Protein Kinase C ζ Plays a Central Role in Activation of the p42/44 Mitogen-Activated Protein Kinase by Endotoxin in Alveolar Macrophages¹

Endotoxin or LPS is an essential component of the cell wall of Gram-negative bacteria. It can interact with LPS-binding protein (LBP),³ allowing binding to CD14 and association with at least one other cell membrane receptor which contains an intracellular signaling domain (the most likely candidate being one of the Toll receptors) (1). The Toll 2 receptor has been shown to associate with CD14. Mutant cells lacking the C terminus of Toll 2 are unable to transmit LPS-induced signals (2). Binding of LPS to these receptors results in the activation of a number of signaling cascades, resulting in the production of inflammatory mediators (3, 4, 5). The mechanism by which LPS triggers the release of inflammatory mediators is unclear.

Alveolar macrophages play a central role in the response of the lung to bacterial infection. They are specialized cells, which respond to infection by phagocytosis and killing of bacteria (6, 7, 8). They also respond to LPS by releasing large amounts of inflammatory mediators (5, 9). We and others have shown that activation of various mitogen-activated protein kinase (MAP kinase) pathways is necessary for expression of cytokine genes in alveolar macrophages in response to LPS (10, 11).

Three major MAP kinase cascades have been described in mammalian cells, the extracellular signal-related kinase (ERK), p38, and c-Jun N-terminal kinase pathways. All three of these pathways have been linked to activation by LPS and subsequent cytokine gene expression. In addition to LPS, the ERK pathway is activated by growth and differentiation signals (12). Activation of the p38 and c-Jun N-terminal kinase pathways is often linked to cell stress (13, 14). In this study, we focus on pathways that are immediately upstream of LPS-induced ERK activation. The best described pathway leading to ERK activation is the Ras-Raf-1-mitogen-activated protein/ERK kinase (MEK)-ERK kinase cascade (12). Another possible activator of MEK is the “atypical” protein kinase C (PKC) isoform, PKC ζ. PKC ζ is one of a large family of serine/threonine kinases and is characterized by a lack of Ca²⁺ dependence or 1,2-diacylglycerol responsiveness. It has been linked to a number of mitogenic signals (15).

In previous studies from this laboratory, we evaluated some of the early signaling events associated with LPS activation of ERK in alveolar macrophages. We showed that LPS activates a phosphatidylcholine-specific phospholipase (PC-PLC), resulting in the generation of ceramide. We also showed that LPS activates PKC ζ and that an inhibitor of PC-PLC inhibits activation of PKC ζ and ERK. These studies, however, did not establish a direct link between activation of PKC ζ and activation of ERK kinase. In this study, we show that LPS activates ERK without any demonstrable activation of Raf-1. LPS activates PKC ζ in a time-dependent manner, which matches the time course of LPS-induced ERK activation. We also show that PKC ζ can be linked to MEK/ERK activation in two ways. First, LPS induces formation of a complex between PKC ζ and MEK. In addition, a PKC ζ-specific inhibitory peptide blocks LPS-induced MEK and ERK activation and the in vivo phosphorylation of MEK. We also demonstrate LPS-induced activation of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway and PKC ζ/PI 3-kinase-dependent kinase (PDK-1) complex formation. PDK-1 (downstream of PI 3-kinase), a constitutively active kinase, will, when in proximity to PKC ζ, phosphorylate Thr⁴¹⁰ in the activation loop of PKC ζ. This initial activation event is thought to allow autophosphorylation of Thr⁵⁶⁰, resulting in an active kinase (15, 16, 17). These studies clearly demonstrate that PKC ζ is important for LPS-induced activation of ERK in human alveolar macrophages.

Alveolar macrophages were obtained from bronchoalveolar lavage as previously described (18). Briefly, normal volunteers with a lifetime nonsmoking history, no acute or chronic illness, and no current medications underwent bronchoalveolar lavage. The lavage procedure used five 20-ml aliquots of sterile, warmed saline in each of three segments of the lung. The lavage fluid was filtered through two layers of gauze and centrifuged at 1500 × g for 5 min. The cell pellet was washed twice in HBSS without Ca²⁺ and Mg²⁺ and suspended in complete medium, RPMI tissue culture medium (Life Technologies, Gaithersburg, MD) with 100 ng/ml LBP (a gift from Peter Tobias, The Scripps Research Institute, La Jolla, CA), and added gentamicin (80 μg/ml). Differential cell counts were determined with a Wright-Giemsa-stained cytocentrifuge preparation. All cell preparations had between 90 and 100% alveolar macrophages. This study was approved by the Committee for Investigations Involving Human Subjects at the University of Iowa.

Alveolar macrophages were cultured in complete medium with or without LPS (1 μg/ml, Sigma, St. Louis, MO). In some cases, inhibitory peptides (PKC ζ: myr-SIYRRGARRWRKL-OH; PKC αβ: myr-RFARKGALRQKNV-OH; nonsense: myr-LRISRAGRYRANWYRKR-OH; the myristate on the N terminus of these peptides allows for membrane permeability) were added 30 min before the LPS. After culture, cells were lysed on ice for 20 min in 500 μl lysis buffer (0.05 M Tris (pH 7.4), 0.15 M NaCl, 1% Nonidet P-40, 0.5 M PMSF, 50 μg/ml aprotinin, 10 μg/ml leupeptin, 50 μg/ml pepstatin, 0.4 mM sodium orthovanadate, 10 mM sodium fluoride, and 10 mM sodium pyrophosphate, all from Boehringer Mannheim, Indianapolis, IN). The lysates were then sonicated for 20 s and spun at 15,000 × g for 10 min, and the supernatant was saved. Protein was measured, and 200–600 μg from each sample were removed for immunoprecipitation. The samples were cleared by incubating for 2 h with 1 μg/sample rabbit IgG and 10 μl/sample GammaBind Sepharose (Pharmacia, Piscataway, NJ). After centrifugation, the supernatants were transferred to a tube containing 3 μg/sample kinase Ab (all of the Abs used in this paper, except for the phospho-ERK Ab (Sigma), were obtained from Santa Cruz Biotechnology, Santa Cruz, CA) bound to GammaBind Sepharose, and rotated at 4^oC overnight. The beads were subsequently washed three times with high salt buffer (0.5 M Tris (pH 7.4), 0.50 M NaCl, and 1% Nonidet P-40) and three times with lysis buffer without protease inhibitors. The immunoprecipitated complexes were either released with 2× sample buffer for Western analysis or used to determine kinase activity. In the case of Western analysis, the samples were sometimes divided, and two separate gels were run to evaluate the formation of protein complexes.

After immunoprecipitating the relevant kinase from alveolar macrophages, the protein-containing pellet was washed twice with kinase buffer (20 mM MgCl₂, 25 mM HEPES, 20 mM β-glycerophosphate, 20 mM p-nitrophenylphosphate, 20 mM sodium orthovanadate, and 2 mM DTT). The pellet was then suspended in 20 μl kinase buffer, and the following was added: 20 μM ATP, 5 μCi [γ-³²P]ATP (BLU 002Z, DuPont/NEN, Boston MA) and 10 μg myelin basic protein (MBP, Sigma) or 5 μg MEK-1 or 5 μg ERK (polyhistidine-tagged fusion proteins from Santa Cruz Biotechnology). The total volume of sample plus additions at this point was 25 μl. The reaction was continued for 15 min to 1 h, depending on the kinase, at 25°C, and then stopped by the addition of 25 μl/sample 2× sample buffer. The samples were boiled for 5 min and run on a 12% SDS-PAGE gel. The gel was dried, and autoradiography was performed to visualize the ³²P-labeled MBP, MEK-1, or ERK. Densitometry was performed on films and fold increase calculated as experimental sample/control sample.

Western analysis (all Abs from Santa Cruz Biotechnology) was performed on three different types of samples: 1) whole cell protein, for the phosphorylated form of ERK; 2) immunoprecipitated proteins from the kinase activity assay to determine equal loading of the proteins in the assay; and 3) samples divided into two fractions after immunoprecipitation to monitor intracellular complex formation. For straight Westerns, 50–100 μg of protein were mixed 1:1 with 2× sample buffer (20% glycerol, 4% SDS, 10% 2-ME, 0.05% bromphenol blue, and 1.25 M Tris (pH 6.8); all chemicals from Sigma), loaded onto a 10% SDS-PAGE gel, and run at 30 mA for 3 h. Cell proteins were transferred to nitrocellulose (ECL, Amersham, Arlington Heights, IL) overnight at 30 V. The nitrocellulose was then blocked with 5% milk in TTBS (Tris-buffered saline with 0.1% Tween 20) for 1 h, washed, and then incubated with the primary Ab (anti-phosphorylated ERK, Sigma) overnight. The blots were washed four times with TTBS and incubated for 1 h with HRP-conjugated anti-rabbit IgG Ab (Amersham, at 1:20,000 dilution). Immunoreactive bands were developed using a chemiluminescent substrate (SuperSignal West Femto; Pierce, Rockford, IL). To determine equal loading in the kinase activity assays, 20% of the total immunoprecipitated protein was mixed 1:1 with 2× sample buffer and Western analysis performed as described above. For complex formation assays, immunoprecipitated samples were divided in two after the last immunoprecipitation wash, and two gels were run. After transfer, one blot was analyzed for the immunoprecipitated protein (MEK or PKC ζ), and the other blot was analyzed for PKC ζ or PDK-1.

Alveolar macrophages were labeled with 1.25 mCi ³²P_i/group (NEN Life Science Products, Boston, MA) in phosphate-free RPMI without serum for 3 h at 37°C. The cells were harvested and placed in RPMI with 100 ng/ml LBP and treated with peptides for 30 min. After the peptide incubation, the cells were stimulated with LPS for 15 min at 37°C. The cells were harvested, resuspended in lysis buffer (1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M Na₃PO₄ (pH 7.2), 2 mM Na₃VO₄, 1 μM okadaic acid, 100 μg/ml PMSF, 50 μg/ml aprotinin, 10 μg/ml leupeptin, and 50 μg/ml pepstatin, all from Boehringer Mannheim), and sonicated. MEK was immunoprecipitated from the lysate, and the sample separated on a 10% SDS-PAGE discontinuous gel as described above.

After culture, whole cell lysates were obtained and PI 3-kinase was immunoprecipitated using an Ab to the p85-regulatory subunit of PI 3-kinase (Santa Cruz Biotechnology). Activity was assayed by measuring the formation of PI 3-[³²P]phosphate (19, 20). After overnight incubation with Ab-coated beads (see Immunoprecipitation), the bound protein was washed three times with buffer I (PBS containing 1% Nonidet P-40 and 100 μM Na₃VO₄), three times with buffer II (100 mM Tris-HCl (pH 7.5), 500 mM LiCl, and 100 μM Na₃VO₄), and finally three times with buffer III (Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA and 100 μM Na₃VO₄). After washing, immunoprecipitates were resuspended in 50 μl buffer III with the addition of 10 μl 100 mM MgCl₂ and 10 μl PI (2 μg/ml). The samples sat at room temperature for 5 min before the addition of 10 μl ATP (ATP 440 μM with 30 μCi/10 μl [γ-³²P]ATP). The samples were then shaken at room temperature for 10 min. The reaction was stopped by the addition of 20 μl 8 N HCl and 160 μl chloroform-methanol (1:1). The lipids were extracted by standard methods, dried down, resuspended in 20 μl chloroform-methanol (1:1), and separated on thin layer silica gel plates (pretreated with 10% w/v potassium oxalate) in a solvent system of chloroform-methanol-water-NH₄OH (60:47:11:2.2, v/v/v/v). Incorporation of ³²P into PI 3-phosphate was detected by autoradiography, and activity was quantified on a Bio-Rad Molecular Imager FX.

