Stimulation of PPARα Promotes Epidermal Keratinocyte Differentiation In Vivo  László G. Kömüves, Karen Hanley, Anne-Marie Lefebvre, Mao-Qiang Man, Dean C. Ng, Daniel D. Bikle, Mary L. Williams, Peter M. Elias, Johan Auwerx, Kenneth R. Feingold  Journal of Investigative Dermatology  Volume 115, Issue 3, Pages 353-360 (September 2000) DOI: 10.1046/j.1523-1747.2000.00073.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Effect of topical treatment with PPARα activators on the epidermis. Skin of adult hairless mice (a, untreated control) were treated twice a day with vehicle (b, 3 d treatment with acetone; c, 3 d treatment with propylene glycol-ethanol; d, 6 d treatment with propylene glycol-ethanol), or with Wy-14,643 (e, 3 d treatment with Wy-14,643 dissolved in propylene glycol-ethanol), or with clofibrate (f, 6 d treatment with clofibrate dissolved in ethanol; g, 3 d treatment with clofibrate dissolved in propylene glycol-ethanol; h, 6 d treatment with clofibrate dissolved in propylene glycol-ethanol). Hematoxylin and eosin stained sections. Measurements of the epidermal thickness of these samples are presented in Table 1. Scale bars: 25 μm. Journal of Investigative Dermatology 2000 115, 353-360DOI: (10.1046/j.1523-1747.2000.00073.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Immunohistochemical and in situ hybridization analysis of epidermal keratinocyte differentiation following topical treatment with PPARα activators. Adult hairless mice were treated for 3 d, twice a day, with vehicle or Wy-14,643 (b, e, h, k), or clofibrate (c, f, i, l). Profilaggrin-filaggrin (a-c) and loricrin (d-f) proteins were detected with rabbit antipeptide antibodies (brown stain) and counterstained with methyl green. Profilaggrin (g-i) and loricrin (j-k) mRNAs were detected with DIG-labeled antisense riboprobes (black stain). Notice that the proteins are localized in the stratum granulosum and in the stratum corneum, whereas the mRNAs are expressed in the upper stratum spinosum and in the stratum granulosum but are not detectable in the stratum corneum. Scale bars: 50 μm for panels (a)-(f); 25 μm for panels (g)-(l). Journal of Investigative Dermatology 2000 115, 353-360DOI: (10.1046/j.1523-1747.2000.00073.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Epidermal cell proliferation and apoptotic cell death following topical clofibrate treatment. Adult hairless mice were treated twice with vehicle (a, d), Wy-14,643 (b, e), or clofibrate (c, f) for 1 d (a, b, c), or for 3 d (d, e, f). The sections were counterstained with Sytox Red to detect nuclei (red color). To identify proliferating cells, the animals were injected with BrdU and tissue samples were collected 1 h later. Proliferating cells (yellow nuclei, a result of overlayed green and red signals) were identified by immunohistochemical detection of BrdU incorporation (a, b, c) with a confocal microscope. Apoptotic cells (green or yellowish-green signal) were identified by TUNEL assay and visualized by laser scanning confocal microscopy (d, e, f). The dotted line indicates the epidermis-dermis border. Scale bars: (a–c) 50 μm; (d–f) 25 μm. Journal of Investigative Dermatology 2000 115, 353-360DOI: (10.1046/j.1523-1747.2000.00073.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Effect of topical clofibrate treatment on the epidermis of PPARα knockout mice. Shaved areas of the dorsal skin of PPARα+/+ mice (wild-type congenic C57BL/6 mice, left columns) and PPARα–/– mice were treated with vehicle or with clofibrate for 3 d. Hematoxylin and eosin stained sections (a-d). The arrows indicate the thickness of the nucleated cellular layers in the epidermis. The framed areas in PPARα–/– stratum corneum are displaying focal parakeratosis (c, d). Localization of profilaggrin mRNA (e-h) and loricrin (j-l) mRNA (black stain) was detected by in situ hybridization. Notice the slightly decreased staining in PPARα–/– epidermis compared with PPARα+/+ epidermis (e vs g, and i vs k), and the lack of increased staining following clofibrate treatment (f vs h, and j vs l). Loricrin protein was detected with rabbit antipeptide antibody (brown stain), and counterstained with methyl green (m-p). In PPARα–/– epidermis there is a slightly weaker loricrin signal than in PPARα+/+ epidermis (o vs m). Furthermore, topical clofibrate treatment did not increase loricrin staining in PPARα–/– epidermis whereas in PPARα+/+ epidermis the signal increased (n vs p). Scale bars: 25 μm. Journal of Investigative Dermatology 2000 115, 353-360DOI: (10.1046/j.1523-1747.2000.00073.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Effects of PPARα activators on reporter activity in keratinocytes transfected with involucrin promoter-luciferase constructs. (A) Schematic representation of involucrin promoter constructs used. (B) Keratinocytes were transiently transfected with 2.0 μg of the corresponding construct together with 0.2 μg RSV-β-galactosidase, and were then incubated 24 h in the presence of vehicle (veh), clofibric acid (200 μM), or Wy-14,643 (10 μM) as described in Materials and Methods. Data presented here (mean ± SEM) represent the average of three separate experiments; *p < 0.01. Journal of Investigative Dermatology 2000 115, 353-360DOI: (10.1046/j.1523-1747.2000.00073.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Mutagenesis of the AP-1 site at -2117 bp to -2111 bp prevents the increase in involucrin transcription by PPARα activators. Keratinocytes were transfected with an involucrin-luciferase construct containing either a wild-type (WT) or a mutated (mutAP-1) AP-1 site as described in Materials and Methods. Data presented here represent the average of three independent experiments; *p < 0.01. Journal of Investigative Dermatology 2000 115, 353-360DOI: (10.1046/j.1523-1747.2000.00073.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions