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Potentiation of Tumor Necrosis Factor α-induced Secreted Phospholipase A2 (sPLA2)-IIA Expression in Mesangial Cells by an Autocrine Loop Involving sPLA2 and Peroxisome Proliferator-activated Receptor α Activation

2003; Elsevier BV; Volume: 278; Issue: 32 Linguagem: Inglês

10.1074/jbc.m211763200

ISSN

1083-351X

Autores

Sabine Gehrke-Beck, Gérard Lambeau, Kristen Scholz-Pedretti, Michael H. Gelb, Marcel J.W. Janssen, S.H. Edwards, David C. Wilton, Josef Pfeilschifter, Marietta Kaszkin,

Tópico(s)

Metabolism, Diabetes, and Cancer

Resumo

In rat mesangial cells, exogenously added secreted phospholipases A2 (sPLA2s) potentiate the expression of pro-inflammatory sPLA2-IIA first induced by cytokines like tumor necrosis factor-α (TNFα) and interleukin-1β. The transcriptional pathway mediating this effect is, however, unknown. Because products of PLA2 activity are endogenous activators of peroxisome proliferator-activated receptor α (PPARα, we postulated that sPLA2s mediate their effects on sPLA2-IIA expression via sPLA2 activity and subsequent PPARα activation. This study shows that various sPLA2s, including venom enzymes, human sPLA2-IIA, and wild-type and catalytically inactive H48Q mutant of porcine pancreatic sPLA2-IB, enhance the TNFα-induced sPLA2-IIA expression at the mRNA and protein levels. In cells transfected with luciferase sPLA2-IIA promoter constructs, sPLA2s are active only when the promoter contains a functional PPRE-1 site. The effect of exogenous sPLA2s is also blocked by the PPARα inhibitor MK886. Interestingly, the expression of sPLA2-IIA induced by TNFα alone is also attenuated by MK886, by the sPLA2-IIA inhibitor LY311727, by heparinase, which prevents the binding of sPLA2-IIA to heparan sulfate proteoglycans, and by the specific cPLA2-α inhibitor pyrrolidine-1. Together, these data indicate that sPLA2-IIA released from mesangial cells by TNFα stimulates its own expression via an autocrine loop involving cPLA2 and PPARα. This signaling pathway is also used by exogenously added sPLA2s including pancreatic sPLA2-IB and is distinct from that used by TNFα. In rat mesangial cells, exogenously added secreted phospholipases A2 (sPLA2s) potentiate the expression of pro-inflammatory sPLA2-IIA first induced by cytokines like tumor necrosis factor-α (TNFα) and interleukin-1β. The transcriptional pathway mediating this effect is, however, unknown. Because products of PLA2 activity are endogenous activators of peroxisome proliferator-activated receptor α (PPARα, we postulated that sPLA2s mediate their effects on sPLA2-IIA expression via sPLA2 activity and subsequent PPARα activation. This study shows that various sPLA2s, including venom enzymes, human sPLA2-IIA, and wild-type and catalytically inactive H48Q mutant of porcine pancreatic sPLA2-IB, enhance the TNFα-induced sPLA2-IIA expression at the mRNA and protein levels. In cells transfected with luciferase sPLA2-IIA promoter constructs, sPLA2s are active only when the promoter contains a functional PPRE-1 site. The effect of exogenous sPLA2s is also blocked by the PPARα inhibitor MK886. Interestingly, the expression of sPLA2-IIA induced by TNFα alone is also attenuated by MK886, by the sPLA2-IIA inhibitor LY311727, by heparinase, which prevents the binding of sPLA2-IIA to heparan sulfate proteoglycans, and by the specific cPLA2-α inhibitor pyrrolidine-1. Together, these data indicate that sPLA2-IIA released from mesangial cells by TNFα stimulates its own expression via an autocrine loop involving cPLA2 and PPARα. This signaling pathway is also used by exogenously added sPLA2s including pancreatic sPLA2-IB and is distinct from that used by TNFα. During the last decade, increasing evidence has been obtained that secreted phospholipases A2 (sPLA2) 1The abbreviations used are: sPLA2, secreted phospholipase A2; cPLA2, cytosolic phospholipase A2; TNFα, tumor necrosis factor-α; IL, interleukin; PPARα, peroxisome proliferator-activated receptor α; PPRE, peroxisome proliferator-responsive element; kbp, kilobase pair(s); HSPG, heparan sulfate proteoglycan; 5-LOX, 5-lipoxygenase; FLAP, 5-lipoxygenase-activating protein; BSA, bovine serum albumin; PBS, phosphate-buffered saline; RXRα, 9-cis-retinoic acid receptor-α; EMSA, electrophoretic mobility shift assay; RT, reverse transcription. are important players in inflammatory diseases. Among the various sPLA2s that have now been identified (1Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1220) Google Scholar, 2Valentin E. Lambeau G. Biochim. Biophys. Acta. 2000; 1488: 59-70Crossref PubMed Scopus (314) Google Scholar, 3Gelb M.H. Valentin E. Ghomashchi F. Lazdunski M. Lambeau G. J. Biol. Chem. 2000; 275: 39823-39826Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), group IIA sPLA2 (sPLA2-IIA) has been found to be expressed at very high levels in various acute and chronic inflammatory diseases like sepsis (4Guidet B. Piot O. Masliah J. Barakett V. Maury E. Bereziat G. Offenstadt G. Infection. 1996; 24: 103-108Crossref PubMed Scopus (46) Google Scholar), asthma (5Calabrese C. Triggiani M. Marone G. Mazzarella G. Allergy. 2000; 55: 27-30Crossref PubMed Scopus (43) Google Scholar), and rheumatoid arthritis (6Bidgood M.J. Jamal O.S. Cunningham A.M. Brooks P.M. Scott K.F. J. Immunol. 2000; 165: 2790-2797Crossref PubMed Scopus (101) Google Scholar). sPLA2-IIA is also highly expressed in the kidney during experimental and human acute pancreatitis (7Nevalainen T.J. Hietaranta A.J. Gronroos J.M. Hepatogastroenterology. 1999; 46: 2731-2735PubMed Google Scholar), which can lead to post-injury multiple organ failure (8Partrick D.A. Moore E.E. Silliman C.C. Barnett C.C. Kuypers F.A. Crit. Care Med. 2001; 29: 989-993Crossref PubMed Scopus (33) Google Scholar). In rat renal mesangial cells, which are used as a cell model system to study inflammatory processes, it was shown earlier that cAMP-elevating agents and proinflammatory cytokines such as interleukin-1β and TNFα stimulate the gene expression and secretion of sPLA2-IIA by different transcriptional activation pathways (9Pfeilschifter J. Mühl H. Pignat W. Märki F. van den Bosch H. Eur. J. Clin. Pharmacol. 1993; 44: S7-S9Crossref PubMed Scopus (21) Google Scholar, 10Walker G. Kunz D. Pignat W. van den Bosch H. Pfeilschifter J. FEBS Lett. 1995; 364: 218-222Crossref PubMed Scopus (35) Google Scholar, 11Scholz-Pedretti K. Eberhardt W. Rupprecht G. Beck K.F. Spitzer S. Pfeilschifter J. Kaszkin M. Br. J. Pharmacol. 2000; 130: 1183-1190Crossref PubMed Scopus (13) Google Scholar). The functional role of the sPLA2-IIA released from mesangial cells is, however, less clear. In rat mesangial cells, it has been shown that exogenously added sPLA2-IIA acts as a growth factor mediating the action of IL-1β on cell proliferation, and that this effect is mimicked by lysophospholipids (12Wada A. Tojo H. Sugiura T. Fujiwara Y. Kamada T. Ueda N. Okamoto M. Biochim. Biophys. Acta. 1997; 1345: 99-108Crossref PubMed Scopus (23) Google Scholar). Exogenous sPLA2-IIA and lysophospholipids were also found to rapidly stimulate the mitogen activated protein kinase cascade in mesangial cells, leading to early activation of cPLA2 (13Sugiura T. Wada A. Itoh T. Tojo H. Okamoto M. Imai E. Kamada T. Ueda N. FEBS Lett. 1995; 370: 141-145Crossref PubMed Scopus (25) Google Scholar, 14Huwiler A. Staudt G. Kramer R.M. Pfeilschifter J. Biochim. Biophys. Acta. 1997; 1348: 257-272Crossref PubMed Scopus (82) Google Scholar). It is, however, unclear whether sPLA2-IIA released by mesangial cells after cytokine induction acts like exogenously added sPLA2-IIA and, in particular, whether sPLA2-IIA can exert a positive feedback amplification loop on its own gene expression by activating one of the above signaling pathways and/or other pathways (see below). High levels of another sPLA2 subtype, the so-called pancreatic-type sPLA2-IB, are also found in kidney during acute pancreatitis (7Nevalainen T.J. Hietaranta A.J. Gronroos J.M. Hepatogastroenterology. 1999; 46: 2731-2735PubMed Google Scholar, 15Uhl W. Schrag H.J. Schmitter N. Nevalainen T.J. Aufenanger J. Wheatley A.M. Buchler M.W. Gut. 1997; 40: 386-392Crossref PubMed Scopus (50) Google Scholar), suggesting that sPLA2-IB may also contribute to the pathophysiological effects in such conditions. In rat mesangial cells, exogenously added pancreatic sPLA2-IB can stimulate the mRNA and protein expression of sPLA2-IIA, as well as prostaglandin biosynthesis (16Kishino J. Ohara O. Nomura K. Kramer R.M. Arita H. J. Biol. Chem. 1994; 269: 5092-5098Abstract Full Text PDF PubMed Google Scholar, 17Kishino J. Kawamoto K. Ishizaki J. Verheij H.M. Ohara O. Arita H. J. Biochem. (Tokyo). 1995; 117: 420-424Crossref PubMed Scopus (32) Google Scholar). This effect is thought to involve binding of sPLA2-IB to the M-type sPLA2 receptor expressed in mesangial cells (17Kishino J. Kawamoto K. Ishizaki J. Verheij H.M. Ohara O. Arita H. J. Biochem. (Tokyo). 1995; 117: 420-424Crossref PubMed Scopus (32) Google Scholar). This view is strengthened by the fact that a catalytically inactive mutant of sPLA2-IB, which still binds to the M-type receptor, has effects similar to those of the wild-type enzyme (16Kishino J. Ohara O. Nomura K. Kramer R.M. Arita H. J. Biol. Chem. 1994; 269: 5092-5098Abstract Full Text PDF PubMed Google Scholar). Interestingly, sPLA2-IB from different species including rat, but not rat sPLA2-IIA, binds to the M-type receptor expressed in rat mesangial cells (17Kishino J. Kawamoto K. Ishizaki J. Verheij H.M. Ohara O. Arita H. J. Biochem. (Tokyo). 1995; 117: 420-424Crossref PubMed Scopus (32) Google Scholar, 18Cupillard L. Mulherkar R. Gomez N. Kadam S. Valentin E. Lazdunski M. Lambeau G. J. Biol. Chem. 1999; 274: 7034-7051Abstract Full Text Full Text PDF Scopus (124) Google Scholar, 19Hanasaki K. Arita H. Arch. Biochem. Biophys. 1999; 372: 215-223Crossref PubMed Scopus (69) Google Scholar), suggesting that sPLA2-IIA acts through binding to a different cell membrane target. When exogenously added to other cell types, sPLA2-IB was also found to activate the expression of a number of pro-inflammatory genes including cyclooxygenase-2 (20Tohkin M. Kishino J. Ishizaki J. Arita H. J. Biol. Chem. 1993; 268: 2865-2871Abstract Full Text PDF PubMed Google Scholar, 21Yuan C.J. Mandal A.K. Zhang Z. Mukherjee A.B. Cancer Res. 2000; 60: 1084-1091PubMed Google Scholar), sphingomyelinase, and ceramidase (22Mandal A.K. Zhang Z. Chou J.Y. Mukherjee A.B. FASEB J. 2001; 15: 1834-1836Crossref PubMed Scopus (22) Google Scholar). On the other hand, exogenously added sPLA2-IIA can induce the activation of cPLA2 and cyclooxygenase-2 (3Gelb M.H. Valentin E. Ghomashchi F. Lazdunski M. Lambeau G. J. Biol. Chem. 2000; 275: 39823-39826Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 23Hernandez M. Fuentes L. Fernandez Aviles F.J. Crespo M.S. Nieto M.L. Circ. Res. 2002; 90: 38-45Crossref PubMed Scopus (42) Google Scholar, 24Tada K. Murakami M. Kambe T. Kudo I. J. Immunol. 1998; 161: 5008-5015PubMed Google Scholar), the release of elastase (25Zallen G. Moore E.E. Johnson J.L. Tamura D.Y. Barkin M. Stockinger H. Silliman C.C. Arch. Surg. 1998; 133: 1229-1233Crossref PubMed Scopus (30) Google Scholar) and β-glucuronidase (26Triggiani M. Granata F. Oriente A. De Marino V. Gentile M. Calabrese C. Palumbo C. Marone G. J. Immunol. 2000; 164: 4908-4915Crossref PubMed Scopus (74) Google Scholar), and the expression of Mac-1 (27Takasaki J. Kawauchi Y. Yasunaga T. Masuho Y. J. Leukoc. Biol. 1996; 60: 174-180Crossref PubMed Scopus (16) Google Scholar), IL-6 (26Triggiani M. Granata F. Oriente A. De Marino V. Gentile M. Calabrese C. Palumbo C. Marone G. J. Immunol. 2000; 164: 4908-4915Crossref PubMed Scopus (74) Google Scholar), CD-69 (28Urasaki T. Takasaki J. Nagasawa T. Ninomiya H. Inflamm. Res. 2000; 49: 177-183Crossref PubMed Scopus (10) Google Scholar), inducible nitricoxide synthase (29Baek S.H. Lim J.H. Park D.W. Kim S.Y. Lee Y.H. Kim J.R. Kim J.H. Eur. J. Immunol. 2001; 31: 2709-2717Crossref PubMed Scopus (27) Google Scholar), and Fas ligand (23Hernandez M. Fuentes L. Fernandez Aviles F.J. Crespo M.S. Nieto M.L. Circ. Res. 2002; 90: 38-45Crossref PubMed Scopus (42) Google Scholar) on different cell types. The nature of the sPLA2-IIA cellular target involved in these biological effects remains, however, to be clearly identified. Although human sPLA2-IIA does not bind to the human M-type receptor (18Cupillard L. Mulherkar R. Gomez N. Kadam S. Valentin E. Lazdunski M. Lambeau G. J. Biol. Chem. 1999; 274: 7034-7051Abstract Full Text Full Text PDF Scopus (124) Google Scholar), it has been proposed that this receptor or a related receptor may be involved, whereas the sPLA2 activity may not play a major role. Heparan sulfate proteoglycans (HSPG) including glypican-1 may also contribute to the effects of sPLA2-IIA (30Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 31Murakami M. Kudo I. J. Biochem. (Tokyo). 2002; 131: 285-292Crossref PubMed Scopus (436) Google Scholar). Besides activation of p38, p42/44, and c-Jun N-terminal kinase kinases by sPLA2-IB or -IIA (13Sugiura T. Wada A. Itoh T. Tojo H. Okamoto M. Imai E. Kamada T. Ueda N. FEBS Lett. 1995; 370: 141-145Crossref PubMed Scopus (25) Google Scholar, 23Hernandez M. Fuentes L. Fernandez Aviles F.J. Crespo M.S. Nieto M.L. Circ. Res. 2002; 90: 38-45Crossref PubMed Scopus (42) Google Scholar, 32Kinoshita E. Handa N. Hanada K. Kajiyama G. Sugiyama M. FEBS Lett. 1997; 407: 343-346Crossref PubMed Scopus (37) Google Scholar), little is known about the transcriptional pathways activated by exogenously added sPLA2s. It has been shown that sPLA2-IB enhances the expression of COX-2 through activation of the transcription factor CCAT/enhancer-binding protein β in NIH3T3 and MC3T3E1 cells (21Yuan C.J. Mandal A.K. Zhang Z. Mukherjee A.B. Cancer Res. 2000; 60: 1084-1091PubMed Google Scholar). Because sPLA2s can produce lipid mediators such as free fatty acids and prostaglandins that are peroxisome proliferator-activated receptors (PPARs) ligands (33Corton J.C Anderson S.P. Stauber A. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 491-518Crossref PubMed Scopus (292) Google Scholar), another attractive hypothesis is that sPLA2s induce the expression of genes containing PPAR-responsive promoters by activating PPAR nuclear receptors. Interestingly, a recent work has shown that sPLA2-IB may exert its proliferative effects via hydrolysis of nuclear phospholipids and activation of PPARα (34Specty O. Pageaux J.F. Dauca M. Lagarde M. Laugier C. Fayard J.M. FEBS Lett. 2001; 490: 88-92Crossref PubMed Scopus (9) Google Scholar). More recently, we and others found that the rat sPLA2-IIA promoter contains peroxisome proliferator-responsive elements (PPRE) (35Scholz-Pedretti K. Gans A. Beck K.F. Pfeilschifter J. Kaszkin M. J. Am. Soc. Nephrol. 2002; 13: 611-620Crossref PubMed Google Scholar, 36Couturier C. Brouillet A. Couriaud C. Koumanov K. Bereziat G. Andreani M. J. Biol. Chem. 1999; 274: 23085-23093Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), suggesting that PPAR activation in rat mesangial cells might be involved in the induction of sPLA2-IIA by exogenously added sPLA2s including sPLA2-IB, and also by rat sPLA2-IIA endogenously produced after cytokine treatment. The purpose of this study was to analyze the role of PPARα in the induction of sPLA2-IIA transcription by cytokines and by exogenous sPLA2s in rat mesangial cells. To determine the role of PPARα and the sPLA2 signaling pathways, we used rat sPLA2-IIA promoter constructs containing functional or mutated PPARα binding sites, as well as PPARα and various PLA2 inhibitors. Altogether, our data indicate that sPLA2-IIA released by mesangial cells after treatment with cytokines potentiates its own expression in a positive feedback loop via activation of cPLA2 and PPARα. Exogenously added sPLA2s including sPLA2-IB also use this transcriptional mechanism to enhance sPLA2-IIA gene expression, although the pathways used at the plasma membrane may differ among sPLA2s. Materials—TNFα was a generous gift from Knoll AG (Ludwigshafen, Germany). IL-1β was obtained from Cell Concept (Umkirch, Germany). [1-14C]Oleic acid, [γ-32P]ATP (185 TBq/mmol), and [α-32P]dCTP (110 TBq/mmol) were from Amersham Biosciences (Freiburg, Germany). Kidneys of rats with Thy-1 glomerulonephritis were a generous gift from Dr. T. Ostendorf (Rheinisch-Westfälische Technische Hochschule, Aachen, Germany). Immobilon-PVDF membranes were purchased from Millipore (Eschborn, Germany), and nylon membranes (GeneScreen) were purchased from PerkinElmer Life Sciences (Köln, Germany). 18 S RNA probe from mouse as well as specific antibodies against PPARα and 9-cis-retinoic acid receptor-α (RXRα) were purchased from Ambion (Wiesbaden, Germany). Pyrrolidine-1 was prepared as described by Ghomashchi et al. (37Ghomashchi F. Stewart A. Hefner Y. Ramanadham S. Turk J. Leslie C.C. Gelb M.H. Biochim. Biophys. Acta. 2001; 1513: 1-7Crossref PubMed Scopus (89) Google Scholar). LY311727 was a generous gift from Eli Lilly (Indianapolis, IN). MK886 was from Biomol (Hamburg, Germany), heparinase-1 was from Sigma (Deisenhofen, Germany), and all other chemicals used were from Sigma, Biomol, or Calbiochem (Bad Soden, Germany). All cell culture media and nutrients were from Invitrogen (Eggenstein, Germany). sPLA2s used for the treatment of mesangial cells are listed in Table I. All enzymes were endotoxin-free as tested by the Limulus amebocyte assay from BioWhittaker (Walkersville, MD). Human sPLA2-IIA was a generous gift of Prof. Tibes, Roche Diagnostics (Penzberg, Germany). sPLA2s from Taipan snake venom, bee venom, Naja mossambica mossambica venom, and porcine pancreas were obtained as described (18Cupillard L. Mulherkar R. Gomez N. Kadam S. Valentin E. Lazdunski M. Lambeau G. J. Biol. Chem. 1999; 274: 7034-7051Abstract Full Text Full Text PDF Scopus (124) Google Scholar, 38Lambeau G. Ancian P. Nicolas J.P. Beiboer S.H. Moinier D. Verheij H. Lazdunski M. J. Biol. Chem. 1995; 270: 5534-5540Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The construction of the H48Q mutant of porcine sPLA2-IB, and the H48Q and H48N mutants of human sPLA2-IIA have been described elsewhere (39Janssen M.J.W. van de Wiel W.A.E.C. Beiboer S.H.W. van Kampen M.D. Verheij H.M. Slotboom A.J. Egmond M.R. Protein Eng. 1999; 12: 497-503Crossref PubMed Scopus (44) Google Scholar, 40Edwards S. Thompson D. Baker S.F. Wood S.P. Wilton D.C. Biochemistry. 2002; 41: 15468-15476Crossref PubMed Scopus (43) Google Scholar).Table IsPLA2s used for treatment of rat mesangial cellsAbbreviationTypeSourceBinding to the rat M-type sPLA2 receptor (K 0.5 values)aK 0.5 values were determined by competition binding assays with labeled OS1 on rat mesangial cell membranes as described (38).nmWT-IBWild-type porcine sPLA2-IBPorcine pancreas0.5IB-H48QCatalytically inactive porcine sPLA2-IB mutantRecombinant0.5hIIAWild-type human sPLA2-IIARecombinant>500hIIA-H48NMutant human sPLA2-IIA with 0.2 % residual catalytic activityRecombinant>500RatIIARat sPLA2-IIARecombinant>500BeeBee venom sPLA2-IIIBee venom>500NajaSnake venom sPLA2N. mossambica mossambica>500OS1Snake venom sPLA2O. scutellatus sc.0.3OS2Snake venom sPLA2O. scutellatus sc.0.1a K 0.5 values were determined by competition binding assays with labeled OS1 on rat mesangial cell membranes as described (38Lambeau G. Ancian P. Nicolas J.P. Beiboer S.H. Moinier D. Verheij H. Lazdunski M. J. Biol. Chem. 1995; 270: 5534-5540Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Open table in a new tab Cell Culture—Rat mesangial cells were cultured and characterized as described (41Pfeilschifter J. Vosbeck K. Biochem. Biophys. Res. Commun. 1991; 175: 372-379Crossref PubMed Scopus (163) Google Scholar). Cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 μg/ml), and bovine insulin (0.66 units/ml). Twenty-four hours prior to stimulation and during the experiments, cells were incubated in Dulbecco's modified Eagle's medium containing 0.1 mg/ml fatty acid-free BSA. Northern Blot Analysis—Confluent mesangial cells were cultured in 100-mm diameter culture dishes. After stimulation for 24 h, cells were washed with PBS and harvested using a rubber policeman. Total cellular RNA was extracted from the cell pellet using the guanidinium isothiocyanate/phenol/chloroform method. Ten μg of total RNA was separated on a 1.4% agarose/formaldehyde gel, transferred to GeneScreen membranes, and hybridized with the radiolabeled cDNA probes for sPLA2-IIA or 18 S RNA. For quantification the signals of the filters were scanned and evaluated densitometrically using a BAS 1500 phosphorimager from Fuji (Raytest, Straubenhardt, Germany). The signal obtained with the sPLA2-IIA probe was normalized to that obtained with the 18 S RNA probe. Western Blot Analysis—sPLA2-IIA protein secreted by mesangial cells was measured by precipitating 500 μl of the culture supernatant with 200 μl of 20% trichloroacetic acid. SDS-PAGE using a 15% polyacrylamide gel was performed under nonreducing conditions. The proteins were transferred to Immobilon-PVDF membranes for 30 min at 0.7 mA/cm2. Nonspecific binding was blocked with 2% BSA in PBS plus 0.05% Tween 20 for 1 h at room temperature, followed by incubation with a mouse monoclonal antibody against rat sPLA2-IIA (generous gift from Prof. Henk van den Bosch, Utrecht, The Netherlands) at a 1:100 dilution in 0.01% milk powder in PBS. This rat sPLA2-IIA antibody cross-reacts with neither the human recombinant sPLA2-IIA nor the other sPLA2s used in this study. Indeed, Fig. 1B shows that the antibody detected the exogenously added recombinant rat enzyme (100 nm) as a thick band, but does not recognize the human sPLA2-IIA and the other sPLA2s. This clearly demonstrates that this antibody can be specifically used to detect the sPLA2-stimulated release of rat sPLA2-IIA from mesangial cells. Blots were incubated with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (Amersham Biosciences, Freiburg, Germany) at a 1:15,000 dilution in blocking buffer for 1 h at room temperature. The washing steps were performed in 0.05% Tween 20 in PBS. After washing, peroxidase activity was detected using ECL (Amersham Biosciences). sPLA 2 Assay—sPLA2 activity in supernatants of mesangial cells was determined with [1-14C]oleate-labeled Escherichia coli membranes as substrate (42Märki F. Franson R. Biochim. Biophys. Acta. 1986; 879: 149-156Crossref PubMed Scopus (100) Google Scholar). Briefly, assay mixtures (1 ml) contained 100 mm Tris-HCl (pH 7.0), 10 mm CaCl2, [1-14C]oleate-labeled E. coli (≈10,000 cpm), and 5 μl of cell supernatants, which produces less than 5% of substrate hydrolysis. Reaction mixtures were incubated for 30 min at 37 °C in a thermomixer. The extraction of the lipids was performed by Dole's method exactly as described (42Märki F. Franson R. Biochim. Biophys. Acta. 1986; 879: 149-156Crossref PubMed Scopus (100) Google Scholar). Free [1-14C]oleate was measured in a β-counter. Construction of Reporter Gene Fusions—A BamHI/KpnI fragment (2.67 kbp) of the rat sPLA2-IIA promoter (accession no. AF375595) was fused to the luciferase gene by cloning this fragment to the respective sites in the pGL3 basic vector (Promega, Mannheim, Germany). Unidirectional nested deletions of this construct were performed with the Erase-a-Base system (Promega) as described previously (35Scholz-Pedretti K. Gans A. Beck K.F. Pfeilschifter J. Kaszkin M. J. Am. Soc. Nephrol. 2002; 13: 611-620Crossref PubMed Google Scholar). Site-directed Mutagenesis—Mutations within the putative PPAR binding site –909 to –888 (5′-AGGTTGTCCTCTGAACTCCACA-3′ in the rat sPLA2-IIA promoter fragment were introduced by PCR-based site-directed mutagenesis according to the instructions from the manufacturer (Stratagene) as described previously (35Scholz-Pedretti K. Gans A. Beck K.F. Pfeilschifter J. Kaszkin M. J. Am. Soc. Nephrol. 2002; 13: 611-620Crossref PubMed Google Scholar); the changes in the obtained sequence 5′-AGGTTGTGTTCTGCGCTCCACA-3′ are underlined. Transfection and Luciferase Reporter Gene Assay—For transfection, cells were seeded in 35-mm culture dishes and incubated for 24 h at 37 °C in RPMI containing 10% fetal calf serum. The cells were then incubated in Dulbecco's modified Eagle's medium containing 0.1 mg/ml BSA and transfected with 400 ng of plasmid DNA and 40 ng of Renilla luciferase DNA (pRL-TK vector) per well using the Effectene transfection reagent from Qiagen (Hilden, Germany). After 16 h, cells were stimulated with the different effectors for another 24 h. The cells were then washed with ice-cold PBS, lysed in 250 μl of lysis buffer from the dual luciferase reporter assay system (Promega), scraped with a rubber policeman, and transferred into 1.5-ml vials. The cell lysates were subjected to two freeze/thaw cycles for complete lysis of cells. After short centrifugation, the assays for firefly luciferase activity and Renilla luciferase activity were performed sequentially by using a luminometer (Autolumat from Berthold, Wildbad, Germany). Values for the sPLA2-IIA promoter activity were divided by those obtained from Renilla luciferase activity. The mean values ± S.D. obtained for the control cells were set as 1. Values obtained with treated cells are expressed as -fold increase in luciferase activity (relative units) compared with control. Electrophoretic Mobility Shift Assay (EMSA)—The sequences of the double-strand oligonucleotides used to detect the DNA binding activities of PPAR were chosen as described previously (35Scholz-Pedretti K. Gans A. Beck K.F. Pfeilschifter J. Kaszkin M. J. Am. Soc. Nephrol. 2002; 13: 611-620Crossref PubMed Google Scholar). The complementary DNA strands were labeled with T4 polynucleotide kinase using [γ-32P]ATP. Nuclear extracts from stimulated cells were isolated as described previously (35Scholz-Pedretti K. Gans A. Beck K.F. Pfeilschifter J. Kaszkin M. J. Am. Soc. Nephrol. 2002; 13: 611-620Crossref PubMed Google Scholar). Binding reactions with radioactive oligonucleotides were performed for 30 min at room temperature with 5 μg of total protein in 25 μl of 10 mm Tris-HCl (pH 7.5), 50 mm NaCl, 1 mm EDTA, 10% glycerol, 1 μg of acetylated bovine serum albumin, 2 μg of poly(dI-dC), 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and 50,000 dpm of 32P-labeled oligonucleotides. For competition experiments, nuclear extracts were pre-incubated with a 100-fold excess of cold PPRE-1 oligonucleotide or with antibodies against PPARα (4 μg/onset) and RXRα (3 μg/onset) for 30 min at room temperature before addition of the labeled oligonucleotides. DNA-protein complexes were separated from unbound DNA probe on native 8% polyacrylamide gels at 20 mA in 34 mm Tris-HCl (pH 7.5), 17 mm sodium acetate, and 0.5 mm EDTA (pH 8.0). Gels were vacuumdried and analyzed with a phosphorimager. RT-PCR Analysis of 5-Lipoxygenase (5-LOX) and 5-LOX-activating Protein (FLAP)—Expression of mRNA for 5-LOX and FLAP was analyzed by RT-PCR using a total of 5 μg of RNA. As positive controls, RNA extracts from kidneys of rats sacrificed at 6 and 24 h after induction of Thy-1 nephritis were used (43Lianos E.A. Bresnahan B.B. Wu S. J. Lipid Mediat. 1993; 6: 333-342PubMed Google Scholar). First strand cDNA was transcribed with Superscript II RNase H-RT obtained from Invitrogen and oligo(dT)15 primer (Promega). PCR was performed on a PerkinElmer Thermal Cycler with specific primers as follows: arachidonate 5-lipoxygenase (Alox5), sense (5′-CTGGTAGCCCATGTGAGGTT-3′) and antisense (5′-GCACAGGGAGGAATAGGTCA-3′) (product, 162 bp); rat FLAP sense (5′-CGTAGATGCGTACCCCACTT-3′) and antisense (5′-CGCTTCCGAAGAAGAAGATG-3′) (product, 245 bp); 18 S RNA, sense (5′-GCGGTAATTCCAGCTCCAATAG-3′) and antisense (5′-CCCTCTTAATCATGGCCTCAGT-3′) (product, 289 bp). The different cDNA probes were amplified in a prepared Mastermix containing dNTPs, specific primers, and Red Taq polymerase (Sigma) in the corresponding PCR buffer. For the PCR reactions, the following sequences were performed. For 5-LOX, sequence was 95 °C for 4 min (1 cycle) followed immediately by 95 °C for 50 s, 55 °C for 30 s, and 72 °C for 20 s (36 cycles) and a final extension phase at 72 °C for 10 min. For FLAP, sequence was 95 °C for 4 min (1 cycle) followed immediately by 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 20 s (36 cycles) and a final extension phase at 72 °C for 10 min. For 18 S RNA, sequence was 95 °C for 4 min (1 cycle) followed immediately by 95 °C for 1 min, 60 °C for 1 min, and 72 °C for 2 min (24 cycles) and a final extension phase at 72 °C for 10 min. Amplified PCR products were separated on 1% agarose gels containing 0.5 μg/ml ethidium bromide. The PCR products from the rat kidneys were purified with the QIAquick PCR purification kit (Qiagen) for sequencing using a kit based on the dye terminator technology (PerkinElmer Applied Biosystems, Weiterstadt, Germany) in combination with the automated sequence analyzer A310 (PerkinElmer Applied Biosystems). Statistical Analysis—Data are represented as means ± S.D. (n = 3; in transfection experiments, n = 6) showing one representative experiment of three with similar results. Statistical analysis was performed by Student's t test. A probability < 0.05 was considered as significant. Effect of Exogenous sPLA 2 s on sPLA 2 -IIA Expression in Rat Mesangial Cells—The scope of this study was to identify the transcriptional regulatory mechanisms of sPLA2-IIA expression in rat mesangial cells triggered by exogenous sPLA2s. For this purpose, we treated rat mesangial cells with various sPLA2s from mammalian and venom origins in the absence or presence of TNFα (Table I). The effects of a catalytically inactive mutant of the porcine pancreatic sPLA2-IB containing a single amino acid mutation at position 48 (H48Q) was also studied. This mutant has less than 0.02% of wild-type PLA2 activity (39Janssen M.J.W. van de Wiel W.A.E.C. Beiboer S.H.W. van Kampen M.D. Verheij H.M. Slotboom A.J. Egmond M.R. Protein Eng. 1999; 12: 497-503Crossref PubMed Scopus (44) Google Scholar)

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