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1
Lai, Yun-Ju, Jei-Hwa Yu, Braden C. McFarland, and Etty N. Benveniste. "The role of Rac proteins in glioblastoma stem cells." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): e13011-e13011. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.e13011.
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e13011 Background: Glioblastoma is the grade 4 astrocytoma which is notorious for its highly invasive phenotype, very low survival rate and generally poor responses to conventional therapies. Glioblastoma stem-like cells (GSC), which are usually more resistant to therapeutic treatment, may account for the poor prognosis of this disease. Rac (Ras-related C3 botulinum toxin substrate) is a subfamily of Rho small GTPase which function is regulation of actin cytoskeleton rearrangement. While Rac1 is expressed ubiquitous in different tissues and cells, Rac2 is highly expressed in the mesenchymal subtype of glioblastoma according to the TCGA (the Cancer Genome Atlas) database, and Rac3 is mainly expressed in the brain. Methods: We used Rac proteins overexpressing-glioblastoma cellines derived GSC and Rac proteins specific siRNA harboring-GSC to perform colony formation assay and migration assay. Results: Here we report that Rac proteins overexpressing glioblastoma stem-like cells derived from glioblastoma cell lines have higher proliferation rate and stronger responses to LPA-induced cell migration. Knocking-down their expression by specific siRNA reduces the proliferation and migration of these cells. Instead of Rac1, Rac2 and Rac3 are more effective on promoting proliferation and migration of glioblastoma stem-like cells. Moreover, Rac proteins promote glioblastoma progression is associated with activation of JAK-STAT and ERK pathway. Conclusions: Although Rac1 is the most studied one in the Rac family, and has also been implicated in the progression of different cancers, however, it is homogeneously expressed in all different tissues, and plays important roles in normal cellular functions involving cell movement, such as wound healing, make it not a good candidate for specific drug targeting. According to our results, Rac2 or Rac3 serve as a better potential therapeutic targets for glioblastoma treatment.
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Kalfa, Theodosia A., Suvarnamala Pushkaran, Jose A. Cancelas, James F. Johnson, Deidre Daria, Hartmut Geiger, David A. Williams, and Yi Zheng. "Rac GTPases Regulate Erythropoiesis Both in the Early Steps of Differentiation and in Enucleation." Blood 110, no. 11 (November 16, 2007): 1714. http://dx.doi.org/10.1182/blood.v110.11.1714.1714.
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Abstract Rac GTPases (i.e. Rac1, Rac2 and Rac3), a subfamily of Rho GTPases, control actin organization and have overlapping as well as distinct roles in cell survival, proliferation, and differentiation in various hematopoietic cell lineages (Gu et al, Science 2003, Cancelas et al, Nature Med 2005). Using conditional gene-targeting in mice, we have previously demonstrated that Rac1 and Rac2 deficiency causes anemia with abnormal erythrocyte cytoskeleton and decreased deformability (Kalfa et al, Blood 2006). In the present studies, we found by colony assays that although bone marrow (BM) BFU-E activity was unaltered from that of the wild type (WT) mice, Rac1−/−;Rac2−/− erythroid bursts had a strikingly different morphology appearing as round, small, dense colonies, likely a manifestation of motility defects associated with Rac GTPase deficiency. Total CFU-Es recovered from Rac1−/−;Rac2−/− BM were as low as 25% of that in WT mice (p<0.05). To further assess erythroblast differentiation, BM cells were immunostained with fluorescent label-conjugated anti-CD71 and anti-Ter119, as previously described (Socolovski et al. Blood 2001). Flow cytometry analysis revealed that proerythroblasts and basophilic erythroblasts in the BM were significantly decreased in Rac1−/−;Rac2−/− (∼30–50% of WT content) while the terminal differentiation to orthochromatic erythroblasts was comparable. In vivo BrdU labeling and flow cytometry with 7-AAD and annexin-V in combination with staining for CD71 and Ter119 revealed no difference in proliferation or survival between WT and Rac1−/−;Rac2−/− erythroid cells after the proerythroblast stage. These data suggest that deficiency of Rac1 and Rac2 GTPases affect erythropoiesis mainly at the early stages of BFU-E and CFU-E formation but not during terminal differentiation to orthochromatic erythroblasts. Given the prominent role of Rac GTPases in regulating actin structure, we next evaluated the possible involvement of Rac GTPases in enucleation, the terminal step of erythropoiesis that likely requires significant actin remodeling. We performed quantitative analysis in ex vivo erythropoiesis cultures, by flow cytometry, using SYTO16, a cell-permeable nucleic acid-staining dye. The frequency of enucleated red cells (SYTO16-low, Ter119-positive population) was similar in the WT and the Rac1−/−;Rac2−/− erythroid cultures. However, application of a Rac GTPase inhibitor, NSC23766, to the WT or the Rac1−/−;Rac2−/− erythroid cultures during the enucleation phase resulted in an inhibition of enucleation up to 80% dose-dependently (figure 1). Rac1 and Rac2 deficiency led to a compensatory elevation of Rac3 activity that was effectively suppressed by NSC23766, as demonstrated by immunoblotting in the Rac1−/−;Rac2−/− erythroblasts and effector-domain pull-down studies. Moreover, NSC23766 inhibited Rac1, Rac2, and Rac3 activities as well as actin polymerization of the erythroblasts. Thus, Rac1, Rac2, and Rac3 have redundant but essential roles in supporting actin dynamics necessary for the nucleus extrusion during the enucleation process. Figure Figure
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Engers, R., S. Ziegler, M. Mueller, A. Walter, R. Willers, and H. E. Gabbert. "Prognostic relevance of increased Rac GTPase expression in prostate carcinomas." Endocrine-Related Cancer 14, no. 2 (June 2007): 245–56. http://dx.doi.org/10.1677/erc-06-0036.
