Activation of the JAK-STAT pathway by reactive oxygen species

Amy R. Simon1, Usha Rai2, Barry L. Fanburg1, and Brent H. Cochran2

1 Pulmonary and Critical Care Division, Tupper Research Institute, New England Medical Center, Boston 02111; and 2 Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Reactive oxygen species (ROS) play an important role in the pathogenesis of many human diseases, including the acute respiratory distress syndrome, Parkinson's disease, pulmonary fibrosis, and Alzheimer's disease. In mammalian cells, several genes known to be induced during the immediate early response to growth factors, including the protooncogenes c-fos and c-myc, have also been shown to be induced by ROS. We show that members of the STAT family of transcription factors, including STAT1 and STAT3, are activated in fibroblasts and A-431 carcinoma cells in response to H2O2. This activation occurs within 5 min, can be inhibited by antioxidants, and does not require protein synthesis. STAT activation in these cell lines is oxidant specific and does not occur in response to superoxide- or nitric oxide-generating stimuli. Buthionine sulfoximine, which depletes intracellular glutathione, also activates the STAT pathway. Moreover, H2O2 stimulates the activity of the known STAT kinases JAK2 and TYK2. Activation of STATs by platelet-derived growth factor (PDGF) is significantly inhibited by N-acetyl-L-cysteine and diphenylene iodonium, indicating that ROS production contributes to STAT activation in response to PDGF. These findings indicate that the JAK-STAT pathway responds to intracellular ROS and that PDGF uses ROS as a second messenger to regulate STAT activation.

hydrogen peroxide; platelet-derived growth factor; TYK2

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

OXIDATIVE DAMAGE contributes to the pathogenesis of several human diseases, including atherosclerosis, acute respiratory distress syndrome (ARDS), Alzheimer's disease, and pulmonary fibrosis (4, 28, 42). Virtually every known organism has evolved specific mechanisms to protect itself from oxidative damage. Mechanisms for reaction to specific oxidative conditions have been identified in bacteria, yeast, and mammalian cells. An important part of the cellular defense to oxidative stress is the specific induction of new gene expression in response to specific oxidative stressors. Some of the genes induced in response to oxidative stress such as superoxide dismutase and glutathione peroxidase are directly involved in the neutralization of oxygen radicals and their precursors (42, 61). Others, such as p21CIP1/WAF1, c-fos, and the caspases, are involved in the control of cell proliferation and apoptosis in response to oxidative stress (81). The cell must be able to detect these oxidative stressors and then specifically activate transcription factors to upregulate the expression of these genes. Indeed, the molecular detection and response of cells to reactive oxygen species (ROS) are likely to be among the earliest evolved second-messenger systems. Moreover, there is substantial evidence that ROS are not only pathological but, in many instances, are utilized as second messengers by the cell in response to growth factors and cytokines (72, 85, 87, 89).

Thus it is not surprising that some of the transcription factors known to be involved in immediate early gene expression are also regulated by ROS. One of the best characterized examples of this is nuclear factor-kappa B (NF-kappa B). Before activation, NF-kappa B is held in the cytoplasm through a complex with another factor called I-kappa B (3). In response to peroxide or ultraviolet (UV) treatment of many cell types, the I-kappa B factor is degraded, releasing NF-kappa B to translocate to the nucleus, where it can specifically activate transcription (35, 56, 57, 72). Moreover, ROS or peroxides appear to be the second messenger used by cells to activate NF-kappa B, inasmuch as antioxidants prevent the activation of NF-kappa B in response to a variety of cytokines (72). Recent data indicate that ROS are also important mediators of the response to the platelet-derived growth factor (PDGF) (85).

The signal transducer and activator of transcription (STAT) factors were originally described as growth factor- and interferon-inducible DNA binding complexes (32, 51). Subsequently, the STAT factors have been shown to be induced by a wide variety of growth factors and cytokines (39). These factors participate in the regulation of many genes, including the c-fos protooncogene, caspases, and the cell cycle regulator p21CIP1/WAF1, which can also respond to oxidative stress (11, 12, 30, 49, 75, 94, 100). The STAT factors are unique, in that they are phosphorylated on tyrosine residues in response to a variety of growth factors and cytokines (25, 55, 66, 68, 69, 77, 80). On phosphorylation, STATs dimerize via SH2-phosphotyrosine interactions and become competent to bind DNA (76). Before phosphorylation the STATs are found in the cytoplasm and translocate to the nucleus on activation (69, 78). In response to interferon treatment, the Janus kinase (JAK) family of kinases is required for STAT phosphorylation (59, 91, 95). Here we report that STAT1 and STAT3 factors are activated on treatment of Rat-1 fibroblasts with H2O2 and that this activation is sensitive to antioxidants. Moreover, H2O2 stimulates the activity of the STAT kinases JAK2 and TYK2. Finally, we find that ROS play an important role in the activation of STATs by PDGF.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell culture. Rat-1 fibroblasts were maintained in DMEM with 10% fetal bovine calf serum (FCS). NIH/3T3 and A-431 cells were maintained in DMEM-10% bovine calf serum (CS). For experiments, cells were made quiescent by growth to confluence and incubation in DMEM-0.5% FCS or CS for 48 h. Iron-free medium was used for all stimulations to minimize ROS generated by the Haber-Weiss reaction (52). For stimulations, epidermal growth factor (EGF) and PDGF-BB were obtained from Collaborative Biomedical (Waltham, MA). Catalase (bovine liver), glucose oxidase, H2O2, buthionine sulfoximine (BSO), diphenyl iodinium (DPI) chloride, N-acetyl-L-cysteine (NAC), pyrrolidine dithiocarbamate (PDTC), N-nitro-L-arginine, vanadate, vanadyl sulfate, nitrosoglutathione, isosorbide dinitrite, sodium nitroprusside, and reduced glutathione were obtained from Sigma Chemical (St. Louis, MO).

Antibodies. The anti-STAT1C rabbit antiserum was made against a peptide with the unique COOH-terminal 37 amino acids of murine STAT1 protein. The anti-NH2-terminal STAT1 antibody was obtained from Transduction Laboratories. STAT3 antibody was a polyclonal rabbit antiserum made against another unique sequence in the COOH-terminal region of human STAT3 (amino acids 688-700, QEHPEADPGSAAP). The antiphosphotyrosine STAT3 antibody was obtained from New England Biolabs (Beverly, MA). The JAK1, JAK2, TYK2, and phosphotyrosine (4G10) antibodies were obtained from Upstate Biotechnology (Lake Placid, NY). The anti-beta -PGDF receptor antibody was from Santa Cruz Biotechnology (Santa Cruz, CA).

Preparation of nuclear extracts. After the indicated treatments, cells were rinsed three times with ice-cold PBS. PBS containing 1 mM Na3VO4 and 5 mM NaF was added to each dish, and the cells were scraped from the dish and pelleted at 1,500 rpm for 10 min at 4°C. Cells were resuspended in the same buffer and pelleted as described above. Then the pellet was resuspended in 0.8 ml of ice-cold hypotonic buffer, transferred to microfuge tubes, and allowed to swell on ice for 15-30 min. The lysate was vortexed vigorously for 1 min, and the nuclei were pelleted (14,000 g for 30 s). The nuclear pellets were resuspended in 100-150 µl of high-salt buffer and rotated at 4°C for 30 min. The extracted proteins were separated from residual nuclei (14,000 g for 10 min), and the supernatant was quick frozen in a dry ice-methanol bath. The buffer compositions were as follows: 1) 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM Na4P2O7, 20 mM NaF, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 1 µg/ml each of leupeptin, antipain, pepstatin, and chymostatin (hypotonic buffer) and 2) 20 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 25% glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM Na4P2O7, 20 mM NaF, 1 mM DTT, 0.5 mM PMSF, and 1 µg/ml each of leupeptin, antipain, pepstatin, and chymostatin (high-salt buffer).

Electrophoretic mobility-shift assays. Nuclear extracts (12 µg) were added to 32P-end-labeled oligonucleotide m67-SIE probe (94) (~30,000 cpm, ~5 fmol), and the mixture was incubated in binding buffer for 30 min at 30°C. Final binding reactions (20 µl) contained 14 mM HEPES, pH 7.9, 85 mM NaCl, 10 mM KCl, 0.3 mM MgCl2, 1.25 mM DTT, 0.25 mM EDTA, 0.2 mM EGTA, 15% glycerol, 50 µg/ml poly(dI-dC) · poly(dI-dC), and 250 µg/ml acetylated BSA. Binding reactions were electrophoresed through 5% polyacrylamide gels (39:1 acrylamide-bis) containing 2.5% glycerol in 0.5× Tris-borate-EDTA buffer at room temperature. The gel was then dried and exposed to X-ray film.

Transient transfections. Murine NIH/3T3 fibroblasts were grown in DMEM with 10% CS. For transient transfection assays, the calcium phosphate method was used (calcium phosphate transfection kit, 5 Prime 3 Prime). The reporter construct had six copies of the high-affinity SIE site upstream of a minimal TK-luciferase gene and was a gift of Richard Jove (88). For reporter assays, NIH/3T3 cells were maintained for 30-40 h in medium containing 0.5% CS after transfection and stimulated with H2O2 for 6 h before harvest. One microgram of reporter construct and 100 ng of pRL-TK normalization plasmid were used per 35-mm dish. Dual luciferase assay was carried out following the manufacturer's recommendation (Promega). All transfection experiments were performed in duplicate, and results were normalized to the expression of the Renilla luciferase transfection control.

Immunoprecipitation and Western blotting. Quiescent Rat-1 cells were treated for 10 min with 1 mM H2O2, 100 ng/ml interferon-gamma , or 100 ng/ml EGF, lysed in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 2 mM EGTA, 2 mM Na3VO4, 1 mM PMSF, and 5 µg/ml of the protease inhibitors leupeptin, pepstatin, and antipain), and incubated on ice for 30 min. The lysate was then centrifuged for 10 min at 1,000 g at 4°C. The supernatant was removed, and the protein concentration was determined via a Bradford assay. Lysate (500 µg of protein) was incubated with JAK2, 4G10, or anti-PDGF-beta -receptor antibodies as specified (UBI) at 4°C overnight. The immune complexes were then captured with 100 µl of a slurry of protein A-agarose beads for 2 h. The beads were collected by centrifugation, resuspended in 2× Laemmli sample buffer, and boiled before they were run on an 8% SDS polyacrylamide gel and transferred to nitrocellulose. The membrane was then blotted with anti-JAK or 4G10 antibodies, depending on the experiment, and processed via chemiluminescence (Amersham).

Kinase assays. Whole cell extracts of quiescent Rat-1 cells that were treated with H2O2 for 5 min were immunoprecipitated as outlined above on 100 µl of a slurry of protein A-agarose beads with anti-JAK2 antibody. The beads were then washed with kinase buffer (50 mM NaCl, 5 mM MgCl2, 5 mM MnCl2, 0.1 mM Na3VO4, and 10 mM HEPES) before they were incubated with 0.25 mCi/ml of [gamma -32P]ATP for 30 min at room temperature. The beads were washed with PBS and processed as described above before they were run on an SDS polyacrylamide gel. The gel was dried, exposed to X-ray film, and quantitated on a phosphorimager.

Cell permeabilization. Quiescent Rat-1 fibroblasts were permeabilized by a modification of the method of Bernier et al. (5). Cells were incubated for 20 min in a digitonin permeabilization buffer (15 µg/ml digitonin, 20 mM Tris, pH 7.5, 125 mM KCl, 5 mM NaCl, 10 mM MgCl2, 11 mM glucose, and 0.1% albumin) and the specified reagents, such as vanadate. Permeabilization was assayed by trypan blue staining. Cells were then processed as outlined above for nuclear extracts, and band shift assays were performed. The permeabilization buffer was modified from the method of Bernier et al. by decreasing the digitonin and using a 1:4 dilution for the final concentration.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Activation of STATs by ROS. To investigate whether members of the STAT family of transcription factors are activated by ROS, Rat-1 fibroblasts were treated with 1 mM H2O2 for various lengths of time. Rat-1 fibroblasts were initially examined because they have a relatively low background of constitutive STAT binding activity and have a robust activation of STAT factors in response to PDGF. Nuclear extracts were prepared from the H2O2-treated cells, and the extracts were analyzed by electrophoretic mobility shift assay, with the high-affinity c-fos SIE site as a probe. This sequence binds with high affinity to most STATs (73, 94). Within 5 min of exposure to H2O2, there is a rapid activation of DNA binding activity (Fig. 1A). The induction is biphasic, with a decrease in activity observed at 15 min and a return to peak levels by 30 min. By 2 h the levels of H2O2-induced DNA binding activity returned to near-basal levels. The biphasic response was observed in repeated time course experiments, although the timing of the first decrease in binding varies from 15 to 30 min. A dose-response experiment indicates that STAT activation by H2O2 is first detected at 100 µM (see complex B) and increases with doses up to 1 mM (Fig. 1B). Three distinct complexes (A, B, and C) are induced, with the lowermost band (complex C) being the faintest. The magnitude of induction is as much as 14-fold, as measured by phosphorimage analysis. For comparison, the induction of STATs by PDGF is shown in Fig. 1. At optimum doses of PDGF and H2O2, PDGF gives a two- to threefold greater induction of STAT DNA binding than does H2O2. As would be expected, generation of H2O2 by glucose and glucose oxidase in the culture medium also induces the STAT DNA binding (Fig. 2A). The rapid induction of STAT binding by H2O2 is inhibited by catalase treatment and does not require new protein synthesis, as it is not blocked by cycloheximide (Fig. 2B). These results indicate that H2O2 is the active agent for STAT induction and that the induction is not secondary to an earlier transcriptional response to H2O2.


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Fig. 1.   H2O2 activates signal transducer and activator of transcription (STAT) in Rat-1 cells. A: time course of STAT activation by H2O2 in Rat-1 cells. Confluent, quiescent Rat-1 cells were treated with 1 mM H2O2 for indicated periods of time or with 25 ng/ml platelet-derived growth factor (PDGF) for 15 min and then lysed, and nuclear proteins were extracted and assayed for band shift with an SIE probe. B: dose response of STATs to H2O2 treatment for Rat-1 fibroblasts. Indicated concentrations of H2O2 were added to confluent, quiescent fibroblasts for 10 min, and extracts were prepared for band shift gels. Probe was high-affinity SIE (94).


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Fig. 2.   Activation of STATs is specific for H2O2. A: glucose oxidase (GO) activates STATs. Confluent, quiescent (Q) Rat-1 fibroblasts were treated for 10 min with indicated concentrations of glucose oxidase, then extracts were prepared for band shift analysis by using a high-affinity SIE probe. FP, free probe. B: catalase inhibits H2O2 activation of STATs. Confluent, quiescent Rat-1 fibroblasts were preincubated with 10 µg/ml cycloheximide (CH) or 3,000 U/ml catalase before addition of 1 mM H2O2 for 10 min. Extracts were then prepared for band shift analysis by using a high-affinity SIE probe.

The activation of the SIE binding activity by H2O2 is rapid and specific and has the binding and specificity properties characteristic of the STAT proteins. Inclusion of 100-fold molar excess of oligonucleotides of the interferon-stimulated response element, the cAMP response element, or high-affinity c-fos SIE STAT binding site indicates that only the high-affinity SIE site is able to compete for all three bands (Fig. 3). At least three distinct STAT binding complexes are observed, sometimes referred to as bands A, B, and C (C being the fastest-migrating and, in this case, the faintest complex) (68). In the case of PDGF, there is evidence that the uppermost band (A) is primarily composed of an STAT3 homodimer, the lowermost band (C) of an STAT1 homodimer, and the middle band (B) of a heterodimer of the two (78). Thus, by the position of migration through the gel, H2O2 induces in fibroblasts complexes likely to be STAT1 and STAT3, with the two STAT3-containing complexes predominating. To confirm the identity of these SIE binding complexes, antibody supershift analysis was performed (Fig. 3). The addition of anti-STAT3 antiserum to the binding reaction supershifts and diminishes the upper complex and slightly diminishes band B but leaves the lower complex unaltered. The addition of anti-STAT1 antibody, made against a unique COOH-terminal peptide of STAT1, supershifts the lowermost and weakest SIE binding complex (band C) and diminishes the middle band (band B). Thus H2O2 induces at least STAT3 and weakly STAT1 in Rat-1 fibroblasts. Because anti-STAT3 antibody only weakly diminishes band B but strongly reacts with band A, it is likely that there are other STATs induced by H2O2 or that alternate forms of STAT3 are present in complex B. STAT3-beta , which has a truncated COOH terminus, would not be recognized by this antibody (9). Alternatively, STAT3 may be poorly recognized by the antibody when it is in the heterodimeric complex. STAT5 and STAT6 can be induced by PDGF in fibroblasts and run near the same position in the gel (62, 90). However, antibodies to STAT5b and STAT6 failed to shift or disrupt the complexes (data not shown).


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Fig. 3.   Induction of STAT1 and STAT3 by H2O2 in Rat-1 fibroblasts. Extracts of quiescent Rat-1 cells or cells treated for 10 min with 1 mM H2O2 were assayed using a high-affinity SIE probe on band shift gels. Binding reactions were performed in presence or absence of indicated antibodies or competitor DNAs. Indicated antisera or normal rabbit serum (NRS) was incubated with 10 µl (12 µg protein) of nuclear extracts for 30 min on ice at a dilution of 1:10 before binding reaction. Then probe was added, resulting in a final volume of 20 µl. Competitor DNA sequences for the SIE, interferon-stimulated response element (ISRE) (51) and cAMP response element (CRE) (22) were used at a concentration 100 times greater than that of 32P-labeled probe.

To further confirm the activation of STAT3 by H2O2, we examined the tyrosine phosphorylation state of tyrosine-705 of STAT3 with an anti-STAT3 phosphotyrosine-specific antibody by Western blotting after H2O2 treatment. Phosphorylation of tyrosine-705 is known to be responsible for STAT3 dimerization and activation of DNA binding (97, 103). This antibody recognizes phospho-Y705-STAT3 as opposed to STAT3 or phosphotyrosine alone. There is an increase in STAT3 tyrosine phosphorylation after H2O2 (Fig. 4), consistent with the induction of STAT3 DNA binding activity. The same blot was stripped and reblotted with an anti-STAT3 antibody. This experiment confirmed that the increases in tyrosine phosphorylation observed are not due to increases in the levels of STAT3 protein. The two STAT3 bands observed are likely due to differential phosphorylation of STAT3 on serine-727 residue, which regulates STAT3 transactivation activity (13, 96, 97).


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Fig. 4.   STAT3 is phosphorylated on tyrosine in response to H2O2. Quiescent Rat-1 cells were treated with 1 mM H2O2 for indicated times. Total cellular extract was obtained and run on an 8% SDS polyacrylamide gel. STAT3 proteins [phosphorylated (P-STAT3) and nonphosphorylated (STAT3)] were detected using a PhosphoPlus STAT3 (Tyr-705) antibody (New England Biolabs) or an STAT3 antibody and chemiluminescence detection.

To further characterize the activation of STATs by ROS, we attempted to induce STAT activity in Rat-1 cells by using other types of ROS (data not shown). When cells were treated for as long as 1 h with superoxide-generating stimuli, such as xanthine and xanthine oxidase (0.6 U/ml), menadione (100 µM), or paraquat (200 µM), no or very little induction of STAT complexes was observed. Thus STAT activation in Rat-1 cells appears analogous to the activation of NF-kappa B, in that it is activated by peroxides and not superoxides (71). Although superoxide is broken down into H2O2, the concentrations of H2O2 generated are apparently not sufficient for STAT induction. Consistent with this hypothesis, it has been found previously and we have confirmed that xanthine oxidase and menadione are less efficient generators of H2O2 than glucose oxidase (10, 24). Very high levels of menadione and xanthine/xanthine oxidase will induce small but detectable levels of STAT DNA binding (data not shown).

Other types of ROS also failed to activate STATs. Stimulation of Rat-1 cells with nitric oxide donors, such as isosorbide dinitrite (5 mM), sodium nitroprusside (0.5 mM), or nitrosoglutathione (0.2 mM), failed to activate STAT DNA binding (data not shown). Heavy metals such as 52Fe can produce hydroxyl radicals via the Haber-Weiss reaction but failed to induce the JAK-STAT pathway. In addition, UV irradiation (60 J/m2), previously shown to cause oxidative stress and activate Jun kinase and the EGF receptor (21, 36, 67), fails to stimulate activation of STAT DNA binding in Rat-1 fibroblasts. Heat shock, a more general cell stressor, also failed to induce the STAT pathway (data not shown). Thus the STAT pathway is activated by specific types of ROS and is not a general response to cellular stress. The failure of these cellular stresses to induce STATs is not due to acute toxicity of these agents. Although some of these treatments will lead to toxicity or apoptosis after several hours of treatment, the cells remained intact during the short time of exposure needed to induce the STAT pathway.

