Results
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).
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).
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.
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.
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.
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.
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.
|
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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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