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Rac proteins of the Rho-like GTPase family, including the ubiquitous Rac1, the hematopoiesis-specific Rac2, and the least-characterized Rac3 play a major role in oncogenic transformation, tumor invasion and metastasis. However, the prognostic relevance of Rac expression in human tumors has not been investigated yet. In the present study, Rac protein expression was analyzed in benign secretory epithelium, high-grade prostatic intraepithelium neoplasia (HG-PIN), and prostate carcinomas of 60 R0-resected radical prostatectomy specimens by semiquantitative immunohistochemistry. Thus, Rac proteins were significantly strongly expressed in HG-PIN (P < 0.001) and prostate carcinomas (P < 0.001) when compared with benign secretory epithelium. Accordingly, all tumor tissues analyzed by isoform-specific real-time PCR (n = 7) exhibited significantly higher RNA expression levels of Rac (i.e. sum of Rac1 and Rac3 expression levels) than the respective benign counterparts (P = 0.018) and this appeared to result mainly from increased expression of the Rac3 isoform as verified by immunoblotting. Univariate analyses showed statistically significant associations of increased Rac protein expression in prostate cancer (P = 0.045), preoperative prostate-specific antigen levels (P = 0.044), pT stage (P = 0.002), and Gleason score (P = 0.001) with decreased disease-free survival (DFS). This prognostic effect of increased protein expression of Rac remained significant even in a multivariate analysis including all these four factors (relative risk = 3.22, 95% confidence interval = 1.04–10.00; P = 0.043). In conclusion, our data suggest that increased Rac protein expression in prostate cancer relative to the corresponding benign secretory epithelium is an independent predictor of decreased DFS and appears to result mainly from increased expression of the Rac3 isoform.
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Cho, Young Jin, Bin Zhang, Vesa Kaartinen, Leena Haataja, Ivan de Curtis, John Groffen, and Nora Heisterkamp. "Generation of rac3 Null Mutant Mice: Role of Rac3 in Bcr/Abl-Caused Lymphoblastic Leukemia." Molecular and Cellular Biology 25, no. 13 (July 1, 2005): 5777–85. http://dx.doi.org/10.1128/mcb.25.13.5777-5785.2005.
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ABSTRACT Numerous studies indirectly implicate Rac GTPases in cancer. To investigate if Rac3 contributes to normal or malignant cell function, we generated rac3 null mutants through gene targeting. These mice were viable, fertile, and lacked an obvious external phenotype. This shows Rac3 function is dispensable for embryonic development. Bcr/Abl is a deregulated tyrosine kinase that causes chronic myelogenous leukemia and Ph-positive acute lymphoblastic leukemia in humans. Vav1, a hematopoiesis-specific exchange factor for Rac, was constitutively tyrosine phosphorylated in primary lymphomas from Bcr/Abl P190 transgenic mice, suggesting inappropriate Rac activation. rac3 is expressed in these malignant hematopoietic cells. Using lysates from BCR/ABL transgenic mice that express or lack rac3, we detected the presence of activated Rac3 but not Rac1 or Rac2 in the malignant precursor B-lineage lymphoblasts. In addition, in female P190 BCR/ABL transgenic mice, lack of rac3 was associated with a longer average survival. These data are the first to directly show a stimulatory role for Rac in leukemia in vivo. Moreover, our data suggest that interference with Rac3 activity, for example, by using geranyl-geranyltransferase inhibitors, may provide a positive clinical benefit for patients with Ph-positive acute lymphoblastic leukemia.
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Thomas, Emily K., Jose A. Cancelas, Heedon Chae, Adrienne D. Cox, Patricia J. Keller, Danilo Perrotti, Paolo Neviani, et al. "Rac GTPases Are Potential Therapeutic Targets in p210-BCR-ABL-Induced Myeloproliferative Disease (MPD)." Blood 110, no. 11 (November 16, 2007): 465. http://dx.doi.org/10.1182/blood.v110.11.465.465.
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Abstract The p210-BCR-ABL fusion protein is a constitutively active tyrosine kinase that is necessary and sufficient for the development of chronic myelogenous leukemia (CML). ABL-kinase inhibitors such as imatinib mesylate (Gleevec, STI571) potently block BCR-ABL activation, but the continued presence of leukemic stem cells and the emergence of imatinib-resistant BCR-ABL mutants suggest that ABL kinase inhibitors alone cannot completely eradicate disease. Rac GTPases have been implicated in BCR-ABL-mediated proliferation in cell lines and regulate many of the same signaling pathways as BCR-ABL, suggesting that these proteins could be additional therapeutic targets in CML. We have found that Rac1, Rac2, and, to a lesser extent, Rac3 were hyperactivated in CD34+ cells purified from the peripheral blood of two CML patients. To better study the role of Rac in BCR-ABL disease development, murine hematopoietic stem cells (HSC) genetically deficient in Rac1 and/or Rac2 were transduced with a retroviral vector expressing p210-BCR-ABL. Wild type (WT) and Rac1−/− mice experienced similar disease progression [median survival 23 ± 6 days (n=30) and 22 ± 4 days (n=8), respectively], Rac2−/− mice exhibited significantly attenuated development of BCR-ABL-mediated MPD [median survival 43 ± 27 days (n=18); p<0.001], and Rac1−/−;Rac2−/− animals showed markedly prolonged survival [median survival 92 ± 34 days (n=19); p<0.001]. p210-BCR-ABL WT, Rac1−/−, and Rac2−/− mice had elevated circulating myeloblasts 30 days post-transplant, while Rac1−/−;Rac2−/− mice had normal peripheral blood morphology. Attenuation of disease in Rac2- and Rac1/Rac2-deficient animals correlated with severely diminished activation of BCR-ABL-induced signaling pathways, including p44/42 and p38 ERK, JNK, CrkL, and Akt. The leukemogenesis impairment induced by Rac deficiency did not appear to be due to loss of p210-BCR-ABL vector integration, as clonal analysis of leukemic bone marrow from mice in each genotype by LAM-PCR showed similar, oligoclonal reconstitution of p210-BCR-ABL expressing cells. Interestingly, bone marrow cells obtained from Rac1/Rac2-deficient animals that developed late leukemia showed marked hyperactivation of Rac3 and initiated disease in recipients with a latency of three weeks, suggesting that leukemia-initiating cells were able to engraft, in spite of Rac1/Rac2 deficiency. Treatment of BCR-ABL-expressing murine HSC with NSC23766, a rationally-designed Rac-specific small molecule antagonist, potently inhibited cell proliferation in vitro and increased the survival of leukemic animals treated in vivo, compared to PBS control-treated animals (p<0.05). NSC23766 also inhibited the growth of an imatinib-resistant p210-BCR-ABL-T315I-expressing Ba/F3 leukemic cell line by 90%, compared to <5% by imatinib alone, blocked the growth of primary human chronic phase Rac-hyperactivated CML blast colonies by 80% in vitro, and inhibited survival of these cells in NOD-SCID mice. These results suggest that individual Rac proteins play both unique and combinatorial roles in stem cell transformation and may represent unique targets for therapy of BCR-ABL-persistent and imatinib-resistant CML.
