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An essential role of the JAK-STAT pathway in ischemic preconditioning.

ABSTRACT: The goal of this study was to determine the role of the Janus tyrosine kinase (JAK)-signal transducers and activators of transcription (STAT) pathway in the late phase of ischemic preconditioning (PC). A total of 230 mice were used. At 5 min after ischemic PC (induced with six cycles of 4-min coronary occlusion/4-min reperfusion), immunoprecipitation with anti-phosphotyrosine (anti-pTyr) antibodies followed by immunoblotting with anti-JAK antibodies revealed increased tyrosine phosphorylation of JAK1 (+257 +/- 53%) and JAK2 (+238 +/- 35%), indicating rapid activation of these two kinases. Similar results were obtained by immunoblotting with anti-pTyr-JAK1 and anti-pTyr-JAK2 antibodies. Western analysis with anti-pTyr-STAT antibodies demonstrated a marked increase in nuclear pTyr-STAT1 (+301 +/- 61%) and pTyr-STAT3 (+253 +/- 60%) 30 min after ischemic PC, which was associated with redistribution of STAT1 and STAT3 from the cytosolic to the nuclear fraction and with an increase in STAT1 and STAT3 gamma-IFN activation site DNA-binding activity (+606 +/- 64%), indicating activation of STAT1 and STAT3. No nuclear translocation or tyrosine phosphorylation of STAT2, STAT4, STAT5A, STAT5B, or STAT6 was observed. Pretreatment with the JAK inhibitor AG-490 20 min before the six occlusion/reperfusion cycles blocked the enhanced tyrosine phosphorylation of JAK1 and JAK2 and the increased tyrosine phosphorylation, nuclear translocation, and enhanced DNA-binding activity of STAT1 and STAT3. The same dose of AG-490 abrogated the protection against myocardial infarction and the concomitant up-regulation of inducible NO synthase (iNOS) protein and activity observed 24 h after ischemic PC. Taken together, these results demonstrate that ischemic PC induces isoform-selective activation of JAK1, JAK2, STAT1, and STAT3, and that ablation of this response impedes the up-regulation of iNOS and the concurrent acquisition of ischemic tolerance. This study demonstrates that the JAK-STAT pathway plays an essential role in the development of late PC. The results reveal a signaling mechanism that underlies the transcriptional up-regulation of the cardiac iNOS gene and the adaptation of the heart to ischemic stress.

SUBMITTER: Xuan YT

PROVIDER: S-EPMC55371 | biostudies-literature | 2001 Jul

REPOSITORIES: biostudies-literature

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An essential role of the JAK-STAT pathway in ischemic preconditioning.

Xuan Y T YT Guo Y Y Han H H Zhu Y Y Bolli R R

Proceedings of the National Academy of Sciences of the United States of America 20010701 16

The goal of this study was to determine the role of the Janus tyrosine kinase (JAK)-signal transducers and activators of transcription (STAT) pathway in the late phase of ischemic preconditioning (PC). A total of 230 mice were used. At 5 min after ischemic PC (induced with six cycles of 4-min coronary occlusion/4-min reperfusion), immunoprecipitation with anti-phosphotyrosine (anti-pTyr) antibodies followed by immunoblotting with anti-JAK antibodies revealed increased tyrosine phosphorylation of ...[more]

PMID: 11481471

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Project description:BackgroundThe JAK/STAT pathway transduces signals from multiple cytokines and controls haematopoiesis, immunity and inflammation. In addition, pathological activation is seen in multiple malignancies including the myeloproliferative neoplasms (MPNs). Given this, drug development efforts have targeted the pathway with JAK inhibitors such as ruxolitinib. Although effective, high costs and side effects have limited its adoption. Thus, a need for effective low cost treatments remains.Methods & findingsWe used the low-complexity Drosophila melanogaster pathway to screen for small molecules that modulate JAK/STAT signalling. This screen identified methotrexate and the closely related aminopterin as potent suppressors of STAT activation. We show that methotrexate suppresses human JAK/STAT signalling without affecting other phosphorylation-dependent pathways. Furthermore, methotrexate significantly reduces STAT5 phosphorylation in cells expressing JAK2 V617F, a mutation associated with most human MPNs. Methotrexate acts independently of dihydrofolate reductase (DHFR) and is comparable to the JAK1/2 inhibitor ruxolitinib. However, cells treated with methotrexate still retain their ability to respond to physiological levels of the ligand erythropoietin.ConclusionsAminopterin and methotrexate represent the first chemotherapy agents developed and act as competitive inhibitors of DHFR. Methotrexate is also widely used at low doses to treat inflammatory and immune-mediated conditions including rheumatoid arthritis. In this low-dose regime, folate supplements are given to mitigate side effects by bypassing the biochemical requirement for DHFR. Although independent of DHFR, the mechanism-of-action underlying the low-dose effects of methotrexate is unknown. Given that multiple pro-inflammatory cytokines signal through the pathway, we suggest that suppression of the JAK/STAT pathway is likely to be the principal anti-inflammatory and immunosuppressive mechanism-of-action of low-dose methotrexate. In addition, we suggest that patients with JAK/STAT-associated haematological malignancies may benefit from low-dose methotrexate treatments. While the JAK1/2 inhibitor ruxolitinib is effective, a £43,200 annual cost precludes widespread adoption. With an annual methotrexate cost of around £32, our findings represent an important development with significant future potential.

Dataset Information

An essential role of the JAK-STAT pathway in ischemic preconditioning.

Publications

An essential role of the JAK-STAT pathway in ischemic preconditioning.

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