Sun A. Ham, Eun S. Kang, Hanna Lee, Jung S. Hwang, Taesik Yoo, Kyung S

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PPARδ Inhibits UVB-Induced Secretion of MMP-1 through MKP-7-Mediated Suppression of JNK Signaling Sun A. Ham, Eun S. Kang, Hanna Lee, Jung S. Hwang, Taesik Yoo, Kyung S. Paek, Chankyu Park, Jin-Hoi Kim, Dae-Seog Lim, Han G. Seo Journal of Investigative Dermatology Volume 133, Issue 11, Pages 2593-2600 (November 2013) DOI: 10.1038/jid.2013.202 Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Peroxisome proliferator–activated receptor (PPAR) δ inhibits UVB-induced secretion of matrix metalloproteinase (MMP)-1. (a–c) Human dermal fibroblasts transfected without (a) or with (b) small interfering RNA (siRNA) or pretreated with GSK0660 for 30minutes (c) were treated with GW501516 for 24hours and then exposed to UVB. After incubation for 48hours, conditioned media were subjected to ELISA, zymography, or immunoblot analysis. (d) Cells pretreated with GW501516 for 24hours were exposed to UVB. Following incubation for 24hours, a methyl thiazolyl tetrazolium assay was performed. GW501516 and GSK0660 were dissolved in DMSO. Results are expressed as the means±SD (n=6). *P<0.01, **P<0.05 versus untreated group; #P<0.01, ##P<0.05 versus UVB-exposed group; ††P<0.05 versus UVB plus GW501516-treated group. Journal of Investigative Dermatology 2013 133, 2593-2600DOI: (10.1038/jid.2013.202) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Peroxisome proliferator–activated receptor (PPAR) δ suppresses UVB-induced reactive oxygen species (ROS) generation. Cells transfected with or without indicated small interfering RNA (siRNA) were pretreated with GSK0660 or N-acetyl-L-cysteine (NAC) for 30min and then treated with GW501516 for 24hours. After exposure to UVB, the cells were incubated for 30minutes and then intracellular ROS levels were detected by (a) confocal laser fluorescence microscopy and (b, c) quantified. (d) Cells pretreated with NAC for 30minutes were treated with GW501516 for 24hours and exposed to UVB. After incubation for 48hours, conditioned media were subjected to ELISA, zymography, or immunoblot analysis. Results are expressed as the means±SD (n=3 or 5). *P<0.01 versus untreated group; #P<0.01 versus UVB-exposed group; †P<0.01 versus UVB plus GW501516-treated group. MMP-1, matrix metalloproteinase 1. Journal of Investigative Dermatology 2013 133, 2593-2600DOI: (10.1038/jid.2013.202) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 c-Jun N-terminal kinase (JNK) signaling cascade is responsible for the peroxisome proliferator–activated receptor-mediated inhibition of matrix metalloproteinase (MMP)-1 in human dermal fibroblasts (HDFs) exposed to UVB. (a) HDFs were exposed to UVB for the indicated times. (b) Cells pretreated with GW501516 for 24hours were exposed to UVB for 30minutes. An aliquot of protein was immunoblotted with activation-specific antibodies, and parallel immunoblots were analyzed for total kinase levels. (c) Cells were pretreated with SP600125, PD98059, or SB203580 for 30minutes and then exposed to UVB. (d) Cells pretreated with SP600125 for 30minutes were treated with GW501516 for 24hours and then exposed to UVB. After incubation for 48hours, conditioned media were subjected to ELISA, zymography, or immunoblot analysis. Results shown are representative of three or four independent experiments. ERK, extracellular signal–regulated kinase. Journal of Investigative Dermatology 2013 133, 2593-2600DOI: (10.1038/jid.2013.202) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Peroxisome proliferator–activated receptor δ attenuates UVB-induced activation of c-Jun N-terminal kinase (JNK) through the stabilization of mitogen-activated protein kinase phosphatase (MKP)-7. Human dermal fibroblasts (HDFs) pretreated with indicated doses of GW501516 for 24hours were exposed to UVB irradiation for 30minutes. Cell extracts were separated by electrophoresis, (a) immunoblotted with the indicated antibodies, and (b) quantified. HDFs pretreated with 50nM GW501516 (GW) for 24hours were exposed to UVB (40mJcm−2) for the indicated times in the presence of actinomycin D (Act D) (5μgml−1). (c) Total messenger RNA was extracted and subjected to northern blot analysis. (d) The radioactivity of the signals was quantified using an image analyzer; data are expressed as ratios of MKP-7 to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), normalized against the ratio at time zero. Journal of Investigative Dermatology 2013 133, 2593-2600DOI: (10.1038/jid.2013.202) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Peroxisome proliferator–activated receptor δ prevents breakdown of type I and III collagen in UVB-irradiated human dermal fibroblasts (HDFs). HDFs pretreated with a range of GW501516 for 24hours were exposed to UVB irradiation. Following incubation for 48hours, conditioned media or cell extracts were harvested and analyzed by (a) zymography or immunoblot analysis. Cells pretreated with (b) N-acetyl-L-cysteine (NAC) or (c) SP600125 for 30minutes were incubated with GW501516 for 24hours. Following exposure to UVB for 30minutes, the cells were incubated for 48hours. Cell extracts were analyzed by western blotting with the indicated antibodies. Representative images from three independent experiments are shown. MMP-1, matrix metalloproteinase 1. Journal of Investigative Dermatology 2013 133, 2593-2600DOI: (10.1038/jid.2013.202) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Peroxisome proliferator–activated receptor δ prevents UVB-induced skin deformity in HR-1 hairless mice. (a–e) Representative cross-sections of the dorsal skin of UVB-exposed HR-1 hairless mice treated with or without GSK0660 and/or GW501516. Wrinkle formation was detected by silicon replica assay (a). Paraffin tissue sections were subjected to hematoxylin and eosin (H&E) staining and quantitative analysis of epidermal and dermal thickness was performed (a, b). Bars=100μm. (c) Tissue extracts of dorsal skin from mice were subjected to immunoblot analysis. (d, e) Relative band intensities were quantified using an image analyzer and represented as a fold change relative to the untreated control. *P<0.01 versus untreated group; #P<0.01 versus UVB-exposed group; †P<0.01 versus UVB plus GW501516-treated group. JNK, c-Jun N-terminal kinase; MKP, mitogen-activated protein kinase phosphatase; MMP-1, matrix metalloproteinase 1. Journal of Investigative Dermatology 2013 133, 2593-2600DOI: (10.1038/jid.2013.202) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions