From the Department of Neurosciences, The Lerner
Research Institute, The Cleveland Clinic Foundation, Cleveland,
Ohio 44195, § Institut Pasteur, INSERM U 276, Paris 75724 Cedex 15, France, and ¶ Department of Medical Genetics & Microbiology, University of Toronto, Toronto, Ontario
M5S 3E2, Canada
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
The Interferons (IFNs)1
elicit multiple biological responses including antiproliferative and
immunomodulatory activities, which are mediated by the proteins encoded
by IFN-stimulated genes (ISGs) (1-4). The structurally related
superfamily of type I IFNs (in humans, 14 expressed subspecies of
IFN- Interaction of IFN with cognate receptor initiates JAK-STAT signaling,
commencing with tyrosine phosphorylation and activation of two
receptor-associated Janus kinases (JAK), JAK1 and TYK2, with subsequent
phosphorylation of cytoplasmic tyrosine residues of the IFN-receptor
subunits, IFNAR1 and IFNAR2c. The major signaling output of the
ligand-stimulated IFN receptor consists of activated cytoplasmic
transcription factors called STATs (signal transducers and activators
of transcription). Type I IFNs activate STAT1, STAT2, and STAT3.
Activated STAT1 and STAT2 homo/heterodimerize and translocate to the
nucleus. The majority of ISGs are activated by the transcription factor
IFN-stimulated gene factor 3 (ISGF3), consisting of STAT1/STAT2
heterodimers in association with a 48-kDa DNA-binding protein. ISGF3
binds to IFN-stimulated response elements (ISREs) in promoters of ISGs
(7, 8).
JAK-STAT signaling is clearly essential for IFN-mediated biological
responses (9). However, several lines of evidence indicate that this
central pathway does not solely account for the biological effects of
IFNs. In structure-activity studies of the type I IFN receptor,
antiviral function was dissociated from antiproliferative action
through selective receptor mutations. In these experiments, low
affinity interaction between IFN- Clarifying the accessory signaling that is required for full biological
responses to IFNs may help elucidate the observed distinctions between
type I IFNs in clinical and antiviral protocols. In this regard, recent
reports suggest that interactions of the different IFN subtypes with
receptor may generate distinct signaling outputs. For example, IFNAR2c
co-immunoprecipitated with IFNAR1 in cells stimulated with IFN- We recently reported studies of the regulation of a gene designated
The signaling pathway whereby IFN- Unexpectedly, U1 cells that lacked TYK2 expression failed to express
TYK2 is a 135-kDa cytosolic protein characterized by the presence of a
C-terminal protein tyrosine kinase (TK) domain and an adjacent
kinase-like (KL) domain. Five further domains of substantial amino acid
similarity with other JAKs extend to the N terminus of the protein and
are designated JAK homology domains (Fig. 2A) (20, 21).
The functions of receptor-associated tyrosine kinases TYK2 and JAK1 in
type I IFN signaling pathway have been well established in part through
the study of IFN- Prior reports delineated various functions for TYK2 in IFN signaling.
First, TYK2 protein is required to sustain expression of IFNAR1; the
complete failure of U1 cells to respond to IFN- In this report, we describe further investigation of the role of TYK2
in the induction of Cells and Interferons
Human fibrosarcoma 2fTGH cells were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% calf serum (18). U1.wt-5, U1.KR930, U1.YYFF1054-55, U1. Cloning the Promoter of A human genomic library (CLONTECH) was
screened using the partial Transient Transfection Assay
Plasmids--
A 303-bp upstream putative
A 102-bp promoter-reporter construct containing the ISRE from the p56
ISG was provided by Dr. Ganes Sen (Lerner Research Institute, Cleveland
Clinic Foundation, Cleveland, OH).
A simian virus 40 promoter- Assays--
2fTGH cells, U1.wt-5, and U1.KR930 cells were grown
to 70-80% confluency in 100-mm plates, co-transfected with 10 µg of
test plasmid DNA and 1.5 µg of pCH110 Cell Extracts and EMSA
Cells were treated with IFNs or reserved as controls, harvested,
and lysed for 15 min on ice in hypotonic buffer containing 20 mM HEPES (pH 7.9), 20 mM NaF, 1 mM
Na3VO4, 1 mM
Na4P2O7, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, leupeptin (1 µg/ml),
aprotinin (1 µg/ml), and pepstatin (1 µg/ml). The extract was
centrifuged at high speed for 1 min. The pellet was discarded and 0.1 M NaCl added to the supernatant and microcentrifuged for 10 min and adjusted to 10% glycerol. Protein concentration was measured
by the method of Bradford by using protein dye reagent (Bio-Rad).
Extracts were stored at For binding reactions, extracts (10 µg of protein) were incubated
with a 32P-labeled oligonucleotide probe (0.5 ng,
20,000-40,000 cpm) in buffer containing 6 mM HEPES, 1 mM dithiothreitol, 6% glycerol, and 5 µg of poly(dI-dC)
for 20 min at room temperature. An oligonucleotide probe corresponding
to ISRE sequence of the 6-16 gene,
5'CCTTCTGGGAAAATGAAACTCA3' was used. The reaction products were
resolved on 8% non-denaturing polyacrylamide gels which were dried and
analyzed by autoradiography.
RNase Protection Assay
Total RNA was prepared from IFN-treated cells using the TRIzol
method (32), and protection experiments were performed as described
(16). The probes used were Selective Induction of
The differential regulation of Induction of
IFN-
To address the possibility that inability to induce
Induction of 6-16 by either IFN- Overexpression of TYK2 Selectively Augments Induction of IFN-induced Generation of ISGF3 Is Not Defective in U1.KR930
Cells--
Failure of IFN-
Normalized transient transfection analysis was used to determine the
function of this element in U1.wt-5 and U1.KR930 cells. p Our current results suggest that catalytically active TYK2 is
required for accessory signaling that results in transcription of
Furthermore, IFN- The failure of IFN- A 303-bp In summary, data described in this report demonstrate that the pathway
utilized by IFN--R1/I-TAC
(interferon-inducible T-cell
-chemoattractant) gene encodes an
-chemokine that is a potent chemoattractant for activated T-cells.
We previously reported that
-R1 was selectively induced
by interferon (IFN)-
compared with IFN-
and that the canonical
type I IFN transcription factor interferon-stimulated gene factor 3 (ISGF3) was necessary but not sufficient for
-R1 induction by IFN-
. These findings
suggested that
-R1 induction by IFN-
required an accessory component. To begin characterizing this signaling
pathway, we examined the function of TYK2 protein in the
IFN-
-mediated induction of
-R1. This
study was motivated by the observation that
-R1 could
not be induced in TYK2-deficient U1 cells by IFN-
(Rani, M. R. S., Foster, G. R., Leung, S., Leaman, D., Stark, G. R., and Ransohoff, R. M. (1996) J. Biol. Chem.
271, 22878-22884), an unexpected result because IFN-
evokes
substantial expression of IFN-stimulated genes (ISGs) in U1 cells
through a TYK2-independent pathway. We now report
-R1
expression patterns in U1 cells complemented with wild-type or mutant
TYK2 proteins. Complementation with wild-type TYK2 rescued
IFN-
-inducible expression of
-R1. Cells expressing
kinase-deficient deletion or point mutants of TYK2 were refractory to
induction of
-R1 by IFN-
despite robust expression of
other ISGs. Transient transfection analysis of a
-R1
promoter-reporter confirmed that transcriptional activation of
-R1 by IFN-
required competent TYK2 kinase. These
studies indicate that the catalytic function of TYK2 is required for
IFN-
-mediated induction of
-R1. Catalytic TYK2 is the
first identified component in an accessory signaling pathway that
supplements ISGF3/interferon-stimulated response element signaling for
gene induction by type I IFNs.
INTRODUCTION
Top
Abstract
Introduction
References
and 1 expressed IFN-
and IFN-
) shares a common receptor
and signal transduction apparatus (5). Type I IFN subspecies exert
varying antiviral, antiproliferative, and clinical effects, but the
bases for these differences are not understood at the biochemical level
(6).
and receptor supported ISGF3
activation and antiviral response, without IFN-inducible growth
inhibition; an additional component encoded on human chromosome 21 was
required for high affinity binding and growth inhibition (10).
Analogous observations in the type II IFN system were made by preparing
cells that expressed kinase-deficient JAK1; these cells expressed
JAK-STAT-dependent gene-regulatory and antiviral responses
to IFN-
, without demonstrating IFN-
-mediated growth inhibition
(11).
but
not IFN-
2 (12-15). This result suggested that IFN-
engagement
generated a more stable signaling complex than IFN-
2. Recently, it
has been shown that IFN-
2 and IFN-
require distinct
intracytoplasmic regions of the IFNAR-2 chain of the receptor to elicit
an antiviral response (15).
-R1, which was selectively induced by IFN-
compared with IFN-
in astrocytoma and fibrosarcoma cells (16). Sequence analysis of cDNAs indicated that
-R1, which was
initially cloned by differential display, was predicted to encode a
chemokine-like peptide with an N-terminal CXC motif.
Independently, Neote and colleagues (17) isolated a chemokine
designated I-TAC from cytokine-treated fetal astrocytes. Initial
comparisons indicated that
-R1 and I-TAC were highly
related, and sequence analysis of multiple independent cDNA
isolates has established identity between the two chemokines.
-R1/I-TAC possesses potent chemoattractant activity
toward activated T-lymphocytes (17).
induces the expression of
-R1 appears to be novel. Our studies, conducted in
fibrosarcoma cell lines that were deleted for individual constituents
of the IFN signaling pathway, established the following: 1)
-R1 was selectively induced by IFN-
in comparison to
IFN-
2, IFN-
CON, mixtures of IFN-
subtypes, or IFN-
8; 2)
cellular components needed to generate transcription factor ISGF3 were
essential but not sufficient for induction of
-R1 by
IFN-
; 3) the ISRE-binding protein p48 was essential for
-R1 expression in response either to IFN-
or IFN-
,
implying that transcription was regulated by an ISRE-like element
(16).
-R1 in response to IFN-
; this result was confirmed in
three unrelated lines of U1 cells obtained from two independent mutagenesis experiments (16). This finding was surprising as U1 cells
are responsive to IFN-
for ISGs, through a TYK2-independent pathway
(18, 19). Furthermore, when U1 cells were immunoselected for high
efficiency response to IFN-
using major histocompatibility complex
class I induction to monitor the IFN response,
-R1
induction was not rescued (16). These results suggested that TYK2
protein mediated an essential structural or catalytic role in the
induction of
-R1 by IFN-
.
unresponsive human fibrosarcoma mutant cell lines
U1 and U4 lacking TYK2 and JAK1, respectively (18, 22, 23). Both
kinases interact in ligand-independent fashion with type I IFN receptor
components, TYK2 associated with IFNAR1 (24-26) and JAK1 with IFNAR2c
(27, 28). JAK1 catalytic activity is absolutely essential for responses
to type I IFNs (22). In contrast, IFN-
signaling for induction of
ISGs can proceed at reduced efficiency in the absence of TYK2, and
kinase-deficient TYK2 protein augments responses to type I IFNs in U1
cells (18, 29).
species correlated
with the inability to bind ligand (18). The contribution of TYK2 to the
ligand binding activity of type I IFN receptor does not require
catalytic activity. Indeed, ligand-binding function for IFN-
is
restored by expression of kinase-deficient TYK2 (20). Second, TYK2 is
required for generating STAT-docking sites on IFNAR1. Docking occurs
through mutual phosphotyrosine/src-homology-2 (SH2)
interactions between Tyr-466 on the activated IFNAR1 cytoplasmic tail
and STAT-2 which is preassociated with IFNAR2c (30). This SH2-phosphotyrosine interaction determines signaling specificity through the type I IFN receptor complex (31). The role of TYK2 in this
process was demonstrated by showing that TYK2 phosphorylates IFNAR1
in vitro and that IFNAR1-Tyr-466-containing phosphopeptides block signaling (25, 26).
-R1, using U1 cells complemented with
TYK2 deletion and substitution mutants or with wild-type TYK2.
Kinase-deficient TYK2 mutant proteins restored IFN-
binding and weak
response to IFN-
; responses to IFN-
were augmented (20). However,
none of the cells complemented with mutant TYK2 proteins expressed
-R1in response to IFN-
, indicating that TYK2 catalytic
activity was required for the induction of
-R1.
MATERIALS AND METHODS
TK, and U1.
KL were
maintained in Dulbecco's modified Eagle's medium with 10% calf serum
in the presence of 250 µg/ml hygromycin and 450 µg/ml G418 (20).
Purified recombinant IFN-
2 (1 × 105 units/ml) was
obtained from Wellcome, and recombinant IFN-
-1b (2 × 108 units/mg protein) was from Berlex Biosciences
(Richmond, CA). IFNs were used at a final concentration of 1000 units/ml unless stated otherwise.
-R1
-R1 cDNA obtained by RNA
fingerprinting (16). Seven clones were obtained. Southern blot analysis
of phage DNA digested with restriction enzymes EcoRI and
SalI revealed a 3-kilobase pair fragment that hybridized
with a 100-bp probe to the extreme N terminus of the cDNA. The DNA
from one clone was cleaved with EcoRI, and fragments were
randomly cloned into pBS vector. A clone containing a 3-kilobase pair
insert was determined by sequence analysis to contain 300 bp of
sequence content upstream of the putative transcription start site of
-R1, by alignment with cDNA products.
-R1
promoter element including the transcription start site was amplified
by polymerase chain reaction using F
-R1 containing the
SacI restriction site (5'AGGCGAGCTCTCCGCTGCCC3') and
R
-R1 containing the BglII restriction site
(5'TGGAAGATCTAGTAGAAAGT3') incorporated in the primers. The
PCR-amplified product was excised with BglII and
SacI and was subcloned into the promoterless pGL3-basic vector (Promega Corp., Madison, WI). One clone
(p
-R1-300-luc) was sequenced to verify the nucleotide
sequence and has the 5'-flanking region of the
-R1 gene
from
305 to +1 (transcriptional start site), and DNA from this clone
was used for transfection experiments.
-galactosidase reporter plasmid (pCH110,
Amersham Pharmacia Biotech) was co-transfected with experimental plasmids as an internal control to normalize for transfection efficiency.
-galactosidase plasmid DNA
using Polybrene (10 µg/ml) for 6 h at 37 °C. After
incubation, the cells were subjected to Me2SO shock for
90 s (30% Me2SO in Dulbecco's modified Eagle's
medium), washed, and allowed to recover overnight from
Me2SO shock. The following morning, cells were pooled and equally redistributed in several plates and reserved as controls or
treated with IFN-
(1000 units/ml) for 16 h. Lysates were
prepared, and luciferase activity was assayed using a luciferase assay
kit (Promega Corp, Madison, WI), and measurements made using a
Luminometer (Dynatech Laboratories, Chantilly, VA).
-Galactosidase
activity was assayed using Galacto-Light Plus assay (Tropix Inc.,
Bedford, MA). Equal amounts of protein were assayed for enzyme
expression, and luciferase activity was normalized to
-galactosidase activity.
70 °C until use.
-R1 (protects 500 bases), 6-16 (protects 190 bases), and 9-27 (protects 160 bases) (16, 33).
-Actin probe protects 130 bases, and IRF-1 probe
protects 175 bases; these are as described by Muller et al.
(34). The hybridization signal was quantitated with storage phosphor
technique, using a PhosphorImager (Molecular Dynamics, Sunnyville, CA).
RESULTS
-R1 by IFN-
as Compared with IFN-
in HT1080 Cells--
The role of TYK2 in
-R1 induction
was examined by studying U1 cell lines complemented with wild-type or
mutant TYK2. The previously reported characteristics of these cell
lines, all derived from HT1080 fibrosarcoma cells, are summarized in
Table I. In CRT astrocytoma cells (from
which
-R1 was originally cloned),
-R1 was
induced to an equivalent extent by 10 units/ml IFN-
or by 2500 units/ml IFN-
2 (16). To initiate our studies in cells of
the HT1080 background, we examined differential regulation of
-R1 by IFN-
compared with IFN-
in these cells.
Dose-response experiments in the HT1080 cells indicated that selective
induction of
-R1 by IFN-
was maintained over a range
of IFN concentrations studied (Fig. 1 and
Table II). In order to compare
-R1 induction by IFN-
2 with IFN-
, we normalized the
IFN response to induction of the ISG 6-16 (Table II, see
legend). By this analysis, IFN-
was an average of 8-9-fold more
efficient for induction of
-R1 than IFN-
at
concentrations of 800-6400 units/ml. The differential response to
IFN-
was not observed for 6-16, a well characterized type
I IFN-induced gene (Fig. 1).
Characteristics of U1 cells complemented with various TYK2 proteins
View larger version (73K):
[in a new window]
Fig. 1.
Induction of -R1 by
recombinant IFN-
2 and IFN-
in HT1080 cells. The figure shows
an autoradiogram derived from RNase protection assay of total RNA (20 µg) from untreated cells (C) or cells treated with the
indicated amounts of recombinant IFN-
2 or IFN-
for 16 h. The
dried gel was exposed to film (Kodak XAR5) for 16 h at
70 °C
for the
-R1 and 8 h for 6-16 and
-actin. Results from one of two experiments are shown.
Selective induction of -R1 by IFN-
in HT1080 cells
-R1, normalized to
6-16/
-actin at different concentrations of IFN (Fig. 1),
was determined after the protected fragments from the RPA assay were
quantitated on a PhosphorImager (Molecular Dynamics). To compare the
response to IFN-
with IFN-
,
-R1 induction was
normalized to the induction of ISG, as shown.
-R1 induction ratio (IFN-
/IFN-
):
-R1 mRNA by IFN-
as
compared with IFN-
could be determined at the level of
transcriptional or post-transcriptional events. In order to begin
addressing this issue, we examined the stability of
-R1
mRNA induced by IFN-
2 or IFN-
. A high dose of IFN-
2 (2500 units/ml) was used to induce
-R1 and was compared with
2500 units/ml IFN-
. After cells were treated with IFNs for 6 h,
actinomycin D (5 µg/ml) was added to arrest transcription, and
-R1 mRNA levels were analyzed by normalized nuclease
protection assays at hourly intervals up to 8 h (the time point of
maximal accumulation of the message).
-R1 mRNA was
equally stable up to 8 h after arrest of transcription in cells
treated with either IFN-
2 or IFN-
, arguing that differential stability of
-R1 message did not account for the
accumulation of
-R1 mRNA in cells treated with
IFN-
compared with IFN-
2 (results not shown).
-R1 in Cells Expressing TYK2 Deletion and
Substitution Mutants--
To determine the role of catalytic TYK2 in
the induction of
-R1, we examined regulation of this and
other ISGs in U1 cell lines expressing wild-type TYK2 or mutants that
are schematically depicted in Fig.
2A. Deletion mutant
TK
lacks the tyrosine kinase domain, whereas
KL lacks the kinase-like
domain but retains an intact tyrosine kinase domain. Both deletion
mutants lack kinase function in vitro (20). The mutant KR930
was constructed by substituting lysine for arginine in the ATP-binding
site that results in the generation of a kinase-inactive TYK2 protein
that retains weak ligand-dependent phosphorylation on
tyrosine (Table I) (29). In substitution mutant YYFF1054-55, two
conserved tyrosines are mutated to phenylalanine in the putative
activation loop. Phosphorylation of these tyrosines is required for
ligand-dependent activation of TYK2. Therefore, the
YYFF1054-55 mutant retains basal kinase activity that is not enhanced
upon ligand binding (Table I) (29). The wild-type cells expressed the
full-length catalytically active TYK2. Cell clones used in these
experiments expressed comparable levels of TYK2 (approximately 5-fold
higher than the endogenous TYK2 in 2fTGH cells), by Western analysis (20, 29).
View larger version (32K):
[in a new window]
Fig. 2.
A, schematic of the structure
of TYK2 mutant proteins. Names of the proteins are on the
left. wt, wild-type; KL, kinase-like
domain; TK, tyrosine kinase domain. B, induction
of -R1 by IFN-
in cells expressing wild-type or
TYK2-deleted/substituted forms of protein. The figure shows an
autoradiogram derived from one representative experiment (out of five)
using RNase protection assay of total RNA (20 µg) from untreated
U1.wt-5 cells (
) and cells treated with 1000 units/ml recombinant
IFN-
for 16 h.
-mediated induction of IFN-responsive
-R1 and
6-16 genes in these cell lines was analyzed by nuclease
protection assay. The 6-16 gene was induced by IFN-
in
cell lines expressing both wild-type and mutant TYK2 (Fig.
2B). Strikingly,
-R1 was not induced by
IFN-
treatment in either deletion mutants (U1.
TK and U1.
KL) or
substitution mutants (U1.YYFF1054-55 and U1.KR930) of TYK2 in response
to IFN-
(Fig. 2B).
-R1 by IFN-
in cells expressing mutant TYK2
proteins could result from decreased signaling efficiency, cells were
exposed to higher concentrations of IFN-
. At the highest
concentration of IFN-
tested (10,000 units/ml), no induction of
-R1 by IFN-
in U1.KR930 and U1.YYFF1054-55 cells was
detected on prolonged exposure of autoradiograms (Fig. 3).
View larger version (63K):
[in a new window]
Fig. 3.
Induction of -R1, ISGs 6-16, 9-27 in U1.wt-5, U1.KR930 and U1.YYFF1054-55 cells by 10,000 units/ml
recombinant IFN-
for 16 h. RNase protection assay of total
RNA (20 µg) was performed, and the densitometric ratio of
-R1, 6-16, and 9-27 to
-actin was
determined after protected fragments were quantitated by NIH image
analysis. Results from one of two experiments are shown.
2 or IFN-
was
equally robust in cells expressing either kinase-deficient or wild-type
TYK2. ISG 9-27 also accumulated in U1.KR930 and
U1.YYFF1054-55 in response to either IFN-
2 or IFN-
(Fig.
3). These results supported a specific role for inducible TYK2
catalytic activity in regulating expression of
-R1 in
response to IFN-
.
-R1 by
IFN-
--
Cells expressing endogenous or increased levels of TYK2
(2fTGH and U1.wt) were compared for inducible expression of
-R1 or other ISGs (Fig. 4).
-R1 mRNA accumulated approximately 9-fold more in
U1.wt-5 cells than in 2fTGH, in response to IFN-
(Fig. 4). The
induction of other ISGs (6-16 and 9-27) varied at
most by 2-fold in U1.wt-5 cells, compared with 2fTGH.
View larger version (43K):
[in a new window]
Fig. 4.
Augmented -R1 induction by
IFN-
in U1.wt-5 cells.
RNase protection assay of total RNA (20 µg) from untreated U1.wt-5
cells (C) or cells treated with 1000 units/ml recombinant
IFN-
for 16 h and hybridized to
-R1, 6-16, 9-27, and
-actin probes was done. The following cell lines were
examined as indicated: 2fTGH, U1A, and U1.wt-5. The densitometric ratio
of
-R1, 6-16, and 9-27 to
-actin
was determined after protected fragments were quantitated by NIH image
analysis (representative results from one of five
experiments).
-R1 induction in TYK2 deficient
cells could result from impaired generation of transcription factor
ISGF3, whose components we previously showed to be essential for
transcription of
-R1 (16). Expression of ISGs strongly
suggested the formation of ISGF3 in U1-derived cells complemented with
kinase-deficient TYK2; however, the presence of ISGF3 had not been
formally demonstrated. To address this issue, electrophoresis mobility
shift assay (EMSA) was used to monitor generation of ISGF3 after IFN
treatment. The composition of this complex has been previously
confirmed with supershift analysis, using anti-STAT antibodies (35). In
2fTGH cells, the major type I IFN subtypes (IFN-
2 or IFN-
)
generated approximately equal abundance of ISGF3 complex, indicating
that the presence of ISGF3 was not sufficient for induction of
-R1 (Fig. 5, lanes
2 and 3). After IFN-
treatment, 2fTGH cells and U1.KR930 cells contained equivalent amounts of ISGF3, yet only 2fTGH
cells expressed
-R1, further supporting this
interpretation (Fig. 5, lanes 3 and 7). In U1
cells, ISGF3 was generated weakly in response to IFN-
but not
IFN-
2, consistent with induction of ISGs by IFN-
in these cells
(results not shown).
View larger version (38K):
[in a new window]
Fig. 5.
Generation of ISGF-3 is not defective in
U1.KR930 cells. EMSA using whole cell extracts from 2fTGH,
U1.wt-5, and U1.KR930 cells treated with or without (C) 2500 units/ml recombinant IFN- 2 or IFN-
for 15 min and incubated with
a oligodeoxynucleotide probe containing the ISRE of 6-16 gene. DNA-protein complexes were separated by native polyacrylamide gel
electrophoresis (8%) and analyzed by autoradiography.
Induces
-R1 Transcription in U1.wt-5 but Not U1.KR930
Cells--
To investigate the mechanism by which IFN-
induces
-R1 expression, a 303-bp fragment of the human
-R1 gene (Fig. 6), upstream of the transcription start site, was subcloned into the promoterless pGL3 plasmid, producing the promoter/reporter construct
p
-R1-300-luc, in which expression of the luciferase
reporter gene was controlled by cloned
-R1 sequence. The
sequence content of this putative promoter element was examined for
potential regulatory elements; two GAS sites and one binding site each
for AP-1, NF-AT, and NF
B, and one ISRE-like element were identified
(Fig. 6).
View larger version (31K):
[in a new window]
Fig. 6.
-R1 upstream sequence. A 303-bp
fragment of
-R1 was cloned into pGL3-luciferase vector.
Putative regulatory motifs are underlined. See text for
expansion of abbreviations. These sequence data have been submitted to
GenBankTM/EBI Data Bank with accession number AF113846.
-R1-300-luc was significantly induced by IFN-
in
U1.wt-5 but completely inert in U1.KR930 cells, demonstrating that this
fragment contained a functional
-R1 promoter that was
dependent on catalytic TYK2 for its induction (Fig.
7). A promoter-reporter from the 561 ISG
was induced by IFN-
approximately twice as efficiently in U1.wt-5 as
in U1.KR930 cells and retained significant activity in cells expressing
kinase-deficient TYK2 (Fig. 7). These results reflected the relative
IFN-
-mediated induction of endogenous
-R1 and ISGs in
U1.wt-5 and U1.KR930 (compare Figs. 3 and 4 with Fig. 7) and indicated
that the differential accumulation of the respective mRNAs was
regulated at the transcriptional level.
View larger version (29K):
[in a new window]
Fig. 7.
-R1 transcription is defective in
U1.KR930 cells. Transient transfection analysis of
p
-R1-300-luc construct and p561-luc construct in U1.wt-5
cells and U1.KR930 cells exposed to 1000 units/ml recombinant IFN-
for 16 h. The histograms show means (± S.D.) for five experiments
with p
-R1-luc 300 and three experiments with p561-luc.
Fold induction by IFN-
of p
-R1 300-luc in U1.wt-5 and
U1.KR930 cells differed significantly (p < 0.001, paired t test), and expression of p
-R1 300-luc
in U1.KR930 cells did not differ significantly from untreated
controls.
DISCUSSION
-R1 in IFN-
-treated cells. Addressing this point most
directly,
-R1 was not inducible in U1 cell lines
complemented with various deletion or substitution mutants of TYK2,
each of which were catalytically inactive.
-mediated transcription of
-R1
requires inducible TYK2 kinase activity, as shown by failure of
induction in cell lines expressing U1.KR930 and U1.YYFF1054-55
mutants. Both proteins lack ligand-inducible kinase activity, whereas
the U1.YYFF1054-55 mutant retains basal kinase function, indicating that this residual enzymatic action is insufficient to mediate
-R1 induction. Recent reports also suggest that
SH2-phosphotyrosine interactions are important for association between
STAT substrates and JAKs in type I IFN signaling (36). Our results
suggest that the docking function of the TYK2 protein (absent inducible
kinase activity) may not be sufficient for
-R1
expression, since IFN-
could not induce this gene in U1.KR930,
expressing a TYK2 mutant that undergoes phosphorylation in
trans on tyrosine residues (29).
to induce
-R1 in cells expressing
kinase-deficient TYK2 contrasted with IFN-
-mediated expression of other ISGs, activation of ISGF3, and establishment of the antiviral state (not shown) in U1 cells complemented with catalytically inactive
TYK2. These results extend prior demonstrations that IFN-
can
utilize a TYK2-independent pathway (19) for induction of ISGs. Despite
the availability of TYK2-independent IFN-
-mediated signaling,
generation of docking sites on IFNAR1 (a TYK2-dependent function) clearly enhances the efficiency of IFN signaling. However, we
did not detect
-R1 induction by IFN-
in cells
expressing kinase-deficient forms of TYK2 at high concentrations of
ligand that strongly induced expression of other ISGs and efficient
antiviral responses in U1.KR930 cells (not shown). This observation
suggested a specific role for TYK2 in the signaling pathway for
-R1 transcription. As compared with 2fTGH cells, IFN-
treatment of U1.wt-5 cells that overexpressed TYK2 resulted in
selective increase in accumulation of
-R1 mRNA,
further supporting a specific role for TYK2 in
-R1 induction.
-R1 genomic fragment was isolated for these
studies, and its function was examined in transient transfection
assays. The p
-R1-300-luc plasmid directed luciferase
expression in response to IFN-
in U1.wt-5 but not U1.KR930 cells,
precisely reiterating the pattern of expression observed for the
endogenous gene and indicating that TYK2-dependent
regulation of
-R1 by IFN-
occurs at the
transcriptional level.
for inducing the
-R1 gene requires catalytically active TYK2. Elucidation of the components and mechanisms of this accessory pathway will provide insights into biological functions of IFNs for which JAK-STAT signaling is essential but not sufficient.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Ian Kerr (Imperial Cancer Research Fund Laboratories, London, UK), Ed Croze (Berlex Biosciences Inc., Richmond, CA), George R. Stark, and Ganes Sen (Lerner Research Institute, Cleveland, OH) for advice.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant PPG IPOICAG2220 (to G. R. Stark) (R. M. R., Project leader, Project 3), Berlex Biosciences, and the Williams Family Fund for Multiple Sclerosis. Work at the Institut Pasteur was supported by a grant from the Association pour la Recherche Sur le Cancer (to S. P).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF113846.
To whom correspondence should be addressed: Dept.
of Neurosciences, The Lerner Research Institute, NC30, Cleveland Clinic Foundation, Cleveland, OH 44195. Tel.: 216-444-0627; Fax: 216-444-7927; E-mail: ransohr{at}cesmtp.ccf.org.
The abbreviations used are: IFNs, interferons; ISGs, IFN-stimulated genes; ISGF, interferon-stimulated gene factor; JAK, Janus kinases; STATs, signal transducers and activators of transcription; ISREs, IFN-stimulated response elements; bp, base pair(s); TK, tyrosine kinase; KL, kinase-like; EMSA, electrophoretic mobility shift assay.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|