Induction of beta -R1/I-TAC by Interferon-beta Requires Catalytically Active TYK2*

M. R. Sandhya RaniDagger , Cristina Gauzzi§, Sandra Pellegrini§, Eleanor N. Fish, Tao WeiDagger , and Richard M. RansohoffDagger parallel

From the Dagger  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
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Abstract
Introduction
References

The beta -R1/I-TAC (interferon-inducible T-cell alpha -chemoattractant) gene encodes an alpha -chemokine that is a potent chemoattractant for activated T-cells. We previously reported that beta -R1 was selectively induced by interferon (IFN)-beta compared with IFN-alpha and that the canonical type I IFN transcription factor interferon-stimulated gene factor 3 (ISGF3) was necessary but not sufficient for beta -R1 induction by IFN-beta . These findings suggested that beta -R1 induction by IFN-beta required an accessory component. To begin characterizing this signaling pathway, we examined the function of TYK2 protein in the IFN-beta -mediated induction of beta -R1. This study was motivated by the observation that beta -R1 could not be induced in TYK2-deficient U1 cells by IFN-beta (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-beta evokes substantial expression of IFN-stimulated genes (ISGs) in U1 cells through a TYK2-independent pathway. We now report beta -R1 expression patterns in U1 cells complemented with wild-type or mutant TYK2 proteins. Complementation with wild-type TYK2 rescued IFN-beta -inducible expression of beta -R1. Cells expressing kinase-deficient deletion or point mutants of TYK2 were refractory to induction of beta -R1 by IFN-beta despite robust expression of other ISGs. Transient transfection analysis of a beta -R1 promoter-reporter confirmed that transcriptional activation of beta -R1 by IFN-beta required competent TYK2 kinase. These studies indicate that the catalytic function of TYK2 is required for IFN-beta -mediated induction of beta -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

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-alpha and 1 expressed IFN-omega and IFN-beta ) 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).

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-beta 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-gamma , without demonstrating IFN-gamma -mediated growth inhibition (11).

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-beta but not IFN-alpha 2 (12-15). This result suggested that IFN-beta engagement generated a more stable signaling complex than IFN-alpha 2. Recently, it has been shown that IFN-alpha 2 and IFN-beta require distinct intracytoplasmic regions of the IFNAR-2 chain of the receptor to elicit an antiviral response (15).

We recently reported studies of the regulation of a gene designated beta -R1, which was selectively induced by IFN-beta compared with IFN-alpha in astrocytoma and fibrosarcoma cells (16). Sequence analysis of cDNAs indicated that beta -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 beta -R1 and I-TAC were highly related, and sequence analysis of multiple independent cDNA isolates has established identity between the two chemokines. beta -R1/I-TAC possesses potent chemoattractant activity toward activated T-lymphocytes (17).

The signaling pathway whereby IFN-beta induces the expression of beta -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) beta -R1 was selectively induced by IFN-beta in comparison to IFN-alpha 2, IFN-alpha CON, mixtures of IFN-alpha subtypes, or IFN-alpha 8; 2) cellular components needed to generate transcription factor ISGF3 were essential but not sufficient for induction of beta -R1 by IFN-beta ; 3) the ISRE-binding protein p48 was essential for beta -R1 expression in response either to IFN-beta or IFN-gamma , implying that transcription was regulated by an ISRE-like element (16).

Unexpectedly, U1 cells that lacked TYK2 expression failed to express beta -R1 in response to IFN-beta ; 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-beta for ISGs, through a TYK2-independent pathway (18, 19). Furthermore, when U1 cells were immunoselected for high efficiency response to IFN-beta using major histocompatibility complex class I induction to monitor the IFN response, beta -R1 induction was not rescued (16). These results suggested that TYK2 protein mediated an essential structural or catalytic role in the induction of beta -R1 by IFN-beta .

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-alpha 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-beta 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).

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-alpha 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-alpha 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).

In this report, we describe further investigation of the role of TYK2 in the induction of beta -R1, using U1 cells complemented with TYK2 deletion and substitution mutants or with wild-type TYK2. Kinase-deficient TYK2 mutant proteins restored IFN-alpha binding and weak response to IFN-alpha ; responses to IFN-beta were augmented (20). However, none of the cells complemented with mutant TYK2 proteins expressed beta -R1in response to IFN-beta , indicating that TYK2 catalytic activity was required for the induction of beta -R1.

    MATERIALS AND METHODS

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.Delta TK, and U1.Delta 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-alpha 2 (1 × 105 units/ml) was obtained from Wellcome, and recombinant IFN-beta -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.

Cloning the Promoter of beta -R1

A human genomic library (CLONTECH) was screened using the partial beta -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 beta -R1, by alignment with cDNA products.

Transient Transfection Assay

Plasmids-- A 303-bp upstream putative beta -R1 promoter element including the transcription start site was amplified by polymerase chain reaction using Fbeta -R1 containing the SacI restriction site (5'AGGCGAGCTCTCCGCTGCCC3') and Rbeta -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 (pbeta -R1-300-luc) was sequenced to verify the nucleotide sequence and has the 5'-flanking region of the beta -R1 gene from -305 to +1 (transcriptional start site), and DNA from this clone was used for transfection experiments.

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-beta -galactosidase reporter plasmid (pCH110, Amersham Pharmacia Biotech) was co-transfected with experimental plasmids as an internal control to normalize for transfection efficiency.

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 beta -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-beta (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). beta -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 beta -galactosidase activity.

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 -70 °C until use.

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 beta -R1 (protects 500 bases), 6-16 (protects 190 bases), and 9-27 (protects 160 bases) (16, 33). gamma -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

Selective Induction of beta -R1 by IFN-beta as Compared with IFN-alpha in HT1080 Cells-- The role of TYK2 in beta -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 beta -R1 was originally cloned), beta -R1 was induced to an equivalent extent by 10 units/ml IFN-beta or by 2500 units/ml IFN-alpha 2 (16). To initiate our studies in cells of the HT1080 background, we examined differential regulation of beta -R1 by IFN-beta compared with IFN-alpha in these cells. Dose-response experiments in the HT1080 cells indicated that selective induction of beta -R1 by IFN-beta was maintained over a range of IFN concentrations studied (Fig. 1 and Table II). In order to compare beta -R1 induction by IFN-alpha 2 with IFN-beta , we normalized the IFN response to induction of the ISG 6-16 (Table II, see legend). By this analysis, IFN-beta was an average of 8-9-fold more efficient for induction of beta -R1 than IFN-alpha at concentrations of 800-6400 units/ml. The differential response to IFN-beta was not observed for 6-16, a well characterized type I IFN-induced gene (Fig. 1).

                              
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Table I
Characteristics of U1 cells complemented with various TYK2 proteins
U1 cells are deleted for TYK2; complementation with mutant TYK2 has revealed structure-function relation, as indicated below and described by Pellegrini and colleagues (18, 20, 23, 29, 37).


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Fig. 1.   Induction of beta -R1 by recombinant IFN-alpha 2 and IFN-beta 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-alpha 2 or IFN-beta for 16 h. The dried gel was exposed to film (Kodak XAR5) for 16 h at -70 °C for the beta -R1 and 8 h for 6-16 and gamma -actin. Results from one of two experiments are shown.

                              
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Table II
Selective induction of beta -R1 by IFN-beta in HT1080 cells
The densitometric ratio of beta -R1, normalized to 6-16/gamma -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-beta with IFN-alpha , beta -R1 induction was normalized to the induction of ISG, as shown.

beta -R1 induction ratio (IFN-beta /IFN-alpha ): <FR><NU><IT>6-16</IT>(<IT>&agr;</IT>)<IT>/&ggr;-actin</IT></NU><DE><IT>6-16</IT>(<IT>&bgr;</IT>)<IT>/&ggr;-actin</IT></DE></FR><IT> × </IT><FR><NU><IT>&bgr;-R1</IT>(<IT>&bgr;</IT>)</NU><DE><IT>&bgr;-R1</IT>(<IT>&agr;</IT>)</DE></FR>

The differential regulation of beta -R1 mRNA by IFN-beta as compared with IFN-alpha could be determined at the level of transcriptional or post-transcriptional events. In order to begin addressing this issue, we examined the stability of beta -R1 mRNA induced by IFN-alpha 2 or IFN-beta . A high dose of IFN-alpha 2 (2500 units/ml) was used to induce beta -R1 and was compared with 2500 units/ml IFN-beta . After cells were treated with IFNs for 6 h, actinomycin D (5 µg/ml) was added to arrest transcription, and beta -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). beta -R1 mRNA was equally stable up to 8 h after arrest of transcription in cells treated with either IFN-alpha 2 or IFN-beta , arguing that differential stability of beta -R1 message did not account for the accumulation of beta -R1 mRNA in cells treated with IFN-beta compared with IFN-alpha 2 (results not shown).

Induction of beta -R1 in Cells Expressing TYK2 Deletion and Substitution Mutants-- To determine the role of catalytic TYK2 in the induction of beta -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 Delta TK lacks the tyrosine kinase domain, whereas Delta 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).


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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 beta -R1 by IFN-beta 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-beta for 16 h.

IFN-beta -mediated induction of IFN-responsive beta -R1 and 6-16 genes in these cell lines was analyzed by nuclease protection assay. The 6-16 gene was induced by IFN-beta in cell lines expressing both wild-type and mutant TYK2 (Fig. 2B). Strikingly, beta -R1 was not induced by IFN-beta treatment in either deletion mutants (U1.Delta TK and U1.Delta KL) or substitution mutants (U1.YYFF1054-55 and U1.KR930) of TYK2 in response to IFN-beta (Fig. 2B).

To address the possibility that inability to induce beta -R1 by IFN-beta in cells expressing mutant TYK2 proteins could result from decreased signaling efficiency, cells were exposed to higher concentrations of IFN-beta . At the highest concentration of IFN-beta tested (10,000 units/ml), no induction of beta -R1 by IFN-beta in U1.KR930 and U1.YYFF1054-55 cells was detected on prolonged exposure of autoradiograms (Fig. 3).


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Fig. 3.   Induction of beta -R1, ISGs 6-16, 9-27 in U1.wt-5, U1.KR930 and U1.YYFF1054-55 cells by 10,000 units/ml recombinant IFN-beta for 16 h. RNase protection assay of total RNA (20 µg) was performed, and the densitometric ratio of beta -R1, 6-16, and 9-27 to gamma -actin was determined after protected fragments were quantitated by NIH image analysis. Results from one of two experiments are shown.

Induction of 6-16 by either IFN-alpha 2 or IFN-beta 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-alpha 2 or IFN-beta (Fig. 3). These results supported a specific role for inducible TYK2 catalytic activity in regulating expression of beta -R1 in response to IFN-beta .

Overexpression of TYK2 Selectively Augments Induction of beta -R1 by IFN-beta -- Cells expressing endogenous or increased levels of TYK2 (2fTGH and U1.wt) were compared for inducible expression of beta -R1 or other ISGs (Fig. 4). beta -R1 mRNA accumulated approximately 9-fold more in U1.wt-5 cells than in 2fTGH, in response to IFN-beta (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.


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Fig. 4.   Augmented beta -R1 induction by IFN-beta 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-beta for 16 h and hybridized to beta -R1, 6-16, 9-27, and gamma -actin probes was done. The following cell lines were examined as indicated: 2fTGH, U1A, and U1.wt-5. The densitometric ratio of beta -R1, 6-16, and 9-27 to gamma -actin was determined after protected fragments were quantitated by NIH image analysis (representative results from one of five experiments).

IFN-induced Generation of ISGF3 Is Not Defective in U1.KR930 Cells-- Failure of beta -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 beta -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-alpha 2 or IFN-beta ) generated approximately equal abundance of ISGF3 complex, indicating that the presence of ISGF3 was not sufficient for induction of beta -R1 (Fig. 5, lanes 2 and 3). After IFN-beta treatment, 2fTGH cells and U1.KR930 cells contained equivalent amounts of ISGF3, yet only 2fTGH cells expressed beta -R1, further supporting this interpretation (Fig. 5, lanes 3 and 7). In U1 cells, ISGF3 was generated weakly in response to IFN-beta but not IFN-alpha 2, consistent with induction of ISGs by IFN-beta in these cells (results not shown).


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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-alpha 2 or IFN-beta 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.

IFN-beta Induces beta -R1 Transcription in U1.wt-5 but Not U1.KR930 Cells-- To investigate the mechanism by which IFN-beta induces beta -R1 expression, a 303-bp fragment of the human beta -R1 gene (Fig. 6), upstream of the transcription start site, was subcloned into the promoterless pGL3 plasmid, producing the promoter/reporter construct pbeta -R1-300-luc, in which expression of the luciferase reporter gene was controlled by cloned beta -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 NFkappa B, and one ISRE-like element were identified (Fig. 6).


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Fig. 6.   beta -R1 upstream sequence. A 303-bp fragment of beta -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.

Normalized transient transfection analysis was used to determine the function of this element in U1.wt-5 and U1.KR930 cells. pbeta -R1-300-luc was significantly induced by IFN-beta in U1.wt-5 but completely inert in U1.KR930 cells, demonstrating that this fragment contained a functional beta -R1 promoter that was dependent on catalytic TYK2 for its induction (Fig. 7). A promoter-reporter from the 561 ISG was induced by IFN-beta 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-beta -mediated induction of endogenous beta -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.


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Fig. 7.   beta -R1 transcription is defective in U1.KR930 cells. Transient transfection analysis of pbeta -R1-300-luc construct and p561-luc construct in U1.wt-5 cells and U1.KR930 cells exposed to 1000 units/ml recombinant IFN-beta for 16 h. The histograms show means (± S.D.) for five experiments with pbeta -R1-luc 300 and three experiments with p561-luc. Fold induction by IFN-beta of pbeta -R1 300-luc in U1.wt-5 and U1.KR930 cells differed significantly (p < 0.001, paired t test), and expression of pbeta -R1 300-luc in U1.KR930 cells did not differ significantly from untreated controls.


    DISCUSSION

Our current results suggest that catalytically active TYK2 is required for accessory signaling that results in transcription of beta -R1 in IFN-beta -treated cells. Addressing this point most directly, beta -R1 was not inducible in U1 cell lines complemented with various deletion or substitution mutants of TYK2, each of which were catalytically inactive.

Furthermore, IFN-beta -mediated transcription of beta -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 beta -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 beta -R1 expression, since IFN-beta could not induce this gene in U1.KR930, expressing a TYK2 mutant that undergoes phosphorylation in trans on tyrosine residues (29).

The failure of IFN-beta to induce beta -R1 in cells expressing kinase-deficient TYK2 contrasted with IFN-beta -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-beta can utilize a TYK2-independent pathway (19) for induction of ISGs. Despite the availability of TYK2-independent IFN-beta -mediated signaling, generation of docking sites on IFNAR1 (a TYK2-dependent function) clearly enhances the efficiency of IFN signaling. However, we did not detect beta -R1 induction by IFN-beta 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 beta -R1 transcription. As compared with 2fTGH cells, IFN-beta treatment of U1.wt-5 cells that overexpressed TYK2 resulted in selective increase in accumulation of beta -R1 mRNA, further supporting a specific role for TYK2 in beta -R1 induction.

A 303-bp beta -R1 genomic fragment was isolated for these studies, and its function was examined in transient transfection assays. The pbeta -R1-300-luc plasmid directed luciferase expression in response to IFN-beta 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 beta -R1 by IFN-beta occurs at the transcriptional level.

In summary, data described in this report demonstrate that the pathway utilized by IFN-beta for inducing the beta -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.

parallel 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
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Abstract
Introduction
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