- Email: [email protected]
Effects of dehydroepiandrosterone on collagen and collagenase gene expression by skin fibroblasts in culture
Journal of Dermatological Science 23 (2000) 103 – 110 www.elsevier.com/locate/jdermsci
Effects of dehydroepiandrosterone on collagen and collagenase gene expression by skin fibroblasts in culture Kyu Suk Lee *, Kwang Youl Oh, Byung Chun Kim Department of Dermatology, School of Medicine, Keimyung Uni6ersity, 194 Dong san-dong, Jung gu, Taegu 700 -310, South Korea Received 9 June 1999; received in revised form 28 July 1999; accepted 28 July 1999
Abstract Dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) are the most abundant steroids in humans whose low levels are related to aging, greater incidence of various cancers, immune dysfunction, atherosclerosis, and osteoporosis. It has been shown that collagen and collagenase gene expression decreases in fibroblasts taken from more aged donors. In this paper, to investigate the relationship between DHEA and skin aging, we examined the effects of DHEA on the regulation of collagen, collegians and stromelysin-1 genes in cultured human skin fibroblasts. In collagen assay, DHEA slightly increased collagen production in a dose-related fashion, its maximal effect occurred at 10 − 5 M DHEA (P \0.05). In the presence of DHEA, steady-state levels of a1 (I) procollagen mRNA increased to 1.6-fold of the non-treated group, while those of fibronectin were not. Interestingly, DHEA differently regulated collagenase and stromelysin-1 gene expression. The steady-state levels of collagenase mRNA decreased in response to DHEA by 40%, whereas those of stromelysin-1 mRNA increased up to 2.4-fold, compared to controls. Similar results were obtained for chloramphenicol acetyltransferase assay (CAT); maximal promoter activation of stromelysin-1 gene occurred at 10 − 6 M DHEA, 4.5-fold higher than control. CAT assay revealed that treatment with 10 − 5 M DHEA resulted in a strong ( : 70%) inhibition of the collagenase promoter activity. In our experiments, the effects of DHEA on these gene expressions were higher at pharmacologic concentration ( ]10 − 5 M) than those at physiologic concentration (10 − 8 –10 − 6 M). This study suggests that the level of DHEA may be related to the process of skin aging through the regulation of production and degradation in extracellular matrix. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: DHEA; Collagen; MMP; In vitro
1. Introduction
Abbre6iations: CAT, chloramphenicol acetyltransferase; DHEA, dehydroepiandrosterone; MMP, matrix metalloproteinase. * Corresponding author. Tel.: + 82-53-250-7625; fax: +8253-250-7626.
Dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) are the most abundant steroids in the human plasma, having serum concentrations of the order of 10 − 8 and 10 − 6 M, respectively. The concentrations in serum reach a peak be-
0923-1811/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 9 2 3 - 1 8 1 1 ( 9 9 ) 0 0 0 9 4 - 8
104
K.S. Lee et al. / Journal of Dermatological Science 23 (2000) 103–110
tween the ages of 25 and 30 years and thereafter decline steadily, so that by age 60, serum concentrations are only 5– 10% of corresponding values in young adults [1]. This age-dependent decline has recently been clinically linked to age-related illnesses such as atherosclerosis [2], obesity, diabetes [3], aging [4], and even some forms of cancer [5]. Unfortunately, the biologic functions and molecular mechanisms of these predominant steroid hormones have yet to be clarified. Cutaneous aging consists of two distinct processes: chronological or intrinsic aging, and solar aging. It has been shown that collagen and collagenase gene expression decreases in fibroblasts taken from more aged donors [6]. Histopathological studies of photodamaged skin have revealed reduced amounts of collagen, accumulation of abnormal elastic fibers (elastotic material), and increased quantities of glycosaminoglycans in the upper and mid dermis [7 – 9]. It is well known that oxygen-derived species including free radicals, formed in living cells by normal metabolism and exogenous influences such as ultraviolet light, are related to aging [10]. Recently, Nickman et al. [11] showed that DHEA-S reduced damage associated with elevated oxidation due to aging and retrovirus infection. We presumed that DHEA may have an anti-skin aging effect and examined the effects of DHEA on collagen, collagenase, and stromelysin-1 gene expression by collagen assay, Northern blot analyses, and chloramphenicol acetyltransferase (CAT) assay in cultured human skin fibroblasts.
left over from cosmetic surgery and subcultivated in plastic culture dishes in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100 mg/ml), and amphotericin B (1 mg/ ml). The cells were maintained in a humidified 5:95 CO2/air (%) incubator at 37°C. Analyses of confluent fibroblast cultures were carried out after 3–6 passages of subcultivation.
2.3. Assay of collagen production Assay of collagen production was carried out as described previously [12]. Collagen production was determined by measuring the incorporation of [3H]proline into bacterial collagenase sensitive protein synthesized by confluent fibroblast monolayers. The relative rate of collagen production as a percentage of total protein production was calculated by the following formula: Percentage of total protein production =
dpm in collagen× 100 (dpm in non-collagenous protein)× 5.4+ dpm in collagen
where disintegrations per min (dpm) in non-collagenous protein was determined by subtracting the dpm in collagen from the dpm in total protein [13]. Cellular DNA contents were measured by the method of Labarca and Paigen and used as the basis of protein synthesis [14]. All experiments were carried out in triplicate.
2.4. cDNA preparation 2. Materials and methods
DHEA was purchased from Sigma (St Louis, MO) and was used after dissolution in 95% ethanol. We treated cultured fibroblasts with different doses of DHEA from 10 − 8 to 10 − 5 M.
The following human-sequence-specific cDNAs were used in this study: for a1 (I) collagen: a 1.5-kb a1 (I) collagen cDNA [15]; for collagenase: a 2.3-kb collagenase cDNA [16]; for stromelysin1: a 1.2-kb stromelysin-1 cDNA [17]; for fibronectin: a 1.8-kb fibronectin cDNA [18]; for glyceraldehyde-3-phosphate dehydrogenase (GAPDH): a 1.3-kb GAPDH cDNA [19].
2.2. Fibroblasts culture
2.5. Northern analyses
Primary cultures of dermal fibroblasts were established from adult skin (n =3; mean age, 23)
Total RNA was isolated by the methods of Chomczynski and Sacchi [20] from cultured nor-
2.1. DHEA
K.S. Lee et al. / Journal of Dermatological Science 23 (2000) 103–110
mal skin fibroblasts. Extracted RNA was analyzed by Northern hybridization with various 32Plabeled cDNA probes. The [32P]cDNA – mRNA hybrids were visualized by autoradiography, and the steady-state levels of mRNA were quantitated by laser densitometry (LKB Instruments, Bromma, Sweden). All quantitated mRNA levels were standardized to GAPDH mRNA levels in the same samples. All experiments were run in triplicate.
105
the cells were harvested, as described previously [24]. For CAT activity determination, an aliquot of the samples containing the same amount of b galactosidase activity was used with [14C]chloramphenicol as a substrate, and the acetylated and non-acetylated forms of radioactive chloramphenicol were separated by thin-layer chromatography [24]. The CAT activity in the cell cultures was determined by the radioactivity in the acetylated forms as a percentage of the total radioactivity in each sample.
2.6. Transient transfection and CAT assay Human skin fibroblasts in late logarithmic growth phase were transfected with 10 mg of plasmid DNA pCLCAT3, which contains − 3.8 kb of 5%-flanking DNA of collagenase gene linked to the CAT reporter gene [21], with 10 mg of DNA plasmid (pST560/CAT), which contains a segment from − 560 to +6 in the 5%-flanking DNA of human stromelysin gene [22]. pCLCAT3 was a gift from Dr J. Uitto (Thomas Jefferson University, Philadelphia, PA) and pST560/CAT was a gift from Dr D. Sawamura (Hirosaki University, Hirosaki, Japan). The transfections were carried out with the calcium phosphate/DNA coprecipitation method [23], followed by a 1-min (15%) glycerol shock. Parallel transfections were carried out with an SV2CAT plasmid. These constructs were cotransfected with an RSV-b galactosidase construct for determination of transfection efficiency. After 24 h of incubation, Table 1 Effect of DHEA on the collagen production in cultured human skin fibroblastsa Concentaration of DHEA (M)
Relative rate of colla- Fold induction gen production (%)
0 10−8 10−7 10−6 10−5
10.79 9 0.24 11.50 90.52* 12.04 90.42* 11.87 90.29* 11.81 90.44*
1.00 1.07 1.12 1.10 1.09
The values are mean 9S.D. of triplicate samples and calculated from the formula [3H]collagen dpm/([3H]non-collagen dpm×5.4+[3H]collagen dpm). * Not significantly different from control at P\0.05 with Student’s t-test. a
3. Results
3.1. Effect of DHEA on collagen synthesis DHEA slightly increased production of collagen by dermal fibroblasts which was maximized at a concentration of 10 − 5 M. In comparison with non-DHEA treated controls, the increase in collagen production by DHEA was 1.07-fold at the concentration of 10 − 8 M, 1.09-fold at 10 − 7 M, 1.10-fold at 10 − 6 M, and 1.12-fold at 10 − 5 M. There was no statistically significant difference between DHEA-treated fibroblasts and control (Table 1).
3.2. Effect of DHEA on the steady-state le6els of type I collagen, collagenase and stromelysin-1 mRNA To examine the type I collagen, collagenase and stromelysin-1 mRNA expression, we treated cultured fibroblasts with different doses of DHEA, and the corresponding mRNA levels were determined by Northern hybridizations. In DHEAtreated and non-treated cell cultures, a1 (I) procollagen revealed two mRNA transcripts, 5.8 and 4.8 kb (Fig. 1), whereas collagenase and stromelysin-1 revealed one transcript, with sizes of 2.4 and 1.8 kb, respectively (Fig. 2). Steady-state levels of a1 (I) procollagen mRNA in relation to the GAPDH were increased in a dose-dependent manner up to 1.6-fold in DHEA-treated fibroblasts; on the other hand, those of fibronectin mRNA were not altered appreciably (Table 2). DHEA caused marked alteration in the collage-
106
K.S. Lee et al. / Journal of Dermatological Science 23 (2000) 103–110
nase and stromelysin-1 promoter activity, transient transfections with human collagenase promoter-CAT construct and stromelysin-1 promoter-CAT construct were carried out in the cultured skin fibroblasts and transfected fibroblasts were incubated with DHEA in concentrations varying from 10 − 7 to 10 − 5 M for 24 h after glycerol shock. The presence of DHEA resulted in a decrease of collagenase promoter activity, as measured by CAT activity (Fig. 3). Maximal promoter inhibition decreased by 70% at a concentration of 10 − 5 M. The relative CAT activity was 1.0 in the non-treated control, 0.9 at a concentration of 10 − 7 M, 0.6 at 10 − 6 M, and 0.3 at 10 − 5 M.
Fig. 1. Effects of DHEA on a1 (I) procollagen and fibronectin (FN) mRNA expression in human skin fibroblast cultures. Fibroblast cultures were treated with varying doses of DHEA (10 − 8 – 10 − 5 M) for 24 h and compared with culture medium alone (control). Upper panel: Total RNA was isolated and the a1 (I) procollagen, FN and GAPDH mRNAs were detected by northern hybridizations with corresponding cDNAs. Lower panel: The relative levels of a1 (I) procollagen and FN mRNAs were presented as fold induction of non-treated control group. Data are represented as the mean of three experiments9 S.D.
nase and stromelysin-1 mRNA expression in a dose-related fashion. The collagenase mRNA levels in relation to the GAPDH were decreased by 40%, whereas stromelysin-1 mRNA levels were increased up to 2.4-fold (Table 3). The effects of DHEA on collagenase and stromelysin-1 mRNA levels were blocked by cycloheximide (0.1 mM), an inhibitor of protein synthesis (data not shown).
3.3. E6idence for transcriptional regulation of the collagenase and stromelysin-1 gene at promoter le6el To examine the effects of DHEA on collage-
Fig. 2. Effects of DHEA on collagenase and stromelysin-1 mRNA expression in human skin fibroblast cultures. Fibroblast cultures were treated with varying doses of DHEA (10 − 8 – 10 − 5 M) for 24 h and compared with culture medium alone (control). Upper panel: Total RNA was isolated and the collagenase, stromelysin-1 and GAPDH mRNAs were detected by northern hybridizations with corresponding cDNAs. Lower panel: The relative levels of collagenase and stromelysin-1 mRNAs were presented as fold induction of non-treated control group. Data are represented as the mean of three experiments 9S.D.
K.S. Lee et al. / Journal of Dermatological Science 23 (2000) 103–110 Table 2 Quantification of a1 (I) procollagen, fibronectin, and GAPDH mRNA levels in cultured human skin fibroblasts treated with different doses of DHEAa Concentration of DHEA (M) 0 10−8 10−7 10−6 10−5
a1 (I) procollagen/ Fibronectin/ GAPDH GAPDH 2.3790.24 (1) 3.2390.19 (1.36) 3.45 90.17 (1.45)* 3.80 90.21 (1.60)* 3.869 0.30 (1.63)*
2.87 90.34 (1) 2.8590.29 (0.91) 2.8190.12 (0.87) 2.7790.18 (0.81) 2.7490.17 (0.79)
a The values are mean 9 S.D. and expressed as densitometric absorbance units which are the percentage of the value of GAPDH; fold difference in parentheses. * PB0.05, significantly different from control with Student’s t-test.
107
4. Discussion Several clinical studies [2–5,11] suggest that DHEA(-S) is protective against many age-related illnesses. In this study, we focused on the relationship between DHEA and skin aging, demonstrated that DHEA regulates collagen synthesis and degradation in cultured skin fibroblasts by up-regulation of the human a1 (I) collagen and stromelysin-1 gene expression and down-regulation of collagenase gene expression at transcriptional level. Out of accordance with our findings, previous studies have revealed that DHEA-S stimulated collagenase and gelatinase production in rabbit [25,26] and human cervical tissue [27], accordingly, collagen content of cervices was decreased in the DHEA-S treated rabbit. Sakyo et al. [28] reported that DHEA-S increases collagenase activity of cervical fibroblasts in rabbits, while DHEA does not. The divergence of results
Table 3 Quantification of collagenase mRNA, stromelysin-1 and GAPDH mRNA levels in cultured human skin fibroblasts treated with different doses of DHEAa Concentartion of DHEA (M) 0 10−8 10−7 10−6 10−5
Collagenase/ GAPDH
Stromelysin-1/ GAPDH
4.459 0.21 (1) 4.29 90.19 (0.95) 4.1790.11 (0.93) 3.8390.12 (0.86) 2.6490.15 (0.59)*
1.759 0.34 (1) 1.919 0.29 (1.09) 2.71 9 0.12 (1.54) 3.55 9 0.18 (2.03)* 4.02 9 0.17 (2.36)*
a The values are mean 9 S.D. and expressed as densitometric absorbance unit which are the percentage of the value of GAPDH; fold difference in parentheses. * PB0.05, significantly different from control with Student’s t-test.
DHEA caused a marked increase in stromelysin-1 promoter activity. The relative CAT activity was 1.0 in the non-treated control, 2.7 at a concentration of 10 − 7 M, 4.5 at 10 − 6 M, and 4.1 at 10 − 5 M (Fig. 4).
Fig. 3. Dose-dependent decrease of collagenase promoter activity in transient transfections of cultured fibroblasts by DHEA treatment. Cells were transfected with the pCLCAT3 and incubated with or without DHEA (10 − 7 – 10 − 5 M). After 24 h of incubation, the CAT activity was determined by measuring the distribution of radioactivity between the acetylated (AC) and nonacetylated (C) forms of [14C]chloramphenicol.
108
K.S. Lee et al. / Journal of Dermatological Science 23 (2000) 103–110
Fig. 4. Upregulation of stromelysin-1 promoter activity in transient transfections of cultured skin fibroblasts. Cells were transfected with pST560/CAT and exposed to different doses of DHEA (10 − 7 – 10 − 5 M); 24 h following treatment with DHEA, the CAT activity was determined by the radioactivity in the acetylated (AC) and nonacetylated (C) forms of [14C]chloramphenicol.
may be due to tissue and/or species specific effects of DHEA or different biological effects between DHEA-S and DHEA. Collagen metabolism is under tight control and equilibrium between accumulation and degradation of extracellular proteins is essential to the maintenance of the extracellular matrix. Several studies indicated that the matrix metalloproteinase (MMP) collagenase is crucial to initiate the degradation of collagen [29,30]. Collagenase is secreted into the extracellular spaces as a proenzyme form and then activated extracellularly [31]. It has been shown that stromelysin secreted together with collagenase from connective-tissue cells in culture is considered as an activator for collagenase. The presence of stromelysin is important for the expression of full collagenase activity [32]. These enzymes are thought to be important for tissue remodeling, wound healing, cervical ripening and aging. In the present study, DHEA increased the steady-state levels of a1 (I) procolla-
gen and stromelysin-1 mRNA; on the other hand, collagenase mRNA levels were decreased. Collagenase and stromelysin are regulated in parallel by many agents, but independently regulated by pharmacologic doses of interferon-g [32]. It is difficult to explain the mechanism of independent regulation of MMPs by DHEA. Although MMP synthesis is regulated at the transcriptional level, the responsible enzymes are secreted from cells in inactive forms (proMMP). Thus, the activation process of proMMPs in extracellular spaces is a key step in the regulation of connective tissue matrix breakdown. Accordingly, although we did not measured the activity of these enzyme activities, we cannot exclude the possibility that collagenase activity or collagenolytic activity in the DHEA-treated fibroblasts may be increased by strongly enhanced (2.4-fold) stromelysin-1 mRNA expression which is required for maximal activation of collagenase in spite of moderately inhibited (40%) collagenase mRNA expression. The increase in collagen protein production was less than the augmentation of a1 (I) procollagen mRNA; it may be due to increased collagenolytic activity or changes in the process of translation in response to DHEA. Further studies are required to explain the independent regulation of collagenase and stromelysin-1 gene expression and different augmentations of a1 (I) procollagen mRNA and of its protein level. The results of CAT assay showed regulation of collagenase and stromelysin gene expression by DHEA is occurring at the transcriptional level. Recent studies have shown that DHEA(-S) has regulatory effects on several cytokines such as IL-6 [33], IL-10 [34], and IL-8 [35]. Accordingly, additional studies are necessary to elucidate whether the effects of DHEA on collagen and MMPs are directly or indirectly due to cytokines. In conclusion, DHEA had up-regulatory effects on stromelysin-1 gene expression, whereas DHEA had down-regulatory effects on collagenase gene expression. However, the increase of collagen protein production and steady-state level of a1 (I) procollagen was not appreciable. As a result, although the results of the present study do not provide any clear evidence of the relationship between DHEA and skin aging, DHEA may be
K.S. Lee et al. / Journal of Dermatological Science 23 (2000) 103–110
involved in the physiologic or pathologic process of collagen metabolism such as connective tissue disease, wound healing, and skin aging. Further studies are required to clarify the effects and mechanisms of DHEA on the extracellular matrix besides collagen and MMP.
Acknowledgements This study was supported by the special research fund (1998) of the Dong san Medical Center.
