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• W1997445760 abstract "A number of chemical mediators can induce human keratinocytes and epidermal-derived carcinomas to undergo apoptosis, or programmed cell death. Recent evidence suggests pro-inflammatory cytokines, such as interleukin-1β or transforming growth factor α, protects carcinomas from numerous pro-apoptotic stimuli. Platelet-activating factor (1-alkyl-2-acetyl-3-glycerophosphocholine; PAF) is a lipid mediator with pro-inflammatory effects on numerous cell types. Although PAF can be metabolized to other bioactive lipids, the majority of PAF effects occur through activation of a G protein-coupled receptor. Using a model system created by retroviral transduction of the PAF receptor (PAF-R) into the PAF-R-negative human epidermal cell line KB and the PAF-R-expressing keratinocyte cell line HaCaT, we now demonstrate that activation of the epidermal PAF-R results in protection from apoptosis induced by tumor necrosis factor (TNF) α or TNF-related apoptosis-inducing ligand. The PAF-mediated protection was inhibited by PAF-R antagonists, and protection did not occur in PAF-R-negative KB cells. Additionally, we show protection from TNFα- or TRAIL-induced apoptosis by PAF-R activation is dependent on the transcription factor nuclear factor (NF)-κB, because PAF-R activation-induced NF-κB and epidermal cells transduced with a super-repressor form of inhibitor κB were not protected by the PAF-R. These studies provide a mechanism whereby the epidermal PAF-R, and possibly other G protein-coupled receptors, can exert anti-apoptotic effects through an NF-κB-dependent process. A number of chemical mediators can induce human keratinocytes and epidermal-derived carcinomas to undergo apoptosis, or programmed cell death. Recent evidence suggests pro-inflammatory cytokines, such as interleukin-1β or transforming growth factor α, protects carcinomas from numerous pro-apoptotic stimuli. Platelet-activating factor (1-alkyl-2-acetyl-3-glycerophosphocholine; PAF) is a lipid mediator with pro-inflammatory effects on numerous cell types. Although PAF can be metabolized to other bioactive lipids, the majority of PAF effects occur through activation of a G protein-coupled receptor. Using a model system created by retroviral transduction of the PAF receptor (PAF-R) into the PAF-R-negative human epidermal cell line KB and the PAF-R-expressing keratinocyte cell line HaCaT, we now demonstrate that activation of the epidermal PAF-R results in protection from apoptosis induced by tumor necrosis factor (TNF) α or TNF-related apoptosis-inducing ligand. The PAF-mediated protection was inhibited by PAF-R antagonists, and protection did not occur in PAF-R-negative KB cells. Additionally, we show protection from TNFα- or TRAIL-induced apoptosis by PAF-R activation is dependent on the transcription factor nuclear factor (NF)-κB, because PAF-R activation-induced NF-κB and epidermal cells transduced with a super-repressor form of inhibitor κB were not protected by the PAF-R. These studies provide a mechanism whereby the epidermal PAF-R, and possibly other G protein-coupled receptors, can exert anti-apoptotic effects through an NF-κB-dependent process. interleukin platelet-activating factor PAF receptor 1-O-hexadecyl-2-N-methylcarbamoyl-glycerophosphocholine tumor necrosis factor α, TRAIL, TNF-related apoptosis-inducing ligand G protein-coupled receptor nuclear factor-κB 7-amino-4-methylcoumarin inhibitor of apoptosis proteins inhibitor κB 2-Val-Ala-Asp-FMK Apoptosis, or programmed cell death, is a fundamental physiological process enabling the removal of damaged or infected cells and in the control of cell populations (1Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4084) Google Scholar). Apoptosis can occur during embryogenesis, induction, and maintenance of immune tolerance, development of the nervous system, and endocrine-dependent tissue atrophy (2Jacobson M.D. Weil M. Raff M.C. Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (2383) Google Scholar). In the skin, apoptosis is involved in epidermal development and growth and deletion of UV-damaged keratinocytes (3Woodcock A. Magnus I.A. Br. J. Dermatol. 1976; 95: 459-468Crossref PubMed Scopus (105) Google Scholar). In addition to normal physiological conditions, essentially all chemotherapeutic agents exert their effects by induction of apoptosis (4Reed J.C. Am. J. Pathol. 2000; 157: 1415-1430Abstract Full Text Full Text PDF PubMed Google Scholar). Thus, regulation of apoptosis can have important consequences both during development and in the treatment of cancer. Recent evidence suggests an association between cancer and inflammation, with inflammatory mediators affecting the growth and survival of tumors (5Marks F. Muller-Decker K. Furstenberger G. Toxicology. 2000; 153: 11-26Crossref PubMed Scopus (86) Google Scholar, 6Bours V. Bonizzi G. Bentires-Alj M. Bureau F. Piette J. Lekeux P. Merville M. Toxicology. 2000; 153: 27-38Crossref PubMed Scopus (92) Google Scholar, 7Balkwill F. Mantovani A. Lancet. 2001; 357: 539-545Abstract Full Text Full Text PDF PubMed Scopus (5803) Google Scholar). Pro-inflammatory cytokines, such as IL-1β1 or transforming growth factor α, have been shown to protect human keratinocytes and epidermal-derived carcinomas against numerous pro-apoptotic stimuli (8Kothny-Wilkes G. Kulms D. Poppelmann B. Luger T.A. Kubin M. Schwarz T. J. Biol. Chem. 1998; 273: 29247-29253Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 9Kothny-Wilkes G. Kulms D. Luger T.A. Kubin M. Schwarz T. J. Biol. Chem. 1999; 274: 28916-28921Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 10Reinartz J. Bechtel M.J. Kramer M.D. Exp. Cell. Res. 1996; 228: 334-340Crossref PubMed Scopus (33) Google Scholar, 11Qin J.Z. Chaturvedi V. Denning M.F. Choubey D. Diaz M.O. Nickoloff B.J. J. Biol. Chem. 1999; 274: 37957-37964Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Thus the anti-apoptotic effects exerted by pro-inflammatory mediators may provide a mechanism for decreased effectiveness of chemotherapy. Furthermore, the protective effect of these cytokines depends upon the de novo synthesis of protective proteins (10Reinartz J. Bechtel M.J. Kramer M.D. Exp. Cell. Res. 1996; 228: 334-340Crossref PubMed Scopus (33) Google Scholar, 11Qin J.Z. Chaturvedi V. Denning M.F. Choubey D. Diaz M.O. Nickoloff B.J. J. Biol. Chem. 1999; 274: 37957-37964Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) implicating the activation of epidermal transcription factors. One potential transcription factor that may mediate anti-apoptotic effects in epithelial cells is NF-κB. NF-κB proteins are sequence-specific transcription factors induced in response to inflammatory and other stressful stimuli (12Foo S.Y. Nolan G.P. Trends Genet. 1999; 15: 229-235Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 13Kaufman C.K. Fuchs E. J. Cell Biol. 2000; 149: 999-1004Crossref PubMed Scopus (102) Google Scholar). Indeed, exposure to IL-1β or interferon-γ has been shown to activate NF-κB in primary cultures of human keratinocytes and in transformed keratinocytes (8Kothny-Wilkes G. Kulms D. Poppelmann B. Luger T.A. Kubin M. Schwarz T. J. Biol. Chem. 1998; 273: 29247-29253Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar,14Chaturvedi V. Qin J.Z. Denning M.F. Choubey D. Diaz M.O. Nickoloff B.J. J. Biol. Chem. 1999; 274: 23358-23367Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). These studies determined that activation of NF-κB mediated the observed anti-apoptotic activity exerted by IL-1β against TRAIL-induced apoptosis (8Kothny-Wilkes G. Kulms D. Poppelmann B. Luger T.A. Kubin M. Schwarz T. J. Biol. Chem. 1998; 273: 29247-29253Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Coincident to the protection from apoptosis, recent evidence indicates that activation of epidermal NF-κB enhances the expression of the inhibitor of apoptosis proteins (IAP), such as c-IAP1 and c-IAP2, which interfere with apoptosis (15Wang C.Y. Mayo M.W. Baldwin Jr., A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2499) Google Scholar,16Seitz C.S. Freiberg R.A. Hinata K. Khavari P.A. J. Clin. Invest. 2000; 105: 253-260Crossref PubMed Scopus (95) Google Scholar). Expression of IAPs and other anti-apoptotic proteins have been shown to inhibit apoptosis in various cell types (17Wang C.Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin Jr., A.S. Science. 1998; 281: 1680-1683Crossref PubMed Scopus (2553) Google Scholar, 18Chu Z.L. McKinsey T.A. Liu L. Gentry J.J. Malim M.H. Ballard D.W. Proc. Natl. Acad. Sci. 1997; 94: 10057-10062Crossref PubMed Scopus (819) Google Scholar, 19Erl W. Hansson G.K. Martin R. Draude G. Weber K. Weber C. Circ. Res. 1999; 84: 668-677Crossref PubMed Scopus (146) Google Scholar). Accumulating evidence suggests platelet-activating factor (PAF)-mediated pathways are involved in cutaneous inflammation and keratinocyte stress responses (20Prescott S.M. Zimmerman G.A. Stafforini D.M. McIntyre T.M. Annu. Rev. Biochem. 2000; 69: 419-445Crossref PubMed Scopus (578) Google Scholar). PAF (1-alkyl-2-acetyl glycerophosphocholine) is a glycerophosphocholine-derived lipid mediator implicated in numerous inflammatory processes. Keratinocytes synthesize PAF and related 1-acyl-PAF-like species in response to various stimuli including ionophores, growth factors, PAF agonists, the pro-oxidative stressor, tert-butyl hydroperoxide, ultraviolet light irradiation, or acute thermal damage (21Travers J.B. Huff J.C. Rola-Plaeszczynski M. Gelfand E.W. Morelli J.G. Murphy R.C. J. Invest. Dermatol. 1995; 105: 816-823Abstract Full Text PDF PubMed Scopus (65) Google Scholar, 22Travers J.B. Harrison K.A. Johnson C.A. Clay K.L. Morelli J.G. Murphy R.C. J. Invest. Dermatol. 1996; 107: 88-94Abstract Full Text PDF PubMed Scopus (43) Google Scholar, 23Dy L.C. Pei Y. Travers J.B J. Biol. Chem. 1999; 274: 26917-26922Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 24Alappatt C. Johnson C.A. Clay K.L. Travers J.B. Arch. Dermatol. Res. 2000; 292: 256-259Crossref PubMed Scopus (54) Google Scholar). Although PAF can be metabolized to other biologically active lipids, the majority of PAF effects appear to be mediated by interaction with a G protein-coupled receptor (GPCR), the PAF receptor (PAF-R). In addition to producing PAF, keratinocytes also express the PAF-R (21Travers J.B. Huff J.C. Rola-Plaeszczynski M. Gelfand E.W. Morelli J.G. Murphy R.C. J. Invest. Dermatol. 1995; 105: 816-823Abstract Full Text PDF PubMed Scopus (65) Google Scholar), and activation of the epidermal PAF-R leads to the production and release of PAF, IL-6, IL-8, TNFα, and eicosanoids (23Dy L.C. Pei Y. Travers J.B J. Biol. Chem. 1999; 274: 26917-26922Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 25Pei Y. Barber L.A. Murphy R.C. Johnson C.A. Kelley S.A. Dy L.C. Fertel R.H. Nguyen T.M. Williams D.A. Travers J.B. J. Immunol. 1998; 161: 1954-1961PubMed Google Scholar). Although the ability of cytokines to protect keratinocytes from apoptosis has been established, and potential mechanisms for this protection have been suggested, it is unclear whether PAF has anti-apoptotic effects. However, several lines of evidence suggest that epidermal PAF may inhibit apoptosis. First, PAF can be produced under the same conditions that induce apoptosis and thus may have a role in the regulation of apoptosis. Exposure to chemotherapeutic agents, such as cisplatin, gentamycin, or 5-fluorouracil have been shown to induce PAF production (26Dos Santos O.F. Boim M.A. Barros E.J. Schor N. Kidney Int. 1991; 40: 742-747Abstract Full Text PDF PubMed Scopus (43) Google Scholar, 27Denizot Y. Dupuis F. Comte L. Dulery C. Praloran V. Cancer Lett. 1995; 88: 185-189Crossref PubMed Scopus (2) Google Scholar). Second, activation of the PAF-R expressed in Chinese hamster ovary cells has been shown to stimulate NF-κB and induce gene expression (28Kravchenko V.V. Pan Z. Han J. Herbert J.M. Ulevitch R.J. Ye R.D. J. Biol. Chem. 1995; 270: 14928-14934Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Thus, activation of the keratinocyte PAF-R may induce NF-κB expression and protect keratinocytes in a manner similar to cytokines. Of significance, activation of the PAF-R has been shown to rescue B lymphocytes from apoptotic stimuli through an unknown mechanism (29Toledano B.J. Bastein Y. Noya F. Mazer B. Cell. Immunol. 1999; 191: 60-68Crossref PubMed Scopus (16) Google Scholar). Because PAF is produced during inflammation and oxidative stress and activates signal transduction pathways similar to the anti-apoptotic effects of growth factors and cytokines, we sought to determine whether apoptosis in epidermal cells can be modulated by PAF. In this paper, we report that activation of the epidermal PAF-R protects from apoptosis induced by either TNFα or TRAIL. This protective effect was inhibited by pretreatment with PAF-R antagonists. Stimulation of epidermal cells with a PAF receptor agonist resulted in a rapid degradation of IκB and subsequent increase in NF-κB binding. Furthermore, the protective effect induced by PAF-R activation was abolished in epidermal cells expressing a super-repressor form of IκB, indicating that the PAF-R-induced protective effect was mediated by activation of an NF-κB-dependent pathway. Thus, these results demonstrate that, in addition to the anti-apoptotic actions induced by growth factors and cytokines, activation of GPCRs such as PAF-R may also protect epithelial cells from pro-apoptotic agents. All chemicals were obtained from Sigma unless indicated otherwise. Recombinant human TRAIL/APO2L was purchased from Chemicon (Temecula, CA). Recombinant human IL-1β and TNFα were from PeproTech (Rocky Hill, NJ), the broad-spectrum caspase inhibitor 2-Val-Ala-Asp-FMK (2-VAD-FMK) was purchased from Alexis Biochemicals (San Diego, CA). The PAF-R antagonist WEB-2086 was kindly provided by Boehringer Ingelheim (Ridgefield, CT) and A-85783 was a gift from Dr. James Summers (Abbot Pharmaceuticals, Abbott Park, IL). The human epidermoid cell line KB and human keratinocyte cell line HaCaT cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) were supplemented with 10% fetal bovine serum (Intergen, Purchase, NY). A KB PAF-R model system was created by transduction of PAF-R-negative KB cells with the MSCV2.1 retrovirus encoding the human leukocyte PAF-R as described previously (25Pei Y. Barber L.A. Murphy R.C. Johnson C.A. Kelley S.A. Dy L.C. Fertel R.H. Nguyen T.M. Williams D.A. Travers J.B. J. Immunol. 1998; 161: 1954-1961PubMed Google Scholar, 30Travers J.B. J. Invest. Dermatol. 1999; 112: 279-283Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). KB cells transduced with the PAF-R (KBP) or with control MSCV2.1 retrovirus (KBM) were characterized by Southern and Northern blot analysis and by radioligand binding and calcium mobilization studies to demonstrate that the PAF-R was functional (25Pei Y. Barber L.A. Murphy R.C. Johnson C.A. Kelley S.A. Dy L.C. Fertel R.H. Nguyen T.M. Williams D.A. Travers J.B. J. Immunol. 1998; 161: 1954-1961PubMed Google Scholar, 30Travers J.B. J. Invest. Dermatol. 1999; 112: 279-283Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). All experiments were replicated with at least two separate KBM and KBP clones. The IκBM containing S32A and S36A mutation of IκBα has previously been described (31Newton T.R. Patel N.M. Bhat-Nakshatri P. Stauss C.R. Goulet Jr., R.J. Nakshatri H. J. Biol. Chem. 1999; 274: 18827-18835Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), and the retroviral DNA vector MIEG3, which uses enhanced green fluorescent protein as the selectable marker, was a kind gift of Dr. David Williams (Indiana University) (32Williams D.A. Tao W. Yang F. Kim C. Gu Y. Mansfield P. Levine J.E. Petryniak B. Derrow C.W. Harris C. Jia B. Zheng Y. Ambruso D.R. Lowe J.B. Atkinson S.J. Dinauer M.C. Boxer L. Blood. 2000; 96: 1646-1654PubMed Google Scholar). To create KBP cells stably expressing IκBM, the IκBM cDNA was subcloned into the EcoRI site of MIEG3, and orientation was assessed by restriction endonuclease mapping and sequencing. Infectious amphotropic retroviruses were produced from both MIEG-IκBM and control MIEG backbone by transient transfection using standard protocols (33Grignani F. Kinsella T. Mencarelli A. Valtieri M. Riganelli D. Grignani F. Lanfrancone L. Peschle C. Nolan G.P. Pelicci P.G. Cancer Res. 1998; 58: 14-19PubMed Google Scholar). Briefly, the Phoenix amphotropic packaging cell line was transfected with the DNA constructs using Fugene-6, and the supernatants collected 48 h later containing infectious virions were then used to infect KBP cells. Transduced cells were fluorescence-activated cell sorter-sorted on the basis of enhanced green fluorescent protein expression 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 Molecular Biochemicals) and 10 mm sodium orthovanadate for 20 min on ice. IκB degradation was determined by immunoblotting with polyclonal anti-IκBα antibody (Santa Cruz Biotechnology) and enhanced chemiluminescence (Amersham Pharmacia Biotech). Cells were plated at a density of 1.0 × 104 cells/well in 96-well plates and allowed to stabilize for 1 day. Cells were then treated for 24 h with fresh medium containing the indicated agents. Cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Roche Molecular Biochemicals) and analyzed using a microplate reader (Molecular Devices, Sunnyvale, CA). The effects of treatment are expressed as a percentage of viable cells using the untreated cells as the maximum cell viability. Low molecular weight DNA was extracted from KB cells after TNFα or TRAIL treatment at the indicated times using the Stratagene DNA extraction kit (Stratagene, La Jolla, CA). 40 μg of total genomic DNA was separated on a 2.0% agarose gel, and the gel was stained with ethidium bromide. Apoptosis was determined quantitatively using a cell death detection enzyme-linked immunosorbent assay (Roche Molecular Biochemicals) according to the manufacturer's instructions. The kit measures the enrichment of mono- and oligonucleosomes released into the cytoplasm of apoptotic cells as a result of DNA degradation. The absorption was measured at 405 nm using a microplate reader (Molecular Devices, Sunnyvale, CA). The enrichment factor was calculated using the following formula: absorbance of apoptotic cells/absorbance of control cells. The activation of the caspase proteolytic cascade was measured by the direct assay of caspase-3 enzyme activity in cell lysates using a synthetic fluorogenic substrate (caspase-3 substrate, Ac-Asp-Glu-Val-Asp-AMC (Ac-DEVD-AMC); Alexis Biochemicals, San Diego, CA) as described previously (34Kuhn C. Hurwitz S.A. Kumar M.G. Cotton J. Spandau D.F. Int. J. Cancer. 1999; 80: 431-438Crossref PubMed Scopus (101) Google Scholar). Reactions were performed for 1 h at 37 °C. Release of the fluorogenic AMC moiety was measured using a Hitachi F2000 spectrophotometer (excitation, 380 nm; detection, 460 nm). The fluorescent intensity was converted to pmol of AMC released by comparison to standards of AMC (Molecular Probes, Eugene, OR). The specific activity of caspase-3 in cell lysates was determined following quantitation of the total protein in the cell lysates (NanoOrange protein quantitation reagent; Molecular Probes, Eugene, OR). Whole cell extracts were prepared and subjected to electrophoretic mobility shift assay as described previously (31Newton T.R. Patel N.M. Bhat-Nakshatri P. Stauss C.R. Goulet Jr., R.J. Nakshatri H. J. Biol. Chem. 1999; 274: 18827-18835Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Protein-DNA binding reactions were performed for 30 min at room temperature using 6 μg of whole cell extract and32P-labeled oligonucleotide probes for NF-κB consensus binding sites (Promega, Madison, WI). Complexes were separated by electrophoresis on nondenaturing 6% acrylamide gel and assayed by autoradiography. Cells were plated at a density of 1.5 × 106 cells in a 10-cm dish and allowed to stabilize for 1 day. Cells were then transfected using Fugene6 (Roche Molecular Biochemicals) with 10 μg of NF-κB-luciferase reporter plasmid (pNF-κB-luc) and 10 μg of pCMV-β-galactosidase as an internal control for the transfection efficiency. 24 h after transfection, cells were treated with CPAF and IL-1β or were mock-treated and then harvested in reporter lysis buffer (Promega) following an additional 6-h incubation. 20-μl aliquots of the lysates were assayed for β-galactosidase and luciferase activities using an LB9501 luminometer (Lumat). Luciferase activities were normalized for each transfection using the control β-galactosidase activities. Because PAF may have both receptor-dependent and -independent effects (secondary to the formation of biologically active metabolites), our laboratory has previously created a model system by transduction of the PAF-R into a PAF-R-deficient epidermal cell line to study the role of the PAF-R in keratinocyte cell biology. The human epidermal cell line KB, originally obtained from a patient with an oral squamous cell carcinoma (36Eagle H. Proc. Soc. Exp. Biol. Med. 1955; 89: 362-366Crossref PubMed Scopus (298) Google Scholar), does not express functional PAF-Rs, unlike normal human keratinocytes and the human keratinocyte-derived cell line HaCaT (21Travers J.B. Huff J.C. Rola-Plaeszczynski M. Gelfand E.W. Morelli J.G. Murphy R.C. J. Invest. Dermatol. 1995; 105: 816-823Abstract Full Text PDF PubMed Scopus (65) Google Scholar, 22Travers J.B. Harrison K.A. Johnson C.A. Clay K.L. Morelli J.G. Murphy R.C. J. Invest. Dermatol. 1996; 107: 88-94Abstract Full Text PDF PubMed Scopus (43) Google Scholar). A PAF-R-positive KB cell line, KBP, was created by transducing KB cells with a replication-deficient MSCV2.1 retrovirus containing the human PAF-R cDNA. KB cells were also transduced with the retrovirus backbone alone to establish a vector control cell line, KBM. Expression of the PAF-R protein was verified by binding studies using radiolabeled PAF-R antagonist WEB-2086 (25Pei Y. Barber L.A. Murphy R.C. Johnson C.A. Kelley S.A. Dy L.C. Fertel R.H. Nguyen T.M. Williams D.A. Travers J.B. J. Immunol. 1998; 161: 1954-1961PubMed Google Scholar). Calcium mobilization studies demonstrated that the KB PAF-R was functionally active (25Pei Y. Barber L.A. Murphy R.C. Johnson C.A. Kelley S.A. Dy L.C. Fertel R.H. Nguyen T.M. Williams D.A. Travers J.B. J. Immunol. 1998; 161: 1954-1961PubMed Google Scholar, 30Travers J.B. J. Invest. Dermatol. 1999; 112: 279-283Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Therefore, this in vitroepidermoid system consists of both PAF-R-negative (KBM) and -positive (KBP) cells. In initial experiments, the dose-response and time-response of TNFα- and TRAIL-induced apoptosis were determined using activation of caspase-3, a cell death detection enzyme-linked immunosorbent assay, and DNA fragmentation as three distinct markers of apoptosis in epidermal cells. Exposing KBP cells to 100 ng/ml TNFα or 40 ng/ml TRAIL for various times was found to induce caspase-3-specific activity (Fig. 1 A), enrich the release of mono- and oligonucleosomes into the cytoplasm (Fig. 1 B), and enhance DNA fragmentation as shown by DNA laddering (Fig.1 C). Inasmuch as these markers of apoptosis determined that TNFα and TRAIL treatment resulted in a maximal apoptotic effect after 24 h, subsequent experiments therefore examined apoptosis at the 24-h time point. In addition to KBP cells, treatment with TNFα- or TRAIL-induced apoptosis in KBM and HaCaT cells was similar to that observed in KBP cells (data not shown). To determine whether PAF-R activation affects TNF family-mediated apoptosis in epidermal cells, KB cells were pre-treated with the metabolically stable PAF agonist, CPAF. Pre-treatment of KBP cells for 1 h with 100 nm CPAF resulted in a decrease in the TRAIL- and TNFα-induced apoptosis (Fig.2 A, black bars), shown as a decrease in caspase-3-specific activity and decreased enrichment of nucleosomes in the cytoplasm (Fig. 2 B); however, CPAF had no effect on apoptosis in the PAF-R-deficient KBM cells (Fig. 2 A, gray bars). In contrast to the actions of CPAF, pre-treating cells for 1 h with 25 ng/ml IL-1β protected both KBP and KBM cells from TRAIL- and TNFα-induced apoptosis. These results confirm the studies of Schwartz and co-workers (8Kothny-Wilkes G. Kulms D. Poppelmann B. Luger T.A. Kubin M. Schwarz T. J. Biol. Chem. 1998; 273: 29247-29253Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 9Kothny-Wilkes G. Kulms D. Luger T.A. Kubin M. Schwarz T. J. Biol. Chem. 1999; 274: 28916-28921Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) that IL-1β protects KB cells from TRAIL-induced apoptosis. Furthermore these results suggest that the anti-apoptotic effects of PAF are mediated by activation of the PAF-R, because CPAF inhibited apoptosis in KBP, but not KBM, cells. To further confirm whether the protective effects of CPAF were mediated through a PAF-R-dependent mechanism, we determined whether the selective PAF-R antagonists A-85783 (ABT) or WEB2086 (WEB) could block the protective effects of PAF. As shown in Fig.3 A, exposure to WEB or ABT abolished the anti-apoptotic effects of PAF. Neither ABT nor WEB had any direct effect alone on TRAIL-induced apoptosis. In addition, pre-treatment with WEB or ABT did not affect the anti-apoptotic effects of IL-1β in KBP cells (Fig. 3 B). Taken together, these results indicate that activation of the PAF-R mediates the protective effects of PAF on epidermal cells. To address whether the protective effects induced by CPAF were secondary to the overexpression of PAF-R in KBP cells, we also examined the protective role of physiological levels of the PAF-R in the immortalized keratinocyte cell line, HaCaT. Our laboratory has demonstrated previously that HaCaT cells express functional PAF-Rs (21Travers J.B. Huff J.C. Rola-Plaeszczynski M. Gelfand E.W. Morelli J.G. Murphy R.C. J. Invest. Dermatol. 1995; 105: 816-823Abstract Full Text PDF PubMed Scopus (65) Google Scholar,25Pei Y. Barber L.A. Murphy R.C. Johnson C.A. Kelley S.A. Dy L.C. Fertel R.H. Nguyen T.M. Williams D.A. Travers J.B. J. Immunol. 1998; 161: 1954-1961PubMed Google Scholar). As illustrated in Fig. 4, treatment with 100 nm CPAF or 25 ng/ml IL-1β for 1 h prior to exposure to TRAIL or TNFα protected HaCaT cells from TNFα- and TRAIL-induced apoptosis in a manner similar to the protective effect observed with KBP cells. Taken together, these results demonstrate that activation of the PAF-R, a GPCR, protects keratinocyte and epidermal carcinoma cells from apoptosis. To determine whether the PAF-R-mediated protection from apoptosis would affect the survival of epidermal cells, we examined the viability of cells treated with either TRAIL or TNFα in the presence or absence of CPAF, IL-1β, or the nonspecific caspase inhibitor z-VAD-fmk. TRAIL and TNFα treatment decreased the number of viable KBP cells by 41.6 ± 1.8 and 49.5 ± 8.3%, respectively (Fig.5). Pre-treatment of KBP cells for 1 h with 100 nm CPAF or 25 ng/ml IL-1β protected cells, shown as a 25–30% increase in cell viability. Treatment with 10 μm of the caspase inhibitor z-VAD-fmk also blocked the decrease in cell viability induced by TRAIL and TNFα. Thus, the cytoprotective actions induced by the PAF-R exerted a survival effect on TRAIL- and TNFα-treated epidermal cells, presumably by inhibiting apoptosis. Because pro-inflammatory cytokines and growth factors can induce anti-apoptotic effects through activation of the transcription factor NF-κB, we next examined whether the PAF-R protective effects were similarly mediated through an NF-κB-dependent pathway. Exposure to CPAF or IL-1β induced a time-dependent degradation of IκBα in KBP cells resulting in the disappearance of immunoreactive IκB within 15–20 min (Fig.6 A). Secondary to the degradation of IκBα, NF-κB binding activity was increased and reached maximal activity ∼1 h after treatment (Fig. 6 B). However, CPAF did not induce IκB degradation nor increase NF-κB binding activity in control KBM cells unlike IL-1β, which induced NF-κB in both KBP and KBM cells (data not shown). To establish that the PAF-R is functionally coupled to a pathway leading to activation of NF-κB, KBP cells were transfected with a luciferase reporter containing NF-κB consensus binding sites and were then stimulated with CPAF or IL-1β. Exposure to CPAF or IL-1β resulted in a 6.2 ± 1.0 (n = 6)- or 5.1 ± 0.5 (n = 6)-fold increase in NF-κB-dependent gene transcription over unstimulated cells, respectively (Fig.6 C). Thus, activation of the PAF-R is functionally coupled to a biochemical pathway that results in NF-κB activation and gene transcription. To determine whether the PAF-R protective effects were dependent on the NF-κB pathway, KBP cells were transduced by retroviral infection to express a super-repressor IκB protein (KBP-MIEG-IκBM). Whereas both CPAF and IL-1β induced IκB degradation and increased NF-κB binding activity in KBP cells transfected with the vector backbone (KBP-MIEG), the stimulated IκB degradation (Fig.7 A) and NF-κB binding activity (Fig. 7 B) were abolished in KBP-MIEG-IκBM cells. Furthermore, the CPAF- and IL-1β-induced protection from TNFα- and TRAIL-induced apoptosis were decreased in KBP-MIEG-IκBM cells (Fig.7, C and D), indicating that the anti-apoptotic activity induced by PAF-R is mediated by an NF-κB-dependent pathway. In addition to abolishing the" @default.
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