Erythropoietin promotes cell proliferation in Neuro-2A mouse neuroblastoma cells

Erythropoietin (Epo) is a glycoprotein hormone primarily known for regulating red blood cell production. Beyond erythropoiesis, Epo has been reported to exert tissue-protective effects, including neuroprotective and neuroregenerative properties. While its erythropoietic activity is mediated by the Epo receptor (EpoR), its tissue-protective function has been suggested to involve alternative receptor(s), the existence of which remains unclear. To investigate the effects of Epo on neuronal cell growth and explore potential tissue-protective receptors in the future, we stably expressed the human <i>EPO</i> gene in the mouse neuroblastoma cell line Neuro-2A (N2A) and hippocampal neuronal cell line HT22 and investigated the effects of Epo overexpression on neuronal cell proliferation. ELISA and Western blot analyses confirmed successful Epo overexpression in both cell lines. Notably, N2A cells overexpressing Epo (N2A^+Epo) exhibited a rapid increase in cell number compared to vector-only controls, suggesting Epo-stimulated cell proliferation. Consistent with this observation, N2A^+Epo cells showed increased BrdU incorporation and elevated levels of cell proliferation markers, including proliferating cell nuclear antigen (PCNA) and nucleophosmin (NPM1), as revealed by Western blot analysis, compared to the vector-only controls. These findings indicate increased DNA synthesis and cell proliferation in N2A^+Epo cells. Additionally, CRISPR-Cas9-mediated knockdown of <i>EPO</i> resulted in the abrogation of cell proliferation, whereas <i>EPOR</i> knockdown showed no effect, suggesting that Epo promotes N2A cell proliferation through an EpoR-independent mechanism. Collectively, these findings highlight the potential of the N2A^+Epo cell line as a model for identifying alternative tissue-protective Epo receptors.