Statistical analysis of the densitometric data was performed by determining the fold increase of all the samples as they relate to the control. Statistical comparisons were performed using a paired t test with a probability value of p < 0.05 considered to be significant.

We initially wanted to confirm that LPS activated ERK in alveolar macrophages. To do this, we cultured alveolar macrophages in complete RPMI for various time points. At the end of the experiment, whole cell lysates were obtained, and Western analysis was performed. The blots were probed with an Ab specific for the phosphorylated form of ERK (phospho-Thr¹⁸³ and Tyr¹⁸⁵) and one for total ERK. We found that LPS caused a time-dependent increase in the amount of phosphorylated ERK in alveolar macrophages (Fig. 1). The best described pathway for ERK activation is the Ras-Raf-1-MEK-ERK phosphorylation cascade. The next two experiments evaluate the feasibility of this scenario in LPS-induced ERK activation.

To evaluate the activation of Raf-1 in LPS-treated alveolar macrophages, we performed kinase activity assays using immunoprecipitated ERK and Raf-1 from LPS-treated cells. In Fig. 2, we show that LPS strongly activates ERK kinase, while having little effect on the activity of Raf-1 in alveolar macrophages. In addition, we show composite data from three experiments, using densitometry to calculate fold increase. Control values are designated as 1. To ensure that we were not missing possible Raf-1 activation, we performed a kinase activity assay on Raf-1 compared with ERK immunoprecipitated from various time points (Fig. 3). This experiment demonstrates that at multiple time points when there is demonstrable ERK activation, there is no Raf-1 activity. To validate our experimental system, we performed one more experiment. Alveolar macrophages were treated with LPS or PMA, and then kinase activity assays were performed for ERK 2 or Raf-1. Fig. 4 shows that LPS activated ERK without activating Raf-1. PMA, however, a strong activator of the conventional and novel PKCs known to activate Raf-1, does activate Raf-1 in alveolar macrophages.

Our previous work demonstrated that LPS can activate an atypical PKC isoform, ζ, and that this is linked to LPS-induced activation of a PC-PLC. We also showed that LPS generates ceramide in alveolar macrophages. Because of this finding and previous work by other authors showing that PKC ζ could be activated by ceramide and phosphatidic acid, we evaluated the hypothesis that PKC ζ might be the upstream kinase responsible for MEK activation in alveolar macrophages (21, 22). In Fig. 5, kinase activity assays, using relevant substrates (MEK for PKC ζ, ERK for MEK, and MBP for ERK), show that LPS causes a substantial activation of PKC ζ, MEK, and ERK. In Fig. 6, we show that LPS activates PKC ζ over an extended time course. We also evaluated the response of PKC to various amounts of LPS and found that PKC ζ was activated by a range of LPS doses (10 ng/ml to 1 μg/ml) (data not shown). These experiments show that LPS activates PKC ζ and that the results of activation assays are consistent with a PKC ζ-MEK-ERK pathway.

An important step in the activation of PKC ζ is the removal of the pseudosubstrate region from the kinase core (Fig. 7,A). In these experiments, we use a myristolated peptide specific for the ζ pseudosubstrate region to inhibit PKC ζ activity in vivo. Fig. 8 shows that the PKC ζ-specific peptide blocks LPS-induced ERK activation, whereas a peptide specific for PKC α and β has no effect. Fig. 8 (bottom) shows data from three separate experiments, confirming the kinase activity assay shown. In Fig. 9, using a kinase activity assay, we show that the PKC ζ-specific peptide also blocks MEK activation, whereas the α- and β-specific peptide and a peptide made from scrambled ζ-amino acids had no effect. These experiments directly link LPS-induced ERK activation to PKC ζ, by showing that a PKC ζ-specific inhibitor can inhibit ERK activation by LPS.

