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Keratinocyte Differentiation in Hyperproliferative Epidermis: Topical Application of PPARα Activators Restores Tissue Homeostasis

2000; Elsevier BV; Volume: 115; Issue: 3 Linguagem: Inglês

10.1046/j.1523-1747.2000.00076.x

1523-1747

László G. Kömüves, Karen Piper Hanley, Mao‐Qiang Man, Peter M. Elias, Mary L. Williams, Kenneth R. Feingold,

NF-κB Signaling Pathways

SummaryWe recently showed that topically applied PPARα activators promote epidermal differentiation in intact adult mouse skin. In this study we determined the effect of clofibrate and Wy-14,643, activators of PPARα, on hyperproliferative epidermis in hairless mice, induced either by repeated barrier abrogation (subacute model) or by essential fatty acid deficiency (chronic model). The hyperproliferative epidermis was characterized by an increased number of proliferating cells expressing proliferating cell nuclear antigen. Topical treatment with PPARα activators resulted in a substantial decrease in epidermal hyperplasia in both the subacute and chronic models of hyperproliferation. Following topical treatment, proliferating cell nuclear antigen-expressing cells were restricted to the basal layer, similar to normal epidermis. In hyperproliferative epidermis there was decreased expression of involucrin, profilaggrin-filaggrin, and loricrin as assayed by in situ hybridization and immunohistochemistry. Following topical treatment with PPAR activators staining for these mRNAs and proteins increased towards normal levels. Finally, topically applied clofibrate also increased apoptosis. This study demonstrates that topical PPAR activators have profound effects on epidermal gene expression in hyperproliferative skin disorders. Treatment with PPARα activators normalizes cell proliferation and promotes epidermal differentiation, correcting the cutaneous pathology. This study identifies PPARα activators as potential skin therapeutic agents. We recently showed that topically applied PPARα activators promote epidermal differentiation in intact adult mouse skin. In this study we determined the effect of clofibrate and Wy-14,643, activators of PPARα, on hyperproliferative epidermis in hairless mice, induced either by repeated barrier abrogation (subacute model) or by essential fatty acid deficiency (chronic model). The hyperproliferative epidermis was characterized by an increased number of proliferating cells expressing proliferating cell nuclear antigen. Topical treatment with PPARα activators resulted in a substantial decrease in epidermal hyperplasia in both the subacute and chronic models of hyperproliferation. Following topical treatment, proliferating cell nuclear antigen-expressing cells were restricted to the basal layer, similar to normal epidermis. In hyperproliferative epidermis there was decreased expression of involucrin, profilaggrin-filaggrin, and loricrin as assayed by in situ hybridization and immunohistochemistry. Following topical treatment with PPAR activators staining for these mRNAs and proteins increased towards normal levels. Finally, topically applied clofibrate also increased apoptosis. This study demonstrates that topical PPAR activators have profound effects on epidermal gene expression in hyperproliferative skin disorders. Treatment with PPARα activators normalizes cell proliferation and promotes epidermal differentiation, correcting the cutaneous pathology. This study identifies PPARα activators as potential skin therapeutic agents. essential fatty acid deficiency proliferating cell nuclear antigen peroxisome-proliferator-activated receptor retinoic X-activated receptor TdT-mediated dUTP nick end-labeling Regulation of keratinocyte differentiation is still not well understood (Fuchs, 1990Fuchs E. Epidermal differentiation. The bare essentials.J Cell Biol. 1990; 111: 2807-2814Crossref PubMed Scopus (589) Google Scholar; Eckert et al., 1997Eckert R.L. Crish J.F. Robinson N.A. The epidermal keratinocyte as a model for the study of gene regulation and function.Physiol Rev. 1997; 77: 397-424Crossref PubMed Scopus (343) Google Scholar). Recently, however, it has become clear that ligands of certain nuclear hormone receptors (Mangelsdorf et al., 1995Mangelsdorf D.J. Thummel C. Beator M. et al.The nuclear receptor superfamily, the second decade.Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6064) Google Scholar) that form heterodimers with retinoic-X-activated receptor (RXR) are regulators of keratinocyte differentiation. Treatment with ligands, i.e., activators of these receptors (such as vitamin D and retinoic acid), regulate keratinocyte differentiation (Eichner et al., 1996Eichner R. Gendimenico G.J. Kahn M. et al.Effects of long-term retinoic acid treatment on epidermal differentiation in vivo, specific modification in the program of terminal differentiation.Br J Dermatol. 1996; 135: 687-695Crossref PubMed Scopus (27) Google Scholar; Fisher and Voorhees, 1996Fisher G.J. Voorhees J.J. Molecular mechanisms of retinoid actions in skin.FASEB J. 1996; 10: 1002-1013Crossref PubMed Scopus (355) Google Scholar; Kang et al., 1996Kang S. Li X.-Y. Voorhees J.J. Pharmacology and molecular action of retinoids and vitamin D in skin.J Invest Dermatol Symp Proceedings of The. 1996; 1: 15-21Crossref PubMed Google Scholar; Hanley et al., 1997Hanley K. Jiang Y. Crumvine D. et al.Activation of the nuclear hormone receptors PPARα and FXR accelerate the development of the fetal epidermal permeability barrier.J Clin Invest. 1997; 100: 705-712Crossref PubMed Scopus (141) Google Scholar). Furthermore, transgenic mice expressing dominant-negative RXR or retinoic-acid-activated receptor in the epidermis display defective keratinocyte proliferation and differentiation (Imakado et al., 1995Imakado S. Bickenbach J.R. Buudman D.S. et al.Targeting expression of a dominant-negative retinoic acid receptor mutant in the epidermis of transgenic mice results in loss of barrier function.Genes Dev. 1995; 9: 317-329Crossref PubMed Scopus (127) Google Scholar; Saitou et al., 1995Saitou M. Sugai S. Tawaka T. et al.Inhibition of skin development by targeted expression of a dominant-negative retinoic acid receptor.Nature. 1995; 374: 159-162Crossref PubMed Scopus (161) Google Scholar; Feng et al., 1997Feng X. Peng Z.H. Di W. et al.Suprabasal expression of a dominant-negative RXRα mutant in transgenic mouse epidermis impairs regulation of gene transcription and basal keratinocyte proliferation by RAR-selective retinoids.Genes Dev. 1997; 11: 59-71Crossref PubMed Scopus (59) Google Scholar). Peroxisome-proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that forms heterodimers with RXR (Isseman and Green, 1990Isseman I. Green S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators.Nature. 1990; 347: 645-650Crossref PubMed Scopus (3037) Google Scholar; Schoonjans et al., 1996Schoonjans K. Staels B. Auwerx J. Role of the peroxisome proliferator-activated receptor in mediating the effects of fibrates and fatty acids on gene expression.J Lipid Res. 1996; 37: 907-925Abstract Full Text PDF PubMed Google Scholar). The three PPAR isoforms, PPARα, PPARδ, and PPARγ, are expressed in cultured keratinocytes (Rivier et al., 1998Rivier M. Satonova I. Lebrun P. et al.Differential expression of peroxisome proliferator-activated receptor subtypes during the differentiation of human keratinocytes.J Invest Dermatol. 1998; 111: 1116-1121Crossref PubMed Scopus (147) Google Scholar). Moreover, PPARα and PPARδ are expressed both in fetal and adult rodent epidermis (Braissant et al., 1996Braissant O. Foutelle F. Scotto C. et al.Differential expression of peroxisome proliferator-activated receptors, PPA, Rs, Tissue distribution of, PPAR- & alpha;-β and-γ in the adult rat.Endocrinology. 1996; 137: 354-366Crossref PubMed Scopus (1723) Google Scholar; Braissant and Wahli, 1998Braissant O. Wahli W. Differential expression of peroxisome proliferator activated receptor-α -β and -γ during rat embryonic development.Endocrinology. 1998; 139: 2748-2754Crossref PubMed Scopus (376) Google Scholar). Studies in our laboratory have demonstrated that ligands of PPARα (Schoonjans et al., 1996Schoonjans K. Staels B. Auwerx J. Role of the peroxisome proliferator-activated receptor in mediating the effects of fibrates and fatty acids on gene expression.J Lipid Res. 1996; 37: 907-925Abstract Full Text PDF PubMed Google Scholar) stimulate differentiation and inhibit proliferation in cultured human keratinocytes (Hanley et al., 1997Hanley K. Jiang Y. Crumvine D. et al.Activation of the nuclear hormone receptors PPARα and FXR accelerate the development of the fetal epidermal permeability barrier.J Clin Invest. 1997; 100: 705-712Crossref PubMed Scopus (141) Google Scholar). We also showed that PPARα activators accelerate stratum corneum development when added to fetal rat skin explants (Hanley et al., 1997Hanley K. Jiang Y. Crumvine D. et al.Activation of the nuclear hormone receptors PPARα and FXR accelerate the development of the fetal epidermal permeability barrier.J Clin Invest. 1997; 100: 705-712Crossref PubMed Scopus (141) Google Scholar; Kömüves et al., 1998Kömüves L.G. Hawley K. Jiang Y. et al.Ligands and activators of nuclear hormone receptors regulate epidermal differentiation during fetal rat skin development.J Invest Dermatol. 1998; 111: 429-433Crossref PubMed Scopus (95) Google Scholar). Moreover, we recently demonstrated that PPARα activators (clofibrate and Wy-14,643) modulate keratinocyte differentiation and gene expression in intact normal skin following topical application (Kömüves et al., 2000Kömüves L.G. Hanley K. Lefebvre A.-M. et al.Stimulation of PPARα promotes epidermal keratinocyte differentiation in vivo.J Invest Dermatol. 2000; 115: 353-360Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Topical treatment of adult mice with PPARα activators (clofibrate and Wy-14,643) resulted in a decrease in epidermal thickness and increased expression of structural proteins of the upper spinous/granular layers (involucrin, profilaggrin-filaggrin, loricrin). Furthermore, topically applied PPARα activators also increased apoptosis, decreased cell proliferation, and accelerated recovery of barrier function following acute barrier abrogation. PPARα activators did not elicit these epidermal responses in PPARα knockout mice, however (Lee et al., 1995Lee S.S. Pineau T. Drager J. et al.Targeted disruption of the alpha isoform of the peroxisome-proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators.Molec Cell Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1498) Google Scholar), indicating that PPARα mediates these effects (Kömüves et al., 2000Kömüves L.G. Hanley K. Lefebvre A.-M. et al.Stimulation of PPARα promotes epidermal keratinocyte differentiation in vivo.J Invest Dermatol. 2000; 115: 353-360Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Furthermore, the epidermis of PPARα–/– mice displayed slightly decreased expression of involucrin, profilaggrin-filaggrin, and loricrin, and also focal parakeratosis, indicative of impaired differentiation (Kömüves et al., 2000Kömüves L.G. Hanley K. Lefebvre A.-M. et al.Stimulation of PPARα promotes epidermal keratinocyte differentiation in vivo.J Invest Dermatol. 2000; 115: 353-360Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). These observations suggest that PPARα activation is one of the pathways that regulate keratinocyte differentiation in normal epidermis. The aim of this study was to determine whether PPARα activation modulates keratinocyte differentiation in pathologic epidermis, characterized by aberrant cell differentiation and hyperproliferation. We show here that topical applications of PPARα activators, clofibrate and Wy-14,643, result in a normalization of gene expression, together with a normalization of the epidermal morphology, both in subacute and chronic models of hyperproliferative skin. These results suggest that PPARα activators play a role in maintaining tissue homeostasis by modulating cell proliferation, keratinocyte differentiation, and apoptosis in the epidermis. We used adult male hairless mice (Simonsen, Gilroy, CA, or Charles River, Wilmington, MA) in this study. Essential fatty acid deficiency (EFAD) mice were produced by feeding an isocaloric essential fatty acid free diet (Dyets, Bethlehem, PA) after weaning for 2 to 3 mo (Man et al., 1993Man M.Q. Elias P.M. Feingold K.R. Fatty acids are required for epidermal barrier function.J Clin Invest. 1993; 92: 791-798Crossref PubMed Scopus (152) Google Scholar). Subacute epidermal hyperproliferation was induced by repeated barrier abrogation with acetone treatment (Denda et al., 1996Denda M. Wood L.C. Emami S. et al.The epidermal hyperplasia associated with repeated barrier disruption by acetone treatment or tape stripping cannot be attributed to increased water loss.Arch Dermatol Res. 1996; 288: 230-238Crossref PubMed Scopus (131) Google Scholar). Animals were treated twice a day with acetone until the transepidermal water loss reached 8–10 mg per cm2 per h (measured on four or five different spots) as determined with an electrolytic water analyzer (Meeco, Warrington, PA), for 7 d (Denda et al., 1996Denda M. Wood L.C. Emami S. et al.The epidermal hyperplasia associated with repeated barrier disruption by acetone treatment or tape stripping cannot be attributed to increased water loss.Arch Dermatol Res. 1996; 288: 230-238Crossref PubMed Scopus (131) Google Scholar). To test the effect of PPARα activators on the hyperproliferative skin, EFAD or acetone-treated mice were treated on one side of the flank with 40 μl per cm2 of 1 m M clofibrate (Sigma, St. Louis, MO) or with 0.5 m M Wy-14,643 (Sigma), dissolved in propylene glycol:ethanol (7:3 ratio). Animals were treated twice a day, for 3 d. Control animals were treated with vehicle only. Tissue samples were collected for histology, in situ hybridization, or immunohistochemistry as described earlier (Kömüves et al., 1998Kömüves L.G. Hawley K. Jiang Y. et al.Ligands and activators of nuclear hormone receptors regulate epidermal differentiation during fetal rat skin development.J Invest Dermatol. 1998; 111: 429-433Crossref PubMed Scopus (95) Google Scholar). Affinity-purified rabbit antibodies specific for mouse involucrin, profilaggrin-filaggrin, and loricrin were from BabCo (Richmond, CA); affinity-purified biotinylated goat antirabbit IgG was purchased from Vector (Burlingame, CA). The immunohistochemical staining was detected by ABC-peroxidase (Vector) and diaminobenzidine substrate (Vector) as described earlier (Kömüves et al., 1998Kömüves L.G. Hawley K. Jiang Y. et al.Ligands and activators of nuclear hormone receptors regulate epidermal differentiation during fetal rat skin development.J Invest Dermatol. 1998; 111: 429-433Crossref PubMed Scopus (95) Google Scholar). Omission of the first antibodies or incubation with the substrate solution resulted in no signal, showing the specificity of the detection. The biotinylated anti-proliferating cell nuclear antigen mouse monoclonal antibody was from CalTag Laboratories (Burlingame, CA). The binding of this antibody was detected by Avidin-Alexa (Molecular Probes, Eugene, OR), followed by Sytox Green nuclear counterstain. Sections were analyzed with Leica (Deerfield, IL) TCS laser-scanning confocal microscope. Digoxigenin-labeled RNA probes to detect loricrin (3′ noncoding region, 200 bases) (Mehrel et al., 1990Mehrel T. Hohl D. Rothnagel J.A. Longley M.A. Bundman D. Identification of a major keratinocyte cell envelope protein, loricrin.Cell. 1990; 61: 1103-1112Abstract Full Text PDF PubMed Scopus (381) Google Scholar) and profilaggrin (coding region, 300 bases) (Yuspa et al., 1989Yuspa S.H. Kilkenny A.E. Steinert P.M. Roop D.R. Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro.J Cell Biol. 1989; 109: 1207-1217Crossref PubMed Scopus (513) Google Scholar) mRNAs were made from linearized cDNA sequences (a gift from S. Yuspa, NIH) using reagents supplied by Boehringer-Mannheim (Indianapolis, IN). In situ hybridization was performed as described earlier (Kömüves et al., 1998Kömüves L.G. Hawley K. Jiang Y. et al.Ligands and activators of nuclear hormone receptors regulate epidermal differentiation during fetal rat skin development.J Invest Dermatol. 1998; 111: 429-433Crossref PubMed Scopus (95) Google Scholar). Hybridization of DIG-labeled probes to the endogenous mRNA was detected by anti-DIG-alkaline phosphatase (Boehringer-Mannheim), using 5-bromo-4-chloro-3-indolyl phosphate/nitrotetrazolium blue substrate. Hybridization with DIG-labeled sense control probes, omission of antisense probes from the hybridization, or incubation with substrate only resulted in no staining. Deparaffinized sections were treated first with 0.5% sodium tetraborohydrate for 30 min. TUNEL assay was carried out using the in situ cell death detection kit from Boehringer-Mannheim, following the instructions of the manufacturer. Following the TUNEL reaction the sections were counterstained with Sytox Orange (Molecular Probes, Eugene, OR) nuclear stain. The incorporation of FITC-labeled dUTP was visualized with a Leica (Deerfield, IL) TCS laser-scanning confocal microscope. Epidermal thickness was measured on hematoxylin and eosin stained sections using a 20× objective and an ocular micrometer (final magnification 100×). Epidermal thickness was defined as the distance between the basement lamina and the apical surface of the uppermost nucleated keratinocytes, i.e., the border between the stratum granulosum and stratum corneum. Photographs were taken with a Zeiss Axiophot microscope on Kodak (Rochester, NY) Gold 100 print film. Printed images (4 × 5 inches) were digitized with a flatbed scanner, and assembled using Photoshop 3.0 (Adobe Systems, Mountain View, CA) on a Macintosh G3 platform (Apple, Cupertino, CA). Data are presented as mean ± SEM. Statistical differences were determined by the Student's t test using Excel (Microsoft, Redmond, WA). To determine the effects of PPARα activators on hyperproliferative skin, we employed two well-characterized murine models of epidermal hyperproliferation (Man et al., 1993Man M.Q. Elias P.M. Feingold K.R. Fatty acids are required for epidermal barrier function.J Clin Invest. 1993; 92: 791-798Crossref PubMed Scopus (152) Google Scholar; Denda et al., 1996Denda M. Wood L.C. Emami S. et al.The epidermal hyperplasia associated with repeated barrier disruption by acetone treatment or tape stripping cannot be attributed to increased water loss.Arch Dermatol Res. 1996; 288: 230-238Crossref PubMed Scopus (131) Google Scholar). In the subacute model of epidermal hyperproliferation (Denda et al., 1996Denda M. Wood L.C. Emami S. et al.The epidermal hyperplasia associated with repeated barrier disruption by acetone treatment or tape stripping cannot be attributed to increased water loss.Arch Dermatol Res. 1996; 288: 230-238Crossref PubMed Scopus (131) Google Scholar), repeated barrier abrogation, twice a day for 7 d, led to an 80% increase in epidermal thickness compared with untreated, control animals (Figure 1, Table 1). Similarly, when hairless mice were kept on an essential fatty acid free diet for 2 mo (Man et al., 1993Man M.Q. Elias P.M. Feingold K.R. Fatty acids are required for epidermal barrier function.J Clin Invest. 1993; 92: 791-798Crossref PubMed Scopus (152) Google Scholar), the epidermal thickness doubled (Figure 1, Table 1). The epidermis in both of these models also displayed extensive acanthosis and hyperkeratosis Figure 1. Following topical treatments with clofibrate Figure 1 or Wy-14,643 (not shown) we noted a substantial decrease in epidermal thickness in both models of epidermal hyperproliferation. No necrotic cells were seen by histologic evaluation of the tissues, however, following these treatments. Whereas in the subacute model a complete recovery was observed Table 1, the epidermis in the EFAD mice remained slightly thicker than the normal control despite a 66% decrease in epidermal thickness Table 1. Epidermal thickness did not change in the untreated side of clofibrate-treated animals and it did not differ from vehicle-treated or untreated hyperproliferative skin Table 1, suggesting that clofibrate exerts its effects locally.Table 1Topical application of a PPARα activator (clofibrate) decreases epidermal thickness in hyperproliferative epidermisAnimalsTreatmentEpidermal thickness(μm)aData are presented as mean ± SEM. The total length of the epidermis analyzed varied between 6 and 15 mm for each sample.NormalUntreated (n = 5)14 ± 2EFADUntreated (n = 3)50 ± 5Vehicle (n = 4)53 ± 4100%Clofibrate (n = 6)22 ± 5bSignificantly different (p < 0.001) as compared with the vehicle-treated mice.44%Untreated side (n = 6)50 ± 2Repeated barrier disruptionUntreated (n = 3)31 ± 3Vehicle (n = 4)33 ± 2100%Clofibrate (n = 4)b16 ± 6bSignificantly different (p < 0.001) as compared with the vehicle-treated mice.52%Untreated side (n = 4)30 ± 5a Data are presented as mean ± SEM. The total length of the epidermis analyzed varied between 6 and 15 mm for each sample.b Significantly different (p < 0.001) as compared with the vehicle-treated mice. Open table in a new tab We next determined whether the decrease in epidermal hyperplasia could result from decreased cell proliferation. Proliferative cells were identified with an antibody specific for proliferating cell nuclear antigen (PCNA). In normal control hairless mice skin PCNA-positive cells were confined to the basal layer Figure 2a. In contrast, an expansion of proliferative keratinocytes was seen in both subacute and acute hyperproliferative epidermis (Table 2, Figure 2b, e). In both hyperproliferative models topical clofibrate treatment decreased the number of PCNA-positive cells (Table 2, Figure 2c, f). Moreover, PCNA-positive cells were found mainly in the basal layer following topical clofibrate treatment Figure 2c, f. Similar results were seen following topical Wy-14,643 treatment Figure 2d, g. These observations indicate that topically applied PPARα activators downregulate cell proliferation in hyperproliferative epidermis and restore a normal cell proliferation pattern that is restricted to the basal cell layer.Table 2Topical application of a PPARα activator (clofibrate) decreases cell proliferation in hyperproliferative epidermisAnimalsTreatmentPCNA-positive cellsper mmaData are presented as mean number of PCNA-positive cells ± SEM per mm. The total length of the epidermis analyzed varied between 10 and 16 mm for each sample.NormalUntreated (n = 5)80 ± 12EFADVehicle (n = 4)164 ± 11100%Clofibrate (n = 6)77 ± 15bSignificantly different (p < 0.001) as compared to vehicle-treated control.,cNo significant difference (p > 0.01) as compared to normal untreated control.47%Repeated barrier disruptionVehicle (n = 4)150 ± 21100%Clofibrate (n = 4)98 ± 16bSignificantly different (p < 0.001) as compared to vehicle-treated control.,cNo significant difference (p > 0.01) as compared to normal untreated control.65%a Data are presented as mean number of PCNA-positive cells ± SEM per mm. The total length of the epidermis analyzed varied between 10 and 16 mm for each sample.b Significantly different (p < 0.001) as compared to vehicle-treated control.c No significant difference (p > 0.01) as compared to normal untreated control. Open table in a new tab The altered distribution of proliferative cells in murine hyperproliferative epidermis observed in this study suggested that keratinocyte differentiation could be disturbed. Therefore, we next determined the expression of genes characteristic for terminal keratinocyte differentiation (Fuchs, 1990Fuchs E. Epidermal differentiation. The bare essentials.J Cell Biol. 1990; 111: 2807-2814Crossref PubMed Scopus (589) Google Scholar; Eckert et al., 1997Eckert R.L. Crish J.F. Robinson N.A. The epidermal keratinocyte as a model for the study of gene regulation and function.Physiol Rev. 1997; 77: 397-424Crossref PubMed Scopus (343) Google Scholar). Involucrin, filaggrin, and loricrin mRNA and protein are localized to the upper layers of epidermis in normal hairless mice. This expression and distribution pattern, however, was altered in hyperproliferative epidermis. Whereas involucrin staining was seen in the upper layers Figure 3a, g, profilaggrin-filaggrin Figure 3b, h and loricrin Figure 3c, i stained weakly in the hyperproliferative epidermis, especially in EFAD mice. Following topical clofibrate treatment involucrin staining increased somewhat in individual keratinocytes Figure 3d, j, and the granular nature of the staining was more obvious. Moreover, staining both for profilaggrin-filaggrin Figure 3e, k and loricrin Figure 3f, l increased in the granular layer following clofibrate treatment, and the staining intensity was similar to that observed in control hairless mice epidermis. Next we used in situ hybridization to analyze the changes in profilaggrin and loricrin gene expression. Whereas both profilaggrin mRNA Figure 4a, e and loricrin mRNA Figure 4b, f were present in the upper layers of the stratum granulosum in hyperproliferative epidermis, this staining was weak, particularly for loricrin, compared with normal epidermis. Following topical clofibrate treatment a more proximal expression of these genes was observed Figure 4c, d, g, h. The staining intensity in individual cells was also increased greatly, especially for profilaggrin mRNA (Figure 4a vs 4c, and 4e vs 4g), suggesting increased expression of these genes. These results show that topical clofibrate treatment normalizes the distribution of involucrin, profilaggrin-filaggrin, and loricrin proteins, and increases profilaggrin and loricrin gene expression in both models of epidermal hyperproliferation. Apoptosis plays a major role in the elimination of injured or abnormal cells in other tissues as well as in the epidermis (Haake and Polakowska, 1993Haake A.R. Polakowska R.R. Cell death by apoptosis in epidermal biology.J Invest Dermatol. 1993; 101: 107-112Abstract Full Text PDF PubMed Google Scholar). We used the TUNEL method, which detects DNA fragmentation, a hallmark of apoptosis, to identify apoptotic cells in the hyperproliferative skin in the models. In the epidermis of untreated normal hairless mice apoptotic cells were occasionally encountered Figure 5a. Likewise, apoptotic cells were found at the stratum granulosum-stratum corneum interface in the hyperproliferative epidermis of EFAD mice Figure 5b or following repeated barrier abrogation Figure 5e. The number of TUNEL-positive cells increased following topical clofibrate Figure 5c, f or Wy-14,643 Figure 5d, g treatments, however. Moreover, apoptotic cells not only occurred at the stratum granulosum-stratum corneum interface, but were encountered in the deeper epidermal layers Figure 5c, d and in the dermis. These observations indicate a strong increase in apoptosis following topical clofibrate treatment of acute and chronic hyperproliferative skin. Several studies have shown that the pattern of keratinocyte differentiation is abnormal in hyperproliferative epidermis in human skin. In psoriasis, a hyperproliferative skin disease, cell proliferation is increased whereas the onset of epidermal differentiation is delayed (Bernerd et al., 1992Bernerd F. Magnaldo T. Darmon M. Delayed onset of epidermal differentiation in psoriasis.J Invest Dermatol. 1992; 98: 902-910Abstract Full Text PDF PubMed Scopus (83) Google Scholar). The mRNA and protein levels for several genes (such as keratin 15, filaggrin, and loricrin), which are normally expressed in terminally differentiating granular keratinocytes, are decreased (Watanabe et al., 1991Watanabe S. Wagatsuma K. Ichikawa E. Takahashi H. Abnormal distribution of epidermal protein antigens in psoriatic epidermis.J Dermatol. 1991; 18: 143-151Crossref PubMed Scopus (56) Google Scholar; Bernerd et al., 1992Bernerd F. Magnaldo T. Darmon M. Delayed onset of epidermal differentiation in psoriasis.J Invest Dermatol. 1992; 98: 902-910Abstract Full Text PDF PubMed Scopus (83) Google Scholar). Moreover, the gene expression pattern of transglutaminase 1 (Nonomura et al., 1993Nonomura K. Yamanishi K. Hosokawa Y. et al.Localization of transglutaminase 1 mRNA in normal and psoriatic epidermis by non-radioactive in situ hybridization.Br J Dermatol. 1993; 128: 23-28Crossref PubMed Scopus (24) Google Scholar) and involucrin (Ishida-Yamamoto and Izuka, 1995Ishida-Yamamoto A. Izuka H. Differences in involucrin immunolabeling within cornified cell envelopes in normal and psoriatic epidermis.J Invest Dermatol. 1995; 104: 391-395Crossref PubMed Scopus (70) Google Scholar) is also disturbed, resulting in abnormal cornified envelope formation (Ishida-Yamamoto and Izuka, 1995Ishida-Yamamoto A. Izuka H. Differences in involucrin immunolabeling within cornified cell envelopes in normal and psoriatic epidermis.J Invest Dermatol. 1995; 104: 391-395Crossref PubMed Scopus (70) Google Scholar). Furthermore, in transgenic mice expressing a mutant desmosomal cadherin, which exhibit epidermal hyperplasia, the expression of filaggrin and loricrin decreases (Allen et al., 1996Allen E. Yu Q.-C. Fuchs E. Mice expressing a mutant desmosomal cadherin exhibit abnormalities in desmosomes, proliferation, and epidermal differentiation.J Cell Biol. 1996; 133: 1367-1382Crossref PubMed Scopus (135) Google Scholar). As we have described in this study, in two mouse models of pathologic epidermal hyperproliferation, i.e., induced by EFAD or by repeated barrier abrogation, several features of abnormal epidermal keratinocyte differentiation (increased cell proliferation, decreased mRNA levels for profilaggrin and loricrin) also occur. As these features are hallmarks of human hyperproliferative skin diseases such as psoriasis, these murine models may be useful to analyze pathologic mechanisms in hyperproliferative skin disorders. It should be recognized, however, that these murine models do not perfectly mimic psoriasis or other human disorders. In these studies we have elected to use two animal models that are likely to develop epidermal hyperproliferation by different mechanisms. It is hoped that by studying two different pathogenic models the observations reported in this paper will be more likely to be applicable to human disorders. This study expands the role of PPARα in skin biology as we demonstrate here that epidermal hyperproliferation can be corrected by topical application of clofibrate or WY14,643, PPARα activators (Isseman and Green, 1990Isseman I. Green S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators.Nature. 1990; 347: 645-650Crossref PubMed Scopus (3037) Google Scholar; Schoonjans et al., 1996Schoonjans K. Staels B. Auwerx J. Role of the peroxisome proliferator-activated receptor in mediating the effects of fibrates and fatty acids on gene expression.J Lipid Res. 1996; 37: 907-925Abstract Full Text PDF PubMed Google Scholar). Topical treatment with PPARα activators not only decreased cell proliferation in hyperproliferative epidermis but increased transformation of the terminally differentiated keratinocytes into corneocytes, as witnessed by the elevated expression of involucrin, filaggrin, and loricrin genes and by an increased incidence of apoptosis. Apoptosis was induced in all layers of the epidermis by topical treatment with PPARα activators. Moreover, the pool of proliferative cells returned to normal, as, following topical PPARα activator treatment, PCNA-positive cells were almost exclusively restricted to basal keratinocytes. These pronounced changes in PCNA immunostaining are probably due to the short half-life of these proteins. Cyclins (recognized by the PCNA antibody) are rapidly degraded as cells cease proliferation (King et al., 1996King R.W. Deshaies R.J. Peters J.-M. Kirschner M.W. How proteolysis drives the cell cycle.Science. 1996; 274: 1652-1659Crossref PubMed Scopus (1117) Google Scholar). Clofibrate treatment also increased the expression of genes required for terminal keratinocyte differentiation (involucrin, profilaggrin, and loricrin). Interestingly, however, the clofibrate-induced changes were more pronounced at mRNA levels than at protein levels. These observations indicate that coordinate regulation of a set of genes (including involucrin, filaggrin, and loricrin) is specifically modulated with PPARα activators. These findings also suggest that the changes induced by PPARα activators are probably not due to nonspecific, metabolic effects, but are mediated by not yet identified PPARα-response elements. The clofibrate-induced changes in gene expression coupled with the increased apoptosis resulted in a marked decrease in epidermal thickness as a consequence of increased cellular turnover. Fatty acids, particularly polyunsaturated fatty acids, are ligands of PPARα (Schoonjans et al., 1996Schoonjans K. Staels B. Auwerx J. Role of the peroxisome proliferator-activated receptor in mediating the effects of fibrates and fatty acids on gene expression.J Lipid Res. 1996; 37: 907-925Abstract Full Text PDF PubMed Google Scholar). One could therefore postulate that a decrease in essential fatty acids in the EFAD mice results in a reduction of ligands for PPARα, thereby leading to hyperproliferation. This is very unlikely to be the case, however, because our studies of PPARα-deficient mice have not demonstrated a hyperproliferative phenotype (Kömüves et al., 2000Kömüves L.G. Hanley K. Lefebvre A.-M. et al.Stimulation of PPARα promotes epidermal keratinocyte differentiation in vivo.J Invest Dermatol. 