            gerona      J Gerontol A Biol Sci Med Scigerona      The Journals of Gerontology Series A: Biological Sciences and Medical Sciences      J Gerontol A Biol Sci Med Sci      1079-5006      1758-535X              Oxford University Press                    9902310.1093/gerona/55.5.B220                        Journal of Gerontology: Biological Sciences                            Increase of Oxidatively Modified Protein Is Associated With a Decrease of Proteasome Activity and Content in Aging Epidermal Cells                                          Petropoulos            Isabelle                                a                                                Conconi            Mariangela                                a                                                Wang            Xin                                b                                                Hoenel            Batya                                b                                                Brégégère            François                                a                                b                                                Milner            Yoram                                b                                                Friguet            Bertrand                                a                          aLaboratoire de Biologie et Biochimie Cellulaire du Vieillissement, Université Denis Diderot, Paris, France        bDepartment of Biological Chemistry, The Hebrew University, Jerusalem, Israel                    Bertrand Friguet, Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement, Universit\|[eacute]\| Denis Diderot-Paris 7, CC 7128, 2 place Jussieu, 75251 Paris Cedex 05, France E-mail: bfriguet@paris7.jussieu.fr.                    1        5        2000            55      5      B220      B227                        20          9          1999                          1          3          1999                            The Gerontological Society of America        2000                    For the process of aging in epidermal cells to be characterized, the status of oxidized and damaged protein accumulation and removal by the proteasome has been investigated. Modified protein content and proteasome activity were assayed in lysates of epidermal cells from donors of different ages. Increased levels of oxidized proteins, glycated proteins, and proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal were observed in cells from old donors. At the same time, a decline of chymotrypsin-like and peptidylglutamyl-peptide hydrolase activities of the proteasome was found in aging keratinocytes. This age-related decline of the proteasome peptidase activities can be explained, at least in part, by a decreased proteasome content as observed by immunoblotting and enzyme-linked immunosorbent assay. In keratinocyte cultures, a decrease of proteasome activity and content was observed upon serial passaging. In cultures, as well as in skin, an inverse relationship was found between the aging marker β-galactosidase and the proteasome content. These results suggest that proteasome is downregulated during replicative senescence as well as in aged cells in vivo, possibly resulting in the accumulation of modified proteins.                              hwp-legacy-fpage          B220                          hwp-legacy-dochead          RESEARCH ARTICLE                            Decision Editor: Jay Roberts, PhD            KERATINOCYTES, which are the main cell type in the epidermis, not only constitute the physicochemical barrier that protects the body from environmental assaults (e.g., by pathogens and toxins) but also have more complex immune functions, such as the expression of class II antigens and interaction with T lymphocytes during inflammation (1). The epidermis is also very sensitive to age-related alterations leading to various major pathologies, including hyperproliferative and autoimmune diseases (2)(3)(4). Numerous theories have been proposed to explain the mechanisms of cellular aging. Among them, the free-radical hypothesis suggests that oxygen free radicals are causal factors in aging, inflicting molecular damages, some of which are not repaired and accumulate with age (5). Indeed, it has been shown that aging cells do accumulate damaged DNA, lipids, and proteins, leading to cellular functional alterations that are strongly believed to contribute to the aging process (6)(7).    Among biological macromolecules, proteins constitute a particular target for the reactive oxygen species attacks that generate protein carbonyl derivatives. There is an age-related increase in the carbonyl content of protein in the human brain (8), gerbil brain (9), rat hepatocyte (10), human red blood cells (11), and in the whole body of flies (12). Moreover, the carbonyl content of protein in cultured human fibroblasts increases exponentially as a function of donor age (11). There are other protein modifications, such as glycation, that cause alterations to cellular and organ function (13). Aging is also characterized by a decline of the specific activity and thermostability of enzymes, which is due to oxidative damage as well as to other modifications (11)(13)(14)(15). The accumulation of modified proteins reflects a dysregulation of the steady-state level of damaged proteins in aged tissues, which can be explained by a higher modification rate, or a lower degradation rate of these proteins, or both. The half-lives of proteins in senescent animals have been shown to be significantly extended as compared with those in younger ones (16).    It is now well established that the main proteolytic system, responsible for the intracellular degradation of oxidized or misfolded proteins, is the multicatalytic proteinase (MCP), also called 20 S proteasome (17)(18)(19). The 20 S proteasome is a high-molecular-mass (700 kDa) proteolytic complex, made of 28 subunits arranged as four stacked rings (see ref. (20) for a review). The two outer rings are formed by α subunits and the two inner rings are formed by the β subunits that carry the proteolytic activities (21)(22). Recently, we and other investigators have shown that the peptidylglutamyl-peptide hydrolase (PGPH) activity of rat liver proteasome exhibited the most remarkable reduction with age (23)(24)(25)(26). These results strongly suggest a potential participation of the MCP in the slower turnover of proteins, thus contributing to the age-related accumulation of altered proteins.    In this study, we present evidence for an impaired proteasome function in human keratinocytes upon aging. We have found a decrease of proteasome peptidase activities in epidermis extracts from old donors as compared with young ones and, as expected, an age-related increase of damaged proteins. We also found a similar decrease in senescent keratinocyte cultures. This can be explained, at least in part, by the decrease of proteasome content found in old cells.          Materials and Methods              Reagents and Chemicals        Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (LLVY-AMC), N-Cbz-Leu-Leu-Glu-β-naphthylamine (LLE-NA), 7-aminomethylcoumarin, and β-naphthylamine were purchased from Sigma-Aldrich (L'Isle d'Abeau, France). N-Cbz-Leu-Leu-Leucinal (MG 132) was kindly supplied by Dr. F. Arenzana-Seisdedos. The rabbit anti-glycation products polyclonal antibodies (anti-GP) (27), a gift from Dr. H. Bakala, were raised against advanced glycated endproducts conjugated RNase. Rabbit immune sera raised against pure rat MCP and against HNE (4-hydroxynonenal)-modified keyhole limpet hemocyanin (anti-HNE) were obtained from Assay Research (Silver Spring, MD) as previously described (23)(28).                    Epidermis and Keratinocyte Extracts Preparations        Epidermal cells were prepared from skin biopsies or plastic surgery procedures (mammary gland reductions) from healthy donors from 17 to 67 years of age. Skin samples were incubated 2 hours at 4°C in 2 M of NaCl and 20 mM of ethylenediamine tetra-acetic acid (EDTA). Then, the epidermis was separated from the dermis and homogenized with a Dounce homogenizer (Prolabo, Fontenay, France). Primary cultures of keratinocytes were derived from newborn foreskins and cultivated in Dulbecco's modified eagle medium nutrient mixture F-12 ham (1:1) at 37°C and 5% CO2 by using a 3T3 fibroblast feeder layer, as previously described (29). Cells were usually passaged up to six times. Cells were collected with a rubber policeman and homogenized in an extraction buffer (20 mM of Tris, pH 7.4, 250 mM of sucrose, 1 mM of EDTA, and 5 mM of 2-mercaptoethanol) with a Dounce homogenizer. Cell debris and organelles were removed from the crude extracts by centrifugation for 1 hour at 10,000 × g at 4°C.                    Protein and Peptidase Assays        Protein concentrations were determined by a bicinchoninic acid assay (Pierce). Chymotrypsin-like and PGPH activities of the MCP in crude extracts were assayed with fluorogenic peptides LLVY-AMC and LLE-NA, respectively, as previously described (30). Typically, assay mixtures contained 75-μg proteins of epidermal extract, or 50-μg proteins of keratinocyte extract, in extraction buffer with the appropriate peptide substrate (10 μM of LLVY-AMC or 200 μM of LLE-NA) in a final volume of 200 μl. The mixtures were incubated 30 minutes at 37°C and analyzed by spectrofluorometry for release of aminomethylcoumarin (AMC) with excitation and emission wavelengths at 350 nm and 440 nm, respectively, or for release of β-naphthylamine (NA) with excitation and emission wavelengths at 333 nm and 410 nm, respectively. MCP proteolytic activity was determined as the difference between the total activity of crude extracts and the remaining activity in the presence of 20-μM proteasome inhibitor MG 132 (31).                    