From the a Ottawa Regional Cancer Center Research Laboratories, Ottawa, Ontario K1H 8L6, the b Department of Biochemistry, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada, the d Department of Molecular Biology, University of Geneva, 30 Quai Ernest-Ansermet, CH-1211 Geneva, Switzerland, the g Division of Hematology, Ottawa General Hospital, Ottawa, Ontario K1H 8L6, the f Department of Oncology and Medicine, Lady Davis Institute for Medical Research, Montreal, Quebec H3T 1E2, the h Department of Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1H 8M5, and the e Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada
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ABSTRACT |
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The interferon-inducible, double-stranded
RNA-dependent protein kinase PKR has been implicated in
anti-viral, anti-tumor, and apoptotic responses. Others have attempted
to examine the requirement of PKR in these roles by targeted disruption
at the amino terminal-encoding region of the Pkr gene. By
using a strategy that aims at disruption of the catalytic domain of
PKR, we have generated mice that are genetically ablated for functional
PKR. Similar to the other mouse model of Pkr disruption, we
have observed no consequences of loss of PKR on tumor suppression.
Anti-viral response to influenza and vaccinia also appeared to be
normal in mice and in cells lacking PKR. Cytokine signaling in the type I interferon pathway is normal but may be compromised in the
erythropoietin pathway in erythroid bone marrow precursors. Contrary to
the amino-terminal targeted Pkr mouse, tumor necrosis
factor The interferon
(IFN)1-inducible protein
kinase PKR is a well characterized effector of anti-viral responses in
mammals (1, 2). Activation by double-stranded RNA (dsRNA) or stem loop RNA structures results in autophosphorylation and subsequent
phosphorylation of the PKR overexpression in HeLa cells induces apoptosis (28) by mechanisms
that are inhibitable by Bcl-2 (27). Antisense ablation of PKR conferred
resistance to tumor necrosis factor Much of the work elucidating the role of PKR in growth control and
apoptosis utilized mutated PKR in tissue culture settings. Previous
work on targeted disruption of Pkr by homologous
recombination in mice focused on interruption of two exons including
one that encodes the initiating methionine (25). Surprisingly, analysis of this PKR-defective mouse model revealed no evidence of tumors and
normal anti-viral responses in untreated animals. The mice were
defective in IRF-1 and NF- Construction of the PKR Gene-targeting Vector--
A 28.8-kb
region of isogenic DNA encoding murine PKR was isolated and mapped. A
2.9-kb SacI fragment that encodes exons 10 and 11 (39) as
determined by sequence analysis and a 1.9-kb XhoI/BamHI fragment that bears intron XII
sequence just distal to exon 12 were cloned into the MC1 neo poly(A)
vector (Stratagene). The herpes simplex virus thymidine kinase
expression cassette was introduced into the 3' end of the vector at the
NotI site to produce the PKR catalytic domain replacement
vector designated pTV65TK. Insertion of the neomycin (neo) cassette
results in replacement of a 3.7-kb BamHI to XhoI
fragment of the Pkr gene including complete removal of the
180-nucleotide exon 12.
Generation of PKR-deficient Mice--
The targeting vector was
introduced into the J1 embryonic stem (ES) cell line (strain 129/terSv
(40)) by electroporation, and cells were selected with neomycin (200 µg/ml) and
1-(2'-deoxy-2'-fluoro-1-
Chimeric mice were generated by microinjection of ES cells with a
disrupted Pkr gene into Balb/c donor blastocysts and
subsequent implantation into CD-1 foster mothers. Chimeric animals were
tested for germ-line transmission of the agouti coat phenotype of
129/Sv-derived ES cells by crossing with Balb/C mice. Heterozygote
individuals were identified by genotyping and then intercrossed to
generate homozygous lines.
Northern Blot Analysis--
Total RNA was isolated from various
ES cells that were untreated or treated with 500 units/ml interferon
Derivation of Primary Mouse Embryo Fibroblasts
(MEFs)--
Primary MEF cultures were established from E13 embryos as
described previously (40). Cells were genotyped as above.
Immunoblot and Immune Complex Kinase Analysis of Pkr-null
Cells--
Wild-type and homozygous-null ES and MEF cells were lysed
in Lysis buffer (10 mM Tris (pH 7.5), 150 mM
sodium chloride, 5 mM EDTA, 1% Triton X-100), lysates
denatured, resolved by SDS-PAGE, and blotted onto nitrocellulose
membranes. PKR polypeptides were analyzed by immunoblotting with
antisera (anti-TikMAP2) raised against a multiple antigen peptide (MAP)
bearing mPKR residues 101-114 and visualized by enhanced
chemiluminescence. Alternatively, cell lysates were incubated with
poly(I)·poly(C) (pIC)-agarose (Amersham Pharmacia Biotech) at 4 °C
with rotation to allow binding, washed in lysis buffer, eluted and
resolved by SDS-PAGE, and blotted. dsRNA binding PKR polypeptides were
visualized with antisera (anti-Tik100) raised against His-tagged mPKR
residues 1-98 and ECL.
Immune complex kinase reactions were performed by incubating lysates
from various cells with the anti-Tik100A antisera and recovering the
immune complex with protein A-Sepharose beads. The complexes were
resuspended in reaction mixtures of kinase buffer with 5 µCi of
[ Clonogenic Assays and Hematopoietic Development--
Bone marrow
from age-matched mice was harvested by flushing tibias under sterile
conditions. An aliquot of cells was obtained, and erythrocytes were
removed by lysis in hypotonic solution, and viable nucleated marrow
cells were counted using a hemocytometer and trypan blue. Cells were
resuspended in Iscove's modified Dulbecco's medium/methyl cellulose
supplemented with 20% fetal calf serum, 0.09% bovine serum albumin,
and cytokines as noted below. 8 × 104 cells were
plated in 1 ml of media in 35-mm2 Petri dishes and
incubated at 37 °C and 5% CO2 for 10 days. Erythroid and myeloid colonies were counted, and colony-forming units (CFU) were
determined after 10 days. Recombinant human erythropoietin (Epo) was
used at 2 units/ml, recombinant murine IL-3 at 10 µg/ml, human Kit
ligand (KL) from conditioned media at 3% (v/v), and human
granulocyte-colony-stimulating factor at 100 ng/ml.
Vaccinia Virus Growth in Cell Lines--
CV-1,
L-929, wild-type, or Pkr-null cells were plated
in duplicate at 2 × 105 cells per well in 6-well
tissue culture plates. The next day, cells were infected with
trypsin-treated wild-type vaccinia virus (WR strain) at an m.o.i. of 10 for 1 h at 37 °C. Following incubation, monolayers were rinsed
and the inoculum replaced with media. At 4, 8, 12, 15, and 18 h
after infection, cells were frozen at Influenza Infection of PKR-null Mice--
Five-week-old
Pkr+/O heterozygous control,
Clk1-null,2 and
Pkr-null mice were anesthetized with halothane and infected
by intra-nasal inoculation with 50 µl of serial dilutions of the W29
strain of influenza in phosphate-buffered saline. Each group contained
8-12 animals that were monitored daily over a 10-day period. The W29 strain is pneumovirulent for mice due to mutations selected on mouse
adaptation of the prototype human influenza A strain, A/FM/1/47 (43).
Influenza virus stocks were prepared in chicken embryo allantoic
cavity, and infectivity titers were assessed by plaque assay on
Madin-Darby canine kidney cells (43). The LD50 value of the
virus in the control and Pkr-null animals was assessed by
Karber-Spearman analysis. Influenza virus growth in mouse lung was
determined from pools of three mice from each group.
Influenza-mediated Apoptosis in Cell Lines--
Wild-type and
Pkr-null MEFs were plated at 2 × 105 cells
per well in 6-well tissue culture plates and either left untreated or
treated with 500 units/ml IFN- TNF- eIF-2 Generation of Mice with Mutated Pkr Gene--
We designed a
targeting vector such that it would replace exon 12 of PKR (following
the nomenclature of Kuhen et al. (39)) with an HSV
TK gene promoter-driven neomycin resistance cassette in an
orientation opposite to the Pkr promoter (Fig.
1A). Exon 12 encodes
subdomains V and VI of the catalytic domain and is essential for enzyme
activity (30). Transfected ES cells were grown in selection media,
picked, and screened for true homologous recombinants (Fig.
1B and data not shown) by Southern blot analysis. Targeted
cell lines showed the predicted sized fragment for single site
integration as detected by the internal neomycin probe (Fig. 1B). Selected ES cells were microinjected into blastocysts
from BALB/C mice, implanted into foster mothers, and gave rise to
chimeric animals. These were bred with wild-type BALB/C mice to produce heterozygotes which were then interbred to yield progeny with wild-type, heterozygous, and homozygous null genotypes. The frequency of wild-type, heterozygous, and homozygous animals was 0.24, 0.57, and
0.19, respectively (Table I). As with the
amino-terminal targeted Pkr mouse (25), no tumors have
arisen in homozygous mice targeted at the PKR catalytic domain after a
year.
Characterization of Targeted Disruption of the PKR Catalytic
Domain--
An ES cell line (7A8.3.7) that was homozygous
Pkr0/0 was selected from the heterozygous mutant
cell line, 7A8.3, as described previously (42), using higher dose G418
selection (Fig. 1B). These cell lines and primary mouse
embryo fibroblasts (MEFs) derived from homozygous null animals were
assessed for PKR activity. Northern blot analysis of
poly(A)+-selected RNA from wild-type ES cells showed the
normal pattern of murine PKR transcripts of 6, 4, and 2.5 kb that are
induced 2.5-3-fold in IFN-treated cells (Fig.
2 (44)). The 4- and 6-kb and larger minor
mPKR transcripts appear to be incompletely spliced since they hybridize
to an intron-spanning riboprobe (data not shown). Heterozygous
Pkr+/0 cells showed similar expression of
transcripts. Homozygous Pkr0/0 cells completely
lacked the fully spliced mature 2.5-kb transcript as well as the
incompletely spliced 6-kb transcript (Fig. 2). However, a transcript of
about 4 kb and which appeared IFN-inducible was found in all cells.
Reprobing of this blot with the neomycin probe showed this transcript
in Pkr0/0 cells bore neomycin sequence (Fig. 2,
right panel) not found in the 4-kb transcript in wild-type
cells and thus represented a novel transcript that was derived from the
targeted allele. Since the 4-kb transcript in wild-type cells does not
hybridize with the neomycin probe, the transcript seen in
Pkr0/0 cells arises from the targeted allele and
coincidentally comigrates with the 4-kb wild-type transcript. The HSV
TK promoter-derived neomycin transcript is also detected by the
neomycin probe as a smaller transcript of about 900 base pairs. The IFN
inducibility of the 4-kb novel transcript in
Pkr0/0 cells suggests it is likely
Pkr promoter-derived with read-through into the neomycin
cassette. This was confirmed by reverse transcriptase-polymerase chain
reaction analysis which detects the proximal intron XI and strand-specific riboprobe hybridization which identifies the distal intron XII in this transcript (data not shown). If splicing variants that splice over the neomycin cassette exist, such transcripts would
encounter frameshifts in all downstream exons (39, 45). The above
observations show that targeted disruption of Pkr with our
strategy generates alleles that do not produce the normal PKR
transcripts and could at most produce message with premature stop
codons in the region encoding the catalytic domain of PKR.
