Age-Associated Decrease in Proteasome Content and Activities in Human Dermal Fibroblasts: Restoration of Normal Level of Proteasome Subunits Reduces Aging Markers in Fibroblasts From Elderly Persons

Abstract

We measured proteasome activities and the levels of proteasome subunits in dermal fibroblasts from individuals aged 20–82 years. Proteasome activities changed with age in a biphasic manner, decreasing significantly up to 50 years of age and showing no significant change between 50 and 78 years of age. Similarly, proteasome activities in replicatively senescent dermal fibroblasts showed a passage-dependent biphasic change. We confirmed that the decreases in proteasome activities were accompanied by the accumulation of oxidized and ubiquitinated proteins. The decline in proteasome activities in aging fibroblasts was associated with a decrease in the expression of proteasome subunits. We found that the restoration of the normal level of proteasome catalytic subunits, using a lentivirus gene-delivery system, decreased the severity of the aging markers in dermal fibroblasts from elderly donors. These findings suggest that proteasome malfunction may contribute to the aging process in human skin and that the maintenance of normal proteasome activities could delay skin aging.

THE proteasome is the multicatalytic proteinase complex that plays a central role in protein degradation (1). In particular, the proteasome is known to be responsible for the removal of abnormal and oxidatively damaged proteins as well as normal proteins degraded as part of basic cellular processes (2). The 20S proteolytic core of the proteasome is a 700 kd complex composed of four stacked rings of seven subunits each. The two outside rings are composed of the α subunits, and the two inner rings are composed of the β subunits (PSMB6, PSMB7, and PSMB5) that have catalytic activities, such as peptidylglutamyl–peptide hydrolase (PGPH), trypsin-like (T-L), and chymotrypsin-like (CT-L) activities, respectively. In mammalian cells, the 20S core complex can bind to the 19S regulatory complex or 11S (PSME) activator to form the 26S proteasome or the 11S-20S proteasome complex, respectively. The 26S proteasome is an essential component of adenosine triphosphate (ATP)– and ubiquitin-dependent protein degradation (3–5). The 11S-20S proteasome complex mediates the degradation of oxidized and nonubiquitinated substrates (6).

A reduction in proteasome activity has been observed in aged cells of diverse origins and in replicatively senescent cells (7). In parallel, the accumulation of oxidized and damaged cellular proteins has been shown to be one of the characteristics of cellular senescence in mammals (8,9).

In addition, a reduction in protein levels of proteasome subunits has been observed in aged cells of different origins (10,11). These findings suggest that the ubiquitin–proteasome pathway of protein degradation is involved in intrinsic aging processes, as well as in aging-related degenerative diseases.

In the field of skin aging research, a decrease in proteasome activity and subunit content has been observed in human epidermal keratinocytes from elderly persons (12,13) and in replicatively senescent fibroblasts (14). Although these investigations have suggested a relationship between the proteasome and aging of the skin, most studies on proteasome subunits at the molecular level have been limited to an in vitro replicative-senescence model and have not been intensively performed with intrinsically aged dermal fibroblasts obtained from elderly persons.

In this study, we measured proteasome activity in dermal fibroblasts from individuals ranging from 20 to 78 years old and compared the data obtained with the results from the in vitro replicative-senescence model. We also investigated the relationships between proteasome activity, expression levels of proteasome subunits (PSMB6, PSMB5, and the 19S regulatory subunit), and aging markers in the dermal fibroblasts from individuals of different ages. Finally, we analyzed the effects of the restoration of PSMB6 or PSMB5 gene expression on the aging phenotypes of dermal fibroblasts obtained from elderly persons.

Materials and Methods

Antibodies and Reagents

Dulbecco's modified Eagle's medium (DMEM), trypsin–EDTA, and penicillin–streptomycin antibiotic solution were obtained from Life Technologies (Gaithersburg, MD). Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (LLVY-AMC), MG132 (PI-102), and primary antibodies against PSMA1 (PW8100), PSMB6 (PW8140), PSMB5 (PW8895), PSMC4 (PW8765), PSME1 (PW8185), and PSMD8 (PW8835) were obtained from BIOMOL (Plymouth Meeting, PA). N-t-Boc-Leu-Ser-Thr-Arg-7-amido-4-methylcoumarin (LSTR-AMC) and Z-Leu-Leu-Glu-7-amido-4-methylcoumarin (LLE-AMC) were purchased from Sigma (St. Louis, MO). Primary antibodies against β-actin and ubiquitinated proteins and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against p21 was obtained from Cell Signaling Technology (Beverly, MA).

