RESUMO
SWAP-70 and DEF6, two proteins that feature similar domain and motif arrangements, are mainly known for their functions in differentiated hematopoietic cells. Both proteins interact with and regulate RhoGTPases and F-actin dynamics, yet their role in hematopoietic stem and precursor cells (HSPCs) remained unexplored. Here, the role of the SWEF proteins SWAP-70 and DEF6 in HSPCs was examined. Both SWEF proteins are expressed in HSCs. HSCs and different precursor populations were analyzed in mice deficient for SWAP-70, DEF6, SWAP-70 and DEF6 (double knockout, DKO), and wild-type controls. HSPCs isolated from these strains were used for competitive adoptive transfer into irradiated wild-type mice. Reconstitution of the myeloid and lymphoid lineages in the recipient mice was determined. The numbers of HSPCs in the bone marrow of Swap-70-/- and Swap-70-/-Def6-/- mice were >3-fold increased. When transplanted into lethally irradiated wild-type recipients, the reconstitution potential of Swap-70-/- HSPCs was intrinsically impaired in competing with wild-type HSPCs for contribution to hematopoiesis. Def6-/- HSPCs show wild type-like reconstitution potential under the same transplantation conditions. DKO HSPCs reconstituted to only 25% of wild-type levels, indicating a partial rescue by DEF6 deficiency in the Swap-70-/- background. Our study reveals the two SWEF proteins as important contributors to HSPC biology. Despite their similarity these two proteins regulate HSC/progenitor homeostasis, self-renewal, lineage contributions and repopulation in a distinct and mostly antagonistic manner.
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Hematopoese , Células-Tronco Hematopoéticas/citologia , Antígenos de Histocompatibilidade Menor/genética , Proteínas Nucleares/genética , Actinas/metabolismo , Animais , Linfócitos B/citologia , Medula Óssea/metabolismo , Transplante de Medula Óssea , Linhagem da Célula , Proliferação de Células , Separação Celular , Feminino , Citometria de Fluxo , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T/citologiaRESUMO
Pentatransmembrane glycoprotein prominin-1 (CD133) is expressed at the cell surface of multiple somatic stem cells, and it is widely used as a cell surface marker for the isolation and characterization of human hematopoietic stem cells (HSCs) and cancer stem cells. CD133 has been linked on a cell biological basis to stem cell-fate decisions in human HSCs and emerges as an important physiological regulator of stem cell maintenance and expansion. Its expression and physiological relevance in the murine hematopoietic system is nevertheless elusive. We show here that CD133 is expressed by bone marrow-resident murine HSCs and myeloid precursor cells with the developmental propensity to give rise to granulocytes and monocytes. However, CD133 is dispensable for the pool size and function of HSCs during steady-state hematopoiesis and after transplantation, demonstrating a substantial species difference between mouse and man. Blood cell numbers in the periphery are normal; however, CD133 appears to be a modifier for the development of growth-factor responsive myeloerythroid precursor cells in the bone marrow under steady state and mature red blood cells after hematopoietic stress. Taken together, these studies show that CD133 is not a critical regulator of hematopoietic stem cell function in mouse but that it modifies frequencies of growth-factor responsive hematopoietic progenitor cells during steady state and after myelotoxic stress in vivo.
Assuntos
Antígenos CD/metabolismo , Células da Medula Óssea/metabolismo , Glicoproteínas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células Progenitoras Mieloides/metabolismo , Peptídeos/metabolismo , Antígeno AC133 , Animais , Células da Medula Óssea/citologia , Primers do DNA/genética , Citometria de Fluxo , Imunofluorescência , Fluoruracila , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Estimativa de Kaplan-Meier , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Especificidade da Espécie , Imagem com Lapso de TempoRESUMO
UNLABELLED: Background The regulation of normal and stress-induced erythropoiesis is incompletely understood. Integrin-dependent adhesion plays important roles in erythropoiesis, but how integrins are regulated during erythropoiesis remains largely unknown. DESIGN AND METHODS: To obtain novel insights into the regulation of erythropoiesis, we used cellular and molecular approaches to analyze the role of SWAP-70 and the control of integrins through SWAP-70. In addition, mice deficient for this protein were investigated under normal and erythropoietic stress conditions. RESULTS: We show that SWAP-70, a protein involved in cytoskeletal F-actin rearrangements and integrin regulation in mast cells, is expressed in hematopoietic stem cells and myeloid-erythroid precursors. Although Swap-70(-/-) mice are not anemic, erythroblastic differentiation is perturbed, and SWAP-70 is required for an efficient erythropoietic stress response to acute anemia and for erythropoietic recovery after bone marrow transplantation in irradiated mice. SWAP-70 deficiency impairs colony-forming unit erythroid development, while burst-forming unit erythroid development is normal, and significantly affects development of late erythroblasts in the spleen and bone marrow. The α(4) integrin is constitutively hyper-activated in Swap-70(-/-) colony-forming unit erythroid cells, which hyper-adhere to fibronectin. Blocking α(4) and ß(1) integrin chains in vivo restored erythroblastic differentiation and the erythropoietic stress response in Swap-70(-/-) mice. Conclusions Our study reveals that SWAP-70 is a novel regulator of integrin-mediated red blood cell development and stress-induced erythropoiesis.