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The Journal of Immunology, 2006, 176: 5934-5942.
Copyright © 2006 by The American Association of Immunologists

Annexin V Associates with the IFN-{gamma} Receptor and Regulates IFN-{gamma} Signaling1

Carlos Leon2,*,{dagger}, Devki Nandan2,*, Martin Lopez*, Alireza Moeenrezakhanlou* and Neil E. Reiner3,*,{dagger}

* Department of Medicine, Division of Infectious Diseases, and {dagger} Department of Microbiology and Immunology, University of British Columbia Faculties of Medicine and Science, and Vancouver Coastal Health Research Institute, Vancouver, British Columbia, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Many of the biological activities of IFN-{gamma} are mediated through the IFN-{gamma}R3-linked Jak-Stat1{alpha} pathway. However, regulation of IFN-{gamma} signaling is not fully understood, and not all responses to IFN-{gamma} are Stat1{alpha} dependent. To identify novel elements involved in IFN-{gamma} cell regulation, the cytoplasmic domain of the R2 subunit of the human IFN-{gamma}R was used as bait in a yeast two-hybrid screen of a human monocyte cDNA library. This identified annexin A5 (AxV) as a putative IFN-{gamma}R binding protein. The interaction was confirmed in pull-down experiments in which a GST-R2 cytoplasmic domain fusion protein was incubated with macrophage lysates. Furthermore, immunoprecipitation using anti-IFN-{gamma}R2 Abs showed that AxV interacted with IFN-{gamma}R2 to form a stable complex following incubation of cells with IFN-{gamma}. In 293T cells with reduced expression of AxV, brought about by small interfering RNA targeting, activation of Jak2 and Stat1{alpha} in response to IFN-{gamma} was enhanced. Inhibition of cell proliferation, a hallmark of the IFN-{gamma} response, also was potentiated in HeLa cells treated with small interfering RNA directed at AxV. Taken together, these results suggest that through an inducible association with the R2 subunit of the IFN-{gamma}R, AxV modulates cellular responses to IFN-{gamma} by modulating signaling through the Jak-Stat1 pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IFN-{gamma} has pleiotropic immunomodulatory and proinflammatory activities and is pivotally involved in host defense against infection and malignancy (1, 2). The current paradigm of IFN-{gamma} signaling was assembled during the 1990s (3, 4, 5). The IFN-{gamma}R is a heterotetramer comprised of two {alpha}-chains (R1)4 and two beta-chains (R2) that are constitutively associated with Jak1 and Jak2, respectively. Upon ligand binding to IFN-{gamma}R1, activation of Jak1 and Jak2 ensues leading to phosphorylation of tyrosine440 on IFN-{gamma}R1, thereby generating a docking site for the transcription factor Stat1{alpha}. Once at the activated receptor complex, Stat1{alpha} monomers homodimerize and are phosphorylated on tyrosine701. The phosphorylated homodimer disassociates from the receptor and translocates to the nucleus where it binds to IFN-{gamma} activated sequences (GAS) elements in IFN-{gamma}-regulated genes (6). Stat1{alpha} also undergoes phosphorylation on serine727 in the cytosol (7), before moving to the nucleus, an event that significantly potentiates its ability to activate gene transcription (8). The importance of this phosphorylation event is illustrated by the finding that the transactivation potential of a S727A mutant of Stat1{alpha} was reduced by 80% (9).

Multiple mechanisms have been shown to negatively regulate the activity of Stat1{alpha}, including dephosphorylation by dual specificity phosphatases (10), arginine methylation (11), ubiquitination and consequent degradation by the proteasome (12), and binding of PIASy, a protein inhibitor of the activated Stat1{alpha}, which represses Stat1{alpha}-mediated gene activation (13). The Jak-Stat1{alpha} signaling pathway also is regulated by contraints on activation of Jak1 and Jak2 imposed by their association with tyrosine phosphatases, such as SHP-1 (14, 15) and SHP-2 (16, 17). In addition, the IFN-{gamma}-inducible tyrosine phosphorylation of Jak2 on residue tyrosine1007, leads to the recruitment of suppressor of cytokine signaling 1 and polyubiquitination of Jak2, resulting in its rapid degradation through the proteasome (18). Signaling through the Jak-Stat1{alpha} pathway also has been shown to be negatively regulated via the association of Jak2 with the human DnaJ protein, hTid-1 (19).

Although many biologic responses to IFN-{gamma} are regulated by Stat1{alpha}, recent evidence indicates that IFN-{gamma} signaling uses elements other than or in addition to Stat1{alpha}. For example, microarray data generated using cells derived from Stat1{alpha} null mice has shown that there are at least 200 genes regulated by IFN-{gamma} in the absence of this transcriptional activator (20). In this regard, a diverse group of signaling elements distinct from the Jak-Stat1{alpha} pathway have been implicated in regulating IFN-{gamma} signaling (reviewed in Refs. 21 and 22), and it is likely that others are yet to be characterized.

To identify novel components involved in IFN-{gamma} signaling, we designed a yeast two-hybrid screen using the intracellular domain of R2 subunit of the IFN-{gamma}R as bait to screen a monocyte cDNA library. This approach resulted in the identification of annexin V (AxV) as an IFN-{gamma}R2 interacting protein. To address the role of AxV in IFN-{gamma} signaling, small interfering RNA (siRNA) was used to silence the AxV gene. Cells with reduced AxV expression showed increased responses to IFN-{gamma} for activation of Jak2 and Stat1{alpha}, and IFN-{gamma}-induced growth arrest also was potentiated under these conditions. These findings are consistent with a role for AxV in modulating signaling through the Jak-Stat1{alpha} pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents and chemicals

Anti-phosphotyrosine mAb 4G10 and rabbit polyclonal sera to Jak2 were obtained from Upstate Biotechnology; anti-IFN-{gamma}R2 was from PBL Biochemical Laboratories. Abs to Gal binding domain, GST, AxV, AxII, abl, Stat1{alpha}, actin, and IFN-{gamma}R1 were purchased from Santa Cruz Biotechnology. Ab to phosphoJak2 was from BioSource International. The rhIFN-{gamma} was a gift from Genentech. RPMI 1640, DMEM, HBSS, PBS, and protease inhibitors PMSF, pepstatin A, aprotinin, leupeptin, and Ab to phosphoserine were obtained from Sigma-Aldrich. BCECF-AM and BAPTA-AM were from Molecular Probes.

Cell culture

THP-1 and human embryonic kidney 293T cells were obtained from the American Type Culture Collection. THP-1 cells were cultured in RPMI 1640 supplemented with 10% FCS (HyClone), 100 µg/ml penicillin, 100 µg/ml streptomycin, and 10 mM HEPES. The 293T cells were grown in DMEM supplemented with 10% FCS, 0.1 mM minimal nonessential amino acids, 10 mM L-glutamine, 20 mM HEPES, and 100 µg/ml each of penicillin and streptomycin.

Electrophoresis and immunoblotting

Protein samples were separated by SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad), and blocked with either 3% BSA (Sigma-Aldrich) or 5% milk in 1x TBS with 0.1% Tween 20 for 1–2 h and then probed with the indicated primary Ab diluted in blocking solution for 2 h or as otherwise specified. Membranes were washed three times, incubated with the appropriate secondary Ab coupled to peroxidase, and proteins were detected by ECL (Amersham Biosciences).

Cloning of the intracellular domain of the R2 subunit of the IFN-{gamma}R as bait for yeast two-hybrid screening

The intracellular domain of the IFN-{gamma}R2 subunit (residues 265–334) was cloned by PCR into the EcoRI and BglII restriction sites of the pYTH9 shuttle vector (provided by GlaxoSmithKline) as a fusion with Gal-4 binding domain (GBD-R2). The cytoplasmic domain of the IFN-{gamma}R1 subunit (residues 262–489) was cloned in an identical fashion (GBD-R1) and used as a control for specificity. Both constructs were checked by DNA sequencing to confirm the correct frame and the absence of any PCR errors. The constructs and the vector alone, which also contained a Trp gene, were linearized with XbaI and used to stably transform yeast strain SDY191 (SDY191/R1, SDY191/R2, and SDY191/GBD) (23). The Saccharomyces cerevisiae strain SDY191 is auxotrophic for the selectable markers tryptophan, leucine, and histidine and encodes Escherichia coli LacZ and yeast HIS3 genes as reporters under the control of the Gal-4 transcription factor. Integration of the construct into the yeast strain was assessed by PCR of yeast genomic DNA, and expression of the fusion protein was determined by Western blot using anti-GBD Ab (data not shown).

Construction and screening of a cDNA library for yeast two-hybrid screening

An expression library in the HybriZap vector (Stratagene) was constructed according to the manufacturer’s specifications using mRNA extracted from human monocytes treated with IFN-{gamma} (provided by Dr. A. Mui, University of British Columbia). The quality of the library was assessed by the number of clones, the sizes and percentage of inserts, and the presence of IFN-{gamma}-inducible genes as determined by PCR (data not shown). Screening of the library used 1 million clones and was conducted in the presence of 15 mM 3-aminotriazole to prevent leakage of histidine expression. Candidate interacting clones were examined further by for beta-galactosidase activity. Double-positive clones were amplified for plasmid extraction, and the latter were used to transform bacteria in selective medium. Plasmids isolated from these bacteria were used to retransform yeast cells recombinant for the bait construct to confirm the interaction. Negative controls consisted of transforming recombinant yeast strain SDY191/R2 with the Gal-4 activator domain (GAD) alone and by transformation of yeast recombinant for the GBD alone with the putative interacting clone. Plasmids with the correct phenotype were sent for DNA sequencing (NAPS Unit, University of British Columbia).

Cloning of the cytoplasmic domain of IFN-{gamma}R2 as a GST fusion protein and GST pull-down experiments

The cytoplasmic domain of IFN-{gamma}R2 (residues 269–334) was cloned into pGEX4T using the EcoRI and XhoI restriction sites. As a specificity control, the cytoplasmic domain of IFN-{gamma}R1 (residues 267 to 489) also was cloned in the same vector using the BamHI and SalI restriction sites. Clones containing inserts were sequenced, and those in frame with GST and without mutations were grown at 37°C in 2x YTA medium with 100 µg/ml ampicillin up to an OD of 0.6. Isopropyl beta-D-thiogalactoside was added to a final concentration of 0.1 mM, and bacteria were grown at 28°C for 1.5 h, after which they were pelleted and disrupted in PBS with 1 mM EDTA by sonication. Sonicates were incubated with 0.1% Triton X-100 and 0.1 mM PMSF on ice for 5 min and cleared by centrifugation at a speed of 10,000 x g at 4°C for 15 min. Fusion protein was purified using glutathione beads as suggested by the manufacturer and analyzed by SDS-PAGE and immunoblotting with anti-GST Ab. For GST pull-downs, purified fusion protein bound to glutathione beads was incubated with whole cell lysates from THP-1 cells at 4°C for 2 h. Beads were then washed with PBS/0.5% Triton X-100 three times, and bound material was released by boiling coated beads in 2x SDS-Laemmli buffer. Eluted samples were analyzed by SDS-PAGE and immunoblotting with an anti-AxV Ab.

Coimmunoprecipitation of IFN-{gamma}R2 and AxV

Five million THP-1 cells grown in RPMI 1640 complete medium were differentiated overnight (12–14 h) using 10 nM PMA in complete medium. After differentiation, cells (70% confluent) were washed with HBSS three times and left to rest for 4 h in complete medium. Cells were then either untreated or treated with IFN-{gamma} (100 U/ml) for 15 min and then washed with HBSS and solubilized in digitonin buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 0.1% digitonin, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM PMSF, 10 µg/ml pepstatin, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) for 30 min on ice. Lysates were cleared by centrifugation at 10,000 x g at 4°C for 15 min, and supernatants were incubated with mAbs (both mouse IgG1) specific for either IFN-{gamma}R2 or c-abl for 2 h. Protein G (Amersham Biosciences) was then added, and the incubation was continued for 45 min after which immune complexes were recovered by centrifugation at 5,000 x g. Immunoprecipitates were washed in digitonin buffer three times and solubilized in Laemmli buffer without 2-mercaptoethanol (nonreducing conditions). After transfer to nitrocellulose, membranes were immunoblotted for AxV, IFN-{gamma}R2, and c-abl.

Modulation of annexin expression in 293T cells using siRNA

To examine the role of AxV in IFN-{gamma} signaling, siRNA constructs were designed using a web based platform (<http://katahdin.cshl.org>). The regions chosen for targeting were specific to either AxV and or AxII (used as a specificity control) DNA sequences as determined by a BLAST search. Two pairs of partially complementary oligonucleotides ("a" and "b") were used to create the pSHAG constructs expressing short hairpin RNA’s (shRNA) targeting AxV.

The following sequences were used to create the pSHAG-AV2.1 construct: AV2a, 5'-TCCCCAGATGTATCTCCCTTAATCATGGGAAGCTTGCTATGATTAAGGGGGATACGTCTGGGGATTATTTTTT-3'; and AV2b, 5'-GATCAAAAAATAATCCCCAGACGTATCCCCCTTAATCATAGCAAGCTTCCCATGATTAAGGGAGATACATCTGGGGACG-3'.

The following sequences used to create the pSHAG-AV3.1 construct: AV3a, 5'-CATCAGGGTCTCTGTTAGCCTGAAGGAGGAAGCTTGCTCCTTCGGGCTAGCAGAGGCCCTGGTGCTGTTTTTT-3'; and AV3b, 5'-GATCAAAAAACAGCACCAGGGCCTCTGCTAGCCCGAAGGAGCAAGCTTCCTCCTTCAGGCTAACAGAGACCCTGATGCG-3'. Only the pSHAG AV2.1 construct effectively reduced cellular levels of AxV protein, and the pSHAG AV3.1 construct was used as a control for nonspecific effects.

A single pair of oligos was designed to generate a shRNA for targeting AxII. The following sequences were used to create the pSHAG AxII construct: AxIIa, 5'-TGGGGCACGCTCCGCTCGGTCATGATGCGAAGCTTGGTATTATGACCGAGCGGAGTGTGCCTCACCTTTTTTT-3'; and AxIIb, 5'-GATCAAAAAAAGGTGAGGCACACTCCGCTCGGTCATAATACCAAGCTTCGCATCATGACCGAGCGGAGCGTGCCCCACG-3'.

The oligonucleotide pairs were annealed and cloned into the BseRI/BamHI cloning site of the pSHAG vector that directs the in vivo synthesis of shRNA molecules using a U6 promoter (24). Positive clones were selected by the acquisition of both kanamycin resistance and a new HindIII site. Clones containing the inserts of interest (pSHAG-AV2.1, pSHAG AV3.1, and pSHAG AxII) were grown, purified using an endotoxin-free Maxiprep kit (Qiagen), and used to transfect 293T cells. Transfection of the pSHAG constructs was conducted using Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer’s instructions. Cells were transfected when they reached 95% confluency and were incubated with transfection reagent containing plasmid DNA diluted in OptiMEM medium for 24 h. Medium was then replaced by complete DMEM, and cells were cultured for an additional 48 h to allow for expression of siRNA and down-regulation of either AxV or AxII. Transfection efficiency was >95% as determined by cotransfection with a pCMV beta-galactosidase expression construct combined with measurement of beta-galactosidase activity in situ. The extent to which either AxV or AxII expression was reduced by the siRNA constructs was assessed by immunoblotting with AxV or AxII Abs coupled with densitometric analysis using UN-SCAN-IT software (Silk Scientific). Actin was used as a protein loading control.

Western blotting

The 293T cells transfected with either pSHAG (empty vector), pSHAG AV2.1 (referred as AV2.1), or pSHAG AxII plasmids for 24 h were washed with HBSS and suspended in complete medium for an additional 48 h. Cells were then either untreated or incubated with IFN-{gamma} (50–100 U/ml) for 4 h, followed by washing with HBSS and solubilization in 2x SDS-Laemmli sample buffer by boiling for 5 min. After separation of lysates by SDS-PAGE and transfer to nitrocellulose membranes, Western blotting was conducted with Abs directed at AxV, AxII, Stat1{alpha}, SHP-2, and actin. In addition, Stat1{alpha} was immunoprecipitated from pSHAG- and pSHAG AV2.1-transfected cells to analyze the effect of AxV down-regulation on Stat1{alpha} tyrosine phosphorylation. IFN-{gamma}-induced activation of Jak2 was measured by immunoblotting of cell lysates using anti-phosphoJak2 pYpY1007/1008, which recognizes the activated form of Jak2 (25).

Growth arrest assays

To examine the influence of AxV levels on IFN-{gamma}-mediated growth arrest, HeLa cells transfected with pSHAG, pSHAG AxII, or pSHAG AV2.1 constructs were seeded in 12-well plates (0.5 x 104 cells per well) and left to rest for 24 h. The cells were then treated or not with 100 U IFN-{gamma} for 48 h. Cells were washed with HBSS, dislodged using a cell dissociation solution (Sigma-Aldrich), and viable cells were counted in a hematocytometer using trypan blue dye. Alternatively, for assessment of cell proliferation based on thymidine incorporation, HeLa cells transfected with either pSHAG or pSHAG AV2.1 constructs were seeded in 6-well plates (1.0 x 104 cells per well) and left to rest for 24 h. The cells were then treated or not with 100 U IFN-{gamma} for 16–18 h. [Methyl-3H]Thymidine (PerkinElmer) was added to a final concentration of 1 µCi/ml, and the cells were further incubated for 8 h. At the end of metabolic labeling, the cells were washed with PBS and incubated with 5% TCA for 30 min at 4°C. TCA was removed by washing with PBS, and cells were solubilized in lysis solution (0.5 M NaOH/0.5% SDS), and incorporation of 3H was measured by liquid scintillation counting.

For statistical analysis, one-way ANOVA was performed to compare results between experimental groups, followed by Tukey test for multiple comparisons. A p value of <0.05 was considered significant. All statistics were performed using GraphPad Prism software, version 3.0 (GraphPad).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Yeast two-hybrid screening identified AxV as an IFN-{gamma}R2 interacting protein

Screening of the human monocyte cDNA library in yeast strain SDY191/R2, recombinant for the cytoplasmic domain of IFN-{gamma}R2, identified a clone designated D6d with the required phenotype consisting of the ability to grow in auxotrophic medium combined with a positive beta-galactosidase assay. The plasmid from this interacting clone was amplified and used to re-transform SDY191/R2 and the interaction was confirmed. To control for specificity of the phenotype, a series of control transfections were done as outlined in Fig. 1A. Neither growth of yeast nor beta-galactosidase activity was observed when SDY191/R2 was transformed with the pACT2 vector expressing only the GAD. Clone D6d also was transformed into yeast host SDY191 recombinant for the GBD, and no yeast growth was detected. In addition, when SDY191 recombinant for the cytoplasmic domain of IFN-{gamma}R1 was transformed with pACT clone D6d, although limited yeast growth was detected, the beta-galactosidase cracking assay was consistently negative. Together, these results suggest that the interaction between D6d and the intracellular domain of IFN-{gamma}R2 was specific.


Figure 1
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  		FIGURE 1. Specificity of the interaction between the intracellular domain of IFN-{gamma}R2 chain and AxV in the yeast two-hybrid system. A, To screen for IFN-{gamma}R2 chain interacting proteins, the sequence of human intracellular domain of IFN-{gamma}R2 chain was cloned into GBD vector and recombined into the genome of yeast strain SDY 191 (GBD-R2). The integration of the construct into the yeast strain was assessed by PCR of yeast genomic DNA and the expression of the fusion protein was determined by Western blot using an anti GBD Ab (data not shown). In parallel, the sequence of human intracellular domain of IFN-{gamma}R1 chain also was cloned into GBD and introduced into yeast strain SDY 191 (GBD-R1). Both of these yeast clones expressing fusion proteins were transformed with GAD plasmid containing the sequence of human AxV. Double transformants were tested for evidence of an interaction using two independent assays (histidine auxotropic growth and the expression of beta-galactosidase). B, Schematic representation of a IFN-{gamma}R2 interacting clone from the monocyte cDNA library. Clone D6d which showed a strong interaction with R2 was sequenced (826 nucleotides) and identified as AxV. This figure shows the region present in clone D6d, (aa 157–319), including the C-terminal region of AxV and the 3' untranslated region. The AxV sequence present in this clone encompassed domains three and four, which are sufficient to form a binding site for calcium and phospholipids. This truncated AxV chain was apparently also sufficient for the interaction with the intracellular domain of IFN-{gamma}R2 receptor.

 
The insert in clone D6d was sequenced and was identified as AxV. Fig. 1B shows that the region of AxV contained within this clone consisted of 826 nucleotides, corresponding to aa 157–319 plus the 3' untranslated region in frame with the GAD sequence. Residues 157–319 encompass domains three and four of AxV, including the C terminus, and these domains are sufficient to form a binding site for calcium and phospholipids.

GST-IFN-{gamma}R2 cytoplasmic domain protein specifically interacts with AxV

To seek independent evidence to support the yeast two-hybrid results, the cytoplasmic domains of IFN-{gamma}R2 and IFN-{gamma}R1 were cloned and expressed as GST fusion proteins. After purification using glutathione beads, the purity of the preparations was assessed by SDS-PAGE and Coomassie brilliant blue staining (Fig. 2A; n = 3) and equivalent amounts of GST, GST-R1 and GST-R2 fusion proteins were used in pull-down experiments. When a lysate of IFN-{gamma}-treated THP-1 cells was incubated with the fusion proteins, AxV bound to the GST-R2 fusion protein (Fig. 2B, lane 2), but not to either GST alone or the GST-R1 fusion (Fig. 2B, lanes 1 and 3; n = 3). The absence of AxV binding to either GST or GST-R1 provided additional evidence to support the specificity of the interaction of AxV with the cytoplasmic domain of IFN-{gamma}R2.


Figure 2
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  		FIGURE 2. Association of the intracellular domain of IFN-{gamma}R2 chain with AxV in vitro. The intracellular domain of R2 was amplified using RT-PCR from total RNA of THP-1 cells and cloned into pGEX4T expression vector. IPTG-induced GST-R2 fusion protein was purified using glutathione-agarose beads. In addition, the intracellular domain of IFN-{gamma}R1 chain also was expressed and purified as a GST fusion protein to use as a specificity control. A, Affinity-purified GST-fusion proteins. Lane 1, GST; lane 2, GST-R1; and lane 3, GST-R2. B, Interaction of GST-R2 with AxV. PMA differentiated THP-1 cell lysates containing a mixture of protease and phosphatase inhibitors were incubated with GST-fusion proteins (R1 or R2) immobilized on glutathione-Sepharose. After washings, complexes were released by boiling in SDS sample buffer, electrophoresed on 10% SDS-PAGE, and subjected to immunoblotting with anti-AxV Ab. The data shown are from one of three independent experiments that yielded similar results.

 
Confirmation of the interaction between IFN-{gamma}R2 and AxV in vivo by coimmunoprecipitation

Based on the findings from both the yeast two-hybrid screen and the GST pull-down experiment, we sought evidence to determine whether AxV and IFN-{gamma}R2 associate in vivo. To examine this question, THP-1 cells were either untreated or incubated with IFN-{gamma} and then subjected to immunoprecipitation using an Ab directed at IFN-{gamma}R2. As can be seen in Fig. 3A, AxV coimmunoprecipitated with IFN-{gamma}R2, but this was observed only when using lysates from cells that had been IFN-{gamma} treated. When the same membrane was stripped and reprobed, the results showed that similar amounts of receptor were brought down from lysates of both IFN-{gamma}-treated and control cells (Fig. 3B, lanes 6 and 7), suggesting that the interaction is ligand inducible. To examine further the specificity of the interaction, the membrane containing the proteins from the immunoprecipitation was stripped and reprobed with an anti-AxII Ab (Fig. 3C, lane 1). This Ab only recognized AxII in the whole cell lysates and not in the immunoprecipitates, indicating that the association between AxV and IFN-{gamma}R2 was specific.


Figure 3
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  		FIGURE 3. Association of the intracellular domain of IFN-{gamma}R2 chain with AxV in vivo. A, PMA differentiated THP-1 cells were treated with IFN-{gamma} (100 U/ml for 15 min) or left untreated. After stimulation, cells were lysed in digitonin containing buffer supplemented with a mixture of protease and phosphatase inhibitors. Cell lysates were incubated with anti-IFN-{gamma}R2 Abs for 2 h at 4°C. Immune complexes were recovered using protein G-Sepharose and released into boiling SDS-sample buffer without mercaptoethanol (nonreduced). Soluble proteins in parallel with an aliquot of total cell lysate were separated on 7.5% SDS-PAGE and immunoblotted with AxV Ab. Lane 1, total cell lysate; lanes 2 and 4, immunoprecipitations of lysates from untreated cells conducted using two different irrelevant Abs; lanes 3 and 5, immunoprecipitations of lysates from IFN-{gamma}-treated cells conducted using the same two irrelevant Abs; lane 6, immunoprecipitation using anti-IFN-{gamma}R2 incubated with lysate from control cells; lane 7, anti-IFN-{gamma}R2 incubated with lysates from IFN-{gamma}-treated cells. The same blot was stripped and reprobed with anti-IFN-{gamma}R2 (B) and anti-AxII Abs (C). The data are from one of three independent experiments that yielded similar results.

 
Specific down-regulation of AxV expression using siRNA

To examine the role of AxV in IFN-{gamma} signaling, siRNA was used to down-regulate AxV expression in 293T cells. Before undertaking these experiments, we verified that 293T cells respond to IFN-{gamma} as expected by analyzing ligand-inducible tyrosine phosphorylation of Stat1{alpha}, Jak1, Jak2, and IFN-{gamma}R1 (data not shown). Transfection of 293T cells with siRNA construct pSHAG AV2.1 brought about a marked reduction in AxV (70–80% reduction), as determined by immunoblotting. Specificity of the AV2.1 construct was determined by analyzing AxII expression that was unaffected (Fig. 4D).


Figure 4
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  		FIGURE 4. AxV gene silencing in 293T cells using siRNA. A, Two AxV-specific nucleotide sequences (21 mers) were selected using a web-based platform for the design of shRNA constructs. Chemically synthesized oligonucleotides were cloned into the pSHAG vector that directs the in vivo synthesis of siRNA using a U6 promoter. Plasmids were purified from positive clones and used for transfecting 293T cells. After 72 h of transfection, cells were solubilized in boiling SDS-PAGE sample buffer and separated on SDS-PAGE, followed by immunoblotting using anti-AxV Ab. B, To verify equal loading, immunoblotting also was performed with an anti-actin Ab. C, Histogram of densitometric analysis of AxV levels in control and siRNA down-regulated 293T cells. D, The same blot was stripped and reprobed with Ab to AxII. Values are mean ± SD of five independent experiments that yielded similar results.

 
Reduced expression of AxV in 293T cells is associated with enhanced tyrosine phosphorylation of Stat1{alpha} in response to IFN-{gamma} treatment

To examine whether AxV regulates cell signaling in response to IFN-{gamma}, either control (pSHAG) or pSHAG AV2.1 (AV2.1)-transfected 293T cells were incubated with IFN-{gamma}, and whole cell lysates were subjected to immunoprecipitation with an Ab directed at Stat1{alpha}. Immune complexes were then separated by SDS-PAGE, transferred to nitrocelullose membranes and probed with anti-phosphotyrosine Ab. As shown in Fig. 5, A and D, the amount of tyrosine phosphorylated Stat1{alpha} recovered from pSHAG AV2.1 transfected 293T cells after 4 h of IFN-{gamma} treatment was increased two-fold, compared with that recovered from IFN-{gamma}-treated control (pSHAG)-transfected cells (n = 3). These findings suggested that AxV may regulate IFN-{gamma} signaling pathway by controlling the level of tyrosine phosphorylation of Stat1{alpha}.


Figure 5
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  		FIGURE 5. Down-regulation of AxV promotes enhanced tyrosine phosphorylation of Stat1{alpha} in response to IFN-{gamma}. A, 293T cells were transfected with pSHAG or pSHAG AV2.1 plasmids. After 24 h of transfection, cells were washed and rested for another 48 h in complete medium. Cells were then either untreated or incubated with IFN-{gamma} (100 U/ml) for either 15 min or 4 h. Cells were then lysed and immunoprecipitated with anti-Stat1{alpha} Ab. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to a nitrocellulose, and probed with anti-phosphotyrosine Abs. The same membrane was stripped and probed with anti-Stat1{alpha} (B) and anti-AxV Abs (C). D, Results of densitometric scanning of the results in A. The data are from one of three independent experiments that yielded similar results.

 
Influence of AxV on activation of Jak2 in response to IFN-{gamma}

Because Jak2 has been identified as a kinase involved in the tyrosine phosphorylation of Stat1{alpha}, experiments were done to examine whether activation of Jak2 is influenced by reduced level of AxV. To investigate this possibility, we used an anti-phosphoJak2 Ab that recognizes the activated kinase. As shown in Fig. 6, when compared with control cells transfected with pSHAG and incubated with IFN-{gamma}, cells in which AxV was down-regulated showed an approximate twofold increase in Jak2 activation in response to IFN-{gamma}. Protein loading between samples was controlled for by immunoblotting for actin.


Figure 6
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  		FIGURE 6. Down-regulation of AxV promotes IFN-{gamma}-induced tyrosine phosphorylation of Jak2. A, 293T cells were transfected with pSHAG or pSHAG AV2.1 plasmids. After 24 h of transfection, cells were washed and left for another 48 h in complete medium and subsequently either untreated or incubated with IFN-{gamma} (100 U/ml) for 4 h. Cells were then lysed directly in SDS sample buffer and separated on SDS-PAGE, followed by immunodetection using anti-phosphoJak2. B, To verify equal loading, immunoblotting was performed with an anti-actin Ab. C, AxV levels in treatment groups by immunoblotting. D, Densitometric scanning of the results in A. The data are from one of three independent experiments that yielded similar results.

 
AxV regulates growth arrest induced by IFN-{gamma}

To identify a phenotype associated with enhanced Jak-Stat activation in cells expressing reduced levels of AxV, the antiproliferative effect of IFN-{gamma} was examined by two independent methodologies. In the first, the number of viable cells after 48 h of IFN-{gamma} treatment were counted directly by microscopy. As expected, in both pSHAG-transfected control cells and pSHAG-AxII-irrelevant control-transfected cells (expressing reduced levels of AxII and control levels of AxV), IFN-{gamma} treatment brought about partial growth arrest (Refs. 26, 27, 28 and Fig. 7A). Of interest was the finding that the antiproliferative effect of IFN-{gamma} was significantly potentiated in cells transfected with pSHAG AV2.1, which expressed reduced levels of AxV (n = 3). It should be pointed out that down-regulation of AxV per se, in the absence of IFN-{gamma}, did not induce growth arrest, compared with untreated control cells (Fig. 7A). In the second proliferation assay, we used thymidine incorporation into DNA to assess the effect of AxV down-regulation on the antiproliferative action of IFN-{gamma}. As shown in Fig. 7, E and F, based on thymidine incorporation, AxV down-regulation was again observed to bring about enhanced growth arrest mediated by IFN-{gamma}, compared with pSHAG-transfected cells treated with IFN-{gamma}.


Figure 7
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  		FIGURE 7. Reduced levels of AxV potentiate IFN-{gamma} growth arrest in HeLa cells. HeLa cells were transfected with pSHAG, pSHAG AxII, or pSHAG AV2.1 plasmids. After 24 h, cells were washed and left for another 48 h in complete medium and subsequently were seeded in 12-well plates at 0.5 x 105 cells per well. After overnight incubation, cells were either untreated or incubated with IFN-{gamma} (100 U/ml) for 48 h. Cells were then dislodged and counted in a hematocytometer. A, The growth of the cells in the presence of IFN-{gamma} normalized in respect to the growth of corresponding untreated control cells. The values shown are the mean ± SD of results obtained in three independent experiments. For [3H]thymidine incorporation into DNA, control and AxV down-regulated cells were seeded into six-well plates at 1.0 x 105 cells per well. After overnight incubation, cells were either untreated or incubated with IFN-{gamma} (100 U/ml) for16–18 h followed by labeling with [3H]thymidine for 8 h, and incorporated [3H]activity was measured by liquid scintillation counting (E and F). B–D, Results of Western blotting to confirm the specific down-regulation of AxII (B) or AxV (C). Actin was used as a loading control. The values shown are the mean + SD of results obtained in three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Given its central role in both immune and inflammatory responses, regulation of IFN-{gamma} action is a subject of considerable interest. Intuitively, modulation of IFN-{gamma} signaling proximally at the level of the IFN-{gamma}R per se would appear to be an ideal locus to exert control. Whereas there is considerable knowledge about negative regulation of IFN-{gamma} signaling at the level of receptor-associated proteins such as Jak1, Jak2, and Stat1{alpha} (29), information about regulatory molecules that target receptor subunits directly is limited. Previously, the human DnaJ protein hTid-1 was identified as a Jak2 binding protein in a yeast two-hybrid screen (19). hTid-1 also was shown to bind directly to IFN-{gamma}R2 in transfected COS cells and to form a trimolecular complex with Jak2 and IFN-{gamma}R2 in COS cells simultaneously transfected with DNA constructs corresponding to all three proteins (19). Importantly, these associations were found not to require prior incubation of cells with IFN-{gamma}. In the present study, AxV was identified as a novel IFN-{gamma}R2 binding protein in a yeast two-hybrid screen,. This interaction was confirmed in GST-IFN-{gamma}R2 fusion protein pull-down experiments (Fig. 2) and by coimmunoprecipitation of IFN-{gamma}R2 and AxV from IFN-{gamma}-treated THP-1 cells (Fig. 3). The finding from the coimmunoprecipitation indicated that the interaction was ligand inducible, and this suggested that AxV may be involved in regulating IFN-{gamma} signaling. To the best of our knowledge, this is the first identification of an inducible association between IFN-{gamma}R2 and another cellular protein.

Whereas the biochemical data clearly showed that the interaction was ligand inducible (Fig. 3), the yeast two-hybrid screen suggested that the interaction between AxV and R2 was ligand independent. Although these findings may appear to be contradictory, there are other reports in the literature of interactions detected by yeast two- or three-hybrid screening that were found to be ligand inducible under native conditions (30, 31, 32). There are several potential mechanisms to explain this apparent paradox. The observation that a protein-protein interaction is induced by ligand in intact cells may reflect conformational changes, changes in subcellular localization, or a posttranslational modification in one or both proteins. For example, under native conditions, there is the possibility that a conformational constraint in either one or both of the interacting partners may limit the opportunity for an interaction to take place. In the case of AxV and IFN-{gamma}R2, IFN-{gamma} may induce changes in the cell leading to removal of a conformational restriction, thus promoting an interaction between the binding partners. In contrast, in the yeast two-hybrid system, proteins are imported into the nucleus where they reside ectopically, and if an interaction between bait and prey proteins occurs, a functional transcription factor is reconstituted, leading to transcription of reporter genes. Ectopic localization may result in a conformational change in one or both binding partners that favors an interaction. Ectopic expression also may overcome constraints related to subcellular localization that exist under native conditions. Notably, the interaction between AxV and IFN-{gamma}R2 observed in the two-hybrid screen involved an AxV clone containing only domains III and IV, including the C terminus. Because of this, we favor the possibility that, under normal conditions, the presence of N-terminal domains I and II may prevent binding to IFN-{gamma}R2 and that this constraint is removed upon exposure to ligand. However, we cannot exclude the possibility that ligand dependency was related to effects on subcellular localization or to a posttranslational modification.

Of interest, both in the case of hTid-1 mentioned above (19) and AxV (Figs. 5–7), the evidence suggested that these associations with IFN-{gamma}R2 negatively regulated IFN-{gamma} action. The discovery of this novel interaction between IFN-{gamma}R2 and AxV raises important questions about the basis for and biological consequences of the inducible association.

Annexins are a family of 13 structurally related calcium-dependent phospholipid binding proteins (33). They share a conserved 70-aa repeat sequence and have variable N-terminal domains. In general, annexins have been implicated in the maintenance of calcium homeostasis, signal transduction, receptor-mediated endocytosis, cell proliferation and growth, ion channel formation, and the secretion of neurotransmitters and hormones (34, 35). AxV, in particular, has been implicated in regulating cell proliferation (36) and as a protein kinase C inhibitor (37, 38).

Recently, AxV was shown to bind to and regulate signaling through the vascular endothelial growth factor receptor (VEGFR) (39). The finding that AxV bound to the IFN-{gamma}R through an association with the R2 subunit suggests the possibility of a previously unrecognized role for this protein in regulating signal transduction in response to diverse agonists. At present, it is not clear what accounted for the inducible association of the AxV with R2 and whether this was influenced by changes in levels of cytosolic free calcium or by the phosphorylation status of AxV. Tyrosine phosphorylation of AxV also could potentially facilitate its recruitment to IFN-{gamma}R2 by stabilizing its interaction with the receptor complex through interactions with other signaling components, such as Grb2 or other SH2 adaptor proteins. In this regard, AxV has been shown to be tyrosine phosphorylated in response to cell treatment with VEGF (39). Nevertheless, when AxV was shown to be a VEGFR-binding protein, neither the yeast two-hybrid screen nor the vitro translation system used to demonstrate this interaction (39) would have allowed for tyrosine phosphorylation of AxV to have taken place. In fact, in the present study, we were unable to find any evidence for a change in tyrosine phosphorylation of AxV in response to IFN-{gamma} (data not shown). Therefore, this posttranslational modification of the protein would not seem to explain its interaction with IFN-{gamma}R2 in ligand-treated cells.

Another possibility to consider is that the interaction of R2 with AxV could have been influenced by changes in the serine phosphorylation status of AxV. It has been reported that serine phosphorylation of AxV promotes its recruitment to the plasma membrane (40) and the induction of serine phosphorylation of AxI promoted its association with the glucocorticoid receptor in response to ligand (41). However, this would appear unlikely to explain the inducible association of R2 with AxV, because we were unable to detect any change in the serine phosphorylation status of AxV in IFN-{gamma}-treated cells (data not shown).

It is known that cellular responses to IFN-{gamma} can be critically influenced by internalization of R2 (42, 43, 44, 45, 46). Because annexins have been implicated in receptor-mediated endocytosis, it was of interest to investigate whether down-regulation of AxV induced cell surface accumulation of R2. This was examined by flow cytometry, and the results showed that cell surface expression of R2 was equivalent in control and AxV down-regulated cells (data not shown). This finding ruled out the possibility that the observed differences in IFN-{gamma} signaling were due to the accumulation of R2 in the face of reduced levels of AxV.

Given that calcium levels are known to influence the association of annexins with other proteins (47, 48, 49), it also is possible that a conformational change in AxV, the R2 subunit or both, due to an increase in intracellular free calcium induced by IFN-{gamma} (50), could have influenced the association of these proteins. However, this possibility seems unlikely because buffering intracellular free calcium with the known calcium chelator BAPTA-AM (51) failed to block this event (data not shown). We expect that a more detailed understanding of the molecular basis for the interaction between R2 and AxV will be facilitated by identifying the amino acid residues involved in this interaction.

To examine the role of AxV in regulating IFN-{gamma} signaling, we transfected 293T cells with a pSHAG vector expressing siRNA-targeting AxV sequences. The pSHAG system expressing siRNA has been shown to efficiently silence both endogenous and exogenous genes in 293T cells (24). The 293T cells also were selected for this purpose both because of the ease with which they are transfected and because they respond to IFN-{gamma} with activation of the Jak-Stat1{alpha} pathway (Refs. 10 and 52 and Figs. 5 and 6). A phenotype of hyperresponsiveness to IFN-{gamma} in cells with reduced levels of AxV was initially suggested by antiphosphotyrosine blots of whole cell lysates. These showed increased tyrosine phosphorylation of a 90-kDa protein in IFN-{gamma}-treated pSHAG AV2.1-transfected cells, compared with pSHAG vector-transfected control cells (data not shown). The size of this protein suggested that it might have been Stat1{alpha} and prompted investigation of the phosphorylation status of this IFN-{gamma}-regulated transcription factor. In fact, after immunoprecipitation and anti-phosphotyrosine blotting, we found direct evidence for enhanced tyrosine phosphorylation of Stat1{alpha} in IFN-{gamma}-treated cells with reduced content of AxV (Fig. 5). Increased tyrosine phosphorylation of Stat1{alpha} was not explained by increased protein abundance (Fig. 5B) but did correlate with enhanced activation of Jak2 in response to IFN-{gamma} (Fig. 6). Of interest, enhanced phosphorylation of Stat1{alpha} was observed in cells that had been exposed to ligand for 4 h, but not in cells treated for only 15 min. One potential model to explain these findings is that AxV may recruit a protein tyrosine phosphatase to the vicinity of the receptor that acts to modulate the phosphorylation status of Stat1{alpha}. Abnormal phosphatase recruitment in cells deficient in AxV could result in sustained and heightened tyrosine phosphorylation of Stat1{alpha}.

Recent reports indicate that a nonspecific IFN response to siRNA may occur with a frequency of ~30%, although dose-related effects can drive this higher (53, 54, 55). Such a response could have complicated interpretation of the phenotype that we obtained in cells with reduced levels of AxV. Three lines of evidence from our analysis argue strongly against a nonspecific IFN response to siRNA in our system. First, whereas inhibition of cell growth is a hallmark of the IFN response, introduction of the AxII siRNA control construct into cells did not have an antiproliferative effect (Fig. 7). Moreover, we independently confirmed that this siRNA construct was functional leading to reduced expression of the AxII gene (Fig. 7). Second, in this same experiment, when siRNA construct AV2.1 directed at AxV was expressed in HeLa cells in the absence of IFN-{gamma} treatment, no inhibition of cell proliferation was observed. Third, induction of IFN responsive genes such as Stat1 has been used to monitor nonspecific IFN responses to siRNA (54). However, when we transfected cells with the AV2.1 siRNA construct, no induction of Stat1{alpha} expression was observed (Fig. 5B). Based on these complementary controls, we concluded that the impact of silencing the AxV gene was not complicated by a nonspecific IFN response.

Taken together, these results suggested that the interaction of AxV with R2 controls activation levels of Jak2 and Stat1{alpha}, and they are consistent with a model in which AxV normally regulates signaling through this pathway. One possible mechanism to explain this could involve the association of AxV with R2 interfering with Jak2 binding because of either overlapping binding sites or steric hindrance. In this model, reduced levels of AxV would increase access of Jak2 to R2 and enhance its activation, and secondarily that of Stat1{alpha}, in response to IFN-{gamma}. Another possibility is that AxV may inhibit Jak2 activation either directly or indirectly, as discussed above in the context of Stat1{alpha}, by promoting the recruitment of a phosphotyrosine phosphatase to the vicinity of the receptor.

Although the data available do not allow discrimination between these various alternatives, the model does allow for predictions to be made. For example, if AxV normally controls the level of Jak2-Stat1 activation, then removing this influence should lead to enhanced cellular responses to IFN-{gamma}. To investigate the impact of enhanced activation of Jak2 and Stat1{alpha} in cells with reduced levels of AxV, we evaluated IFN-{gamma}-induced growth inhibition. Inhibition of cell proliferation is a hallmark of the IFN response (26, 27, 28), and the antiproliferative response to IFN-{gamma} has been shown to be dependent on activated Stat1{alpha} (26). In the latter study, cells transfected with either wild-type Stat1{alpha} and/or Stat1{alpha} mutants were examined. The introduction of wild-type Stat1{alpha} enhanced the antiproliferative effect of IFN-{gamma}, whereas Stat1{alpha} mutants reversed IFN-{gamma}-induced growth arrest. Consistent with this model, we observed that, compared with control cells, the antiproliferative effect of IFN-{gamma} was enhanced in cells expressing reduced levels of AxV, but not in those with reduced levels of AxII (Fig. 7). These results provide direct evidence to show modulation of biologic responses to IFN-{gamma} specifically by AxV and suggest that this may be related to effects on Jak-Stat1{alpha} activation.

In summary, the results of the present study identify a novel role for AxV as a ligand-inducible IFN-{gamma}R2-binding protein. The predominant impact of this interaction is negative regulation of IFN-{gamma} signaling, and this may represent a physiologic mechanism to control signaling through this pathway (Fig. 8). As such, these findings add to what is already known about the complex regulation of IFN-{gamma} signaling through other control elements such as SOCS proteins (18, 56), SHP-1 and SHP-2 (14, 17, 57, 58, 59, 60), hTid-1 (19), and regulated expression of the IFN-{gamma}R2 chain itself (61). Finally, although the model shown in Fig. 8 suggests that AxV modulates signaling through the IFN-{gamma}-activated Jak-Stat1 pathway, it does not preclude the possibility that this interaction also may position AxV to influence Stat1-independent signaling elements that are activated downstream of the IFN-{gamma}R.


Figure 8
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  		FIGURE 8. Proposed model for the role of AxV in regulating IFN-{gamma} cell signaling. A, In the absence of ligand, AxV is not associated with the IFN-{gamma}R. B, Upon exposure of cells to IFN-{gamma}, AxV is recruited to the R2 receptor subunit. The association of AxV with R2 controls the phosphorylation and activation states of Jak2 and Stat1{alpha}, thereby regulating downstream responses. C, Under conditions where the abundance of AxV is limiting, diminished association of regulator with R2 leads to enhanced tyrosine phosphorylation of Jak2 and Stat1{alpha} and to augmented cellular responses to IFN-{gamma}.

 

   Acknowledgments
 
We thank Dr. Alice Mui (University of British Columbia) for providing cells and monocyte RNA, and Dr. Martin Sims (GlaxoSmithKline) for providing the pYTH9 shuttle vector and yeast strain SDY191. The technical assistance of Richard Ly and Chrystal Lapinsky is gratefully acknowledged.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Canadian Institutes of Health Research Operating Grants MOP-8633 (to N.E.R.) and FRN-38005 (to D.N.). Back

2 C.L. and D.N. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Neil E. Reiner, Division of Infectious Diseases, University of British Columbia, Room 452D, 2733 Heather Street, Vancouver, BC, Canada, V5Z 3J5. E-mail address: ethan{at}interchange.ubc.ca Back

4 Abbreviations used in this paper: R1, {alpha}-chain of IFN-{gamma}R; R2, beta-chain of IFN-{gamma}R; GAS, {gamma}-activated sequence; AxV, annexin V; GAD, Gal-4 activator domain; GBD, Gal-4 binding domain; SH2, Src homology 2; siRNA, small interfering RNA; shRNA, short hairpin RNA; VEGFR, vascular endothelial growth factor receptor. Back

Received for publication September 7, 2005. Accepted for publication February 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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