Cell Cultures

Human skin-punch biopsies were obtained from the buttocks of individuals aged 20–82 years after written informed consent. Normal human dermal fibroblasts (HDFs) were isolated as previously described (15). The isolated cells were incubated in DMEM supplemented with 10% fetal bovine serum and penicillin-streptomycin at 100 U/mL in a 37°C, humidified, 5% CO₂ incubator. The fibroblasts were harvested at passage 5. For replicatively senescent fibroblasts, HDFs taken from the foreskins of three 21-year-old men were subcultured at a split ratio of 1:2 when the cells reached 80%–90% confluence, until they entered senescence at about passage 30. These cells were harvested at passage 5, passage 16, and passage 30, referred to as early, middle, and late stages, respectively.

Protein Extraction and Western Blot Analysis

Cells were harvested on ice using RIPA buffer (25 mM Tris, pH 7.4, 150 mM KCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate [SDS]) and protease inhibitor cocktail at 10 μL/mL (Sigma). The cells were disrupted by vortexing for 20 seconds on ice three times and centrifuged at 15,000 g for 10 minutes at 4°C, to collect the supernatants. Protein concentrations were determined by the bicinchoninic acid method (Pierce, Rockford, IL) using bovine serum albumin (BSA) as the standard. Protein samples (30 μg) were resolved on 4%–10% SDS–polyacrylamide gradient gels. After the proteins were electrotransferred to nitrocellulose membranes, the blots were blocked with 5% nonfat milk in TBST (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.15% Tween-20) for 1 hour at room temperature and probed overnight at 4°C with anti-PSMB6, anti-PSMB5, anti-PSMC4, anti-PSMD8, or anti-PSME1 antibodies (1:1000 dilution in TBST containing 5% nonfat milk). Following three 10-minute washings with TBST, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase in 5% nonfat milk in TBST for 50 minutes at room temperature and then washed three times for 10 minutes each with TBST. Detection was performed with an enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Piscataway, NJ). The relative protein levels were analyzed using ImageMaster 2D Elite software (Amersham Biosciences, Buckinghamshire, U.K.). β-actin was used as a loading control.

RNA Preparation, Reverse Transcription, and Real-Time Polymerase Chain Reaction

Cells from individuals aged 20–82 years were washed twice with Dulbecco's phosphate-buffered saline (PBS), and total RNA was isolated from cells using TRIzol reagent (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. One microgram of total RNA was reverse-transcribed in 25 μL of reaction mixture, containing Moloney Murine Leukemia Virus (M-MuLV) reverse transcriptase (2.5 U), RNAse inhibitor (1 U), 5 mM MgCl₂, 50 mM KCl, 10 mM Tris–HCl (pH 8.3), 2.5 μM oligo-(dT) primer, and 1 mM deoxyribonucleotide triphosphates (dNTPs). The reaction mixture was heated to 42°C for 60 minutes and then denatured at 85°C for 5 minutes. Complementary DNA (cDNA) was amplified with ICycler (Bio-Rad, Hercules, CA) in 50 μL of reaction mixture containing AmpliTaq DNA polymerase (1 U, Perkin Elmer, Shelton, CT), 50 mM Tris (pH 8.3), BSA at 0.25 mg/mL, 3 mM MgCl₂, 0.25 mM dNTPs, 1:50,000 dilution of SYBR green I (Molecular Probes, Eugene, OR), and 0.25 μM of appropriate sense and antisense polymerase chain reaction (PCR) primers. The sequences of the primers were as follows: PSMA1 forward 5′-TGT ATT CGA TAG ACC ACT GCC T-3′, reverse 5′-GCA ATA AGG AGA CCA ACA CCA TA-3′; PSMB6 forward 5′-CGA TTT TCG CCC TAC GTT TTC A-3′, reverse 5′-CCG CTG CAT CCA ATG ACT GT-3′; PSMB4 forward 5′-CCT CAG TCC TCG GCG TTA AG-3′, reverse 5′-GCA TGG TAC TGT TGT TGA CTC G-3′; PSMB5 forward 5′-GTG AAG GGA ACC GGA TTT CAG-3′, reverse 5′-CTC GAC GGG CCA GAT CAT AG-3′; PSMC4 forward 5′-GGT GCA GGA GGA ATA CAT CAA A-3′, reverse 5′-CTC CAG AAA TTG TCC GAT GAC C-3′; PSMD8 forward 5′-CTC CCC GAG TCA GCC TAT ATG-3′, reverse 5′-GCT GCC CTC CAT CAG GTA TTG-3′; PSME1 forward 5′-AGC GTG GTG ATG CAG TGA C-3′, reverse 5′-AGG GGA GAA ACA AAG GGA ATG A-3′; PSME2 forward 5′-GGA ATG AGA AAG TCC TGT CCC T-3′, reverse 5′-CTC CTG GAT TGC TAC CCC AAA-3′; beta-2microglobulin (B2M) forward 5′-ACC CCC ACT GAA AAA GAT GA-3′, reverse 5′-ATC TTC AAA CCT CCA TGA TG-3′. The following cycling conditions were used: one denaturing cycle at 95°C for 5 minutes, followed by 30 cycles of 95°C for 30 seconds, 60°C for 45 seconds, and 72°C for 1 minute. Relative RNA levels were determined by analyzing the changes in SYBR green I fluorescence during PCR, according to the manufacturer's instructions. B2M was amplified in parallel, and the results were used for normalization. The correctly sized PCR products were confirmed by electrophoresis on a 2% agarose gel stained with ethidium bromide. Purity of the PCR products was determined by melting-point analysis, using the ICycler software.

Oxidized Protein Detection

Protein–carbonyl content was measured with an Oxyblot protein oxidation detection kit (Invitrogen), according to the manufacturer's instructions. Briefly, protein extraction was performed as described above, and 5 μg of protein from cell lysates was mixed with an equal volume of 12% SDS. The mixtures were incubated with 2,4-dinitrophenylhydrazine (DNPH) solution for 15 minutes at room temperature. Following derivatization, the reaction was immediately terminated using 2 M Tris base/30% glycerol containing 2-mercaptoethanol. The samples were subjected to 10% SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes. Blots were blocked with 5% nonfat milk in TBST for 1 hour at room temperature and probed with antidinitrophenyl (DNP) antibody (1:1000 dilution in TBST containing 5% nonfat milk) for 1 hour at room temperature. After three 10-minute washings with TBST, the membranes were incubated with horseradish peroxidase–conjugated goat antirabbit immunoglobulin G (IgG) in 5% nonfat milk in TBST for 50 minutes at room temperature and then washed three times for 10 minutes each with TBST. The immunoreactive bands were detected with the ECL system.

Senescence-Associated β–Galactosidase Assays

Senescence-associated β–galactosidase (SA-β-gal) staining was performed with a senescent cell staining kit (Sigma) according to the manufacturer's instructions. Briefly, 1 × 10⁵ cells were plated in 35-nm culture dishes. After 24 hours, the cells were washed twice with PBS and fixed with 0.2% glutaraldehyde and 2% formaldehyde for 7 minutes. The cells were rinsed three times with PBS and incubated with 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside at 1 mg/mL of in a buffer containing 40 mM citric acid/phosphate (pH 6.0), 5 mM K₃FeCN₆, 5 mM K₄FeCN₆, 150 mM NaCl, and 2 mM MgCl₂ for 24 hours at 37°C in the absence of CO₂. The blue-stained cells were counted under light microscopy, and their percentages were calculated.

Proteasome Activity Assays

Cells from individuals aged 20–78 years were washed twice with cold PBS, harvested on ice using a buffer containing 20 mM Tris-HCl (pH 7.8), 5 mM MgCl₂, 10 mM KCl, and 1 mM dithiothreitol (DTT), and briefly sonicated. The lysates were centrifuged at 15,000 g for 10 minutes at 4°C, and the supernatants were collected. Protein concentrations were determined by the bicinchoninic acid method using BSA as the standard. Proteasome activities were assayed using fluorogenic substrates: 100 μM LLVY-AMC for the CT-L activity, 50 μM LSTR-AMC for the T-L activity, and 100 μM LLE-AMC for the PGPH activity. Protein (30 μg) from the supernatants was mixed with the fluorogenic substrates in a final volume of 100 μL. The mixtures were incubated for 30 minutes at 37°C in the dark. The fluorescence spectra were measured using a Wallac spectrofluorometer (PerkinElmer Life Sciences, Boston, MA). The samples were excited at 380 nm, and the fluorescence emission spectra were collected at 460 nm. Proteasome activity was determined as the difference between the total activity in the lysates and the activity remaining in the presence of 20 μM of the proteasome inhibitor MG132.

Construction of Lentiviral Vectors

PSMB6 and PSMB5 cDNAs were obtained by PCR from the Jurkat cDNA library by using the following primers: PSMB6 forward 5′-GGG GTA CCA TGT TGT CCT CTA CAG CC-3′ and reverse 5′-CCG CTC GAG TCA GTC CTT CCT TAA G-3′ and PSMB5 forward 5′-GGG GTA CCA TGG CGC TTG CCA GCG TG-3′ and reverse 5′-CCG CTC GAG TCA GGG GGT AGA GCC-3′. For the construction of the PSMB6- and PSMB5-expressing lentiviruses, KpnI-digested, T4 polymerase–blunted PSMB6 and PSMB5 cDNA fragments were cloned into the EcoR V-XhoI site of the lentiviral vector (VectorCore A, Daejeon, Korea). This vector contains the human cytomegalovirus (hCMV) promoter and expresses puromycin from the internal ribosome entry site (IRES) system.

Overexpression of Proteasome Subunits in Dermal Fibroblasts

HDFs were transduced at a multiplicity of infection (MOI) of 20 with lentiviral vectors expressing PSMB6 or PSMB5, together with polybrene at 6 μg/mL. After a 20-hour incubation, DMEM supplemented with 10% fetal bovine serum was added. To achieve stable transduction, cells were incubated for 4 days in medium containing puromycin at 1 μg/mL.

Statistical Analysis

Data were expressed as means ± standard error of the mean (SEM). Data were analyzed by one-way analysis of variance (ANOVA) with Tukey's post hoc test using the SPSS version 10.0 statistical package for Windows (SPSS, Chicago, IL). Differences were considered significant at p <.05.

Results

SA-β-Gal Activity and p21 Protein Levels Are Higher in Dermal Fibroblasts From Elderly Persons

We measured SA-β-gal activity and p21 levels as senescence markers in HDFs from individuals of different ages (16). The percentages of SA-β-gal–positive cells were 6-fold higher in old dermal fibroblasts than in young dermal fibroblasts (Figure 1A), and the level of p21 protein was also higher in old dermal fibroblasts than in young cells (Figure 1B). Similarly, the percentage of SA-β-gal–positive cells increased during replicative senescence, and p21 levels were already elevated in middle-passage fibroblasts (Figure 1). HDFs from elderly persons showed senescence-like phenotypes similar to those of replicatively senescent cells.

Proteasome Activities Are Lower in Dermal Fibroblasts From Elderly Persons

The study of CT-L, T-L, and PGPH proteasomal activities in dermal fibroblasts from individuals ranging from 20 to 78 years old showed an age-associated decrease in the proteasome activities. All three proteasome activities dramatically decreased in HDFs from 20- to 50-year-old donors, but remained stable in HDFs from donors aged 50–78 years (Figure 2A–C). Similarly, proteasome activities decreased during replicative senescence. CT-L activity was lower in middle- and late-stage cells than in early-stage cells (43% and 55% lower than that of early-stage cells, respectively) (Figure 2D). PGPH activity also decreased in the middle- and late-stage cells to 37% and 21% of that in early-stage cells, respectively (Figure 2E). T-L activity showed similar changes (19% in the middle-stage cells and 14% in the late-stage cells, compared with the early-stage cells) (Figure 2F).

Accumulation of Oxidized Proteins and Ubiquitinated Proteins in Dermal Fibroblasts of Elderly Persons

We measured the change in the oxidized protein content of aged cells by protein–carbonyl content detection. An increase in oxidized protein content was observed in dermal fibroblasts from elderly persons (2.2 ± 0.3-fold) and in late-stage fibroblasts (1.9 ± 0.5-fold) (Figure 3A). These results were consistent with the lower proteasome activities of these cells. The ubiquitinated protein content also increased in the aging dermal fibroblasts and replicatively senescent cells (Figure 3B).

Levels of Proteasome Subunits Are Lower in Dermal Fibroblasts From Elderly Persons

To investigate whether the decline in proteasome activities was related to the proteasome content in aging HDFs, we examined the levels of the proteasome catalytic subunits (PSMB6 and PSMB5), the 19S regulatory subunits (PSMC4 and PSMD8), and the 11S activator (PSME1) by Western blotting. We found decreased levels of the catalytic β-subunits PSMB6 and PSMB5, which have PGPH and CT-L activity, respectively, and the 19S regulatory subunits PSMC4 and PSMD8 in aging dermal fibroblasts (Figure 4). The differences in the relative expression levels between young- and old-donor HDFs were more striking for PSMB6 and PSMB5 than for PSMC4 and PSMD8. Declines in the protein levels of the catalytic and 19S regulatory subunits were also observed in replicatively senescent fibroblasts. However, the levels of PSME1 subunits were higher in old-donor HDFs and late-stage fibroblasts.

mRNA Levels of Proteasome Subunits Are Lower in Dermal Fibroblasts From Elderly Persons

Next, we examined mRNA levels of the α subunit (PSMA1), β subunits (PSMB6, PSMB5, and PSMB4), 19S regulatory subunits (PSMC4 and PSMD8), and the 11S activators (PSME1 and PSME2) by real-time PCR analysis. We found decreased mRNA levels for the α subunit (PSMA1), β subunits (PSMB6, PSMB5, and PSMB4), and the 19S regulatory subunits PSMC4 and PSMD8 in aging dermal fibroblasts (Figure 5). The expression level of the PSMA1 subunit mRNA was 2.56 ± 0.36-fold in the five young-donor cultures, but only 1.00 ± 0.17-fold in the five elderly-donor cultures, as compared to a common reference sample, which was set at 1.00. The mRNA levels for the PSMB subunits in young-donor cultures were 2.33-fold for PSMB6, 2.06-fold for PSMB4, and 2.04-fold for PSMB5, as compared to a common reference sample. However, the levels in old-donor cultures were 0.91-fold for PSMB6, 0.96-fold for PSMB4, and 0.89-fold fold for PSMB5, as compared to the reference sample. The difference in the relative expression levels between young- and old-donor HDFs was the most striking for PSMC4. The mRNA levels for PSMC4 in young-donor cultures were 6.5-fold higher than those in old-donor cultures. The mRNA levels of the PSME1 and PSME2 subunits did not differ between the young- and old-donor HDFs.

Restoration of Normal Level of PSMB6 or PSMB5 Increases Proteasome Activity and Decreases Accumulation of Ubiquitinated and Oxidized Proteins in Dermal Fibroblasts from Elderly Persons

Because the PSMB6 and PSMB5 subunits were found to be significantly reduced in dermal fibroblasts from elderly persons in this study, and because previous reports showed that the restoration of normal PSMB5 expression reduced the damage from oxidative stress and increased survival rates in replicatively senescent lung fibroblasts (17), we evaluated the effects of restoring normal levels of the PSMB6 or PSMB5 subunits in dermal fibroblasts from elderly persons. Western blot analysis with anti-PSMB6 and anti-PSMB5 antibodies showed that the levels of these subunits were higher in cells transduced with vectors expressing PSMB6 or PSMB5 subunits than in cells transduced with the vector alone (Figure 6A). Also, cells transduced with the PSMB6 or PSMB5 subunits exhibited elevated expression of the PSMA1 subunit. To investigate whether the increased levels of the catalytic subunits were accompanied by an increase in proteasome activities, we measured PGPH activity in the PSMB6-transduced cells and CT-L activity in the PSMB5-transduced cells. The proteasome activities were increased in the subunit-transduced cells as compared to the vector-transduced cells (Figure 6B).

We also investigated whether the increased proteasome activities observed after the restoration of normal PSMB6- or PSMB5-subunit levels reduced the accumulation of oxidized proteins and ubiquitinated proteins in the fibroblasts from aged skin. Estimation of the protein–carbonyl content showed that the accumulation of oxidized proteins was significantly lower in the cells with restored subunit expression than in the control cells (Figure 7A). The level of ubiquitinated proteins also decreased in skin fibroblasts when the expression of PSMB6 and PSMB5 was restored to the normal level (Figure 7B).

Restoration of Normal Levels of PSMB6 or PSMB5 Delays Senescence in Dermal Fibroblasts From Elderly Persons

To study the effects of proteasome activation on skin aging, we measured the senescence markers, SA-β-gal activity and p21 content, in old-donor HDFs with restored normal levels of the PSMB6 or PSMB5 subunits. The percentage of SA-β-gal activity–positive cells was lower in cell populations with restored expression of PSMB6 and PSMB5 (11 ± 1% and 7 ± 2% of total cells, respectively) as compared to cell populations transduced with the vector alone (26 ± 3% of total cells) (Figure 8A). As expected, the expression of p21 in elderly cells was decreased by re-establishing the normal level of expression of these subunits (Figure 8B).

Discussion

Previous studies have shown that malfunctions in the proteasome machinery is a causal factor in age-related degenerative diseases and that the decline in proteasome activity is related to senescence-like phenotypes in mammalian cells (6,7,10,11,18,19). However, most studies at the molecular level have been limited to studies in replicatively senescent cell models using established cell lines. Here, we studied fibroblasts from humans ranging from 20 to 82 years old to understand the involvement of the proteasome in intrinsic skin aging.

Our results showed a biphasic change in three major proteasome activities in dermal fibroblasts with aging: a steady decline in activities was observed until age 50, after which the activities remained at a low level until age 78. This finding indicates that old cells maintain minimal levels of proteasome activity, which might be essential for cell survival. In previous studies, three major activities of the proteasome were shown to be differentially affected in different regions of the ageing rodent brain (20). In the present study, however, all three proteasome activities showed a similar pattern of age-associated decline. The discrepancies between our results and those of previous studies might be due to genetic differences between rodent and human or to the different characteristics of brain and skin tissues. We confirmed that this age-associated loss of proteasome activity was accompanied by the accumulation of oxidized and ubiquitinated proteins.

The model system of replicative senescence also showed a biphasic change in proteasome activities as the number of cell passages increased: The activities of the proteasome were dramatically lower in middle-passage fibroblasts, but there was no major difference in activity between middle- and late-passage fibroblasts. This result is similar to previous results in human lung fibroblasts (14) and human keratinocytes (13). As observed previously (14,17), we also found that oxidized and ubiquitinated proteins accumulated with increasing cell passages. Interestingly, oxidized proteins accumulated only in late-passage fibroblasts, not in middle-passage fibroblasts where the most drastic drop in proteasome activity was observed. Although the accumulation of oxidized proteins was originally attributed to reduced proteasome activity (19), repair systems for eliminating oxidized proteins such as peptide methionine sulfoxide reductase (21) could also affect the accumulation of oxidized proteins. Previous studies showed that the activity and expression of the repair system significantly decreased in late-passage fibroblasts (22). The time-lag between the decrease in proteasomal activities and accumulation of oxidized protein could be ascribed to delayed impairment of the repair system for eliminating oxidized proteins.

There are many factors that could contribute to decreased proteasome activity during aging, such as proteasome content, localization, modification, and the presence of cross-linked proteins (18).

Impaired proteasome activity in replicatively senescent human fibroblasts has been shown to be associated with decreases in several proteasome subunits (14). Therefore, we measured the levels of the proteasome subunits PSMB6, PSMB5, PSMC4, PSMD8, and PSME1 in old dermal fibroblasts and late-stage fibroblasts. The protein levels of PSMB6, PSMB5, PSMC4, and PSMD8 were lower in old dermal fibroblasts and late-stage fibroblasts. The 20S catalytic subunits, PSMB6 and PSMB5, were significantly lower in old dermal fibroblasts, similar to previous observations made in replicative senescence models (14,17). Of the various proteasome subunits, the 20S catalytic subunit might be critically important for proteasome activity. In addition to the protein levels, the mRNA levels of proteasome subunits were lower in aging HDFs. These results indicated that the age-associated decline in protein levels of proteasome subunits was correlated with similar reductions in their mRNA levels. Interestingly, in contrast to the other subunits, the PSME1 protein level increased in old dermal fibroblasts and late-stage fibroblasts. However, the PSME1 mRNA level was not affected by age. Although the cause of the increase in the PSME subunit level in aged HDFs is not clear, signals elicited during the aging process could act at the posttranscriptional level, the translational level, and/or during posttranslational modification of PSME subunits. Impaired proteasome activity in aged HDFs might be a causal factor in the decreased degradation and subsequent accumulation of PSME subunits. Further studies on the role and regulation of PSME in the aging process will be necessary to resolve this issue.

