No evidence for protective erythropoietin alpha signalling in rat hepatocytes.
BACKGROUND
Recombinant human erythropoietin alpha (rHu-EPO) has been reported to protect the liver of rats and mice from ischemia-reperfusion injury. However, direct protective effects of rHu-EPO on hepatocytes and the responsible signalling pathways have not yet been described. The aim of the present work was to study the protective effect of rHu-EPO on warm hypoxia-reoxygenation and cold-induced injury to hepatocytes and the rHu-EPO-dependent signalling involved.


METHODS
Loss of viability of isolated rat hepatocytes subjected to hypoxia/reoxygenation or incubated at 4 degrees C followed by rewarming was determined from released lactate dehydrogenase activity in the absence and presence of rHu-EPO (0.2-100 U/ml). Apoptotic nuclear morphology was assessed by fluorescence microscopy using the nuclear fluorophores H33342 and propidium iodide. Erythropoietin receptor (EPOR), EPO and Bcl-2 mRNAs were quantified by real time PCR. Activation of JAK-2, STAT-3 and STAT-5 in hepatocytes and rat livers perfused in situ was assessed by Western blotting.


RESULTS
In contrast to previous in vivo studies on ischemia-reperfusion injury to the liver, rHu-EPO was without any protective effect on hypoxic injury, hypoxia-reoxygenation injury and cold-induced apoptosis to isolated cultured rat hepatocytes. EPOR mRNA was identified in these cells but specific detection of the EPO receptor protein was not possible due to the lack of antibody specificity. Both, in the cultured rat hepatocytes (10 U/ml for 15 minutes) and in the rat liver perfused in situ with rHu-EPO (8.9 U/ml for 15 minutes) no evidence for EPO-dependent signalling was found as indicated by missing effects of rHu-EPO on phosphorylation of JAK-2, STAT-3 and STAT-5 and on the induction of Bcl-2 mRNA.


CONCLUSIONS
Together, these results indicate the absence of any protective EPO signalling in rat hepatocytes. This implies that the protection provided by rHu-EPO in vivo against ischemia-reperfusion and other causes of liver injury is most likely indirect and does not result from a direct effect on hepatocytes.
Bramey
Thorsten
T
Institut für Physiologische Chemie, Universitätsklinikum Essen, Hufelandstrasse 55, D-45122 Essen, Germany. thorsten.bramey@uni-duisburg-essen.de
Freitag
Patricia
P
Fandrey
Joachim
J
Rauen
Ursula
U
Pamp
Katja
K
Erhard
Jochen
J
Frede
Stilla
S
de Groot
Herbert
H
Petrat
Frank
F
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015687:Cell Hypoxia / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D003080:Cold Temperature / Q000009:adverse effects; D004921:Erythropoietin / Q000378:metabolism / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D053614:Janus Kinase 2 / Q000378:metabolism; D008297:Male; D023421:Models, Animal; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins; D015427:Reperfusion Injury / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D050796:STAT3 Transcription Factor / Q000378:metabolism; D050799:STAT5 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*
D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D011994:Recombinant Proteins; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; C494088:Stat3 protein, rat; D004921:Erythropoietin; D053614:Janus Kinase 2