Preconditioning mediated by sublethal oxygen-glucose deprivation-induced cyclooxygenase-2 expression via the signal transducers and activators of transcription 3 phosphorylation

Abstract

The signal transducers and activators of transcription (STATs) were found to be essential for cardioprotection. However, their role in preconditioning (PC) neuroprotection remains undefined. Previously, our studies showed that PC mediated a signaling cascade that involves activation of epsilon protein kinase C (varepsilonPKC), extracellular signal-regulated kinase (ERK1/2), and cyclooxygenase-2 (COX-2) pathways. However, the intermediate pathway by which ERK1/2 activates COX-2 was not defined. In this study, we investigated whether the PC-induced signaling pathway requires phosphorylation of STAT isoforms for COX-2 expression. To mimic PC or lethal ischemia, mixed cortical neuron/astrocyte cell cultures were subjected to 1 and/or 4 h of oxygen-glucose deprivation (OGD), respectively. The results indicated serine phosphorylation of STAT3 after PC or varepsilonPKC activation. Inhibition of either varepsilonPKC or ERK1/2 activation abolished PC-induced serine phosphorylation of STAT3. Additionally, inhibition of STAT3 prevented PC-induced COX-2 expression and neuroprotection against OGD. Therefore, our findings suggest that PC signaling cascade involves STAT3 activation after varepsilonPKC and ERK1/2 activation. Finally, we show that STAT3 activation mediates COX-2 expression and ischemic tolerance.

Figure 1

Experimental design. Control; OGD: cell cultures were subjected for 4 h of OGD; PC: cell cultures were subjected for 1 h of OGD and 48 h of reperfusion and then 4 h of OGD was induced; PC + inhibitor: either MAPK-K inhibitor (PD98059, 10 µmol/L) or εPKC-specific inhibitory peptide (εV1-2, 100 nmol/L) or STAT3 inhibitory peptide (PpYLKTK, 100 nmol/L, 1 and 5 µmol/L) was administered to cell cultures during PC and 48 h of reperfusion and then 4 h of OGD was induced; εPKC activator: εPKC-specific activator peptide (ψεRACK, 100 nmol/L) was administered to cell cultures for 2 h without PC.

Figure 2

Preconditioning induced phosphorylation of STAT3 at the serine residue. (A) Cells were lysed immediately after 1 h of PC and at 15 mins, 30 mins, 1 h, and 2 h of reperfusion after 1 h of PC. Western blotting for p-serine 727-STAT3 was performed with cytosolic protein extracts (A) or nuclear protein extracts (B) and the membrane was reprobed with the STAT3 (A) or nuclear lamin B (B) antibody. The histogram depicts densitometric analysis of western blots of p-serine 727-STAT3 in cytosolic protein extracts compared with STAT3 (A). *P < 0.05 compared with control (n = 5). (C) Confocal microscopic images of mixed cortical neuron/astrocyte cell cultures depicting colocalization of immunoreactivities for neuronal specific antibody NeuN (red) or astrocyte-specific antibody GFAP (glial fibrillary acidic protein; red) and p-serine 727-STAT3 (pSTAT, green). At 30 mins of reperfusion after PC, p-serine 727-STAT3-positive cells expressed NeuN (arrows) (a–c) and GFAP (arrows) (d–f). Bar: 20 µmol/L.

Figure 3

Preconditioning induced phosphorylation of STAT3 at the tyrosine residue, but not STAT1 at the serine residue. Cells were lysed immediately after 1 h of PC and at 15 mins, 30 mins, 1 h, and 2 h of reperfusion after 1 h of PC. Western blotting for p-tyrosine 705-STAT3 (A) or p-serine 727-STAT1 (B) was performed with cytosolic protein extracts and the membrane was reprobed with STAT3 (A) or STAT1 (B).

Figure 4

Activation of εPKC is involved in the phosphorylation of STAT3 at the serine residue after PC. (A) Inhibition of εPKC reduced PC-induced serine phosphorylation of STAT3. Cells were treated with εPKC inhibitor (εPKCi, εV1-2, 100 nmol/L) or vehicle during PC and reperfusion (time indicated). Cells were isolated at 15 and 30 mins of reperfusion after 1 h of PC. Western blotting for p-serine 727-STAT3 or p-tyrosine 705-STAT3 was performed and the membrane was reprobed with STAT3 antibody. The histogram depicts densitometric analysis of western blots of p-serine 727-STAT3 compared with STAT3. *P < 0.05 compared with control, ^#P < 0.05 15 min versus 15 mins with εPKC inhibitor, ^&P < 0.05 30 versus 30 mins with εPKC inhibitor (n = 3). (B) The activation of εPKC induced serine phosphorylation of STAT3. Cells were lysed immediately at 5 mins, 15 mins, 30 mins, 1 h, and 2 h after εPKC activator (ψεRACK, 100 nmol/L) treatment. Western blotting for p-serine 727-STAT3 was performed and the membrane was reprobed with STAT3 antibody. The histogram depicts densitometric analysis of western blots of p-serine 727-STAT3 compared with STAT3. *P < 0.05 compared with control (n = 5).

Figure 5

Preconditioning induced ERK1/2 phosphorylation and inhibition of ERK1/2 activation reduced PC-induced phosphorylation of STAT3 at the serine residue. (A) Cells were lysed immediately after 1 h of PC and at 15 mins, 30 mins, 1 h, and 2 h of reperfusion after 1 h of PC. Western blotting for phospho-ERK1/2 was carried out and the blot was reprobed with ERK1/2 antibody. The histogram depicts densitometric analysis of western blots of pERK1/2 compared with ERK1/2. *P < 0.05 compared with control (n = 3). (B) Cells were treated with PD98059 (MAPK-K inhibitor, 10 µmol/L) or vehicle during PC and reperfusion (time as indicated). Cells were lysed at 15 and 30 mins of reperfusion after 1 h of PC. Western blotting for p-serine 727-STAT3 or p-tyrosine 705-STAT3 was performed and the membrane was reprobed with STAT3 antibody. The histogram depicts densitometric analysis of western blots of p-serine 727-STAT3 compared with STAT3. *P < 0.05 compared with control, ^#P < 0.05 15 versus 15 mins with PD98059, ^&P < 0.05 30 versus 30 mins with PD98059 (n = 3).

Figure 6

Neither ERK1/2 nor εPKC directly interacted with STAT3. Cells were lysed immediately at 5, 15, and 30 mins of reperfusion after 1 h of PC. (A) Cell lysates were immunoprecipitated (IP) with phospho-ERK1/2 antibody and then pulled down phospho-ERK1/2 (pellet) and the supernatant were analyzed by western blotting (WB) with p-serine 727-STAT3 and pERK1/2 antibody. (B) Cell lysates were immunoprecipitated with εPKC antibody and then pulled down εPKC (pellet) and the supernatant were analyzed by western blotting with p-serine 727-STAT3 and εPKC antibody.

Figure 7

Inhibition of STAT3 activation reduced PC-induced COX-2 expression and neuroprotection. (A) Cells were treated with a STAT3 inhibitory peptide (PpYLKTK, 1 µmol/L) or vehicle during 24 h of reperfusion after 1 h of PC. Cells were lysed at 24 h of reperfusion and analyzed by western blotting with COX-2 antibody. The membrane was reprobed with β-actin antibody. The histogram depicts densitometric analysis of western blots of COX-2 compared with β-actin. **P < 0.01 compared with control, ^#P < 0.05 compared with PC (n = 3). (B) Histogram representing cell death measured by LDH release at 48 h of reperfusion after OGD (4 h) injury. Note that neuroprotection was blocked with the inhibition of STAT3 activity using the STAT3 inhibitory peptide (PpYLKTK, 1 and 5 µmol/L) during 48 h after PC. The STAT3 inhibitory peptide alone (PpYLKTK, 100 nmol/L, 1 and 5 µmol/L) had no effect on cell death. **P < 0.01 compared with control, ^#P < 0.05 compared with OGD, and ^&P < 0.05 compared with PC (n = 20).