rho gtpases in hematopoiesis and hemopathies · mammalian rho gtpases are a family of small...

Review article

Rho GTPases in hematopoiesis and hemopathiesJames C. Mulloy,1 Jose A. Cancelas,1 Marie-Dominique Filippi,1 Theodosia A. Kalfa,1 Fukun Guo,1 and Yi Zheng1

1Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, OH

Rho family GTPases are intracellular sig-naling proteins regulating multiple path-ways involved in cell actomyosin organi-zation, adhesion, and proliferation. Ourknowledge of their cellular functionscomes mostly from previous biochemicalstudies that used mutant overexpressionapproaches in various clonal cell lines.Recent progress in understanding RhoGTPase functions in blood cell develop-ment and regulation by gene targeting ofindividual Rho GTPases in mice has al-lowed a genetic understanding of their

physiologic roles in hematopoietic pro-genitors and mature lineages. In particu-lar, mouse gene–targeting studies haveprovided convincing evidence that indi-vidual members of the Rho GTPase fam-ily are essential regulators of cell type–specific functions and stimuli-specificpathways in regulating hematopoieticstem cell interaction with bone marrowniche, erythropoiesis, and red blood cellactin dynamics, phagocyte migration andkilling, and T- and B-cell maturation. Inaddition, deregulation of Rho GTPase fam-

ily members has been associated withmultiple human hematologic diseasessuch as neutrophil dysfunction, leuke-mia, and Fanconi anemia, raising thepossibility that Rho GTPases and down-stream signaling pathways are of thera-peutic value. In this review we discussrecent genetic studies of Rho GTPasesin hematopoiesis and several blood lin-eages and the implications of RhoGTPase signaling in hematologic malig-nancies, immune pathology. and ane-mia. (Blood. 2010;115:936-947)

Introduction

Mammalian Rho GTPases are a family of small GTP-bindingproteins containing 22 members that are involved in many impor-tant cellular functions, including gene transcription, survival,adhesion, and cytoskeleton reorganization.1-3 They are closelyrelated to Ras and share considerable structure–function similari-ties with Ras and other Ras-related small GTPases. As shown inFigure 1, similar to Ras, most Rho GTPases switch between theinactive GDP-bound form and the active GTP-bound form by amechanism that is strictly regulated by the upstream guaninenucleotide exchange factors (GEFs). Once activated, the GTP-bound forms are capable of interacting with multiple downstreameffector proteins in a spatially and temporally controlled manner.Conversely, GTPase-activating proteins (GAPs) stimulate the hy-drolysis of bound GTP to GDP, switching Rho GTPases back to theinactive state. Rho GDP-dissociation inhibitors can sequesterinactive GDP-bound Rho proteins in the cytosol and prevent theiractivation and intracellular trafficking. Further adding to thecomplexity of this tightly regulated mechanism, multiple RhoGEFs, Rho GAPs and Rho GDIs can activate or inactivate the sameRho GTPase, and each Rho GTPase may activate multipledownstream effectors, initiating a network of signals that affectscell functions.4,5

The role of Rho GTPase signaling in blood cell developmentand function was first implicated through studies of Rho regulatorsand effectors.5-7 GEFs such as Vav1, GAPs such as Bcr, andeffectors such as WASP, are among the best documented ones.Although the use of dominant negative or constitutively active RhoGTPase mutant overexpression has led to some important observa-tions in several blood lineages (Table 1), the inferred functions ofRho GTPases in blood development remained unclear because ofthe nonspecific and dose-dependent nature of this approach. Recentmouse gene–targeting studies have extended these studies to

members of the Rho GTPases themselves,3,5,6 further showingmany unique and redundant functions that each Rho GTPase playsin different blood lineages (Table 1). In this review we focus on ourcurrent understanding of Rho GTPases, mainly the Rac, Cdc42,Rho, and RhoH subfamilies that have been most extensivelystudied, in hematopoietic stem/progenitor cell engraftment, erythro-poiesis and myelopoiesis, lymphopoiesis, and phagocyte regula-tion, with an emphasis on their contributions to both physiologicand pathologic conditions.

Rho GTPases in hematopoietic stem andprogenitor cell regulation

Hematopoietic stem and progenitor cell (HSC/P) fate is finelyregulated to initiate the production of billions of blood cells everyday. The endosteal space of adult bone marrow (BM) containsputative “niches” where a specialized microenvironment supportsand nurtures HSC/Ps.8-10 There has been a tremendous advance-ment in knowledge about the molecular pathways involved inHSC/P regulation over the past few years, including that oftrafficking to and from the BM niche.11 The cell functions involvedin HSC/P homing, engraftment, and mobilization include cytoskel-eton rearrangements, migration, transcription activation, survival,and cell-cycle progression. These events are regulated by multipleRho GTPases, particularly Rac1, Rac2, Cdc42, RhoA, and RhoH(Figure 2).

HSC/P homing, trafficking, and interaction with the BMmicroenvironment

HSC/P homing and migration activities require Rac GTPases(Table 1). Rac1 and Rac2 integrate signals from �1-integrins and

Submitted August 31, 2009; accepted October 27, 2009. Prepublished onlineas Blood First Edition paper, November 24, 2009; DOI 10.1182/blood-2009-09-198127.

