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• W2019606302 abstract "Platelet-activating factor acetylhydrolases are a family of specialized phospholipase A2 enzymes. They serve an anti-inflammatory function by converting the proinflammatory autocoid, PAF, into biologically inactive lyso-PAF, by the removal of the sn-2 acetyl group of this glycerophospholipid. Similarly, platelet-activating factor acetylhydrolases can also degrade oxidatively modified sn-2 polyunsaturated-fatty-acid-containing phospholipids, which are toxic to cells. Platelet-activating factor acetylhydrolase II is a recently cloned member of this family of specialized phospholipases. Consistent with a potential role of this intracellular enzyme in protecting membrane phospholipids against oxidative stress, platelet-activating factor acetylhydrolase II has been shown to translocate from cytosol to membranes in response to pro-oxidative stressors, and overexpression of this enzyme decreases the cytotoxic effects of these agents. The objective of this study was to assess whether platelet-activating factor acetylhydrolase II is involved in protecting skin against oxidative stress. Platelet-activating factor acetylhydrolase II protein was demonstrated in human skin by immunohistochemistry, with the highest levels of the enzyme found in sebaceous glands and lesser amounts in epidermal keratinocytes. Treatment of epidermal cells with t-butylhydroperoxide or ultraviolet B radiation resulted in platelet-activating factor acetylhydrolase II translocation from cytosol to membranes. To assess the role of this enzyme in epidermal function, a recombinant retroviral strategy was used to overexpress platelet-activating factor acetylhydrolase II in the human keratinocyte-derived cell line HaCaT. Overexpression of platelet-activating factor acetylhydrolase II protected HaCaT cells against apop tosis induced by oxidative stressors t-butylhydroperoxide and ultraviolet B radiation. Similar levels of apoptosis, however, were seen in both control and platelet-activating-factor-acetylhydrolase-II-over expressing HaCaT cells in response to C2 ceramide. These studies demonstrate the presence of platelet-activating factor acetylhydrolase II in a restricted pattern in human skin, and provide evidence that this specialized phospholipase is involved in protecting this organ against oxidative stress through the degradation of oxidatively modified bioactive phospholipids. Platelet-activating factor acetylhydrolases are a family of specialized phospholipase A2 enzymes. They serve an anti-inflammatory function by converting the proinflammatory autocoid, PAF, into biologically inactive lyso-PAF, by the removal of the sn-2 acetyl group of this glycerophospholipid. Similarly, platelet-activating factor acetylhydrolases can also degrade oxidatively modified sn-2 polyunsaturated-fatty-acid-containing phospholipids, which are toxic to cells. Platelet-activating factor acetylhydrolase II is a recently cloned member of this family of specialized phospholipases. Consistent with a potential role of this intracellular enzyme in protecting membrane phospholipids against oxidative stress, platelet-activating factor acetylhydrolase II has been shown to translocate from cytosol to membranes in response to pro-oxidative stressors, and overexpression of this enzyme decreases the cytotoxic effects of these agents. The objective of this study was to assess whether platelet-activating factor acetylhydrolase II is involved in protecting skin against oxidative stress. Platelet-activating factor acetylhydrolase II protein was demonstrated in human skin by immunohistochemistry, with the highest levels of the enzyme found in sebaceous glands and lesser amounts in epidermal keratinocytes. Treatment of epidermal cells with t-butylhydroperoxide or ultraviolet B radiation resulted in platelet-activating factor acetylhydrolase II translocation from cytosol to membranes. To assess the role of this enzyme in epidermal function, a recombinant retroviral strategy was used to overexpress platelet-activating factor acetylhydrolase II in the human keratinocyte-derived cell line HaCaT. Overexpression of platelet-activating factor acetylhydrolase II protected HaCaT cells against apop tosis induced by oxidative stressors t-butylhydroperoxide and ultraviolet B radiation. Similar levels of apoptosis, however, were seen in both control and platelet-activating-factor-acetylhydrolase-II-over expressing HaCaT cells in response to C2 ceramide. These studies demonstrate the presence of platelet-activating factor acetylhydrolase II in a restricted pattern in human skin, and provide evidence that this specialized phospholipase is involved in protecting this organ against oxidative stress through the degradation of oxidatively modified bioactive phospholipids. platelet-activating factor platelet-activating factor acetylhydrolase II tertiary butyl hydroperoxide Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) is a potent phospholipid-derived mediator implicated in a broad spectrum of physiologic and pathologic activities, particularly inflammation and allergy (reviewed byPinckard et al., 1994Pinckard R.N. Woodard D.S. Showell H.J. Conklyn M.J. McManus M.J. Structural and (patho) physiological diversity of PAF.Clin Rev Allergy. 1994; 12: 329-339PubMed Google Scholar;Prescott et al., 2000Prescott S.M. Zimmerman G.A. Stafforini D.M. MacIntyre T.M. Platelet-activating factor and related lipid mediators.Ann Rev Biochem. 2000; 69: 419-445Crossref PubMed Scopus (561) Google Scholar). A variety of human cell types including polymorphonuclear leukocytes, macrophages, endothelial cells, and keratinocytes both synthesize PAF and express functional PAF receptors (PAF-R). The PAF-R is a G-protein-linked transmembrane protein that can signal through many second messenger systems including phospholipases A2, C, and D, as well as by activation of kinases including protein kinase C and mitogen-activated protein kinases (reviewed byIzumi and Shimizu, 1995Izumi T. Shimizu T. Platelet-activating factor receptor: gene regulation and signal transduction.Biochim Biophys Acta. 1995; 1259: 317-333Crossref PubMed Scopus (206) Google Scholar). Accumulating evidence suggests that the PAF system is involved in keratinocyte function and skin inflammation. Significant levels of PAF are not found in normal skin, but can be measured in various inflammatory dermatoses ranging from psoriasis to urticaria (Grandel et al., 1985Grandel K.E. Farr R.S. Wanderer A.A. Eisenstaldt T.C. Wasserman S.I. Association of platelet-activating factor with primary acquired cold urticaria.New Engl J Med. 1985; 313: 405-409Crossref PubMed Scopus (133) Google Scholar; Mallet and Cunningham, 1985Mallet A.I. Cunningham F.M. Structural identification of platelet-activating factor in psoriatic scale.Biochem Biophys Res Commun. 1985; 126: 192-196Crossref PubMed Scopus (89) Google Scholar). Injection of PAF into skin results in cutaneous inflammation within 1 h, which clinically and histologically resembles an urticarial lesion (Archer et al., 1984Archer C.B. Page C.P. Morley J.P. MacDonald D.M. Inflammatory characteristics of platelet activating factor (paf acether) in human skin.Br J Dermatol. 1984; 110: 45-49Crossref PubMed Scopus (111) Google Scholar;Michel et al., 1987Michel L. Mencia-Huerta J.M. Benveniste J. Dubertret L. Biological properties of LTB4 and PAF acether in vivo in human skin.J Invest Dermatol. 1987; 88: 675-681Abstract Full Text PDF PubMed Google Scholar;Hellewell and Williams, 1989Hellewell P.G. Williams T.J. Antagonism of PAF-induced oedema formation in rabbit skin: a comparison of different antagonists.Br J Pharmacol. 1989; 97: 171-180Crossref PubMed Scopus (22) Google Scholar; Travers et al., 1998Travers J. Pei Y. Morin S.M. Hood A.F. Antiinflammatory activity of the platelet-activating factor receptor antagonist A-85783.Arch Dematol Res. 1998; 290: 569-573Crossref PubMed Scopus (12) Google Scholar). Human keratinocytes express functional PAF-Rs, which can trigger the production of other pro-inflammatory mediators including eicosanoids, interleukins IL-6, IL-8, and tumor necrosis factor α (Pei et al., 1998Pei Y. Barber L.A. Murphy R.C. et al.Activation of the epidermal platelet-activating factor receptor results in cytokine and cyclooxygenase-2 biosynthesis.J Immunol. 1998; 161: 1954-1961PubMed Google Scholar;Dy et al., 1999Dy L.C. Pei Y. Travers J.B. Augmentation of ultraviolet B radiation-induced tumor necrosis factor production by the epidermal platelet-activating factor receptor.J Biol Chem. 1999; 274: 26917-26921Crossref PubMed Scopus (45) Google Scholar). Other pro-oxidative stimuli such as tertiary butyl hydroperoxide (t-BuOOH) and ultraviolet B (UVB) irradiation can also induce the production of PAF or PAF-like species in epidermal cells (Dy et al., 1999Dy L.C. Pei Y. Travers J.B. Augmentation of ultraviolet B radiation-induced tumor necrosis factor production by the epidermal platelet-activating factor receptor.J Biol Chem. 1999; 274: 26917-26921Crossref PubMed Scopus (45) Google Scholar;Travers, 1999Travers J.B. Oxidative stress can activate the epidermal platelet-activating factor receptor.J Invest Dermatol. 1999; 112: 279-283Crossref PubMed Scopus (30) Google Scholar). Of significance, recent studies have implicated PAF in UVB-mediated immunosuppression via epidermal-produced IL-10 (Watersheid et al., 2002Watersheid J.P. Ullrich S.E. Ngheim D.X. Platelet-activating factor, a molecular sensor for cellular damage, activates systemic immune suppression.J Exp Med. 2002; 195: 171-179Crossref PubMed Scopus (193) Google Scholar). The pathway for the acute synthesis of PAF is the sequential actions of phospholipase A2 and PAF acetyltransferase upon 1-alkyl glycerophosphocholines with a long chain unsaturated fatty acid (often arachidonic acid) at the sn-2 position. The PAF-R not only recognizes native PAF, but also 1-acyl 2-acetyl glycerophosphocholine analogs as well as sn-2 short-chained glycerophosphocholines created by free-radical-mediated fragmentation of arachidonic acid or other unsaturated fatty acid (Murphy, 1996Murphy R.C. Free radical-induced oxidation of glycerophosphocholine lipids and formation of biologically active products.Adv Exp Med Biol. 1996; 416: 51-58Crossref PubMed Google Scholar;Marathe et al., 1999Marathe G.K. Davies S.S. Harrison K.A. et al.Inflammatory platelet-activating factor-like phospholipids in oxidized low density lipoproteins are fragmented alkyl phosphatidylcholines.J Biol Chem. 1999; 274: 28395-28404Crossref PubMed Scopus (154) Google Scholar). This production of PAF-like lipids in response to oxidative stress differs from the tightly controlled enzymatic pathway. PAF is inactivated by removal of the sn-2 acetate moiety through the actions of a family of specific phospholipases termed platelet-activating factor acetylhydrolases (PAF-AH) (reviewed byStafforini et al., 1997Stafforini D.M. McIntyre T.M. Zimmerman G.A. Prescott S.M. Platelet-activating factor acetylhydrolases.J Biol Chem. 1997; 272: 17895-17898Crossref PubMed Scopus (232) Google Scholar). Unlike phospholipase A2 enzymes, which hydrolyze phospholipids with long chain acyl groups at the sn-2 position, PAF-AHs selectively hydrolyze short-chained fatty acids at the sn-2 position of glycerophosphocholines. PAF-AH enzymes can also inactivate short-chained PAF-like lipids produced as a consequence of oxidative stress. In addition to their unusual specificity, PAF-AHs typically are in a fully activated state (Yanoshita et al., 1988Yanoshita R. Kudo I. Ikizawa K. et al.Hydrolysis of platelet-activating factor and its methylated analogs by acetylhydrolases.J Biochem. 1988; 103: 815-819PubMed Google Scholar;Stafforini et al., 1991Stafforini D.M. Prescott M. Zimmerman G.A. McIntyre T.M. Platelet-activating factor acetylhydrolase activity in human tissues and blood cells.Lipids. 1991; 26: 279-985Crossref Scopus (86) Google Scholar). Both extracellular or plasma associated and intracellular or tissue associated types of PAF-AHs have been described (Stafforini et al., 1997Stafforini D.M. McIntyre T.M. Zimmerman G.A. Prescott S.M. Platelet-activating factor acetylhydrolases.J Biol Chem. 1997; 272: 17895-17898Crossref PubMed Scopus (232) Google Scholar). Extracellular PAF-AH is a 43 kDa monomeric enzyme, which is thought to regulate plasma PAF levels in the plasma. Approximately 4% of the Japanese population have a mutation in PAF-AH abolishing the activity of this enzyme. The presence of this mutation has been reported to be a severity factor for the development of severe asthma (Miwa et al., 1988Miwa M. Miykate T. Yamaoka T. et al.Characterization of serum platelet-activating factor (PAF) acetylhydrolase: correlation between deficiency of serum PAF acetylhydrolase and respiratory symptoms in asthmatic children.J Clin Invest. 1988; 82: 1983-1991Crossref PubMed Scopus (271) Google Scholar). Two separate intracellular types of PAF-AHs have been described (types I and II). Type I PAF-AH has two isoforms, tentatively named Ia and Ib. Isoform Ib is a heterotrimeric enzyme restricted to brain, the lack of which results in malformation of the cerebral cortex (Miller-Dieker lissencephaly) (Hattori et al., 1994Hattori M. Adachi H. Tsujimoto M. Arai H. Inoue K. Miller-Dieker lissencephaly gene encodes a subunit of brain platelet-activating factor.Nature. 1994; 370: 216-218Crossref PubMed Scopus (435) Google Scholar). The intracellular isoform II (PAF-AHII) is a 40 kDa monomer most abundantly expressed in bovine liver and kidney (Hattori et al., 1995Hattori K. Hattori M. Adachi H. Tisujimoto M. Arai H. Inoue K. Purification and characterization of platelet-activating factor acetylhydrolase II from bovine liver cytosol.J Biol Chem. 1995; 38: 23308-23315Google Scholar;Hattori et al., 1996Hattori K. Adachi H. Matusawa A. et al.cDNA cloning and expression of intracellular platelet-activating acetylhydrolase II.J Biol Chem. 1996; 270: 33032-33038Crossref Scopus (100) Google Scholar). Recent studies using MDBK cells have found that PAF-AHII translocates from cytosol to membranes within 20 min following exposure of the cells to pro-oxidative stressors. Conversely, this enzyme translocates back to cytosol following treatment with antioxidants (Matsuzawa et al., 1997Matsuzawa A. Hattori K. Aoki J. Arai H. Inoue K. Protection against oxidative stress-induced cell death by intracellular platelet-activating factor-acetylhydrolase II.J Biol Chem. 1997; 272: 32315-32320Crossref PubMed Scopus (114) Google Scholar). In addition, overexpression of PAF-AHII in Chinese hamster ovary cells inhibited apoptosis induced by the pro-oxidant t-BuOOH (Matsuzawa et al., 1997Matsuzawa A. Hattori K. Aoki J. Arai H. Inoue K. Protection against oxidative stress-induced cell death by intracellular platelet-activating factor-acetylhydrolase II.J Biol Chem. 1997; 272: 32315-32320Crossref PubMed Scopus (114) Google Scholar). These findings suggest that this enzyme acts to degrade oxidatively fragmented phospholipids that are toxic to the cell. The objective of our studies was to assess whether human skin expresses PAF-AHII. Because this enzyme has been hypothesized to protect cells against oxidative stress, we tested whether overexpression of the enzyme in an epidermal cell line could protect against the cytotoxic effects of pro-oxidative stressors including t-BuOOH and UVB. PAF, t-BuOOH, and routine chemicals were obtained from Sigma (St. Louis, MO). Because of its chemical instability, t-BuOOH bottles were discarded within 48 h after opening. Growth media and supplements were purchased from Life Technologies (Gaithersburg, MD) and fetal bovine serum from Intergen (Purchase, NY). Primary cultures of neonatal-foreskin-derived human keratinocytes and fibroblasts were harvested and cultured as previously outlined (Travers et al., 1996Travers J.B. Harrison K.A. Johnson C.A. Clay K.L. Morelli J.G. Murphy R.C. Platelet-activating factor biosynthesis induced by various stimuli in human HaCaT keratinocytes.J Invest Dermatol. 1996; 107: 88Crossref PubMed Scopus (43) Google Scholar). The immortalized sebaceous gland cell line SZ95 was grown in Sebomed medium as described previously (Zouboulis et al., 1999Zouboulis C.C. Seltmann H. Neitzel H. Orfanos C.E. Establishment and characterization of an immortalized human sebaceous gland cell line (SZ95).J Invest Dermatol. 1999; 113: 1011-1020Crossref PubMed Scopus (257) Google Scholar). The cDNA for the PAF-AHII (Hattori et al., 1996Hattori K. Adachi H. Matusawa A. et al.cDNA cloning and expression of intracellular platelet-activating acetylhydrolase II.J Biol Chem. 1996; 270: 33032-33038Crossref Scopus (100) Google Scholar) was ligated into the NdeI/HindIII sites of the pET21a vector (pET system, Novagen). After the plasmid had been introduced into Escherichia coli strain BL21 (DE3), protein was expressed as His-tagged protein by induction with 1 mM isopropyl-b-D-thiogalactopyranoside. The protein was purified using nickel column chromatography (Novagen) according to the manufacturer's protocol. Balb/c mice were immunized with the purified protein and monoclonal antibodies were produced using the PAI myeloma cell line. Hybridoma cell lines were established according to the standard protocol. The presence of PAF-AHII protein in skin was assessed by immunofluorescence studies using the monoclonal antibody against human PAF-AHII whose synthesis is described above. Biopsies of human normal skin were collected and immediately frozen in Tissue-Tek, optimal cutting temperature (OCT), obtained from Sakura Finetek Europe BV (Zauterwoude, The Netherlands). The frozen block subsequently was stored at -80°C until used. Six to eight micrometer cryostat sections were obtained and stained with the monoclonal antibody against PAF-AHII. Briefly, the sections were fixed in ethanol for 1 min, washed in ddH2O for 20–30 s, then washed with phosphate-buffered saline (PBS) for 5 min, and then incubated with anti-PAF-AHII monoclonal antibody diluted at 1:100 in PBS containing 3% bovine serum albumin (BSA) for 1 h at room temperature. The sections were washed three times with PBS, and then incubated with a fluorescein-conjugated goat antimouse IgG polyclonal antibody diluted at 1:100 in PBS containing 3% BSA for 1 h at room temperature, in the dark. After the incubation with the conjugated fluorescent antibody the sections were washed three times with PBS and mounted using Fluoromount-G and cover slides. Immunostaining patterns were visualized using an Olympus fluorescent microscope. As a control, extra sections from the same specimen, following all the staining procedures, substituting the PAF-AHII antibody with mouse IgG (Sigma) in the incubation with 3% BSA, were stained at the same time. The immortalized human keratinocyte derived cell line HaCaT (Boukamp et al., 1988Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J Cell Biol. 1988; 106: 761-778Crossref PubMed Scopus (3276) Google Scholar) was a gift from Dr. Petra Boukamp (German Cancer Research Center, Heidelberg, Germany) and was cultured as previously reported (Travers et al., 1996Travers J.B. Harrison K.A. Johnson C.A. Clay K.L. Morelli J.G. Murphy R.C. Platelet-activating factor biosynthesis induced by various stimuli in human HaCaT keratinocytes.J Invest Dermatol. 1996; 107: 88Crossref PubMed Scopus (43) Google Scholar). To create HaCaT cells stably overexpressing PAF-AHII, the entire human PAF-AHII cDNA was cloned into the MSCV 2.1 retroviral vector and high titer replication-deficient retrovirus was produced as previously described (Pei et al., 1998Pei Y. Barber L.A. Murphy R.C. et al.Activation of the epidermal platelet-activating factor receptor results in cytokine and cyclooxygenase-2 biosynthesis.J Immunol. 1998; 161: 1954-1961PubMed Google Scholar). HaCaT cells were transduced with MSCV 2.1PAF-AHII or empty MSCV 2.1 retrovirus (as control) using polybrene, and infected cells were screened by using G418 as previously reported (Pei et al., 1998Pei Y. Barber L.A. Murphy R.C. et al.Activation of the epidermal platelet-activating factor receptor results in cytokine and cyclooxygenase-2 biosynthesis.J Immunol. 1998; 161: 1954-1961PubMed Google Scholar). PAF-AHII protein was assessed by immunoblotting using the same monoclonal PAF-AHII antibody used in the immunofluorescence studies. Briefly, confluent 10 cm dishes of HaCaTM PAF-AHII and HaCaTM were washed twice with PBS and lyzed with 1.5 ml of lysis buffer (RIPA) for 30 min, disrupted by sonication in a vessel surrounded by ice for three 30 s periods at 45 s intervals using a sonic dismembrator (Fischer Scientific, Pittsburgh, PA). The cell lysate was centrifuged at 35,000 rpm for 10 min; protein in the resultant supernatant was quantitated using a commercial kit (Bio-Rad), and aliquots were placed in Eppendorf tubes and stored at -80°C. The samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis on a 12% acrylamide gel and blotted onto a nitrocellulose membrane. The membrane was blocked with Tris-buffered saline (TBS) containing 5% skim milk overnight at 4°C and then washed three times with TBS containing 2% Tween 20 (T-TBS), incubated with the anti-PAF-AHII monoclonal antibody diluted 1:3000 in TBS containing 5% skim milk for 1 h, and incubated with a horseradish-peroxidase-conjugated goat antimouse antibody diluted 1:3000 in TBS containing 5% skim milk. After washing the membrane four times with T-TBS, peroxidase was detected by an enhanced chemiluminescence method using an ECL Western Blotting Detection Set from Hyperfilm ECL (Amersham Life Science). PAF-AHII distribution in membrane versus cytosolic fractions of HaCaT cells was assessed as previously described (Matsuzawa et al., 1997Matsuzawa A. Hattori K. Aoki J. Arai H. Inoue K. Protection against oxidative stress-induced cell death by intracellular platelet-activating factor-acetylhydrolase II.J Biol Chem. 1997; 272: 32315-32320Crossref PubMed Scopus (114) Google Scholar). PAF-AH enzymatic activity of HaCaT cells was measured as previously described (Narahara and Johnson, 1993Narahara H. Johnson J.M. Effects of endotoxins and cytokines on the secretion of platelet activating factor acetylhydrolase by human decidual macrophages.Am J Obstet Gynecol. 1993; 169: 531-537Abstract Full Text PDF PubMed Scopus (60) Google Scholar). Briefly, 4–6 × 106 cells were trypsinized, washed with PBS, and pelleted; the supernatant was aspirated and the pellets were frozen (-80°C) until analyzed. The samples were thawed on ice and 500 µl of cold sucrose (0.25 M) was added to each tube. They were then sonicated in a vessel surrounded by ice for five periods of 10 s at 20 s intervals using an ultrasonic device (Ultrasonic, Planeview, NY), and then centrifuged at 12,000 × g at 4°C. The resultant supernatant was removed and assayed for PAF-AH enzymatic activity using 1-O-alkyl-2-[3H]acetate-glycerophosphocholine as previously described (Narahara and Johnson, 1993Narahara H. Johnson J.M. Effects of endotoxins and cytokines on the secretion of platelet activating factor acetylhydrolase by human decidual macrophages.Am J Obstet Gynecol. 1993; 169: 531-537Abstract Full Text PDF PubMed Scopus (60) Google Scholar). Samples were run in triplicate. HaCaTM or HaCaTM PAF-AHII cells were plated at 8 × 105 cells in 10 cm dishes for 24 h. Following washing with medium, the cells were treated with 200 µM t-BuOOH or 100 µM of C2 ceramide, or irradiated with 400 J per m2 of UVB (all in regular medium) as previously described (Barber et al., 1998Barber L.A. Spandau D.F. Rathman S.C. et al.Expression of the platelet-activating factor receptor results in enhanced ultraviolet B radiation-induced apoptosis in a human epidermal cell line.J Biol Chem. 1998; 273: 18891-18897Crossref PubMed Scopus (67) Google Scholar). The irradiator was an unfiltered Philips F20T12/UVB source (270–390 nm; containing 2.6% UVC, 43.6% UVB, 53.8% UVA). The intensity of the UV source was routinely measured using an International Light radiometer equipped with a UVB detector. At various time points following treatment, the cells were harvested by collection of supernatant and trypsinization of adherent cells and pelleted. Caspase 3 proteolytic activity in cell lysates was measured using a synthetic fluorogenic substrate (DEVD-AMC, Alexid Biochemicals, San Diego, CA;Hurwitz and Spandau, 2000Hurwitz S.A. Spandau D.F. Quantitative analysis of UVB-induced apoptosis in human epidermis.Exp Dematol. 2000Crossref Scopus (19) Google Scholar). Briefly, the cells were suspended in lysis buffer (50 mM PIPES, pH 7.0, 50 mM KCl, 5 mM ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 2 mM MgCl2, 1 mM dithiothreitol) for 30 min on ice. Following removal of cellular debris by centrifugation, an aliquot of the cell lysate was added to caspase 3 reaction buffer (100 mM HEPES, pH 7.5, 10% sucrose, 0.1% CHAPS, 10 mM dithiothreitol, 0.1 mg per ml bovine serum albumin, and 50 µM CPP32-AMC substrate) and incubated at 37°C for 1 h. Release of the fluorescent AMC moiety was measured using a Hitachi F2000 Spectrophotometer (excitation, 380 nm; emission, 460 nm). The fluorescent intensity was converted to moles of AMC by comparison to the fluorescent intensity of standards of AMC (7-amino-4 methylcoumarin; Molecular Probes, Eugene, OR). The specific activity of caspase 3 in cell lysates was then determined after the total protein concentration of the cell lysates was measured (Nano Orange Protein Quantification Reagent, Molecular Probes) and was expressed as pmoles AMC per h per µg protein. Our first studies assessed whether PAF-AHII protein was expressed in human skin. Immunofluorescence studies with a PAF-AHII specific monoclonal antibody revealed expression of this protein in normal human skin. As shown in Figure 1, sebaceous glands exhibited the highest levels of PAF-AHII expression with lesser amounts found in the epidermis. Weak PAF-AHII immunoreactivity was expressed throughout the epidermis, yet significant levels of PAF-AHII immunoreactivity were not seen in dermal fibroblasts or blood vessels. Examination of PAF-AHII protein levels in primary cultures of human keratinocytes, fibroblasts, and the immortalized sebaceous gland cell line SZ95 (Zouboulis et al., 1999Zouboulis C.C. Seltmann H. Neitzel H. Orfanos C.E. Establishment and characterization of an immortalized human sebaceous gland cell line (SZ95).J Invest Dermatol. 1999; 113: 1011-1020Crossref PubMed Scopus (257) Google Scholar) revealed the highest levels of immunoreactive protein in the sebaceous gland cell line (Figure 2). These studies indicate that human skin expresses PAF-AHII protein in a restricted pattern with the highest amounts of protein associated with sebaceous glands/sebocytes.Figure 2Measurement of PAF-AHII protein in keratinocytes, fibroblasts, and a sebaceous cell line. Protein (40 µg) was isolated from primary cultures of neonatal-foreskin-derived human keratinocytes (NHK), human fibroblasts (NHF), and SZ95 sebocytes, and run on a 15% sodium dodecyl sulfate polyacrylamide gel. PAF-AHII immunoreactivity was determined using a monoclonal antibody. The membranes were stripped and re-probed with an antibody against β-actin.View Large Image Figure ViewerDownload (PPT) Recent studies have found that PAF-AHII translocates from cytosol to membranes following exposure of the cells to the pro-oxidative stressor t-BuOOH (Matsuzawa et al., 1997Matsuzawa A. Hattori K. Aoki J. Arai H. Inoue K. Protection against oxidative stress-induced cell death by intracellular platelet-activating factor-acetylhydrolase II.J Biol Chem. 1997; 272: 32315-32320Crossref PubMed Scopus (114) Google Scholar). The next studies assessed whether the PAF-AHII expressed in epidermal cells translocates in response to oxidative stressors. HaCaT keratincoytes were treated with 100 µM t-BuOOH or irradiated with 600 J per m2 UVB and the levels of PAF-AHII protein in cytosol versus membranes was evaluated. As shown in Figure 3, resting HaCaT cells express approximately equal amounts of PAF-AHII protein in membrane versus cytosolic fractions. Treatment with UVB or t-BuOOH resulted in increased levels of protein in the membrane over cytosolic fractions. Through its ability to recognize and inactivate biologically active PAF as well as PAF-like lipids created in response to oxidative stress, PAF-AHII is thought to play a protective role in the skin. Consistent with the notion that PAF-AHII could protect cells against oxidative stress, overexpression of this protein in Chinese hamster ovary cells inhibits t-BuOOH-induced apoptosis (Matsuzawa et al., 1997Matsuzawa A. Hattori K. Aoki J. Arai H. Inoue K. Protection against oxidative stress-induced cell death by intracellular platelet-activating factor-acetylhydrolase II.J Biol Chem. 1997; 272: 32315-32320Crossref PubMed Scopus (114) Google Scholar). As immunofluorescence studies suggest that human keratinocytes express PAF-AHII, we created a model system using the human keratinocyte-derived cell line HaCaT, which was genetically engineered to overexpress PAF-AHII using a replication-deficient retrovirus. Northern blotting of HaCaT cells transduced with PAF-AHII (HaCaTM PAF-AHII) or control transduced (HaCaTM) demonstrated that both cell populations contained PAF-AHII mRNA, with less than a 2-fold increase found in the HaCaTM PAF-AHII cells (data not shown). Immunoblotting studies revealed increased levels of PAF-AHII protein in HaCaTM PAF-AHII over HaCaTM cells. Enzyme activity studies demonstrated a greater than 3-fold increase in PAF-AH activity in the HaCaTM PAF-AHII over HaCaTM cells (Figure 4). Using this model system, the role of moderate overexpression of PAF-AHII in a keratinocyte-derived cell line could be assessed. The next experiments investigated whether overexpression of PAF-AHII in HaCaT cells protects against apoptosis induced by oxidative stressors such as t-BuOOH and UVB irradiation. Apoptosis was assessed by measurement of caspase 3 enzymatic activity using a fluorescent substrate as outlined in Materials and Methods. Our laboratory has previously demonstrated that caspase 3 enzymatic levels correlate with other apoptotic measurements in epithelial cells (Southall et al., 2001Southall M.D. Isenberg J.S. Nakshatri H. Yi Q. Pei Y. Spandau D.F. Travers J.B. The platelet-activating factor receptor protects epidermal cells from tumor necrosis factor and TNF-related apoptosis-inducing ligand-induced apoptosis through an NFκB-dependent process.J Biol Chem. 2001; 276: 45548-45554Crossref PubMed Scopus (39) Google Scholar). HaCaTM and HaCaTM PAF-AHII cells were treated with 100 µM t-BuOOH and incubated at 37°C, and caspase 3 enzymatic activity was assessed at various times. As shown in Figure 5, t-BuOOH treatment resulted in increased caspase 3 specific activity by 8 h in HaCaTM cells. Maximal levels of caspase 3 induction were seen by 12 h. HaCaTM PAF-AHII cells did not exhibit increased caspase 3 enzymatic activity until 24 h, however. Consistent with the cytotoxic effects described in other cell types, HaCaTM cells treated with t-BuOOH began to detach from the plate by 8 h. In contrast, the majority of HaCaTM PAF-AHII cells remained attached to the plate and viable until 24 h. These studies indicate that overexpression of PAF-AHII protein is protective against cellular cytotoxicity induced by the oxidative stressor t-BuOOH. Recent studies suggest that UVB acts as a potent pro-oxidative stimulus in human keratinocytes (Peus et al., 1998Peus D. Vasa R.A. Meves A. Pott M. Beyerle A. Squillace K. Pitterkow M.R. H202 is an important mediator of UVB-induced EGF-receptor phosphorylation in cultured keratinocytes.J Invest Dermatol. 