IFN-γ is an important immunoregulatory protein with tightly controlled expression in activated T and NK cells. Three potential STAT binding regions have been recognized within the IFN-γ promoter: 1) an IL-12-mediated STAT4 binding site at −236 bp; 2) a newly identified IL-2-induced STAT5 binding element at −3.6 kb; and 3) CD2-mediated STAT1 and STAT4 binding to an intronic element in mucosal T cells. However, functional activation of these sites remains unclear. In this study we demonstrate CD2-mediated activation of the newly characterized −3.6-kb IFN-γ STAT5 binding region. CD2 signaling of human PBMC results in activation of the −3.6-kb IFN-γ promoter, whereas mutation of the −3.6-kb STAT5 site attenuates promoter activity. Functional activation is accompanied by STAT5A but little STAT5B nucleoprotein binding to the IFN-γ STAT5 site, as determined by competition and supershift assays. STAT5 activation via CD2 occurs independent of IL-2. Western and FACS analysis shows increased phospho-STAT5 following CD2 signaling. AG490, a tyrosine kinase inhibitor affecting Jak proteins, inhibits CD2-mediated IFN-γ mRNA expression, secretion, and nucleoprotein binding to the IFN-γ STAT5 site in a dose-dependent fashion. This report is the first to describe CD2-mediated activation of STAT5 and supports STAT5 involvement in regulation of IFN-γ expression.
Interferon-γ is a prominent immunoregulatory protein vital to immune development and function. The regulation of IFN-γ secretion by Th1 cells plays an important role in protective immunity. Activation of T cells is initiated by engagement of the TCR, leading to differentiation and cytokine production. Alternatively, T cell activation may also occur through signaling of other cell surface receptors such as the CD2 receptor (1, 2).
CD2 is a transmembrane cell surface adhesion protein expressed on nearly all T cells, thymocytes, and NK cells. CD2 signaling fosters physical cell-to-cell interaction between T cells and APC expressing the CD58 ligand (3). Although a close correlation exists between TCR and CD2 signaling, distinct differences have been noted. The dissociation rate for the CD2-CD58 interaction is far more rapid than that observed for the TCR-MHC interaction (4), and is believed to promote specific but short-lived binding interactions, allowing T cell-MHC interactions even when a small number of complexes are displayed on the surface of APC. Activation of T cells does not increase the amount of CD2 receptor expressed on the cell surface, although it has been noted that there is redistribution and clustering of receptors to lipid rafts, which results in enhanced binding avidity (5, 6). Moreover, CD2-CD58 interactions play a unique role in reversing anergy (7). Interestingly, mucosal T cells, characterized by a relatively anergic state, are unresponsive to activation via the TCR pathway, yet exhibit heightened responsiveness to CD2 signaling (8, 9).
Expression of IFN-γ is controlled primarily on a transcriptional level. Early studies identified potential regions of transcriptional regulation based on DNA methylation and DNase I hypersensitive sites located within the intronic and 5′ flanking region of the IFN-γ gene (10). Subsequently, several functionally important elements have been defined that contain binding sites for jun/fos, AP-1, CREB/activating transcription factor, and NFAT nucleoprotein complexes (11). The molecular mechanisms involved in regulation of increased IFN-γ production following CD2 signaling are poorly defined, which is in contrast to the relative wealth of information available regarding TCR-mediated IFN-γ expression. We have previously identified CD2-specific response elements within the IFN-γ promoter (12). In PBL, CD2 activation elements reside mainly within the −108- to +64-bp region, encompassing the proximal and distal AP-1 binding sites. In fact, an increase in AP-1 driven TPA response element promoter-reporter activity is detected following CD2 activation, supporting a role for AP-1 in CD2-mediated expression. CD28 coligation further enhances promoter activity, although a CD28 transcriptional binding region has not been defined for IFN-γ.
Recently published reports have suggested that STAT proteins contribute to optimal gene transactivation. Three potential STAT binding regions have been identified within the IFN-γ promoter. Activation of these sites involves complex regulatory mechanisms. IL-12 mediates STAT4 binding to a site at the −236-bp region, and requires cooperative interaction with a flanking AP-1 element (13). STAT binding regions, which are able to bind recombinant members of the STAT family in response to IL-12 treatment (14), have also been identified within the first intron. We have subsequently shown that CD2 signaling mediates activation of STAT1 and STAT4 proteins and binding to the intronic IFN-γ region in mucosal T cells (15). A recent study, using comparative sequence analysis, has identified conserved T-bet, AP-1, and NFAT binding regions at a distal site −5 kb upstream of the start of transcription (16). This region is consistent with a previously reported DNase I hypersensitive site. Adjoining that region, a novel functional IL-2-responsive STAT5 binding element located at −3.6 kb has been identified (17). This region undergoes chromatin remodeling and plays a role in IL-2-induced transcription of IFN-γ expression. In this article, we demonstrate that CD2 mediates activation of this novel −3.6-kb STAT5 binding enhancer region.
Materials and Methods
PBMC isolation and stimulation
PBMC were isolated from normal healthy volunteers by separation on Ficoll-Hypaque gradients. PBMC were stimulated with 0.1 μg of anti-CD2 Abs (clones CB6 and GD10; a gift from C. Benjamin, Biogen, Cambridge, MA) per 106 cells at 37°C for the times indicated for each experiment. Stimulation of PBMC resulted in the secretion of 1400 ng/ml IFN-γ/106 cells. Cells treated with the indicated amounts of AG490 (Calbiochem, San Diego, CA) or 20–80 nM damnacanthal (IC50, 17 nM) (BIOMOL, Plymouth Meeting, PA) were preincubated for 18 h before activation.
Gel mobility EMSA
Double-stranded oligonucleotide was end-labeled with adenosine 5′-triphosphate, γ-32P, and T4 polynucleotide kinase. A total of 3–6 μg of nuclear extract protein was incubated at 25°C with 0.25 mg/ml poly(dI-dC), in 20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, and 50 mM Tris (pH 7.5) for 10 min. The oligonucleotide was then added (20,000 cpm), and the binding reactions were incubated for an additional 30 min. Specificity was determined by the addition of 100-fold excess unlabeled oligonucleotide as competitor. The DNA-protein complexes were separated from unbound probe on a prerun native 6% polyacrylamide gel in low ionic strength buffer (22.3 mM Tris (pH 7.4), 22.3 mM borate, 0.5 mM EDTA (pH 8.0)). After 2 h, the gel was dried under vacuum and exposed to x-ray film. The −3.6-kb IFN-γ STAT5 element wild-type (in bold) or mutant oligonucleotide (lower case) used were: 5′-AGT TAT TAG AAA TTT CAA GGA AGT GAC AAC AGA G, STAT5 wild type; 5′-AGT TAT TAG AAA TTT CAA cct tGT GAC AAC AGA G-3′, STAT5 mutant.
Consensus STAT oligonucleotides obtained from Santa Cruz Biotechnology (Santa Cruz, CA) were as follows: STAT1, 5′-CATGTTATGCATATTCCTGTAAGTG-3′; STAT5, 5′-AGATTTCTAGGAATTCAATCC-3′; mutant STAT5, 5′-AGATTTagtttAATTCAATCC-3′. Rabbit anti-STAT5A and STAT5B Abs used for supershift assays were obtained from R&D Systems (Minneapolis, MN).
Promoter cloning and reporter constructs
Genomic DNA (200 ng) was used as a template for all PCR in 10- to 50-μl volumes. The full −3.662-kb to +38-bp IFN promoter was amplified in 10 μl PCR containing 10 mM Tris-HCl (pH 8.3); 50 mM KCl; 1.5 mM MgCl2; 200 μM each of dATP, dGTP, and dTTP; 80 ng of each oligonucleotide primer; and 1.5 U High Fidelity Taq polymerase (Invitrogen Life Technologies, Rockville, MD). The primers used to amplify the −3.662 to +38 human IFNG promoter are as follows with the MluI linkers underlined: 5′–GTA CGC GTG TGG AAA CAA GTG CAA AAC G–3′ and 5′–GTA CGC GTC TAA TAG CTG ATC TTC AGA TG–3′.
For amplification of the −3.6-kb fragment, the conditions were as follows: 95°C for 5 min, then 30 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 60 s, and extension at 68°C for 5 min. These PCR were done with a Hybaid Touchdown thermal cycler (Thermo Hybaid, Middlesex, U.K.).
The −3.6-kb IFNG promoter was amplified from genomic DNA with MluI linkers and ligated into the TA vector. Following MluI and PvuI double digestion, the −3.6-kb insert was extracted from the 0.8% agarose gel, purified, and ligated into the PGL3 Basic Luciferase vector (Promega, Madison, WI). Restriction digests revealed clones with a 5′-3′ orientation and sequencing confirmed the Stat element. Site-directed mutatgenesis was performed on the −3.6-kb IFNG-Luc construct to disrupt the Stat5 motif from TTCAAGGAA to TTCAACCTT. DNA sequencing confirmed the mutagenesis. The mutant −3.6-kb IFN-γ STAT5 construct possessed a 4-bp substitution identical with that used in EMSA experiments.
Freshly isolated PBMC were transfected following overnight culture in RPMI 1640 medium containing 10% FCS. Cells were then washed and resuspended in 250 μl of fresh medium at 2 × 107 cells/ml and electroporated in the presence of 50 μg of reporter construct (600 V, for 9 pulses of 500 microseconds, with 100 microseconds between pulses) using 40-mm (gap width) cuvettes in a BTX Electro Square Porator ECM 830 (Genetronics, San Diego, CA). A control plasmid containing the β-actin promoter driving a Renilla luciferase (provided by Dr. C. Wilson, University of Washington, Seattle, WA) was cotransfected as an internal standard and values were normalized to correct for transfection efficiency. After electroporation, the cells were diluted in fresh medium, allowed to rest for 1 h before plating, and then stimulated with anti-CD2 mAbs for 4 h. Luminescence was measured using a Promega luciferase assay kit and counted on a 6-detector PerkinElmer Life Sciences (Gaithersburg, MD) 1450 Microbeta liquid scintillation counter with coincidence counting deactivated.
PBMC were lysed and separated on a NuPAGE 7% Tris-acetate gel (Invitrogen Life Technologies) transferred to PVDF membrane (Invitrogen Life Technologies) and analyzed for immunoreactivity with antisera specific to anti-phospho-STAT5 or anti-phospho-STAT3 (Cell Signaling Technology, Beverly, MA) and detected with an ECL detection system (Pierce, Rockford, IL). Blots were then stripped of the phospho-specific Ab and reprobed for equal loading of STAT protein
Flow cytometric staining of intracellular phosphorylated STAT
PBMC were stimulated with anti-CD2 Abs and analyzed for the presence of phosphorylated STAT5 by flow cytometry as previously described (18, 19). Briefly, freshly isolated PBMC or lamina propria mononuclear cells were rested overnight and then stimulated with anti-CD2 mAbs and cultured in 300 μl PBS with 2% FCS for the indicated periods of time. Cells were then fixed with 300 μl of fixation reagent (reagent A, Fix and Perm; Caltag Laboratories, Burlingame, CA) for 2–3 min at room temperature followed by the addition of 3 ml of cold methanol. The tubes were kept on ice for 10 min, centrifuged, washed with PBS, and resuspended in permeabilization medium (reagent B). Cells were then incubated at room temperature for 30 min with rabbit anti-human phospho-STAT5 (Cell Signaling Technology), or control rabbit IgG (Caltag Laboratories). Cells were washed and stained with FITC-conjugated F(ab′)2 goat-anti-rabbit IgG (Caltag Laboratories) for 30 min at room temperature. Following a final wash, the cells were analyzed by flow cytometry.
Northern blot analysis
Total cellular RNA was extracted using the RNeasy mini kit (Qiagen, Chatsworth, CA). RNA was electrophoretically separated on a denaturing 1% agarose gel containing 7% formaldehyde. Gels were transferred to nylon membrane (Amersham, Arlington Heights, IL) and hybridized to 32P-labeled DNA probe. Isolated cDNA insert was labeled by random priming and used at 106 cpm/ml of hybridization buffer. Blots were prehybridized (50% formamide, 0.75 M NaCl, 75 mM sodium citrate, 1× Denhardt solution, 25 mM sodium phosphate (pH 6.5), and 100 μg/ml sheared salmon sperm DNA) at 42°C for 2 h and hybridized overnight in prehybridization solution containing labeled probe and 10% dextran sulfate.
IFN-γ was measured by an amplified ELISA (20). Greiner Bio-One (Longwood, FL) ELISA plates were coated overnight with 100 μl of 5 μg/ml monoclonal anti-IFN-γ (BD Biosciences, Woburn, MA). Samples and standards were added for 24 h followed by addition of 100 μl of 2.5 μg/ml polyclonal biotinylated rabbit anti-IFN-γ (BD Biosciences) for 2 h. This was followed by addition of 100 μl of 1/1000 diluted alkaline phosphatase-conjugated steptavidin (Jackson ImmunoResearch Laboratories, West Grove, PA) for 2 h. Substrate, 0.2 mM NADP (Sigma-Aldrich, St. Louis, MO) was added for 30 min followed by addition of amplifier (3% 2-propanol, 1 mM iodonitrotetrazolium violet, 75 μg/ml alcohol dehydrogenase, and 50 μg/ml diaphorase; Sigma-Aldrich) for 30 min. Plates were read at 490 nm using an E max plate reader (Molecular Devices, Sunnyvale, CA). All data acquisition and reduction was performed using the ELISA Master program for Macintosh computers, developed by R. L. Deem.
Tests for statistical significance was determined by Wilcoxon sign-rank using JMP Statistical Software (SAS Institute, Cary, NC).
The −3.6-kb STAT5 binding region participates in CD2-mediated activation of the IFN-γ promoter
We have previously demonstrated that CD2 signaling of PBMC is followed by activation of STAT proteins with enhanced levels of phosphotyrosine-STAT1 (15). Recently, a novel STAT5 binding element was characterized within the distal −3.6-kb IFN-γ promoter region that is involved in IL-2-mediated regulation of IFN-γ production (17) To investigate whether the −3.6-kb STAT5 binding region was involved in CD2-mediated regulation of IFN-γ gene expression, promoter/reporter constructs containing the −3.6-kb IFN-γ were transfected into human PBMC. As seen in Fig. 1, CD2 signaling resulted in enhanced promoter activity of the −3.6-kb IFN-γ promoter region. Enhancement in CD2-mediated expression was detected in response to IL-2 treatment, reminiscent of what had been reported following PMA/ionomycin activation (17). In contrast, mutation of the STAT5 binding region impaired CD2-mediated expression, resulting in a marked reduction of promoter activity even in the presence of IL-2 (Fig. 1). These results suggest that CD2 mediates functional activation of the −3.6-kb STAT binding region.
CD2 stimulation results in up-regulation of nuclear protein binding to the −3.6-kb STAT5 site
To confirm functional activation of STAT5 following CD2 signaling, nuclear proteins were analyzed for DNA-binding activity to the IFN-γ −3.6-kb STAT5 element (Fig. 2,A). CD2 activation resulted in up-regulation of trans-acting factors binding to the −3.6-kb IFN-γ STAT5 element, which was sustained up to 360 min. A similar up-regulation of binding was detected to a consensus STAT5 oligonucleotide (Fig. 2,A). No changes in the pattern of binding to the IFN-γ −3.6-kb STAT5 element were detected in PBMC treated with IL-2 (Fig. 2,B) or anti-IL-2 before and during CD2 activation (Fig. 2 C).
Binding to this site was specific for STAT5 as demonstrated by cold competition experiments. Nuclear extracts were prepared from PBMC stimulated for 30 min, and nucleo-protein binding was conducted in the presence of excess competing oligonucleotides to the −3.6-kb IFN-γ STAT5 or with identical probes generated with base pair substitutions mutating either the STAT5 binding element or the immediate flanking region. As seen in Fig. 3,A, the addition of 5-fold excess −3.6-kb IFN-γ STAT5 oligonucleotide successfully competed for binding of the complex. Mutation of the putative STAT5 binding site abolished its capability to compete for the binding of the protein complex, whereas mutation of the flanking region did not affect its ability to compete for binding. Competition experiments were likewise conducted using a consensus STAT5 binding element (Fig. 3,B). Five-fold excess cold-consensus STAT5 oligonucleotide competed successfully for the DNA-protein binding, whereas addition of excess cold −3.6-kb IFN-γ STAT required 100-fold excess oligonucleotide to successfully compete, suggesting a weaker binding avidity of STAT5 to the −3.6-kb IFN-γ promoter region than to the consensus STAT5 site. In contrast, no competition was detected even with 100-fold excess mutant STAT oligonucleotide (Fig. 3,B). The presence of STAT5 was further confirmed through supershift assays. Nuclear protein extracts were preincubated with Abs specific to STAT5A or STAT4B. As seen in Fig. 3 C, a pronounced supershift complex binding to the −3.6-kb IFN-γ oligonucleotide was detected following addition of Abs specific to STAT5A while addition of Abs specific to STAT5B were barely able to supershift the complex.
CD2 signaling results in phosphorylation of STAT5
Phosphorylation of STAT proteins is essential for activation and binding to promoter sequences. Flow cytometry was conducted to detect the presence of phospho-STAT5. As seen in Fig. 4 A, there is a shift in the cell population staining for the presence of phospho-STAT5 within 15 min of CD2 activation, which was sustained for 60 min.
In addition, Western blot analysis was used to confirm these findings. Basal levels of phospho-STAT5 in unstimulated cells were minimal, whereas a significant increase in phospho-STAT5 was detected within 15 min following CD2 activation, which was sustained for 30 min (Fig. 4 B). Phosphorylation was specific for STAT5 in that no change in the level of phospho-STAT3 was detected. In each case, the blots were stripped of the phospho-specific Ab and reprobed to check for equal loading of STAT protein.
AG490, a Jak kinase inhibitor, suppresses IFN-γ expression
Activation of STAT proteins is strongly associated with phosphorylation of members of the Jak kinase family. AG-490 is a tyrosine kinase inhibitor specific for Jak2 (21) and Jak3 activity (22) but not Jak1, tyk2, or lck (21). To study the contribution of Jak activation on regulation of IFN-γ production, PBMC were cultured with increasing concentrations of AG490. As seen in Fig. 5 A, AG490 inhibits both CD2 and PMA/ionomycin-mediated IFN-γ production in a dose-dependent manner. The level of cytokine in control DMSO treated cells remained unchanged. AG490 was not merely a toxic agent, because removal of AG490 before activation by CD2 restored cytokine levels. In addition, the lck inhibitor damnacanthal failed to inhibit IFN-γ production at concentrations up to four times the IC50 (data not shown).
To determine the molecular events associated with inhibition of IFN-γ production, IFN-γ mRNA levels were measured following treatment with AG490. Northern blot analysis of IFN-γ mRNA revealed a marked reduction in IFN-γ mRNA levels following treatment with as little as 50 μM AG490, whereas the β-actin mRNA was significantly less affected (Fig. 5 B). Once again, washout experiments confirmed that the inhibition was reversible.
Nuclear proteins were analyzed for DNA-binding activity to the IFN-γ −3.6-kb STAT5 element following CD2 activation. As seen in Fig. 5,C, AG490 inhibited STAT5 nuclear protein binding to the IFN-γ −3.6-kb STAT5 element in a dose-dependent manner. Moreover, inhibition of nuclear complex formation following treatment with AG490 was selective for STAT5, whereas binding of STAT1 nuclear protein complex remained unchanged (Fig. 5 D).
In the present study we show that signaling through the CD2 pathway in T cells leads to activation of STAT5, up-regulation of IFN-γ promoter activity, and binding to a recently identified STAT5 sequence located at a distal region −3.6-kb upstream of the transcriptional start site. Promoter activity was greatly reduced by disruption of the STAT5 binding sequence. Activation of STAT5 following signaling through the CD2 receptor occurs independent of IL-2. CD2 signaling results in binding of a complex comprised of STAT5A but little or no STAT5B. Following CD2 stimulation there is a significant increase in the level of phosphorylation of STAT5. Furthermore, the JAK inhibitor, tyrophostin AG490, inhibits IFN-γ secretion, mRNA levels, and binding to the −3.6-kb STAT5 promoter region, suggesting that activation of the Jak/STAT pathway might play an important role in CD2-mediated regulation of IFN-γ expression.
The STAT pathway was originally considered to be a cytokine/growth receptor-mediated pathway. However, more recently, TCR signaling has been linked with STAT activation. TCR signaling of Th cell line, D10, has been reported to activate STAT5, which, in turn, triggers proliferation (23). Nevertheless, other groups failed to detect phosphorylation of STAT5 following TCR signaling of primary human or murine lymphocytes (24, 25). This discrepancy was attributed to differences in the cell systems examined. Indeed, CD2 signaling of highly activated human cultured T cells results in delayed tyrosine phosphorylation of STAT1 while CD2 stimulation of NK cells induces functional activation in the absence of any STAT phosphorylation (26). This finding is in contrast to what is seen following TCR signaling of long-term cultured T cells, which results in delayed serine phosphorylation of STAT1 in the absence of tyrosine phosphorylation. A further link between TCR ligation and STAT5 activation was detected in a transgenic animal model. There was a predisposition to development of lymphoblastic lymphoma in mice overexpressing STAT5A and STAT5B (20). Introduction of TCR transgenes or Ag stimulation dramatically increased the rate of lymphoma, suggesting a contributing role for TCR stimulation in up-regulating STAT5 expression. Furthermore, TCR stimulation of cell cycle progression was abolished in STAT5 knockout mice even in the presence of IL-2, whereas STAT5B expression in common cytokine receptor γc knockout mice was able to enhance TCR-mediated proliferation (27, 28). Interestingly, a strong association exists between TCR ligation and expression of CIS, a negative modulator of STAT5 activity (29). Moreover, the simultaneous activation of STAT in conjunction with TCR-mediated signaling is able to allow progression of cell division and release of anergy (30). A recent study proposed a role for STAT5 in differentiation and maintenance of the IFN-γ secreting Th1 phenotype (31). The experiments reported in this study suggest a role for CD2 receptor signaling in STAT5 activation.
Our EMSA data suggest that CD2 signaling results in preferential binding of STAT5A to the −3.6-kb STAT binding sequence. Preferential activation of a specific STAT5 isoform has been previously described in other systems. STAT5A and STAT5B are two highly conserved proteins displaying >90% homology on a protein level, but encoded by two separate genes that are linked head to head on chromosome 17 (32, 33). Both proteins contain a conserved tyrosine residue in the C terminus that undergoes phosphorylation following activation. Although differences exist in the amino acid sequence in the DNA binding domain of these proteins, both can bind to identical DNA binding sites containing the IFN-γ-activated site binding motif (34). Despite the similarities between STAT5A and STAT5B, distinct differences in cellular functions have been attributed to them. To a great extent these differences have been defined using animal model systems. STAT5A is required for effective prolactin signaling and mammary gland development (35), and has recently been shown to participate in ischemic heart disease (36). STAT5B participates in growth hormone receptor signaling and sexual dimorphic growth (36). STAT5A knockout mice display decreased proliferation of splenocytes in response to low-dose IL-2, which could be rescued by increasing the dose of IL-2. In contrast, high dose IL-2 failed to rescue proliferation in STAT5B knockout mice (27, 37). A role for STAT5B, but not STAT5A, has been suggested in regulation of gene expression in diabetes associated vascular disease (38). Similarly, increased expression and phosphorylation of STAT5B but not STAT5A has been proposed as a contributing factor in squamous cell tumorigenesis (39). Selective involvement of STAT5 isotypes has also been noted in other cell model systems. Binding of fms-related tyrosine kinase 3 ligand to its tyrosine kinase receptor fms-related tyrosine kinase 3 in hemopoietic cells leads to selective activation of STAT5A but not STAT5B (40). Likewise, IFN-α and IFN-γ can selectively activate STAT5A (41), whereas STAT5B is preferentially activated by GM-CSF (42). STAT5B can enhance expression of adipose genes but is not adipogenic on its own (43). In contrast, expression of STAT5A alone is sufficient to confer adipogenesis. Furthermore, transcriptional regulation of the estrogen receptor ERβ, but not ERα, was dependent entirely on STAT5B (44). It is interesting to note that in NK cells and highly activated T cells, IL-2-mediated activation of the −3.6-kb IFN-γ STAT5 promoter region leads to a STAT5A/STAT5B heterodimer complex. However, in our system using resting T cells, selective activation of STAT5A was observed following CD2 signaling, arguing that differences in the specificity of STAT activation probably reflect selective gene activation dependent on cell type as well as mode of activation.
Inhibition by AG490 of CD2 mediated IFN-γ secretion and mRNA levels as well as binding to the −3.6-kb IFN STAT5 element is consistent with a participatory role for the Jak pathway. Although it was not the aim of this study to identify the specific Jak involved, unlike most kinase inhibitors, which broadly inhibit multiple tyrosine kinases, inhibition by AG490 is largely restricted to particular members of the Jak kinase family. AG490 is a member of the tyrphostin family of tyrosine kinase inhibitors. AG490 was originally described as a specific inhibitor of Jak2 (21), which was subsequently extended to inhibit Jak3 activity as well (22). Although one cannot rule out the possibility that AG490 may act on other kinases and recent studies suggest that AG490 may causes growth arrest of cells in G1 phase (45) and inhibit epidermal growth factor receptor autophosphorylation (46), nevertheless AG490 is undoubtedly a potent inhibitor of Jak2 and Jak3 and a powerful tool for studying inhibition of Jak/STAT pathway. Furthermore, AG490 does not inhibit Jak1, tyk2, or other lymphocyte tyrosine kinases such as lck, lyn, Btk, Syk, or Src (20, 21, 47).
A rapid activation of STAT has been observed following cytokine gene receptor signaling whereas our data suggests that activation of STAT5 following CD2 ligation is relatively slow and sustained. Delayed and sustained activation of STAT1 has previously been reported following CD2 or TCR activation of highly activated T cells. STAT1 phosphorylation following TCR ligation was resistant to cycloheximide and could not be triggered by adding conditioned medium from cells activated by CD2 ligation arguing against an autocrine-mediated mechanism of activation (26). Likewise, our data suggest that STAT5 activation occurs in an IL-2-independent fashion in as much as pretreatment with IL-2 or anti-IL-2 Abs did not alter the kinetics of CD2-mediated binding of STAT5 to the −3.6-kb STAT5 IFN-γ promoter region. Moreover binding to the −3.6-kb STAT5 IFN-γ promoter region was not transferred by preconditioned medium from CD2 activated cells (data not shown). Interestingly, even within the cytokine/growth receptor-mediated pathway, a delayed and sustained activation of STAT3 by Jak2 has been reported in response to basic fibroblast growth factor and platelet-activating factor (48). Although the mechanism involved in this delayed response remains to be determined it should be taken into consideration that to achieve effective functional activation of lymphocytes, sustained Ag receptor signaling is required. It is conceivable therefore that delayed activation of STAT may be a common hallmark to TCR-mediated activation of this pathway.
We thank Stephanie Cha and Jaclyn Zhou for providing cultured lamina propria mononuclear cells and PBMC.
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.
This work was supported by U.S. Public Health Service Grants DK-43211 and DK-46763 and Cedars Sinai Medical Center Inflammatory Bowel Disease Research Funds.