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Regulation of type I procollagen and MMP-1 expression after single or repeated exposure to infrared radiation in human skin
Mechanisms of Ageing and Development 127 (2006) 875–882 www.elsevier.com/locate/mechagedev
Regulation of type I procollagen and MMP-1 expression after single or repeated exposure to infrared radiation in human skin Mi-Sun Kim, Yeon Kyung Kim, Kwang Hyun Cho, Jin Ho Chung * Department of Dermatology, Seoul National University College of Medicine, Laboratory of Cutaneous Aging Research, Clinical Research Institute, Seoul National University Hospital, and Institute of Dermatological Science, Medical Research Center, Seoul National University, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Republic of Korea Received 27 March 2006; accepted 27 September 2006 Available online 25 October 2006
Abstract Human skin is daily exposed to infrared (IR) radiation from natural sunlight. However, the effects of IR irradiation on collagen metabolism have not been investigated in human skin in vivo. Here, we examined whether single or repeated (three times a week for 4 weeks) exposure to IR irradiation changes the expressions of type I procollagen and interstitial collagenase (MMP-1). By using immunostaining, Western blotting, and semi-quantitative RT-PCR, we analyzed the protein and mRNA levels of type I procollagen and MMP-1 in young buttock skin. A single dose of IR to human skin increased the expression of type I procollagen within 24 h, but did not change the expression of MMP-1. On the other hand, multiple IR doses reduced the expression of type I procollagen and increased the expression of MMP-1. We also found that TGF-bs may mediate type I procollagen synthesis in IR-irradiated human skin. Our results demonstrate that the regulations of the expressions of type I procollagen and MMP-1 differ in acute and chronically IR-irradiated skin. In particular, decreased collagen levels and increased MMP-1 levels in chronic IR-irradiated skin may be associated with connective tissue damage. Thus, we suggest that repeated exposure to IR irradiation might induce premature skin aging (photoaging) in human skin in vivo. # 2006 Published by Elsevier Ireland Ltd. Keywords: Infrared; Type I procollagen; MMP-1; TGF-b; Photoaging
1. Introduction Ultraviolet (UV) irradiation from the sun damages human skin, which causes it to age prematurely. This premature aging process is termed photoaging (extrinsic aging) and is cumulatively affected by sun exposure (Chung et al., 2003). Photoaging induces marked cutaneous alterations, which are clinically characterized by fine and coarse wrinkles, roughness, sallowness, mottled dyspigmentation, and telangiectasia. Histologic and ultrastructural studies have revealed that the major alterations in photoaged skin are Abbreviations: IR, Infrared; UV, Ultraviolet; ECM, Extracellular matrix; MMP, Matrix metalloproteinase; MHD, Minimal heating dose; TGF-b, Transforming growth factor-b * Corresponding author. Tel.: +82 2 2072 2414; fax: +82 2 742 7344. E-mail address: [email protected] (J.H. Chung). 0047-6374/$ – see front matter # 2006 Published by Elsevier Ireland Ltd. doi:10.1016/j.mad.2006.09.007
localized in dermal connective tissue (Bernstein and Uitto, 1996). Collagen accounts for roughly 90% of the protein in human dermis, and collagen alterations have been considered to be a primary cause of skin aging and wrinkle formation. Furthermore, collagen is crucial during connective tissue remodeling, e.g., wound healing and fibrosis. The matrix metalloproteinases (MMPs) are a large family of zinc-dependent endoproteases with a broad range of substrate specificities, and are capable of degrading all extracellular matrix proteins (ECMs). MMP-1, interstitial collagenase, initiates the degradation of type I and III collagens (Pilcher et al., 1998), and it has been established that single or repeated exposure to UV reduces type I procollagen levels and increases MMP-1 levels in human skin in vivo (Fisher et al., 2000, 1997). However, although the effects of UV radiation on human skin have been at the focus of photobiological research, little effort has been
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devoted to the study of the effects of infrared (IR) radiation on the expression of type I procollagen and MMP-1 in human skin in vivo. Human skin is frequently exposed to IR from natural sunlight as well as from artificial devices, which are being increasing used for phototherapeutic or cosmetic purposes. The IR spectrum may be arbitrarily divided by wavelength into three regions, IR-A (760–400 nm), IR-B (1400– 3000 nm) and IR-C (3000 nm–1 mm), or alternatively into near-IR (760–3000 nm), middle-IR (3000–30000 nm) and far-IR (30000 nm–1 mm). Solar IR reaching the earth’s surface is predominantly near-IR, and in actuality 54.3% of incident solar energy is composed of IR, whereas the energy contributions of UV and visible radiation are 6.8 and 38.9%, respectively (Kochevar et al., 1999). As was shown in a recent overview, IR exposure is considered to have biologic effects on human skin and some molecular mechanisms associated with its effects have been identified (Schieke et al., 2003). Repeated exposure to sources of heat and IR may result in a skin lesion described as erythema ab igne, which is clinically characterized by a reticular hyperpigmentation and telangiectasia, and histologically by dermal fiber alterations (Hurwitz and Tisserand, 1987). Most interestingly, chronic IR exposure can cause pronounced elastosis in mouse skin, which mimics UV-induced damage (Kligman, 1982). Recently, we demonstrated that chronic near-IR contributes to the induction of wrinkles in a hairless mouse (Kim et al., 2005a). These findings provide evidence that IR does induce ECM changes in skin. We have proposed that the minimal heating dose (MHD) be used as a novel standard unit to measure IR energy incident on human skin (Lee et al., 2006). When we irradiated volunteers’ skins with IR, skin temperatures rose up to a certain time point and then equilibrated. The biological unit ‘MHD’ corresponds to 1317.3 + 144.8 J/cm2 and was applied during the present study. Although many studies have been undertaken to elucidate the effects of UV irradiation on collagen metabolism, the effects of IR irradiation on collagen metabolism in human skin remain unclear. Therefore here, we examined whether a single (3 MHD) or repeated (3 MHD, three times a week for 4 weeks) exposure of human skin to IR irradiation changes the expressions of type I procollagen and MMP-1. We also investigated whether TGF-bs are associated with changes in type I procollagen expression in IR-irradiated skin.
Daekyoung Co., Kyungki, Korea) with an emission spectrum over 600 nm (maximum intensity at 1100–1120 nm) as described previously (Lee et al., 2006. The irradiance of the IR generated was measured using a 320-D Instrument System (Hioki E.E. Co., Nagano, Japan). The minimal heating dose (MHD) for each subject was defined as the minimal dose of IR required reaching a steady skin temperature (Lee et al., 2006). The average value of 1 MHD was 1.317 + 0.145 kJ/cm2 for Korean skin. For acute IR treatment, a single dose of 3 MHD was irradiated to buttock skin (n = 13). Skin samples were obtained from each subject by punch biopsy at 4, 24, and 48 h after IR irradiation. For chronic IR treatment, buttock skin was irradiated with 3 MHD of IR on alternate days, three times a week, for 4 weeks (n = 7). Skin samples were obtained from each subject by punch biopsy 24 h after the last IR treatment. The contralateral buttock skin of each subject served as nonirradiated control. All procedures involving human subjects were approved by the Institutional Review Board at the Seoul National University Hospital, and all subjects provided written informed consent.
2. Materials and methods
2.3. Semi-quantitative RT-PCR
2.1. IR exposure and skin samples
Total RNA was extracted from whole skin tissues using TRIZOL reagent (Invitrogen Life Technologies, Carlsbad, CA). RNA (1 (mg) was converted to cDNA using reverse transcriptase at 37 8C for 1 h (First Strand cDNA Synthesis Kit, Fermentas, Hanover, MD). PCR reactions were performed using 0.5–2 ml of cDNA and a pre-aliquoted ReddyMixTM PCR master mix (Abgene, Surrey, UK). Optimal semi-quantitative conditions were set to fall in the
Korean adult volunteers whose buttock skins had not been exposed to sunlight for at least 6 months, and with no history of disease associated with hypersensitivity to sunlight, were included in this study (20 men, mean age 27.0 + 3.9 years, age range 20–33 years). Human skin was exposed to IR using an IR lamp (IR 300,
2.2. Immunohistochemical and immunofluorescence staining Frozen human skin tissues were examined for type I procollagen expression as described previously (Shin et al., 2005). Briefly, snapfrozen tissues were placed immediately into a cryomatrix (Shandon, Pittsburgh, PA), stored at 70 8C, sectioned at 4 mm, mounted onto silane-coated slides (Dako, Glostrup, Denmark), and acetone fixed at 20 8C for 15 min. Then the sections were rehydrated and endogenous peroxidase activity was quenched using 3% hydrogen peroxide for 10 min. Sections were blocked with blocking solution (Zymed, San Francisco, CA) for 30 min, and incubated with monoclonal anti-al(I) procollagen aminoterminal extension peptide (SP1.D8) antibody (Developmental Studies Hybridoma Bank, Iowa City, IA) or with monoclonal anti-human procollagen type I C-peptide (PIC) antibody (Takara, Shiga, Japan) in a humidified chamber at 4 8C overnight. They were then incubated with biotinylated anti-mouse IgG antibody for 15 min and incubated with horseradish-streptoavidin conjugate for 15 min. After washing in PBS, color was developed using AEC (3-amino-9-ethylcarbazole; Zymed) for 5 to 10 min. The sections were then washed in running tap water for 5 min, and cell nuclei were counterstained with Mayer’s hematoxylin (Dako). For immunofluorescence staining, sections prepared as described above were incubated with anti-human TGF-b1, -b2, or -b3 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) in a humidified chamber at 4 8C overnight. Then the sections were incubated with secondary TRITC-conjugated goat anti-rabbit IgG (Dako) for 1 h at room temperature. After washing in PBS, nuclei were counterstained with DAPI. Immunofluorescent staining was monitored with confocal laser-scanning microscope (LSM 510, Carl Zeiss Co., Germany).
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linear PCR product range (data not shown). PCR consisted of 28 amplification cycles (94 8C, 1 min; 60 8C, 1 min; 72 8C, 1 min) for human al(I) procollagen and 30 amplification cycles for MMP-1 using the oligonucleotide primer sets detailed below. In parallel, the GAPDH house-keeping gene was amplified in each RNA sample. The sequences of the primers used were as follows: al(I) procollagen (forward, 50 -CTC GAG GTG GAC ACC ACC CT-30 ; reverse, 50 -CAG CTG GAT GGC CAC ATC GG-30 ), MMP-1 (forward, 50 ATT CTA CTG ATA TCG GGG CTT TGA-30 ; reverse, 50 -ATG TCC TTG GGG TAT CCG TGT AG-30 ), and internal control GAPDH (forward, 50 -ATT GTT GCC ATC AAT GAC CC-30 ; reverse, 50 -AGT AGA GGC AGG GAT GAT GT-30 ). Reaction products were electroporesed in 2% agarose gels and visualized with ethidium bromide. The primer sets used yielded PCR products of 366, 409 and 565 bp for al(I) procollagen, MMP-1 and GAPDH, respectively. Signal strengths were quantified using a densitometric program (Bio ID; Vilber Lourmat, Torcy Z.I., France). 2.4. Western blot analysis Western blot analyses of skin extracts were performed as described previously (Kim et al., 2005b). Briefly, samples were loaded and separated on 10% SDS-polyacrylamide gels, and proteins were electrotransferred onto nitrocellulose membranes (Amersham Pharmacia) at 4 8C. Membranes were then blocked with 5% nonfat dry milk in PBS containing 0.1% Tween-20 (PBST) for 1 h, and membranes were probed with primary antibody for 1 h. A monoclonal anti-al(I) procollagen aminoterminal extension peptide (SP1.D8) antibody (Developmental Studies Hybridoma Bank), and a monoclonal anti-MMP-1 antibody (Oncogene Research Products, San Diego, CA) were used as primary antibodies. Membranes were treated with anti-mouse IgG-HRP conjugates, and blots were detected using ECL detection reagent (Amersham Biosciences, Piscataway, NJ). Proteins were visualized by fluorography using Agfa X-ray film blue.
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2.5. Statistical analysis Significances of differences between nonirradiated skin and irradiated skin were analyzed using the Wilcoxon Signed-Ranks test, p-values of < 0.05 were considered statistically significant. All data were analyzed using SPSS for Windows (SPSS Inc., Chicago, IL).
3. Results 3.1. The effects of single IR irradiation on type I procollagen and MMP-1 expression in human skin in vivo To investigate the effects of acute IR irradiation on type I procollagen expression in human skin, we exposed human buttock skin to a single dose of near-IR irradiation (3 MHD). Skin sections of volunteers were immunostained with two types of antibody for type I procollagen. As described previously (Chung et al., 2001), anti-al(I) procollagen aminoterminal extension peptide (SP1.D8) antibody stains extracellular procollagen below the dermo-epidermal junction (Fig. 1a), and anti-procollagen type I C-peptide (PIC) antibody stains intracellular procollagen (Fig. 1b). Here, we found that the expression of type I procollagen was increased versus nonirradiated control skin at 24 h after this single dose of IR (n = 6, Fig. 1). To confirm that IR-induced type I procollagen, we performed semi-quantitative RT-PCR and Western blot analyses. Procollagen al(I) mRNA expressions in skins treated with a single dose of IR were significantly higher by 157.2 + 24.9% (n = 4, p < 0.05) at 4 h post-IR than in
Fig. 1. Type I procollagen expressions were increased in skin sections exposed to a single IR. Skin samples were obtained from each subject by punch biopsy at 24 h after a single IR irradiation (3 MHD, n = 6). The extracellular expression of type I procollagen was detected by immunohistochemical staining using (a) SP1.D8 antibody and (b) PIC antibody.
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Fig. 2. Levels of type I procollagen mRNA and protein were increased after a single IR irradiation. Skin samples were obtained from each subject by punch biopsy at 4, 24, and 48 h after a single IR irradiation (3 MHD). (a) Levels of procollagen al(I) mRNA were determined by RT-PCR (n = 4). Data are expressed as ratios of procollagen al(I) to GAPDH. (b) Levels of type I procollagen protein were determined by Western blotting (n = 7). Data are expressed as ratios of type I procollagen to b-actin. *p < 0.05 vs. nonirradiated control skin.
nonirradiated control skins (Fig. 2a). This increased mRNA expression returned to basal level at 48 h post-IR. The levels of type I procollagen protein in single IR-irradiated skin were significantly increased by 200.9 + 42.6% (n = 7, p < 0.05) at 24 h post-IR versus nonirradiated control skins (Fig. 2b). We next examined the expression of MMP-1, which is believed to initiate the degradation of type I collagen, its expressions were unchanged at the mRNA and protein levels by a single IR irradiation (n = 4, Fig. 3). 3.2. The effects of repeated IR irradiation on type I procollagen and MMP-1 expression in human skin in vivo To investigate the effects of chronic IR exposure on type I procollagen expression in human skin, human buttock skin was irradiated with 3 MHD of near-IR three times a week for
4 weeks. Skin samples were obtained from each subject by punch biopsy at 24 h after the last IR irradiation. Immunostaining with SP1.D8 antibody revealed greater reduction in type I procollagen expression after repeated IR irradiation (n = 3, Fig. 4a). Moreover, the intracellular expression of type I procollagen was also decreased in repeated IR-irradiated skin versus nonirradiated control skin (n = 3, Fig. 4b). To further confirm these different effects of multiple IR and single IR irradiation, we determined the expressions of type I procollagen and MMP-1 by RT-PCR and Western blotting. Repeated IR irradiation significantly reduced procollagen a1(I) mRNA expression (48.3 + 19.4%; n = 3, p < 0.05), but increased MMP-1 mRNA expression (175.5 + 37.5%; n = 3, p < 0.05) (Fig. 5a). The levels of type I procollagen protein in repeated IR-irradiated skin were significantly decreased by 40.8 10.1% (n = 7,
Fig. 3. MMP-1 mRNA and protein expressions were unchanged after a single IR irradiation. Skin samples were obtained from subjects by punch biopsy at 4, 24, and 48 h after a single IR irradiation (3 MHD). (a) The levels of MMP-1 mRNA were measured by RT-PCR (n = 4). Data are expressed as ratios of MMP-1 to GAPDH. (b) The levels of MMP-1 protein were determined by Western blotting (n = 4). Data are expressed as ratios of MMP-1 to b-actin.
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Fig. 4. Type I procollagen expressions in skin sections reduced after multiple IR irradiations. Skin samples were obtained from subjects by punch biopsy at 24 h after the final IR treatments (3 MHD, three times a week for 4 weeks, n = 3). Extracellular expressions of type I procollagen were detected by immunohistochemical staining using (a) SP1.D8 antibody and (b) PIC antibody.
p < 0.05), whereas MMP-1 protein levels increased by 164.4 32.4% (n = 7, p < 0.05) versus nonirradiated control skins (Fig. 5b). These results indicate that chronic exposure to IR can cause collagen deficiency by reducing collagen synthesis and inducing collagen degradation.
3.3. The effects of IR irradiation on TGF-bs expression in human skin in vivo TGF-bs are major profibrotic cytokines, which positively regulate type I and type III procollagen production
Fig. 5. Type I procollagen expression was decreased and MMP-1 expression was increased after multiple IR irradiations. Skin samples were obtained from each subject by punch biopsy at 24 h after final IR treatments (3 MHD, three times a week for 4 weeks). (a) procollagen al(I) and MMP-1 mRNA levels were measured by RT-PCR (n = 3). Densitometric data are expressed as ratios of target gene to GAPDH levels, (b) type I procollagen and MMP-1 protein levels were measured by Western blotting (n = 7). Data are expressed as ratios of target genes to b-actin. *p < 0.05 vs. nonirradiated control skin.
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Fig. 6. The expressions of TGF-b family members changed in accord with type I procollagen expression changes in IR-irradiated skin. Samples were obtained by punch biopsy at 24 h after single or multiple IR irradiations. Immunofluorescent staining was performed using anti-human TGF-b1, -b2, or -b3 antibodies in (a) singly or (b) multiply IR-irradiated skin.
(Massague, 1998). We investigated whether IR-induced type I procollagen expression is associated with TGF-b expression in human skin in vivo. Immunofluorescence staining was performed using anti-human TGF-b1, -b2, or -b3 antibodies at 24 h post-IR in single or multiple IR-irradiated skins. TGF-b1 was found to stain through the entire epidermis and some dermal cells, whereas TGF-b2 and TGF-b3 were primarily expressed in the lower epidermis (Fig. 6). We found that a single IR treatment increased the expressions of TGF-bs at 24 h after exposure (Fig. 6a), but that multiple IR treatment reduced their expressions at 24 h after last exposure (Fig. 6b). These results suggest that TGFb may mediate changes in type I procollagen synthesis in IR exposed human skin.
4. Discussion The effects of UV on collagen metabolism have been widely studied, and it has been well established that the UV irradiation reduces the expression of procollagen and induces the expression of MMPs in human skin in vitro and in vivo (Fisher et al., 2002). Moreover, alterations in collagen, the major structural component of skin, have been suggested to cause clinical changes, such as, wrinkles and elastin loss observed in aged skin. Although human skin is frequently exposed to IR radiation, little is known about the biological effects of IR on collagen metabolism in human skin. In this study, we investigated for the first time the effects of acute and chronic IR exposure on type I procollagen expression. It was found by immunohistochemical staining, Western blotting and RTPCR that a single IR irradiation to the human skin increased type I procollagen expression, but that multiple IR irradiations reduced its expression. The procollagen
increases induced by a single IR irradiation are in accordance with the views from previous studies, which found that IR promotes the wound healing process (Danno et al., 2001; Schramm et al., 2003). However, although they suggested that near-IR has potential therapeutic effects on the cutaneous wound healing process in animal models, no direct changes in collagen synthesis were reported. Wound healing is a complex process that can be viewed as a sequential process involving inflammation, proliferation and maturation of newly formed tissues (Chodorowska and Rogus-Skorupska, 2004). Once the tissue within the wound is occurred the synthesis of collagens and other ECM components. A recent report demonstrated that lasers generating heat can play a role in the healing process with a coordinated expression of TGF-b in skin (Capon and Mordon, 2003). In the present study, a single IR irradiation on human skins increased the expression of type I procollagen, and multiple IR irradiations reduced the expression of type I procollagen. Thus, we investigated whether IR-induced type I procollagen expression is associated with TGF-bs expression in human skin in vivo. TGF-b is a multifunctional cytokine that plays an important role in biosynthesis of extracelluar connective tissue (Massague, 1990). It is well known that TGF-b potently stimulates the proliferation of fibroblasts in the dermis and induces the synthesis and secretion of procollagen (Massague, 1998; Varga et al., 1987). The actions of TGF-b are mediated by specific cell surface receptors, TbRI, TbRII, and TbRIII. The binding of TGF-bs to their receptors results in the activations of the transcription factors Smad2 and Smad3, which combine with Smad4 to enter the nucleus and regulate target gene expression, such as, type I procollagen. UV exposure has been shown to impair the TGF-b signaling pathway by reducing TpRII expression and increasing Smad7, the
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inhibitory Smad (Quan et al., 2001). Thus, UV impairs the initial step of the TGF-b/Smad signaling cascade. In the present study, we found that a single IR irradiation increased the expression of type I procollagen and the expressions of TGF-b1, -b2, and -b3 in human skin in vivo. In addition, we also found that repeated IR irradiation reduced the expressions of both type I procollagen and these TGF-bs. These results suggest that TGF-b may mediate type I procollagen synthesis in IR exposed human skin. However, further study is required to determine how IR regulates the expressions of TGF-bs and how IR-induced TGF-bs affect the collagen metabolism. The exposure of human skin to UV radiation induces MMP expression, which results in the cleavage of fibrillar collagen, and thus impairs the structural integrity of the dermis. Thus, repeated exposure to UV radiation and insufficient repair lead to accumulated connective tissue damage; the key pathophysiological factor in the photoaging process (Fisher et al., 1997; Kang et al., 2001). Recently, it was reported a single treatment of human dermal fibroblasts with IR-A induced MMP-1, and that this effect was not mediated by heat generation by IR-A (Schieke et al., 2002). In our study, MMP1 was not induced by single IR irradiation in human skin (Fig. 3). One noticeable difference between Schieke’s study and the present study concerns the IR sources used. They used a water-filtered IR-A source which generated no heat and no increase in temperature, whereas we used a near-IR lamp which generated heat and increased skin temperature (Kim et al., in press). The molecular effects and biological consequences of IR irradiation can be mediated by the production of heat via the internal conversion of electromagnetic energy into vibrational energy. Another difference between the two studies is that they used human dermal fibroblsts and an in vitro approach, whereas we used human skin samples and an in vivo approach. In contrast to the effects of a single IR irradiation, multiple IR irradiations significantly increased MMP-1 expression. It has been demonstrated that increased procollagen degradation by MMP may contribute to the observed reductions in type I procollagen in photodamaged skin (Talwar et al., 1995). The balance between the synthesis of procollagen and degradation of newly synthesized procollagen is critical in determination of net collagen synthesis in skin. The present study clearly demonstrates that multiple IR irradiation increases MMP-1 levels and reduces type I procollagen levels like in the same manner as UV. In conclusion, our results indicate that the regulations of type I procollagen and MMP-1 differ in human skin acutely and chronically exposed to IR. In particular, the observed reduction in collagen levels and increase in MMP-1 levels in chronically exposed skins might be associated with connective tissue damage. Thus, we suggest that repeated exposure to IR irradiation might induce premature skin aging (photoaging) in human skin in vivo and that the protection of skin from incident electromagnetic radiation should include considerations of protection from IR.
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Acknowledgements This study was supported by the Korea Science and Engineering Foundation (KOSEF) through the Center for Ageing and Apoptosis Research at Seoul National University (Rl 1-2002-097-06001-0) and by a research agreement with the Amore-Pacific Corporation. We thank Ae Kyung Woo and Ju Mi Shim for their technical assistance.
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Schramm, J.M., Warner, D., Hardesty, R.A., Oberg, K.C., 2003. A unique combination of infrared and microwave radiation accelerates wound healing. Plast. Reconstr. Surg. 111, 258–266. Shin, M.H., Rhie, G.E., Park, C.H., Kim, K.H., Cho, K.H., Eun, H.C., Chung, J.H., 2005. Modulation of collagen metabolism by the topical application of dehydroepiandrosterone to human skin. J. Invest. Dermatol. 124, 315–323. Talwar, H.S., Griffiths, C.E., Fisher, G.J., Hamilton, T.A., Voorhees, J.J., 1995. Reduced type I and type III procollagens in photodamaged adult human skin. J. Invest. Dermatol. 105, 285–290. Varga, J., Rosenbloom, J., Jimenez, S.A., 1987. Transforming growth factor beta (TGF beta) causes a persistent increase in steady-state amounts of type I and type III collagen and fibronectin mRNAs in normal human dermal fibroblasts. Biochem. J. 247, 597–604.
Regulation of type I procollagen and MMP-1 expression after single or repeated exposure to infrared radiation in human skin Mi-Sun Kim, Yeon Kyung Kim, Kwang Hyun Cho, Jin Ho Chung * Department of Dermatology, Seoul National University College of Medicine, Laboratory of Cutaneous Aging Research, Clinical Research Institute, Seoul National University Hospital, and Institute of Dermatological Science, Medical Research Center, Seoul National University, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Republic of Korea Received 27 March 2006; accepted 27 September 2006 Available online 25 October 2006
Abstract Human skin is daily exposed to infrared (IR) radiation from natural sunlight. However, the effects of IR irradiation on collagen metabolism have not been investigated in human skin in vivo. Here, we examined whether single or repeated (three times a week for 4 weeks) exposure to IR irradiation changes the expressions of type I procollagen and interstitial collagenase (MMP-1). By using immunostaining, Western blotting, and semi-quantitative RT-PCR, we analyzed the protein and mRNA levels of type I procollagen and MMP-1 in young buttock skin. A single dose of IR to human skin increased the expression of type I procollagen within 24 h, but did not change the expression of MMP-1. On the other hand, multiple IR doses reduced the expression of type I procollagen and increased the expression of MMP-1. We also found that TGF-bs may mediate type I procollagen synthesis in IR-irradiated human skin. Our results demonstrate that the regulations of the expressions of type I procollagen and MMP-1 differ in acute and chronically IR-irradiated skin. In particular, decreased collagen levels and increased MMP-1 levels in chronic IR-irradiated skin may be associated with connective tissue damage. Thus, we suggest that repeated exposure to IR irradiation might induce premature skin aging (photoaging) in human skin in vivo. # 2006 Published by Elsevier Ireland Ltd. Keywords: Infrared; Type I procollagen; MMP-1; TGF-b; Photoaging
1. Introduction Ultraviolet (UV) irradiation from the sun damages human skin, which causes it to age prematurely. This premature aging process is termed photoaging (extrinsic aging) and is cumulatively affected by sun exposure (Chung et al., 2003). Photoaging induces marked cutaneous alterations, which are clinically characterized by fine and coarse wrinkles, roughness, sallowness, mottled dyspigmentation, and telangiectasia. Histologic and ultrastructural studies have revealed that the major alterations in photoaged skin are Abbreviations: IR, Infrared; UV, Ultraviolet; ECM, Extracellular matrix; MMP, Matrix metalloproteinase; MHD, Minimal heating dose; TGF-b, Transforming growth factor-b * Corresponding author. Tel.: +82 2 2072 2414; fax: +82 2 742 7344. E-mail address: [email protected] (J.H. Chung). 0047-6374/$ – see front matter # 2006 Published by Elsevier Ireland Ltd. doi:10.1016/j.mad.2006.09.007
localized in dermal connective tissue (Bernstein and Uitto, 1996). Collagen accounts for roughly 90% of the protein in human dermis, and collagen alterations have been considered to be a primary cause of skin aging and wrinkle formation. Furthermore, collagen is crucial during connective tissue remodeling, e.g., wound healing and fibrosis. The matrix metalloproteinases (MMPs) are a large family of zinc-dependent endoproteases with a broad range of substrate specificities, and are capable of degrading all extracellular matrix proteins (ECMs). MMP-1, interstitial collagenase, initiates the degradation of type I and III collagens (Pilcher et al., 1998), and it has been established that single or repeated exposure to UV reduces type I procollagen levels and increases MMP-1 levels in human skin in vivo (Fisher et al., 2000, 1997). However, although the effects of UV radiation on human skin have been at the focus of photobiological research, little effort has been
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devoted to the study of the effects of infrared (IR) radiation on the expression of type I procollagen and MMP-1 in human skin in vivo. Human skin is frequently exposed to IR from natural sunlight as well as from artificial devices, which are being increasing used for phototherapeutic or cosmetic purposes. The IR spectrum may be arbitrarily divided by wavelength into three regions, IR-A (760–400 nm), IR-B (1400– 3000 nm) and IR-C (3000 nm–1 mm), or alternatively into near-IR (760–3000 nm), middle-IR (3000–30000 nm) and far-IR (30000 nm–1 mm). Solar IR reaching the earth’s surface is predominantly near-IR, and in actuality 54.3% of incident solar energy is composed of IR, whereas the energy contributions of UV and visible radiation are 6.8 and 38.9%, respectively (Kochevar et al., 1999). As was shown in a recent overview, IR exposure is considered to have biologic effects on human skin and some molecular mechanisms associated with its effects have been identified (Schieke et al., 2003). Repeated exposure to sources of heat and IR may result in a skin lesion described as erythema ab igne, which is clinically characterized by a reticular hyperpigmentation and telangiectasia, and histologically by dermal fiber alterations (Hurwitz and Tisserand, 1987). Most interestingly, chronic IR exposure can cause pronounced elastosis in mouse skin, which mimics UV-induced damage (Kligman, 1982). Recently, we demonstrated that chronic near-IR contributes to the induction of wrinkles in a hairless mouse (Kim et al., 2005a). These findings provide evidence that IR does induce ECM changes in skin. We have proposed that the minimal heating dose (MHD) be used as a novel standard unit to measure IR energy incident on human skin (Lee et al., 2006). When we irradiated volunteers’ skins with IR, skin temperatures rose up to a certain time point and then equilibrated. The biological unit ‘MHD’ corresponds to 1317.3 + 144.8 J/cm2 and was applied during the present study. Although many studies have been undertaken to elucidate the effects of UV irradiation on collagen metabolism, the effects of IR irradiation on collagen metabolism in human skin remain unclear. Therefore here, we examined whether a single (3 MHD) or repeated (3 MHD, three times a week for 4 weeks) exposure of human skin to IR irradiation changes the expressions of type I procollagen and MMP-1. We also investigated whether TGF-bs are associated with changes in type I procollagen expression in IR-irradiated skin.
