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• W2022279037 abstract "Heme oxygenase-1 (HO-1) is a key enzyme in the cellular response to tissue injury and oxidative stress. HO-1 enzymatic activity results in the formation of the cytoprotective metabolites CO and biliverdin. In the skin, HO-1 is strongly induced after long wave ultraviolet radiation (UVA-1). Here we show that UVA-1 irradiation generates oxidized phospholipids derived from 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) that mediate the expression of HO-1 in skin cells. Using EO6 antibodies that recognize oxidized phospholipids, we show that UVA-1 irradiation of dermal fibroblasts generates oxidation-specific epitopes. Irradiation of arachidonate-containing phospholipids with UVA-1 led to formation of defined lipid oxidation products including epoxyisoprostane-phosphatidylcholine that induced HO-1 expression in dermal fibroblasts, in keratinocytes, and in a three-dimensional epidermal equivalent model. In addition, we demonstrate that the oxidation of PAPC by UVA-1 is a singlet oxygen-dependent mechanism. Together, we present a novel mechanism of UVA-1-induced HO-1 expression that is mediated by the generation of biologically active phospholipid oxidation products. Because UVA-1 irradiation is a mainstay treatment of several inflammatory skin diseases, structural identification of UVA-1-generated biomolecules with HO-1-inducing capacity should lead to the development of drugs that could substitute for irradiation. Heme oxygenase-1 (HO-1) is a key enzyme in the cellular response to tissue injury and oxidative stress. HO-1 enzymatic activity results in the formation of the cytoprotective metabolites CO and biliverdin. In the skin, HO-1 is strongly induced after long wave ultraviolet radiation (UVA-1). Here we show that UVA-1 irradiation generates oxidized phospholipids derived from 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) that mediate the expression of HO-1 in skin cells. Using EO6 antibodies that recognize oxidized phospholipids, we show that UVA-1 irradiation of dermal fibroblasts generates oxidation-specific epitopes. Irradiation of arachidonate-containing phospholipids with UVA-1 led to formation of defined lipid oxidation products including epoxyisoprostane-phosphatidylcholine that induced HO-1 expression in dermal fibroblasts, in keratinocytes, and in a three-dimensional epidermal equivalent model. In addition, we demonstrate that the oxidation of PAPC by UVA-1 is a singlet oxygen-dependent mechanism. Together, we present a novel mechanism of UVA-1-induced HO-1 expression that is mediated by the generation of biologically active phospholipid oxidation products. Because UVA-1 irradiation is a mainstay treatment of several inflammatory skin diseases, structural identification of UVA-1-generated biomolecules with HO-1-inducing capacity should lead to the development of drugs that could substitute for irradiation. Cells of the skin express HO-1 2The abbreviations used are: HO-1, heme oxygenase-1; ESI, electrospray ionization; MS, mass spectrometry; PAPC, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine; OxPAPC, PAPC oxidized with atmospheric oxygen; UV-PAPC, PAPC oxidized with UVA-1; POPC, 1-palmitoyl-2-oleoyl-sn-3-glycerophosphorylcholine; PLPC, 1-palmitoyl-2-linoleyl-sn-3-glycerophosphorylcholine; KC, keratinocyte; FB, fibroblast; qPCR, quantitative real time PCR; HPLC, high pressure liquid chromatography; POVPC, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine; lyso-PC, 1-palmitoyl-2-lyso-sn-glycero-3-phosphorylcholine; ROS, reactive oxygen species; PEIPC, 1-palmitoyl-2-(epoxy-isoprostane-E2)-sn-glycero-3-phosphorylcholine; RB, Rose Bengal. 