N-(3-Oxo-acyl)homoserine Lactones Signal Cell Activation through a Mechanism distinct from the Canonical Pathogen-associated Molecular Pattern Recognition Receptor Pathways*

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 have shown 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 induced distension of mitochondria and the endoplasmic reticulum as well as c-jun gene transcription. These effects occurred in a wide variety of cell types including alveolar macrophages and bronchial epithelial cells, requiring 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). 4 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-B 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 2␣ (eIF2␣) (3,12).
PAMPs are exclusive to bacterial or viral pathogens and do not exist in mammalian organisms. Moreover, they are essen-tial to the pathogenesis of the microbe, and structurally 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-acylhomoserine lactones (AHLs), that are produced by a large number of Gram-negative bacteria (14). AHLs are a structurally highly conserved group of bacteria-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)(16)(17)(18). A prototypic member of the AHL family, N-(3oxo-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-B (15,25). These observations suggest that C12 may function as a PAMP. However, no studies have directly addressed this possibility.
We 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 knock-out 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 eIF2␣ (P-eIF2␣) are two major biochemical markers indicative of the C12 responsiveness of diverse cell types. Evidence is provided here that the N-(3-oxo-acyl)homoserine lactone ring motif of C12 is required for the induction of phosphorylated p38 and eIF2␣ and that 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 of canonical PRR-mediated mechanisms.
Northern Blot-Total RNA was isolated by using TRizol reagent (Invitrogen). Total RNA (10 g/lane) was analyzed by Northern blot as described previously (29). Blots were hybridized with specific antisense oligonucleotides end-labeled by T4 polynucleotide kinase using [␥-32 P]ATP.
Electron Microscopy-Cells were plated in 35-mm dishes as described above. After treatment of cells with a compound or 0.5% Me 2 SO as a control, samples for electron microscopy analysis were prepared as described (30). Images were doc-umented on a Philips CM100 electron microscope (FEI, Hillsborough, OR).
Data Presentation-Data shown in each figure are representative of three or more experiments

Phosphorylated p38 and eIF2␣ Are Two Biochemical Markers
Indicative of Homoserine Lactone-mediated Signaling-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 eIF2␣, a biochemical marker indicative of TRIF-dependent signaling. Therefore, we reasoned that the 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 eIF2␣ phosphorylation ( Fig. 1, A and B, P-p38 and P-eIF2␣).
These observed biochemical effects were highly specific for the naturally occurring S-stereoisomer of C12, because no biochemical effects were observed after the addition of the unnatural R-stereoisomer of C12 (Fig. 1C, C12R). Interestingly, induction of P-p38 and P-eIF2␣ could also be detected after treatment with other members of the 3-oxo-AHL family such as C10 and C14 (Fig. 1, D and E). However, BHL, another AHL 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. 1, D and 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-eIF2␣.
In aqueous conditions at neutral pH ϳ7.4, 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 ϳ7.4, both C12-hyd and C12-TA could be generated under our experimental conditions after the addition of C12. Indeed, HPLC and mass spectrometry analysis revealed that C12-hyd is rapidly generated after the 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 10 times more C12-hyd than C12, whereas C12 was predominantly distributed in the supernatant (Fig. 2B). Notably, a 30-s treatment of macrophages with C12 was sufficient to induce P-p38 as well as P-eIF2␣, 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-eIF2␣, because C12-hyd, C12-TA, and C12-LM all lost this ability (Fig. 2D). Because previous studies had shown that prolonged incubation of transformed human cells with C12 induces the cleavage of PARP (23), a biochemical marker indicative of apoptosis (31), investigations were warranted to determine whether the integ-rity 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 C12-mediated induction of apoptotic pathways as well as phosphorylation of p38 and eIF2␣.
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 eIF2␣ 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-eIF2␣ are characteristic markers of the general cellular responses to C12 as observed in various cell types, including primary alveolar macrophages and

. N-(3-Oxo-acyl)homoserine lactones induce phosphorylation of p38 and eIF2␣ in BMDM.
A, cells were treated with C12 (50 M) for the indicated period of time, and cellular extracts were examined by Western blot analysis for the phosphorylated forms of p38 (P-p38), eIF2␣ (P-eIF2␣), and actin (as a loading control). Expression levels of p38 and eIF2-␣ were also analyzed as an additional loading control. B, Western blot analysis shows the induction of P-p38 and P-eIF2␣ as well as expression levels of p38, eIF2-␣, 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, eIF2␣, P-eIF2␣, and actin. D, cells were treated with 50 M of BHL, C10, C14, or C12-LM for 30 min, and cellular extracts were examined by Western blot analysis for p38, P-p38, eIF2␣, P-eIF2␣, and actin. E, chemical structures of the AHLs and analogs used in this study.
normal lung epithelial cells (Fig. 3A). Importantly, C12-induced phosphorylation of p38 and eIF2␣ 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. 3, B 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/TI-RAP, 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; and 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 eIF2␣ 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-eIF2␣ was identical in the BMDM from the adaptor protein-deficient and control mice (Fig. 4A). Thus, neither MyD88 nor TRIF is 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 because both receptors have the ability to recognize molecules containing long-chain fatty acid moieties. Comparison of the C12-mediated biochemical changes in TLR2 Ϫ/Ϫ , TRL4 Ϫ/Ϫ , or control BMDM failed to reveal any differences in the response to C12 in this panel of BMDM (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, eIF2␣, and PARP cleavage was identical in the BMDM from Nod1/2deficient 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 C12 affects signaling pathways in a manner distinct from TLR-specific PAMPs. LPS is a major TLR-specific PAMP present in P. aeruginosa, and 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 signaling events examined, 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). Although 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 were further investigated by the examination of mRNA levels of tumor necrosis factor (TNF), macrophage inflammatory protein 2 (MIP-2), interferon ␤ (IFN-␤), 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-␤, and Cox-2 mRNAs were FIGURE 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 the 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 nmol. The data shown are representative of three independent experiments. C, cells were treated with C12 (50 M) for 30 s; then the C12containing medium was removed. The cellular extracts were prepared 15 min later and analyzed by Western blot for P-p38 and P-eIF2␣ as well as actin (as a loading control). D, cells were treated with 50 M 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-eIF2␣, PARP, and actin (as a loading control). Expression levels of p38 and eIF2-␣ were also analyzed as additional loading controls.
increased after LPS treatment, whereas C12 failed to induce these transcripts (Fig. 5B). Additionally, although 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), the 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 the 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. 5, B-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 the C12-induced Apoptotic Pathway-Mitochondria and the 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 proapoptotic 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 eIF2␣ (41). Observations that treatment of myeloid cells with C12 resulted in the induction of these biochemical markers (Fig. 6A) prompted the investigation of 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 were treated with C12 as indicated. The cellular extracts were analyzed by Western blot for P-p38, P-eIF2␣, PARP, and actin. Expression levels of p38 and eIF2-␣ were also analyzed as additional loading controls (data not shown). B, C12 affects the viability of BMDM (macrophages) and MEFs (fibroblasts). Cells (ϳ10 5 cells/well) were plated in 30-mm dishes, and viable cells were detected after 24 h of incubation in growth medium containing the indicated concentrations of C12. CTL, control. C, viability of BMDM, MEF, or NHBE cells was examined after 24-h 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. 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 proapoptotic 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 the lungs of patients suffering from cystic fibrosis and other chronic obstructive pulmonary diseases (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 M) or as a part of biofilms (100 -600 M) (46,47). Our observations revealed that treatment of macrophages with C12 at a concentration of 25 M significantly reduces the viability of mammalian cells, whereas concentrations of 10 M 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-␤, 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 LPSinduced production of TNF in macrophages and human monocytes (22,48) are in accordance with this suggestion. However, the nature of biochemical mechanisms responsible for C12mediated effects on mammalian cells has been unclear.
We have demonstrated that N-(3-oxo-acyl)homoserine lactones induce the phosphorylation of MAPK p38 and eIF2␣ 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 to 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 PRRdependent signaling in response to canonical PAMPs, we have clearly demonstrated that MyD88, TRIF, TLR2, TLR4, Nod1, and Nod2, key molecules of PRR pathways (3)(4)(5)(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 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 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, although 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)(54)(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 the 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 the 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-induci- ble expression of the 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 eIF2␣ is consistent with previous observations that P-eIF2␣ 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 proinflammatory cytokines including TNF (66). The efficiency of the translation initiation depends on the dynamic exchange between the GTP-and GDP-bound states of eIF2␣. Specific phosphorylation of eIF2␣ 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 eIF2␣ 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 relevant also in that cystic fibrosis is associated with ER dysfunction (68). Hence, our findings provide evidence that the product of the Gram-negative bacterium P. aeruginosa, C12, affects host cells through eIF2␣-dependent mechanisms.
In total, the data presented here grant 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.