Reactive Oxygen Species Regulate Heat-Shock Protein 70 via the JAK/STAT Pathway
Abstract—Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) activate intracellular signal transduction pathways implicated in the pathogenesis of cardiovascular disease. H2O2 is a mitogen for rat vascular smooth muscle cells (VSMCs), and protein tyrosine phosphorylation is a critical event in VSMC mitogenesis. Therefore, we investigated whether the mitogenic effects of H2O2, such as stimulation of extracellular signal–regulated kinase (ERK)2, are mediated via activation of cytoplasmic Janus tyrosine kinases (JAKs). JAK2 was activated rapidly in VSMCs treated with H2O2, and signal transducers and activators of transcription (STAT) STAT1 and STAT3 were tyrosine-phosphorylated and translocated to the nucleus in a JAK2-dependent manner. Inhibition of JAK2 activity with AG-490 partially inhibited H2O2-induced ERK2 activity, suggesting that JAK2 is upstream of the Ras/Raf/mitogen-activated protein kinase–ERK/ERK mitogenic pathway. Because heat-shock proteins (HSPs) can protect cells from ROS, we investigated the effect of H2O2 on HSP expression. H2O2 stimulated HSP70 expression in a time-dependent manner, and AG-490 abolished H2O2-induced HSP70 expression. H2O2 activated the HSP70 promoter via enhanced binding of STATs to cognate binding sites in the promoter. Regulation of chaperones such as HSP70 via activation of the JAK/STAT pathway suggests that in addition to its growth-promoting effects, this pathway may help VSMCs adapt to oxidative stress.
This work was supported by National Institutes of Health grants HL57352 and HL61656.
- Received October 12, 2000.
- Accepted December 15, 2000.
Accumulating evidence supports a critical role for oxidative stress in the pathogenesis of atherosclerosis, cancer, and other human diseases.1 High levels of reactive oxygen species (ROS) damage DNA and inactivate proteins,2 resulting in chronic cellular dysfunction. Many cell types have also harnessed ROS, albeit in lower concentrations, as intracellular signaling molecules to mediate growth factor and cytokine responses.3 4 Modulation of growth responses by ROS has been demonstrated in a number of cell types, including vascular smooth muscle cells (VSMCs).5 6 Stimulation of VSMC proliferation by ROS is thought to be a critical step in atherosclerotic lesion formation.7
Tyrosine phosphorylation of cellular proteins and the consequent induction of transcription of early-response genes are key determinants of cell growth and differentiation in response to mitogenic signaling.8 VSMC mitogens such as platelet-derived growth factor and epidermal growth factor activate receptor protein tyrosine kinases on binding, which stimulate intracellular signaling pathways that result in mitogen-activated protein kinase activation.9 10 Other mitogens, such as thrombin and angiotensin II, activate G protein–coupled receptors that do not possess intrinsic tyrosine kinase activity but require tyrosine phosphorylation events to induce mitogenesis.11 12 13
The necessary role for protein tyrosine phosphorylation in mitogenesis elicited by thrombin and ROS indicates that these mitogens may utilize cytoplasmic protein tyrosine kinases in their signaling cascade. Forming 1 such group of tyrosine kinases are Janus kinases (JAKs), which along with their substrates, signal transducers and activators of transcription (STATs), have hitherto been characterized as essential mediators of cytokine and polypeptide hormone-induced signaling.8 14 Members of the JAK/STAT pathway mediate at least some biological effects of angiotensin II,15 platelet-derived growth factor-BB,15 16 and endothelial growth factor.17 Activation of the JAK/STAT pathway has also been observed in response to generation of intracellular ROS18 and exogenous hydrogen peroxide (H2O2).19 On phosphorylation by JAKs of tyrosine residues, activated STAT dimers translocate to the nucleus to transactivate target gene expression.20
The 70- and 90-kDa heat-shock proteins (HSPs) are among the subset of stress-responsive proteins known to be transactivated by STATs.21 22 Elevated levels of HSPs are rapidly synthesized within the cell in response to environmental stresses.23 Under physiological conditions, constitutively expressed HSPs function as molecular chaperones, whereas under stress conditions, HSPs prevent protein aggregation.24 In addition, HSPs may directly regulate specific stress-responsive signaling pathways and may antagonize signaling cascades that result in apoptosis.25
Because ROS do not directly activate receptor protein tyrosine kinases to exert their mitogenic effects on VSMCs, we investigated the effect of H2O2 on the activation of cytoplasmic tyrosine kinases, JAKs, and their substrates, STATs. We show here that H2O2 causes activation of JAK2 and subsequent tyrosine phosphorylation and nuclear translocation of STAT1 and STAT3. Our results also indicate that JAK2 partially regulates extracellular signal–regulated kinase (ERK)2 activity and thus, contributes to VSMC proliferation. Furthermore, we demonstrate that the JAK/STAT pathway mediates H2O2-induced HSP70 expression, which may help cells adapt to oxidative stress.