Western blot analysis of cell lysates from wild-type and
Pkr0/0 ES cells and primary MEFs using the
antibody raised against murine PKR residues 101-114 (anti TikMAP2)
show no mature murine PKR of 65 kDa (Fig.
3A) detectable in the
homozygous targeted cell lines. No truncated PKR polypeptide is
apparent either, even with 10-fold excess loadings (data not shown). We
attempted to increase the sensitivity of detection by enriching for PKR
polypeptides with intact RNA binding capacity by incubating lysates
with pIC-agarose then immunoblotting for bound PKR polypeptides.
Wild-type and Pkr+/0 cells show murine PKR
binding to dsRNA that is detectable by another antibody raised to
murine PKR residues 1-98 (anti-Tik100; Fig. 3B).
Pkr0/0 cells, however, have no detectable mature
or truncated PKR polypeptides. An immune complex kinase
autophosphorylation assay using this same antibody, with dsRNA as an
activator, was performed on cell extracts from
Pkr0/0 cells. No functional PKR activity was
detectable in Pkr0/0 cells of either ES or MEF
origin (Fig. 4). Analysis by cell extract kinase assay without use of our antibodies also revealed no
IFN-inducible, dsRNA-activated 65-kDa phosphoprotein in
Pkr-null cells (data not shown). These results provide
convincing evidence that the strategy of catalytic disruption of
Pkr by targeting exon 12 leads to a null allele with no
catalytic function retained.
IFN and Cytokine Signaling in PKR0/0
Cells--
Increasing evidence points to a role for PKR in
transcription control and cytokine signaling (20-24). PKR is
implicated in regulating IRF-1 and NF-
We assessed other cytokine signaling pathways by determining whether
hematopoietic development in Pkr0/0 animals was
affected. Bone marrow from animals of wild-type and homozygous null
backgrounds were isolated, normalized, and plated in colony forming
assays in the presence of the indicated cytokines (Fig.
5). Colony-forming units (CFU) were
scored 10 days later for color and counted to determine the response of
each hematopoietic precursor population to cytokine stimulation.
Myeloid bone marrow precursors showed comparable response to IL-3, Kit
ligand (KL), and granulocyte-colony-stimulating factor treatment (data
not shown). Erythroid CFU growth from PKR0/0 animals,
however, demonstrated a reproducible diminished response to
erythropoietin (Epo). IL-3 and KL co-treatment appear to be able to
overcome this PKR-dependent Epo block in erythroid
precursors from Pkr0/0 animals (Fig. 5). These
precursors show a higher fold stimulation of CFU in response to
IL-3 and KL than in wild-type bone marrow. This is consistent with a
study implicating PKR in IL-3 stimulation of protein synthesis in an
IL-3-dependent cell line (46). This block appears to have
little physiological impact since hematocrit volumes from
Pkr-null animals were unaffected (data not shown).
Pkr0/0 Animals Retain Anti-viral
Responsiveness--
We wished to determine if loss of PKR would
affect the ability of null mice to counter viral challenge. Vaccinia
maintains resistance to IFN despite producing large amounts of dsRNA in late infection (47). This is thought to be due to the production of K3L
and E3L, two virally encoded PKR inhibitors (48, 49). The growth curve
of vaccinia (WR strain) in wild-type and Pkr-null primary
MEF cells in culture was examined (Fig.
6). The production of infectious vaccinia
virus particles in control CV-1 and L-929 cells closely
matched the kinetics of growth of this virus strain in HeLa cells (50).
The virus replicated equally well in wild-type primary MEFs and,
surprisingly, showed no significant difference in growth curve in
PKR-deficient MEFs (Fig. 6 and inset). No enhancement of
vaccinia yield was evident in the absence of PKR, in contrast to the
repressive effect of overexpression of PKR (50).
In order to determine if replication of an RNA virus might be more
affected by the loss of PKR, we examined the LD50 of the W29 mouse-adapted strain of influenza virus by intranasal infection of
control and Pkr0/0 animals. As shown in Table
II, heterozygous and Pkr-null
animals showed an LD50 of 104.3 and
104.0 infectious particles, respectively. This 2-fold
difference in apparent susceptibility of Pkr0/0
animals does not seem significant when compared, for example, to the
consequences of loss of STAT1 function upon anti-viral response (51,
52). STAT1-deficient mice completely succumbed to doses of vesicular
stomatitis virus challenge that were 104 to 106
lower than doses that were sublethal to wild-type animals. Furthermore, these animals had elevated susceptibility to a bacterial pathogen, Listeria, and opportunistic infections by murine hepatitis
virus. We also measured the level of influenza growth in mouse lung 2 days post-infection for pools of three mice from each group.
Pkr-null mice had 3.8 × 107
versus 9.0 × 107 pfu/ml for control mice.
To date, we have found no sign of impaired anti-viral response or
opportunistic infections in these animals. Our data demonstrate that
PKR is not essential for IFN type I responses or for countering
vaccinia or influenza infections and may be redundant in function.
Virus-induced Apoptosis in PKR0/0 Cells Is
Unimpaired--
The role of PKR in regulating apoptotic responses has
been shown by various groups ((26-28, 36, 53) see Introduction), and we wished to determine if PKR may curtail viral infections by inducing apoptosis.
To this end, Pkr0/0 MEFs were challenged with a
strain of influenza A/HK/1/68 (H3N2) (54) at various m.o.i. Cells
undergoing apoptosis were monitored using Annexin V-FITC (Boehringer
Mannheim) staining, an early apoptosis marker (55). FACS analysis of
MEFs infected with virus (Fig. 7) show a
dose-dependent increase in cells undergoing apoptosis as
determined by the elevated number of FITC-positive cells. The
percentage of these cells also showing propidium iodide uptake,
indicating incidental necrotic death, was small (typically 10-20% of
total cells) (data not shown). Pkr0/0 cells were
indistinguishable from wild-type cells in triggering apoptosis upon
influenza infection. This contrasts with the effect of
dominant-negative mutants of PKR that reduced influenza-mediated cell
death (26). IFN- Stress-induced Apoptotic Response in Pkr0/0 Cells Is
Normal--
Apoptosis may be induced by a number of signals including
cellular stress. PKR itself has been implicated in responses to various
forms of stress (56, 57). Der et al. (38) describe the
abrogation of stress-induced apoptosis in the Pkr-null cells they generated (25). The cells had impaired apoptotic responses to
TNF- Intact eIF-2 Targeted disruption of a Pkr, catalytic domain exon was
performed to explore fully the consequences of loss of PKR function at
the organismal level. Our targeting strategy inactivates the catalytic
domain by replacing an exon which encodes 60 amino acid residues
including 6 amino acids that are known to be required for catalytic
function (30). If any splicing variants exist that bypass the disrupted
exon and splice into any of the distal exons 13-16 (66), all such
transcripts will encounter frameshifts that terminate prematurely
within the catalytic domain. PKR cDNA hybridizes to two mRNA
species in humans (2.5 and 6 kb) (67), one transcript in rats (4.4 kb)
(68), and three transcripts in mouse (2.5, 4, and 6 kb) (44). The 4-kb
transcript derived from the targeted allele is coincidentally of a
similar size to the 4-kb partially spliced transcript in normal cells.
However, our data show that this 4-kb transcript contains intron
sequence and antisense neomycin sequence that would terminate the
reading frame. No mature or truncated polypeptide was observed in
Pkr0/0 cells, and PKR catalytic activity was
undetectable. Mice homozygous for this Pkr disruption were
bred and appeared to have no obvious defects in gross anatomy and were
fertile with average sized litters. No evidence of susceptibility to
opportunistic infections or signs of increased tumorigenesis have been observed.
The maintenance of proper control of translation is critical to
cellular growth control. Indeed, it has been reported that translation
restrictive elements are found in a disproportionate number of the
untranslated regions of transcripts of proto-oncogenes, growth factors,
hormone receptors, and transcription factors (69). The absence of any
tumorigenic phenotype in either our Pkr-null mice or that of
Yang et al. (25) was puzzling given the evidence of the
consequences of translation deregulation. For instance, when the
restriction of translation at the Cap binding step is circumvented by
overexpression of the Cap-binding protein, eIF-4E, cellular
transformation occurs (70). PKR restricts growth when expressed in
yeast and causes morphological transformation when inactive mutated
versions are expressed in mammalian cells. Furthermore, expression of
non-phosphorylatable eIF-2 The anti-viral function of PKR has been studied with analyses of virus
mutants lacking anti-PKR inhibitors as well as overexpression of
Pkr. Strategies utilized by viruses to overcome PKR
activation include inactivating RNAs, pseudosubstrates, endogenous
inhibitor mobilization, Pkr expression down-regulation, and
dsRNA sequestration. Certain viruses have also developed means to
prevent cell death responses to infection such as cowpox
crmA (73) and baculovirus IAP (74). A few studies now
implicate PKR in controlling apoptosis in response to some triggers.
Pkr is itself a candidate "death gene" where
overexpression causes apoptosis (28). This has been shown to require
the third basic region implicated in PKR autoregulatory regulation
(53). PKR is located upstream of Bcl-2 function since Bcl-2 abrogates
PKR-mediated apoptosis (27). Expression of inactivated PKR suppresses
influenza-mediated apoptosis (26) and antisense ablation of PKR
provides immunity to TNF- Both viral growth and virus-induced apoptosis is normal in
Pkr0/0 cells further supporting the idea that
the biological roles of PKR are assisted by parallel pathways.
Recently, Taniguchi and co-workers (75) have made similar observations
with null fibroblasts derived from the Pkr-null mouse
engineered by Yang et al. (25).
We did not find any evidence in Pkr0/0 cells of
defective cytokine signaling (20) or apoptotic response to TNF- The discrepancy between our results and those of Yang et al.
(25) may be accounted for by mouse strain differences. The mice with
targeted Pkr deletion in exons 2 and 3 have a 129/Sv(ev) × C57BL/6J background (25), whereas our Pkr-null mice have a 129/terSv × BALB/C background. Indeed, strain differences in p53 targeted mice (76, 77) have been shown to account for phenotypic differences. Alternatively, our divergent observations could be resolved if the knock-out model described by Yang et al.
(25) in fact encodes a truncated gene product that functions as a
transdominant protein. Interestingly, Barber et al. (32, 78)
describe a dsRBD I deletion mutation of human PKR which would closely
resemble the putative polypeptide encoded by the Yang et al.
(25) PKR knock-out mouse. High level expression of the dsRBD I deletion mutant is an effective dominant-negative polypeptide that causes both
cellular transformation and reduced eIF-2 The nature of PKR redundancy is not known but could be attributed to
the recently discovered human homolog of the yeast GCN2 gene. Indeed, Zhu and Wek (79) have shown that yeast GCN2 contains a
previously unidentified motif that functions to target this kinase to
ribosomes independently from its response to amino acid starvation. We
are pursuing the disruption of this putative eIF-2-induced apoptosis and the anti-viral apoptosis response to
influenza is not impaired in catalytic domain-targeted
Pkr-null cells. The observation of intact eukaryotic
initiation factor-2
phosphorylation in these Pkr-null
cells provides proof of rescue by another eukaryotic initiation
factor-2
kinase(s).
INTRODUCTION
Top
Abstract
Introduction
References
subunit of eukaryotic initiation factor 2 (eIF-2
) (3-8). This phosphorylation causes sequestration of the
guanine nucleotide exchange factor eIF-2B which prevents the exchange
of GDP for GTP on eIF-2 and thereby inhibits translation initiation (9, 10). The decline in protein synthesis rates is deleterious to virus
replication, and various viral mechanisms exist to circumvent inhibition of translation by PKR (11-18). Although there has been a
considerable focus on its anti-viral role, PKR has been implicated in
regulating other cellular functions such as differentiation (19),
transcription (20-24), signal transduction (25), apoptotic response
(26-28), and cell growth (29-32). For example, in yeast expression of
PKR results in a slow growth phenotype (29), whereas expression of
catalytically inactive PKR results in malignant transformation of NIH
3T3 cells (30-32). Interestingly, the p58ipk inhibitor of
PKR (33), the TAR RNA-binding protein inhibitor of PKR (34), and a
non-phosphorylatable variant of eIF-2
(35) also transform NIH 3T3
cells underscoring the importance of translation initiation control and
the regulation of cell growth.
(TNF-
)-induced apoptosis in
U937 cells (36) indicating the requirement for PKR in the apoptotic
response to TNF-
in these cells. It is thought that dsRNA is a
trigger for apoptosis in vaccinia virus-infected cells (37), and
influenza-mediated apoptosis is suppressed in cells expressing
inactivated PKR (26).
B signaling and showed diminished stress-induced apoptotic responses (20, 25, 38). To examine whether
PKR is essential in anti-viral response, anti-proliferative functions
of cellular growth control, and in apoptotic response to various
stimuli, we generated mice devoid of PKR function by targeted
disruption of the PKR catalytic domain using homologous recombination
that interrupts exon 12. Mice homozygous for Pkr disruption
(Pkr 0/0) develop normally and are fertile with
average sized litters. IFN-
and -
induction of transcription is
intact, and the mice show normal hematopoiesis. Pkr
0/0 mice show responses to vaccinia and influenza
infection comparable to control animals or cells. Apoptotic response to
influenza infection or TNF-
was not impaired. Our data indicate that
catalytic disruption of Pkr is not sufficient to ablate
eIF-2
phosphorylation and that unappreciated members of the eIF-2
kinase family must compensate for loss of PKR function.
MATERIALS AND METHODS
-D-arabinofuranosyl)-5-iodouracil (0.2 µM; kindly provided by Dr. Michael A. Rudnicki) as
described previously (41). Targeted disruption of the Pkr
gene was determined by EcoRI or PstI digestion
followed by Southern hybridization with probe A or probe B,
respectively (see Fig. 1). To verify that only one copy of each
construct was integrated in each targeted ES clone,
XhoI-digested DNA was probed with the Neo probe. 3 out of
750 ES clones screened were true homologous recombinants. ES cells that
were homozygous for the targeted Pkr allele were obtained by
selection of the heterozygous targeted clone 7A8.3 in high neomycin
selection (2 mg/ml) as described previously (42). Of 20 resistant
colonies picked, one clone (7A8.3.7) was homozygous mutant.
and
(Cytimmune) for 16 h. Poly(A)+ RNA was
selected on an oligo(dT)-cellulose (Life Technologies, Inc.) column. 6 µg of mRNA was resolved on an agarose-formaldehyde gel, blotted,
and hybridized with a murine PKR cDNA
StuI/SspI fragment corresponding to nucleotides
290-1621 that encode residues 98-515. The blot was washed at high
stringency and imaged on a Molecular Dynamics PhosphorImager. The blot
was stripped and reprobed with the mouse glyceraldehyde phosphate
dehydrogenase cDNA, washed, and exposed. After stripping the blot
again, it was hybridized with an MluI/BamHI
fragment of the neomycin gene, washed, and exposed as above. IFN
inducibility of 2',5'-oligoadenylate synthetase was assessed by
rehybridizing this blot with the 2',5'-oligoadenylate synthetase cDNA.
-32P]ATP and 10 ng/µl reovirus dsRNA and incubated
at 25 °C for 30 min. Autophosphorylation activity of PKR was
visualized by SDS-PAGE and phosphorimaging.
80 °C. Cells were scraped
off in 0.3 ml of serum-free media and freeze-thawed twice before
trypsinization and serial dilution. Plaque assays were performed, and
after 2 days of incubation, plates were fixed, stained with crystal
violet, and plaques counted to determine the number of pfus/ml of cell
lysate as a function of time after infection.
and -
overnight. Monolayers were
rinsed in phosphate-buffered saline and then infected with influenza
A/HK/1/68 (H3N2) virus in 100 µl of phosphate-buffered saline at
multiplicity of infection (m.o.i.) of 5, 10, or 25 versus mock-infected for 30 min at 37 °C. Twenty-four hours later, cells were harvested and assayed for apoptosis by Annexin V-FITC staining (CLONTECH). After incubation of cells with the
Annexin V reagent for 10 min, stained cells were fixed in 2%
paraformaldehyde and analyzed on a Becton Dickinson FACS instrument for
percentage cells with elevated FITC fluorescence indicating apoptosis.
, Lipopolysaccharide (LPS), and dsRNA-mediated Apoptosis
in PKR-null Cells--
4 × 105 MEFs were plated in
60-mm2 tissue culture plates and treated the next day with
50-2500 ng/ml actinomycin D for 24 h to establish minimal
cytostatic dose and ensure Pkr-null cells responded
comparably to control cells. Cell viability was determined by
trypsinizing cells and trypan blue staining. Actinomycin D toxicity was
not as profound as described previously (38) and was comparable in the
two cell types. TNF-
, LPS, and poly(I)·poly(C) synthetic dsRNA
(pIC) induction of apoptosis in wild-type MEFs was examined by treating
these cells as above with 20 ng/ml TNF-
, 100 ng/ml LPS, and 100 µg/ml pIC with increasing amounts of actinomycin D for 24 h.
Cell viability was measured by trypan blue staining and verified by
TUNEL assay (Boehringer Mannheim) or morphology under Hoechst staining.
TNF-
-induced apoptosis in these MEFs required actinomycin D
co-treatment. The apoptotic dose response to TNF-
was determined in
PKR-null cells by treating cells with actinomycin D (500 ng/ml) and
between 0.1 to 20 ng/ml TNF-
for 24 h. Experiments were
conducted at least in triplicate.
Phosphorylation State in Pkr-null
Cells--
Immortalized pools of Pkr-null and wild-type
cells were washed and lysed in RIPA buffer with protease and
phosphatase inhibitors. After clearing the lysate, 20 µg of protein
were resolved by SDS-PAGE and transferred to nitrocellulose.
Phosphorylation of eIF-2
was determined by immunoblot with a
polyclonal anti-eIF-2
phosphoserine 51 antibody, whereas protein
levels of eIF-2
were determined with a monoclonal antibody to
eIF-2
. Normalization of protein levels was with anti-actin antibody.
RESULTS
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Fig. 1.
Targeted inactivation of the Pkr
gene in ES cells and mice. A, schematic drawing
of wild-type Pkr locus, replacement-type targeting
vector pTV65TK, and targeted PKR locus after homologous
recombination (not drawn to scale). Shaded boxes represent
Pkr exons. pTV65TK contains an HSVTK-neo cassette in the
opposite transcriptional orientation to the Pkr gene and an
HSVTK-tk expression cassette on the 3' end of the construct. Homologous
recombination results in introduction of new EcoRI and
PstI sites in the targeted allele. Probe A was
used in initial screens with EcoRI digestion. Wild-type and
recombinant alleles produce 9.4- and 6.9-kb fragments, respectively,
with this screen. Established mouse lines were screened with
PstI digestion and probe B. This screen produces
4.6- and 2.3-kb fragments from wild-type and recombinant alleles,
respectively. The Neo probe was used with an XhoI
digest to screen for integration of the targeting vector. R,
EcoRI; B, BamHI; S,
ScaI; P, PstI; Xh,
XhoI; neo, neomycin expression cassette;
HSVTK, herpes simplex virus-thymidine kinase promoter;
tk, herpes simplex virus-thymidine kinase expression
cassette. B, ES cell clones bearing homologous recombinant
targeted alleles. Electroporated ES cell colonies were picked and
analyzed by Southern screening with EcoRI digest and
probe A (left panel). 3 in 750 G418-1-(2'-deoxy-2'-fluoro-1- -D-arabinofuranosyl)-5-iodouracil-resistant
cell lines were homologous recombinants. Single copy integration of the
targeting vector was checked with a XhoI digest with the
Neo probe showing the predicted 16-kb fragment (right
panel). The homozygous Pkr-null cell line 7A8.3.7 was
derived by high dose G418 selection. +/+, wild-type; +/0, heterozygous
for Pkr-null allele; 0/0, homozygous for Pkr-null
allele.
Genotype of progeny of heterozygote and homozygote self-crosses
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Fig. 2.
Characterization of the
Pkr-targeted allele. Poly(A)+
Northern blot analysis of targeted Pkr allele. mRNA was
isolated from ES cells untreated or treated with IFN- and -
as
indicated. +/+, wild-type J1 cells; +/0, heterozygous 7A8.3 cells; 0/0,
homozygous 7A8.3.7 cells. Left panel, hybridization with
murine PKR (mPKR) cDNA probe spanning exons 4-16, from
nucleotides 290 to 1621; bottom panel, hybridization with
glyceraldehyde phosphate dehydrogenase (GAPDH) cDNA;
right panel, hybridization with neomycin (Neo)
cDNA. Arrows indicate the 4-kb novel transcript and the
0.9-kb HSVTK-neo derived transcript from the targeted allele.
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Fig. 3.
Immunoblot analysis of
Pkr-null ES and MEF cells demonstrate ablation of PKR
protein. A, Western analysis. Total cell lysates from
wild-type (+/+) or homozygous Pkr-null (0/0) cells were
extracted, separated by SDS-PAGE, immunoblotted with anti-mPKR antibody
(affinity purified anti-TikMAP2 antiserum), directed against residues
101-114, and visualized with ECL. Lane 1 loaded with 25 µg of total protein; lanes 2-5 loaded with 100 µg of
total protein. B, dsRNA affinity binding enrichment
analysis. Total protein from the indicated ES cell lines were incubated
with pIC-agarose; washed and bound protein were analyzed by immunoblot
analysis. Anti-mPKR antiserum (anti-Tik100A) raised against
purified His-mPKR residues 1-98 show mature PKR polypeptide in
wild-type (+/+) and heterozygous (+/0) cells but no immunoreactive PKR
polypeptides in homozygous (0/0) cells. Lane 1, 1.5 µg of total protein; lane 2, 15 µg of total protein; and
lanes 3-5, 150 µg of total protein incubated with dsRNA.
Molecular mass markers (kDa) are indicated on the left, and
the 65-kDa murine PKR polypeptide (mPKR) is indicated on the
right of each panel.
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Fig. 4.
Immune complex kinase assay demonstrates loss
of PKR catalytic activity in Pkr-null cells. PKR
protein was recovered by incubating lysates from the indicated
wild-type and homozygous Pkr-null ES and MEF cells with
anti-mPKR antiserum (anti-Tik100A). Immune complexes were incubated in
reaction buffer with reovirus dsRNA and [ -32P]ATP and
following autophosphorylation of the kinase, resolved by SDS-PAGE and
visualized. Lanes 1, 2, 4, 5, 7, and 8, 200 µg
of total protein; lanes 3 and 6, 10 µg of total
protein. Lane 1, protein A-Sepharose beads alone; lane
2, preimmune antiserum and protein A-Sepharose beads; lanes
3-8, anti-Tik100A and protein A-Sepharose beads. Molecular mass
markers (kDa) are indicated on the left and the 65-kDa
murine PKR polypeptide (mPKR) indicated on the right.
B activation (20, 21) and is
found in complex with STAT1 in an inverse relation to STAT1 activation (24). We examined the responsiveness of Pkr0/0
cells to the type I IFNs by assessing the induction of the
2',5'-oligoadenylate synthetase transcript in these cells. IFN-
/
signaling through STAT1 appears to be unimpaired since induction of
2',5'-oligoadenylate synthetase was normal in
Pkr0/0 cells (data not shown). Furthermore, IFN
induction of the 4-kb transcript in Pkr-null cells (Fig. 2)
supports this conclusion.
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Fig. 5.
Hematopoietic development in
Pkr-null animals appears normal. Erythroid bone
marrow precursors were assessed for cytokine responsiveness by
clonogenic assay in the presence of the indicated cytokines. Six
animals of each genotype were analyzed with triplicate platings of each
cytokine treatment. CFU were scored 10 days after plating with
cytokine. Error bars represent standard error of the mean.
G-CSF, granulocyte-colony-stimulating factor.
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Fig. 6.
Vaccinia growth in Pkr-null
cells is normal. Growth curve of vaccinia virus (WR strain) in
MEFs and control cells were examined. Cells were infected at m.o.i. 10, washed, then monitored over 18 h for virus production by plaque
assay of cell lysates. Error bars represent standard error
of the mean. Main panel, vaccinia growth in control cells
and MEFs; inset, vaccinia growth in wild-type (+/+) and
Pkr-null (0/0) MEFs.
Influenza LD50 in normal and Pkr-null animals
/
pretreatment partially abrogated
influenza-induced apoptosis in both normal and Pkr-null
cells up to an m.o.i. of 10 (Fig. 7). At higher m.o.i., IFN
pretreatment was unable to rescue cells from virally induced apoptotic
pathways. These data indicate that PKR is not essential for influenza
to trigger apoptosis pathways in cells and that type I IFNs can mount
anti-apoptotic responses at low m.o.i. independent of PKR.
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Fig. 7.
Apoptotic response to influenza infection is
unimpaired in Pkr-null cells. Untreated or
IFN-pretreated MEFs were mock-infected or infected with influenza
A/HK/1/68 virus at increasing m.o.i. After 24 h, cells were
isolated, stained with Annexin V-FITC, fixed, and analyzed by FACS for
cells undergoing apoptosis. Histograms showing distribution of
fluorescence of cells of each genotype and treatment are shown.
Left panels, wild-type (+/+) MEFs; right panels,
Pkr-null (0/0) MEFs. Black trace, cells without
IFN pretreatment; gray trace, IFN- - and -
-pretreated
cells.
, pIC, and lipopolysaccharide (LPS). We addressed the role of
PKR in these pathways by determining whether our Pkr-null cells were likewise impaired. We first assessed the dose response of
our wild-type and Pkr0/0 MEFs to actinomycin D
(Fig. 8A) which is required to
prime cells to respond to these stimuli (38, 58, 59) (Fig.
8B). By having found no significant differences, we then
optimized TNF-
, LPS, and pIC treatments of wild-type MEFs to trigger
cell death using trypan blue exclusion (Fig. 8B) as
described by Der et al. (38) and verified by TUNEL assay
(data not shown). We found that at the cytostatic dose of actinomycin D
(50 ng/ml) described previously (38), none of these treatments elicited
much response from the MEF cells, even at the maximal doses of TNF-
,
LPS, or pIC (Fig. 8B). It required higher doses of
co-treatment with actinomycin D (500 ng/ml) before any effect was seen
on cell viability, and this was restricted to TNF-
treatment only.
Finally, we determined the dose response of wild-type and
Pkr-null MEFs to TNF-
-induced apoptosis and found that
Pkr0/0 cells with disruption of the catalytic
domain had no impairment of cell death response (Fig. 8C) as
described in the other Pkr-null mouse model (38). This was
apparent throughout the dose range of TNF-
(0.1-20 ng/ml) used
(Fig. 8C and inset).
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Fig. 8.
TNF- -mediated
apoptosis is normal in Pkr-null cells.
A, comparable actinomycin D dose response in MEFs. Since
actinomycin D co-treatment is required to trigger apoptosis with
various stimuli, wild-type (+/+) and PKR Pkr-null (0/0) MEFs
were first assessed for toxicity response to actinomycin D. Cells were
treated with increasing doses for 24 h and stained with trypan
blue to determine viability. These determinations were performed in
triplicate, and error bars represent standard error of the
mean. B, the optimal actinomycin D dose for various
apoptosis triggers was determined. Wild-type MEFs were co-treated with
TNF-
, LPS, or pIC and increasing amounts of actinomycin D for
24 h. Cell viablity was determined as above and confirmed with
TUNEL assay and morphology with Hoechst staining. LPS and pIC fail to
show apoptosis above actinomycin D alone levels. Actinomycin D
co-treatment is required to potentiate TNF-
-induced cell death, and
the minimal actinomycin D dose for such effect is 500 ng/ml.
C, TNF-
dose response of Pkr-null cells
indicates unimpaired apoptotic response in the absence of PKR.
Wild-type (+/+) and Pkr-null (0/0) MEFs were co-treated with
actinomycin D (500 ng/ml) and increasing amounts of TNF-
for 24 h. Cell viability was determined as above and confirmed with TUNEL
assay and morphology with Hoechst staining. TNF-
treatment induces
cell death in a dose-dependent manner that is unaffected by
the absence of PKR. Error bars represent standard error of
the mean. Inset, TNF-
dose response plotted on a log
scale.
Phosphorylation in Pkr0/0 Cells May
Indicate the Presence of a Pkr Homolog--
The best known mammalian
Pkr homolog, the heme-regulated inhibitor, is restricted to
cells of erythroid lineage (60-62). The only other known homolog of
Pkr is the yeast GCN2 kinase that is activated by uncharged
tRNA and regulates amino acid biosynthesis by control of translation of
the GCN4 transcript (63). Despite apparent specialization in function
with unique regulatory domains, both mammalian eIF-2
kinases,
heme-regulated inhibitor, and PKR can rescue GCN2-defective yeast (64).
Since our knock-out strategy effectively renders the PKR catalytic
domain inactive, any eIF-2
phosphorylation in homozygous null cells
must result from a previously unappreciated kinase activity. We used an
antibody specific to phosphorylated eIF-2
(Fig.
9) (65) and determined the
phosphorylation status of eIF-2
in both wild-type and
Pkr-null animals. As shown in Fig. 9, both cell populations
contain similar levels of eIF-2
, and there is little or no
difference in eIF-2
phosphorylation status between wild-type and
Pkr-null cells.
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Fig. 9.
Lysates derived from wild-type and
Pkr-null MEFs were assessed for the degree of
eIF-2 phosphorylation. Top
panel shows immunoblot with anti-eIF-2
phosphoserine 51;
middle panel represents total eIF-2
as determined using
anti-eIF-2
; and lower panel is an immunoblot with
anti-actin.
DISCUSSION
mutated at the serine 51 position
transforms cells (35) as does overexpression of the PKR
inhibitors (p58ipk (71, 72)) and TAR-binding
protein (33, 34). Thus, the surprising absence of tumors in
Pkr-null mice suggests a redundancy of PKR function that has
been previously unappreciated.
(36).
(38). This contrasts with the observation that cells expressing the
catalytically inactive PKR
6 mutant are unable to induce a
STAT1-inducible gene in response to IFN-
/
(24). The defect in
erythropoietin-induced erythroid CFU development may indicate one
developmental signaling pathway that requires PKR, although this defect
appears readily compensated by other cytokines in vitro and
in vivo.
phosphorylation (32, 78).
We speculate that low level expression of a potent transdominant mutant
could produce the mouse phenotype observed by Yang et al.
(20, 25, 38). In contrast, we would argue that our Pkr
knock-out model produces a null allele that can be compensated for by
redundant pathways. This is consistent with our observation of
unimpaired eIF-2
phosphorylation in null fibroblasts. It will be of
interest to determine the in vivo state of eIF-2
phosphorylation in cells derived from the Yang et al. (25)
mouse with Pkr targeted at the 5' end.
protein kinase in
the mouse to examine its function in mammals. Studying the mouse GCN2
homolog would allow further elucidation of the importance of the family
of eIF-2
protein kinases, and their role in control of cell growth
and apoptosis.
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ACKNOWLEDGEMENTS |
---|
We acknowledge Ricardo Marius, Simon Ginsberg, Scot Kelso, and Heidi K. Gruber for valuable technical assistance. We also thank Dr. Michel Tremblay, Dr. Michael Rudnicki, and Dr. Dan Skup for providing reagents.
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FOOTNOTES |
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* This work was supported in part by funds from the National Cancer Institute of Canada and Canadian Cancer Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
c Supported by National Cancer Institute of Canada scholarship.
i National Cancer Institute of Canada senior scientist. To whom correspondence should be addressed: Tel.: 613-737-7700 (ext. 6893); Fax: 613-247-3524; E-mail: jbell{at}med.uottawa.ca.
2 P. I. Duncan and J. C. Bell, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
IFN, interferon;
PKR, double-stranded RNA-dependent protein kinase;
Epo, erythropoietin;
TNF-, tumor necrosis factor
;
eIF, eukaryotic initiation factor;
ds, double-stranded;
kb, kilobase pair;
ES, embryonic stem;
MEF, mouse
embryo fibroblasts;
PAGE, polyacrylamide gel electrophoresis;
CFU, colony-forming units;
KL, Kit ligand;
m.o.i., multiplicity of
infection;
pfu, plaque-forming units;
LPS, lipopolysaccharide;
IL-3, interleukin-3;
MAP, multiple antigen peptide;
pIC, poly(I)·poly(C);
FITC, fluorescein isothiocyanate;
FACS, fluorescence-activated cell
sorter;
TUNEL, terminal deoxynucleotide
transferase-mediated deoxyuridine 5'triphosphate
nick end labeling.
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REFERENCES |
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