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Increased expression of 14-3-3ɛ protein in intrinsically aged and photoaged human skin in vivo
Mechanisms of Ageing and Development 126 (2005) 629–636 www.elsevier.com/locate/mechagedev
Increased expression of 14-3-3e protein in intrinsically aged and photoaged human skin in vivo Kyung-Chul Choia, Serah Leeb, Sun Young Kwaka, Mi-Sun Kimb, Hyo Kyoung Choia, Kyu Han Kimb, Jin Ho Chungb,*, Seok Hee Parka,* a
b
Department of Pathology, Inha University College of Medicine, Incheon 400-712, Republic of Korea Department of Dermatology, Seoul National University College of Medicine, Seoul 110-744, Republic of Korea Accepted 10 November 2004 Available online 21 January 2005
Abstract Skin aging is a complicated process associated with the passage of time and environmental exposure, especially to UV light. This aging phenomenon is related to alterations in various cellular mechanisms, such as changes in apoptosis, perturbations to cellular signaling, and an increased genetic instability. In this study, we investigated changes of proteins involved in intrinsic aging by the proteomic analysis of human sun-protected (upper inner arm) young and aged dermis. One of the proteins upregulated in aged dermis was identified as 14-3-3e. This protein is an isoform of 14-3-3 protein, which is involved in cellular processes like signal transduction, cell cycle arrest, and apoptosis. 14-3-3e is consistently found to be upregulated in the sun-protected dermis of aged skin, by Western blotting and immunohistochemical staining. In addition, we demonstrate that the expression of 14-3-3e is further upregulated in the sun-exposed (photodamaged) dermis, and that the UV irradiation of young skin significantly upregulates 14-3-3e in vivo. Our results suggest the possibility that the cellular processes related to 143-3e protein play an important role in the photoaging and intrinsic aging of human skin. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Skin aging; Proteomics; 14-3-3e; UV irradiation
1. Introduction Skin aging is a complex phenomenon that affects different constituents of human skin. The aging process of human skin can be considered to be due to intrinsic aging and photoaging (Gilchrest, 1982, 1989; Jenkins, 2002; Ma et al., 2001). The intrinsic aging is due to chronologic damage caused by slow, irreversible tissue degeneration whereas the photoaging is primarily the results of UV exposure (Jenkins, 2002; Ma et al., 2001). Clinically, chronologically aged skin is smooth, pale, and finely Abbreviations: 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; IPG, immobilized pH gradient; MALDI-TOF, matrix associated laser desorption/ionization time-of-flight * Corresponding authors. Tel.: +82 32 890 0942 (S.H. Park)/ 82 2 760 2414 (J.H. Chung); fax: +82 32 890 0944 (S.H. Park). E-mail addresses: [email protected] (J.H. Chung), [email protected] (S.H. Park).
wrinkled. In contrast, photoaged skin is coarsely wrinkled and associated with dyspigmentation and telagiectasia (Gilchrest, 1989; Lavker, 1979; Lavker and Kligman, 1988). The morphological and histological features of chronologically aged and photoaged skin are distinct. However, emerging evidence indicates that these processes have partly overlapping, biological, biochemical, and molecular mechanisms (Jenkins, 2002; Oikarinen, 1990; Rittie and Fisher, 2002). The basic principles underlying intrinsic aging are believed to be generally relevant in age-related diseases. Therefore, the skin represents an excellent and accessible model organ for the study of intrinsic aging. The molecular mechanisms underlying intrinsic skin aging are described by two processes: cellular senescence (Campisi, 1996; Dimri et al., 1995; West et al., 1989) and oxidative stress (Harman, 1956, 1981; Sohal and Allen, 1990; Sohal and Weindruch, 1996). Cellular senescence associated with intrinsic skin
0047-6374/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2004.11.013
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aging includes irreversible growth arrest (Cristofalo and Pignolo, 1993; Dimri et al., 1994; Hara et al., 1994; Hayflick, 1965) and the resistance of skin-derived cells to apoptosis (Wang, 1995), decreased matrix synthesis in the dermis, and the upregulation of enzymes that degrade the collagenous matrix (Chung et al., 2001; Millis et al., 1992). In contrast, oxidative stress concerns the contribution made by cumulative oxidative damage (Muscari et al., 1996; Sohal and Allen, 1990; Sohal and Weindruch, 1996). This is supported by a large body of experimental evidence and is particularly relevant in the skin given its high exposure to environmental agents. Both processes, however, have at least in part overlapping, biological, biochemical and molecular mechanisms (Oikarinen, 1990). Although studies have begun to reveal the molecular mechanisms underlying intrinsic skin aging, no study has been undertaken to compare the protein expressions in aged and young human skin. In recent years, protein expression profiling has been used successfully to identify proteins associated with the diseases, particularly cancers (Ahram et al., 2002; Bernard et al., 2003; Wulfkuhle et al., 2001). Thus, to reveal the candidate proteins involved in skin aging, we decided to analyze protein expressions in aged skin tissues by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry. In this study, we have investigated the changes in protein expressions in the dermis of naturally aged and young skin, using conventional proteomics, and identified 14-3-3e as a candidate protein by proteomic analysis. We here demonstrate that comparative proteomics is relevant for the analysis of intrinsic skin aging, and suggest the involvement of 14-3-3e in the skin aging process.
2. Materials and methods
(n = 3), provided both buttock and facial (crow’s feet area) skin samples. Each group was composed of three volunteers. The preparation of skin samples was approved by the Institutional Review Board at Seoul National University Hospital. 2.2. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) Immobilized pH gradient (IPG) strips, pH 3–10 (Amersham Biosciences, Uppsala, Sweden), were rehydrated in ES solution (7 M urea, 2 M thiourea, 2% CHAPS, 65 mM dithiothreitol, 0.01% bromophenol blue, 2 mM tributyl phosphine). The 200 mg of the protein extract derived from dermis was loaded and isoelectric focused for 32,000 Vh on an IPGphore unit (Amersham Biosciences). Two-dimensional SDS-PAGE was performed using 8–18% gels using the Ettan Dalt system (Amersham Biosciences). Proteins were visualized by silver nitrate staining. 2.3. Protein identifications by MALDI-TOF mass spectrometry The stained gels were scanned and analyzed using the Melanine IV image analysis program (Genbio, Geneva, Switzerland). Spots with consistently elevated intensities in the sun-protected (upper inner arm) dermis of aged people were excised, in-gel digested with 12.5 mg/ml trypsin and extracted according to instructions provided by Applied Biosystems (Foster City, USA). The eluted peptides were analyzed using a matrix associated laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometer (Applied Biosystems, Foster City, USA). Peptide masses were searched for in the Swiss-Prot and TrEMBL protein databases.
2.1. Skin samples and protein extractions 2.4. Western blot analysis Six young Koreans (age range 19–26 years) and six elderly Koreans (age range 60–87 years), without current or prior skin disease, provided both sun-protected and sunexposed skin samples. The specimens for proteomic analysis and Western blot were snap frozen in liquid nitrogen and the dermal proteins of each specimen were extracted as described by Chung et al. (2001). Briefly, the defrosted skin samples were heated at 55 8C for 2 min to separate the dermis from the epidermis with forceps. Each sample of dermis was homogenized in lysis buffer (containing 1% Triton X-100, 150 mM NaCl, 5 mM ethylenediamine tetraacetic acid, 0.1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 protein inhibitor mixtures from Promega). Lysates were rotated at 4 8C for 15 min and centrifuged at 130,000 g for 15 min, and the supernatant was used for proteomic and Western blot analysis. Another group of volunteers in three different age groups, representing ages 20–39 (n = 3), 40–59 (n = 3), and 60–79
About 50 mg of dermal proteins were electrophoresed on 10% SDS-PAGE gels and the separated proteins were detected using 14-3-3e antibody (Santa Cruz Biotechnology, Santa Cruz, USA) and horseradish peroxidase-conjugated secondary antibody according to the manufacturer’s protocol. Protein bands were visualized by enhanced chemiluminescence (Amersham Biosciences). 2.5. Immunohistochemical staining The frozen sections (8 mm) were mounted onto silane coated slides (Dako, Glostrup, Denmark), fixed in acetone, and stained with 14-3-3e antibody, which was applied for 1 h at room temperature. After rinsing in phosphate-buffered saline, sections were visualized using an LSAB kit (Dako, Glostrup, Denmark), which employed a biotinylated secondary antibody and horseradish-streptavidin conjugate.
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Table 1 Examples of increased proteins in the sun-protected (upper inner arm) dermis of aged and young skin Identified proteins
OID/YIDa (fold increase)
Frequencyb
14-3-3e Serum amyloid P Component (SAP) Mitochondrial malate dehydrogenase (mMDH) Mitochondrial L-lactate dehydrogenase (mLDH) Carbonic anhydrase 1 (CA1)c Apolipoprotein I Glutathione S transferase Annexin V
3.5 8.5
3/3 3/3
2.6 3.6 0.24 2.3 2.1 2.1
3/3 3/3 2/3 2/3 2/3 2/3
a b c
OID: old inner arm dermal protein; YID: young inner arm dermal protein. The sample numbers showing the same pattern of protein expression/the analyzed sample numbers. CA1 represents a protein down-regulated in the upper inner arm dermis of aged subjects.
3-Amino-9-ethylcarbazole was used as chromogenic substrate. Sections were counterstained briefly in Mayer’s hematoxylin. The negative control, prepared using mouse IgG showed no immunoreactivity (data not shown). 2.6. UV irradiation and skin samples Healthy adult Korean subjects (three subjects, age range 20–29 years), were irradiated with F75/85W/UV21 fluorescent sunlamps, as described previously (Chung et al., 2002; Seo et al., 2001). The minimal erythema dose (MED) for each subject was determined 24 h after UV irradiation. Irradiated and non-irradiated skin samples were obtained from each subject by punch-biopsy and used for immunohistochemical staining and Western blot analysis. The preparations of skin samples were approved by the
Institutional Review Board at Seoul National University Hospital. NIH3T3 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamate, and antibiotics. After starvation for 24 h in DMEM without FBS, NIH3T3 cells were irradiated with UV, as previously described (Chung et al., 2002), and incubated for the indicated times. At the end of the incubation, protein extracts were prepared and used for Western blot analysis against 14-3-3e protein. 2.7. Statistical analysis Statistical analyses were performed using the Mann– Whitney U-test. A p-value of less than 0.05 was considered statistically significant.
Fig. 1. The maps of proteins of young-aged and old-aged sun-protected (upper inner arm) dermis by two-dimensional polyacrylamide gel electrophoresis (2DPAGE). Total dermal proteins were prepared from the punch-biopsied specimens of the sun-protected (upper inner arm) skin of both young and elderly subjects. The proteomic map shown in this figure is typical of experiments using samples of young (n = 3) and old (n = 3) upper inner arm. The lower panel shows 14-3-3e and serum amyloid P component spots, which were found to be consistently elevated in the aged dermis vs. young dermis. These spots were identified by MALDI-TOF peptide mass fingerprinting.
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3. Results 3.1. The upregulation of 14-3-3e protein was identified by proteomic analysis of aged human skin dermis To identify proteins related to intrinsic skin aging, total dermal proteins were prepared from the punch-biopsied specimens of the sun-protected (upper inner arm) skins of young and elderly subjects. Three pairs of young and aged sunprotected (upper inner arm) skin samples were taken by punchbiopsy from each subject, and used for proteomic analysis. These dermal proteins were separated by 2D-PAGE using pH 3–10 IPG in the first dimension. Protein expression changes between the young and aged dermis were determined using the Melanine IVimage analysis program. Differentially expressed proteins, showing consistent increases in all three elderly subjects,were excisedfrom silver-stainedgels andanalyzedby MALDI-TOF mass spectrometry (Table 1). Of these proteins, 14-3-3e protein was found to be significantly upregulated in the aged dermis (Fig. 1). 14-3-
3e protein is an isoform of the 14-3-3 protein family. Members of the 14-3-3 family are ubiquitous proteins that are highly conserved in bacteria, plants, and humans, and are involved in important cellular processes, such as, signal transduction, cell cycle control, apoptosis, and stress responses (Fu et al., 2000; Muslin and Xing, 2000; Tzivion and Avruch, 2002). Serum amyloid P component (SAP) was also consistently increased in the aged dermis. This has previously been reported to be increased in human renal glomeluli and testis in an age-dependent manner (Herriot and Walker, 1989; Rantala et al., 1997), which demonstrates the relevance of proteomic analysis. 3.2. 14-3-3e protein is upregulated in the dermis of aged human skin in vivo To verify the increased expression of 14-3-3e protein in the aged dermis, Western blot analysis was performed using dermal proteins extracted from human skin. As shown in
Fig. 2. Increased expression of 14-3-3e protein in the sun-protected (upper inner arm) dermis of aged human skin. (A) Western blot analysis of 14-3-3e protein was performed by using dermal proteins derived from young (n = 3) and old (n = 3) inner arm skin tissues. Dermal proteins were separated by SDS-PAGE and probed with the antibody against 14-3-3e protein (32 kDa). YI: young inner arm, OI: old inner arm. (B) The expression of 14-3-3e protein in human skin was detected by immunohistochemical staining. The frozen sections of 8 mm thickness were mounted onto silane coated slides, fixed in acetone, and stained with 143-3e antibody, as described in Section 2. The sun-protected (upper inner arm) young skins (n = 3) and sun-protected (upper inner arm) and sun-exposed (forearm) old skins (n = 3) were used for immunohistochemical staining. Immunohistochemical stainings were performed under the same experimental conditions. The insets show representative results of at least three independent experiments.
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Fig. 3. Intrinsic aging and photoaging effects on 14-3-3e protein expression in human skin in vivo. The grading of photoaging in Korean subjects has been described previously (Chung et al., 2001). Grades 1 and 2 represent the 20 to 39-year-old age group, and Grades 3 and 4 and Grade 5 and 6 indicate groups from 40 to 59 and 60 to 79 years. The expression of 14-3-3e protein was detected using 14-3-3e antibody by immunohistochemical staining. Immunohistochemical staining of human skin samples derived from each group was performed under the same experimental conditions. Three subjects in each group were used for these experiments. The inset shows representative immunohistochemical staining results from three subjects.
Fig. 2A, 14-3-3e proteins in the dermis of three different elderly subjects, were significantly upregulated versus three young subjects. Theses results indicate that 14-3-3e or a 143-3e-related function plays an important role in intrinsic aging. Next, we immunohistochemical stained the inner arm skin tissues of both young (n = 3) and old (n = 3) subjects to compare their expressions of 14-3-3e protein in vivo. The results obtained showed significantly higher expression of 14-3-3e protein in the aged dermis (Fig. 2B). We also observed that 14-3-3e protein appeared to be upregulated in the epidermis of aged skin versus young skin (Fig. 2B). Although intrinsic aging and photoaging are considered to be distinct entities during skin aging, emerging data suggest that UV irradiation, the most important factor in photoaging, accelerates the intrinsic aging process (Jenkins, 2002; Rittie and Fisher, 2002). Thus, we examined the expression of 14-3-3e protein in the photoaged dermis of the same old-aged people; interestingly, the expression of 14-3-3e was significantly increased in the photoaged versus the intrinsically aged dermis (upper inner arm) (Fig. 2B). These results strongly suggest that 14-3-3e protein is specifically upregulated in the human dermis in an age-dependent manner, and raise the possibility that 14-3-3e-related cellular signaling may
be modulated in both the photoaging and intrinsic aging processes. 3.3. Expression of 14-3-3e protein is significantly affected by photoaging and by the intrinsic aging To confirm that the photoaging process affects the expression of 14-3-3e protein, we investigated changes in 14-3-3e protein expression caused by intrinsic aging and photoaging over a lifetime. Sun-exposed (crow’s feet area) and sun-protected (buttock) skin samples (n = 3) were taken from individuals belonging to three different age groups and immunohistochemically stained for 14-3-3e protein (Fig. 3). 14-3-3e protein showed higher expression in the buttock dermis of the elderly group >60 years than in young buttock dermis, supporting our previous result that 14-3-3e is upregulated during intrinsic aging (Fig. 3). Interestingly, the expression of 14-3-3e protein was significantly upregulated in sun-exposed dermis in all aged groups, compared with the corresponding sun-protected dermis (Fig. 3). Moreover, facial (photoaged) skin >60 years showed higher dermal expression than the other age groups (Fig. 3). These results indicate that the expression of 14-3-3e is affected by both the intrinsic and photoaging process, and suggest the possibility that photoaging may accelerate 14-3-3e accumulation in the dermis.
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Fig. 4. Expression of 14-3-3e protein following UV irradiation in human skin in vivo and in NIH3T3 cells. (A) Human buttock skin (n = 3) was irradiated with 2 MED of UV. Immunohistochemical staining against 14-3-3e protein was performed using human skin samples obtained by punch-biopsy at the indicated times after UV irradiation. Three subjects (n = 3) were irradiated in these experiments. The inset shows representative immunohistochemical staining from three different subjects. The control is of a non-irradiated sample. (B) Western blot analysis against the 14-3-3e protein was performed on non-irradiated and irradiated skin samples. Buttock skins were obtained from young subjects (n = 3) at 24, 48, and 72 h after UV irradiation (2 MED). Total proteins were prepared from non-irradiated and irradiated human skin samples taken at the indicated times and used for Western blot analysis against the 14-3-3e protein. The result is representative of the results obtained in three independent subjects. Data are expressed as percentage changes in 14-3-3e protein levels relative to levels in nonirradiated control skin. Data are normalized vs. a b-actin level used as an internal control for quantitation purposes. Control (C) indicates a non-irradiated skin sample. * p < 0.05 vs. non-irradiated control skin, n = 3. (C) NIH3T3 cells were irradiated with UV, 30 mJ/cm2, and incubated for the indicated times following UV irradiation. Total proteins were prepared from non-irradiated and irradiated samples and used for Western blot analysis against the 14-3-3e protein. b-actin was used as an internal control. The inset shows a Western blot which is representative of three independent experiments.
3.4. UV irradiation induces the expression of 14-3-3e protein in human skin in vivo Photoaging is caused by repeated sun exposure, and primarily by solar UV irradiation, and is of considerable importance in skin aging. The finding that 14-3-3e is increased in all of sun-exposed (facial) skins prompted us to examine the effect of UV irradiation on 14-3-3e gene expression in human skin in vivo. Thus, buttock skin was obtained from young subjects (n = 3) at 24, 48, and 72 h after UV irradiation (2 MED). Immunohistochemical staining revealed that the expression of 14-3-3e protein gradually upregulated at 24, 48, and 72 h post-UV irradiation versus non-irradiated control skin (Fig. 4A). Western blot analysis of irradiated buttock skin showed that the expression of 14-3-3e protein was significantly elevated 48 h after irradiation (Fig. 4B). In addition, the expression of 14-3-3e was induced by UV irradiation in NIH3T3 cell lines, thus supporting the in vivo results (Fig. 4C). The above findings demonstrate that 14-3-3e protein is induced and then accumulated after UV irradiation in human skin, and suggest the possibility that
accumulated 14-3-3e plays an important role in UVinduced and intrinsic aging.
4. Discussion We here investigated changes in the proteomic patterns of both young and aged dermis directly derived from sunprotected (upper inner arm) human skins by 2D-PAGE and mass spectrometry. This approach primarily focused on identifying proteins involved in intrinsic aging, and our results indicate that 14-3-3e protein is one such protein by proteomic analysis, Western blot, and immunohistochemical staining. Interestingly, 14-3-3e protein was induced by UV irradiation and its expression was up-regulated in sunexposed human skin in vivo, indicating that the enhanced expression of 14-3-3e could be related to both the photoaging and intrinsic aging processes in human skin. To our knowledge, this is the first report to identify a protein related to human skin aging by proteomic analysis. During the last decade, our understanding of the molecular mechanisms underlying skin aging has improved
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(Jenkins, 2002; Ma et al., 2001; Rittie and Fisher, 2002; Sohal and Weindruch, 1996). Emerging information reveals that although the process of skin aging can be considered to be due to intrinsic aging and photoaging, these two aging processes share fundamental molecular pathways. In particular, the process of intrinsic skin aging has been thought to be similar to the aging process in most internal organs, and to accompany the senescence of skin-derived cells (Campisi, 1996; Cristofalo and Pignolo, 1993), resistance to apoptotic death (Wang, 1995), altered signal transduction (Rittie and Fisher, 2002), and cumulative oxidative damage (Sohal and Weindruch, 1996). 14-3-3e protein is an isoform of the 14-3-3 protein family, members of which have been found in all eukaryotic organisms. In particular, seven isoforms have been found in humans (Fu et al., 2000). These proteins play critical roles in the cell signaling events that control cellular events through the cell cycle, transcriptional alterations in response to environmental cues, and programmed cell death (Fu et al., 2000; Tzivion and Avruch, 2002; van Hemert et al., 2001). In particular, 14-3-3 proteins bind to phosphoserine/phosphothreonine motifs in a sequence-specific manner (Muslin et al., 1996; Yaffe et al., 1997). And, it has been reported that 14-3-3e is essential for normal brain development and neuronal migration in mouse and this function is performed at least by binding to CDK5/p35 phosphorylated NUDEL (Toyo-oka et al., 2003). Thus it appears that 14-3-3e plays important roles in essential biological processes. The findings of the present study are of significance. First, the results demonstrate the possibility that the proteomics approach using human skin tissues can be used to identify age-related proteins. Although we report here upon the identification of 14-3-3e protein, several other proteins showing statistically significance are currently being analyzed. Second, it has not been previously reported that 14-3-3e is related to skin aging process. The functions of 143-3e are less clear than those of other 14-3-3 protein family members. Among its reported functions, 14-3-3e protein has been demonstrated to be involved in neuronal development (Toyo-oka et al., 2003), cellular signaling associated with the actin cytoskeleton (Gohla and Bokoch, 2002), the modulation of human DNA topoisomerase IIa function (Kurz et al., 2000), and the modulation of Ca2+-activated Cl channels (Chan et al., 2000). However, other isoforms of the 14-3-3e proteins have an overall inhibitory effect on cell cycle progression and apoptosis, and they may have dual roles as positive or negative factors during signal transduction (reviewed in van Hemert et al., 2001). Finally, we here present evidences that 14-3-3e is related to the photoaging and intrinsic aging of human skin. Our study shows that the expression of 14-3-3e is upregulated in the aged dermis versus young dermis, and further enhanced in the sunexposed dermis more than in the sun-protected dermis of the same elderly subjects (Figs. 2 and 3). In addition, sunexposed (facial) skin showed higher 14-3-3e expression than sun-protected (buttock) skin, and 14-3-3e was found to be
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induced by UV irradiation (Fig. 4). These results support the hypothesis that UV irradiation accelerates many key aspects of the chronological aging process in human skin. This is the first report of the induction of 14-3-3e by UV irradiation. In summary, our data suggest that altered signal transduction caused by the overexpression of 14-3-3e protein is an important skin aging factor, and imply that 14-3-3e has a novel function in human skin aging, and that its function may be modulated during the aging process.
Acknowledgements We thank Dr. Chang-Hun Lee and Hye-Young Cho for technical assistances of the proteomic analysis at the National Cancer Center, Korea. This study was supported by a grant by the Korea Health 21 R&D Project, Ministry of Health & Welfare of Korea (02-PJ1-PG1-CH10-0001).
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Oikarinen, A., 1990. The aging of skin: chronoaging versus photoaging. Photodermatol. Photoimmunol. Photomed. 7, 3–4. Rantala, I.S., Helin, H.J., Mustonen, J.T., Reunala, T., 1997. Age-related appearance of amyloid P component in human renal glomeruli. Nephron 77, 399–403. Rittie, L., Fisher, G.J., 2002. UV-light-induced signal cascades and skin aging. Ageing Res. Rev. 1, 705–720. Seo, J.Y., Lee, S.H., Youn, C.S., Choi, H.R., Rhie, G.E., Cho, K.H., Kim, K.H., Park, K.C., Eun, H.C., Chung, J.H., 2001. Ultraviolet radiation increases tropoelastin mRNA expression in the epidermis of human skin in vivo. J. Invest. Dermatol. 116, 915–919. Sohal, R.S., Allen, R.G., 1990. Oxidative stress as a causal factor in differentiation and aging: a unifying hypothesis. Exp. Gerontol. 25, 499–522. Sohal, R.S., Weindruch, R., 1996. Oxidative stress, caloric restriction, and aging. Science 273, 59–63. Toyo-oka, K., Shionoya, A., Gambello, M.J., Cardoso, C., Leventer, R., Ward, H.L., Ayala, R., Tsai, L.H., Dobyns, W., Ledbetter, D., Hirotsune, S., Wynshaw-Boris, A., 2003. 14-3-3epsilon is important for neuronal migration by binding to NUDEL: a molecular explanation for MillerDieker syndrome. Nat. Genet. 34, 274–285. Tzivion, G., Avruch, J., 2002. 14-3-3 proteins: active cofactors in cellular regulation by serine/threonine phosphorylation. J. Biol. Chem. 277, 3061–3064. van Hemert, M.J., Steensma, H.Y., vanHeusden, G.P., 2001. 14-3-3 proteins: key regulators of cell division, signaling and apoptosis. Bioessays 23, 936–946. Wang, E., 1995. Senescent human fibroblasts resist programmed cell death, and failure to suppress bcl2 is involved. Cancer Res. 55, 2284–2292. West, M.D., Pereira-Smith, O.M., Smith, J.R., 1989. Replicative senescence of human skin fibroblasts correlates with a loss of regulation and overexpression of collagenase activity. Exp. Cell Res. 184, 138–147. Wulfkuhle, J.D., McLean, K.C., Paweletz, C.P., Sgroi, D.C., Trock, B.J., Steeg, P.S., Petricoin, E.F., 2001. New approaches to proteomic analysis of breast cancer. Proteomics 1, 1205–1215. Yaffe, M.B., Rittinger, K., Volinia, S., Caron, P.R., Aitken, A., Leffers, H., Gamblin, S.J., Smerdon, S.J., Cantley, L.C., 1997. The structural basis for 14-3-3:phosphopeptide binding specificity. Cell 91, 961–971.
Increased expression of 14-3-3e protein in intrinsically aged and photoaged human skin in vivo Kyung-Chul Choia, Serah Leeb, Sun Young Kwaka, Mi-Sun Kimb, Hyo Kyoung Choia, Kyu Han Kimb, Jin Ho Chungb,*, Seok Hee Parka,* a
b
Department of Pathology, Inha University College of Medicine, Incheon 400-712, Republic of Korea Department of Dermatology, Seoul National University College of Medicine, Seoul 110-744, Republic of Korea Accepted 10 November 2004 Available online 21 January 2005
Abstract Skin aging is a complicated process associated with the passage of time and environmental exposure, especially to UV light. This aging phenomenon is related to alterations in various cellular mechanisms, such as changes in apoptosis, perturbations to cellular signaling, and an increased genetic instability. In this study, we investigated changes of proteins involved in intrinsic aging by the proteomic analysis of human sun-protected (upper inner arm) young and aged dermis. One of the proteins upregulated in aged dermis was identified as 14-3-3e. This protein is an isoform of 14-3-3 protein, which is involved in cellular processes like signal transduction, cell cycle arrest, and apoptosis. 14-3-3e is consistently found to be upregulated in the sun-protected dermis of aged skin, by Western blotting and immunohistochemical staining. In addition, we demonstrate that the expression of 14-3-3e is further upregulated in the sun-exposed (photodamaged) dermis, and that the UV irradiation of young skin significantly upregulates 14-3-3e in vivo. Our results suggest the possibility that the cellular processes related to 143-3e protein play an important role in the photoaging and intrinsic aging of human skin. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Skin aging; Proteomics; 14-3-3e; UV irradiation
1. Introduction Skin aging is a complex phenomenon that affects different constituents of human skin. The aging process of human skin can be considered to be due to intrinsic aging and photoaging (Gilchrest, 1982, 1989; Jenkins, 2002; Ma et al., 2001). The intrinsic aging is due to chronologic damage caused by slow, irreversible tissue degeneration whereas the photoaging is primarily the results of UV exposure (Jenkins, 2002; Ma et al., 2001). Clinically, chronologically aged skin is smooth, pale, and finely Abbreviations: 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; IPG, immobilized pH gradient; MALDI-TOF, matrix associated laser desorption/ionization time-of-flight * Corresponding authors. Tel.: +82 32 890 0942 (S.H. Park)/ 82 2 760 2414 (J.H. Chung); fax: +82 32 890 0944 (S.H. Park). E-mail addresses: [email protected] (J.H. Chung), [email protected] (S.H. Park).
wrinkled. In contrast, photoaged skin is coarsely wrinkled and associated with dyspigmentation and telagiectasia (Gilchrest, 1989; Lavker, 1979; Lavker and Kligman, 1988). The morphological and histological features of chronologically aged and photoaged skin are distinct. However, emerging evidence indicates that these processes have partly overlapping, biological, biochemical, and molecular mechanisms (Jenkins, 2002; Oikarinen, 1990; Rittie and Fisher, 2002). The basic principles underlying intrinsic aging are believed to be generally relevant in age-related diseases. Therefore, the skin represents an excellent and accessible model organ for the study of intrinsic aging. The molecular mechanisms underlying intrinsic skin aging are described by two processes: cellular senescence (Campisi, 1996; Dimri et al., 1995; West et al., 1989) and oxidative stress (Harman, 1956, 1981; Sohal and Allen, 1990; Sohal and Weindruch, 1996). Cellular senescence associated with intrinsic skin
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aging includes irreversible growth arrest (Cristofalo and Pignolo, 1993; Dimri et al., 1994; Hara et al., 1994; Hayflick, 1965) and the resistance of skin-derived cells to apoptosis (Wang, 1995), decreased matrix synthesis in the dermis, and the upregulation of enzymes that degrade the collagenous matrix (Chung et al., 2001; Millis et al., 1992). In contrast, oxidative stress concerns the contribution made by cumulative oxidative damage (Muscari et al., 1996; Sohal and Allen, 1990; Sohal and Weindruch, 1996). This is supported by a large body of experimental evidence and is particularly relevant in the skin given its high exposure to environmental agents. Both processes, however, have at least in part overlapping, biological, biochemical and molecular mechanisms (Oikarinen, 1990). Although studies have begun to reveal the molecular mechanisms underlying intrinsic skin aging, no study has been undertaken to compare the protein expressions in aged and young human skin. In recent years, protein expression profiling has been used successfully to identify proteins associated with the diseases, particularly cancers (Ahram et al., 2002; Bernard et al., 2003; Wulfkuhle et al., 2001). Thus, to reveal the candidate proteins involved in skin aging, we decided to analyze protein expressions in aged skin tissues by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry. In this study, we have investigated the changes in protein expressions in the dermis of naturally aged and young skin, using conventional proteomics, and identified 14-3-3e as a candidate protein by proteomic analysis. We here demonstrate that comparative proteomics is relevant for the analysis of intrinsic skin aging, and suggest the involvement of 14-3-3e in the skin aging process.
2. Materials and methods
(n = 3), provided both buttock and facial (crow’s feet area) skin samples. Each group was composed of three volunteers. The preparation of skin samples was approved by the Institutional Review Board at Seoul National University Hospital. 2.2. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) Immobilized pH gradient (IPG) strips, pH 3–10 (Amersham Biosciences, Uppsala, Sweden), were rehydrated in ES solution (7 M urea, 2 M thiourea, 2% CHAPS, 65 mM dithiothreitol, 0.01% bromophenol blue, 2 mM tributyl phosphine). The 200 mg of the protein extract derived from dermis was loaded and isoelectric focused for 32,000 Vh on an IPGphore unit (Amersham Biosciences). Two-dimensional SDS-PAGE was performed using 8–18% gels using the Ettan Dalt system (Amersham Biosciences). Proteins were visualized by silver nitrate staining. 2.3. Protein identifications by MALDI-TOF mass spectrometry The stained gels were scanned and analyzed using the Melanine IV image analysis program (Genbio, Geneva, Switzerland). Spots with consistently elevated intensities in the sun-protected (upper inner arm) dermis of aged people were excised, in-gel digested with 12.5 mg/ml trypsin and extracted according to instructions provided by Applied Biosystems (Foster City, USA). The eluted peptides were analyzed using a matrix associated laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometer (Applied Biosystems, Foster City, USA). Peptide masses were searched for in the Swiss-Prot and TrEMBL protein databases.
2.1. Skin samples and protein extractions 2.4. Western blot analysis Six young Koreans (age range 19–26 years) and six elderly Koreans (age range 60–87 years), without current or prior skin disease, provided both sun-protected and sunexposed skin samples. The specimens for proteomic analysis and Western blot were snap frozen in liquid nitrogen and the dermal proteins of each specimen were extracted as described by Chung et al. (2001). Briefly, the defrosted skin samples were heated at 55 8C for 2 min to separate the dermis from the epidermis with forceps. Each sample of dermis was homogenized in lysis buffer (containing 1% Triton X-100, 150 mM NaCl, 5 mM ethylenediamine tetraacetic acid, 0.1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 protein inhibitor mixtures from Promega). Lysates were rotated at 4 8C for 15 min and centrifuged at 130,000 g for 15 min, and the supernatant was used for proteomic and Western blot analysis. Another group of volunteers in three different age groups, representing ages 20–39 (n = 3), 40–59 (n = 3), and 60–79
About 50 mg of dermal proteins were electrophoresed on 10% SDS-PAGE gels and the separated proteins were detected using 14-3-3e antibody (Santa Cruz Biotechnology, Santa Cruz, USA) and horseradish peroxidase-conjugated secondary antibody according to the manufacturer’s protocol. Protein bands were visualized by enhanced chemiluminescence (Amersham Biosciences). 2.5. Immunohistochemical staining The frozen sections (8 mm) were mounted onto silane coated slides (Dako, Glostrup, Denmark), fixed in acetone, and stained with 14-3-3e antibody, which was applied for 1 h at room temperature. After rinsing in phosphate-buffered saline, sections were visualized using an LSAB kit (Dako, Glostrup, Denmark), which employed a biotinylated secondary antibody and horseradish-streptavidin conjugate.
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Table 1 Examples of increased proteins in the sun-protected (upper inner arm) dermis of aged and young skin Identified proteins
OID/YIDa (fold increase)
Frequencyb
14-3-3e Serum amyloid P Component (SAP) Mitochondrial malate dehydrogenase (mMDH) Mitochondrial L-lactate dehydrogenase (mLDH) Carbonic anhydrase 1 (CA1)c Apolipoprotein I Glutathione S transferase Annexin V
3.5 8.5
3/3 3/3
2.6 3.6 0.24 2.3 2.1 2.1
3/3 3/3 2/3 2/3 2/3 2/3
a b c
OID: old inner arm dermal protein; YID: young inner arm dermal protein. The sample numbers showing the same pattern of protein expression/the analyzed sample numbers. CA1 represents a protein down-regulated in the upper inner arm dermis of aged subjects.
3-Amino-9-ethylcarbazole was used as chromogenic substrate. Sections were counterstained briefly in Mayer’s hematoxylin. The negative control, prepared using mouse IgG showed no immunoreactivity (data not shown). 2.6. UV irradiation and skin samples Healthy adult Korean subjects (three subjects, age range 20–29 years), were irradiated with F75/85W/UV21 fluorescent sunlamps, as described previously (Chung et al., 2002; Seo et al., 2001). The minimal erythema dose (MED) for each subject was determined 24 h after UV irradiation. Irradiated and non-irradiated skin samples were obtained from each subject by punch-biopsy and used for immunohistochemical staining and Western blot analysis. The preparations of skin samples were approved by the
Institutional Review Board at Seoul National University Hospital. NIH3T3 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamate, and antibiotics. After starvation for 24 h in DMEM without FBS, NIH3T3 cells were irradiated with UV, as previously described (Chung et al., 2002), and incubated for the indicated times. At the end of the incubation, protein extracts were prepared and used for Western blot analysis against 14-3-3e protein. 2.7. Statistical analysis Statistical analyses were performed using the Mann– Whitney U-test. A p-value of less than 0.05 was considered statistically significant.
Fig. 1. The maps of proteins of young-aged and old-aged sun-protected (upper inner arm) dermis by two-dimensional polyacrylamide gel electrophoresis (2DPAGE). Total dermal proteins were prepared from the punch-biopsied specimens of the sun-protected (upper inner arm) skin of both young and elderly subjects. The proteomic map shown in this figure is typical of experiments using samples of young (n = 3) and old (n = 3) upper inner arm. The lower panel shows 14-3-3e and serum amyloid P component spots, which were found to be consistently elevated in the aged dermis vs. young dermis. These spots were identified by MALDI-TOF peptide mass fingerprinting.
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3. Results 3.1. The upregulation of 14-3-3e protein was identified by proteomic analysis of aged human skin dermis To identify proteins related to intrinsic skin aging, total dermal proteins were prepared from the punch-biopsied specimens of the sun-protected (upper inner arm) skins of young and elderly subjects. Three pairs of young and aged sunprotected (upper inner arm) skin samples were taken by punchbiopsy from each subject, and used for proteomic analysis. These dermal proteins were separated by 2D-PAGE using pH 3–10 IPG in the first dimension. Protein expression changes between the young and aged dermis were determined using the Melanine IVimage analysis program. Differentially expressed proteins, showing consistent increases in all three elderly subjects,were excisedfrom silver-stainedgels andanalyzedby MALDI-TOF mass spectrometry (Table 1). Of these proteins, 14-3-3e protein was found to be significantly upregulated in the aged dermis (Fig. 1). 14-3-
3e protein is an isoform of the 14-3-3 protein family. Members of the 14-3-3 family are ubiquitous proteins that are highly conserved in bacteria, plants, and humans, and are involved in important cellular processes, such as, signal transduction, cell cycle control, apoptosis, and stress responses (Fu et al., 2000; Muslin and Xing, 2000; Tzivion and Avruch, 2002). Serum amyloid P component (SAP) was also consistently increased in the aged dermis. This has previously been reported to be increased in human renal glomeluli and testis in an age-dependent manner (Herriot and Walker, 1989; Rantala et al., 1997), which demonstrates the relevance of proteomic analysis. 3.2. 14-3-3e protein is upregulated in the dermis of aged human skin in vivo To verify the increased expression of 14-3-3e protein in the aged dermis, Western blot analysis was performed using dermal proteins extracted from human skin. As shown in
Fig. 2. Increased expression of 14-3-3e protein in the sun-protected (upper inner arm) dermis of aged human skin. (A) Western blot analysis of 14-3-3e protein was performed by using dermal proteins derived from young (n = 3) and old (n = 3) inner arm skin tissues. Dermal proteins were separated by SDS-PAGE and probed with the antibody against 14-3-3e protein (32 kDa). YI: young inner arm, OI: old inner arm. (B) The expression of 14-3-3e protein in human skin was detected by immunohistochemical staining. The frozen sections of 8 mm thickness were mounted onto silane coated slides, fixed in acetone, and stained with 143-3e antibody, as described in Section 2. The sun-protected (upper inner arm) young skins (n = 3) and sun-protected (upper inner arm) and sun-exposed (forearm) old skins (n = 3) were used for immunohistochemical staining. Immunohistochemical stainings were performed under the same experimental conditions. The insets show representative results of at least three independent experiments.
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Fig. 3. Intrinsic aging and photoaging effects on 14-3-3e protein expression in human skin in vivo. The grading of photoaging in Korean subjects has been described previously (Chung et al., 2001). Grades 1 and 2 represent the 20 to 39-year-old age group, and Grades 3 and 4 and Grade 5 and 6 indicate groups from 40 to 59 and 60 to 79 years. The expression of 14-3-3e protein was detected using 14-3-3e antibody by immunohistochemical staining. Immunohistochemical staining of human skin samples derived from each group was performed under the same experimental conditions. Three subjects in each group were used for these experiments. The inset shows representative immunohistochemical staining results from three subjects.
Fig. 2A, 14-3-3e proteins in the dermis of three different elderly subjects, were significantly upregulated versus three young subjects. Theses results indicate that 14-3-3e or a 143-3e-related function plays an important role in intrinsic aging. Next, we immunohistochemical stained the inner arm skin tissues of both young (n = 3) and old (n = 3) subjects to compare their expressions of 14-3-3e protein in vivo. The results obtained showed significantly higher expression of 14-3-3e protein in the aged dermis (Fig. 2B). We also observed that 14-3-3e protein appeared to be upregulated in the epidermis of aged skin versus young skin (Fig. 2B). Although intrinsic aging and photoaging are considered to be distinct entities during skin aging, emerging data suggest that UV irradiation, the most important factor in photoaging, accelerates the intrinsic aging process (Jenkins, 2002; Rittie and Fisher, 2002). Thus, we examined the expression of 14-3-3e protein in the photoaged dermis of the same old-aged people; interestingly, the expression of 14-3-3e was significantly increased in the photoaged versus the intrinsically aged dermis (upper inner arm) (Fig. 2B). These results strongly suggest that 14-3-3e protein is specifically upregulated in the human dermis in an age-dependent manner, and raise the possibility that 14-3-3e-related cellular signaling may
be modulated in both the photoaging and intrinsic aging processes. 3.3. Expression of 14-3-3e protein is significantly affected by photoaging and by the intrinsic aging To confirm that the photoaging process affects the expression of 14-3-3e protein, we investigated changes in 14-3-3e protein expression caused by intrinsic aging and photoaging over a lifetime. Sun-exposed (crow’s feet area) and sun-protected (buttock) skin samples (n = 3) were taken from individuals belonging to three different age groups and immunohistochemically stained for 14-3-3e protein (Fig. 3). 14-3-3e protein showed higher expression in the buttock dermis of the elderly group >60 years than in young buttock dermis, supporting our previous result that 14-3-3e is upregulated during intrinsic aging (Fig. 3). Interestingly, the expression of 14-3-3e protein was significantly upregulated in sun-exposed dermis in all aged groups, compared with the corresponding sun-protected dermis (Fig. 3). Moreover, facial (photoaged) skin >60 years showed higher dermal expression than the other age groups (Fig. 3). These results indicate that the expression of 14-3-3e is affected by both the intrinsic and photoaging process, and suggest the possibility that photoaging may accelerate 14-3-3e accumulation in the dermis.