Antioxidants inhibit STAT induction by ROS and PDGF. To assess whether the activation of the STAT pathway by H2O2 was secondary to oxidative stress, we performed experiments with Rat-1 cells using H2O2 and various antioxidants (Fig. 5A). By themselves, the antioxidants NAC, PDTC, and glutathione fail to induce STAT activity in quiescent cells. However, the activation of the STATs by H2O2 was inhibited by each of these antioxidants. In contrast, BSO, a glutathione-depleting agent, led to the induction of STAT DNA binding (Fig. 5B). Figure 5C shows that the DNA binding activity induced by BSO is specifically competed away by unlabeled high-affinity STAT binding oligonucleotide. These results indicate that specific types of oxidative stress lead to the activation of STAT proteins.


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Fig. 5.   Effects of antioxidants and buthionine sulfoximine (BSO) on induction of STAT pathway by H2O2. A: confluent, quiescent Rat-1 fibroblasts were treated with 20 mM N-acetyl-L-cysteine (NAC), 100 µM pyrrolidine dithiocarbamate (PDTC), or 1 mM glutathione (GSH) alone or in combination with 1 mM H2O2 before nuclear extracts were prepared and analyzed as described in Fig. 1 legend. When cells were treated with H2O2, antioxidants were added for 90 min before incubation with H2O2 for 15 min. B: quiescent Rat-1 cells were treated for 5 h with 2 mM BSO or solvent (DMSO), then extracts were prepared for band shift analysis by using a high-affinity SIE probe. C: extracts from BSO-treated lanes from B were run on band shift gels with and without a 100× excess of unlabeled SIE oligonucleotides as a competitor DNA (comp). D: activation of STATs in A-431 cells by oxidative stress. Confluent, quiescent A-431 cells were treated with 100 ng/ml epidermal growth factor (EGF) for 15 min or indicated concentrations of H2O2 for 30 min before extracts were prepared for band shift analysis by using a high-affinity SIE probe. In indicated lane, cells were treated with 2 mM BSO for 12 h before harvest.

The induction of STATs by oxidative stress is not limited to Rat-1 cells. Other fibroblasts such as NIH/3T3 and Balb/c 3T3 cells activate STATs in response to H2O2 (data not shown). In addition, H2O2 and BSO activate STATs in the epidermally derived A-431 cell line (Fig. 5D). However, in these cells the activation requires higher concentrations of H2O2, and the response is less robust. This may indicate that A-431 cells have higher levels of antioxidant enzymes than fibroblasts. This would not be surprising, since many tumor cells have greater resistance to oxidative stress (63).

Interestingly, antioxidants reduce the level of STAT activation by PDGF. NAC reduced the activation of STATs by PDGF by 40%, as measured by phosphorimager analysis (Fig. 6A). This suggests that intracellular oxidation in response to PDGF contributes to the overall levels of STAT induction. Intracellular generation of ROS in response to ligands such as PDGF and tumor growth factor-beta is believed to be mediated by the activity of a membrane-bound NADH oxidase (41, 48, 54, 87). This enzyme complex is known to be inhibited by DPI. Therefore, we tested whether STAT activation by PDGF could be inhibited by DPI (Fig. 6B). DPI inhibited STAT activation in a dose-responsive manner by PDGF to >80% at 100 µM, as determined by densitometry. DPI was consistently a better inhibitor than NAC, suggesting that the targets of the reactive oxygen products produced in response to PDGF may be colocalized with the NADH oxidase. Because DPI is a flavoprotein inhibitor and not completely specific for the NADH oxidase, we investigated whether inhibitors of other ROS-generating reactions might inhibit STAT activation by PDGF. Inhibitors of nitric oxide synthase (NG-nitro-L-arginine methyl ester), xanthine oxidase (allopurinol), mitochondrial electron transport (KCN), and microsomal P-450 (methoxypsoralen) were much less effective than DPI in inhibiting STAT induction by PDGF (Fig. 6C). KCN did inhibit 25% of the STAT induction, probably by virtue of reducing the overall ROS load on the cell by inhibiting respiration. These findings suggest that the NADH oxidase is an important intermediary enzyme for the generation of ROS and subsequent activation of STATs by PDGF.


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Fig. 6.   Antioxidants inhibit PDGF activation of STATs. A: confluent, quiescent Rat-1 fibroblasts were pretreated for 6.5 h with 20 mM NAC before addition of indicated concentrations of PDGF. Extracts were then prepared for band shift analysis by using a high-affinity SIE probe. B: quiescent Rat-1 cells were treated for 15 min with 25 ng/ml of PDGF with or without 30 min of pretreatment with indicated concentrations of diphenylene iodonium (DPI). Extracts were then prepared for band shift analysis by use of high-affinity SIE probe. C: specific inhibition of STAT activation by 20 µM DPI. Confluent, quiescent Rat-1 fibroblasts were pretreated with indicated flavoprotein inhibitors [250 µM methoxypsoralen (MePs), 200 µM allopurinol (AP), 1 mM N-monomethyl-L-arginine (LMNA), and 1 mM KCN] for 30 min and treated with 25 ng/ml PDGF for 15 min, then extracts were prepared for band shift analysis by use of a high-affinity SIE probe.

