Protein Kinase C-α Modulates Lipopolysaccharide-induced Functions in a Murine Macrophage Cell Line

Abstract

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. 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. Med. 1997; 185: 1661-1670Crossref PubMed Scopus (322) Google Scholar), studies with pharmacological inhibitors revealed that their activity is essential for the expression of LPS-induced macrophage functions (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,6Dong Z. O'Brian C.A. Fidler I.J. J. Leukoc. Biol. 1993; 53: 53-60Crossref PubMed Scopus (79) Google Scholar, 7Weinstein S.L. June C.H. DeFranco A.L. J. Immunol. 1993; 151: 3829-3938PubMed Google Scholar, 8Novogrodsky A. Vanichkin A. Patya M. Gazit A. Osherov N. Levitzki A. Science. 1994; 264: 1319-1322Crossref PubMed Scopus (332) Google Scholar, 9Shapira L. Takashiba S. Champagne C. Amar S. Van Dyke T. J. Immunol. 1994; 153: 1818-1824PubMed Google Scholar). In addition to protein tyrosine kinases, exposure of macrophages to LPS activates protein kinases, C (PKC) (9Shapira L. Takashiba S. Champagne C. Amar S. Van Dyke T. J. Immunol. 1994; 153: 1818-1824PubMed Google Scholar, 10Fujihara M. Connolly N. Ito N. Suzuki T. J. Immunol. 1994; 152: 1898-1906PubMed Google Scholar, 11Shinji H. Akagawa K.S. Yoshida T. J. Immunol. 1994; 153: 5760-5771PubMed Google Scholar, 12Aderem A.A. Albert K.A. Keum M.M. Wang J.K. Greengard P. Cohn Z.A. Nature. 1988; 332: 362-364Crossref PubMed Scopus (153) Google Scholar, 13Novotney M. Chang Z.L. Uchiyama H. Suzuki T. Biochemistry. 1991; 30: 5597-5604Crossref PubMed Scopus (50) Google Scholar, 14Sweet M.J. Hume D.A. J. Leukocyte Biol. 1996; 60: 8-26Crossref PubMed Scopus (710) Google Scholar, 15Herrera-Velit P. Knutson K.L. Reiner N.E. J. Biol. Chem. 1997; 272: 16445-16452Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 16Liu M.K. Herrera-Velit P. Brownsey R.W. Reiner N.E. J. 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, The of this mutant was by of the in the inactive mutant D. Z. K. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus (79) Google Scholar, F. S. K. F. M. G. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, F. F. H. H. G. A. G. Biol. 1996; PubMed Google Scholar). DN PKC-α cDNA was the of the expression S. J. J. S. S. 1996; PubMed Scopus Google Scholar) and the was The murine macrophage cell line RAW 264.7 Type Culture by D. was in a with in with with and were as described Hume D.A. Immunol. Cell Biol. 1993; PubMed Google Scholar). were in and were and examined for PKC-α by cells were with in and and protein were determined using the protein proteins were in and was by isoenzyme were from and degradation of was with the from phosphorylation of the p38 kinase was determined with the p38 and phosphorylation of the Jun N-terminal kinase was with the from dibutyrate was determined as described S. 141: PubMed Scopus Google Scholar). in were with and in the of at for were with with and was by experiments were in in the or of was the and and were as described A. G. J. Immunol. 1990; Google Scholar), with the that were by Biochem. PubMed Scopus Google Scholar). The TNF-α was the from L. R. A. J. Van J. E. A. R. Van H. Van A. M.R. W. Nucleic Acids Res. 13: PubMed Scopus Google Scholar), by W. the was the from U. M. K. R. Nature. PubMed Scopus Google Scholar), obtained from the American Type Culture the inducible nitric oxide synthase was the from J. R.A. A. T. C. Science. PubMed Scopus Google Scholar) by D. were in the or of LPS for and the of nitrite was determined with the as described C.A. J. Immunol. 