Protein Kinase C-α Modulates Lipopolysaccharide-induced Functions in a Murine Macrophage Cell Line
1998; Elsevier BV; Volume: 273; Issue: 49 Linguagem: Inglês
10.1074/jbc.273.49.32787
ISSN1083-351X
AutoresAnik St‐Denis, Frédéric Chano, Pierre Tremblay, Yves St‐Pierre, Albert Descoteaux,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoLipopolysaccharide (LPS), a potent modulator of macrophage functional activity, binds to CD14 and triggers the activation of several protein kinases, leading to the secretion of variety of immunomodulatory molecules such as nitric oxide and proinflammatory cytokines. In this study, we have examined the role of the α isoenzyme of protein kinase C (PKC) in the regulation of LPS-initiated signal transduction in macrophages. To this end, we have stably overexpressed a dominant-negative (DN) version of PKC-α (DN PKC-α) in the murine macrophage cell line RAW 264.7. Clones overexpressing DN PKC-α were indistinguishable from the parental line with respect to morphology and growth characteristics. At the functional level, DN PKC-α overexpression strongly inhibited LPS-induced interleukin-1α mRNA accumulation, and to a lesser extent inducible nitric oxide synthase and tumor necrosis factor-α expression. DN-PKC-α overexpression did not cause a general unresponsiveness to LPS, as secretion of the matrix metalloproteinase-9 was up-regulated in our DN PKC-α-overexpressing clones. Moreover, LPS-induced phosphorylation and degradation of IκBα, NF-κB activation, as well as p38 mitogen-activated protein kinase and Jun N-terminal kinase phosphorylation, were not affected by DN PKC-α overexpression. Collectively, these data provide evidence that PKC-α regulates selective LPS-induced macrophage functions involved in host defense and inflammation. Lipopolysaccharide (LPS), a potent modulator of macrophage functional activity, binds to CD14 and triggers the activation of several protein kinases, leading to the secretion of variety of immunomodulatory molecules such as nitric oxide and proinflammatory cytokines. In this study, we have examined the role of the α isoenzyme of protein kinase C (PKC) in the regulation of LPS-initiated signal transduction in macrophages. To this end, we have stably overexpressed a dominant-negative (DN) version of PKC-α (DN PKC-α) in the murine macrophage cell line RAW 264.7. Clones overexpressing DN PKC-α were indistinguishable from the parental line with respect to morphology and growth characteristics. At the functional level, DN PKC-α overexpression strongly inhibited LPS-induced interleukin-1α mRNA accumulation, and to a lesser extent inducible nitric oxide synthase and tumor necrosis factor-α expression. DN-PKC-α overexpression did not cause a general unresponsiveness to LPS, as secretion of the matrix metalloproteinase-9 was up-regulated in our DN PKC-α-overexpressing clones. Moreover, LPS-induced phosphorylation and degradation of IκBα, NF-κB activation, as well as p38 mitogen-activated protein kinase and Jun N-terminal kinase phosphorylation, were not affected by DN PKC-α overexpression. Collectively, these data provide evidence that PKC-α regulates selective LPS-induced macrophage functions involved in host defense and inflammation. nitric oxide tumor necrosis factor interleukin lipopolysaccharide protein kinase C dominant-negative inducible nitric oxide synthase phorbol dibutyrate matrix metalloproteinase mitogen-activated protein kinase electrophoretic mobility shift assay. Mononuclear phagocytes are multipotential cells that can be modulated to perform a variety of functions including secretion of nitric oxide (NO)1 and proinflammatory cytokines, which are important mediators in host defense and inflammation. In this regards, LPS, a major component of the cell wall of Gram-negative bacteria, is one of the most potent and best characterized modulator of macrophage function. Binding of LPS to the cell surface CD14 molecule triggers multiple intracellular biochemical cascades, including the phosphorylation of several proteins by either tyrosine or serine/threonine kinases (1Wright S.D. Ramos R.A. Tobias P.S. Ulevitch R.J. Mathison J.C. Science. 