Gel Electrophoresis and Western Blots        Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) was done by using the Laemmli method (32) on a 12% acrylamide (wt/vol) separating gel. Immunoblot experiments using anti-MCP, anti-GP, or anti-HNE antibodies were performed after SDS/PAGE separation of 20 μg of total cellular proteins from keratinocyte cultures or epidermis followed by electrotransfer onto Hybond nitrocellulose membrane (Amersham-Pharmacia-Biotech, Les Ulis, France). Immunoblot detection of carbonyl groups was performed with the OxyBlot oxidized protein detection kit (Appligene-Oncor, Strasbourg, France), according to the manufacturer. Briefly, 10 μg of proteins were incubated for 15 minutes at room temperature with 2,4-dinitrophenylhydrazine (DNPH) to form the carbonyl derivative dinitrophenylhydrazone before SDS/PAGE separation. After transfer onto nitrocellulose, modified proteins were revealed by anti-DNP antibodies. The detection of bands in immunoblots was carried out with the ECL (Enhanced ChemiLuminescence) Western blotting analysis system of Amersham (Les Ulis, France), using peroxidase conjugated anti-rabbit, secondary antibodies. The densitometric analyses of the autoradiographies were performed by using Lecphor software on a Biocom system (Biocom, Les Ulis, France).                    Enzyme-linked Immunosorbent Assays and β-Galactosidase Assay        A competitive enzyme-linked immunosorbent assay (ELISA) was performed to measure the amount of modified glycated products in skin epidermis upon aging. Microplates were coated with 1 μg/ml of modified glycated bovine serum albumin (BSA) diluted in a buffer carbonate (20 mM, pH 9.6) for 14 hours at 4°C. After they were washed with 0.05% Tween in phosphate-buffered saline (PBS) and blocked with 6% milk (wt/vol) in PBS, epidermis extracts from various donors were added and incubated with polyclonal anti-GP antibodies (27) for 2 hours at room temperature. After washing, peroxidase-conjugated secondary antibodies were added and incubated for 2 hours at room temperature. After washing, the substrate 2, 2′Azino-bis-3-ethylbenz-thiazoline-6-sulfonic acid was added and the absorbance of the reaction product was measured at 405 nm in a SLT Rainbow microplate reader (SLT Labinstruments, Salzburg, Austria). A calibration curve was obtained by using glycated BSA. For the proteasome amount to be determined, cell suspensions were obtained by trypsinization of skin epidermis from donors of different ages, or cultured keratinocytes after different passages. For ELISA tests, 5 × 105 suspended cells were fixed in 3% paraformaldehyde in Eppendorf tubes for 15 minutes at room temperature, then incubated for 15 minutes with 0.1 M of glycine (pH 7.0), and permeabilized by 1% Triton X-100 for 30 minutes at room temperature. Suspended cells were pretreated for 10 minutes with 3% H2O2 and blocked with 5% BSA in PBS for 2 hours at 37°C. They were incubated with anti-MCP antibodies, properly diluted in PBS containing 3% BSA and 0.05% NaN3, overnight at 4°C, and then washed four times with 0.05% Tween in PBS for 10 minutes each. Peroxidase-conjugated secondary antibodies were diluted in PBS, 3% BSA, 0.05% NaN3, and added to the cells for 2 hours at room temperature. Finally, the cells were washed and incubated with 0.4 mg/ml of tetramethyl benzidine and 1.3 mM of H2O2 in 0.1 M of citric acid Na2HPO4 buffer (pH 5.5). The reaction was stopped in 0.33 M of H3PO4 and the absorbance was measured at 450 nm in a BIO-TEK (BIO-TEK Instruments, Winooski, VT) microplate reader (33)(34). For β-galactosidase activity to be measured, 5 × 105 suspended cells were fixed with 3% paraformaldehyde in PBS for 5 minutes, washed, and incubated in 0.5 ml of the following substrate solution: 0.4 mg/ml of p-nitrophenyl-β-galactopyranoside, 2 mM of MgCl2, 150 mM of NaCl, and 0.1% NaN3 in 50 mM of Tris buffer (pH 8.0) supplemented with a mixture of protease inhibitors at 37°C. The reaction product (p-nitrophenol) was measured spectrophotometrically at 405 nm.                    Statistical Analysis        Differences for proteasome and β-galactosidase activities and proteasome antigen data were evaluated by using analysis of variance (ANOVA) procedures with the StatView software (Abacus Concepts, Berkeley, CA). A p value of <.05 was considered significant.                    