Finally, we determined whether increased expression of PSMB6 and PSMB5 contributed to a decrease in skin aging. In fibroblasts from elderly persons, the restoration of normal levels of these proteasome subunits increased proteasome activities and decreased the levels of damaged proteins and aging markers. The protein level of the PSMA1 subunit increased in PSMB6- and PSMB5-transduced cells. These results indicated that the induction of one catalytic subunit can stimulate the expression of other core subunits, resulting in a general increase in proteasome subunit content and enhanced proteasome proteolytic activity. The increased proteasome activity accelerated the proteasome-mediated degradation of damaged proteins, resulting in less severe senescence phenotypes in aged dermal fibroblasts.

To our knowledge, this study is the first attempt to correct age-related defects by restoring normal levels of proteasome subunits in cells from elderly persons. Further studies will be directed toward determining whether increased proteasome activities delay or prevent the accumulation of advanced aging markers, such as lipofuscin and ceroids, in vitro and in vivo.

Decision Editor: Huber R. Warner, PhD

Figure 1.

Characterization of aging human dermal fibroblasts and replicatively senescent fibroblasts. Senescence-associated β-galactosidase (SA-β-gal) activity (A) and levels of p21 protein (B) were determined in human dermal fibroblasts from three young (ages 20, 25, and 30) and three old (ages 68, 70, and 77) donors at passage four and in replicatively senescent fibroblasts from three young (age 21) donors at passages 5 (early), 16 (middle), and 30 (late). SA-β-gal activity is represented by the percentage of blue-stained cells. Protein levels of p21 were quantified by densitometer and normalized to the expression of β-actin. Results represent relative levels of p21 protein. Mean value of p21 levels in young donor cells and early-stage cells was set to 100%. Representative microscopic images and Western blots were selected from three independent experiments. Results are presented as means ± standard error of the man (SEM). *, Differences as compared with the young or early-stage groups. *p <.05. a, b, and c: values that do not share the same superscript letter are statistically significant (p <.05)

Figure 2.

Proteasome activity in different ages of human dermal fibroblasts and replicatively senescent fibroblasts. Chymotrypsin (CT)-like (A and D), peptidylglutamyl-peptide hydrolase (PGPH) (B and E), and trypsin (T)-like (C and F) activities of the proteasomes were assayed in dermal fibroblasts from donors of different ages (A–C) and replicatively senescent fibroblasts (D–F) from three young (age 21) donors at passages 5 (early), 16 (middle), and 30 (late), as described in the Materials and Methods section. Results from replicatively senescent fibroblasts are means ± standard error of the mean (SEM) of three independent experiments. a, b, c: values that do not share the same superscript letter are statistically significant (p <.05). AMC, 7-amido-4-methylcoumarin

Figure 3.

Levels of oxidized and ubiquitinated proteins in aging human dermal fibroblasts and replicatively senescent fibroblasts. Oxidized proteins (A) and ubiquitinated proteins (B) in primary-cultured dermal human fibroblasts from three young (ages 20, 25, and 30) and three old (ages 68, 70, and 77) donors at passage 4 were analyzed by Oxyblot and Western blotting. In addition, these proteins were analyzed in replicatively senescent fibroblasts from three young (age 21) donors at passages 5 (early), 16 (middle), and 30 (late). Oxidized and ubiquitinated proteins were quantified by densitometer and normalized to the expression of β-actin. Relative contents of oxidized and ubiquitinated proteins are presented as means ± standard error of the mean (SEM). Mean values of the levels in young and early-stage cells were set at 100%. Representative blots were selected from three independent experiments. *, Differences as compared with the young or early-stage groups; *p <.05. a, b, c: values that do not share the same superscript letter are statistically significant (p <.05)

Figure 4.

Levels of proteasome subunits in aging human dermal fibroblasts and replicatively senescent fibroblasts. Protein levels of representative 20S catalytic subunits (PSMB6, PSMB5), 19S regulatory subunits (PSMC4, PSMD8), and the 11S activator PSME1 were analyzed by Western blotting in primary cultured dermal human fibroblasts from three young (ages 20, 25, and 30) and three old (ages 68, 70, and 77) donors at passage 4 and in replicatively senescent fibroblasts from three young (age 21) donors at passages 5 (early), 16 (middle), and 30 (late). Levels of proteasome subunits were quantified by densitometer and normalized to the expression of β-actin. Relative levels of proteasome subunits are presented as means ± standard error of the mean (SEM). Mean values of protein levels in young and early-stage cells were set at 100%. Representative Western blots were selected from three independent experiments. *, Differences as compared with the young or early-stage groups; *p <.05. a, b, c: values that do not share the same superscript letter are statistically significant (p <.05)

Figure 5.

Analysis of proteasome subunit messenger RNA (mRNA) by real-time polymerase chain reaction (PCR). Relative mRNA expression levels of the α subunit (PSMA1), β subunits (PSMB6, PSMB5, and PSMB4), 19S regulatory subunits (PSMC4 and PSMD8), and the 11S activator (PSME1 and PSME2) were measured in cells from five young donors (ages 20, 24, 27, 28, and 29 years) and five elderly donors (ages 51, 57, 70, 74, and 82 years). Total RNA was isolated from passages 4–6 cultures from all samples. RNAs were reverse-transcribed, PCR co-amplified along with β₂-microglobulin (B2M), and quantified as described in the Materials and Methods section. Results are normalized to B2M mRNA expression and presented as the fold-differences (means ± standard error of the mean [SEM]). Values for each mRNA in a common reference sample were set at 1. *, Differences as compared with the young-donor group; *p <.05, **p <.01

Figure 6.

Effects of the restoration of PSMB6 or PSMB5 protein levels and proteasome activities in fibroblasts from elderly donors. Levels of PSMB6 and PSMB5 were confirmed by Western blotting (A). Equal protein loading was verified by stripping the membrane and reprobing with anti-β-actin antibody. Proteasome activities (B) in fibroblasts from three elderly donors (ages 60, 67, and 72 years) after lenti-PSMB6 or lenti-PSMB5 transduction were assayed. Protein levels of PSMB6 and PSMB5 was quantified by densitometry and normalized to the expression of β-actin. Relative levels of PSMB6 and PSMB5 are presented as means ± standard error of the mean (SEM). Mean protein-level values in lenti-vector-transduced cells (Vec) were set at 100%. Representative Western blots were selected from three independent experiments. Proteasome activities are expressed as the percentage of peptidylglutamyl-peptide hydrolase (PGPH) or chymotrypsin (CT)-like activities in the vector control (Vec). Mean activity values in the vector control cells were set at 100%. Results are means ± SEM of three independent experiments. *, Differences as compared with the vector control; *p <.05

Figure 7.

Levels of oxidized and ubiquitinated proteins in aged fibroblasts with PSMB6 and PSMB5 expression restored to normal levels. Levels of oxidized proteins (A) and ubiquitinated proteins (B) were determined by Oxyblot and Western blotting, respectively, in fibroblasts from three elderly donors (ages 60, 67, and 72 years) after lenti-PSMB6 or lenti-PSMB5 transduction. Oxidized and ubiquitinated proteins were quantified by densitometry and normalized to the expression of β-actin. Relative levels of oxidized and ubiquitinated proteins are presented as means ± standard error of the mean (SEM). Mean protein-level values of in vector control cells (Vec) were set at 100%. Representative blots were selected from three independent experiments. *, Differences as compared with the vector group; *p <.05

Figure 8.

Levels of senescence markers in old fibroblasts with restored normal level expression of PSMB6 and PSMB5. Senescence-associated β-galactosidase (SA-β-gal) activity (A) and levels of p21 protein (B) were determined in fibroblasts from three elderly donors (ages 60, 67, and 72 years) after lenti-PSMB6 or lenti-PSMB5 transduction. Protein level of p21 was quantified by densitometry and normalized to the expression of β-actin. Relative protein levels of p21 are presented as means ± standard error of the mean (SEM). Mean p21 levels and SA-β-gal activities in the vector control cells (Vec) were set at 100%. Representative microscopic images and Western blots were selected from three independent experiments. *, Differences as compared with the vector alone; *p <.05, **p <.01

The research of Dr. Jae Sung Hwang was supported in part by a grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (A050432).

We thank the reviewers for providing helpful comments that greatly improved this article.