Cell Culture and Reagents
VSMCs were isolated from the thoracic aortas of 200- to 250-g male Sprague-Dawley rats. Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% (vol/vol) calf serum as described previously.12 VSMCs were growth-arrested by incubation in Dulbecco’s modified Eagle’s medium containing 0.1% calf serum for 72 hours. Antibodies used were as follows: anti-phosphotyrosine (4G10), anti-JAK1, anti-JAK2, anti-TYK2, anti-STAT1, anti-STAT3, and anti-ERK2 (UBI and Santa Cruz Biotechnology); anti-phosphospecific JAK2 (Biosource International); anti-phosphospecific and anti-nonphosphospecific ERK1/2 (New England Biolabs); and anti-HSP70 (Affinity BioReagents). AG-490 was obtained from Calbiochem.
Immunoprecipitation, ERK2 Activity Assay, and Western Blotting
Growth-arrested VSMCs were treated with 200 μmol/L H2O2 in the presence or absence of inhibitors for the specified time periods at 37°C. Cell lysates were prepared, and immunoprecipitation, ERK2 activity assay, and Western blotting were performed as described previously.26
Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared from growth-arrested VSMCs that were either left untreated or treated with 200 μmol/L H2O2.27 DNA binding was performed by incubating 5 μg of nuclear protein with 100 000 counts per minute of 32P-labeled, double-stranded HSP70 oligonucleotide (−122GATCCGGCGAAACCCCTGGAATATTCCCCGACCT−90) for 20 minutes at room temperature. Canonical double-stranded oligonucleotides for SP1 (5′-ATTCGATCGG-GGCGGGGCGAGC-3′) and STAT3 (5′-TGATTACGGGAAATG-3′) and a high-affinity, double-stranded STAT1 binding sequence SIEm67 (5′-GATCTGATTACGGGAAATG-3′)28 were used in competition studies. For identifying the STAT1-specific band, samples were incubated with STAT1 or STAT3 antibody for 30 minutes before the DNA binding reaction was performed. Protein-DNA complexes were resolved on a 4% polyacrylamide gel.
Transient Transfection and CAT Assay
The HSP70 chloramphenicol acetyltransferase (CAT) reporter constructs LSN (−188 to +1) and LSNP (−100 to +1) were kindly provided by Richard Morimoto (Northwestern University, Evanston, Ill). VSMCs at 70% to 80% confluence were transfected with 10 μg of reporter plasmid DNA by using the calcium precipitate method.26 Cells were cotransfected with β-galactosidase expression vector to normalize for transfection efficiency and were quiesced for 16 hours after transfection. After quiescence was maintained for 36 hours, cells were either left untreated or treated with 200 μmol/L H2O2 for 6 hours. In experiments with AG-490, cells were treated with the inhibitor for 16 hours before H2O2 treatment. Cell lysates were prepared,26 and CAT activity was measured by the method of Gorman et al.29 The β-galactosidase assay was performed by following the manufacturer’s protocol (Promega).
Differences were analyzed with 1-way ANOVA, and post hoc analysis was performed with the Student-Newman-Keuls method. Values of P<0.05 were considered statistically significant.
H2O2 Causes Activation of Kinases JAK2 and TYK2 in Rat VSMCs
To test the hypothesis that JAK/STAT pathway activation contributes to the mitogenic effects of ROS on VSMCs, we treated growth-arrested VSMCs with 200 μmol/L H2O2, a physiological concentration of this representative ROS that elicits VSMC proliferation.6 H2O2 caused rapid tyrosine phosphorylation of JAK2, which was sustained for 60 minutes (Figure 1A⇓), but it had no effect on JAK1 phosphorylation (data not shown). JAK2 phosphorylation was biphasic, with peaks at 5 minutes (9.10±2.97-fold increase) and 30 minutes (5.0±1.52-fold increase; Figures 1A⇓, 1B⇓ [top], and 1C). A similar biphasic stimulation of various kinases by ROS has been reported, and the second peak has been attributed to the secretion of autocrine factors.30 Tyrosine phosphorylation of JAK2 by H2O2 was concentration dependent, and maximum phosphorylation was observed with 200 μmol/L H2O2 at 5 minutes (please see http://www.atvb.ahajournals.org). In contrast to JAK2, peak TYK2 tyrosine phosphorylation was observed after a 15-minute exposure to H2O2, and TYK2 phosphorylation (3.86±0.64-fold increase) was not as pronounced as that of JAK2 (please see http://atvb.ahajournals.org). H2O2 altered tyrosine phosphor- ylation without affecting steady-state protein levels of JAK2 (Figure 1B⇓, bottom) and TYK2 (please see http://atvb.ahajournals.org).
H2O2 Induces Tyrosine Phosphorylation and Nuclear Translocation of STAT1 and STAT3 in Rat VSMCs
To determine whether activation of JAK2 and TYK2 by H2O2 leads to activation of STAT proteins, H2O2-treated VSMC lysates were immunoprecipitated with an anti-phosphotyrosine antibody, and immunoprecipitates were immunoblotted with antibodies against STAT1α/β or STAT3. Constitutive phosphorylation was lower for STAT1α than for STAT1β in untreated VSMCs (Figure 2A⇓), but STAT1α phosphorylation was greater than that for STAT1β after 10 minutes of H2O2 treatment. Tyrosine-phosphorylated STAT1α/β were not detectable 15 and 30 minutes after H2O2 treatment, and phosphorylated STAT1α/β levels at 60 minutes were much lower than in untreated cells. In broad terms, the biphasic STAT1 phosphorylation corresponded to the biphasic JAK2 phosphorylation, suggesting that the former is dependent on JAK2 activation. STAT1 was rapidly translocated to the nucleus within 5 minutes after H2O2 treatment (Figure 2B⇓, top), whereas no detectable change was observed in cytosolic STAT1 protein levels (Figure 2B⇓, bottom). Peak nuclear translocation of STAT1 was observed at 10 minutes (4.03±0.32-fold increase), and in contrast to the tyrosine phosphorylation in immunoprecipitation studies (Figure 2A⇓), was sustained for 60 minutes (Figure 2C⇓). The reason for this discrepancy could be that nuclear fractions may contain some dephosphorylated STAT1. Constitutive tyrosine phosphorylation of STAT3 was observed in growth-arrested VSMCs, and phosphorylation increased within 5 minutes of H2O2 treatment and was sustained for 60 minutes (Figure 3A⇓). As with STAT1, rapid nuclear translocation of STAT3 was observed in H2O2-treated VSMCs (Figure 3B⇓, top), with no detectable change in protein levels in the cytosolic fractions (Figure 3B⇓, bottom). Peak nuclear STAT3 protein levels were observed at 15 minutes (2.90±0.49-fold increase; Figure 3C⇓) and were sustained for 60 minutes. These experiments demonstrate the phosphorylation and nuclear translocation of STATs in H2O2-stimulated VSMCs.
JAK2 Contributes to ERK2 Activation in Response to H2O2 in VSMCs
Because ERK1/2 activation is associated with H2O2-induced mitogenesis,31 32 we investigated the relationship between JAK2 stimulation by H2O2 and ERK2 activity. Pretreatment of VSMCs with 50 μmol/L AG-490, a specific JAK2 inhibitor, inhibited H2O2-induced JAK2 tyrosine phosphorylation and the consequent phosphorylation of STAT1 and STAT3, whereas it had no effect on c-Src, a non-JAK cytosolic tyrosine kinase (data not shown). Peak ERK2 activity, as measured by an immunocomplex kinase assay, was observed 15 minutes after treatment with H2O2 (Figure 4⇓). This increase in ERK2 activation by H2O2 is consistent with 1 previous report32 but is contradictory to another33 ; the reasons for this discrepancy are not clear. H2O2-induced ERK2 activation was blocked partially by AG-490 (4.5±1.4-fold increase with H2O2 at 15 minutes vs 2.7±0.7 and 1.9±0.4-fold for H2O2 in the presence of 10 and 50 μmol/L AG490, respectively), suggesting that JAK2 activation is necessary for H2O2-induced ERK2 activation. Partial inhibition of ERK2 activity by a JAK2 antagonist indicates that, along with JAK2-mediated stimulation, there may be an alternative pathway for ERK1/2 activation.
H2O2 Induces HSP70 Expression Through Activation of the JAK/STAT Pathway
Accumulation of RNA for HSPs has been reported during conditions known to produce ROS.34 The 5′-flanking sequences of the HSP70 and HSP90β genes contain functional binding sites for STATs.21 Hence, we investigated whether H2O2 causes accumulation of these proteins in rat VSMCs. H2O2 stimulated expression of both HSP70 (Figure 5⇓) and HSP90 (data not shown). Because of more pronounced upregulation, we further investigated the expression of HSP70. HSP70 protein levels increased in VSMCs in a time-dependent manner, with a 5.90±0.97-fold increase at 24 hours after 200 μmol/L H2O2 treatment (Figures 5A⇓ and 5B⇓). Pretreatment of VSMCs with 50 μmol/L AG-490 abolished the increase in HSP70 protein levels induced by H2O2 at 24 hours (Figures 5C⇓ and 5D⇓; 5.70±0.85 vs 1.40±0.50, P<0.05).
To investigate whether HSP70 expression in VSMCs exposed to H2O2 is mediated by the binding of STATs to their responsive element (−122 to −90 bp of the HSP70 promoter), we measured the ability of nuclear proteins from H2O2-treated cells to bind an HSP70-STAT oligonucleotide (please see http://www.atvb.ahajournals.org). Nuclear extracts from H2O2-treated VSMCs produced 2 shifted bands that were competed with an excess of unlabeled specific oligonucleotide but not with a nonspecific sequence. The faster migrating band was competed with an unlabeled, high-affinity STAT1 binding site (SIEm67 oligonucleotide) from the c-fos promoter, but not with an unlabeled STAT3 consensus sequence, and was abolished by preincubation of the complexes with an anti-STAT1 antibody (but not by an anti-STAT3 antibody), demonstrating the presence of STAT1 protein in this complex. The slower migrating band was partially competed by STAT1 and STAT3 oligonucleotides and partially abolished by anti-STAT1 and anti-STAT3 antibodies, suggesting that it contains a STAT1/STAT3 heterodimer.
H2O2 activates the HSP70 promoter in a JAK2-dependent manner. To investigate whether H2O2-induced HSP70 expression was mediated via a direct effect on its promoter, VSMCs were transfected with HSP70 promoter-reporter constructs either containing (LSN, −188 to +1) or lacking (LSNP, −100 to +1) a functional STAT binding site. The reporter construct LSN was activated 2.5-fold by 200 μmol/L H2O2, whereas transactivation in response to H2O2 was lost by deletion of the STAT binding site (Figure 6A⇓). AG-490 inhibited the H2O2-induced increase in HSP70 promoter activity, suggesting that STATs require JAK2 activation for activity (Figure 6B⇓). Taken together with the results shown in Figure 5⇑, these findings indicate that H2O2 activates the JAK/STAT pathway in VSMCs, which has functional consequences on the expression of physiologically relevant proteins such as HSP70.
We have shown that H2O2 stimulates specific members of the JAK/STAT pathway, an effect that participates in stress-responsive gene expression. Rapid activation of different JAKs and STATs in response to H2O2 has recently been reported in fibroblasts and lymphocytes.18 19 35 In this report, we link JAK2 activation by H2O2 with STAT phosphorylation and specific transactivation of a STAT-responsive gene promoter.
H2O2 stimulates ERK2 activity in VSMCs in a time-dependent manner, with peak activity occurring at 15 to 30 minutes. Inhibition of JAK2 activity by AG-490 pretreatment partially inhibits H2O2-stimulated ERK2 activity (Figure 4⇑). In contrast, a temporal shift in ERK1/2 activation was observed in fibroblasts treated with H2O2 and AG-490.35 The formation of a Ras/JAK2/Raf-1 complex after JAK2 phosphorylation, resulting in Ras and Raf-1 activation, was initially thought to be a cytokine-induced response.36 Emerging evidence indicates that JAK2 activation is also required for angiotensin II– and platelet-derived growth factor–induced JAK2/Raf-1 complex formation, Raf-1 tyrosine phosphorylation, and ERK1/2 activation.15 Recently it was reported that H2O2-mediated Ras and ERK1/2 activations are partially dependent on JAK2 activity. Based on those findings, it is proposed that JAK2 is upstream of Ras in the Ras/Raf/mitogen-activated protein kinase–ERK/ERK pathway and thus, regulates activation of early growth–response genes and cell proliferation, although a parallel H2O2-stimulated pathway independent of JAK2 must also exist to activate ERK1/2.35 Cross-talk between the JAK/STAT and ERK pathways suggests that inhibition of JAK2 activity not only blocks direct tyrosine phosphorylation but can also indirectly inhibit serine phosphorylation of STATs by ERK1/2. Maximal transactivation by STATs requires serine phosphorylation by mitogen-activated protein kinase besides tyrosine phosphorylation.37 On the contrary, ERK1/2 activation inhibits interleukin-6–induced JAK/STAT signaling.38 Further experiments will define the role of ERK1/2 in the activation of STATs by H2O2.
Increased HSP70 protein synthesis is seen in cardiovascular tissues exposed to various stressors39 and in VSMCs surrounding the necrotic zones of atherosclerotic plaques.40 Enhanced HSP70 protein levels protect against lethal heat stress and ischemia.41 ROS such as H2O2 have pleiomorphic effects on cells, eliciting apoptosis in some cell types42 while stimulating early growth–response gene expression and proliferation in others.5 The cell types that exhibit proliferative responses likely have adaptive mechanisms to overcome the adverse effects of oxidants. Because HSPs modulate the stress response, we hypothesized that they might be induced in VSMCs exposed to H2O2, especially because HSP promoters contain functional STAT binding sites.21 Our results demonstrate a time-dependent increase in HSP70 protein levels in VSMCs treated with H2O2, which is blocked by pretreatment with AG-490, indicating that this protein is indeed regulated via activation of the JAK/STAT pathway. HSP70 has been reported to promote cell proliferation.43 Oxidative stress–induced HSP90α and cyclophilin may promote VSMC growth.37 These observations suggest that regulation of HSP70 could be 1 of the mechanisms by which the H2O2-induced JAK/STAT pathway promotes VSMC growth. Further studies will determine the growth-promoting versus protective effects of HSP70 on H2O2-induced VSMC growth.
Our results also indicate that the HSP70 promoter construct containing a functional STAT-binding site is activated by H2O2, which is blocked by pretreatment with AG-490. JAK2-mediated phosphorylation of STATs is therefore required for HSP70 promoter activation by H2O2. STAT1 is the major protein binding the STAT-responsive element in VSMCs, although it is possible that STAT3 also participates, as STAT3 is rapidly phosphorylated after H2O2 treatment and an anti-STAT3 antibody partially competes for H2O2-induced binding activity. In addition to STAT binding sites, the HSP70 promoter (LSN, −188 to +1) also has overlapping binding sites for another transcription factor, heat-shock factor-1.21 Although we do not specifically address this point, it is possible that heat-shock factor-1 may also be involved in H2O2-mediated HSP70 expression in VSMCs. Indeed, significant heat-shock factor-1 activation has been observed in heart tissue perfused with H2O2.44 Stephanou et al21 have shown that overexpression of STAT1 and heat-shock factor-1 has additive effects on HSP70 promoter activity in HepG2 cells and suggest that protein-protein interactions may play a role in transcriptional activity. The demonstration of enhanced HSP70 expression in VSMCs treated with H2O2 via the JAK/STAT pathway suggests that, in addition to its growth-promoting effects, this pathway may play a key role in the adaptive response to oxidative stress.
We are thankful to Joann Aaron and Chris Horaist for editorial assistance.
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