2000; 115: 353-360Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Additionally, this deficiency of fatty acids could not explain the effect of PPARα activators in normalizing the epidermis of animals in which the pathology was induced by repeated barrier abrogation. In an earlier study (Kömüves et al., 2000Kömüves L.G. Hanley K. Lefebvre A.-M. et al.Stimulation of PPARα promotes epidermal keratinocyte differentiation in vivo.J Invest Dermatol. 2000; 115: 353-360Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) we found that topical clofibrate treatment decreased cell proliferation and increased apoptosis in normal epidermis. Moreover, PPARα ligands inhibit cell proliferation in human cultured keratinocytes (Hanley et al., 1998Hanley K. Jiang Y. He S.S. et al.Keratinocyte differentiation is stimulated by activators of the nuclear hormone receptor PPARα.J Invest Dermatol. 1998; 110: 368-375Crossref PubMed Scopus (169) Google Scholar) and breast cancer cells (Elstner et al., 1998Elstner E. Muller C. Koshizuka K. et al.Ligands for peroxisome-proliferator-activated receptor and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cell in vitro and in BNX mice.Proc Natl Acad Sci. 1998; 95: 8806-8811Crossref PubMed Scopus (760) Google Scholar). Therefore, in general, fatty acids and other PPARα ligands have a cytostatic effect (Pineau et al., 1996Pineau T. Hudgins W.R. Liu L. et al.Activation of a human peroxisome proliferator-activated receptor by the antitumor agent phenylacetate and its analogs.Biochem Pharmacol. 1996; 52: 659-667Crossref PubMed Scopus (116) Google Scholar; Van den Heuvel, 1999Van den Heuvel J.P. Peroxisome proliferator activated receptors: a critical link among fatty acids, gene expression and carcinogenesis.J Nutr. 1999; 129: 575S-580SPubMed Google Scholar), and induce apoptosis (Chinetti et al., 1998Chinetti G. Griglio S. Antonucci M. et al.Activation of peroxisome proliferator-activated receptors -α and -γ induces apoptosis of human monocyte-derived macrophages.J Biol Chem. 1998; 273: 25573-25580Crossref PubMed Scopus (849) Google Scholar). In extra-hepatic tissues (including the epidermis) PPARα activation regulates tissue homeostasis. In contrast, in rodent liver prolonged treatment with PPARα activators results in hepatic carcinogenesis as a consequence of increased cell proliferation and suppressed apoptosis (Bayly et al., 1994Bayly A.C. Roberts R.A. Dive C. Suppression of liver cell apoptosis in vitro by the non-genotoxic hepatocarcinogen and peroxisome proliferator nafenopin.J Cell Biol. 1994; 125: 197-203Crossref PubMed Scopus (188) Google Scholar; Gonzalez et al., 1998Gonzalez F.J. Peters J.M. Cattley R.C. Mechanism of action of the nongenotoxic peroxisome proliferators: role of the peroxisome proliferator-activated receptor α.J Natl Cancer Inst. 1998; 90: 1702-1709Crossref PubMed Scopus (264) Google Scholar). These opposing effects of PPARα ligands in hepatic versus nonhepatic tissues suggest the existence of tissue-specific PPARα-response elements, which regulate cell proliferation and apoptosis in a cell- and/or differentiation-specific manner. The epidermis is a continuously renewing tissue in which homeostasis is maintained by a tightly regulated balance between cell proliferation, cell differentiation, and programmed cell death. Proliferating cells, terminally differentiating cells, and apoptotic cells are spatially and temporally separated in the epidermis. In the normal epidermis proliferating cells are confined to the basal layer, whereas apoptosis occurs at the border of the stratum granulosum and stratum corneum (Haake and Polakowska, 1993Haake A.R. Polakowska R.R. Cell death by apoptosis in epidermal biology.J Invest Dermatol. 1993; 101: 107-112Abstract Full Text PDF PubMed Google Scholar; Steiner, 1995). In a plethora of skin diseases (Cerio et al., 1998Cerio R. Histopathology of the skin: general principles.in: Champion R.H. Burton J.L. Burns D.A. Breathnach S.M. Textbook of Dermatology. Blackwell Science, London1998: 159-203Google Scholar), such as localized benign epidermal tumors (keratoderma, epidermal naevi), in papillomas (viral warts, molluscum contagiosum), and in certain secondary inflammatory conditions (psoriasis and lichen planus) normal tissue homeostasis is disturbed, resulting in epidermal hyperproliferation. The data presented here show that PPARα activation is able to counteract epidermal hyperproliferation, suggesting that PPARα activators could be used effectively as therapeutics to treat a variety of skin conditions and skin diseases. We thank Dr. S. Yuspa (NIH), who provided profilaggrin and loricrin cDNAs. This work was supported by NIH grants HD 29706, AR 39639, AR 29706, and PO 039448, and by VA Research funding.