Heat Modulation of Tropoelastin, Fibrillin-1, and Matrix Metalloproteinase-12 in Human Skin In Vivo  Zhou Chen, Jin Young Seo, Yeon Kyung Kim, Se Rah Lee, Kyu Han Kim, Kwang Hyun Cho, Hee Chul Eun, Jin Ho Chung  Journal of Investigative Dermatology  Volume 124, Issue 1, Pages 70-78 (January 2005) DOI: 10.1111/j.0022-202X.2004.23550.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Heat increased tropoelastin expression in human skin in vivo. The buttock skin of young subjects (n=4) was heated at 43°C for 90 min. Specimens of heated and non-heated skins were obtained from each subject at 24, 48, and 72 h after heat treatment. Epidermis was completely separated from dermis, as described in Materials and Methods. (a, b) The level of tropoelastin mRNA was determined by RT-PCR, using total RNA extracted from epidermis and dermis, respectively. (c, d) Tropoelastin protein was measured by western blotting soluble proteins extracted from the epidermis and dermis, respectively. The bands are representative of findings in four subjects. Values are means±SEM of four subjects. TE, tropoelastin. *p<0.05 versus non-heated control skin. Journal of Investigative Dermatology 2005 124, 70-78DOI: (10.1111/j.0022-202X.2004.23550.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Heat increased fibrillin-1 expression in the epidermis, but decreased this expression in the dermis of human skin in vivo. The buttock skin of young subjects (n=3) was heat treated, as described in Materials and Methods. (a, b) The level of fibrillin-1 mRNA was determined by RT-PCR, using total RNA extracted from epidermis and dermis, respectively. (c, d) The fibrillin-1 protein was measured in soluble proteins extracted from the epidermis and dermis, respectively, by western blotting. Bands are representative of the findings of three subjects. Values are the means±SEM of three subjects *p<0.05 versus non-heated control skin. Journal of Investigative Dermatology 2005 124, 70-78DOI: (10.1111/j.0022-202X.2004.23550.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Immunohistochemical and immunofluorescence staining of tropoelastin and fibrillin-1 protein in heat-treated human skin in vivo. The buttock skin of young subjects (n=5) was heat treated as described in Materials and Methods. (a) Immunohistochemical and (b) immunofluorescent staining were performed using anti-human tropoelastin and fibrillin-1 antibodies. The total areas of tropoelastin-positive and fibrillin-1-positive fibers were determined by image analysis. The figures shown are representative of the findings of five subjects. Values are the means±SEM of five subjects. *p<0.05 versus non-heated control skin. Journal of Investigative Dermatology 2005 124, 70-78DOI: (10.1111/j.0022-202X.2004.23550.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 The antioxidants, genistein, and N-acetyl cysteine (NAC), inhibited the heat-induced expression of tropoelastin mRNA but not of fibrillin-1 mRNA in human skin epidermis in vivo. (a, c) 1% genistein or (b, d) 20% NAC were pre-treated topically on the buttock skin of young volunteers (n=5), under occlusion, for 24 h prior to heat treatment. Buttock skin was then heated as described in Materials and Methods. Heated and non-heated skin samples were taken at 48 h post-heat treatment by punch biopsy, and epidermis was completely separated from dermis. The levels of (a, b) tropoelastin and (c, d) fibrillin-1 mRNA were determined by RT-PCR, in total RNA extracted from epidermis. The bands shown are representative of the findings in five subjects. Values are the means±SEM of five subjects. TE, tropoelastin. *p<0.05 versus non-heated control skin, §p<0.05 versus heat-treated skin. Journal of Investigative Dermatology 2005 124, 70-78DOI: (10.1111/j.0022-202X.2004.23550.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Heat-induced matrix metalloproteinase (MMP)-12 expression in human skin in vivo. The buttock skin of young subjects (n=3) was heat treated as described in Materials and Methods. (a) The level of MMP-12 mRNA was determined by RT-PCR, using the total RNA extracted from dermis. (b) MMP-12 protein was measured in soluble proteins extracted from the dermis, by western blotting. The bands shown are representative of the findings of three subjects. Values are the means±SEM of three subjects *p<0.05 versus non-heated control skin. Journal of Investigative Dermatology 2005 124, 70-78DOI: (10.1111/j.0022-202X.2004.23550.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Heat modulation of tropoelastin, fibrillin-1, and MMP-12 in human skin in vivo. Journal of Investigative Dermatology 2005 124, 70-78DOI: (10.1111/j.0022-202X.2004.23550.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions