1

N-(3-OXO-ACYL) HOMOSERINE LACTONES SIGNAL CELL ACTIVATION THROUGH A MECHANISM DISTINCT FROM THE CANONICAL PATHOGEN-ASSOCIATED

MOLECULAR PATTERN RECOGNITION RECEPTOR PATHWAYS* Vladimir V. Kravchenko1‡, Gunnar F. Kaufmann1,2,4‡, John C. Mathison1, David A. Scott1, Alexander Z. Katz1, Malcolm R. Wood3, Andrew P. Brogan2,4, Mandy Lehmann1, Jenny M. Mee2,4, Kazunori Iwata1, Qilin Pan1, Colleen Fearns1, Ulla G. Knaus1, Michael M. Meijler2,4,

Kim D. Janda1,2,4,5 & Richard J. Ulevitch1 From 1Department of Immunology and 2Department of Chemistry, 3The Core Microscopy

Facility, The Scripps Research Institute; 4The Skaggs Institute for Chemical Biology; 5Worm Institute of Research and Medicine; 10550 North Torrey Pines Road, La Jolla, CA 92037

Running title: Homoserine Lactone Signaling Address correspondence to: Richard J. Ulevitch, Tel. 858-784 8219; Fax. 858-784 8333; E-mail: ulevitch@scripps.edu or Kim D. Janda, Tel. 858-784 2516; Fax. 858-784 2590; E-mail: kdjanda@scripps.edu

Innate immune system receptors function as sensors of infection and trigger the immune responses through ligand-specific signaling pathways. These ligands are pathogen-associated products, such as components of bacterial walls and viral nuclear acids. A common response to such ligands is the activation of mitogen-activated protein kinase p38, whereas double-stranded viral RNA additionally induces the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α). Here we show that p38 and eIF2α phosphorylation represent two biochemical markers of the effects induced by N-(3-oxo-acyl) homoserine lactones, the secreted products of a number of Gram-negative bacteria, including the human opportunistic pathogen Pseudomonas aeruginosa. Furthermore, N-(3-oxo-dodecanoyl) homoserine lactone induces distension of mitochondria and the endoplasmic reticulum as well as c-jun gene transcription. These effects occur in a wide variety of cell types including alveolar macrophages and bronchial epithelial cells and require the structural integrity of the lactone ring motif and its natural stereochemistry. These findings suggest that N-(3-oxo-acyl) homoserine lactones might be recognized by receptors of the innate immune system. However, we provide evidence that N-(3-oxo-dodecanoyl) homoserine lactone-mediated signaling does not require the presence of the canonical innate immune system receptors, Toll-like receptors or two members of the NLR/Nod/Caterpillar family, Nod1 and Nod2. These data offer a new understanding of the effects of N-(3-oxo-dodecanoyl) homoserine lactone on host cells

and its role in persistent airway infections caused by P. aeruginosa.

The innate immune system has evolved

to recognize a wide variety of structurally highly conserved microbial products, also referred to as pathogen-associated molecular patterns (PAMPs)6. These signals are recognized by membrane and cytosolic receptors termed pattern recognition receptors (PRRs) and trigger the host response (1,2). Examples of canonical PRRs in mammalians are the Toll-like receptors (TLRs) and the NLR/Nod/Caterpillar proteins (3,4). TLRs signal through adaptor protein complexes that possess Toll/interleukin-1 receptor (TIR) domains (5). Although TIR domain-containing adaptors, known as MyD88, TIRAP/MAL, TRAM, and TRIF/TICAM-1, may form homo- and heterodimers, the signal transduction of most known PAMPs requires the involvement of MyD88, TRIF or both (6-11). For example, both adaptors are essential for signaling mediated by the Gram-negative bacterial product lipopolysaccharide (LPS) which is a TLR4 specific PAMP, whereas MyD88 is required for the TLR9 specific PAMPs, and TRIF is used in TLR3 specific manner. Common characteristics of MyD88- and TRIF-dependent pathways include activation of the transcription factor NF-κB and mitogen-activated protein kinases (MAPK), including p38. An additional unique feature of TRIF-dependent signaling is the attenuation of protein synthesis via phosphorylation of the eukaryotic translation initiation factor 2α (eIF2a) (3,12).

PAMPs are exclusive to bacterial or

viral pathogens and do not exist in mammalian organisms. Morevover, they are essential to the pathogenesis of the microbe, and structurally

http://www.jbc.org/cgi/doi/10.1074/jbc.M606613200The latest version is at JBC Papers in Press. Published on August 7, 2006 as Manuscript M606613200

Copyright 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on July 17, 2018http://w

ww

.jbc.org/D

ownloaded from

2

invariant PAMPs are shared by groups of pathogens. Examples of bacterial PAMPs include LPS, peptidoglycan, unmethylated CpG motifs present in DNA and viral nucleic acids (13). Here we have examined a class of secreted molecules, the N-acyl homoserine lactones (AHLs), that are produced by a large number of Gram-negative bacteria (14). AHLs are a structurally highly conserved group of bacterial-derived molecules. They are very important in the life cycle of bacteria by being linked to virulence and thus are often essential for chronic infection in the host (15-18). A prototypic member of the AHL family, N-(3-oxo-dodecanoyl) homoserine lactone (C12), was originally identified in the opportunistic human pathogen Pseudomonas aeruginosa (19). The abilities of this Gram-negative bacterium to survive and establish chronic infections in the host appear to be associated with functions of C12 (20,21). Interestingly, C12 has also been reported to exhibit effects on eukaryotic cells (15,21) including inhibition of cell proliferation (22,23), induction of apoptosis, (23,24) and nuclear translocation of NF-κB (15,25). These observations suggest that C12 may function as a PAMP. However, no studies have directly addressed this possibility.

We have performed biochemical and

genetic analyses to identify signaling pathways and cellular organelles responsive to C12 with respect to PRR-dependent mechanisms by using primary cells and cell lines of different origin, including a panel of single and double knockout bone marrow derived macrophages. Our results reveal that C12 treatment of myeloid or non-myeloid cells results in the morphological alteration of mitochondria and especially endoplasmic reticulum (ER), and that the phosphorylated forms of p38 (P-p38) and eIF2α (P-eIF2α) are two major biochemical markers indicative of the C12 responsiveness of diverse cell types. Evidence is provided that the N-(3-oxo-acyl) homoserine lactone ring motif of C12 is required for the induction of P-p38 and P-eIF2α, and similar phosphorylation of both proteins is also activated by other members of the AHL family containing this structural motif. We also examined these responses in macrophages obtained from mice bearing targeted gene deletions in components that are essential for signaling induced by canonical PAMPs of Gram-negative and Gram-positive

bacteria. We demonstrate that MyD88, TRIF, TLR2, TLR4, Nod1, and Nod2 are dispensable for the C12-mediated signaling. These observations unveil C12 as a paradigm for a new class of PAMPs acting independently from canonical PRR-mediated mechanisms.

EXPERIMENTAL PROCEDURES

Mice, bone marrow-derived macrophages and other cells — TLR2-/-, TLR4-/-, MyD88-/- mice were received from S. Akira; TRIF-deficient lps2 mice – from B. Beutler; and Nod1/2-/- double knockout mice were generated by crossing Nod1-/- mice (from T. Mak) with Nod2-/-mice (from R. Flavell). C57BL/6 mice were purchased from the Jackson Laboratory. All knockout mice were of the C57BL/6 background. Normal human bronchial epithelial cells (NHBE) were obtained from Cambrex and cultured as recommended by the manufacturer. L929/NCTC clone 929 (connective tissue, mouse), THP-1 (monocyte, human), RAW 264.7 (monocyte-macrophage, mouse) and WI-38 (SV40transformed human lung fibroblasts; CCL-75.1) cell lines were purchased from ATCC. Bone marrow-derived macrophages (BMDM) and murine embryonic fibroblasts (MEFs) were prepared by using standard protocols. Alveolar macrophages (AM) were isolated from C57BL/6 mice as described (26). MEFs and cell lines were maintained in growth medium (GM): DMEM medium (4.5 g/L glucose) supplemented with 10% FBS (HyClone), L-glutamine, penicillin/streptomycin and nonessential amino acids (Invitrogen). BMDM were cultured in 70% GM and 30% L929 conditioned medium. NHBE were cultured in SABM (Cambrex) supplemented with 30 g/ml bovine pituitary extract, 0.5 µg/ml hydrocortisone, 0.5 ng/ml human recombinant epidermal growth factor, 0.5 µg/ml epinephrine, 10 µg/ml transferrin, 5 µg/ml insulin, 0.1 ng/ml retinoic acid, 6.5 ng/ml triiodothyronine, 50µg/ml Gentamicin, 50ng/ml Amphotericin-B and 0.5mg/ml bovine serum albumin-fatty acid free at 37°C in a 5% CO2 incubator. In general, cells (~ 60-70 % confluency) were incubated in corresponding fresh medium for 12-14 h before stimulation. Reagents and antibodies — N-(3-oxododecanoyl) -S- and -R-homoserine lactone (C12, C12R respectively), N-butyryl-S-homoserine lactone (BHL), 3-oxo-dodecanoic acid (2-oxo-pyrrolidin -3-yl)-amide (lactam, C12-LM), (S)-3-(1-

by guest on July 17, 2018http://w

ww

.jbc.org/D

ownloaded from

3

hydroxydecylidene)-5-(2-hyroxyethyl) pyrrolidine-2,4-dione (C12-TA), N-(3-oxo decanoyl)-S-homoserine lactone (C10) and N-(3-oxotetradecanoyl)-S-homoserine lactone (C14) were synthesized and purified as previously described (27). The purity of all these synthetic compounds was greater than 99% and was confirmed by HPLC/mass spectrometry analysis. All synthetic molecules were dissolved in DMSO at 200x the desired concentration and aliquots were stored at -20 oC. In addition, a quantitative QCL-1,000 chromogenic Limulus amoebocyte lysate assay (BioWhittaker, Walkersville, MD) demonstrated that preparations of C10, C12, C12R, C12-LM, C12-TA, C14 and BHL were endotoxin free. 4-hydroxy-2-(3-oxo-dodecanoylamino)-butyric acid (C12-hyd) was prepared by incubation of C12 in aqueous solution (pH 10) at 37 oC for 24 h; under these conditions greater then 85% of C12 was converted to C12-hyd, which was confirmed by HPLC/mass spectrometry analysis. S. minnesota Re595 LPS was prepared as previously described (28). Anti-PARP, phospho-p38 (P-p38), P-eIF2α, P-JNK, P-ERK1/2, ERK1/2, caspase-9, cleaved form of caspase-3, and actin (from Sigma) antibodies were purchased from Cell Signaling and used in Western blot analysis as recommended by the manufacturer. Northern blot — Total RNA was isolated by using TRizol reagent (Invitrogen). Total RNA (10 µg/lane) were analyzed by Northern blot as previously described (29). Blots were hybridized with specific anti-sense oligonucleotides end-labelled by T4 polynucleotide kinase using γ-[32P]-ATP. Electron microscopy — Cells were plated in 35-mm dishes as described above. After treatment of cells with a compound or 0.5% DMSO as a control, samples for electron microscopy analysis were prepared as described (30); images were documented on a Philips CM100 electron microscope (FEI, Hillsborough, OR). Data presentation — Data depicted in figures represent one of three or more experiments with each graph reflecting findings typical of multiple studies.

RESULTS

Phosphorylation of p38 and eIF2α are two biochemical markers indicative of C12-

mediated signaling: Structure-activity relationship studies of C12.

Macrophages are a key effector cell type

of the innate immune system. Treatment of these cells with PAMPs induces several signaling events including the phosphorylation of MAPK p38, a shared marker of Nod-dependent as well as MyD88- and TRIF-dependent signaling pathways, and/or the phosphorylation of the translation initiation factor eIF2α, a biochemical marker indicative of TRIF-dependent signaling. Therefore, we reasoned that determination of the effects of C12 on the phosphorylation state of p38 and eIF2a would be appropriate markers for our initial studies to investigate possible PAMP-like signaling of C12 in macrophages. Exposure of BMDM to C12 resulted in time- and dose-dependent induction of p38 and eIF2α phosphorylation (P-p38 and P-eIF2α) (Fig. 1A and B).

These observed biochemical effects

were highly specific for the naturally occurring S-stereoisomer of C12 since no biochemical effects were observed after addition of the unnatural R-stereoisomer of C12 (C12R) (Fig. 1C). Interestingly, induction of P-p38 and P-eIF2α could also be detected after treatment with other members of 3-oxo-AHL family such as N-3-oxo-decanoyl homoserine lactone (C10) and N-(3-oxo-tetradecanoyl) homoserine lactone (C14) (Fig. 1D, E). However, another AHL, N-butyryl homoserine lactone (BHL), also synthesized by P. aeruginosa was found to be inactive (Fig. 1D). Additionally, the lactam congener of the C12 lactone, C12-LM, failed to induce these biochemical changes (see Fig. 1D, E). Taken together, these data suggest that the integrity of the homoserine lactone ring motif is required to induce distinct cellular events leading to P-p38 and P-eIF2α.

In aqueous conditions at neutral pH ~

7.4 the C12 lactone ring can undergo two different non-enzymatic reactions: 1) reversible hydrolysis generating the corresponding carboxylic acid (C12-hyd); 2) an irreversible Claisen-like reaction resulting in the conversion of C12 to a tetramic acid (C12-TA) (Fig. 2A). As the cell culture media usually has a pH ~ 7.4, thus both C12-hyd and C12-TA may be generated under our experimental conditions after addition of C12. Indeed, HPLC and mass

by guest on July 17, 2018http://w

ww

.jbc.org/D

ownloaded from

4

spectrometry analysis revealed that C12-hyd is rapidly generated after addition of C12 to the cells, and its presence is seen in both supernatant and cellular fractions (Fig. 2B). Interestingly, the cellular fractions contained approximately ten times more C12-hyd than C12, whereas C12 was predominantly distributed in the supernatant (Fig. 2B). Notably, a 30 second treatment of macrophages with C12 was sufficient to induce P-p38 as well as P-eIF2α as detected 15 min later (Fig. 2C), suggesting that association of C12, C12-hyd or both with the cells is required for these signaling events. However, the initial integrity of C12 is a prerequisite for the induction of P-p38 and P-eIF2α, because C12-hyd, C12-TA, as well as C12-LM all lost this ability (Fig. 2D). Since previous studies had shown that prolonged incubation of transformed human cells with C12 induces the cleavage of poly(ADP-ribose) polymerase (PARP) (23), a biochemical marker indicative of apoptosis (31), investigations were warranted to determine whether the integrity of the homoserine lactone ring motif is also required for induction of PARP cleavage in macrophages. As expected, Western blot analysis showed that C12, but not C12-hyd, C12-TA or C12-LM induces the cleavage of PARP (see Fig. 2D). Thus, the structural integrity of the homoserine lactone ring motif is required for the C12-mediated induction of apoptotic pathways as well as phosphorylation of p38 and eIF2α.

Comparison of C12-induced effects on myeloid and non-myeloid cells.

Our observations that treatment of

BMDM with C12 induces the cleavage of PARP as well as phosphorylation of p38 and eIF2α prompted investigations of whether C12 has the ability to activate similar signaling events in other cell types. Based on the clinical observation that P. aeruginosa is a common causative agent of chronic lung infections in humans, alveolar macrophages and epithelial cells were examined for their response to C12. These experiments confirmed that the induction of PARP cleavage, P-p38 and P-eIF2α are characteristic markers of the general cellular responses to C12 as observed in various cell types, including the primary alveolar macrophages and normal lung epithelial cells (Fig. 3A). Importantly, C12-induced phosphorylation of p38 and eIF2α was very

rapid and evident in all cell types. However, significantly delayed kinetics and reduction of the extent of PARP cleavage were observed in cells of non-hematopoietic origin (see Fig. 3A). Additionally, dose titration experiments revealed that C12 significantly affects the viability of macrophages when compared with non-myeloid cells such as epithelial cells and fibroblasts (Fig. 3B and D).

MyD88, TRIF, TLR2, TLR4 Nod1, and Nod2 are not required for C12-mediated signaling in macrophages

Macrophages are activated though a

variety of PAMPs recognized by specific PRRs. Four adaptor proteins, MyD88, TRIF, MAL/TIRAP and TRAM, are involved in TLR-dependent signal transduction events. However, there are very specific patterns of adaptor protein usage - TLR2 uses MyD88 and TIRAP, TLR3 signals through TRIF, TLR4 utilizes all four; the rest of the known TLRs, including TLR5 and TLR7-9, appear to require only MyD88. In keeping with our observations that the effects of C12 mimic the activation of MyD88- and TRIF-dependent pathways, experiments were conducted to determine whether MyD88 or TRIF are required for C12-mediated phosphorylation of p38 and eIF2α as well as induction of PARP cleavage. Examination of the responses in BMDM from mice with genetic defects in MyD88 or TRIF function revealed that C12 induction of PARP cleavage, P-p38 and P-eIF2α was identical in the BMDM from the adaptor protein-deficient and control mice (Fig. 4A). Thus, neither MyD88 nor TRIF are required for the C12-mediated biochemical signaling events. Additionally, these findings suggest that TLR3, TLR5, TLR7, TLR8, and TLR9 are not involved in the recognition and transduction of the C12 signal. We cannot exclude the possibility that MyD88 and TRIF are both required for the full C12 response, but at present we consider this possibility very unlikely.

We also asked whether TLR2 or TLR4

are required for C12-mediated signaling since both receptors have the ability to recognize molecules containing long-chain fatty acid moieties. Comparison of the C12-mediated biochemical changes in TLR2-/- , TRL4-/- or control BMDM failed to reveal any differences in the response to C12 in this panel of BMDM

by guest on July 17, 2018http://w

ww

.jbc.org/D

ownloaded from

5

(Fig. 4B). The intracellular proteins Nod1 and Nod2 have been implicated in innate immune responses distinct from those mediated by the TLRs (2-4). Thus, we also examined BMDM from mice lacking both Nod1 and Nod2 and observed that C12-induced phosphorylation of p38, eIF2α, and PARP cleavage were identical in the BMDM from Nod1/2-deficient and control mice (Fig. 4C). These data indicate that the Nod1 and Nod2 proteins are not required for the initial recognition and signaling events of C12 in macrophages.

In total, these observations suggest that C12 signals through TLR- and NLR-independent pathways and raise the possibility that it affects signaling pathways in a manner distinct from TLR-specific PAMPs. LPS is a major TLR-specific PAMP present in P. aeruginosa, hence, we compared the responses of macrophages to C12 and LPS. As a well-known TLR4 agonist, LPS induces signaling through MyD88- and TRIF-dependent pathways (5,6). Among the examined signaling events, the phosphorylation of p38 was the only response induced by both LPS and C12, although with different kinetic patterns (Fig. 5A). Particularly interesting is the opposite effect of these stimuli on the MAPKs p44 and p42, also known as extracellular signal regulated kinases 1 and 2 (ERK1/2). While LPS treatment resulted in the induction of ERK1/2 phosphorylation (P-ERK1/2), the basal levels of P-ERK1/2 were down-regulated in response to C12.

The consequences of these differences between LPS- and C12-mediated signaling events was further investigated by the examination of mRNA levels of tumor necrosis factor (TNF), macrophage inflammatory protein 2 (MIP-2), interferon β (IFN-β), cyclooxygenase 2 (Cox-2) and cellular proto-oncogene c-jun, genes that are induced in LPS-stimulated cells (11,29,32,33). The levels of TNF, MIP-2, IFN-β and Cox-2 mRNAs were increased after LPS treatment, whereas C12 failed to induce these transcripts (Fig. 5B). Additionally, while LPS treatment resulted in weak and transient induction of c-jun mRNA, a robust and prolonged increase in the level of c-jun mRNA was observed after C12 treatment, indicating that C12-mediated signaling results in the transcriptional activation of the gene encoding

the c-Jun protein, a key regulator of proliferation and apoptosis (34-36).

To confirm and extend the findings obtained using BMDM we also examined C12 or LPS induction of c-jun and genes encoding the inflammatory modulators MCP-1, IP-10, Cox-2 and IL-8 in non-myeloid cell types. As was expected for LPS responsiveness in MEFs (29,32), levels of MCP-1, IP-10 and Cox-2 mRNAs were significantly up-regulated, and the level of c-jun mRNA was slightly increased (Fig. 5C). Importantly, although C12 treatment did not induce MCP-1, IP-10 and Cox-2 mRNAs, c-jun mRNA was strongly induced after C12 treatment of MEFs (Fig. 5C). Similar results were observed when C12 or TNF induction of mRNAs for IL-8, Cox-2 and c-Jun was examined in primary human bronchial epithelial cells (Fig. 5D). However, transformed human lung fibroblasts (WI-38) showed an increase in levels of IL-8 and Cox-2 mRNAs after treatment with C12 (Fig. 5E). Interestingly, the kinetics and levels of C12- and TNF-induced Cox-2 mRNAs were comparable, whereas extremely weak and delayed IL-8 induction was found in C12-treated WI-38 cells (Fig. 5E, upper panel). Nevertheless, the C12-induced level of c-jun mRNA was quite similar in all cell types including WI-38 cells (Fig. 5B-E). Thus, cells of different origin appear to possess a shared TLR/Nod-independent pathway that responds to C12 with the activation of c-jun, a response normally induced by mitogens and tumor promoters (34,37) as well as by some genotoxic agents (38).

Mitochondria and the endoplasmic reticulum are cellular targets for C12-induced apoptotic pathway

Mitochondria and the endoplasmic

reticulum (ER) are cellular organelles involved in the regulation of a variety of intracellular processes including the stress response to genotoxic shock (39). Mitochondrial stress triggers pro-apoptotic events, including the activation of caspase-9 and caspase-3, and subsequent proteolytic cleavage of PARP (31,40). The ER often responds to local stress stimuli with a change in the protein synthesis rate through phosphorylation-dependent inactivation of eIF2α (41). Observations that treatment of myeloid cells with C12 resulted in

by guest on July 17, 2018http://w

ww

.jbc.org/D

ownloaded from

6

the induction of these biochemical markers (Fig. 6A) prompted investigations whether mitochondria and ER are indeed cellular targets of C12. Electron microscopic analysis of C12-treated BMDM revealed dramatic morphological alterations in both organelles including mitochondrial swelling and distension of the ER (Fig. 6B). The effects seen are specific for the naturally occurring S-stereoisomer of C12, as no morphological changes were observed after addition of C12R (see Fig. 6B). Additionally, ultrastructural alterations of these organelles were observed in other mammalian cells treated with C12 (Fig. 6C), indicating that diverse cell types of either human or murine origin are affected by C12 in a qualitatively similar manner. However, when compared with macrophages, the C12-induced morphological alterations of mitochondria were less pronounced in fibroblasts and epithelial cells, consistent with our previous observations that macrophages are more sensitive to the pro-apoptotic effects of C12. In contrast, the C12-induced ER distention was identical in macrophages and non-myeloid cells, suggesting that the ER is centrally involved in the response of different cell types to C12.

DISCUSSION

In response to PAMPs, macrophages

rapidly produce and secrete cytokines that are essential for host defense and strong adaptive immune response resulting in the elimination of most environmental bacteria (1). The Gram-negative bacterium P. aeruginosa produces prototypic PAMPs including LPS; however, it also possesses the ability to establish a persistent state of infection, especially in lungs of patients suffering from cystic fibrosis and other chronic obstructive pulmonary diseases (COPD) (42,43). The establishment of chronic P. aeruginosa infections correlates with the formation of biofilms (20,44), which are polymeric matrices of bacterial growth (45). In turn, biofilm formation and maintenance is a complex process regulated by C12 (20,21,44). Previous studies revealed that concentrations of C12 in vivo vary to a large extent depending on the growth status of P. aeruginosa, i.e. whether the bacteria are growing as planktonic culture (1-5 µM) or as a part of biofilms (100-600 µM) (46,47). Our observations revealed that treatment of macrophages with C12 at a concentration of 25

µM significantly reduces the viability of mammalian cells, while concentrations of 10 µM are sufficient to induce biochemical changes in macrophages. Moreover, we also found that in contrast to LPS, C12 does not induce the activation of genes encoding the immune modulators TNF, IFN-β and MIP-2. Although further work is needed to characterize the direct effects of C12-containing P. aeruginosa biofilms on macrophages, these results are consistent with the hypothesis that C12 contributes to the establishment and maintenance of chronic infection with P. aeruginosa through its effects on macrophages. Previous findings that C12 accelerates apoptosis (24) and inhibits LPS-induced production of TNF in macrophages and human monocytes (22,48) are in accordance with this suggestion. However, the nature of biochemical mechanisms responsible for C12-mediated effects on mammalian cells has been unclear.

We have demonstrated that N-(3-oxo-

acyl) homoserine lactones induce the phosphorylation of MAPK p38 and eIF2α in different cell types, including alveolar macrophages, lung fibroblasts, and bronchial epithelial cells. These cell types are most likely to be exposed to P. aeruginosa and subsequently C12 as the airways are the main point of entry of this pathogen into the human body. Although similar biochemical changes of these two proteins were previously noted as markers of PRR-dependent signaling in response to canonical PAMPs, we have clearly demonstrated that MyD88, TRIF, TLR2, TLR4, Nod1, and Nod2, key molecules of PRR pathways (3-6), are not required for C12-mediated signaling events. These observations suggest that C12 might represent a new class of PAMP that signals through pathways distinct from those engaged by TLR and Nod1/Nod2 proteins.

The mammalian stress response is activated by a broad array of environmental factors such as bacterial infections and short wavelength ultraviolet (UV) radiation (38,49). A hallmark of these cellular responses is the induction of immediate-early proto-oncogenes such as c-jun and c-fos (38,50). Remarkably, transcription of c-jun is activated not only in cells exposed to environmental agents including UV and bacterial LPS but also in response to cellular products, such as growth factors and

by guest on July 17, 2018http://w

ww

.jbc.org/D

ownloaded from

7

cytokines (33,38,51,52). However, striking stimulus-specific differences are noted in the kinetics and levels of c-jun induction, which are correlated with functional effects of the c-Jun protein on cell cycle progression (51,53,54). For example, while the transient character of c-jun stimulation promotes this process, the prolonged c-jun induction by UV and other genotoxic agents causes cell growth arrest, rather than induction of cell proliferation (53-55). Here we found that C12 treatment of various cell types results in the prolonged induction of c-jun, suggesting a UV-like effect of C12 on cell growth. This assumption is consistent with observations by others that C12 inhibits cell proliferation (22,23). The c-Jun protein is a key component of the transcription factor AP-1 (activator protein-1) that is involved in the regulation of cell growth, transformation and apoptosis (56). Studies of the stress-mediated effects in cells lacking or constitutively expressing c-Jun revealed that cellular functions of c-Jun in the regulation of these processes are p53 dependent (53,54). The p53 gene is the most frequently inactivated tumor suppressor identified in human cancer, and its product acts as the transcription factor (57,58). This transcription factor regulates genes that function in diverse cellular processes, including stress responses, cell proliferation and apoptosis (57). It was reported that p53 is also involved in the regulation of Cox-2 gene transcription (59,60). Consistent with the observation that SV40 large T antigen alters the functions of p53 (57,61), we found that C12 induces Cox-2 mRNA in SV40 transformed WI-38 cells, but not in primary cells. Analogous to C12-mediated transcriptional response in WI-38 cells, Cox-2 and IL-8 induction was previously observed after C12 treatment of immortalized or transformed human epithelial cells and lung fibroblasts (25,62). Although integrity of p53 was not tested in these cells, immortalization and transformation may be a result of mutations in the p53 gene (57,61). Thus, we suggest that C12 affects the gene induction programs through the c-Jun/p53 mechanisms (53,54). This suggestion is consistent with recent finding that the induction of c-Jun plays a role in the regulation of Cox-2 gene expression (63,64). Although further work is needed to identify and characterize specific subsets of C12-regulated

genes, the effect on c-jun gene expression further emphasizes differences between signaling in response to pro-inflammatory stimuli and C12. Additionally, C12 inducible expression of proto-oncogene c-jun may be relevant to P. aeruginosa infections in cancer patients (65).

Other unique characteristics of the

cellular responses to C12 are the rapid ultrastructural alterations of ER and mitochondria. We provide evidence that mitochondrial damage accompanies activation of caspase-9, a key biochemical event indicative of mitochondria-mediated apoptosis (40). In turn, the ability of C12 to induce ER stress and phosphorylate eIF2α is consistent with previous observations that P-eIF2α is involved in the cellular response to failure of the structural and functional integrity of the ER (41). Normal function of ER is essential for coupling transcriptional inflammatory response with high translation rates of newly transcribed mRNA encoding pro-inflammatory cytokines including TNF (66). The efficiency of the translation initiation depends on the dynamic exchange between GTP- and GDP-bound states of eIF2α. Specific phosphorylation of eIF2α at serine 51 disrupts this balance and thereby significantly reduces the initiation of translation resulting in the inhibition of protein biosynthesis (41,67). Thus, C12-mediated induction of eIF2α phosphorylation during P. aeruginosa infection may represent a key signaling event that undermines the host defense response by blocking translation of the secreted immune response regulatory proteins. In addition, it appears to be also relevant in that cystic fibrosis is associated with ER dysfunction (68). Hence, our findings provide evidence that the product of Gram-negative bacterium P. aeruginosa, C12, affects host cells through eIF2α-dependent mechanisms.

In total, the data presented grants us a new insight into the effects of C12 on mammalian cells and will be essential for the understanding of the molecular mechanisms of C12 on host immunity. Furthermore, our report highlights the relevance of C12 in the establishment and maintenance of persistent airway infections caused by P. aeruginosa.

by guest on July 17, 2018http://w

ww

.jbc.org/D

ownloaded from

8

REFERENCES 1. Medzhitov, R., and Janeway, C. (2000) N. Engl. J. Med. 343, 338-344 2. Meylan, E., Tschopp, J., and Karin, M. (2006) Nature 442, 39-44 3. Janeway, C. A., and Medzhitov, R. (2002) Annu. Rev. Immunol. 20, 197-216 4. Inohara, Chamaillard, McDonald, C., and Nunez, G. (2005) Annu. Rev. Biochem. 74, 355-383 5. Yamamoto, M., Takeda, K., and Akira, S. (2004) Mol. Immunol. 40, 861-868 6. Beutler, B. (2004) Nature 430, 257-263 7. Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H., Matsumoto, M.,

Hoshino, K., Wagner, H., Takeda, K., and Akira, S. (2000) Nature 408, 740-745 8. Yamamoto, M., Sato, S., Hemmi, H., Sanjo, H., Uematsu, S., Kaisho, T., Hoshino, K.,

Takeuchi, O., Kobayashi, M., Fujita, T., Takeda, K., and Akira, S. (2002) Nature 420, 324-328

9. Horng, T., Barton, G. M., Flavell, R. A., and Medzhitov, R. (2002) Nature 420, 329-333 10. Hoebe, K., Du, X., Georgel, P., Janssen, E., Tabeta, K., Kim, S. O., Goode, J., Lin, P., Mann,

N., Mudd, S., Crozat, K., Sovath, S., Han, J., and Beutler, B. (2003) Nature 424, 743-748 11. Yamamoto, M., Sato, S., Hemmi, H., Hoshino, K., Kaisho, T., Sanjo, H., Takeuchi, O.,

Sugiyama, M., Okabe, M., Takeda, K., and Akira, S. (2003) Science 301, 640-643 12. Hsu, L., Park, J. M., Zhang, K., Luo, J., Maeda, S., Kaufman, R. J., Eckmann, L., Guiney, D.

G., and Karin, M. (2004) Nature 428, 341-345 13. Aderem, A., and Ulevitch, R. J. (2000) Nature 406, 782-787 14. Lazdunski, A. M., Ventre, I., and Sturgis, J. N. (2004) Nat. Rev. Microbiol. 2, 581-592 15. Shiner, E. K., Rumbaugh, K. P., and Williams, S. C. (2005) FEMS Microbiol. Rev. 29, 935-

947 16. Jones, S., Yu, B., Bainton, N. J., Birdsall, M., Bycroft, B. W., Chhabra, S. R., Cox, A. J. R.,

Golby, P., Reeves, P. J., Stephens, S., Winson, M. K., Salmond, G. P. C., Stewart, G. S. A. B., and Williams, P. (1993) EMBO J. 12, 2477-2482

17. Rumbaugh, K. P., Griswold, J. A., Iglewski, B. H., and Hamood, A. N. (1999) Infect. Immun. 67, 5854-5862

18. Erickson, D. L., Endersby, R., Kirkham, A., Stuber, K., Vollman, D. D., Rabin, H. R., Mitchell, I., and Storey, D. G. (2002) Infect. Immun. 70, 1783-1790

19. Pearson, J. P., Gray, K. M., Passador, L., Tucker, K. D., Eberhard, A., Iglewski, B. H., and Greenberg, E. P. (1994) Proc. Natl. Acad. Sci. USA 91, 197-201

20. Davies, D. G., Parsek, M. R., Pearson, J. P., Iglewski, B. H., Costerton, J. W., and Greenberg, E. P. (1998) Science 280, 295-298

21. Smith, R. S., and Iglewski, B. H. (2003) J. Clin. Invest. 112, 1460-1465 22. Telford, G., Wheeler, D., Williams, P., Tomkins, P. T., Appleby, P., Sewell, H., Stewart, G.

S. A. B., Bycroft, B. W., and Pritchard, D. I. (1998) Infect. Immun. 66, 36-42 23. Li, L., Hooi, D., Chhabra, S. R., Pritchard, D., and Shaw, P. E. (2004) Oncogene 23, 4894-

4902 24. Tateda, K., Ishii, Y., Horikawa, M., Matsumoto, T., Miyairi, S., Pechere, J. C., Standiford, T.

J., Ishiguro, M., and Yamaguchi, K. (2003) Infect. Immun. 71, 5785-5793 25. Smith, R. S., Fedyk, E. R., Springer, T. A., Mukaida, N., Iglewski, B. H., and Phipps, R. P.

(2001) J. Immunol. 167, 366-374 26. Steinhauser, M. L., Hogaboam, C. M., Kunkel, S. L., Lukacs, N. W., Strieter, R. M., and

Standiford, T. J. (1999) J. Immunol. 162, 392-399 27. Kaufmann, G. F., Sartorio, R., Lee, S. H., Rodgers, C. J., Meijler, M. M., Moss, J. A.,

Clapham, B., Brogan, A. P., Dickerson, T. J., and Janda, K. D. (2005) Proc. Natl. Acad. Sci. USA 102, 309-314

28. Mathison, J. C., Virca, G. D., Wolfson, E., Tobias, P. S., Glaser, K., and Ulevitch, R. J. (1990) J. Clin. Invest. 85, 1108-1118

29. Kravchenko, V. K., Mathison, J. C., Schwamborn, K., Mercurio, F., and Ulevitch, R. J. (2003) J. Biol. Chem. 278, 26612-26619

30. Tan, S., Wood, M., and Maher, P. (1998) J. Neurochem. 71, 96-105

by guest on July 17, 2018http://w

ww

.jbc.org/D

ownloaded from

9

31. Nicholson, D. W., Ali, A., Thornberry, N. A., Vaillancourt, J. P., Ding, C. K., Gallant, M., Gareau, Y., Griffin, P. R., Labelle, M., Lazebnik, Y. A., Munday, N. A., Raju, S. M., Smulson, M. E., Yamin, T., Yu, V. L., and Miller, D. K. (1995) Nature 376, 37-43

32. Covert, M. W., Leung, T. H., Gaston, J. E., and Baltimore, D. (2005) Science 309, 1854-1857 33. Han, J., Jiang, Y., Li, Z., Kravchenko, V. V., and Ulevitch, R. J. (1997) Nature 386, 296-299 34. Angel, P., and Karin, M. (1991) Biochim. Biophys. Acta 1072, 129-157 35. Ham, J., Babij, C., Whitfield, J., Pfarr, C. M., Lallemand, D., Yaniv, M., and Rubin, L. L.

(1995) Neuron 14, 927-939 36. Bossy-Wetzel, E., Bakiri, L., and Yaniv, M. (1997) EMBO J. 16, 1695-1709 37. Angel, P., Imagawa, M., Chiu, R., Stein, B., Imbra, R. J., Rahmsdorf, H. J., Jonat, C.,

Herrlich, P., and Karin, M. (1987) Cell 49, 729-739 38. Herrlich, P., Blattner, C., Knebel, A., Bender, K., and Rahmsdorf, H. J. (1997) Biol. Chem.

378, 1217-1229 39. Ferri, K. F., and Kroemer, G. (2001) Nat. Cell Biol. 3, 255-263 40. Green, D. R., and Reed, J. C. (1998) Science 281, 1309-1312 41. Clemens, M. J., Bushell, M., Jeffrey, I. W., Pain, V. M., and Morley, S. J. (2000) Cell Death

Differ. 7, 603-615 42. Lyczak, J. B., Cannon, C. L., and Pier, G. B. (2002) Clin. Microbiol. Rev. 15, 194-222 43. Lieberman, D., and Lieberman, D. (2003) Am. J. Respire. Med. 2, 459-468 44. Singh, P. K., Schaefer, A. L., Parsek, M. R., Moninger, T. O., Welsh, M. J., and Greenberg,

E. P. (2000) Nature 407, 762-764 45. Costerton, J. W., Cheng, K.-J., Geesey, G. G., Ladd, T. I., Nickel, J. C., Dasgupta, M., and

Marrie, T. J. (1987) Annu. Rev. Microbiol. 41, 435-464 46. Pearson, J. P., Passador, L., Iglewski, B. H., and Greenberg, E. P. (1995) Microbiology 92,

1490-1494 47. Charlton, T. S., Nys, R., Netting, A., Kumar, N., Hentzer, M., Givskov, M., and Kjelleberg, S.

(2000) Environ. Microbiol. 2, 530-541 48. Hooi, D. S. W., Bycroft, B. W., Chhabra, S. R., Williams, P., and Prichard, D. I. (2004)

Infect. Immun. 72, 6463-6470 49. Pahl, H. L. (1999) Oncogene 18, 6853-6866 50. Buscher, M., Rahmsdorf, H. J., Litfin, M., Karin, M., and Herrlich, P. (1988) Oncogene 3,

301-311 51. Brenner, D. A., O'Hara, M., Angel, P., Chojkier, M., and Karin, M. (1989) Nature 337, 661-

663 52. Munoz, E., Zubiaga, A. M., and Huber, B. T. (1992) Eur. J. Immunol. 22, 2101-2106 53. Schreiber, M., Kolbus, A., Piu, F., Szabowski, A., Mohle-Steinlein, U., Tian, J., Karin, M.,

Angel, P., and Wagner, E. F. (1999) Genes Dev. 13, 607-619 54. Shaulian, E., Schreiber, M., Piu, F., Beeche, M., Wagner, E. F., and Karin, M. (2000) Cell

103, 897-907 55. Son, D. S., Arai, K. Y., Roby, K. F., and Terranova, P. F. (2004) Endocrinology 145, 1218-

1226 56. Shaulian, E., and Karin, M. (2002) Nat. Cell Biol. 4, E131-136 57. Levine, A. J. (1997) Cell 88, 323-331 58. Vogelstein, B., Lane, D., and Levine, A. J. (2000) Nature 408, 307-310 59. Subbaramaiah, K., Altorki, N., Chung, W. J., Mestre, J. R., Sampat, A., and Dannenberg, A.

J. (1999) J. Biol. Chem. 274, 10911-10915 60. Han, J. A., Kim, J. I., Ongusaha, P. P., Hwang, D. H., Ballou, L. R., Mahale, A., Aaronson, S.

A., and Lee, S. W. (2002) EMBO J. 21, 5635-5644 61. Johnson, R., Spiegelman, B., Hanahan, D., and Wisdom, R. (1996) Mol. Cell. Biol. 16, 4504-

4511 62. Smith, R. S., Kelly, R., Iglewski, B. H., and Phipps, R. P. (2002) J. Immunol. 169, 2636-2642 63. Chen, L. C., Chen, B. K., Chang, J. M., and Chang, W. C. (2004) Biochim. Biophys. Acta

1683, 38-48 64. Yamaguchi, K., Lantowski, A., Dannenberg, A. J., and Subbaramaiah, K. (2005) J. Biol.