Along with activation by phosphorylation, one mechanism, which regulates kinase activity, is the formation of signaling complexes. For activation to occur, kinases must be brought into proximity with each other. We investigated the effect of LPS on PKC ζ/MEK complex formation. Alveolar macrophages were treated with LPS and then MEK was immunoprecipitated from the lysates. After immunoprecipitation, the sample was divided in half and Western analysis for MEK (equal loading) and PKC ζ (association) was performed. Fig. 10 shows that LPS causes an increase in MEK/PKC ζ association.

To further evaluate the link between PKC ζ and MEK, we performed the following assay. Alveolar macrophages were phosphate loaded with ³²P and then treated with a PKC ζ-specific peptide or a nonsense peptide before activation with LPS. The cells were then lysed and MEK was immunoprecipitated from the lysate. A 10% SDS gel was run and dried, and an autoradiograph was obtained. Fig. 11 shows that LPS causes an increased phosphorylation of MEK that is blocked by the PKC ζ-specific peptide and not by the nonsense peptide. This experiment places PKC ζ upstream of MEK in LPS-activated alveolar macrophages.

Activation of PI 3-kinase results in the conversion of PI 4-phosphate and PI 4,5-bisphosphate to PI 3,4-bisphosphate and PI 3,4,5-trisphosphate, respectively. These lipid species interact with the pleckstrin homology domains on a number of proteins bringing them to the membrane where they become activated (16, 17). The initial downstream event after PI 3-kinase activation is activation of the kinase PDK-1. This kinase is known to phosphorylate a threonine in the activation loop of a number of kinases. Phosphorylation by PDK-1 is the initial event in a sequence of phosphorylations of these kinases that result in activation. PDK-1 has been shown to phosphorylate protein kinase A, Akt (protein kinase B), PKC ζ (on Thr⁴¹⁰; see Fig. 7,B), and some of the “novel” PKCs (15, 16, 17, 23). One defining feature of PDK-1 is that it is a constitutively active kinase, which is regulated by binding to its pleckstrin homology domain and proximity to substrate. To investigate the role of PI 3-kinase on PKC ζ activation in alveolar macrophages, we performed the following experiments. Initially, we found that LPS activated PI 3-kinase in a time-dependent manner (Fig. 12). LPS-treated alveolar macrophages were lysed at various time points, and PI 3-kinase was immunoprecipitated. Kinase activity was determined using PI as a substrate and then separating the phosphorylated species on a TLC plate. We then evaluated whether or not PDK-1 might be involved in PKC ζ activation by examining complex formation. We found that LPS caused a significant increase in the association of PDK-1 with PKC ζ (Fig. 13). These experiments suggest that LPS activates PKC ζ via activation of PI 3-kinase and PKC ζ/PDK-1 complex formation.

This study evaluates the role of PKC ζ in LPS activation of ERK. LPS causes a rapid and time-dependent activation of ERK. This is not matched by an increase in the activation of Raf-1, the best described activator of MEK (the MAP kinase kinase for ERK). Based on studies in the literature and previous studies in our laboratory, we evaluated the hypothesis that PKC ζ, rather than Raf-1, was the MAP kinase kinase involved in MEK activation in LPS-treated alveolar macrophages. We demonstrated that this was so in a number of ways. First we showed activation of PKC ζ by LPS. To link this to MEK activation, we used a PKC ζ pseudosubstrate-specific peptide to show that in vivo blocking of PKC ζ resulted in decreased MEK and ERK activation. Finally, we showed that LPS induced the physical association of MEK and PKC ζ and that in vivo phosphorylation of MEK by LPS was blocked by a PKC ζ-specific peptide. As further support for the activation of PKC ζ by LPS, we showed that LPS activated the lipid kinase, PI 3-kinase, and caused an association between PKC ζ and PDK-1 (an activation loop kinase). The various kinase activity assays shown in this study show slightly different time frames. Because of the difficulties in using primary cells and in freezing cellular activity at short time frames, we do not make any conclusions about the relative time frames of these events. We have shown, however, that ERK, PKC ζ, and PI 3-kinase are all activated shortly after endotoxin exposure. In conclusion, these studies show that LPS activates ERK in alveolar macrophages, at least in part, through a Raf-1-independent pathway involving PKC ζ (Fig. 14).

ERK becomes activated by phosphorylation of Thr¹⁸³ and Tyr¹⁸⁵ in the activation loop by the dual active kinase MEK (12). The early descriptions of ERK activation showed that ligation of many receptors led to activation of Ras-Raf-1-MEK-ERK (12). Our experiments in alveolar macrophages found little or no activation of Raf-1, suggesting an alternative upstream kinase as an activator of MEK. This is not the first description of a possible Raf-1-independent pathway. Guthridge et al. (24) have demonstrated that in Raw 264.7 cells, Raf-1 is not a part of the LPS signaling pathway regulating ERK. An early study by Winston et al. showed that in mouse macrophages, TNF activated ERK independently of either c-Raf-1 or Raf B (25). Interestingly, they found activity in an unknown MEK kinase with a time course very similar to that of LPS-activated PKC ζ. Insulin also has been shown to activate ERK in a Raf-1-independent manner (26). Zheng et al. (27) showed in Swiss 3T3 cells that EGF activated ERK without any demonstrable Raf-1 activation. More recently, Kartha et al. (28) have shown that in myocytes, platelet-derived growth factor stimulates a MEK kinase that is distinct from all members of the Raf family. In an inflammation model, IL-8 has been shown to activate ERK independently of Ras and Raf-1 (29). Directly applicable to our hypothesis is a study by Takeda et al. (30), who showed that lysophosphatidic acid activated ERK in a Ras-independent manner and that ERK activation could be blocked by inhibitors of PKC ζ and PI 3-kinase.

Previous work in our laboratory suggests that ceramide is involved in PKC ζ activation in LPS-treated cells (31). We found that LPS increased amounts of ceramide in alveolar macrophages and that the addition of exogenous ceramide resulted in ERK activation. Studies in other laboratories have also found that PKC ζ is activated by ceramide (22, 32). A number of studies have documented that LPS induces ceramide and that increases in ceramide can be linked to a Raf-1-independent activation of ERK (33, 34, 35). Further, a recent study demonstrated that ceramide inhibited Raf-1 activity (36). A study by Muller et al. (37) also showed that whereas TNF and ceramide both increased Ras-Raf-1 complexes, the ceramide bound to the catalytic domain of Raf-1, preventing activity.

Ceramide may play a second role in LPS activation of ERK, because it has been linked to PKC ζ activation. Ceramide has been shown to bind and activate PKC ζ in a number of studies. Recently, Wang et al. (32) have shown that PKC ζ activation by ceramide is very concentration dependent; low doses activate and high doses inhibit. Varying amounts of ceramide induction by LPS might be one way alveolar macrophages regulate the magnitude of their response to LPS.

Another possible pathway involved in the activation of PKC ζ and subsequently ERK by LPS in alveolar macrophages is the PI 3-kinase pathway. PDK-1, a kinase that is downstream of PI 3-kinase is known to phosphorylate PKC ζ in the activation loop (Thr⁴¹⁰) (38, 39). Sajan et al. (40) have linked PDK-1 and PKC ζ to ERK activation by LPS in rat adipocytes. In a model closer to ours, Herrara-Velit et al. (41) have shown that in LPS-treated monocytes, PKC ζ activation is PDK-1 dependent. PKC ζ activation requires autophosphorylation, phosphorylation of Thr⁴⁰² in the activation loop (probably by PDK-1), removal of the pseudosubstrate from the catalytic domain, and subcellular localization near a substrate (42). It is possible that both ceramide and PI 3-kinase play a role in the activation of PKC ζ by LPS. Additional studies will be necessary to delineate exactly how PKC ζ is activated in alveolar macrophages. These studies, however, clearly show that PKC ζ plays a central role in ERK activation in LPS-stimulated human alveolar macrophages.

We thank Shawn Roach for graphics assistance and Pam Robeff for technical support.