Cell Death and Differentiation (2006) 13, 53–60 & 2006 Nature Publishing Group All rights reserved 1350-9047/06 $30.00 www.nature.com/cdd PPARb/d selectively induces differentiation and inhibits cell proliferation DJ Kim1,2, MT Bility1, AN Billin3, TM Willson3, FJ Gonzalez4 and described including the regulation of keratinocyte differentia- JM Peters*,1,2 tion,1–3 apoptosis4 and cell proliferation,3,5 inflammation2 and wound healing.6 However, the data supporting these roles for 1 Department of Veterinary Science and The Center for Molecular Toxicology PPARb in the skin remain highly controversial, as there is and Carcinogenesis, The Pennsylvania State University, University Park, PA inconsistent evidence in the literature based on results from 16802, USA 2 the skin, keratinocyte and other cell/tissue models. Recent Graduate Program in Molecular Toxicology, The Huck Institutes of the Life observations suggest that activation of PPARb may be of Sciences, The Pennsylvania State University, University Park, PA 16802, USA 7,8 3 Discovery Research, GlaxoSmithKline, Research Triangle Park, Bethesda, benefit for the treatment of dyslipidemias and studies in MD 20892, USA PPARb-null mice have suggested a possible role for this 9,10 4 Laboratory of Metabolism, National Cancer Institute, Bethesda, MD 20892, receptor in cancer, thus illustrating the need to accurately USA determine the specific biological functions mediated by * Corresponding author: JM Peters, Department of Veterinary Science and PPARb ligands so that the safety of these potentially Center for Molecular Toxicology and Carcinogenesis, 312 Life Sciences therapeutic drugs can be assessed. The use of highly specific Building, The Pennsylvania State University, University Park, PA 16802, USA. ligands for PPARb in conjunction with the PPARb-null mouse Tel: þ 1 814 863 1387; Fax: þ 1 814 863 1696; E-mail: [email protected] model is the most definitive approach for these purposes, and Received 19.1.05; revised 16.5.05; accepted 06.6.05; published online 15.7.05 was used for these studies. Edited by JA Cidlowski Abstract Results Peroxisome proliferator-activated receptor (PPAR) b-null Application of the tumor promoter 12-O-tetradecanoyl- mice exhibit exacerbated epithelial cell proliferation and phorbol-13-acetate (TPA) to mouse skin results in increased enhanced sensitivity to skin carcinogenesis, suggesting that expression of PPARb that is coincident with enhanced expression of mRNAs encoding proteins that are markers ligand activation of PPARb will inhibit keratinocyte prolifera- for keratinocyte differentiation.11 However, increased expres- tion. By using of a highly specific ligand (GW0742) and the sion of differentiation marker mRNAs in skin occurs in vivo PPARb-null mouse model, activation of PPARb was found to in response to TPA in the absence of PPARb expression.5 selectively induce keratinocyte terminal differentiation and Similar to results obtained in vivo, treatment of primary inhibit keratinocyte proliferation. Additionally, GW0742 was keratinocytes derived from either wild-type or PPARb-null found to be anti-inflammatory due to inhibition of myeloper- mice in vitro with TPA results in morphological changes oxidase activity, independent of PPARb. These data suggest indicative of differentiation (Figure 1a), coupled with increased that ligand activation of PPARb could be a novel approach to expression of mRNAs encoding both early and late markers of selectively induce differentiation and inhibit cell proliferation, differentiation (Figure 1b). In particular, a 2–6-fold induction of thus representing a new molecular target for the treatment of mRNAs encoding TG-I, involucrin, small proline-rich protein skin disorders resulting from altered cell proliferation such as (SPR)1A, SPR2H was observed after 6 h of treatment, while induction of loricrin did not occur until after 48 h of treatment; a psoriasis and cancer. pattern of early and late markers of differentiation typically Cell Death and Differentiation (2006) 13, 53–60. observed in primary keratinocytes.12,13 Using the classic doi:10.1038/sj.cdd.4401713; published online 15 July 2005 approach of increasing culture medium Ca2 þ to induce keratinocyte differentiation, similar results were obtained as Keywords: peroxisome proliferator-activated receptor-b; differ- morphological changes indicative of differentiation (Figure 1c) entiation; cell proliferation; anti-inflammatory; null mouse; ligand and increased expression of mRNAs encoding both early and activation late markers of differentiation was observed in keratinocytes derived from either wild-type or PPARb-null mice (Figure 1d). Abbreviations: ADRP, adipocyte differentation-related protein; Despite the increase in PPARb expression coincident with