JAK3, STAT, and MAPK Signaling Pathways as Novel Molecular Targets for the Tyrphostin AG-490 Regulation of IL-2-Mediated T Cell Response

Li Hua Wang, Robert A. Kirken, Rebecca A. Erwin, Cheng-Rong Yu and William L. Farrar
J Immunol April 1, 1999, 162 (7) 3897-3904;

Abstract

AG-490 is a member of the tyrphostin family of tyrosine kinase inhibitors. While AG-490 has been considered to be a Janus kinase (JAK)2-specific inhibitor, these conclusions were primarily drawn from acute lymphoblastic leukemia cells that lack readily detectable levels of JAK3. In the present study, evidence is provided that clearly demonstrates AG-490 potently suppresses IL-2-induced T cell proliferation, a non-JAK2-dependent signal, in a dose-dependent manner in T cell lines D10 and CTLL-2. AG-490 blocked JAK3 activation and phosphorylation of its downstream counterpart substrates, STATs. Inhibition of JAK3 by AG-490 also compromised the Shc/Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathways as measured by phosphorylation of Shc and extracellular signal-related kinase 1 and 2 (ERK1/2). AG-490 effectively inhibited tyrosine phosphorylation and DNA binding activities of several transcription factors including STAT1, -3, -5a, and -5b and activating protein-1 (AP-1) as judged by Western blot analysis and electrophoretic mobility shift assay. These data suggest that AG-490 is a potent inhibitor of the JAK3/STAT, JAK3/AP-1, and JAK3/MAPK pathways and their cellular consequences. Taken together, these findings support the notion that AG-490 possesses previously unrecognized clinical potential as an immunotherapeutic drug due to its inhibitory effects on T cell-derived signaling pathways.

Normal immune function requires activation of the TCR, which drives cells from G0-G1 transition and subsequent expression of IL-2 and its receptor (1). IL-2, a potent T lymphocyte growth factor, is then responsible for promoting G1-S phase transition and subsequent clonal expansion and differentiation of T cells (2). To date, effective clinical immune management has been molecularly localized to the inhibition of IL-2 synthesis and secretion. The blockade of IL-2 transcription, by either cyclosporin A or corticosteroids, has been demonstrated to clearly dampen immunological responsiveness (3, 4, 5, 6). Only recently have the general aspects of the biochemical signal transduction process of IL-2 been realized, thus making IL-2 signaling pathways a potential target for pharmaceutical intervention that can alter the progression of a broad range of T cell-mediated disease.

The propagation of IL-2-mediated signal transduction occurs following binding of the ligand to the high affinity IL-2 receptor complex consisting of two members of the hematopoietin receptor superfamily, designated IL-2Rβ and IL-2Rγ (7, 8, 9). IL-2-induced heterodimerization of IL-2Rβ and IL-2Rγ results in intermolecular transphosphorylation of their corresponding receptor-associated Janus kinase 1 (JAK1)4 and JAK3, respectively (10, 11). The JAKs often catalyze the activation of two distinct signaling pathways; for IL-2 it includes the Shc/Ras/Raf/mitogen-activated protein kinase (MAPK) cascade, while the other is comprised of a family of gene-regulating transcription factors, known as STATs (12, 13, 14, 15). These signaling proteins regulate gene transcription, ultimately controlling cell growth, differentiation, and immune responsiveness (12). Moreover, JAK3 provides a key role in T cell development and activation. Patients unable to activate this kinase, as well as JAK3-deficient mice, display SCID (16).

AG-490 is a recent addition to the synthetically derived tyrphostin family of tyrosine kinase inhibitors (17, 18, 19). At relatively low concentrations, AG-490 has been shown to block hyperactive forms of JAK2 found in B cell precursors of acute lymphoblastic leukemia (ALL) patients (19) and in genetically constructed variants (9). AG-490 has also been shown to inhibit cytokine-induced activation of JAK2 in eosinophils stimulated with granulocyte-macrophage CSF (20) and in vascular smooth muscle cells and cardiac myocytes activated by angiotensin II (21, 22). A recent study has demonstrated that AG-490 effectively blocks STAT3 activation in mycosis fungoides-derived T cell lymphoma cells (23). While many kinase inhibitors are often promiscuous in the enzymes they target, AG-490 is unique in that it does not inhibit other lymphocytic tyrosine kinases such as Lck, Lyn, Btk, Syk, Src, JAK1, or Tyk2 (19).

In this study, our results suggest that this tyrphostin potently and effectively inhibits IL-2-mediated T cell responses. To define the mechanism of action for this inhibitor, we investigated putative molecular targets in the IL-2 signal transduction pathway. We provide evidence that JAK3 and its downstream substrates, STAT and MAPK signaling pathways may indeed be targets of AG-490. Taken together, this data suggests that AG-490 or its analogues may represent previously unrecognized therapeutic agents that can be used to treat a variety of IL-2-derived T cell disorders by inhibiting JAK3, STATs, and MAPK signaling pathways.

Materials and Methods

Materials

AG-490 was obtained from Calbiochem (San Diego, CA). Tissue culture materials were purchased from Life Technologies (Gaithersburg. MD). IL-1 was obtained from PeproTech (Rock Hill, NJ), and IL-2 was from Hoffmann-LaRoche (Nutley, NJ). Con A was purchased from Sigma (St. Louis. MO). STAT1 Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and STAT3, -5a, and -5b from R&D Systems (Minneapolis, MN). The rabbit anti-Shc, anti-JAK1, -2, -3, and Tyk2 and monoclonal antiphosphotyrosine Abs were obtained from Upstate Biotechnology (Lake Placid, NY), rabbit antiphospho-extracellular signal-related kinase 1 and 2 (ERK1/2) were from New England BioLabs (Beverly, MA), and the monoclonal pan-ERK from Transduction Laboratories (Lexington, KY). Protein A-Sepharose beads were purchased from Pharmacia (Piscataway, NJ). Immobilion-P (polyvinylidene difluoride (PVDF)) membrane was purchased from Millipore (Bedford, MA). TRIzol reagent was purchased from Life Technologies. Phycoerythrin-labeled anti-CD25 for IL-2Rα, anti-CD122 for IL-2Rβ, anti-common γ (γc)-chain or control mAb, and all probes for ribonuclease protection assays (RPA) were purchased from PharMingen (San Diego, CA).

Cell culture and treatment

The IL-2-dependent T cell line, D10, kindly provided by Dr. Li-Weber (DKFZ Cancer Research Center, Heidelberg, Germany), was maintained in RPMI 1640 medium containing 10% FCS, 2 mM l-glutamine and penicillin-streptomycin (50 IU/ml and 50 μg/ml, respectively), IL-1 (2 U/ml), recombinant human IL-2 (25 U/ml), Con A (2 μg/ml), 35 μM β-mercaptoethanol, and 6 mM HEPES. CTLL-2 cells were maintained in RPMI 1640 medium containing 10% FCS, 2 mM l-glutamine and penicillin-streptomycin (50 IU/ml and 50 μg/ml, respectively), and 25 U/ml of recombinant human IL-2 (25 U/ml). Cells were treated with varying concentrations of AG-490, as described in figure legends. All reactions with AG-490 were kept in the dark to avoid inactivation of the tyrphostin. Cells were stimulated with 100 nM of IL-2 at 37°C, as indicated in the corresponding figure legends. Cell pellets were frozen at −70°C.

Proliferation assays

Quiescent cells (50 × 103/well) were plated in flat-bottom 96-well microtiter plates in 200 μl of growth medium (described above), employing 5% FCS in the presence or absence of IL-2 (1 nM). Cells were treated for 16 h with AG-490 as above, pulsed for the remaining 4 h of the assay with [3H]thymidine (0.5 μCi/200 μl), and harvested onto glassfiber filters. [3H]thymidine incorporation was analyzed by liquid scintillation counting (24).

Flow cytometric analysis

D10 cells were stained with phycoerythrin-labeled anti-CD25 for IL-2Rα, anti-CD122 for IL-2Rβ, anti-γc-chain or control mAb, respectively. FACS analysis of indirect cell immunofluorescence of IL-2 receptor chains was performed on a flow cytometer (Becton Dickinson, San Jose, CA). Data are presented as histograms of cell number (y-axis) vs fluorescence intensity (log scale, x-axis), as corrected mean fluorescence after subtraction of nonbinding isotype-matched control mAb.

Solubilization of membrane proteins, immunoprecipitation, and Western blot analysis

Frozen cell pellets were thawed on ice and solubilized in lysis buffer (108 cells/ml) containing 10 mM Tris-HCl (pH 7.6), 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 200 mM sodium ortho-vanadate, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 1 μg/ml pepstatin A, and 2 μg/ml leupeptin. Cell lysates were rotated end over end at 4°C for 60 min, and insoluble material was pelleted at 12,000 × g for 20 min. Depending on the experiment, supernatants were incubated with 5 μg/ml polyclonal rabbit antisera raised against JAK3, STAT5a, STAT5b, anti-Shc, or monoclonal antiphosphotyrosine Abs for 2 h at 4°C. Abs were captured by incubation for 30 min with protein A-Sepharose beads. Precipitated material was eluted by boiling in SDS-sample buffer for 4 min and subjected to 7.5% SDS-PAGE under reducing conditions. For MAPK analysis, ∼20 μg of total cell lysate was dissociated in SDS-sample buffer and separated on SDS-PAGE, as described above. All proteins were transferred to PVDF membrane as previously described (10). Blots were Westerned with above Abs, rabbit antiphospho-ERK1/2 and monoclonal pan-ERK were diluted 1:1000 in blocking buffer, as previously described (10).

Electrophoretic mobility shift assay (EMSA)

AG-490 and control (DMSO)-treated D10 cells were washed with 5 vol of the hypotonic buffer (10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, and 0.5 mM DTT), then lysed in the same buffer supplemented with 1% Nonidet P-40 and incubated for 20 min on ice. The nuclei-containing pellet was resuspended in equal volumes of low-salt buffer (10 mM HEPES, 25% glycerol, 1.5 mM MgCl2, 20 mM KCl, 0.5 mM DTT, and 0.2 mM EDTA) and high-salt buffer (high-salt buffer containing 800 mM KCl). The nuclear extract was centrifuged at 4°C for 10 min. Supernatants were saved as nuclear protein extract and stored at −70°C. For the EMSA, end-labeled [32P]-oligonucleotides probes corresponding to the β-casein gene sequence (5′-AGATTTCTAGGAATTCAATCC-3′) were used to detect STAT5a and STAT5b binding, m67 serum-inducible response element (SIE) gene sequence (5′-AGCTTGTCGACATTTCCCGTAAATCGTCGAG-3′) to identify STAT3 and STAT1, and activating protein-1 (AP-1) oligonucleotide (5′-CGCTTGATGAGTCAGCCGGAA-3′) to monitor Fos/Jun binding. Each probe was then incubated with 5 μg of nuclear extracted proteins in 15 μl of binding mixture (50 mM Tris-Cl (pH 7.4), 25 mM MgCl2, 0.5 mM DTT, and 50% glycerol) at 4°C for 2 h. For supershift assays, nuclear extracts were preincubated with 1 μg of either normal rabbit serum or antisera specific to STAT1, -3, -5a, -5b, Jun, or Fos (Figs. 4⇓, 6) at 4°C for 20 min. Samples were then incubated for an additional 15 min at room temperature. The DNA-protein complexes were resolved on a 5% polyacrylamide gels containing 0.25× TBE that were prerun in 0.25× TBE buffer for 1 h at 100 V. After loading of samples, gels were electrophoresed at room temperature for ∼2 h at 140 V. Gels were then dried by heating under vacuum and exposed to x-ray film at −70°C overnight.

RPA

D10 cells were treated as described above and then pelleted by centrifugation (20,000 × g) for 1 min at 4°C and subsequently washed in PBS. Total RNA was isolated using TRIzol from parallel sets of treated cells. Receptor RNA-message was examined by RPA using 20 μg RNA hybridized with 1 × 105 cpm of each 33P-labeled probes corresponding to mCR-1 probes set overnight at 56°C. Unhybridized RNA probes were digested with RNase T1 and RNase A for 45 min at 30°C. The RNase-treated samples were then digested with proteinase K for 15 min at 37°C. After phenol/chloroform extraction and sodium acetate/ethanol precipitation, hybridized RNA probes were denatured at 90°C for 3 min and electrophoresed on a 5% polyacrylamide gel. The dried gels were exposed to x-ray film.

Results

AG-490 blocks IL-2-mediated T cell proliferation

Utilizing a murine IL-2-dependent T cell line, D10, we first sought to identify an effective pharmacological agent that could block IL-2-mediated cell proliferation. The results demonstrating the efficacy of AG-490 are presented in Fig. 1⇓ (upper panel). D10 cells were cocultured with increasing concentrations of AG-490 for 16 h. AG-490 abolished IL-2-inducible [3H]thymidine incorporation in a dose-dependent manner, displaying an IC50 of 25 μM. Overall, these cells were judged to be >85% viable based on trypan blue dye exclusion and FACS analysis using propidium iodide and Annexin V staining as apoptotic indicators (data not shown). This effect was not limited to D10 cells since similar results were also observed for the IL-2-dependent T cell line, CTLL-2 (Fig. 1⇓, lower panel). These findings suggest that AG-490 potently inhibits IL-2-mediated proliferation in T cells, results distinct from previous studies that showed this agent induced apoptosis in ALL cells while exerting apparently no effects on the growth of mitogen-stimulated normal T cells (19). To identify putative targets of AG-490, we next investigated known IL-2R-activated signaling proteins.

FIGURE 1.
FIGURE 1.

AG-490 inhibits proliferation of the IL-2-dependent T cell lines D10 and CTLL-2 in a dose-dependent manner. Proliferation of quiescent D10 cells or CTLL-2 cells (50 × 103/well) were examined following treatment with DMSO (0 μM AG-490) or increasing concentrations of AG-490 (ordinate) for 16 h at 37°C in the presence (filled box) or absence of 1 nM IL-2 (hatched box). Cells were then pulsed with [3H]thymidine (0.5 μCi/200 μl) for 4 h and incorporation of radiolabeled probe plotted on the abscissa expressed as total cpm (n = 6).

AG-490 does not alter IL-2R chain expression

Since the initial step in IL-2 signaling requires competent activation of its cognate receptor chains, we examined whether AG-490-mediated inhibitory effects were due to reduced expression of IL-2Rα, -β, or -γ chains. For this analysis, total mRNA was isolated from two different sets of control (DMSO) or AG-490-treated cells and hybridized against 33P-labeled receptor probes (Fig. 2⇓A). RNase-protected probes were electrophoretically separated by PAGE, dried, and subjected to autoradiography. Receptor message for DMSO (lanes a and b) or AG-490 (lanes c and d)-treated D10 cells failed to show a significant change in IL-2Rα, -β, and -γ mRNA expression, compared with the control housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Next, we addressed whether surface expression of receptor proteins was compromised by AG-490. FACS analysis for IL-2Rα, -β, and -γ chains showed no change following drug treatment (Fig. 2⇓B). From this data, it could be concluded that loss of IL-2-mediated cell growth by AG-490 was not due to a significant loss of IL-2R expression and suggested that its site of action was distal to the receptor.

FIGURE 2.
FIGURE 2.

AG-490 does not affect mRNA or protein expression of IL-2Rα, -β, or -γ chains. A, RPA of AG-490-treated D10 cells does not alter mRNA expression of IL-2R subunits. cDNA was generated from freshly isolated mRNA obtained from two sets of D10 cells (see Materials and Methods) treated in the absence (lanes a and b) or presence (lanes c and d) of 100 μM AG-490 for 16 h. D10 cells mRNA was then hybridized with 33P-labeled RNA probes corresponding to transcripts for individual murine γc-receptors (mCR-1) according to PharMingen Protocol (see Materials and Methods). The autoradiograph of the RNase protected fragments were separated on 5% PAGE is shown. B, Cell surface expression of IL-2R subunit on D10 cells was assessed by FACS analysis. Each panel shows a histogram of receptor expression of DMSO (dashed line) or AG-490 (solid line) treated D10 cells stained for either IL-2Rα (upper), -β (middle) and -γc-chain (lower) after 16 h of at 37°C of drug treatment.

AG-490 ablates JAK3 and STAT5a and STAT5b tyrosine phosphorylation in vivo

Following cytokine-induced oligomerization of the receptor chains, JAKs are believed to phosphorylate themselves, their respective receptor, and signaling molecules that are directed to the activated complex (14). Since one cytokine can often show a unique pattern of JAK activation in a cell-dependent manner, we investigated which JAKs were activated by IL-2 in D10 cells. The results shown in Fig. 3⇓A suggest that JAK3 is the most potently activated JAK based on tyrosine phosphorylation (lanes c and d), similar to previously published findings that were observed for many other cell lines (10, 24, 25). We did not observe detectable levels of activated JAK1, JAK2, or Tyk2, which is weakly expressed in this cell line. We next examined the dose-dependent inhibition of JAK3 by AG-490 over a 16-h period. As shown in Fig. 3⇓B, AG-490 indeed blocked IL-2 activation of JAK3 at concentrations as low as 50 μM (lane d) and showed nearly complete inhibitory at 100 μM (lane f). Reblots of JAK3 (lower panel) confirmed equivalent loading (Fig. 3⇓B).

FIGURE 3.
FIGURE 3.

IL-2-induced tyrosine phosphorylation of JAK3 and STAT5a and STAT5b is inhibited by AG-490. A, D10 cells were stimulated with 100 nM IL-2 at 37°C for 10 min. Cells were lysed and immunoprecipitated with anti-Tyk2 (lanes a and b), anti-JAK3 (lanes c and d), anti-JAK2 (lanes e and f), or anti-JAK1 (lanes g and h) Abs separated by SDS-PAGE, transferred to PVDF membrane and immunoblotted with antiphosphotyrosine. Lower panel, the same membrane was stripped and reprobed with Abs to JAK1, JAK2, JAK3, or Tyk2 to ensure equivalent loading of protein each lane. Tyk2 is weakly expressed in D10 cells. In both panels, arrows denote the positions of JAK enzymes. Molecular size markers are indicated on the left. B, AG-490 inhibits IL-2-induced tyrosine phosphorylation of JAK3 and STAT5a and STAT5b in D10 cells. D10 cells were treated as described in A with varying concentrations of AG-490. Cells were then lysed and immunoprecipitated with either anti-JAK3 (upper panel), anti-STAT5a (middle panel), or anti-STAT5b (lower panel) and Westerned with αPY (upper panel) or reprobed with either αJAK3, αSTAT5a, or αSTAT5b (indicated beneath phosphorylation blots). Arrow indicates migration location of either JAK3, STAT5a, or STAT5b. C, Tyrosine phosphorylated proteins were normalized against total protein. Ratio of phosphorylated/unphosphorylated protein is plotted on the abscissa (arbitrary units) against increasing concentration of AG-490 (ordinate).

Several groups have shown that expression of catalytically competent JAK3 is required for IL-2 activation of JAK1, STAT5, and cell proliferation (7, 11, 26, 27, 28, 29). STAT5 was originally identified as a prolactin-responsive mammary gland factor (MGF) (30, 31), but has since been found to be regulated by a variety of cytokines, including IL-2 (12, 14, 32). To assess AG-490 inhibitory effects on IL-2-modulated STAT5a and STAT5b activity, D10 cells were treated with increasing AG-490. STAT5a and STAT5b displayed similar sensitivity as compared with JAK3 (Fig. 3⇑B). Reblotting for both STAT5a and STAT5b showed equivalent loading and also showed that changes in tyrosine phosphorylation were not due to a loss in protein expression (lower panel). Quantification of decreased tyrosine phosphorylation levels was performed by normalizing densitometric traces against total JAK3, STAT5a, or STAT5b protein (Fig. 3⇑C). AG-490 clearly inhibited tyrosine phosphorylation of STAT5a, as well as STAT5b, at approximately IC50 (50–75 μM) following drug treatment. STAT1 and STAT3 also displayed a similar dose-dependent sensitivity to AG-490 (data not shown). The reason AG-490 displayed a greater inhibitory effect on STAT5a compared with STAT5b is not readily apparent, since reprobing of these blots with anti-STAT5a and -STAT5b confirmed equivalent loading. However, we have previously demonstrated in PHA-activated human T cells and IL-2Rβ-transfected murine BA/F3 cells that IL-2 preferentially induces more pronounced tyrosine and serine phosphorylation of STAT5b. This may be due to additional phosphorylation sites not present in STAT5a (20), thus accounting for the differences observed in Fig. 3⇑, B and C.

AG-490 inhibits IL-2-induced STAT1, -3, -5a, and -5b DNA binding

JAK-regulated STAT tyrosine phosphorylation is a prerequisite for STAT dimerization, nuclear translocation, and gene transcription (12, 14, 33). Since AG-490 blocked tyrosine phosphorylation of STATs, we used gel EMSAs to confirm a predicted concomitant loss in STAT-DNA binding activity. For this analysis, STAT5a and STAT5b was examined for its ability to bind to the prolactin-response element of the β-casein gene promoter, while STAT1/STAT3 binding was assessed using an oligonucleotide probe corresponding to the SIE-element (Fig. 4⇓). Nuclear extracts obtained from AG-490-treated cells (Fig. 4⇓A, lanes g-l) showed reduced β-casein DNA binding activity as compared with equivalent protein samples (5 μg) obtained from DMSO control-treated cells (Fig. 4⇓A, lanes a-f). Moreover, these IL-2-inducible DNA complexes could be partially supershifted with anti-STAT5a (lane c), STAT5b (lane d), or completely supershifted with both Abs (lane e) confirming their identity. Densitometric analysis of DMSO and AG-490-treated cells showed IL-2-inducible DNA binding was reduced by 78, 65, and 65% for STAT5a and STAT5b, STAT1, and STAT3, respectively. The findings above suggest that AG-490 can act as a potent inhibitor of JAK3 and its downstream gene-regulating transcription factors STAT1, -3, -5a, and -5b.

FIGURE 4.
FIGURE 4.

Pretreatment of D10 cells with AG-490 inhibits IL-2-induced STAT1, -3, -5a, and -5b DNA binding as demonstrated by EMSA analysis. A, β-casein EMSA. D10 cells were treated with DMSO-control (lanes a-f) or 100 μM AG-490 (lanes g-l) and incubated with medium (−) or 100 nM IL-2 (+) for 10 min at 37°C. Nuclear extracts corresponding to 5 μg of protein were incubated in the absence of Ab (lanes a, b, g,and h), anti-STAT5a (α-STAT5a; lanes c and i), α-STAT5b (lanes d and j), α-STAT5b, and α-STAT5a (lanes e and k) or normal rabbit serum (lanes f and l) in combination with a 32P-labeled oligonucleotide probe corresponding to the PRL response element of the β-casein gene promoter. B, SIE EMSA. D10 cells were treated as above in the absence (lanes a-e) or presence (lanes f-j) of AG-490. Nuclear extracts corresponding to 5 μg of protein were incubated in the absence of Ab (lanes a, b, f, and g), α-STAT1 (lanes c and h), α-STAT3 (lanes d and i), or normal rabbit serum (lanes e and j) in combination with a 32P-labeled oligonucleotide probe corresponding to the SIE gene promoter. Arrow indicates migrational location of each nonsupershifted STAT-DNA complex or free probe.

AG-490 inhibits Shc and ERK1/2 phosphorylation in a dose-dependent manner

It is generally accepted that activation of the MAPK cascade represents a key signal required for cellular proliferation. IL-2 potently activates the Shc/Ras/Raf/MAPK pathway via the adapter protein, Shc, which binds to Tyr338 of the IL-2R β-chain (13). Mutations or deletions of this residue result in a significant loss of cytokine-inducible growth (13, 34) and strengthen the hypothesis that this pathway is at least partially responsible for IL-2-mediated cell proliferation. Thus, we investigated whether the loss in T cell growth was due to disruption of the Shc/Ras/Raf/MAPK cascade.

To answer this question we first examined activation of the Shc/ERK1/2 pathway using Abs that recognize activated forms of these signaling molecules. Shc tyrosine phosphorylation was completely blocked at ∼50 μM AG-490 (Fig. 5⇓A), while Abs to activated phospho-ERK1/2 (Thr202/Tyr204) displayed a parallel dose-dependent inhibition. Immunoblotting of Shc and ERK1/2 (indicated beneath phosphorylation blots) verified equivalent loading. Densitometric analysis indicated that loss of tyrosine phosphorylation, normalized to nonphosphorylated protein, displayed an IC50 of ∼50 μM for all three proteins (Fig. 5⇓B). These data support our earlier conclusions that demonstrated deletional mutation of the acidic domain of the IL-2R β-chain, Tyr338, or inactivation of Shc/MAPK inhibits IL-2-mediated cell growth (35).

FIGURE 5.
FIGURE 5.

AG-490 inhibits Shc and p44/42 ERK1/2 phosphorylation in a dose-dependent manner. A, Quiescent D10 cells were treated with DMSO-control (0 μM AG-490) or increasing concentrations of AG-490 up to 100 μM for 16 h and then stimulated in the presence of 100 nM IL-2 at 37°C for 10 min. Cells were lysed and immunoprecipitated with anti-Shc (αShc) and Westerned with αPY (upper most panel) or reprobed with αShc (second panel). For detection of phospho-MAPK, total cell lysate was Westerned with anti-phospho-p44/42 MAPK (third panel) or pan ERK (bottom panel). Arrows indicate location of Shc or p44/42 MAPK. B, Densitometric analysis of Shc and MAPK. Tyrosine phosphorylated proteins (above) were normalized against total Shc or p44/p42 ERK. Ratios are plotted on the abscissa (arbitrary units) against increasing concentration of AG-490 (ordinate).

AG-490 inhibits IL-2-induced AP-1 DNA binding

AP-1 is a heterodimeric complex comprised of fos and jun oncogene family members (36). Moreover, it is generally accepted that ERKs activate AP-1, which promotes gene transcription (37). Therefore, we examined whether AG-490 treatment of T cells modified AP-1 DNA binding activity as measured by gel mobility shift assays. As shown in Fig. 6⇓, nuclear extracts obtained from AG-490-treated cells (lanes f-j) showed reduced DNA binding as compared with equivalent protein samples (5 μg) obtained from DMSO-treated cells (lanes a-e). Densitometric analysis revealed an inhibition of >83% in AP-1 DNA binding of AG-490-treated cells (lane g, 10.1) in comparison to DMSO treatment (lane b, 59.7). In our hands, we observed a significant reduction of AP-1 complex with both anti-Jun and Fos Abs as demonstrated by the densitometric relative units in each lane (lane b, 59.7; lane c, 50.5; and lane d, 28.2). Furthermore, the anti-Jun Ab induced supershift of the band. Interestingly, AG-490 greatly reduced basal levels of AP-1 DNA binding (lane f, 10.8), as well as its IL-2-induction, as compared with control samples (lane a, 34.8). This data suggests that the inability of T cells to adequately respond to IL-2 may be due to the inhibitory effects of AG-490 on the JAK3/STAT1, -3, -5a, -5b, JAK3/AP-1, and JAK3/Shc/Ras/Raf/MAPK cascades.

FIGURE 6.
FIGURE 6.

Pretreatment of D10 cells with AG-490 inhibits IL-2-induced AP-1 DNA binding as demonstrated by EMSA analysis. D10 cells were treated as above in the absence (lanes a-e) or presence (lanes f-j) AG-490 100 μM (see Materials and Methods for details). Nuclear extracts corresponding to 5 μg of protein were incubated in the absence of Ab (lanes a, b, e,and f), Jun, Fos (lanes g and c), or normal rabbit serum (nrs; lane dand h) and then with a 32P-labeled oligonucleotide probe corresponding to the AP-1 gene promoter. Arrow indicates migrational location of each nonsupershifted Jun/Fos-DNA complex or free probe.

Discussion

The present study demonestrates that AG-490, one member of the tyrphostin family of tyrosine kinase inhibitors, potently blocks IL-2-mediated T cell proliferation in D10 and CTLL-2 cells. This evidence suggests that AG-490 can inhibit a critical signal required for driving T cell proliferation and expansion. To characterize the molecular and biochemical events affected by AG-490, we examined the effects of this inhibitor on known signaling effectors belonging to the IL-2 signaling pathway.

JAK3 is recognized to be predominantly expressed in leukocytes, including monocytes, T, B, and NK cells, and is required for their development and function (16). The importance of JAK3 in IL-2 signaling pathways has been substantiated by recent findings that demonstrate that homozygous-point or -deletion mutations in JAK3 are found in autosomal recessive T-B+ severe combined immunodeficiency patients (38, 39). Moreover, JAK3 knockout mice are also immunodeficient (40) and have served as an excellent model to define the signaling pathways necessary for discerning T and B cell development and their function. Current models for IL-2 signaling suggest that JAK3 functions to phosphorylate specific tyrosine residues within its cognate IL-2R β-chain, thereby allowing for the recruitment of substrates to the activated receptor complex. In this model, JAK3 would then phosphorylate other proteins, including STATs, that would then disengage from the receptor and mediate gene transcription (41).

In D10 cells, the inhibition of IL-2-mediated growth observed with AG-490 suggests that it is due to inhibition of JAK3. We demonstrated in Fig. 3⇑ that IL-2 only activates JAK3 in these cells and not JAK1, JAK2, or Tyk2. JAK3 also displays dose-dependent sensitivity to AG-490 (Fig. 3⇑). Likewise, STAT5a and STAT5b tyrosine phosphorylation were also blocked by AG-490 (Fig. 3⇑), presumably due to inactive JAK3, which would be required to catalyze this event. Similarly, AG-490 effectively inhibited STAT1, -3, -5a, and -5b DNA binding (Fig. 4⇑). In addition to tyrosine phosphorylation of STATs, serine phosphorylation has also been proposed to be required for nuclear localization and DNA binding activity (42, 43). Emerging evidence suggests that serine phosphorylation of STATs may be mediated by MAPK-dependent and -independent pathways (32, 44). Since we showed in Fig. 5⇑ that activation of MAPK pathway (p44/p42 ERK1/2) was greatly suppressed by AG-490 in a dose-dependent manner, we cannot rule out the possibility that STAT serine phosphorylation was also disrupted by AG-490 and may account for this loss in DNA binding. Similarly, reduced AP-1 DNA binding activity (Fig. 6⇑) may also be due to inhibition of its upstream activator, MAPK. Collectively, these findings suggest that inhibition of JAK3 activity by AG-490 leads to a loss in STAT and AP-1 transcriptional activity and their consequent ability to transcribe proliferative response elements, as was clearly observed in the thymidine-incorporation assay for D10 and CTLL-2 cells (Fig. 1⇑). It is of interest to note that AG-490 reduced basal levels of STAT/AP-1 DNA binding (Figs. 4⇑A and 6). Moreover, we have found no change in basal levels of DNA binding components not believed to be activated by the JAK3 signaling pathway, including c-Maf or C/EBPβ in AG-490-treated cells (data not shown). These suggest that AG-490 may have a therapeutic function in constitutively activated JAK3 signaling pathways, such as those reported by Meydan et al. (19). Ongoing studies in the laboratory are exploring such issues discussed as below.

While AG-490 has previously been considered to be a JAK2-specific inhibitor, these conclusions were primarily drawn from ALL cells that lack readily detectable levels of JAK3 (19). Herein, we have provided evidence that suggests AG-490 effectively inhibits IL-2 signaling pathway in D10 cells that fail to activate JAK1, JAK2, or Tyk2. Interestingly, this drug has been shown not to affect other lymphocytic tyrosine kinases (i.e., Lck and Src) nor the more ubiquitously expressed JAKs, JAK1 and Tyk2 (19). This may also illustrate why AG-490 is well-tolerated by mice, findings that support the notion that it is not a general kinase inhibitor (19). The ability of AG-490 to selectively block JAK2 and JAK3 may be explained by the high level of homology shared between these two closest related members of the JAK family. Indeed, AG-490 displays similar micromolar inhibitory concentrations required to block JAK2 and JAK3 activity (19). It seems plausible to expect that generation of AG-490 analogues may refine and discriminate between not only JAK2 and JAK3 kinases, but JAK1 and Tyk2 as well.

From these findings, it therefore seems reasonable to expect that AG-490 may ablate or diminish certain lymphoid-dependent pathologies such as graft-vs-host disease or HTLV-1 based adult T cell leukemia, which is characterized by hyperactivated forms of JAK3 and constitutively active STAT proteins (45). Similarly, recent work by Nielson et al. (23) has shown that AG-490 can block constitutive activation of STAT3 and growth of mycosis fungoides-derived T cell lymphoma cells. Moreover, we have also observed that AG-490 effectively inhibits constitutive growth of the NK-like tumor line, YT, completely blocking tyrosine phosphorylated forms of JAK3 and STAT5a and STAT5b (R.A.K. and W.L.F., unpublished observation) suggesting that certain tumors may be more (or less) susceptible to AG-490. Recently, we have found that AG-490 can also block activation of JAK3/STAT6 phosphorylation induced by IL4 and IL13 (L.H.W. and W.L.F., unpublished observation), cytokines that may play a pivotal role in various type 2 disease states including Leishmaniasis, leprosy, allergy, and viral infection (46). Interestingly, AG-490 reduced the production of the Th2 cytokine, IL4, yet had no effect on other Th2 cytokine. Whether or not AG-490 may also impair lymphocyte recruitment and activation that characterizes a number of autoimmune diseases awaits further investigation.

In conclusion, we have provided evidence that AG-490 effectively inhibits IL-2-mediated T cell growth and JAK3, STAT, AP-1, and MAPK activation by IL-2. These findings suggest that this tyrphostin may prove therapeutically beneficial by suppressing immunopathological states such as graft-vs-host disease, autoimmune disorders and perhaps viral infections that compromise T cell function and may have far-reaching applications in addition to treating ALL patients with hyperactive forms of JAK2.

Acknowledgments

We thank Dr. Min Li-Weber for generously providing D10 cell line, Jennifer Brown for skillful preparation of the figures, and Dr. Joost Oppenheim for critical review of the manuscript.

Footnotes

  • ↵1 This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contact NO1-CO-56000.

  • ↵2 The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government. By acceptance of this article, the publisher or recipient acknowledge the right of the U.S. government to retain nonexclusive, royalty-free license in and to any copyright covering the article.

  • ↵3 Address correspondence and reprint requests to Dr. William L. Farrar, National Cancer Institute, P. O. Box B, Building 560, Room 31-68, Frederick, MD 21702. E-mail address: farrar{at}mail.ncifcrf.gov

  • ↵4 Abbreviations used in this paper: JAK, Janus kinase; ALL, acute lymphoblastic leukemia; AP-1, activating protein-1; PVDF, polyvinylidene difluoride; γc, common γ; EMSA, electrophoretic mobility shift assay; MAPK, mitogen-activated protein kinase; RPA, ribonuclease protection assay; ERK, extracellular signal-related kinase; SIE, serum-inducible response element.

  • Received September 16, 1998.
  • Accepted December 28, 1998.

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The Journal of Immunology: 162 (7)
The Journal of Immunology
Vol. 162, Issue 7
1 Apr 1999
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