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c-kit in HSC/Ps.12,13 CXCL12-induced chemoattraction is mediatedby both Rac1 and Rac2 and is at least partly mediated by the GEFTiam-1.14 c-Kit signals to Rac1 and Cdc42 at least in part throughthe GEF Vav1.15 Initial studies in Rac2�/� mice have shown thatRac2 is important for HSC/P adhesion and migration, and Rac2�/�

mice showed increased numbers of circulating HSC/Ps and in-creased migration,16 suggesting an important role for Rac2 inretaining HSC/Ps in the BM niche. Further, Rac2 can affect Rac1and Cdc42 activities, and such a cross talk between Rac2 and otherRho GTPases is involved in regulating HSC/P migration.16,17

Although Rac1 and Rac2 are highly homologous, they canregulate distinct HSC/P functions. Rac1 is required for engraftmentof HSC/Ps into the BM,12 attributable specifically to its role inhoming. Rac1 deficiency caused defective homing and localizationof HSC/Ps in the BM endosteal space after transplantation ofRac1�/� HSC/Ps into wild-type recipients, whereas Rac2 defi-ciency decreased the retention of HSC/Ps in the BM.16,18 Deletionof both Rac1 and Rac2 led to a mobilization of HSC/Ps from BM toperipheral blood because of decreased adhesion and retention in theBM.12,18 These data strongly suggest that at the molecular levelHSC/P homing and retention in the BM are not mirror imageprocesses of each other, and both activities are subject to Rac1 andRac2 regulation. In this aspect, Rac1, but not Rac2, appears tocontrol fetal HSC/P trafficking. In the absence of Rac1, the numberof circulating HSC/Ps in the blood of E10.5 embryos is severelydiminished; consequently, embryonic hematopoiesis is signifi-cantly impaired in Rac1-deficient embryos as manifested by theabsence of intraaortic clusters and near complete absence of fetalliver hematopoiesis.19 These deficiencies correlate with a decreasedmigration in response to CXCL12 and impaired interaction withthe microenvironment-derived stromal cells.19

Cdc42 also uniquely regulates HSC/P trafficking and residencein the BM niche. Gene-targeted mice deficient in Cdc42 GAP,a negative regulator of Cdc42, exhibited disorganized F-actinstructure, reduced adhesion and directional migration, and defec-tive engraftment in HSC/P population.20 Cdc42�/� mice showedincreased numbers of circulating HSC/Ps and hyperactivecell-cycle profiles, as well as profound defects in adhesion,

migration, and homing activities (Table 1).21 Interestingly, agedHSC/Ps contain increased Cdc42 activity, and adhesion ofHSC/Ps to the BM microenvironment is reduced in aged micesimilar to that in Cdc42GAP�/� mice. Granulocyte colony-stimulating factor–induced HSC/P mobilization is also in-creased in aged mice, and this effect may be a consequence ofthe increased Cdc42 activity.22

RhoH constitutes a distinct subtype of Rho GTPases that isGTPase deficient and hematopoietic specific; it has been shown tomodulate Rac1 signaling and function in primary HSC/Ps.23-25

RhoH gene targeting led to increased HSC/P chemotaxis andchemokinesis toward the chemoattractant CXCL12. This migratoryresponse is due to increased Rac1 activity and translocation ofRac1 protein to the cell membrane, where it colocalizes withcortical F-actin and lipid rafts. Conversely, overexpression ofRhoH in HSC/Ps blocks the membrane translocation of Rac1 andimpairs cortical F-actin assembly and chemotaxis in response toCXCL12 stimulation (Table 1). It is possible that RhoH plays anopposite role from Rac1 and Rac2 in regulating HSC/P BMresidency.25

Proliferation, survival, and self-renewal of HSC/Ps

Rac and Cdc42 GTPases are also required for proliferation andsurvival of HSC/Ps. Deletion of Cdc42 in the BM resulted in a lossof quiescence of long-term repopulating–HSCs and a transientaccumulation and mobilization of short-term repopulating–HSCs,effects that are associated with transcription changes of �1-integrin, p21Cip, and c-Myc.21 Gain of function of Cdc42, in theCdc42GAP knockout mice, caused elevated c-Jun N-terminalkinase (JNK) activity and increased apoptosis in the HSC/Pcompartment.20 However, Rac1-deficient HSC/Ps showed defec-tive proliferative signaling by the c-Kit receptor tyrosine kinase,whereas loss of Rac2 activity led to a proapoptotic phenotype.12

Double-deficient Rac1�/�;Rac2�/� HSC/Ps showed a combinationof reduced proliferation and increased apoptosis. It remains to beseen if any of the previously implicated Rac1 effectors, includingPAK, POR1, IQGAP, and WAVE, and downstream transcriptionregulators such as p42/p44 extracellular signal-regulated kinases,p38, JNK, Akt, and/or �-catenin12,16,26-28 are directly involved inmediating Rac1/Rac2 proliferative signals in maintaining thehealthy state of HSC/Ps. Interestingly, Rac1 and a GAP termedMcgRacGAP are also required for nuclear localization of Stat5athrough direct interaction,29 suggesting a role for Rac in regulationof transcription factor translocation to the nucleus and control ofgene expression.

