Erythropoietin mediates tissue protection through an erythropoietin and common beta-subunit heteroreceptor

Abstract

The cytokine erythropoietin (Epo) is tissue-protective in preclinical models of ischemic, traumatic, toxic, and inflammatory injuries. We have recently characterized Epo derivatives that do not bind to the Epo receptor (EpoR) yet are tissue-protective. For example, carbamylated Epo (CEpo) does not stimulate erythropoiesis, yet it prevents tissue injury in a wide variety of in vivo and in vitro models. These observations suggest that another receptor is responsible for the tissue-protective actions of Epo. Notably, prior investigation suggests that EpoR physically interacts with the common beta receptor (betacR), the signal-transducing subunit shared by the granulocyte-macrophage colony stimulating factor, and the IL-3 and IL-5 receptors. However, because betacR knockout mice exhibit normal erythrocyte maturation, betacR is not required for erythropoiesis. We hypothesized that betacR in combination with the EpoR expressed by nonhematopoietic cells constitutes a tissue-protective receptor. In support of this hypothesis, membrane proteins prepared from rat brain, heart, liver, or kidney were greatly enriched in EpoR after passage over either Epo or CEpo columns but covalently bound in a complex with betacR. Further, antibodies against EpoR coimmunoprecipitated betacR from membranes prepared from neuronal-like P-19 cells that respond to Epo-induced tissue protection. Immunocytochemical studies of spinal cord neurons and cardiomyocytes protected by Epo demonstrated cellular colocalization of Epo betacR and EpoR. Finally, as predicted by the hypothesis, neither Epo nor CEpo was active in cardiomyocyte or spinal cord injury models performed in the betacR knockout mouse. These data support the concept that EpoR and betacR comprise a tissue-protective heteroreceptor.

Fig. 1.

Despite having no affinity for CEpo, EpoR is bound to a CEpo affinity column but within a complex. (A) Rat brain membrane proteins sequentially purified over a lentil lectin column and Epo or CEpo affinity columns were subjected to EpoR Western blotting in either a reduced or nonreduced state. Bands (≈64 kDa) consistent with the EpoR were visualized only under reduced conditions. S, soluble EpoR-positive control (29 kDa); E, Epo affinity column; C, CEpo affinity column. (B) Membranes prepared from heart, kidney (kid), and liver show results similar to those for brain membranes but as a distinct doublet. (C) In contrast, cell membranes obtained from TF-1 cells and run under nonreducing conditions show the doublet EpoR.

Fig. 2.

EpoR and βcR are present as a complex in neuronal lysates. Immunoprecipitation of membranes prepared from P19 mouse neuroblastoma cells demonstrate that either anti-EpoR or anti-βcR pulls down a protein consistent with βcR (≈130 kDa), as well as a smaller molecular species. Equivalent results were obtained in the presence or absence of Epo.

Fig. 3.

Cells exhibiting tissue protection express both the EpoR and βcR subunit. (A) Spinal cord neurons within the central gray obtained from normal mice show prominent staining of the somata for both proteins. (B) Choroid plexus (arrow), as well as the ependymal cell layer (arrowhead), exhibit prominent staining for both EpoR and βcR. EpoR staining appears punctate. Radial glia (double arrow) are also doubly immunoreactive. (C) Purkinje cells of the cerebellum stain densely for EpoR in a punctate manner (Left), whereas anti-βcR stains somata, as well as the proximal dendrites (arrow), more diffusely. Note that neuronal somata in the molecular layer, as well as granule cells, are also immunopositive for both proteins. Right illustrates negative control (primary antibody omitted). (D) Sections of myocardium obtained from normal mice show prominent EpoR and βcR immunostaining. Sections obtained from the βcR knockout mouse exhibit prominent EpoR immunoreactivity. CTL EpoR is a negative control in which the primary antibody was omitted.

Fig. 4.

CEpo restores motor function after spinal compression in wild-type mice but not in strain-matched βcR knockout mice. (A) Spinal cord morphology is normal in the βcR knockout mouse (hematoxylin/eosin-stained). (B) βcR knockout mice subjected to spinal cord compressive injury do not respond to either Epo or CEpo (10 μg/kg of body weight) given i.p. as a single dose immediately after injury.

Fig. 5.

rhEpo is tissue-protective of staurosporine-induced apoptosis for cardiomyocytes derived from wild-type cells but not identically prepared cells from βcR knockout mice. rhEpo was added (100 ng/ml) 30 min before the addition of staurosporine (0.1 μg/ml). Each condition corresponds to between four and eight replications. ^***, P < 0.001 vs. staurosporine alone.