The Rho guanosine triphosphatases (GTPases) Rac1 and Rac2 regulate actin cytoskeleton, microtubule dynamics, gene transcription, proliferation, and survival in multiple cell types. They have been shown to have overlapping as well as distinct roles in actin organization, cell survival, and proliferation in various hematopoietic cell lineages. However their role in erythroid precursors and erythrocytes has not been evaluated. Using gene-targeted mice we have found that deficiency of Rac1 and Rac2 GTPases causes a significant phenotype in erythroid lineage: mild anemia with relative reticulocytosis, both hemolytic (abnormal structure of the erythrocyte cytoskeleton with decreased mechanical stability) and dyserythropoietic in nature. Erythropoiesis is affected mainly at the early stages, when stem cell factor (SCF) plays a critical role. We therefore hypothesize that Rac GTPases (1) dynamically regulate the structure and function of the erythrocyte cytoskeleton, and (2) play a critical role in homing, proliferation, survival, and differentiation of erythroid precursors, likely intersecting signaling pathways activatedvia the SCF receptor, c-Kit. To test our hypotheses, we propose a systematic characterization of the effects of Rac GTPase deficiency on the actin polymerization mechanics in the RBC cytoskeleton, with analysis of the actin-associated proteins and evaluation of possible cross-talk of Rac with other Rho GTPases and signaling molecules. In parallel, we will investigate the perturbations of erythropoiesis in Rac1"/";Rac2"/" mice, seeking the mechanism/s by which Rac GTPases, integrating multiple signals from one or more of the erythropoietic cytokines, affect erythropoiesis. These two proposed aims intent to explore and test how Rac GTPases regulate erythroid development, morphogenesis, and structure-function relation of the RBC cytoskeleton and its components. The central goal is to define critical molecular signaling pathways that participate in normal erythroid development and RBC structural membrane biology. In the long-term, this research is expected to offer new perspectives on the pathogenesis of hemolytic anemia and provide potential therapeutic targets in human disease characterized by abnormal erythropoiesis and decreased RBC survival.
|Konstantinidis, Diamantis G; Pushkaran, Suvarnamala; Giger, Katie et al. (2014) Identification of a murine erythroblast subpopulation enriched in enucleating events by multi-spectral imaging flow cytometry. J Vis Exp :|
|George, Alex; Pushkaran, Suvarnamala; Konstantinidis, Diamantis G et al. (2013) Erythrocyte NADPH oxidase activity modulated by Rac GTPases, PKC, and plasma cytokines contributes to oxidative stress in sickle cell disease. Blood 121:2099-107|
|Konstantinidis, Diamantis G; Pushkaran, Suvarnamala; Johnson, James F et al. (2012) Signaling and cytoskeletal requirements in erythroblast enucleation. Blood 119:6118-27|
|Mizukawa, Benjamin; George, Alex; Pushkaran, Suvarnamala et al. (2011) Cooperating G6PD mutations associated with severe neonatal hyperbilirubinemia and cholestasis. Pediatr Blood Cancer 56:840-2|
|Hammill, Adrienne M; Risinger, Mary A; Joiner, Clinton H et al. (2011) Compound heterozygosity for two novel mutations in the erythrocyte protein 4.2 gene causing spherocytosis in a Caucasian patient. Br J Haematol 152:780-3|
|George, Alex; Pushkaran, Suvarnamala; Li, Lina et al. (2010) Altered phosphorylation of cytoskeleton proteins in sickle red blood cells: the role of protein kinase C, Rac GTPases, and reactive oxygen species. Blood Cells Mol Dis 45:41-5|
|Kalfa, Theodosia A; Pushkaran, Suvarnamala; Zhang, Xiaoling et al. (2010) Rac1 and Rac2 GTPases are necessary for early erythropoietic expansion in the bone marrow but not in the spleen. Haematologica 95:27-35|