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Fig. 4. Expression of 14-3-3e protein following UV irradiation in human skin in vivo and in NIH3T3 cells. (A) Human buttock skin (n = 3) was irradiated with 2 MED of UV. Immunohistochemical staining against 14-3-3e protein was performed using human skin samples obtained by punch-biopsy at the indicated times after UV irradiation. Three subjects (n = 3) were irradiated in these experiments. The inset shows representative immunohistochemical staining from three different subjects. The control is of a non-irradiated sample. (B) Western blot analysis against the 14-3-3e protein was performed on non-irradiated and irradiated skin samples. Buttock skins were obtained from young subjects (n = 3) at 24, 48, and 72 h after UV irradiation (2 MED). Total proteins were prepared from non-irradiated and irradiated human skin samples taken at the indicated times and used for Western blot analysis against the 14-3-3e protein. The result is representative of the results obtained in three independent subjects. Data are expressed as percentage changes in 14-3-3e protein levels relative to levels in nonirradiated control skin. Data are normalized vs. a b-actin level used as an internal control for quantitation purposes. Control (C) indicates a non-irradiated skin sample. * p < 0.05 vs. non-irradiated control skin, n = 3. (C) NIH3T3 cells were irradiated with UV, 30 mJ/cm2, and incubated for the indicated times following UV irradiation. Total proteins were prepared from non-irradiated and irradiated samples and used for Western blot analysis against the 14-3-3e protein. b-actin was used as an internal control. The inset shows a Western blot which is representative of three independent experiments.
3.4. UV irradiation induces the expression of 14-3-3e protein in human skin in vivo Photoaging is caused by repeated sun exposure, and primarily by solar UV irradiation, and is of considerable importance in skin aging. The finding that 14-3-3e is increased in all of sun-exposed (facial) skins prompted us to examine the effect of UV irradiation on 14-3-3e gene expression in human skin in vivo. Thus, buttock skin was obtained from young subjects (n = 3) at 24, 48, and 72 h after UV irradiation (2 MED). Immunohistochemical staining revealed that the expression of 14-3-3e protein gradually upregulated at 24, 48, and 72 h post-UV irradiation versus non-irradiated control skin (Fig. 4A). Western blot analysis of irradiated buttock skin showed that the expression of 14-3-3e protein was significantly elevated 48 h after irradiation (Fig. 4B). In addition, the expression of 14-3-3e was induced by UV irradiation in NIH3T3 cell lines, thus supporting the in vivo results (Fig. 4C). The above findings demonstrate that 14-3-3e protein is induced and then accumulated after UV irradiation in human skin, and suggest the possibility that
accumulated 14-3-3e plays an important role in UVinduced and intrinsic aging.
4. Discussion We here investigated changes in the proteomic patterns of both young and aged dermis directly derived from sunprotected (upper inner arm) human skins by 2D-PAGE and mass spectrometry. This approach primarily focused on identifying proteins involved in intrinsic aging, and our results indicate that 14-3-3e protein is one such protein by proteomic analysis, Western blot, and immunohistochemical staining. Interestingly, 14-3-3e protein was induced by UV irradiation and its expression was up-regulated in sunexposed human skin in vivo, indicating that the enhanced expression of 14-3-3e could be related to both the photoaging and intrinsic aging processes in human skin. To our knowledge, this is the first report to identify a protein related to human skin aging by proteomic analysis. During the last decade, our understanding of the molecular mechanisms underlying skin aging has improved
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(Jenkins, 2002; Ma et al., 2001; Rittie and Fisher, 2002; Sohal and Weindruch, 1996). Emerging information reveals that although the process of skin aging can be considered to be due to intrinsic aging and photoaging, these two aging processes share fundamental molecular pathways. In particular, the process of intrinsic skin aging has been thought to be similar to the aging process in most internal organs, and to accompany the senescence of skin-derived cells (Campisi, 1996; Cristofalo and Pignolo, 1993), resistance to apoptotic death (Wang, 1995), altered signal transduction (Rittie and Fisher, 2002), and cumulative oxidative damage (Sohal and Weindruch, 1996). 14-3-3e protein is an isoform of the 14-3-3 protein family, members of which have been found in all eukaryotic organisms. In particular, seven isoforms have been found in humans (Fu et al., 2000). These proteins play critical roles in the cell signaling events that control cellular events through the cell cycle, transcriptional alterations in response to environmental cues, and programmed cell death (Fu et al., 2000; Tzivion and Avruch, 2002; van Hemert et al., 2001). In particular, 14-3-3 proteins bind to phosphoserine/phosphothreonine motifs in a sequence-specific manner (Muslin et al., 1996; Yaffe et al., 1997). And, it has been reported that 14-3-3e is essential for normal brain development and neuronal migration in mouse and this function is performed at least by binding to CDK5/p35 phosphorylated NUDEL (Toyo-oka et al., 2003). Thus it appears that 14-3-3e plays important roles in essential biological processes. The findings of the present study are of significance. First, the results demonstrate the possibility that the proteomics approach using human skin tissues can be used to identify age-related proteins. Although we report here upon the identification of 14-3-3e protein, several other proteins showing statistically significance are currently being analyzed. Second, it has not been previously reported that 14-3-3e is related to skin aging process. The functions of 143-3e are less clear than those of other 14-3-3 protein family members. Among its reported functions, 14-3-3e protein has been demonstrated to be involved in neuronal development (Toyo-oka et al., 2003), cellular signaling associated with the actin cytoskeleton (Gohla and Bokoch, 2002), the modulation of human DNA topoisomerase IIa function (Kurz et al., 2000), and the modulation of Ca2+-activated Cl channels (Chan et al., 2000). However, other isoforms of the 14-3-3e proteins have an overall inhibitory effect on cell cycle progression and apoptosis, and they may have dual roles as positive or negative factors during signal transduction (reviewed in van Hemert et al., 2001). Finally, we here present evidences that 14-3-3e is related to the photoaging and intrinsic aging of human skin. Our study shows that the expression of 14-3-3e is upregulated in the aged dermis versus young dermis, and further enhanced in the sunexposed dermis more than in the sun-protected dermis of the same elderly subjects (Figs. 2 and 3). In addition, sunexposed (facial) skin showed higher 14-3-3e expression than sun-protected (buttock) skin, and 14-3-3e was found to be
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induced by UV irradiation (Fig. 4). These results support the hypothesis that UV irradiation accelerates many key aspects of the chronological aging process in human skin. This is the first report of the induction of 14-3-3e by UV irradiation. In summary, our data suggest that altered signal transduction caused by the overexpression of 14-3-3e protein is an important skin aging factor, and imply that 14-3-3e has a novel function in human skin aging, and that its function may be modulated during the aging process.
Acknowledgements We thank Dr. Chang-Hun Lee and Hye-Young Cho for technical assistances of the proteomic analysis at the National Cancer Center, Korea. This study was supported by a grant by the Korea Health 21 R&D Project, Ministry of Health & Welfare of Korea (02-PJ1-PG1-CH10-0001).
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