Role of protein kinase C isoforms in the regulation of interleukin-13-induced 15-lipoxygenase gene expression in human monocytes.

We reported previously that interleukin-13 (IL-13) induces tyrosine phosphorylation/activation of Jak2 and Tyk2 kinases and Stats 1, 3, 5, and 6 in primary human monocytes. We recently revealed that p38 MAPK-mediated serine phosphorylation of both Stat1 and Stat3 is required for the induction of 15-lipoxygenase (15-LO) expression by IL-13. In this study, we present data indicating that another serine/threonine kinase, PKCdelta, is also required for IL-13-induced 15-LO expression. PKCdelta, a member of the novel protein kinase C (PKC) subclass, was rapidly phosphorylated and activated upon exposure to IL-13. Treatment of cells with rottlerin, a PKCdelta inhibitor, blocked IL-13-induced 15-LO mRNA and protein expression, whereas Go6976, an inhibitor of the conventional PKC subclass, had no inhibitory effects. Down-regulation of cellular PKCdelta protein levels by PKCdelta-specific antisense oligodeoxyribonucleotides also inhibited 15-LO expression markedly. IL-13-induced 15-LO expression resulted in significant inhibition of synthesis of the potent chemotactic factor leukotriene B4, and that process was reversed by rottlerin, presumably through the blockage of PKCdelta-dependent 15-LO expression. Furthermore, our data demonstrate that IL-13-mediated activation of PKCdelta and p38 MAPK are independent pathways, because inhibition of one kinase activity had no effect on the other, suggesting that the two pathways act in parallel to regulate the downstream targets necessary for 15-LO expression. Inhibition of PKCdelta activation by rottlerin also markedly attenuated IL-13-induced Stat3 DNA binding activity. Our findings indicate that PKCdelta plays an important role in regulating IL-13-induced 15-LO expression in human monocytes and subsequently modulates the inflammatory responses mediated by 15-LO products.

Th2 lymphocytes secrete IL-4 1 and IL-13, which have the unique ability to induce the expression of the lipid-oxidizing enzyme 15-LO in primary human monocytes (1)(2)(3). To date, no monocytic cell lines have been shown to respond in a similar fashion. 15-LO dioxygenates polyenoic fatty acids to their corresponding hydroperoxide derivatives. These molecules are potent mediators of inflammatory responses and are found in atherosclerotic lesions. It is believed that 15-LO products play an important role in the pathogenesis of atherosclerosis and other inflammatory diseases (4 -7). In addition to its pro-inflammatory actions, 15-LO has also been shown to suppress the inflammatory responses by inhibiting the production of the potent chemotactic factor LTB 4 . This process has been observed in several cell types, including human monocytes (8 -10).
Previously, we demonstrated that Jak2 and Tyk2 are upstream tyrosine kinases involved in regulating IL-13-induced expression of . We have further defined the functional IL-13 receptor complex, the association of Jaks with the receptor constituents, and the tyrosine phosphorylation and activation of Stats in response to IL-13 (11). Tyrosine phosphorylation of Stat molecules facilitates the formation of dimerized Stat complexes, which are then translocated to the nucleus, bind DNA, and regulate target gene expression (12). In addition to tyrosine phosphorylation, the serine phosphorylation of Stat molecules is also necessary for the maximal activation of transcription (13,14). Several lines of evidence suggest the role of different Ser/Thr kinases in serine phosphorylation of Stat molecules and their transactivation (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). Our recent studies demonstrated that p38 MAPK regulates serine 727 phosphorylation on Stat1 and Stat3 and that p38 MAPK activity is required for IL-13-induced 15-LO expression (27).
PKC is a heterogenous family of serine/threonine kinases mediating important intracellular signaling pathways (28 -30). PKC isoforms are classified into three major categories, depending on their activation mechanisms as well as Ca 2ϩ and lipid requirements. The conventional PKCs (cPKCs; ␣, ␤, and ␥) require both Ca 2ϩ and diacylglycerol. The novel PKCs (nPKCs; ⑀, ␦, , and ) are Ca 2ϩ independent but require diacylglycerol, whereas the atypical PKC isoforms (aPKCs; , , and ) are independent of both Ca 2ϩ and diacylglycerol. Usually, PKCs are activated in response to the engagement of G protein-coupled receptors, tyrosine kinase receptors, and nontyrosine kinase receptors. The activation of PKC␦ as evidenced by tyrosine and serine/threonine phosphorylation is induced in response to several stimuli, such as H 2 O 2 (31), IL-6 (19,24), or the proinflammatory mediator thrombin (32). Upon activation, PKC␦ phosphorylates a variety of proteins and has been shown to activate various transcription factors (24,25,(32)(33)(34).
In the present study, we investigated the role of PKC isoforms, particularly PKC␦, in IL-13-induced 15-LO expression in primary human monocytes. We provide evidence that activation of PKC␦ by IL-13 is required for the induction of 15-LO in human monocytes and is independent of the p38 MAPK pathway.

EXPERIMENTAL PROCEDURES
Reagents-Recombinant human IL-13 was purchased from BIO-SOURCE. Antibodies against 15-LO were kindly provided by Dr. Joseph Cornicelli, Parke-Davis. Antibodies to phospho-p38 MAPK, p38 MAPK, phosphoserine 643-PKC␦, and phosphothreonine 505-PKC␦ were purchased from Cell Signaling Technology (Beverly, MA). Antibodies to PKC␦, p47 phox , and ␤-tubulin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The general PKC inhibitor calphostin C, the PKC␦-selective inhibitor rottlerin, and the cPKC inhibitor Go 6976 were purchased from Biomol (Butler Pike, PA). The inhibitors were dissolved in Me 2 SO and stored at Ϫ20°C as stock solutions.
Isolation of Human Monocytes-Human peripheral blood monocytes were isolated from whole blood by the separation of mononuclear cells followed by adherence to bovine calf serum (BCS)-coated flasks as described earlier (3). Adherent cells were collected from the flask after release with 5 mM EDTA and plated in six-well cell culture plates (Nunclon; Nalge Nunc International, Roskilde, Denmark). The cell preparations were routinely Ͼ95% monocytes and were maintained in Dulbecco's modified Eagle's medium containing 10% BCS at 37°C in the presence of 10% CO 2 . For some experiments, monocytes were purified from human peripheral blood by Ficoll-Hypaque sedimentation followed by countercurrent centrifugal elutriation (35,36). Monocyte preparations purified in this manner are consistently Ͼ95% CD14ϩ. We observed no differences in results with monocytes obtained by these two protocols.
RNA Extraction and Real Time RT-PCR-Monocytes were plated in six-well culture plates at 5 ϫ 10 6 cells/well in 2 ml of medium. Two hours after plating, cells were treated with 500 pM recombinant IL-13 for 4 h. In some experiments, monocytes were pretreated with inhibitors for 30 min followed by IL-13 treatment for 4 h. Total cellular RNA was extracted using the RNeasy mini-kit from Qiagen (Valencia, CA), and real time quantitative RT-PCR was performed according to established protocols (27).
Immunoprecipitation and Immunoblotting-Monocytes were plated at 2 ϫ 10 6 /ml in six-well plates, pretreated with the inhibitors (30 min) or antisense ODN whenever required and then treated with IL-13 (500 pM) for different time intervals as indicated. After protein concentration was determined using the Bio-Rad protein assay reagent (Hercules, CA), lysates (50 g/well) were loaded on an 8% SDS-PAGE gel. The proteins were transferred to a polyvinylidene difluoride membrane and blocked with 5% nonfat milk in phosphate-buffered saline with 0.1% Tween 20 and blotted with appropriate antibodies. The hybridization signal was detected using enhanced chemiluminescence (Pierce). For immunoprecipitation experiments, the lysates were incubated with anti-PKC␦ antibodies for 2 h at 4°C with constant rotation. Immune complexes were collected using pre-washed protein A-Sepharose beads (Sigma) at 4°C overnight. Immunoprecipitates were washed three times with lysis buffer. Immune complexes were released by boiling the beads in SDS sample buffer and then subjected to Western blot analysis as described above. In several experiments, immunoblots were stripped and reprobed to assess equal loading. Quantification of all Western blot results was conducted using the software program NIH Image as indicated previously (11).
Treatment of Cells with PKC␦ Antisense ODN-Human monocytes were plated (5 ϫ 10 6 /well) as described above in six-well plates. PKC␦ antisense or sense ODNs were boiled for 1 min and then cooled on ice before being added to the cells. For PKC␦ ODN treatment, human monocytes were treated with equivalent molar concentrations of two different PKC␦ antisense or sense ODN sequences to yield final concentrations of 5 or 10 M for 72 h with one re-feed at 24 h prior to the addition of IL-13. Cells were then lysed or utilized for further experimentation. The PKC␦ antisense ODN sequences were 5Ј-GAAG-GCGATGCGCAGGAA-3Ј (PKC␦4) and 5Ј-AGGAACGGCGCCATGGTG-GG3-Ј (PKC␦5). Complementary PKC␦ sense ODN sequences were used as controls. The antisense ODN sequences were selected based on prior literature (37) and our recently described protocol for identifying optimal mRNA regions for antisense ODN design (38,39).
PKC␦ Kinase Assay-The immune complex PKC␦ kinase assay was performed as described previously (19,25,32). Briefly, monocytes treated with different reagents for various time points were lysed in a phosphorylation lysis buffer (50 mM HEPES, 150 mM NaCl, 200 M sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 1 mM EDTA, 1.5 mM, MgCl 2 , 10% glycerol, 0.5% Triton X-100, ) as determined by migration relative to recombinant standards and molecular mass markers. The blots were then stripped and reprobed with p47 phox, a constitutively produced monocyte protein, to assess protein loading (lower panels).

FIG. 2. Activation of PKC␦ in IL-13-treated monocytes.
Monocytes were treated with IL-13 for various times as indicated. Cells were lysed, and 100 g of total protein was immunoprecipitated (IP) with an anti-PKC␦ antibody and subjected to Western blot analysis with antiphospho-PKC␦ antibodies. Arrows indicate phospho-PKC␦ (upper and middle panels) and total PKC␦ (lower panel) (A). PKC␦ kinase activity was assessed using cell lysates from cells either untreated or treated with IL-13 for 15 min (B). 100 g of total protein was immunoprecipitated with anti-PKC␦ antibody. PKC␦ activity was measured using the immune complex protein kinase assay with histone H1 as the substrate as described under "Experimental Procedures." The upper panel is the phosphorimage; the lower panel shows the Western blot result using anti-PKC␦ antibody.
1 mM phenylmethylsulfonyl fluoride, and 1ϫ protease inhibitor mixture (Sigma). Cell lysates were immunoprecipitated with an antibody against PKC␦ and collected onto protein A-Sepharose beads. The immune complexes were washed three times with the phosphorylation lysis buffer, twice with kinase assay buffer, and then resuspended in 30 l of kinase assay buffer. Histone H1 (10 g) was added as a substrate for PKC␦ and incubated at 30°C for 10 min before adding 20 M ATP, 10 Ci of [␥-32 P]ATP, and 20 g of phosphatidylserine. The incubation was continued at 30°C for an additional 30 min. The reactions were terminated by adding 5ϫ sample buffer. After boiling for 5 min, the reaction mixtures were separated by 8% SDS-PAGE and transferred to a nitrocellulose membrane. The phosphorylated form of histone H1 was detected using a PhosphorImager. The membranes were also subjected to Western blot analysis to assess the amount of PKC␦ in the immune complexes.
LTB 4 Production by Monocytes-Monocytes (1 ϫ 10 6 cells/ml) were plated in duplicate in 12-well culture plates and either pretreated with 5 M rottlerin for 30 min or left without rottlerin before IL-13 treatment for 72 h. Cells were then challenged with the Ca 2ϩ -ionophore A23187, at a final concentration of 5 M for 15 min. At the end of the incubation, cell supernatants were collected and assayed for LTB 4 using the LTB 4 Biotrak enzyme immunoassay (EIA) system (Amersham Biosciences) following the manufacturer's protocol.
Nuclear Protein Extraction and Electrophoretic Mobility Shift Assay (EMSA)-To assess the role of PKC␦ in IL-13-induced Stat3 DNA binding activity, EMSA was performed using nuclear extracts and a specific Stat3 probe (Santa Cruz Biotechnology). Monocytes were pretreated with 5 M rottlerin for 30 min followed by the addition of IL-13 for another 30 min. Nuclear proteins were extracted, and the EMSA was performed following our previously published protocol (11,27).

Role of PKC Isoforms in Regulating IL-13-induced 15-LO
Expression-Our previous studies indicated that, in addition to protein tyrosine kinases, serine/threonine kinases are also required in the IL-13 induction of 15-LO expression (27). PKCs are a major class of serine/threonine kinases and are involved in regulating the expression of a variety of different genes. We, therefore, assessed the possible involvement of PKC in IL-13-mediated 15-LO expression. Monocytes were treated with calphostin C, a general inhibitor for PKC (40), before exposure to IL-13. Calphostin C substantially inhibited IL-13-induced 15-LO protein expression in a dose-dependent fashion (Fig. 1A). In contrast, a cPKC-selective inhibitor, Go 6976, had no effect on the IL-13-mediated 15-LO expression at the doses shown to be specific for inhibiting PKC␣ and PKC␤ activity (41) (Fig. 1B). These data suggest that PKC isoform(s) other than cPKCs are required for IL-13-induced 15-LO expression.
IL-13 Induces Phosphorylation and Activation of PKC␦ in Human Monocytes-We recently reported that IL-13-induced 15-LO gene expression requires p38 MAPK activity (27). Another study suggests that thrombin-induced intercellular adhesion molecule-1 (ICAM-1) gene expression in endothelial cells involves PKC␦-dependent activation of p38 MAPK (32). To determine whether PKC␦ is involved in the regulation of IL-13-induced 15-LO expression, we first examined PKC␦ activation following IL-13 treatment utilizing antibodies that specifically recognize phosphoserine at position 643 or phosphothreonine at position 505 of PKC␦. After treatment with IL-13 for different times, the cells were lysed, and the whole cell lysates were immunoprecipitated with anti-PKC␦ antibody followed by immunoblot analysis using the anti-phospho-PKC␦ antibodies. As shown in Fig. 2A, IL-13 induced both serine (643) and threonine (505) phosphorylation in a time-dependent manner. The phosphorylated forms of PKC␦ were detected as early as 5 min, and the peak of phosphorylation was at 10 min after IL-13 treatment, with the signal diminishing afterward. To further determine whether phosphorylation of PKC␦ resulted in increased kinase activity, we performed a PKC␦ kinase assay in which PKC␦ from monocytes was immunoprecipitated using an anti-PKC␦ antibody, and the phosphorylation of the substrate histone H1 was assessed. IL-13 treatment for 15 min resulted in a remarkable induction of PKC␦ kinase activity as evidenced by increased phosphorylation of its substrate, histone H1 (Fig. 2B). These results suggest that the activation of PKC␦ in IL-13-treated cells is one of the early  3A). The mRNA levels of glyceraldehyde-3-phosphate dehydrogenase were nearly identical in all the samples, indicating specificity of the response. Rottlerin was also effective in inhibiting IL-13-induced 15-LO protein expression as assessed by Western blot analysis (Fig. 3B). In contrast, Go 6976 had no effect on 15-LO mRNA or protein expression.
To further test the functional role of PKC␦ in regulating 15-LO gene expression, we treated cells with the PKC␦-specific antisense or control sense ODN before IL-13 addition. Total cellular RNA or proteins were extracted for real time RT-PCR or Western blot analysis, respectively. Our results, presented in Fig. 4, A and B, indicate that PKC␦ antisense ODN inhibited both 15-LO mRNA and protein expression by ϳ50%, whereas PKC␦ sense ODN caused no inhibition of 15-LO expression. These data confirm the results presented in Fig. 3, A and B, where pharmacological inhibitors were employed.

Induction of 15-LO Expression Blocks LTB 4 Production-Previous studies suggest that LTB 4 production in human monocytes is inhibited by IL-4-induced 15-LO expression (10).
We reproduced this observation with IL-13-induced 15-LO expression and tested whether a blockade of IL-13-induced 15-LO expression by the PKC␦ inhibitor rottlerin could restore LTB 4 production. For these studies, monocytes were either left untreated or treated with 5 M rottlerin for 30 min, followed by IL-13 for 72 h. Cells were then challenged by the Ca 2ϩ ionophore A23187, and LTB 4 production in supernatants was determined. As presented in Fig. 5, treatment of monocytes with A23187 induced LTB 4 production by Ͼ3-fold, and IL-13 pretreatment significantly inhibited the A23187-induced LTB 4 production (p ϭ 0.0302). Furthermore, when cells were treated with rottlerin at the dose that almost completely inhibited IL-13-induced 15-LO expression, it blocked the IL-13 inhibitory effects on LTB 4 production (p ϭ 0.0215 when compared with IL-13/A23187 group). These data suggest the potential importance and biological function of PKC␦ in regulating inflammatory responses via 15-LO expression induced by Th2 cytokines.
PKC␦ Does Not Regulate p38 MAPK Phosphorylation/Activation-We recently found that, in human monocytes, IL-13 induces p38 MAPK activation, which regulates the Ser-727 phosphorylation of both Stat1 and Stat3 (27). As mentioned previously, a prior study suggests that PKC␦ may be one of the upstream kinases regulating p38 MAPK activation (32). To determine whether PKC␦ activity is required for p38 MAPK activation in IL-13-treated monocytes, we treated cells with rottlerin for 30 min before IL-13 stimulation. Total cell lysates were extracted, and p38 MAPK phosphorylation/activation was evaluated. As presented in Fig. 6A, pretreatment with rottlerin at a dose (5 M) that substantially inhibited PKC␦ activation (42) and 15-LO expression (Fig. 3) had no detectable effect on IL-13-induced p38 MAPK phosphorylation. These data suggest that p38 MAPK is not the downstream target of PKC␦ in the IL-13-induced signaling pathway in human monocytes. To determine whether p38 MAPK was upstream of PKC␦, we treated cells with the p38 MAPK inhibitor SB202190 prior to IL-13 stimulation and evaluated PKC␦ phosphorylation. Pretreatment with SB202190 at a dose that significantly inhibited p38 MAPK activity and IL-13-induced 15-LO expression (27,43) had no effect on IL-13-induced serine 643 and threonine 505 phosphorylation of PKC␦ (Fig. 6B). These results indicate that the PKC␦ and p38 MAPK regulation of downstream targets are independent from each other and the two kinases act in parallel in regulating IL-13-induced 15-LO expression in human monocytes.
PKC␦ Is Involved in Stat3 DNA Binding Activity-PKC␦ has been shown to regulate the expression of a variety of genes through the activation of various transcription factors (24,25,(32)(33)(34)44). In our previous report, we demonstrated a predominant role of Stat3 in IL-13-induced 15-LO expression (27). To investigate whether PKC␦ activity is required for Stat3 DNA binding, EMSA was performed using nuclear extracts from IL-13-treated monocytes and a 32 P-labeled probe specific for Stat3. As shown in Fig. 7, pretreatment of cells with rottlerin profoundly inhibited the IL-13-induced Stat3 DNA binding activity. These results thus suggest that the activation of PKC␦ by IL-13 is required for maximal Stat3 DNA binding activity. DISCUSSION Our previous studies indicated that the induction of 15-LO expression by IL-13 requires activation of Jak2 and Tyk2 tyrosine kinases (3) and that IL-13 induces tyrosine phosphorylation of selective Stat proteins (11). We recently reported that IL-13-induced 15-LO expression is also modulated through Ser/ Thr kinases, particularly through p38 MAPK regulation of Stat1 and Stat3 Ser 727 phosphorylation (27). In this study, we provide new insights of co-stimulatory signals for IL-13-induced 15-LO expression and provide evidence that one of the PKC isoforms, PKC␦, is required for 15-LO gene expression and modulates the production of inflammatory mediators.
The activation of PKC␦ requires the phosphorylation of residues at Thr-505 and Ser-643 (31,45). Although PKC␦ can be activated by a variety of agonists, our results demonstrate for the first time that IL-13 is able to induce PKC␦ activation in primary human monocytes. The induction of PKC␦ activity represents one of the early events in IL-13-initiated signaling pathways, suggesting an important role of PKC␦ in IL-13induced gene expression. We employed multiple approaches to establish the functional role of PKC␦ in IL-13-induced 15-LO gene expression. Pretreatment of cells with rottlerin or PKC␦specific antisense ODN inhibited IL-13-induced 15-LO mRNA and protein expression, whereas Go 6976, which inhibits only cPKCs (19) or PKC␦ sense ODN were ineffective, suggesting an isoform-specific role in the regulation of the IL-13-mediated signaling pathways.
Our current studies also showed that, in human monocytes, IL-13-induced 15-LO expression is accompanied by a significant reduction of LTB 4 production upon challenge with A23187, and this inhibitory effect can be reversed by the PKC␦ inhibitor rottlerin. It has been suggested that the inhibition of LTB 4 synthesis by Th2 cytokines is not due to changes in the expression of the key enzymes such as 5-LO nor due to the newly expressed 15-LO competing for common substrates with 5-LO (10). Instead, 15-LO products such as 15(S)-HETE may be responsible for the inhibition of LTB 4 production. Because 15(S)-HETE and other 15-LO products exert various immunoregulatory functions, our findings regarding the role of PKC␦ in IL-13-induced 15-LO expression may prove to be physiologically relevant to the pathogenesis of atherosclerosis and asthma.
The early activation of PKC␦ suggests that it may be one of the upstream Ser/Thr kinases providing a co-stimulatory signal for IL-13-induced gene expression. There is increasing evidence supporting the critical role of PKC␦-mediated Stat1 and Stat3 Ser-727 phosphorylation in cytokine-induced gene expression (19,24,32). Because IL-13 treatment induces p38 MAPK activation and subsequent Stat1 and Stat3 Ser-727 phosphorylation in human monocytes (27), we investigated whether p38 MAPK was related to PKC␦ in the IL-13-mediated signal transduction pathways. Our results with pharmacological inhibitors reveal that IL-13-induced p38 MAPK phosphorylation/activation is neither a downstream event nor an upstream kinase of PKC␦. Hence, these two different pathways leading to phosphorylation of downstream targets are independent, parallel pathways, but both are indispensable for IL-13-induced 15-LO expression. The finding that two distinct Ser/Thr kinases regulate Stat3 DNA binding activity is novel and intriguing (27). We propose that, although neither of PKC␦ or p38 MAPK regulates the phosphorylation/activation of the other, they may be present in a signaling complex that is required for and controls the important phosphorylation events of their downstream targets like Stat3. Current studies in the laboratory are focused on delineating the details of this unique process.
In summary, our current study indicates that activation of PKC␦ by IL-13 plays an important role in IL-13-induced 15-LO expression in human monocytes. Although PKC␦-mediated 15-LO expression is independent of p38 MAPK, the two Ser/ Thr kinases may provide co-stimulatory signals to downstream targets regulating 15-LO expression and subsequently modulate the inflammatory responses mediated by 15-LO products. FIG. 7. PKC␦ Regulates Stat3 DNA Binding Activity. Monocytes were either pretreated with 5 M rottlerin for 30 min or left untreated, followed by the addition of IL-13 for an additional 30 min. Nuclear proteins were extracted, and 5 g was subjected to EMSA analysis using 32 P-labeled Stat3-specific probes. In some reactions, 50ϫ amounts of either cold Stat3 or nonspecific (NS) probes were used for competitive inhibition. Arrows point to the positions of Stat protein and DNA complexes as well as free probes.