Daekyoung Co., Kyungki, Korea) with an emission spectrum over 600 nm (maximum intensity at 1100–1120 nm) as described previously (Lee et al., 2006. The irradiance of the IR generated was measured using a 320-D Instrument System (Hioki E.E. Co., Nagano, Japan). The minimal heating dose (MHD) for each subject was defined as the minimal dose of IR required reaching a steady skin temperature (Lee et al., 2006). The average value of 1 MHD was 1.317 + 0.145 kJ/cm2 for Korean skin. For acute IR treatment, a single dose of 3 MHD was irradiated to buttock skin (n = 13). Skin samples were obtained from each subject by punch biopsy at 4, 24, and 48 h after IR irradiation. For chronic IR treatment, buttock skin was irradiated with 3 MHD of IR on alternate days, three times a week, for 4 weeks (n = 7). Skin samples were obtained from each subject by punch biopsy 24 h after the last IR treatment. The contralateral buttock skin of each subject served as nonirradiated control. All procedures involving human subjects were approved by the Institutional Review Board at the Seoul National University Hospital, and all subjects provided written informed consent.
2. Materials and methods
2.3. Semi-quantitative RT-PCR
2.1. IR exposure and skin samples
Total RNA was extracted from whole skin tissues using TRIZOL reagent (Invitrogen Life Technologies, Carlsbad, CA). RNA (1 (mg) was converted to cDNA using reverse transcriptase at 37 8C for 1 h (First Strand cDNA Synthesis Kit, Fermentas, Hanover, MD). PCR reactions were performed using 0.5–2 ml of cDNA and a pre-aliquoted ReddyMixTM PCR master mix (Abgene, Surrey, UK). Optimal semi-quantitative conditions were set to fall in the
Korean adult volunteers whose buttock skins had not been exposed to sunlight for at least 6 months, and with no history of disease associated with hypersensitivity to sunlight, were included in this study (20 men, mean age 27.0 + 3.9 years, age range 20–33 years). Human skin was exposed to IR using an IR lamp (IR 300,
2.2. Immunohistochemical and immunofluorescence staining Frozen human skin tissues were examined for type I procollagen expression as described previously (Shin et al., 2005). Briefly, snapfrozen tissues were placed immediately into a cryomatrix (Shandon, Pittsburgh, PA), stored at 70 8C, sectioned at 4 mm, mounted onto silane-coated slides (Dako, Glostrup, Denmark), and acetone fixed at 20 8C for 15 min. Then the sections were rehydrated and endogenous peroxidase activity was quenched using 3% hydrogen peroxide for 10 min. Sections were blocked with blocking solution (Zymed, San Francisco, CA) for 30 min, and incubated with monoclonal anti-al(I) procollagen aminoterminal extension peptide (SP1.D8) antibody (Developmental Studies Hybridoma Bank, Iowa City, IA) or with monoclonal anti-human procollagen type I C-peptide (PIC) antibody (Takara, Shiga, Japan) in a humidified chamber at 4 8C overnight. They were then incubated with biotinylated anti-mouse IgG antibody for 15 min and incubated with horseradish-streptoavidin conjugate for 15 min. After washing in PBS, color was developed using AEC (3-amino-9-ethylcarbazole; Zymed) for 5 to 10 min. The sections were then washed in running tap water for 5 min, and cell nuclei were counterstained with Mayer’s hematoxylin (Dako). For immunofluorescence staining, sections prepared as described above were incubated with anti-human TGF-b1, -b2, or -b3 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) in a humidified chamber at 4 8C overnight. Then the sections were incubated with secondary TRITC-conjugated goat anti-rabbit IgG (Dako) for 1 h at room temperature. After washing in PBS, nuclei were counterstained with DAPI. Immunofluorescent staining was monitored with confocal laser-scanning microscope (LSM 510, Carl Zeiss Co., Germany).
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linear PCR product range (data not shown). PCR consisted of 28 amplification cycles (94 8C, 1 min; 60 8C, 1 min; 72 8C, 1 min) for human al(I) procollagen and 30 amplification cycles for MMP-1 using the oligonucleotide primer sets detailed below. In parallel, the GAPDH house-keeping gene was amplified in each RNA sample. The sequences of the primers used were as follows: al(I) procollagen (forward, 50 -CTC GAG GTG GAC ACC ACC CT-30 ; reverse, 50 -CAG CTG GAT GGC CAC ATC GG-30 ), MMP-1 (forward, 50 ATT CTA CTG ATA TCG GGG CTT TGA-30 ; reverse, 50 -ATG TCC TTG GGG TAT CCG TGT AG-30 ), and internal control GAPDH (forward, 50 -ATT GTT GCC ATC AAT GAC CC-30 ; reverse, 50 -AGT AGA GGC AGG GAT GAT GT-30 ). Reaction products were electroporesed in 2% agarose gels and visualized with ethidium bromide. The primer sets used yielded PCR products of 366, 409 and 565 bp for al(I) procollagen, MMP-1 and GAPDH, respectively. Signal strengths were quantified using a densitometric program (Bio ID; Vilber Lourmat, Torcy Z.I., France). 2.4. Western blot analysis Western blot analyses of skin extracts were performed as described previously (Kim et al., 2005b). Briefly, samples were loaded and separated on 10% SDS-polyacrylamide gels, and proteins were electrotransferred onto nitrocellulose membranes (Amersham Pharmacia) at 4 8C. Membranes were then blocked with 5% nonfat dry milk in PBS containing 0.1% Tween-20 (PBST) for 1 h, and membranes were probed with primary antibody for 1 h. A monoclonal anti-al(I) procollagen aminoterminal extension peptide (SP1.D8) antibody (Developmental Studies Hybridoma Bank), and a monoclonal anti-MMP-1 antibody (Oncogene Research Products, San Diego, CA) were used as primary antibodies. Membranes were treated with anti-mouse IgG-HRP conjugates, and blots were detected using ECL detection reagent (Amersham Biosciences, Piscataway, NJ). Proteins were visualized by fluorography using Agfa X-ray film blue.
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2.5. Statistical analysis Significances of differences between nonirradiated skin and irradiated skin were analyzed using the Wilcoxon Signed-Ranks test, p-values of < 0.05 were considered statistically significant. All data were analyzed using SPSS for Windows (SPSS Inc., Chicago, IL).
3. Results 3.1. The effects of single IR irradiation on type I procollagen and MMP-1 expression in human skin in vivo To investigate the effects of acute IR irradiation on type I procollagen expression in human skin, we exposed human buttock skin to a single dose of near-IR irradiation (3 MHD). Skin sections of volunteers were immunostained with two types of antibody for type I procollagen. As described previously (Chung et al., 2001), anti-al(I) procollagen aminoterminal extension peptide (SP1.D8) antibody stains extracellular procollagen below the dermo-epidermal junction (Fig. 1a), and anti-procollagen type I C-peptide (PIC) antibody stains intracellular procollagen (Fig. 1b). Here, we found that the expression of type I procollagen was increased versus nonirradiated control skin at 24 h after this single dose of IR (n = 6, Fig. 1). To confirm that IR-induced type I procollagen, we performed semi-quantitative RT-PCR and Western blot analyses. Procollagen al(I) mRNA expressions in skins treated with a single dose of IR were significantly higher by 157.2 + 24.9% (n = 4, p < 0.05) at 4 h post-IR than in
Fig. 1. Type I procollagen expressions were increased in skin sections exposed to a single IR. Skin samples were obtained from each subject by punch biopsy at 24 h after a single IR irradiation (3 MHD, n = 6). The extracellular expression of type I procollagen was detected by immunohistochemical staining using (a) SP1.D8 antibody and (b) PIC antibody.
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Fig. 2. Levels of type I procollagen mRNA and protein were increased after a single IR irradiation. Skin samples were obtained from each subject by punch biopsy at 4, 24, and 48 h after a single IR irradiation (3 MHD). (a) Levels of procollagen al(I) mRNA were determined by RT-PCR (n = 4). Data are expressed as ratios of procollagen al(I) to GAPDH. (b) Levels of type I procollagen protein were determined by Western blotting (n = 7). Data are expressed as ratios of type I procollagen to b-actin. *p < 0.05 vs. nonirradiated control skin.
nonirradiated control skins (Fig. 2a). This increased mRNA expression returned to basal level at 48 h post-IR. The levels of type I procollagen protein in single IR-irradiated skin were significantly increased by 200.9 + 42.6% (n = 7, p < 0.05) at 24 h post-IR versus nonirradiated control skins (Fig. 2b). We next examined the expression of MMP-1, which is believed to initiate the degradation of type I collagen, its expressions were unchanged at the mRNA and protein levels by a single IR irradiation (n = 4, Fig. 3). 3.2. The effects of repeated IR irradiation on type I procollagen and MMP-1 expression in human skin in vivo To investigate the effects of chronic IR exposure on type I procollagen expression in human skin, human buttock skin was irradiated with 3 MHD of near-IR three times a week for
4 weeks. Skin samples were obtained from each subject by punch biopsy at 24 h after the last IR irradiation. Immunostaining with SP1.D8 antibody revealed greater reduction in type I procollagen expression after repeated IR irradiation (n = 3, Fig. 4a). Moreover, the intracellular expression of type I procollagen was also decreased in repeated IR-irradiated skin versus nonirradiated control skin (n = 3, Fig. 4b). To further confirm these different effects of multiple IR and single IR irradiation, we determined the expressions of type I procollagen and MMP-1 by RT-PCR and Western blotting. Repeated IR irradiation significantly reduced procollagen a1(I) mRNA expression (48.3 + 19.4%; n = 3, p < 0.05), but increased MMP-1 mRNA expression (175.5 + 37.5%; n = 3, p < 0.05) (Fig. 5a). The levels of type I procollagen protein in repeated IR-irradiated skin were significantly decreased by 40.8 10.1% (n = 7,
Fig. 3. MMP-1 mRNA and protein expressions were unchanged after a single IR irradiation. Skin samples were obtained from subjects by punch biopsy at 4, 24, and 48 h after a single IR irradiation (3 MHD). (a) The levels of MMP-1 mRNA were measured by RT-PCR (n = 4). Data are expressed as ratios of MMP-1 to GAPDH. (b) The levels of MMP-1 protein were determined by Western blotting (n = 4). Data are expressed as ratios of MMP-1 to b-actin.
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Fig. 4. Type I procollagen expressions in skin sections reduced after multiple IR irradiations. Skin samples were obtained from subjects by punch biopsy at 24 h after the final IR treatments (3 MHD, three times a week for 4 weeks, n = 3). Extracellular expressions of type I procollagen were detected by immunohistochemical staining using (a) SP1.D8 antibody and (b) PIC antibody.
p < 0.05), whereas MMP-1 protein levels increased by 164.4 32.4% (n = 7, p < 0.05) versus nonirradiated control skins (Fig. 5b). These results indicate that chronic exposure to IR can cause collagen deficiency by reducing collagen synthesis and inducing collagen degradation.
3.3. The effects of IR irradiation on TGF-bs expression in human skin in vivo TGF-bs are major profibrotic cytokines, which positively regulate type I and type III procollagen production
Fig. 5. Type I procollagen expression was decreased and MMP-1 expression was increased after multiple IR irradiations. Skin samples were obtained from each subject by punch biopsy at 24 h after final IR treatments (3 MHD, three times a week for 4 weeks). (a) procollagen al(I) and MMP-1 mRNA levels were measured by RT-PCR (n = 3). Densitometric data are expressed as ratios of target gene to GAPDH levels, (b) type I procollagen and MMP-1 protein levels were measured by Western blotting (n = 7). Data are expressed as ratios of target genes to b-actin. *p < 0.05 vs. nonirradiated control skin.
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Fig. 6. The expressions of TGF-b family members changed in accord with type I procollagen expression changes in IR-irradiated skin. Samples were obtained by punch biopsy at 24 h after single or multiple IR irradiations. Immunofluorescent staining was performed using anti-human TGF-b1, -b2, or -b3 antibodies in (a) singly or (b) multiply IR-irradiated skin.
(Massague, 1998). We investigated whether IR-induced type I procollagen expression is associated with TGF-b expression in human skin in vivo. Immunofluorescence staining was performed using anti-human TGF-b1, -b2, or -b3 antibodies at 24 h post-IR in single or multiple IR-irradiated skins. TGF-b1 was found to stain through the entire epidermis and some dermal cells, whereas TGF-b2 and TGF-b3 were primarily expressed in the lower epidermis (Fig. 6). We found that a single IR treatment increased the expressions of TGF-bs at 24 h after exposure (Fig. 6a), but that multiple IR treatment reduced their expressions at 24 h after last exposure (Fig. 6b). These results suggest that TGFb may mediate changes in type I procollagen synthesis in IR exposed human skin.
4. Discussion The effects of UV on collagen metabolism have been widely studied, and it has been well established that the UV irradiation reduces the expression of procollagen and induces the expression of MMPs in human skin in vitro and in vivo (Fisher et al., 2002). Moreover, alterations in collagen, the major structural component of skin, have been suggested to cause clinical changes, such as, wrinkles and elastin loss observed in aged skin. Although human skin is frequently exposed to IR radiation, little is known about the biological effects of IR on collagen metabolism in human skin. In this study, we investigated for the first time the effects of acute and chronic IR exposure on type I procollagen expression. It was found by immunohistochemical staining, Western blotting and RTPCR that a single IR irradiation to the human skin increased type I procollagen expression, but that multiple IR irradiations reduced its expression. The procollagen
increases induced by a single IR irradiation are in accordance with the views from previous studies, which found that IR promotes the wound healing process (Danno et al., 2001; Schramm et al., 2003). However, although they suggested that near-IR has potential therapeutic effects on the cutaneous wound healing process in animal models, no direct changes in collagen synthesis were reported. Wound healing is a complex process that can be viewed as a sequential process involving inflammation, proliferation and maturation of newly formed tissues (Chodorowska and Rogus-Skorupska, 2004). Once the tissue within the wound is occurred the synthesis of collagens and other ECM components. A recent report demonstrated that lasers generating heat can play a role in the healing process with a coordinated expression of TGF-b in skin (Capon and Mordon, 2003). In the present study, a single IR irradiation on human skins increased the expression of type I procollagen, and multiple IR irradiations reduced the expression of type I procollagen. Thus, we investigated whether IR-induced type I procollagen expression is associated with TGF-bs expression in human skin in vivo. TGF-b is a multifunctional cytokine that plays an important role in biosynthesis of extracelluar connective tissue (Massague, 1990). It is well known that TGF-b potently stimulates the proliferation of fibroblasts in the dermis and induces the synthesis and secretion of procollagen (Massague, 1998; Varga et al., 1987). The actions of TGF-b are mediated by specific cell surface receptors, TbRI, TbRII, and TbRIII. The binding of TGF-bs to their receptors results in the activations of the transcription factors Smad2 and Smad3, which combine with Smad4 to enter the nucleus and regulate target gene expression, such as, type I procollagen. UV exposure has been shown to impair the TGF-b signaling pathway by reducing TpRII expression and increasing Smad7, the
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inhibitory Smad (Quan et al., 2001). Thus, UV impairs the initial step of the TGF-b/Smad signaling cascade. In the present study, we found that a single IR irradiation increased the expression of type I procollagen and the expressions of TGF-b1, -b2, and -b3 in human skin in vivo. In addition, we also found that repeated IR irradiation reduced the expressions of both type I procollagen and these TGF-bs. These results suggest that TGF-b may mediate type I procollagen synthesis in IR exposed human skin. However, further study is required to determine how IR regulates the expressions of TGF-bs and how IR-induced TGF-bs affect the collagen metabolism. The exposure of human skin to UV radiation induces MMP expression, which results in the cleavage of fibrillar collagen, and thus impairs the structural integrity of the dermis. Thus, repeated exposure to UV radiation and insufficient repair lead to accumulated connective tissue damage; the key pathophysiological factor in the photoaging process (Fisher et al., 1997; Kang et al., 2001). Recently, it was reported a single treatment of human dermal fibroblasts with IR-A induced MMP-1, and that this effect was not mediated by heat generation by IR-A (Schieke et al., 2002). In our study, MMP1 was not induced by single IR irradiation in human skin (Fig. 3). One noticeable difference between Schieke’s study and the present study concerns the IR sources used. They used a water-filtered IR-A source which generated no heat and no increase in temperature, whereas we used a near-IR lamp which generated heat and increased skin temperature (Kim et al., in press). The molecular effects and biological consequences of IR irradiation can be mediated by the production of heat via the internal conversion of electromagnetic energy into vibrational energy. Another difference between the two studies is that they used human dermal fibroblsts and an in vitro approach, whereas we used human skin samples and an in vivo approach. In contrast to the effects of a single IR irradiation, multiple IR irradiations significantly increased MMP-1 expression. It has been demonstrated that increased procollagen degradation by MMP may contribute to the observed reductions in type I procollagen in photodamaged skin (Talwar et al., 1995). The balance between the synthesis of procollagen and degradation of newly synthesized procollagen is critical in determination of net collagen synthesis in skin. The present study clearly demonstrates that multiple IR irradiation increases MMP-1 levels and reduces type I procollagen levels like in the same manner as UV. In conclusion, our results indicate that the regulations of type I procollagen and MMP-1 differ in human skin acutely and chronically exposed to IR. In particular, the observed reduction in collagen levels and increase in MMP-1 levels in chronically exposed skins might be associated with connective tissue damage. Thus, we suggest that repeated exposure to IR irradiation might induce premature skin aging (photoaging) in human skin in vivo and that the protection of skin from incident electromagnetic radiation should include considerations of protection from IR.
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Acknowledgements This study was supported by the Korea Science and Engineering Foundation (KOSEF) through the Center for Ageing and Apoptosis Research at Seoul National University (Rl 1-2002-097-06001-0) and by a research agreement with the Amore-Pacific Corporation. We thank Ae Kyung Woo and Ju Mi Shim for their technical assistance.
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Schramm, J.M., Warner, D., Hardesty, R.A., Oberg, K.C., 2003. A unique combination of infrared and microwave radiation accelerates wound healing. Plast. Reconstr. Surg. 111, 258–266. Shin, M.H., Rhie, G.E., Park, C.H., Kim, K.H., Cho, K.H., Eun, H.C., Chung, J.H., 2005. Modulation of collagen metabolism by the topical application of dehydroepiandrosterone to human skin. J. Invest. Dermatol. 124, 315–323. Talwar, H.S., Griffiths, C.E., Fisher, G.J., Hamilton, T.A., Voorhees, J.J., 1995. Reduced type I and type III procollagens in photodamaged adult human skin. J. Invest. Dermatol. 105, 285–290. Varga, J., Rosenbloom, J., Jimenez, S.A., 1987. Transforming growth factor beta (TGF beta) causes a persistent increase in steady-state amounts of type I and type III collagen and fibronectin mRNAs in normal human dermal fibroblasts. Biochem. J. 247, 597–604.