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.