The Cbl E3 Ubiquitin Ligase Negatively Regulates Erythropoiesis through Expression of the Foxo3a Transcription Factor.
Authors: Richmond Terri D , Bailey Monica L , Langdon Wallace Y , Barber Dwayne 
Published: April 2020

Abstract
Erythropoietin (EPO) is the primary cytokine regulator of red blood cell (RBC) progenitor growth, survival and differentiation. EPO stimulation is regulated by EPO binding to its cognate ligand, the EPO receptor (EPO-R), and activating the primary associated tyrosine kinase, JAK2. The critical importance of EPO, EPO-R and JAK2 to erythropoiesis is demonstrated by the fatal embryonic anemia that develops upon EPO, EPO-R or JAK2 deletion. Intracellular signal transduction pathways regulating growth, survival and differentiation downstream of the EPO-R and JAK2 are well documented. However, relatively little is known about down-regulation of EPO-R signal transduction pathways at this time. Our laboratory has previously demonstrated that EPO stimulation leads to Cbltyrosine phosphorylation and subsequent recruitment of Crk-C3G, leading to Rap1activation. In addition, Cbl serves as an adaptor protein linking to PI 3 kinase and Rasand targets receptor tyrosine kinases for ubiquitination and proteasomal degradation. Cbl knockout mice have been generated and have defects in stem and T cell signaling pathways. Elevated platelet numbers and splenomegaly was observed, suggesting that Cbl −/− mice may have defects in megakaryocyte/erythroid progenitors or more committed cells in each lineage. The objective of this studyis to determine whether Cbl affects erythropoiesis and EPO-dependent signaling. Resting Cbl −/− mice (in the C57Bl/6 background) have increased numbers of Burst Forming Unit-Erythroid and Colony Forming Unit-Erythroid (CFU-E) cells. Furthermore, there is a 3-fold elevation of splenic CFU-E numbers. Erythroid differentiation was monitored via expression of the Transferrin Receptor (CD71) and Ter119. Cbl-deficient mice have delayed differentiation in the bone marrow with diminished CD71-Ter119+ cells. Increased apoptosis is observed in Ter119+ erythroid cells isolated from Cbl −/− mice as determined by Annexin V staining and confirmed by increased PARP cleavage. Interestingly, reactive oxygen species in wild type and Cbl-deficient mice remain unchanged. Despite normal resting hematologic parameters, serum EPO concentrations are elevated in Cbl knockout mice. Serum VEGF levels are comparable between wild type and Cbl −/− mice, suggesting that the EPO effect is specific to the erythroid lineage and not an effect of hypoxia. Notable differences in wild type and Cbl −/− mice were observed when stress erythropoiesiswas induced by phenylhydrazine-mediated anemia. Cbl-deficient mice respond with enhanced hematocrit recovery and increased reticulocyte production. EPO-dependent Aktphosphorylation is hypersensitive in Cbl −/− splenic erythroblasts. Interestingly, expression ofFoxo3a was stabilized in Cbl −/− splenic erythroblasts, suggesting that Cbl degrades Foxo3a in a direct or indirect manner. Given the importance of Foxo3a in regulating erythropoiesis, we are currently determining whether Cbl targets Foxo3a for ubiquitin-mediated degradation. These data demonstrate the remarkable homeostatic ability of the mouse to retain normal RBC concentrations in the peripheral blood despite elevated erythroid progenitors and cell signaling. Importantly, these studies are the first to phenotypically explore the effects of genetic ablation of an EPO-responsive E3 ubiquitin ligase in erythropoiesis.