The effect of RhoA GTPase on hematopoiesis has been lessstudied compared with Rac1, Rac2, and Cdc42. RhoA activationcan induce stress fiber formation and cell shape changes,decrease expression of Cdk inhibitors, and promote S phasecell-cycle progression in fibroblasts.1-3 However, the effect ofRhoA on cell cycle and proliferation may be both cell type andagonist specific. Thus far, the role of RhoA in HSC/P functionshas only been indirectly studied (Table 1). Inhibition of RhoAactivity through expression of the dominant-negative mutantRhoAN19 by retrovirus-mediated gene transfer of murine BMcells was associated with a significant enhancement of HSC/Pengraftment in vivo.30 More recently, HSC/Ps from p190-B-RhoGAP–deficient mice, which exhibit elevated RhoA activity,were found to have increased HSC self-renewal activity in serialtransplantation experiments.31 This activity was independent ofcell-cycle control; rather, p190-B seemed to affect HSC fatedecision during cell division. These studies suggest that HSC

Receptor

Plasma membrane

Effectors

Rho-GTP

RhoGDI

Ligand

Rho-GDP

RhoGEFRhoGAP

Cytoskeleton organization AdhesionCell cycle Survival Transcription

Figure 1. Intracellular signaling model of Rho GTPases in receptor-initiatedpathways. Most Rho GTPases cycle between the GDP-bound, inactive state and theGTP-bound, active state. The GTP binding and GTP hydrolysis cycle is tightlyregulated by Rho guanine nucleotide exchange factors (GEFs), Rho GTPase-activating proteins (GAPs), and Rho GDIs (GDP dissociation inhibitors), which alsocontrol their intracellular localization patterns. On activation by cytokine, chemokine,growth factor, or adhesion molecules, the GTP-bound Rho GTPases can transientlyinteract with a large panel of effector proteins to transduce signals that affect cellcycle, survival, transcription, adhesion, and cytoskeleton machineries.

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Table 1. Effects of dominant negative mutant expression or gene targeting of Rho GTPases on hematopoietic cell functions. Please see thefollowing additional references: Akbar et al127; Flevaris et al128; McCarty et al129; Savina et al130; Zhang et al131; Fulkerson et al132; Yoshida et al133;Lammermann et al134; Xu et al135; Subauste et al136. NA indicates data not available. **Only Rho GTPase gene-targeted mouse models are included.

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maintenance probably requires a tightly regulated RhoA activ-ity. Further analysis of RhoA function awaits the development ofa RhoA knockout model.

Rho GTPases in myelopoiesis anderythropoiesis

The production of mature blood cells of the different lineagesrequires the differentiation of HSC/Ps through a successive seriesof increasingly lineage-restricted intermediate progenitors. Theearliest step of commitment occurs at the level of the commonlymphoid progenitor (CLP), and the common myeloid progenitor(CMP). CLPs differentiate into T, B, and natural killer cells,32

whereas CMPs give rise to the granulocyte-macrophage progenitor(GMP) and the megakaryocyte-erythroid progenitor (MEP).33 Thisdifferentiation program is regulated by an intertwining transcrip-tion factor network, tightly controlled by both dosage and timing.34-36

The role of Rho GTPases in regulating specific steps ofmyelopoiesis and erythropoiesis has just begun to be appreciated.The involvement of Rac in myelopoiesis has been examined invitro with the use of isolated HSC/Ps from conditional knockoutmice, and it appears that Rac1 and Rac2 GTPases regulate uniqueaspects of hematopoietic development. Rac1�/�;Rac2�/� as well asRac1�/� HSC/Ps displayed reduced proliferation in response tostem cell factor, and this is associated with reduced cyclin D1 anddecreased p42/p44 extracellular signal-regulated kinase phosphor-ylation.12 Increased apoptosis was noted in Rac1�/�;Rac2�/� andRac2�/� progenitors, which was associated with reduced AKTactivation after stem cell factor stimulation.12 How such observa-tions relate to in vivo myeloid cell development remains unclear,because Rac2�/� and Rac1�/�;Rac2�/� mice show neutrophiliaand defective mast cell functions.12,27,37

In erythropoiesis, Rac GTPases were shown to be essential forenucleation using constitutively active or dominant-negative mu-tants of Rac1 and Rac2 in cultured mouse fetal liver erythroblasts,in which either excessive activation or inhibition of Rac GTPases

inhibits enucleation.38 Pharmacologic inhibition of Rac GTPasesby NSC23766, a specific Rac inhibitor,39 was also shown to inhibitenucleation by disruption of the interaction of Rac with thediaphanous related-formin mDia2, leading to a disappearance ofcontractile actin ring in enucleating erythroblasts.38 However,enucleation is not decreased in Rac1�/�;Rac2�/� erythroblasts, inwhich up-regulated Rac3 may compensate for Rac1 and Rac2deficiency. It is possible that the redundant activities of Rac1/Rac2/Rac3 GTPases collectively regulate the enucleation process.

Combined Rac1 and Rac2 deficiency in mice caused hemolyticanemia as evidenced by concurrent reticulocytosis, fragmented redblood cells (RBCs), and decreased deformability under shearstress, indicating an erythrocyte cytoskeleton defect. Indeed,Rac1�/�;Rac2�/� RBCs showed disturbed actin distribution in thecytoskeleton, with gaps and aggregates of F-actin and irregularclumping of band 3.40 Adducin, the capping protein of the actinoligomers at the rapidly exchanging barbed ends, is hyperphospho-rylated on serine-724, a target of protein kinase C,41 thus facilitat-ing uncapping with a potential promotion of actin polymerizationas well as actin dissociation from spectrin40 (Figure 3). Althoughthis pathology may be related to abnormal assembly of thecytoskeleton during erythropoiesis, a dynamic regulation of theactin oligomers by Rac GTPases during the life of the mature RBCsin the blood stream is an attractive possibility in explaining theremarkable deformability shown by normal erythrocytes undershear stress. The reticulocytosis that provides partial compensationof hemolytic anemia observed in Rac1�/�;Rac2�/� mice is pro-duced by increased erythroid progenitors and precursors in thespleen, whereas MEPs along with erythroid colony-forming unit(CFU-E) activity are significantly decreased in the BM.42 Theseresults indicate that Rac1 and Rac2 are essential for homeostaticerythropoiesis in the BM but are dispensable for stress erythropoi-esis in the spleen.

Cdc42 has emerged as an essential regulator of the balancebetween myelopoiesis and erythropoiesis. Mice with deletion ofCdc42GAP (and therefore increased Cdc42 activity) displayed areduction of BM cellularity with decreased granulocyte, erythroid,

Figure 2. Role of Rho GTPases in HSC/P homing,engraftment, and mobilization. Rac1, Rac2, Cdc42,and Rho control different hematopoietic stem and progeni-tor cell (HSC/P) functions. Signals required for HSCself-renewal are mediated by Rho, Cdc42, and Rac1.Although Rac1 appears to be required for proliferation,Rac2 controls survival. Cdc42 is necessary for cell-cycleentry of quiescent (G0) HSC/Ps in the bone marrow (BM)microenvironment, proliferation, and aging of HSC/Ps.Rac1 and Cdc42 control homing and interaction with theBM niche, whereas combined Rac1 and Rac2 activitiesand Cdc42 are necessary for the retention of HSC/Ps inthe BM.





macrophage, megakaryocyte CFU; erythroid burst-forming unit,and CFU-E activities and anemia.20 This decrease in erythroid andmyeloid progenitors correlated with increased apoptosis associatedwith increased JNK activity. Conditional deletion of Cdc42 in micealtered the frequency and number of primitive committed progeni-tors, leading to an increase in the GMP population and a decrease inthe MEP population and significantly reduced erythroid burst-forming unit and CFU-E activities in Cdc42�/� BM cells.43

Hematopoietic-specific Cdc42�/� mice develop a fatal myeloprolif-erative disorder manifested by significant leukocytosis with neutro-philia in the peripheral blood along with myeloid hyperprolifera-tion in the BM and spleen and myeloid cell infiltration of otherorgans, including lungs and liver.43 In parallel, severe anemia isnoted in these mice, caused by a block in the early stages oferythropoiesis and associated with both proliferation and survivalof Gr1�/Mac1� cells. Because survival and proliferation in theerythroblast stages appeared normal, it is likely that Cdc42 deletionaffects the erythroid differentiation program at a stage beforeproerythroblasts, such as the MEP level. These effects in bothmyeloid and erythroid lineages are probably caused by a biasedgene transcription program in the Cdc42-deficient progenitors,including up-regulation of promyeloid genes (PU.1, C/EBP1�, andGfi-1) in CMP, GMP, and MEP populations and down-regulation ofproerythroid transcription factor GATA-2 in short-term repopulating-HSC, CMP, and MEP populations.43 Addressing how Cdc42 elicitssignaling events to affect the key transcription factor network is animportant subject for future studies.

Rho GTPases in lymphopoiesis

The role of Rho GTPases in lymphocyte biology was firstappreciated by overexpressing dominant-negative and/or activeforms of Rho GTPases in clonal cell lines (Table 1). With the use ofthis approach, Rho GTPases were suggested to mediate T-cellpolarity, cytotoxic responses, spreading, migration, cytokine secre-tion, and T-cell receptor (TCR) and B-cell receptor (BCR) signal-ing.44-48 Analysis of Rac1 transgenic mice has shown that Rac1regulates T-cell development by promoting the transition fromCD4�CD8� double-negative stage III (DN3) to DN4 and bydiverting CD4�CD8� double-positive thymocytes from positive tonegative selection.49,50 Examination of Rac2 and Cdc42 transgenicmice showed a role for Rac2 and Cdc42 in T-cell survival,51,52

whereas studies of a RhoA transgenic model showed that RhoApromotes positive selection of double-positive thymocytes and

enhanced TCR signaling with increased proliferation on TCRcross-linking.53-55

Rho GTPases in T-cell development and activation

Gene targeting of either Rac1 or Rac2 found no apparent abnormali-ties in T-cell development.56-58 However, when both Rac1 and Rac2are removed, T-cell development is severely impaired at the CLPstage and at checkpoints of � selection and positive selection. Thedevelopmental block is associated with defects in thymocyteproliferation, survival, adhesion, and migration. Further, simulta-neous loss of Rac1 and Rac2 suppresses TCR-mediatedinterleukin-2 production and Akt activation and leads to hyperacti-vation of Notch signaling.57,58 Thus, Rac1 and Rac2 play aredundant but essential role in T-cell development. Rac2�/� miceshow attenuated T helper type 1 differentiation of peripheral T cellsassociated with impaired production of interferon-�.59 Moreover,Rac2 deficiency causes a disruption of actin polymerization andTCR clustering. It also produces a defect in calcium influx and Erkand p38 activation.56 Nonetheless, these defects are minor. Whetherthe defects are attenuated because of a compensatory effect of Rac1remains to be elucidated.

In a case of cross talk among Rho GTPases, overexpression ofRhoH inhibits Rac1- and RhoA-mediated activation of nuclearfactor-�B, as well as p38 mitogen-activated protein kinase signal-ing, in Jurkat T cells.60 Thus, RhoH may function as an antagonistfor the classic Rho GTPases. Physiologic functions of RhoH inT cells have recently been investigated in RhoH knockout mice.These mice display a defective T-cell maturation during transitionfrom DN3 to DN4 and during positive selection.61,62 RhoHdeficiency leads to defective TCR signaling manifested by de-creased activation of CD3�, LAT, PLC�, Vav1, and Erk, and byreduced calcium influx.61,62 Interestingly, RhoH was found tointeract directly with tyrosine kinase Zap70. In the absence ofRhoH, translocation of Zap70 to the immunologic synapse isimpaired.62 RhoH is clearly involved in the early signaling steps ofT cells, and this may be associated with its potential role inlymphocyte malignancies (see “Rho GTPases in hematologicabnormalities”).

Although the Rho family member RhoG shares high homologywith Rac GTPases,63 it has been shown to uniquely regulate genetranscription in both T and B cells and to mediate cytoskeletalreorganization in T cells in response to fibronectin binding.64 RhoGknockout mice did not show a developmental defect in T cells;however, depletion of RhoG resulted in a moderate increase inT-cell proliferation in response to antigen receptor engagement.65

Figure 3. In erythrocytes that lack Rac1 and Rac2 GTPases, there is increased phosphorylation of adducin by protein kinase C, leading to decreased F-actincapping at the barbed ends, dissociation of spectrin from actin, and increased fragility of the RBC cytoskeleton.





Rho GTPases in B-cell development and activation

Our understanding of Rho GTPases in B cells is limited in scope.Rac2-deficient mice show a reduction in B1a and marginal zoneB cells associated with impaired proliferation and calcium influx.66

Furthermore, Rac2 deficiency leads to a decrease in immunoglobu-lin M–secreting plasma cells and hence an attenuated humoralimmune response. In contrast, Rac1 deficiency has minimal effectson B cells.67 However, simultaneous ablation of Rac1 and Rac2 inB cells causes a more severe developmental block than the onecaused by Rac2 deficiency alone.67 The B-cell developmentaldefects in Rac1/Rac2 double knockout mice are likely due to asurvival defect resulting from impaired activation of Akt and areduced expression of antiapoptotic Bcl-xL and blockade ofB cell–activating factor receptor.67 These findings are reminiscentof the overlapping and critical role of Rac1 and Rac2 in T-celldevelopment.57,58

A recent characterization of mice bearing B cell–specific dele-tion of Cdc42 showed that, similar to Rac1 and Rac2 simultaneousloss, Cdc42 deficiency leads to reductions in all splenic B-cellsubpopulations and B1a cells, possibly because of defects inproliferation and B cell–activating factor receptor–mediated sur-vival.68 In contrast to the Rac2 knockout effect, Cdc42 deficiencyhas no effect on B-cell migration.68 Therefore, although bothCdc42 and Rac1/Rac2 are important in late B-cell development inthe spleen by mediating B-cell survival and proliferation, Rac1/Rac2, but not Cdc42, are required for B-cell migration.

Combining the results of overexpression and gene-targetingstudies, the functional requirement of individual members of theRho family in the critical steps of lymphopoiesis is becoming clear(Figure 4).

Rho GTPases in phagocyte regulation

Neutrophils provide the first line of defense by eradicatinginvading microbial pathogens. This is achieved through a series ofhighly coordinated responses of migration, ingestion, and ulti-mately killing of invading microbes. By regulating cytoskeletonrearrangements, Rho GTPases have been implicated as centralregulators of specialized phagocyte functions69,70 (Table 1; Figure 5).

Migration

Directional neutrophil migration in response to a gradient ofchemoattractant allows neutrophils to rapidly arrive at sites ofinfection. This process requires the establishment of a front–rearasymmetry, with a front enriched in filamentous actin and a rearmade of actomyosin filaments. In general, Rac is the crucialregulator of actin polymerization, RhoA promotes myosin cytoskel-eton organization at the rear, whereas Cdc42 is essential in organizingcell polarity, although it is not required for motility per se.69-72

Genetic models have shown that actin polymerization is specifi-cally dependent on Rac2 rather than on Rac1 or Cdc42. WhereasRac2�/� neutrophils exhibit a dramatic defect in polymerized actin,Rac1�/� neutrophils and neutrophils with deregulated Cdc42exhibit normal F-actin polymerization.12,37,73-75 Instead, Rac1 hasbeen shown to play significant roles in neutrophil shape and tailretraction.12,71 Recent work that used primary neutrophils fromCdc42GAP�/� and Cdc42�/� mice further confirm the involvementof Cdc42 in neutrophil polarity.76,77 Mechanistic studies suggest anunexpected mechanism in which CD11b may serve as a secondary

Figure 4. Scheme of Rho GTPase involvements in lymphocyte development. Rac1 and Rac2 are important for common lymphoid progenitor (CLP) differentiation fromhematopoietic stem cells (HSCs) in the BM. Rac1 and Rac2, as well as RhoH, regulate T-cell development in the thymus by affecting �-selection and positive selection. Rac2 isalso required for Th1 differentiation in peripheral lymphoid tissues. Rac1, Rac2, and Cdc42 are critical for multiple stages of B-cell development in the spleen, whereas Rac2,Cdc42, and RhoG also regulate antibody production. DN indicates CD4�CD8� double-negative thymocytes; DP, CD4�CD8� double-positive thymocytes; SP, CD4� or CD8�

single-positive T cells; Th, helper T cells; Tc, cytotoxic T cells; NFB, newly formed B cells; MZB, marginal zone B cells; and FOB, follicular B cells.





signal of Cdc42-mediated polarity to determine the site of actomyo-sin assembly. Whereas the phenotypes of RhoA genetic deletion inphagocytes remain unknown, RhoA distributes at the rear ofprimary neutrophils and may regulate actomyosin contraction.73,78

Closely related to directional movement, gradient sensing,which is necessary to orient the cells toward high chemokineconcentrations, also appears to depend on Rho GTPases but not onphosphatidylinositol 3-kinase, as initially proposed.79 Neutrophilsdeficient in the PAK-associated Rac GEF, PIX�, which exhibitslow Cdc42 activity, fail to orient toward high chemokine (ie, C5a)concentration.80 Interestingly, this is also the case for Rac1�/�

neutrophils, at least under defined experimental conditions.75

The mechanisms enabling macrophage migration are differentfrom those used by neutrophils. For example, Rac1 and Rac2appear to be largely dispensable for macrophage migration.81 It isnot clear how macrophages migrate without Rac1 and Rac2. RhoBmay contribute to this activity, because the speed of RhoB�/�

macrophage migration is significantly impaired compared withwild-type macrophage migration.82

Phagocytosis

The uptake of bacteria by phagocytosis is the first step of bacterialkilling. This process involves either integrin (CD11b or comple-ment receptor 3 [cr3])–mediated or Fc� receptor–mediated internal-ization. Because phagocytosis requires actin rearrangement be-neath the phagocytic cup, Rho GTPases are implicated in thisprocess.83,84 The prevailing view was that Rac and Cdc42, but notRhoA, activities were necessary for Fc� receptor–mediated internal-ization. In contrast, RhoA seemed to be the main regulator ofCR3-mediated uptake, also by controlling actin remodeling, al-though through a distinct structure than the one regulated by Racand Cdc42.85 Recent work that used Rac1/Rac2-null macrophageshave challenged this view.86 The genetic deletion of Rac1 and Rac2prevents both CR3-mediated and Fc� receptor–mediated phagocy-tosis (through defective actin rearrangement). In contrast, studiesthat used C3 transferase to inhibit RhoA in primary macrophagessuggest that RhoA may also affect both Fc�R and CR3-mediatedphagocytosis, but independently of actin polymerization.86 Be-

cause other studies that used Rac2-null macrophages have sug-gested that Rac2 controls Fc� receptor–mediated, but not CR3-mediated, phagocytosis,87 it will be important to further analyze thespecific roles Rho GTPases play in phagocytosis in neutrophils ormacrophages or both.

Bacteria killing

Once bacteria are internalized, the microbicidal function of phago-cytes is largely ascribed to their ability to release reactive oxygenspecies (ROS) and hydrogen peroxide on activation of the reducednicotinamide adenine dinucleotide phosphate (NADPH) oxidasecomplex. This function has been attributed to Rac activity.69,70,88

Under cell-free conditions, both Rac1 and Rac2 can stimulateNADPH oxidase activity. However, genetic deletion of Rac2, butnot Rac1, in neutrophils results in severe defects in ROS produc-tion, although in an agonist-dependent fashion.12,37,76,89,90 Impor-tantly, ectopic expression of Rac1 in Rac2-null neutrophils cannotcomplement for the lack of Rac2 in superoxide production.89,91

Interestingly, Rac2-null macrophages exhibited defects in NADPHoxidase activation in a similar agonist-dependent fashion.87 RhoGhas also been implicated in ROS production. RhoG�/� neutrophilsexhibited a marked impairment in ROS production; however, thiseffect may be through Rac2 protein.92 The roles of other RhoGTPases in NADPH oxidase regulation remain to be analyzed ingene-targeted models. Cdc42 might be an important counter-regulator of Rac2-induced NAPH activation, because inactivationof Cdc42 by the introduction of the Cdc42/Rac interactive bindingdomain of WASP caused a dramatic increased in ROS production.93

Release of neutrophil granule content can also contribute tomicrobicidal activity. Both Rac1 and Rac2 are probably importantduring this process. Rac2�/� neutrophils failed to release primarygranule content in response to chemoattractant stimulation, andRac1 has been shown to play a role in myeloperoxidase release.94,95

The use of mouse infectious or inflammatory models has shownthe importance of Rho GTPases in innate immunity. Rac2�/� miceshow an increased mortality to invasive aspergillosis.37 Mice withRac1�/� neutrophils are protected against neutrophilic lung inflam-mation.73 These analyses of Rho GTPase functions in primary cells

Figure 5. Phagocyte functions in re-sponse to bacterial infections. After sens-ing chemokines released by invading micro-organism, leukocytes rapidly move towardthe site of infection. This process involvesinitial changes in adhesive properties of thecells to the vessel endothelium followed byan extravasation step out of the blood vesseland directed migration into tissue. Once atthe site of infection, cells phagocytize mi-crobes and kill them by degranulation andreactive oxygen species (ROS) release.





derived from genetic models show a more complex picture than theoriginal paradigm (Table 1).

Rho GTPases in hematologic abnormalities

Given their critical role in gene transcription, cell survival,adhesion, and cytoskeleton organization, it is no surprise thatseveral Rho GTPases and their signaling pathways have beenimplicated in hematologic pathogenesis.

Hematologic abnormalities associated with decreased RhoGTPase activity

The first clearly defined mutation of a Rho GTPase family proteinassociated with a hematologic disease was described nearly adecade ago with the observation that an infant with severe immunedefects harbored a missense mutation on 1 allele of the Rac2 gene,resulting in an asparagine residue in place of aspartic acid in theguanine nucleotide binding site.96,97 The patient had recurrentbacterial infections and a severe neutrophilia, phenotypic character-istics closely mimicking that of Rac2�/� mice.37 Biochemical andcell biology experiments showed that the Rac2 D57N mutant behavedas a dominant negative toward both Rac1 and Rac2, effectively ablatingRac activity in blood cells, causing growth and survival defects as wellas engraftment and migration deficiencies.12,18,98

Recently, an association between decreased activity of Cdc42and the hematologic disease Fanconi anemia (FA) has beendescribed.99 FA is an inherited BM failure syndrome caused bymutations of a family of FA genes. Mouse model studies of FA haveconcluded that the defect is at the level of the HSC/P, but themechanisms responsible for the HSC/P failure remain elusive.100-102 Itwas shown that aberrant down-regulation of Cdc42 activity by the lossof the Fanc-A gene could explain the defective homing, adhesion, andmigration phenotype associated with patient-derived BM cells. Onexpression of an active form of Cdc42, the adhesion defect could becorrected.99 These studies implicate Cdc42 in the functional deficiencyof FA and highlight the importance of this Rho GTPase in a hematopoi-etic stem/progenitor deficiency syndrome.