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Shutes, Adam, Cercina Onesto, Virginie Picard, Bertrand Leblond, Fabien Schweighoffer, and Channing J. Der. "Specificity and Mechanism of Action of EHT 1864, a Novel Small Molecule Inhibitor of Rac Family Small GTPases." Journal of Biological Chemistry 282, no. 49 (October 11, 2007): 35666–78. http://dx.doi.org/10.1074/jbc.m703571200.
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There is now considerable experimental evidence that aberrant activation of Rho family small GTPases promotes the uncontrolled proliferation, invasion, and metastatic properties of human cancer cells. Therefore, there is considerable interest in the development of small molecule inhibitors of Rho GTPase function. However, to date, most efforts have focused on inhibitors that indirectly block Rho GTPase function, by targeting either enzymes involved in post-translational processing or downstream protein kinase effectors. We recently determined that the EHT 1864 small molecule can inhibit Rac function in vivo. In this study, we evaluated the biological and biochemical specificities and biochemical mechanism of action of EHT 1864. We determined that EHT 1864 specifically inhibited Rac1-dependent platelet-derived growth factor-induced lamellipodia formation. Furthermore, our biochemical analyses with recombinant Rac proteins found that EHT 1864 possesses high affinity binding to Rac1, as well as the related Rac1b, Rac2, and Rac3 isoforms, and this association promoted the loss of bound nucleotide, inhibiting both guanine nucleotide association and Tiam1 Rac guanine nucleotide exchange factor-stimulated exchange factor activity in vitro. EHT 1864 therefore places Rac in an inert and inactive state, preventing its engagement with downstream effectors. Finally, we evaluated the ability of EHT 1864 to block Rac-dependent growth transformation, and we determined that EHT 1864 potently blocked transformation caused by constitutively activated Rac1, as well as Rac-dependent transformation caused by Tiam1 or Ras. Taken together, our results suggest that EHT 1864 selectively inhibits Rac downstream signaling and transformation by a novel mechanism involving guanine nucleotide displacement.
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Pai, Sung-Yun, Chaekyun Kim, and David A. Williams. "Rac GTPases in Human Diseases." Disease Markers 29, no. 3-4 (2010): 177–87. http://dx.doi.org/10.1155/2010/380291.
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Rho GTPases are members of the Ras superfamily of GTPases that regulate a wide variety of cellular functions. While Rho GTPase pathways have been implicated in various pathological conditions in humans, to date coding mutations in only the hematopoietic specific GTPase,RAC2, have been found to cause a human disease, a severe phagocytic immunodeficiency characterized by life-threatening infections in infancy. Interestingly, the phenotype was predicted by a mouse knock-out ofRAC2and resembles leukocyte adhesion deficiency (LAD). Here we review Rho GTPases with a specific focus on Rac GTPases. In particular, we discuss a new understanding of the unique and overlapping roles of Rac2 in blood cells that has developed since the generation of mice deficient in Rac1, Rac2 and Rac3 proteins. We propose that Rac2 mutations leading to disease be termed LAD type IV.
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Heyworth, P. G., U. G. Knaus, J. Settleman, J. T. Curnutte, and G. M. Bokoch. "Regulation of NADPH oxidase activity by Rac GTPase activating protein(s)." Molecular Biology of the Cell 4, no. 11 (November 1993): 1217–23. http://dx.doi.org/10.1091/mbc.4.11.1217.
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Activation of the NADPH oxidase of phagocytic cells requires the action of Rac2 or Rac1, members of the Ras superfamily of GTP-binding proteins. Rac proteins are active when in the GTP-bound form and can be regulated by a variety of proteins that modulate the exchange of GDP for GTP and/or GTP hydrolysis. The p190 Rac GTPase Activating Protein (GAP) inhibits human neutrophil NADPH oxidase activity in a cell-free assay system with a K1 of approximately 100 nM. Inhibition by p190 was prevented by GTP gamma S, a nonhydrolyzable analogue of GTP. Similar inhibition was seen with a second protein exhibiting Rac GAP activity, CDC42Hs GAP. The effect of p190 on superoxide (O2-) formation was reversed by the addition of a constitutively GTP-bound Rac2 mutant or Rac1-GTP gamma S but not by RhoA-GTP gamma S. Addition of p190 to an activated oxidase produced no inhibitory effect, suggesting either that p190 no longer has access to Rac in the assembled oxidase or that Rac-GTP is not required for activity once O2- generation has been initiated. These data confirm the role of Rac in NADPH oxidase regulation and support the view that it is the GTP form of Rac that is necessary for oxidase activation. Finally, they raise the possibility that NADPH oxidase may be regulated by the action of GAPs for Rac proteins.
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Pestonjamasp, Kersi N., Carol Forster, Chunxiang Sun, Elisabeth M. Gardiner, Ben Bohl, Orion Weiner, Gary M. Bokoch, and Michael Glogauer. "Rac1 links leading edge and uropod events through Rho and myosin activation during chemotaxis." Blood 108, no. 8 (October 15, 2006): 2814–20. http://dx.doi.org/10.1182/blood-2006-01-010363.
