Pharmacological preconditioning with tumor necrosis factor-α activates signal transducer and activator of transcription-3 at reperfusion without involving classic prosurvival kinases (Akt and extracellular signal-regulated kinase)

Background - We previously reported that tumor necrosis-factor-alpha (TNF-alpha) can mimic classic ischemic preconditioning (IPC) in a dose- and time-dependent manner. Because TNF-alpha activates the signal transducer and activator of transcription-3 (STAT-3), we hypothesized that TNF-alpha-induced preconditioning requires phosphorylation of STAT-3 rather than involving the classic prosurvival kinases, Akt and extracellular signal - regulated kinase (Erk) 1/2, during early reperfusion. Methods and Results - Isolated, ischemic/reperfused rat hearts were preconditioned by either IPC or low-dose TNF-alpha (0.5 ng/mL). Western blot analysis confirmed that IPC phosphorylated Akt and Erk 1/2 after 5 minutes of reperfusion ( Akt increased by 34 +/- 6% and Erk, by 105 +/- 28% versus control; P < 0.01). Phosphatidylinositol 3-kinase/Akt inhibition ( wortmannin) or mitogen-activated protein kinase - Erk 1/2 kinase inhibition (PD-98059) during early reperfusion abolished the infarct-sparing effect of IPC. In contrast, TNF-alpha preconditioning did not phosphorylate these kinases (Akt increased by 7 +/- 7% and Erk, by 17 +/- 14% versus control; P = NS). Neither wortmannin nor PD-98059 inhibited TNF-alpha-mediated cardioprotection. However, TNF-alpha and IPC both phosphorylated STAT- 3 and the proapoptotic protein Bcl-2 antagonist of cell death ( BAD) ( STAT- 3 increased by 58 +/- 17% with TNF-alpha or by 68 +/- 12% with IPC; BAD increased by 75 +/- 8% with TNF-alpha or by 205 +/- 20% with IPC; P < 0.01 versus control), thereby activating the former and inactivating the latter. The STAT- 3 inhibitor AG 490 abolished cardioprotection and BAD phosphorylation with both preconditioning stimuli. Conclusions - Activation of the classic prosurvival kinases ( Akt and Erk 1/2) is not essential for TNF-alpha-induced preconditioning in the early reperfusion phase. We show the existence of an alternative protective pathway that involves STAT- 3 activation specifically at reperfusion in response to both TNF-alpha and classic IPC. This novel prosurvival pathway may have potential therapeutic significance.