Physical Interaction between the Erythropoietin and the Glucocorticoid Receptors during the Differentiation of Primary Human Erythroblasts.
Authors: Stellacci E. , Di Noia A. , Migliaccio A.R. , Battistini A. , Migliaccio G. 
Published: April 2020

Abstract
Glucocorticoids are capable to modulate erythroid differentiation in vivo and in vitro. The reversible inhibition exerted by these factors on the maturation of CD36high CD235alow pro-erythroblasts was exploited to establish HEMA, a culture system that allows massive production (108−10 cells) of erythroblasts from as little as 10 mL of adult peripheral blood (G. Migliaccio et al. Blood Cells Mol Dis28:169, 2002). How glucocorticoids block erythroid differentiation is not known. We hypothesized that this effect could be due to physical interaction of the glucocorticoid receptor (GR) either with the erythropoietin receptor (EPO-R), or with one of its downstream signalling partners. To test this hypothesis, CD36high CD235alow proerythroblasts were generated in HEMA culture, growth factor starved for up to 4 hrs and then stimulated either with EPO (3U/mL), Dexamethasone (DXM, 10−6M), or the combination of both, up to 4 hrs. Next, we undertook a series of immunoprecipitation (IP), followed by appropriate western blots (WB), of lysates from cells exposed to the different conditions. Both EPO and DXM rapidly (within 15 min) phosphorylated STAT-5, as shown by IP for STAT-5 followed by WB for P-STAT-5. In contrast, stimulation of the cells with both EPO and DXM for up to 4 hrs did not result in STAT-5 phosphorylation. On the other hand, IP with EPO-R was positive for EPO-R WB under all the conditions, but positive for P-STAT-5 WB and GR WB when cells were stimulated either with EPO or with DXM. The amount of GR detected by WB in EPO-R IP from cells stimulated with both EPO and DXM was greatly reduced (see Figure). In contrast, in STAT-5 IP, EPO-R was WB only in cells stimulated with EPO, P-STAT-5 was WB in cells treated with EPO or DXM, and GR was WB mainly in cells treated with DXM. In agreement with these results, IP for P-Tyr were positive for EPO-R only from cells treated with EPO, but were WB for GR with all the conditions. The fact that P-STAT-5 was WB in EPO-R IP from cells treated with EPO or with DXM, while EPO-R was WB in STAT-5 IP only from cells treated with EPO, is consistent with a model according to which, since EPO-R IP is positive for GR by WB, P-STAT-5 is IP with EPO as part of the EPO-R/GR/P-STAT-5 complex. These results suggest that EPO-R and GR are present in CD36high CD235alow cells stimulated with either EPO or DXM as a multicomplex, P-STAT-5 becames part of this complex in association with either EPO-R or GR, depending on the stimulus, EPO or DXM, to which the cells are exposed. Stimulation with both factors, leads to dissociation of the EPO-R/GR complex and neither of the two receptors is capable to stimulate phosphorylation of STAT-5. We propose that glucocorticoids interfere with erythroid differentiation by a non-genomic mechanism that involves physical interaction with EPO-R.
Figure 1: Summary of the IP with EPO-R, STAT-5 and P-Tyr specific antibody processed by WB for EPO-R, P-STAT-5 and GR, as indicated. A model summarizing the results is described on the top. Figure 1:. Summary of the IP with EPO-R, STAT-5 and P-Tyr specific antibody processed by WB for EPO-R, P-STAT-5 and GR, as indicated. A model summarizing the results is described on the top.