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Abstract Chemotactic responsiveness is crucial to neutrophil recruitment to sites of infection. During chemotaxis, highly divergent cytoskeletal programs are executed at the leading and trailing edge of motile neutrophils. The Rho family of small GTPases plays a critical role in cell migration, and recent work has focused on elucidating the specific roles played by Rac1, Rac2, Cdc42, and Rho during cellular chemotaxis. Rac GTPases regulate actin polymerization and extension of the leading edge, whereas Rho GTPases control myosin-based contraction of the trailing edge. Rac and Rho signaling are thought to crosstalk with one another, and previous research has focused on mutual inhibition of Rac and Rho signaling during chemotaxis. Indeed, polarization of neutrophils has been proposed to involve the activity of a negative feedback system where Rac activation at the front of the cell inhibits local Rho activation, and vice versa. Using primary human neutrophils and neutrophils derived from a Rac1/Rac2-null transgenic mouse model, we demonstrate here that Rac1 (and not Rac2) is essential for Rho and myosin activation at the trailing edge to regulate uropod function. We conclude that Rac plays both positive and negative roles in the organization of the Rhomyosin “backness” program, thereby promoting stable polarity in chemotaxing neutrophils.
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Miyano, Kei, Hirofumi Koga, Reiko Minakami, and Hideki Sumimoto. "The insert region of the Rac GTPases is dispensable for activation of superoxide-producing NADPH oxidases." Biochemical Journal 422, no. 2 (August 13, 2009): 373–82. http://dx.doi.org/10.1042/bj20082182.
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Rac1 and Rac2, which belong to the Rho subfamily of Ras-related GTPases, play an essential role in activation of gp91phox/Nox2 (cytochrome b-245, β polypeptide; also known as Cybb), the catalytic core of the superoxide-producing NADPH oxidase in phagocytes. Rac1 also contributes to activation of the non-phagocytic oxidases Nox1 (NADPH oxidase 1) and Nox3 (NADPH oxidase 3), each related closely to gp91phox/Nox2. It has remained controversial whether the insert region of Rac (amino acids 123–135), unique to the Rho subfamily proteins, is involved in gp91phox/Nox2 activation. In the present study we show that removal of the insert region from Rac1 neither affects activation of gp91phox/Nox2, which is reconstituted under cell-free and whole-cell conditions, nor blocks its localization to phagosomes during ingestion of IgG-coated beads by macrophage-like RAW264.7 cells. The insert region of Rac2 is also dispensable for gp91phox/Nox2 activation at the cellular level. Although Rac2, as well as Rac1, is capable of enhancing superoxide production by Nox1 and Nox3, the enhancements by the two GTPases are both independent of the insert region. We also demonstrate that Rac3, a third member of the Rac family in mammals, has an ability to activate the three oxidases and that the activation does not require the insert region. Thus the insert region of the Rac GTPases does not participate in regulation of the Nox family NADPH oxidases.
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Korhonen, Matti, Haibo Zhao, Roberta Faccio, F. Patrick Ross, Tracy M. Hopkins, Jose A. Cancelas, Steven L. Teitelbaum, and David A. Williams. "Rac1 and Rac2 GTPases Play Distinct Roles and Are Essential for Full Osteoclast Differentiation." Blood 106, no. 11 (November 16, 2005): 67. http://dx.doi.org/10.1182/blood.v106.11.67.67.
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Abstract Bone-resorbing osteoclasts play a central role in bone remodeling, which occurs throughout life. Many skeletal diseases such as osteoporosis, Paget’s disease and the lytic lesions of multiple myeloma, display excess osteoclast activity. Thus, in addition to basic biological questions, there is considerable clinical interest in the control of osteoclast differentiation and function. Previously we have demonstrated that the small GTPases Rac1 and Rac2 have specific roles in the control of hematopoietic stem cell and neutrophil functions (Gu and Filippi et al., Science 2003; Filippi et al., Nat Immunol 2004; Cancelas et al., Nat Med 2005). During these studies, we noted differences in the bone structure of Rac-deficient mice, suggesting alterations in osteoclast activity. Furthermore we found that in hematopoietic stem cells Rac proteins regulate signaling pathways that are also known to control osteoclastogenesis. In this study, we have employed a genetic approach to analyze the roles of Rac proteins in osteoclast differentiation. We utilized constitutively Rac2-null mice in combination with cre-induced deletion of floxed Rac1 sequences to effect the loss of both Rac GTPases in hematopoietic cells. Macrophages from Rac2−/− mice generated normal numbers of osteoclasts in vitro. However, the full differentiation of these cells, as assayed by emergence of differentiation markers, was perturbed. Expression the TRAP (tartrate-resistant acid phosphatase) enzyme was delayed (12 +/−3% vs. 88 +/−8%, Rac2−/− vs. wt, n= 5, p<0.001) and the expression of the β3 integrin subunit was decreased (16% vs. 76%, Rac2−/− vs. wt, n=5). The number of cells having podosomes was reduced (8 +/−3 vs. 206 +/−48 cells with podosomes/well Rac2−/− vs. wt, p<0.001). Cell fusion, which accompanies osteoclastogenesis, was also reduced. In contrast Rac1−/− macrophages produced severely reduced numbers of osteoclasts in vitro (13 +/−8/well vs. 272 +/−52 Rac1−/− vs. wt, n=2, p<0.001). Rac1−/−Rac2−/− double knock-out cells essentially developed no osteoclasts in vitro. The p44/42, JNK (jun N-terminal kinase), Akt and p38 intracellular kinase signaling pathways have all been shown to be important for osteoclastogenesis. Activation of the p44/42 and JNK (jun N-terminal kinase) pathways in response to stimulation with M-CSF (macrophage colony stimulating factor) and RANKL (receptor activator of NF-κB ligand), cytokines critically involved in osteoclast differentiation, was reduced in the Rac2−/− macrophages. When Rac1−/− cells were stimulated with M-CSF, decreased activation of the Akt and JNK pathways was observed. To study the effect of Rac deficiency on bone mass in vivo, we generated Rac1−/−Rac2−/− double knock-out mice. These mice had significantly increased bone mass (bone volume/tissue volume 0.33 +/−0.03 vs. 0.13 +/−0.02 Rac1−/−Rac2−/− vs. wild-type; p<0.001). These results indicate that 1) Rac GTPases are critical to the differentiation of macrophages into osteoclasts, 2) in the absence of Rac2 osteoclastogenesis is perturbed while inhibition of Rac1 function leads to nearly complete inhibition osteoclastogenesis, 3) specific alterations in intracellular signaling pathways are seen in Rac-deficient osteoclast precursors, and 4) inhibition of Rac function in vivo leads to an increase in bone mass.
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Troeger, Anja, Pascal-David Johann, Mumine Senturk, Michael D. Milsom, and David A. Williams. "Intact Rac Signaling Is Important for Leukemia Cell Survival." Blood 116, no. 21 (November 19, 2010): 2885. http://dx.doi.org/10.1182/blood.v116.21.2885.2885.
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Abstract Abstract 2885 Rho GTPases, Ras-related small G proteins, regulate multiple cell processes in hematopoietic cells. There is growing evidence that acute myeloid leukemia (AML) blasts and particularly MLL-rearranged AML blasts, rely on Rac activity (Mulloy JC et al, Blood, 2010). However, little is known about the role of these GTPases in acute lymphoblastic leukemia (ALL) and particularly precursor B cell ALL. To investigate the role of Rac and potential compensation by other GTPases in ALL, we first assessed the protein expression and activation of Rac in a number of B-ALL cell lines (SEM; RS4,11; REH; Nalm 6; Raji), compared with a T-ALL cell line (Jurkat) and several AML cell lines (ML2; MV4,11). Of these cell lines SEM; RS4,11; ML2 and MV4,11 are characterized by MLL-fusion genes. Jurkat and MLL-rearranged AML cell lines show higher expression of Rac proteins compared to B cell leukemia lines (Table 1). Overall, B-ALL cell lines exhibit highly variable levels of Rac expression and activity with no obvious correlation to the presence of MLL-fusion proteins. We then investigated proliferation and apoptosis in cell lines treated with the small molecule inhibitor NSC23766 (NSC), which blocks interaction of a subset of guanine exchange factors (GEFs) with Rac and thus inhibits its activation. Treatment with NSC led to ∼2-fold increase in cells arrested at G0/G1 and induced apoptosis in a dose-dependent fashion at NSC concentrations previously demonstrated to be non-toxic in normal hematopoietic cells (Muller LUW et al., Leukemia, 2008) (Table 2). The lymphoid cell lines Jurkat, Raji and SEM appeared less responsive to NSC with no increased apoptosis at 40μM NSC. There was no correlation between NSC response and baseline expression or activation status of Rac. However, cell lines resistant to NSC exhibited a paradoxical and transient early increase in Rac activation, suggesting the existence of compensatory activation mechanisms. To determine if the relative resistance observed in some cell lines was related to dependence on GEFs not targeted by NSC and to validate that the inhibitory effect of NSC was specifically due to Rac inhibition in sensitive cells, shRNAs were utilized to knock-down different members of the Rac subfamily. Effective shRNA-mediated knockdown was validated by western blot. Knockdown of Rac1 or Rac2 consistently induced apoptosis compared to non-targeting vector controls in NSC sensitive cell lines ML2 and Nalm6, with ML2 cells appearing slightly more sensitive to knock-down of Rac2 (Table 3). Knock-down of either Rac1 or Rac2 had little effect upon Jurkat cells which are resistant to NSC treatment. These data suggest that Jurkat cells are not dependent upon Rac signaling for survival; however we cannot discount the possibility that some compensation may occur between Rac1 and Rac2. These experiments demonstrate the importance of intact Rac signaling pathways for the survival of the majority of leukemia cell lines tested and demonstrate that dependence on Rac signaling is not restricted to leukemias characterized by MLL-rearrangements. Our observations also suggest that activation of different Rac isoforms may influence sensitivity towards pharmacological Rac inhibition. Table 1: Baseline Expression of Rac assessed by Western blot Cell line Jurkat ML-2 MV-4,11 RS-4,11 SEM Nalm 6 REH Raji Rac/b-actin expression* 1.6 2.5 1.7 0.5 0.7 0.8 1.0 1.0 (*arbitrary units, italics indicate cell lines carrying MLL-rearrangements) Table 2: % AnnexinV+ cells after treatment of the different cell lines with increasing doses the Rac-specific inhibitor NSC Cell line Jurkat ML-2 MV-4,11 RS-4,11 SEM Nalm 6 REH Raji control 6%+1.4 6%+1.3 9%+0.3 12%+3.6 9%+1.9 7%+1.5 9%+2 13%+2.3 20uM NSC 6%+1.4 9%+1.3 15%+0.3** 21%+8.5 8%+1.5 6%+1.9 25%+6.4 16%+3 40uM NSC 7%+1.8 24%+9.1 60%+4** 52%+11* 10%+1.3 10%+3.4 39%+11 16%+1.9 80uM NSC 15%+3.5* 73%+14.7** 97%+0.4** 80%+4** 17%+1.2* 46%+10.5** 62%+12.3* 22%+4 (Mean±SEM; n=5; * p<0.05; ** p<=0.01 versus control, bolded columns indicate increased NSC sensitivity) Table 3: % AnnexinV+ cells 7 days after lentiviral transduction of the different cell lines with Rac1 and Rac2-specific shRNA Cell line Jurkat ML-2 Nalm 6 non targeting control 4.3%+0.3 14.2%+8 11.4%+2.2 Rac1 shRNA* 8.0%+3.5 26.3%+7.9 36.8%+8.5 non targeting control 9.6%+4.2 8.1%+4.0 16.2%+3.1 Rac2 shRNA* 18.7%+4.5 35.5%+12.9 43.7%+7.1 (Mean±SEM; n=6; * second set of Rac1 and Rac2 shRNAs gave comparable results) Disclosures: No relevant conflicts of interest to declare.