References [1] Orentreich N, Brind JL, Rizer RL, Vogelman JH. Age changes and sex differences in serum dehydroepiandrosterone sulfate concentrations throughout adulthood. J Clin Endocrinol 1984;59:551–5. [2] Alexandersen P, Haarbo J, Christiansen C. The relationship of natural androgens to coronary heart disease in males: a review. Atherosclerosis 1996;125:1–13. [3] Coleman DL, Schweitzer 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. [4] Watson RR, Hulls A, Araghi-Niknam M, Chung S. Dehydroepiandrosterone (DHEA) supplementation to prevent symptoms of aging. Drugs Aging 1996;9:274–91. [5] Ebeling P, Koivisto VA. Physiological importance of dehydroepiandrosterone. Lancet 1994;343:1479–81. [6] Chen YQ, Mauviel A, Uitto J. Age-related changes in the expression and cytokine response of extracellular matrix genes in human dermal fibroblast cultures. J Geriatr Dermatol 1994;2:163–9. [7] Bernstein EF, Underhill CB, Haun PJ, Brown DB, Uitto J. Chronic sun exposure alters both the content and distribution of dermal glycosaminoglycans. Br J Dermatol 1996;135:255 – 62. [8] Sams WM Jr, Smith JG. The histochemistry of chronically sun-damaged skin. J Invest Dermatol 1961;37:509– 16. [9] Sams WM Jr. Sun-inducing aging: clinical and laboratory observations in man. Dermatol Clin 1986;4:509–16. [10] Gilchrest BA, Bohr VA. Aging process, DNA damage, and repair. FASEB J 1997;11:322–30. [11] Nickman MA, Ardestani SK, Molitor M, Inserra P, Eskelson CD, Watson RR. Dehydroepiandrosterone (DHEA) sulfate prevents reduction in tissue vitamin E and increased lipid peroxidation due to murine retrovirus infection of aged mice. Proc Soc Exp Biol Med 1998;218:210 – 7.
109
[12] Peterkofsky B, Diegelmann R. Use of mixture of proteinase-free ss proteinase for the specific assay of radioactive collagen in the presence of other proteins. Biochemistry 1971;10:988 – 94. [13] Peterkofsky B, Prather WB. Increased collagen synthesis in Kirsten sarcoma virus transformed BALB 3T3 cells grown in the presence of dibutiryl cyclic AMP. Cell 1974;3:291 – 9. [14] Labarca C, Paigen K. A simple, rapid, and sensitive DNA assay procedure. Anal Biochem 1980;102:344 – 52. [15] Chu ML, Meyers JC, Bernard MP, Ding JF, Ramirez F. Cloning and characterization of five overlapping cDNAs specific for the human pro alpha1 (I) collagen chain. Nucleic Acids Res 1982;10:5925 – 34. [16] Goldberg GI, Wilhelm SM, Kronberger A, Bauer EA, Grant GA, Eisen AZ. Human fibroblast collagenase: complete primary structure and homology to an oncogene transformation-induced rat protein. J Biol Chem 1986;261:6600– 5. [17] Saus J, Quinones S, Otani Y, Nagase H, Harris Jr ED, Kurkinen M. The complete primary structure of human matrix metalloproteinase-3. J Biol Chem 1988;263:6742– 5. [18] Schwarzbauer JE, Tamkun JW, Lemischka IR, Hynes RO. Three different fibronectin mRNAs arise by alternative splicing within the coding region. Cell 1983;35:421 – 31. [19] Fort P, Marty L, Piechaczyk M, El SS, Danz C, Jeanteur P, Blanchard JM. Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3phosphate-dehydrogenase multigene family. Nucleic Acids Res 1985;13:1431 – 42. [20] Chomczynski P, Sacchi N. Single step method of RNA isolation by guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156 – 9. [21] Frisch SM, Reich R, Collier IE, Genrich LT, Martin G, Goldberg GI. Adenovirus E1A represses protease gene expression and inhibits metastasis of the tumor cells. Oncogene 1990;5:75 – 83. [22] Sawamura D, Ohta T, Hanada K, Ishikawa H, Tamai K, Yazima H, Meng X, Nomura K, Hashimoto I, Mauviel A, Uitto J. Involvement of the AP-1 site within the 5%-flanking region of the stromelysin-1 gene in induction of the gene expression by UVA irradiation. Arch Dermatol Res 1996;288:628 – 32. [23] Graham F, Van der EBA. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 1973;52:456 – 67. [24] Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratories, 1989. [25] Sakyo K, Ito A, Mori Y. Effects of dehydroepiandrosterone sulfate on the production of collagenase and gelatinolytic metalloproteinase by rabbit uterine cervical cells in primary cultures. J Pharmaco-biodyn 1986;9:276 – 86. [26] Maradny E, Kanayama N, Maehara K, Kobayashi T, Terao T. Dehydroepiandrosterone sulfate potentiates the
110
[27]
[28]
[29] [30]
[31]
K.S. Lee et al. / Journal of Dermatological Science 23 (2000) 103–110 effect of interleukin-8 on the cervix. Gynecol Obstet Invest 1996;42:191 – 5. Yoshida K, Tahara R, Nakayama T, Yanaihara T. Effect of dehydroepiandrosterone sulfate, oestrogens and prostaglandins on collagen metabolism in human cervical tissue in relation to cervical ripening. J Int Med Res 1993;21:26 – 35. Sakyo K, Ito A, Hirakawa S, Mori Y. Specific binding of dehydroepiandrosterone sulfate to cytoplasmic macromolecules in human fetal membrane. Chem Pharm Bull 1986;34:2126 – 32. Harris ED Jr, Welgus HG, Krane SM. Regulation of the mammalian collagenases. Coll Relat Res 1984;4:493–512. Delany AM, Brinckerhoff CE. Post-translational regulation of collagenase and stromelysin gene expression by epidermal growth factor and dexamethasone in cultured human skin fibroblasts. J Cell Biochem 1992;50:400–10. Murphy G, McAlpine CG, Poll CT, Reynolds JJ. Purification and characterization of a bone metalloproteinase that degrades gelatin type IV and V collagen. Biochim Biophys Acta 1985;831:49–58.
.
[32] Unemori EN, Bair MJ, Bauer EA, Amento EP. Stromelysin expression regulates collagenase activation in human fibroblasts: dissociable control of two metalloproteinases by interferon-g. J Biol Chem 1991;266:23477– 82. [33] Straub RH, Konecna L, Hrach S, Rothe G, Kreutz M, Scho¨lmerich J, Falk W, Lang B. Serum dehydroepiandrosterone (DHEA) and DHEA sulfate are negatively correlated with serum interleukin-6 (IL-6), and DHEA inhibits IL-6 secretion from mononuclear cells in man in vitro: possible link between endocrinosenescence and immunosenescence. J Clin Endocrinol Metab 1998;83:2012 – 7. [34] Spencer NFL, Norton SD, Harrison LL, Li GZ, Daynes RA. Dysregulation of IL-10 production with aging: possible linkage to the age-associated decline in DHEA and its sulfated derivative. Exp Gerontol 1996;31:393 – 408. [35] Kanayama N, Maradny EE, Goto J, Terao T. Effect of dehydroepiandrosterone sulfate on interleukin-8 receptor during cervical ripening. Eur J Endocrinol 1998;138:587 – 93.
Effects of dehydroepiandrosterone on collagen and collagenase gene expression by skin fibroblasts in culture Kyu Suk Lee *, Kwang Youl Oh, Byung Chun Kim Department of Dermatology, School of Medicine, Keimyung Uni6ersity, 194 Dong san-dong, Jung gu, Taegu 700 -310, South Korea Received 9 June 1999; received in revised form 28 July 1999; accepted 28 July 1999
Abstract Dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) are the most abundant steroids in humans whose low levels are related to aging, greater incidence of various cancers, immune dysfunction, atherosclerosis, and osteoporosis. It has been shown that collagen and collagenase gene expression decreases in fibroblasts taken from more aged donors. In this paper, to investigate the relationship between DHEA and skin aging, we examined the effects of DHEA on the regulation of collagen, collegians and stromelysin-1 genes in cultured human skin fibroblasts. In collagen assay, DHEA slightly increased collagen production in a dose-related fashion, its maximal effect occurred at 10 − 5 M DHEA (P \0.05). In the presence of DHEA, steady-state levels of a1 (I) procollagen mRNA increased to 1.6-fold of the non-treated group, while those of fibronectin were not. Interestingly, DHEA differently regulated collagenase and stromelysin-1 gene expression. The steady-state levels of collagenase mRNA decreased in response to DHEA by 40%, whereas those of stromelysin-1 mRNA increased up to 2.4-fold, compared to controls. Similar results were obtained for chloramphenicol acetyltransferase assay (CAT); maximal promoter activation of stromelysin-1 gene occurred at 10 − 6 M DHEA, 4.5-fold higher than control. CAT assay revealed that treatment with 10 − 5 M DHEA resulted in a strong ( : 70%) inhibition of the collagenase promoter activity. In our experiments, the effects of DHEA on these gene expressions were higher at pharmacologic concentration ( ]10 − 5 M) than those at physiologic concentration (10 − 8 –10 − 6 M). This study suggests that the level of DHEA may be related to the process of skin aging through the regulation of production and degradation in extracellular matrix. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: DHEA; Collagen; MMP; In vitro
1. Introduction
Abbre6iations: CAT, chloramphenicol acetyltransferase; DHEA, dehydroepiandrosterone; MMP, matrix metalloproteinase. * Corresponding author. Tel.: + 82-53-250-7625; fax: +8253-250-7626.
Dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) are the most abundant steroids in the human plasma, having serum concentrations of the order of 10 − 8 and 10 − 6 M, respectively. The concentrations in serum reach a peak be-
0923-1811/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 9 2 3 - 1 8 1 1 ( 9 9 ) 0 0 0 9 4 - 8
104
K.S. Lee et al. / Journal of Dermatological Science 23 (2000) 103–110
tween the ages of 25 and 30 years and thereafter decline steadily, so that by age 60, serum concentrations are only 5– 10% of corresponding values in young adults [1]. This age-dependent decline has recently been clinically linked to age-related illnesses such as atherosclerosis [2], obesity, diabetes [3], aging [4], and even some forms of cancer [5]. Unfortunately, the biologic functions and molecular mechanisms of these predominant steroid hormones have yet to be clarified. Cutaneous aging consists of two distinct processes: chronological or intrinsic aging, and solar aging. It has been shown that collagen and collagenase gene expression decreases in fibroblasts taken from more aged donors [6]. Histopathological studies of photodamaged skin have revealed reduced amounts of collagen, accumulation of abnormal elastic fibers (elastotic material), and increased quantities of glycosaminoglycans in the upper and mid dermis [7 – 9]. It is well known that oxygen-derived species including free radicals, formed in living cells by normal metabolism and exogenous influences such as ultraviolet light, are related to aging [10]. Recently, Nickman et al. [11] showed that DHEA-S reduced damage associated with elevated oxidation due to aging and retrovirus infection. We presumed that DHEA may have an anti-skin aging effect and examined the effects of DHEA on collagen, collagenase, and stromelysin-1 gene expression by collagen assay, Northern blot analyses, and chloramphenicol acetyltransferase (CAT) assay in cultured human skin fibroblasts.
left over from cosmetic surgery and subcultivated in plastic culture dishes in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100 mg/ml), and amphotericin B (1 mg/ ml). The cells were maintained in a humidified 5:95 CO2/air (%) incubator at 37°C. Analyses of confluent fibroblast cultures were carried out after 3–6 passages of subcultivation.
2.3. Assay of collagen production Assay of collagen production was carried out as described previously [12]. Collagen production was determined by measuring the incorporation of [3H]proline into bacterial collagenase sensitive protein synthesized by confluent fibroblast monolayers. The relative rate of collagen production as a percentage of total protein production was calculated by the following formula: Percentage of total protein production =
dpm in collagen× 100 (dpm in non-collagenous protein)× 5.4+ dpm in collagen
where disintegrations per min (dpm) in non-collagenous protein was determined by subtracting the dpm in collagen from the dpm in total protein [13]. Cellular DNA contents were measured by the method of Labarca and Paigen and used as the basis of protein synthesis [14]. All experiments were carried out in triplicate.
2.4. cDNA preparation 2. Materials and methods
DHEA was purchased from Sigma (St Louis, MO) and was used after dissolution in 95% ethanol. We treated cultured fibroblasts with different doses of DHEA from 10 − 8 to 10 − 5 M.
The following human-sequence-specific cDNAs were used in this study: for a1 (I) collagen: a 1.5-kb a1 (I) collagen cDNA [15]; for collagenase: a 2.3-kb collagenase cDNA [16]; for stromelysin1: a 1.2-kb stromelysin-1 cDNA [17]; for fibronectin: a 1.8-kb fibronectin cDNA [18]; for glyceraldehyde-3-phosphate dehydrogenase (GAPDH): a 1.3-kb GAPDH cDNA [19].
2.2. Fibroblasts culture
2.5. Northern analyses
Primary cultures of dermal fibroblasts were established from adult skin (n =3; mean age, 23)
Total RNA was isolated by the methods of Chomczynski and Sacchi [20] from cultured nor-
2.1. DHEA
K.S. Lee et al. / Journal of Dermatological Science 23 (2000) 103–110
mal skin fibroblasts. Extracted RNA was analyzed by Northern hybridization with various 32Plabeled cDNA probes. The [32P]cDNA – mRNA hybrids were visualized by autoradiography, and the steady-state levels of mRNA were quantitated by laser densitometry (LKB Instruments, Bromma, Sweden). All quantitated mRNA levels were standardized to GAPDH mRNA levels in the same samples. All experiments were run in triplicate.
105
the cells were harvested, as described previously [24]. For CAT activity determination, an aliquot of the samples containing the same amount of b galactosidase activity was used with [14C]chloramphenicol as a substrate, and the acetylated and non-acetylated forms of radioactive chloramphenicol were separated by thin-layer chromatography [24]. The CAT activity in the cell cultures was determined by the radioactivity in the acetylated forms as a percentage of the total radioactivity in each sample.
2.6. Transient transfection and CAT assay Human skin fibroblasts in late logarithmic growth phase were transfected with 10 mg of plasmid DNA pCLCAT3, which contains − 3.8 kb of 5%-flanking DNA of collagenase gene linked to the CAT reporter gene [21], with 10 mg of DNA plasmid (pST560/CAT), which contains a segment from − 560 to +6 in the 5%-flanking DNA of human stromelysin gene [22]. pCLCAT3 was a gift from Dr J. Uitto (Thomas Jefferson University, Philadelphia, PA) and pST560/CAT was a gift from Dr D. Sawamura (Hirosaki University, Hirosaki, Japan). The transfections were carried out with the calcium phosphate/DNA coprecipitation method [23], followed by a 1-min (15%) glycerol shock. Parallel transfections were carried out with an SV2CAT plasmid. These constructs were cotransfected with an RSV-b galactosidase construct for determination of transfection efficiency. After 24 h of incubation, Table 1 Effect of DHEA on the collagen production in cultured human skin fibroblastsa Concentaration of DHEA (M)
Relative rate of colla- Fold induction gen production (%)
0 10−8 10−7 10−6 10−5
10.79 9 0.24 11.50 90.52* 12.04 90.42* 11.87 90.29* 11.81 90.44*
1.00 1.07 1.12 1.10 1.09
The values are mean 9S.D. of triplicate samples and calculated from the formula [3H]collagen dpm/([3H]non-collagen dpm×5.4+[3H]collagen dpm). * Not significantly different from control at P\0.05 with Student’s t-test. a
3. Results
3.1. Effect of DHEA on collagen synthesis DHEA slightly increased production of collagen by dermal fibroblasts which was maximized at a concentration of 10 − 5 M. In comparison with non-DHEA treated controls, the increase in collagen production by DHEA was 1.07-fold at the concentration of 10 − 8 M, 1.09-fold at 10 − 7 M, 1.10-fold at 10 − 6 M, and 1.12-fold at 10 − 5 M. There was no statistically significant difference between DHEA-treated fibroblasts and control (Table 1).
3.2. Effect of DHEA on the steady-state le6els of type I collagen, collagenase and stromelysin-1 mRNA To examine the type I collagen, collagenase and stromelysin-1 mRNA expression, we treated cultured fibroblasts with different doses of DHEA, and the corresponding mRNA levels were determined by Northern hybridizations. In DHEAtreated and non-treated cell cultures, a1 (I) procollagen revealed two mRNA transcripts, 5.8 and 4.8 kb (Fig. 1), whereas collagenase and stromelysin-1 revealed one transcript, with sizes of 2.4 and 1.8 kb, respectively (Fig. 2). Steady-state levels of a1 (I) procollagen mRNA in relation to the GAPDH were increased in a dose-dependent manner up to 1.6-fold in DHEA-treated fibroblasts; on the other hand, those of fibronectin mRNA were not altered appreciably (Table 2). DHEA caused marked alteration in the collage-
106
K.S. Lee et al. / Journal of Dermatological Science 23 (2000) 103–110
nase and stromelysin-1 promoter activity, transient transfections with human collagenase promoter-CAT construct and stromelysin-1 promoter-CAT construct were carried out in the cultured skin fibroblasts and transfected fibroblasts were incubated with DHEA in concentrations varying from 10 − 7 to 10 − 5 M for 24 h after glycerol shock. The presence of DHEA resulted in a decrease of collagenase promoter activity, as measured by CAT activity (Fig. 3). Maximal promoter inhibition decreased by 70% at a concentration of 10 − 5 M. The relative CAT activity was 1.0 in the non-treated control, 0.9 at a concentration of 10 − 7 M, 0.6 at 10 − 6 M, and 0.3 at 10 − 5 M.
Fig. 1. Effects of DHEA on a1 (I) procollagen and fibronectin (FN) mRNA expression in human skin fibroblast cultures. Fibroblast cultures were treated with varying doses of DHEA (10 − 8 – 10 − 5 M) for 24 h and compared with culture medium alone (control). Upper panel: Total RNA was isolated and the a1 (I) procollagen, FN and GAPDH mRNAs were detected by northern hybridizations with corresponding cDNAs. Lower panel: The relative levels of a1 (I) procollagen and FN mRNAs were presented as fold induction of non-treated control group. Data are represented as the mean of three experiments9 S.D.
nase and stromelysin-1 mRNA expression in a dose-related fashion. The collagenase mRNA levels in relation to the GAPDH were decreased by 40%, whereas stromelysin-1 mRNA levels were increased up to 2.4-fold (Table 3). The effects of DHEA on collagenase and stromelysin-1 mRNA levels were blocked by cycloheximide (0.1 mM), an inhibitor of protein synthesis (data not shown).
3.3. E6idence for transcriptional regulation of the collagenase and stromelysin-1 gene at promoter le6el To examine the effects of DHEA on collage-
Fig. 2. Effects of DHEA on collagenase and stromelysin-1 mRNA expression in human skin fibroblast cultures. Fibroblast cultures were treated with varying doses of DHEA (10 − 8 – 10 − 5 M) for 24 h and compared with culture medium alone (control). Upper panel: Total RNA was isolated and the collagenase, stromelysin-1 and GAPDH mRNAs were detected by northern hybridizations with corresponding cDNAs. Lower panel: The relative levels of collagenase and stromelysin-1 mRNAs were presented as fold induction of non-treated control group. Data are represented as the mean of three experiments 9S.D.
K.S. Lee et al. / Journal of Dermatological Science 23 (2000) 103–110 Table 2 Quantification of a1 (I) procollagen, fibronectin, and GAPDH mRNA levels in cultured human skin fibroblasts treated with different doses of DHEAa Concentration of DHEA (M) 0 10−8 10−7 10−6 10−5
a1 (I) procollagen/ Fibronectin/ GAPDH GAPDH 2.3790.24 (1) 3.2390.19 (1.36) 3.45 90.17 (1.45)* 3.80 90.21 (1.60)* 3.869 0.30 (1.63)*
2.87 90.34 (1) 2.8590.29 (0.91) 2.8190.12 (0.87) 2.7790.18 (0.81) 2.7490.17 (0.79)
a The values are mean 9 S.D. and expressed as densitometric absorbance units which are the percentage of the value of GAPDH; fold difference in parentheses. * PB0.05, significantly different from control with Student’s t-test.
