Lysophosphatidic Acid Activates NF-κB in Fibroblasts
1999; Elsevier BV; Volume: 274; Issue: 6 Linguagem: Inglês
10.1074/jbc.274.6.3828
ISSN1083-351X
AutoresMandana Shahrestanifar, Xiaomin Fan, David R. Manning,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoLysophosphatidic acid (LPA) is a growth factor that exerts a number of well characterized biological actions on fibroblasts and other cells. In the present study, we investigated the possibility that LPA activates the transcription factor NF-κB. NF-κB is a target of cytokines, but its activation by other classes of agonists has raised considerable interest in the control of processes such as inflammation and wound healing through varied mechanisms. We find that LPA causes a marked activation of NF-κB in Swiss 3T3 fibroblasts as determined by the degradation of IκB-α in the cytosol and the emergence of κB binding activity in nuclear extracts. The EC50 for activation of NF-κB is 1–5 μm, a range similar to that reported for reinitiation of DNA synthesis and activation of the serum response element. Activation of NF-κB is attenuated by pertussis toxin and inhibitors of protein kinase C, and it is completely blocked by the Ca2+ chelator BAPTA-AM. The combination of phorbol ester and thapsigargin promotes an activation comparable with that of LPA. Activation by LPA is additionally inhibited by tyrphostin A25 but not genistein or AG1478, indicating a selective utilization of protein-tyrosine kinases, and by certain antioxidants, implying a role for reactive oxygen species. The activation is also inhibited by tricyclodecan-9-yl-xanthogenate (D609), implying a requirement for hydrolysis of phosphatidylcholine. The data demonstrate the utilization of multiple pathways in the activation of NF-κB by LPA, not inconsistent with the relevance of several families of GTP-binding regulatory proteins. Lysophosphatidic acid (LPA) is a growth factor that exerts a number of well characterized biological actions on fibroblasts and other cells. In the present study, we investigated the possibility that LPA activates the transcription factor NF-κB. NF-κB is a target of cytokines, but its activation by other classes of agonists has raised considerable interest in the control of processes such as inflammation and wound healing through varied mechanisms. We find that LPA causes a marked activation of NF-κB in Swiss 3T3 fibroblasts as determined by the degradation of IκB-α in the cytosol and the emergence of κB binding activity in nuclear extracts. The EC50 for activation of NF-κB is 1–5 μm, a range similar to that reported for reinitiation of DNA synthesis and activation of the serum response element. Activation of NF-κB is attenuated by pertussis toxin and inhibitors of protein kinase C, and it is completely blocked by the Ca2+ chelator BAPTA-AM. The combination of phorbol ester and thapsigargin promotes an activation comparable with that of LPA. Activation by LPA is additionally inhibited by tyrphostin A25 but not genistein or AG1478, indicating a selective utilization of protein-tyrosine kinases, and by certain antioxidants, implying a role for reactive oxygen species. The activation is also inhibited by tricyclodecan-9-yl-xanthogenate (D609), implying a requirement for hydrolysis of phosphatidylcholine. The data demonstrate the utilization of multiple pathways in the activation of NF-κB by LPA, not inconsistent with the relevance of several families of GTP-binding regulatory proteins. Lysophosphatidic acid (LPA 1The abbreviations used are: LPA, lysophosphatidic acid; ERK, extracellular signal-regulated kinase; G protein, GTP-binding regulatory protein; GPCR, G protein-coupled receptor; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; PTX, pertussis toxin; TNFα, tumor necrosis factor-α; EGF, epidermal growth factor; D609, tricyclodecan-9-yl-xanthogenate; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester.1The abbreviations used are: LPA, lysophosphatidic acid; ERK, extracellular signal-regulated kinase; G protein, GTP-binding regulatory protein; GPCR, G protein-coupled receptor; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; PTX, pertussis toxin; TNFα, tumor necrosis factor-α; EGF, epidermal growth factor; D609, tricyclodecan-9-yl-xanthogenate; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester.; 1-acyl-2-lyso-sn-glycero-3-phosphate) is a naturally occurring, water-soluble glycerophospholipid that exhibits striking hormone- and growth factor-like activities (1Moolenaar W.H. J. Biol. Chem. 1995; 270: 12949-12952Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar, 2Moolenaar W.H. Kranenburg O. Postma F.R. Zondag G.C.M. Curr. Opin. Cell Biol. 1997; 9: 168-173Crossref PubMed Scopus (472) Google Scholar). Synthesized and released by platelets, LPA represents a major bioactive constituent of serum, and its actions on fibroblasts, endothelial cells, and smooth muscle cells in particular suggest roles in wound healing among other events. Indeed, LPA acts on a large number of cells to achieve a broad range of immediate and long lasting effects. Specific responses to LPA include changes in cell shape and tension, chemotaxis, proliferation, and differentiation. The molecular actions of LPA have been characterized best in rodent fibroblasts, where at low concentrations (i.e. 10–100 nm) LPA stimulates phosphoinositide hydrolysis (3Jalink K. van Corven E.J. Moolenaar W.H. J. Biol. Chem. 1990; 265: 12232-12239Abstract Full Text PDF PubMed Google Scholar) and promotes the Rho-dependent formation of stress fibers and focal adhesions (4Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3806) Google Scholar). The stimulation of phosphoinositide hydrolysis is thought to occur through the GTP-binding regulatory protein (G protein) Gq. The formation of stress fibers and focal adhesions is consistent with activation of Rho through G12 or G13 (5Buhl A.M. Johnson N.L. Dhanasekaran N. Johnson G.L. J. Biol. Chem. 1995; 270: 24631-24634Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). One or a combination of these G proteins is also responsible for the protein tyrosine phosphorylation elicited by LPA (6Kumagai N. Morii N. Fujisawa K. Nemoto Y. Narumiya S. J. Biol. Chem. 1993; 268: 24535-24538Abstract Full Text PDF PubMed Google Scholar, 7Hordijk P.L Verlaan I. van Corven E.J. Moolenaar W.H. J. Biol. Chem. 1994; 269: 645-651Abstract Full Text PDF PubMed Google Scholar, 8Seufferlein T. Rozengurt E. J. Biol. Chem. 1994; 269: 9345-9351Abstract Full Text PDF PubMed Google Scholar). LPA additionally inhibits adenylyl cyclase, an action achieved through the pertussis toxin (PTX)-sensitive G protein Gi(9van Corven E.J. Groenink A. Jalink K. Eicholtz T. Moolenaar W.H. Cell. 1989; 59: 45-54Abstract Full Text PDF PubMed Scopus (679) Google Scholar). LPA uses Gi, moreover, to activate Ras, Raf, and the extracellular signal-regulated kinases (ERKs) ERK1 and ERK2 (10van Corven E.J. Hordijk P.L. Medema R.H. Bos J.L. Moolenaar W.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1257-1261Crossref PubMed Scopus (337) Google Scholar, 11Howe L.R. Marshall C.J. J. Biol. Chem. 1993; 268: 20717-20720Abstract Full Text PDF PubMed Google Scholar). The activation of ERKs (11Howe L.R. Marshall C.J. J. Biol. Chem. 1993; 268: 20717-20720Abstract Full Text PDF PubMed Google Scholar), and presumably the inhibition of adenylyl cyclase (3Jalink K. van Corven E.J. Moolenaar W.H. J. Biol. Chem. 1990; 265: 12232-12239Abstract Full Text PDF PubMed Google Scholar), occurs at concentrations of LPA comparable with those stimulating phosphoinositide hydrolysis and cytoskeletal changes. At higher concentrations (i.e. 5–70 μm), LPA promotes reinitiation of DNA synthesis in quiescent fibroblasts (9van Corven E.J. Groenink A. Jalink K. Eicholtz T. Moolenaar W.H. Cell. 1989; 59: 45-54Abstract Full Text PDF PubMed Scopus (679) Google Scholar). Whether G proteins