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Published ahead of print on August 9, 2007, doi:10.1165/rcmb.2007-0010OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 38, pp. 26-31, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0010OC

PKR Regulates TLR2/TLR4-Dependent Signaling in Murine Alveolar Macrophages

Maciej Cabanski1, Mirko Steinmüller1, Leigh M. Marsh1, Ewa Surdziel1, Werner Seeger1 and Jürgen Lohmeyer1

1 University of Giessen Lung Center, Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine and Infectious Diseases, Justus-Liebig-University, Giessen, Germany

Correspondence and requests for reprints should be addressed to Maciej Cabanski, University of Giessen Lung Center, Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine and Infectious Diseases, Justus-Liebig-University, Klinikstrasse 36, Giessen 35392, Germany. E-mail: maciej.cabanski{at}uglc.de


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The double-stranded RNA (dsRNA)-activated serine/threonine kinase R (PKR) is well characterized as an essential component of the innate antiviral response. Recently, PKR has been implicated in Toll-like receptor (TLR) signal transduction in response to bacterial cell wall components. Its contribution to pulmonary immunity, however, has not yet been elucidated. In this report we investigated whether PKR is involved in TLR2/TLR4-mediated immune responses of primary alveolar macrophages (AM). We found that both TLR2 (Pam3CSK4) and TLR4 (LPS) ligands induced rapid phosphorylation of PKR. Moreover, this activation was strictly dependent on the functionality of the respective TLR. Pharmacologic inhibition of PKR activity using 2-aminopurine (2-AP) and PKR gene deletion was found to reduce the TLR2/TLR4-induced activation of the JNK signaling pathway (MKK4/JNK/c-Jun), but did not affect p38 and extracellular signal–regulated kinase 1/2 activation. Moreover, inhibition of PKR phosphorylation severely impaired TNF-{alpha} and IL-6 production by AM in response to LPS and Pam3CSK4. In addition, we found that PKR phosphorylation plays a major role in LPS- but not Pam3CSK4-induced activation of the p65 subunit of NF-{kappa}B. Collectively, these results indicate that functional PKR is critically involved in inflammatory responses of primary AM to gram-positive as well as gram-negative bacterial cell wall components.

Key Words: PKR • 2-aminopurine • alveolar macrophages • LPS • Pam3CSK4



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Our results demonstrate that serine/threonine kinase R (PKR) is involved in the control of Toll-like receptor (TLR)2/TLR4-mediated inflammatory response of alveolar macrophages to bacterial pathogen-associated molecular patterns. This suggests that PKR may also contribute to host defense mechanisms in vivo against bacterial infections.

 
Alveolar macrophages (AM) are highly specialized mononuclear phagocytes broadly distributed in the alveolar space. They function as a first host defense line against inhaled particles and act as sentinels for pathogens and their components called PAMPs (pathogen-associated molecular patterns). This early detection of pathogens by host cell pattern recognition receptors expressed on AM activates various signaling pathways that initiate and control pulmonary immune responses (1, 2). Toll-like receptors (TLRs) are one of the major receptor classes involved in microbial recognition (3, 4). Among the TLRs, TLR4 was shown to be a specific receptor for LPS, a major cell wall component of gram-negative bacteria, whereas TLR2 is involved in the recognition of bacterial lipoproteins from both gram-negative and gram-positive bacteria (5–7). Both TLR4 and TLR2 receptors are expressed and functionally active on AM (8, 9). Binding of bacterial PAMPs to their respective TLRs activates signaling pathways that require adaptor proteins such as myeloid differentiation primary-response gene 88 (MyD88) and Toll/IL-1 receptor domain–containing adapter protein (TIRAP) (4, 10). These engage downstream signaling cascades that culminate in the increased production of inflammatory cytokines, including TNF-{alpha} and IL-6, which are key players of the host inflammatory response to bacterial infections. Two major TLR-mediated signaling pathways have been described in detail: the mitogen-activated protein kinases (MAPKs) family and the Rel family transcription factor NF-{kappa}B (2, 10–16).

Another protein suggested to play a role in TLR signaling is double-stranded RNA (dsRNA)-activated serine/threonine kinase R (PKR), a well-characterized component of the antiviral response. Upon binding to dsRNA, PKR is activated by autophosphorylation and phosphorylates the {alpha} subunit of the protein synthesis eukaryotic initiation factor 2 (eIF-2{alpha}), resulting in rapid inhibition of translation that effectively limits virion production (17–19). More recently, several studies have reported a potential role for PKR in signal transduction in response to bacterial LPS, where it was found to be involved in the activation of MAPKs pathways and of transcription factors such as NF-{kappa}B or signal transducer and activator of transcription-1 (STAT1) (20–24). These in turn regulate the expression of inflammatory genes such as TNF-{alpha} and IL-6. For example, PKR-deficient mouse embryonic fibroblasts (MEFs) were found to be unable to activate MAPKs and further cytokine production in response to LPS (23). However, there are no reports addressing PKR function in lung innate immunity in response to bacterial PAMPs. Thus, in the present study we examined the contribution of PKR to TLR signaling elicited by PAMPs in primary murine AM. We found that both the synthetic TLR2 ligand Pam3CSK4 and the TLR4 ligand LPS rapidly induced PKR phosphorylation and activated p38, extracellular signal–regulated protein kinases (ERK1/2) and c-Jun NH2-terminal kinase (JNK) MAPKs and also NF-{kappa}B. After pharmacologic inhibition of PKR by 2-aminopurine (2-AP) in wild-type AM or in the absence of a functional PKR gene in AM derived from PKR-KO mice, we observed reduced activation of the JNK pathway, and impaired secretion of TNF-{alpha} and IL-6 in AM supernatants in response to TLR2 or TLR4 ligands. Moreover, we showed that LPS- but not Pam3CSK4-induced activation of NF-{kappa}B transcription factor p65 depends on PKR activity. Collectively, these results indicate that PKR is critically involved in the inflammatory response of AM to bacterial cell wall components.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Animals
Wild-type BALB/c mice were purchased from Charles River (Sulzfeld, Germany). CC3TLR/J BALB/c (TLR4-mutated) mice were purchased from Jackson Laboratories (Bar Harbor, ME) and TLR2-KO C57BL/6 mice from Oriental Yeast Co. (Tagata Shizouka, Tokyo, Japan). PKR-KO mice generated on a C57BL/6 background (25) were a kind gift of Jovan Pavlovic (University of Zurich, Zurich, Switzerland). All animals were kept under specific pathogen–free (SPF) conditions and used throughout the study between 8 and 12 weeks of age. Experimental protocols involving animals were approved by institutional and local government comities.

Reagents
LPS (Escherichia coli 0111: B4) was purchased from Calbiochem (Darmstadt, Germany). 2-AP was purchased from Sigma-Aldrich (St. Louis, MO) and was dissolved in PBS/acetic acid mixture (1:200) by heating at 70°C. Synthetic bacterial lipoprotein analog Pam3-Cys-Ser-Lys-Lys-Lys-Lys-OH (Pam3CSK4) was purchased from EMC Microcollections (Tubingen, Germany). Polyriboinosinic:polyribocytydylic acid (Poly I:C) was obtained from Alexis Biochemicals (Grueberg, Germany). Antibodies against Thr-446 phosphorylated PKR and total PKR were from Abcam (Cambridge, UK). Antibodies against phosphorylated p38, JNK, ERK1/2, MKK4, and total p38, JNK, ERK1/2 were obtained from Cell Signaling Technology (Beverly, MA). Antibody against p65 subunit of NF-{kappa}B and c-Jun were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Isolation and Culture of Murine Primary AM
To isolate AM, mice were killed with an overdose of isoflurane (Forene; Abbott, Wiesbaden, Germany) and bronchoalveolar lavage fluid (BALF) was collected. In short, the trachea was exposed and a small incision was made to insert a shortened 21-gauge cannula connected to a 1-ml insulin syringe. BAL was performed until a total volume of 5 ml was recovered. BALF was spun for 10 minutes at 1,400 rpm at 4°C. AM were cultured in RPMI 1640 medium containing 10% fetal bovine serum, L-glutamine, and penicillin/streptomycin at 2.5 to 3 x 105 cells/well in 24-well tissue culture plates for Western blot and TransAM assay, at 8 x 104 cells/well in 96-well tissue culture plates for enzyme-linked immunosorbent assay (ELISA), or at 6 x 104 cells/well on glass slides for immunofluorescence.

Stimulation and 2-AP Treatment
AM were incubated with 4 mM 2-AP or medium alone for 30 minutes, and then left untreated or stimulated with Pam3CSK4 (2 µg/ml), poly(I:C) (50 µg/ml), or LPS (200 ng/ml) for indicated time intervals.

Cell Extracts and Western Blotting
Cells were washed twice with cold PBS and lysed with lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1 mM EGTA (pH 8.0), 0.5% NP-40, 2 mM sodium orthovanadate (pH 10.0), and protease inhibitor cocktail (Roche, Mannheim, Germany). The lysates were kept on ice for 30 minutes, followed by centrifugation for 15 minutes at 13,000 rpm at 4°C. Proteins were separated by electrophoresis on 10% SDS PAGE under reducing and denaturating conditions. Proteins were transferred to polyvinylidene difluoride membranes (Micron Separations, Westborough, MA). After transfer membranes were incubated in blocking buffer (5% nonfat milk in PBS, 0.05% Tween 20) at room temperature for 1 hour. Primary antibodies (1:1,000) were added and membranes were incubated overnight at 4°C, washed three times, and incubated with horseradish peroxidase–conjugated anti-rabbit secondary antibody (Pierce, Rockford, IL). Enhanced chemiluminescence system was used to visualize immune complexes (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK). To remove bound antibodies, membranes were incubated at room temperature for 1 hour in stripping buffer containing PBS/10% glycine/1% HCl.

ELISA
The quantification of murine TNF-{alpha} and IL-6 proteins in culture supernatants was performed by commercially available ELISA kits following the instructions of the manufacturer (R&D Systems, Minneapolis, MN).

Immunofluorescence
Cells were washed twice with PBS and fixed with cold (–20°C) methanol:acethone mixture (1:1) for 10 minutes. After washing, cells were blocked overnight with 10% bovine serum albumin (BSA) in PBS, washed twice, and then incubated overnight with rabbit anti-p65 NF-{kappa}B antibody (1:50) diluted in PBS/1% BSA at 4°C. Immune complexes were detected with Alexa Fluor 488 goat anti-rabbit secondary antibody (Invitrogen, Eugene, OR) diluted in PBS/1% BSA (1:1,000) at room temperature for 1 hour in darkness. After washing, cells were fixed in 4% paraformaldehyde for 10 minutes. Nuclei were stained with DAPI (1:100) diluted in PBS/1% BSA. The coverslips were mounted on slides in mounting medium (Dako, Hamburg, Germany). Cells were imaged by conventional fluorescence microscopy at a magnification of x65 (Leica, Wetzlar, Germany).

TransAM Assay
Whole cell extract (5 µg) was used to monitor activation of the p65 subunit of NF-{kappa}B by the commercially available TransAM p65 NF-{kappa}B assay kit purchased from Active Motif (Rixensart, Belgium) following the instructions of the manufacturer.

Statistical Analysis
All data are given as mean ± SD. Statistical significance was estimated by two-tailed, paired t test. A value of P < 0.05 was considered significant.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
TLR2 and TLR4 Ligands Induce PKR Phosphorylation in AM
Recently, the TLR4 ligand LPS has been reported to activate PKR in mouse tissue macrophages (26, 27). Accordingly, we first examined whether the TLR4 ligand LPS or the TLR2 ligand synthetic bacterial lipopeptide Pam3CSK4 were able to activate PKR in murine primary AM. AM were stimulated in vitro with Pam3CSK4 (2 µg/ml) or LPS (200 ng/ml) for 0, 15, 30, 60, 90, and 120 minutes and PKR phosphorylation was evaluated by Western blotting. PKR was found to be activated by both TLR2 (Pam3CSK4) and TLR4 (LPS) ligands within 15 minutes, reaching maximum phosphorylation levels at 30 minutes (Figure 1A). Pretreatment of AM with the PKR inhibitor, 2-AP for 30 minutes before stimulation with the respective TLR agonist, significantly reduced PKR phosphorylation (Figure 1B).


Figure 1
Figure 1
Figure 1
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  		Figure 1. Serine/threonine kinase R (PKR) phosphorylation induced by Toll-like receptor (TLR)2 (Pam3CSK4) and TLR4 (LPS) ligands. (A) Time-dependent PKR phosphorylation in alveolar macrophages (AM) stimulated with 2 µg/ml Pam3CSK4 or 200 ng/ml LPS for the indicated time intervals. (B) Pharmacologic inhibition of TLR-ligand induced PKR activation by 2-AP. Cells were pretreated with 4 mM 2-AP for 30 minutes or left untreated, followed by stimulation with 2 µg/ml Pam3CSK4 or 200 ng/ml LPS for 30 minutes. (C) Activation of PKR by Pam3CSK4 and LPS is TLR2 and TLR4 receptor dependent. AM from wild-type (WT), TLR2-KO, and CC3TLR/J mice were left untreated or stimulated with 200 ng/ml LPS, 2 µg/ml Pam3CSK4, or 50 µg/ml Poly I:C for 30 minutes. Cell lysates were resolved on SDS-PAGE and probed with anti–phospho PKR and anti-PKR antibodies. The data are representative of three independent experiments.

 
To elucidate whether Pam3CSK4- and LPS-induced activation of PKR was dependent on specific binding to the respective pattern recognition receptors TLR2 and TLR4, we isolated AM from TLR2-deficient (TLR2-KO) and TLR4-mutated mice (CC3TLR/J). AM were stimulated with LPS, Pam3CSK4, or poly I:C (synthetic dsRNA) for 30 minutes, and PKR phosphorylation was assessed by Western blotting. No activation of PKR was observed after stimulation with LPS and Pam3CSK4 in TLR4 mutated and TLR2-KO cells, respectively (Figure 1C). As expected, the synthetic dsRNA poly I:C triggered PKR activation in wild-type (WT), TLR2-KO, and TLR4-mutated macrophages. These are the first studies to show that not only TLR4 but also TLR2 ligands are capable of inducing PKR phosphorylation.

PKR Is Involved in PAMP-Induced Release of Inflammatory Cytokines
Induction of TLR signaling by PAMP binding ultimately leads to inflammatory cytokine secretion (10). To evaluate whether PKR activation is required for TLR2- and TLR4-mediated cytokine release, WT AM in the presence or absence of the PKR inhibitor 2-AP and PKR-KO AM were stimulated with LPS or Pam3CSK4 for 6 hours. As shown in Figure 2, both 2-AP pretreated WT cells and PKR-KO cells displayed a significantly reduced PAMP-induced release of TNF-{alpha} and IL-6 as assessed by ELISA in the supernatant. These data indicate that PKR phosphorylation is critically involved in TLR2- and TLR4-mediated cytokine secretion by AM.


Figure 2
Figure 2
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  		Figure 2. TLR2 (Pam3CSK4) and TLR4 (LPS) ligand-induced inflammatory cytokine production by AM requires PKR phosphorylation. AM from WT mice incubated with medium alone or with 4 mM 2-AP for 30 minutes or AM derived from PKR-KO animals were stimulated with 2 µg/ml Pam3CSK4 or 200 ng/ml LPS for 6 hours and supernatants were assayed for TNF-{alpha} (A) and IL-6 (B) concentrations by enzyme-linked immunosorbent assay (ELISA). Data are mean ± SD of three different experiments (**P < 0.01; ***P < 0.001).

 
LPS-Induced Activation of NF-{kappa}B Is PKR Dependent
PAMP-induced production of TNF-{alpha} and IL-6 is dependent on the activation of transcription factors such as NF-{kappa}B (16). To assess whether the activation of NF-{kappa}B after TLR2 or TLR4 signaling depends on PKR phosphorylation, WT AM in the presence or absence of 2-AP and PKR-KO AM were stimulated with LPS or Pam3CSK4. Activation of the p65 subunit of NF-{kappa}B was determined with a NF-{kappa}B p65 ELISA-based assay. p65 activation induced by LPS (200 ng/ml) peaked at 2 hours and was found to be significantly reduced after pretreatment with 2-AP. Correspondingly, LPS-induced p65 NF-{kappa}B signaling was found to be significantly attenuated in PKR-KO AM (Figure 3A). In contrast, p65 activation elicited by the TLR2 agonist Pam3CSK4 peaked after 1 hour of stimulation and was only slightly impaired by pretreatment of WT AM with 2-AP or in PKR-KO cells (Figure 3B). To confirm this differential effect of PKR phosphorylation inhibition on TLR2- versus TLR4-mediated NF-{kappa}B activation, we examined PAMP-induced nuclear translocation of p65 by immunofluorescence microscopy (Figure 3C). In untreated AM, p65 was localized exclusively in the cytoplasm; after stimulation with LPS or Pam3CSK4, p65 translocated to the nucleus. 2-AP pretreatment caused a significant reduction of p65 nuclear translocation after LPS but not after Pam3CSK4 stimulation. Together, these results suggest that PKR phosphorylation plays a major role in LPS- but not Pam3CSK4-induced activation of the p65 NF-{kappa}B subunit.


Figure 3
Figure 3
Figure 3
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  		Figure 3. LPS-induced activation of NF-{kappa}B is PKR dependent. AM from WT mice incubated with medium alone or with 4 mM 2-AP for 30 minutes, or AM derived from PKR-KO animals, were stimulated with 2 µg/ml Pam3CSK4 for 1 hour or with 200 ng/ml LPS for 2 hours. Activation of p65 subunit of NF-{kappa}B was monitored in whole cell extracts (5 µg) using the ELISA-based TransAM NF-{kappa}B kit assay (A, B) or by immunofluorescence microscopy (C). Results for TransAM assay are presented as mean ± SD of three different experiments (*P < 0.05; **P < 0.01). For immunofluorescence microscopy, AM were treated as indicated, stained with primary rabbit antibody against p65, followed by incubation with a secondary Alexa fluor 488–labeled goat anti-rabbit Ig antibody (green). Nuclei were counterstained with DAPI (blue). The cells were imaged by conventional microscopy. Images are representative of three independent experiments.

 
PKR Mediates the JNK Signaling Pathway in Pam3CSK4- and LPS-Stimulated AM
Previous studies have demonstrated that PKR mediates activation of MAPKs in response to inflammatory stimuli in various cell types (23, 28). Therefore we analyzed the phosphorylation of three major MAP kinases (p38, JNK, and ERK1/2) by Western blotting after Pam3CSK4 or LPS stimulation of WT AM in the presence or absence of 2-AP and in PKR-KO AM (Figures 4A and 4B). All three kinases were found to be activated in AM in response to Pam3CSK4 and LPS stimulation, but only JNK phosphorylation was affected by pharmacologic suppression of PKR activity and in PKR-KO AM. To further confirm the role of PKR in the JNK signaling pathway, we tested the effect of 2-AP on the regulation of MKK4, a kinase upstream of JNK, and on the downstream JNK target c-Jun. Inhibition of PKR markedly reduced phosphorylation of MKK4 and strongly decreased c-Jun phosphorylation as assessed by mobility shift (Figure 4C). Collectively, our data indicate that PKR regulates the JNK signaling pathway (PKR-MKK4-JNK-c-Jun) in AM activated by TLR2 or TLR4 ligands.


Figure 4
Figure 4
Figure 4
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  		Figure 4. PKR regulates the TLR2 (Pam3CSK4) and TLR4 (LPS) ligand-induced JNK signaling pathway. AM from WT mice incubated with medium alone or with 4 mM 2-AP for 30 minutes, or AM derived from PKR-KO animals, were stimulated with 2 µg/ml Pam3CSK4 or 200 ng/ml LPS for 30 minutes. Cell lysates were resolved on SDS-PAGE and analyzed for phosphorylation of JNK, p38, ERK1/2 (A, B), and MKK4, c-Jun (C). Actin was used as protein loading control. The arrows indicate the mobility shift of phosphorylated c-Jun. Data are representative of three independent experiments.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The antiviral function of PKR is well characterized. However, its contribution to host defense mechanisms against bacterial infections is still poorly understood. In this study, we investigated whether PKR is involved in innate immune responses of resident AM to gram-positive and gram-negative bacterial pattern molecules. We found that PKR was consistently activated in vitro after interaction of TLR2 and TLR4 with their respective ligands, Pam3CSK4 and LPS. Using both the specific PKR phosphorylation inhibitor 2-AP and AM devoid of functional PKR (PKR-KO), we additionally revealed that PKR activation is critically involved in Pam3CSK4- and LPS-induced activation of the JNK signaling pathway as well as in the secretion of crucial inflammatory cytokines like TNF-{alpha} and IL-6. In addition, we found that LPS-induced activation of NF-{kappa}B was PKR dependent, whereas activation of NF-{kappa}B in response to Pam3CSK4 was not significantly attenuated by pharmacologic PKR inhibition or PKR gene deletion. Together, our studies demonstrate that PKR differentially regulates TLR-mediated responses and plays an essential role in the activation of innate immunity in lung AM.

Prior studies have suggested that PKR is involved in LPS signaling in various cell types. For example, PKR-deficient MEFs were found to be unable to activate p38 and JNK MAP kinases in response to LPS, dsRNA, and proinflammatory cytokines. Moreover, IL-6 and IL-12p40 production after intraperitoneal LPS challenge was impaired in PKR-KO mice, suggesting a major role for PKR in TLR4-mediated inflammatory responses (23). In contrast, PKR-KO bone marrow–derived macrophages showed normal activation of JNK, p38, and ERK1/2 MAP kinases as well as I{kappa}B kinases (IKKs) after stimulation with LPS, suggesting PKR-independent TLR4-mediated signaling (27). Thus, the requirement of functional PKR in TLR signaling seems to vary with the cell type. Our experiments have demonstrated for the first time that in AM not only LPS-induced TLR4 signaling but also Pam3CSK4-induced TLR2 signaling stimulated PKR phosphorylation. Activation of PKR by LPS and Pam3CSK4 was strictly dependent on the cellular expression of the respective pattern recognition receptor molecules. These data imply potential involvement of PKR in signal transduction during both gram-negative and gram-positive bacterial infections. Our results are in agreement with previous reports placing PKR downstream of TIRAP and MyD88, which are essential adaptor molecules for TLR2 and TLR4 signaling (29).

