Ischemic preconditioning via epsilon PKC induces ERK1/2 and STAT3 pathways in mixed cortical neurons/astrocytes

Joo Kim Eun, Jesus Purroy, Anna M. Planas, Miguel Perez-Pinzon

Research output: Contribution to journalArticle

Abstract

Ischemic preconditioning (IPC) is a protective mechanism against ischemia/reperfusion injury in brain and heart. Our previous studies have shown that IPC via epsilon PKC activation induced the phosphorylation of extracelluar signal regulated kinase1/2 (ERK1/2) and cyclooxygenase-2 (COX-2) leading to the neuroprotection (1). However, transcriptional signal pathways by which IPC regulates COX-2 in brain remain undefined. The signal transducers and activators of transcription (STATs) upregulate COX-2 expression during cardioprotection (2). A previous study with epsilon PKC knock out mouse model suggests that serine-727-STAT3 phosphorylation was mediated by epsilon PKC activation (3). Therefore, in this study, we tested the hypothesis that IPC via epsilon PKC activates STATs transcriptional signaling pathways. Methods: Mixed cortical neuron/astrocyte cell cultures are prepared from rat embryo/neonatal rat (18-19 days old/ 1-2 days old) respectively. Cell cultures were exposed to 1 h of IPC and 48 h of reperfusion later. Epsilon PKC specific activating peptide (100 nM) was administrated to cell culture for 1 h. Cell lysates were isolated from different intervals of reperfusion after IPC or epsilon PKC specific activating peptide treatment for immunoblotting with antibody against p-ERK1/2 and P- ser727-STAT3. Results: Ischemic preconditioning induced the serine-727 phosphorylation of STAT3 from 15 min and was sustained for 2 h of reperfusion. STAT3 phosphorylation was accompanied by the phosphorylation of ERK1/2 (15 min-1 h of reperfusion). Similarly, epsilon PKC specific activating peptide (100 nM) also induced the phosphorylation of STAT3 and ERK1/2. To test whether IPC-induced STAT3 activation is mediated by ERK1/2 activation or by directly PKC activation, PD98059 (MAPK-K inhibitor,0.01 mM) was administrated to cell cultures during and after IPC. PD98059 prevented the IPC-induced phosphorylation of STAT3. Our previous studies showed that Inhibition of ERK1/2 prevented IPC-induced COX-2 expression (1). Based on our findings in future studies, we will further define whether STAT3 activation induces COX-2 expression after IPC. Conclusion: Our findings suggest that IPC via epsilon PKC activation is mediated by ERK1/2->STAT3 pathway and the signaling pathways of epsilon PKC->ERK1/2->STAT3 might be involved COX-2 expression following IPC.

Original languageEnglish
JournalJournal of Cerebral Blood Flow and Metabolism
Volume27
Issue numberSUPPL. 1
StatePublished - Nov 13 2007

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Protein Kinase C-epsilon
Ischemic Preconditioning
Astrocytes
Neurons
Cyclooxygenase 2
Phosphorylation
Reperfusion
Cell Culture Techniques
Transducers
Serine
Peptides
Brain
Reperfusion Injury

ASJC Scopus subject areas

  • Endocrinology
  • Neuroscience(all)
  • Endocrinology, Diabetes and Metabolism

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Ischemic preconditioning via epsilon PKC induces ERK1/2 and STAT3 pathways in mixed cortical neurons/astrocytes. / Eun, Joo Kim; Purroy, Jesus; Planas, Anna M.; Perez-Pinzon, Miguel.

In: Journal of Cerebral Blood Flow and Metabolism, Vol. 27, No. SUPPL. 1, 13.11.2007.

Research output: Contribution to journalArticle

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abstract = "Ischemic preconditioning (IPC) is a protective mechanism against ischemia/reperfusion injury in brain and heart. Our previous studies have shown that IPC via epsilon PKC activation induced the phosphorylation of extracelluar signal regulated kinase1/2 (ERK1/2) and cyclooxygenase-2 (COX-2) leading to the neuroprotection (1). However, transcriptional signal pathways by which IPC regulates COX-2 in brain remain undefined. The signal transducers and activators of transcription (STATs) upregulate COX-2 expression during cardioprotection (2). A previous study with epsilon PKC knock out mouse model suggests that serine-727-STAT3 phosphorylation was mediated by epsilon PKC activation (3). Therefore, in this study, we tested the hypothesis that IPC via epsilon PKC activates STATs transcriptional signaling pathways. Methods: Mixed cortical neuron/astrocyte cell cultures are prepared from rat embryo/neonatal rat (18-19 days old/ 1-2 days old) respectively. Cell cultures were exposed to 1 h of IPC and 48 h of reperfusion later. Epsilon PKC specific activating peptide (100 nM) was administrated to cell culture for 1 h. Cell lysates were isolated from different intervals of reperfusion after IPC or epsilon PKC specific activating peptide treatment for immunoblotting with antibody against p-ERK1/2 and P- ser727-STAT3. Results: Ischemic preconditioning induced the serine-727 phosphorylation of STAT3 from 15 min and was sustained for 2 h of reperfusion. STAT3 phosphorylation was accompanied by the phosphorylation of ERK1/2 (15 min-1 h of reperfusion). Similarly, epsilon PKC specific activating peptide (100 nM) also induced the phosphorylation of STAT3 and ERK1/2. To test whether IPC-induced STAT3 activation is mediated by ERK1/2 activation or by directly PKC activation, PD98059 (MAPK-K inhibitor,0.01 mM) was administrated to cell cultures during and after IPC. PD98059 prevented the IPC-induced phosphorylation of STAT3. Our previous studies showed that Inhibition of ERK1/2 prevented IPC-induced COX-2 expression (1). Based on our findings in future studies, we will further define whether STAT3 activation induces COX-2 expression after IPC. Conclusion: Our findings suggest that IPC via epsilon PKC activation is mediated by ERK1/2->STAT3 pathway and the signaling pathways of epsilon PKC->ERK1/2->STAT3 might be involved COX-2 expression following IPC.",
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T1 - Ischemic preconditioning via epsilon PKC induces ERK1/2 and STAT3 pathways in mixed cortical neurons/astrocytes

AU - Eun, Joo Kim

AU - Purroy, Jesus

AU - Planas, Anna M.

AU - Perez-Pinzon, Miguel

PY - 2007/11/13

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N2 - Ischemic preconditioning (IPC) is a protective mechanism against ischemia/reperfusion injury in brain and heart. Our previous studies have shown that IPC via epsilon PKC activation induced the phosphorylation of extracelluar signal regulated kinase1/2 (ERK1/2) and cyclooxygenase-2 (COX-2) leading to the neuroprotection (1). However, transcriptional signal pathways by which IPC regulates COX-2 in brain remain undefined. The signal transducers and activators of transcription (STATs) upregulate COX-2 expression during cardioprotection (2). A previous study with epsilon PKC knock out mouse model suggests that serine-727-STAT3 phosphorylation was mediated by epsilon PKC activation (3). Therefore, in this study, we tested the hypothesis that IPC via epsilon PKC activates STATs transcriptional signaling pathways. Methods: Mixed cortical neuron/astrocyte cell cultures are prepared from rat embryo/neonatal rat (18-19 days old/ 1-2 days old) respectively. Cell cultures were exposed to 1 h of IPC and 48 h of reperfusion later. Epsilon PKC specific activating peptide (100 nM) was administrated to cell culture for 1 h. Cell lysates were isolated from different intervals of reperfusion after IPC or epsilon PKC specific activating peptide treatment for immunoblotting with antibody against p-ERK1/2 and P- ser727-STAT3. Results: Ischemic preconditioning induced the serine-727 phosphorylation of STAT3 from 15 min and was sustained for 2 h of reperfusion. STAT3 phosphorylation was accompanied by the phosphorylation of ERK1/2 (15 min-1 h of reperfusion). Similarly, epsilon PKC specific activating peptide (100 nM) also induced the phosphorylation of STAT3 and ERK1/2. To test whether IPC-induced STAT3 activation is mediated by ERK1/2 activation or by directly PKC activation, PD98059 (MAPK-K inhibitor,0.01 mM) was administrated to cell cultures during and after IPC. PD98059 prevented the IPC-induced phosphorylation of STAT3. Our previous studies showed that Inhibition of ERK1/2 prevented IPC-induced COX-2 expression (1). Based on our findings in future studies, we will further define whether STAT3 activation induces COX-2 expression after IPC. Conclusion: Our findings suggest that IPC via epsilon PKC activation is mediated by ERK1/2->STAT3 pathway and the signaling pathways of epsilon PKC->ERK1/2->STAT3 might be involved COX-2 expression following IPC.

AB - Ischemic preconditioning (IPC) is a protective mechanism against ischemia/reperfusion injury in brain and heart. Our previous studies have shown that IPC via epsilon PKC activation induced the phosphorylation of extracelluar signal regulated kinase1/2 (ERK1/2) and cyclooxygenase-2 (COX-2) leading to the neuroprotection (1). However, transcriptional signal pathways by which IPC regulates COX-2 in brain remain undefined. The signal transducers and activators of transcription (STATs) upregulate COX-2 expression during cardioprotection (2). A previous study with epsilon PKC knock out mouse model suggests that serine-727-STAT3 phosphorylation was mediated by epsilon PKC activation (3). Therefore, in this study, we tested the hypothesis that IPC via epsilon PKC activates STATs transcriptional signaling pathways. Methods: Mixed cortical neuron/astrocyte cell cultures are prepared from rat embryo/neonatal rat (18-19 days old/ 1-2 days old) respectively. Cell cultures were exposed to 1 h of IPC and 48 h of reperfusion later. Epsilon PKC specific activating peptide (100 nM) was administrated to cell culture for 1 h. Cell lysates were isolated from different intervals of reperfusion after IPC or epsilon PKC specific activating peptide treatment for immunoblotting with antibody against p-ERK1/2 and P- ser727-STAT3. Results: Ischemic preconditioning induced the serine-727 phosphorylation of STAT3 from 15 min and was sustained for 2 h of reperfusion. STAT3 phosphorylation was accompanied by the phosphorylation of ERK1/2 (15 min-1 h of reperfusion). Similarly, epsilon PKC specific activating peptide (100 nM) also induced the phosphorylation of STAT3 and ERK1/2. To test whether IPC-induced STAT3 activation is mediated by ERK1/2 activation or by directly PKC activation, PD98059 (MAPK-K inhibitor,0.01 mM) was administrated to cell cultures during and after IPC. PD98059 prevented the IPC-induced phosphorylation of STAT3. Our previous studies showed that Inhibition of ERK1/2 prevented IPC-induced COX-2 expression (1). Based on our findings in future studies, we will further define whether STAT3 activation induces COX-2 expression after IPC. Conclusion: Our findings suggest that IPC via epsilon PKC activation is mediated by ERK1/2->STAT3 pathway and the signaling pathways of epsilon PKC->ERK1/2->STAT3 might be involved COX-2 expression following IPC.

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