Effect of erythropoietin on the gene expression of Xenopus tropicalis peripheral blood cells.
The regulatory system of erythropoiesis through erythropoietin (EPO) and its receptor (EPOR) is essential for vertebrates' life. In humans, EPO affects the erythroid progenitors and stimulates proliferation and differentiation. The EPO-EPOR axis is mainly mediated by the JAK2-STAT5 pathway. After terminal maturation, erythrocytes lost EPOR expression; however, erythrocytes of amphibian Xenopus tropicalis maintain EPOR expression even after their terminal maturation. Because erythrocytes of vertebrates except for adult mammals are nucleated, we hypothesized that EPO can alter its transcriptional state. To explore the effects of EPO on Xenopus mature erythrocytes, we performed RNA-Seq analysis on EPO-stimulated Xenopus peripheral blood cells. The EPO-stimulated group showed increased expression of direct target genes of STAT such as cish, socs3, and socs1 indicating a functional signal transduction system. Furthermore, we observed several EPO-responsive genes that were not reported in mammalian erythroid progenitors. These findings suggest the functional difference between enucleated erythrocytes in adult mammals and nucleated erythrocytes in fetal mammals and non-mammalian vertebrates. Overall design: Xenopus peripheral blood was corrected from the heart. Recombinant Xenopus tropicalis was EPO expressed in E. coli. The control group was cultured in 7/9 x IMDM with 10% fetal calf serum. EPO added group was cultured in the same condition supplemented with 1 ng/mL EPO. Fresh blood cells before culture and cells cultured for 1.5 or 4.0 hours were harvested and their total RNA was extracted by the acid guanidine phenol -chloroform method. Three biological replicates were made for each experimental group. We then performed gene expression profiling analysis using the data obtained from 3' RNA-Seq analysis.