(Received for publication, December 16, 1996, and in revised form, June 5, 1997)
From the Molecular Genetics and Development Group,
Institute of Reproduction and Development, Monash University, Monash
Medical Centre, Clayton, Victoria 3168, Australia and the
§ Department of Pathology, University of Tennessee Health
Science Center, Memphis, Tennessee, 38163
The type I interferons (IFNs) are a family of
cytokines, comprising at least 17 subtypes, which exert pleiotropic
actions by interaction with a multi-component cell surface receptor and at least one well characterized signal transduction pathway involving JAK/STAT (Janus kinase/signal
transducer and activator of
transcription) proteins. In a previous report, we showed
that a signaling factor, encoded by a gene located on the distal
portion of chromosome 21, distinct from the IFNAR-1 receptor, was
necessary for 2-5
-oligoadenylate synthetase activity and antiviral
responses, but not for high affinity ligand binding. In the present
studies using hybrid Chinese hamster ovary cell lines containing
portions of human chromosome 21, we show that the type I IFN signaling
molecule, designated herein as ISF21, is distinct from the second
receptor component, IFNAR-2, which is expressed in signaling and
non-signaling cell lines. The location of the gene encoding ISF21 is
narrowed to a region between the 10;21 and the r21 breakpoints,
importantly eliminating the Mx gene located at 21q22.3 (the product of
which is involved in IFN-induced antiviral responses) as a candidate for the signaling factor. To characterize the action of this factor in
the type I IFN signaling pathway, we show that it acts independently of
receptor down-regulation following ligand binding, both of which occur
equally in the presence or absence of the factor. In addition, we
demonstrate that ISF21 is necessary for transcriptional activation of
2
-5
-oligoadenylate synthetase, 6-16, and guanylate-binding protein
gene promoter reporter constructs, which are mediated by several
signaling pathways. ISF21 represents a novel factor as the localization
to chromosome 21, and the data presented in this study exclude any of
the known type I IFN signal-transducing molecules.
The type I interferons
(IFNs)1 are a family of
species-specific, multifunctional cytokines, which in humans include 15 subtypes of IFN with 75-98% amino acid identity, IFN
with 70%
identity to consensus IFN
, and the least related IFN
with 35%
identity to IFN
(1). Despite quantitative differences in biological specific activities among type I IFN subtypes (2) and differences in
antigenicity (3), they all induce similar biological functions in human
cells (4) and compete for binding to cell surface receptors (5).
The first cloned component of the human type I IFN receptor (designated
as IFNAR-1), when expressed in mouse BTG9A cells, appeared to
selectively mediate responses to a restricted range of type I IFNs:
only IFNB but not IFN
2 nor IFN
(6). The inability of
IFNAR-1-transfected cells to respond to all IFNs may have been due to
the absences of other human receptor components and a difference in the
ability of these subtypes to interact with (other) murine receptor
components. Indeed, the definition of the role of IFNAR-1 in ligand
binding has been complicated by the differences in results obtained
when the receptor was expressed in different types of host cells.
Recently, a second IFN receptor component (encoded by a gene designated
as IFNAR-2) was identified and shown to exist as a soluble form
(IFNAR-2a) and a transmembrane form with a short cytoplasmic domain
(IFNAR-2b) (7). This component was shown to bind type I IFNs
B,
2,
C, and
by cross-linking experiments, and when co-expressed
with IFNAR-1 in murine cells bound 125I-IFN
2 with an
affinity of ~300 pM. However, the function of IFNAR-2b in
signal transduction was unclear (7). Recently, it has been shown that
the IFNAR-2 gene encodes a third form with a longer cyotoplasmic
domain, designated as IFNAR-2c, which mediates signaling when
co-expressed with IFNAR-1 in murine L929 cells (8, 9).
IFNAR-1 has been localized to human chromosome 21 in the region 21q22.1
(5, 6, 10). We recently showed using a panel of CHO-human chromosome 21 hybrid cells that there is a gene(s) encoded in the region 21q22.2-3,
and therefore distinct from IFNAR-1, that is necessary for type I IFN
signal transduction (11). Cells containing human chromosome 21 proximal
to the 8;21 breakpoint (21q+) expressed the mRNA for IFNAR-1 and
bound IFNs B,
2, and
with an affinity of approximately 200 pM, indicating that the region 21q22.1 contained factors,
in addition to IFNAR-1, required for ligand binding. However, unlike
cells that contained the entire chromosome 21, the 21q+ cell line did
not signal as measured by induction of 2
-5
-oligoadenylate synthetase
enzyme activity and antiviral responses.