The effector of Cdc42, WASP, has been known to be involved inthe rare immune deficiency disease, Wiskott-Aldrich syndrome.103

Mutations in the WASP gene result in loss of function of the WASp

protein, and the defect seems to be at the level of the HSCs and isassociated with defective migration.104 Clinically, this syndromepresents as an immune disorder with variable susceptibility toopportunistic infections. This is consistent with the idea that loss ofCdc42 effector function in hematopoietic cells can be causal foraberrant development and function of the immune system withassociated immune deficiency and susceptibility to infection.

Hematologic abnormalities associated with increased GTPaseactivity

In contrast to the phenotypes associated with loss of Rho GTPaseactivity, the disease spectrum that is associated with increased RhoGTPase activity is often linked to enhanced proliferation andsurvival in diverse cancers of the blood. In non-Hodgkin lym-phoma and multiple myeloma, transcriptional up-regulation ofRhoH, a constitutively GTP-bound family member that is notregulated by the classic GEF/GAP molecular switch, is effected byrecurrent chromosomal translocations t(3;4) and t(4;14) as well asthrough somatic hypermutation.105,106 The precise result of thesegenetic changes as they relate to the tumor phenotype is not clear,but studies from genetic models and biochemical analyses suggestthat deregulated RhoH signaling may have profound effects on thesurvival, adhesion, and migration properties of cells.107,108 Interest-ingly, RhoH expression was recently found to be elevated in B cellchronic lymphocytic leukemia109 and suppressed in B hairy cellleukemia.110 In these diseases, RhoH depletion or RhoH overexpres-sion, respectively, were effective in reversing the transformingphenotype, suggesting that RhoH may be distinctly involved in theinitiation or maintenance of these B cell lymphoproliferativedisorders.

The role of Rac GTPase signaling in myeloid-associated diseaseis well documented, especially in the context of the p210-BCR-ABL fusion protein and chronic myeloid leukemia. This particularsubject has been the topic of a recent review.111 Rac1 and Rac2appear to function downstream of BCR-ABL and are essential forthe transforming ability of this oncogene (Figure 6). Rac3 has alsobeen shown to play a role downstream of p190 BCR-ABL in thegeneration of lymphoblastic leukemia.112 Although the precisemechanism of activation and the interplay of Rac family membersin the initiation, maintenance, or dissemination of BCR-ABLdisease is not known, the importance of Rac GTPases in chronic

Figure 6. A biochemical model of RacGTPase involvement in hematopoietic celltransformation. Rac1 has unique as well asredundant roles with Rac2 and Rac3 assignal transducers in multiple oncogene ortumor suppressor gene–mediated leukemo-genesis or lymphomagenesis, in addition totheir physiologic roles in mediating receptor-stimulated signaling. They are required forp210 BCR-ABL–induced HSC transforma-tion, and MLL-AF9–induced HSC/GMP trans-formation. Targeting of Rac GTPases hasbeen suggested as a potential therapeuticmeans in the blood malignancies.





myeloid leukemia is clearly documented in mice with the use of agenetic approach and confirmed in human cells with the use of aRac inhibitor.113

Rho GTPase activation in acute myeloid leukemia (AML) hasrecently received significant scrutiny. It has been known for almosta decade that specific translocations in AML target signalingmolecules involved in Rho GTPase activation. For example, atranslocation involving the mixed lineage leukemia (MLL) gene onchromosome 11 was found to target a Rho GEF, LARG.114 TheMLL-LARG fusion protein was shown to activate RhoA and play amechanistic role in the development of the leukemia, implicatingthe constitutive Rho GTPase activity in the pathogenesis ofAML.115 MLL has also been found in a translocation with GRAF, aGAP that is involved in “turning off” RhoA activity.116 TheMLL-GRAF translocation inactivates the GAP activity of GRAF,resulting in constitutive activation of RhoA. A follow-up studyanalyzing GRAF expression in AML samples determined that 38%of samples showed CpG methylation of the GRAF promoter,implicating loss of expression of GRAF in AML development.117 Inaddition to RhoA activation in AML, loss of RhoA activity mayalso be associated with T-cell malignancy. T cell–specific expres-sion of the bacterial toxin C3 transferase, a Rho inhibitor, promotedaggressive thymic lymphoblastic lymphoma in mice.118 Whetherthese differences are due to specific cell lineage requirements or tothe different approaches used to generate the models remains to beexperimentally determined.

It has also been reported that the Rac and Cdc42 GTPases areup-regulated in murine AML cells using a retroviral model ofMLL-AF9 leukemia.119 These observations were subsequentlyconfirmed in another model of this type of leukemia, using primaryhuman rather than murine cells to model AML.120 Inhibition of Racactivity using either a pharmacologic or a knockdown approachwas effective in inducing apoptosis in the human MLL-AF9leukemia cells and showed a significant delay of the progression ofAML induced by a human leukemia cell line.120,121 In addition,primary AML patient samples have been shown to be highlysensitive to Rac GTPase inhibition in vitro, potentially through amechanism requiring stroma cell interaction.122

In addition to these blood malignancies, Rho GTPase activitieshave been suggested to play an important role in the hematologicpathologies associated with certain viral infections, including HIVand � herpesviruses. The HIV protein v-Nef binds to Vav1 and

activates the Rac signaling pathway.123 Down-regulation of Rhoactivity using statins or a geranylgeranyl transferase inhibitor, butnot a farnesyl transferase inhibitor, specifically blocked entry ofHIV-1–pseudotyped viruses, implicating Rho as a potential targetfor antiretroviral therapy.124 In the � herpesvirus MHV-68, the viralprotein M2 is necessary for activating the Vav1/Rac signalingcascade and promoting viral latency in B lymphocytes, a processcritical in the life cycle of this virus and essential for effectivecolonization of the host.125,126

Although much remains to be studied about the specificmechanistic contributions of Rho GTPase signaling that areimportant in blood malignancies, the principle of pharmacologictargeting of Rho GTPase pathways, such as Rac signaling, presentsan exciting new opportunity for future therapy (Figure 6). To thisend, the development of lead inhibitors of this subfamily of RhoGTPases has shown promise, suggesting pharmacologic targetingof aberrant Rac activities may be a valid approach in reversing themalignant phenotypes of leukemia and lymphoma. As a demonstra-tion of this principle, based on the crystal structure of Rac1 and thestructure–function data of Rac1 interaction with GEFs, a smallmolecule inhibitor, NSC23766, was identified that showed specific-ity for a subset of GEF binding to Rac GTPases and blocked Racactivation by several GEFs.39 Although this lead inhibitor could inpart mimic the Rac1 and Rac2 knockout phenotype of HSC/Pmobilization from the BM,18 it displayed potent efficacy insuppressing the elevated Rac1 and Rac2 activities in BCR-ABL orMLL-AF9 oncogene–induced myeloproliferative disorder or AML-like syndrome and significantly slowing the disease progres-sions.113,120,121 These studies strongly imply that pharmacologic agentscan be developed to tackle various pathologic conditions associated withmalregulated activation states of individual Rho GTPases.

Conclusions and perspectives

The mouse gene–targeting approach has been invaluable in under-standing Rho GTPase signaling functions in mammalian hematopoi-esis and has led to our current understanding of some of the uniqueversus overlapping roles of individual Rho GTPases in differentblood cells (Table 1). It is becoming clear that Rho GTPases Rac1,Rac2, Cdc42, RhoA, RhoH, and RhoG, as well as several

Figure 7. A summary of the known in-volvement of Rho GTPases in the regula-tion of hematopoiesis based on mousegene–targeting studies. Cdc42, Rac1,Rac2, RhoH, RhoG, and RhoA are amongthe best understood Rho GTPase familymembers for which mouse gene–targetingmodels have been characterized in variousblood lineages. LTR indicates long-term re-populating; and STR, short-term repopulat-ing. Other abbreviations of various progeni-tors are described in the text.





regulators and effectors, are involved in multiple steps of hemato-poiesis, including stem cell interaction with the BM niche,erythropoiesis, myelopoiesis, and lymphopoiesis, and it is likelythat considerable cross talk may exist between the family membersin defined functions (Figure 7). An emerging theme is that,although individual Rho GTPases may regulate unique aspects ofcell proliferation, survival, or differentiation in defined blood celltypes, they collectively play an essential role in regulating cytoskeletonorganization, adhesion, and migration for most blood cell types.

Because Rho GTPases are capable of mediating signal transduc-tion in multiple pathways, a continuing effort is to define blood celltype– and pathway-specific signaling functions using cell type–restricted knockout models. Inducible knockout or knockin mousemodels under the control of endogenous promoters, as well ascross-breeding among various Rho GTPase or Rho GTPaseregulator knockouts and other signaling component-gene–targetedmice, will help clarify the relationship between the Rho GTPasesand other signaling networks. The small interfering RNA technol-ogy will be complementary to the mouse gene–targeting studies inanalyzing Rho GTPase functions in human blood cells and may beespecially suitable in determining the contributions of overex-pressed or elevated levels of individual Rho GTPases in humanblood malignancies. It can be expected that the mouse geneticstudies will produce useful animal models to implicate specific RhoGTPase–mediated signaling pathways in multiple human blood

abnormalities, including neutropenia, anemia, leukemia, lym-phoma, and immune deficiency. With our growing understandingof the molecular mechanisms of Rho GTPase signaling importantin various physiologic processes and disease states, the signaltransduction pathways involving Rho GTPases and the cascades ofprotein–protein interactions as well as enzymatic activities can bepursued as drug targets for future therapeutic benefits.

Acknowledgments

We thank Dr David A. Williams for a critical reading of themanuscript, and Mr Xuan Zhou for a draft of the figures.

This work was supported by grants from the National Institutesof Health.

Authorship

Contribution: J.C.M., J.A.C., M.-D.F., T.A.K., F.G., and Y.Z.surveyed the literature, generated the figures, and wrote the paper.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Yi Zheng, Division of Experimental Hematol-ogy and Cancer Biology, Children’s Hospital Medical Center,3333 Burnet Ave, Cincinnati, OH 45229; e-mail: [email protected].

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online November 24, 2009 originally publisheddoi:10.1182/blood-2009-09-198127

2010 115: 936-947

ZhengJames C. Mulloy, Jose A. Cancelas, Marie-Dominique Filippi, Theodosia A. Kalfa, Fukun Guo and Yi Rho GTPases in hematopoiesis and hemopathies

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