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Wei, Junping, Mark Wunderlich, Chad Harris, Benjamin Mizukawa, Yi Zheng, David A. Williams, and James C. Mulloy. "Rac GTPases Are Required for MLL-AF9-Induced Mixed Lineage Leukemia." Blood 112, no. 11 (November 16, 2008): 3368. http://dx.doi.org/10.1182/blood.v112.11.3368.3368.
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Abstract The t(9;11) translocation fusion gene MLL-AF9 (MA9) is commonly found in acute myeloid and lymphoid leukemia and is associated with intermediate to poor outcome. The specific signaling pathways downstream of MA9 are still poorly understood. It has recently been reported that MA9 leukemia cells express higher levels of the small GTPase protein Rac and CDC42 when compared to in vitro MA9 immortal cells in a murine model. To determine the importance of Rac GTPase signaling in MA9-induced transformation, we used an MA9 leukemia model we recently established involving MA9 fusion gene expression in human CD34+ cells. Treatment with the Rac specific inhibitor NSC23766, or transient knockdown of Rac expression by RNAi, induced rapid apoptosis in MA9 cells but not in normal cord blood or t(8;21) translocation fusion gene AML-ETO expressing cells. These data demonstrate that the Rac signaling pathway plays a critical role in the growth and survival of MA9 leukemia cells. To extend this work to an in vivo genetic model, we used mice deficient in Rac2 (Rac2-KO) or with floxed alleles of Rac1 in mice transgenic for Mx-Cre. Leukemia development was compared in mice transplanted with MA9-transduced wild type, Rac1 or Rac2 deficient low density bone marrow cells. Poly I:C injections were performed 2 weeks after transplantation to delete Rac1. Rac deletion was confirmed by PCR and western blot analysis. Mice that received either WT or Rac1−/ − MA9 expressing cells uniformly developed AML and died at 3 to 5 months. Mice transplanted with Rac2-KO cells expressing MA9 showed a decreased incidence and increased latency (6 to 10 months) of AML development despite the persistent engraftment of MA9-expressing cells. All three groups of mice maintained MA9 EGFP+ cells in peripheral blood over the entire experiment, and eventually gave rise to similar end stage AML with a Gr-1+/Mac-1+/Kit+/B220-/CD3- phenotype and myelomonocytic blast morphology. Combined with our observation in human CD34+ cord blood cells transduced with MA9, these in vitro and in vivo data indicate that MA9-mediated transformation and survival requires Rac and their downstream effectors. Rac2 signaling appears to be particularly important in the murine MA9 AML model. Therapeutic targeting of Rac could be a unique and important approach to treating MLL leukemia.
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Korhonen, Matti, Haibo Zhao, Roberta Faccio, Patric F. Ross, Tracy M. Hopkins, Jose A. Cancelas, and David A. Williams. "Rac1 and Rac2 GTPases Play Distinct Roles and Are Essential for Full Osteoclast Differentiation." Blood 108, no. 11 (November 16, 2006): 4231. http://dx.doi.org/10.1182/blood.v108.11.4231.4231.
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Abstract Bone-resorbing osteoclasts play a central role in bone remodeling, which occurs throughout life. Many skeletal diseases such as osteoporosis, Paget’s disease and the lytic lesions of multiple myeloma, display excess osteoclast activity. Thus, in addition to basic biological questions, there is considerable clinical interest in the control of osteoclast differentiation and function. Previously we have demonstrated that the small GTPases Rac1 and Rac2 have specific roles in the control of hematopoietic stem cell and neutrophil functions (Gu and Filippi et al., Science 2003; Filippi et al., Nat Immunol 2004; Cancelas et al., Nat Med 2005). During these studies, we noted differences in the bone structure of Rac-deficient mice, suggesting alterations in osteoclast activity. Furthermore we found that in hematopoietic stem cells Rac proteins regulate signaling pathways that are also known to control osteoclastogenesis. In this study, we have employed a genetic approach to analyze the roles of Rac proteins in osteoclast differentiation. We utilized constitutively Rac2-null mice in combination with cre-induced deletion of floxed Rac1 sequences to effect the loss of both Rac GTPases in hematopoietic cells. Macrophages from Rac2−/− mice generated normal numbers of osteoclasts in vitro. However, the full differentiation of these cells, as assayed by emergence of differentiation markers, was perturbed. Expression the TRAP (tartrate-resistant acid phosphatase) enzyme was delayed (12 +/−3% vs. 88 +/−8%, Rac2−/− vs. wt, n= 5, p<0.001) and the expression of the β3 integrin subunit was decreased (16% vs. 76%, Rac2−/− vs. wt, n=5). The number of cells having podosomes was reduced (8 +/−3 vs. 206 +/−48 cells with podosomes/well Rac2−/− vs. wt, p<0.001). Cell fusion, which accompanies osteoclastogenesis, was also reduced. In contrast Rac1−/− macrophages produced severely reduced numbers of osteoclasts in vitro (13 +/−8/well vs. 272 +/−52 Rac1−/− vs. wt, n=2, p<0.001). Rac1−/−Rac2−/− double knock-out cells essentially developed no osteoclasts in vitro. The p44/42, JNK1 (jun N-terminal kinase 1), Akt and p38 intracellular kinase signaling pathways have all been shown to be important for osteoclastogenesis. Activation of the p44/42 pathway in response to stimulation with M-CSF (macrophage colony stimulating factor) and RANKL (receptor activator of NF-κB ligand), cytokines critically involved in osteoclast differentiation, was reduced in the Rac2−/− macrophages. When Rac1−/− cells were stimulated with RANKL, decreased activation of NF-κB and JNK1 was observed. Interestingly, the combination of Rac1 and Rac2 deficiencies induces a significant increase of bone mass in vivo (bone volume/tissue volume 0.33 ± 0.03 vs. 0.13 ± 0.02 Rac1−/−Rac2−/− vs. wild-type; p<0.001), similar to osteopetrosis models. These results indicate that Rac GTPases are critical to the differentiation of macrophages into osteoclasts, Rac2 deficiency perturbs osteoclast differentiation while in the absence of Rac1 it is severely inhibited, specific alterations in intracellular signaling pathways are seen in Rac-deficient osteoclast precursors, and inhibition of Rac function in vivo leads to an increase in bone mass due to, at least in part, deficient osteoclast function.
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Mizukawa, Benjamin, Junping Wei, Mahesh Shrestha, Mark Wunderlich, Fu-Sheng Chou, Andrea Griesinger, Chad E. Harris, et al. "Inhibition of Rac GTPase signaling and downstream prosurvival Bcl-2 proteins as combination targeted therapy in MLL-AF9 leukemia." Blood 118, no. 19 (November 10, 2011): 5235–45. http://dx.doi.org/10.1182/blood-2011-04-351817.
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Abstract 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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Cancelas, Jose A., Andrew W. Lee, Rethinasamy Prabhakar, Michael Jansen, YI Zheng, and David A. Williams. "Rac1 and Rac2 Rho GTPases Distinctly Regulate Stem Cell Engraftment and Mobilization and Are Novel Targets for Pharmacologically-Induced Progenitor Mobilization." Blood 104, no. 11 (November 16, 2004): 115. http://dx.doi.org/10.1182/blood.v104.11.115.115.
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Abstract Rac members of the Ras-related Rho GTPase family regulate mammalian cell cytoskeleton, survival and proliferation. We have recently implicated Rac1 in short-term hematopoietic stem/progenitor (HSC/P) cell engraftment and Rac2 in HSC/P mobilization (Gu Y et al., Science 2003). Indeed, Rac proteins are activated via β1-integrins, CXCR4 and c-kit, all receptors implicated in homing and mobilization. Recent data examining the function of CXCR4 have been interpreted to show that mobilization and engraftment are mirror image processes. Using both a genetic and a pharmacological approach, we examined the role of Rac proteins in mobilization, engraftment and steady-state hematopoiesis. Here we demonstrate that whereas Rac1−/− HSC/P fail to engraft after transplantation, deletion of Rac1 by Cre-mediated deletion of floxed Rac1 after engraftment does not significantly affect either blood formation or HSC/P mobilization. Rac1−/−;Rac2−/− HSC/P dramatically fail to sustain steady-state hematopoiesis (over 95% reduction at 6 months) leading to a replacement of hematopoiesis by cells expressing Rac1. By infusing 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester labeled Lin-/c-kit+ BM cells into the blood followed by in situ fixative perfusion after 16 hours, we observed that the spatial distribution of transplanted marrow cells in the endosteal space (defined as less than 15 cells away from either endosteum in μm-longitudinal sections) was defective in Rac1−/− cells 4 (25%) versus wild-type (39%), suggesting defective retention of Rac1−/− HSC in the “stem cell niche” after engraftment. Rac1−/−;Rac2−/− lin-/c-kit+ cells showed a more severe defect in spatial localization to the endosteum (19% vs 39% in wild-type). In vitro, Rac1−/− HSC/P also showed a severely decreased cobblestone area formation ability (>95% reduction in CAFC frequency) but had normal transendothelial migration. In contrast, Rac2−/− HSC/P demonstrated normal short-term engraftment and only mild defects in these assays. Induction of combined Rac1 and Rac2 deficiency induces a striking mobilization of progenitor cells (Gu Y et al., Science 2003) while Rac1 re-expression by retrovirus-mediated gene transfer into these mobilized HSC is sufficient to effect engraftment of HSC/P into the BM (9-fold increase in engraftment ability in a competitive repopulation assay). Altogether these data suggest distinct roles for Rac1 versus Rac2 in retention of HSC/P in the BM endosteal space implying that engraftment and mobilization are not mirror image processes. To further exploit the identification of Rac as a regulator of HSC retention in BM, we employed a newly identified compound, NSC23776, specifically designed to block the interaction of Rac proteins with activating GTPase exchange factors (GEFs). When injected into the poorly mobilizing C57Bl/6 mouse strain, NSC23776 (2.5 mg/Kg i.p.) induced a 2-fold increase in circulating progenitors at 3–6 h after injection and additional trafficking (5-fold increase over wild-type mice) of these cells in Rac2−/− mice which show increased mobilization at baseline. The mobilization induced by a Rac inhibitory compound demonstrates that Rac is a novel target to induce mobilization of HSC/P.
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Chuang, T. H., X. Xu, L. A. Quilliam, and G. M. Bokoch. "SmgGDS stabilizes nucleotide-bound and -free forms of the Rac1 GTP-binding protein and stimulates GTP/GDP exchange through a substituted enzyme mechanism." Biochemical Journal 303, no. 3 (November 1, 1994): 761–67. http://dx.doi.org/10.1042/bj3030761.
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The Rac proteins, Rac1 and Rac2, are essential components of the NADPH oxidase system of phagocytes and regulate the actin assembly associated with membrane ruffling. These functions are controlled by the GTP-bound form of Rac. The biochemical interaction between Rac and its only known GDP-dissociation stimulator (termed smgGDS) was characterized. SmgGDS was able to stimulate the incorporation of guanosine 5′-[gamma-thio]-triphosphate GTP[gamma S] into the RhoA, Rac2, Rac1, Rap1A and CDC42Hs GTP-binding proteins, but the activity was greatest toward RhoA and Rac2. Isoprenoid modification of these proteins was not absolutely required for the interaction with smgGDS. Interestingly, the activity of smgGDS toward Rac1 could not be observed in a [3H]GDP/GTP exchange assay under conditions where it stimulated incorporation of GTP[gamma S] into Rac1. We determined that smgGDS prevented the loss of Rac1 activity during the [3H]GDP/GTP exchange assay by demonstrating the ability of smgGDS to inhibit the loss of Rac1 GTP[gamma S]-binding during incubations at 30 degrees C. This stabilizing effect was exactly counterbalanced by the ability of smgGDS to stimulate the release of [3H]GDP from Rac1, thereby producing no net observable effect in the exchange assay. SmgGDS was able to effectively stimulate the release of GDP but not GTP[gamma S] from Rac1. SmgGDS maintains Rac1 in a nucleotide-free form after release of GDP, indicating that the reaction between Rac1 and smgGDS involves a substituted enzyme mechanism.
