Results

Effects of DHEA on the expressions of type I procollagen, MMP-1, and TIMP-1 in cultured human dermal fibroblasts

We studied the effects of DHEA on the expressions of type I procollagen, MMP-1, and TIMP-1 proteins in cultured human dermal fibroblasts. Human dermal fibroblasts were treated with 10 M DHEA for 24, 48, and 72 h, and the expression levels of these proteins were determined in the culture media by western blot. We found that DHEA increased the expression of type I procollagen and TIMP-1 protein, and decreased MMP-1 protein maximally at 72 h post-treatment (data not shown). Human dermal fibroblasts were then treated with various doses of DHEA for 72 h to investigate its concentration-dependent effects. DHEA significantly increased type I procollagen protein expression (Figure 1a), and concentration-dependently decreased MMP-1 protein expression, versus the vehicle-treated controls (Figure 1b). The expression of TIMP-1 protein was increased in a concentration-dependent manner, compared with the non-DHEA-treated controls (Figure 1c).

Figure 1.

The effects of dehydroepiandrosterone (DHEA) on the expressions of type I procollagen, matrix metalloprotease (MMP)-1, tissue inhibitor of metalloprotease (TIMP)-1 proteins in human dermal fibroblasts. Human dermal fibroblast cultures were treated with 10, 50, and 100 M DHEA for 72 h. The expression levels of (a) type I procollagen, (b) MMP-1, and (c) TIMP-1 were analyzed by western blotting, as described in "Material and Methods". Results shown are meansSEM. n=5, ^*p<0.05, ^**p<0.01.

Full figure and legend (45K)

DHEA prevented UV-induced changes of MMP-1 and type I procollagen expression and inhibited UV-induced AP-1 binding activity in human dermal fibroblasts

Previously it has been shown that UVR decreases procollagen synthesis (^{Fisher et al, 2000}) and induces the synthesis of MMP, such as MMP-1, MMP-3, and MMP-9, in human skin in vivo (^{Fisher et al, 1997}). To determine whether DHEA has any inhibitory effect on the UV-induced changes of procollagen, MMP-1, and TIMP-1 protein expression, human dermal fibroblasts were irradiated with 100 mJ per cm² of UV light with or without DHEA (10–50 M) treatment. UVR decreased type I procollagen expression (Figure 2a, p<0.01, n=5) and induced the expression of MMP-1 protein (Figure 2b, p<0.01, n=5). DHEA treatment (50 M) significantly prevented the down-regulation of type I procollagen by UV. On the other hand, UV-induced MMP-1 protein expression was inhibited significantly by DHEA pretreatment (10 M and 50 M) (p<0.05, n=5). The expression of TIMP-1 protein was increased by UV, and pretreatment with DHEA (10 and 50 M) augmented the UV-induced TIMP-1 expression in cultured fibroblasts in a concentration-dependent manner (Figure 2c).

Figure 2.

Dehydroepiandrosterone (DHEA) prevented UV-induced expression of matrix metalloprotease (MMP)-1 and type I procollagen, and AP-1 binding activity in cultured human dermal fibroblasts. Cultured fibroblast cells were exposed to 100 mJ per cm² of UV irradiation, as described in "Material and Methods". After UV irradiation, cells were treated with 10 or 50 M DHEA for 72 h. The expression levels of (a) type I procollagen (n=5), (b) MMP-1 (n=5), and (c) tissue inhibitor of metalloprotease (TIMP)-1 (n=4) were analyzed by western blotting. Results shown are meansSEM. ^*p<0.05, ^**p<0.01, vehicle versus UV irradiation. §p<0.05, UV irradiation versus DHEA treated groups. (d) After UV irradiation, cells were treated with 10, 50, or 100 M DHEA for 12 h. AP-1 binding activity was analyzed by electrophoretic mobility shift assays. The photograph is representative of three identical experiments. NS, non-specific binding.

Full figure and legend (75K)

It is known that activated AP-1 stimulates MMP-1 production (^{Fisher et al, 1997}), but inhibits type I procollagen expression transcription (^{Chung et al, 1996b}). Therefore, we examined whether DHEA has any suppressive effect on UV-induced AP-1 DNA binding activity in human dermal fibroblasts. Cultured fibroblasts were irradiated with 100 mJ per cm² of UV light, and DHEA (10, 50, and 100 M) was treated immediately after this irradiation for 12 h. UVR induced AP-1 binding activity, and DHEA inhibited this UV-induced AP-1 DNA binding activity concentration-dependently (Figure 2d).