Chem. 280, 32569-32577

by guest on July 17, 2018http://w

ww

.jbc.org/D

ownloaded from

10

65. Rolston, K. V., and Bodey, G. P. (1992) Cancer Invest. 10, 43-59 66. Chen, Y., and Lin-Shiau, S. (2000) J. Biomed. Sci. 7, 122-127 67. Pain, V. M. (1996) Eur. J. Biochem. 236, 747-771 68. Aridor, M., and Balch, W. E. (1999) Nat. Med. 5, 745-751 FOOTNOTES

*This work was supported by grants from the National Institutes of Health (5P01GM037696 to R.J.U., AI055781 to K.D.J.) and the Skaggs Institute for Chemical Biology to K.D.J. The authors are grateful to S. Akira, B. Beutler, R. Flavell, and T. Mak for providing mice used in our experiments. ‡These authors contributed equally to this work.

6The abbreviations used are: PAMP, pathogen-associated molecular pattern; PRR, pattern recognition receptor; TLRs, Toll-like receptors; TIR, Toll/interleukin-1 receptor; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; eIF2α, eukaryotic translation initiation factor 2α; AHL, N-acyl homoserine lactone; C12, N-(3-oxo-dodecanoyl)-S-homoserine lactone; NHBE, normal human bronchial epithelial cells; BMDM, bone-marrow-derived macrophages; MEF, mouse embryonic fibroblasts; DMEM, Dulbecco‘s modified Eagle’s medium; C12R, N-(3-oxododecanoyl)-R-homoserine lactone; BHL, N-butyryl-S-homoserine lactone; C12-LM, 3-oxo-dodecanoic acid (2-oxo-pyrrolidin -3-yl)-amide; C12-TA, (S)-3-(1-hydroxydecylidene)-5-(2-hyroxyethyl) pyrrolidine-2,4-dione; C10, N-(3-oxo decanoyl)-S-homoserine lactone; C14, N-(3-oxotetradecanoyl)-S-homoserine lactone; HPLC, high performance liquid chromatography, DMSO, Dimethylsulfoxide; C12-hyd, 4-hydroxy-2-(3-oxo-dodecanoylamino)-butyric acid; PARP, poly(ADP-ribose) polymerase; ER, endoplasmic reticulum; TNF, tumor necrosis factor α.

FIGURE LEGENDS Fig. 1. N-(3-oxo-acyl) homoserine lactones induce phosporylation of p38 and eIF2α in BMDM. A, Cells were treated with C12 (50 µM) for the indicated period of time and cellular extracts were examined by Western blot analysis for the phosphorylated forms of p38 (P-p38), eIF2α (P-eIF2α) and actin (as a loading control); expression levels of p38 and eIF2-α were also analyzed as an additional loading control. B, Western blot analysis shows the induction of P-p38 and P-eIF2α as well as expression levels of p38, eIF2-α and actin at 30 min after treatment of cells with the indicated doses of C12. C, Stereospecificity of the biochemical changes induced by C12. Cells were treated with C12 or its unnatural stereoisomer, C12R, as indicated, and cellular extracts were examined by Western blot analysis for p38, P-p38, eIF2α, P-eIF2α and actin. D, Cells were treated with 50 µM of BHL, C10, C14 or C12-LM for 30 min, and cellular extracts were examined by Western blot analysis for p38, P-p38, eIF2α, P-eIF2α and actin. E, Chemical structures of the AHLs and analogs used in this study. Fig. 2. The structural integrity of the homoserine lactone ring motif is essential for C12-mediated effects in macrophages. A, Generation of C12-TA and C12-hyd is the result of rearrangement and hydrolysis of C12 (3-oxo-C12-AHL), respectively. B, C12-hyd is rapidly generated after addition of C12 to the cells. Kinetics of C12 hydrolysis and distribution of C12-hyd and C12 between the supernatant and the cellular fractions were estimated by HPLC/mass spectrometry analysis after extraction of cell culture supernatants and pellets by ethyl acetate. The initial amount of C12 added to the cells was 100 nmoles. The data shown is representative of three independent experiments. C, Cells were treated with C12 (50 µM) for 30 sec; then the C12-containing media was removed. The cellular extracts were prepared 15 min later and analyzed by Western blot for the phosphorylated forms of p38 (P-p38) and eIF2α (P-eIF2α) as well as actin (as a loading control). D, Cells were treated with 50 µM of C12-hyd, C12-TA, C12-LM or C12 (as a positive control) for the indicated times, and cellular extracts were examined by Western blot analysis for P-p38, P-eIF2α, PARP and actin (as a loading control). Expression levels of p38 and eIF2-α were also analyzed as additional loading controls.

by guest on July 17, 2018http://w

ww

.jbc.org/D

ownloaded from

11

Fig. 3. C12-induced biochemical changes are qualitatively independent of the cell type. A, Murine alveolar macrophages (AM), murine embryonic fibroblasts (MEFs), human monocytes (THP-1), normal human bronchial epithelial cells (NHBE), and human lung fibroblasts (WI-38) were treated with C12 as indicated. The cellular extracts were analyzed by Western blot for P-p38, P-eIF2α, PARP and actin. Expression levels of p38 and eIF2-α were also analyzed as additional loading controls (data not showm). B, C12 affects the viability of BMDM (macrophages) and MEFs (fibroblasts). Cells (~105 cells per well) were plated in 30 mm dishes, and viable cells were detected after 24 hours of incubation in growth media containing the indicated concentrations of C12. C, Viability of BMDM, MEFs or NHBE cells was examined after 24 hr incubation in media containing the indicated doses of C12. Viable cells remaining after the treatment were determined as a function of mitochondrial activity of living cells according to XTT based toxicology assay kit (Sigma), and are shown as a percentage of viable untreated cells. Fig. 4. C12 activates cells though a mechanism distinct from the canonical PPR-mediated signaling pathways. A, C12-induced cellular responses are independent of MyD88 and TRIF. Western blot analysis shows cleavage of PARP, phosphorylation of p38 and eIF2α, as well as levels of actin expression in the extracts of MyD88-/-, TRIF-/- or control (WT) BMDM treated by C12 as indicated. B, Both TLR2 and TLR4 are not required for C12-mediated PARP cleavage and phosphorylation of p38 and eIF2α. The extracts from TLR2-/-, TLR4-/- and control BMDM were examined by Western blot. C, Neither Nod1 nor Nod2 is required for C12-mediated PARP cleavage or phosphorylation of p38 and eIF2α. Western blot analysis shows cleavage of PARP, phosphorylation of p38 and eIF2α, and levels of actin expression in the extracts of Nod1 and Nod2 double knockout (Nod-/-) and wild type BMDM treated with C12 as indicated. In all panels, expression levels of p38 and eIF2-α were also analyzed as additional loading controls (data not showm) Fig. 5. Comparison of mammalian cells responsiveness to C12, LPS or TNF. A, Comparison of the biochemical changes induced by LPS or C12 in BMDM. At the indicated times, cells were harvested and cellular extracts were analyzed by Western blot with antibodies specific for the phosphorylated forms of p-38 (P-p38), JNK (P-JNK), eIF2α (P-eIF2α), ERK1/2 (P-ERK) and total ERK1/2 and eIF2α as loading controls. Expression levels of actin and p38 were also analyzed as additional loading controls (data not showm). B, Northern blot analysis of TNF, MIP-2, IFN-β, Cox-2, c-jun and GAPDH (as a loading control) mRNAs prepared at indicated times after treatment of BMDM with LPS (100 ng/ml) or C12 (50 µM). C, Northern blot analysis of MCP-1, IP-10, Cox-2, c-jun and GAPDH mRNAs prepared after treatment of MEFs with C12 (50 µM) for 2 hr. D, Northern blot analysis of IL-8, Cox-2, c-jun and GAPDH mRNAs prepared at indicated times after treatment of primary NHBE cells with TNF (50 ng/ml) or C12 (50 µM). E, Northern blot analysis of IL-8, Cox-2, c-jun and GAPDH mRNAs prepared at indicated times after treatment of WI-38 cells with TNF (50 ng/ml) or C12 (50 µM). Two autoradiograph films for IL-8 mRNA are shown (after 24 hr on the top and after 1 hr on the bottom). It should be noted that no additional signals were detected after the prolonged exposition of the films shown in all other panels, including panels B-D. Fig. 6. C12 induces morphological alterations in mitochondria and the endoplasmic reticulum accompanied by activation of apoptotic markers and phosphorylation of eIF2α. A, C12 treatment of BMDM results in rapid activation of pro-apoptotic events and phosphorylation of eIF2α. BMDM cells were treated with C12 (50 µΜ) for the indicated times, and the cellular extracts were analyzed by Western blot with antibodies specific for caspase-9, the cleaved form of caspase-3, PARP, P-eIF2α or actin (as a loading control). B, C12 induces morphological alterations in the mitochondria and endoplasmic reticulum (ER). Electron micrographs of BMDM after 1 h treatment with DMSO (control), C12R or C12. The organelles are marked by arrows. C,. Electron micrographs of macrophages (RAW 264.7), fibroblasts (WI-38) or epithelial cells (NHBE) treated with DMSO (control) or 50 µM C12 for 1 h. The organelles are marked by arrows; mitochondria (m), endoplasmic reticulum (ER).

by guest on July 17, 2018http://w

ww

.jbc.org/D

ownloaded from

Figure 1

E

0 1 5 10 25 50 (µM)

actin

P-p38

P-eIF2α

BA0 5 15 30 60 120 (min)

P-p38

P-eIF2α

actin

p38

eIF2α

p38

eIF2α

0 ¼ ½ 1 0 ¼ ½ 1 0 ¼ ½ 1 (h)

C12, 25 µM

C12R, 25 µM

C12R, 50 µM

P-eIF2α

P-p38

actin

C

eIF2α

p38

C12-

LM

- BHL

C10

C14

P-p38

P-eIF2α

actin

D

eIF2α

p38

12

by guest on July 17, 2018http://w

ww

.jbc.org/D

ownloaded from

Figure 2

Time (min)15 30 45 60

20

15

10

5

0

Cellular fraction (%)

15 30 45 60

100

75

50

25

0

C12C12-hyd

Supernatant (%) B C12 : - +

P-p38

P-eIF2α

actin

C

A

Time (h): 0 ½2 ½2 ½2 ½ 2

C12-

hyd

C12-

TAC1

2-LM

C12

D

actin

P-eIF2α

P-p38

PARPp38

eIF2α

13

by guest on July 17, 2018http://w

ww

.jbc.org/D

ownloaded from

Figure 3

BAM MEFs

0 45 0 45 0 45 0 45 15 120 15 120 15 120 15 120 min

25 µM 50 µM 25 µM 50 µM C12

PARPP-p38

P-eIF2α

actin

0 15 60 0 15 60 0 15 60 5 30 120 5 30 120 5 30 120 min

THP-1 NHBE WI38

PARPP-p38P-eIF2αactin

25 µM

10 µM

1 µM

CTL

50 µM

Macrophages FibroblastsA

0 20 40 60C12 (µM)

80100

60

40

20

BMDMMEFsNHBE

% V

iabl

e ce

lls

C

14

by guest on July 17, 2018http://w

ww

.jbc.org/D

ownloaded from

C12 (µM): 0 5 10 25 50 0 5 10 25 50 0 5 10 25 50

MyD88-/- WT TRIF-/-

actin

PARP

P-eIF2αP-p38

0 ¼½1 2 0¼½1 2 0½¼1 2 hr MyD88-/- WT TRIF-/-A

B

0 ¼ ¾ 3 0 ¼ ¾ 3 0¼ ¾ 3 hr TLR2-/- WT TLR4-/-

actin

PARPP-eIF2α

P-p38

C12 (µM): 0 5 10 25 50 0 5 10 25 50 0 5 10 25 50

TLR2-/- WT TLR4-/-

C12 (µM): 0 5 10 25 50 0 5 10 25 50 0 10 30 60 90 0 10 30 60 90 min

Wild type Nod-/- Wild type Nod-/-

P-eIF2α

PARP

P-p38actin

C

Figure 4

15

by guest on July 17, 2018http://w

ww

.jbc.org/D

ownloaded from

Figure 5

0 1 2 4 1 2 4 hrLPS C12

TNFMIP-2

c-junGAPDH

B

IFN-βCox-2

- C12

LPS

MCP-1IP-10

Cox-2c-jun

GAPDH

C

0 1 2 4 1 2 4 hrC12 TNF

GAPDHc-junCox-2

IL-8

D 0 1 2 4 1 2 4 hrC12 TNF

Cox-2

GAPDHc-jun

IL-8

E

(min):Time

P-p38

P-JNK

P-eIF2α

P-ERK

0 15 60 0 15 605 30 120 5 30 120

LPS C12

ERK

A

eIF2α

16

by guest on July 17, 2018http://w

ww

.jbc.org/D

ownloaded from

A Control C12R(60 min) C12 (60 min)

Mito

chon

dria

ER

0 ¼ ¾ 2 hr

actin

Casp-9

Casp-3PARP

P-eIF2α

B

C12

CTL

Macrophages Fibroblasts Epithelial cells

ER

m

m

m

m

m

m

ER

ER

ER

ER

ER

C

Figure 6

17

by guest on July 17, 2018http://w

ww

.jbc.org/D

ownloaded from

D. Janda and Richard J. UlevitchMee, Kazunori Iwata, Qilin Pan, Colleen Fearns, Ulla G. Knaus, Michael M. Meijler, Kim

Alexander Z. Katz, Malcolm R. Wood, Andrew B. Brogan, Mandy Lehmann, Jenny M. Vladimir V. Kravchenko, Gunnar F. Kaufmann, John C. Mathison, David A. Scott,

receptor pathwaysdistinct from the canonical pathogen-associated molecular pattern recognition

N-(3-OXO-ACYL) homoserine lactones signal cell activation through a mechanism

published online August 7, 2006J. Biol. Chem. 

  10.1074/jbc.M606613200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

by guest on July 17, 2018http://w

ww

.jbc.org/D

ownloaded from