Jak2 Tyrosine Kinase Mediates Oxidative Stress-induced Apoptosis in Vascular Smooth Muscle Cells*

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 33, Issue of August 13, pp. 34547 34552, 2004 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Jak2 Tyrosine Kinase Mediates Oxidative Stress-induced Apoptosis in Vascular Smooth Muscle Cells* Received for publication, May 6, 2004 Published, JBC Papers in Press, May 24, 2004, DOI 10.1074/jbc.M405045200 Eric M. Sandberg and Peter P. Sayeski From the Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, Florida 32610 In vascular smooth muscle cells, Jak2 tyrosine kinase becomes activated in response to oxidative stress in the form of hydrogen peroxide. Although it has been postulated that hydrogen peroxide-induced Jak2 activation promotes cell survival, this has never been tested. We therefore examined the role that Jak2 plays in vascular smooth muscle cell apoptosis following hydrogen peroxide treatment. Here, we report that Jak2 tyrosine kinase activation by hydrogen peroxide is required for apoptosis of vascular smooth muscle cells. Upon treatment of primary rat aortic smooth muscle cells with hydrogen peroxide, we observed laddering of genomic DNA and nuclear condensation, both hallmarks of apoptotic cells. However, apoptosis was prevented by either the expression of a dominant negative Jak2 protein or by the Jak2 pharmacological inhibitor AG490. Moreover, expression of the proapoptotic Bax protein was induced following hydrogen peroxide treatment. Again, expression of a dominant negative Jak2 protein or treatment of cells with AG490 prevented this Bax induction. Following Bax induction by hydrogen peroxide, mitochondrial membrane integrity was compromised, and caspase-9 became activated. In contrast, in cells expressing a Jak2 dominant negative we observed that mitochondrial membrane integrity was preserved, and no caspase-9 activation occurred. These data demonstrate that the activation of Jak2 tyrosine kinase by hydrogen peroxide is essential for apoptosis of vascular smooth muscle cells. Furthermore, this report identifies Jak2 as a potential therapeutic target in vascular diseases in which vascular smooth muscle cell apoptosis contributes to pathological progression. Jak2 tyrosine kinase is a non-receptor tyrosine kinase known primarily as a mediator of cytokine signal transduction (1, 2). It is, though, potently activated in response to oxidative stress in the form of hydrogen peroxide (3). Although the signaling cascade responsible for hydrogen peroxide-induced Jak2 activity was at least partially elucidated in vascular smooth muscle cells, no physiological role has been described for this pathway (4, 5). Runge and co-workers demonstrated that Jak2 activation by hydrogen peroxide induces the expression of heat-shock protein 70, a protein that can protect cells from oxidative stress. Based on these data, they suggested that Jak2 might help vascular smooth muscle cells adapt to oxidative stress (5). Oxidative stress in vascular smooth muscle cells can cause proliferation, contraction, or apoptosis (6 8). How the same stimulus can result in such opposing endpoints is unknown but is probably dependent on the concentration of the oxidant. Furthermore, the signaling proteins that mediate these different responses are unknown. During vascular conditions such as atherosclerosis, circulating macrophages release large amounts of hydrogen peroxide on vascular smooth muscle cells. Apoptosis that occurs as a result of this release contributes to the progression of these pathologies (9, 10). Identifying the mediators of oxidant-induced apoptosis may therefore uncover novel therapeutic targets. Interestingly, both pro- and anti-apoptotic roles have been ascribed to Jak2 tyrosine kinase activity in a variety of signaling systems (11 13). We therefore sought to determine what role, if any, Jak2 plays in oxidative stress-induced apoptosis in vascular smooth muscle cells. For this purpose we used primary isolated rat aortic smooth muscle (RASM) 1 cells. From these cells, two cell types were created. RASM-control cells were created by transfecting the RASM cells with a neomycin-resistant cassette. RASM-DN cells were created by stably transfecting RASM cells with a Jak2 dominant negative mutant to inhibit the function of endogenously expressed Jak2. The creation and characterization of these cells has been described previously (14). Here, these cells were treated with hydrogen peroxide, and indicators of apoptosis were measured. We found that the suppression of Jak2 function by either the dominant negative protein or the Jak2 pharmacological inhibitor AG490 protected cells against apoptosis. Collectively, the data show that Jak2 is essential for hydrogen peroxide-induced apoptosis in vascular smooth muscle cells. * This work was supported by a Biomedical Research Support Program for Medical Schools Award to the University of Florida College of Medicine from the Howard Hughes Medical Institute, American Heart Association National Scientist Development Grant 0130041N, and National Institutes of Health Grants K01-DK60471 and R01-HL67277. 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. Supported by a University of Florida Medical Guild fellowship and a predoctoral fellowship from the Florida/Puerto Rico affiliate of the American Heart Association. To whom correspondence should be addressed: Dept. of Physiology and Functional Genomics, University of Florida College of Medicine, P. O. Box 100274, Gainesville, FL 32610. Tel.: 352-392-1816; Fax: 352-846-0270; E-mail: Psayeski@phys.med.ufl.edu. This paper is available on line at http://www.jbc.org 34547 EXPERIMENTAL PROCEDURES Cell Culture Creation and characterization of the RASM-control and RASM-DN cells has been described previously (14). These cells were cultured in Dulbecco s modified Eagle s medium containing 4.5 g/liter glucose and 10% fetal bovine serum and growth-arrested in serum-free Dulbecco s modified Eagle s medium for 48 h prior to the experiments. Cell culture reagents were obtained from Invitrogen. AG9, AG490, and hydrogen peroxide were from Calbiochem. All other reagents were purchased from Sigma or Fisher Scientific. Immunoprecipitation Cells were washed with two volumes of ice- 1 The abbreviations used are: RASM, rat aortic smooth muscle; DN, dominant negative; STAT, signal transducers and activators of transcription; pab, polyclonal antibody.

34548 Role of Jak2 in Oxidative Stress-induced Apoptosis FIG. 1.Hydrogen peroxide-induced Jak2 activity is suppressed in RASM-DN cells. RASM-control and RASM-DN cells were treated with 0.2, 0.5, or 1 mm hydrogen peroxide for 0, 5, or 10 min. Cellular lysates were immunoprecipitated with anti-phosphotyrosine-monoclonal antibody and immunoblotted with anti-jak2-pab to detect tyrosine phosphorylated Jak2 (Jak2(P); top). An aliquot from each lysate was Western blotted with anti-jak2-pab to demonstrate equal Jak2 expression among all samples (bottom). Shown is one of three representative experiments. cold phosphate-buffered saline containing 1 mm Na 3 VO 4 and lysed in 1 ml of ice-cold radioimmune precipitation assay buffer containing protease inhibitors (15). The samples were immunoprecipitated exactly as described (16). The immunoprecipitating anti-phosphotyrosine antibody (PY20) was from BD Transduction Laboratories. Western Blotting Proteins were detected using enhanced chemiluminescence exactly as described (16). The anti-jak2, anti-stat1 (signal transducers and activators of transcription 1), anti-bcl2, and anti-bax blotting antibodies were from Santa Cruz Biotechnology. The anti-cleaved-caspase-9 antibody was from Cell Signaling Technology. Analysis of Genomic DNA Laddering Genomic DNA was isolated using the Easy-DNA kit from Invitrogen. 20 g of DNA was separated on a 1.8% agarose gel and stained with ethidium bromide. Laddering was analyzed under ultraviolet light using the GelDoc system from Bio-Rad. Hoechst 33342 Nuclear Staining Cells were grown on microscope slides, serum-starved for 48 h, and treated with 1 mm hydrogen peroxide for 24 h. Cells were washed twice with phosphate-buffered saline and incubated for 30 min at room temperature with 50 g/ml Hoechst 33342 nuclear stain. Cells were then washed, fixed, and mounted as described previously (17). The cells were visualized using a fluorescent microscope with the appropriate filter. Apoptotic cells were counted as those showing condensed and/or fragmented nuclei. A minimum of 100 cells from each of four replicates were counted for each of the six treatment conditions. Analysis of Mitochondrial Membrane Integrity Mitochondrial membrane integrity was analyzed using the MitoCapture apoptosis detection kit from BioVision. Cells were treated with 1 mm hydrogen peroxide for 2 h and stained according to manufacturer s protocol. Live cells were then visualized using appropriate filters on a confocal microscope. The pixel intensity of photomicrographs of the cells was determined using morphometric software. RESULTS To determine what role, if any, Jak2 plays in oxidative stress-induced apoptosis, we used two cell types derived from primary RASM cells. RASM-control cells were created by transfecting primary RASM cells with a neomycin-resistant cassette, whereas RASM-DN cells were created by stably transfecting RASM cells with a Jak2 dominant negative mutant in order to inhibit the function of endogenously expressed Jak2. These cells have been characterized previously (14). To determine whether expression of the Jak2 dominant negative could inhibit Jak2 tyrosine phosphorylation in response to hydrogen peroxide, the two cell types were treated with 0.2, 0.5, or 1 mm hydrogen peroxide for 0, 5, or 10 min. Cellular lysates were immunoprecipitated with an anti-phosphotyrosine antibody and immunoblotted with an anti-jak2 antibody to detect tyrosine-phosphorylated Jak2 (Fig. 1, top). The results demonstrate that, in RASM-control cells, hydrogen peroxide potently induces Jak2 tyrosine phosphorylation and that tyrosine phosphorylation levels are highest following treatment with 0.5 or 1 FIG. 2. Jak2 is essential for hydrogen peroxide-induced apoptosis of vascular smooth muscle cells. A, RASM-DN and RASMcontrol cells were either left untreated, treated with 1 mm hydrogen peroxide for 24 h, or treated with 1 mm hydrogen peroxide for 24 h following 16 h of pretreatment with 100 M AG490. Genomic DNA was isolated and separated on a 1.8% agarose gel. The gel was stained with EtBr and visualized under UV light to detect genomic DNA laddering. B, RASM-DN cells were either left untreated or treated with 5 M staurosporine for 4 h. Genomic DNA was isolated and separated on a 1.8% agarose gel. The gel was stained and visualized to detect genomic DNA laddering. C, RASM-control and RASM-DN cells were treated with 0.2, 0.5, or 1 mm hydrogen peroxide for 24 h. Genomic DNA was isolated and separated on a 1.8% agarose gel. The gel was stained with EtBr and visualized under UV light to detect genomic DNA laddering. Shown is one of four (A and B) or one of three (C) representative experiments for each cell type. mm hydrogen peroxide. In contrast, Jak2 tyrosine phosphorylation is suppressed in RASM-DN cells. We believe, therefore, that these cells are a good model for delineating the role of Jak2 tyrosine kinase in response to exogenous hydrogen peroxide treatment. To demonstrate equal Jak2 expression among the samples, an aliquot of each sample was Western blotted with anti-jak2 antibody (Fig. 1, bottom). We next used these cells to determine the role that Jak2

Role of Jak2 in Oxidative Stress-induced Apoptosis 34549 FIG. 3. Quantification of apoptosis in RASM-control and RASM-DN cells treated with hydrogen peroxide. A, RASM-control and RASM-DN cells were grown on microscope slides and treated with 1 mm hydrogen peroxide for 0 or 24 h or with 1 mm hydrogen peroxide for 24 h following 16 h of pretreatment with 100 M AG490. The cells were then stained with 50 g/ml Hoechst 33342 nuclear stain, fixed, mounted, and visualized using a florescent microscope to detect nuclear condensation. B, a minimum of 100 cells from each of four replicates for each of the six treatment conditions represented in panel A were counted. Apoptotic cells were counted as those showing condensed and/or fragmented nuclei. Data are presented as the percentage of cells undergoing apoptosis S.D. *, p 0.001. Statistical analysis was performed using Student s t test. plays in hydrogen peroxide-induced apoptosis. For this purpose, the two cell types were treated with 1 mm hydrogen peroxide for 24 h. We assessed apoptosis by analyzing genomic DNA isolated from each cellular condition (Fig. 2A). Genomic DNA from RASM-DN cells treated with hydrogen peroxide showed no evidence of DNA fragmentation. In contrast, genomic DNA isolated from RASM-control cells that were treated with hydrogen peroxide was fragmented into 200 base pair bands, an occurrence that is characteristic of apoptosis. Furthermore, the Jak2 pharmacological inhibitor AG490 prevented genomic DNA fragmentation in RASM-control cells treated with hydrogen peroxide. The data in Fig. 2A demonstrate that Jak2 is essential for hydrogen peroxideinduced apoptosis in vascular smooth muscle cells. To demonstrate that RASM-DN cells could undergo apoptosis in response to another proapoptotic stimulus, genomic DNA was isolated from RASM-DN cells treated with 5 M staurosporine, a potent activator of the intrinsic apoptosis pathway (Fig. 2B). The banding pattern characteristic of fragmented DNA was clearly seen in these cells, demonstrating that they are capable of undergoing apoptosis. Finally, to determine whether hydrogen peroxide-induced apoptosis of the RASM cells was dependent on the dose of hydrogen peroxide used, we treated RASM-DN and RASMcontrol cells with 0.2, 0.5, and 1 mm hydrogen peroxide for 24 h. Genomic DNA was then analyzed for fragmentation (Fig. 2C). As expected, the RASM-DN cells showed no evidence of DNA fragmentation. Treatment of RASM-control cells with a dose of 1mM hydrogen peroxide was essential for the induction of DNA fragmentation. We next quantified the amount of apoptosis occurring in RASM-control and RASM-DN cells treated with hydrogen peroxide. The cells were grown on microscope slides and treated with 1 mm hydrogen peroxide for either 0 or 24 h. The cells were then stained with the nucleus-specific Hoechst 33342 dye. Four replicate experiments were performed for each condition, and a minimum of 100 cells were counted from each replicate. Cells clearly showing condensed and/or fragmented nuclei were counted as apoptotic. Representative photographs of RASM-control and RASM-DN cells either left untreated, treated with hydrogen peroxide, or treated with hydrogen peroxide following pretreatment with the Jak2 inhibitor AG490 are shown (Fig. 3A). RASM-control cells treated with hydrogen peroxide had shrunken nuclei that fluoresced more intensely than those in untreated cells, which is indicative of apoptotic cells. In contrast, RASMcontrol cells treated with AG490 prior to hydrogen peroxide treatment appeared similar to untreated cells. Similarly, RASM-DN cells treated either with hydrogen peroxide alone or with both AG490 and hydrogen peroxide showed nuclear staining akin to that of untreated cells. Cells were counted, and the data are presented as the percentage of cells undergoing apoptosis (Fig. 3B). These data demonstrate that untreated RASM-control and RASM-DN cells showed very low levels of apoptosis (2.4 0.54% and 2.9 0.85%, respectively). RASM-control cells treated with hydrogen peroxide showed 54.1 3.71% of total cells undergoing apoptosis, whereas only 12.3 1.93% of RASM-DN cells treated with hydrogen peroxide were apoptotic. Finally, 5.7 0.81% of RASM-control cells and 4.4 0.98% of RASM-DN cells that were pretreated with AG490 prior to hydrogen peroxide treatment were apoptotic. Collectively, the data in Figs. 2

34550 Role of Jak2 in Oxidative Stress-induced Apoptosis FIG. 4. Jak2 mediates hydrogen peroxide-induced up-regulation of Bax expression. A, RASM-control and RASM-DN cells were treated with 1 mm hydrogen peroxide for 0, 0.5, 1, 2, or 3 h. Cellular lysates were Western blotted with anti-bax-pab (top). The membrane was stripped and reprobed with anti-bcl2-monoclonal antibody (middle). Finally, the membrane was stripped and reprobed with anti- STAT1-pAb to demonstrate equal loading among all samples (bottom). B, RASM-control cells were pretreated with 100 M AG9 or 100 M AG490 for 16 h followed by treatment with 1 mm hydrogen peroxide for 0, 0.5, 1, 2, or 3 h. Cellular lysates were Western blotted with anti-baxpab (top). The membrane was stripped and reprobed with anti-stat1- pab to demonstrate equal loading among all samples (bottom). Shown is one of three representative experiments for each cell type. and 3 demonstrate that tyrosine phosphorylation of Jak2 is essential for hydrogen peroxide-induced apoptosis of vascular smooth muscle cells, as either the expression of a Jak2 dominant negative protein or the pretreatment of cells with the Jak2 inhibitor AG490 reduces the percentage of cells undergoing hydrogen peroxide-induced apoptosis. We hypothesized that Jak2 was promoting apoptosis by regulating expression of a proapoptotic protein. Because Bax is a proapoptotic protein required for oxidative stress-induced apoptosis, we used Western blot analysis to determine whether Jak2 was required for hydrogen peroxide-mediated induction of Bax expression. For this purpose, RASM-control and RASM-DN cells were treated with 1 mm hydrogen peroxide for the indicated times. Cellular lysates were Western blotted with anti-bax antibody (Fig. 4A, top). The data show that in RASMcontrol cells hydrogen peroxide induced a rapid and transient induction of Bax expression that peaked at 1 2 h. In contrast, hydrogen peroxide did not induce Bax expression in RASM-DN cells. To determine expression of the anti-apoptotic BCL2 protein, the membrane was stripped and reprobed with an anti- BCL2 antibody (Fig. 4A, middle). The data show that hydrogen peroxide caused a slight induction of BCL2 expression in both RASM-control and RASM-DN cells that was similar between the two cell types. The BCL2:Bax expression ratio is an important indicator of apoptosis; apoptotic cells often have a lower ratio than do non-apoptotic cells. Because hydrogen peroxide induced a similar BCL2 induction in both RASM-control and RASM-DN cells but preferentially induced strong Bax induction in RASM-control cells, the BCL2:Bax expression ratio is lower in RASM-control cells than in RASM-DN cells. This result is consistent with our finding that hydrogen peroxide preferentially induces apoptosis in RASM-control cells. To demonstrate equal protein loading among all samples, the membrane was stripped and reprobed with an anti-stat1 antibody (Fig. 4A, bottom). Thus, these data demonstrate that Jak2 activation is required for up-regulation of the proapoptotic Bax protein and a subsequent alteration of the BCL2: Bax ratio in response to hydrogen peroxide. To demonstrate this result an alternative way, RASM-control cells were pretreated with either the Jak2 inhibitor AG490 or its inactive analog, AG9, and then treated with hydrogen peroxide for the indicated times. Cellular lysates were again Western blotted with anti-bax antibody (Fig. 4B, top). The data demonstrate that AG490 attenuates hydrogen peroxide-mediated Bax induction in RASM-control cells when compared with the AG9-treated cells. The membrane was stripped and reprobed with anti-stat1 antibody to demonstrate equal loading among all samples (Fig. 4B, bottom). Collectively, the data in Fig. 4 demonstrate that Jak2 activation by hydrogen peroxide causes up-regulation of the proapoptotic Bax protein. During oxidative stress, the proapoptotic Bax protein is induced, localizes to the outer mitochondrial membrane, and increases mitochondrial membrane permeability (18 20). This is an essential step in the intrinsic apoptosis pathway, as it leads to activation of the downstream initiator caspase, caspase-9 (21). Because we demonstrated in Fig. 4 that Jak2 tyrosine kinase activity induces Bax expression, we hypothesized that activation of Jak2 by hydrogen peroxide may also contribute to mitochondrial dysfunction and subsequent caspase-9 cleavage. To examine mitochondrial membrane integrity, we used the MitoCapture reagent to stain live cells treated with 1 mm hydrogen peroxide for 0 or 2 h and visualized the cells using confocal microscopy (Fig. 5A). In healthy, nonapoptotic cells, the dye accumulates predominantly in the mitochondria, where it forms aggregates and fluoresces red. In contrast, in apoptotic cells, the dye is unable to enter the mitochondria because of altered mitochondrial transmembrane potential and therefore remains predominantly in the cytosol as a monomer and fluoresces green. The data demonstrate that in untreated RASM-control and RASM-DN cells the MitoCapture dye fluoresces predominantly red, indicating that these cells are healthy. Following hydrogen peroxide treatment, the RASM-control cells show a decrease in red staining and a dramatic increase in green staining, indicative of mitochondrial dysfunction in these cells. In contrast, the RASM-DN cells treated with hydrogen peroxide actually show increased red staining and only marginally increased green staining as compared with the RASM-control cells. To quantitate the results, the pixel intensity for both green and red staining of four photographs of each sample was determined. The ratio of green to red staining was calculated (Fig. 5B). The data show that the increase in green to red intensity ratio was greater in the RASM-control cells treated with hydrogen peroxide than in similarly treated RASM-DN cells. Collectively, these data indicate that hydrogen peroxide-induced Jak2 activation promotes mitochondrial dysfunction in vascular smooth muscle cells and that this result can be prevented by expression of a Jak2 dominant negative protein. Mitochondrial dysfunction is an essential step leading to cleavage of caspase-9. Caspase-9 is the primary initiator caspase of the intrinsic apoptosis pathway and is involved in hydrogen peroxide-induced apoptosis (22, 23). We therefore examined caspase-9 cleavage following hydrogen peroxide treatment in RASM-control and RASM-DN cells. For this purpose, the two cell types were treated with 1 mm hydrogen

Role of Jak2 in Oxidative Stress-induced Apoptosis 34551 FIG. 5. Jak2 is essential for hydrogen peroxide-induced mitochondrial membrane dysfunction and caspase-9 cleavage. A, RASM-control and RASM-DN cells were grown on microscope slides and treated with 1 mm hydrogen peroxide for 0 or 2 h. Cells were stained with the MitoCapture reagent and visualized using a confocal microscope. Predominant red staining is indicative of healthy cells, whereas predominant green staining is indicative of apoptotic cells. B, pixel intensity of each of four photographs from each condition shown in panel A was determined, and the intensity of green and red staining was determined for each. Data are presented as average ratio of green to red pixel intensity S.D. for each condition. *, p 0.01. Statistical analysis was performed using Student s t test. C, RASMcontrol and RASM-DN cells were treated with 1 mm hydrogen peroxide for 0, 0.5, 1, 2, or 3 h. Cellular lysates were Western blotted with anti-cleaved-caspase-9-pab to detect caspase-9 activation (top). The membrane was stripped and reprobed with anti-stat1-pab to demonstrate equal loading among all samples (bottom). Shown is one of three representative experiments for each. peroxide for the indicated times, and cellular lysates were Western blotted with anti-cleaved-caspase-9 antibody (Fig. 5C). The data demonstrate that in RASM-control cells hydrogen peroxide treatment caused the accumulation of cleaved caspase-9. In contrast, little caspase-9 cleavage occurred in RASM-DN cells. Furthermore, AG490 prevented cleavage of caspase-9 in RASM-control cells treated with hydrogen peroxide (data not shown). Jak2 tyrosine kinase activity is therefore required for cleavage and activation of caspase-9 in vascular smooth muscle cells during oxidative stress. Collectively, the data in Fig. 5 demonstrate that Jak2 activation by hydrogen peroxide is required for both mitochondrial membrane dysfunction and cleavage of the initiator caspase, caspase-9. Both of these events occur downstream of Bax induction in the intrinsic apoptosis pathway. DISCUSSION Although Jak2 is traditionally considered a mediator of cytokine signaling, other ligands and stimuli can activate this signaling pathway (3, 24). Oxidative stress is one such stimulus; Jak2 is potently activated by hydrogen peroxide in a number of cell types, yet no physiological end point has been attributed to hydrogen peroxide-induced Jak2 activity (3 5). Additionally, hydrogen peroxide induces apoptosis in vascular smooth muscle cells, yet few intracellular mediators of hydrogen peroxide-induced apoptosis have been identified (25, 26). Here, we report that Jak2 activation by oxidative stress in the form of hydrogen peroxide mediates apoptosis of vascular smooth muscle cells. We demonstrated laddering of genomic DNA, a characteristic of apoptosis, in control rat aortic smooth muscle cells treated with hydrogen peroxide (RASM-control), but we also showed that laddering was non-existent in the same cells expressing a Jak2 dominant negative protein (RASM-DN) or in RASM-control cells treated with the Jak2 inhibitor AG490. We observed a significant decrease in the percentage of cells undergoing apoptosis in response to hydrogen peroxide in the RASM-DN cells as compared with RASMcontrol cells. Moreover, pretreatment with AG490 reduced the percentage of cells undergoing hydrogen peroxide-induced apoptosis nearly to the level of untreated cells in both cell types, again suggesting a critical role for Jak2 in apoptosis. Finally, we provided evidence of the mechanism by which this occurs. In RASM-control cells, expression of the proapoptotic Bax protein was rapidly induced. There was no such Bax induction in the RASM-DN cells. Furthermore, in RASM-control cells, AG490 attenuated hydrogen peroxide-mediated Bax induction. This is the first evidence that Jak2 mediates Bax protein expression in response to hydrogen peroxide. Because Jak2 mediates changes in gene expression via activation of STAT proteins, we presume that Jak2 mediates Bax induction via activation of one or more STAT proteins. Moreover, we found that mitochon-

34552 Role of Jak2 in Oxidative Stress-induced Apoptosis drial membrane integrity was compromised and that caspase-9 was cleaved in RASM-control cells but not in RASM-DN cells, indicating an essential role for Jak2 in these events. This report has therefore identified apoptosis as a physiological end point of Jak2 activation by hydrogen peroxide. Moreover, this work demonstrates that Jak2 is a novel mediator of hydrogen peroxide-induced apoptosis in vascular smooth muscle cells. These results could have profound consequences for the treatment of a number of vascular diseases in which oxidative stress-mediated cell death plays a prominent role. As such, this work identifies Jak2 as a potential therapeutic target in vascular diseases associated with oxidative stress. Acknowledgment We thank Tim Vaught of the Optical Microscopy Suite at the University of Florida McKnight Brain Institute for training and assistance with confocal microscopy. REFERENCES 1. Neubauer, H., Cumano, A., Muller, M., Wu, H., Huffstadt, U., and Pfeffer, K. (1998) Cell 93, 397 409 2. Paragnas, E., Wang, D., Stravopodis, D., Topham, D. J., Marine, J.-C., Teglund, S., Vanin, E. F., Bodner, S., Colamonici, O. R., van Deursen, J. M., Grosveld, G., and Ihle, J. (1998) Cell 93, 385 395 3. Simon, A. R., Rai, U., Fanburg, B. L., and Cochran, B. H. (1998) Am. J. Physiol. 275, C1640 C1652 4. Frank, G. D., Mifune, M., Inagami, T., Ohba, M., Sasaki, T. Higashiyama, S., Dempsey, P. J., and Eguchi, S. (2003) Mol. Cell. Biol. 23, 1581 1589 5. Madamanchi, N. R., Li, S., Patterson, C., and Runge, M. S. (2001) Arterioscler. Thromb. Vasc. Biol. 21, 321 326 6. Rao, G. N., and Berk, B. C. (1992) Circ. Res. 70, 593 599 7. Sotnikova, R. (1998) Gen. Pharmacol. 31, 115 119 8. Li, P. F., Dietz, R., and von Harsdorf, R. (1997) Circulation 96, 3602 3609 9. Geng, Y.J., and Libby, P. (1995) Am. J. Pathol. 147, 251 266 10. Isner, J. M., Kearney, M., Bortman, S., and Passeri, J. (1995) Circulation 91, 2703 2711 11. Hattori, R., Maulik, N., Otani, H., Zhu, L., Cordis, G., Engleman, R. M., Siddiqui, M. A., and Das, D. K. (2001) J. Mol. Cell. Cardiol. 33, 1929 1936 12. El-Adawi, H., Deng, L., Tramontano, A., Smith, S., Mascareno, E., Ganguly, K., Castillo, R., and El-Sherif, N. (2003) Cardiovasc. Res. 57, 129 138 13. Mascareno, E., El-Shafei, M., Mualik, N., Sato, M., Guo, Y., Das, D. K., and Siddiqui, M. A. (2001) Circulation 104, 325 329 14. Sayeski, P. P., Ali, M. S., Safavi, A., Lyles, M., Kim, S. O., Frank, S. J., and Bernstein, K.E. (1999) J. Biol. Chem. 274, 33131 33142 15. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1997) Current Protocols in Molecular Biology, pp. 18.2.1 18.2.8, John Wiley & Sons, Inc., New York 16. Sayeski, P. P., Ali, M. S., Harp, J. B., Marrero, M. B., and Bernstein, K.E. (1998) Circ. Res. 82, 1279 1288 17. Sandberg, E. M., Ma, X., VonDerLinden, D., Godeny, M. D., and Sayeski, P.P. (2004) J. Biol. Chem. 279, 1956 1967 18. Naderi, J., Hung, M., and Pandey, S. (2003) Apoptosis 8, 91 100 19. Sarosi, L. A., Rieber, M. S., and Rieber, M. (2003) Biochem. Biophys. Res. Commun. 312, 355 359 20. Nechushtan, A., Smith, C. L., Hsu, Y. T., and Youle, R. J. (1999) EMBO J. 18, 2330 2341 21. Desagher, S., and Martinou, J.-C. (2000) Trends Cell Biol. 10, 369 377 22. Katoh, I., Tomimori, Y., Ikawa, Y., and Kurata, S. I. (2004) J. Biol. Chem. 279, 15515 15523 23. Yamakawa, H., Ito, Y., Naganawa, T., Banno, Y., Nakashima, S., Yoshimura, S., Sawada, M., Nishimura, Y., Nozawa, Y., and Sakai, N. (2000) Neurol. Res. 22, 556 562 24. Marrero, M. B., Scheiffer, B., Paxton, W. G., Heerdt, L., Berk, B. C., Delafontaine, P., and Bernstein, K. E. (1995) Nature 375, 247 250 25. Li, J., Li, W., Liu, W., Altura, B. T., and Altura, B. M. (2003) Brain Res. Bull. 62, 101 106 26. Li, P. F., Maasch, C., Haller, H., Dietz, R., and von Harsdorf, R. (1999) Circulation 100, 967 973