The profound decrease of PAMP-induced TNF-{alpha} and IL-6 production after PKR inhibition suggests that PKR activity may also determine the extent of alveolar inflammation in vivo. In our study we investigated the role of PKR using both pharmacologic inhibition and gene knockout approaches. Although the specificity of 2-AP as a PKR inhibitor has been broadly reported, additional nonspecific effects were observed (26, 30–32). However, using PKR-KO AM we provided data that confirm specificity of 2-AP as a valuable PKR inhibitor in our system.

It is known that bacterial components activate several transcription factors, such as NF-{kappa}B, which is one of the major players responsible for TNF-{alpha} and IL-6 cytokine production (16). The NF-{kappa}B family of proteins includes p50, p105, p52, p65, Rel B, and c-Rel, which under physiologic conditions are kept in the cytoplasm in an inactive state, by inhibitors of {kappa}B (I{kappa}B). Upon stimulation, I{kappa}B proteins are phosphorylated by I{kappa}B kinases (IKKs), and NF-{kappa}B proteins are released and translocated to the nucleus (2, 33). It has been previously proposed that PKR is a component of the IKK complex and plays an important role in NF-{kappa}B activation by dsRNA (19, 34). Our work supports this requirement for PKR in NF-{kappa}B activation in response to LPS. In contrast, TLR2-mediated activation of NF-{kappa}B was found to be largely PKR independent. This finding suggests that the molecular steps regulating NF-{kappa}B activity differ in TLR2- and TLR4-mediated signaling.

MAP kinases represent a second major signaling pathway activated by bacterial components (10, 12, 15, 35). In our study, we observed phosphorylation of three major MAP kinases (p38, JNK, and ERK1/2) in response to Pam3CSK4 and LPS in AM. However, after inhibition of PKR phosphorylation by 2-AP and in PKR-KO AM, we observed only reduced JNK phosphorylation. JNK has been reported to be an important mediator of inflammatory processes induced by LPS (15, 36) and regulates AP-1 transcription factor activity and the production of inflammatory cytokines (2, 37). A major substrate for JNK kinase is c-Jun, a central component of the AP-1 heterodimer. Correspondingly, we observed reduced phosphorylation of c-Jun/AP-1 when PKR was inhibited. In addition, phosphorylation of MKK4, a direct activator of JNK, was also found to be dependent on PKR activity. Collectively, these data indicate that PKR specifically regulates the JNK signaling pathway (PKR-MKK4-JNK-c-Jun) in AM activated by TLR2 and TLR4 bacterial ligands (Figure 5).


Figure 5
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  		Figure 5. Model of PKR transducer function in TLR2/4-mediated signaling in AM.

 
In summary, our study shows that the secretion of TNF-{alpha} and IL-6 by primary AM in response to cell wall components of gram-positive as well as gram-negative bacteria in vitro is critically dependent on PKR phosphorylation. Activated PKR was found to act as upstream component of the Pam3CSK4- and LPS-activated JNK pathway. PKR also regulates TLR4- but not TLR2-induced activation of NF-{kappa}B in AM. These data identify PKR phosphorylation as important signaling step in the TLR2/4-mediated inflammatory response of AM to bacterial PAMPs.


   Acknowledgments
 
The authors thank Jovan Pavlovic (University of Zurich, Switzerland) for kindly providing PKR-KO mice and for helpful comments on the manuscript. The authors acknowledge the expert assistance of Petra Janssen and Emma Braun.


   Footnotes
 
This study was supported by the German Research Foundation, grant 547 "Cardiopulmonary Vascular System," and by the National Network on Community-Acquired Pneumonia, CAPNETZ.

Originally Published in Press as DOI: 10.1165/rcmb.2007-0010OC on August 9, 2007

Conflict of Interest Statement: W.S. received/receives grant and contract support from the following companies: Schering AG, Pfizer Ltd., Altana Pharma AG, Lung Rx, Myogen. None of the other authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form January 16, 2007

Accepted in final form July 16, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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