Epo Modulation of Cell Cycle Regulatory Genes Via PY343/STAT5 and PI3K Pathways in Primary Erythroblasts.

Erythropoietin’s essential role in late erythropoiesis is ascribed to survival effects on colony forming unit-erythroid-derived erythroblasts. However, several studies have pointed to possible Epo regulation of cell cycle factors, and proliferation. To investigate this issue, developmentally-staged Kit^posCD71^high erythroblasts were prepared from murine marrow; were shown to proliferate in response to low-dose Epo (0.1U/ml); and were used in the Affymetrix 430 2.0 array-based discovery of Epo-modulated cell cycle regulatory genes. In keeping with certain prior studies, myelocytomatosis oncogene (c-Myc) was induced while cyclin-dependent kinase inhibitor 1B (p27) was inhibited several-fold. Beyond this, four select cell cycle progression factors were rapidly up-modulated up to five-fold by Epo: nuclear protein 1 (Nupr1), G1 to S phase transition 1 (Gspt1), early growth response 1 (Egr1) and Ngfi-A binding protein 2 (Nab2). B-cell leukemia 6 (Bcl6) in contrast, was down-modulated several-fold. Finally, among cyclins, D2 was up-modulated ~5-fold while G2 was selectively down-modulated 4- to 5-fold (All findings were confirmed by RT-PCR). Studies in erythroblasts expressing knocked-in minimal erythropoietin receptor (EpoR) alleles indicated EpoR proximal tyrosine-343 (PY343) regulation of cyclin D2 induction, and EpoR C-terminal regulation of cyclin G2 inhibition. Despite the capacity of a PY-null EpoR-HM allele to support steady-state erythropoiesis, EpoR-HM erythroblasts proved to be skewed in cell cycle phases, especially in G2/M (0.01% vs ~5% for wt-EpoR and EpoR-H alleles). For cyclin D2, cyclin G2, p27 and Bcl6, in silico models for promoter regulation are outlined which highlight Stat and Forkhead action. In summary, specific new evidence is provided for erythroblast cell cycle entry modulation by Epo (especially as elevated during anemia) via discrete EpoR subdomains, and nine selectively modulated cell cycle regulatory genes.