Mika Murai1, Gaku Tsuji2, Akiko Hashimoto-Hachiya3, Yoshihito Kawakami4, Masutaka Furue5, Chikage Mitoma6. 1. Department of Dermatology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. Electronic address: mika-m@dermatol.med.kyushu-u.ac.jp. 2. Department of Dermatology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. Electronic address: gakku@dermatol.med.kyushu-u.ac.jp. 3. Department of Dermatology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. Electronic address: ahachi@dermatol.med.kyushu-u.ac.jp. 4. Research and Clinical Center for Yusho and Dioxin, Kyushu University Hospital, Fukuoka 812-8582, Japan. Electronic address: yosihito@dermatol.med.kyushu-u.ac.jp. 5. Department of Dermatology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan; Research and Clinical Center for Yusho and Dioxin, Kyushu University Hospital, Fukuoka 812-8582, Japan; Division of Skin Surface Sensing, Department of Dermatology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. Electronic address: furue@dermatol.med.kyushu-u.ac.jp. 6. Department of Dermatology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan; Research and Clinical Center for Yusho and Dioxin, Kyushu University Hospital, Fukuoka 812-8582, Japan. Electronic address: mchikage@dermatol.med.kyushu-u.ac.jp.
Abstract
BACKGROUND: Persistent ultraviolet (UV) radiation in the form of sunlight causes photo-aging of the skin by reducing the production of type I collagen, the major constituent of the extracellular matrix of the dermis. Transforming growth factor (TGF)-β transforms dermal fibroblasts into α2-smooth muscle actin (ACTA2)-expressing myofibroblasts. Myofibroblasts produce a precursor form of type I collagen, type I procollagen (collagen I), consisting of pro-alpha1 (produced by the COL1A1 gene) and pro-alpha2 chains (produced by the COL1A2 gene). Smad2/3 is a key downstream molecule of TGF-β signaling. The mechanisms through which UV inhibits collagen I synthesis are not fully understood. 6-Formylindolo[3,2-b]carbazole (FICZ) is an endogenous tryptophan photo-metabolite generated by UV irradiation. FICZ is well known as a high-affinity ligand for aryl hydrocarbon receptor (AHR). However, the physiological roles of FICZ in photo-aging have yet to be addressed. OBJECTIVE: To evaluate the effects of FICZ on the TGF-β-mediated ACTA2 and collagen I expression in normal human dermal fibroblasts (NHDFs). METHODS: Quantitative real-time polymerase chain reaction and western blot analysis were performed to determine the expression of ACTA2, COL1A1, and COL1A2 in NHDFs with or without FICZ and TGF-β. The phosphorylated Smad2/3 (pSmad2/3) protein levels in cytoplasmic or nuclear portions were investigated by western blot analysis. Immunofluorescence staining was conducted to evaluate pSmad2/3 localization, and F-actin staining with phalloidin was performed to visualize actin polymerization in myofibroblasts. The actions of FICZ on the TGF-β-mediated collagen I expression and nuclear translocation of pSmad2/3 were analyzed in the presence of selective AHR antagonists or in AHR-knockdown NHDFs. RESULTS: We found that FICZ significantly inhibited the TGF-β-induced upregulation of mRNA and protein levels of ACTA2 and collagen I and actin polymerization in myofibroblasts. FICZ did not disturb the phosphorylation of Smad2/3. Notably, FICZ reduced the expression of pSmad2/3 in the nucleus, while it increased that in the cytoplasm, suggesting that it inhibits the nuclear translocation of pSmad2/3 induced by TGF-β. The inhibitory actions of FICZ on the TGF-β-mediated collagen I expression and nuclear translocation of pSmad2/3 were independent of AHR signaling. Another endogenous AHR agonist, kynurenine, also inhibited the TGF-β-mediated ACTA2 and collagen I upregulation in NHDFs in an AHR-independent manner; however, its effects were insignificant in comparison with those of FICZ. CONCLUSIONS: These findings suggest that the endogenous photo-product FICZ may be a key chromophore that involves in photo-aging. Downregulation of FICZ signaling is thus a potential strategy to protect against photo-aging.
BACKGROUND: Persistent ultraviolet (UV) radiation in the form of sunlight causes photo-aging of the skin by reducing the production of type I collagen, the major constituent of the extracellular matrix of the dermis. Transforming growth factor (TGF)-β transforms dermal fibroblasts into α2-smooth muscle actin (ACTA2)-expressing myofibroblasts. Myofibroblasts produce a precursor form of type I collagen, type I procollagen (collagen I), consisting of pro-alpha1 (produced by the COL1A1 gene) and pro-alpha2 chains (produced by the COL1A2 gene). Smad2/3 is a key downstream molecule of TGF-β signaling. The mechanisms through which UV inhibits collagen I synthesis are not fully understood. 6-Formylindolo[3,2-b]carbazole (FICZ) is an endogenous tryptophan photo-metabolite generated by UV irradiation. FICZ is well known as a high-affinity ligand for aryl hydrocarbon receptor (AHR). However, the physiological roles of FICZ in photo-aging have yet to be addressed. OBJECTIVE: To evaluate the effects of FICZ on the TGF-β-mediated ACTA2 and collagen I expression in normal human dermal fibroblasts (NHDFs). METHODS: Quantitative real-time polymerase chain reaction and western blot analysis were performed to determine the expression of ACTA2, COL1A1, and COL1A2 in NHDFs with or without FICZ and TGF-β. The phosphorylated Smad2/3 (pSmad2/3) protein levels in cytoplasmic or nuclear portions were investigated by western blot analysis. Immunofluorescence staining was conducted to evaluate pSmad2/3 localization, and F-actin staining with phalloidin was performed to visualize actin polymerization in myofibroblasts. The actions of FICZ on the TGF-β-mediated collagen I expression and nuclear translocation of pSmad2/3 were analyzed in the presence of selective AHR antagonists or in AHR-knockdown NHDFs. RESULTS: We found that FICZ significantly inhibited the TGF-β-induced upregulation of mRNA and protein levels of ACTA2 and collagen I and actin polymerization in myofibroblasts. FICZ did not disturb the phosphorylation of Smad2/3. Notably, FICZ reduced the expression of pSmad2/3 in the nucleus, while it increased that in the cytoplasm, suggesting that it inhibits the nuclear translocation of pSmad2/3 induced by TGF-β. The inhibitory actions of FICZ on the TGF-β-mediated collagen I expression and nuclear translocation of pSmad2/3 were independent of AHR signaling. Another endogenous AHR agonist, kynurenine, also inhibited the TGF-β-mediated ACTA2 and collagen I upregulation in NHDFs in an AHR-independent manner; however, its effects were insignificant in comparison with those of FICZ. CONCLUSIONS: These findings suggest that the endogenous photo-product FICZ may be a key chromophore that involves in photo-aging. Downregulation of FICZ signaling is thus a potential strategy to protect against photo-aging.
Authors: Christian Vogeley; Charlotte Esser; Thomas Tüting; Jean Krutmann; Thomas Haarmann-Stemmann Journal: Int J Mol Sci Date: 2019-11-28 Impact factor: 5.923