1990; 249: 1431-1433Crossref PubMed Scopus (3406) Google Scholar, 2Ulevitch R.J. Tobias P.S. Annu. Rev. Immunol. 1995; 13: 437-457Crossref PubMed Scopus (1323) Google Scholar, 3Weinstein S.L. Gold M.R. DeFranco A.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4148-4152Crossref PubMed Scopus (302) Google Scholar). Although the identity of the protein tyrosine kinases that mediate LPS-initiated signal transduction remains to be determined with certainty (4English B.K. Ihle J.N. Myracle A. Yi T. J. Exp. Med. 1993; 178: 1017-1022Crossref PubMed Scopus (112) Google Scholar, 5Meng F. Lowell C.A. J. Exp. 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Immunol. 1994; 153: 2642-2652PubMed Google Scholar), and experiments using various PKC inhibitors indicated that PKC activity is required for the expression of several macrophage functions, including TNF-α and IL-1 secretion, NO production, and tumoricidal activity (9Shapira L. Takashiba S. Champagne C. Amar S. Van Dyke T. J. Immunol. 1994; 153: 1818-1824PubMed Google Scholar, 13Novotney M. Chang Z.L. Uchiyama H. Suzuki T. Biochemistry. 1991; 30: 5597-5604Crossref PubMed Scopus (50) Google Scholar, 17Kovacs E.J. Radzioch D. Young H.A. Varesio L. J. Immunol. 1988; 141: 3101-3105PubMed Google Scholar,18Taniguchi H. Sakano T. Hamasaki T. Kashiwa H. Ueda K. Immunology. 1989; 67: 210-215PubMed Google Scholar). PKC was first characterized as a Ca2+-dependent and phospholipid-dependent protein serine/threonine kinase that requires diacylglycerol for activity (19Parker P.J. Coussens L. Totty N. Rhee L. Young S. Chen E. Stabel S. Waterfield M.D. Ullrich A. Science. 1986; 233: 853-859Crossref PubMed Scopus (592) Google Scholar). Subsequently, it has been established that PKC is not a single entity, but rather a family of closely related isoenzymes comprising at least 12 different members (20Nishizuka Y. Nature. 1988; 334: 661-665Crossref PubMed Scopus (3536) Google Scholar). Differences in their structure, requirement for activity, subcellular localization, and substrate specificity suggest that in a given cell, the various PKC isoenzymes may exert specific functions (20Nishizuka Y. Nature. 1988; 334: 661-665Crossref PubMed Scopus (3536) Google Scholar, 21Dekker L.V. Parker P.J. Trends Biochem. Sci. 1994; 19: 73-77Abstract Full Text PDF PubMed Scopus (920) Google Scholar). Macrophages and monocytic cells express the Ca2+-dependent isoenzymes α, βI, and βII, the Ca2+-independent isoenzymes δ and ε, and the atypical isoenzyme ζ (10Fujihara M. Connolly N. Ito N. Suzuki T. J. Immunol. 1994; 152: 1898-1906PubMed Google Scholar, 16Liu M.K. Herrera-Velit P. Brownsey R.W. Reiner N.E. J. Immunol. 1994; 153: 2642-2652PubMed Google Scholar, 22Zheng L. Zomerdijk T.P. Aarnoudse C. van Furth R. Nibbering P.H. J. Immunol. 1995; 155: 776-784PubMed Google Scholar, 23Mischak H. Kolch W. Goodnight J. Davidson W.F. Rapp U. Rose J.S. Mushinski J.F. J. Immunol. 1991; 147: 3981-3987PubMed Google Scholar). However, our current knowledge on their respective contribution to the regulation of macrophage function is limited and mainly concerns the regulation of nitric oxide production. In one study, differential down-regulation of PKC isoenzymes induced by phorbol ester treatment revealed that PKC-βII participates in LPS-induced iNOS gene expression and nitrite production in the J774 macrophage cell line (10Fujihara M. Connolly N. Ito N. Suzuki T. J. Immunol. 1994; 152: 1898-1906PubMed Google Scholar). More recently, transient PKC isoenzymes transfection studies in the RAW 264.7 macrophage cell line showed that iNOS gene expression is also regulated by PKC-ε, but in contrast to the pathway regulated by PKC-βII, the PKC-ε-dependent pathway is apparently not involved in the LPS response (24Diaz-Guerra M.J.M. Bodelon O.G. Velasco M. Whelan R. Parker P.J. Bosca L. J. Biol. Chem. 1996; 271: 32028-32033Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Elucidation of the role of a particular PKC isoenzyme in cellular regulation is complicated by the concomitant expression of several isoenzymes and by the lack of isoenzyme-specific activators or inhibitors. In the present study, we have investigated the role of PKC-α in the regulation of LPS-induced functions by overexpressing a kinase-deficient mutant of this isoenzyme in the murine macrophage line RAW 264.7. Such catalytically inactive mutants, which behaves as a dominant-negative molecule, acts by competing with the corresponding endogenous isoenzyme (21Dekker L.V. Parker P.J. Trends Biochem. Sci. 1994; 19: 73-77Abstract Full Text PDF PubMed Scopus (920) Google Scholar, 25Jaken S. Curr. Opin. Cell Biol. 1996; 8: 168-173Crossref PubMed Scopus (407) Google Scholar). Using this approach, we obtained evidence that PKC-α activity regulates selective LPS-induced macrophage functions involved in host defense and inflammation. The wild type human PKC-α cDNA (26Finkenzeller G. Marme D. Hug H. Nucleic Acids Res. 1990; 18: 2183Crossref PubMed Scopus (55) Google Scholar) was obtained from the American Type Culture Collection (Rockville, MD). A dominant-negative version of the gene, DN PKC-α (K368D), was created by site-directed mutagenesis using the Transformer System with the mutagenic primer AD-5 (5′-GTATGCAATCGATATCCTGAAGAAGG-3′), as described by the manufacturer (CLONTECH, Palo Alto, CA). The sequence of this mutant was confirmed by sequence analysis. Replacement of the conserved lysine residue in the ATP-binding domain yields an enzymatically inactive trans-dominant mutant (27Rosson D. O'Brien T.G. Kampherstein J.A. Szallasi Z. Bogi K. Blumberg P.M. Mullin J.M. J. Biol. Chem. 1997; 272: 14950-14953Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 28Uberall F. Giselbrecht S. Hellbert K. Fresser F. Bauer B. Gschwendt M. Grunicke H.H. Baier G. J. Biol. Chem. 1997; 272: 4072-4078Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 29Baier B.G. Uberall F. Bauer B. Fresser F. Wachter H. Grunicke H. Utermann G. Altman A. Baier G. Mol. Cell. Biol. 1996; 16: 1842-1850Crossref PubMed Google Scholar). DN PKC-α cDNA was cloned into the EcoRI site of the expression vector pCIN-4 (30Rees S. Coote J. Stables J. Goodson S. Harris S. Lee M.G. BioTechniques. 1996; 20 (; 106, 108–110): 102-104Crossref PubMed Scopus (263) Google Scholar) and the resulting construct was designated pCIN-DN PKC-α. The murine macrophage cell line RAW 264.7 (American Type Culture Collection, kindly provided by D. Oth) was cultured in a 37 °C incubator with 5% CO2 in Dulbecco's modified Eagle's medium with glutamine (Life Technologies Inc., ON, Canada), supplemented with 10% heat-inactivated fetal calf serum (Hyclone, Logan, UT), 10 mm Hepes pH 7.3, and antibiotics (complete medium). Stable transfections were performed as described (31Stacey K.J. Ross I.L. Hume D.A. Immunol. Cell Biol. 1993; 71: 75-85Crossref PubMed Google Scholar). Transfectants were selected in complete medium containing 500 μg/ml G418 (Life Technologies Inc.) and individual clones were harvested, expanded, and examined for PKC-α levels by Western blot analysis. Adherent cells were washed once with phosphate-buffered saline, homogenized in lysis buffer (10 mm Tris-HCl, pH 7.5, 1 mm EGTA, 1% Triton X-100) containing protease and phosphatase inhibitors, and protein concentrations were determined using the BCA protein assay kit (Pierce, Rockford, IL). Total proteins (15 μg) were fractionated in 10% SDS-polyacrylamide gels, electroblotted onto Hybond-ECL membranes (Amersham Life Science Inc., ON, Canada) and immunodetection was achieved by chemiluminescence (ECL, Amersham Life Science). Anti-PKC isoenzyme monoclonal antibodies were from Transduction Laboratories (Lexington, KY). Phosphorylation and degradation of IκBα was analyzed with the PhosphoPlus IκBα (Ser32) Antibody kit from New England BioLabs (Beverly, MA), phosphorylation of the p38 MAP kinase was determined with the PhosphoPlus p38 MAPK (Tyr182Antibody kit, New England Biolabs), and phosphorylation of the Jun N-terminal kinase was analyzed with the Anti-Active JNK pAb from Promega (Madison, WI). [3H]Phorbol dibutyrate (PDBu) binding was determined as described (32Jaken S. Methods Enzymol. 1987; 141: 275-287Crossref PubMed Scopus (49) Google Scholar). Cells plated in 24-well plates were washed twice with binding buffer (Dulbecco's modified Eagle's medium, 1 mg/ml bovine serum albumin, 10 mm Hepes pH 7.0) and incubated in the presence of 10 nm [3H]PDBu (DuPont NEN, ON, Canada) at 37 °C for 30 min. Cells were then washed three times with ice-cold phosphate-buffered saline, lysed with 0.1n NaOH, and bound [3H]PDBu was measured by liquid scintillation counting. All experiments were done in triplicate determinations, in the presence (nonspecific binding) or absence (total binding) of 10 μm unlabeled PDBu (Sigma). Specific binding was the difference between total binding and nonspecific binding. RNA preparation and Northern blot analysis were performed as described previously (33Descoteaux A. Matlashewski G. J. Immunol. 1990; 145: 846-853PubMed Google Scholar), with the exception that probes were radiolabeled by random priming (34Feinberg A.P. Vogelstein B. Anal. Biochem. 1983; 132: 6-13Crossref PubMed Scopus (16653) Google Scholar). The TNF-α probe was the 1.5-kilobase PstI fragment from pmTNF-1 (35Fransen L. Muller R. Marmenout A. Tavernier J. Van der Heyden J. Kawashima E. Chollet A. Tizard R. Van Heuverswyn H. Van Vliet A. Ruysschaert M.R. Fiers W. Nucleic Acids Res. 1985; 13: 4417-4429Crossref PubMed Scopus (224) Google Scholar), provided by W. Fiers, the IL-1α probe was the 2.0-kilobase BamHI-HindIII fragment from pmIL1A (36Lomedico P.T. Gubler U. Hellmann C.P. Dukovich M. Giri J.G. Pan Y.C. Collier K. Semionow R. Chua A.O. Mizel S.B. Nature. 1984; 312: 458-462Crossref PubMed Scopus (598) Google Scholar), obtained from the American Type Culture Collection, the inducible nitric oxide synthase (iNOS) probe was the 817-base pairHincII-EcoRI fragment from piNOSL3 (37Xie Q.W. Cho H.J. Calaycay J. Mumford R.A. Swiderek K.M. Lee T.D. Ding A. Troso T. Nathan C. Science. 1992; 256: 225-228Crossref PubMed Scopus (1740) Google Scholar) (provided by D. Radzioch). Cells were incubated in the absence or presence of LPS (Escherichia coli, strain 0127:B8, Sigma) for 18 h and the amount of nitrite released into supernatants was determined with the Griess reagent as described (38Green S.J. Meltzer M.S. Hibbs J.J. Nacy C.A. J. Immunol. 1990; 144: 278-283PubMed Google Scholar). Adherent cells were incubated in the absence or presence of LPS for 18 h and the amounts of TNF-α and IL-1α in cell supernatants were determined by enzyme-linked immunosorbent assay. For TNF-α levels, a rat anti-mouse TNF-α monoclonal antibody and a biotin-labeled rat anti-mouse TNF-α monoclonal antibody (both from Cedarlane Laboratories, ON, Canada) were used. For IL-1α, a hamster anti-mouse IL-1α monoclonal antibody (Genzyme, Cambridge, MA), a rabbit anti-mouse IL-1α polyclonal serum (Cedarlane, ON, Canada), and alkaline phosphatase-conjugated anti-rabbit IgG antibodies (Calbiochem, San Diego, CA) were used. Secretion of MMP-9 in cell supernatants was determined by gelatin zymography, as described previously (39Tremblay P. Houde M. Arbour N. Rochefort D. Masure S. Mandeville R. Opdenakker G. Oth D. Cytokine. 1995; 7: 130-136Crossref PubMed Scopus (29) Google Scholar). Briefly, aliquots from cell supernatants were fractionated by electrophoresis in a 8% SDS-polyacrylamide gel containing 1% gelatin (Sigma). Gels were washed to remove SDS and incubated for 18 h at 37 °C in renaturing buffer (50 mm Tris, 5 mmCaCl2, 0.02% NaN3, 1% Triton X-100). MMP-9 activity was visualized following staining/destaining of the gel with Coomassie Brilliant Blue G-250 and was quantitated by computerized image analysis (Bio-Rad, model GS-670 Densitometer). Results were expressed as arbitrary scanning units. Adherent cells (107 per 100-mm tissue culture dishes) were stimulated with LPS for various time points, washed, and scraped into 1.5 ml of cold phosphate-buffered saline. The cell suspensions were transferred to microcentrifuge tubes, pelleted, and the nuclear protein extracts were prepared essentially as described (40Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2211) Google Scholar). Protein contents were determined using the BCA protein assay kit (Pierce) and the extracts were stored at −70 °C. EMSA were performed by incubating 32P-labeled NF-κB consensus oligonucleotide (5′-AGTTGAGGGGACTTTCCCAGG-3′, obtained from Promega) with 10 μg of nuclear extracts for 20 min at room temperature. The incubation mixture contained 3 μg of poly(dI-dC) in a binding buffer (10 mm Tris-HCl, 1 mm MgCl2, 0.5 mm EDTA, 0.5 mm dithiothreitol, 20 mm NaCl, 4% glycerol). The DNA-protein complexes were separated from free oligonucleotide by electrophoresis under nondenaturing conditions in 4% native polyacrylamide gels in a buffer containing 44.5 mm Tris, 44.5 mm borate, pH 8.0, and 1 mm EDTA. The specificity of binding was determined by competition with excess unlabeled oligonucleotide. After electrophoresis, gels were exposed to films at −70 °C. Stable transfectants from two independent populations of RAW 264.7 macrophages electroporated with pCIN-DN PKC-α were selected in the presence of 500 μg/ml G418. Western blot analyses were performed on three clones selected from each independent populations of transfectants to determine their PKC-α expression levels. The three clones from the first population (clones DN PKC-α B1, C2, and D1), and one clone from the second population (clone DN PKC-α A2) expressed immunoreactive PKC-α above endogenous levels (not shown). To determine whether DN PKC-α overexpression had any effect on LPS-induced responses, we measured the ability of these four DN PKC-α-overexpressing clones to secrete nitrite in response to LPS. As shown in Table I, LPS-induced nitrite secretion was inhibited in the four DN PKC-α-overexpressing clones (clones DN PKC-α B1, D1, A2, C2) with respect to the RAW 264.7 cells transfected with the empty vector. Inhibition of LPS-induced nitrite production was likely a consequence of DN PKC-α overexpression, as LPS-induced nitrite secretion in three clones of RAW 264.7 cells transfected with a construct containing the wild-type murine PKC-ζ cDNA (41Goodnight J. Kazanietz M.G. Blumberg P.M. Mushinski J.F. Mischak H. Gene (Amst.). 1992; 122: 305-311Crossref PubMed Scopus (50) Google Scholar) was similar to that of RAW 264.7 cells transfected with the empty vector (clones PKC-ζ A1, A2, B1) (Table I).Table IDN PKC-α inhibits LPS-induced nitrite secretionCellsTreatment (ng/ml LPS)010100μm1-aThese values represent the mean ± S.E. of one experiment performed in triplicate samples.Exp. 1 pCIN-42.1 ± 0.544.5 ± 2.848.0 ± 2.4 DN PKC-α A23.0 ± 0.612.3 ± 1.017.5 ± 3.2 DN PKC-α C22.1 ± 0.15.0 ± 0.88.1 ± 0.9 DN PKC-α B12.3 ± 0.25.0 ± 0.68.1 ± 1.9 DN PKC-α D11.7 ± 0.110.6 ± 0.515.6 ± 3.6Exp. 2 pCIN-42.9 ± 0.714.5 ± 4.319.4 ± 2.5 PKC-ζ A13.2 ± 0.215.6 ± 1.920.4 ± 5.2 PKC-ζ A23.0 ± 0.510.4 ± 1.117.4 ± 2.9 PKC-ζ B13.2 ± 0.211.8 ± 1.219.1 ± 1.4Adherent cells (2.5 × 105 per well) were incubated in the absence or presence of either 10 or 100 ng/ml LPS for 18 h. Nitrite levels were measured as described under “Experimental Procedures.” In Experiment 1, clones of RAW 264.7 cells overexpressing DN PKC-α were compared to RAW 264.7 cells transfected with the empty vector (pCIN-4) for nitrite production. In Experiment 2, clones of RAW 264.7 cells transfected with a PKC-ζ construct were compared to RAW 264.7 cells transfected with the empty vector.1-a These values represent the mean ± S.E. of one experiment performed in triplicate samples. Open table in a new tab Adherent cells (2.5 × 105 per well) were incubated in the absence or presence of either 10 or 100 ng/ml LPS for 18 h. Nitrite levels were measured as described under “Experimental Procedures.” In Experiment 1, clones of RAW 264.7 cells overexpressing DN PKC-α were compared to RAW 264.7 cells transfected with the empty vector (pCIN-4) for nitrite production. In Experiment 2, clones of RAW 264.7 cells transfected with a PKC-ζ construct were compared to RAW 264.7 cells transfected with the empty vector. Clone DN PKC-α A2, with a 2-fold increase in immunoreactive PKC-α levels, and clone DN PKC-α C2, with a 10-fold increase in immunoreactive PKC-α levels (Fig. 1 A), were selected for further analyses. Increased DN PKC-α levels in these clones was also demonstrated by measuring [3H]PDBu binding levels (42Knopf J.L. Lee M.H. Sultzman L.A. Kriz R.W. Loomis C.R. Hewick R.M. Bell R.M. Cell. 1986; 46: 491-502Abstract Full Text PDF PubMed Scopus (455) Google Scholar), which were higher (1.3-fold for clone A2 and 2-fold for clone C2) than in the parental line (RAW 264.7 transfected with pCIN-4) (Fig. 1 B). Finally, clones A2 and C2 were similar to the parental cells with respect to their growth characteristics and morphology (not shown). Of note, we were unsuccessful, despite several attempts, in generating stable G418-resistant clones overexpressing a wild type PKC-α construct, suggesting that elevated levels of wild type PKC-α is toxic for the RAW 264.7 cells. Exposure of macrophages to LPS induces TNF-α, IL-1α, and iNOS mRNA accumulation. To assess the contribution of PKC-α in this process, we determined the levels of TNF-α, IL-1α, and iNOS mRNA in RAW 264.7 control cells (transfected with the empty vector) and in the DN PKC-α-overexpressing clones A2 and C2 after LPS stimulation (10 and 100 ng/ml) for 6 h. In control RAW 264.7 cells, LPS induced the expression of these three genes in a dose-dependent manner (Fig. 2, lanes 1–3). DN PKC-α overexpression had a minor inhibitory effect on the induction of TNF-α mRNA accumulation (20–25% reduction in clone A2 and 45 to 55% in clone C2 with respect to control cells) (Fig. 2, top panel). In contrast, LPS-induced IL-1α mRNA accumulation was reduced by 50–70% in clone A2 (Fig. 2, second panel, lanes 5 and 6) with respect to control cells (lanes 2 and 3), and abolished in clone C2 (lanes 8and 9). Finally, iNOS mRNA accumulation was reduced by 50–60% in clones A2 (Fig. 2, third panel, lanes 5 and 6) and by 65–75% in clone C2 (lanes 8 and 9) with respect to iNOS mRNA levels present in control cells (lanes 2 and 3). This inhibition can be correlated with DN PKC-α expression levels. We next compared the ability of control RAW 264.7 cells and clones A2 and C2, to produce TNF-α, IL-1α, and nitrite. In the presence of 10 and 100 ng/ml LPS, RAW 264.7 cells secreted high levels of TNF-α, IL-1α, and nitrite in a dose-dependent manner (Fig. 3, A-C). Secretion of immunoreactive TNF-α (Fig. 3 A) by clone A2 was similar to that of RAW 264.7 cells and was reduced by 40–50% in clone C2. Similarly, secretion of TNF-α was reduced by 55% in clone B1 and by 40% in clone D1 in response to 100 ng/ml LPS (data not shown). Consistent with the inhibition of IL-1α mRNA accumulation, clones A2 and C2 failed to produce significant IL-1α levels in response to 10 ng/ml LPS (Fig. 3 B). At 100 ng/ml LPS, IL-1α secretion was slightly increased for clone A2 and was barely above basal levels for clone C2. Similar results were obtained with clones B1 and D1, both of which failed to secrete IL-1α in response to 100 ng/ml LPS (data not shown). Nitrite secretion (Fig. 3 C) was reduced by approximately 60–70% for clone A2, and by 80–90% for clone C2 with respect to RAW 264.7 cells. Thus, DN PKC-α overexpression had a major inhibitory effect on LPS-induced IL-1α and NO production. In addition to inflammatory cytokines and nitrite, LPS stimulates macrophages to secrete various hydrolases, including the matrix metalloproteinase MMP-9 (43Van Ranst M. Norga K. Masure S. Proost P. Vandekerckhove F. Auwerx J. Van Damme J. Opdenakker G. Cytokine. 1991; 3: 231-239Crossref PubMed Scopus (40) Google Scholar). Based on data obtained with PKC inhibitors, it has been proposed that PKC exerts both positive and negative regulation on LPS-induced MMP-9 secretion in macrophages (39Tremblay P. Houde M. Arbour N. Rochefort D. Masure S. Mandeville R. Opdenakker G. Oth D. Cytokine. 1995; 7: 130-136Crossref PubMed Scopus (29) Google Scholar). To determine the role of PKC-α in this process, we measured the secretion of MMP-9 in the supernatants of control cells and of clones A2 and C2 after stimulation with either 10 or 100 ng/ml LPS for 24 h. As shown in Fig. 4, MMP-9 levels were significantly higher in the supernatants of clones A2 (2-fold,lanes 7–10) and C2 (4-fold, lanes 12–15) than in the supernatants of normal cells (lanes 2–5). Of note, the reduced MMP-9 secretion by normal cells stimulated with 100 ng/ml LPS (lanes 4 and 5) was not observed with clones A2 (lanes 9 and 10) and C2 (lanes 14and 15). Thus, increased LPS-stimulated MMP-9 secretion in DN PKC-α-overexpressing RAW 264.7 cells suggests that PKC-α negatively regulates MMP-9 secretion. Treatment of macrophages with LPS rapidly induces the dissociation of NF-κB from IκB and its translocation to the nucleus where it binds to specific DNA sequences (14Sweet M.J. Hume D.A. J. Leukocyte Biol. 1996; 60: 8-26Crossref PubMed Scopus (710) Google Scholar, 44Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4599) Google Scholar). This process is initiated with IκB phosphorylation by the IκB kinase, IKK-α, on specific serine residues (45Régnier 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 (1072) Google Scholar, 46DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1913) Google Scholar), followed by its ubiquitination and degradation. To investigate whether DN PKC-α overexpression affected this pathway, we measured the kinetics of LPS-induced IκBα phosphorylation and degradation by immunoblotting analysis. In both control cells (Fig. 5 A, lanes 1–5) and clone C2 (lanes 6–10), IκBα phosphorylation was maximal within 10–20 min following the addition of LPS. Decline in phosphorylated IκBα levels was observed between 20 and 30 min post-stimulation. Kinetics of IκBα degradation were also similar in control cells (Fig. 5 B, lanes 1–5) and in clone C2 (lanes 6–10), with a sharp decline occurring between 10 and 20 min after LPS stimulation. Consistently, the kinetics of NF-κB nuclear translocation were similar in LPS-stimulated control cells and in clone C2 as determined by electrophoretic mobility shift assay (Fig. 6). Similar to clone C2, both clones B1 and D1 showed normal kinetics of LPS-induced NF-κB nuclear translocation (data not shown). Thus, DN PKC-α overexpression did not interfere with LPS-induced IκBα phosphorylation and degradation and with NFκB nuclear translocation in RAW 264.7 cells.Figure 6Effect of DN PKC-α overexpression on LPS-induced NF-κB activation. Adherent cells (vector alone,lanes 1–3; and clone C2, lanes 4–6) were incubated in the presence of 100 ng/ml LPS for 15 and 30 min. Cell extracts were prepared and EMSA for NF-κB were performed as described under “Experimental Procedures.” In lane 7, 50-fold excess cold probe was co-incubated with the reaction mixture containing nuclear extracts from control cells incubated with LPS for 30 min prior to EMSA. 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