TaiHao Quan, TianYuan He, Sewon Kang, John J. Voorhees, Gary J

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Ultraviolet Irradiation Alters Transforming Growth Factor β/Smad Pathway in Human Skin In Vivo TaiHao Quan, TianYuan He, Sewon Kang, John J. Voorhees, Gary J. Fisher, Dr Journal of Investigative Dermatology Volume 119, Issue 2, Pages 499-506 (August 2002) DOI: 10.1046/j.1523-1747.2002.01834.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 UV irradiation alters TGF-β1, TGF-β2, and TGF-β3 mRNA expression in human skin in vivo– northern analysis. Human skin biopsies were obtained at the indicated times following exposure to UVB (2 MED, UVB source). mRNAs for TGF-β1, TGF-β2, and TGF-β3 were analyzed by northern analysis and quantitated by STORM PhosphorImager. Data were normalized to mRNA for 36B4, a ribosomal protein used as an internal control for quantitation. The insets in each panel show representative northern blots. Bar heights represent means ± SEM. Data are expressed as fold change in mRNA levels relative to levels in nonirradiated control skin. *p <0.05 vs nonirradiated control skin, n = 6–7. Journal of Investigative Dermatology 2002 119, 499-506DOI: (10.1046/j.1523-1747.2002.01834.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Localization of TGF-β1, TGF-β2, and TGF-β3 mRNA expression following UV irradiation in human skin in vivo–in situ hybridization. Full-thickness 4 mm biopsies were obtained 72 h post UV (2 MED) irradiation. Cryostat sections were hybridized with TGF-β1, TGF-β2, and TGF-β3 sense or antisense ribo probes, as described in Materials and Methods. (a) TGF-β1 antisense probe for nonirradiated skin; (b) TGF-β1 antisense probe for UV-irradiated skin; (c) UV-irradiated skin with TGF-β1 sense probe; (d) TGF-β2 antisense probe for non irradiated skin; (e) TGF-β2 antisense probe for UV-irradiated skin; (f) UV-irradiated skin with TGF-β2 sense probe; (g) TGF-β3 antisense probe for nonirradiated skin; (h) TGF-β3 antisense probe for UV-irradiated skin; and (i) UV-irradiated skin with TGF-β3 sense probe. Panels show results for a single individual, but are representative of six individuals. Journal of Investigative Dermatology 2002 119, 499-506DOI: (10.1046/j.1523-1747.2002.01834.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Localization of TGF-β1, TGF-β2, and TGF-β3 mRNA expression following solar-simulated UV irradiation in human skin in vivo–in situ hybridization. Full- thickness 4 mm biopsies were obtained 72 h post UV (2 MED, solar simulator) irradiation. Cryostat sections were hybridized with TGF-β1, TGF- β2, and TGF-β3 sense or antisense riboprobes, as described in Materials and Methods. (a) TGF-β1 antisense probe for nonirradiated skin; (b) TGF-β1 antisense probe for UV-irradiated skin; (c) UV-irradiated skin with TGF-β1 sense probe; (d) TGF-β2 antisense probe for nonirradiated skin; (e) TGF-β2 antisense probe for UV-irradiated skin; (f) UV-irradiated skin with TGF-β2 sense probe; (g) TGF-β3 antisense probe for non irradiated skin; (h) TGF-β3 antisense probe for UV-irradiated skin; and (i) UV-irradiated skin with TGF-β3 sense probe. Panels show results for a single individual, but are representative of six individuals. Journal of Investigative Dermatology 2002 119, 499-506DOI: (10.1046/j.1523-1747.2002.01834.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 UV irradiation alters TGF-β1, TGF-β2, and TGF-β3 protein expression in human skin in vivo– immunohistology. Biopsied skin samples were obtained 72 h after exposure to UV (2 MED), and were localized by immunohistology, as described in Materials and Methods. (a) TGF-β1 nonirradiated skin; (b) TGF-β1 UV-irradiated skin; (c) UV-irradiated skin with TGF-β1 blocking peptide; (d) TGF-β2 nonirradiated skin; (e) TGF-β2 UV-irradiated skin; (f) UV-irradiated skin with TGF-β2 blocking peptide; (g) T GF-β3 nonirradiated skin; (h) TGF-β3 UV-irradiated skin; (i) UV-irradiated skin with TGF-β3 blocking peptide. Panels show results for a single individual, but are representative of six individuals. Journal of Investigative Dermatology 2002 119, 499-506DOI: (10.1046/j.1523-1747.2002.01834.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 UV irradiation inhibits TβRII mRNA but not TβRI mRNA in human skin in vivo– northern analysis. Human skin biopsies were obtained at the indicated times following exposure to UV (2 MED). mRNAs for TβRI and TβRII were analyzed by northern analysis and quantitated by STORM PhosphorImager. Data were normalized to mRNA for 36B4, a ribosomal protein used as an internal control for quantitation. The insets show representative northern blots. Bar heights represent means ± SEM. Data are expressed as percentage of change in mRNA levels relative to levels in nonirradiated control skin. *p <0.05 vs nonirradiated control skin, n = 6. Journal of Investigative Dermatology 2002 119, 499-506DOI: (10.1046/j.1523-1747.2002.01834.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 UV irradiation inhibits TβRII mRNA in human skin in vivo–in situ hybridization. Full-thickness 4 mm skin biopsies were obtained from nonirradiated skin and 8 h following exposure to UV (2 MED, UVB source) irradiation. Cryostat sections (5 µm) were hybridized with TβRII sense or antisense riboprobes, as described in Materials and Methods. (a) TβRII antisense probe for nonirradiated skin; (b) TβRII antisense probe for UV-irradiated skin; (c) UV-irradiated skin with TβRII sense probe. Panels show results for a single individual, but are representative of six individuals. Journal of Investigative Dermatology 2002 119, 499-506DOI: (10.1046/j.1523-1747.2002.01834.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Solar-simulated UV irradiation inhibits TβRII mRNA in human skin in vivo–in situ hybridization. Full-thickness 4 mm skin biopsies were obtained from nonirradiated skin and 8 h following exposure to solar-simulated UV (2 MED) irradiation. Cryostat sections (5 µm) were hybridized with TβRII sense or antisense riboprobes, as described in Materials and Methods. (a) TβRII antisense probe for nonirradiated skin; (b) TβRII antisense probe for UV-irradiated skin; (c) UV-irradiated skin with TβRII sense probe. Panels show results for a single individual, but are representative of six individuals. Journal of Investigative Dermatology 2002 119, 499-506DOI: (10.1046/j.1523-1747.2002.01834.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 UV irradiation induces Smad7 mRNA expression in human skin in vivo- northern analysis. Human skin biopsy specimens were obtained at the indicated times following exposure to UV (2 MED). mRNA for Smad7 was analyzed by northern analysis and quantitated by STORM PhosphorImager. Data were normalized to mRNA for 36B4, a ribosomal protein used as an internal control for quantitation. The inset shows representative northern analysis at each time point. Bar heights represent means ± SEM. Data are expressed as fold increase in mRNA levels relative to levels in nonirradiated control skin. *p <0.05 vs nonirradiated control skin, n = 6. Journal of Investigative Dermatology 2002 119, 499-506DOI: (10.1046/j.1523-1747.2002.01834.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 Localization of Smad7 mRNA expression following UV irradiation in human skin in vivo–in situ hybridization. Full-thickness 4 mm skin biopsies were obtained following exposure to UV (2 MED). Cryostat sections (5 µm) were hybridized with Smad7 sense or antisense riboprobes, as described in Materials and Methods. (a) Smad7 antisense probe for nonirradiated skin; (b) Smad7 antisense probe for UV-irradiated skin (4 h); (c) Smad7 antisense probe for UV-irradiated skin (8 h); (d) Smad7 antisense probe for UV-irradiated skin (24 h); (e) UV-irradiated skin (8 h) with Smad7 sense probe. Panels show results for a single individual, but are representative of six individuals. Journal of Investigative Dermatology 2002 119, 499-506DOI: (10.1046/j.1523-1747.2002.01834.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 10 Localization of Smad7 mRNA expression following solar-simulated UV irradiation in human skin in vivo–in situ hybridization. Full-thickness 4 mm skin biopsies were obtained following exposure to solar-simulated UV (2 MED). Cryostat sections (5 µm) were hybridized with Smad7 sense or antisense riboprobes, as described in Materials and Methods. (a) Smad7 antisense probe for nonirradiated skin; (b) Smad7 antisense probe for UV-irradiated skin (8 h); (c) UV-irradiated skin (8 h) with Smad7 sense probe. Panels show results for a single individual, but are representative of six individuals. Journal of Investigative Dermatology 2002 119, 499-506DOI: (10.1046/j.1523-1747.2002.01834.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 11 UV irradiation inhibits Smad/DNA complex formation in human skin in vivo– EMSA. Sun-protected human skin keratome biopsies were taken at 4, 8, 16, 24 h following UV irradiation (2 MED) and then whole cell extracts were extracted. Samples were analyzed by EMSA using well-characterized Smad3/4 binding oligonucleotides as a probe. Closed triangle indicates specific retarded complexes. Open triangles indicate nonspecific bands (NS). n = 3. Journal of Investigative Dermatology 2002 119, 499-506DOI: (10.1046/j.1523-1747.2002.01834.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions