdottir H.
        • Østergaard M.
        • Gromov P.
        • Primdahl H.
        • Orntoft T.F.
        • Wolf H.
        • Celis A.
        • Gromova I.
        Proteomic strategies to reveal tumor heterogeneity among urothelial papillomas.
        Mol. Cell. Proteomics. 2002; 1: 269-279
        • Honore B.
        • Leffers H.
        • Madsen P.
        • Celis J.E.
        Interferon-γ up-regulates a unique set of proteins in human keratinocytes. Molecular cloning and expression of the cDNA encoding the RGD-sequence-containing protein IGUP I-5111.
        Eur. J. Biochem. 1993; 218: 421-430
        • Rasmussen H.H.
        • Celis J.E.
        Evidence for an altered protein kinase C (PKC) signaling pathway in psoriasis.
        J. Investig. Dermatol. 1993; 101: 560-566
        • Macmillan-Crow L.A.
        • Cruthirds D.L.
        Invited review: manganese superoxide dismutase in disease.
        Free Radic. Res. 2001; 34: 325-336
        • Wallace D.C.
        Animal models for mitochondrial disease.
        Methods Mol. Biol. 2002; 197: 3-54
        • Li Y.
        • Huang T.T.
        • Carlson E.J.
        • Melov S.
        • Ursell P.C.
        • Olson J.L.
        • Noble L.J.
        • Yoshimura M.P.
        • Berger C.
        • Chan P.H.
        • Wallace D.C.
        • Epstein C.J.
        Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase.
        Nat. Genet. 1995; 11: 376-381
        • Kokoszka J.E.
        • Coskun P.
        • Esposito L.A.
        • Wallace D.C.
        Increased mitochondrial oxidative stress in the Sod2 (+/−) mouse results in the age-related decline of mitochondrial function culminating in increased apoptosis.
        Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2278-2283
        • Halestrap A.P.
        • McStay G.P.
        • Clarke S.J.
        The permeability transition pore complex: another view.
        Biochimie (Paris). 2002; 84: 153-166
        • Poswig A.
        • Wenk J.
        • Brenneisen P.
        • Wlaschek M.
        • Hommel C.
        • Quel G.
        • Faisst K.
        • Dissemond J.
        • Briviba K.
        • Krieg T.
        • Scharffetter-Kochanek K.
        Adaptive antioxidant response of manganese-superoxide dismutase following repetitive UV irradiation.
        J. Investig. Dermatol. 1999; 112: 13-18
        • Warner B.B.
        • Stuart L.
        • Gebb S.
        • Wispe J.R.
        Redox regulation of manganese superoxide dismutase.
        Am. J. Physiol. 1996; 271: L150-L158
        • Hirokawa K.
        Age-related changes of signal transduction in T cells.
        Exp. Gerontol. 1999; 34: 7-18
        • Sakata-Kaneko S.
        • Wakatsuki Y.
        • Matsunaga Y.
        • Usui T.
        • Kita T.
        Altered Th1/Th2 commitment in human CD4+ T cells with ageing.
        Clin. Exp. Immunol. 2000; 120: 267-273
        • Bandres E.
        • Merino J.
        • Vazquez B.
        • Inoges S.
        • Moreno C.
        • Subira M.L.
        • Sanchez-Ibarrola A.
        The increase of IFN-γ production through aging correlates with the expanded CD8(+high)CD28(−)CD57(+) subpopulation.
        Clin. Immunol. 2000; 96: 230-235
        • Eylar E.H.
        • Lefranc C.E.
        • Yamamura Y.
        • Baez I.
        • Colon-Martinez S.L.
        • Rodriguez N.
        • Breithaupt T.B.
        HIV infection and aging: enhanced interferon- and tumor necrosis factor-α production by the CD8+ CD28− T subset.
        BMC Immunol. 2001; 2: 10-21
        • McNerlan S.E.
        • Rea I.M.
        • Alexander H.D.
        A whole blood method for measurement of intracellular TNF-α, IFN-γ and IL-2 expression in stimulated CD3+ lymphocytes: differences between young and elderly subjects.
        Exp. Gerontol. 2002; 37: 227-234
        • Cantley L.C.
        The phosphoinositide 3-kinase pathway.
        Science. 2002; 296: 1655-1657
        • Weng L.
        • Brown J.
        • Eng C.
        PTEN induces apoptosis and cell cycle arrest through phosphoinositol-3-kinase/Akt-dependent and -independent pathways.
        Hum. Mol. Genet. 2001; 10: 237-242
        • Zhang Q.S.
        • Maddock D.A.
        • Chen J.P.
        • Heo S.
        • Chiu C.
        • Lai D.
        • Souza K.
        • Mehta S.
        • Wan Y.S.
        Cytokine-induced p38 activation feedback regulates the prolonged activation of AKT cell survival pathway initiated by reactive oxygen species in response to UV irradiation in human keratinocytes.
        Int. J. Oncol. 2001; 19: 1057-1061
        • del Peso L.
        • Gonzalez-Garcia M.
        • Page C.
        • Herrera R.
        • Nunez G.
        Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt.
        Science. 1997; 278: 687-689
        • Datta S.R.
        • Dudek H.
        • Tao X.
        • Masters S.
        • Fu H.
        • Gotoh Y.
        • Greenberg M.E.
        Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery.
        Cell. 1997; 91: 231-241
        • Lee H.C.
        • Wei Y.H.
        Mitochondrial alterations, cellular response to oxidative stress and defective degradation of proteins in aging.
        Biogerontology. 2001; 2: 231-244
        • Squier T.C.
        Oxidative stress and protein aggregation during biological aging.
        Exp. Gerontol. 2001; 36: 1539-1550
        • Grune T.
        Oxidative stress, aging and the proteasomal system.
        Biogerontology. 2000; 1: 31-40
        • Mehlhase J.
        • Grune T.
        Proteolytic response to oxidative stress in mammalian cells.
        Biol. Chem. 2002; 383: 559-567
        • Khan S.
        • van den Broek M.
        • Schwarz K.
        • de Giuli R.
        • Diener P.A.
        • Groettrup M.
        Immunoproteasomes largely replace constitutive proteasomes during an antiviral and antibacterial immune response in the liver.
        J. Immunol. 2001; 167: 6859-6868
        • Rivett A.J.
        • Bose S.
        • Brooks P.
        • Broadfoot K.I.
        Regulation of proteasome complexes by γ-interferon and phosphorylation.
        Biochimie (Paris). 2001; 83: 363-366
        • Griffin T.A.
        • Nandi D.
        • Cruz M.
        • Fehling H.J.
        • Kaer L.V.
        • Monaco J.J.
        • Colbert R.A.
        Immunoproteasome assembly: cooperative incorporation of interferon γ (IFN-γ)-inducible subunits.
        J. Exp. Med. 1998; 187: 97-104
        • Brophy V.A.
        • Tavare J.M.
        • Rivett A.J.
        Treatment of COS-7 cells with proteasome inhibitors or γ-interferon reduces the increase in caspase 3 activity associated with staurosporine-induced apoptosis.
        Arch. Biochem. Biophys. 2002; 397: 199-205
        • Bulteau A.-L.
        • Petropoulos I.
        • Friguet B.
        Age-related alterations of proteasome structure and function in aging epidermis.
        Exp. Gerontol. 2000; 35: 767-777
        • Haller O.
        • Frese M.
        • Kochs G.
        MX proteins: mediators of innate resistance to RNA viruses.
        Rev. Sci. Tech. 1998; 17: 220-230
        • Brod S.A.
        • Nelson L.
        • Jin R.
        • Wolinsky J.S.
        Ingested interferon-α induces Mx mRNA.
        Cytokine. 1999; 11: 492-499
        • Yamada T.
        • Horisberger M.A.
        • Kawaguchi N.
        • Moroo I.
        • Toyoda T.
        Immunohistochemistry using antibodies to α-interferon and its induced protein, MxA, in Alzheimer’s and Parkinson’s disease brain tissues.
        Neurosci. Lett. 1994; 181: 61-64
        • Mibayashi M.
        • Nakad K.
        • Nagata K.
        Promoted cell death of cells expressing human MxA by influenza virus infection.
        Microbiol. Immunol. 2002; 46: 29-36
        • Li Y.
        • Youssoufian H.
        MxA overexpression reveals a common genetic link in four Fanconi anemia complementation groups.
        J. Clin. Investig. 1997; 100: 2873-2880
        • Chieux V.
        • Hober D.
        • Harvey J.
        • Lion G.
        • Lucidarme D.
        • Forzy G.
        • Duhamel M.
        • Cousin J.
        • Ducoulombier H.
        • Wattre P.
        The MxA protein levels in whole blood lysates of patients with various viral infections.
        J. Virol. Methods. 1998; 70: 183-191
        • Fernandez M.
        • Quiroga J.A.
        • Martin J.
        • Herrero M.
        • Pardo M.
        • Horisberger M.A.
        • Carreno V.
        In vivo and in vitro induction of MxA protein in peripheral blood mononuclear cells from patients chronically infected with hepatitis C virus.
        J. Infect. Dis. 1999; 180: 262-267
        • Kracke A.
        • von Wussow P.
        • Al-Masri A.N.
        • Dalley G.
        • Windhagen A.
        • Heidenreich F.
        Mx proteins in blood leukocytes for monitoring interferon β-1b therapy in patients with MS.
        Neurology. 2000; 54: 193-199
        • Wakasugi K.
        • Slike B.M.
        • Hood J.
        • Otani A.
        • Ewalt K.L.
        • Friedlander M.
        • Cheresh D.A.
        • Schimmel P.
        A human aminoacyl-tRNA synthetase as a regulator of angiogenesis.
        Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 173-177
        • Wakasugi K.
        • Schimmel P.
        Two distinct cytokines released from a human aminoacyl-tRNA synthetase.
        Science. 1999; 284: 147-151
        • Frolova L.Y.
        • Grigorieva A.Y.
        • Sudomoina M.A.
        • Kisselev L.L.
        The human gene encoding tryptophanyl-tRNA synthetase: interferon-response elements and exon-intron organization.
        Gene (Amst.). 1993; 128: 237-245
        • Xue H.
        • Wong J.T.
        Interferon induction of human tryptophanyl-tRNA synthetase safeguards the synthesis of tryptophan-rich immune-system proteins: a hypothesis.
        Gene (Amst.). 1995; 165: 335-339
        • Yang D.
        • Nakada-Tsukui K.
        • Ohtani M.
        • Goto R.
        • Yoshimura T.
        • Kobayashi Y.
        • Watanabe N.
        Identification and cloning of genes associated with the guinea pig skin delayed-type hypersensitivity reaction.
        J. Biochem. 2001; 129: 561-568
        • Ivery M.T.
        Immunophilins: switched on protein binding domains?.
        Med. Res. Rev. 2000; 20: 452-458
        • Bukrinsky M.I.
        Cyclophilins: unexpected messengers in intercellular communications.
        Trends Immunol. 2002; 23: 323-325
        • Ivery M.T.
        A proposed molecular model for the interaction of calcineurin with the cyclosporin A-cyclophilin A complex.
        Bioorg. Med. Chem. 1999; 7: 1389-1402
        • Nomura T.
        • Yamamoto H.
        • Mimata H.
        • Shitashige M.
        • Shibasaki F.
        • Miyamoto E.
        • Nomura Y.
        Enhancement by cyclosporin A of Taxol-induced apoptosis of human urinary bladder cancer cells.
        Urol. Res. 2002; 30: 102-111
        • Capano M.
        • Virji S.
        • Crompton M.
        Cyclophilin-A is involved in excitotoxin-induced caspase activation in rat neuronal B50 cells.
        Biochem. J. 2002; 363: 29-36
        • Jin G.
        • Melaragno M.G.
        • Liao D.F.
        • Yan C.
        • Haendeler J.
        • Suh Y.A.
        • Lambeth J.D.
        • Berk B.C.
        Cyclophilin A is a secreted growth factor induced by oxidative stress.
        Circ. Res. 2000; 87: 789-796
        • Allain F.
        • Vanpouille C.
        • Carpentier M.
        • Slomianny M.C.
        • Durieux S.
        • Spik G.
        Interaction with glycosaminoglycans is required for cyclophilin B to trigger integrin-mediated adhesion of peripheral blood T lymphocytes to extracellular matrix.
        Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2714-2719
        • Caraglia M.
        • Marra M.
        • Giuberti G.
        • D’Alessandro A.M.
        • Budillon A.
        • del Prete S.
        • Lentini A.
        • Beninati S.
        • Abbruzzese A.
        The role of eukaryotic initiation factor 5A in the control of cell proliferation and apoptosis.
        Amino Acids (Vienna). 2001; 20: 91-104
        • Chen K.Y.
        • Liu A.Y.
        Biochemistry and function of hypusine formation on eukaryotic initiation factor 5A.
        Biol. Signals. 1997; 6: 105-109
        • Tome M.E.
        • Fiser S.M.
        • Payne C.M.
        • Gerner E.W.
        Excess putrescine accumulation inhibits the formation of modified eukaryotic initiation factor 5A (eIF-5A) and induces apoptosis.
        Biochem. J. 1997; 328: 847-854
        • Kregel K.C.
        Heat shock proteins: modifying factors in physiological stress responses and acquired thermotolerance.
        J. Appl. Physiol. 2002; 92: 2177-2186
        • Ranford J.C.
        • Henderson B.
        Chaperonins in disease: mechanisms, models and treatments.
        Mol. Pathol. 2002; 55: 209-213
        • Cabiscol E.
        • Belli G.
        • Tamarit J.
        • Echave P.
        • Herrero E.
        • Ros J.
        Mitochondrial Hsp60, resistance to oxidative stress and the labile iron pool are closely in Saccharomyces cerevisiae.
        J. Biol. Chem. 2002; 277: 44531-44538
        • Mitsumoto A.
        • Takeuchi A.
        • Okawa K.
        • Nakagawa Y.
        A subset of newly synthesized polypeptides in mitochondria from human endothelial cells exposed to hydroperoxide stress.
        Free Radic. Biol. Med. 2002; 32: 22-37
        • Kirchhoff S.R.
        • Gupta S.
        • Knowlton A.A.
        Cytosolic heat shock protein 60, apoptosis, and myocardial injury.
        Circulation. 2002; 105: 2899-2904
        • Gerke V.
        • Moss S.E.
        Annexins: from structure to function.
        Physiol. Rev. 2002; 82: 331-371
        • Flower R.J.
        • Rothwell N.J.
        Lipocortin-1: cellular mechanisms and clinical relevance.
        Trends Pharmacol. Sci. 1994; 15: 71-76
        • Solito E.
        • de Coupade C.
        • Canaider S.
        • Goulding N.J.
        • Perretti M.
        Transfection of annexin I in monocytic cells produces a high degree of spontaneous and stimulated apoptosis associated with caspase-3 activation.
        Br. J. Pharmacol. 2001; 133: 217-228
        • Andreasen P.A.
        • Egelund R.
        • Petersen H.H.
        The plasminogen activation system in tumor growth, invasion, and metastasis.
        Cell. Mol. Life Sci. 2000; 57: 25-40
        • Hartsough M.T.
        • Steeg P.S.
        Nm23/nucleoside diphosphate kinase in human cancers.
        J. Bioenerg. Biomembr. 2000; 32: 301-308
        • Otero A.S.
        NM23/nucleoside diphosphate kinase and signal transduction.
        J. Bioenerg. Biomembr. 2000; 32: 269-275
        • Postel E.H.
        • Berberich S.J.
        • Flint S.J.
        • Ferrone C.A.
        Human c-myc transcription factor PuF identified as nm23-H2 nucleoside diphosphate kinase, a candidate suppressor of tumor metastasis.
        Science. 1993; 261: 478-480
        • Henriksson M.
        • Selivanova G.
        • Lindstrom M.
        • Wiman K.G.
        Inactivation of Myc-induced p53-dependent apoptosis in human tumors.
        Apoptosis. 2001; 6: 133-137
        • Nasi S.
        • Ciarapica R.
        • Jucker R.
        • Rosati J.
        • Soucek L.
        Making decisions through Myc.
        FEBS Lett. 2001; 490: 153-162
        • Madsen P.
        • Rasmussen H.H.
        • Leffers H.
        • Honore B.
        • Dejgaard K.
        • Olsen E.
        • Kii J.
        • Walbum E.
        • Andersen A.H.
        • Basse B.
        • Lauridsen J.B.
        • Ratz G.P.
        • Celis A.
        • Vandekerckhove J.
        • Celis J.E.
        Molecular cloning, occurrence, and expression of a novel partially secreted protein “psoriasin” that is highly up-regulated in psoriatic skin.
        J. Investig. Dermatol. 1991; 97: 701-712
        • Shtrichman R.
        • Samuel C.E.
        The role of γ interferon in antimicrobial immunity.
        Curr. Opin. Microbiol. 2001; 4: 251-259
        • Ikeda H.
        • Old L.J.
        • Schreiber R.D.
        The roles of IFN γ in protection against tumor development and cancer immunoediting.
        Cytokine Growth Factor Rev. 2002; 13: 95-109
        • Wei Y.P.
        • Kita M.
        • Shinmura K.
        • Yan X.Q.
        • Fukuyama R.
        • Fushiki S.
        • Imanishi J.
        Expression of IFN-γ in cerebrovascular endothelial cells from aged mice.
        J. Interferon Cytokine Res. 2000; 20: 403-409
        • Bonner R.F.
        • Emmert-Buck M.
        • Cole K.
        • Pohida T.
        • Chuaqui R.
        • Goldstein S.
        • Liotta L.A.
        Laser capture microdissection: molecular analysis of tissue.
        Science. 1997; 278: 1481-1483
        • Bichsel V.E.
        • Petricion III, E.F.
        • Liotta L.A.
        The state of the art microdissection and its downstream applications.
        J. Mol. Med. 2000; 78: B20