Protein Profiling of the Human Epidermis from the Elderly Reveals Up-regulation of a Signature of Interferon-
-induced Polypeptides That Includes Manganese-superoxide Dismutase and the p85ß Subunit of Phosphatidylinositol 3-Kinase*
Pavel Gromov
,
,¶,
Gunhild Lange Skovgaard||,
Hildur Palsdottir
,**,
Irina Gromova
,
,
Morten Østergaard
and
Julio E. Celis
,
,
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
|| 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
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ABSTRACT
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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 (2130 and 7592 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 p85ß subunit of phosphatidylinositol 3-kinase, and proteasomal proteins PA28-
and SSP 0107) that is induced by interferon-
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-
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. 1 and 2 and references therein). UV radiation generates reactive oxygen species (ROS),1 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. 25 and references therein).
Currently there is mounting evidence indicating that the aging process of cells and organs is associated with increased oxidative stress (6) as well as alterations in apoptosis (Ref. 7 and references therein), a homeostatic mechanism that is exacerbated by increased production of ROS (810). 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. 6, 7, 11, and 12 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 (1318). 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 (1921) and mouse brain (22, 23) and liver (24, 25). 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 (26). 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 (26).
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. 2729; 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-
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. 6 and 7 and references therein).
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EXPERIMENTAL PROCEDURES
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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 Dulbeccos modified Eagles 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 (30) and kept at -20 °C until use. Whole protein lysates were then subjected to both IEF and NEPHGE 2D PAGE as described previously (31). 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 (32). Cells were labeled for 14 h in Dulbeccos modified Eagles medium lacking methionine and containing 1% dialyzed fetal calf serum, 500 µCi/ml [35S]methionine, and 50 units/ml recombinant cytokines: IFN-
, IFN-
, or TNF-
. After labeling, the medium was aspirated, and the cells were resuspended in lysis solution for 2D PAGE (see above and Ref. 30). 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 (33). 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. 2729; proteomics.cancer.dk). One or a combination of procedures that included Edman degradation (34), mass spectrometry (35), and 2D PAGE Western immunoblotting (36) 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 (35, 37, 38). 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 (36).
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 (39). 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.
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RESULTS
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To monitor the structural disorganization and fragmentation of the elastic fiber meshwork, which is a hallmark of skin aging (1, 2), we performed immunohistochemistry of skin biopsies using a monoclonal antibody prepared in our laboratory (monoclonal antibody b9) that specifically decorates these fibers (40). 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)).

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FIG. 1. Immunofluorescence 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 (x200). 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.
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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 (41). 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) (2729). 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.

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FIG. 2. 2D 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 Table I. Protein levels were normalized with respect to the level of actin (SSP 6310) and annexin II (SSP 0210, IEF; and SSP 4205, NEPHGE).
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TABLE I Levels of [35S]methionine-labeled proteins in normal human skin biopsies obtained from young (2130-year-old) and old (7592-year-old) individuals
hnRNP, heterogeneous nuclear riboprotein; GDI, guanine nucleotide dissociation inhibitor; PA-FABP, psoriasis-associated fatty acid-binding protein; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; GST, glutathione S-transferase; SCC A1, squamous cell carcinoma antigen; CCT, chaperonin containing TCP-1 complex; NKCEF-A, natural killer cell enhancing factor A.
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FIG. 3. Master 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.
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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 (
= 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 p85ß subunit of phosphatidylinositol 3-kinase (PI3K,
2.1), the proteasome regulator PA28-
(
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 (p85ß subunit of PI3K, tryptophanyl-tRNA synthetase, and Mx-A; Fig. 5) and mass spectrometry (PA28-
, Mn-SOD, and eIF-5A; Fig. 6).