1990; Google Scholar). cells were in the or of LPS for and the of TNF-α and in cell were determined by assay. TNF-α a TNF-α and a TNF-α from were a a and were of in cell was determined by as described P. M. N. D. S. R. G. D. 1995; PubMed Scopus Google Scholar). from cell were by in a were to and for at in activity was of the with and was by were as cells were with LPS for various and of The cell were to and the protein were as described Nucleic Acids Res. 1991; 19: PubMed Scopus Google Scholar). were determined using the protein and the were at were by NF-κB obtained from with of for at The of in a The were from by in in a and The specificity of was determined by with were to at from of RAW 264.7 macrophages with PKC-α were in the of were on from of to their PKC-α expression The from the first DN PKC-α and and one from the DN PKC-α PKC-α endogenous To DN PKC-α overexpression on LPS-induced we the of these DN PKC-α-overexpressing to nitrite in response to in LPS-induced nitrite secretion was inhibited in the DN PKC-α-overexpressing DN PKC-α with respect to the RAW 264.7 cells with the of LPS-induced nitrite production was a of DN PKC-α as LPS-induced nitrite secretion in of RAW 264.7 cells with a the murine cDNA J. Mushinski J.F. H. PubMed Scopus (50) Google Scholar) was to that of RAW 264.7 cells with the PKC-α LPS-induced nitrite the of one in DN PKC-α DN PKC-α DN PKC-α DN PKC-α cells were in the or of either or LPS for were as described In of RAW 264.7 cells overexpressing DN PKC-α were to RAW 264.7 cells with the for nitrite production. In of RAW 264.7 cells with a were to RAW 264.7 cells with the the of one in in a cells were in the or of either or LPS for were as described In of RAW 264.7 cells overexpressing DN PKC-α were to RAW 264.7 cells with the for nitrite production. In of RAW 264.7 cells with a were to RAW 264.7 cells with the DN PKC-α with a in PKC-α and DN PKC-α with a in PKC-α were for DN PKC-α in these was also by R.W. 1986; Full Text PDF PubMed Scopus Google Scholar), which were for and for in the parental line 264.7 with and were to the parental cells with respect to their growth and morphology we were several in overexpressing a wild type PKC-α that of wild type PKC-α is for the RAW 264.7 of macrophages to LPS and iNOS mRNA To the contribution of PKC-α in this we determined the of and iNOS mRNA in RAW 264.7 cells with the and in the DN PKC-α-overexpressing and LPS and for In RAW 264.7 LPS induced the expression of these in a DN PKC-α overexpression a on the of TNF-α mRNA in and to in with respect to In LPS-induced mRNA was by in and with respect to cells and and in iNOS mRNA was by in and and by in and with respect to iNOS mRNA present in cells and can be with DN PKC-α expression the of RAW 264.7 cells and and to and In the of and LPS, RAW 264.7 cells of and nitrite in a of TNF-α by was to that of RAW 264.7 cells and was by in secretion of TNF-α was by in and by in in response to LPS not with the of mRNA accumulation, and to in response to LPS At LPS, secretion was for and was for were obtained with and of which to in response to LPS not secretion was by for and by for with respect to RAW 264.7 DN PKC-α overexpression a major on LPS-induced and NO production. In addition to and LPS macrophages to various including the matrix metalloproteinase M. K. S. P. F. J. Van J. G. 1991; PubMed Scopus Google Scholar). on data obtained with PKC it has been that PKC and regulation on LPS-induced secretion in macrophages P. M. N. D. S. R. G. D. 1995; PubMed Scopus Google Scholar). To the role of PKC-α in this we the secretion of in the of cells and of and with either or LPS for in were in the of and in the of cells the secretion by cells with LPS and was not with and and secretion in DN PKC-α-overexpressing RAW 264.7 cells that PKC-α regulates of macrophages with LPS the of NF-κB from and to the it binds to specific M.J. Hume D.A. J. Leukocyte Biol. 1996; 60: 8-26Crossref PubMed Scopus (710) Google Scholar, T. Annu. Rev. Immunol. 1994; PubMed Scopus Google Scholar). is with phosphorylation by the on specific C.H. Z. M. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, M. E. M. Nature. 1997; PubMed Scopus Google Scholar), by and To DN PKC-α overexpression affected this we the of LPS-induced phosphorylation and degradation by In cells and phosphorylation was the addition of in was and of degradation were also in cells and in with a and LPS the of NF-κB were in cells and in as determined by electrophoretic mobility shift to and showed of LPS-induced NF-κB not DN PKC-α overexpression did not with LPS-induced phosphorylation and degradation and with in RAW 264.7 of DN PKC-α overexpression on LPS-induced NF-κB cells and were in the of LPS for and Cell were and for NF-κB were as described In was with the from cells with LPS for to were obtained in different LPS the leading to the activation of the mitogen-activated protein kinases and J. L. Ulevitch R.J. Science. 1994; PubMed Scopus Google Scholar, J.C. S. D. D. M.J. M.M. Young Nature. 1994; PubMed Scopus Google Scholar, J. S.L. L. DeFranco A.L. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, J. M. DeFranco A.L. J. Exp. Med. 1995; PubMed Scopus Google Scholar, P. Med. 1994; PubMed Google Scholar). LPS-induced p38 and activation is required for the expression of IL-1 and TNF-α J.C. S. D. D. M.J. M.M. Young Nature. 1994; PubMed Scopus Google Scholar, Biol. 1997; PubMed Google Scholar), we have determined these were affected by DN PKC-α overexpression. To this end, we have the of LPS-induced phosphorylation of and p38 by In cells and phosphorylation was at and a at p38 phosphorylation was in cells and at a at and was at LPS-induced phosphorylation of p38 and was not inhibited by DN PKC-α overexpression. A role for PKC in the regulation of in macrophages has been from the that exposure of macrophages to LPS activates PKC (9Shapira L. Takashiba S. Champagne C. Amar S. Van Dyke T. J. Immunol. 1994; 153: 1818-1824PubMed Google Scholar, 10Fujihara M. Connolly N. Ito N. Suzuki T. J. 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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, H. Sakano T. Hamasaki T. Kashiwa H. Ueda K. Immunology. 1989; 67: 210-215PubMed Google Scholar, P. M. N. D. S. R. G. D. 1995; PubMed Scopus Google Scholar). However, our knowledge on the contribution of particular isoenzymes in the regulation of specific macrophage functions is limited and mainly on The present was at the role of PKC-α in the regulation of LPS-induced macrophage To this end, we have stably overexpressed a dominant-negative mutant of this isoenzyme in the murine macrophage line RAW 264.7. is that overexpression of DN PKC-α selective on LPS-initiated signal that PKC-α activity is required for the of specific macrophage function by In and NO production were inhibited in DN PKC-α-overexpressing studies on the iNOS gene revealed that at least PKC isoenzymes expression. on the differential down-regulation of PKC isoenzymes by phorbol it has been that PKC-βII may in LPS-induced iNOS gene expression and nitrite production (10Fujihara M. Connolly N. Ito N. Suzuki T. J. Immunol. 1994; 152: 1898-1906PubMed Google Scholar). More recently, transient transfection studies in RAW 264.7 cells evidence that regulates a pathway that iNOS gene expression in response to phorbol (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). this PKC-ε-dependent pathway is apparently not involved in the LPS that DN PKC-α overexpression inhibited LPS-induced nitrite secretion and iNOS mRNA indicated that PKC-α also regulates NO production. Collectively, these the that iNOS expression is regulated by multiple which may be by the multiple of regulation for iNOS expression C. J. Leukocyte Biol. 1994; PubMed Scopus Google Scholar, J. C. Annu. Rev. Immunol. 1997; PubMed Scopus Google Scholar), it is that particular PKC isoenzymes at the intracellular leading to NO production. The regulation of and TNF-α production in macrophages by particular PKC isoenzymes is not well E.J. Radzioch D. Young H.A. Varesio L. J. Immunol. 1988; 141: 3101-3105PubMed Google Scholar) that of murine macrophages with the PKC in a the expression of mRNA with The potent of LPS-induced mRNA in cells overexpressing DN PKC-α that PKC-α is one of the PKC isoenzymes that gene expression. The of DN PKC-α overexpression on LPS-induced TNF-α production was important as TNF-α protein secretion and TNF-α mRNA were by in which the of DN on the that inhibited LPS-induced TNF-α gene expression in murine macrophages E.J. Radzioch D. Young H.A. Varesio L. J. Immunol. 1988; 141: 3101-3105PubMed Google Scholar), our data suggest that PKC PKC-α LPS-induced TNF-α expression. Macrophages various matrix function is the of H. 1996; PubMed Google Scholar). of M. K. S. P. F. J. Van J. G. 1991; PubMed Scopus Google Scholar) in RAW 264.7 cells is inducible by LPS and is to and regulation by PKC P. M. N. D. S. R. G. D. 1995; PubMed Scopus Google Scholar). was of to overexpression of DN PKC-α production. In contrast to and nitrite secretion, we that LPS-induced secretion was in DN PKC-α overexpressing that PKC-α is one of the isoenzyme that regulates expression. regulation of gene expression by particular PKC isoenzymes has been described in the cell line overexpression of either PKC-α or and inhibited activity and Z. P. C. J. J. Immunol. 1997; Google Scholar). of production that DN PKC-α overexpression did not LPS in RAW 264.7 is one of the major intracellular mediators of LPS-induced M.J. Hume D.A. J. Leukocyte Biol. 1996; 60: 8-26Crossref PubMed Scopus (710) Google Scholar, T. Annu. Rev. Immunol. 1994; PubMed Scopus Google Scholar). In NF-κB are to a of the family of proteins which the NF-κB cell is on specific by C.H. Z. M. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, M. E. M. Nature. 1997; PubMed Scopus Google Scholar), and NF-κB to to the and to T. Annu. Rev. Immunol. 1994; PubMed Scopus Google Scholar). A role for PKC in the regulation of NF-κB activation pathway has been by the that with kinase activity and that overexpression of a dominant-negative mutant of NF-κB activation E. M.M. L. J. J. F. J. Biol. 1993; 13: PubMed Google Scholar, L. P. J. M.M. E. J. J. 1994; 13: PubMed Scopus Google Scholar, L. M.M. J. J. 1996; PubMed Google Scholar). LPS-induced phosphorylation and degradation and NF-κB activation in DN PKC-α-overexpressing it is that PKC-α is not involved in the activation of this that NF-κB important role in the activation of TNF-α gene expression M.J. Hume D.A. J. Leukocyte Biol. 1996; 60: 8-26Crossref PubMed Scopus (710) Google Scholar), this be with the of DN PKC-α overexpression on LPS-induced TNF-α expression. the regulation of iNOS is evidence that activation of NF-κB is not for LPS can NF-κB in macrophages from either or of iNOS or TNF-α in macrophages from A. S. J. Leukocyte Biol. 1995; PubMed Scopus Google Scholar). J. Biol. 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The that LPS-induced p38 and phosphorylation in our DN PKC-α-overexpressing that activation of these kinase not PKC-α Moreover, this data evidence that DN PKC-α overexpression did not cause a of LPS-induced in RAW 264.7 studies are required to the identity of the LPS-induced and regulated by In this regards, LPS activates the kinase pathway in macrophages and evidence was that may in the of and TNF-α gene expression J. M. DeFranco A.L. J. Exp. Med. 1995; PubMed Scopus Google Scholar, P. Med. 1994; PubMed Google Scholar, T. D. R.A. S. M. J. Immunol. 1994; 153: Google Scholar). on the of activation in and cells revealed that PKC including are activators of in W. G. G. R. H. H. G. Marme D. Rapp Nature. 1993; PubMed Scopus Google Scholar, H. U. J. Rapp U. Biol. 1997; PubMed Scopus Google Scholar). be of to PKC-α is required for activation in macrophages. for are to S. for the to the expression and P. D. and H. for and