In the present study, we show that the type I IFN signaling factor encoded on human chromosome 21, now designated as ISF21, is distinct from IFNAR-2 as well as IFNAR-1 receptor components, both of which are expressed in the hybrid cell lines, including those that do not signal. The Mx gene was a candidate for the signaling factor by virtue of it exhibiting properties of a signaling molecule and its ability to induce an antiviral state and its location on human chromosome 21q22.3. In the present study, the location of the gene encoding ISF21 is narrowed to a 400-kb region between the 10;21 and r21 breakpoints which is proximal to the Mx locus. Despite the lack of signaling, hybrid cells containing the IFNAR-1 and -2 genes but not containing ISF21 are shown to bind a range of IFN subtypes, which compete with each other and undergo ligand-dependent down-regulation of IFN binding sites. Thus the signaling factor ISF21 acts independently of ligand-receptor interaction, processing, and receptor down-regulation. Importantly, we also demonstrate, using a range of IFN-sensitive reporter constructs, that the signaling factor ISF21 is necessary for signaling pathways prior to activation of IFN-responsive genes.
The parental CHO-K1 cell line
was obtained form the American Type Culture Collection. The following
CHO-human chromosome 21 containing hybrid cell lines were obtained from
D. Patterson (Eleanor Roosevelt Institute, Denver, CO): 21q+, MRC 2G,
10;21 (9542C-5a), 6918-8a1, R2-10W, 21;22 (RAJ-5), 643C-13 (7;21),
and 72532x6. The human chromosomal complement of the hybrids has been
described elsewhere (12) and is summarized in Fig. 2. The CHO-K1 cell line was stably transfected with the human IFNAR-1 cDNA contained in an expression vector controlled by the sheep metallothionein promoter (pTV2, 13), by electroporation at 960 microfarads and 270 V. Several independent clones were expanded, and expression was confirmed
by RT-PCR (data not shown). All cell lines were grown in RPMI 1640 medium supplemented with 5% dialyzed fetal calf serum, 100 units/ml
penicillin, 100 µg/ml streptomycin, except CHO-K1 and 21q+ cultures,
which were also supplemented with 2.3 mg/ml proline. HeLa cells were
grown in Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin.
The IFNs used in this study were huIFN2a (Hoffman La Roche, Basel,
Switzerland), huIFN
B (Ciba Geigy, Basel, Switzerland),
huIFN
ser (Berlex Laboratories, Alameda, CA),
huIFN
con1 (Amgen, Thousand Oaks, CA), and Wellferon (human
lymphoblastoid IFN, Wellcome Laboratories, UK). IFN
4 was transcribed
using SP6 polymerase and translated using rabbit reticulocyte lysate as described previously (14); control experiments using rabbit reticulocyte lysate only had no effects on gene induction in the hybrid
cell lines (data not shown). For receptor binding studies, the IFNs
were iodinated using modified chloramine-T procedures to a specific
activity of ~100 µCi/µg and the integrity of 125I-IFN
was monitored as described previously (11, 15).
Receptor Binding and Down-regulation
Receptor binding
assays were performed essentially as described previously (11).
Scatchard analysis of binding curves was performed using the LIGAND
program and was found to be statistically significant
(p < 0.05) only when resolved by a "one-site" fit. Competitive binding experiments were performed using 400,000 cpm of
125I-IFNB and 1-, 3-, 10-, 30-, 100-, and 300-fold
excess IFN
B,
2 and
, essentially as described previously
(16).
The down-regulation of ligand binding sites on the cell surface was
determined using 125I-IFNcon1 essentially as described
previously (17). The number of receptors per cell was determined by a
conventional ligand binding assay and Scatchard analysis before and
after incubation with 20,000 IU of this IFN/ml for 18 h. A similar
study was undertaken using an 125I-4B1 monoclonal
anti-IFNAR-1 antibody (17) to measure the number of IFNAR-1 chains.
Primers were generated using the published IFNAR-2 cDNA sequence (7), spanning the regions 219-240 bp and 1203-1222 bp, which encompass the ATG and TGA codons, respectively. Reverse transcription was carried out using 3 µg of total RNA derived from human Daudi cells using avian myeloblastosis virus reverse transcriptase (Promega) at 42 °C and the antisense primer. PCR was subsequently performed on the cDNA under the following conditions: 93.5 °C for 60 s, 56 °C for 60 s, and 72 °C for 90 s for 35 cycles. The PCR product was electrophoresed on a 1% agarose gel, and a band of the expected size of 1003 bp was observed. The PCR product was cloned into pGEM-T (Promega) and sequenced, using an automated DNA sequencer (Applied Biosystems).
Northern BlotsCells were grown to mid-log phase, harvested, and poly(A)+ mRNA extracted as described previously (18). Approximately 9 µg of RNA in 50% formamide was electrophoresed on 1% agarose-formaldehyde gels, transferred to Hybond C membranes (Amersham) in 20 × SSC overnight. Filters were then baked at 80 °C for 2 h and prehybridized at 42 °C for 2-3 h.The filters were hybridized with a 32P-labeled IFNAR-2 cDNA probe as described previously (11), stripped, and reprobed overnight at 42 °C with a 32P-labeled 1.1-kb fragment of the glyceraldehyde-3-phosphate dehydrogenase cDNA as a control for RNA loading. After hybridization and washing in 0.1 × SSPE, 0.1% SDS at 65 °C, signals were visualized by autoradiography onto Kodak BioMax film.
Analysis of IFN-stimulated Gene Promoter Activity in Hybrid Cell LinesTo construct a plasmid containing the 2-5
-OAS
promoter-CAT reporter (25A-CAT), a human 2
-5
-oligoadenylate
synthetase gene promoter fragment corresponding to residues 525-1435
in the published sequence (19) was generated by PCR using
oligonucleotides 5
-GAACTCTCTGCACATTCAGC-3
and
5
-GGAAACACGTGTCTGGCAAC-3
and cloned into the pCRII vector (Invitrogen Corp.). A SpeI restriction site was then created
18 bp 5
of the ATG by PCR. A XbaI-SpeI fragment
encompassing
834 to +29 of the 2
-5
-OAS promoter was then cloned
into the XbaI site of pCATBasic (Promega). Reporter
constructs containing a human 6-16 promoter fragment (fragment no. 3 in Ref. 20) were constructed by first digesting the p30X plasmid (gift
from P. Rathjen, Department of Biochemistry, University of Adelaide)
with HindIII and then end-filling with Klenow (Promega).
After digestion with BglII, the fragment was ligated into
the vector pGL3-Basic (Promega) that had been digested with
SmaI and BglII, to give the p30XLuc construct.
The vector had also been modified to contain a neomycin resistance
gene, derived form pMCINeo, that had been inserted into the unique
SalI site of pGL3-Basic. The GBP promoter-luciferase construct (GBP-LUC) was a gift from B. R. G. Williams
(Cleveland Clinic Foundation, Cleveland, OH).
For analysis of reporter activity, cells were diluted to 1 × 107 cells/ml in electroporation buffer (20 mM
Hepes, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM
glucose, 0.1 mM 2-mercaptoethanol, pH 7.0) and 0.5 ml was
incubated with 5 µg of 25A-CAT, 10 µg of 30x.Luc or 10 µg of
GBP-Luc, and 5 µg of pSV--galactosidase Control vector (Promega)
before electroporation at 960 microfarads and 270 V in a Gene Pulser
(Bio-Rad). Cells were plated in 10-cm dishes and allowed to recover
overnight before incubation with 100 IU/ml various type I IFNs (as
indicated) for 16 h. Cells were then harvested, sonicated in 250 mM Tris, pH 8, and 20 µl of clarified lysate incubated as
described previously for CAT enzyme activity (21). For luciferase
assays, cells were lysed directly in reporter lysis buffer (Promega)
and luciferase light units measured using a Promega Luciferase kit and
a Berthold luminometer.
As a control for transfection efficiency, -galactosidase enzyme
activity was determined by incubation of 30 µl of clarified lysate,
prior to heat inactivation, with 2 ×
-galactosidase buffer (200 mM NaPO4, pH 7.3, 2 mM
MgCl2, 100 mM
-mercaptoethanol, 1.33 mg/ml
o-nitrophenyl-
-D-galactopyranoside) in a
total volume of 100 µl, at 37 °C for 30 min, and the absorbance at
415 nm read using a microplate reader (Bio-Rad). The CAT activity was
determined as the percent of substrate converted to product, then
expressed relative to the
-galactosidase activity for the same
sample. Results were shown as -fold induction by IFN relative to
untreated controls. Luciferase light units were determined relative to
-galactosidase enzyme activity and expressed as -fold induction by
interferon relative to untreated controls.
Cells
were incubated with 0 or 1000 IU/ml IFN for 48 h before being
harvested and lysed. Enzyme activity was determined by the
incorporation of [-32P]ATP into alkaline
phosphatase-resistant 2
-5
-oligoadenylate-resistant "cores," as
described previously (22).
To determine whether the lack of signaling observed
previously in the 21q+ cells was due to the absence of IFNAR-2, we
performed Northern blot analysis (Fig.
1). A full-length cDNA probe for IFNAR-2a (7) was generated by RT-PCR using human Daudi cell total RNA.
It is noteworthy that the sequence of the IFNAR-2a cDNA was
identical to the published sequence (7) except for 3 nucleotides. At
nucleotide 700, a change from C to T would result in an amino acid
change from Pro to Ser at amino acid residue 212; at nucleotide 859, an
A to G change would result in a change from Thr to Ala at residue 265, while a change from T to C at nucleotide 501 would be silent. Using
this cDNA as a probe, IFNAR-2 mRNA transcripts were detected in
all of the human chromosome 21 hybrid cells, which had previously been
reported to also express IFNAR-1 (11), but not in the parental CHO
cells. As an example, Fig. 1 shows a Northern blot analysis of poly(A)+
mRNA from one signaling (72532x6) and one non-signaling (21q+) cell
line and the parental CHO-K1 cells. Two transcripts of approximately
4.5 and 1.5 kb were observed, consistent with published data (7). This
result indicates that the IFNAR-2 gene is encoded on human chromosome
21, in the region 21p-q22.1, as is IFNAR-1, and both are expressed even
in the hybrid cell lines which do not signal (see below, and Ref.
11).
Location of the Gene Encoding the Signaling Factor ISF21 to a 400-kb Region on Human Chromosome 21q22.2
Previous studies had
described the location of a gene encoding a type I IFN signaling factor
to be on the distal third of chromosome 21, distal to the 8;21
breakpoint. This region of human chromosome 21 contained the Mx genes,
which possess GTPase activity and contain Zn finger motifs
characteristic of signaling molecules, and are necessary for some
antiviral responses to type I IFNs. Therefore it was important to
determine whether ISF21 could be distinguished from Mx and at the same
time, to narrow down the region containing this gene to facilitate
further cloning studies. We therefore examined an extended panel of
CHO-human chromosome 21 hybrid cell lines which contained smaller
chromosomal deletions (Fig. 2). Induction of 2-5
-OAS
enzyme activity was observed after IFN treatment in cell lines
containing human chromosome 21 fragments which extended further than
the r21 breakpoint, namely R2-10W, RAJ 5, 643C-13, and 72532x6.
However, no induction of 2
-5
-OAS was observed in hybrid cell lines
which contained only human chromosome 21 sequences proximal to the
10;21 breakpoint, namely 6918-8a1, MRC 2G, and 21q+. This was despite
the observation that these non-signaling cell lines expressed the genes
encoding both known type I IFN receptor components. Therefore, the gene encoding the signaling factor ISF21 is located between the 10;21 and
r21 breakpoints, and is thus distinct from the IFN receptor locus which
lies in the region between the 6918 and 8;21 breakpoints (9).
Furthermore, the signaling factor designated ISF21 is not the Mx gene,
which would be absent from the R2-10W cell line, whereas this cell
line does transduce signals.
Recently an arginine methyltransferase, termed IR1B4, has been shown to associate with the type I IFN receptor and was implicated in IFN signaling (23); interestingly, a related arginine methly transferase, hHMT 1, was localized to human chromosome 212 (GenBankTM accession no. X99209). We generated a probe for the latter gene for Southern blot analysis of the panel of hybrid cell lines. This gene was detected in the 643C-13 cell line, but not in 21q+, MRC-2G, 6918-8a1, or RAJ 5 (data not shown) and therefore did not fit the pattern of expression of ISF21.
Signaling Factor ISF21 Acts Independently of Receptor Down-regulation following Ligand-Receptor InteractionSince both
IFNAR-1 and IFNAR-2 receptor components are known to be expressed in
signaling and non-signaling cell lines, it was important to determine
whether both components contributed to the IFN ligand binding process,
which had been previously shown to be normal in all hybrid cell lines.
Studies of binding to the 21q+ cell line showed the
125I-IFNB can bind in a dose-dependent,
saturable manner (Kd of 201 pM, 488 binding sites/cell) (Fig. 3A).
However, after CHO-K1 cells were stably transfected with human IFNAR-1
cDNA, no specific binding of 125I-IFN
B could be
detected in several independent, transfected cell lines (Fig.
3A). IFNAR-1 expression was confirmed by Northern blot (data
not shown) analysis. Thus, multiple components of the type I IFN
receptor encoded by genes on chromosome 21 are required for high
affinity binding, presumably IFNAR-1 and IFNAR-2.
Next, binding competition studies were performed using a non-signaling
cell line, deficient in the signaling factor ISF21, to ascertain
whether the signaling factor might have a more subtle influence on the
ability of the receptor complex to bind multiple type I IFN ligands.
Competitive binding experiments show that a range of type I IFNs,
namely B,
2, and
, all compete for binding to the 21q+ cells
(Fig. 3B). The different slopes reflect the different
binding affinities of type I IFNs, particularly the relatively high
binding affinity of IFN
(consistent with previous studies; see Refs.
16 and 24). However, insufficient "cold" IFNs were available to
achieve complete inhibition. The concentration for 50% inhibition of
binding is 344 pM for IFN
B, 629 pM for
IFN
2, and <102 pM for IFN
. The important result for this study was that the absence of the signaling factor did not affect
the ability of all these type I IFNs to compete for receptor interaction.
An early event in signal transduction that follows ligand-receptor
engagement is internalization of the complex and subsequent down-regulation of cell surface components of the type I IFN receptor. In many receptor systems, this step depends on phosphorylation, an
early signal-transducing event. Furthermore, the inability to
down-regulate cell surface receptors correlates with insensitivity to
IFN action (17). It was therefore possible that the signaling factor,
ISF21, could be required for receptor down-regulation. We therefore
examined whether the hybrid cell lines, which bound IFNs but had
previously been shown not to signal, could down-regulate the
ligand-receptor complex. As shown in Table
I both the 21q+ non-signaling and 72532x6
signaling cell lines had similar numbers of binding sites for
IFNcon1 and an anti-IFNAR-1 monoclonal antibody on the cell surface.
Furthermore, both cell lines showed equivalent down-regulation of
receptors 18 h after incubation with IFN, whether determined by
ligand binding (~50% each) or binding of monoclonal antibodies to
the receptor (~20% each). Thus the proposed signaling factor, ISF21,
which is absent from the 21q+ cell line, must not be required for this
step in the processing of the ligand-receptor complex.
|
Previous studies had
indicated that the type I IFN signaling factor was required for
induction of 2-5
-OAS enzyme activity (11), but it was not known at
which stage in the IFN-dependent increase of this enzyme
this factor acted. To better define the nature of this factor, we set
out to determine at what stage of IFN signaling the factor acted and
whether it was involved in the induction of other IFN-responsive genes.
First, a 910-bp fragment from the 2
-5
-OAS promoter region, which
contains all the elements necessary for the induction of transcription
of this gene, was ligated upstream of a CAT reporter gene (25A-CAT).
The CHO-K1, 21q+, and 72532x6 cell lines were transiently transfected
with 25A-CAT and cotransfected with a
-galactosidase construct as a
control for transfection efficiency. After treatment with various human
type I IFNs, the parental CHO and 21q+ cell lines showed no significant
induction of CAT activity, apart from a weak induction with huIFN
due to a low level of reactivity with hamster cells (Fig.
4A). All type I IFNs tested, namely
2,
4,
B, Wellferon, and
, induced CAT activity in the 72532x6
cell line but not in the 21q+ cell line (Fig. 4A), again
emphasizing that ISF21 is necessary for signaling in response to a
broad range of type I IFNs. Treatment of the same three cell lines with
murine IFN
4 resulted in induction of the reporter to a similar
extent in all three cell lines consistent with this IFN acting through
the hamster receptors. This result importantly demonstrates that all of
the components necessary for transcriptional activation of the
2
-5
-OAS-reporter are present in these cells; but they cannot be
activated through the human type I IFN receptor in the absence of
ISF21.
To determine if ISF21 was also necessary for the transcriptional
activation of other IFN-responsive genes, two other ISGs were analyzed
using this system. The 6-16 gene promoter was shown to be responsive
to huIFNB in the 72532x6 cells, which contain ISF21, but not in
21q+, which lack this factor (Fig. 4B). Interestingly, the
level of induction of 2
-5
-OAS and 6-16 reporter constructs was
similar, namely 4-5-fold, and both genes are known to be inducible via
ISGF3 binding to ISRE elements. The third promoter construct used in
this study, the GBP-LUC, was chosen because GBP is reportedly induced
independently of ISGF3 binding to the ISRE, but instead through the
IRF-1 and NF-
B transcription factors (25). Although only a low level
of induction was observed in the 72532x6 cell line by IFN
B, it was
similar to that detected in the human HeLa cell line (Fig.
4C). Importantly, no induction was observed in the 21q+ nor
the CHO K1 cell lines (Fig. 4C), indicating that the
signaling pathway responsible for the induction of the GBP promoter
in these cells is also dependent on ISF21.
The data presented herein establish several important points about
the type I IFN signaling molecule, designated as ISF21. 1) It is
distinct from the receptor component IFNAR-2 as well as IFNAR-1. 2) It
acts independently of down-regulation of the functional receptor
subsequent to ligand binding. 3) It is localized to a 400-kb region on
human chromosome 21 between the 10;21 and r21 breakpoints and thus
distinguished from the Mx gene, which encodes an IFN inducible
antiviral molecule, and from an arginine methyltransferase gene related
to a proposed IFN signaling molecule. 4) It is essential for the
induction of the interferon-inducible genes 2-5
-oligoadenylate
synthetase, 6-16, and guanylate-binding protein and therefore probably
involved early in signal transduction for activation of several
pathways for induction of IFN-responsive genes.
Human IFNAR-2 cDNA was generated by RT-PCR using RNA from Daudi
cells and used to demonstrate that hybrid cell lines containing portions of chromosome 21 contained the IFNAR-2 gene and
expressed both mRNA transcripts observed for this gene. We had
previously shown by direct binding studies that these cells bind human
type I IFN ligands with an affinity of approximately 200 pM
and that the affinity and number of binding sites are not affected by
the presence or absence of the signaling factor. Interestingly, hamster cells containing a yeast artificial chromosome expressing both IFNAR-1 and IFNAR-2 (26), or murine cells containing cDNA
for human IFNAR-1 and -2 (27) also bind type I IFNs with affinities of
200-300 pM. In CHO cells, we found that IFNAR-1 alone was
insufficient to enable any detectable binding of human type I IFNs.
Therefore the products of these two genes are necessary and may account for all the receptor components necessary for binding to the type I IFN
ligands. The conflicting data previously reported on the necessity of
huIFNAR-1 for the binding of type I IFN ligands (6, 8, 9, 28) probably
reflect the requirement for more than one component for binding and the
variable ability of different type I IFNs to interact with other
endogenous receptor components in non-human cells. The fact that IFNs
2,
B, and
(the least homologous type I IFN) compete for
binding to the 21q+ cells, albeit with different affinities, indicates
that they share at least one, probably both of these chromosome
21-encoded binding components, and that competition between type I IFNs
is not influenced by the signaling factor ISF21.
Although hybrid cells containing IFNAR-1 and -2 genes bind type I IFNs, they do not signal unless the distal portion of chromosome 21 is present as evidenced by our studies on three independent non-signaling cell lines. There have been reports of similar situations in human cell lines that bind IFN but are insensitive to the biological actions of IFNs; these cell lines did not efficiently down-regulate the receptor-ligand complex (29). It was therefore possible that the signaling factor described herein might be involved in down-regulation of receptors. Our data clearly show that this was not the case, since a signaling (72532x6) and non-signaling (21q+) hybrid cell line both down-regulated the type I IFN receptor complex to similar levels, using two different methods to measure this phenomenon. Thus ISF21 is not required for the down-regulation process, and acts either at a step in signal transduction that occurs after the down-regulation of the ligand-receptor complex or independently of it.
Our previous data showed that ISF21 is necessary for induction of
2-5
-OAS enzyme activity by IFNs
2,
B, and
(11). However, it
was not clear whether this signaling factor was necessary for
transcriptional activation of this IFN-responsive gene or if it was
involved in post-transcriptional regulation of enzyme activity. The
data presented herein using the 2
-5
-OAS promoter-CAT reporter
construct demonstrated that the ISF21 was necessary for transcriptional
activation of this ISG in response to many type I IFNs, indicating its
importance in signaling by probably all type I IFNs. Our results may
seem to be in apparent contradiction to other reports using murine (8)
or human (9) cells, wherein a combination of human IFNAR-1 and IFNAR-2
products is sufficient for binding and signaling in response to IFN (8,
9). An explanation for this discrepancy could be that the hamster ISF21 does not interact with human IFN receptors, whereas the murine ISF21
does. This explanation is consistent with observations on human IFN
binding to human IFNAR-1, which occurs when expressed in murine cells
but not in CHO cells (see below and Ref. 26), suggesting a species
specificity for facilitating ligand binding in mouse but not hamster
cells. It is important to note that signal tranduction through the
endogenous hamster components could be detected since murine IFN
induced the 2
-5
-OAS-CAT reporter (and 6-16 as well) in CHO-K1 as
well as 21q+ and 72532x6 hybrid cell lines. This demonstrates that all
of the "downstream" components required for transcriptional
activation are present in the hybrid cell lines, but that human ISF21
is necessary, in a species-specific manner, for transactivation of
IFN-responsive genes.
To examine the scope of ISF21 action in IFN signaling, we examined the
responsiveness of the two other IFN-responsive genes, 6-16 and GBP.
The 6-16 gene promoter, like the 2-5
OAS contains multiple
IFN-responsive elements, ISRE, interferon response element, and
activated sequence (25), but are mainly inducible by type I IFNs via
ISGF3 binding to the ISRE (25). The GBP promoter, although more weakly
inducible by IFN
than by IFN
, is regulated by IRF-1 and NF-
B
rather than ISGF3 (25). Importantly, all three promoter constructs were
dependent on ISF21 by virtue of their induction in 72532x6 cells but
not in 21q+ or CHO K1 cells. These data indicate that ISF21 is
necessary for several signaling pathways activated by type I IFNs. This
conclusion is supported by the necessity of ISF21 for an antiviral
response in these cells, which could be achieved via multiple signaling
pathways or ISGs.
Mx genes, located on the distal portion of human chromosome 21 were candidates for ISF21, since these are known to be induced by type I IFN, are involved in signal transduction. In these studies we have narrowed the chromosomal localization of ISF21 to the region on chromosome 21 between the 10;21 and the r21 breakpoint, indicating that the Mx gene is not ISF21. There was also circumstantial evidence for an arginine methyltransferase involvement in IFN signaling (23), but this was also excluded on the basis of gene mapping in signaling and non-signaling cell lines. Furthermore, since none of the well characterized IFN signaling molecules such as JAKs (Janus kinase) and STATs (signal transducer and activator of transcription) are encoded by genes on chromosome 21, ISF21 is likely to be a novel IFN signaling factor. Its cloning will be facilitated by the narrowing of the location of its gene reported herein.
Thus, as shown in Fig. 2, there is a cluster of genes associated with IFN response found on human chromosome 21, which include IFNAR-1, IFNAR-2, CRF2-4, IFNGR-2, ISF21, MX-1 and MX-2. The factors encoded by these genes are essential for biological responses to IFNs including the regulation of cell proliferation and differentiation, and immune responses. It is interesting to note that in Down syndrome, where there is trisomy of chromosome 21, there is retarded growth and perturbations of the immune system, which may be in part due to altered regulation of the various IFN response genes located on this chromosome.
We acknowledge Lerna Gulluyan for technical assistance and D. Patterson, Eleanor Roosevelt Institute, Denver, CO for the hybrid cell lines.