To examine whether STAT activation by H2O2 results in transcriptional activation by STATs as well, we determined whether H2O2 could activate transcription of a reporter gene with only STAT binding sites upstream of the promoter. Thus a TK-luciferase reporter gene with or without six high-affinity SIE/STAT binding sites upstream was transfected into NIH/3T3 cells and stimulated for 6 h with 250 µM H2O2. H2O2 produced a ninefold induction of transcription of the reporter that was dependent on the presence of the STAT binding sites (Fig. 7). Thus H2O2 activates STAT DNA binding activity and transcriptional activation.


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Fig. 7.   Activation of a STAT-dependent promoter by H2O2. NIH/3T3 cells were transfected with TK-luciferase (TK-Luc) or 6XSIE/TK-luciferase (SIE-Luc) reporter constructs. Cells were serum starved for 36 h in 0.5% calf serum, then stimulated for 6 h with 250 µM H2O2 in DMEM. Cell extracts were then processed for luciferase activity. Relative luciferase activities are from an average of 2 experiments. All transfections were performed in duplicate for each experiment.

Activation of JAK2 and TYK2 by H2O2. Phosphorylation of the STATs on a specific tyrosine residue is required for the dimerization of the STATs via an SH2 domain-phosphotyrosine interaction and appears to be sufficient to activate STAT DNA binding (76, 77). Thus H2O2 activates a cellular tyrosine kinase that phosphorylates the STAT proteins or inactivates a STAT phosphatase. H2O2 has previously been shown to stimulate tyrosine phosphorylation in some cells as well as to inhibit tyrosine phosphatases (26, 33, 60). Although the JAK family of kinases have been shown to be necessary for STAT activation by interferons, recent data from our laboratory and others indicate that other tyrosine kinases may also activate STATs (50, 102).

We initially investigated whether any of the JAK family of kinases is activated by H2O2 treatment. Quiescent Rat-1 fibroblasts were treated for various times with H2O2, and cellular extracts were immunoprecipitated with antiphosphotyrosine monoclonal antibody (4G10) and then processed for Western blotting with an anti-JAK2 antibody. Figure 8A shows an increase in tyrosine phosphorylation of JAK2 detectable at 1 min of stimulation, with a peak of autophosphorylation at 5'. To determine whether the tyrosine phosphorylation of JAK2 was indicative of increased kinase activity of the enzyme, the activity of the enzyme was measured by immune complex kinase assay. JAK2 was immunoprecipitated from extracts of H2O2-treated quiescent cells and incubated in the presence of [gamma -32P]ATP for 5 min. The resulting extracts were then analyzed on SDS polyacrylamide gels for autophosphorylation of JAK2 and total 32P incorporation into protein. Figure 8B shows that H2O2 caused an increase in in vitro autophosphorylation activity of the JAK2. Quantitation of total 32P incorporation by phosphorimage analysis indicates a fourfold increase in JAK2 kinase activity (Fig. 8C). Thus, not only is JAK2 phosphorylated on tyrosine in response to H2O2 treatment, but JAK2 kinase activity is increased as well. Given the strong evidence that JAK kinases are in vivo STAT kinases, this result would suggest that STAT activation by H2O2 is mediated at least in part by JAK2. We also examined the activation of JAK1 and TYK2 by H2O2. We found no evidence of JAK1 activation, but TYK2 does become phosphorylated on tyrosine in response to H2O2 (Fig. 8D), and its kinase activity is activated sixfold (data not show). The expression of the other JAK family member, JAK3, is limited to hematopoietic cells and was not investigated (98). Thus JAK2 and TYK2 respond to H2O2.


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Fig. 8.   Activation of JAK2 and TYK2 by H2O2. A: quiescent Rat-1 cells were treated with 1 mM H2O2 for times indicated, and whole cell extracts were prepared. Immunoprecipitation with antiphosphotyrosine antibody (4G10) was performed, and extracts were resolved on an SDS polyacrylamide gel. A Western blot analysis was performed using anti-JAK2 antibody as indicated and processed via chemiluminescence. B: quiescent Rat-1 cells were treated for indicated times with 1 mM H2O2, and extracts were immunoprecipitated on protein A-agarose beads with anti-JAK2 antibody. Immunoprecipitates were then incubated with [gamma -32P]ATP, electrophoresed through an SDS polyacrylamide gel, and exposed to X-ray film. C: quantitation of kinase assay in B by phosphorimager analysis. D: whole cell extracts from quiescent Rat-1 cells were treated with 1 mM H2O2 for 5 min, extracts were precipitated with anti-TYK2 or JAKI antibody, and immunoprecipitates were processed for Western blots with antiphosphotyrosine antibody (4G10) and chemiluminescence detection.

There is one report that treatment of mesangial cells with 5 mM H2O2 activates the PDGF receptor, and it is known that PDGF activates JAK kinases as well as STATs (27). Therefore, we investigated whether H2O2 might activate the JAK-STAT pathway through activation of the PDGF receptor in Rat-1 cells. We have found that Rat-1 cells treated with 1 mM H2O2 fail to induce phosphorylation of the PDGF-beta receptor on tyrosine (data not shown). We have also found that EGF only very weakly induces STATs in Rat-1 cells (probably because of low receptor number), so that activation of the EGF receptor does not account for the induction of STATs or JAKs by H2O2.

