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, 35 (5), 525-37

Mechanism of cyclooxygenase-2 Upregulation in Late Preconditioning

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Mechanism of cyclooxygenase-2 Upregulation in Late Preconditioning

Yu-Ting Xuan et al. J Mol Cell Cardiol.

Abstract

Although the cardioprotection of late preconditioning (PC) is known to be mediated by both inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), the signaling mechanism responsible for COX-2 upregulation and the interaction between iNOS and COX-2 remain unknown. A total of 122 mice were used to address this issue. In wild-type mice preconditioned with six cycles of 4-min coronary occlusion-4-min reperfusion, ischemic PC resulted in rapid activation of nuclear STAT1/3 through tyrosine phosphorylation (STAT1: 339 +/- 48% of control; STAT3: 389 +/- 46% of control) and increased STAT1/3-DNA binding activity (687 +/- 58% of control) at 30 min after PC, with subsequent upregulation of COX-2 protein (373 +/- 60% of control) and activity(increased myocardial levels of PGE2, PGF(2alpha), and 6-keto-PGF(1alpha)) at 24 h. However, COX-1 protein was not changed 24 h after ischemic PC. Pretreatment with the Janus tyrosine kinase (JAK) inhibitor AG-490 before the six occlusion-reperfusion cycles blocked both the tyrosine phosphorylation of STAT1/3 and the subsequent upregulation of COX-2 protein, demonstrating a necessary role of the JAK-STAT pathway in the induction of COX-2. Targeted disruption of the iNOS gene (iNOS-/-) did not block the increased expression of COX-2 protein 24 h after ischemic PC but completely blocked the increase in COX-2 activity, whereas targeted disruption of the COX-2 gene (COX-2-/-) did not alter ischemic PC-induced iNOS induction. Immunoprecipitation of preconditioned heart tissues with anti-COX-2 antibodies followed by immunoblotting with anti-iNOS antibodies revealed that the increased iNOS protein co-precipitated with COX-2. We conclude that (i) the upregulation of COX-2 protein expression after ischemic PC is mediated by a JAK1/2-STAT1/3-signaling cascade; (ii) COX-2 activity requires upregulated iNOS and iNOS-derived NO; and (iii) COX-2 forms complexes with iNOS, supporting a direct interaction between these two proteins. To our knowledge, this is the first evidence that myocardial COX-2 is upregulated via a JAK1/2-STAT1/3 pathway.

Figures

Fig. 1
Fig. 1
Diagram of the experimental protocol for phase I. O: indicates 4-min coronary occlusion and R: 4-min reperfusion.
Fig. 2
Fig. 2
Diagram of the experimental protocol for phase II. O: indicates 4-min coronary occlusion and R: 4-min reperfusion.
Fig. 3
Fig. 3
Effect of ischemic PC on tyrosine phosphorylation of nuclear STAT1 and STAT3. Nuclear extracts of myocardial samples were prepared from mice that underwent a sham operation (1 h of open-chest state without coronary occlusion-reperfusion) without treatment (group I, control) or after receiving AG-490 (group V, AG-490 control) or from the ischemic-reperfused region of preconditioned mice that received either no treatment (group IV, PC-30′) or AG-490 20 min before ischemic PC (group VI, AG-490 + PC-30′). All mice were euthanized 30 min after the sham operation or 30 min after ischemic PC (i.e. after the sixth occlusion), and nuclear extracts of myocardial samples were prepared as described in Section 2. Tyrosine-phosphorylated forms of STAT1 and STAT3 in the nuclear extracts were analyzed by immunoblotting using specific anti-pTyr(701)-STAT1 or anti-pTyr(705)-STAT3 antibodies. (A,C) The immunoreactivity of tyrosine-phosphorylated STAT1 and STAT3 in the nuclear fraction increased markedly 30 min after ischemic PC, and this increase was inhibited by AG-490. (B,D) Densitometric analysis of tyrosine-phosphorylated STAT1 and STAT3. Data are mean ± S.E.M.
Fig. 4
Fig. 4
Effect of ischemic PC on STAT1/3-DNA binding activity. Nuclear extracts were prepared from mice that underwent a sham operation without treatment (group I, control) or after receiving AG-490 (group V, AG-490 control), or from the ischemic-reperfused region of untreated preconditioned mice that were euthanized 0 min (group II, PC-0′), 15 min (group III, PC-15′), or 30 min (group IV, PC-30′) after the sixth occlusion, or from the ischemic-reperfused region of preconditioned mice that received AG-490 20 min before ischemic PC and were euthanized 30 min after the sixth occlusion (group VI, AG-490 + PC-30′). The nuclear extracts were subjected to EMSA for analysis of STAT1/3-DNA binding activity using the 32P-labeled GAS probe. (A) Representative EMSA showing that the shifted band corresponding to the STAT-GAS complex (indicated by the arrow) increased markedly 15 and 30 min after ischemic PC in the nuclear extracts. (C) The increased activity of STAT1/3-DNA binding was inhibited by AG-490. (B,D) Densitometric analysis of the STAT1/3-GAS complexes. Data are mean ± S.E.M.
Fig. 5
Fig. 5
Effects of ischemic PC on myocardial COX-2 and COX-1 expression. Samples were obtained from wild-type mice that underwent a sham operation without treatment (group VII, control) or after receiving AG-490 (group IX, AG-490 control) or from the ischemic-reperfused region of preconditioned mice that received no treatment (group VIII, PC–24 h) or AG-490 (group X, AG-490 + PC – 24 h) 20 min before the first occlusion. All mice were euthanized 24 h after the sixth reperfusion. (A) Representative immunoblots of COX-2 and COX-1 expression. (B,C) Densitometric analysis of immunoreactive COX-2 and COX-1 signals, respectively. COX-2 was significantly increased 24 h after ischemic PC, and this increase was prevented by AG-490. Data are mean ± S.E.M.
Fig. 6
Fig. 6
Effect of targeted disruption of the iNOS gene on myocardial levels of PGE2, PGF, and 6-keto-PGF 24 h after ischemic PC. Myocardial samples were obtained from wild-type and iNOS−/− mice that underwent a sham operation (control) or from the nonischemic (NIZ) and the ischemic-reperfused (IZ) zones of wild-type and iNOS−/− mice that were preconditioned with six coronary occlusion-reperfusion cycles. Myocardial levels of PGE2, PGF, and 6-keto-PGF were analyzed with EIA kits as described in Section 2. Data are mean ± S.E.M.
Fig. 7
Fig. 7
Effect of targeted disruption of the iNOSgene on ischemic PC-induced expression of COX-2 and eNOS protein. Samples were obtained from wild-type (group XI, wild-type control) and iNOS−/− (group XIII, iNOS−/− control) mice that underwent a sham operation or from the ischemic-reperfused region of both wild-type (group XII, wild-type PC) and iNOS−/− (group XIV, iNOS−/− PC) mice that were preconditioned with six coronary occlusion-reperfusion cycles 24 h earlier. (A) Western blot confirming that the immunoreactivity of iNOS protein was upregulated 24 h after ischemic PC in the wild-type mice and was completely ablated in control and preconditioned iNOS−/− mice. (C) Immunoblotting indicating that COX-2 expression was increased 24 h after ischemic PC in both wild-type and iNOS−/− mice. (E) Immunoblotting showing that eNOS expression was not affected in control and preconditioned mice with targeted disruption of the iNOS gene (iNOS−/−). (B,D,F) Densitometric analysis of immunoreactive iNOS, COX-2, and eNOS signals. Data are mean ± S.E.M.
Fig. 8
Fig. 8
Effect of targeted disruption of the COX-2 gene on iNOS protein expression and activity. Samples were obtained from wild-type (group XI, wild-type control) and COX-2−/− (group XVII, COX-2−/− control) mice that underwent a sham operation or from the ischemic-reperfused region of wild-type (group XVI, wild-type PC) and COX-2−/− (group XVIII, COX-2−/− PC) mice that were preconditioned with six coronary occlusion-reperfusion cycles. All mice were euthanized 24 h after the sixth reperfusion. (A) Western blot confirming that the immunoreactivity of COX-2 was completely ablated in COX-2−/− mice. (C) Representative immunoblots showing that iNOS expression was upregulated 24 h after ischemic PC in wild-type mice, and that this increase was not affected by deletion of the COX-2 gene. (B,D) Densitometric analysis of immunoreactive COX-2 and iNOS signals. (E) iNOS activity in the cytosolic fraction was increased in both preconditioned wild-type and preconditioned COX-2−/− mice. Data are mean ± S.E.M.
Fig. 9
Fig. 9
Immunoprecipitation of iNOS with COX-2 in preconditioned myocardium. Samples were taken from wild-type mice that underwent a sham operation (group XV, control) or from the ischemic-reperfused region of the mice that were preconditioned with six coronary occlusion-reperfusion cycles (group XIX, wild-type PC). All mice were euthanized 24 h after the sixth reperfusion and the homogenates were immunoprecipitated either with anti-COX-2 antibodies followed by immunoblotting with anti-COX-2 and anti-iNOS antibodies (A) or with anti-COX-1 antibodies followed by immunoblotting with anti-COX-1 and anti-iNOS antibodies (C). (A,C) Immunoblotting showing that increased iNOS co-immunoprecipitated with COX-2 (A) but not with COX-1 (C). Positive (P) iNOS control: LPS-treated macrophages (C). (B,D) Densitometric analysis of COX-2, iNOS, and COX-1 signals. Data are mean ± S.E.M.

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