107
4. Discussion Several clinical studies [2–5,11] suggest that DHEA(-S) is protective against many age-related illnesses. In this study, we focused on the relationship between DHEA and skin aging, demonstrated that DHEA regulates collagen synthesis and degradation in cultured skin fibroblasts by up-regulation of the human a1 (I) collagen and stromelysin-1 gene expression and down-regulation of collagenase gene expression at transcriptional level. Out of accordance with our findings, previous studies have revealed that DHEA-S stimulated collagenase and gelatinase production in rabbit [25,26] and human cervical tissue [27], accordingly, collagen content of cervices was decreased in the DHEA-S treated rabbit. Sakyo et al. [28] reported that DHEA-S increases collagenase activity of cervical fibroblasts in rabbits, while DHEA does not. The divergence of results
Table 3 Quantification of collagenase mRNA, stromelysin-1 and GAPDH mRNA levels in cultured human skin fibroblasts treated with different doses of DHEAa Concentartion of DHEA (M) 0 10−8 10−7 10−6 10−5
Collagenase/ GAPDH
Stromelysin-1/ GAPDH
4.459 0.21 (1) 4.29 90.19 (0.95) 4.1790.11 (0.93) 3.8390.12 (0.86) 2.6490.15 (0.59)*
1.759 0.34 (1) 1.919 0.29 (1.09) 2.71 9 0.12 (1.54) 3.55 9 0.18 (2.03)* 4.02 9 0.17 (2.36)*
a The values are mean 9 S.D. and expressed as densitometric absorbance unit which are the percentage of the value of GAPDH; fold difference in parentheses. * PB0.05, significantly different from control with Student’s t-test.
DHEA caused a marked increase in stromelysin-1 promoter activity. The relative CAT activity was 1.0 in the non-treated control, 2.7 at a concentration of 10 − 7 M, 4.5 at 10 − 6 M, and 4.1 at 10 − 5 M (Fig. 4).
Fig. 3. Dose-dependent decrease of collagenase promoter activity in transient transfections of cultured fibroblasts by DHEA treatment. Cells were transfected with the pCLCAT3 and incubated with or without DHEA (10 − 7 – 10 − 5 M). After 24 h of incubation, the CAT activity was determined by measuring the distribution of radioactivity between the acetylated (AC) and nonacetylated (C) forms of [14C]chloramphenicol.
108
K.S. Lee et al. / Journal of Dermatological Science 23 (2000) 103–110
Fig. 4. Upregulation of stromelysin-1 promoter activity in transient transfections of cultured skin fibroblasts. Cells were transfected with pST560/CAT and exposed to different doses of DHEA (10 − 7 – 10 − 5 M); 24 h following treatment with DHEA, the CAT activity was determined by the radioactivity in the acetylated (AC) and nonacetylated (C) forms of [14C]chloramphenicol.
may be due to tissue and/or species specific effects of DHEA or different biological effects between DHEA-S and DHEA. Collagen metabolism is under tight control and equilibrium between accumulation and degradation of extracellular proteins is essential to the maintenance of the extracellular matrix. Several studies indicated that the matrix metalloproteinase (MMP) collagenase is crucial to initiate the degradation of collagen [29,30]. Collagenase is secreted into the extracellular spaces as a proenzyme form and then activated extracellularly [31]. It has been shown that stromelysin secreted together with collagenase from connective-tissue cells in culture is considered as an activator for collagenase. The presence of stromelysin is important for the expression of full collagenase activity [32]. These enzymes are thought to be important for tissue remodeling, wound healing, cervical ripening and aging. In the present study, DHEA increased the steady-state levels of a1 (I) procolla-
gen and stromelysin-1 mRNA; on the other hand, collagenase mRNA levels were decreased. Collagenase and stromelysin are regulated in parallel by many agents, but independently regulated by pharmacologic doses of interferon-g [32]. It is difficult to explain the mechanism of independent regulation of MMPs by DHEA. Although MMP synthesis is regulated at the transcriptional level, the responsible enzymes are secreted from cells in inactive forms (proMMP). Thus, the activation process of proMMPs in extracellular spaces is a key step in the regulation of connective tissue matrix breakdown. Accordingly, although we did not measured the activity of these enzyme activities, we cannot exclude the possibility that collagenase activity or collagenolytic activity in the DHEA-treated fibroblasts may be increased by strongly enhanced (2.4-fold) stromelysin-1 mRNA expression which is required for maximal activation of collagenase in spite of moderately inhibited (40%) collagenase mRNA expression. The increase in collagen protein production was less than the augmentation of a1 (I) procollagen mRNA; it may be due to increased collagenolytic activity or changes in the process of translation in response to DHEA. Further studies are required to explain the independent regulation of collagenase and stromelysin-1 gene expression and different augmentations of a1 (I) procollagen mRNA and of its protein level. The results of CAT assay showed regulation of collagenase and stromelysin gene expression by DHEA is occurring at the transcriptional level. Recent studies have shown that DHEA(-S) has regulatory effects on several cytokines such as IL-6 [33], IL-10 [34], and IL-8 [35]. Accordingly, additional studies are necessary to elucidate whether the effects of DHEA on collagen and MMPs are directly or indirectly due to cytokines. In conclusion, DHEA had up-regulatory effects on stromelysin-1 gene expression, whereas DHEA had down-regulatory effects on collagenase gene expression. However, the increase of collagen protein production and steady-state level of a1 (I) procollagen was not appreciable. As a result, although the results of the present study do not provide any clear evidence of the relationship between DHEA and skin aging, DHEA may be
K.S. Lee et al. / Journal of Dermatological Science 23 (2000) 103–110
involved in the physiologic or pathologic process of collagen metabolism such as connective tissue disease, wound healing, and skin aging. Further studies are required to clarify the effects and mechanisms of DHEA on the extracellular matrix besides collagen and MMP.
Acknowledgements This study was supported by the special research fund (1998) of the Dong san Medical Center.
References [1] Orentreich N, Brind JL, Rizer RL, Vogelman JH. Age changes and sex differences in serum dehydroepiandrosterone sulfate concentrations throughout adulthood. J Clin Endocrinol 1984;59:551–5. [2] Alexandersen P, Haarbo J, Christiansen C. The relationship of natural androgens to coronary heart disease in males: a review. Atherosclerosis 1996;125:1–13. [3] Coleman DL, Schweitzer 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. [4] Watson RR, Hulls A, Araghi-Niknam M, Chung S. Dehydroepiandrosterone (DHEA) supplementation to prevent symptoms of aging. Drugs Aging 1996;9:274–91. [5] Ebeling P, Koivisto VA. Physiological importance of dehydroepiandrosterone. Lancet 1994;343:1479–81. [6] Chen YQ, Mauviel A, Uitto J. Age-related changes in the expression and cytokine response of extracellular matrix genes in human dermal fibroblast cultures. J Geriatr Dermatol 1994;2:163–9. [7] Bernstein EF, Underhill CB, Haun PJ, Brown DB, Uitto J. Chronic sun exposure alters both the content and distribution of dermal glycosaminoglycans. Br J Dermatol 1996;135:255 – 62. [8] Sams WM Jr, Smith JG. The histochemistry of chronically sun-damaged skin. J Invest Dermatol 1961;37:509– 16. [9] Sams WM Jr. Sun-inducing aging: clinical and laboratory observations in man. Dermatol Clin 1986;4:509–16. [10] Gilchrest BA, Bohr VA. Aging process, DNA damage, and repair. FASEB J 1997;11:322–30. [11] Nickman MA, Ardestani SK, Molitor M, Inserra P, Eskelson CD, Watson RR. Dehydroepiandrosterone (DHEA) sulfate prevents reduction in tissue vitamin E and increased lipid peroxidation due to murine retrovirus infection of aged mice. Proc Soc Exp Biol Med 1998;218:210 – 7.
109
[12] Peterkofsky B, Diegelmann R. Use of mixture of proteinase-free ss proteinase for the specific assay of radioactive collagen in the presence of other proteins. Biochemistry 1971;10:988 – 94. [13] Peterkofsky B, Prather WB. Increased collagen synthesis in Kirsten sarcoma virus transformed BALB 3T3 cells grown in the presence of dibutiryl cyclic AMP. Cell 1974;3:291 – 9. [14] Labarca C, Paigen K. A simple, rapid, and sensitive DNA assay procedure. Anal Biochem 1980;102:344 – 52. [15] Chu ML, Meyers JC, Bernard MP, Ding JF, Ramirez F. Cloning and characterization of five overlapping cDNAs specific for the human pro alpha1 (I) collagen chain. Nucleic Acids Res 1982;10:5925 – 34. [16] Goldberg GI, Wilhelm SM, Kronberger A, Bauer EA, Grant GA, Eisen AZ. Human fibroblast collagenase: complete primary structure and homology to an oncogene transformation-induced rat protein. J Biol Chem 1986;261:6600– 5. [17] Saus J, Quinones S, Otani Y, Nagase H, Harris Jr ED, Kurkinen M. The complete primary structure of human matrix metalloproteinase-3. J Biol Chem 1988;263:6742– 5. [18] Schwarzbauer JE, Tamkun JW, Lemischka IR, Hynes RO. Three different fibronectin mRNAs arise by alternative splicing within the coding region. Cell 1983;35:421 – 31. [19] Fort P, Marty L, Piechaczyk M, El SS, Danz C, Jeanteur P, Blanchard JM. Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3phosphate-dehydrogenase multigene family. Nucleic Acids Res 1985;13:1431 – 42. [20] Chomczynski P, Sacchi N. Single step method of RNA isolation by guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156 – 9. [21] Frisch SM, Reich R, Collier IE, Genrich LT, Martin G, Goldberg GI. Adenovirus E1A represses protease gene expression and inhibits metastasis of the tumor cells. Oncogene 1990;5:75 – 83. [22] Sawamura D, Ohta T, Hanada K, Ishikawa H, Tamai K, Yazima H, Meng X, Nomura K, Hashimoto I, Mauviel A, Uitto J. Involvement of the AP-1 site within the 5%-flanking region of the stromelysin-1 gene in induction of the gene expression by UVA irradiation. Arch Dermatol Res 1996;288:628 – 32. [23] Graham F, Van der EBA. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 1973;52:456 – 67. [24] Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratories, 1989. [25] Sakyo K, Ito A, Mori Y. Effects of dehydroepiandrosterone sulfate on the production of collagenase and gelatinolytic metalloproteinase by rabbit uterine cervical cells in primary cultures. J Pharmaco-biodyn 1986;9:276 – 86. [26] Maradny E, Kanayama N, Maehara K, Kobayashi T, Terao T. Dehydroepiandrosterone sulfate potentiates the
110
[27]
[28]
[29] [30]
[31]
K.S. Lee et al. / Journal of Dermatological Science 23 (2000) 103–110 effect of interleukin-8 on the cervix. Gynecol Obstet Invest 1996;42:191 – 5. Yoshida K, Tahara R, Nakayama T, Yanaihara T. Effect of dehydroepiandrosterone sulfate, oestrogens and prostaglandins on collagen metabolism in human cervical tissue in relation to cervical ripening. J Int Med Res 1993;21:26 – 35. Sakyo K, Ito A, Hirakawa S, Mori Y. Specific binding of dehydroepiandrosterone sulfate to cytoplasmic macromolecules in human fetal membrane. Chem Pharm Bull 1986;34:2126 – 32. Harris ED Jr, Welgus HG, Krane SM. Regulation of the mammalian collagenases. Coll Relat Res 1984;4:493–512. Delany AM, Brinckerhoff CE. Post-translational regulation of collagenase and stromelysin gene expression by epidermal growth factor and dexamethasone in cultured human skin fibroblasts. J Cell Biochem 1992;50:400–10. Murphy G, McAlpine CG, Poll CT, Reynolds JJ. Purification and characterization of a bone metalloproteinase that degrades gelatin type IV and V collagen. Biochim Biophys Acta 1985;831:49–58.
.
[32] Unemori EN, Bair MJ, Bauer EA, Amento EP. Stromelysin expression regulates collagenase activation in human fibroblasts: dissociable control of two metalloproteinases by interferon-g. J Biol Chem 1991;266:23477– 82. [33] Straub RH, Konecna L, Hrach S, Rothe G, Kreutz M, Scho¨lmerich J, Falk W, Lang B. Serum dehydroepiandrosterone (DHEA) and DHEA sulfate are negatively correlated with serum interleukin-6 (IL-6), and DHEA inhibits IL-6 secretion from mononuclear cells in man in vitro: possible link between endocrinosenescence and immunosenescence. J Clin Endocrinol Metab 1998;83:2012 – 7. [34] Spencer NFL, Norton SD, Harrison LL, Li GZ, Daynes RA. Dysregulation of IL-10 production with aging: possible linkage to the age-associated decline in DHEA and its sulfated derivative. Exp Gerontol 1996;31:393 – 408. [35] Kanayama N, Maradny EE, Goto J, Terao T. Effect of dehydroepiandrosterone sulfate on interleukin-8 receptor during cervical ripening. Eur J Endocrinol 1998;138:587 – 93.