2The abbreviations used are: HO-1, heme oxygenase-1; ESI, electrospray ionization; MS, mass spectrometry; PAPC, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine; OxPAPC, PAPC oxidized with atmospheric oxygen; UV-PAPC, PAPC oxidized with UVA-1; POPC, 1-palmitoyl-2-oleoyl-sn-3-glycerophosphorylcholine; PLPC, 1-palmitoyl-2-linoleyl-sn-3-glycerophosphorylcholine; KC, keratinocyte; FB, fibroblast; qPCR, quantitative real time PCR; HPLC, high pressure liquid chromatography; POVPC, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine; lyso-PC, 1-palmitoyl-2-lyso-sn-glycero-3-phosphorylcholine; ROS, reactive oxygen species; PEIPC, 1-palmitoyl-2-(epoxy-isoprostane-E2)-sn-glycero-3-phosphorylcholine; RB, Rose Bengal. in response to the oxidative stress imposed by long wavelength UV radiation (UVA) (1Keyse S.M. Tyrrell R.M. J. Biol. Chem. 1987; 262: 14821-14825Abstract Full Text PDF PubMed Google Scholar, 2Keyse S.M. Tyrrell R.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 99-103Crossref PubMed Scopus (1100) Google Scholar, 3Tyrrell R.M. Antioxid. Redox. Signal. 2004; 6: 835-840Crossref PubMed Scopus (72) Google Scholar). Evidence suggests that the generation of singlet oxygen (1O2) by UVA-1 (340–390 nm) and subsequent oxidation of intracellular membrane lipids (4Basu-Modak S. Tyrrell R.M. Cancer Res. 1993; 53: 4505-4510PubMed Google Scholar, 5Basu-Modak S. Luscher P. Tyrrell R.M. Free Radic. Biol. Med. 1996; 20: 887-897Crossref PubMed Scopus (62) Google Scholar) are involved in this process. However, neither the mechanisms nor the structures of responsible lipid oxidation products have been described so far. HO-1 plays a general role in cutaneous wound repair, and the resolution of inflammation is strongly up-regulated after skin injury and declines to basal levels after completion of wound healing (6Hanselmann C. Mauch C. Werner S. Biochem. J. 2001; 353: 459-466Crossref PubMed Scopus (117) Google Scholar). HO-1 converts free heme and heme moieties of proteins to carbon monoxide, iron, and biliverdin, which is rapidly converted to bilirubin by biliverdin reductase. CO and bilirubin have well described antioxidant and anti-inflammatory properties (7Otterbein L.E. Soares M.P. Yamashita K. Bach F.H. Trends Immunol. 2003; 24: 449-455Abstract Full Text Full Text PDF PubMed Scopus (1000) Google Scholar, 8Ryter S.W. Otterbein L.E. Morse D. Choi A.M. Mol. Cell Biochem. 2002; 234–235: 249-263Crossref PubMed Scopus (440) Google Scholar), suggesting that induction of HO-1 is a general mechanism that protects the cell against oxidative damage. Accordingly, the adaptive response of skin fibroblasts after repeated UVA irradiation, which protects them against further membrane damage, is mediated by induction of HO-1 (9Vile G.F. Basu-Modak S. Waltner C. Tyrrell R.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2607-2610Crossref PubMed Scopus (461) Google Scholar). The phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) is a major component of cell membranes and lipoproteins. Its oxidation products are found in cells during inflammation (10Subbanagounder G. Wong J.W. Lee H. Faull K.F. Miller E. Witztum J.L. Berliner J.A. J. Biol. Chem. 2002; 277: 7271-7281Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar), in membranes of apoptotic cells (11Huber J. Vales A. Mitulovic G. Blumer M. Schmid R. Witztum J.L. Binder B.R. Leitinger N. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 101-107Crossref PubMed Scopus (240) Google Scholar), as well as in oxidized low density lipoprotein (12Watson A.D. Leitinger N. Navab M. Faull K.F. Horkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar). Polyunsaturated fatty acyl residues at the sn-2 position of the glycerol backbone, such as the arachidonoyl moiety in PAPC, are especially prone to oxidative modification. Thus, the oxidation of PAPC (OxPAPC) leads to the addition of oxygen atoms as well as to fragmentation of the arachidonate moiety. The type of oxidative modification determines the biological activity of these oxidized phospholipids (13Furnkranz A. Leitinger N. Curr. Pharm. Des. 2004; 10: 915-921Crossref PubMed Scopus (32) Google Scholar). OxPAPC was shown to induce HO-1 expression in several cell types of the vasculature and the immune system (14Ishikawa K. Navab M. Leitinger N. Fogelman A.M. Lusis A.J. J. Clin. Investig. 1997; 100: 1209-1216Crossref PubMed Scopus (254) Google Scholar). Here we report that photooxidation of PAPC or irradiation of skin cells results in formation of biologically active lipid oxidation products that induce HO-1 expression. Using mass spectrometry and EO6 antibodies that detect oxidation products of PAPC, we identify epoxyisoprostane-phosphatidylcholine, a known inducer of HO-1, among these oxidation products, and demonstrate their intracellular formation. Because UVA-1 phototherapy is successfully used for treatment of inflammatory skin diseases (15Mang R. Krutmann J. Photodermatol. Photoimmunol. Photomed. 2005; 21: 103-108Crossref PubMed Scopus (31) Google Scholar), identification of UV-generated biomolecules with protective effects will lead to the development of novel drugs that could be used to substitute for UVA-1 phototherapy. Cell Culture—Neonatal human epidermal keratinocytes (KCs) derived from foreskin were obtained from Clonetics (San Diego, CA). Keratinocytes were cultured in keratinocyte growth medium up to the fifth passage. Human neonatal skin fibroblasts (FB) were obtained from Cascade Biologics (Portland, OR) and grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum and penicillin/streptomycin (1000 units/ml; Invitrogen) to subconfluence. Skin Equivalents—In vitro reconstructed skin equivalents were generated as described previously (16Rendl M. Ban J. Mrass P. Mayer C. Lengauer B. Eckhart L. Declerq W. Tschachler E. J. Investig. Dermatol. 2002; 119: 1150-1155Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Briefly, 1.3 × 106 KCs were added on top of a collagen gel containing fibroblasts. After overnight incubation, the medium from the upper chamber was removed, thus putting the KC at air-liquid interface. Afterward, skin equivalents were cultured in serum-free KC-defined medium, which is KC growth medium without bovine pituitary extract, supplemented with 1.3 mm calcium, 10 μg/ml transferrin, 50 μg/ml ascorbic acid, and 0.1% bovine serum albumin. Quantitative Real Time PCR (qPCR)—RNA was isolated using TRIzol reagent (Invitrogen). 900 ng of total RNA were reverse-transcribed with murine leukemia virus reverse transcriptase (Applied Biosystems, Foster City, CA) and oligo(dT) primers. The following forward (F) and reverse (R) primers were used: HO-1: F, 5′-AAGATTGCCCAGAAAGCCCTGGAC-3′; R, 5′-AACTGTCGCCACCAGAAAGCTGAG-3′; β2-microglobulin; F, 5′-GATGAGTATGCCTGCCGTGTG-3′; and R, 5′-CAATCCAAATGCGGCATCT-3′. qPCR was performed using LightCycler technology and the Fast Start SYBR Green I kit (Roche Applied Science). In all assays, cDNA was amplified using a standardized program (10-min denaturing step; 55 cycles of 5 s at 95°C, 15 s at 65°C, and 15 s at 72 °C; melting point analysis in 0.1 °C steps). Quantification of target gene expression was performed using a mathematical model by Pfaffl (17Pfaffl M.W. Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (24586) Google Scholar). The expression of the target molecule was normalized to the expression of β2-microglobulin. Western Blot Analysis—Bound HO-1 antibodies (SPA-896; Stressgen, Victoria, Canada) were detected by anti-IgG conjugated with peroxidase and subsequent chemiluminescent detection. UV Irradiation—UV irradiation was carried out as described previously (18Mildner M. Weninger W. Trautinger F. Ban J. Tschachler E. Photochem. Photobiol. 1999; 70: 674-679Crossref PubMed Scopus (55) Google Scholar). As a light source for UVA-1, a Mutzhas Supersun 5000-type solar simulator (Mutzhas, Munich, Germany) filtered for the emission of UVA-1 (340–390 nm) was used. UVB (280–320 nm) irradiation was performed with a Waldmann F15 T8 tube (Waldmann, Villingen, Germany). The cells were irradiated with 10 to 40 J/cm2 of UVA-1 or with 20 mJ/cm2 of UVB under a thin layer of phosphate-buffered saline at 25 °C. The maximal amount of UVA-1 radiation used in this study on cells (60 J/cm2) would reach the surface of the skin during 168 min of sunlight exposure at noon at a northern latitude of 35°; 30% would reach the dermis (19Vile G.F. Tyrrell R.M. Free Radic. Biol. Med. 1995; 18: 721-730Crossref PubMed Scopus (250) Google Scholar). This is within the fluency range used for UVA-1 phototherapy (15Mang R. Krutmann J. Photodermatol. Photoimmunol. Photomed. 2005; 21: 103-108Crossref PubMed Scopus (31) Google Scholar). Immunofluorescence/Immunohistochemistry—Dermal fibroblasts were grown on 8-well Permanox slides (Nalgene, Rochester, NY). The cells were fixed with 80% methanol (5 min, 4 °C) and labeled with EO6 (kindly provided by J. L. Witztum, San Diego, CA) and control IgM antibody as described previously (20Chang M.K. Bergmark C. Laurila A. Horkko S. Han K.H. Friedman P. Dennis E.A. Witztum J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6353-6358Crossref PubMed Scopus (389) Google Scholar). The cells were counterstained using an antibody against active Caspase 3 (rabbit IgG, 0.5 mg/ml, R & D Systems, Minneapolis, MN) and control IgG. Formalin-fixed paraffin sections of skin equivalents were stained for HO-1 (SPA-896; Stressgen). Lipid Oxidation—PAPC was oxidized by exposure of the dry lipid to air for 72 h to generate OxPAPC. PAPC dried to a thin film on a glass support was irradiated with UVA-1 with 80 J/cm2 to generate UV-PAPC. For treatment with singlet oxygen, all lipids were vortexed in phosphate-buffered saline containing 90 μm (final concentration in the culture medium was 9 μm) Rose Bengal (Sigma-Aldrich) and irradiated with 10 J/cm2 UVA-1 (see Fig. 5A) or under a commercial 35 W halogen lamp at a distance of 30 cm for 30 min (see Fig. 5, B and C). The extent of oxidation was monitored by ESI-MS as described previously (12Watson A.D. Leitinger N. Navab M. Faull K.F. Horkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar). Lipid Extraction—Total lipids were extracted from dermal fibroblasts using chloroform/methanol (2:1, v/v) in the presence of 0.01% butylated hydroxytoluene and 0.17 m formic acid as described (12Watson A.D. Leitinger N. Navab M. Faull K.F. Horkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar). Thin Layer Chromatography—TLC analysis of lipids was performed on Silica gel 60 TLC plates (Merck) using a mixture of chloroform-methanol-water (100:50:10, v/v/v) as a developing solvent. Lipid spots were visualized after treatment with 10% copper sulfate in an 8.5% aqueous solution of orthophosphoric acid and subsequent heating at 180 °C. Mass Spectrometry—Mass spectrometry was performed on a PE Sciex API 365 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA) equipped with an electrospray source. Flow injection experiments were performed by injecting 20-μl aliquots of lipid samples dissolved in 200 μl of methanol-water-formic acid (80:20:0.1, v/v/v) into a stream of the same solvent mixture, delivered by an HPLC system (HP1100; Agilent Technologies, Waldbronn, Germany). Spectra were acquired in the positive mode in the range of 400–900 Da. Liquid chromatography-MS and liquid chromatography-MS/MS was performed using an Agilent Zorbax Eclipse XDB-C8 column (150 × 4.6 mm, 5 μm). Samples were dissolved in mobile phase A (10 mm ammonium acetate in methanol-water, 80:20, v/v). Analytes were eluted using a gradient from 25% mobile phase B (10 mm ammonium acetate in methanol) to 100% B in 30 min, followed by an isocratic step at 100% B for 30 min. MS and tandem MS detection was performed in the scan mode (400–900 Da) or the multiple reaction monitoring mode detecting the phosphatidylcholine-specific fragment at 184.1 Da produced from various precursor ions (m/z 594, 610, 782, 814, 828, 846, 878 [MH]+) at a collision energy of 35 eV. Quantification of the peak areas for the multiple reaction monitoring transition m/z 828 > 184 in the elution time range of 7–13 min was performed using Analyst software (version 1.4, Applied Biosystems). OxPAPC Induces HO-1 Expression in KCs and Dermal FB—We and others have recently shown that HO-1 expression in vascular endothelial and smooth muscle cells is strongly up-regulated by OxPAPC, which had been generated by air exposure (14Ishikawa K. Navab M. Leitinger N. Fogelman A.M. Lusis A.J. J. Clin. Investig. 1997; 100: 1209-1216Crossref PubMed Scopus (254) Google Scholar, 22Kadl A. Huber J. Gruber F. Bochkov V.N. Binder B.R. Leitinger N. Vascul. Pharmacol. 2002; 38: 219-227Crossref PubMed Scopus (90) Google Scholar, 23Kronke G. Bochkov V.N. Huber J. Gruber F. Bluml S. Furnkranz A. Kadl A. Binder B.R. Leitinger N. J. Biol. Chem. 2003; 278: 51006-51014Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Here we examined whether OxPAPC had similar effects on skin cells. As it has been reported previously (5Basu-Modak S. Luscher P. Tyrrell R.M. Free Radic. Biol. Med. 1996; 20: 887-897Crossref PubMed Scopus (62) Google Scholar), treatment of FB with UVA-1 (40 J/cm2), but not UVB (20 mJ/cm2), leads to a strong induction of HO-1 mRNA expression. This effect was mimicked by the addition of OxPAPC (100 μg/ml) (Fig. 1A). When OxPAPC was added at different concentrations to cultures of primary human epidermal KC and FB, HO-1 mRNA and protein expression were strongly induced in both cell types in a dose-dependent manner (Fig. 1, B and C). Strong induction of HO-1 mRNA and protein was also observed when epidermal equivalent cultures, which mimic more closely the in vivo situation, (16Rendl M. Ban J. Mrass P. Mayer C. Lengauer B. Eckhart L. Declerq W. Tschachler E. J. Investig. Dermatol. 2002; 119: 1150-1155Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) were exposed to OxPAPC for 4 consecutive days (Fig. 1D). HO-1 protein expression was most pronounced in the epidermal layers immediately below the stratum corneum. These data demonstrate that like UVA-1, OxPAPC is able to strongly stimulate HO-1 expression in skin cells. HO-1 Inducing Activity Is Contained in “Long Chain” but Not in “Short Chain” Oxidized Phospholipids—OxPAPC contains several oxidation products of PAPC that can be grouped into long chain oxidation products, which result from insertion of oxygen into the arachidonic acid moiety, and short chain oxidation products, which result from oxidative fragmentation (21Birukov K.G. Bochkov V.N. Birukova A.A. Kawkitinarong K. Rios A. Leitner A. Verin A.D. Bokoch G.M. Leitinger N. Garcia J.G. Circ. Res. 2004; 95: 892-901Crossref PubMed Scopus (135) Google Scholar). Distinct biological properties have been attributed to compounds of either group (24Leitinger N. Tyner T.R. Oslund L. Rizza C. Subbanagounder G. Lee H. Shih P.T. Mackman N. Tigyi G. Territo M.C. Berliner J.A. Vora D.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12010-12015Crossref PubMed Scopus (226) Google Scholar, 25Subbanagounder G. Watson A.D. Berliner J.A. Free Radic. Biol. Med. 2000; 28: 1751-1761Crossref PubMed Scopus (90) Google Scholar). To identify HO-1-inducing compounds within OxPAPC, we tested long chain, short chain, and a polar lipid fraction that had been separated by thin layer chromatography. As shown in Fig. 2A, HO-1 was strongly induced in fibroblasts by the long chain fraction, whereas the other fractions had virtually no effect. This was confirmed when the synthetic short chain compounds 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine, as well as 1-palmitoyl-2-lyso-sn-glycero-3-phosphorylcholine (lyso-PC) failed to induce HO-1 expression, as did unoxidized PAPC and di-myristoyl-phosphorylcholine (Fig. 2B). These data demonstrate that the capacity to induce HO-1 resides within the long chain oxidation products of PAPC. To examine a possible contribution of lipid hydroperoxides, which are present in the OxPAPC preparation, to the induction of HO-1 expression, we incubated fibroblasts with the ROS scavengers β-carotene, vitamin C, and vitamin E prior to exposure to OxPAPC. This treatment did not significantly reduce OxPAPC-mediated HO-1 induction (supplemental Fig. S1), suggesting that secondary peroxidation of cellular lipids caused by OxPAPC was not involved in the induction of HO-1 expression. UVA-1 Irradiation of PAPC (UV-PAPC) Leads to Formation of Long Chain Oxidation Products That Induce HO-1 Expression—PAPC is abundant among phospholipids of cellular membranes (26Williams S.D. Hsu F.F. Ford D.A. J. Lipid Res. 2000; 41: 1585-1595Abstract Full Text Full Text PDF PubMed Google Scholar). Because it contains a polyunsaturated fatty acyl side chain (20:4) at the sn-2 position, it is prone to oxidation by UV light, singlet oxygen, or free radicals (27McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 1999; 274: 25189-25192Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 28Girotti A.W. J. Photochem. Photobiol. B. 2001; 63: 103-113Crossref PubMed Scopus (462) Google Scholar). For the skin, UVA irradiation is an important inducer of oxidative stress and results in the expression of HO-1 in fibroblasts (2Keyse S.M. Tyrrell R.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 99-103Crossref PubMed Scopus (1100) Google Scholar). To investigate the direct effects of UVA-1 irradiation on PAPC oxidation, we irradiated synthetic PAPC in vitro. PAPC was dried to a film on a glass support and irradiated with fluencies up to 80 J/cm2 UVA-1. The irradiated PAPC (UV-PAPC) and the sham treated PAPC were analyzed by ESI-MS (Fig. 3, A and B). Fig. 3C shows the formation of mono-, di-, and tri-hydroperoxides of the arachidonic acid moiety of PAPC (m/z 814 (peak b), 846 (peak d), and 878 (peak e)) as well as ions with m/z 828 (peak c). The relative intensity of the nonoxidized [MNa]+ ion (peak a) was set as 100%. Generation of these oxidation products was UVA-1 fluency-dependent, whereas irradiation with corresponding fluencies of UVB (up to 120 mJ/cm2) did not lead to formation of these oxidation products (supplemental Fig. S2). The ion with m/z 828 (c) corresponds to 1-palmitoyl-2-(epoxy-isoprostane-E2)-sn-glycero-3-phosphorylcholine (PEIPC), a strong inducer of HO-1 expression (14Ishikawa K. Navab M. Leitinger N. Fogelman A.M. Lusis A.J. J. Clin. Investig. 1997; 100: 1209-1216Crossref PubMed Scopus (254) Google Scholar). PEIPC was also present in the long chain TLC fraction of OxPAPC (Fig. 2A), as evidenced by ESI-MS and recently described by us (21Birukov K.G. Bochkov V.N. Birukova A.A. Kawkitinarong K. Rios A. Leitner A. Verin A.D. Bokoch G.M. Leitinger N. Garcia J.G. Circ. Res. 2004; 95: 892-901Crossref PubMed Scopus (135) Google Scholar). Ions (b), (d), and (e) correspond to the mono-, di-, and trihydroperoxides of PAPC. To investigate the respective biological activities, sham treated PAPC and UVA-1-treated (20, 40, and 80 J/cm2) PAPC were added to cultures of FB, and HO-1 mRNA expression was measured after 4 h. UV-PAPC, but neither sham treated nor UVB-treated PAPC (supplemental Fig. S2), induced HO-1 expression (Fig. 3D), confirming that UVA-1-mediated oxidation of PAPC leads to the formation of biologically active lipid mediators. UV-PAPC consists of a molecule with m/z 828 (PEIPC), hydroperoxides, and trace amounts of hydroxides of PAPC. To investigate which of the oxidation products present in UV-PAPC induce HO-1 expression, we treated dermal FB with synthetic hydroperoxides and hydroxides of PAPC as well as with PEIPC that had been purified from OxPAPC. Although purified PEIPC strongly induced HO-1 expression, neither the hydroperoxides nor the hydroxides were biologically active (supplemental Fig. S3). Thus, we conclude that PEIPC (m/z 828) is the oxidized phospholipid species present in UV-PAPC, which induces HO-1 expression. UVA-1 Irradiation Generates Oxidation-specific Phospholipid Epitopes in Human Fibroblasts and Leads to Formation of PEIPC—To investigate whether UVA-1 would induce phospholipid oxidation in living cells, we irradiated FB for different times and analyzed them for the presence of PAPC oxidation products. In addition to ESI-MS, we used the murine monoclonal IgM antibody EO6, which binds to oxidized phosphorylcholine-containing phospholipids present in OxPAPC, in oxidized low density lipoprotein, and in the cell membranes of apoptotic cells. For instance, EO6 recognizes POVPC and PEIPC, but not lyso-PC (10Subbanagounder G. Wong J.W. Lee H. Faull K.F. Miller E. Witztum J.L. Berliner J.A. J. Biol. Chem. 2002; 277: 7271-7281Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 30Palinski W. Horkko S. Miller E. Steinbrecher U.P. Powell H.C. Curtiss L.K. Witztum J.L. J. Clin. Investig. 1996; 98: 800-814Crossref PubMed Scopus (496) Google Scholar, 31Horkko S. Bird D.A. Miller E. Itabe H. Leitinger N. Subbanagounder G. Berliner J.A. Friedman P. Dennis E.A. Curtiss L.K. Palinski W. Witztum J.L. J. Clin. Investig. 1999; 103: 117-128Crossref PubMed Scopus (466) Google Scholar). Irradiation of FB with UVA-1 resulted in a dose-dependent increase of intracellular EO6 immunoreactivity, demonstrating the formation of phospholipid oxidation products within 10 min after UVA-1 exposure (Fig. 4, A–D). Staining with antibodies detecting active caspase 3 (aC-3) showed that EO6 immunoreactivity was not confined to apoptotic cells (EO6 positive: 45.5%, aC-3 positive: 15.6%, double positive: 8.5% after irradiation with 60J/cm2) (supplemental Fig. S4). At lower fluencies of UVA-1, apoptosis (as detected with aC-3) in FB did not occur at all (supplemental Fig. S4), whereas EO6 immunoreactivity was found on 12% of cells as compared with 3% of sham treated cells. Among the lipids present in UV-PAPC, only PEIPC (m/z 828) was reported to be recognized by the EO6 antibody (10Subbanagounder G. Wong J.W. Lee H. Faull K.F. Miller E. Witztum J.L. Berliner J.A. J. Biol. Chem. 2002; 277: 7271-7281Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). To confirm the structure of the ion with m/z 828 as PEIPC (12Watson A.D. Leitinger N. Navab M. Faull K.F. Horkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar), we performed HPLC-tandem MS analysis of OxPAPC and UV-PAPC (Fig. 4, E and F). Multiple reaction monitoring revealed an identical HPLC elution profile for m/z 828 in both OxPAPC and UV-PAPC. In addition, retention times were identical, and relative abundances of individual peaks were highly comparable. Thus, we conclude that the molecule with m/z 828 that is present in UV-PAPC is identical to PEIPC. To investigate whether PEIPC is formed in dermal FB upon irradiation, we analyzed total lipid extracts from nonirradiated and UVA-1-irradiated (40 J/cm2) dermal fibroblasts. In Fig. 4G we show that PEIPC is strongly increased in extracts of UVA-1-irradiated cells. To quantify the amounts of PEIPC formed in the cells after irradiation, we measured the peak areas for the multiple reaction monitoring transition 828 > 184 in the elution time range of 7–13 min (shaded gray in the diagram in Fig. 4) using Analyst software (version 1.4, Applied Biosystems). We generated a calibration curve (Fig. 4H) where we plotted the area counts versus the amount of OxPAPC loaded onto the column. This was used to calculate the amount of PEIPC in the cell extracts. For this calculation we estimated the cell volume of a human dermal foreskin fibroblast to be 3.5 pl, a cell number of 3.2 × 106 cells/mg of protein (32Dall'Asta V. Rossi P.A. Bussolati O. Gazzola G.C. J. Biol. Chem. 1994; 269: 10485-10491Abstract Full Text PDF PubMed Google Scholar) and about 20 mol % of OxPAPC corresponding to the isomers of PEIPC. We found that the intracellular concentration of PEIPC was 0.8 μm in nonirradiated cells and increased to 7.7 μm after irradiation with 40 J/cm2 of UVA-1. It was recently shown that PEIPC-induced gene expression in endothelial cells at concentrations as low as 0.1 μm (33Li R. Mouillesseaux K.P. Montoya D. Cruz D. Gharavi N. Dun M. Koroniak L. Berliner J.A. Circ. Res. 2006; 98: 642-650Crossref PubMed Scopus (100) Google Scholar, 34Subbanagounder G. Leitinger N. Schwenke D.C. Wong J.W. Lee H. Rizza C. Watson A.D. Faull K.F. Fogelman A.M. Berliner J.A. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2248-2254Crossref PubMed Scopus (197) Google Scholar). These data demonstrate that UVA-1 irradiation leads to formation of oxidized phospholipids in cells that remain viable and that PEIPC is formed in sufficient amounts to induce expression of HO-1. Generation of HO-1-inducing Oxidized Phospholipids by UVA-1 Involves Singlet Oxygen and Requires an sn-2 Arachidonate Moiety—Singlet oxygen (1O2) is an important ROS that mediates photooxidation by UVA-1 (35Ryter S.W. Tyrrell R.M. Free Radic. Biol. Med. 1998; 24: 1520-1534Crossref PubMed Scopus (181) Google Scholar). To investigate the role of singlet oxygen in UVA-1-induced oxidation of PAPC, the 1O2 generator Rose Bengal (RB) was added before irradiation. To test whether generation of 1O2 would enhance the formation of biologically active phospholipids and whether this activity is confined to phospholipids containing an arachidonic acid moiety, we used 1-palmitoyl-2-oleoyl-sn-3-glycerophosphorylcholine (POPC) and 1-palmitoyl-2-linoleyl-sn-3-glycerophosphorylcholine (PLPC), which differ from PAPC in their length and number of double bonds of the sn-2 acyl chain: a single double bond in" @default.
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• W2022279037 title "Photooxidation Generates Biologically Active Phospholipids That Induce Heme Oxygenase-1 in Skin Cells" @default.
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