Activation of NF-kappaB and MAP kinase cascades by hypothermic stress in endothelial cells.
Signal transduction pathways and transcription factors are likely to be important mediators of stress responses to ischaemia and reperfusion injury following renal transplantation. We have investigated the activation of the transcription factor nuclear factor kappaB (NF-kappaB) and the mitogen activated protein kinases (MAPK), p44/42 (ERK 1/2), p38 and c-Jun N-terminal kinase (JNK) during cold stress at 4 degrees C. Human umbilical vein endothelial cells (HUVECs) were subjected to 72 h of hypothermia in a renal preservation solution. NF-kappaB activation was assessed by electromobility shift assays and MAPK activation by immunoblotting. Cell viability and apoptosis was assessed. Hypothermia activated the NF-kappaB complex, ERK 1/2 and p38 MAPK pathway. There was a 6-fold increase in NF-kappaB in the nucleus within minutes of hypothermia, correlating with p38 (p = 0.01) and ERK 1/2 activation (p = 0.03). A significant relationship was found between ERK 1/2, p38 and NF-kappaB throughout the 72 h time course (p = 0.01). In contrast, hypothermia had no effect on JNK phosphorylation. Inhibition of MAPK with an MEK inhibitor (PD098059) blocked the activation of NF-kappaB but a specific p38 inhibitor (SB203580) had no effect on NF-kappaB. Increased lactate production after 48 h indicated a switch towards anaerobic metabolism during prolonged hypothermia. Endothelial cells had a high viability and no DNA fragmentation throughout the experiment. Activation of stress pathways during organ procurement may be important in the quality of stored grafts.
Roberts
Jonathan R
JR
Department of Molecular Medicine, Postgraduate Medical School, University of Plymouth, Plymouth, UK.
Rowe
Peter A
PA
Demaine
Andrew G
AG
D017209:Apoptosis; D002470:Cell Survival; D002478:Cells, Cultured; D003080:Cold Temperature; D003451:Cryoprotective Agents; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D007036:Hypothermia, Induced; D048031:JNK Mitogen-Activated Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism*; D009926:Organ Preservation; D015398:Signal Transduction; D012996:Solutions; D048051:p38 Mitogen-Activated Protein Kinases
D003451:Cryoprotective Agents; D016328:NF-kappa B; D012996:Solutions; D048031:JNK Mitogen-Activated Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases