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JAK-STAT pathway in cardiac ischemic stress
Vascular Pharmacology 43 (2005) 353 – 356 www.elsevier.com/locate/vph
JAK-STAT pathway in cardiac ischemic stress Radha Ananthakrishnan, Kellie Hallam, Qing Li, Ravichandran Ramasamy * Division of Surgical Science, Department of Surgery, College of Physicians and Surgeons 17-401, Columbia University, 630 West 168th street, New York, NY 10032, United States Received 1 June 2005; accepted 1 August 2005
Abstract In our quest for comprehensive protection of ischemic myocardium, both basic and clinical studies have lead us to examine signal transduction pathways involved in ischemia – reperfusion injury for potential therapeutic targets. In this review, we have highlighted the importance of the JAKSTAT pathway in modulating ischemia – reperfusion injury. The mechanisms linking glucose metabolism, angiotensin II, with JAK-STAT pathway in ischemic injury are explored in this review. Clearly, the studies discussed in this review provide rationale for the design and synthesis of selective blockers of JAK-STAT pathway as potential therapeutic adjuncts in protecting ischemic myocardium. D 2005 Elsevier Inc. All rights reserved. Keywords: JAK-STAT pathway; Ischemic stress; Ischemia – reperfusion
1. Introduction The cellular and molecular events during ischemia limit the effectiveness of reperfusion therapy in patients with acute myocardial infarction. A comprehensive protection of the ischemic myocardium against tissue injury has been elusive so far. Basic and clinical investigators have continued in their quest for an adjunctive approach to protecting ischemic myocardium with limited success. These limitations arise from our incomplete understanding of the molecular events that modulate the severity of myocardial ischemia – reperfusion injury. However, recent studies have demonstrated that numerous complex signaling pathways are initiated by the ischemic myocardium and that these signaling can either mediate protective responses, or activate cell death events. In this review, our focus will be on one such signal transduction pathway — the JAK-STAT pathway. 2. JAK-STAT pathway The Janus kinases (JAKs) are a family of cytosolic tyrosine kinases that are associated with membrane receptors and play a
* Corresponding author. Fax: +1 212 305 5337. E-mail address: [email protected] (R. Ramasamy). 1537-1891/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.vph.2005.08.020
vital role in transduction of signal from the cell surface to the nucleus (Imada and Leonard, 2000). JAKs consist of the JH1 domain (the kinase domain) and the JH2 domain (the pseudokinase domain). The JH1 domain is responsible for the catalytic activity of JAKs, while the JH2 domain has been postulated to be a docking site for signal transducers and activators of transcription (STATs) (Imada and Leonard, 2000). Four JAKs (JAK1, JAK2, JAK3, and TYK2) have been identified in mammals, and their roles in signal transduction have been studied in various cell types. The JAK-STAT signaling pathway is initiated upon binding of interferon or cytokines to a transmembrane receptor. Since these receptors do not possess catalytic activity, they associate constitutively with tyrosine kinases of JAKs forming a receptor –kinase complex. Ligand (cytokine/interferon) binding induces dimerization of the receptor– kinase complex leading to activation of kinases to phosphorylate each other and the receptor (Imada and Leonard, 2000; Schindler and Darnell, 1995; Horvath, 2000). This latter phosphorylation provides a pair of docking sites for the SH2 domain of STATs. STATs are a unique family of transcription factors with seven identified members—STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6. Upon docking in the receptor –kinase complex, the STATs undergo phosphorylation by JAKs, dimerize, and translocate to the nucleus (Imada and Leonard, 2000; Schindler and Darnell, 1995; Horvath, 2000). In the
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nucleus the STATs bind to the g-IFN activation site (GAS), resulting in transcription of downstream target genes (Horvath, 2000; Igarshi et al., 1994; Darnell, 1997). Extensive research has demonstrated that the JAK-STAT pathway plays a critical role in regulating the expression of stress responsive genes in various cells and organs (Imada and Leonard, 2000; Schindler and Darnell, 1995; Horvath, 2000; Igarshi et al., 1994; Darnell, 1997). In the heart, studies have demonstrated the critical role played by the JAK-STAT pathway in ischemia – reperfusion injury, cardiac hypertrophy, apoptosis, and heart transplant. 3. Renin –angiotensin system (RAS) and JAK-STAT signaling in ischemic hearts In isolated perfused hearts it was demonstrated that ischemia – reperfusion selectively activates STAT5A and STAT6 and that these changes were accompanied by increases in the mRNA level of angiotensinogen (ANG) (Mascareno et al., 2001). These changes in ANG mRNA expression were shown to be mediated by the enhanced binding of STATs to ANG promoter (Mascareno et al., 2001). Blockade of angiotensin receptor-1 (AT1) by losartan or JAK2 by AG490 effectively inhibited functional activation of STATs and attenuated changes in ANG mRNA expression (Mascareno et al., 2001). It was shown that inhibition of the AT1 receptor by losartan resulted in loss of STAT/St-domain complex formation and reduction in ANG mRNA level (Mascareno et al., 2001). JAK2 inhibitor AG490 perfusion resulted in similar changes (i.e. loss of STAT/St-domain complex formation and reduction in ANG mRNA level) (Mascareno et al., 2001). Furthermore, AG490 inhibition of JAK2 not only blocked STAT signaling, but also resulted to improved cardiac function in hearts after ischemia –reperfusion. In a rat model of LAD ligation, phosphorylation of JAK2, STAT1, STAT3, STAT5a, and STAT6 was observed as early as 5 min post myocardial infarction (MI) (El-Adawi et al., 2003). STATs 1, 3, and 5a remained activated for 7 days post MI (ElAdawi et al., 2003). In this LAD occlusion model, increased binding of STATs to the St-domain of the ANG gene promoter was observed (El-Adawi et al., 2003). Furthermore, AT1 receptor and JAK2 blockade decreased protein phosphatase activity and normalized expression of p16-phospholamban post MI (El-Adawi et al., 2003). Thus, these studies support the role for angiotensin II autocrine loop in JAK-STAT signaling and ischemia –reperfusion injury.
enzymes aldose reductase or sorbitol dehydrogenase {FASEB paper}. Furthermore, it was shown that lowering of cytosolic NADH/NAD+ is an important mechanism by which inhibition of aldose reductase or sorbitol dehydrogenase blocks JAK2, STAT5 phosphorylation. Niacin, known to lower cytosolic NADH/NAD+ independent of aldose reductase pathway, was also shown to block JAK-STAT activation (Hwang et al., 2005). These data clearly indicated that changes in cytosolic NADH/NAD+ are an important event by which ischemia influences JAK-STAT signaling. Our study further delineated the sequence of events by which glucose flux via polyol pathway influences JAK-STAT signaling. Since earlier studies had shown that ischemia increases membrane protein kinase C activity and expression (Strasser et al., 1999; Inagaki et al., 2003; Hahn et al., 2002), and that inhibition of certain PKC isoform reduces myocardial injury due to ischemia –reperfusion (Strasser et al., 1999; Inagaki et al., 2003; Hahn et al., 2002), we probed this axis as a key event in JAK-STAT signaling in ischemic hearts. We demonstrated that inhibitors of aldose reductase or sorbitol dehydrogenase reduced membrane bound protein kinase C activity during ischemia and were associated with changes in diacylglycerol and cytosolic NADH/NAD+ (Hwang et al., 2005). Furthermore, it was also demonstrated that inhibitors of protein kinase C (a/h) block JAK-STAT activation in ischemia hearts (Hwang et al., 2005). Consistent with our observations, an earlier study demonstrated involvement of aldose reductase and PKC axis in activating JAK-STAT signaling in vascular smooth muscle cell culture (Shaw et al., 2003). These results indicate that PKC-(a/h) plays a critical role by which flux via aldose reductase pathway mediates JAK-STAT signaling. The beneficial effects of blocking JAK2 or PKC-(a/h) or aldose reductase or sorbitol dehydrogenase clearly demonstrated the signaling events that underlie ischemic injury (Fig. 1). Since diabetic patients have higher flux via polyol pathway, the above findings have greater impact in our understanding of diabetic cardiovascular complications. Increased Aldose reductase Pathway Activity
Increased cytosolic NADH/NAD+
α /β) activity Increased Protein kinase C-(α
4. Glucose and JAK-STAT signaling in ischemic hearts Glucose flux via the polyol pathway has been demonstrated to play a central role in modulating myocardial ischemia – reperfusion injury (Hwang et al., 2002, 2003, 2004). In our recent study we demonstrated the impact of glucose flux via polyol pathway in JAK-STAT signaling in ischemic hearts (Hwang et al., 2005). We showed that ischemia in rats and mice hearts induces JAK-2 and STAT-5 phosphorylation and that this activation can be blocked by inhibiting the polyol pathway
JAK2-STAT5 activation
Increased Ischemic Injury
Fig. 1. Pathway describing the impact of glucose flux via aldose reductase pathway on JAK-STAT activation and ischemic injury. (Reprinted from Hwang et al., 2005 with permission from FASEB Journal).
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5. JAK-STAT signaling in ischemic preconditioning
6. Allograft rejection and JAK3
Using a mouse in-vivo model of late ischemic preconditioning (PC), it was demonstrated that exposure of the heart to brief episodes of ischemia followed by reperfusion induces phosphorylation of JAK1 and JAK2 (Xuan et al., 2001). This was accompanied by rapid translocation of STAT1 and STAT3 from the cytosol to the nucleus concomitant with increased phosphorylation and DNA-binding activity of these two STATs (Xuan et al., 2001). The other STATs (STAT2, STAT4, STAT5A, STAT5B, and STAT6) were unaffected by late PC. Treatment of the mice with JAK inhibitor AG490 prior to the PC ischemia blocked the activation of JAK1, JAK2, STAT1, and STAT3. Furthermore, it was shown that JAK-STAT activation was essential for recruitment of iNOS, a key feature in late PC (Xuan et al., 2001). In early PC, Hattori et al have shown activation of JAK2 and STAT3 as well as recruitment of iNOS and reduction in number of apoptotic cardiomyocytes (Hattori et al., 2001). The authors also showed that PC induced upregulation of antiapoptotic gene bcl-2 and downregulation of proapoptotic gene bax (Hattori et al., 2001) and that JAK2 inhibition with AG490 ablated the protection against infarction and apoptosis. In mice, protective effects of PC were not observed in STAT3 deficient mice (Smith et al., 2004). In contrast to the wild type mice, STAT3 mice exhibited increased infarct size, apoptosis, and vulnerability to developing heart failure after ischemia –reperfusion (Liao et al., 2002; Stephanou et al., 1998; Hilfiker-Kleiner et al., 2004; Jacoby et al., 2003). Activation of STAT3 by cardiotropin-1 reduced apoptotic cell death and enhanced cell survival (Liao et al., 2002; Stephanou et al., 1998; Hilfiker-Kleiner et al., 2004). STAT1 activation, unlike STA3, has been shown to be detrimental for cell survival. STAT1 activation after ischemia – reperfusion has been shown to induce expression of proapoptotic caspase-1, Fas and FasL genes leading to cardiac cell death (Jacoby et al., 2003; Stephanou et al., 2001; Stephanou et al., 2000b) as well as inhibiting promoters of genes encoding antiapoptotic Bcl2 and Bcl-x (Stephanou et al., 2000a). Together, these studies demonstrate that activation of JAK2 and STAT3 protect the myocardium from ischemic injury.
Cytokine receptors require the cytoplasmic JAK3 for signal transduction. Clinical studies have shown that patients lacking JAK3 expression exhibit severe combined immunodeficiency phenotype (Macchi et al., 1995; Russell et al., 1995). Changelian et al. (2003) developed inhibitors for JAK3 as a potential therapy for immunosuppression. In their study it was demonstrated that JAK3 inhibition prolonged survival in a mouse model of heart transplant and in a monkey model of kidney transplant (Changelian et al., 2003). The protection afforded by JAK3 inhibition has immense potential for human organ transplantation.
A
Phosphorylation of STATs 1, 3, 5, and 6 were observed in human hearts with dilated cardiomyopathy (DCM) (Ng et al., 2003), while increased STAT1 and 5 phosphorylation were observed in ischemic heart disease (IHD) (Ng et al., 2003). The study by Ng et al. (2003) compared the general phosphorylation status between control and failing (DCM, IHD) hearts and observed marked phosphorylation of high molecular weight proteins corresponding to the size of cytokine receptors in failing hearts (Ng et al., 2003). Interestingly, the study identified hyperphosphorylation of the STAT3h isoform, and not of the STAT3a, in failing hearts. Earlier non-cardiac studies have shown that STAT3h binding to STAT binding consensus sites in regulatory DNA of STAT responsive genes failed to activate transcription (Caldenhoven et al., 1996). These observations suggest that, perhaps, STAT3h is functioning in the dominant negative manner towards others STATs. Nevertheless, emergence of these data provides a compelling case for investigation of JAK-STAT axis in heart failure. 8. Conclusions Understanding of cellular and molecular signaling events underlying ischemia – reperfusion injury is critical to developing new therapeutic approaches to protect patients with myocardial infarction. It is clear that JAK-STAT pathway is a critical player in modulating ischemic injury. Activation of
B PC Followed by Ischemia-Reperfusion
Activators
7. STATs in human heart failure
Ischemia-Reperfusion
STAT1, STAT5 Activation
JAK2-STAT3 Activation
Protection of Ischemic Myocardium
Apoptosis
Ischemic Injury
Fig. 2. Illustration summarizing the impact of STAT activation on cardiac ischemic injury after myocardial infarction. Activation of STAT3 by preconditioning (PC) (A) protects ischemic myocardium, while activation of STATs 1 and 5 facilitate apoptosis and injury to the myocardium after ischemia – reperfusion (B).
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STAT 3 has been shown to protect ischemic myocardium, while activation of STATs 1 and 5 increases injury to the myocardium (Fig. 2). The studies discussed in this review provide rationale for the design and synthesis of selective blocker of STATs 1 and 5 as potential therapeutic adjuncts in protecting ischemic myocardium. Acknowledgments This work was supported by grants from the National Institutes of Health (HL61783, HL68954, HL60901), the Juvenile Diabetes Research Foundation, and the Surgical Research Fund. RR is an Established Investigator of the American Heart Association. References Caldenhoven, E., van Dijk, T.B., Solari, R., Armstrong, J., Raaijmakers, J.A., Lammers, J.W., Koenderman, L., de Groot, R.P., 1996. STAT3beta, a splice variant of transcription factor STAT3, is a dominant negative regulator of transcription. J. Biol. Chem. 271 (22), 13221 – 13227 (May. 31). Changelian, P.S., Flanagan, M.E., Ball, D.J., Kent, C.R., Magnuson, K.S., Martin, W.H., Rizzuti, B.J., Sawyer, P.S., Perry, B.D., Brissette, W.H., McCurdy, S.P., Kudlacz, E.M., Conklyn, M.J., Elliott, E.A., Koslov, E.R., Fisher, M.B., Strelevitz, T.J., Yoon, K., Whipple, D.A., Sun, J., Munchhof, M.J., Doty, J.L., Casavant, J.M., Blumenkopf, T.A., Hines, M., Brown, M.F., Lillie, B.M., Subramanyam, C., Shang-Poa, C., Milici, A.J., Beckius, G.E., Moyer, J.D., Su, C., Woodworth, T.G., Gaweco, A.S., Beals, C.R., Littman, B.H., Fisher, D.A., Smith, J.F., Zagouras, P., Magna, H.A., Saltarelli, M.J., Johnson, K.S., Nelms, L.F., Des Etages, S.G., Hayes, L.S., Kawabata, T.T., Finco-Kent, D., Baker, D.L., Larson, M., Si, M.S., Paniagua, R., Higgins, J., Holm, B., Reitz, B., Zhou, Y.J., Morris, R.E., O’Shea, J.J., Borie, D.C., 2003. Prevention of organ allograft rejection by a specific Janus kinase 3 inhibitor. Science 302 (5646), 875 – 878. Darnell Jr., J.E., 1997. STATs and gene regulation. Science 277, 1630 – 1635. El-Adawi, H., Deng, L., Tramontano, A., Smith, S., Mascareno, E., Ganguly, K., Castillo, R., El-Sherif, N., 2003. The functional role of the JAK-STAT pathway in post-infarction remodeling. Cardiovasc. Res. 57, 129 – 138. Hahn, H.S., Yussman, M.G., Toyokawa, T., Marreez, Y., Barrett, T.J., Hilty, K.C., Osinska, H., Robbins, J., Dorn II, G.W., 2002. Ischemic protection and myofibrillar cardiomyopathy: dose-dependent effects of in vivo deltaPKC inhibition. Circ. Res. 91 (8), 741 – 748. Hattori, R., Maulik, N., Otani, H., Zhu, L., Cordis, G.R., Engelman, M.M., Siddiqui, A.Q., Das, D.K., 2001. Role of STAT3 in ischemic preconditioning. J. Mol. Cell. Cardiol. 33, 1929 – 1936. Hilfiker-Kleiner, D., Hilfiker, A., Fuchs, M., Kaminski, K., Schaefer, A., Schieffer, B., Hillmer, A., Schmiedl, A., Ding, Z., Podewski, E., Podewski, E., Poli, V., Schneider, M.D., Schulz, R., Park, J.K., Wollert, K.C., Drexler, H., 2004. Signal transducer and activator of transcription 3 is required for myocardial capillary growth, control of interstitial matrix deposition, and heart protection from ischemic injury. Circ. Res. 95 (2), 187 – 195. Horvath, C.M., 2000. STAT proteins and transcriptional responses to extracellular signals. Trends Biochem. Sci. 25, 496 – 502. Hwang, Y.C., Sato, S., Tsai, J.Y., Bakr, S., Yan, S.D., Oates, P.J., Ramasamy, R., 2002. Aldose reductase activation is a key component of myocardial response to ischemia. FASEB J. 16, 243 – 245. Hwang, Y.C., Bakr, S., Ellery, C.R., Oates, P.J., Ramasamy, R., 2003. Sorbitol dehydrogenase: a novel target for adjunctive protection of ischemic myocardium. FASEB J. 17, 2331 – 2333. Hwang, Y.C., Kaneko, M., Bakr, S., Liao, H., Lu, Y., Lewis, E.R., Yan, S.D., Ii, S., Itakura, M., Rui, L., Skopicki, H., Homma, S., Schmidt, A.M., Oates, P.J., Szabolcs, M., Ramasamy, R., 2004. Central role for aldose reductase
pathway in myocardial ischemic injury. Central role for aldose reductase in myocardial ischemic injury. FASEB J. 18, 1192 – 1199. Hwang, Y.C., Shaw, S., Kaneko, M., Redd, H., Marerro, M., Ramasamy, R., 2005. Aldose reductase pathway mediates JAK-STAT signaling: a novel axis in myocardial ischemic injury. FASEB J. 19, 795 – 797. Igarshi, K., Garotta, G., Ozmen, L., Zeimecki, A., Wilks, A.F., Harpur, A.G., Lerner, A.C., Finbloom, D.S., 1994. J. Biol. Chem. 269, 14333 – 14336. Imada, K., Leonard, W.J., 2000. The JAK-STAT pathway. Mol. Immunol. 37, 1 – 11. Inagaki, K., Hahn, H.S., Dorn II, G.W., Mochly-Rosen, D., 2003. Additive protection of the ischemic heart ex vivo by combined treatment with deltaprotein kinase C inhibitor and epsilon-protein kinase C activator. Circulation 108, 869 – 875. Jacoby, J.J., Kalinowski, A., Liu, M.G., Zhang, S.S., Gao, Q., Chai, G.X., Ji, L., Iwamoto, Y., Li, E., Schneider, M., Russell, K.S., Fu, X.Y., 2003. Cardiomyocyte-restricted knockout of STAT3 results in higher sensitivity to inflammation, cardiac fibrosis, and heart failure with advanced age. Proc. Natl. Acad. Sci. U. S. A. 100 (22), 12929 – 12934. Liao, Z., Brar, B.K., Cai, Q., Stephanou, A., O’Leary, R.M., Pennica, D., Yellon, D.M., Latchman, D.S., 2002. Cardiotrophin-1 (CT-1) can protect the adult heart from injury when added both prior to ischemia and at reperfusion. Cardiovasc. Res. 53 (4), 902 – 910. Macchi, P., Villa, A., Giliani, S., Sacco, M.G., Frattini, A., Porta, F., Ugazio, A.G., Johnston, J.A., Candotti, F., O’Shea, J.J., et al., 1995. Mutations of Jak-3 gene in patients with autosomal severe combined immune deficiency (SCID). Nature 377 (6544), 65 – 68. Mascareno, E., El-Shafei, M., Maulik, N., Sato, M., Guo, Y., Das, D.K., Siddiqui, M.A., 2001. JAK/STAT signaling is associated with cardiac dysfunction during ischemia and reperfusion. Circulation 104, 325 – 329. Ng, D.C., Court, N.W., dos Remedios, C.G., Bogoyevitch, M.A., 2003. Activation of signal transducer and activator of transcription (STAT) pathways in failing human hearts. Cardiovasc. Res. 57 (2), 333 – 346. Russell, S.M., Tayebi, N., Nakajima, H., Riedy, M.C., Roberts, J.L., Aman, M.J., Migone, T.S., Noguchi, M., Markert, M.L., Buckley, R.H., O’Shea, J.J., Leonard, W.J., 1995. Mutation of Jak3 in a patient with SCID: essential role of Jak3 in lymphoid development. Science 270 (5237), 797 – 800. Schindler, C., Darnell, J., 1995. Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. Annu. Rev. Biochem. 64, 621 – 651. Shaw, S., Wang, X., Redd, H., Alexander, G.D., Isales, C.M., Marrero, M.B., 2003. High glucose augments the angiotensin II-induced activation of JAK2 in vascular smooth muscle cells via the polyol pathway. J. Biol. Chem. 278, 30634 – 30641. Smith, R.M., Suleman, N., Lacerda, L., Opie, L.H., Akira, S., Chien, K.R., Sack, M.N., 2004. Genetic depletion of cardiac myocyte STAT-3 abolishes classical preconditioning. Cardiovasc. Res. 63 (4), 611 – 616. Stephanou, A., Brar, B., Heads, R., Knight, R.D., Marber, M.S., Pennica, D., Latchman, D.S., 1998. Cardiotrophin-1 induces heat shock protein accumulation in cultured cardiac cells and protects them from stressful stimuli. J. Mol. Cell. Cardiol. 30 (4), 849 – 855. Stephanou, A., Brar, B.K., Knight, R.A., Latchman, D.S., 2000a. Opposing actions of STAT-1 and STAT-3 on the Bcl-2 and Bcl-x promoters. Cell Death Differ. 7 (3), 329 – 330. Stephanou, A., Brar, B.K., Scarabelli, T.M., Jonassen, A.K., Yellon, D.M., Marber, M.S., Knight, R.A., Latchman, D.S., 2000b. Ischemia-induced STAT-1 expression and activation play a critical role in cardiomyocyte apoptosis. J. Biol. Chem. 275 (14), 10002 – 10008. Strasser, R.H., Simonis, G., Schlon, S.P., Braun, M.U., Ihl-Vahl, R., Weinbrenner, C., Marquetant, R., Kubler, W., 1999. Two distinct mechanisms mediate a differential regulation of protein kinase C isozymes in acute and prolonged myocardial ischemia. Circ. Res. 85, 77 – 87. Xuan, Y.-T., Guo, Y., Han, H., Zhu, Y., Bolli, R., 2001. An essential role of the JAK-STAT pathway in ischemic preconditioning. Proc. Natl. Acad. Sci. U. S. A. 98, 9050 – 9055.
JAK-STAT pathway in cardiac ischemic stress Radha Ananthakrishnan, Kellie Hallam, Qing Li, Ravichandran Ramasamy * Division of Surgical Science, Department of Surgery, College of Physicians and Surgeons 17-401, Columbia University, 630 West 168th street, New York, NY 10032, United States Received 1 June 2005; accepted 1 August 2005
Abstract In our quest for comprehensive protection of ischemic myocardium, both basic and clinical studies have lead us to examine signal transduction pathways involved in ischemia – reperfusion injury for potential therapeutic targets. In this review, we have highlighted the importance of the JAKSTAT pathway in modulating ischemia – reperfusion injury. The mechanisms linking glucose metabolism, angiotensin II, with JAK-STAT pathway in ischemic injury are explored in this review. Clearly, the studies discussed in this review provide rationale for the design and synthesis of selective blockers of JAK-STAT pathway as potential therapeutic adjuncts in protecting ischemic myocardium. D 2005 Elsevier Inc. All rights reserved. Keywords: JAK-STAT pathway; Ischemic stress; Ischemia – reperfusion
1. Introduction The cellular and molecular events during ischemia limit the effectiveness of reperfusion therapy in patients with acute myocardial infarction. A comprehensive protection of the ischemic myocardium against tissue injury has been elusive so far. Basic and clinical investigators have continued in their quest for an adjunctive approach to protecting ischemic myocardium with limited success. These limitations arise from our incomplete understanding of the molecular events that modulate the severity of myocardial ischemia – reperfusion injury. However, recent studies have demonstrated that numerous complex signaling pathways are initiated by the ischemic myocardium and that these signaling can either mediate protective responses, or activate cell death events. In this review, our focus will be on one such signal transduction pathway — the JAK-STAT pathway. 2. JAK-STAT pathway The Janus kinases (JAKs) are a family of cytosolic tyrosine kinases that are associated with membrane receptors and play a
* Corresponding author. Fax: +1 212 305 5337. E-mail address: [email protected] (R. Ramasamy). 1537-1891/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.vph.2005.08.020
vital role in transduction of signal from the cell surface to the nucleus (Imada and Leonard, 2000). JAKs consist of the JH1 domain (the kinase domain) and the JH2 domain (the pseudokinase domain). The JH1 domain is responsible for the catalytic activity of JAKs, while the JH2 domain has been postulated to be a docking site for signal transducers and activators of transcription (STATs) (Imada and Leonard, 2000). Four JAKs (JAK1, JAK2, JAK3, and TYK2) have been identified in mammals, and their roles in signal transduction have been studied in various cell types. The JAK-STAT signaling pathway is initiated upon binding of interferon or cytokines to a transmembrane receptor. Since these receptors do not possess catalytic activity, they associate constitutively with tyrosine kinases of JAKs forming a receptor –kinase complex. Ligand (cytokine/interferon) binding induces dimerization of the receptor– kinase complex leading to activation of kinases to phosphorylate each other and the receptor (Imada and Leonard, 2000; Schindler and Darnell, 1995; Horvath, 2000). This latter phosphorylation provides a pair of docking sites for the SH2 domain of STATs. STATs are a unique family of transcription factors with seven identified members—STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6. Upon docking in the receptor –kinase complex, the STATs undergo phosphorylation by JAKs, dimerize, and translocate to the nucleus (Imada and Leonard, 2000; Schindler and Darnell, 1995; Horvath, 2000). In the
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nucleus the STATs bind to the g-IFN activation site (GAS), resulting in transcription of downstream target genes (Horvath, 2000; Igarshi et al., 1994; Darnell, 1997). Extensive research has demonstrated that the JAK-STAT pathway plays a critical role in regulating the expression of stress responsive genes in various cells and organs (Imada and Leonard, 2000; Schindler and Darnell, 1995; Horvath, 2000; Igarshi et al., 1994; Darnell, 1997). In the heart, studies have demonstrated the critical role played by the JAK-STAT pathway in ischemia – reperfusion injury, cardiac hypertrophy, apoptosis, and heart transplant. 3. Renin –angiotensin system (RAS) and JAK-STAT signaling in ischemic hearts In isolated perfused hearts it was demonstrated that ischemia – reperfusion selectively activates STAT5A and STAT6 and that these changes were accompanied by increases in the mRNA level of angiotensinogen (ANG) (Mascareno et al., 2001). These changes in ANG mRNA expression were shown to be mediated by the enhanced binding of STATs to ANG promoter (Mascareno et al., 2001). Blockade of angiotensin receptor-1 (AT1) by losartan or JAK2 by AG490 effectively inhibited functional activation of STATs and attenuated changes in ANG mRNA expression (Mascareno et al., 2001). It was shown that inhibition of the AT1 receptor by losartan resulted in loss of STAT/St-domain complex formation and reduction in ANG mRNA level (Mascareno et al., 2001). JAK2 inhibitor AG490 perfusion resulted in similar changes (i.e. loss of STAT/St-domain complex formation and reduction in ANG mRNA level) (Mascareno et al., 2001). Furthermore, AG490 inhibition of JAK2 not only blocked STAT signaling, but also resulted to improved cardiac function in hearts after ischemia –reperfusion. In a rat model of LAD ligation, phosphorylation of JAK2, STAT1, STAT3, STAT5a, and STAT6 was observed as early as 5 min post myocardial infarction (MI) (El-Adawi et al., 2003). STATs 1, 3, and 5a remained activated for 7 days post MI (ElAdawi et al., 2003). In this LAD occlusion model, increased binding of STATs to the St-domain of the ANG gene promoter was observed (El-Adawi et al., 2003). Furthermore, AT1 receptor and JAK2 blockade decreased protein phosphatase activity and normalized expression of p16-phospholamban post MI (El-Adawi et al., 2003). Thus, these studies support the role for angiotensin II autocrine loop in JAK-STAT signaling and ischemia –reperfusion injury.
enzymes aldose reductase or sorbitol dehydrogenase {FASEB paper}. Furthermore, it was shown that lowering of cytosolic NADH/NAD+ is an important mechanism by which inhibition of aldose reductase or sorbitol dehydrogenase blocks JAK2, STAT5 phosphorylation. Niacin, known to lower cytosolic NADH/NAD+ independent of aldose reductase pathway, was also shown to block JAK-STAT activation (Hwang et al., 2005). These data clearly indicated that changes in cytosolic NADH/NAD+ are an important event by which ischemia influences JAK-STAT signaling. Our study further delineated the sequence of events by which glucose flux via polyol pathway influences JAK-STAT signaling. Since earlier studies had shown that ischemia increases membrane protein kinase C activity and expression (Strasser et al., 1999; Inagaki et al., 2003; Hahn et al., 2002), and that inhibition of certain PKC isoform reduces myocardial injury due to ischemia –reperfusion (Strasser et al., 1999; Inagaki et al., 2003; Hahn et al., 2002), we probed this axis as a key event in JAK-STAT signaling in ischemic hearts. We demonstrated that inhibitors of aldose reductase or sorbitol dehydrogenase reduced membrane bound protein kinase C activity during ischemia and were associated with changes in diacylglycerol and cytosolic NADH/NAD+ (Hwang et al., 2005). Furthermore, it was also demonstrated that inhibitors of protein kinase C (a/h) block JAK-STAT activation in ischemia hearts (Hwang et al., 2005). Consistent with our observations, an earlier study demonstrated involvement of aldose reductase and PKC axis in activating JAK-STAT signaling in vascular smooth muscle cell culture (Shaw et al., 2003). These results indicate that PKC-(a/h) plays a critical role by which flux via aldose reductase pathway mediates JAK-STAT signaling. The beneficial effects of blocking JAK2 or PKC-(a/h) or aldose reductase or sorbitol dehydrogenase clearly demonstrated the signaling events that underlie ischemic injury (Fig. 1). Since diabetic patients have higher flux via polyol pathway, the above findings have greater impact in our understanding of diabetic cardiovascular complications. Increased Aldose reductase Pathway Activity
Increased cytosolic NADH/NAD+
α /β) activity Increased Protein kinase C-(α
4. Glucose and JAK-STAT signaling in ischemic hearts Glucose flux via the polyol pathway has been demonstrated to play a central role in modulating myocardial ischemia – reperfusion injury (Hwang et al., 2002, 2003, 2004). In our recent study we demonstrated the impact of glucose flux via polyol pathway in JAK-STAT signaling in ischemic hearts (Hwang et al., 2005). We showed that ischemia in rats and mice hearts induces JAK-2 and STAT-5 phosphorylation and that this activation can be blocked by inhibiting the polyol pathway
JAK2-STAT5 activation
Increased Ischemic Injury
Fig. 1. Pathway describing the impact of glucose flux via aldose reductase pathway on JAK-STAT activation and ischemic injury. (Reprinted from Hwang et al., 2005 with permission from FASEB Journal).
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5. JAK-STAT signaling in ischemic preconditioning
6. Allograft rejection and JAK3
Using a mouse in-vivo model of late ischemic preconditioning (PC), it was demonstrated that exposure of the heart to brief episodes of ischemia followed by reperfusion induces phosphorylation of JAK1 and JAK2 (Xuan et al., 2001). This was accompanied by rapid translocation of STAT1 and STAT3 from the cytosol to the nucleus concomitant with increased phosphorylation and DNA-binding activity of these two STATs (Xuan et al., 2001). The other STATs (STAT2, STAT4, STAT5A, STAT5B, and STAT6) were unaffected by late PC. Treatment of the mice with JAK inhibitor AG490 prior to the PC ischemia blocked the activation of JAK1, JAK2, STAT1, and STAT3. Furthermore, it was shown that JAK-STAT activation was essential for recruitment of iNOS, a key feature in late PC (Xuan et al., 2001). In early PC, Hattori et al have shown activation of JAK2 and STAT3 as well as recruitment of iNOS and reduction in number of apoptotic cardiomyocytes (Hattori et al., 2001). The authors also showed that PC induced upregulation of antiapoptotic gene bcl-2 and downregulation of proapoptotic gene bax (Hattori et al., 2001) and that JAK2 inhibition with AG490 ablated the protection against infarction and apoptosis. In mice, protective effects of PC were not observed in STAT3 deficient mice (Smith et al., 2004). In contrast to the wild type mice, STAT3 mice exhibited increased infarct size, apoptosis, and vulnerability to developing heart failure after ischemia –reperfusion (Liao et al., 2002; Stephanou et al., 1998; Hilfiker-Kleiner et al., 2004; Jacoby et al., 2003). Activation of STAT3 by cardiotropin-1 reduced apoptotic cell death and enhanced cell survival (Liao et al., 2002; Stephanou et al., 1998; Hilfiker-Kleiner et al., 2004). STAT1 activation, unlike STA3, has been shown to be detrimental for cell survival. STAT1 activation after ischemia – reperfusion has been shown to induce expression of proapoptotic caspase-1, Fas and FasL genes leading to cardiac cell death (Jacoby et al., 2003; Stephanou et al., 2001; Stephanou et al., 2000b) as well as inhibiting promoters of genes encoding antiapoptotic Bcl2 and Bcl-x (Stephanou et al., 2000a). Together, these studies demonstrate that activation of JAK2 and STAT3 protect the myocardium from ischemic injury.
Cytokine receptors require the cytoplasmic JAK3 for signal transduction. Clinical studies have shown that patients lacking JAK3 expression exhibit severe combined immunodeficiency phenotype (Macchi et al., 1995; Russell et al., 1995). Changelian et al. (2003) developed inhibitors for JAK3 as a potential therapy for immunosuppression. In their study it was demonstrated that JAK3 inhibition prolonged survival in a mouse model of heart transplant and in a monkey model of kidney transplant (Changelian et al., 2003). The protection afforded by JAK3 inhibition has immense potential for human organ transplantation.
A
Phosphorylation of STATs 1, 3, 5, and 6 were observed in human hearts with dilated cardiomyopathy (DCM) (Ng et al., 2003), while increased STAT1 and 5 phosphorylation were observed in ischemic heart disease (IHD) (Ng et al., 2003). The study by Ng et al. (2003) compared the general phosphorylation status between control and failing (DCM, IHD) hearts and observed marked phosphorylation of high molecular weight proteins corresponding to the size of cytokine receptors in failing hearts (Ng et al., 2003). Interestingly, the study identified hyperphosphorylation of the STAT3h isoform, and not of the STAT3a, in failing hearts. Earlier non-cardiac studies have shown that STAT3h binding to STAT binding consensus sites in regulatory DNA of STAT responsive genes failed to activate transcription (Caldenhoven et al., 1996). These observations suggest that, perhaps, STAT3h is functioning in the dominant negative manner towards others STATs. Nevertheless, emergence of these data provides a compelling case for investigation of JAK-STAT axis in heart failure. 8. Conclusions Understanding of cellular and molecular signaling events underlying ischemia – reperfusion injury is critical to developing new therapeutic approaches to protect patients with myocardial infarction. It is clear that JAK-STAT pathway is a critical player in modulating ischemic injury. Activation of
B PC Followed by Ischemia-Reperfusion
Activators
7. STATs in human heart failure
Ischemia-Reperfusion
STAT1, STAT5 Activation
JAK2-STAT3 Activation
Protection of Ischemic Myocardium
Apoptosis
Ischemic Injury
Fig. 2. Illustration summarizing the impact of STAT activation on cardiac ischemic injury after myocardial infarction. Activation of STAT3 by preconditioning (PC) (A) protects ischemic myocardium, while activation of STATs 1 and 5 facilitate apoptosis and injury to the myocardium after ischemia – reperfusion (B).
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