A Novel Protein Kinase C (PKCϵ) Is Required for fMet-Leu-Phe-induced Activation of NF-κB in Human Peripheral Blood Monocytes

Abstract

We have reported that the chemoattractant, fMet-Leu-Phe (fMLP), induces the activation of NF-κB in human peripheral blood monocytes and that this requires the activity of small GTPase, RhoA (Huang, S., Chen, L.-Y., Zuraw, B. L., Ye, R. D., and Pan, Z. K. (2001) J. Biol. Chem. 276, 40977-40981). Here we showed that the novel protein kinase C isozyme, PKCϵ, associates functionally with RhoA in fMLP-stimulated monocytes and that PKCϵ acted as a signaling component downstream of the GTPase RhoA during fMLP-induced activation of NF-κB. Stimulation of monocytes with fMLP resulted in activation of both PKCϵ and NF-κB. This latter activation was largely blocked by specific inhibitors of PKCϵ by transient expression of a dominant-negative form of PKCϵ and by PKCϵ-specific short interfering RNA. These findings demonstrate, for the first time, that fMLP-induced activation of NF-κB utilizes a signaling pathway, which requires activity of PKCϵ, and that PKCϵ acts as a signaling component downstream of RhoA in cytokine gene transcription stimulated by a chemoattractant. The specificity of this response suggests an important role for the Rho GTPase-PKCϵ-NF-κB pathway in host defense and represents a novel and potentially important mechanism through which fMLP not only attracts leukocytes but may also contribute directly to inflammation. We have reported that the chemoattractant, fMet-Leu-Phe (fMLP), induces the activation of NF-κB in human peripheral blood monocytes and that this requires the activity of small GTPase, RhoA (Huang, S., Chen, L.-Y., Zuraw, B. L., Ye, R. D., and Pan, Z. K. (2001) J. Biol. Chem. 276, 40977-40981). Here we showed that the novel protein kinase C isozyme, PKCϵ, associates functionally with RhoA in fMLP-stimulated monocytes and that PKCϵ acted as a signaling component downstream of the GTPase RhoA during fMLP-induced activation of NF-κB. Stimulation of monocytes with fMLP resulted in activation of both PKCϵ and NF-κB. This latter activation was largely blocked by specific inhibitors of PKCϵ by transient expression of a dominant-negative form of PKCϵ and by PKCϵ-specific short interfering RNA. These findings demonstrate, for the first time, that fMLP-induced activation of NF-κB utilizes a signaling pathway, which requires activity of PKCϵ, and that PKCϵ acts as a signaling component downstream of RhoA in cytokine gene transcription stimulated by a chemoattractant. The specificity of this response suggests an important role for the Rho GTPase-PKCϵ-NF-κB pathway in host defense and represents a novel and potentially important mechanism through which fMLP not only attracts leukocytes but may also contribute directly to inflammation. The bacterial tripeptide fMet-Leu-Phe (fMLP) 1The abbreviations used are: fMLP, fMet-Leu-Phe; PKC, protein kinase C; TNF, tumor necrosis factor; NF-κB, nuclear factor κB; siRNA, short interfering RNA; IKK, IκB kinase. 1The abbreviations used are: fMLP, fMet-Leu-Phe; PKC, protein kinase C; TNF, tumor necrosis factor; NF-κB, nuclear factor κB; siRNA, short interfering RNA; IKK, IκB kinase. is one of the most powerful leukocyte chemoattractants. It binds to seven transmembrane-spanning G-protein-coupled receptors on monocytes and neutrophils and induces a variety of functional responses that are associated with inflammation. The inflammatory response produced by infecting bacteria involves a leukocyte gene expression that is tightly regulated by the activities of transcription factors such as NF-κB, NF-IL-6, and AP-1. NF-κB is of considerable importance in immune cell function because of its ability to activate the transcription of many proinflammatory immediate early genes, and we have shown that it can be activated by chemoattractants such as fMLP (1Pan Z. Kravchenko V.V. Ye R.D. J. Biol. Chem. 1995; 270: 7787-7790Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 2Kravchenko 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, 3Browning D.D. Pan Z.K. Prossnitz E.R. Ye R.D. J. Biol. Chem. 1997; 272: 7995-8001Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 4Pan Z.K. Biochim. Biophys. Acta. 1998; 1443: 90-98Crossref PubMed Scopus (39) Google Scholar). Indeed, the fMLP-induced activation of NF-κB appears essential for proinflammatory cytokine synthesis in human peripheral blood monocytes (5Pan Z.K. Chen L-Y. Cochrane C.G. Zuraw B.L. J. Immunol. 2000; 164: 404-411Crossref PubMed Scopus (59) Google Scholar). Protein kinase C (PKC) represents a family of serine/threonine kinases mediating intracellular signaling events (6Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-292Crossref PubMed Scopus (1345) Google Scholar, 7Newton A.C. Curr. Opin. Cell Biol. 1997; 9: 161-167Crossref PubMed Scopus (843) Google Scholar). PKC isoforms have been classified into three groups on the basis of their molecular structure and activation mechanisms: 1) conventional PKCs (cPKCα, βI, βII, and γ), 2) novel PKCs (nPKCδ, ϵ, μ, θ, and η), and 3) atypical PKCs (aPKCζ, and λ/ι). A number of in vitro and in vivo studies have implicated a role for PKCs in the regulation of host defense and inflammation (8Ivaska J. Bosca L. Parker P.J. Nat. Cell Biol. 2003; 5: 363-369Crossref PubMed Scopus (63) Google Scholar, 9Castrillo A. Pennington D.J. Otto F. Parker P.J. Owen M.J. Boscá L. J. Exp. Med. 2001; 194: 1231-1242Crossref PubMed Scopus (202) Google Scholar, 10Lin X. O'Mahony A. Mu Y. Geleziunas R. Greene W.C. Mol. Cell. Biol. 2000; 20: 2933-2940Crossref PubMed Scopus (230) Google Scholar, 11Rahman A. Anwar K.N. Uddin S. Xu N. Ye R.D. Platanias L.C. Malik A.B. Mol. Cell. Biol. 2001; 21: 5554-5565Crossref PubMed Scopus (147) Google Scholar, 12Dang P.M.-C. Fontayne A. Hakim J. El Benna J. Périanin A. J. Immunol. 2001; 166: 1206-1213Crossref PubMed Scopus (198) Google Scholar), and it has also been reported that PKCβ may modulate the respiratory burst of both neutrophils and HL-60 cells (13Majumdar S. Kane L.H. Rossi M.W. Volpp B.D. Nauseef W.M. Korchak H.M. Biochim. Biophys. Acta. 1993; 1176: 276-286Crossref PubMed Scopus (121) Google Scholar, 14Korchak H.M. Rossi M.W. Kilpatrick L.E. J. Biol. Chem. 1998; 273: 27292-27299Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). However, the contribution of each PKC isoform to the regulation of monocyte activation has not yet been elucidated. We have demonstrated that fMLP stimulates increased activity in RhoA, a small GTPase (15Huang S. Chen L-Y. Zuraw B.L. Ye R.D. Pan Z.K. J. Biol. Chem. 2001; 276: 40977-40981Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), and that this activity in human peripheral blood monocytes is mediated by the guanine nucleotide exchange factor Lbc (16Chen L-Y. Zuraw B.L. Ye R.D. Pan Z.K. J. Biol. Chem. 2004; 279: 7208-7212Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). We therefore investigated the role of PKC in the fMLP-stimulated signaling events that lead to the activation of NF-κB. We report that exposure of monocytes to fMLP causes increased activity of an nPKC isoform, PKCϵ, and that inhibition of PKCϵ activity blocks fMLP-stimulated activation of NF-κB. We also report that RhoA associates specifically with the PKCϵ in fMLP-stimulated monocytes and that the fMLP-induced PKCϵ activity is blocked by an inhibitor of RhoA. The results demonstrate that PKCϵ is a novel signal transducer for RhoA-mediated activation of NF-κB leading to proinflammatory cytokine gene expression in human peripheral blood monocytes stimulated with bacterial products. Reagents—fMLP and 1,25(OH)2D3 were obtained from Sigma. Staurosporine, Gö 6976, and PKCϵ inhibitor peptide were purchased from Calbiochem. Recombinant human TNFα was obtained from R & D Systems Inc (Minneapolis, MN). The cDNAs encoding the constitutively active mutant of PKCϵ and the dominant-negative mutants of PKCϵ and PKCα were cloned into the eukaryotic expression vector pEFneo. The dominant-negative IKKβ mutant and the recombinant Clostridium botulinum C3 transferase exoenzyme were obtained as described previously (17Chen L-Y. Zuraw B.L. Liu F.-T. Huang S. Pan Z.K. J. Immunol. 2002; 169: 3934-3939Crossref PubMed Scopus (55) Google Scholar). The polyclonal antibody against phospho-PKCϵ and monoclonal antibodies (mAbs) against RhoA, PKCϵ, and PKCα were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The polyclonal antibody against phospho-PKCα/β was purchased from Cell Signaling Technology (Beverly, MA). Preparation of Monocytes from Peripheral Blood—Heparinized human peripheral blood from healthy donors was fractionated on Percoll (Amersham Biosciences) density gradients. Mononuclear cells and neutrophils were obtained by centrifugation through a 55/74% discontinuous Percoll gradient. Monocytes were further separated from this population using gelatin/plasma-coated flasks (1Pan Z. Kravchenko V.V. Ye R.D. J. Biol. Chem. 1995; 270: 7787-7790Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Monocytes were resuspended in RPMI 1640 medium (Irvine Scientific, Santa Ana, CA) containing 10% (v/v) heat-inactivated fetal bovine serum, penicillin (100 units/ml), streptomycin (100 μg/ml), and l-glutamine (2 mm; Irvine Scientific). Electrophoretic Mobility Shift Assay (EMSA)—Nuclear extracts were prepared from human peripheral blood monocytes using the method of Dignam et al. (18Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9131) Google Scholar). The electrophoretic mobility shift assay was performed using 2.5 μg of the nuclear extract as described previously (2Kravchenko 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). Immunoprecipitation and Immunoblotting—Monocyte lysates were incubated with an appropriate amount of antibody for 3 h and then precipitated following absorption onto protein A-Sepharose (Amersham Biosciences). Precipitates were washed three times, separated by SDS-PAGE, and transferred to Hybond-ECL nitrocellulose (Amersham Biosciences). Filter strips were incubated with primary antibody for 30 min at room temperature, followed by addition of peroxidase-conjugated IgG (1:10,000 dilution, 30 min) and then analyzed for peroxidase using enhanced chemiluminescence reagents (PerkinElmer Life Science). Detection of Phosphorylated PKCs—Monocytes were incubated in medium ± fMLP then lysed in Laemmli buffer. Equal volumes of the lysates from each condition were resolved by SDS-PAGE using 8% gels and transferred to Hybond-ECL nitrocellulose membranes. The membranes were initially probed with a polyclonal anti-phospho PKC antibody (Cell Signaling Technology) that at a dilution of 1:2000 detects activated forms of cPKCα/β. A 1:200 dilution of anti-PKCϵ antibody was used at the same time to detect the nPKCϵ isoform. Membranes then were stripped and incubated with a murine anti-PKCα or murine anti-PKCϵ mAb. Transfection of Cells with PKC-specific siRNA and Luciferase Activity Assay—Short interfering RNA was used to reduce PKC levels in THP1 cells. 21-mer siRNA sequences with a dTdT-3′ overhang (Qiagen-Xeragon, Germantown, MD) specific for PKCα (5′-AAGCACAAGTTCAAAATCCAC-3′) or PKCϵ (5′-AAGCCCCTAAAGACAATGAAG-3′) were purchased. THP1 cells were co-transfected with either a PKCα-siRNA or a PKCϵ-siRNA together with the NF-κB-dependent luciferase reporter plasmid (pNF-κB-LUC). The reporter construct NF-κB-LUC was generated as described (19Shapiro V.S. Mollenauer M.N. Greene W.C. Weiss A. J. Exp. Med. 1996; 184: 1663-1669Crossref PubMed Scopus (68) Google Scholar). The plasmid pCMVβ (Clontech) was used as a control for transfection efficiency, and this was monitored via the expression of β-galactosidase. THP1 cells were differentiated by incubation with 1,25(OH)2D3 for 3 days (20Mackman N. Brand K. Edgington T.S. J. Exp. Med. 1991; 174: 1517-1526Crossref PubMed Scopus (329) Google Scholar) and then transiently transfected with plasmids using diethyl aminoethyl (DEAE)-dextran. The transfected cells were cultivated for 48 h before a 6-h incubation in medium ± 100 nm fMLP. Luciferase activity was determined by using the luciferase assay kit (Promega) and a Monolight 2010 luminometer (Analytical Luminescence, San Diego, CA). fMLP-induced NF-κB Activation Is Blocked by a PKC Inhibitor—To assess the role of PKC in fMLP-induced NF-κB activation, we examined the consequences of preincubating cells with specific PKC inhibitors. Staurosporine is a specifically inhibitor of PKC. It inhibits cPKC and nPKC but not aPKC isozymes (21Marte B.M. Meyer T. Stabel S. Standke G.J.R. Jaken S. Fabbro D. Hynes N.E. Cell Growth & Differ. 1994; 5: 239-247PubMed Google Scholar, 22Seynaeve C.M. Kazanietz M.G. Blumberg P.M. Sausville E.A. Worland P.J. Mol. Pharmacol. 1994; 45: 1207-1214PubMed Google Scholar). Following pretreatment ±50 nm staurosporine for 15 min, human monocytes were stimulated with fMLP or TNFα for 60 min, and the activation of NF-κB was assessed by electrophoretic mobility shift assay. In the absence of an inhibitor, the DNA binding activity of NF-κB was strongly induced by both fMLP and TNFα (Fig. 1A, lanes 2 and 3). This activation was largely prevented in those monocytes pretreated with staurosporine that were subsequently exposed to fMLP (Fig. 1A, lane 5). In contrast, the staurosporine treatment only marginally affected TNFα-induced activation of NF-κB (Fig. 1A, lane 6). Because staurosporine inhibits both cPKC and nPKC activity, we next examined which of these was required for fMLP-induced activation of NF-κB. Although pretreatment of monocytes with Gö 6976, an inhibitor of cPKC (PKCα and -β), not NF-κB activation (Fig. lanes and the pretreatment with a PKCϵ peptide the activation of NF-κB in fMLP-stimulated monocytes (Fig. lane a role for In contrast, a peptide a control peptide not fMLP Activation of PKCϵ and in inhibition of PKCϵ activity fMLP-induced NF-κB activation, we next examined fMLP induced PKCϵ activation, as PKCs a role in intracellular signal to PKCϵ is activated through of a which the kinase activity to D. K. K. Parker P.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), we were to an antibody that PKCϵ to detect the activation of Following with fMLP, monocytes were and the lysates were analyzed for of PKC by using a to fMLP to a in of PKCϵ that was min and min (Fig. This was not as as that that for the fMLP-induced of which was largely min (Fig. PKCϵ but PKCα Is for fMLP-induced Activation of of PKCϵ for fMLP-induced activation of NF-κB was further demonstrated by a dominant-negative form of PKCϵ in THP1 cells. Cells were co-transfected with expression encoding either a dominant-negative PKCα or a dominant-negative PKCϵ together with the NF-κB-dependent luciferase reporter plasmid and then exposed to fMLP. protein expression was by using antibodies not 3 that expression of the dominant-negative PKCϵ mutant protein was associated with of luciferase activity by NF-κB (Fig. lane 6). In contrast, the expression of a dominant-negative PKCα not have this (Fig. lane further the role of PKCα and PKCϵ in fMLP-induced NF-κB The of siRNA on PKC expression was by cells h Although of the PKC siRNA sequences affected the expression of PKCα expression was by PKCα-siRNA to ± and PKCϵ-siRNA expression of PKCϵ to ± of the in cells transfected with control RNA (Fig. The cells were then exposed to fMLP. PKCϵ-siRNA NF-κB activity, PKCα siRNA (Fig. These results that PKCϵ activity was required for fMLP-induced activation of NF-κB. Although fMLP exposure to increased PKCα activity, this not to be required for the activation of NF-κB. PKCα activity may be in the respiratory burst or both of which be induced by fMLP (13Majumdar S. Kane L.H. Rossi M.W. Volpp B.D. Nauseef W.M. Korchak H.M. Biochim. Biophys. Acta. 1993; 1176: 276-286Crossref PubMed Scopus (121) Google Scholar, 14Korchak H.M. Rossi M.W. Kilpatrick L.E. J. Biol. Chem. 1998; 273: 27292-27299Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, J. Biochem. Biophys. Res. PubMed Scopus Google Scholar, Cell. 1991; PubMed Scopus Google Scholar). PKCϵ fMLP-induced Activation NF-κB Activation via IκB previously that fMLP induced the of in human blood monocytes (5Pan Z.K. Chen L-Y. Cochrane C.G. Zuraw B.L. J. Immunol. 2000; 164: 404-411Crossref PubMed Scopus (59) Google Scholar). This is required for the activation NF-κB and requires to be by We therefore to the of IKKβ in the fMLP-induced activation of NF-κB. THP1 cells were co-transfected with NF-κB-LUC in with a plasmid encoding a dominant-negative mutant of which was by San Diego, CA). This dominant-negative mutant largely prevented the activation of NF-κB (Fig. lane a for IKKβ in the We also the function of IKKβ in activation of NF-κB. Although expression of the constitutively active form of PKCϵ induces NF-κB activity in the absence of fMLP, this NF-κB activity was not these cells were also co-transfected with a plasmid encoding the dominant-negative IKKβ (Fig. lane 3). it appears that PKCϵ its via the activation of PKCϵ with RhoA in fMLP-stimulated previously demonstrated that fMLP stimulates the activity of RhoA, a small GTPase and that this activity in monocytes is required for the activation of NF-κB induced by fMLP (15Huang S. Chen L-Y. Zuraw B.L. Ye R.D. Pan Z.K. J. Biol. Chem. 2001; 276: 40977-40981Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). We therefore the functional PKCϵ and RhoA was important for this fMLP-induced activation of NF-κB. We examined the activity of the PKCϵ in human peripheral blood monocytes was on its ability to with molecular and PKCϵ was functional binding to RhoA GTPase in Monocytes were exposed to medium ± 100 nm fMLP min) and then antibody was used to the RhoA GTPase from the and the of the were by with antibodies specific for PKCϵ or PKCϵ was as a with RhoA (Fig. 5). In control we the antibody with before it to cell The the of an containing PKCϵ (Fig. lanes 3). These showed that PKCϵ binds to the RhoA PKCϵ as a Signaling of The results demonstrated that was an PKCϵ and RhoA in monocytes their exposure to fMLP. We next examined RhoA mediated the activation of NF-κB via a downstream on This was by using botulinum C3 which is an that inhibits the Rho and but not K. J. 1997; PubMed Scopus (147) Google Scholar). Following incubation with or recombinant C3 transferase for human monocytes were stimulated with fMLP for min, and PKCϵ activity was shown in the PKCϵ activity was by C3 that PKCϵ is a signaling component that activated downstream of RhoA. The RhoA activity and PKCϵ activation was further investigated using THP1 cells that been co-transfected with expression encoding a constitutively active form of RhoA (Fig. lanes and either a dominant-negative PKCϵ 3) or a dominant-negative PKCα together with an NF-κB-dependent luciferase reporter The constitutively active RhoA induced a luciferase activity, but this activity was by of the dominant-negative PKCϵ (Fig. lane of the dominant-negative PKCα (Fig. lane These results that RhoA activity is required for the activation of PKCϵ induced by fMLP and that PKCϵ acts as a signaling component downstream of RhoA. We previously demonstrated that fMLP stimulates small GTPase RhoA activity in and this activity is required for the activation of NF-κB that is induced by fMLP (15Huang S. Chen L-Y. Zuraw B.L. Ye R.D. Pan Z.K. J. Biol. Chem. 2001; 276: 40977-40981Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In the we have demonstrated that PKCϵ is a signaling component downstream of RhoA and the fMLP-induced activation of NF-κB in human peripheral blood We to the role of PKCϵ in fMLP-induced activation of NF-κB in monocytes as PKCϵ has not previously been implicated in the signaling of monocytes to fMLP stimulated a in of PKCϵ and of the monocytes with staurosporine or PKCϵ peptide the fMLP-induced activation of NF-κB. The for of PKCϵ was then by THP1 cells with either plasmids encoding dominant-negative forms of PKCϵ and PKCα or short interfering RNA to reduce the expression of PKCϵ and Because was only inhibition of NF-κB activity, both incubation with PKCϵ peptide and by expression of dominant-negative PKCϵ, it is that nPKC such as and are also in fMLP-induced because and can activate NF-κB X. O'Mahony A. Mu Y. Geleziunas R. Greene W.C. Mol. Cell. Biol. 2000; 20: 2933-2940Crossref PubMed Scopus (230) Google Scholar, 11Rahman A. Anwar K.N. Uddin S. Xu N. Ye R.D. Platanias L.C. Malik A.B. Mol. Cell. Biol. 2001; 21: 5554-5565Crossref PubMed Scopus (147) Google Scholar). The that PKCϵ was to the activation of NF-κB to its role in fMLP-induced signaling in an role of the Rho in cytokine gene is the intracellular signaling RhoA activation to cytokine gene transcription in The Rho family of small has been shown to activate serine/threonine which can downstream These to on the and A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) reported that kinase the immediate downstream of and and activated leading to of IκB and the binding of NF-κB to DNA in cells by et al. S. M.J. R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) showed that a constitutively active kinase stimulated NF-κB activity in and and that activation of kinase was required for the of NF-κB activity both by in and following exposure of to These results that kinase may a role in the activation of NF-κB and the of inflammatory cytokine expression mediated by both and but not by RhoA. RhoA has been shown to be in activation of PKC in a murine cell and cells H. A. Parker P.J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). studies have the role of PKCϵ in protein kinase activation and in NF-κB activation in activated by bacterial A. Pennington D.J. Otto F. Parker P.J. Owen M.J. Boscá L. J. Exp. Med. 2001; 194: 1231-1242Crossref PubMed Scopus (202) Google Scholar, Z. S. F. J. Immunol. 2002; PubMed Scopus Google Scholar, J. A. J. Immunol. 2000; 164: PubMed Scopus Google Scholar). that fMLP PKCϵ in human peripheral blood and this activation a signal downstream of GTPase RhoA that fMLP-induced NF-κB We determined the by which PKCϵ and its downstream IκB to NF-κB have shown that NF-κB activity is regulated at the of IκB which is through of IκB and by Chen Y. PubMed Scopus Google Scholar, D. F. PubMed Scopus Google Scholar). We that expression of a dominant-negative form of IKKβ prevented the of NF-κB activity induced both by fMLP and by the expression of a constitutively active form of Although we that IKKβ is required for NF-κB activation induced by fMLP, the mechanism is yet to be mechanism is that PKCϵ may IKKβ This is by the that an atypical isoform of PKC, directly IKKβ in vitro and associates with IKKβ in cells M.J. J. Mol. Cell. Biol. PubMed Google Scholar). However, we have been to detect a PKCϵ and the not is that PKCϵ may activate through one or such signaling be which has been implicated in the cell signaling pathway to the activation of NF-κB through activation of IKKβ T.S. J. Immunol. Google Scholar). In we have shown that fMLP stimulates the activation of PKCϵ in both specific inhibitors as as transient expression of a dominant-negative PKCϵ mutant or PKCϵ siRNA, we that the fMLP-induced activation of NF-κB required PKCϵ we have shown that PKCϵ associates functionally with the RhoA in fMLP-stimulated monocytes and that the PKCϵ activation induced by fMLP is by a specific RhoA inhibitor, C3 that RhoA of PKCϵ in the signaling These findings that fMLP-induced activation of NF-κB utilizes a signaling pathway that requires the activity of The RhoA GTPase-PKCϵ-NF-κB pathway appears to a role in the leukocyte response to and is to be important in host defense and inflammation. We J. F. and for DNA and for their of this