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David A. Williams - One of the best experts on this subject based on the ideXlab platform.
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inhibition of rac gtpase signaling and downstream prosurvival bcl 2 proteins as combination targeted therapy in mll af9 leukemia
Blood, 2011Co-Authors: Benjamin Mizukawa, Yi Zheng, Chad E Harris, Junping Wei, Mahesh Shrestha, Mark Wunderlich, Fusheng Chou, Andrea Griesinger, Ashish Kumar, David A. WilliamsAbstract:The Rac family of small Rho GTPases coordinates diverse cellular functions in hematopoietic cells including adhesion, migration, cytoskeleton rearrangements, gene transcription, proliferation, and survival. The integrity of Rac signaling has also been found to critically regulate cellular functions in the initiation and maintenance of hematopoietic malignancies. Using an in vivo gene targeting approach, we demonstrate that RAC2, but not Rac1, is critical to the initiation of acute myeloid leukemia in a retroviral expression model of MLL-AF9 leukemogenesis. However, loss of either Rac1 or RAC2 is sufficient to impair survival and growth of the transformed MLL-AF9 leukemia. RAC2 is known to positively regulate expression of Bcl-2 family proteins toward a prosurvival balance. We demonstrate that disruption of downstream survival signaling through antiapoptotic Bcl-2 proteins is implicated in mediating the effects of RAC2 deficiency in MLL-AF9 leukemia. Indeed, overexpression of Bcl-xL is able to rescue the effects of RAC2 deficiency and MLL-AF9 cells are exquisitely sensitive to direct inhibition of Bcl-2 family proteins by the BH3-mimetic, ABT-737. Furthermore, concurrent exposure to NSC23766, a small-molecule inhibitor of Rac activation, increases the apoptotic effect of ABT-737, indicating the Rac/Bcl-2 survival pathway may be targeted synergistically.
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RAC2 GTPase deficiency depletes BCR-ABL+ leukemic stem cells and progenitors in vivo
Blood, 2010Co-Authors: Amitava Sengupta, David A. Williams, Jorden Arnett, Susan K. Dunn, Jose A. CancelasAbstract:Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disease (MPD) initiated by p210-BCR-ABL–mediated transformation of hematopoietic stem cells (HSCs). Inhibition of the ABL kinase alone is not sufficient to eradicate leukemic stem cells (LSCs). We have previously shown that the deficiency of RAC2 GTPase signaling, but not Rac1, in p210-BCR-ABL–transduced hematopoietic cells prolonged survival of mice with MPD. Here we demonstrate that absence of RAC2 GTPase prolongs survival of HSC-initiated, inducible Scl/p210-BCR-ABL (Scl/p210) binary transgenic mice, it induces apoptosis, and, unlike in normal HSC and progenitor (HSC/P), impairs LSC and progenitor (LSC/P) proliferation in vivo. As a result, RAC2 deficiency causes functional exhaustion of the LSC pool in vivo. This defect is not due to impaired interaction with the hematopoietic microenvironment as reflected by its unaltered adhesion, migration, and homing to recipient organs. In summary, RAC2 deficiency exhausts the LSC pool in vivo through impairment of oncogene-induced proliferation and survival signals.
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Rac1 and RAC2 GTPases are necessary for early erythropoietic expansion in the bone marrow but not in the spleen
Haematologica, 2010Co-Authors: Theodosia A. Kalfa, Jose A. Cancelas, David A. Williams, Suvarnamala Pushkaran, James F. Johnson, Xiaoling Zhang, Dao Pan, Deidre Daria, Hartmut Geiger, Yi ZhengAbstract:Background The small Rho GTPases Rac1 and RAC2 have both overlapping and distinct roles in actin organization, cell survival, and proliferation in various hematopoietic cell lineages. The role of these Rac GTPases in erythropoiesis has not yet been fully elucidated. Design and Methods Cre-recombinase-induced deletion of Rac1 genomic sequence was accomplished on a RAC2 -null genetic background, in mouse hematopoietic cells in vivo . The erythroid progenitors and precursors in the bone marrow and spleen of these genetically engineered animals were evaluated by colony assays and flow cytometry. Apoptosis and proliferation of the different stages of erythroid progenitors and precursors were evaluated by flow cytometry. Results Erythropoiesis in Rac1−/−;RAC2−/− mice is characterized by abnormal burst-forming unit-erythroid colony morphology and decreased numbers of megakaryocyte-erythrocyte progenitors, erythroid colony-forming units, and erythroblasts in the bone marrow. In contrast, splenic erythropoiesis is increased. Combined Rac1 and RAC2 deficiency compromises proliferation of the megakaryocyte-erythrocyte progenitor population in the bone marrow, while it allows increased survival and proliferation of megakaryocyte-erythrocyte progenitors in the spleen. Conclusions These data suggest that Rac1 and RAC2 GTPases are essential for normal bone marrow erythropoiesis but that they are dispensable for erythropoiesis in the spleen, implying different signaling pathways for homeostatic and stress erythropoiesis.
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vav3 and RAC2 activation are required for b progenitor transformation induced by p190 bcr abl
Blood, 2009Co-Authors: Kyunghee Chang, David A. Williams, Amitava Sengupta, Jorden Arnett, Susan K. Dunn, Abel Sanchezaguilera, Jose A. CancelasAbstract:Abstract 449 The Philadelphia chromosome-positive (Ph + ) hematologic malignancies are characterized by the reciprocal translocation between the BCR serine/threonine kinase and the ABL tyrosine kinase genes. Fifty percent of adult B-cell Ph+ acute lymphoblastic leukemia (B-ALL) and most cases of pediatric Ph+ B-ALL express the p190 BCR-ABL isoform, which causes abnormal expansion of lymphoid progenitors and a poor prognosis despite the administration of tyrosine kinase inhibitors. A major signaling pathway activated by BCR-ABL is the Rac/Rho GTPase signaling pathway. We had previously demonstrated that p210-BCR-ABL activates Rac GTPases and the deficiency of RAC2, or even more, the deficiency of both Rac1 and RAC2, impairs myeloid leukemogenesis induced by p210 BCR-ABL in vitro and in vivo (Thomas EK et al., Cancer Cell 2007). The p190-BCR-ABL isoform also activates Rac GTPases and the deficiency of RAC2 induces resistance to develop proB-ALL (Sanchez-Aguilera et al., ASH 2008). Since p190-BCR-ABL is devoid of guanine nucleotide exchange factor (GEF) activity through the absence of the BCR Dbl-homology (DH) domain, it probably requires the recruitment of intermediate GEFs to activate Rac GTPases. The Vav family of GEF has been shown to bind the SH3/SH2 domains of ABL and become activated. Three isoforms of Vav (Vav1, Vav2 and Vav3) are expressed in hematopoietic cells. Several studies have indicated that Vav3 has a role distinct from Vav1 or Vav2. Here, we hypothesized that the hematopoietic specific Rho GTPase, RAC2, may mediate key signals in the transformation of B-cell progenitors upon Vav-mediated activation. We have found that both Vav1 and Vav3 are overexpressed and hyperphosphorylated in p190 BCR-ABL expressing human B-ALL cells as well as in BCR-ABL transduced murine proB cells. While the combined deficiency of Vav1 and Vav2 does not impair leukemogenesis in vivo, Vav3 deficiency reverses p190-BCR-ABL induced transformation phenotypes. Specifically, Vav3 deficiency in p190-BCR-ABL expressing proB-ALL cells associated with a significant reduction of Rho and Rac activation (75% and 56%), apoptosis induction (4-fold) and impaired ex-vivo expansion (33% reduction). In addition, p190-BCR-ABL-induced adhesion-to-fibronectin defect and hypermigratory response to CXCL12 were corrected by deletion of Vav3 (2.1-fold increase and 60% decrease, respectively), suggesting that Vav3 deficiency reverses the transformation-associated phenotype associated to BCR-ABL expression. Collectively, our work identifies a critical role of RAC2 and its activator Vav3 for p190 BCR-ABL induced B-cell progenitor leukemic transformation and may define a novel therapeutic target for this poor prognosis disease. Disclosures: Cancelas: CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.
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RAC2 gtpase activation is necessary for development of p190 bcr abl induced b cell acute lymphoblastic leukemia
Blood, 2008Co-Authors: Abel Sanchezaguilera, David A. Williams, Amitava Sengupta, Joseph P Mastin, Kyung H Chang, Jose A. CancelasAbstract:The fusion gene BCR-ABL, resulting from t(9;22) reciprocal chromosomal translocations, encodes a constitutively active tyrosine kinase. Two different isoforms of BCR-ABL, p190 and p210, are associated to two completely different diseases. In the tyrosine kinase inhibitor (TKI) era, while p210-BCR-ABL-induced CML is highly responsive to TKI, p190-BCR-ABL still induces a poor prognosis B-cell acute lymphoblastic leukemia (B-ALL). The only difference between these two forms of BCR-ABL is the existence of a DH/Cdc24/PH domain in p210-BCR-ABL, which acts as a guanine nucleotide exchange factor (GEF) able to activate Rho GTPases. Rac is a subfamily of Rho GTPases with regulatory activity on hematopoietic stem cell and progenitor (HSC/P) functions. We have previously shown that RAC2 and further the combination of Rac1 and RAC2 mediate downstream signals in p210 BCR-ABL-induced myeloproliferation (Thomas EK, et al. , Cancer Cell, 2007). Interestingly, despite the absence of a GEF domain in p190-BCR-ABL, Rac is activated, suggesting the activation of other GEF(s). Here we have analyzed whether Vav and Rac family members are involved in p190-BCR-ABL-induced B-ALL. We have used a combination of in vitro (Ba/F3 pro-B cells transduced with p190 or p210 BCR-ABL) and in vivo (murine transduction-transplantation model of p190 BCR-ABL-induced B-ALL) approaches. In Ba/F3 cells, both p190 BCR-ABL and p210 BCR-ABL activated Rac and the Rac effector p21 activated kinase (PAK), and their proliferation and survival appeared severely decreased in response to the Rac activation inhibitor NSC23766. Stat3, Stat5 and Jnk, but not ERK, p38 or NF- k B, were constitutively hyperactivated in p190 BCRABL-expressing Ba/F3 cells and primary murine B-ALL cells. Intracellular flow cytometry analysis demonstrated that Stat5 was specifically activated in the pro/pre-B leukemic cell population, compared to normal B cells. In the murine model of B-ALL, loss of RAC2, but not Rac3, prolonged survival and impaired leukemia development. Like in Ba/F3 cells, primary B-CFU and outgrowth in Witte-Whitlock assays of leukemic primary cells from mice was severely decreased by the addition of NSC23766 to the culture. Although Vav was activated by both p190- and p210-BCR-ABL, since NSC23766 does not block the activation by Vav1, we hypothesized that other GEFs were involved. Indeed, the loss of Vav1 or even combined loss of Vav1 and Vav2 did not impair BCR-ABL-mediated lymphoid leukemogenesis in vivo . Vav3, another member in the Vav family which uses a different mechanism of activation of Rac GTPases was a likely candidate. In fact, loss of Vav3 alone was able to significantly prolong the survival and attenuate development of p190 BCR-ABL-driven B-ALL. In conclusion, the results of this study indicate that Rac activation is necessary for the development of B-ALL induced by p190-BCR-ABL in vitro and in vivo, and validate a new signaling pathway as a therapeutic target for BCR-ABL-induced B-ALL.
Michael Glogauer - One of the best experts on this subject based on the ideXlab platform.
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RAC2 is required for the formation of neutrophil extracellular traps
Journal of leukocyte biology, 2011Co-Authors: Michael Byung H. Lim, Jan W. P. Kuiper, Ashley Katchky, Hailey Goldberg, Michael GlogauerAbstract:Neutrophils play a critical role as a first line of defense against invading pathogens. Recently, a new defense strategy of neutrophils was described, in which pathogens are trapped and killed by NETs. However, the exact underlying mechanisms leading to the formation of NETs remain elusive. Here, we explored the role of the Rac small GTPases in the formation of NETs using neutrophils that lack Rac1, RAC2, or both isoforms. Efficient NET formation was observed in WT and Rac1null neutrophils. In contrast, NET formation was markedly impaired in cells lacking RAC2 or both RAC2 and Rac1. The defect in NET formation in RAC2null cells was rescued by exogenous ROS sources, suggesting that RAC2-mediated ROS generation is required for NET formation. In addition, we assessed the role of NO in NET formation in mouse neutrophils. Blocking NO production with the NOS inhibitor L-NAME significantly reduced NET formation. Moreover, we show that RAC2null cells produce significantly less NO than Rac1null cells or their WT counterparts. Our data suggest that RAC2 is essential for NET formation via pathways involving ROS and NO. J. Leukoc. Biol. 90: 771–776; 2011.
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sbds is required for RAC2 mediated monocyte migration and signaling downstream of rank during osteoclastogenesis
Blood, 2011Co-Authors: Roland Leung, Yongqiang Wang, Karl Cuddy, Johanna M Rommens, Michael GlogauerAbstract:Shwachman-Diamond syndrome (SDS) results from mutations in the SBDS gene, characterized by exocrine pancreatic insufficiency and hematologic and skeletal abnormalities. Neutropenia and neutrophil dysfunction are hallmark features of SDS; however, causes for the bone defects are unknown. Dysfunction of boneresorbing osteoclasts, formed by the fusion of monocytic progenitors derived from the same granulocytic precursors as neutrophils, could be responsible. We report that Sbds is required for in vitro and in vivo osteoclastogenesis (OCG). Sbds-null murine monocytes formed osteoclasts of reduced number and size because of impaired migration and fusion required for OCG. Phenotypically, Sbdsnull mice exhibited low-turnover osteoporosis consistent with findings in SDS patients. Western blotting of Rho GTPases that control actin dynamics and migration showed a 5-fold decrease in RAC2, whereas Rac1, Cdc42, and RhoA were unchanged or only mildly reduced. Although migration was rescued on RAC2 supplementation, OCG was not. This was attributed to impaired signaling downstream of receptor activator of nuclear factor-B (RANK) and reduced expression of the RANK-ligand-dependent fusion receptor DC-STAMP. We conclude that Sbds is required for OCG by regulating monocyte migration via RAC2 and osteoclast differentiation signaling downstream of RANK. Impaired osteoclast formation could disrupt bone homeostasis, resulting in skeletal abnormalities seen in SDS patients. (Blood. 2011;117(6): 2044-2053)
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pivotal advance phospholipids determine net membrane surface charge resulting in differential localization of active rac1 and RAC2
Journal of Leukocyte Biology, 2010Co-Authors: Marco A. O. Magalhaes, Michael GlogauerAbstract:In this investigation, we used primary murine neutrophils to demonstrate that local changes in membrane phospholipid composition alter the net cytoplasmic membrane surface charge, which results in selective recruitment of Rac1 or RAC2 based on the net charge of their respective C-terminal domains. Murine neutrophils undergoing chemotaxis or carrying out phagocytosis were transfected with K-ras4B-derived membrane charge biosensors and lipid markers, which allowed us to simultaneously monitor the levels of PIP2, PIP3, and PS and net membrane charge of the newly developing phagosome membrane and plasma membrane. Our results indicate that the combination of PIP2, PIP3, and PS generates a high negative charge (–8) at the plasma membrane of actin-rich pseudopods, where active Rac1 preferentially localizes during phagosome formation. The lipid metabolism that occurs during phagosome maturation results in the localized depletion of PIP2, PIP3, and partial decrease in PS. This creates a moderately negative net charge that correlates with the localization of active RAC2. Conversely, the accumulation of PIP3 at the leading-edge membrane during chemotaxis generates a polarized accumulation of negative charges that recruits Rac1. These results provide evidence that alterations in membrane lipid composition and inner-membrane surface charge are important elements for the recruitment of differentially charged proteins and localization of signaling pathways during phagocytosis and chemotaxis in neutrophils. J. Leukoc. Biol. 87: 000–000; 2010.
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Rac1 and RAC2 in Osteoclastogenesis: A Cell Immortalization Model
Calcified Tissue International, 2009Co-Authors: Yongqiang Wang, Denise D. Belsham, Michael GlogauerAbstract:Cell lines generated from primary cells with a particular gene deletion are useful for examining the function of the specific deleted genes and provide the opportunity to genetically rescue the lost genes using standard gene transfection techniques. In the present study, bone marrow monocytes from wild-type (WT), Rac1 null, and RAC2 null mice were primed with macrophage colony-stimulating factor and soluble receptor activator of NF-κB ligand to generate preosteoclasts. This was followed by transduction of a retrovirus containing simian virus 40 large T-antigen and a neomycin-resistant cassette. Seven to 19 immortalized cell lines from each genotype were established. Among them, WT2, Rac1 null-D9, and RAC2 null-A2 were characterized to verify that osteoclastogenesis and osteoclast functions were identical to the parental primary cells. Results showed that immortalized WT2 cells were able to differentiate into mature, multinucleated, functional, tartrate-resistant acid phosphatase-positive osteoclasts. Immortal Rac1 null cells, as with their primary cell counterparts, displayed a severe defect in osteoclastogenesis and function. Transfection of the Rac1 gene into Rac1 null cells was sufficient to rescue osteoclastogenesis. We believe this method of generating immortalized preosteoclasts will provide a key tool for studying the signaling mechanisms involved in osteoclastogenesis.
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Human Neutrophils Coordinate Chemotaxis by Differential Activation of Rac1 and RAC2
Journal of immunology (Baltimore Md. : 1950), 2009Co-Authors: Hui Zhang, Michael Glogauer, Chunxiang Sun, Gary M BokochAbstract:Rac1 and RAC2, members of the small Rho GTPase family, play essential roles in coordinating directional migration and superoxide production during neutrophil responses to chemoattractants. Although earlier studies in Rac1 and RAC2 knockout mice have demonstrated unique roles for each Rac isoform in chemotaxis and NADPH oxidase activation, it is still unclear how human neutrophils use Rac1 and RAC2 to achieve their immunological responses to foreign agent stimulation. In the current study, we used TAT dominant-negative Rac1-T17N and RAC2-T17N fusion proteins to acutely alter the activity of Rac1 and RAC2 individually in human neutrophils. We demonstrate distinct activation kinetics and different roles for Rac1 and RAC2 in response to low vs high concentrations of fMLP. These observations were verified using neutrophils from mice in which Rac1 or RAC2 was genetically absent. Based on these results, we propose a model to explain how human neutrophils kill invading microbes while limiting oxidative damage to the adjacent surrounding healthy tissue through the differential activation of Rac1 and RAC2 in response to different concentrations of chemoattractant.
Dianqing Wu - One of the best experts on this subject based on the ideXlab platform.
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lack of a significant role of p rex1 a major regulator of macrophage rac1 activation and chemotaxis in atherogenesis
Prostaglandins & Other Lipid Mediators, 2008Co-Authors: Zhenglong Wang, Xuemei Dong, Jonathan D. Smith, Zhong Li, Dianqing WuAbstract:Abstract Background Rho GTPases are known to play important roles in regulating multiple cellular processes that include cell polarization and migration. Among these Rho GTPases, Rac has been shown to be essential for F actin formation and cell migration. P-Rex1 is a guanine nucleotide exchange factor (GEF) that was previously found to mediate the activation of RAC2, but not Rac1, in mouse neutrophils. Objectives Here we examined the role of P-Rex1 in mouse macrophages and atherogenesis. Methods and results PBD (p21 binding domain) pull down assay was performed to compare the Rac1 activation in WT and P-Rex1-deficient macrophage. In addition, transwell assay was conducted to compare chemotaxis of WT and P-Rex1-deficient macrophage. We found that P-Rex1 is a major Rac1 regulator in mouse macrophages as its deficiency significantly compromises macrophage chemotaxis, superoxide production (SOD), and Rac1 activation in response to chemoattractants. The potential role of P-Rex1 in atherogenesis is also investigated by transferring P-Rex1-deficient bone marrow cells to LDLR deficient mice. Contrary to our prediction, P-Rex1 deficiency did not alter atherogenesis, suggesting chemoattractant-induced macrophage migration may not have a significant role in atherogenesis. Conclusions P-Rex1 is one of the major GEFs in macrophage regulating Rac1 activation and chemotaxis.
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Lack of a significant role of P-Rex1, a major regulator of macrophage Rac1 activation and chemotaxis, in atherogenesis.
Prostaglandins & other lipid mediators, 2008Co-Authors: Zhenglong Wang, Xuemei Dong, Jonathan D. Smith, Zhong Li, Dianqing WuAbstract:Rho GTPases are known to play important roles in regulating multiple cellular processes that include cell polarization and migration. Among these Rho GTPases, Rac has been shown to be essential for F actin formation and cell migration. P-Rex1 is a guanine nucleotide exchange factor (GEF) that was previously found to mediate the activation of RAC2, but not Rac1, in mouse neutrophils. Here we examined the role of P-Rex1 in mouse macrophages and atherogenesis. PBD (p21 binding domain) pull down assay was performed to compare the Rac1 activation in WT and P-Rex1-deficient macrophage. In addition, transwell assay was conducted to compare chemotaxis of WT and P-Rex1-deficient macrophage. We found that P-Rex1 is a major Rac1 regulator in mouse macrophages as its deficiency significantly compromises macrophage chemotaxis, superoxide production (SOD), and Rac1 activation in response to chemoattractants. The potential role of P-Rex1 in atherogenesis is also investigated by transferring P-Rex1-deficient bone marrow cells to LDLR deficient mice. Contrary to our prediction, P-Rex1 deficiency did not alter atherogenesis, suggesting chemoattractant-induced macrophage migration may not have a significant role in atherogenesis. P-Rex1 is one of the major GEFs in macrophage regulating Rac1 activation and chemotaxis.
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p rex1 is a primary RAC2 guanine nucleotide exchange factor in mouse neutrophils
Current Biology, 2005Co-Authors: Xuemei Dong, Zhicheng Mo, Zhong Li, Gary M Bokoch, Dianqing WuAbstract:Summary Leukocyte chemoattractants regulate many leukocyte functions, including leukocyte chemotaxis, via the Rho family of small GTPases that include RhoA, Cdc42, and Rac [1–4]. Previous work has revealed mechanisms by which chemoattractants regulate RhoA and Cdc42 in mouse neutrophils [5–8], but the mechanisms for regulation of Rac remain unclear even though Rac is important for neutrophil functions [9–13]. Here, we characterized P-Rex1, a Gβγ and PIP 3 -regulated guanine nucleotide exchange factor that was initially identified as a Rac activator in response to chemoattractants [14], for its roles in the regulation of Rac activity and neutrophil functions. We generated a mouse line in which the P-Rex1 gene is disrupted and found that P-Rex1 deficiency did not significantly affect Rac1 activation but diminished RAC2 activation in response to a chemoattractant fMLP in mouse neutrophils. This preference for RAC2 may partially result from the apparent higher affinity of P-Rex1 for RAC2 than for Rac1 because P-Rex1 was more readily immunoprecipitated with RAC2 S17N than Rac1 S17N . In addition, P-Rex1 deficiency significantly attenuated fMLP-induced F actin formation and superoxide production without affecting LPS- or PMA-induced production. Furthermore, P-Rex1 deficiency caused a chemotactic defect that is primarily attributed to a reduction in the migration rate rather than directionality.
Mary C. Dinauer - One of the best experts on this subject based on the ideXlab platform.
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The Dual Effect of RAC2 on Phospholipase D2 Regulation That Explains both the Onset and Termination of Chemotaxis
Molecular and cellular biology, 2011Co-Authors: Hong-juan Peng, Christophe C. Marchal, Mary C. Dinauer, Karen M. Henkels, Madhu Mahankali, Paula A. Bubulya, Julian Gomez-cambroneroAbstract:We document a biphasic effect of RAC2 on the activation and inhibition of PLD2. Cells overexpressing RAC2 and PLD2 simultaneously show a robust initial ( 30 min) greatly diminished over PLD2-only controls. The first phase is due to the presence of a RAC2-PLD2 positive-feedback loop. To explain the mechanism for the RAC2-led PLD2 inhibition (the second phase), we used leukocytes from wild-type (WT) and RAC2 / knockout mice. RAC2 / cells displayed an enhanced PLD2 (but not PLD1) enzymatic activity, confirming the inhibitory role of RAC2. Late inhibitory responses on PLD2 due to RAC2 were reversed in the presence of phosphatidylinositol 4,5-bisphosphate (PIP 2) both in vitro (purified GST-PH-PLD2, where GST is glutathione S-transferase and PH is pleckstrin homology) and in vivo. Coimmunoprecipitation and immunofluorescence microscopy indicated that PLD2 and RAC2 remain together. The presence of an “arc” of RAC2 at the leading edge of leukocyte pseudopodia and PLD2 physically posterior to this wave of RAC2 was observed in late chemotaxis. We propose Rac-led inhibition of PLD2 function is due to sterical interference of Rac with PLD2’s PH binding site to the membrane and deprivation of the PIP 2. This work supports the importance of functional interactions between PLD and Rac in the biological response of cell migration.
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Evidence for Two CRIB Domains in Phospholipase D2 (PLD2) That the Enzyme Uses to Specifically Bind to the Small GTPase RAC2
The Journal of biological chemistry, 2011Co-Authors: Hong-juan Peng, Mary C. Dinauer, Karen M. Henkels, Madhu Mahankali, Julian Gomez-cambroneroAbstract:Abstract Phospholipase D (PLD) and small GTPases are vital to cell signaling. We report that the RAC2 and the PLD2 isoforms exist in the cell as a lipase-GTPase complex that enables the two proteins to elicit their respective functionalities. A strong association between the two molecules was demonstrated by co-immunoprecipitation and was confirmed in living cells by FRET with CFP-RAC2 and YFP-PLD2 fluorescent chimeras. We have identified the amino acids in PLD2 that define a specific binding site to RAC2. This site is composed of two CRIB (Cdc42-and Rac-interactive binding) motifs that we have named “CRIB-1” and “CRIB-2” in and around the PH domain in PLD2. Deletion mutants PLD2-ΔCRIB-1/2 negate co-immunoprecipitation with RAC2 and diminish the FRET signal in living cells. The PLD2-RAC2 association was further confirmed in vitro using affinity-purified recombinant proteins. Binding was saturable with an apparent Kd of 3 nm and was diminished with PLD2-ΔCRIB mutants. Furthermore, PLD2 bound more efficiently to RAC2-GTP than to RAC2-GDP or to a GDP-constitutive RAC2-N17 mutant. Increasing concentrations of recombinant RAC2 in vitro and in vivo during cell adhesion inhibit PLD2. Conversely, RAC2 activity is increased in the presence of PLD2-WT but not in PLD2-ΔCRIB. We propose that in activated cells PLD2 affects RAC2 in an initial positive feedback, but as RAC2-GTP accumulates in the cell, this constitutes a “termination signal” leading to PLD2 inactivation.
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The Role of RAC2 in Regulating Neutrophil Production in the Bone Marrow and Circulating Neutrophil Counts
The American journal of pathology, 2008Co-Authors: John C. Gomez, Mary C. Dinauer, Jindrich Soltys, Keiichi Okano, Claire M. DoerschukAbstract:Circulating neutrophils are persistently higher in mice deficient in the small GTPase RAC2 than in wild-type (WT) mice. Therefore, we examined the mechanisms through which the small GTPase RAC2 regulates neutrophil production and release. Lethally irradiated WT mice reconstituted with a 50:50 mixture of WT and RAC2−/− fetal liver cells were protected from neutrophilia, suggesting that neutrophilia is primarily because of extrinsic defects that can be corrected by WT leukocytes. However, the differential counts and numbers of leukocyte subtypes differed between RAC2−/− and WT cells, suggesting that RAC2 modulates leukocyte lineage distribution. Kinetic studies suggest RAC2 modulates the release of neutrophils into the circulation and does not prolong their circulating half life. The percentage of bone marrow cells that expressed the neutrophil marker Gr-1 in lethally irradiated WT or RAC2−/− recipients of RAC2−/− stem cells was greater than in recipients of WT stem cells; however, circulating neutrophil counts were higher only in RAC2−/− recipients of RAC2−/− stem cells. RAC2 mRNA was expressed in the bone marrow of WT recipients of RAC2−/− stem cells and in human mesenchymal stem cells. The data presented here suggest that RAC2 in hematopoietic cells regulates leukocyte lineage distribution and RAC2 in nonhematopoietic cells might contribute to regulating circulating neutrophil counts.
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impaired nadph oxidase activity in RAC2 deficient murine neutrophils does not result from defective translocation of p47phox and p67phox and can be rescued by exogenous arachidonic acid
Journal of Leukocyte Biology, 2006Co-Authors: Chaekyun Kim, Mary C. DinauerAbstract:RAC2 is a hematopoietic-specific Rho-GTPase that plays a stimulus-specific role in regulating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and other functional responses in neutrophils. In this study, RAC2-/- neutrophils were shown to have significantly decreased NADPH oxidase activity and actin remodeling in response to exogenous arachidonic acid (AA), as previously observed for phorbol 12-myristate 13-acetate (PMA) or formyl-Met-Leu-Phe (fMLP) as agonists. PMA-, fMLP-, or AA-induced translocation of p47 p h o x and p67 p h o x to the plasma membrane was not impaired in RAC2-/- neutrophils. Combined stimulation of RAC2-/- neutrophils with exogenous AA and PMA had a synergistic effect on NADPH oxidase activity, and superoxide production increased to a level that was at least as high as wild-type cells and had no effect on fMLP-elicited enzyme activity. Membrane translocation of p47 and p67 p h o x as well as Rac1 activation was not increased further by combined PMA and AA stimulation. Inhibitor studies were consistent with important roles for phorbol ester-activated protein kinase C (PKC) isoforms and an atypical isoform, PKCζ, in superoxide production by wild-type and RAC2-/- neutrophils stimulated with AA and PMA. In addition, PMA-stimulated release of AA and cytoplasmic phospholipase A 2 expression in RAC2-/- neutrophils were similar to wild-type, suggesting that deficient AA production by PMA-stimulated RAC2-/- neutrophils does not explain the effect of exogenous AA on oxidase activity. Although not required for translocation of p47 p h o x and p67 p h o x , RAC2 is necessary for optimal activity of the assembled oxidase complex, an effect that can be replaced by exogenous AA, which may act directly or via an exogenous AA-induced mediator.
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Arachidonic Acid Rescues Defects on PMA-Elicited NADPH Oxidase Activity in RAC2-Deficient Neutrophils.
Blood, 2004Co-Authors: Chaekyun Kim, Mary C. DinauerAbstract:RAC2 is a hematopoietic-specific Rho-GTPase implicated as an important constituent of the NADPH oxidase complex. We previously showed that RAC2 plays a stimulus-specific role in regulating NADPH oxidase activation and other functional responses in neutrophils [Kim and Dinauer, JI 166, 2001]. Here we investigate the effect of arachidonic acid (AA) on RAC2-regulated NADPH oxidase activity. Superoxide production in RAC2-/- neutrophils was significantly lower (~4-fold) than that of wild-type when stimulated with PMA or AA alone. However, exogenously added AA (10 μM) fully restored the defect in PMA-elicited NADPH oxidase activity in RAC2 − / − neutrophils, while having no effect on FMLP-elicited superoxide production. Impaired PMA- or AA-induced F-actin polymerization in RAC2 − / − neutrophils was also not restored by co-stimulation with PMA and AA. Taken together, these observations suggest that there are agonist- and pathway-specific differences in the underlying basis of functional defects in RAC2 − / − neutrophils. To further investigate possible mechanisms of AA-mediated rescue of PMA-stimulated NADPH oxidase activation in RAC2 − / − neutrophils, we measured protein expression and activity of cytosolic phospholipase A 2 (cPLA 2 ) and protein kinase C (PKC). The expression of cPLA 2 and PMA-stimulated release of AA was similar between wild-type and RAC2 − / − neutrophils, suggesting that defects in AA production by PMA-stimulated RAC2 − / − neutrophils do not account for the effect of exogenous AA on oxidase activity. The neutrophil expression of PKC isoforms (α, β, δ, ζ) was also similar between genotypes. The cytosolic p47 phox and p67 phox components of NADPH oxidase were translocated to the plasma membrane upon stimulation with PMA in both genotypes, and no additional translocation in either wild-type or RAC2 − / − neutrophils was detected upon co-stimulation with AA. The level of activated Rac1-GTP was similar between genotypes following stimulation, and was not increased by co-stimulation with PMA and AA. These studies indicate that the addition of exogenous AA reconstitutes PMA-elicited superoxide production in RAC2 − / − neutrophils independent of the effects on translocation of p47 phox and p67 phox and activation of Rac1 GTPase. We hypothesize that the effect of AA is exerted through conformational changes of the assembled NADPH oxidase.
Chaekyun Kim - One of the best experts on this subject based on the ideXlab platform.
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opposing roles of hematopoietic specific small gtpase RAC2 and the guanine nucleotide exchange factor vav1 in osteoclast differentiation
Scientific Reports, 2020Co-Authors: In Soon Kang, Jin Sun Jang, Chaekyun KimAbstract:Vav1 regulates Rac activation as a hematopoietic-specific Rho/Rac-family guanine nucleotide exchange factor. Rac is a subfamily of Rho GTPases that regulates the bone-resorbing capacity of osteoclasts (OCs). In this study, we show that hematopoietic-specific RAC2 and Vav1 play opposing roles by enhancing or attenuating OC differentiation, respectively. This was demonstrated by higher and lower bone density in the femurs from RAC2-deficient (RAC2-/-) and Vav1-deficient (Vav1-/-) mice, respectively, compared to the wild-type (WT) mice. Accordingly, RAC2-/- cells displayed low numbers of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (41%) compared to WT cells, whereas, Vav1-/- cells showed high TRAP-positive cell numbers (150%), and the double-knockout RAC2-/-Vav1-/- mice nullified the effects on OC numbers achieved by the individual knockouts. These reciprocal roles of RAC2 and Vav1 in OC differentiation were confirmed by reduced and increased levels of OC-specific markers, such as TRAP, calcitonin receptor, cathepsin K, and DC-STAMP in the RAC2-/- and Vav1-/- OCs, respectively. Our findings of decrease and increase in actin ring formation and αvβ3 integrin-mediated adhesion in RAC2-/- and Vav1-/- mice, respectively, suggest that Vav1 and its downstream GTPase, RAC2, may counteract to fine-tune OC differentiation and bone resorption.
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impaired nadph oxidase activity in RAC2 deficient murine neutrophils does not result from defective translocation of p47phox and p67phox and can be rescued by exogenous arachidonic acid
Journal of Leukocyte Biology, 2006Co-Authors: Chaekyun Kim, Mary C. DinauerAbstract:RAC2 is a hematopoietic-specific Rho-GTPase that plays a stimulus-specific role in regulating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and other functional responses in neutrophils. In this study, RAC2-/- neutrophils were shown to have significantly decreased NADPH oxidase activity and actin remodeling in response to exogenous arachidonic acid (AA), as previously observed for phorbol 12-myristate 13-acetate (PMA) or formyl-Met-Leu-Phe (fMLP) as agonists. PMA-, fMLP-, or AA-induced translocation of p47 p h o x and p67 p h o x to the plasma membrane was not impaired in RAC2-/- neutrophils. Combined stimulation of RAC2-/- neutrophils with exogenous AA and PMA had a synergistic effect on NADPH oxidase activity, and superoxide production increased to a level that was at least as high as wild-type cells and had no effect on fMLP-elicited enzyme activity. Membrane translocation of p47 and p67 p h o x as well as Rac1 activation was not increased further by combined PMA and AA stimulation. Inhibitor studies were consistent with important roles for phorbol ester-activated protein kinase C (PKC) isoforms and an atypical isoform, PKCζ, in superoxide production by wild-type and RAC2-/- neutrophils stimulated with AA and PMA. In addition, PMA-stimulated release of AA and cytoplasmic phospholipase A 2 expression in RAC2-/- neutrophils were similar to wild-type, suggesting that deficient AA production by PMA-stimulated RAC2-/- neutrophils does not explain the effect of exogenous AA on oxidase activity. Although not required for translocation of p47 p h o x and p67 p h o x , RAC2 is necessary for optimal activity of the assembled oxidase complex, an effect that can be replaced by exogenous AA, which may act directly or via an exogenous AA-induced mediator.
