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Originally published In Press as doi:10.1074/jbc.M409795200 on October 29, 2004

J. Biol. Chem., Vol. 280, Issue 1, 73-79, January 7, 2005
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Epidermal Peroxisome Proliferator-activated Receptor {gamma} as a Target for Ultraviolet B Radiation*

Qiwei Zhang{ddagger}§, Michael D. Southall{ddagger}§, Steven M. Mezsick{ddagger}, Christopher Johnson¶, Robert C. Murphy¶, Raymond L. Konger{ddagger}||, and Jeffrey B. Travers{ddagger}§**{ddagger}{ddagger}§§

From the Departments of {ddagger}Dermatology, ||Pathology, and §Pediatrics, the Herman B. Wells Center for Pediatric Research, and the Department of **Pharmacology and Toxicology, Indiana University School of Medicine, and the {ddagger}{ddagger}Richard L. Roudebush Veterans Affairs Medical Center, Indianapolis, Indiana 46202 and the Department of Pharmacology, University of Colorado Health Sciences Center, Aurora, Colorado 80045

Received for publication, August 26, 2004 , and in revised form, October 28, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ultraviolet B radiation (UVB) is a pro-oxidative stressor with profound effects on skin in part through its ability to stimulate cytokine production. Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) has been shown to regulate inflammatory processes and cytokine release in various cell types. Since the oxidized glycerophospholipid 1-hexadecyl-2-azelaoyl glycerophosphocholine (azPC) has been shown to be a potent PPAR{gamma} agonist, this study was designed to assess whether the PPAR{gamma} system is a target for UVB irradiation and involved in UVB-induced inflammation in epidermal cells. The present studies demonstrated the presence of PPAR{gamma} mRNA and functional protein in human keratinocytes and epithelial cell lines HaCaT, KB, and A431. The treatment of epidermal cells with the PPAR{gamma}-specific agonist ciglitazone or azPC augmented cyclooxygenase-2 expression and enzyme activity induced by phorbol 12-myristate-13-acetate or interleukin-1{beta}. Lipid extracts from the cell homogenate of UVB-irradiated, but not control, cells contained a PPAR{gamma}-agonistic activity identified by reporter assay, and this activity up-regulated cyclooxygenase-2 expression induced by phorbol 12-myristate-13-acetate. Subjecting purified 1-hexadecyl-2-arachidonoyl-glycerophosphocholine to UVB irradiation generated a PPAR{gamma}-agonistic activity, among which the specific PPAR{gamma} agonist azPC was identified by mass spectrometry. These findings suggested that UVB-generated PPAR{gamma}-agonistic activity was due to the free radical mediated non-enzymatic cleavage of endogenous glycerophosphocholines. Treatment with the specific PPAR{gamma} antagonist GW9662 or expression of a dominant-negative PPAR{gamma} mutant in KB cells inhibited UVB-induced epidermal cell prostaglandin E2 production. These findings suggested that UVB-generated PPAR{gamma} activity is necessary for the optimal production of epidermal prostaglandins. These studies demonstrated that epithelial cells contain a functional PPAR{gamma} system, and this system is a target for UVB through the production of novel oxidatively modified endogenous phospholipids.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ultraviolet B (UVB,1 290–320 nm) irradiation has profound effects on human skin. These effects include DNA damage, apoptosis, and cytokine production (1, 2). UVB irradiation induces many cytokines including tumor necrosis factor-{alpha}, interleukin (IL)-1{beta}, IL-6, IL-8, IL-10, as well as prostaglandins (reviewed in Refs. 3 and 4). The exact mechanisms by which UVB irradiation leads to cytokine release in keratinocytes are still unknown. One possible mechanism is related to the ability of UVB to act as a potent pro-oxidative stressor (5). Acute short term UVB absorption by keratinocytes results in oxidative stress and DNA damage (6). It has been shown that exposure of epidermal homogenates to UV light-induced lipid peroxide formation via lipid radicals (7). The hydroxyl radical was found to generate lipid radicals as a result of ·OH-mediated hydrogen atom abstraction (8).

Oxidatively modified phospholipids, especially those arising from alkyl glycerophosphocholines (GPC), can exert potent biological activities (reviewed in Refs. 9–11). For example, oxidation of low density lipoprotein generates a series of lipid inflammatory mediators among which some fragmented phospholipids could potently activate inflammatory cells through the platelet-activating factor receptor (12, 13). Davies et al. (14) showed that oxidatively modified phosphatidylcholines from oxidized low density lipoprotein are high affinity ligands and agonists for peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}). To date, 1-hexadecyl 2-azelaoyl phosphatidylcholine (azPC) is the only oxidized GPC with PPAR{gamma}-agonistic activity described (14). This oxidized GPC is a potent PPAR{gamma} agonist, exerting effects in the nanomolar range.

The PPARs are ligand-dependent transcription factors that regulate target gene expression by binding to specific peroxisome proliferator-response elements (PPREs) (reviewed in Ref. 15). Three isoforms, encoded by separate genes, have been identified so far: PPAR{alpha}, PPAR{gamma}, and PPAR{delta} (reviewed in Ref. 16). PPARs have been demonstrated to be key regulators of lipid metabolism and associated with genes that affect insulin action. Recent studies have suggested that PPAR{gamma} also plays an important role in the control of cytokine release and inflammatory mediator expression (17–19). For example, it was demonstrated that challenging obese and diabetic db/db mice with the PPAR{gamma} agonist thiazolidinedione resulted in higher blood levels of tumor necrosis factor-{alpha} and IL-6 (20). Pontsler et al. (21) have demonstrated that cyclooxygenase-2 (COX-2) and prostaglandin (PGE2) secretion were induced in monocytes treated by PPAR{gamma} agonists including the oxidized alkyl phospholipid azPC. The regulatory effects of PPAR{gamma} on epidermal inflammation and cytokine release have not been examined.

Since UVB has potent pro-oxidative effects, and oxidative fragmentation of phospholipids could result in the non-enzymatic production of potent PPAR{gamma} agonists, the objective of the present study was to assess whether UVB irradiation of keratinocytes could result in a PPAR{gamma}-agonistic activity. The present studies provide evidence that UVB irradiation triggers the production of novel oxidized GPCs, including azPC, which appears to be involved in UVB-mediated PGE2 production. These findings suggested that the epidermal PPAR{gamma} system is a novel target for UVB.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—All chemicals were obtained from Sigma unless otherwise indicated. PMA (phorbol-12-myristate-13-acetate) was obtained from Sigma. Ciglitazone and (4-choloro-6-(2,3-xylidino)-2-pyrimidinylthio)acetic acid (WY14643) were from Alexis Biochemicals (San Diego, CA). Recombinant human IL-1{beta} was from PeproTech (Rocky Hill, NJ). azPC and 1-hexadecyl-2-arachidonoyl-glycerophosphocholine (HAPC) were purchased from Avanti%20Polar%20Lipids">Avanti Polar Lipids, Inc. (Alabaster, AL). The antioxidant 6-hydroxy-2,5,7,8-tertamethylchroman-2-carboxylic acid (Trolox®) was purchased from Calbiochem. The specific PPAR{gamma} antagonist 2-chloro-5-nitrobenzanilide (GW9662) was purchased from Cayman Chemical (Ann Arbor, MI). The selective COX-2 inhibitor N-(2-(cyclohexyloxy)-4-nitrophenyl)methanesulfonamide (NS-398) was from Sigma. Calcium ionophore A23187 [GenBank] was purchased from Roche Applied Science.

Cell Culture—KB, A431, and HaCaT cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Intergen, Purchase, NY), as described previously (2). The KB/PPAR{gamma} dominant-negative model was created by transfection of KB cells with the PPAR{gamma} dominant-negative construct encoding the mutant hPPAR{gamma} (human PPAR{gamma}) with two point mutations in the AF-2 domain, L466A and E469A (22); control cells were transfected with mock plasmid pcDNA3.1. The PPAR{gamma} dominant-negative plasmid was a kind gift from Dr. V. K. K. Chatterjee (University of Cambridge, Cambridge, UK).

RNA Isolation and Reverse Transcription-PCR—Total RNA was first isolated from cultured cells and purified using Tripure (Roche Diagnostics), according to the manufacturer's protocol. An additional phenol (pH 4.2) extraction was then performed, followed by ethanol precipitation. The mRNA was reversed-transcribed into cDNA using Superscript II reverse transcriptase (Superscript II RNase H- reverse transcriptase kit, Invitrogen). Briefly, 5 µg of total RNA and 0.5 µg of oligo(dT)12–18 primer were heated to 70 °C for 10 min and briefly chilled on ice. After primer annealing, the following were added: 50 mM Tris-HCl, pH 8.8, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 1 mM dNTP, and 40 units of RNasin ribonuclease inhibitor. The reaction was incubated for 50 min at 42 °C and then for 15 min at 70 °C. An aliquot of each reaction was subsequently used as template for a PCR reaction. Primer sequences for human PPAR{gamma} were as follows: PPAR{gamma} sense, 5'-tctctccgtaatggaagacc-3'; PPAR{gamma} antisense, 5'-gcattatgagacatccccac-3' (23). The PCR mixture contained a cDNA template derived from total RNA, 1 unit of recombinant Taq DNA polymerase (Invitrogen), 50 pmol each of 5' and 3' primers (Invitrogen), 0.2 mM dNTP, in a buffer containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgSO4 in a volume of 50 µl. The PCR reaction for PPAR{gamma} was performed using a Hybaid PCRExpress thermocycler as follows: 94 °C for 120 s and then 30 cycles of 94 °C for 45 s, 55 °C for 60 s, and 72 °C for 120 s followed by 74 °C for 10 min. Samples were applied on 1% agarose gel prestained with ethidium bromide.

Immunoblotting—Cells were washed twice with ice-cold phosphate-buffered saline and lysed with radioimmune precipitation buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40) containing 0.5 mM Pefabloc SC (Roche Diagnostics) and 10 mM sodium orthovanadate for 20 min on ice. Total cellular protein of 40 µg was separated on 10% SDS-PAGE; PPAR{gamma} and COX-2 protein expression was determined by immunoblotting with PPAR{gamma} polyclonal antibody and COX-2 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), respectively, and enhanced chemiluminescence (Amersham Biosciences). The expression of COX-2 protein was normalized to the levels of housekeeping protein actin. The arbitrary optical densities were measured by Scion Image Software (Scion Co., Frederick, MD).

PPRE-Luciferase Reporter Assay—Cells were plated at a density of 1.5 x 106 cells/plate in 100-mm dishes and allowed to stabilize for 1 day. Cells were then transfected with 10 µg of PPAR{lambda}-luciferase reporter plasmid (pPPAR-luc) (14) and 10 µg of p-cytomegalovirus (pCMV)-{beta}-galactosidase plasmid as an internal control for the transfection efficiency using FuGENE 6 according to the manufacturer's instructions (Roche Diagnostics). After the 24-h transfection, cells were treated with various PPAR{gamma} agonists or antagonists for 24 h and harvested by lysis with 900 µl of reporter lysis buffer (Promega, Madison, WI). A 20-µl aliquot of the lysate was used in the {beta}-galactosidase assay, and 20 µl of the lysate was assayed using a luciferase assay kit (Promega) and counted for 10 s in a FB12 luminometer (Zylux, Oak Ridge, TN) with the data represented as the relative light unit/second.

COX-2 Activity Assay—COX-2 activity assay kit (Cayman Chemical, Ann Arbor, MI) was used according to the manufacturer's protocol. Briefly, cells were collected by scraping and then sonicated in cold buffer (0.1 M Tris-HCl, pH 7.8, containing 1 mM EDTA). The supernatant was obtained by centrifugation at 10,000 x g for 15 min. 10 µl of supernatant was mixed with 140 µl of assay buffer, 10 µl of heme, and 10 µl of COX-1 inhibitor SC-560 in triplicate wells of a 96-well plate. The plate was shaken and incubated for 5 min at 25 °C. 20 µl of colorimetric substrate and 20 µl arachidonic acid solution were then added sequentially, shaken, and incubated at 25 °C for 5 min. The absorbance was read at 590 nm using the enzyme-linked immunosorbent assay multiplate reader.

PGE2 Immunoassay—First, 2 x 106 cells/well were seeded in a 6-well dish. After 1 day of serum starvation, cells were treated variously as described in the figure legends. The cell supernatant was extracted after an 8-h treatment. The PGE2 levels in the supernatant were quantified using an immunoassay kit (R&D Systems, Minneapolis, MN), according to the manufacturer's protocol. Briefly, 100 µl of supernatant was mixed with 50 µl of PGE2 conjugate and 50 µl of PGE2 antibody solution in each well of 96-well enzyme-linked immunosorbent assay plate. After 2 h of incubation on a horizontal orbital microplate shaker at 500 rpm at room temperature, each well was aspirated and washed four times with washing solution. Then, 200 µl of pNPP substrate was added and incubated at room temperature for 1 h. After adding 50 µl of stop solution, the optical density of each well was measured at 405 nm with wavelength correction set at 580 nm.

UV Irradiation Study—Cells were irradiated with UVB as described previously (2). The UV source was a Philips F20T12/UV-B lamp (270–390 nm; containing 2.6% UVC, 43.6% UVB, 53.8% UVA). The intensity of the UVB source was measured prior to each experiment using an IL1700 radiometer and a SED240 UVB detector (International Light, Newburyport, MA) at a distance of 8 cm from the UVB source to the monolayer of cells/purified lipid. KB cells were irradiated, and the lipids were extracted as described previously (24). For the cell-free irradiation of HAPC, 1 mg of compound was dried under N2 gas on a polystyrene tissue culture plate prewashed four times with methanol. Following either sham (non-irradiation, left out on bench top open) or UVB irradiation, the lipid was removed with methanol.

Structural Characterization of UVB-irradiated HAPC—An aliquot of the UVB-irradiated HAPC was analyzed by reversed-phase HPLC coupled to a tandem mass spectrometer. The column used was a Columbus 5-µm C18, 150 x 1 mm (Phenomenex, Torrance, CA). The mobile phase consisted of methanol, 20 mM ammonium acetate, acetonitrile (80:15:5, v/v/v) as solvent A and methanol as solvent B. The HPLC flow rate was 0.05 ml/min, initially isocratic at 20% B for 5 min, then increased by a linear gradient to 90% B over 18 min, and then held at 90% B for 10 min. The mass spectrometer used was an API 3000 (Applied Biosystems, Foster City, CA). Negative ion LC/MS/MS spectra were obtained by collisionally activating the [M-H]- ion for azPC at m/z 650.4 using nitrogen as collision gas and collision energy at -36 eV and then scanning the resultant product ions with the last quadrupole sector.

Data Analysis—Data are presented as mean ± standard deviation (S.D.) of at least three independent experiments. Statistical significance is assessed by the Student's t test, and significance is set as p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Functional PPAR{gamma} Protein in Epidermal Cells—The first studies were designed to confirm that epidermal cells contain the functional PPAR{gamma} activity. RNA isolated from the human keratinocyte-derived cell line HaCaT (25) and human epithelial carcinoma cell lines KB and A431 was subjected to reverse transcription-PCR for human PPAR{gamma}. One 474-bp PCR product identified as PPAR{gamma} was found in epidermal cells (Fig. 1A). Similarly, PPAR{gamma} protein was shown to be present in HaCaT, KB, and A431 cell lines (Fig. 1B). Immunoblotting also demonstrated the presence of PPAR{gamma} protein in primary cultures of human keratinocytes (not shown). To determine whether PPAR{gamma} in epidermal cells was functionally active, a PPAR{gamma} luciferase reporter assay was used (14). The specific PPAR{gamma} agonists, azPC and ciglitazone, activated endogenous PPAR{gamma} and induced PPAR{gamma}-dependent luciferase expression; however, the specific PPAR{alpha} agonist, WY14643, had no effect (Fig. 1C). It should be noted that the PPRE reporter used does not result in high levels of promoter activity (only 2–3-fold increases), findings consistent with the use of this gene reporter system in other cell types (14).



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  		FIG. 1.
Human epidermal cell lines express functionally active PPAR{gamma}. a, RNA isolated from the human keratinocyte cell line (HaCaT), human oral epithelial cell carcinoma (KB), or the epidermoid carcinoma (A431) was subjected to reverse transcription-PCR for human PPAR{gamma}. PCR products were separated on 1% agarose gel and visualized with ethidium bromide. One 474-bp PCR product is identified as PPAR{gamma}. b, 40 µg of cellular protein isolated from HaCaT, KB, and A431 cells was separated on a 10% SDS-PAGE, and PPAR{gamma} immunoreactivity was determined using a polyclonal antibody. MW, molecular mass. c, activation of epidermal PPAR{gamma} induces PPAR{gamma}-dependent gene transcription. KB cells co-transfected with PPAR{gamma}-luciferase reporter and {beta}-galactosidase plasmids were treated variously with vehicle control (CON, 0.4% ethanol), specific PPAR{gamma} agonists 1 µM azPC or 20 µM ciglitazone (CIG), or specific PPAR{alpha} agonist 20 µM WY14643 for 24 h. The cells were harvested for luciferase activity assay and normalized to {beta}-galactosidase activity. The values shown are mean ± S.D. and are representative of three separate experiments (*p < 0.05).

 
Augmentation of Induced COX-2 Expression by PPAR{gamma} Agonist—The biological significance of PPAR{gamma} activation was investigated by examining COX-2 expression in KB cells as this enzyme, catalyzing inflammatory prostaglandin formation, has a PPRE consensus site in its promoter (21). The PPAR{gamma} agonist ciglitazone did not induce COX-2 protein expression alone, but augmented COX-2 expression induced by PMA or IL-1{beta} (Fig. 2A). Consistent with the immunoblotting studies, COX-2 enzyme activity of KB cells pretreated with ciglitazone, in the presence of PMA or IL-1{beta}, was modestly increased (Fig. 2B). Since there was an excellent correlation between augmented protein expression identified by Western blot analysis and COX-2 enzyme activity assay, further studies relied upon the immunoblotting technique. These studies indicated that the epithelial PPAR{gamma} system can modulate induced COX-2 expression and activity.



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  		FIG. 2.
PPAR{gamma} agonist ciglitazone (CIG) augments PMA- or IL-1{beta}-induced COX-2 expression in KB cells. KB cells were pretreated with vehicle or 20 µM ciglitazone for 2 h followed by an 8-h incubation with 100 nM PMA or 25 ng/ml IL-1{beta}. a, 40 µg of cellular protein was isolated and separated on 10% SDS-PAGE; COX-2 protein was detected using a polyclonal antibody. Relative Fold Augmentation represents the ratio of arbitrary optical density of COX-2 normalized to that of actin protein. b, COX-2 enzyme activity was assayed. The values of COX-2 activity shown are mean ± S.D. and are representative of three independent experiments (*, p < 0.05, as compared with control (CON); **, p < 0.05, as compared with PMA or IL-1{beta} treatment, respectively).

 
UV Irradiation of KB Cells Generates a PPAR{gamma}-agonistic Activity—The next studies were designed to assess whether the treatment of KB cells with the physiologically relevant prooxidative stressor UVB could generate a PPAR{gamma}-agonistic activity. To that end, KB cells were treated with various doses of UVB, and 1 h after irradiation, these adherent cells were scraped and sonicated to disrupt cellular membranes (Fig. 3A). Control cells were not irradiated but were scraped and sonicated similarly. The cell homogenates from control unirradiated KB cells did not contain a PPAR{gamma}-agonistic activity as shown by the lack of response when these homogenates were added back to KB cells transfected with PPAR{gamma} reporter. However, UVB irradiation of KB cells resulted in the generation of a PPAR{gamma}-agonistic activity in the homogenate in a dose-dependent manner (Fig. 3B). To define whether the PPAR{gamma}-agonistic activity found in homogenates from UVB-irradiated KB cells resided in the lipid fraction, the lipids from the cell homogenate was extracted (24) and tested for PPAR{gamma}-agonistic activity. As shown in Fig. 3C, the PPAR{gamma}-agonistic activity found in homogenates from UVB-irradiated KB cells resided in the lipid fraction. Consistent with the notion that UVB generates this lipid PPAR{gamma}-agonistic activity via oxidative stress, preincubation of KB cells with the antioxidant 6-hydroxy-2,5,7,8-tertamethylchroman-2-carboxylic acid (Trolox®) before UVB irradiation ablated the activity (Fig. 3D). It was demonstrated that Trolox® does not block the activation of PPAR{gamma} in KB cells treated with ciglitazone (data not shown). Altogether, these studies indicated that UVB irradiation generates a lipid-soluble PPAR{gamma} agonist activity via its ability to act as a prooxidative stressor.



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  		FIG. 3.
Cellular homogenates from UVB-irradiated KB cells contain a PPAR{gamma}-agonistic activity. a, experimental procedure. b, KB cells co-transfected with PPAR{gamma} luciferase reporter and {beta}-galactosidase plasmids were treated with cell homogenate derived from unirradiated KB cells or cells irradiated with various doses of UVB or treated with 20 µM ciglitazone (CIG) as a positive control (CON) for PPAR{gamma} activation. c, PPAR{gamma} activity produced by UVB irradiation of KB cells is lipid-soluble. KB cells co-transfected with PPAR{gamma} luciferase reporter and {beta}-galactosidase plasmids were treated with lipid extracts of cell homogenate derived from KB cells exposed to UVB irradiation (UVBRx, 4,000 J/m2) or non-irradiated cells ({Phi}Rx) or 20 µM ciglitazone as a positive control for PPAR{gamma} activation. d, pretreatment with antioxidant 6-hydroxy-2,5,7,8-tertamethylchroman-2-carboxylic acid (Trolox®) blocks UVB irradiation from producing PPAR{gamma} activity in KB cells. KB cells co-transfected with PPAR{gamma} luciferase reporter and {beta}-galactosidase plasmids were treated with cell homogenate derived from KB cells exposed to UVB irradiation (4,000 J/m2) with or without the pretreatment of 10 mM antioxidant Trolox® or treated with 20 µM ciglitazone as a positive control for PPAR{gamma} activation. Cells were harvested 24 h after treatment for luciferase and {beta}-galactosidase activity assay. The values shown are mean ± S.D. and are representative of three separate experiments (*, p < 0.05).

 
Although a minor constituent of cellular lipids, HAPC is a potential precursor for bioactive lipids produced via direct free radical oxidation of the sn-2 arachidonate (9–11). Among the oxidized GPCs described are the platelet-activating factor receptor agonists, 1-hexadecyl-2-butanoyl-GPC and 1-hexadecyl-2-butenoyl-GPC (7), and the PPAR{gamma} agonist azPC (14). To confirm that a considerable element of UVB-induced PPAR{gamma}-agonistic activity was non-enzymatic in origin, purified HAPC was subjected directly to UVB irradiation. As shown in Fig. 4, the lipid extracts from 2,000 J/m2 UVB-irradiated HAPC but not unirradiated HAPC contained a PPAR{gamma} agonist activity.



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  		FIG. 4.
UVB irradiation (UVBRx) of purified HAPC generates a PPAR{gamma}-agonistic activity. KB cells co-transfected with PPAR{gamma} luciferase reporter and {beta}-galactosidase plasmids were treated with vehicle control (CON), 10 µM UVB (2,000 J/m2)-irradiated or unirradiated purified HAPC, cell homogenate derived from KB cells either unirradiated or exposed to UVB (4,000 J/m2), or 20 µM ciglitazone (CIG) as a positive control for PPAR{gamma} activation. Cells were harvested 24 h after treatment for luciferase and {beta}-galactosidase activity assay. The values shown are mean ± S.D. of duplicate samples and are representative of three separate experiments (*, p < 0.05).

 
UVB-generated PPAR{gamma}-agonistic Activity Augments PMA-induced COX-2 Expression in KB Cells—The next studies examined whether UVB irradiation of KB cells or HAPC could exert PPAR{gamma}-agonistic activity as demonstrated by its ability to augment COX-2 induction. UVB-irradiated HAPC or homogenates from UVB-irradiated KB cells, as well as PPAR{gamma}-specific agonists, azPC and ciglitazone, all augmented PMA-induced COX-2 protein expression in KB cells (Fig. 5). However, none of the above stimuli affected baseline COX-2 levels (not shown).



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  		FIG. 5.
Cellular homogenates from UVB-irradiated KB cells or UVB-irradiated HAPC augment PMA-induced COX-2 expression in KB cells. KB cells were treated variously with 100 nM PMA with or without a 1-h pretreatment of specific PPAR{gamma} agonists 1 µM azPC or 20 µM ciglitazone (CIG), UVB (2,000 J/m2)-irradiated or unirradiated HAPC, or cell homogenate derived from UVB-irradiated (4,000 J/m2) or unirradiated KB cells. After incubation for 8 h, ~40 µg of cellular protein was isolated and separated on 10% SDS-PAGE; COX-2 immunoreactivity was determined using a polyclonal antibody. Relative Fold Augmentation represents the ratio of arbitrary optical density of COX-2 and normalized to that of actin protein. CON, control.

 
To confirm that the augmentation of PMA-induced COX-2 expression in response to UVB-irradiated KB cells or HAPC was due to the production of a PPAR{gamma}-agonistic activity, two separate strategies were undertaken. First, the PPAR{gamma} dominant-negative cell line was created by the stable transfection of KB cells with a PPAR{gamma} dominant-negative mutant construct (22, 26), and a control cell line was transfected with the pcDNA3 plasmid. The stable KB/{Delta}PPAR{gamma} dominant-negative cells were shown to lack PPAR{gamma} activity by PPAR{gamma}-dependent luciferase reporter assay, which demonstrated the lack of luciferase reporter expression in response to ciglitazone, azPC, or PPAR{gamma}-agonistic activity existing in UVB-irradiated purified HAPC or cell homogenates from UVB-irradiated KB cells (data not shown). Neither PPAR{gamma}-specific agonists nor cellular homogenates from UVB-irradiated KB cells or UVB-irradiated HAPC were able to augment PMA-induced COX-2 expression in KB/{Delta}PPAR{gamma} cells expressing the mutant PPAR{gamma} dominant-negative construct (Fig. 6A). The second strategy used the specific PPAR{gamma} inhibitor GW9662 (27). Consistent with the studies using the PPAR{gamma} dominant-negative mutant, pretreatment with the PPAR{gamma} antagonist GW9662 blocked the ability of ciglitazone, as well as cellular homogenates from UVB-irradiated cells or UVB-irradiated HAPC, to augment PMA-induced COX-2 expression in KB cells (Fig. 6B). Altogether, these studies confirm that UVB can generate a PPAR{gamma}-agonistic activity that can modulate COX-2 expression.



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  		FIG. 6.
Inhibition of PPAR{gamma} abolishes augmentation of COX-2 expression in KB cells by cellular homogenates from UVB-irradiated KB cells or UVB-irradiated HAPC. a, KB cells transfected with mutant PPAR{gamma} dominant-negative construct (KB/{Delta}PPAR{gamma}) were treated with the specific PPAR{gamma} agonists 1 µM azPC or 20 µM ciglitazone (CIG), UVB (2,000 J/m2)-irradiated or unirradiated HAPC, or cell homogenate derived from UVB (4,000 J/m2)-irradiated or unirradiated KB cells for 1 h before exposure to 100 nM PMA for 8 h. CON, control. b, specific PPAR{gamma} antagonist GW9662 (GW) blocks PPAR{gamma} agonists or UVB-induced augmentation of COX-2 expression. KB cells were pretreated with vehicle or 1 µM GW9662 for 1 h and then treated with the following PPAR{gamma} activation stimuli: 20 µM ciglitazone, UVB (2,000 J/m2)-irradiated HAPC, or cell homogenate derived from UVB (4,000 J/m2)-irradiated cells; after 1 h, cells were then exposed to 100 nM PMA for 8 h. Approximately 40 µg of cellular protein was isolated and separated on 10% SDS-PAGE, and COX-2 immunoreactivity was determined using a polyclonal antibody. Relative Fold Augmentation represents the ratio of arbitrary optical density of COX-2 and normalized to that of actin protein.

 
UVB-generated PPAR{gamma} Activity Critical for UVB-induced PGE2 Production in KB Cells—The generation of PGE2 is among the profound effects that UVB exerts on human keratinocytes. Previous studies suggested that UVB-induced epidermal PGE2 formation is due to COX-2 (28). Since UVB can generate a PPAR{gamma}-agonistic activity that can modulate COX-2 expression, the next studies examined the role of this PPAR{gamma} activity in UVB-induced epidermal PGE2 production. These studies used the dual strategies of molecular and pharmacological inhibition of PPAR{gamma} with the KB/{Delta}PPAR{gamma} dominant-negative cell line and GW9662, respectively. As shown in Fig. 7A, PGE2 was produced in KB cells after exposure to 600 J/m2 UVB irradiation, which was reduced by the pretreatment of either the selective COX-2 inhibitor NS-398 (29) or specific PPAR{gamma} antagonist GW9662; however, neither NS-398 nor GW9662 affected PGE2 production induced by calcium ionophore A23187 [GenBank] . Examination of the absorbance spectra of NS-398 and GW9662 revealed that these compounds did not absorb UV irradiation from 270–320 nm (data not shown), indicating that these agents did not affect UVB-mediated effects via acting as a chemical sunscreen. Similarly, UVB irradiation-induced PGE2 production was decreased in KB/{Delta}PPAR{gamma} dominant-negative cells in comparison with KB/pcDNA3 control cells (Fig. 7B). In contrast, PGE2 production induced by calcium ionophore A23187 [GenBank] was not affected by the presence of a mutant PPAR{gamma} system (Fig. 7B). These studies provide evidence that the epidermal PPAR{gamma} system is critical for optimal UVB-mediated PGE2 formation.



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  		FIG. 7.
Inhibition of PPAR{gamma} or COX-2 blocked UVB-induced PGE2 production in KB cells. a, KB cells were pretreated with vehicle control, 10 µM NS-398 for 30 min, or 1 µM GW9662 for 1 h and then either directly irradiated with 600 J/m2 UVB or treated with 500 nM calcium ionophore A23187 [GenBank] (ION). CON, control; CIG, ciglitazone. b, KB cells transfected with mutant PPAR{gamma} dominant-negative construct (KB/{Delta}PPAR{gamma}) or control cells transfected with vector vehicle (KB/pcDNA3) were treated with vehicle control or 500 nM calcium ionophore A23187 [GenBank] or irradiated with 600 J/m2 UVB. Cell supernatants were removed 8 h later, and PGE2 production was measured by enzyme-linked immunosorbent assay kit. The values shown are mean ± S.D. of duplicate samples and are representative of three separate experiments.

 
azPC Identified in UVB-irradiated Purified HAPC—The characterization of the lipid having PPAR{gamma}-agonistic activity generated by UVB irradiation was carried out using tandem mass spectrometry. Inasmuch as UVB irradiation of both KB cells and purified HAPC resulted in the production of this activity, structural studies were carried out from product isolated from the latter cell-free model system. HPLC fractions derived from UVB-irradiated HAPC were collected and tested for PPAR{gamma}-agonistic activity using a PPAR{gamma} luciferase reporter assay. HPLC fractions (1 min) from times 8–21 min were assayed, revealing positive PPAR{gamma}-agonistic activity (greater than 150% control) in fractions 8, 10, 13, and 16–21 (Fig. 8). Commercially available azPC eluted at 16 min (Fig. 9) and thus would be expected to reside in fraction 16. The presence of this oxidized GPC with known PPAR{gamma}-agonistic activity in UVB-irradiated HAPC was determined by the analysis of tandem mass spectrometric data. Collision-induced dissociation of synthetic azPC and fraction 16 of UVB-irradiated HAPC yielded nearly identical product ions in terms of mass-to-charge ratio and abundance. Importantly, an abundant ion at m/z 201 was present in both, corresponding to the carboxylate anion of azelaic acid (Fig. 10). It should be noted that unirradiated HAPC did not exert biological activity, nor did it contain this component. These studies indicated that azPC was one component of UVB-generated PPAR{gamma}-agonistic activity.



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  		FIG. 8.
Identification of PPAR{gamma}-agonistic activity in UVB-irradiated HAPC. KB cells co-transfected with PPRE-luciferase reporter and {beta}-galactosidase plasmids were treated with vehicle control (CON), 20 µM ciglitazone (CIG) as a positive control for PPAR{gamma} activation, or 1-min HPLC fractions from UVB (2,000 J/m2)-irradiated purified HAPC. Cells were harvested 24 h after treatment for luciferase and {beta}-galactosidase activity assay.

 



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  		FIG. 9.
Identification of azPC in UVB-irradiated HAPC. Commercially available azPC (A) or UVB-irradiated HAPC (B) was subjected to negative ion LC/MS/MS operated in the multiple reaction monitoring (MRM) mode to identify peaks with this specific transition.

 



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  		FIG. 10.
Collision induced decompisition of azPC. The [M-H]- ions from synthetic azPC (A) and fraction 16 from UVB-irradiated HAPC (B) were collisionally activated using a tandem quadrupole mass spectrometer with the resultant product ion mass spectrum. The mechanism of formation of the carboxylate anion of azelaic acid is indicated (inset). CID, collision-induced dissociation.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
These studies provide the first evidence that the epidermal PPAR{gamma} system is a biological target for UVB radiation. Our findings demonstrated the existence of a functionally active PPAR{gamma} system in epidermal cells. In PPRE-luciferase reporter assays, the specific PPAR{gamma} agonists, ciglitazone and azPC, but not the specific PPAR{alpha} agonist WY14643, activated endogenous PPAR{gamma} activity. The epithelial PPAR{gamma} activation was shown to be biologically significant as it augmented COX-2 expression in response to phorbol ester or IL-1{beta}. These findings fit with the report that a region of the COX-2 promoter contains a PPRE responsible for the enhancement of COX-2 expression (30). Thus, epidermal cells contain a functional PPAR{gamma} system potentially linked to COX-2 expression. However, activation of PPAR{gamma} alone by ciglitazone or azPC treatment did not induce COX-2 expression in KB cells, unlike that in monocytes in which COX-2 expression was induced by rosiglitazone (21).

We found that UVB irradiation of KB cells produced a PPAR{gamma}-agonistic activity, quantitatively similar to the specific PPAR{gamma} agonist ciglitazone. This PPAR{gamma}-agonistic activity exists in the lipid extracts from UVB-irradiated cell homogenates and is ablated by the pretreatment of cells with the antioxidant Trolox®. These findings are compatible with the notion that UVB irradiation, acting as an oxidative stressor, produced a PPAR{gamma} activity in KB cells via oxidation of cellular GPCs. It was also demonstrated that subjecting purified HAPC, a phospholipid found in epidermal cells (18), to UVB irradiation generated a PPAR{gamma}-agonistic activity.

The biological effect of UVB-induced PPAR{gamma}-agonistic activity in KB cells was evaluated by examining its effects on COX-2 expression and PGE2 production. We provide evidence that the PPAR{gamma}-agonistic activity produced in cell homogenates from UVB-irradiated cells or UVB-irradiated HAPC was functionally as active as the specific PPAR{gamma} agonist, ciglitazone, in augmenting PMA-induced COX-2 expression. The use of PPAR{gamma} dominant-negative cells, in which mutant and non-functional PPAR{gamma} is overwhelmingly expressed, as well as the specific PPAR{gamma} antagonist GW9662 confirmed the role of PPAR{gamma} in this process.

UVB is known to induce epidermal COX-2 in vitro as well as in vivo (28). The significance of UVB-induced COX-2 is unclear but has been linked to UVB-mediated systemic immunosuppression (32). It should be noted that a clinically relevant UVB exposure (600 J/m2) produced PGE2 in KB cells, and this was blocked by the selective COX-2 inhibitor NS-398, which is consistent with the notion that COX-2 catalyzes UVB-mediated production of prostaglandins. PGE2 production in response to a physiologic (600 J/m2) dose of UVB was highly dependent on cellular PPAR{gamma} functionality, as shown by our findings that PGE2 production was dramatically decreased by the pretreatment of specific PPAR{gamma} antagonist or in PPAR{gamma} dominant-negative cells. This blockade on UVB-induced PGE2 was shown to be PPAR{gamma}-specific, in light of the evidence that GW9662 had little effect on calcium ionophore A23187 [GenBank] , which does not exert a pro-oxidative stress. According to these studies, it is logical to extrapolate that UVB irradiation, acting as a pro-oxidative stressor, oxidized GPC-containing lipids (HAPC might be the major substrate in epidermal cells) by a free radical-mediated non-enzymatic process, resulting in biologically active lipid PPAR{gamma} agonists.

These studies have begun the process of defining the exact oxidized GPCs that comprise UVB-produced PPAR{gamma}-agonistic activity. Fractionation of UVB-irradiated HAPC by HPLC revealed that several separate fractions exhibited PPAR{gamma}-agonistic activity. One fraction (16) co-eluted with commercially available azPC. As shown in Fig. 9, a compound with the identical mass spectrum as azPC was identified in UVB-irradiated purified HAPC. These findings indicated that the known PPAR{gamma} agonist azPC is one of many as yet uncharacterized oxidatively modified short-chained sn-2 GPCs that have PPAR{gamma}-agonistic activity. It should be noted that although azPC is one component of UVB-generated PPAR{gamma} activity, it is probably a minor species and that potentially many other oxidized GPC species with PPAR{gamma} activity are produced by UVB. Ongoing studies are characterizing these other oxidized GPCs.

The biological effects of UVB on skin are of particular interest because of the importance of UV-induced injury in the development of photoaging, skin cancer, and inflammation and the role of UV irradiation in the therapy of skin disease (reviewed in Refs. 31, 33, and 34). However, the exact mechanisms and targets for UVB are unclear at this time. Determining the cellular and molecular mechanisms and finding novel factors that modulate inflammatory responses to acute UVB absorption in epidermal cells are important for understanding the regulation of these processes. Although many biologically relevant UVB-responsive genes including COX-2 are regulated by PPAR{gamma}, the exact significance of UVB-generated PPAR{gamma}-agonistic activity is still unclear. A better understanding of the role of oxidized GPCs in UVB responses could provide the impetus for novel pharmacological strategies involving PPAR{gamma}.


   FOOTNOTES
 
* This research was supported by grants from the Showalter Memorial Foundation, the Riley Memorial Association, and the National Institutes of Health (Grants AR01993, and HL62996). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§§ To whom correspondence should be addressed: Herman B. Wells Center for Pediatric Research, James Whitcomb Riley Hospital for Children, Rm. 2659, Indiana University School of Medicine, 702 Barnhill Dr., Indianapolis, IN 46202. Tel.: 317-274-7705; Fax: 317-274-5378; E-mail jtravers{at}iupui.edu.

1 The abbreviations used are: UVB, ultraviolet B radiation; PPAR{gamma}, peroxisome proliferator-activated receptor {gamma}; PPREs, peroxisome proliferator-response elements; azPC, 1-hexadecyl-2-azelaoyl glycerophosphocholine; COX-2, cyclooxygenase-2; PMA, phorbol 12-myristate-13-acetate; IL-1{beta}, interleukin-1{beta}; HAPC, 1-hexadecyl-2-arachidonoyl-glycerophosphocholine; GPC, glycerophosphocholine; PAF, platelet-activating factor; PGE2, prostaglandin E2; HPLC, high pressure liquid chromatography; LC, liquid chromatography; MS, mass spectrometry. Back


   ACKNOWLEDGMENTS
 
The technical assistance of Qiaofang Yi and Dr. Mohammed Al-Hassani is gratefully acknowledged. We also thank Drs. Loren J. Field, Anthony B. Firulli, Mark R. Kelley, and Dan F. Spandau for discussion of this manuscript.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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