Results              Increase of Modified Proteins in Aging Epidermis        Protein oxidation has been implicated in different biological processes, both physiological, such as aging or apoptosis, and pathological, such as cancer and neurodegenerative diseases (see ref. 6 for a review). In order to investigate the status of oxidatively modified proteins in keratinocytes during aging, protein carbonyl content was monitored by using the OxyBlot detection kit as described in the Materials and Methods section. Proteins of epidermis extracts from young donors (17, 20, and 26 years old), middle-aged donors (39 and 42 years old), and old donors (50, 60, and 67 years old) were treated with DNPH to derivatize their carbonyl groups into 2,4-dinitrophenylhydrazone. Western blots revealed oxidized polypeptides by using anti-DNP antibodies. As shown in Fig. 1, an age-related increase of carbonyl content was observed in some proteins. In samples from old cells in particular, high-molecular-weight polypeptides appeared on the top of the gel (molecular weights above 100 kDa), possibly resulting from cross-linking reactions. As a way to assess whether other modifications occur and to determine the origin of carbonyl groups in these proteins, Western blots were performed by using anti-HNE and anti-GP polyclonal antibodies. A general increase in modified protein adducts was visible with age, and anti-HNE antibodies recognized modified groups in high-molecular-weight proteins (Fig. 1 and Fig. 1). Because the patterns of glycated proteins did not show qualitative changes, a competitive ELISA was performed to measure their amounts and confirmed their increase with age (Fig. 1). Taken together, these results show that glycation and modification by the lipid peroxidation product HNE are implicated in the age-related accumulation of damaged proteins.                    Age-Related Decrease of Proteasome Activity and Content in Epidermis        There is considerable evidence that the 20 S proteasome is responsible for the degradation of oxidatively modified intracellular proteins (35)(36) and that an age-related accumulation of these proteins may be due to a decline of proteasome peptidase activities (23)(24)(25)(26). For this hypothesis to be examined in epidermal cells, the chymotrypsin-like and PGPH activities of proteasome were monitored in epidermis extracts of various donors of from 17 to 67 years of age. As shown in Fig. 2, both activities decreased gradually with the age of donors.        As a way to determine whether the age-related decline in the proteasome peptidase activities was due to decreased proteasome content, the amounts of proteasome were estimated by Western blot analysis of epidermis extracts from donors of different ages, using anti-rat MCP. Several proteasome subunits from human keratinocytes were recognized by these antibodies. A representative experiment is shown in Fig. 2, where the proteasome content was indeed reduced by ∼10% and 60% in the epidermis from 50- and 67-year-old donors, respectively, when compared with the sample from the 17-year-old donor. ELISA measurements of MCP antigens performed on epidermal cell suspensions from various donors (see Fig. 5 below) showed a similar age-related decrease.                    Increase of Protein Damage and Decrease of Proteasome in Keratinocytes Upon Serial Passaging        Normal diploid cells undergo numerous physiological and biochemical changes during serial passaging in vitro. These changes may reflect aging events that occur in organisms (37)(38). We used keratinocytes in culture to examine the possible modification of proteasome activity and content in relation to protein damage upon aging. To investigate the status of oxidatively modified proteins in keratinocytes during serial passages, we monitored protein carbonyl content by using the OxyBlot detection kit as described in the Materials and Methods section. Cellular extracts were prepared at different passages and treated with DNPH; the derived products were revealed by an anti-DNP antiserum after SDS PAGE separation and Western blotting. As shown in Fig. 3, an increase of high-molecular-weight modified proteins was observed in passages 3 and 4 as compared with passages 1 and 2. Western blots were also performed by using an anti-HNE polyclonal antibody. This antibody also reacted with more proteins of high molecular weight in later passages than in earlier ones (Fig. 3). The accumulation of such modified high-molecular-weight polypeptides presumably results from protein cross-linking events.        Proteasome activity was measured by monitoring the chymotrypsin-like and the PGPH activities in cellular extracts of keratinocytes. The results, summarized in Fig. 4, show a drop in chymotrypsin-trypsin-like activity in passages 4 and 5, compared with passages 1 and 2. A similar profile is observed for the PGPH activity. As a way to determine if the decline of activity can be correlated to a decreased proteasome content in different keratinocytes extracts, a Western blot analysis was performed. Fig. 4 clearly shows a decrease of proteasome level during keratinocyte serial passages. In the passages 3 and 4, the residual level was approximately 30% and 20% respectively, of that observed in the first passage.                    Decrease of Proteasome and Increase of β-Galactosidase in Aging Epidermis and Keratinocyte Cultures        Dimri and colleagues described β-galactosidase as a senescence marker in human fibroblasts and keratinocytes (39). The intracellular β-galactosidase activity was measured in suspensions of keratinocytes prepared from skin epidermis or from cell cultures of different ages, as described in the Materials and Methods section. The amount of proteasome was measured in similar suspensions obtained from the same samples, by ELISA tests using anti-rat MCP antibodies (see the Materials and Methods section). As shown in Fig. 5, β-galactosidase activity increased with serial passages of keratinocytes, while proteasome antigen, detected by ELISA, decreased. An age-related increase of β-galactosidase activity and a decrease of proteasome antigen were also observed in skin epidermal cells of old donors (Fig. 5).                    Discussion      The accumulation of damaged or oxidized protein reflects not only the rate of protein modification but also the rate of damaged or oxidized protein degradation that is dependent, at least in part, on the activity of intracellular proteases that preferentially degrade damaged proteins. The proteasome is the major protease that degrades oxidatively modified cytosolic proteins (35)(36), and it has been reported that an age-related accumulation of damaged or oxidized proteins may be explained by an impaired activity of the proteasome (23)(24)(25)(26). In this work, we have studied the fate of the proteasome in the cellular aging of human epidermis, a tissue very sensitive to aging alterations.      We first showed that there is an age-related increase in the protein carbonyl content in the epidermis and in cultured keratinocytes, the main cellular component of the epidermis. This result corroborates previous reports that have established such a correlation in the mammalian brain (8)(9), in rat hepatocytes (10), and in human fibroblasts (11). As demonstrated in this study, in epidermis, as well as in serial passages of keratinocytes, protein carbonyl groups that can be generated by direct oxidation of amino acids are also produced by protein glycation and reactions with peroxidation products of polyunsaturated fatty acids such as HNE (see Fig. 1 and Fig. 3). Interestingly, these modifications seem to be highly selective, because only a few proteins are the target of oxidative damage. This finding is in agreement with previous observations in aging flies, which showed that two mitochondrial proteins, aconitase and adenine nucleotide translocase, are selectively oxidized during aging (40)(41). Furthermore, among these modified proteins that accumulate with age, some are high-molecular-weight proteins. Such high-molecular-weight species might result from cross-linking reactions that occur upon production of bityrosine by the hydroxyl radical (42), of intermolecular lysyl-HNE-lysyl linkages (43)(44), or of Maillard reaction adducts (45).      We then addressed the question of whether the accumulation of damaged proteins reflects an alteration in the activity of the proteolytic machinery in charge of damaged or oxidized protein degradation, that is, the 20 S proteasome. Modifications of proteasome activities and content have been extensively studied in rat liver during aging by us and by other groups (23)(24)(25)(26). Shibatani and colleagues (24) reported a significant decrease of the SDS-treated proteasome PGPH activity in cytosolic extracts upon aging. Using purified proteasome preparations, we demonstrated that the PGPH activity declined in old animals to ∼50% compared with young animals (23)(25). More recently, Hayashi and Goto (26) also reported an age-related decline of the PGPH activity and of the chymotrypsin-like activity, although to a lesser extent. In addition, Wagner and Margolis showed an age-related decrease in proteasome activity in the bovine lens (46). We assayed both the chymotrypsin-like and the PGPH activities of proteasome in extracts of epidermal cells, and we found that they were notably reduced in aged cells. Using the specific proteasome inhibitor MG 132, we determined that proteasome accounts for ∼90% of both activities (data not shown). Therefore, this decrease represents a net reduction of proteasome activity.      To determine whether the decline of proteasome activity may be due to a decrease in proteasome content upon aging, we performed both ELISA and Western blots by using rabbit polyclonal antibodies raised against rat proteasome. These antibodies recognize the human proteasome and reveal two distinct major bands. The intensities of the two major bands decreased with age (Fig. 2). Similarly, Fig. 4 shows that, in keratinocyte cultures, more than one proteasome subunit decreased with age. However, we cannot yet conclude whether there is a general decline of proteasome content, or a differential reduction in some subunits. It would be of interest to identify among proteasome subunits those that show an age-related decrease. A depletion of proteasome subunits with age has not been detected in rat liver (25)(26), leading us to propose that the activity decline resulted from posttranslational modifications of proteasome subunits. To our knowledge, this is the first time that an age-related decline of proteasome subunit content has been observed. However, it has been shown that the β-subunits X (also called MB 1 or epsilon) and Y (also called delta) are downregulated under pathologic conditions such as infection (47) and after γ-interferon treatment (48). It is interesting to note that these subunits have been involved in chymotrypsin-like and PGPH activities, respectively (49).      