MPO, myeloperoxidase; PPAR, peroxisome proliferator-acti- induction of keratinocyte differentiation, the results in Figure 1 vated receptor; SPR, small proline-rich protein; TG-I, transgluta- suggest that PPARb is not required for differentiation induced minase-I by TPA or Ca2 þ signaling. To further examine this hypothesis, primary keratinocytes were cultured in medium containing GW0742 at a concentration that specifically activates PPARb (see below Figure 3a). Stratified cells that are indicative of Introduction differentiation (Figure 1c) were not found in keratinocytes During the past 5 years, a number of potential roles for cultured in GW0742 in low Ca2 þ medium (Figure 2a), a result peroxisome proliferator-activated receptor (PPAR) b (also that is not unexpected since the terminal differentiation of referred to as PPARd) in epithelial homeostasis have been primary keratinocytes requires Ca2 þ for the formation of the PPARb regulates differentiation and proliferation DJ Kim et al 54 Figure 1 PPARb does not influence TPA or Ca2 þ -induced keratinocyte differentiation. Morphology of differentiating keratinocytes from wild-type ( þ / þ ) or PPARb- null (À/À) mice is similar when cultured in either 25 ng TPA/ml of medium (a) or 0.12 mM Ca2 þ (c). Note the dendritic-like cells indicative of TPA-induced differentiation typically observed in keratinocytes in both genotypes after 6–12 h (a). Note the overt changes in morphology and stratified cells (arrows), indicative of differentiation, in both genotypes in response to high Ca2 þ medium (c). Early and late mRNA markers of differentiation are similar in keratinocytes from ( þ / þ ) and (À/À) mice when differentiation was induced with either 0.12 mM Ca2 þ (b) or 25 ng TPA/ml of medium (d). The fold-induction of normalized hybridization signals is presented below each representative Northern blot. *Significantly different than controls, Pr0.05 cornified envelope.14 However, ligand activation of PPARb cornified envelopes were quantified. Indeed, administration of resulted in a PPARb-dependent increase in SPR1A, SPR2H 50 mM GW0742 caused an increase in the average number and adipocyte differentation-related protein (ADRP) mRNA of cornified envelopes in wild-type mouse skin as compared expression (Figure 2b). Interestingly, the expression of ADRP to controls (Figure 2e). In contrast, the average number of was markedly lower in PPARb-null cells (Figure 2b). In- cornified envelopes was lower in PPARb-null mouse skin, and creased expression of mRNAs encoding TG-I and loricrin treatment with GW0742 had no effect on terminal differentia- was not observed in response to GW0742, while increased tion in the absence of PPARb expression (Figure 2e). expression of involucrin was observed after 24 h of treatment Collectively, these results suggest that the induction of in both genotypes (Figure 2b). To determine whether ligand differentiation mediated by TPA and high culture medium activation of PPARb increases the expression of mRNAs Ca2 þ involves a number of other transcriptional regulators encoding differentiation markers in vivo, a dose response (e.g. AP-1) that modulate similar target genes (e.g. SPRs, experiment was performed to identify a concentration of ADRP) and prevents detection of the functional role of GW0742 that specifically activates PPARb in skin after topical PPARb-dependent transcriptional modulation of differentia- application (Figure 2c). Consistent with results observed tion. Importantly, these results also demonstrate that ligand in vitro, topical administration of GW0742 with a stock con- activation of PPARb can selectively induce the expression of centration that specifically activates PPARb (50 mM), resulted mRNAs encoding proteins that mediate lipid accumulation in increased expression of mRNAs encoding markers of and terminal keratinocyte differentiation. keratinocyte differentiation including, TG-I, involucrin, It is well accepted that the induction of terminal differentia- SPR1A, SPR2H and ADRP in wild-type mouse skin 8 h tion is inversely correlated with cell proliferation,14,15 as post-treatment, and these increases were absent in PPARb- terminal differentiation of cells would in theory reduce the null mouse skin (Figure 2d). To determine whether the number of cells available for proliferation. Additionally, increases in mRNAs encoding proteins required for terminal epithelial cell proliferation is exacerbated in the absence differentiation induced by ligand activation of PPARb caused a of PPARb expression in response to TPA.9 Together, these functional increase in keratinocyte terminal differentiation, observations suggest that ligand activation of PPARb could Cell Death and Differentiation PPARb regulates differentiation and proliferation DJ Kim et al 55 Figure 2 Ligand