Jovencio Borneo

Deficiency of the Hematopoietic Cell-Specific Rho Family GTPase Rac2 Is Characterized by Abnormalities in Neutrophil Function and Host Defense

In mammals, the Rho family GTPase Rac2 is restricted in expression to hematopoietic cells, where it is coexpressed with Rac1. Rac2-deficient mice were created to define the physiological requirement for two near-identical Rac proteins in hematopoietic cells. rac2-/- neutrophils displayed significant defects in chemotaxis, in shear-dependent L-selectin-mediated capture on the endothelial substrate Glycam-1, and in both F-actin generation and p38 and, unexpectedly, p42/p44 MAP kinase activation induced by chemoattractants. Superoxide production by rac2-/- bone marrow neutrophils was significantly reduced compared to wild type, but it was normal in activated peritoneal exudate neutrophils. These defects were reflected in vivo by baseline neutrophilia, reduced inflammatory peritoneal exudate formation, and increased mortality when challenged with Aspergillus fumigatus. Rac2 is an essential regulator of multiple specialized neutrophil functions.

Rac and Cdc42 GTPases control hematopoietic stem cell shape, adhesion, migration, and mobilization

Critical to homeostasis of blood cell production by hematopoietic stem/progenitor (HSC/P) cells is the regulation of HSC/P retention within the bone marrow microenvironment and migration between the bone marrow and the blood. Key extracellular regulatory elements for this process have been defined (cell–cell adhesion, growth factors, chemokines), but the mechanism by which HSC/P cells reconcile multiple external signals has not been elucidated. Rac and related small GTPases are candidates for this role and were studied in HSC/P deficient in Rac2, a hematopoietic cell-specific family member. Rac2 appears to be critical for HSC/P adhesion both in vitro and in vivo, whereas a compensatory increase in Cdc42 activation regulates HSC/P migration. This genetic analysis provides physiological evidence of cross-talk between GTPase proteins and suggests that a balance of these two GTPases controls HSC/P adhesion and mobilization in vivo.

Rac2 Stimulates Akt Activation Affecting BAD/Bcl-XL Expression while Mediating Survival and Actin Function in Primary Mast Cells

Mast cells generated from Rac2-deficient (-/-) mice demonstrated defective actin-based functions, including adhesion, migration, and degranulation. Rac2(-/-) mast cells generated lower numbers and less mast cell colonies in response to growth factors and were deficient in vivo. Rac2(-/-) mast cells demonstrated a significant reduction in growth factor-induced survival, which correlated with the lack of activation of Akt and significant changes in the expression of the Bcl-2 family members BAD and Bcl-XL, in spite of a 3-fold induction of Rac1 protein. These results suggest that Rac2 plays a unique role in multiple cellular functions and describe an essential role for Rac2 in growth factor-dependent survival and expression of BAD/Bcl-XL.

Balanced Interactions between Lyn, the p85α Regulatory Subunit of Class IA Phosphatidylinositol-3-Kinase, and SHIP Are Essential for Mast Cell Growth and Maturation

ABSTRACT The growth and maturation of bone marrow-derived mast cells (BMMCs) from precursors are regulated by coordinated signals from multiple cytokine receptors, including KIT. While studies conducted using mutant forms of these receptors lacking the binding sites for Src family kinases (SFKs) and phosphatidylinositol-3-kinase (PI3K) suggest a role for these signaling molecules in regulating growth and survival, how complete loss of these molecules in early BMMC progenitors (MCps) impacts maturation and growth during all phases of mast cell development is not fully understood. We show that the Lyn SFK and the p85α subunit of class IA PI3K play opposing roles in regulating the growth and maturation of BMMCs in part by regulating the level of PI3K. Loss of Lyn in BMMCs results in elevated PI3K activity and hyperactivation of AKT, which accelerates the rate of BMMC maturation due in part to impaired binding and phosphorylation of SHIP via Lyns unique domain. In the absence of Lyns unique domain, BMMCs behave in a manner similar to that of Lyn- or SHIP-deficient BMMCs. Importantly, loss of p85α in Lyn-deficient BMMCs not only represses the hyperproliferation associated with the loss of Lyn but also represses their accelerated maturation. The accelerated maturation of BMMCs due to loss of Lyn is associated with increased expression of microphthalmia-associated transcription factor (Mitf), which is repressed in MCps deficient in the expression of both Lyn and p85α relative to controls. Our results demonstrate a crucial interplay of Lyn, SHIP, and p85α in regulating the normal growth and maturation of BMMCs, in part by regulating the activation of AKT and the expression of Mitf.

Deficiency of Src family kinases compromises the repopulating ability of hematopoietic stem cells

OBJECTIVE Src family kinases (SFK) have been implicated in regulating growth factor and integrin-induced proliferation, migration, and gene expression in multiple cell types. However, little is known about the role of these kinases in the growth, homing, and engraftment potential of hematopoietic stem and progenitor cells. RESULTS Here we show that loss of hematopoietic-specific SFKs Hck, Fgr, and Lyn results in increased number of Sca-1(+)Lin(-) cells in the bone marrow, which respond differentially to cytokine-induced growth in vitro and manifest a significant defect in the long-term repopulating potential in vivo. Interestingly, a significant increase in expression of adhesion molecules, known to coincide with the homing potential of wild-type bone marrow cells is also observed on the surface of SFK(-/-) cells, although, this increase did not affect the homing potential of more primitive Lin(-)Sca-1(+) SFK(-/-) cells. The stem cell-repopulating defect observed in mice transplanted with SFK(-/-) bone marrow cells is due to the loss of Lyn Src kinase, because deficiency of Lyn, but not Hck or Fgr, recapitulated the long-term stem cell defect observed in mice transplanted with SFK(-/-) bone marrow cells. CONCLUSIONS Taken together, our results demonstrate an essential role for Lyn kinase in positively regulating the long-term and multilineage engraftment of stem cells, which is distinct from its role in mature B cells and myeloid cells.

RAC2 Regulates the activation of akt and the balance between pro- and anti-apoptotic proteins bad and bcl-xl

Abstract We have previously demonstrated that the hematopoietic cell specific Rho GTPase Rac2 plays not only an essential role in regulating multiple actin-based functions of bone marrow derived mast cells, such as adhesion to fibronectin, migration, and degranulation but is also critical to mast cell survival. To determine the extent of involvement of signaling pathways in increased apoptosis due to Rac2 deficiency, we examined the activation of PI-3Kinase and its downstream effector, Akt, in response to SCF-stimulation. Although, PI-3Kinase activation in response to SCF stimulation was identical in Rac2−/− compared with wild-type (wt) mast cells, the activation of Akt was completely abolished in Rac2−/− mast cells. Unexpectedly, the level of the proapoptotic protein, Bad was significantly elevated (∼10-fold) in Rac2−/− cells in comparison to wt. In contrast, the level of anti-apoptotic protein Bcl-Xl was significantly reduced (∼10-fold) in Rac2−/− cells in comparison to wt. To confirm that enhanced apoptosis in mast cells was the direct result of Rac2 deficiency, Rac2−/− mast cells were transduced with a retrovirus expressing a wt Rac2 cDNA. As expected, Rac2−/− cells expressing Rac2 as a transgene demonstrated complete correction of all actin-based functions and normal survival of mast cells in response to SCF-stimulation. In addition, the activation of Akt and the level of Bad and Bcl-Xl protein were restored to wt levels. In summary, these data demonstrate that Rac2 not only plays an essential role in hematopoietic cell actin-mediated functions, but also plays a critical role in maintaining the survival of these cells by regulating the activation of Akt and the balance between pro- and anti-apoptotic proteins, Bad and Bcl-Xl.

The hematopoietic-specific rho gtpase, RAC2, is essential for normal adhesion and controlled movement of hematopoietic progenitor cells In vitro And In vivo

Abstract The mechanism of hematopoietic progenitor cell (HPC) migration and mobilization from bone marrow (BM) to peripheral blood (PB) has not yet been clearly defined. Since we have previously reported that absence of the Rho GTPase Rac2 results in defective movement and adhesion of mast cells and neutrophils, we speculated that the Rac2 protein might play a pivotal role in this process. Since previous reports have implicated the integrin VLA4 in HPC mobilization, we measured adhesion of purified HPC (mature cell lineage antigen negative (Lin−) c-kit+Sa-1+ cells isolated from Rac2−/− and wild type (WT) mice) to the VLA4 binding site, CS-1, on the recombinant HPC to this peptide was observed (21±5% in Rac2−/− cells, vs 61±8% in WT cells, P In vitro analysis of directed movement (modified Boyden chambers) by these cells towards stromal cell-derived factor-1 (SDF-1) demonstrated that, in contrast to the response of differentiated hematopoietic cells, migration of Rac2−/− HPC was increased 4-fold, compared with WT cells (12% versus 3% of cells migrated, respectively, P in vivo was investigated by analysis of mobilization in Rac2−/− versus WT mice. Injection of 250μg of G-CSF/kg/day over 5 days resulted in mobilization of 2.8-fold more progenitors into PB of Rac2−/− mice compared with WT. Simultaneous injection of anti-VLA4 antibody with G-CSF into Rac2−/− mice resulted in significantly reduced augmentation of mobilization, compared with WT mice ( in vitro colony forming cells in PB=50% of WT; day 8 CFU-S in PB=69% of WT), consistent with defective VLA4-mediated function in Rac2−/− HPC. These data demonstrate that Rac2 is essential for normal adhesion and controlled movement of HPC, both in vivo and in vitro , and implicate this GTPase downstream of the integrin VLA-4. The data suggest that lack of Rac 2 in HPC may be associated with increased activity of other GTPases, possibly Cdc42.

Role of rac 2 in hemopoietic stem cell motility

Abstract Rho GTPases (Rho, Rac, and Cdc42) act as molecular switches to regulate cellular functions. We have recently generated a mouse deficient in Rac 2, a GTPase which is expressed only in hematopoietic cells (Roberts et al. Immunity, 1999), and shown defects in PMN movement and other actin-based functions in vivo and in vitro . We now demonstrate that Rac 2 is expressed in purified hematopoietic progenitor/stem cells (HSC) (lin−,c−kit+Sca-1+). Analysis of the function of Rac 2−/− HSC shows multiple abnormalities in vitro and in vivo . Adhesion to the extracellular matrix molecule fibronectin via integrin VLA-4 is significantly reduced. Surprisingly, movement of progenitor/stem cells in response to SDF-1 is increased in both modified Boyden chamber assays and in quantitative time-lapsed video imaging. This increased movement is associated with increased size of filopodia, and increased f-actin content, reminiscent of enhanced activity of Cdc42, suggesting cross-talk between GTPase pathways. Importantly, these abnormalities are mirrored by changes in cell movement in vivo . G-CSF-induced mobilization of hematopoietic progenitor cells is enhanced nearly 3-fold in Rac 2−/− mice, while the augmentation of G-CSF mobilization by concurrent administration of antibodies to VLA-4 is reduced, further supporting the role of Rac 2 in VLA-4 function in stem/progenitor cells localization in the bone marrow microenvironment. These data demonstrate that Rac 2 is critical for normal adhesion and movement of HSC, and suggest that deficiency of Rac 2 in these cells may lead to altered activity of other GTPases, particularly Cdc42.