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Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-κB signalling cascades

Abstract

Erythropoietin, a kidney cytokine regulating haematopoiesis (the production of blood cells), is also produced in the brain after oxidative or nitrosative stress1,2. The transcription factor hypoxia-inducible factor-1 (HIF-1) upregulates EPO following hypoxic stimuli3,4. Here we show that preconditioning with EPO protects neurons in models of ischaemic and degenerative damage due to excitotoxins4,5 and consequent generation of free radicals, including nitric oxide (NO). Activation of neuronal EPO receptors (EPORs) prevents apoptosis induced by NMDA (N-methyl-d-aspartate) or NO by triggering cross-talk between the signalling pathways of Janus kinase-2 (Jak2) and nuclear factor-κB (NF-κB). We show that EPOR-mediated activation of Jak2 leads to phosphorylation of the inhibitor of NF-κB (IκB), subsequent nuclear translocation of the transcription factor NF-κB, and NF-κB-dependent transcription of neuroprotective genes. Transfection of cerebrocortical neurons with a dominant interfering form of Jak2 or an IκBα super-repressor blocks EPO-mediated prevention of neuronal apoptosis. Thus neuronal EPORs activate a neuroprotective pathway that is distinct from previously well characterized Jak and NF-κB functions. Moreover, this EPO effect may underlie neuroprotection mediated by hypoxic–ischaemic preconditioning.

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Figure 1: Neuroprotective effect of EPOR activation on cerebrocortical neurons.
Figure 2: EPO-induced NF-κB activation.
Figure 3: EPO induces nuclear translocation of NF-κB in neurons rather than astrocytes.
Figure 4: EPO-induced Jak2 and NF-κB activities in neurons.
Figure 5: Inhibition of Jak2 function abrogates EPO-mediated neuroprotection.

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Acknowledgements

We thank M. Kaul, N. Moayeri, B. Price, M. Cokol and M. Altinoz for insightful discussions or technical advice, and the Genetics Institute, Cambridge, Massachusetts, for supplying the anti-EPOR monoclonal antibodies. The complementary DNA strands for the IκB super-repressor (Ad5IκB) and kinase-negative mutant Jak2 (JAK2.KE) were the gifts of R. R. Ratan and J. Ihle, respectively. This work was supported in part by grants from the National Institutes of Health and American Heart Association (S.A.L.).

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Correspondence to Stuart A. Lipton.

Supplementary information

We characterized the expression of EPO receptor (EPO-R) protein on rat brain neurons by immunohistochemistry of brain sections with a specific monoclonal antibody (Fig. 1a, left panel). To perform the studies elucidating the signalling pathway that mediates EPO neuroprotection, we sought evidence for EPO-Rs on cultured rat cerebrocortical neurons using immunofluorescence and immunoblotting with the same specific anti-EPO-R mAb that we used in vivo6. Double-labeling of cerebrocortical neurons with anti-EPO-R mAb and an antibody against neuron-specific microtubule-associated protein-2 (MAP-2), revealed complete overlap of the two labels (WebFig. 1a, right panel). This anti-EPO-R mAb recognized a single band in immunoblots prepared from soluble EPO-R (Fig. 1b, right) and from lysates of the erythroid cell line UT-7 and HEK293, both of which express EPO-R, but not from lysates of HeLa cells, which do not express the EPO-R (WebFig. 1b, lower left). Anti-EPO-R also recognized a single band of appropriate size in immunoblotted cell lysates prepared from cerebrocortical cultures containing neurons but not from neuron-depleted cultures (WebFig. 1b, upper left). In neuronal cell lysates, immunoprecipitated EPO-R and Jak2 were tyrosine phosphorylated by EPO (Fig. 1c).

Hitherto, it was known that activation of several cytokine receptors, of which the EPO-R is a member, could trigger the Janus kinase-signal transducers and activators of transcription (Jak-Stat) signalling pathway. It was also known that phosphorylated Stats then translocate into the nucleus to act as transcription factors. Based upon results in the present study, we propose that binding of EPO to EPO-R activates a novel form of cross-talk between the well-known Jak2 and NF-kB pathways, in which Jak2 activation leads to IkB phosphorylation. IkB is then either degraded (after serine phosphorylation) or dissociates without degradation (in the case of tyrosine phosphorylation) from NF-kB, which subsequently translocates into the nucleus to act as a transcription factor (WebFig. 2). In this manner, NF-kB participates in EPO-mediated neuroprotection. This EPO signalling cascade may prevent neuronal apoptosis after cerebral infarcts and other neurodegenerative insults involving excessive NMDA receptor activation and NO generation, and thus may have potential therapeutic value.

Methods

Immunofluorescence and immunoblot studies

Frozen sections (20 µm) of rat hippocampus were labelled with anti-EPO-R mAb (Genetics Institute), visualized with horseradish peroxidase (HRP)/diaminobenzidine (DAB), and counterstained with Giemsa. For cultured cerebrocortical cells, neurons were double labelled with anti-EPO-R and the neuronal markers anti-MAP-2 or anti-NeuN. FITC-conjugated secondary antibody was used to visualize the EPO-R, and Texas-Red-conjugated secondary antibody was used for MAP-2 or NeuN. For localization of NF-kB and Jak2, cells were fixed in 4% paraformaldehyde, incubated overnight with anti-NF-kB (p65 subunit) or anti-Jak2 polyclonal antibodies (Santa Cruz Biotechnology), and visualized with FITC conjugated secondary antibody. Neurons were identified with anti-MAP-2 or anti-NeuN, and astrocytes with anti-glial fibrillary acidic protein (GFAP); these markers were visualized with secondary antibodies conjugated to Texas Red or HRP developed with DAB and H2O2. Incubation in 200 µM NMDA overnight yielded neuron-depleted cultures10. Soluble EPO-R protein was resolved on SDS-PAGE and labelled on immunoblots with anti-EPO-R mAb.

Supplementary Figures

Figure 1

(JPG 56.4 KB)

Erythropoietin receptors (EPO-Rs) on rat primary cerebrocortical neurons. a, EPO-Rs in the hippocampus of embryonal rat brain (E19) (arrows). Frozen sections labeled with monoclonal anti-EPO-R (left panel, 400x). Cultured cerebrocortical neurons double labelled with anti-EPO-R (green) and neuronal marker MAP-2 (red; right panel, 630x). b, On western blots with anti-EPO-R mAb, EPO-R protein was virtually absent in neuron-depleted cultures (labelled "") but detectable in cultures with neurons (+; upper left panel). Anti-EPO-R recognized EPO-R protein expressed in the EPO-dependent cell line UT-7 and in HEK 293 cells, but not in HeLa cells, which are known to lack EPO-Rs (bottom left panel). Increasing amounts of soluble EPO-R protein (sEPO-R) were recognized on western blots with anti-EPO-R mAb (right panel). c, EPO incubation induced tyrosine phosphorylation of EPO-R and Jak2 proteins. Cell lysates of cerebrocortical cultures incubated with increasing concentrations of EPO were immunoprecipitated with anti-EPO-R and immunoblotted with anti-phosphotyrosine antibody (p-Tyr; left panel). The same immunoblot was then stripped and reprobed with the anti-EPO-R or anti-Jak2 (right panel).

Figure 2

(JPG 40.8 KB)

Schematic model of EPO-induced NF-kB activation for neuroprotection. EPO binding to the EPO-R induces receptor dimerization, followed by activation of the tyrosine kinase Jak2. In nonneuronal cells, EPO-induced Jak2 kinase activity is known to phosphorylate/activate Stat5. Here we show in neuronal cells that activated Jak2 may directly or indirectly lead to phosphorylation of IkB at tyrosine or serine residues, respectively. Serine phosphorylation may possibly be mediated via IKK complex and is the predominant pathway for NF-kB activation. Jak2-mediated phosphorylation of IkB represents a novel form of cross-talk between the Jak-Stat and NF-kB signalling pathways. Phosphorylation of IkB results in its degradation (after serine phosphorylation) or non-degradory dissociation (after tyrosine phosphorylation) from NF-kB, which then translocates into the nucleus to transcriptionally activate antiapoptotic genes (box at right; SOD, superoxide dismutase).

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Digicaylioglu, M., Lipton, S. Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-κB signalling cascades. Nature 412, 641–647 (2001). https://doi.org/10.1038/35088074

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