(Received for publication, May 15, 1995; and in revised form, June 8, 1995)
From the
Many genes induced by type I interferons (IFNs) are also induced
by double-stranded (ds) RNA. In this study, we investigated the
mechanism of this induction process. Using cell lines from which the
type I IFN genes have been deleted, we established that induction by
dsRNA of the IFN-inducible 561 gene is direct and not mediated by the
intermediate synthesis of IFN. Unlike 561 mRNA, the IFN-inducible 6-16
mRNA was induced poorly by dsRNA. Transfection studies demonstrated
that the sequence difference between the core IFN-stimulated response
elements (ISREs) of these two genes is not responsible for their
differential inducibility by dsRNA. A point mutation in the 561 ISRE
that abolished its response to IFN- also made it unresponsive to
dsRNA, thus demonstrating that the ISRE is the relevant cis-acting
element for dsRNA signaling. The roles of different known ISRE-binding
protein and tyrosine kinases in transducing the signal elicited by
dsRNA were evaluated in genetically altered cell lines. dsRNA failed to
induce 561 mRNA in cells expressing an antisense RNA for interferon
regulatory factor 1, whereas it was induced strongly in cells
expressing the corresponding sense mRNA. 561 mRNA was also induced
strongly by dsRNA, but not by IFN-
, in mutant cell lines that do
not express functional tyrosine kinases Tyk2 or JAK1 or ISRE binding
protein, p48, or STAT2, all of which are required for IFN-
signaling. However, in cells devoid of functional STAT1, which is also
required for IFN-
signaling, the induction of 561 mRNA by dsRNA
was very low. Expression of transfected STAT1
protein, but not of
STAT1
protein, in these cells greatly enhanced the dsRNA
inducibility of the 561 gene. These studies indicate that the major
ISRE-mediated signaling pathway used by dsRNA requires interferon
regulatory factor 1 and STAT1
. This pathway, however, does not
require the other known cytoplasmic components used for IFN-
signaling.
Double-stranded (ds) RNA, ()which is often produced
in a cell during virus replication, is a strong inducer of
transcription of specific cellular
genes(1, 2, 3) . The mechanism of this
induction process and the identity of the cellular transcription
factors that take part in it remain ill defined. Genes encoding
interferon (IFN)-
and IFN-
are among the most extensively
studied dsRNA-induced genes(4) . They contain multiple positive
and negative cis-acting elements which are required for induction.
Moreover, many of these elements can bind to several protein factors
which have different effects on transcription. As a result, the
induction process is quite complex and activation of several
trans-acting factors by dsRNA is required for the assembly of an active
transcription complex. Of these factors, NFkB is a crucial member whose
participation is necessary but not sufficient to activate transcription
of the human IFN-
gene(4, 5) . How dsRNA
activates the other necessary factors remains to be understood.
Another class of dsRNA-inducible genes is also induced by type I IFNs which act through the IFN-stimulated response element (ISRE) present in these genes(6, 7, 8, 9, 10, 11) . All IFN-inducible genes are, however, not equally induced by dsRNA. We have been studying the mechanism of induction of the 561 gene in human cells. The transcriptional regulatory region of this gene is not complex. It contains ISRE but no kB site or site for binding of any other known transcription factors. Thus, this simply regulated gene is highly suited for studying the mechanism of activation of ISRE-containing genes by dsRNA. We have previously shown that 561 mRNA is strongly induced by dsRNA or by virus infections that produce dsRNA(10, 11) . We have also shown that this induction process is direct and does not require ongoing protein synthesis. It, however, is blocked by 2-aminopurine, an inhibitor of RNA-activated protein kinase(11) . Our studies provided persuasive, but indirect, evidence to indicate that the induction process is not mediated by the synthesis of IFNs as intermediates. In this paper, we present more definitive evidence in support of the above characteristics of 561 mRNA induction by dsRNA.
Since ISREs, which
receive the transcriptional signal generated by IFNs, also appear to be
involved in mediating the signal elicited by dsRNA, we were interested
to determine the extent of overlap between the two signaling pathways.
The major signaling pathway used by IFN- to signal through ISREs
is the JAK-STAT
pathway(12, 13, 14, 15, 16, 17, 18) .
Binding of IFN-
to its receptor on the cell surface leads to
tyrosine phosphorylation of two protein tyrosine kinases, Tyk2 and
JAK1, followed by tyrosine phosphorylation of the STAT1 and STAT2
proteins. Tyrosine phosphorylation of the STAT proteins results in
their nuclear translocation and association with the p48 protein. The
trimeric complex containing STAT1, STAT2, and p48 is IFN-stimulated
gene factor 3 (ISGF3), which binds to ISREs and stimulates
transcription. ISRE containing genes fail to respond to IFN-
in
mutant cell lines defective in any component of the JAK-STAT pathway.
Another ISRE-binding protein is IRF-1(19, 20) .
IRF-1 has a DNA sequence recognition specificity partially overlapping
but distinct from the binding specificity of ISGF3. IRF-1 can bind to
the positive regulatory domain I element of the IFN- gene and is
thought to be involved in its transcriptional induction by
dsRNA(19, 21) . It is not clear whether IRF-1 is
involved in ISRE-mediated signal transduction by IFN-
. The most
compelling indication for such an involvement comes from studies with
cell lines missing functional IRF-1(22) . In these cells,
induction of several genes by IFN-
was partially impaired. Whether
this was due to a direct or indirect effect of IRF-1 remains to be
determined.
Here we report that IRF-1 is essential for transduction of the dsRNA-elicited signal to the ISREs. In contrast, Tyk2, JAK-1, p48, and STAT2, all components of the JAK-STAT pathway, are not absolutely required for this signaling process. 561 mRNA could be induced, albeit weakly, by dsRNA in cells missing STAT1 which suggests that although this component is required for the major pathway of signaling, a STAT1-independent minor pathway may exist.
Cell culture materials and G418 were obtained from Life
Technologies, Inc. The source of pure IFN- has been described
previously(23) . Cycloheximide and poly(I)-poly(C) (dsRNA) were
from Boehringer Mannheim, 2-aminopurine was from Sigma, and luciferase
assay reagents kit were from Promega. All radioactive chemicals were
from DuPont.
Figure 1: Induction of IFN-inducible 561 mRNA by dsRNA in IFN-minus cell lines. Glioma cell lines GRE, M007, and SAN were either untreated or treated with 100 µg/ml dsRNA in 0.5% serum containing medium for 4 h. Twenty µg of total RNA was used for each sample in Northern blot with labeled 561 DNA as probe.
Figure 2: Kinetics of induction of IFN-inducible 561 mRNA by dsRNA in GRE cells. Cells were treated with 100 µg/ml dsRNA for the indicated time. Twenty µg of total RNA was used for each sample in the Northern blot, which was probed with labeled 561 DNA. The relevant portion of the autoradiogram is shown. The same blot was deprobed and hybridized with actin cDNA for normalization. The actin mRNA levels in each lane were within 20% of one another.
Effects of inhibitors on the induction process were examined in the experiment shown in Fig. 3. Cycloheximide, an inhibitor of protein synthesis, did not inhibit 561 induction by dsRNA in any of the three lines. This result indicates that ongoing protein synthesis is not required for the induction and that pre-existing proteins are sufficient for this process. Cycloheximide alone did not induce 561 mRNA (data not shown) but more mRNA accumulated in cells treated with dsRNA and cycloheximide than in cells treated with dsRNA alone (Fig. 3). This was probably due to stabilization of the labile 561 mRNA in the presence of cycloheximide. We have previously shown that 2-aminopurine, a known inhibitor of RNA-activated protein kinase, blocks 561 mRNA induction by dsRNA in HeLaM cells(11) . Similar results were obtained with the glioma cell lines, in which 2-aminopurine almost completely blocked 561 mRNA induction by dsRNA (Fig. 3).
Figure 3: Effects of cycloheximide and 2-aminopurine on the induction of 561 mRNA by dsRNA. GRE, M007, and SAN cells were untreated or treated as indicated for 4 h. Cycloheximide (CHX) and 2-aminopurine (2-AP) were added to the cells 15 min prior to the dsRNA treatment. 561 mRNA levels were determined as described in the legend to Fig. 2.
Figure 4:
Induction of 6-16 mRNA by dsRNA. GRE,
M007, and SAN cells were either untreated or treated with dsRNA or
IFN- for 4 h. The same blot as used for Fig. 3was deprobed
and hybridized again with labeled a 6-16 cDNA probe, washed, and
autoradiographed.
Figure 5:
Induction of 561 CAT and 6-16 CAT
expression in GRE cells. 561 CAT and 6-16 CAT constructs were
transiently transfected into GRE cells. Cells were either untreated or
treated with dsRNA or IFN- for 16 h as indicated. CAT activity was
measured in extracts with equal amount of protein as described under
``Materials and Methods.''
Figure 6: Effects of ISRE mutations on gene induction by dsRNA. 561 luciferase (lanes 1-3), 561 to 6-16 ISRE luciferase (lanes 4-6), and 561 to null ISRE luciferase (lanes 7-9) constructs were transiently transfected into GRE cells. ISRE core sequences of different constructs are shown in the figure with the open letter indicating the mutated nucleotide. The cells were treated as indicated and luciferase activities were measured as described under ``Materials and Methods.''
Whether the ISRE sequence is involved in the induction of 561
luciferase by dsRNA at all was addressed in the next experiment (Fig. 6, lanes 7-9). A specific point mutation,
known to destroy the binding of IRF-1 and ISGF3 to ISREs, was
introduced into the ISRE of 561 luciferase. As expected, the mutated
gene could not respond to IFN-. It was also not induced by dsRNA,
thus indicating that the ISRE sequence is required for response to this
agent as well.
Figure 7:
561 mRNA induction by dsRNA in cell lines
expressing sense or antisense IRF-1 RNA. Cultures of C1 (expressing
only vector), S1 (expressing sense IRF-1), and AS11 (expressing
antisense IRF-1) cells were treated in the presence of cycloheximide
for 4 h with 100 µg/ml dsRNA (lanes 2, 5, and 8)
or with 500 units/ml IFN- (lanes 3, 6, and 9) or
with only medium (lanes 1, 4, and 7). The levels of
mRNA 561 were measured by RNase protection assay using 15 µg of
total RNA for each sample. Lane 10 is the antisense 561 probe
digested with RNase and lane 11 is the probe without RNase
digestion. An antisense actin probe was used in the same experiment for
estimating the levels of actin mRNA (data not shown). The amounts of
actin mRNA in each sample were within 20% of one
another.
Figure 8:
561
mRNA induction by dsRNA in IFN- unresponsive mutant cell lines.
Cellular levels of 561 mRNA were measured by Northern blot analysis of
20 µg of total RNA from 2fTGH and its mutant cell lines. Treatments
were for 4 h as indicated in the figure.
Since the extent of induction
of 561 mRNA by dsRNA was different in different mutant lines, we
quantitated the levels of this mRNA by using an RNase protection assay.
Actin mRNA levels were also measured using the same total RNAs and the
same assay. 561 mRNA levels were normalized using the actin mRNA values
and the normalized level of 561 mRNA in dsRNA-treated 2fTGH cells was
assigned a value of 100 units. All untreated cells contained
undetectable (less than 1 unit) levels of 561 mRNA (Table 1).
IFN- treatment of 2fTGH cells, but not of mutant cells, increased
the level of 561 mRNA to 164 units. In U1A cells, lacking Tyk2, and U4A
cells, lacking Jak1, dsRNA induced 51 units of 561 mRNA. In U2A cells,
lacking p48, 96 units of 561 mRNA was induced by dsRNA and in U6A
cells, lacking STAT2, the corresponding level was 29 units. These
results demonstrated that although their absence affects the level of
induction quantitatively, Tyk2, Jak1, p48, and STAT2 are not required
for 561 mRNA induction by dsRNA. In contrast, the induction level in
U3A cells, which lack STAT1, was quite low (11 units). Expression of
one of the two STAT1 proteins, STAT1
(p91), by complementing U3A
cells with the corresponding expression vector, greatly enhanced the
dsRNA inducibility of 561 mRNA. However, the same was not true for the
STAT1
(p84) protein; 561 mRNA was poorly induced in cells
expressing STAT1
but lacking STAT1
. Thus, among the known
cytoplasmic components of the IFN-
signaling pathway, STAT1
appears to be the most important factor in the ISRE-mediated signaling
pathway used by dsRNA. A mutant STAT1
, in which residue 701 has
been changed from tyrosine to phenylalanine, cannot function in the
signaling pathways used by IFN-
or IFN-
(31) . This
mutant also failed to complement the U3A cells for their ability to
transmit the signal generated by dsRNA (Table 1).
Our previous studies strongly indicated that the induction of
561 mRNA transcription in human cells in response to exogenous dsRNA or
virus infection is a direct process(10, 11) . However,
since type I IFNs are also induced by the same inducers and 561 is an
IFN-inducible gene, it was necessary to rigorously rule out the
possible involvement of IFNs as the proximal inducer. Here, we provide
genetic evidence in favor of the direct induction mechanism. dsRNA
could induce 561 mRNA efficiently in two types of mutant cell lines:
one that cannot produce IFNs in response to dsRNA because the IFN genes
are missing and the second that cannot respond to type I IFN because
components of the IFN-signal transduction pathway are defective. Thus,
it is highly likely that dsRNA activates pre-existing cellular
proteins, leading to enhanced transcription of the 561 gene. The rapid
kinetics of induction and its insensitivity to cycloheximide also
support the above conclusion. Similar conclusions have been made by
other investigators, using cell lines missing IFN genes, about the
mechanism of induction of other IFN-inducible genes by
dsRNA(6, 32) . It is curious to note that, like its
induction by IFN-, induction of 561 mRNA by dsRNA was also
transient.
