Involvement of PPARγ in Oxidative Stress-Mediated Prostaglandin E2 Production in SZ95 Human Sebaceous Gland Cells  Qiwei Zhang, Holger Seltmann, Christos C. Zouboulis, Raymond L. Konger  Journal of Investigative Dermatology  Volume 126, Issue 1, Pages 42-48 (January 2006) DOI: 10.1038/sj.jid.5700028 Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Functionally active PPARγ is present in the human SZ95 cell line. (a) Forty micrograms of cellular protein isolated from KB, primary human keratinocyte (PHK), and SZ95 sebocytes was separated on a 10% SDS-PAGE and PPARγ immunoreactivity was determined using a polyclonal antibody. (b) SZ95 sebocytes co-transfected with PPRE-luciferase reporter and β-galactosidase (β-gal) plasmids were treated variously with vehicle control (CON, 0.4% ethanol), specific PPARγ agonists 1μM azPC or 20μM ciglitazone (CIG) for 24hours. The cells were harvested for luciferase activity assay and normalized to β-gal activity. The values shown are mean±SD and are representative of three separate experiments (*P<0.05). Journal of Investigative Dermatology 2006 126, 42-48DOI: (10.1038/sj.jid.5700028) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 PPARγ agonists induce COX-2 protein expression through activation of PPARγ in SZ95 sebocytes. (a) SZ95 sebocytes were treated with 20μM ciglitazone, 1μM azPC, or 100nM PMA as a positive control for induction of COX-2. Approximately 40μg cellular protein was isolated and separated on 10% SDS-PAGE; COX-2-immunoreactivity was determined using a polyclonal antibody. (b) SZ95 sebocytes were pretreated with vehicle control or 1μM GW9662 (GW) for 1hour, and then treated with 20μM ciglitazone or 100nM PMA. Forty micrograms of cellular protein was isolated and separated on 10% SDS-PAGE and COX-2 immunoreactivity was determined using a polyclonal antibody. Journal of Investigative Dermatology 2006 126, 42-48DOI: (10.1038/sj.jid.5700028) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Cellular homogenates from UVB-irradiated SZ95 cells contain PPARγ agonistic activity. SZ95 cells (a) or KB cells (b) co-transfected with PPARγ luciferase reporter and β-gal plasmids were treated with cell homogenate derived from unirradiated SZ95 cells, or cells irradiated with various doses of UVB, or treated with 20μM ciglitazone (CIG) as a positive control for PPARγ activation. Cells were harvested 24hours after treatment for luciferase and β-gal activity assay. The values shown are mean±SD and are representative of three separate experiments (*P<0.05). Journal of Investigative Dermatology 2006 126, 42-48DOI: (10.1038/sj.jid.5700028) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 PPRE-LUC reporter assay demonstrates that direct-acting oxidative stressor TBH produces PPARγ agonistic activity in SZ95 sebocytes in a dose-dependent manner. SZ95 sebocytes co-transfected with PPARγ luciferase reporter and β-gal plasmids were treated with various doses of TBH or 20μM ciglitazone as a positive control for PPARγ activation. Cells were harvested 24hours after treatment for luciferase and β-gal activity assay. The values shown are mean±SD and are representative of three separate experiments (*P<0.05). Journal of Investigative Dermatology 2006 126, 42-48DOI: (10.1038/sj.jid.5700028) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Specific PPARγ antagonist GW9662 blocks oxidative stress-induced COX-2 mRNA induction. SZ95 sebocytes were pretreated with vehicle control or 1μM GW9662 for 1hour, and then exposed to 20μM ciglitazone, 10μM TBH, or 100nM PMA for 6hours. Total cellular RNA was extracted and quantitative RT-PCR was performed for COX-2 mRNA expression. Cycle threshold units were then converted into concentration units using a log standard curve. Relative COX-2 expression is normalized to 18S ribosomal RNA. Results represent the mean±standard error of mean of two experiments carried out in duplicate or triplicate (*P<0.05; **P<0.01). Journal of Investigative Dermatology 2006 126, 42-48DOI: (10.1038/sj.jid.5700028) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Specific PPARγ antagonist GW9662 blocks oxidative stress-induced PGE2 production. SZ95 sebocytes were pretreated with vehicle control or GW9662 1μM for 1hour, and then treated with 20μM ciglitazone, 10μM TBH, 500nM calcium ionophore A23187, or exposed to 600J/m2 UVB irradiation; PGE2 production was assayed in the tissue culture supernatants after 8hours. The values of PGE2 expression shown are mean±SD and are representative of three independent experiments (*P<0.05). Journal of Investigative Dermatology 2006 126, 42-48DOI: (10.1038/sj.jid.5700028) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Inhibition of PPARγ in SZ95/ΔPPARγ cells blocks oxidative stress-induced PGE2 production. SZ95 sebocytes transfected with mutant PPARγ dominant-negative construct (SZ95/ΔPPARγ) or control cells transfected with vector vehicle (SZ95/pcDNA3) were treated with 20μM ciglitazone, 10μM TBH, 500nM calcium ionophore A23187, or exposed to 600J/m2 UVB irradiation; PGE2 production was assayed after 8hours by a PGE2 ELISA kit. The values of PGE2 expression shown are mean±SD and are representative of three independent experiments (*P<0.05). Journal of Investigative Dermatology 2006 126, 42-48DOI: (10.1038/sj.jid.5700028) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions