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We discovered that inhibitors of phosphatidylcholine-specific phospholipase C (PC-PLC), but not other lipases, blocked the activation of STAT6 by IL-4. The.
The Journal of Immunology

Phosphatidylcholine-Specific Phospholipase C Activity Is Necessary for the Activation of STAT61 Jose Zamorano,2* Maria Dolores Rivas,* Antonio Garcia-Trinidad,* Cheng-Kui Qu,† and Achsah D. Keegan‡ It is well established that Janus kinase (JAK) tyrosine kinases play a key role in the activation of STAT6 by IL-4. In this study, we investigated additional molecules involved in this process. We previously found that IL-4 and TNF-␣ cooperate in the activation of STAT6 and NF-␬B, suggesting that these transcription factors are regulated by common intracellular signaling pathways. To test this hypothesis, we analyzed the effect of known inhibitors of NF-␬B on the activation of STAT6. We discovered that inhibitors of phosphatidylcholine-specific phospholipase C (PC-PLC), but not other lipases, blocked the activation of STAT6 by IL-4. The activation of PC-PLC seems to be an early event in IL-4 signaling, because its inhibition abrogated JAK activation and STAT6 tyrosine phosphorylation. Interestingly, we found that the effects of pervanadate and sodium orthovanadate on STAT6 activation correspond to their effect on PC-PLC. Thus, pervanadate by itself activated PC-PLC, JAK, and STAT6, whereas sodium orthovanadate suppressed PC-PLC, JAK, and STAT6 activation by IL-4. We further found that PC-PLC activation is necessary but not sufficient to promote STAT6 activation, and therefore, additional intracellular pathways regulated by IL-4 and pervanadate may collaborate with PC-PLC to signal STAT6 activation. It has been reported that IL-4 signals PC-PLC activation; in this study, we provide evidence that this phospholipase plays a key role in IL-4 signaling. The Journal of Immunology, 2003, 171: 4203– 4209.

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nterleukin-4 is a cytokine that participates in the regulation of the immune system at multiple levels (1). It can regulate proliferation, differentiation, and apoptosis in several cell types including lymphoid, myeloid, and mast cells (2– 4). One of the most studied effects of IL-4 is its role in regulating T cell differentiation promoting the Th2 phenotype (5). IL-4-driven Th2 cells direct beneficial immune responses against parasitic infections as demonstrated by the severity of helminthic infections in mice lacking IL-4 or its receptor (6). In contrast, IL-4 and its signaling machinery regulate the development of allergic and autoimmune diseases. Several studies indicate that allergic diseases like asthma are dependent on IL-4 signaling Th2 cell activation (7). In contrast, IL-4 could have a protective effect on rheumatoid arthritis (8). Therefore, it is of great interest to dissect the molecular mechanisms involved in cell responses to IL-4. IL-4 mediates its effects by binding to a cell surface receptor complex expressed in most cell types (9). Two types of IL-4R have been found. The type I consists of the common ␥-chain (␥c) and the IL-4R ␣-chain (IL-4R␣) (10). The IL-4R␣ gives specificity for IL-4 binding and signal transduction. In the type II receptor, the ␥c is substituted by the IL-13R␣1 (11). The type II is also a receptor for IL-13, which can explain the biological effects shared by these

*Unidad de Investigacion, Hospital San Pedro de Alcantara, Caceres, Spain; and Departments of †Hematopoiesis and ‡Immunology, Holland Laboratory, American Red Cross, Rockville, MD 20855 Received for publication December 31, 2002. Accepted for publication August 5, 2003. 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 in part by the American Red Cross, U.S. Public Health Service Grant AI38985 (to A.D.K.), Fondo de Investigacion Sanitaria Grant 01/0157, and Junta de Extremadura Grant 2PR01C015 (to J.Z.). J.Z. is supported by the Subdireccion General de Investigacion Sanitaria (Expediente 99/3082). 2

Address correspondence and reprint requests to Dr. Jose Zamorano, Unidad de Investigacion, Hospital San Pedro de Alcantara, Avenida Pablo Naranjo s/n, 10003 Caceres, Spain. E-mail address: [email protected] Copyright © 2003 by The American Association of Immunologists, Inc.

cytokines. The IL-4R lacks enzymatic activity, but the binding of IL-4 provokes the activation of the tyrosine kinases Janus kinase (JAK)31 and JAK3 (12, 13). This results in the tyrosine phosphorylation of the IL-4R ␣-chain, which can recruit intracellular messengers (9). Insulin receptor substrate proteins (14), Shc (15), Src homology 2 domain-containing inositol 5⬘-phosphatase (15), and STAT6 (16) are phosphorylated and activated by IL-4. The transcription factor STAT6 plays a principal role in IL-4 signaling as demonstrated in mice lacking STAT6 that show a similar phenotype as mice lacking the IL-4R␣ (17). The proposed mechanisms involved in STAT6 regulation are similar to those of other STATs (9, 18). STAT6 binds through its Src homology 2 domain to specific phosphotyrosine residues within the IL-4R␣ (19). In this complex, STAT6 is quickly phosphorylated by a JAK-dependent mechanism. After phosphorylation, STAT6 leaves the receptor, dimerizes, and migrates to the nucleus where it binds to specific DNA sequences in the promoter of genes (18). It is believed that STAT6 is tightly regulated, because in the absence of IL-4 stimulation, STAT6 is quickly deactivated (20 –22). Thus, phosphatases (20, 21), by dephosphorylating STAT6, and SOCs (22), by inhibiting the kinase activity associated with the receptor, are inhibitors of STAT6. In addition, serine kinases (23) and proteases (24) have also been implicated in STAT6 regulation. However, the role of these and other pathways activated by IL-4 in the regulation of STAT6 are still under extensive investigation. The basic JAK/STAT paradigm is widely accepted (18, 25); however, the processes involved in STAT6 activation are probably more complex. In this regard, we have recently demonstrated a principal role of Src kinases in the activation of STAT6 by IL-4 and IL-13 (26). Therefore, it is possible that additional molecules and metabolic pathways could participate in the processes that lead to the activation of STAT6. 3 Abbreviations used in this paper: JAK, Janus kinase; PC-PLC, phosphatidylcholinespecific phospholipase C; PI-PLC, phosphatidylinositol-specific phospholipase C; PLD, phospholipase D; Dex, dexamethasone; CsA, cyclosporine A; GAS, IFN-␥ activation site; DAG, 1,2-diacylglycerol.

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4204 Cytokine signaling studies are usually performed by stimulating cells with high levels of cytokine in the absence of additional signals. However, this type of stimulation is not similar to situations in vivo where the presence of additional cytokines and other signals could affect responses of cells to a given cytokine. In a recent report, we have shown that TNF-␣, which does not signal STAT6 activation, increases the activation of STAT6 by IL-4 (27). Similarly, IL-4 enhances the activation of NF-␬B by TNF-␣. The mechanisms underlying this effect were not defined. One possible explanation is that IL-4 and TNF-␣ regulate common intracellular pathways that lead to the activation of these transcription factors. To test this hypothesis, we investigated the effect of known inhibitors of NF-␬B on the activation of STAT6. We found that inhibitors of phosphatidylcholine-specific phospholipase C (PC-PLC) blocked the activation of STAT6 by IL-4. PC-PLC has been shown to be necessary for the activation of NF-␬B induced by several agents including TNF-␣ (28). It was previously shown that IL-4 can induce PC-PLC activation (29); however, the role of this lipase in IL-4 signaling has not yet been defined. In this study, we have found that the activation of PC-PLC by IL-4 is required not only to signal STAT6 activation but also to regulate the activation of JAK1 and JAK3. Our results strongly suggest that the hydrolysis of phosphatidylcholine is an early and important event in cell responses to IL-4.

Materials and Methods Cells and reagents The murine B cell lymphomas WEHI231, CH31, and M12 were maintained in RPMI 1640 culture medium with glutamine, penicillin, streptomycin, 0.05 mM 2-ME, and 10% FCS (complete medium). The murine IL-3-dependent myeloid cell lines 32D and SHP-1 mutant were cultured in the same medium supplemented with 5% WEHI-3-conditioned medium. The murine SHP-1 mutant hemopoietic progenitor cell line was previously generated by Hox11-mediated immortalization of yolk sac cells from viable motheaten mice (mev/mev) (21). D609, U73122, and ET-18-OCH3 were purchased from Biomol (Plymouth Meeting, PA). Anti-JAK1, anti-JAK3, and anti-Shc were obtained from Upstate Biotechnology (Lake Placid, NY), RC20 anti-phosphotyrosine Ab from Transduction Laboratories (Lexington, KY), and anti-STAT6 from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-IgM polyclonal Ab was a kind gift from Dr. D. W. Scott (Holland Laboratory, American Red Cross). Cytokines were from R&D Systems (Minneapolis, MN). All rabiolabeled compounds were obtained from Amersham (Arlington Heights, IL). The rest of the reagents used were purchased from Sigma-Aldrich (St. Louis, MO). Pervanadate was produced by the equimolar combination of Na3VO4 and H2O2 as previously described (30).

EMSA Analysis of STAT6 DNA binding was performed as we previously described (26). Briefly, 1 ␮g of total protein extract was incubated with 1 ng of 32P-labeled oligonucleotide corresponding to the IFN-␥ activation site (GAS) sequence in the C⑀ promoter (5⬘-CACTTCCCAAGAACAGA-3⬘). Samples were loaded into 4.5% polyacrylamide gels and run at 200 V for 2 h. Afterward, gels were dried and exposed to film.

Immunoprecipitation and immunoblotting We performed the experiments as we have previously described (26). Cell pellets were lysed with lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 50 mM NaF, 10 mM pyrophosphate, 1 mM PMSF, and protease inhibitor mixture) and clarified by centrifugation. The soluble fraction was incubated with the specified Ab followed with protein G-agarose. The washed precipitates were separated on 7.5% SDS-polyacrylamide gels before transfer to a polyvinylidene difluoride membrane. Membranes were then probed with the indicated Ab. The bound Ab was detected using ECL (Amersham).

Kinase assays JAK1 and JAK3 were precipitated as described above. Precipitates were washed in kinase buffer (50 mM HEPES (pH 7.5), 50 mM NaCl, 5 mM MgCl2, 5 mM MnCl2, 50 ␮M Na3VO4, and protease inhibitors) and incu-

PC-PLC IN STAT6 ACTIVATION bated in the presence of the mentioned inhibitors for 20 min at room temperature. Enzymatic reaction was initiated with the addition of [␥-32P]ATP (Amersham) and incubated for 20 additional minutes. Samples were separated on polyacrylamide gels, dried, and exposed to film.

Phosphatidylcholine hydrolysis determination Cells were labeled for 48 h with [methyl-3H]choline chloride (1 ␮Ci/ml), extensively washed, and starved for 2 h. Then, cells were stimulated as indicated, and the reaction was stopped by immersion in methanol/dry ice followed by centrifugation in a microfuge at 4°C. Cell pellets were resuspended in CH3OH/CHCl3/H2O (2.5:1.25:1) to separate aqueous and organic phases as previously described (31). The water-soluble fraction containing choline metabolites was loaded on silica gel-60 TLC plates and dried. Chromatography was performed in a glass chamber with CH3OH/ 0.5% NaCl/NH4OH (100:100:2) as solvent. Unlabeled phosphorylcholine was added to each sample, and after chromatography, visualized by staining with iodine vapor. The spots corresponding to phosphorylcholine standards were scraped off and collected, and the reactivity incorporated was determined by liquid scintillation counting.

Results Inhibition of STAT6 activation by PC-PLC inhibitors We have previously shown that IL-4 and TNF-␣ cooperate with each other to enhance the activation of NF-␬B and STAT6, suggesting the possibility that these transcription factors share common intracellular signaling pathways that lead to their activation (27). To test this hypothesis, we analyzed whether inhibitors of NF-␬B affected the IL-4-induced activation of STAT6. To this end, we cultured 32D cells in the presence of several pharmacologic agents known to suppress NF-␬B activation including dexamethasone (Dex), cyclosporine A (CsA), SN50 peptide, and D609 (32–35). We subsequently analyzed the ability of IL-4 to induce the binding of STAT6 to a specific GAS sequence found in the C⑀ promoter. We found that, although Dex, CsA, and SN50 had little effect, the incubation of cells with 50 ␮g/ml D609 resulted in the complete inhibition of STAT6 DNA-binding activity promoted by IL-4 (Fig. 1A). In fact, D609 inhibited the activation of STAT6 in a dose-dependent manner (Fig. 1B). Thus, pretreatment of cells with ⱖ50 ␮g/ml D609 completely blocked the activation of STAT6, and a lower amount of 16 ␮g/ml was still able to promote a significant inhibition. These concentrations of D609 required to inhibit STAT6 corresponded with those previously shown to inhibit NF-␬B activation (28). D609 is a xanthogenate compound that has been shown to inhibit a PC-PLC (28, 35). IL-4 has been reported to promote the activation of a PC-PLC, although its role in IL-4 signaling has not been defined (29). Because the concentration of D609 required to inhibit STAT6 activation corresponded with those reported to block PC-PLC (28, 35), our data suggested that PC-PLC is involved in the activation of STAT6 by IL-4. In addition to PC-PLC, some studies have shown that D609 can inhibit other lipases such as phospholipase D (PLD) (36). Therefore, we tested the effect of several other lipase inhibitors on the activation of STAT6. We found that ET-18-OCH3, an inhibitor of phosphatidylinositol-specific phospholipase C (PI-PLC) (37), and neomycin, which inhibits PC-PLD and PI-PLC (38), had no effect on STAT6 activation (Fig. 2, A and B). Similarly, propranolol, which inhibits phosphatidic acid phosphohydrolase blocking 1,2-diacylglycerol (DAG) formation through PLD (29, 39), had little effect on the activation of STAT6 by IL-4 (Fig. 2C). Conversely, U73122, a less specific inhibitor of lipases (39), was able to inhibit STAT6 activation at concentrations of ⬎3 ␮M, described to block PC-PLC activity (Fig. 2D). Therefore, these data strongly suggest that the activation of PC-PLC, but not other phospholipases, is involved in IL4-induced STAT6 activation. Our data are also in agreement with

The Journal of Immunology

FIGURE 1. Effect of NF-␬B inhibitors on the activation of STAT6. A, 32D cells were precultured with Dex at 10⫺7 M, CsA at 10⫺6 M, SN50 at 20 ␮M, TNF-␣ at 10 ng/ml, and D609 at 40 ␮g/ml before stimulation with IL-4 (10 ng/ml) for 20 min. STAT6 DNA-binding activity in cell extracts was analyzed by EMSA using the GAS sequence contained in the C⑀ promoter. B, 32D cells were cultured with the indicated amount of D609 for 30 min, and then stimulated with IL-4. STAT6 activation was also analyzed by EMSA as above. Arrows indicate STAT6 bands. n.s., Nonspecific bands.

previous studies showing the ability of IL-4 to induce PC-PLC, but not PI-PLC, PLD, or phospholipase A activation (29). The hydrolysis of phosphatidylcholine produces DAG and phosphorylcholine (40). DAG can act as a second messenger in the regulation of several intracellular pathways such as protein kinase C (40). Although described to be mitogenic (41), the role of phosphorylcholine in cell signaling is less well characterized (40). We found that the exogenous addition of phosphorylcholine or various agonists and inhibitors of DAG had no effect in the activation of STAT6 (J. Zamorano, unpublished observations). Like other authors (23), we have detected no effect of PKC and other serine kinases in the IL-4-induced STAT6 DNA-binding activity (J. Zamorano, unpublished observations), suggesting that PC-PLC

4205 could regulate additional pathways involved in the activation of STAT6. IL-4 stimulates the phosphorylation of STAT6 on serine and tyrosine residues (16, 23). Because phosphorylation on tyrosine is necessary for DNA binding (18), we investigated the effect of D609 on the phosphorylation of STAT6 by IL-4. Preincubation of the unrelated cell lines 32D, M12, and WEHI231 with D609 inhibited the phosphorylation of STAT6 by IL-4 (Fig. 3; data not shown). This inhibition correlated with the effect of D609 on STAT6 DNA-binding activity. Thus, 50 ␮g/ml D609 blocked tyrosine phosphorylation of STAT6 by IL-4, and a substantial inhibition was still observed in cells treated with 25 ␮g/ml D609. Furthermore, this effect was not limited to STAT6, because pretreatment of cells with D609 also blocked IL-4-induced tyrosine phosphorylation of Shc (Fig. 3B) and insulin receptor substrate 2 (data not shown). These data suggest that the activity of a PC-PLC is not restricted to the activation of STAT6 and may be required for the activation of tyrosine kinases. To verify that the D609 effect was specific, we investigated the effect of D609 on the activation of Shc induced by anti-IgM Abs on B cells, a signal that induces the activation of a PI-PLC rather than a PC-PLC (40, 42). In this case, treatment of B cells with 50 ␮g/ml D609, a concentration that completely inhibited IL-4-induced STAT6 and Shc phosphorylation, had little effect on the phosphorylation of Shc induced by an anti-IgM Ab (Fig. 3C). Taken together, these results indicate that D609 is inhibiting a PC-PLC that is required for IL-4 signaling. The tyrosine kinases JAK1 and JAK3 play an important role in IL-4 signaling and activation of STAT6 (12, 13). Therefore, we tested the effect of D609 on the IL-4-induced activation of these JAKs (Fig. 4). Treatment of cells with D609 inhibited tyrosine phosphorylation of JAK1 and JAK3 induced by IL-4 (Fig. 4, A and B). The doses required to inhibit JAKs were the same as those previously found to block STAT6. To investigate a direct effect of D609 on JAK, we analyzed the effect of D609 on in vitro activity of JAK1 and JAK3 (Fig. 4, C and D). In contrast with AG490, a known inhibitor of JAKs (43), D609 did not inhibit the autophosphorylation of these kinases. Consequently, D609 is not simply an inhibitor of JAK tyrosine kinases, but rather appears to inhibit a PC-PLC required for the activation of these kinases in vivo.

FIGURE 2. Effect of lipase inhibitors on the activation of STAT6. A, 32D cells were preincubated with the indicated amount of neomycin for 18 h (A), or with ET-18-OCH3 (B), propranolol (C), and U73122 (D) for 30 min before stimulation with IL-4 (10 ng/ml) for 20 min. STAT6 DNA-binding activity was analyzed by EMSA as in Fig. 1. Arrows indicate STAT6 bands.

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FIGURE 3. Inhibition of tyrosine phosphorylation of STAT6 by D609. A, 32D cells were incubated with the indicated amount of D609 for 30 min before stimulation with IL-4 for 20 min. Then, STAT6 was precipitated from cell lysates using anti-STAT6 Ab followed by protein G-agarose. Precipitates were separated by SDS-PAGE, and immunoblotted with an antiphosphotyrosine Ab to detect tyrosine phosphorylated (Py; upper blot). Membranes were stripped and reprobed with anti-STAT6 Ab (Stat6; lower blot). B, Cells were pretreated with 50 ␮g/ml D609 and stimulated with IL-4 as in A. Shc was precipitated using an specific Ab, and filters were probed with antiphosphotyrosine Ab (upper blot) and reprobed with antiShc Ab (lower blot). C, CH31 cells were preincubated with 50 ␮g/ml D609 as in A, and then stimulated with a polyclonal anti-IgM Ab for 20 min. Phosphorylated Shc was determined as in B (upper blot). Membrane was reprobed with anti-Shc Ab (lower blot).

Effect of vanadium compounds on PC-PLC and STAT6 activation In addition to IL-4, the nonspecific tyrosine phosphatase inhibitor pervanadate has been shown to induce STAT6 activation (44). This effect of pervanadate was dependent on IL-4R␣ and JAK1 expression, suggesting pervanadate could stimulate the IL-4 signaling pathway involved in STAT6 activation. Interestingly, treatment of cells with pervanadate results in the activation of phospholipases (30, 45), suggesting this pathway could be involved in the activation of STAT6 by pervanadate. Treatment of 32D cells with pervanadate induced the activation of PC-PLC, in a dosedependent manner, as demonstrated by the production of soluble phosphorylcholine (Fig. 5A). That effect correlated with the ability of pervanadate to induce STAT6 activation as analyzed by EMSA (Fig. 5, B and C) and tyrosine phosphorylation (D). Pervanadate is produced by reacting Na3VO4 with H2O2. Neither H2O2 nor Na3VO4 alone were able to induce STAT6 activation (Fig. 5B). Interestingly, pretreatment of cells with D609 completely abro-

FIGURE 4. D609 inhibits JAK kinases in vivo but not in vitro. A and B, M12 cells were incubated with D609 (40 ␮g/ml) for 30 min before IL-4 stimulation for 5 min. Then, cell lysates were incubated with anti-JAK1 (A) or anti-JAK3 Abs (B) followed by protein G-agarose. Precipitates were separated by SDS-PAGE, and immunoblotted with an antiphosphotyrosine Ab to detect tyrosine phosphorylated kinases (Py; upper blots). Membranes were stripped and reprobed with anti-JAK1 (A) or anti-JAK3 Abs (B) (lower blots). C and D, M12 cell extracts were precipitated with anti-JAK1 (C) or anti-JAK3 (D). Precipitates were then incubated with nothing or D609 (40 ␮g/ml), herbimycin A (HA; 10 ␮M), or AG490 (50 ␮M) for 20 min. The kinase activity of precipitates was then analyzed by autophosphorylation using radioactive ATP.

FIGURE 5. Pervanadate induces PC-PLC, JAK, and STAT6 activation. A, 32D cells were incubated with [methyl-3H]choline chloride for 48 h before stimulation with the indicated amount of pervanadate. Then, cell extracts were obtained, and the water-soluble fraction containing labeled phosphorylcholine was separated by TLC. Bands corresponding to phosphorylcholine were scraped off and counted in a liquid scintillation counter. The values express the mean ⫾ SD of two experiments. B, Cells were stimulated with pervanadate, H2O2, or Na3VO4 for 30 min and then activated STAT6 was analyzed by EMSA using the GAS sequence within the C⑀ promoter. C, Cells were left untreated or incubated with D609 (40 ␮g/ml) for 30 min before stimulation with various concentrations of pervanadate. STAT6 activation was measured by EMSA as above. D, Cells were treated with D609 before stimulation with pervanadate for 30 min. STAT6 phosphorylation was determined as in Fig. 3. E, Cells were treated using the same conditions described in D, but phosphorylation of JAK1 (left blots) and JAK3 (right blots) was analyzed. Upper panels show phosphorylated kinase, and lower panels show total protein.

gated the ability of pervanadate to signal STAT6 DNA-binding activity (Fig. 5C) and tyrosine phosphorylation (D). The activation of STAT6 by pervanadate correlated with the activation of JAK kinases (Fig. 5E). Furthermore, pretreatment of cells with D609 also abrogated the activation of JAK1 and JAK3 by pervanadate (Fig. 5E). These data suggest that the activity of PC-PLC is a universal requirement for the activation of the molecular pathways that participates in the activation of STAT6. The effect of vanadium compounds on cells has been associated with the inhibition of tyrosine phosphatases (46). However, the effect of Na3VO4 on the activation of STAT proteins differs from the effect of pervanadate (46, 47) (Figs. 5–7). Treatment with Na3VO4 alone did not promote STAT1 (47) and STAT6 activation (Fig. 5B). Interestingly, we found that pretreatment of cells with Na3VO4 inhibited the IL-4-induced STAT6 DNA-binding activity in a dose-dependent manner (Fig. 6A), whereas pervanadate cooperated with IL-4 in inducing STAT6 activation (B). Similar effects of vanadium compounds have been described in the activation of STAT1 and IFN-␣-stimulated gene factor 3 complex (47, 48). Furthermore, pretreatment of cells with Na3VO4 not only blocked the IL-4-induced tyrosine phosphorylation of STAT6 (Fig. 6C), but also blocked the tyrosine phosphorylation of JAK1 and JAK3 (D). In addition to its effect on phosphatases, Na3VO4 has been shown to competitively inhibit the enzymatic activity of bacterial PC-PLC (49), suggesting that a mammalian PC-PLC may be the target for

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FIGURE 7. Effect of vanadium compounds in the activation of STAT6 in SHP-1 deficient cells. A, SHP-1mev/mev mutant cells were incubated with Na3VO4 and D609 for 30 min before treatment with IL-4 for 20 min. STAT6 phosphorylation (upper) and total protein (lower) were determined as indicated in Fig. 3. B, SHP-1mev/mev mutant cells were incubated with D609 for 30 min before treatment with pervanadate for 20 min. STAT6 phosphorylation (upper) and total protein (lower) were determined as above.

FIGURE 6. Sodium orthovanadate inhibits the activation of PC-PLC, JAK kinases, and STAT6 by IL-4. A, 32D cells were incubated with the indicated amount of Na3VO4 for 30 min before stimulation with IL-4 for 20 min. Then STAT6 DNA binding activity was analyzed by EMSA as described in Fig. 1. B, Cells were preincubated with several doses of pervanadate for 30 min, and then stimulated with IL-4. STAT6 activation was measured by EMSA as above. C, Cells were treated as in A, but STAT6 phosphorylation was analyzed by immunoprecipitation and immunoblotting as indicated in Fig. 3 (Py; upper panel). Membrane was stripped and reprobed with anti-STAT6 Ab (Stat6; lower panel). D, Cells were preincubated with 1 mM Na3VO4 before treatment with IL-4. Phosphorylation of JAK1 (left) and JAK3 (right) was then analyzed as in Fig. 4. Upper blots show tyrosine phosphorylated kinase, whereas lower blots show total protein. E, Cells were preincubated with Na3VO4 for 30 min before stimulation with IL-4 for 2 min. PC-PLC activation was measured by the detection of phosphorylcholine in the water-soluble fraction as described in Fig. 5A. As control, cells were left untreated (f; left). The values are the means ⫾ SDs of four experiments.

vanadates in regulating STAT6 activation. Interestingly, we found that the effect of vanadium compounds on STAT6 correlated with their effect on PC-PLC activation. In contrast to pervanadate (Fig. 5), pretreatment of 32D cells with Na3VO4 inhibited the activation of PC-PLC by IL-4 as measured by phosphorylcholine production (Fig. 6E). The kinetics of PC-PLC inhibition by Na3VO4 correlated with its inhibitory effect on STAT6 activation (Fig. 6, A and C) and JAKs (D). Although we cannot determine whether Na3VO4 directly acts on a defined PC-PLC using molecular techniques, because it has not been characterized, the data presented suggests that Na3VO4 may inhibit cell responses to IL-4 by targeting this type of lipase. The proposal that vanadium compounds are targeting enzymes other than a tyrosine phosphatase is supported by the findings observed in cells lines expressing a mutated form of the SHP-1 phosphatase, the main phosphatase shown to be involved in regulating STAT6 signaling (21, 44, 50). These cells were derived from mev/

mev embryos that express an anomalous form of SHP-1 that lacks catalytic activity (SHP-1mev/mev) (21). It has already been shown that the loss of STAT6 phosphorylation was delayed in these mutant cells, demonstrating a role for SHP-1 in later events involved in STAT6 regulation (21). However, we investigated the effect of vanadium compounds and D609 in the early phosphorylation response to IL-4. In this case, pretreatment of SHP-1mev/mev cells with Na3VO4 and D609 also inhibited the tyrosine phosphorylation of STAT6 by IL-4 (Fig. 7A). Similar to the results in normal cell lines, pervanadate was able to induce the activation of STAT6 in cells expressing mutated SHP-1. Furthermore, this activation was also blocked by the PC-PLC inhibitor D609 (Fig. 7B). These results clearly indicate that SHP-1 is not the critical target of these agents in these studies, and it is not required for induction of STAT6 tyrosine phosphorylation. It was surprising to find that pretreatment of cells with sodium orthovanadate inhibited the IL-4-induced JAK and STAT6 activation given the fact that treatment of cells with vanadate has been shown to increase STAT6 activation (21, 50). However, in these studies, cells were not pretreated with Na3VO4, and therefore, the observed effect could be due to the inhibition of phosphatases that directly participate in the dephosphorylation of STAT6. Supporting this idea, we found that, in contrast with preincubation, treatment of cells with Na3VO4 after IL-4 stimulation promoted an increase in the activation of STAT6 (Fig. 8). Similar data have been found by analyzing the effect of Na3VO4 in the activation of IFN-␣-stimulated gene factor 3 complex by IFN-␥ (48). Taken together, these data suggest that vanadium compounds may regulate two different enzymes necessary for the activation and regulation of STAT6, a PC-PLC and a phosphatase. PC-PLC could be required during the earliest steps that lead to STAT6 phosphorylation, and the phosphatase could be involved in downstream processes through dephosphorylation of STAT6.

FIGURE 8. Sodium orthovanadate in the activation of STAT6 by IL-4. Na3VO4 was added to cell cultures before and after IL-4 stimulation. 32D cells were stimulated with IL-4 alone (⫺) or pretreated with 1 mM Na3VO4 for 30 min before IL-4 stimulation (⫺30 min), or Na3VO4 was added 5 min after IL-4 stimulation (⫹5 min). Then, STAT6 activation was analyzed by EMSA as indicated in Fig. 1.

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Discussion IL-4 has previously been shown to exclusively activate a PC-specific phospholipase (29). However, the role for this lipase in IL-4 signaling has not been defined. We have found in this study that the activation of PC-PLC may play an important role in IL-4 signaling. Our data indicate that the activation of PC-PLC may be upstream of the activation of JAKs and consequently STAT6. Several studies have established a role for PC-PLC in cell signaling; however, the molecular characterization of mammalian PC-PLC remains elusive (28, 29, 39, 40). PC-PLC activity has been shown to be required for NF-␬B activation (28), and furthermore, bacterial PC-PLC induces a transformed phenotype in transfected NIH3T3 cells (51). The hydrolysis of phosphatidylcholine by PLC produces DAG and phosphorylcholine (40). DAG is involved in the regulation of several intracellular pathways including the activation of protein kinase C isoforms. Although the role of phosphorylcholine in signaling is less characterized, it has been shown to promote cell proliferation and transformation (41). These studies suggest an active role for PC-PLC in cell signaling. However, the addition of exogenous recombinant bacterial PC-PLC, DAG, and/or phosphorylcholine to cells did not elicit an effect on JAK activation or tyrosine phosphorylation of STAT6 induced by IL-4 (J. Zamorano, unpublished observation). This may suggest that additional signals are required for full activation of the JAKs/ STAT6 pathway. Alternatively, it may be possible that the role of PC-PLC in IL-4 signaling is not mediated by DAG or phosphorylcholine. JAKs are believed to be activated by transphosphorylation (25). The binding of factors to their receptor subunits would bring the kinases close enough to facilitate their transphosphorylation and activation. It is reasonable to hypothesize that this process may require changes in the cell membrane that could be facilitated by hydrolysis of membrane phospholipids after IL-4 engagement. The hydrolysis of PC by PC-PLC could provoke cell membrane alterations that would then facilitate JAK1 and JAK3 interaction and consequently activation. The observation that PCPLC activation by IL-4 appears to be upstream of JAK activation supports this model. The effect of Na3VO4 and pervanadate on PC-PLC may explain their opposite roles in STAT6 activation. Vanadium compounds have usually been associated with inhibition of phosphatases (46 – 48). However, the opposite effects of pervanadate, in activating, and orthovanadate, in inhibiting the activation of JAK and STAT6 raises the possibility that they are targeting other proteins involved in IL-4 signaling. It has already been demonstrated that vanadium compounds can also regulate lipases. Thus, pervanadate has been show to promote the activation of phospholipases (30, 45), whereas orthovanadate inhibits the enzymatic activity of bacterial PC-PLC (49). Because we have found an important role of PCPLC on STAT6 activation, their action on this lipase may account for their effect on STAT6. This hypothesis is also supported by the effect of vanadate in the activation of STAT6 in a cell line expressing a mutated inactive form of the SHP-1 phosphatase. These cells are derived from viable motheaten mice that express a form of SHP-1 that lacks catalytic activity (21). As in normal cells, vanadate inhibited and pervanadate promoted the activation of STAT6 in these cells, suggesting they regulate other proteins involved in IL-4 signaling. SHP-1 is the principal phosphatase found to be involved in the regulation of STAT6 through dephosphorylating activated STAT6 (21, 50). This is consistent with the findings that, in contrast with pretreatment, the addition of orthovanadate to cells after IL-4 stimulation increased the activation of STAT6, probably by inhibiting a tyrosine phosphatase. Consequently, we hypothesized that vanadium compounds could regu-

PC-PLC IN STAT6 ACTIVATION late two steps involved in the activation of STAT6, a PC-PLC and a phosphatase. PC-PLC could be required during the earliest steps that lead to JAK and STAT6 phosphorylation, and the phosphatase could be involved in downstream processes dephosphorylating activated STAT6. Interestingly, similar effects of vanadate and pervanadate have been found in the regulation of other STATs (47, 48), suggesting the possibility that PC-PLC belongs to a common pathway in the activation of JAKs and STATs. STAT6 and NF-␬B are latent transcription factors that are quickly activated. In this study, we have shown that they share a requirement for PC-PLC. However, the requirements for STAT6 activation seems to be more complex, because PC-PLC activation is enough to signal NF-␬B but not STAT6 activation (Ref. 28; data not shown). Although PC-PLC inhibition abrogates the activation of STAT6, we have not been able to induce STAT6 activation by inducing the hydrolysis of PC by overexpressing bacterial PC-PLC cDNA or using purified bacterial PC-PLC (J. Zamorano, unpublished observations). This suggests that IL-4 and pervanadate provide additional signals required to activate STAT6. In a recent study, we have shown that, like PC-PLC, Src kinases participate in the earliest steps that lead to the activation of STAT6 (26). Intriguingly, it has been shown that pervanadate but not H2O2 or Na3VO4 can promote the activation of Src kinases in some cell lines (45). Therefore, it is possible that signals through Src kinases and PC-PLC may converge and regulate downstream events involved in STAT6 activation, including JAKs. Our results indicate that, in addition to its well-established role in NF-␬B activation by TNF-␣ (28), PC-PLC participates in the activation of STAT6 induced by IL-4. Given the importance of these cytokines and transcription factors in the development of allergic diseases (7, 52), PC-PLC may be a reasonable target for therapeutic intervention. D609, an inhibitor of PC-PLC, is a xanthogenate compound that was developed as an antiviral and antitumor compound (53). It was later found to block septic shock in mice (54). It will be interesting to determine whether inhibition of PC-PLC by D609, and therefore STAT6 and NF-␬B, will have a protective effect on allergic diseases.

Acknowledgments We thank Dr. Allan Mufson for helpful suggestions during the course of this work.

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