are sufficient for this action is unclear, but the sensitivity of the phenomenon to PTX implies that Girepresents at least one necessary input. The need for high concentrations of LPA in this context may relate to a requirement for more persistent signaling and/or engagement of other receptors or pathways. Micromolar concentrations of LPA also promote arachidonic acid formation, a second phase of inositol phosphate accumulation (PTX-insensitive), and activation of serum response factor (9van Corven E.J. Groenink A. Jalink K. Eicholtz T. Moolenaar W.H. Cell. 1989; 59: 45-54Abstract Full Text PDF PubMed Scopus (679) Google Scholar, 12Perkins L.M. Ramirez R.E. Kumar C.C. Thomson F.J. Clark M.A. Nucleic Acids Res. 1994; 22: 450-452Crossref PubMed Scopus (14) Google Scholar, 13Hill C.S. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1201) Google Scholar). Receptors that recognize LPA are poorly characterized; however, several have been identified that conform to the seven-transmembrane domain motif characteristic of G protein-coupled receptors (GPCRs) (14Hecht J.H. Weiner J.A. Post S.R. Chun J. J. Cell Biol. 1996; 135: 1071-1083Crossref PubMed Scopus (661) Google Scholar, 15Guo Z. Liliom K. Fischer D.J. Bathurst I.C. Tomei L.D. Kiefer M.C. Tigyi G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14367-14372Crossref PubMed Scopus (178) Google Scholar, 16An S. Dickens M.A. Bleu T. Hallmark O.G. Goetzl E.J. Biochem. Biophys. Res. Commun. 1997; 231: 619-622Crossref PubMed Scopus (212) Google Scholar, 17An S. Bleu T. Hallmark O.G. Goetzl E.J. J. Biol. Chem. 1998; 273: 7906-7910Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar). NF-κB (nuclear factor-κB) is the prototype of a family of dimers whose constituents are members of the Rel family of transcription factors (18Thanos D. Maniatis T. Cell. 1995; 80: 529-532Abstract Full Text PDF PubMed Scopus (1217) Google Scholar). In most types of cells, NF-κB is present as a heterodimer comprising p50 (NF-κB1) and p65 (RelA). NF-κB is normally retained in the cytosol in an inactive form through interaction with IκB inhibitory proteins. Release of NF-κB for translocation to the nucleus and interaction with cognate DNA sequences is accomplished through a signal-induced phosphorylation and subsequent degradation of IκB. Originally described as a necessary element for expression of the immunoglobulin κ gene in mature B cells, NF-κB is now recognized to be an important transcriptional regulatory protein in a variety of cell types (19Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2919) Google Scholar). The binding of agonists to certain GPCRs promotes activation of NF-κB. Agonists include serotonin (working through the 5-HT1A receptor) (20Cowen D.S. Molinoff P.B. Manning D.R. Mol. Pharmacol. 1997; 52: 221-226Crossref PubMed Scopus (47) Google Scholar), platelet-activating factor (21Kravchenko 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), thrombin (22Mari B. Imbert V. Belhacene N. Far D.F. Peyron J.-F. Pouysségur J. Van Obberghen-Schilling E. Rossi B. Auberger P. J. Biol. Chem. 1994; 269: 8517-8523Abstract Full Text PDF PubMed Google Scholar), and bradykinin (23Pan Z.K. Zuraw B.L. Lung C.-C. Prossnitz E.R. Browning D.D. Ye R.D. J. Clin. Invest. 1996; 98: 2042-2049Crossref PubMed Scopus (131) Google Scholar). That GPCRs are linked to NF-κB is particularly significant, since these receptors are widely distributed, the actions of NF-κB are notable, and the coincident activation of NF-κB and other GPCR-regulated transcription factors can provide unique forms of transcriptional regulation. Because LPA exerts a wide range of actions in part or entirely through GPCRs, and because NF-κB is especially relevant to inflammation and wound healing, we instituted efforts here to understand whether LPA promotes the activation of NF-κB. We explored the possible relationship between LPA and NF-κB in fibroblasts and the mechanisms by which this relationship is established. l-α-Lysophosphatidic acid (C18:1,[cis]-9), cycloheximide, ascorbic acid, pyrrolidinedithiocarbamate, and dimethyl sulfoxide were obtained from Sigma. Phorbol-12-myristate-13-acetate (PMA), calphostin C, Ro 31-8220, bisindolylmaleimide I, tyrphostins A25 and AG1478, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM), thapsigargin,N-acetylcysteine, and diphenyleneiodonium were obtained from Calbiochem. Dithiothreitol was obtained from Boehringer Mannheim. Tricyclodecan-9-yl xanthogenate (D609) was obtained from Biomol Research Laboratories (Plymouth Meeting, PA) or Sigma. TNFα was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Potassium ethylxanthate was obtained from Aldrich. The double-stranded oligonucleotide conforming to 5′-AGTTGAGGGGACTTTCCCAGGC-3′ was obtained from Promega Corp. (Madison, WI), and those conforming to 5′-AGTTGAGGCGACTTTCCCAGGC-3′ and 5′-ATTCGATCGGGGCGGGGCGAGC-3′ and the antibody toward p65 (RelA) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). [γ-32P]ATP was obtained from NEN Life Science Products. Electrophoretic reagents were obtained from Bio-Rad. Swiss 3T3 mouse embryo fibroblasts (a gift from Dr. E. Rozengurt, Imperial Cancer Research Fund, London, UK) were maintained at 37 °C under a humidified atmosphere of 10% CO2 in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal calf serum, supplemented with penicillin (100 units/ml) and streptomycin (100 μg/ml). For all experiments, 1 × 106 cells were subcultured into 10-cm tissue culture plates (Nunc). After 48 h, the medium was replaced with 6 ml of Dulbecco's modified Eagle's medium containing 1% fetal calf serum and antibiotics, and the cells were incubated for an additional 18 h. LPA (prepared as a stock of 1 mg/ml in phosphate-buffered saline containing 10 mg/ml essentially fatty acid-free bovine serum albumin (Sigma)) and/or other reagents or vehicles were added to achieve the concentrations specified. Nuclear extracts were prepared by the method of Dignam et al. (24Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9149) Google Scholar) with minor modifications. Following incubation with LPA or other reagents, cells were washed twice in ice-cold phosphate-buffered saline, harvested, and resuspended in 400 μl of hypotonic buffer (10 mm HEPES (pH 7.9), 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride). After 10 min on ice, 30 μl of Nonidet P-40 (10% (v/v)) was added with mixing for 2 s. The nuclei were pelleted by centrifugation at 20,000 ×g for 10 s. The supernatant was removed, and the nuclei were resuspended in hypertonic buffer (20 mm HEPES (pH 7.9), 0.4 m NaCl, 1 mm EDTA, 1 mmEGTA, 0.1 mm phenylmethylsulfonyl fluoride) and shaken for 45 min at 4 °C. The samples were centrifuged at 20,000 ×g for 30 s, and the supernatants (nuclear extracts) were saved. Protein concentrations were determined using the Bradford method (Bio-Rad). Electrophoretic mobility shift assays were performed using a double-stranded oligonucleotide containing a consensus κB binding site (5′-AGTTGAGGGGACTTTCCCAGGC-3′; the underlined sequence represents the consensus κB region), which was end-labeled with [γ-32P]ATP and T4 polynucleotide kinase. Nuclear extracts (2.5 μg of protein) were incubated in 10 mmTris-HCl (pH 7.9), 50 mm NaCl, 1 mm EDTA, 10% glycerol, 0.15 mg/ml poly(dI-dC), and 20–30 fmol of32P-labeled oligonucleotide (50,000–100,000 cpm) in a total volume of 15 μl at room temperature for 10 min. The reaction mixture was then subjected to electrophoresis in a 5% polyacrylamide slab gel containing 50 mm Tris, 380 mm glycine, and 2 mm EDTA, pH 8. The gels were dried under vacuum for analysis by autoradiography (overnight exposure) or PhosphorImager analysis. For competition studies, nuclear extracts were incubated prior to the addition of labeled oligonucleotide for 10 min at room temperature with unlabeled oligonucleotide, unlabeled oligonucleotide containing a G → C substitution in the κB binding motif (5′-AGTTGAGGCGACTTTCCCAGGC-3′), or an unlabeled oligonucleotide containing the consensus binding site for Sp1 (5′-ATTCGATCGGGGCGGGGCGAGC-3′). For supershift analysis, nuclear extracts were incubated with approximately 2 μg of antibodies specific for p65 (RelA) or nonimmune goat IgG for 30 min at 4 °C in the presence of radiolabeled oligonucleotide prior to electrophoresis. The results shown in all figures are representative of at least three experiments. Following exposure to LPA with or without cycloheximide as specified, cells were washed with ice-cold phosphate-buffered saline and lysed in 10 mm Tris-HCl (pH 7.6), 5 mm EDTA, 50 mm NaCl, 30 mmsodium pyrophosphate, 50 mm NaF, 100 mmNa3VO4, 0.5% Triton X-100, 10 mg/ml leupeptin, 10 mg/ml aprotinin, and 1 mm phenylmethylsulfonyl fluoride. Lysates were clarified by centrifugation at 20,000 × gfor 15 min at 4 °C. Supernatants were collected and subjected to SDS-polyacrylamide gel electrophoresis (11% acrylamide). Protein was transferred to nitrocellulose membrane and probed with polyclonal rabbit anti-human IκB-α (0.4 mg/ml) detected subsequently by chemiluminescence using a donkey anti-rabbit IgG conjugated with horseradish peroxidase and luminol as recommended by the manufacturer (ECL Western; Amersham Pharmacia Biotech). The possibility that LPA activates the transcription factor NF-κB was investigated in Swiss 3T3 fibroblasts, which have been used extensively in studies of agonists, including LPA, linked to changes in cell morphology and reinitiation of DNA synthesis (4Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3806) Google Scholar, 25Rozengurt E. Science. 1986; 234: 161-166Crossref PubMed Scopus (848) Google Scholar, 26Roche S. Koegl M. Courtneidge S.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9185-9189Crossref PubMed Scopus (262) Google Scholar). We examined first the extent to which LPA promotes degradation of IκB-α, an inhibitory protein whose proteolysis would precede the translocation of NF-κB to the nucleus. As shown in Fig.1 (left panel), LPA caused a transient degradation of this protein. Levels of IκB-α decreased slowly following introduction of LPA, reaching a minimum at 40–60 min, and increased thereafter to near control values. The time-dependent resynthesis of IκB-α is a common finding in cytokine action (27Brown K. Park S. Kanno T. Franzoso G. Sieberlist U. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2532-2536Crossref PubMed Scopus (575) Google Scholar) and appears to reflect activation of the IκB-α gene by NF-κB as part of a feedback loop (28Chiao P.J. Miyamoto S. Verma I.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 28-32Crossref PubMed Scopus (390) Google Scholar). To circumvent resynthesis of IκB-α, we also evaluated degradation of this protein in the presence of cycloheximide. As expected, degradation of IκB-α under this condition, where protein synthesis is blocked, was complete. A more direct evaluation of NF-κB activation was carried out by electrophoretic mobility shift assays. The data in Fig.2 demonstrate that LPA promotes a time- and concentration-dependent appearance of a factor(s) within nuclear extracts that binds to an oligonucleotide probe containing an NF-κB binding site. The proinflammatory cytokine TNFα also promotes the appearance of this factor. The relevant protein-DNA complex was evident as a band of radioactivity (denoted by anarrow) positioned above two less prominent bands. This band, but not the other two, was supershifted with a p65 (RelA)-directed antibody (Fig. 3, top panel), confirming the identity of the induced factor as NF-κB. The nature of the protein-DNA interaction was evaluated further in competition experiments (Fig. 3, bottom panel), where the 32P-labeled oligonucleotide was found to be displaced by unlabeled oligonucleotide. The same unlabeled oligonucleotide, but containing a mutation in the κB site, and an altogether unrelated oligonucleotide (containing an Sp1 binding site) did not displace the 32P-labeled oligonucleotide. The EC50 for LPA based on the intensity of the shifted band was 1–5 μm, and the time required for full development of the response was 40–60 min (Fig. 2). The response was transient, as the level of shifted oligonucleotide began to decrease by 3 h (not shown).Figure 3Identity and specificity of κB binding activity. Swiss 3T3 fibroblasts were incubated with 40 μm LPA (or vehicle) or 30 ng/ml TNFα for 40 min, and nuclear extracts were prepared for electrophoretic mobility shift assays. Upper panel, a supershift assay was performed with a p65 (RelA)-directed or irrelevant (goat IgG) antibody.Lower panel, competition between 32P-labeled 5′-AGTTGAGGGGACTTTCCCAGGC-3′ and 5′-AGTTGAGGGGACTTTCCCAGGC-3′ (same oligonucleotide, but unlabeled), a mutated oligonucleotide 5′-AGTTGAGGCGACTTTCCCAGGC-3′ (which does not bind NF-κB), or a Sp1-binding oligonucleotide (an irrelevant oligonucleotide). The LPA-induced complex of32P-labeled oligonucleotide and NF-κB is designated by the arrow.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The extent to which the G protein Gi might contribute to the activation of NF-κB was assessed with PTX, which suppresses activation of Gi by GPCRs. Pretreatment of cells with PTX attenuated LPA-induced activation of NF-κB by approximately 60% (Fig. 4, top panel). Efforts to enhance the attenuation by manipulating pretreatment time and concentration of PTX were unsuccessful. That the actions of LPA typically assigned to Gi are not completely suppressed by PTX, for example ERK activation and reinitiation of DNA synthesis, is not without precedent (9van Corven E.J. Groenink A. Jalink K. Eicholtz T. Moolenaar W.H. Cell. 1989; 59: 45-54Abstract Full Text PDF PubMed Scopus (679) Google Scholar, 10van Corven E.J. Hordijk P.L. Medema R.H. Bos J.L. Moolenaar W.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1257-1261Crossref PubMed Scopus (337) Google Scholar, 11Howe L.R. Marshall C.J. J. Biol. Chem. 1993; 268: 20717-20720Abstract Full Text PDF PubMed Google Scholar). A downstream target for both Gi and Gq is the phosphoinositide-specific phospholipase C-β, whose activation results in recruitment of PKC and mobilization of Ca2+. Overnight treatment of cells with a high concentration of PMA (1 μm) to induce down-regulation of classical and novel forms of PKC suppressed activation of NF-κB by 70–80% (not shown), as did Ro-31-8220 (Fig. 4, middle panel), which inhibits all forms of PKC. Other inhibitors of PKC, including bisindolylmaleimide I (not shown) and calphostin C, inhibited activation of NF-κB nearly as well. Activation of NF-κB was completely suppressed by pretreatment of cells with the cell-permeable Ca2+ chelator BAPTA-AM (Fig. 4, bottom panel). Given the apparent requirements for PKC and intracellular Ca2+, we tested whether the activation of PKC and/or mobilization of Ca2+ might be sufficient to activate NF-κB. Some degree of activation was achieved with PMA, but not the extent observed with LPA (Fig. 5). Only a small degree of activation was achieved with thapsigargin, moreover, an inhibitor of the endoplasmic reticular Ca2+-ATPase that causes a time-dependent increase in cytosolic Ca2+. However, the combination of thapsigargin and PMA activated NF-κB ultimately to an extent somewhat greater than that achieved with LPA. Because considerable attention has been devoted to the utilization of the EGF receptor and other tyrosine kinases by GPCRs, including the one or more receptors that mediate the actions of LPA (29Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1316) Google Scholar, 30Luttrell L.M. Della Rocca G.J. van Biesen T. Luttrell D.K. Lefkowitz R.J. J. Biol. 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In contrast, tyrphostin A25, like genistein regarded as a general inhibitor of tyrosine kinases, achieved a significant degree of inhibition at 25 and 50 μm and complete inhibition by 150 μm(not shown; 150 μm is a commonly employed concentration (31Gohla A. Harhammer R. Schultz G. J. Biol. Chem. 1998; 273: 4653-4659Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 32Nobes C.D. Hawkins P. Stephens L. Hall A. J. Cell Sci. 1995; 108: 225-233Crossref PubMed Google Scholar)). Chen et al. (33Chen Q. Olashaw N. Wu J. J. Biol. Chem. 1995; 270: 28499-28502Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar) demonstrated that LPA stimulates reactive oxygen species production in HeLa cells and that antioxidants inhibit LPA-stimulated MAP kinase kinase activity. We therefore evaluated the effects of antioxidants on the activation of NF-κB. As shown in Fig.7, N-acetylcysteine completely inhibited LPA-induced activation of NF-κB. Pyrrolidinedithiocarbamate was similarly effective. Dimethyl sulfoxide achieved a less extensive, but still notable, inhibition. Ascorbic acid and dithiothreitol were without effect. The activation of NF-κB was also highly sensitive to diphenyleneiodonium, an inhibitor of flavanoid-containing enzymes such as NADPH oxidase. The tricyclodecan xanthogenate D609 inhibits the hydrolysis of phosphatidylcholine at the level of a phosphatidylcholine-specific phospholipase C-like enzyme and/or phospholipase D (34Cai H. Erhardt P. Troppmair J. Diaz-Meco M.T. Sithanandam G. Rapp U.R. Moscat J. Cooper G.M. Mol. Cell. Biol. 1993; 13: 7645-7651Crossref PubMed Scopus (119) Google Scholar, 35Shütze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Kronke M. Cell. 1992; 71: 765-776Abstract Full Text PDF PubMed Scopus (968) Google Scholar, 36van Dijk M.C.M. Muriana F.J.G. de Widt J. Hilkmann H. van Blitterswijk W.J. J. Biol. 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We found here that D609 inhibits quite effectively the activation of NF-κB by LPA (Fig.8). The EC50 was about 3 μg/ml, and the maximum degree of inhibition was greater than 90%. Potassium ethylxanthate had no effect. D609 was not nearly as potent an inhibitor of TNFα's activation of NF-κB as it was of LPA's. Inhibition in the case of TNFα occurred only at concentrations of D609 exceeding 50 μg/ml. The activation of NF-κB by LPA was thus selectively inhibited by D609. That proinflammatory cytokines such as interleukin-1 and TNFα activate NF-κB has long been appreciated, and the sequence of events by which the activation occurs is now emerging (39Régnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1070) Google Scholar, 40Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1575) Google Scholar). Of perhaps equal significance, however, is the fact that agonists working through GPCRs can also activate NF-κB (20Cowen D.S. Molinoff P.B. Manning D.R. Mol. Pharmacol. 1997; 52: 221-226Crossref PubMed Scopus (47) Google Scholar, 21Kravchenko 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, 22Mari B. Imbert V. Belhacene N. Far D.F. Peyron J.-F. Pouysségur J. Van Obberghen-Schilling E. Rossi B. Auberger P. J. Biol. Chem. 1994; 269: 8517-8523Abstract Full Text PDF PubMed Google Scholar, 23Pan Z.K. Zuraw B.L. Lung C.-C. Prossnitz E.R. Browning D.D. Ye R.D. J. Clin. Invest. 1996; 98: 2042-2049Crossref PubMed Scopus (131) Google Scholar). In the work described here, we have focused on LPA. LPA has a rich and important biology; it induces a number of cells to proliferate and others to differentiate and, at a molecular level, works through one or more G proteins to stimulate MAP kinases, phospholipid metabolism, and cytoskeletal rearrangement. Activation of NF-κB clearly constitutes an additional action of LPA, and one to be reconciled in any setting of transcription relevant to this agonist. LPA triggers a pronounced activation of NF-κB, as ascertained by the degradation of IκB-α and the emergence of κB binding activity in nuclear extracts. Based on supershift experiments, the activated form of N
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