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FIG. 4. Age-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, p85ß 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.
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FIG. 5. Enhanced chemiluminescence 2D PAGE Western blotting analysis of proteins from normal human skin epidermis. A, p85ß 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.
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FIG. 6. Identification of PA28- , 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. BD, 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.
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Careful inspection of the data recorded in the keratinocyte 2D PAGE database (2729) revealed that six of the deregulated proteins, namely Mx-A, Mn-SOD, tryptophanyl-tRNA synthetase, the p85ß subunit of PI3K, and the proteasomal proteins PA28-
and SSP 0107, correspond to a protein signature that is induced by treatment of cultured human keratinocytes with IFN-
(42). To corroborate this observation we treated normal primary human keratinocytes with IFN-
as described under "Experimental Procedures," and the results are presented in Fig. 7. Clearly all six proteins were up-regulated in the IFN-
-treated cells (Fig. 7B) confirming previous studies. Induction of these proteins as a group represents a specific signature for IFN-
as deregulation of the polypeptide set has not been observed in keratinocytes treated with other cytokines (IFN-
(Fig. 7C) and -ß; interleukins 1
, 1ß, 2, 3, 6, 7, and 8; and TNF-
(Fig. 7D)) or growth factors (transforming growth factor-ß and fibroblast growth factor) (proteomics.cancer.dk) (43). IFN-
(Fig. 7C) and -ß up-regulated Mx-A, while interleukins 1
and -ß and TNF-
(Fig. 7D) up-regulated Mn-SOD.
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DISCUSSION
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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 (18) 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 (1926).
Signature of IFN-
-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, p85ß subunit of PI3K, proteasomal proteins PA28-
and SSP 0107, Mx-A, and tryptophanyl-tRNA synthetase) that we have previously shown to be induced by IFN-
in primary human keratinocytes (Ref. 42; proteomics.cancer.dk). These proteins are not induced as a group by other cytokines or growth factors (43) 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 (44, 45). Homologous deletion of sod2 (-/-) causes neonatal lethality in mice highlighting the deleterious effect that severe oxidative stress has on organisms (46). 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 (47). 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 (48). The up-regulation of Mn-SOD in the epidermis from the elderly is most likely triggered by increased ROS (49, 50) 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-
produced by T-cells that undergo activation and selective homing to the skin (Fig. 8). Indeed several groups (5155) have reported increased production of IFN-
in the elderly by subsets of T-cells. In particular, studies of Bandres and colleagues (53) have shown that the increase of IFN-
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.
PI3K
PI3K is an important regulatory component of pathways that control key cellular functions (Ref. 56 and references therein), and there is a growing body of evidence suggesting that it is involved in the control of cell aging (57). 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 (58). 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 (59, 60). The up-regulation of the p85ß subunit of PI3K observed in the epidermis of the elderly is likely triggered by ROS (58) and sustained by the effect of IFN-
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-
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 (61, 62). The proteasome, a multienzymatic proteolytic complex, is the major proteolytic system responsible for the removal of oxidized cytosolic proteins (63, 64). In animal cells there are several distinct molecular forms of proteasomes that contribute to different functions. Exposure of cells to IFN-
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 (65, 66). Immunoproteasomes contain LMP2, LMP7, and MECL1 as well as the proteasome activator PA28 (11S REG), which is composed of two homologous subunits termed PA28-
and -ß (67). Recently it was shown that treatment of COS-7 cells with IFN-
, which increases immunoproteasomes and PA28 complexes, protected these cells from apoptosis (68). The increase in PA28-
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 (69).
Mx-A
Mx-A is a member of the dynamin family that is induced by IFN-
, -ß, and -
and that exhibits antiviral activity against pathogenic RNA viruses (70, 71). Elevated levels of Mx-A and IFN-
have been reported in reactive microglia and white matter microglia, respectively, as well as in the brain of Alzheimers patients (72) 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 (73). Also it has been reported that overexpression of Mx-A in Hep38 cells enhances the sensitivity to mitomycin C and induces apoptosis (74). The levels of Mx-A increase in response to viral infection and uptake of IFN-
/ß (75, 76) 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-ß-treated multiple sclerosis patients the Mx-A protein levels in blood leukocytes increased as compared with untreated patients (77).
Tryptophanyl-tRNA Synthetase
Tryptophanyl-tRNA synthetase (WRS) is an enzyme that catalyzes the ATP-dependent formation of tryptophanyl-tRNA. The IFN-
induction of the gene encoding WRS results in the production of two mRNA species differing in size (78). 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 (79). The WRS-encoding gene contains IFN-response regulatory elements (80), 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 (81). In addition, strong induction of the WRS gene during the delayed-type hypersensitivity reaction suggests its involvement in the immune response in vivo (82).
Other Deregulated Proteins
Among the proteins up-regulated in the epidermis of the elderly that are not induced by IFN-
, 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 (83, 84). 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 (85) that has been reported to play a role in apoptosis (86). Recently it was shown that cyclophilin A participates in the activation of the caspase cascade in neuronal cells (87) 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 (88). 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 (89).
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 (90). eIF-5A is the only cellular protein that contains the basic amino acid hypusine (N
-(4-amino-2-hydroxybutyl)lysine) (91). Tome and coauthors (92) 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 (93). HSP60 is a mitochondrial chaperonin (94) that plays a role in oxidative stress defense (95, 96) and that has been shown to interact with Bax and/or Bak to regulate apoptosis in cardiac myocytes (97). 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. 98 and references therein). Annexin I plays a major role in cell proliferation, differentiation, and neutrophil migration (99). Recently Solito and co-workers (100) 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 (101), while NM23 H2 has been involved in suppression of tumor metastasis (Refs. 102 and 103 and references therein). Postel and co-authors (104) 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. 105 and 106 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 (107) and is expressed in a mosaic-like fashion in differentiated squamous metaplasias (39).
Concluding Remarks
One of the most intriguing observations of the present study is the putative role for IFN-
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 (108, 109). Wei and colleagues (110) have presented evidence indicating that IFN-
may play a role in age-associated changes that take place in mouse brain since they have found increased expression of IFN-
mRNA and protein during aging. Using immunofluorescence they traced the source of IFN-
to the cerebrovascular endothelial cells. Although we have not identified the cellular source of IFN-
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 (111, 112) 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. 6, 7, and 11 and references therein).
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ACKNOWLEDGMENTS
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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.
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FOOTNOTES
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Received, September 3, 2002Published, January 16, 2003
Published, MCP Papers in Press, January 17, 2003, DOI 10.1074/mcp.M200051-MCP200
1 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. 
* This study was supported by grants from the Danish Centre for Molecular Gerontology and the Danish Cancer Society. 
** Present address: Max-Planck Institute of Biophysics, Heinrich-Hoffmann Strasse 7, 60528 Frankfurt am Main, Germany. 
¶ 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; E-mail: psg{at}cancer.dk

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; E-mail: jec{at}cancer.dk
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