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Arachidonic Acid Rescues Defects on PMA-Elicited NADPH Oxidase Activity in RAC2-Deficient Neutrophils.
Blood, 2004Co-Authors: Chaekyun Kim, Mary C. DinauerAbstract:RAC2 is a hematopoietic-specific Rho-GTPase implicated as an important constituent of the NADPH oxidase complex. We previously showed that RAC2 plays a stimulus-specific role in regulating NADPH oxidase activation and other functional responses in neutrophils [Kim and Dinauer, JI 166, 2001]. Here we investigate the effect of arachidonic acid (AA) on RAC2-regulated NADPH oxidase activity. Superoxide production in RAC2-/- neutrophils was significantly lower (~4-fold) than that of wild-type when stimulated with PMA or AA alone. However, exogenously added AA (10 μM) fully restored the defect in PMA-elicited NADPH oxidase activity in RAC2 − / − neutrophils, while having no effect on FMLP-elicited superoxide production. Impaired PMA- or AA-induced F-actin polymerization in RAC2 − / − neutrophils was also not restored by co-stimulation with PMA and AA. Taken together, these observations suggest that there are agonist- and pathway-specific differences in the underlying basis of functional defects in RAC2 − / − neutrophils. To further investigate possible mechanisms of AA-mediated rescue of PMA-stimulated NADPH oxidase activation in RAC2 − / − neutrophils, we measured protein expression and activity of cytosolic phospholipase A 2 (cPLA 2 ) and protein kinase C (PKC). The expression of cPLA 2 and PMA-stimulated release of AA was similar between wild-type and RAC2 − / − neutrophils, suggesting that defects in AA production by PMA-stimulated RAC2 − / − neutrophils do not account for the effect of exogenous AA on oxidase activity. The neutrophil expression of PKC isoforms (α, β, δ, ζ) was also similar between genotypes. The cytosolic p47 phox and p67 phox components of NADPH oxidase were translocated to the plasma membrane upon stimulation with PMA in both genotypes, and no additional translocation in either wild-type or RAC2 − / − neutrophils was detected upon co-stimulation with AA. The level of activated Rac1-GTP was similar between genotypes following stimulation, and was not increased by co-stimulation with PMA and AA. These studies indicate that the addition of exogenous AA reconstitutes PMA-elicited superoxide production in RAC2 − / − neutrophils independent of the effects on translocation of p47 phox and p67 phox and activation of Rac1 GTPase. We hypothesize that the effect of AA is exerted through conformational changes of the assembled NADPH oxidase.
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RAC2-Deficient Murine Macrophages Have Selective Defects in Superoxide Production and Phagocytosis of Opsonized Particles
Journal of immunology (Baltimore Md. : 1950), 2004Co-Authors: Akira Yamauchi, Christophe C. Marchal, Simon J. Atkinson, Chaekyun Kim, Jason Towe, Mary C. DinauerAbstract:The Rho family GTPase Rac is a crucial participant in numerous cellular functions and acts as a molecular switch for signal transduction. Mice deficient in hemopoietic-specific RAC2 exhibited agonist-specific defects in neutrophil functions including chemoattractant-stimulated filamentous actin polymerization and chemotaxis, and superoxide production elicited by phorbol ester, fMLP, or IgG-coated particles, despite expression of the highly homologous Rac1 isoform. In this study, functional responses of RAC2-null murine macrophages were characterized to examine whether RAC2 also has nonredundant functions in this phagocytic lineage. In contrast to murine neutrophils, in which Rac1 and RAC2 are present in similar amounts, Rac1 was ∼4-fold more abundant than RAC2 in both bone marrow-derived and peritoneal exudate macrophages, and macrophage Rac1 levels were unchanged by the absence of RAC2. Accumulation of exudate macrophages during peritoneal inflammation was reduced in RAC2−/− mice. FcγR-mediated phagocytosis of IgG-coated SRBC was also significantly decreased in RAC2-null macrophages, as was NADPH oxidase activity in response to phorbol ester or FcγR stimulation. However, phagocytosis and oxidant production stimulated by serum-opsonized zymosan was normal in RAC2−/− macrophages. Macrophage morphology was also similar in wild-type and RAC2-null cells, as was actin polymerization induced by FcγR-mediated phagocytosis or M-CSF. Hence, RAC2-null macrophages have selective defects paralleling many of the observed functional defects in RAC2-null neutrophils. These results provide genetic evidence that although RAC2 is a relatively minor isoform in murine macrophages, it plays a nonoverlapping role with Rac1 to regulate host defense functions in this phagocyte lineage.
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RAC2 stimulates akt activation affecting bad bcl xl expression while mediating survival and actin function in primary mast cells
Immunity, 2000Co-Authors: Feng Chun Yang, Chaekyun Kim, Reuben Kapur, Alastair J. King, Wen Tao, Jovencio B Borneo, Robert Breese, Mark S. Marshall, Mary C. DinauerAbstract:Abstract 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-X L , 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-X L .
