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

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 mole-cules such as nitric oxide and proinflammatory cytokines. In this study, we have examined the role of the a 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-neg-ative (DN) version of PKC- a (DN PKC- a ) in the murine macrophage cell line RAW 264.7. Clones overexpressing DN PKC- a were indistinguishable from the parental line with respect to morphology and growth characteristics. At the functional level, DN PKC- a overexpression strongly inhibited LPS-induced interleukin-1 a mRNA accumulation, and to a lesser extent inducible nitric oxide synthase and tumor necrosis factor- a expression. DN-PKC- a overexpression did not cause a general unre-sponsiveness to LPS, as secretion of the matrix metallo-proteinase-9 was up-regulated in our DN PKC- a -overex-pressing clones. Moreover, LPS-induced phosphorylation and degradation of I k B a , NF- k B activation, as well as p38 mitogen-activated protein kinase and Jun N-terminal kinase phosphorylation, were not affected by DN PKC- version of the gene, DN PKC- a (K368D), was created by site-directed mutagenesis using the Trans-former System with the mutagenic primer AD-5 (5 9 -GTATGCAATC- GATATCCTGAAGAAGG-3 as described the manufacturer The sequence of this was con- firmed res-*

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 Gramnegative 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 (1)(2)(3). Although the identity of the protein tyrosine kinases that me-diate LPS-initiated signal transduction remains to be determined with certainty (4,5), studies with pharmacological inhibitors revealed that their activity is essential for the expression of LPS-induced macrophage functions (3, 6 -9). In addition to protein tyrosine kinases, exposure of macrophages to LPS activates protein kinases, C (PKC) (9 -16), and experiments using various PKC inhibitors indicated that PKC activity is required for the expression of several macrophage functions, including TNF-␣ and IL-1 secretion, NO production, and tumoricidal activity (9,13,17,18).
PKC was first characterized as a Ca 2ϩ -dependent and phospholipid-dependent protein serine/threonine kinase that requires diacylglycerol for activity (19). 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 (20). 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 (20,21). Macrophages and monocytic cells express the Ca 2ϩ -dependent isoenzymes ␣, ␤I, and ␤II, the Ca 2ϩ -independent isoenzymes ␦ and ⑀, and the atypical isoenzyme (10,16,22,23). 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-␤II participates in LPS-induced iNOS gene expression and nitrite production in the J774 macrophage cell line (10). 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-⑀, but in contrast to the pathway regulated by PKC-␤II, the PKC-⑀-dependent pathway is apparently not involved in the LPS response (24).
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-␣ 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 (21,25). Using this approach, we obtained evidence that PKC-␣ activity regulates selective LPS-induced macrophage functions involved in host defense and inflammation.

EXPERIMENTAL PROCEDURES
cDNAs and Expression Vectors-The wild type human PKC-␣ cDNA (26) was obtained from the American Type Culture Collection (Rockville, MD). A dominant-negative version of the gene, DN PKC-␣ (K368D), was created by site-directed mutagenesis using the Transformer System with the mutagenic primer AD-5 (5Ј-GTATGCAATC-GATATCCTGAAGAAGG-3Ј), as described by the manufacturer (CLONTECH, Palo Alto, CA). The sequence of this mutant was confirmed by sequence analysis. Replacement of the conserved lysine res-* This work was supported by Medical Research Council of Canada Grant MT-12933 (to A. D.) and from the Fonds de la Recherche en Santé du Québec. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  idue in the ATP-binding domain yields an enzymatically inactive transdominant mutant (27)(28)(29). DN PKC-␣ cDNA was cloned into the EcoRI site of the expression vector pCIN-4 (30) and the resulting construct was designated pCIN-DN PKC-␣.
Cell Culture and Stable Transfections-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% CO 2 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 (31). Transfectants were selected in complete medium containing 500 g/ml G418 (Life Technologies Inc.) and individual clones were harvested, expanded, and examined for PKC-␣ levels by Western blot analysis.
Western Blot Analyses-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 g) 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 IB␣ was analyzed with the PhosphoPlus IB␣ (Ser 32 ) Antibody kit from New England BioLabs (Beverly, MA), phosphorylation of the p38 MAP kinase was determined with the PhosphoPlus p38 MAPK (Tyr 182 Antibody kit, New England Biolabs), and phosphorylation of the Jun N-terminal kinase was analyzed with the Anti-Active JNK pAb from Promega (Madison, WI).
[ 3 H]PDBu Binding Assay-[ 3 H]Phorbol dibutyrate (PDBu) binding was determined as described (32). 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 [ 3 H]PDBu (DuPont NEN, ON, Canada) at 37°C for 30 min. Cells were then washed three times with ice-cold phosphatebuffered saline, lysed with 0.1 N NaOH, and bound [ 3 H]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 M unlabeled PDBu (Sigma). Specific binding was the difference between total binding and nonspecific binding.
Determination of Nitrite-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 (38).
Quantitation of Cytokines-Adherent cells were incubated in the absence or presence of LPS for 18 h and the amounts of TNF-␣ and IL-1␣ in cell supernatants were determined by enzyme-linked immunosorbent assay. For TNF-␣ levels, a rat anti-mouse TNF-␣ monoclonal antibody and a biotin-labeled rat anti-mouse TNF-␣ monoclonal antibody (both from Cedarlane Laboratories, ON, Canada) were used. For IL-1␣, a hamster anti-mouse IL-1␣ monoclonal antibody (Genzyme, Cambridge, MA), a rabbit anti-mouse IL-1␣ polyclonal serum (Cedarlane, ON, Canada), and alkaline phosphatase-conjugated anti-rabbit IgG antibodies (Calbiochem, San Diego, CA) were used.
Detection of MMP-9 -Secretion of MMP-9 in cell supernatants was determined by gelatin zymography, as described previously (39). 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 mM CaCl 2 , 0.02% NaN 3 , 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.
Nuclear Protein Extracts and Electrophoretic Mobility Shift Assay (EMSA)-Adherent cells (10 7 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 (40). Protein contents were determined using the BCA protein assay kit (Pierce) and the extracts were stored at Ϫ70°C. EMSA were performed by incubating 32 P-labeled NF-B consensus oligonucleotide (5Ј-AGTTGAGGGGACTT-TCCCAGG-3Ј, obtained from Promega) with 10 g of nuclear extracts for 20 min at room temperature. The incubation mixture contained 3 g of poly(dI-dC) in a binding buffer (10 mM Tris-HCl, 1 mM MgCl 2 , 0.5 mM EDTA, 0.5 mM dithiothreitol, 20 mM NaCl, 4% glycerol). The DNAprotein 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 Ϫ70°C.

RESULTS
Generation of DN PKC-␣ Overexpressing RAW 264.7 Macrophages-Stable transfectants from two independent populations of RAW 264.7 macrophages electroporated with pCIN-DN PKC-␣ were selected in the presence of 500 g/ml G418. Western blot analyses were performed on three clones selected from each independent populations of transfectants to determine their PKC-␣ expression levels. The three clones from the first population (clones DN PKC-␣ B1, C2, and D1), and one clone from the second population (clone DN PKC-␣ A2) expressed immunoreactive PKC-␣ above endogenous levels (not shown).
To determine whether DN PKC-␣ overexpression had any effect on LPS-induced responses, we measured the ability of these four DN PKC-␣-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-␣-overexpressing clones (clones DN PKC-␣ 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-␣ overexpression, as LPS-induced nitrite secretion in three clones of RAW 264.7 cells transfected with a construct containing the wild-type murine PKC-cDNA (41) was similar to that of RAW 264.7 cells transfected with the empty vector (clones PKC-A1, A2, B1) (Table I).
Clone DN PKC-␣ A2, with a 2-fold increase in immunoreactive PKC-␣ levels, and clone DN PKC-␣ C2, with a 10-fold increase in immunoreactive PKC-␣ levels (Fig. 1A), were selected for further analyses. Increased DN PKC-␣ levels in these clones was also demonstrated by measuring [ 3 H]PDBu binding levels (42), 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. 1B). 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-␣ construct, suggesting that elevated levels of wild type PKC-␣ is toxic for the RAW 264.7 cells.
Effect of PKC-␣ Overexpression on LPS-induced TNF-␣, IL-1␣, and iNOS Gene Expression-Exposure of macrophages to LPS induces TNF-␣, IL-1␣, and iNOS mRNA accumulation. To assess the contribution of PKC-␣ in this process, we determined the levels of TNF-␣, IL-1␣, and iNOS mRNA in RAW 264.7 control cells (transfected with the empty vector) and in the DN PKC-␣-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-␣ overexpression had a minor inhibitory effect on the induction of TNF-␣ 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-1␣ 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 8 and 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-␣ expression levels.
Effect of DN PKC-␣ Overexpression on LPS-stimulated Cytokine and Nitrite Production-We next compared the ability of control RAW 264.7 cells and clones A2 and C2, to produce TNF-␣, IL-1␣, and nitrite. In the presence of 10 and 100 ng/ml LPS, RAW 264.7 cells secreted high levels of TNF-␣, IL-1␣, and nitrite in a dose-dependent manner (Fig. 3, A-C). Secretion of immunoreactive TNF-␣ (Fig. 3A) 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-␣ 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-1␣ mRNA accumulation, clones A2 and C2 failed to produce significant IL-1␣ levels in response to 10 ng/ml LPS (Fig. 3B). At 100 ng/ml LPS, IL-1␣ 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-1␣ in response to 100 ng/ml LPS (data not shown). Nitrite secretion (Fig. 3C) 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-␣ overexpression had a major inhibitory effect on LPS-induced IL-1␣ and NO production.
Overexpression of DN PKC-␣ Enhances LPS-stimulated MMP-9 Secretion-In addition to inflammatory cytokines and nitrite, LPS stimulates macrophages to secrete various hydrolases, including the matrix metalloproteinase MMP-9 (43). 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 (39). To determine the role of PKC-␣ 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 14  and 15). Thus, increased LPS-stimulated MMP-9 secretion in DN PKC-␣-overexpressing RAW 264.7 cells suggests that PKC-␣ negatively regulates MMP-9 secretion.
Phosphorylation and Degradation of IB␣ and Nuclear Translocation of NFB Are Normal in DN PKC-␣ Overexpressing RAW 264.7 Cells-Treatment of macrophages with LPS rapidly induces the dissociation of NF-B from IB and its translocation to the nucleus where it binds to specific DNA sequences (14,44). This process is initiated with IB phosphorylation by the IB kinase, IKK-␣, on specific serine residues (45,46), followed by its ubiquitination and degradation. To investigate whether DN PKC-␣ overexpression affected this pathway, we measured the kinetics of LPS-induced IB␣ phosphorylation and degradation by immunoblotting analysis. In both control cells (Fig. 5A, lanes 1-5) and clone C2 (lanes 6 -10), IB␣ phosphorylation was maximal within 10 -20 min following the addition of LPS. Decline in phosphorylated IB␣ levels was observed between 20 and 30 min post-stimulation. Kinetics of IB␣ degradation were also similar in control cells (Fig. 5B,  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-B 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-B nuclear translocation (data not shown). Thus, DN PKC-␣ overexpression did not interfere with LPS-induced IB␣ phosphorylation and degradation and with NFB nuclear translocation in RAW 264.7 cells.

LPS-stimulated Phosphorylation of p38 MAP Kinase and JNK Is Normal in DN PKC-␣ Overexpressing RAW 264.7 Cells-LPS
induces the signaling pathways leading to the activation of the mitogen-activated protein kinases (MAPK) ERK1/2, p38, and JNK (47)(48)(49)(50)(51). Since LPS-induced p38 and JNK activation is required for the expression of IL-1 and TNF-␣ (48, 52), we have determined whether these pathways were affected by DN PKC-␣ overexpression. To this end, we have measured the kinetics of LPS-induced phosphorylation of JNK and p38 by immunoblot analyses. In both control cells and clone C2, JNK phosphorylation (Fig. 7A) was detectable at 10 min post-stimulation and reached a maximum at 30 min. Similarly, p38 phosphorylation was detectable in both control cells and clone C2 at 15 min post-stimulation, reached a peak at 30 min, and was still detectable at 60 min (Fig. 7B). Thus, LPS-induced phosphorylation of p38 and JNK was not inhibited by DN PKC-␣ overexpression. DISCUSSION A role for PKC in the regulation of LPS-inducible events in macrophages has been suggested from the observations that exposure of macrophages to LPS activates PKC (9 -16) and that pretreatment of macrophages with either PKC inhibitors or  [11][12][13][14][15] were incubated in the absence (lanes 1, 6, and 11) or in the presence of either 10 ng/ml (lanes 2, 3, 7, 8, 12, and 13) or 100 ng/ml LPS (lanes 4, 5, 9, 10, 14, and 15) for 18 h. MMP-9 activity was assessed by zymography as described under "Experimental Procedures." The data shown in this figure are representative of two separate experiments. phorbol esters inhibits LPS-induced TNF-␣, IL-1, and MMP-9 secretion, NO production, and tumoricidal activity (9,13,17,18,39). However, our knowledge on the contribution of particular isoenzymes in the regulation of specific macrophage functions is limited and relies mainly on indirect evidence. The present study was aimed at investigating the role of PKC-␣ in the regulation of LPS-induced macrophage functions. To this end, we have stably overexpressed a dominant-negative mutant of this isoenzyme in the murine macrophage line RAW 264.7. Our main finding is that overexpression of DN PKC-␣ had selective effects on LPSinitiated signal transduction, suggesting that PKC-␣ activity is required for the modulation of specific macrophage function by LPS. In particular, IL-1␣ and NO production were significantly inhibited in DN PKC-␣-overexpressing cells.
Previous studies on the iNOS gene revealed that at least two PKC isoenzymes regulate its expression. Based on the differential down-regulation of PKC isoenzymes by phorbol esters, it has been suggested that PKC-␤II may participate in LPSinduced iNOS gene expression and nitrite production (10). More recently, transient transfection studies in RAW 264.7 cells provided evidence that PKC-⑀ regulates a pathway that promotes iNOS gene expression in response to phorbol esters (24). Interestingly, this PKC-⑀-dependent pathway is apparently not involved in the LPS response. Our finding that DN PKC-␣ overexpression inhibited LPS-induced nitrite secretion and reduced iNOS mRNA accumulation indicated that PKC-␣ also regulates NO production. Collectively, these observations raise the possibility that iNOS expression is regulated by multiple PKC-dependent pathways, which may be activated by distinct stimuli. Considering the multiple levels of regulation for iNOS expression (53,54), it is conceivable that particular PKC isoenzymes act at distinct steps along the intracellular cascades leading to NO production.
The regulation of IL-1␣ and TNF-␣ production in macrophages by particular PKC isoenzymes is not well known. Kovacs et al. (17) previously reported that preincubation of murine peritoneal macrophages with the PKC inhibitor H7 reduced in a dose-dependent manner the expression of IL-1␣ mRNA after stimulation with LPS. The potent inhibition of LPS-induced IL-1␣ mRNA accumulation in cells overexpressing DN PKC-␣ suggests that PKC-␣ is one of the PKC isoenzymes that regulate IL-1␣ gene expression. The effect of DN PKC-␣ overexpression on LPS-induced TNF-␣ production was less important as TNF-␣ protein secretion and TNF-␣ mRNA accumulation were reduced by approximately 50% in clone C2, which expresses the highest levels of DN PKC-␣. Thus, based on the previous demonstration that H7 potently inhibited LPSinduced TNF-␣ gene expression in murine macrophages (17), our data suggest that PKC isoenzyme(s) other than PKC-␣ regulate LPS-induced TNF-␣ expression.
Macrophages secrete various matrix metalloproteinases whose function is the remodelling of extracellular matrices (55). Expression of MMP-9 (43) in RAW 264.7 cells is inducible by LPS and is subjected to both positive and negative regulation by PKC (39). It was thus of interest to determine whether overexpression of DN PKC-␣ would affect MMP-9 production. In contrast to IL-1␣, TNF-␣, and nitrite secretion, we found that LPS-induced MMP-9 secretion was significantly enhanced in DN PKC-␣ overexpressing cells. This observation suggests that PKC-␣ is one of the isoenzyme that negatively regulates LPS-stimulated MMP-9 expression. Negative regulation of gene expression by particular PKC isoenzymes has been recently described in the mast cell line RBL-2H3, where overexpression of either PKC-␣ or PKC-⑀ specifically and effectively inhibited receptor-dependent cytosolic phospholipase A 2 activity and arachidonic acid metabolite release (56). Importantly, up-regulation of MMP-9 production demonstrates that DN PKC-␣ overexpression did not inhibit all LPS responses in RAW 264.7 cells.
NF-B, an ubiquitous transcription factor, is one of the major intracellular mediators of LPS-induced responses (14,44). In resting cells, dimeric NF-B are complexed to a member of the IB family of inhibitory proteins which masks the NF-B nuclear localization signal. Upon cell stimulation, IB is phosphorylated on specific serine residues by IKK-␣ (45,46), ubiquitinated, and proteolytically degraded, allowing NF-B dimers to translocate to the nucleus and bind to consensus DNA sequences (44). A role for PKC in the regulation of NF-B activation pathway has been evidenced by the demonstration that PKC-associates with an IB␣ kinase activity and that overexpression of a dominant-negative mutant of PKC-blocked NF-B activation (57)(58)(59). Since LPS-induced IB␣ phosphorylation and degradation and NF-B activation take place normally in DN PKC-␣-overexpressing clones, it is likely that PKC-␣ is not involved in the activation of this pathway. Considering that NF-B plays an important role in the transcriptional activation of TNF-␣ gene expression (14), this suggestion would be consistent with the minor effect of DN PKC-␣ over- expression on LPS-induced TNF-␣ expression. Regarding the regulation of iNOS expression, there is evidence that activation of NF-B alone is not sufficient for its induction. Indeed, while LPS can activate NF-B in macrophages derived from either LPS-responsive or LPS-hyporesponsive mice, induction of iNOS or TNF-␣ takes place only in macrophages from LPSresponsive mice (60). Recently, Xie (61) identified a novel LPSresponse element (LRE AA ) within the iNOS promoter which may work in concert with NF-B in regulating transcriptional activation. It will thus be of interest to determine whether a LRE AA binding activity is induced in our DN PKC-␣ overexpressing RAW 264.7 cells. In contrast to the IL-1␤ promoter region, very little is known on the regulatory elements present upstream the IL-1␣ gene. A recent analysis of the human IL-1␣ promoter region failed to demonstrate the presence of an NF-B-binding site but revealed the presence of a LPS-inducible AP-1-binding site (62), indicating that NF-B does not participate in the transcriptional activation of IL-1␣. This finding is consistent with the previous report that macrophages from mice lacking the p50 subunit of NF-B were normal with respect to their ability to produce IL-1␣ in response to LPS (63). Therefore, identification of the defective LPS-inducible transcriptional activator(s) in our DN PKC-␣ overexpressing RAW 264.7 clones will undoubtedly contribute to our knowledge on the regulation of IL-1␣ expression.
The observation that LPS-induced p38 and JNK phosphorylation takes place normally in our DN PKC-␣-overexpressing clones suggests that activation of these MAP kinase pathways do not require PKC-␣ activity. Moreover, this data provides additional evidence that DN PKC-␣ overexpression did not cause a generalized impairment of LPS-induced responses in RAW 264.7 cells. Further studies are thus required to elucidate the identity of the LPS-induced pathway(s) and transcription factors regulated by PKC-␣. In this regards, LPS activates the Raf-1/MAP kinase pathway in macrophages and evidence was provided that Raf-1 may participate in the induction of IL-1␤ and TNF-␣ gene expression (50,51,64). Studies on the mechanisms of Raf-1 activation in both COS and NIH 3T3 cells revealed that diacylglycerol-regulated PKC isoenzymes, including PKC-␣, are activators of Raf-1 in vivo (65,66). It will be of interest to verify whether PKC-␣ is required for Raf-1 activation in LPS-stimulated macrophages.