title: Erythropoietin is a paracrine mediator of ischemic tolerance in the brain: evidence from an in vitro model.
abstract: In an in vitro model of cerebral ischemia (oxygen glucose deprivation, OGD) we investigated whether erythropoietin (EPO) plays a critical role in ischemic preconditioning. We found that EPO time and dose-dependently induced protection against OGD in rat primary cortical neurons. Protection was significant at 5 min and reached a maximum at 48 hr after EPO application. Protection was blocked by the coapplication of a soluble Epo receptor (sEpoR) or an antibody against EpoR (anti-EpoR). Medium transfer from OGD-treated astrocytes to untreated neurons induced protection against OGD in neurons, which was attenuated strongly by the application of sEpoR and anti-EpoR. In contrast, medium transfer from OGD-treated neurons to untreated neurons induced protection against OGD that did not involve EPO. In astrocytes the OGD enhanced the nuclear translocation of hypoxia-inducible factor 1 (HIF-1), the major transcription factor regulating EPO expression. Consequently, transcription of EPO-mRNA was increased in astrocytes after OGD. Cultured neurons express EpoR, and the Janus kinase-2 (JAK-2) inhibitor AG490 abolished EPO-induced tolerance against OGD. Furthermore, EPO-induced neuroprotection as well as phosphorylation of the proapoptotic Bcl family member Bad was reduced by the phosphoinositide-3 kinase (PI3K) inhibitor LY294002. The results suggest that astrocytes challenged with OGD provide paracrine protective signals to neurons. We provide evidence for the following signaling cascade: HIF-1 is activated rapidly by hypoxia in astrocytes. After HIF-1 activation the astrocytes express and release EPO. EPO activates the neuronal EPO receptor and, subsequently, JAK-2 and thereby PI3K. PI3K deactivates BAD via Akt-mediated phosphorylation and thus may inhibit hypoxia-induced apoptosis in neurons. Our results establish EPO as an important paracrine neuroprotective mediator of ischemic preconditioning.
authors.affiliation: Department of Experimental Neurology, Charité Hospital, Humboldt University, D-10098 Berlin, Germany.
authors.forename: Karsten
authors.initials: K
authors.lastname: Ruscher
authors.forename: Dorette
authors.initials: D
authors.lastname: Freyer
authors.forename: Maria
authors.initials: M
authors.lastname: Karsch
authors.forename: Nikolai
authors.initials: N
authors.lastname: Isaev
authors.forename: Dirk
authors.initials: D
authors.lastname: Megow
authors.forename: Birgit
authors.initials: B
authors.lastname: Sawitzki
authors.forename: Josef
authors.initials: J
authors.lastname: Priller
authors.forename: Ulrich
authors.initials: U
authors.lastname: Dirnagl
authors.forename: Andreas
authors.initials: A
authors.lastname: Meisel
mesh_terms: D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D017209:Apoptosis; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism; D001921:Brain / Q000098:blood supply* / Q000166:cytology / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004921:Erythropoietin / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D005947:Glucose / Q000172:deficiency / Q000378:metabolism; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D020925:Hypoxia-Ischemia, Brain / Q000378:metabolism*; D019194:Ischemic Preconditioning; D053614:Janus Kinase 2; D008954:Models, Biological; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D019899:Paracrine Communication / Q000187:drug effects / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017467:Receptors, Erythropoietin / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014157:Transcription Factors; D051025:bcl-Associated Death Protein
chemical_list: D000906:Antibodies; C494821:Bad protein, rat; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C497444:Hif1a protein, rat; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D017467:Receptors, Erythropoietin; D014157:Transcription Factors; D051025:bcl-Associated Death Protein; D004921:Erythropoietin; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; C507927:Jak2 protein, rat; D053614:Janus Kinase 2; D005947:Glucose