Erythroid Specific RhoA Deficiency Causes Dysplastic and Inefficient Fetal Erythropoiesis with Lethal Embryonic Anemia Due to Defects in Erythroblast Cytokinesis and Enucleation

Abstract 82

RhoA GTPase is known to regulate cell adhesion, actomyosin cytoskeleton, and cytokinesis (Schwartz, J Cell Sci 2004). We recently demonstrated evidence that enucleation, the process through which orthochromatic erythroblasts expel their nucleus during the final stage of mammalian erythropoiesis, is a multi-step process resembling asymmetric cytokinesis. It requires establishment of cell polarity through microtubule function, followed by formation of a contractile actomyosin ring, and coalescence of lipid rafts between reticulocyte and pyrenocyte. We showed that RhoA-related Rac GTPases organize actin in the actomyosin ring and aggregate lipid rafts in the furrow between nascent reticulocyte and pyrenocyte during enucleation (Konstantinidis et al, Blood 2012).

Based on the resemblances between erythroblast enucleation and cytokinesis we hypothesized that RhoA dynamically controls erythroblast enucleation by molecular pathways analogous to the ones involved in cytokinesis. To define the mechanistic contribution of RhoA signaling in erythropoiesis and enucleation, we generated mice with erythroid specific deletion (EpoR^GFPcre/+ driven) of RhoA. Erythroid-specific deletion of RhoA caused severe anemia, which was fatal in utero by E14.5. These embryos as well as their yolk sacs had a pallid, anemic appearance and their peripheral primitive blood cells were large with significant poikilocytosis and anisocytosis, frequently binuclear or multinuclear, and with incomplete clearance of the Golgi network and mitochondria. RhoA^Δ/Δ fetal livers at E12.5 had approximately 33% of the cellularity of WT littermates (n=9, p<0.001). Analysis of definitive erythropoiesis in the RhoA^Δ/Δ fetal liver showed that despite decreased cellularity, the erythroid progenitors BFU-E and CFU-E were not different in total number per fetal liver. In contrast, the erythroblast populations, as determined by CD71-Ter119-FSC flow cytometry as well as by multispectral high-speed cell imaging in flow, demonstrated progressive decline and significantly decreased reticulocyte production (n=3, p<0.05), compatible with a fatal intrauterine anemia. RhoA-deficient fetal liver erythroblasts expressed significantly higher levels of the α4 and α5 integrin subunits, suggesting a compensatory increase of the adhesion receptors due to impaired downstream signaling. We further examined the erythroblastic islands in RhoA^Δ/Δ fetal liver by transmission electron microscopy and found that RhoA-deficient erythroblasts, frequently dysplastic and binucleated, were more loosely associated with the central macrophage and there was a paucity of intracellular vacuoles in comparison to WT erythroblasts, indicating possible defects in vesicular transport, that has also been shown to play a role in erythroblast enucleation (Keerthivasan et al, Blood 2010). Upon evaluation of the RhoA protein level and phosphorylated myosin regulatory light chain (pMRLC) by immunoblotting of RhoA^Δ/Δ fetal liver cells, pMRLC was found significantly decreased, indicating that one of the signaling pathways regulated by RhoA in erythropoiesis involves phosphorylation of MRLC. Moreover, treatment of wild type erythroblasts in in vitro erythropoiesis assays with the pharmacological inhibitor Y27632 of Rho-associated protein kinase (ROCK), a downstream target of RhoA participating in myosin regulation, resulted in a decrease of enucleation by up to 38% (n=3, p<0.05).

Thus, RhoA GTPase regulates erythroblast differentiation and enucleation via regulation of the actomyosin cytoskeleton by ROCK-mediated phosphorylation of MRLC. In addition, RhoA may also regulate erythroblast adhesive interactions with the central macrophage in erythroblastic islands and intracellular vesicular transport. Further exploration of the role of RhoA in erythropoiesis has the potential to reveal targets for therapeutic interventions for anemias due to terminal erythroid maturation defects as well as for improving the efficiency of red blood cell production in vitro.