Our experiments demonstrated that all type I
IFN-sensitive genes are not equally induced by dsRNA. In the three
IFN-minus mutant lines, dsRNA failed to induce the 6-16 mRNA
appreciably, although it was very well induced by IFN-. The
testing of dsRNA inducibility of an IFN-sensitive gene in these cell
lines is more meaningful than in normal cell lines, since intermediate
synthesis of IFN does not complicate the picture. For this reason, much
of the information in the literature regarding dsRNA inducibility of
other IFN-sensitive genes needs to be re-examined in IFN-minus or
IFN-insensitive mutant lines. Our experiments show that the observed
difference in dsRNA inducibility is not due to the single nucleotide
difference in the two core ISREs. It remains possible that the observed
difference is not due to a difference in the ISRE sequences, but due to
the presence of a negative element in the 6-16 regulatory region.
The same point mutation in the 561 ISRE that abolishes its response
to IFN- also abolishes its response to dsRNA, thus establishing a
crucial role for the ISRE in the latter response. This mutation is
known to eliminate binding of ISGF3 and IRF-1 to the ISRE(18) .
Thus, these two factors are attractive candidates for mediating the
response to dsRNA. Our results with the IFN-
unresponsive
2fTGH-derived mutant cell lines clearly demonstrated that ISGF3 does
not mediate the action of dsRNA. Furthermore, these experiments
established that the p48 and STAT2 components of ISGF3 and the Tyk2 and
JAK1 tyrosine kinases are not absolutely required for the
ISGF3-independent pathway of transducing the dsRNA signal. However,
there were noticeable quantitative differences in the extent of 561
mRNA induction in different mutant cell lines missing these proteins.
The biochemical basis of these differences remains to be determined.
The requirement of the STAT1
protein, on the other hand, was more
pronounced. The signal was markedly reduced, although still detectable,
in cells missing STAT1
. Our results suggest that the STAT1
protein may be an ancillary protein for an ISRE-binding factor or its
absence may indirectly affect the function or the synthesis of a
protein which is directly involved in the signal transduction pathway
used by dsRNA. Alternatively, parallel pathways, one of which uses
STAT1
and another which does not, may exist. It appears that the
function of STAT1
in dsRNA signaling cannot be replaced by
STAT1
as is the case for IFN-
, but not IFN-
, signaling
pathway.
In contrast to the lack of a role of ISGF3, our studies
clearly indicate an important role for IRF-1 in ISRE-mediated signal
transduction by dsRNA. This signaling pathway is distinct from
ISRE-mediated IFN-/
signaling and kB-mediated signaling by
dsRNA or virus infection. There is somewhat conflicting information in
the literature about the role of IRF-1 in the latter two pathways. In
cells or mice devoid of functional IRF-1, type I IFN could be
efficiently induced by dsRNA or virus infection and IFN-
could
induce IFN-stimulated genes normally(22, 33) .
However, in another study, overexpression of IRF-1 in a human cell line
enhanced IFN-
mRNA induction by dsRNA and the expression of IRF-1
antisense RNA reduced the level of its induction, thus demonstrating
the need of IRF-1 in IFN-
mRNA induction(21) . Induction
of some genes by IFN-
was also partly affected by the absence of
IRF-1. Results presented here with the antisense IRF-1 RNA-expressing
cells clearly demonstrated that ISRE-mediated signaling by dsRNA also
requires IRF-1.
Although we provide here genetic evidence for the
involvement of IRF-1 and possibly p91 in ISRE-mediated signal
transduction, confirming biochemical evidence is yet to come. Unlike
the report by Daly and Reich(6) , we could not detect any
dsRNA-activated factor which binds to ISREs under conditions in which
dsRNA activation of NFkB was readily detected by electrophoretic
mobility shift assays (data not shown). The observation that the Y701F
mutant of STAT1 cannot support dsRNA signaling suggests that
tyrosine phosphorylation of STAT1
is required for this process.
However, we failed to detect tyrosine phosphorylation of STAT1
in
response to dsRNA although such phosphorylation was apparent in
response to IFN-
or IFN-
treatment (data not shown). This
observation indicates that STAT1
may not be directly involved in
the signaling process since the only known mechanism of its activation
is by tyrosine phosphorylation(12) . However, the formal
possibility remains that STAT1
can be activated by other
alternative means. Our failure to detect a specific dsRNA-stimulated
IRF-1-ISRE complex by mobility shift assays could be due to technical
difficulty. The induced complex may have a mobility very similar to
that of the constitutive complexes. These complexes have indeed been
shown to be highly heterogenous, containing different proteins but
having similar mobilities(34) . On the other hand, it is also
quite possible that the dsRNA-elicited signal does not change the
DNA-binding ability of the relevant factor (such as IRF-1) but changes
its function. This will be similar to the case for cAMP-induced
transcriptional regulation of genes, which contain the cis-acting
cyclic AMP-response element to which the transcription factor
CRE-binding protein binds(35) .