The topical application of DHEA increased the expressions of type I procollagen mRNA and protein in human skin in vivo

To investigate whether DHEA affects collagen metabolism in human skin in vivo, as it does in cultured fibroblasts, the buttock skins of young and aged volunteers were treated with 5% DHEA solution three times per week for 4 wk under occlusion. Vehicle (ethanol:olive oil (1:2))-treated skin served as a control. Using in situ hybridization (Figure 3a) and immunohistochemical staining (Figure 3b), we found that topical DHEA treatment increased the expression of type I procollagen mRNA (Figure 3a) and protein (Figure 3b) significantly in both aged (n=6) and young (n=5) skin in vivo. These results were confirmed by RT-PCR using total RNA isolated from the biopsied skin samples and by western blotting using the soluble protein fraction extracted from the dermis. Topical application of DHEA significantly increased the expression of procollagen 1(I) mRNA in aged (n=4, p<0.01) and young skin (n=5, p<0.05), compared with vehicle-treated control skin (Figure 3c). The expression of type I procollagen protein was also increased in the DHEA-treated aged (n=5, p<0.05) and young (n=5, p<0.05) skin compared with the vehicle-treated control (Figure 3d).

Figure 3.

Topical application of dehydroepiandrosterone (DHEA) increases the expression of type I procollagen mRNA and protein in human skin in vivo. 5% DHEA or its vehicle (ethanol:olive oil (1:2)) were applied to the buttock skin 12 times for 4 wk under occlusion. (a) The expression of type I procollagen mRNA in the dermis of aged (n=6) and young (n=5) skin was detected by in situ hybridization. Areas in boxes are shown as 2.5-fold enlargements. (b) The expression of type I procollagen protein in the dermis was detected by immunohistochemical staining in aged (n=6) and young (n=5) skin. (c) The expressions of type I procollagen and glyceraldehyde-3-phosphate dehydrogenase mRNA were measured in total RNA extracted from skin samples of the control and DHEA-treated subjects, respectively, by RT-PCR. The photographs are representative of aged (n=4) and young (n=5) subjects, respectively. The results shown are meansSEM. ^*p<0.05, ^**p<0.01. (d) The expression of type I procollagen protein was measured in soluble proteins extracted from the dermis of the control and DHEA-treated subjects, respectively, by western blotting. The photographs are representative of aged (n=5) and young (n=5) DHEA-treated subjects. Results shown are meansSEM. ^*p<0.05.

Full figure and legend (58K)

The topical application of DHEA reduced the expressions of MMP-1 mRNA and protein and increased the expression of TIMP-1 protein in human skin in vivo

To investigate the effects of DHEA on the expression of MMP-1 mRNA in human skin in vivo, RT-PCR analysis was performed using total RNA isolated from the biopsied skin samples of elderly volunteers. Topical DHEA treatment significantly reduced MMP-1 mRNA expression (n=5, p<0.01), compared with the vehicle-treated skin (Figure 4a). By western blot analysis, we found that the topical application of DHEA reduced the MMP-1 protein level in the epidermis (n=4, p<0.05) versus vehicle-treated skin (Figure 4b). On the other hand, MMP-1 protein bands in the dermal extracts were too faint to be detected by western blot.

Figure 4.

Topical application of dehydroepiandrosterone (DHEA) decreases the matrix metalloproteinase (MMP)-1 expression and increases the expression of tissue inhibitor of metalloprotease (TIMP)-1 protein in human skin in vivo. The expressions of (a) MMP-1, (c) TIMP-1 mRNA were analyzed by RT-PCR, as described in "Material and Methods". The photographs are representative of five controls and five DHEA-treated subjects. (b) The expression of MMP-1 protein was measured in soluble proteins extracted from the epidermis of control and DHEA-treated subjects, by western blotting. The photographs are representative of four control and four DHEA-treated subjects. (d) The expression of TIMP-1 protein was measured in soluble proteins extracted from the dermis of control and DHEA-treated subjects, by western blotting. The photographs are representative of six control and six DHEA-treated subjects. Results shown are meansSEM. ^*p<0.05, ^**p<0.01.

Full figure and legend (53K)

We demonstrated that topical DHEA treatment did not significantly change the TIMP-1 mRNA expression (n=5, Figure 4c), by RT_PCR using total RNA isolated from punch-biopsied skin samples of elderly volunteers. The topical application of DHEA, however, significantly increased the expression of TIMP-1 protein (n=6, p<0.01) in the dermis, versus vehicle-treated skin (Figure 4d). TIMP-1 protein expression was not detec in epidermal extracts (data not shown).

DHEA increased the expressions of TGF-1 and CTGF mRNA in cultured human dermal fibroblasts and human skin in vivo

To understand the mechanism of the DHEA-induced upregulation of procollagen expression, we investigated the effects of DHEA on the expressions of TGF-1 and CTGF mRNA in cultured human dermal fibroblasts and human skin in vivo. RT-PCR analysis, using total RNA isolated from the fibroblast, showed that DHEA (10 M) treatment significantly increased TGF-1 mRNA level (n=3, p<0.05) at 24 h post-treatment (Figure 5a), and CTGF mRNA expression (n=3, p<0.05) at 48 h post-treatment (Figure 5b). By using total RNA isolated from the biopsied skin samples, we also demonstrated that DHEA treatment significantly increased TGF-1 and CTGF mRNA level (n=4, p<0.05) in human skin in vivo (Figure 5c).

Figure 5.

Dehydroepiandrosterone (DHEA) increased the expressions of transforming growth factor-1 (TGF-1) and connective tissue growth factor (CTGF) mRNA in cultured fibroblasts and aged human skin in vivo. Human dermal fibroblast cultures (n=3) were treated with 10, 50, and 100 M DHEA for 24 and 48 h. The expression levels of (a) TGF-1 mRNA and (b) CTGF mRNA were analyzed by RT-PCR, as described in "Material and Methods". (c) The expressions of TGF-1, CTGF, and GAPDH mRNA in aged human skin (n=4) were analyzed by RT-PCR. The photographs are representative of four control and four DHEA-treated subjects. Results shown are meansSEM. ^*p<0.05.

Full figure and legend (55K)

Discussion

Alterations in collagen, the major structural component of skin, have been suggested to be a cause of the clinical changes observed in photoaged and naturally aged skin (^{Fisher et al, 1997; Varani et al, 2000}). The dermis contains predominantly type I collagen (85%–90%) with lesser amounts of type III collagen (10%–15%). Dermal fibroblasts synthesize the individual polypeptide chains of types I and III collagen as precursor molecules called procollagen.^{Fisher et al (1996, 1997)} showed that UV irradiation induces the synthesis of MMP in human skin in vivo. Moreover, MMP-mediated collagen destruction accounts for a large part of the connective tissue damage that occurs in photoaged skin. Recently, it was suggested that collagen damage might occur during natural skin aging, as it does in photoaging, from elevated MMP expression and a concomitant reduction in collagen synthesis.^{Varani et al (2000)} reported that with increasing age, MMP levels become higher and collagen synthesis lower in sun-protected human skin in vivo.

In this study, we found that DHEA can increase the procollagen synthesis and inhibit collagen degradation by decreasing MMP-1 synthesis and increasing TIMP-1 production in cultured dermal fibroblasts. Several studies have reported the effects of DHEA on the regulation of collagen and MMP gene expressions, and some discrepancies exist among these reports. DHEA increased human 1 (I) procollagen and reduced collagenase (MMP-1) gene expression in cultured skin fibroblasts (^{Lee et al, 2000}). In human cultured chondrocytes, DHEA increased type II collagen and TIMP-1 gene expressions, but decreased type I collagen, MMP-1, and MMP-3 expression (^{Jo et al, 2003}). On the other hand, DHEA-S stimulated collagenase and gelatinase production, but did not alter collagen production in rabbit cervical tissue (^{Sakyo et al, 1987}). It has also been reported that DHEA-S increases the collagenase activity of cervical fibroblasts in rabbits, whereas DHEA does not (^{Sakyo et al, 1986}). This result suggests that the stimulatory effect of DHEA-S on the production of collagenase and gelatinase is due to unchanged DHEA-S, and not to DHEA converted from DHEA-S (^{Sakyo et al, 1986}). In this study, the DHEA treatment of cultured human dermal fibroblasts increased type I procollagen protein expression significantly and reduced the production of MMP-1 protein in a concentration-dependent manner. DHEA also significantly increased the production of TIMP-1 protein in a concentration-dependent manner. These results indicate that DHEA plays an important role in the regulation of the production and degradation of the extracellular matrix. Differences between our results and those of other studies may be due to tissue- and/or species-specific DHEA effects, the different biological effects of DHEA-S and DHEA, and the concentration of DHEA used in the studies.

We also found that DHEA can prevent the UV-induced increase of MMP-1, and the UV-induced decrease of procollagen production in cultured dermal fibroblasts. Therefore, our results strongly suggest the possibility that DHEA is a candidate agent for the prevention and treatment of clinical changes, such as the skin wrinkles and laxity, which typify photoaged and naturally aged skins. Upon the UV irradiation of human skin, c-fos protein heterodimerizes with UV-induced c-jun, and forms an active form of the transcription factor AP-1. This UV-activated AP-1 stimulates the transcription of MMP gene encoding collagenase, 92 kDa gelatinase, and stromelysin-1 in both keratinocytes and fibroblasts (^{Fisher et al, 1996, 1997}) These UV-induced MMPs degrade collagen and other components in the dermal exracellular matrix in human skin. With each intermittent UV exposure, MMP-mediated dermal damage accumulates, and damages the structural integrity of the dermis. Eventually this process manifests itself as clinically observable solar scars (i.e., wrinkles) in chronically photodamaged skin (^{Fisher et al, 1997}). In addition, AP-1 is also known to regulate type I procollagen gene expression negatively (^{Chung et al, 1996b}). In this study, we found that DHEA prevents UV-induced AP-1 DNA binding activity concentration-dependently in cultured dermal fibroblasts. Therefore, DHEA can inhibit UV-induced MMP-1 production and procollagen synthesis, probably by inhibiting UV-induced AP-1 activity. It has been reported that AP-1 stimulates TIMP-1 gene expression (^{Smart et al, 2001; Hall et al, 2003}). In this study, however, DHEA augmented UV-induced TIMP-1 up-regulation, even though DHEA inhibited UV-induced AP-1 activity. These results suggest that DHEA increases the expression of TIMP-1 through an AP-1-independent pathway.

We found that topically applied 5% DHEA on aged human skin in vivo significantly increased the expression of procollagen 1 (I) mRNA and protein. On the other hand, it reduced the basal expressions of MMP-1 mRNA and protein significantly, and increased the expressions of TIMP-1 protein in aged skin. It is well known that TGF- potently stimulates collagen synthesis, and it has been demonstrated that TGF- decreases MMP-1 expression and increases TIMP-1 (^{Edwards et al, 1996; Eickelberg et al, 1999; Hall et al, 2003}). CTGF is constitutively expressed in normal human skin in vivo, and in normal human skin keratinocytes and fibroblasts (^{Quan et al, 2002}). Moreover, CTGF expression is rapidly induced by TGF- in skin fibroblasts, and accumulating evidence indicates that the inductive effects of TGF- on procollagen production are mediated by CTGF (^{Grotendorst, 1997; Duncan et al, 1999; Quan et al, 2002}). CTGF also stimulated procollagen production when injected into mouse skin or when added to fibroblast culture (^{Duncan et al, 1999}). In this study, we found that DHEA significantly increased the expressions of TGF-1 and CTGF mRNA in cultured human fibroblast and aged human skin in vivo. These results suggest that DHEA-induced TGF-1 may be involved in the induction of procollagen and TIMP-1 expression and in the inhibition of MMP-1 expression in cultured human fibroblast and aged human skin in vivo. DHEA may stimulate procollagen synthesis in aged human skin by inducing CTGF by increasing TGF-1 expression. To our knowledge, this is a report regarding the effects of topical DHEA on collagen metabolism in aged human skin in vivo.

DHEA is a multifunctional hormone with beneficial effects, which include an anti-aging effect (^{Yen, 2001}). The adrenal glands of humans synthesize and secrete large amounts of DHEA and DHEA-S that are biotransformed into biologically active androgens and estrogens in peripheral tissues. It is estimated that more than 30% of total androgen in men and over 90% of estrogen in postmenopausal women are derived from the peripheral conversion of DHEA/DHEA-S (^{Labrie et al, 1995}). Thus, the intracellular biotransformation of DHEA to active sex steroids may allow local binding to their specific intracellular nuclear receptors with minimal loss in terms of concentration and time, to exert maximal functional effect (^{Labrie et al, 1995}). In this study, the observed effects of DHEA on collagen metabolism in aged human skin may have been the result of DHEA androgenic or estrogenic action. It has been suggested that the potent stimulatory effect of DHEA on bone formation in ovariectomized rats is mainly due to the local formation of androgens in bone cells, because it was inhibited by antiandrogen treatment but not by antiestrogen treatment (^{Martel et al, 1998}). Further investigation is necessary to understand the molecular mechanisms of the hormonal effects of DHEA on collagen metabolism in aged human skin.

In conclusion, we propose that DHEA has the potential to be used topically for the treatment and prevention of skin aging processes, based on the following; (1) DHEA stimulates procollagen and TIMP-1 protein production and decreases MMP-1 expression in cultured fibroblasts. (2) DHEA prevents UV-induced changes of MMP-1 and procollagen expression by inhibiting UV-induced AP-1 activation. (3) The topical application of DHEA in aged human skin increases procollagen and TIMP-1 levels and reduces MMP-1 expression. (4) Topical application of DHEA increases TGF-1 and CTGF expression to induce procollagen synthesis in aged skin.

Materials and Methods

Fibroblast cell culture

Primary cultures of dermal fibroblasts were established from human adult foreskins in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal calf serum, 2 mM glutamine, penicillin (100 U per mL), and streptomycin (100 g per mL) in a 37°C humidified 5% CO₂ incubator. Ascorbic acid was not added in the culture media, and fibroblasts cultured in this culture media could synthesize collagen normally as observed previously (^{Chung et al, 1996a, 1997}). The fibroblasts were cultured until 90% confluent and then subcultivated. Cells cultured >five passages were used for the experiments.

Human skin samples

A total of six elderly Koreans (six men; mean age, 80 y; age range, 75–87 y) and five young Koreans (five men; mean age, 23 y; age range, 22–24 y), without current or prior skin disease, provided skin samples. To observe the effects of the topical application of DHEA, a 5% DHEA solution and its vehicle (ethanol:olive oil (1:2)) were applied to the buttock skin (3 3 cm) 12 times over 4 wk under occlusion. DHEA treatment did not change the serum levels of DHEA, DHEA-S, and testosterone (data not shown). Skin samples were then obtained from the subjects by punch biopsy, as previously described (^{Seo et al, 2001}). This study was conducted according to the Declaration of Helsinki Principles. All procedures involving human subjects received prior approval from the Seoul National University Institutional Review Board, and all subjects provided written informed consent.

To separate epidermis from dermis, a defrosted whole skin sample was placed dermis-side down on a Petri dish, heated at 55°C for 2 min, and then separated gently with a forceps. The separated epidermis and dermis were then frozen in liquid nitrogen and stored at -80°C until use.

UVR

The UV light source was an F75/85W/UV21 fluorescent sun lamp, with an emission spectrum between 285 and 350 nm (peak at 310–315 nm), as previously described (^{Chung et al, 2003}). A Kodacel filter (TA401/407; Kodak, Rochester, New York) was mounted 2 cm in front of the UV tubes to remove wavelengths <290 nm (UVC).

The fibroblasts were grown in 10 cm culture dishes (Falcon, Lincoln Park, New Jersey) until subconfluent. The cells were then cultured in serum-free medium for 24 h, the medium was replaced by 2 mL of phosphate-buffered saline, and the cells were exposed to UV (0–100 mJ per cm²) light. After this irradiation, the cells were washed with phosphate-buffered saline, and cultured in DMEM with or without DHEA for the indicated times.

DHEA

DHEA was purchased from Sigma (St Louis, Missouri) and was used after dissolution either in dimethyl sulfoxide (DMSO) or ethanol:olive oil (1:2). Cultured fibroblasts were treated with various doses of DHEA dissolved in DMSO. Elderly volunteers were treated on buttock skin with 5% DHEA (12 L per cm²) for 12 times over 4 wk under occlusion. Ethanol:olive oil (1:2) was used as the vehicle.

Western blot analysis

Soluble protein fractions were extracted either from the punch-biopsied skin samples or from cultured fibroblasts using WCE buffer (containing 1% Triton X-100, 150 mM NaCl, 5 mM ethylenediamine tetraacetic acid, 0.1 mM dithiothreitol, 1 mM PMSF, 1 g per mL aprotinin, and 2 g per mL leupeptin). Lysates were centrifuged at 12,000 g for 10 min, and the supernatant thus obtained was used for Western blot analysis, as described previously (^{Chung et al, 2001}). A monoclonal anti-1 (I) procollagen aminoterminal extension peptide (SP1.D8) antibody (Developmental Studies Hybridoma Bank, Iowa City, Iowa), a monoclonal anti-MMP-1 antibody (Oncogen, Boston, Massachusetts), and a polyclonal anti-TIMP-1 antibody (Santa Cruz Biotechnology, Santa Cruz, California) were used as primary antibodies.

Electrophoretic mobility shift assays (EMSA)

EMSA were performed using a commercial kit, according to the manufacturer's instructions (Promega, Madison, Wisconsin). Briefly, AP-1 (5'-CGC TTG ATG CAG CCG GAA-3') consensus oligonucleotides were ³²P end labeled by incubation for 10 min at 37°C with 10 U T4 polynucleotide kinase in a reaction containing 10 Ci (-³²P) ATP (3000 Ci per mmol at 10 mCi per mL, Amersham Pharmacia, Piscataway, New Jersey). Fifteen micrograms of the total proteins were equilibrated for 10 min in a binding buffer (4% glycerol, 1 mM MgCl₂, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), and 50 g per mL poly (dI-dC)). For competition assays, a cold probe (0.35 pM) was added to the reaction and incubated for 20 min at room temperature. The reaction was stopped, loaded on a 6% DNA retardation gel (Invitrogen, Carlsbad, California), and run in 0.5 TBE buffer at 100 V for 120 min. The gel was then dried and detected by autoradiography.

In situ hybridization

The 366 bp XhoI–PvuII frgment of 1 (I) procollagen (corresponding to the coding sequences of 1 (I) procollagen: 3804–4172, GeneBank accession number NM_000088) was cloned into pCR2.1-TOPO. Digoxigenin-containing sense and antisense riboprobes detecting human procollagen 1(I) were synthesized using T7 RNA polymerase. In situ hybridization was performed on 8 m sections as described previously (^{Chung et al, 2001}). All samples were treated with proteinase K and washed in a 0.1 M triethanolamine buffer conatining 0.25% acetic anhydride. After hybridization at 52°C, the slides were washed under stringent conditions, and treated with RNase A to remove unhybridized probe. Hybridization signals were detected immunohistochemically using an alkaline phosphatase-conjugated anti-digoxigenin antibody.

Immunohistochemical staining

Serial sections of 4-m thickness from punch biopsy specimens were mounted onto silane coated slides (Dako, Glostrup, Denmark). Acetone-fixed frozen sections were stained with monoclonal anti-human procollagen type I C-peptide (PIC) antibody (Takara, Shiga, Japan), diluted 1:250 overnight at 4°C. After rinsing in phosphate-buffered saline, the sections were visualized using a Histostain-plus kit (Zymed, San Francisco, California) using a biotinylated secondary antibody and horseradish–streptavidin conjugate. 3-Amino-9-ethylcarbazole was used as a chromogenic substrate, and sections were counterstained briefly in Mayer's hematoxylin. Control staining was performed with normal rabbit and mouse immunoglobulin, which showed no immunoreactivity (data not shown).

RT-PCR

Total RNA was isolated from either the cultured fibroblasts or from biopsied skin samples using Trizol reagent (Life Technologies, Rockville, Maryland). Isolated RNA samples were subjected to electrophoresis on 1% agarose gels to assess their quality and quantity. The extracted total RNA (1 g) was reverse transcribed using a first-strand cDNA synthesis kit for RT-PCR (MBI Fermentas, Hanover, Maryland). The resulting specific cDNA fragments were amplified with 2.5 U of Taq polymerase (Advanced Biotechnologies, Surrey, UK) in the presence of 20 pmol downstream primer and upstream primer, using three thermocycler temperatures (1 min at 94°C, 1 min at 60°C, and 1 min at 72°C). The sequences of specific primers used were as follows: 1 (I) procollagen (5'-CTCGAGGTGGACACCACCCT-3' and 5'-CAGCTGGATGGCCACATCGG-3'), corresponding to the coding sequences of 1 (I) procollagen: 3685–3704 and 4037–4056, respectively (GeneBank accession number: NM_000088), MMP-1 (5'-ATTCTACTGATATCGGGGCTTTGA-3' and 5'-ATGTCCTTGGGGTATCCGTGTAG-3'), corresponding to the coding sequences of MMP-1: 654–677 and 1069–1091, respectively (GeneBank accession number: NM_002421), TIMP-1 (5'-ATTTCCGACCTCGTCATCAGG-3 and 5'-ACTGGAAGCCCTTTTCAGAGC-3'), corresponding to the coding sequences of TIMP-1: 111–129 and 530–550, respectively (GeneBank accession number: NM_003254), TGF-1 (5'-GCCCTGGACACCAACTATTGC-3' and 5'- AGGCTCCAAATGTAGGGGCAGG-3'), corresponding to the coding sequences of TGF-1: 838–858 and 977–998, respectively (GeneBank accession number: NM_000660), CTGF (5'-CCAAGGACCAAACCGTGGT-3' and 5'-TACTCCACAGAATTTAGCTCG-3'), corresponding to the coding sequences of CTGF: 506-524 and 838-858, respectively (GeneBank accession number: NM_001901), and the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5'-ATTGTTGCCATCAATGACCC-3' and 5'-AGTAGAGGCAGGGATGATGT-3'), corresponding to the coding sequences of GAPDH: 88–107 and 614–633, respectively (GeneBank accession number: BC023632).

Reaction products were subjected to electrophoresis on 2.0% agarose gel and visualized with ethidium bromide. Signal strengths were quantified using a densitometric program (TINA; Raytest Isotopenmegerate, Staubenhardt, Germany). No PCR products were obtained in control reactions without reverse transcriptase. After normalizing versus GAPDH intensity, percentage increase or decrease was determined. Each experiment was repeated at least three times.

Statistics

Statistical significance was determined using the Student' t test. Results are presented as meansSEM. All p values quoted are two-tailed and significance was accepted when p was <0.05.

References

1. Arlt, W, Justl, HG, Callies, F, et al: Oral dehydroepiandrosterone for adrenal androgen replacement: Pharmacokinetics and peripheral conversion to androgens and estrogens in young healthy females after dexamethasone suppression. J Clin Endocrinol Metab 1998 83: 1928–1934, 10.1210/jc.83.6.1928 | Article | PubMed | ISI | ChemPort |
2. Baulieu, EE, Thomas, G, Legrain, S, et al: Dehydroepiandrosterone (DHEA), DHEA sulfate, and aging: Contribution of the DHEAge Study to a sociobiomedical issue. Proc Natl Acad Sci USA 2000 97: 4279–4284,  | Article | PubMed | ChemPort |
3. Belanger, A, Candas, B, Dupont, A, Cusan, L, Diamond, P, Gomez, JL, Labrie, F: Changes in serum concentrations of conjugated and unconjugated steroids in 40- to 80-year-old men. J Clin Endocrinol Metab 1994 79: 1086–1090,  | Article | PubMed | ISI | ChemPort |
4. Berdanier, CD, McIntosh, MK: Further studies on the effects of dehydroepiandrosterone on hepatic metabolism in BHE rats. Proc Soc Exp Biol Med 1989 192: 242–247,  | PubMed | ISI | ChemPort |
5. Chung, JH, Han, JH, Hwang, EJ, et al: Dual mechanisms of green tea extract (EGCG)-induced cell survival in human epidermal keratinocytes. Faseb J 2003 17: 1913–1915,  | PubMed | ChemPort |
6. Chung, JH, Seo, JY, Choi, HR, et al: Modulation of skin collagen metabolism in aged and photoaged human skin in vivo. J Invest Dermatol 2001 117: 1218–1224,  | Article | PubMed | ISI | ChemPort |
7. Chung, JH, Youn, SH, Kwon, OS, Cho, KH, Youn, JI, Eun, HC: Regulations of collagen synthesis by ascorbic acid, transforming growth factor-beta and interferon-gamma in human dermal fibroblasts cultured in three-dimensional collagen gel are photoaging- and aging-independent. J Dermatol Sci 1997 15: 188–200,  | Article | PubMed | ISI | ChemPort |
8. Chung, JH, Youn, SH, Kwon, OS, et al: Enhanced proliferation and collagen synthesis of human dermal fibroblasts in chronically photodamaged skin. Photodermatol Photoimmunol Photomed 1996a 12: 84–89,  | ISI | ChemPort |
9. Chung, KY, Agarwal, A, Uitto, J, Mauviel, A: An AP-1 binding sequence is essential for regulation of the human alpha2(I) collagen (COL1A2) promoter activity by transforming growth factor-beta. J Biol Chem 1996b 271: 3272–3278, 10.1074/jbc.271.6.3272 | Article | PubMed | ISI | ChemPort |
10. Clore, JN: Dehydroepiandrosterone and body fat. Obes Res 1995 3(Suppl. 4): 613S–616S,  | PubMed | ChemPort |
11. Coleman, DL, Schwizer, RW, Leiter, EH: Effect of genetic background on the therapeutic effects of dehydroepiandrosterone (DHEA) in diabetes-obesity mutants and in aged normal mice. Diabetes 1984 33: 26–32,  | PubMed | ISI | ChemPort |
12. Duncan, MR, Frazier, KS, Abramson, S, Williams, S, Klapper, H, Huang, X, Grotendorst, GR: Connective tissue growth factor mediates transforming growth factor beta-induced collagen synthesis: Down-regulation by cAMP. FASEB J 1999 13: 1774–1786,  | PubMed | ISI | ChemPort |
13. Edwards, DR, Leco, KJ, Beaudry, PP, Atadja, PW, Veillette, C, Riabowol, KT: Differential effects of transforming growth factor-beta 1 on the expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in young and old human fibroblasts. Exp Gerontol 1996 31: 207–223, 10.1016/0531-5565(95)02010-1 | Article | PubMed | ISI | ChemPort |
14. Eickelberg, O, Kohler, E, Reichenberger, F, et al: Extracellular matrix deposition by primary human lung fibroblasts in response to TGF-beta1 and TGF-beta3. Am J Physiol 1999 276: L814–L824,  | PubMed | ISI | ChemPort |
15. Fisher, GJ, Datta, S, Wang, Z, et al: c-Jun-dependent inhibition of cutaneous procollagen transcription following ultraviolet irradiation is reversed by all-trans retinoic acid. J Clin Invest 2000 106: 663–670,  | PubMed | ISI | ChemPort |
16. Fisher, GJ, Datta, SC, Talwar, HS, Wang, ZQ, Varani, J, Kang, S, Voorhees, JJ: Molecular basis of sun-induced premature skin ageing and retinoid antagonism. Nature 1996 379: 335–339, 10.1038/379335a0 | Article | PubMed | ISI | ChemPort |
17. Fisher, GJ, Wang, ZQ, Datta, SC, Varani, J, Kang, S, Voorhees, JJ: Pathophysiology of premature skin aging induced by ultraviolet light. N Engl J Med 1997 337: 1419–1428, 10.1056/NEJM199711133372003 | Article | PubMed | ISI | ChemPort |
18. Gilchrest, BA: Skin aging and photoaging: An overview. J Am Acad Dermatol 1989 21: 610–613,  | PubMed | ISI | ChemPort |
19. Grotendorst, GR: Connective tissue growth factor: A mediator of TGF-beta action on fibroblasts. Cytokine Growth Factor Rev 1997 8: 171–179, 10.1016/S1359-6101(97)00010-5 | Article | PubMed | ChemPort |
20. Hall, MC, Young, DA, Waters, JG, Rowan, AD, Chantry, A, Edwards, DR, Clark, IM: The comparative role of activator protein 1 and Smad factors in the regulation of Timp-1 and MMP-1 gene expression by transforming growth factor-beta 1. J Biol Chem 2003 278: 10304–10313, 10.1074/jbc.M212334200 | Article | PubMed | ISI | ChemPort |
21. Hinson, JP, Raven, PW: DHEA deficiency syndrome: A new term for old age?J Endocrinol 1999 163: 1–5, 10.1677/joe.0.1630001 | Article | PubMed | ISI | ChemPort |
22. Jo, H, Park, JS, Kim, EM, et al: The in vitro effects of dehydroepiandrosterone on human osteoarthritic chondrocytes. Osteoarthritis Cartilage 2003 11: 585–594, 10.1016/S1063-4584(03)00094-3 | Article | PubMed | ISI | ChemPort |
23. Labrie, F: Intracrinology. Mol Cell Endocrinol 1991 78: C113–C118,  | Article | PubMed | ISI | ChemPort |
24. Labrie, F, Belanger, A, Simard, J, Van, L-T, Labrie, C: DHEA and peripheral androgen and estrogen formation: Intracinology. Ann NY Acad Sci 1995 774: 16–28,  | PubMed | ChemPort |
25. Labrie, F, Diamond, P, Cusan, L, Gomez, JL, Belanger, A, Candas, B: Effect of 12-month dehydroepiandrosterone replacement therapy on bone, vagina, and endometrium in postmenopausal women. J Clin Endocrinol Metab 1997 82: 3498–3505, 10.1210/jc.82.10.3498 | Article | PubMed | ISI | ChemPort |
26. Lee, KS, Oh, KY, Kim, BC: Effects of dehydroepiandrosterone on collagen and collagenase gene expression by skin fibroblasts in culture. J Dermatol Sci 2000 23: 103–110, 10.1016/S0923-1811(99)00094-8 | Article | PubMed | ISI | ChemPort |
27. Martel, C, Sourla, A, Pelletier, G, et al: Predominant androgenic component in the stimulatory effect of dehydroepiandrosterone on bone mineral density in the rat. J Endocrinol 1998 157: 433–442, 10.1677/joe.0.1570433 | Article | PubMed | ChemPort |
28. Nestler, JE, Clore, JN, Blackard, WG: Dehydroepiandrosterone: The "missing link" between hyperinsulinemia and atherosclerosis?FASEB J 1992 6: 3073–3075,  | PubMed | ISI | ChemPort |
29. Orentreich, N, Brind, JL, Rizer, RL, Vogelman, JH: Age changes and sex differences in serum dehydroepiandrosterone sulfate concentrations throughout adulthood. J Clin Endocrinol Metab 1984 59: 551–555,  | PubMed | ISI | ChemPort |
30. Quan, T, He, T, Kang, S, Voorhees, JJ, Fisher, GJ: Connective tissue growth factor: Expression in human skin in vivo and inhibition by ultraviolet irradiation. J Invest Dermatol 2002 118: 402–408, 10.1046/j.0022-202x.2001.01678.x | Article | PubMed | ISI | ChemPort |
31. Sakyo, K, Ito, A, Mori, Y: Effects of dehydroepiandrosterone sulphate on the production of collagenase and gelatinolytic metalloproteinase by rabbit uterine cervical cells in primary cultures. J Pharmacobiodyn 1986 9: 276–286,  | PubMed | ChemPort |
32. Sakyo, K, Ito, A, Mori, Y: Dehydroepiandrosterone sulfate stimulates collagenase synthesis without affecting the rates of collagen and noncollagen protein syntheses by rabbit uterine cervical fibroblasts. Biol Reprod 1987 36: 277–281,  | Article | PubMed | ISI | ChemPort |
33. Seo, JY, Lee, SH, Youn, CS, et al: Ultraviolet radiation increases tropoelastin mRNA expression in the epidermis of human skin in vivo. J Invest Dermatol 2001 116: 915–919, 10.1046/j.1523-1747.2001.01358.x | Article | PubMed | ISI | ChemPort |
34. Smart, DE, Vincent, KJ, Arthur, MJ, Eickelberg, O, Castellazzi, M, Mann, J, Mann, DA: JunD regulates transcription of the tissue inhibitor of metalloproteinases-1 and interleukin-6 genes in activated hepatic stellate cells. J Biol Chem 2001 276: 24414–24421, 10.1074/jbc.M101840200 | Article | PubMed | ISI | ChemPort |
35. Sun, Y, Mao, M, Sun, L, Feng, Y, Yang, J, Shen, P: Treatment of osteoporosis in men using dehydroepiandrosterone sulfate. Chin Med J (Engl) 2002 115: 402–404,  | PubMed | ChemPort |
36. Tagliaferro, AR, Davis, JR, Truchon, S, Van Hamont, N: Effects of dehydroepiandrosterone acetate on metabolism, body weight and composition of male and female rats. J Nutr 1986 116: 1977–1983,  | PubMed | ISI | ChemPort |
37. Varani, J, Warner, RL, Gharaee-Kermani, M, et al: Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin. J Invest Dermatol 2000 114: 480–486, 10.1046/j.1523-1747.2000.00902.x | Article | PubMed | ISI | ChemPort |
38. Yen, SS: Dehydroepiandrosterone sulfate and longevity: New clues for an old friend. Proc Natl Acad Sci USA 2001 98: 8167–8169, 10.1073/pnas.161278698 | Article | PubMed | ChemPort |
39. Yoshida, K, Tahara, R, Nakayama, T, Yanaihara, T: Effect of dehydroepiandrosterone sulphate, oestrogens and prostaglandins on collagen metabolism in human cervical tissue in relation to cervical ripening. J Int Med Res 1993 21: 26–35,  | PubMed | ISI | ChemPort |

Acknowledgments

This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (01-PJ1-PG3-20800-0040) and a research agreement with the Pacific Corporation. We would also like to thank Mi Kyung Lee, Ji Eun Kim, and Ae Kyung Woo for their excellent technical assistance.