Bacterial RNA induces myocyte cellular dysfunction through the activation of PKR
Original Article
Bacterial RNA induces myocyte cellular dysfunction through the activation of PKR
Farag Bleiblo1, Paul Michael1, Danielle Brabant1, Chilakamarti V. Ramana2, TC Tai1, Mazen Saleh1, Joseph E. Parrillo3, Anand Kumar4, Aseem Kumar1
1Department of Chemistry and Biochemistry and the Biomolecular Sciences Programme, Laurentian University, Sudbury, ON, Canada, P3E 2C6; 2Department of Medicine, Yale University, New Haven, CT, USA, 06516; 3Division of Cardiovascular Disease and Critical Care Medicine, Cooper University Hospital, Robert Wood Johnson Medical School, Camden, New Jersey, USA, 08103; 4Section of Critical Care Medicine, University of Manitoba, Winnipeg, MB, Canada, R3A 1R9
Corresponding to: Aseem Kumar, PhD. Department of Chemistry and Biochemistry, Biomolecular Sciences Programme, Laurentian University, 935 Ramsey Lake Rd. Sudbury, ON, Canada, P3E 2C6. Tel: 705-675-1151 ext.2103; Fax: 705-675-4844. Email: akumar@laurentian.ca.
Abstract
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Abstract
Introduction
Materials and methods
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Severe sepsis and the ensuing septic shock are serious life threatening conditions. These diseases are triggered by the host’s over exuberant systemic response to the infecting pathogen. Several surveillance mechanisms have evolved to discriminate self from foreign RNA and accordingly trigger effective cellular responses to target the pathogenic threats. The RNA-dependent protein kinase (PKR) is a key component of the cytoplasmic RNA sensors involved in the recognition of viral double-stranded RNA (dsRNA). Here, we identify bacterial RNA as a distinct pathogenic pattern recognized by PKR. Our results indicate that natural RNA derived from bacteria directly binds to and activates PKR. We further show that bacterial RNA induces human cardiac myocyte apoptosis and identify the requirement for PKR in mediating this response. In addition to bacterial immunity, the results presented here may also have implications in cardiac pathophysiology.
Key words
Pattern recognition receptor; bacterial RNA; PKR; apoptosis
J Thorac Dis 2012;4(2):114-125. DOI: 10.3978/j.issn.2072-1439.2012.01.07
Introduction
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Abstract
Introduction
Materials and methods
Results
Discussion
Acknowledgements
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Sepsis and septic shock represent the systemic immunologic and pathophysiologic response to overwhelming infection. Approximately 800,000 cases of sepsis are admitted every year to hospitals in the North America and despite aggressive antibiotics and supportive care, over 150,000 patients per year succumb to this disorder (1). Sepsis and sepsis-associated multiple organ failure (MOF) are the leading cause of death in intensive care units (ICUs) of the developed world (2,3). The typical human cardiovascular response to septic shock is characterized by hypotension, decreased systemic vascular resistance and elevated cardiac index. In addition, myocardial depression manifested by reversible biventricular dilation and reduction of ejection fraction has been shown to be common in spontaneous human septic shock (4,5). Septic deaths are typically due to early refractory cardiovascular failure (hypotension or shock) or later multiple organ failure. Bacteria induce mammalian cellular dysfunction by releasing bacterial exotoxins such as Toxic Shock Syndrome Toxin-1 and/or bacterial endotoxin (lipopolysaccharide) (3,6). An emerging area of research indicates that bacterial RNA acts as an antigen. Bacterial RNA presents as an antigen due to nucleic acid sequence, differential secondary and tertiary structure and lack of methylation (7-10).
Discriminating self from nonself is crucial to innate immunity that employs pattern recognition receptors (PRRs) to sense molecular signatures associated with pathogenic moieties including nucleic acids (11). Human PKR is a serine/threonine kinase induced by interferon (IFNs) and characterized by a conserved kinase domain in the C-terminal region of the protein and two dsRNA binding domains (dsRBDs) localized to the N-terminal regulatory region (12-14). Binding of viral dsRNA to the dsRBDs causes PKR to undergo autophosphorylation at multiple kinase residues and subsequent activation (15-17). The alpha subunit of eukaryotic initiation factor 2 (eIF2α) is the best characterized cellular substrate that is phosphorylated by PKR. This post-translational modification of eIF2α leads to the inhibition of cellular protein synthesis (18,19). PKR has also been shown to be implicated in regulating physiological and pathological processes such as cellular growth and apoptosis, stress, transcription, and malignancy (20-25).
Recent studies have indicated that other PRRs can recognize distinct subspecies of RNAs released from pathogenic organisms. Of considerable interest are the recent findings that natural bacterial RNA and synthetic oligoribonucleotides can induce antigen specific immunity through the secretion of proinflammatory cytokines and IFNs in a toll like receptor (TLRs)-dependent manner (10,26-29). In addition, the NOD-like receptors (NLRs) have also been identified as an additional quality control mechanism that detects bacterial RNA in a NALP3-dependent manner. However, the NALP3 inflammasome has also been shown to mediate the recognition of different patterns of RNA subtypes such as viral RNA and poly I: C (synthetic dsRNA analogue) (30,31). The retinoic acid-inducible gene I (RIG-I) was first classified as a sensor for dsRNA, however recent reports indicated that singlestranded RNA (ssRNA) containing a 5’-triphosphate end can also function as a ligand for RIG-I (32,33). These observations emphasized the unifying theme in innate immunity whereby the host could use the same immunreceptor to detect various species of foreign RNA. Thus, it is possible that PKR employs a similar strategy to discriminate self from multiple nonself RNA species. For example, it is widely accepted that PKR recognizes viral dsRNA as nonself because it is normally absent in mammalian cells. However, dsRNA and ssRNA ligands with various secondary structures and modifications can also regulate PKR activity (34-36). Although bacterial RNA shares certain structural elements and signaling features with viral RNA, activation of PKR by natural RNA derived from bacteria has not been previously reported.
We reasoned that bacterial RNA could present a distinct pattern to be recognized by PKR as nonself. To address this hypothesis, we examined whether RNAs derived from bacterial or mammalian sources could modulate PKR activity. We determined that PKR recognizes bacterial RNA and we show that bacterial RNA induces apoptosis in a PKR-dependent manner.
Materials and methods
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Introduction
Materials and methods
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Preparation of bacterial total RNA
Pathogenic isolates of E. coli (O18:K1:H7; ATCC #700973) and S. aureus [capsular serotype 8, non-toxic shock toxin (TSST- 1) producer] were used for this study. A modified hot phenol combined with enzymatic lysis was carried out to isolate bacterial total RNA. Exponentially growing bacteria were harvested in ice cold 5% phenol/ethanol solution and then resuspended in a fresh lysis buffer of TE, 2 mg/mL lysozyme and 5 μg/mL lysostaphin (Sigma Alderish, St.Loius, USA). Samples were brought to 2% SDS and a volume of 850 μl water-saturated phenol was added to each tube and incubated for 5 minutes at 64ºC. Following centrifugation at 13000 X g, the aqueous phase was transferred to a fresh tube and equal amounts of chloroform were added and centrifuged for 10 minutes at 4 ºC. The RNA was precipitated by 0.1 vol. 3M sodium acetate and 2.5 vol. ethanol. The RNA pellets were washed with 70% ethanol and resuspended in DEPCtreated water. The integrity and purity of RNA species were determined by electrophoresis on agarose gels and A260/A280 ratios. Extracted crude RNA was treated with RNase-free DNase I (Roche, Indianapolis, USA) to remove contaminant genomic DNA and further cleaned up by the Qiagen method (Qiagen, Valencia, USA) as per the manufacturer’s instructions.
Preparation of mammalian total RNA
Total cellular RNA from human adult cardiac myocytes was prepared with extraction using a silica column-based method, Qiagen RNeasy, according to the manufacturer’s instructions (Qiagen, Valencia, USA). 2×106 human adult cardiac myocytes cells (ScienCell Research Laboratories) were washed three times with ice-cold PBS and then scraped with 1 mL buffer RLT. Prior to the first RPE wash buffer, DNase l incubation mix was added onto RNeasy silica-gel membrane and allowed to sit for 20 minutes to further eliminate any traces of genomic DNA contamination. RNA was quantified by UV spectrometry and electrophorsed on a 1% agarose gel to verify purity and integrity prior to use.
RNA digestion
Aliquots of RNA samples were incubated in the presence of a heterogeneous mixture of ribonucleases [(1 U per 2μL of RNA at 1μg/μL for 1 hr) (Roche, Indianapolis, IN)]. RNA samples were analyzed by denaturing agarose or polyacrylamaide gel electrophoresis for quality assurance. Aliquots of digested total bacterial RNA were added to the media to yield a final concentration of 100 μg/mL or 200 μg/mL.
Cardiac myocytes culture and stimulation
Human adult cardiac myocytes were grown in cardiac myocyte medium supplemented with 10 % fetal bovine serum, growth supplement and 1% of penicillin/streptomycin (ScienCell Research Laboratories). Synthetic dsRNA (polyI: C), and 2-aminopurine (2-AP) were purchased from Sigma Chemical Co. 2×106 cardiac cells were treated with or without a final concentration of 10 mM 2-AP for 1 h and then washed 3 times with Hanks solution (Gibco Laboratories) before stimulation. Cardiac myocytes were stimulated with 100 μg/mL of bacterial total RNA, Poly I:C, digested total RNA, or cardiac myocyte RNA for 24h, or 48h or left untreated as a control.
Immunoprecipitation and Immunoblotting
Western blot was performed as previously described (37). Briefly, the cultured cells were washed with cold PBS and resuspended in RIPA lysis buffer (PBS, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS) plus a protease inhibitor mixture. Cellular lysates were resolved by 10% or 12% SDS-PAGE gels and subjected to immunoblot analysis using antibodies specific for phospho-Ser51 on eIF2α (Cell Signaling), antibodies specific for phospho-Thr451 on PKR (Invitrogen). Caspase-3 antibodies were from Santa Cruz, caspase-8 and caspase-9 antibodies were from Cell Signaling. Membranes were stripped and probed for total eIF2α protein or total human PKR protein (Cell Signaling). For Immunoprecipitation, 500 μg of the cellular lysates were first precleared with the addition of 50 μL of 50% protein G-Sepharose beads (Santa Cruz) for 1 h at 4 °C and then incubated with 5 μL of anti-PKR polyclonal antibody overnight with rotation. The beads were washed twice with RIPA, collected, and immunoprecipitates were subjected to either electrophoresis or in vitro PKR binding assay.
In Vitro PKR Binding Assays
The ability of tested RNAs to bind to and activate PKR was carried out as previously described (21,22). Briefly, immunoprecipitated PKR was incubated in kinase buffer containing 50 mM Tris–HCl, pH 7.5, 2 mM MgCl2, 2 mM MnCl2, 50 mM KCl, 0.1 mM EDTA, 1 μg/mL Aprotinin, 1mM DTT, 20% glycerol, 10 μCi [γ-32P] and 1 μg/mL RNA for 20 min at 30 °C. The beads were washed 6X with the salt buffer and the reaction was terminated by the addition of SDS sample buffer, heated at 95 °C for 5 min, and loaded on a 10% SDS-PAGE gel.
Analysis of Genomic DNA Fragmentation
Isolation of genomic DNA fragments was performed as previously described (37). Briefly, treated cells were collected and resuspended in lysis buffer (1% Nonidet P-40 in 20 mM EDTA, 50 mM Tris-HCl, pH 7.5). The supernatants contained fragmented DNA were brought to 1% SDS and treated with RNase A (0.5 mg/mL) and proteinase K (1 mg/mL). The DNA was precipitated and genomic DNA was fractionated by 1% agarose electrophoresis.
Results
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Bacterial RNA can form pathogen associated molecular patterns (PAMPs) and serve as a danger signal to cells (10,26). To assess the ability of bacterial RNA to modulate PKR activation, cardiac cells were incubated in the presence of 100 μg/mL of RNA derived from bacterial or mammalian origins for 24 h. Total RNA from E. coli and S. aureus were shown to be potent activators of PKR (Figure 1A, lane 5 and 6). Self RNA derived from cardiac cells lacks the ability to activate PKR (Figure 1A, lane 2). Excluding the possibility that the observed effects on PKR activation were induced by potential contaminants in the RNA preparations, bacterial RNA was subjected to digestion with RNase. Digested bacterial RNA did not activate PKR suggesting that intact bacterial RNA is required for PKR phosphorylation (Figure 1A, lanes 3 and 4). As a positive control, poly I:C treatment of cardiac myocytes activated PKR to an extent comparable to that generated by bacterial RNA (Figure 1A, lane 7 vs. lanes 5 and 6).
To investigate the biological significance of PKR activation by bacterial RNA, we tested whether bacterial RNA could induce the phosphorylation of eIF2α. Immunolotting experiments revealed enhanced eIF2α on Ser 51 phosphorylation induced by bacterial RNA (Figure 1B, lanes 5 and 6). RNase treatment of bacterial RNA samples resulted in a reduction of the eIF2α phosphorylation (Figure 1B, lanes 3 and 4). In response to bacterial RNA, the levels of eIF2α phosphorylation were correlated to the PKR activation levels (Figure 1A and Figure 1B, lanes 5 and 6).
Figure 1. Bacterial RNAs activate PKR and induces eIF2α phosphorylation. Cardiac myocytes were stimulated for 24h with 100μg/mL RNAs from various origins (lanes 2-7) or unstimulated (lane1). Western blots were probed with antibodies to (A) pPKR (Thr446), (B) phosphor-eIF2α (Ser51) then stripped, and reprobed with antibodies to total PKR (A, bottom panel) and total eIF2α (B, bottom panel). NT: nontreated cells, CMRNA: cardiac myocytes RNA, ERNA: total RNA isolated from E. coli, SRNA: total RNA isolated from S. aureus, PolyIC: synthetic RNA mimics viral dsRNA. RNA was digested with RNase (+) or left undigested (-) as indicated.
To test whether the activation of PKR by bacterial RNA is dose-dependent, we challenged the cardiac myocytes with bacterial RNA at 100 μg/ml or 200 μg/mL. The data from these experiments indicated that the extent of PKR activation was dependent on the amount of RNA that was added (Figure 2A, lanes 2 and 3 vs. lanes 4 and 5). However, the phosphorylation levels of eIF2α indicated that cardiac cells treated with 200 μg/ mL bacterial RNA were not different from those treated with 100 μg/mL (Figure 2B, lanes 2 and 3 vs. lanes 4 and 5).
Figure 2. Activation of PKR is RNA dose- dependent. Cardiac myocytes were stimulated for 24h with either 100μg/mL RNAs (lanes 2 and 3) or 200μg/mL RNAs (lanes 4 and 5) or left unstimulated (lane1). Cell lysates were separated by 10% SDS-PAGE, and Western blots were probed with antibodies to (A, top panel) pPKR (Thr446) or stripped, and reprobed with antibodies to total PKR (A, bottom panel) phospho-eIF2α (Ser51) (B, top panel) stripped, and reprobed with antibodies to total eIF2α (B, bottom panel). Positions are indicated. NT: nontreated cells, SRNA: total RNA isolated from S. aureus, PolyIC as a positive control for PKR activation.
To further determine the role of PKR signaling in bacterial RNA recognition, we used 2-AP which is widely used as a selective inhibitor for PKR (38-40). Cardiac cells left untreated or treated with 10 mM 2-AP for 1 h and then stimulated without or with bacterial RNA (Figure 3A and 3B). Bacterial RNAinduced phosphorylation of PKR was significantly reduced when PKR was inhibited with 2-AP (Figure 3Aa, lanes 2 and 3 vs. lanes 6 and 7). These results suggested that recognition of bacterial RNA is mediated by PKR. Inhibition of PKR activity by 2-AP also resulted in a significant reduction of eIF2α phosphorylation by bacterial RNA (Figure 3Ba, lanes 2 and 3 vs. 6 and 7). This observation further suggested that PKR is the kinase responsible for induction of eIF2α phosphorylation.
Figure 3. Inhibition of PKR suppresses its RNA-mediated activation and reduces eIF2α phosphorylation. Cardiac cells were pretreated with 10 mM 2-aminopurine (2-AP) for 1h (+) or left untreated (-) and then cells were stimulated with or without RNAs for 24 h. Western blots were probed with antibodies to (Aa) pPKR (Thr446), or stripped, and reprobed with antibodies to total PKR (Ab) phospho-eIF2α (Ser51) (Ba) stripped, and reprobed with antibodies to total eIF2α (Bb). Positions are indicated. NT: nonstimulated cells, ERNA: total RNA isolated from E. coli, SRNA: total RNA isolated from S. aureus, PolyIC: synthetic RNA mimics viral dsRNA used as a positive control for PKR activation.
Although the above results demonstrated that bacterial RNA is a potential activator for PKR, the mechanism of this activation remains undetermined. Therefore, we immunoprecipitated PKR and subsequently performed in vitro PKR binding assay to examine the ability of various RNAs to bind to the purified PKR. The results from this assay revealed that the purified PKR was efficiently activated by total bacterial RNA (Figure 4, lanes 3 and 4). The efficiency of PKR activation by bacterial RNA was comparable to that of poly I: C (Figure 4, lane 5). Cardiac RNA failed to activate PKR (Figure 4, lane 2). These results suggested that bacterial RNA contains structural features that directly bind to PKR. Therefore, PKR is a direct receptor responsible for the recognition of bacterial RNA.
Figure 4. Bacterial RNAs directly binds to and activates PKR. PKR was immunoprecipitated from cardiac myocytes and subjected to in vitro binding assay. Purified PKR was incubated without (lane 1) or 1μg/mL of total RNA from cardiac (lane 2), E. coli (lane 3), S. aureus (lane 4) or PolyI:C (lane 5). After 20 minutes, the reaction was terminated and the samples were subjected to 10% SDS-PAGE. The gel was dried, exposed and analyzed.
Viral and bacterial RNAs share many immunostimulatory potentials such as production of inflammatory cytokines which is considered as a hallmark of the cellular response to nonself RNA. These inflammatory mediators may exert pathological effects and harm the host. It is well established that innate immunity-mediated detection of viral dsRNA can trigger an apoptotic response. However, there have been no reports which describe the role of bacterial RNA as an inducer of apoptosis. Therefore, we tested whether bacterial RNA could provoke an apoptotic response. We observed that cardiac myocytes challenged with total bacterial RNAs for 48 h exhibited a number of morphological changes which are characteristic of apoptosis which were cell shrinkage, membrane blebbing, and apoptotic bodies. (Figure 5, G and H). Digested bacterial RNA failed to trigger these apoptotic responses (Figure 5, B and C). This confirms that intact bacterial RNA is the active inducer of cardiac cell death. We examined next the involvement of activated PKR signaling in bacterial RNA-induced cardiac apoptosis. Resistance to apoptosis was observed when cardiac PKR was inhibited with 2-AP (Figure 5, E and F vs. control D). To assess cardiac cell viability, we performed similar experiments using trypan blue exclusion assay. Bacterial RNA induced an average of 17% apoptosis in the cardiac myocytes (Figure 6). In contrast, only 2% and 3% cardiac apoptosis was detected when the cells were treated with digested bacterial RNA and 2-AP respectively.
Figure 5. Bacterial RNAs induces morphological features of cardiac apoptosis. Cardiac myocytes were treated with 200μg/mL for 48h and evaluated by phase contrast microscopy. Cells were left untreated (A), or incubated with digested RNA from E. coli or S. aureus RNA (images B and C) respectively. Cardiac cells were pretreated with 10 mM 2AP (images D-F) and then stimulated without (image D) or with E. coli RNA (E) or S. aureus RNA (F). Cardiac myocytes were challenged with E. coli RNA or S. aureus RNA (G, H) respectively. Following treatment, micrographs were taken at (100 X) magnification.
Figure 6. Percentage of cardiac myocytes apoptosis. Following treatment with (+) or without (-) digested bacterial RNAs, 10 mM 2-AP, or intact bacterial RNAs as indicated, Trypan Blue was used for viable cell count.
Because DNA fragmentation is considered as a hallmark of apoptosis, we next repeated these experiments and evaluated the potency of bacterial RNA to trigger genomic DNA fragmentation. Consistent with the above assays, we found that stimulation of the cells with E. coli and S. aureus RNA induced cardiac DNA fragmentation (Figure 7, lanes 8 and 9). However, genomic DNA laddering was not apparent in cardiac cells treated with either digested bacterial RNA (Figure 7, lanes 3 and 4) or 2-AP (Figure 7, lanes 6 and 7). Taken together, the above observations revealed that bacterial RNA is an inducer of cardiac myocyte apoptosis and implicates PKR in mediating the apoptotic process.
Figure 7. Bacterial RNAs induce genomic DNA laddering in cardiac myocytes and inhibition of PKR protects from DNA fragmentation. Cardiac myocytes post treated with 200 μg/mL bacterial RNAs for 48 hr or pretreated first with 2-AP for 1hr and then genomic DNA was fractionated by 1% agarose gel electrophoresis. Lanes 1 and 10 DNA marker (M); lane 2 (NT) untreated cells; lanes 3 and 4 cells treated with digested E. coli RNA (ERNA), S. aureus RNA (SRNA) respectively; lanes 5, 6, 7 cells were pretreated with 10 mM 2AP and then stimulated without (lane 5) or with E. coli RNA (ERNA) or S. aureus RNA (SRNA) (lanes 6 and 7 respectively); lanes 8 and 9, cells challenged with E. coli RNA (ERNA) or S. aureus RNA (SRNA).
To understand how bacterial RNA triggers cardiac apoptosis, we tested the cleavage of caspases as key regulators of apoptosis. While bacterial RNA treatment induced the cleavage of caspase 8, caspase 9, and caspase 3 (Figure 8 A-C, lanes 7 and 8), digested RNA suppressed the production of active caspase fragments (Figure 8, lanes 2 and 3). PKR inhibition by 2-AP also prevented production of the caspases fragments (Figure 8, lanes 5 and 6). These results suggested the requirement of PKR for bacterial RNA-induced caspase activation.
Figure 8. Bacterial RNAs induces cleavage of caspase-3, -8 and -9 mediated by PKR. Extracts from cardiac myocytes challenged with 200μg/mL bacterial RNA for 48 h were analyzed by Western blotting using antibodies to caspase-3 (A), caspase-8 (B), or caspase-9 (C). Cardiac Cells untreated (lane 1), or incubated with digested E. coli RNA or S. aureus RNA (lanes 2 and 3) respectively; or pretreated with 10 mM 2AP (lanes 4-6) and stimulated without (lane 4) or with E. coli RNA or S. aureus RNA (lanes 5 and 6) respectively; or challenged with E. coli RNA or S. aureus RNA (lanes 7,8).
Discussion
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We show that bacterial but not mammalian RNA is a novel activator of PKR. This observation suggested that cardiac myocytes are fully able to distinguish between self and foreign RNA. Although the primary function of cardiac myocytes is contraction, accumulating evidence suggested that cardiac myocytes respond to pathogenic insults with a complex inflammatory response. Cardiac myocytes express PRRs and can detect a diverse range of pathogenic patterns including nucleic acids (41-46). Several lines of investigation support the idea that immuoreceptors sense bacterial RNA while ignoring the endogenous RNA. For example, bacterial but not mammalian RNA exerts immunostimulatory potential in TLR-dependent and NALP3-dependent fashion (31,47). These observations raised the question of how the immune sensors discriminate self from foreign RNA. Recent reports have indicated that specific nucleoside modifications and structural elements are differentially represented in either microbial or mammalian RNA and therefore provide a molecular mechanism to discriminate pathogenic from self RNA (9,10,48,49). Importantly, the type and extension of these modifications depends on the evolutionary level of the organism from which the RNA subspecies was derived. In contrast to most cellular species of RNA that are heavily modified, unmodified motifs or low degree of nucleoside modifications are abundant in bacterial and viral RNAs. For example, cellular primary transcripts undergo various modifications to acquire a 7-methylguanosine (7mG) cap structure at its 5’-end while bacterial and viral mRNAs possess a 5’-triphosphate at their terminal end (50-52). It has been recently reported that in vitro transcribed RNA with limited secondary structures activate PKR in a 5′-triphosphate–dependent manner (53). The investigators suggested that PKR uses this feature, which is primarily present in foreign transcripts such as viral and bacterial mRNA, as a part of the RNA-quality mechanism to distinguish between endogenous and pathogenic RNAs. Furthermore, internal nucleosides modifications including nucleobase, sugar, and phosphodiester which mimic their natural modifications modulates PKR activation in RNA structurespecific context (54). In addition to capping, posttranscriptional modifications such as pseudouridine and methylated nucleosides exist unequally in either mammalian or microbial RNA. Unmethylated CpG motifs are found more frequently in bacterial RNA and certain DNA viruses than that of eukaryotic rRNA and mRNA. It has been demonstrated that RNA with unmethylated CpG motifs is immunostimulatory. Methylation of the 5' position of the C in this RNA, mimicks the viral CpG DNA and abrogates its immune activity (55). Internal regions of select RNA viruses such as adenoviruses, influenza, and herpes simplex contain the modification N6-methyladenosine (m6A) to a greater extent than the cellular and bacterial RNA. This modification, which destabilizes RNA duplexes, suppresses the immune activation (10). Pseudouridin is a more frequent modification in human RNA as compared to RNAs of bacterial and viral origins. It has been recently demonstrated that the incorporation of uridine in mRNA activates PKR and enhances translation. In contrast, pseudouridin reduced PKR activation (10,56). Importantly, we show that the potential of bacterial RNA to activate PKR was sensitive to RNase that removes the base-paired secondary structures indicating the dependence Percent Cell Death of Adult Cardiac Myoctyes Treated With Bacterial Percentage of PKR on this feature as a key for the molecular recognition and signaling. Several RNA ligands with various modifications activate PKR to a different extent; however, secondary structure elements are the common theme for these ligands to regulate PKR signaling (34-36,5757). We next determined that bacterial RNA binds to and activates PKR which suggests that bacterial RNA has structural features involved in the direct activation of PKR and thus PKR is a molecular sensor for bacterial RNA. Although dsRNA was first proposed as a ligand and activator for PKR, a diverse range of biological RNA patterns with various structural elements such as dsRNA, bulges, stem- loops, and internal loops can also regulate PKR activity in a sequence-independent manner (57). These secondary structures are also a common characteristic to bacterial RNA. Therefore, it seems likely that these modifications affect the RNA structure which impact on PKR activation.
We also provided evidence that bacterial RNA itself can elicit a lethal response and identified the requirements of PKR for the induction of cardiac apoptosis. Although much progress has been made to understand the PKR signaling in apoptosis induced by different stimuli, the role of bacterial RNA in this response remains unexplored (58-64). Herein, we linked the caspases-3, -8 and -9 activation to bacterial RNA-induced apoptosis and identified PKR-dependent signaling for activation of these caspases and induction of apoptosis. Our findings are in agreement with previous reports which established the PKRdependent activation of caspases 1, -3, -7, -8, and -9 and PKR is essential for different forms of stress-induced apoptosis (62-68). Additionally, it has been shown that PKR is essential for macrophage apoptosis which is induced by bacterial pathogens including Bacillus, Yersinia and Salmonella (69). Collectively, we identified bacterial RNA as a novel inducer of apoptosis in PKRdependent manner.
Bacterial RNA and modif ied ssRNA are potent immunostimulatory agents characterized by the induction of proinflammatory cytokines and interferons (10,26,28,29). Although triggering the immune response is generally considered as protective, the interplay between the pathways that are modulated may harm the host. For example, cytokines can modulate cardiac function and induce apoptosis and thus serve as prognostic markers for morbidity and mortality in patients with microbial infections, septic shock, and cardiac failure (37,70-72). We previously demonstrated that bacterial and viral nucleic acids induce cardiac dysfunction (73). Loss of cardiac myocytes by apoptosis is a serious and frequent complication from microbial infections, septic shock and myocardial infarction (37,71,74,75). Several bacterial pathogens are epidemiologically linked to human heart diseases such as endocarditis and myocarditis. Although it has been established that bacterial infections of cardiac myocytes induces apoptosis and caspases activation, the role that PKR plays in regulating these responses has not been previously described (76,77). We propose that bacterial RNA may contribute to myocardial dysfunction during bacterial sepsis.
Although PKR is considered as a principal player in antiviral protection, its contribution to cellular defenses against bacterial infections has not been previously described. Our studies suggested that activation of PKR by bacterial RNA may play a pivotal role in the innate response to bacterial infections. The results described above also show that the bacterial RNAdriven immune response can provoke an apoptotic response and suggested the potential role of these immunostimulatory agents in cardiac pathophysiology.
Acknowledgements
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The first two authors contributed equally to this paper.
References
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References
  • Annane D, Bellissant E, Cavaillon JM. Septic shock. Lancet 2005;365:63-78.[LinkOut]
  • Parrillo JE. Septic shock--vasopressin, norepinephrine, and urgency. N Engl J Med 2008;358:954-6.[LinkOut]
  • Parrillo JE. Pathogenetic mechanisms of septic shock. N Engl J Med 1993;328:1471-7.[LinkOut]
  • Landgarten MJ, Kumar A, Parrillo JE. Cardiovascular Dysfunction in Sepsis and Septic Shock. Curr Treat Options Cardiovasc Med 2000;2:451-459.[LinkOut]
  • Nduka OO, Parrillo JE. The pathophysiology of septic shock. Crit Care Nurs Clin North Am 2011;23:41-66.[LinkOut]
  • Pitha PM. Unexpected similarities in cellular responses to bacterial and viral invasion. Proc Natl Acad Sci U S A 2004;101:695-6.[LinkOut]
  • Ishii KJ, Akira S. TLR ignores methylated RNA? Immunity 2005;23:111-3.[LinkOut]
  • Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell 2006;124:783-801.[LinkOut]
  • Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, et al. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 2004;303:1526-9.[LinkOut]
  • Karik?K, Buckstein M, Ni H, Weissman D. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 2005;23:165-75.[LinkOut]
  • Bauer S, Pigisch S, Hangel D, Kaufmann A, Hamm S. Recognition of nucleic acid and nucleic acid analogs by Toll-like receptors 7, 8 and 9. Immunobiology 2008;213:315-28.[LinkOut]
  • Williams BRG. The role of the dsRNA-activated kinase, PKR, in signal transduction. Semin Virol 1995;6:191-202.[LinkOut]
  • Feng GS, Chong K, Kumar A, Williams BR. Identification of doublestranded RNA-binding domains in the interferon-induced double-stranded RNA-activated p68 kinase. Proc Natl Acad Sci U S A 1992;89:5447-51.[LinkOut]
  • Tian B, Mathews MB. Functional characterization of and cooperation between the double-stranded RNA-binding motifs of the protein kinase PKR. J Biol Chem 2001;276:9936-44.[LinkOut]
  • Zhang F, Romano PR, Nagamura-Inoue T, Tian B, Dever TE, Mathews MB, et al. Binding of double-stranded RNA to protein kinase PKR is required for dimerization and promotes critical autophosphorylation events in the activation loop. J Biol Chem 2001;276:24946-58.[LinkOut]
  • Su Q, Wang S, Baltzis D, Qu LK, Wong AH, Koromilas AE. Tyrosine phosphorylation acts as a molecular switch to full-scale activation of the eIF2alpha RNA-dependent protein kinase. Proc Natl Acad Sci U S A 2006;103:63-8.[LinkOut]
  • Romano PR, Garcia-Barrio MT, Zhang X, Wang Q, Taylor DR, Zhang F, et al. Autophosphorylation in the activation loop is required for full kinase activity in vivo of human and yeast eukaryotic initiation factor 2alpha kinases PKR and GCN2. Mol Cell Biol 1998;18:2282-97.[LinkOut]
  • Dey M, Cao C, Dar AC, Tamura T, Ozato K, Sicheri F, et al. Mechanistic link between PKR dimerization, autophosphorylation, and eIF2alpha substrate recognition. Cell 2005;122:901-13.[LinkOut]
  • Barber GN, Tomita J, Hovanessian AG, Meurs E, Katze MG. Functional expression and characterization of the interferon-induced double-stranded RNA activated P68 protein kinase from Escherichia coli. Biochemistry 1991;30:10356-61.[LinkOut]
  • Garcí MA, Gil J, Ventoso I, Guerra S, Domingo E, Rivas C, et al. Impact of protein kinase PKR in cell biology: from antiviral to antiproliferative action. Microbiol Mol Biol Rev 2006;70:1032-60.[LinkOut]
  • Kumar A, Haque J, Lacoste J, Hiscott J, Williams BR. Double-stranded RNA-dependent protein kinase activates transcription factor NF-kappa B by phosphorylating I kappa B. Proc Natl Acad Sci U S A 1994;91:6288-92.[LinkOut]
  • Kumar A, Yang YL, Flati V, Der S, Kadereit S, Deb A, et al. Deficient cytokine signaling in mouse embryo fibroblasts with a targeted deletion in the PKR gene: role of IRF-1 and NF-kappaB. EMBO J 1997;16:406-16.[LinkOut]
  • Yang YL, Reis LF, Pavlovic J, Aguzzi A, Schäer R, Kumar A, et al. Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase. EMBO J 1995;14:6095-106.[LinkOut]
  • Nakamura T, Furuhashi M, Li P, Cao H, Tuncman G, Sonenberg N, et al. Double-stranded RNA-dependent protein kinase links pathogen sensing with stress and metabolic homeostasis. Cell 2010;140:338-48.[LinkOut]
  • Gil J, Alcamí J, Esteban M. Induction of apoptosis by double-stranded- RNA-dependent protein kinase (PKR) involves the alpha subunit of eukaryotic translation initiation factor 2 and NF-kappaB. Mol Cell Biol 1999;19:4653-63.[LinkOut]
  • Koski GK, Karikó K, Xu S, Weissman D, Cohen PA, Czerniecki BJ. Cutting edge: innate immune system discriminates between RNA containing bacterial versus eukaryotic structural features that prime for high-level IL- 12 secretion by dendritic cells. J Immunol 2004;172:3989-93.[LinkOut]
  • Lau CM, Broughton C, Tabor AS, Akira S, Flavell RA, Mamula MJ, et al. RNA-associated autoantigens activate B cells by combined B cell antigen receptor/Toll-like receptor 7 engagement. J Exp Med 2005;202:1171-7.[LinkOut]
  • Bourquin C, Schmidt L, Hornung V, Wurzenberger C, Anz D, Sandholzer N, et al. Immunostimulatory RNA oligonucleotides trigger an antigen-specific cytotoxic T-cell and IgG2a response. Blood 2007;109:2953-60.[LinkOut]
  • Hamm S, Heit A, Koffler M, Huster KM, Akira S, Busch DH, et al. Immunostimulatory RNA is a potent inducer of antigen-specific cytotoxic and humoral immune response in vivo. Int Immunol 2007;19:297-304.[LinkOut]
  • Kanneganti TD, Body-Malapel M, Amer A, Park JH, Whitfield J, Franchi L, et al. Critical role for Cryopyrin/Nalp3 in activation of caspase-1 in response to viral infection and double-stranded RNA. J Biol Chem 2006;281:36560-8.[LinkOut]
  • Kanneganti TD, Ozören N, Body-Malapel M, Amer A, Park JH, Franchi L, et al. Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3. Nature 2006;440:233-6.[LinkOut]
  • Hornung V, Ellegast J, Kim S, Brzózka K, Jung A, Kato H, et al. 5'-Triphosphate RNA is the ligand for RIG-I. Science 2006;314:994-7.[LinkOut]
  • Takahasi K, Yoneyama M, Nishihori T, Hirai R, Kumeta H, Narita R,et al. Nonself RNA-sensing mechanism of RIG-I helicase and activation of antiviral immune responses. Mol Cell 2008;29:428-40.[LinkOut]
  • Bevilacqua PC, George CX, Samuel CE, Cech TR. Binding of the protein kinase PKR to RNAs with secondary structure defects: role of the tandem A-G mismatch and noncontiguous helixes. Biochemistry 1998;37:6303-16.[LinkOut]
  • Zheng X, Bevilacqua PC. Straightening of bulged RNA by the doublestranded RNA-binding domain from the protein kinase PKR. Proc Natl Acad Sci U S A 2000;97:14162-7.[LinkOut]
  • Zheng X, Bevilacqua PC. Activation of the protein kinase PKR by short double-stranded RNAs with single-stranded tails. RNA 2004;10:1934-45.[LinkOut]
  • Kumar A, Kumar A, Michael P, Brabant D, Parissenti AM, Ramana CV, et al. Human serum from patients with septic shock activates transcription factors STAT1, IRF1, and NF-kappaB and induces apoptosis in human cardiac myocytes. J Biol Chem 2005;280:42619-26.[LinkOut]
  • Sugiyama T, Fujita M, Koide N, Mori I, Yoshida T, Mori H, et al. 2-aminopurine inhibits lipopolysaccharide-induced nitric oxide production by preventing IFN-beta production.Microbiol Immunol 2004;48:957-63.[LinkOut]
  • Lee JH, Park EJ, Kim OS, Kim HY, Joe EH, Jou I. Double-stranded RNAactivated protein kinase is required for the LPS-induced activation of STAT1 inflammatory signaling in rat brain glial cells. Glia 2005;50:66-79.[LinkOut]
  • Cabanski M, Steinmüller M, Marsh LM, Surdziel E, Seeger W, Lohmeyer J. PKR regulates TLR2/TLR4-dependent signaling in murine alveolar macrophages. Am J Respir Cell Mol Biol 2008;38:26-31.[LinkOut]
  • Frantz S, Kobzik L, Kim YD, Fukazawa R, Medzhitov R, Lee RT, et al. Toll4 (TLR4) expression in cardiac myocytes in normal and failing myocardium. J Clin Invest 1999;104:271-80.[LinkOut]
  • Boyd JH, Mathur S, Wang Y, Bateman RM, Walley KR. Toll-like receptor stimulation in cardiomyoctes decreases contractility and initiates an NFkappaB dependent inflammatory response. Cardiovasc Res 2006;72:384- 93.[LinkOut]
  • Zhu X, Bagchi A, Zhao H, Kirschning CJ, Hajjar RJ, Chao W, et al. Tolllike receptor 2 activation by bacterial peptidoglycan-associated lipoprotein activates cardiomyocyte inflammation and contractile dysfunction. Crit Care Med 2007;35:886-92.[LinkOut]
  • Mitchell JA, Ryffel B, Quesniaux VF, Cartwright N, Paul-Clark M. Role of pattern-recognition receptors in cardiovascular health and disease. Biochem Soc Trans 2007;35:1449-52.[LinkOut]
  • Frantz S, Ertl G, Bauersachs J. Mechanisms of disease: Toll-like receptors in cardiovascular disease. Nat Clin Pract Cardiovasc Med 2007;4:444-54.[LinkOut]
  • Cartwright N, McMaster SK, Sorrentino R, Paul-Clark M, Sriskandan S, Ryffel B, et al. Elucidation of toll-like receptor and adapter protein signaling in vascular dysfunction induced by gram-positive Staphylococcus aureus or gram-negative Escherichia coli. Shock 2007;27:40-7.[LinkOut]
  • Karikó K, Buckstein M, Ni H, Weissman D. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 2005;23:165-75.[LinkOut]
  • Diebold SS, Massacrier C, Akira S, Paturel C, Morel Y, Reis e Sousa C. Nucleic acid agonists for Toll-like receptor 7 are defined by the presence of uridine ribonucleotides. Eur J Immunol 2006;36:3256-67.[LinkOut]
  • Hornung V, Barchet W, Schlee M, Hartmann G. RNA recognition via TLR7 and TLR8. Handb Exp Pharmacol 2008;(183):71-86.[LinkOut]
  • Bieger CD, Nierlich DP. Distribution of 5'-triphosphate termini on the mRNA of Escherichia coli. J Bacteriol 1989;171:141-7.[LinkOut]
  • Doma MK, Parker R. RNA quality control in eukar yotes. Cell 2007;131:660-8.[LinkOut]
  • Maquat LE, Carmichael GG. Quality control of mRNA function. Cell 2001;104:173-6.[LinkOut]
  • Nallagatla SR, Hwang J, Toroney R, Zheng X, Cameron CE, Bevilacqua PC. 5'-triphosphate-dependent activation of PKR by RNAs with short stem-loops. Science 2007;318:1455-8.[LinkOut]
  • Nallagatla SR, Toroney R, Bevilacqua PC. A brilliant disguise for self RNA: 5'-end and internal modifications of primary transcripts suppress elements of innate immunity. RNA Biol 2008;5:140-4.[LinkOut]
  • Sugiyama T, Gursel M, Takeshita F, Coban C, Conover J, Kaisho T, et al. CpG RNA: identification of novel single-stranded RNA that stimulates human CD14+CD11c+ monocytes. J Immunol 2005;174:2273-9.[LinkOut]
  • Anderson BR, Muramatsu H, Nallagatla SR, Bevilacqua PC, Sansing LH, Weissman D, et al. Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation. Nucleic Acids Res 2010;38:5884-92.[LinkOut]
  • Nallagatla SR, Toroney R, Bevilacqua PC. Regulation of innate immunity through RNA structure and the protein kinase PKR. Curr Opin Struct Biol 2011;21:119-27.[LinkOut]
  • Lee SB, Esteban M. The interferon-induced double-stranded RNAactivated protein kinase induces apoptosis. Virology 1994;199:491-6.[LinkOut]
  • Kibler KV, Shors T, Perkins KB, Zeman CC, Banaszak MP, Biesterfeldt J, et al. Double-stranded RNA is a trigger for apoptosis in vaccinia virusinfected cells. J Virol 1997;71:1992-2003.[LinkOut]
  • Srivastava SP, Kumar KU, Kaufman RJ. Phosphorylation of eukaryotic translation initiation factor 2 mediates apoptosis in response to activation of the double-stranded RNA-dependent protein kinase. J Biol Chem 1998;273:2416-23.[LinkOut]
  • Der SD, Yang YL, Weissmann C, Williams BR. A double-stranded RNAactivated protein kinase-dependent pathway mediating stress-induced apoptosis. Proc Natl Acad Sci U S A 1997;94:3279-83.[LinkOut]
  • Scheuner D, Patel R, Wang F, Lee K, Kumar K, Wu J, et al. Double-stranded RNA-dependent protein kinase phosphorylation of the alpha-subunit of eukaryotic translation initiation factor 2 mediates apoptosis. J Biol Chem 2006;281:21458-68.[LinkOut]
  • Yeung MC, Liu J, Lau AS. An essential role for the interferon-inducible, double-stranded RNA-activated protein kinase PKR in the tumor necrosis factor-induced apoptosis in U937 cells. Proc Natl Acad Sci U S A 1996;93:12451-5.[LinkOut]
  • Lee ES, Yoon CH, Kim YS, Bae YS. The double-strand RNA-dependent protein kinase PKR plays a significant role in a sustained ER stress-induced apoptosis. FEBS Lett 2007;581:4325-32.[LinkOut]
  • Balachandran S, Roberts PC, Kipperman T, Bhalla KN, Compans RW, Archer DR, et al. Alpha/beta interferons potentiate virus-induced apoptosis through activation of the FADD/Caspase-8 death signaling pathway. J Virol 2000;74:1513-23.[LinkOut]
  • McAllister CS, Samuel CE. The RNA-activated protein kinase enhances the induction of interferon-beta and apoptosis mediated by cytoplasmic RNA sensors. J Biol Chem 2009;284:1644-51.[LinkOut]
  • Guerra S, Lóez-Fernádez LA, Garcí MA, Zaballos A, Esteban M. Human gene profiling in response to the active protein kinase, interferoninduced serine/threonine protein kinase (PKR), in infected cells. Involvement of the transcription factor ATF-3 IN PKR-induced apoptosis. J Biol Chem 2006;281:18734-45.[LinkOut]
  • Gil J, Garcia MA, Esteban M. Caspase 9 activation by the dsRNAdependent protein kinase, PKR: molecular mechanism and relevance. FEBS Lett 2002;529:249-55.[LinkOut]
  • Hsu LC, Park JM, Zhang K, Luo JL, Maeda S, Kaufman RJ, et al. The protein kinase PKR is required for macrophage apoptosis after activation of Toll-like receptor 4. Nature 2004;428:341-5.[LinkOut]
  • Maass DL, White J, Horton JW. IL-1beta and IL-6 act synergistically with TNF-alpha to alter cardiac contractile function after burn trauma. Shock 2002;18:360-6.[LinkOut]
  • Prabhu SD. Cytokine-induced modulation of cardiac function. Circ Res 2004;95:1140-53.[LinkOut]
  • Brabant D, Michael P, Bleiblo F, Saleh M, Narain R, Tai TC, et al. Septic sera induces apoptosis and DNA fragmentation factor 40 activation in fibroblasts. Biochem Biophys Res Commun 2011;412:260-5.[LinkOut]
  • Paladugu B, Kumar A, Parrillo JE, Der S, Osman J, Mensing J, et al. Bacterial DNA and RNA induce rat cardiac myocyte contraction depression in vitro. Shock 2004;21:364-9.[LinkOut]
  • Lee Y, Gustafsson AB. Role of apoptosis in cardiovascular disease. Apoptosis 2009;14:536-48.[LinkOut]
  • Cinel I, Ark M, Dellinger P, Karabacak T, Tamer L, Cinel L, et al. Involvement of Rho kinase (ROCK) in sepsis-induced acute lung injury. J Thorac Dis 2012;1:30-9.[LinkOut]
  • Londoñ; D, Bai Y, Zükert WR, Gelderblom H, Cadavid D. Cardiac apoptosis in severe relapsing fever borreliosis. Infect Immun 2005;73:7669-76.[LinkOut]
  • Penninger JM, Bachmaier K. Review of microbial infections and the immune response to cardiac antigens. J Infect Dis 2000;181:S498-504.[LinkOut]
Cite this article as: Bleiblo F, Michael P, Brabant D, Ramana CV, Tai TC, Saleh M, Parrillo JE, Kumar A, Kumar A. Bacterial RNA induces myocyte cellular dysfunction through the activation of PKR. J Thorac Dis 2012;4(2):114-125. doi: 10.3978/j.issn.2072-1439.2012.01.07

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