Mortalin-2 (mthsp70/PBP74/Grp75), a Novel Mediator of Erythropoietin Signal.

Erythropoietin (EPO) stimulates erythroid growth by enhancing the proliferation, maturation and survival of late stage erythroid progenitor cells. Intracellular signaling molecules such as Janus kinase 2 (JAK2) and phosphoinositide-3 kinase (PI-3K)/Akt are considered mediators of the EPO signal, however the entire process of EPO stimulation remains undetermined. To further clarify the intracellular mechanism by which EPO affects the growth of erythroid progenitor cells, we analyzed protein obtained from purified human erythroid colony forming cells (ECFCs) cultured with, or without EPO, by 2-dimensional gel electrophoresis. Five protein spots were apparently related to EPO stimuli, and these proteins were then examined by peptide mass fingerprinting. One of the 5 was identified as mortalin-2, which is a member of the heat shock protein 70 family of chaperones. Mortalin-2 is thought to perform multiple functions relevant to stress response, intracellular trafficking, antigen processing, control of cell proliferation and differentiation. When the expression of mortalin-2 was confirmed by Western blotting, the amount of mortalin-2 was found to be greater in ECFCs cultured with EPO. The amount of mortalin-2 mRNA was then analyzed by quantitative real time PCR. In the presence of EPO, the amount of mortalin-2 mRNA gradually increased during erythroid maturation, peaked on day 6, and then decreased in the terminal stage of maturation. When day 6 ECFCs were incubated with different concentrations of EPO, the amount of mortalin-2 mRNA increased in a dose-dependent manner. Next, we designed a small interfering RNA (siRNA) to RNA encoding mortalin-2, and used this in a transient transfection assay. When the siRNA was transfected into day 6 ECFCs, the expression of mortalin-2 mRNA was suppressed to 40–60% of that seen in the control culture. In the presence of different concentrations of EPO (0–1.0 u/ml), the effect of the siRNA on ECFC growth was determined by MTT assay. The siRNA significantly suppressed the growth of ECFCs in the presence of relatively low concentrations of EPO (0.5 u/ml). These data suggest that mortalin-2 mediates the EPO signal to stimulate the growth of erythroid progenitor cells.