Antiapoptotic Effects of Erythropoietin in Differentiated Neuroblastoma SH-SY5Y Cells Require Activation of Both the STAT5 and AKT Signaling Pathways

Moonkyoung Um,Harvey F Lodish

doi:10.1074/jbc.m510943200

Abstract

The hematopoietic cytokine erythropoietin (Epo) prevents neuronal death during ischemic events in the brain and in neurodegenerative diseases, presumably through its antiapoptotic effects. To explore the role of different signaling pathways in Epo-mediated antiapoptotic effects in differentiated human neuroblastoma SH-SY5Y cells, we employed a prolactin receptor (PrlR)/erythropoietin receptor (EpoR) chimera system, in which binding of prolactin (Prl) to the extracellular domain activates EpoR signaling in the cytosol. On induction of apoptosis by staurosporine, Prl supports survival of the SH-SY5Y cells expressing the wild-type PrlR/EpoR chimera. In these cells Prl treatment strongly activates the STAT5, AKT, and MAPK signaling pathways and induces weak activation of the p65 NF-kappaB factor. Selective mutation of the eight tyrosine residues of the EpoR cytoplasmic domain results in impaired or absent activation of either STAT5 (mutation of Tyr(343)) or AKT (mutation of Tyr(479)) or both (mutation of all eight tyrosine residues). Most interestingly, Prl treatment does not prevent apoptosis in cells expressing mutant PrlR/EpoR chimeras in which either the STAT5 or the AKT signaling pathways are not activated. In contrast, ERK 1/2 is fully activated by all mutant PrlR/EpoR chimeras, comparable with the level seen with the wild-type PrlR/EpoR chimera, implying that activation of the MAPK signaling pathway per se is not sufficient for antiapoptotic activity. Therefore, the antiapoptotic effects of Epo in neuronal cells require the combinatorial activation of multiple signaling pathways, including STAT5, AKT, and potentially MAPK as well, in a manner similar to that observed in hematopoietic cells.

Highlights

Indicates that Epo provides neuroprotective effects in the damaged brain during ischemic events and neurodegenerative diseases; the erythropoietin receptor (EpoR) is expressed in several neuronal cell lines and in hippocampal and cortical neurons of rodent and human brains [3,4,5,6]
To verify that the antiapoptotic activity of Epo in SH-SY5Y cells is through prevention of apoptosis, but not through other effects such as promotion of cell growth, apoptosis was induced by staurosporine, a potent
Protein kinase inhibitor that induces apoptosis in many cell types, including SH-SY5Y cells [57, 58]. Using these differentiated SH-SY5Y cells, we showed that Epo through binding to the EpoR significantly alleviates apoptosis induced by staurosporine

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—SH-SY5Y cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum using collagencoated plates (BD Biosciences). SH-SY5Y cells differentiated for 7 days were growth factorstarved for 5 h, harvested with 10 mM EDTA/PBS, treated with Prl (or Epo for the EpoR-overexpressing cells) for 10 min, and lysed in Nonidet P-40 Lysis buffer (5 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40) with protease inhibitors (Roche Applied Science) and 1 mM sodium vanadate. Nuclear Extract Preparation and Electrophoretic Mobility Shift Assay— SH-SY5Y cells differentiated for 7 days were growth factor-starved for 5 h, harvested with 10 mM EDTA/PBS, and treated with Prl (or Epo for the EpoR-overexpressing cells) for the times indicated in the figure legends. Cells were washed three times with PBS, mounted using the Mount media (Vector Laboratories), and analyzed by immunofluorescence microscopy (Nikon Eclipse Inverted TE300) using Openlab (Improvision) software

RESULTS

DISCUSSION

Antiapoptotic activity