1998; 110: 971-996Crossref Scopus (164) Google Scholar). As our previous studies indicate that PAF and PAF-like lipids are produced by epidermal cells in response to UVB (Barber et al., 1998Barber L.A. Spandau D.F. Rathman S.C. et al.Expression of the platelet-activating factor receptor results in enhanced ultraviolet B radiation-induced apoptosis in a human epidermal cell line.J Biol Chem. 1998; 273: 18891-18897Crossref PubMed Scopus (67) Google Scholar;Dy et al., 1999Dy L.C. Pei Y. Travers J.B. Augmentation of ultraviolet B radiation-induced tumor necrosis factor production by the epidermal platelet-activating factor receptor.J Biol Chem. 1999; 274: 26917-26921Crossref PubMed Scopus (45) Google Scholar), we tested whether overexpression of PAF-AHII could protect against UVB-induced apoptosis. Irradiation of HaCaTM and HaCaTM PAF-AHII cells with 400 J per m2 UVB resulted in caspase 3 activation (Figure 6). The levels of caspase 3 were significantly less in HaCaTM PAF-AHII versus HaCaTM cells at 8 and 12 h, however. Examination of the plates for detached cells mirrored the caspase 3 enzymatic assays, with fewer adherent cells in HaCaTM versus HaCaTM PAF-AHII cells until 24 h. These findings indicate that increased levels of PAF-AHII protein delays UVB-induced apoptosis. C2 ceramide is a short-chained ceramide that induces apoptosis in many cell types (Obeid and Hannun, 1995Obeid L. Hannun Y.A. A stress signal and mediator of growth suppression and apoptosis.J Cell Biochem. 1995; 58: 191-198Crossref PubMed Scopus (231) Google Scholar). Although C2 ceramide has been reported to induce oxidative stress in mitochondria (Ballon et al., 1996Ballon L.R. Lautederkind J.T. Roslomiec F.F. Raghow R. C2 ceramide signaling and the immune response.Biochem Biophys Acta. 1996; 1301: 273-287Crossref PubMed Scopus (235) Google Scholar), this toxic lipid does not have the pro-oxidative effects of t-BuOOH or UVB. Treatment of HaCaT cells with 100 µM C2 ceramide resulted in apoptosis by 24 h as measured by enhanced caspase 3 induction (Figure 7). The amount of caspase 3 induction as well as numbers of detached cells were similar in HaCaTM versus HaCaTM PAF-AHII cells, however, suggesting that overexpression of PAF-AHII does not protect against C2-ceramide-induced apoptosis. Keratinocytes are chronically exposed to a powerful oxidant agent UVB, as well as endogenous sources of reactive oxygen species from metabolic processes and also from inflammatory cells such as neutrophils. As both oxidized lipids and the PAF system appear to be involved in epidermal function/cutaneous inflammation, characterization of the enzymes involved in the regulation of these lipids is important. These studies demonstrate that human skin contains the specialized phospholipase PAF-AHII, and provide evidence that this enzyme could be involved in the protection of this organ against the toxic effects of oxidative stress. Accumulating evidence indicates that unsaturated fatty acids are a target for oxidative stress. Indeed, reactive oxygen species have been shown to spontaneously oxidize unsaturated fatty acyl chains in membrane phospholipids (Zimmerman et al., 1995Zimmerman G.A. Stephen M. Prescott S.M. MacIntyre T.M. Oxidatively fragmented phospholipids as inflammatory mediators: the dark side of polyunsaturated lipids.J Nutr. 1995; 125: 1661S-1665SPubMed Google Scholar). Depending upon the structure of the resultant product, oxidized lipids can have biologic activities through activation of the PAF-R, as well as cause cell damage and apoptosis through receptor-independent effects. Thus, oxidized phospholipids need to be rapidly hydrolyzed to prevent their cellular toxicities. PAF-AHII is a recently cloned protein whose enzymatic activity is that of a phospholipase A2, yet restricted to the hydrolysis of short-chained sn-2 acyl groups. Several features of this enzyme suggest that it could be involved in protecting cells against oxidized phospholipids. First, PAF-AHII is constitutively active, unlike other phospholipase A2 enzymes (Blank et al., 1981Blank M.L. Lee T.C. Fitsgerald V. Snyder F. A specific acetylhydrolase for 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (a hypotensive and platelet-activating lipid).J Biol Chem. 1981; 210: 175-178Google Scholar;Stremler et al., 1989Stremler K.E. Stafforini D.M. Prescott S.M. McIntyre T.M. Human plasma platelet-activating factor acetylhydrolase and oxidatively fragmented phospholipids.J Biol Chem. 1989; 266: 11095-11103Google Scholar; Tjoelker et al., 1995Tjoelker L.W. Wilder C. Eberhardt C. et al.Anti-inflammatory properties of a platelet-activating factor acetylhydrolase.Nature. 1995; 374: 549-552Crossref PubMed Scopus (462) Google Scholar). Second, studies using MDBK renal cells and now HaCaT keratinocytes indicate that PAF-AHII translocates from cytosol to membranes following exposure to oxidants. The ability of this active enzyme to rapidly translocate to membranes would ensure that oxidized unsaturated acyl groups in the sn-2 position of phospholipids are rapidly degraded. Finally, overexpression of PAF-AHII in Chinese hamster ovary cells protects against t-BuOOH-induced apoptosis (Matsuzawa et al., 1997Matsuzawa A. Hattori K. Aoki J. Arai H. Inoue K. Protection against oxidative stress-induced cell death by intracellular platelet-activating factor-acetylhydrolase II.J Biol Chem. 1997; 272: 32315-32320Crossref PubMed Scopus (114) Google Scholar). Our studies are also consistent with the notion that this enzyme is involved in protecting cells against oxidized phospholipids. In this paper we present evidence that PAF-AHII is expressed in a restricted pattern in human skin. Immunofluorescence, northern blotting, and enzymatic assays demonstrate that PAF-AHII is expressed in keratinocytes. The highest levels of PAF-AHII expression are found in sebaceous glands, however, including the sebaceous gland cell line SZ95, shown to exhibit major characteristics of human sebocytes in vitro (Zouboulis et al., 1999Zouboulis C.C. Seltmann H. Neitzel H. Orfanos C.E. Establishment and characterization of an immortalized human sebaceous gland cell line (SZ95).J Invest Dermatol. 1999; 113: 1011-1020Crossref PubMed Scopus (257) Google Scholar). This higher level of PAF-AHII expression in sebaceous glands, which have a high lipid content, fits with an increased need of this tissue to protect itself against oxidatively modified phospholipids. It may also indicate a protective action of the sebaceous glands on other neighboring skin structures against oxidative stress via a paracrine pathway. To evaluate potential protective effects of PAF-AHII on pro-oxidative stressors in keratinocytes, a replication-deficient retrovirus was used to stably overexpress this enzyme in HaCaT cells. As shown in Figure 4, HaCaTM PAF-AHII cells had an approximately 300% increase in baseline PAF-AH enzymatic activity over baseline HaCaTM cells. This moderate overexpression of PAF-AHII resulted in protection against the cytotoxic effects of t-BuOOH and UVB irradiation. The protection was manifested as a delay in the onset of apoptosis induced by these oxidative stressors. That PAF-AHII did not fully protect against the cytotoxic effects of these agents is compatible with the notion that other targets for UVB and t-BuOOH that are independent of oxidized phospholipids also play an important role. Increased levels of PAF-AHII, however, did not affect apoptosis induced by C2 ceramide, a nonoxidative stressor. These findings fit with previous studies where overexpression of PAF-AHII was found to be protective against t-BuOOH-induced apoptosis (Matsuzawa et al., 1997Matsuzawa A. Hattori K. Aoki J. Arai H. Inoue K. Protection against oxidative stress-induced cell death by intracellular platelet-activating factor-acetylhydrolase II.J Biol Chem. 1997; 272: 32315-32320Crossref PubMed Scopus (114) Google Scholar). Although all these studies point to a protective effect of overexpression of this enzyme on oxidative stress, we have found that high levels of overexpression (4000%-8000%) in HaCaT or KB cells using a mammalian expression vector result in increased levels of both spontaneous and t-BuOOH-induced apoptosis (data not shown). These findings might be explained by recent studies demonstrating that PAF-AHII has the ability to transfer the short chain sn-2 group to sphingosine, producing short-chained ceramides including C2 ceramide, which has known cytotoxic effects (Karasawa et al., 1999Karasawa K. Qiu X. Lee T.C. Purification and characterization from rat kidney membranes of a novel platelet-activating factor-dependent transacetylase that catalyses the hydrolysis of PAF, formation of PAF analogs and C2-ceramide.J Biol Chem. 1999; 274: 8655-8661Crossref PubMed Scopus (34) Google Scholar). Thus, this enzyme not only can inactivate PAF and related short-chained sn-2 phopsholipids, but can generate other diverse lipid mediators. Both oxidized lipids and the PAF system appear to have the potential to modulate cutaneous inflammation as well as to have cytotoxic effects. Our studies demonstrate that PAF-AHII, a novel phospholipase that serves to regulate levels of both, is found in human skin. Moderate overexpression of PAF-AHII in the human keratinocyte cell line HaCaT protects against oxidative stressors in vitro, suggesting that this enzyme could be involved in protecting skin from oxidative damage in vivo. In addition, this enzyme has anti-inflammatory effects through its ability to degrade PAF. Though genetic mutations with resultant clinical effects have been described for other forms of PAF-AHs in humans, it is not known whether PAF-AHII mutations are associated with disease. Future studies are planned to examine PAF-AHII levels in human skin diseases that are associated with exaggerated responsiveness to oxidative stress, including photosensitivity disorders. The authors wish to acknowledge the technical assistance of Drs Silvio Marques and Dan Spandau (I.U. Department Dermatology). These studies are supported in part by grants from the Showalter Memorial Fundation, the Riley Memorial Association, and the National Institutes of Health grants AR1993 and HL62996. M.M. was supported by Brazilian grants from Fundacao de Amparo a Pesquisa do Estado de Sao Paulo- FAPESP. M.S. was supported by a grant from the Dermatology Foundation. H.S. was supported by a grant from the Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin (BgVV Z 5.1-1328-156)." @default.
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• W2019606302 title "Identification of Platelet-Activating Factor Acetylhydrolase II in Human Skin" @default.
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