STATs are activated by phosphatase inhibitors. There is evidence that JAK kinases are regulated by phosphatases (15, 17, 19, 43, 46, 65, 101). Previously, it was shown that the pervanadate derivative of vanadate, which is a potent phosphatase inhibitor, can induce the STAT pathway in permeabilized cells (31, 38). H2O2 has also been shown to inactivate phosphatases in vitro and in vivo (33, 84). Thus we sought to compare H2O2 induction of STATs with pervanadate induction of STATs. This was done in permeabilized cells to facilitate entry of vanadates into the cells, inasmuch as some reports indicate that vanadate does not readily cross the cell membrane (31, 34). Digitonin-permeabilized cells were treated with H2O2, vanadyl sulfate, orthovanadate, or pervanadate for 30 min and analyzed for STAT activation (Fig. 9). Vanadate induction of STATs was relatively weak under these conditions, even though it is also a phosphatase inhibitor. Pervanadate is a more potent phosphatase inhibitor than other vanadates, since it inhibits phosphatases by a covalent and irreversible mechanism (37). STAT activation by pervanadate is quantitatively and qualitatively different from H2O2 induction of STAT. Figure 9 shows that pervanadate is a very powerful inducer of STAT activation in permeabilized cells (the most potent inducer of STATs we have observed). The same experiments were carried out in unpermeabilized cells with identical results (data not shown), and subsequent reports indicate that vanadate easily enters some cell types (37). However, much more prominent in the pervanadate-treated cell extracts than in the H2O2-treated extracts is the lower STAT1 band. Thus, if H2O2 is working to activate the STAT pathway by inhibition of phosphatases, then it may be acting only on a subset of the phosphatases that are inhibited by pervanadate.


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Fig. 9.   Induction of STATs by pervanadate in permeabilized cells. Rat-1 cells were permeabilized using digitonin and treated with 500 µM orthovanadate (oV) or 500 µM vanadyl sulfate (VS) alone or in combination with indicated concentrations of H2O2. Band shift assays were performed using a high-affinity SIE probe. Lanes 10 and 11 are lighter exposures of lanes 8 and 9.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Recent work in many laboratories has shown the activation of critical signal transduction pathways by ROS, such as the NF-kappa B, the JNK-AP-1, and the ras/rac mitogen-activated protein kinase pathway (23, 36, 41, 72, 100). Although the STAT family of transcription factors is known to be activated by most cytokines and growth factors, we have shown that, in addition to these agonists, STAT family members can also be activated by oxidative stress. In particular, we have shown in Rat-1 fibroblasts that the activation of STAT3 and STAT1 in response to H2O2 occurs within minutes and is independent of new protein synthesis. The activation of STATs by H2O2 also results in STAT-dependent activation of transcription of a luciferase reporter gene. This phenomenon does not appear to be a generalized cell damage response, inasmuch as other stimuli, such as superoxide, UV irradiation, heavy metals, and heat shock, do not activate STATs. In addition, induction of STATs by H2O2 occurs in A-431 epithelial cells as well as in other fibroblasts.

Induction of STATs by H2O2 was observed with extracellular concentrations ranging from 100 µM-1 mM H2O2. These concentrations are similar to the amounts of H2O2 needed to mimic the levels of intracellular H2O2 generated by PDGF treatment (85). Although intracellular H2O2 levels vary significantly between tissues and cellular conditions, some tissues such as the lens of the eye appear to have basal levels of H2O2 as high as 25 µM (29). Thus the response of STATs to H2O2 could well be a physiological response. Moreover, in several pathological states, such as asthma and ARDS, significant increases in the amounts of H2O2 can be measured, and these concentrations can be >1 mM in some cases (20, 53, 70).

The activation of STATs by H2O2 could be inhibited by antioxidants, indicating that activation is dependent on ROS. The importance of cellular redox state is indicated by the observation that depletion of reduced glutathione with BSO also leads to STAT activation. In fact, the types of oxidative stress that lead to STAT activation are very similar to those that lead to activation of NF-kappa B (72, 82).

Not only can exogenous H2O2 activate STATs, but our findings also implicate ROS in growth factor activation of STATs. Recently, it has been shown that PDGF increases intracellular H2O2 concentrations in vascular smooth muscle cells and that treatments with antioxidants such as NAC or catalase transfection can block some downstream PDGF signaling pathways, such as the mitogen-activated protein kinase pathway (85). Here we find that PDGF activation of the STAT pathway can be significantly inhibited by NAC and DPI, indicating that ROS are required for maximal STAT signaling by PDGF. The incomplete inhibition by antioxidants of the induction of STATs by PDGF may be a reflection of the fact that STATs may be phosphorylated by multiple tyrosine kinases that are activated by PDGF, including SRC kinases, JAK kinases, and the PDGF receptor kinase (18, 50, 92). An interesting possibility is that PDGF activation of the STAT pathway requires simultaneous activation of tyrosine kinases and the inhibition of phosphatases.

The mechanism of H2O2 activation of STAT proteins likely involves activation of the JAK2 and TYK2 kinases. The JAK kinases are required for the activation of STATs by interferons (59, 91, 95). We have found that the STATs are phosphorylated in response to H2O2 and that the JAK2 and TYK2 kinases undergo phosphorylation and activation. However, it has previously been shown that H2O2 can also activate other kinases, such as some of the members of the SRC kinase family (60). Thus the activation of STATs by H2O2 is likely to be mediated by multiple kinases as appears to be the case for the response to PDGF (92).

We have found that the induction of STATs by H2O2 follows a biphasic time course. A similar biphasic STAT response has been noted for the response of STATs to interleukin-6, ANG II, and prolactin (47, 83, 99). In none of these cases is the basis or the function of the biphasic response understood. One possibility is that the second induction involves an exchange of repressor forms of STATs for activating ones. This appears to be an important mode of STAT regulation in the slime mold Dictyostelium (44). Likewise, in mammals STAT isoforms that are naturally occurring dominant negatives are expressed (14). The biphasic time course of STAT activation implies the induction of a biphasic kinase cascade. Such a kinase cascade has previously been noted for the S6 ribosomal kinases (86). The activation of JAK2 and TYK2 that we have observed only accounts for the first phase of induction. We have yet to identify the kinases responsible for the second phase of STAT induction.

There are several possibilities for how H2O2 might activate intracellular kinases. One possibility is that H2O2 activates receptors on the outside of the cell, as has been found for UV light activation of the JNK pathway (64). Although this is a possibility since many cytokine and growth factors activate JAKs, UV treatment of Rat-1 cells at least fails to elicit the STAT response. In addition, it appears unlikely that H2O2 directly activates receptors from the outside of the cell or by direct membrane perturbation, inasmuch as we have found that non-cell-permeant peroxides such as cumene hydroperoxide fail to induce STAT activation (93; data not shown). Moreover, we have found that the concentrations of H2O2 used in our experiments are insufficient to activate the PDGF-beta receptor kinase.

Another possible mechanism for activation of JAK2 by H2O2 is inactivation of protein tyrosine phosphatases (PTPs). Tyrosine phosphorylation of proteins is dependent on the balance between kinases and PTPs within a cell. Our data with the PTP inhibitors vanadate and pervanadate suggest that inhibition of PTPs results in STAT activation. The much greater activation of STATs by pervanadate than by vanadate or H2O2 alone is consistent with recent data in the literature (17, 31). Pervanadate is a significantly better inhibitor of PTP, because it irreversibly oxidizes a critical cysteine in the catalytic domain, whereas vanadate inhibits only competitively (37). In addition, in vitro and in vivo data indicate that pervanadate is a more effective inhibitor of phosphatases than ROS alone (33, 34, 37). It is possible that in vivo oxidation of this critical cysteine may, in fact, be an in vivo mechanism of downregulating PTPs. Interestingly, the STAT pathway has been shown to be positively and negatively regulated by phosphatases (16, 19, 46). Identification of the phosphatases involved in STAT regulation in Rat-1 fibroblasts is likely to be crucial to understanding the mechanism of H2O2 induction of STAT signaling.

In contrast, Haque et al. (31) found that cell lines that were mutant for JAK1, JAK2, and TYK2 are still able to activate STATs after pervanadate treatment. This result does not rule out a JAK2- or TYK2-dependent mechanism for H2O2 induction of the STAT pathway, since only a single JAK family kinase is defective in each of these mutant cell lines. Given the intensity of the pervanadate response, it is likely that pervanadate activates multiple JAK kinases or other STAT kinases and that mutations in individual kinases would therefore fail to inhibit STAT activation. This is also the case for STAT activation by growth factors (18, 50, 92).

The biologic roles of the STAT factors are beginning to be understood. It is clear that STATs participate in lactation and mammary gland development, resistance to viral infections, cell cycle regulation, apoptosis, and cellular transformation. STAT3, for instance, has been shown to be important for transformation by v-src (8, 88). The importance of STATs is also evident from knockout mice. Knockouts of STAT2 and STAT3 result in embryonic lethality. STAT1 knockout mice are defective in response to interferons, and STAT5 knockout mice have deficiencies in reproduction (for review see Ref. 14).

The fact that almost all cytokines activate the JAK-STAT pathway suggests that STATs play important roles in the inflammatory process (40). Interestingly, these are the same situations where high levels of ROS are generated. Moreover, the production of H2O2 appears to be an important second messenger in many vital cellular processes, including growth factor signaling, cell proliferation, apoptosis, and cellular transformation (2, 85, 87) in addition to contributing to the development of diseases such as ARDS and Alzheimer's (4, 20, 53). Our data suggest that the STATs are intermediates in ROS-mediated gene expression. Although induction of STATs by H2O2 may not be the only mechanism of STAT induction by PDGF, any ligand that increases intracellular H2O2 could result in the activation of the STAT pathway. Potential target genes for ROS-induced STATs are genes that have known STAT binding sites in their promoters and include genes involved in cell growth regulation, such as c-fos and c-myc (45, 58, 75, 81, 94), genes involved in antioxidant defense (79), such as rat SOD1 (7), and genes involved in apoptosis, such as the caspases (6, 11, 30, 49). We are currently investigating the possibility that the STATs are required for the regulation of these genes by ROS.

    ACKNOWLEDGEMENTS

We thank Jay Lee, E. J. Kulbokas, Deepa Bhavsar, and Susan Spear for help with these experiments.

    FOOTNOTES

This work was supported by National Institutes of Health Grants R01-GM-51551 and R01-GM-54304 (B. H. Cochran) and K08-HL-03547 (A. R. Simon).

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

Address for reprint requests: B. H. Cochran, Dept. of Physiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111.

Received 28 April 1998; accepted in final form 29 August 1998.

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