Furthermore, decreased proteasome content is likely to explain the observed decline in activity but does not rule out the possibility that some subunits are also posttranslationally modified. Moreover, the proteasome activity was tested in crude extracts, and it is possible that the age-related alteration of proteasome activity could also be due to a loss of specific activators, the presence of endogenous inhibitors, or both. In fact, it has been pointed out that HNE cross-linked proteins may act as proteasome inhibitors (43)(50). Thus, further work is needed to ascertain why the proteasome is declining in activity with age.      A decrease of proteasome activity and content was also observed in serial passages of keratinocytes. Cells were tested at various passages for β-galactosidase activity, which was used as a marker of replicative senescence in human fibroblasts, keratinocytes (39), and more recently in endothelial and smooth muscle cells from humans and rabbits (51). The inverse relationship between β-galactosidase activity and proteasome content strongly suggests that proteasome is downregulated in replicative senescence. Interestingly, Bosman and colleagues, in pioneering studies in replicative senescence of WI-38 fibroblasts, have shown a correlated increase of the β-galactosidase activity and a loss of a “critical neutral protease” that might be the proteasome (52).      The accumulation of oxidized and modified proteins and the correlative decrease in proteasome activity and content during epidermial aging take place in the context of complex cellular events leading to skin alterations. The fact that a similar status of protein modification and of proteasome decline was found in senescent keratinocytes in culture and in aged epidermal cells in vivo establishes cell culture as a valuable tool for research on proteasomes in keratinocyte aging. Further investigations will be necessary to elucidate the actual role of proteasomes in the aging process and may lead to an original approach of aging control in skin.                                                  Figure 1.                                 Modification of cytosolic proteins upon epidermal cell aging. A: The OxyBlot was performed with 10 μg of cytosolic protein extracts from epidermal cells from young, middle-aged, and old donors. Extracts were first treated with 2,4-DNPH, then subjected to SDS/PAGE on a 12% polyacrylamide gel, and electrotransferred onto a nitrocellulose membrane. The blots were processed with anti-DNP antibodies as described by the manufacturer. For a densitometric analysis, the measurements corresponding to the 17-year-old donor were taken as 100%. The intensity of the major band was increased to ∼300% in the oldest sample, and the high-molecular-weight bands appeared only in epidermal extracts from middle-aged donors and to a larger extent in those from old-aged donors. B and C: 20 μg of cytosolic protein extracts were subjected to SDS/PAGE on a 12% polyacrylamide gel and electrotransferred onto a nitrocellulose membrane. The blots were processed with anti-HNE (B) or anti-GP (C) polyclonal antibodies at a dilution of 1:2000 or 1:1000, respectively. For a densitometric analysis, the measurements corresponding to the 17-year-old donor were taken as 100%. The intensities of the major bands, reacting with anti-HNE antibodies and ranging from 50 kDa to 80 kDa in molecular weights, were increased to 800% for the 67-year-old donor, and a band of higher molecular weight was only present in the three old samples. The band with a molecular weight ∼80 kDa reacting with anti-GP was increased to 130% for the 50-year-old donor and 160% for the 67-year-old donor, respectively. D: A competitive ELISA was performed as described in the Materials and Methods section to determine the amount of glycated adducts in epidermal extracts. The assay was calibrated by using modified glycated BSA. The increase of glycated adduct is significant (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(F\ =\ 12.073\) \end{document}, p < .05) between the young (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(n\ =\ 3\) \end{document}) and the old (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(n\ =\ 3\) \end{document}) donors.                                                          Figure 2.                                 Effect of age on proteasome peptidase activities and content in epidermal extracts. A: The chymotrypsin-like (▴) and PGPH (○) activities of the proteasome were assayed in epidermal extracts by using LLVY-AMC and LLE-NA as fluorogenic peptide substrates, respectively. The enzymatic assays were performed as described in the Materials and Methods section. For the chymotrypsin-like activity, a significant difference (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(F\ =\ 9.534\) \end{document}, p < .05) was found between the young donors, from 17 to 23 years old (n = 4) and the old donors, from 50 to 67 years old (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(n\ =\ 5\) \end{document}). A similar trend to an age-related decline was observed for the PGPH activity. B: 20 μg of soluble proteins from epidermal extracts from 17, 50, and 67-year-old donors were subjected to SDS/PAGE on a 12% polyacrylamide gel, electrotransferred onto a nitrocellulose membrane, and immunoblotted with rabbit anti-MCP polyclonal antibodies at a dilution of 1:5000, as described in the Materials and Methods section.                                                          Figure 3.                                 Modification of cytosolic proteins in keratinocytes upon serial passages. A: The OxyBlot was achieved with 10 μg of cytosolic protein extracts from passage 1 to passage 4 keratinocyte cultures. Protein extracts were first incubated with 2,4-DNPH followed by SDS/PAGE, electrotransferred, and Western blotted with anti-DNP antibodies as described by the manufacturer. For a densitometric analysis, the measurement corresponding to the first passage was taken as 100%. The intensity of the high-molecular-weight band reacting with anti-DNP antibodies was increased to 180% for passage 4. B: 20 μg of protein cytosolic extracts from passage 1 to passage 4 keratinocyte cultures were subjected to SDS/PAGE, electrotransferred, and Western blotted as described in the Materials and Methods section. The blots were processed with anti-HNE polyclonal antibodies at a dilution of 1:2000. For a densitometric analysis, the measurement corresponding to the first passage was taken as 100%. The intensity of the high-molecular-weight band reacting with anti-HNE antibodies was increased to 620% for passage 4. The experiments, reported in each panel, were done twice with cells obtained from two different cultures.                                                          Figure 4.                                 Effect of serial passaging on the proteasome chymotrypsin-like activity and content in keratinocytes in culture. A: The chymotrypsin-like (♦) and PGPH (○) activities of the proteasome were assayed in cytosolic extracts by using 10 μM of LLVY-AMC and 200 μM of LLE-NA as the peptide substrate with 75 μg of total protein as described in the Materials and Methods section. A significant difference in the chymotrypsin-like activity (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(F\ =\ 18.622\) \end{document}, p < .05) was found between early passages 1 and 2 (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(n\ =\ 3\) \end{document}) and late ones 4 and 5 (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(n\ =\ 3\) \end{document}). A similar trend to a decline of activity in serial passages is observed for the PGPH activity. B: 20 μg of cytosolic protein extracts from keratinocytes at passages 1 to 4 were subjected to SDS/PAGE on a 12% polyacrylamide gel, electrotransferred onto a nitrocellulose membrane, and immunoblotted with rabbit anti-MCP polyclonal antibodies at a dilution of 1:5000, as described in the Materials and Methods section. The experiments, reported in each panel, were done twice with cells obtained from two different cultures.                                                          Figure 5.                                 Proteasome antigen vs β-galactosidase activity in serial keratinocyte cultures and epidermis donors. Cell suspensions were obtained from the epidermis of various age donors or from keratinocytes in serial culture passages. For each sample from skin A or cell culture B, ELISA tests were performed for MCP, and β-galactosidase was assayed as described in the Materials and Methods section. The two series of result values were plotted on a single graph. In A, the symbols used are ♦, young donors; ○, middle-aged donors; and ▴, old donors. In B, the symbols used are ♦, early passages 1 and 2; ○, intermediate passage 3; and ▴, late passages 4 and 5. The decrease of proteasome antigen as well as the increase of β-galactosidase activity are significant between the young donors from 17 to 23 years old (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(n\ =\ 4\) \end{document}) and the old donors from 50 to 67 years old (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(n\ =\ 6\) \end{document}), p < .001 A, and in passages 4 and 5 (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(n\ =\ 5\) \end{document}) as compared with passages 1 and 2 (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(n\ =\ 6\) \end{document}), p < .001 B.                                                    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