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Gavillet, Mathilde, Kimberly Martinod, Denisa D. Wagner, and David A. Williams. "The Role of Rac and Pak in Neutrophil Histone Hypercitrullination and Neutrophil Extracellular Traps Formation." Blood 124, no. 21 (December 6, 2014): 462. http://dx.doi.org/10.1182/blood.v124.21.462.462.
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Abstract Under specific activating conditions, polymorphonuclear neutrophils (PMNs) release neutrophil extracellular traps (NETs) composed of decondensed chromatin lined with microbicidal protein such as neutrophil elastase and myeloperoxidase. NETs contribute to innate immunity but can also foster autoimmune diseases and thrombus formation. NET formation (NETosis) requires reactive oxygen species (ROS) production by NADPH oxidase and histone hypercitrullination by peptidylarginine deiminase 4 (PAD4), allowing for chromatin decondensation. Rac GTPases are expressed in three isoforms: Rac1 is ubiquitously expressed and plays a role in PMN migration and oxidase function; Rac2 is hematopoietic-specific and the major isoform in PMNs and Rac3 is mostly neuronal. Rac1 and Rac2 regulate the cytoskeleton in PMNs, controlling actin polymerization, cell shape, adhesion and migration and are essential components of the NADPH oxidase complex. The present study aimed to explore the role of the Rac pathway on NETosis in PMNs, including the upstream guanosine exchange factor (GEF) activator, Vav, and a downstream effector of Rac, p21 activated kinase, Pak. We developed a flow cytometry-based quantification of H3 hypercitrullination (H3Cit). In response to phorbol myristate ester (PMA) stimulation, H3Cit is increased to 136% of basal in WT cells, compared with 103% in Rac2-/- (P<0.01) (Table). H3Cit levels observed by flow were confirmed in a NET formation assay. Rac2-/- PMNs formed significantly fewer NETs both spontaneously and after PMA stimulation (WT unstimulated 2.79%, Rac2-/- unstimulated 0.72%, WT+PMA 10.84%, Rac2-/-+PMA 1.39%, P< 0.05 for all pair comparisons). Furthermore, Rac2-/- mice demonstrated a trend towards reduced frequency of provoked thrombosis in an in vivo vena cava stenosis model (WT 78% and Rac2-/- 56% of mice with thrombus). Deletion of floxed Rac1 sequences in a Rac2-/- background in vivoallows generation and purification of PMNs lacking both Rac isoforms. Rac1Δ/Δ,Rac2-/- PMNs, which are defective in actin polymerization, had reduced basal H3Cit and a nearly complete lack of PMA-induced increase in H3Cit (136% vs 69%, WT vs Rac1Δ/Δ,Rac2-/-, P<0.01) (Table). Null knockouts of the GEFs Vav1 (hematopoietic-specific), Vav2, Vav3 or both Vav1 and 3 did not impair H3Cit response to PMA (Table). We next studied downstream effectors of Rac. Group A Paks include Pak1, 2 and 3 isoforms. Pretreatment of wild-type PMNs with either PF3758309 or IPA-3, two group A Pak inhibitors with distinct mechanisms of action, led to reduced H3Cit after PMA stimulation (induction reduced from 36% to 11% for both PF3758309 and IPA-3 treated (Table). To validate this in a genetic model, we studied Pak2Δ/Δ PMNs, since we have recently demonstrated the dependence of hematopoietic stem cell migration on Pak2. Pak2Δ/Δ demonstrated a reduced basal level of H3Cit and a significantly reduced PMA-induced increase in H3Cit (136% vs 94%, WT vs Pak2Δ/Δ, P<0.05, Table). In summary, we describe a flow-based assay that quantitates the early processes of NET formation and validated that this assay reliably predicts agonist-induced NET formation in a genetic model. The results establish that both Rac1 and Rac2, and the downstream effector Pak2, regulate histone H3 hypercitrullination and NET formation in PMNs, while suggesting that Vav does not activate the Rac pathway in PMA-induced NET formation. These data further delineate the role of the Rac pathway in NETosis, linking cytoskeleton and oxidase functions. Furthermore, these data indicate Pak could represent a therapeutic target for a wide array of pathological processes related to NETosis such as thrombosis and numerous autoimmune diseases. Table Intensity of H3Cit staining as determined by flow cytometry-based assay. Basal H3Cit level PMA-induced H3Cit PMA-induced change WT PMN 100±2% 136±5% 36% Rac2-/- 82±9% ns 103±15.3% ** 21% Rac1Δ/Δ, Rac2-/- 64±10% *** 69±10% ** 5% Vav1-/- 86±9% ns 145±15% ns 59% Vav2-/- 91±4% ns 134±18% ns 43% Vav3-/- 153±30% * 171±28% ns 18% Vav1,3-/- 125±20% ns 144±24% ns 19% WT+ PF 5nM 76±17% ns 87±8% * 11% WT+ IPA 5µM 100±4% ns 111±10% ns 11% Pak2Δ/Δ 75±7% * 94±11% * 19% Results are expressed as mean±SEM % of the untreated WT control of each experiment. Results are from ≥3 independent experiments. * P<0.05, **P<0.01. P<0.001, ns non-significant, by two-tailed t-test. Disclosures No relevant conflicts of interest to declare.
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Corbetta, Sara, Sara Gualdoni, Chiara Albertinazzi, Simona Paris, Laura Croci, G. Giacomo Consalez, and Ivan de Curtis. "Generation and Characterization of Rac3 Knockout Mice." Molecular and Cellular Biology 25, no. 13 (July 1, 2005): 5763–76. http://dx.doi.org/10.1128/mcb.25.13.5763-5776.2005.
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ABSTRACT Rac proteins are members of the Rho family of GTPases involved in the regulation of actin dynamics. The three highly homologous Rac proteins in mammals are the ubiquitous Rac1, the hematopoiesis-specific Rac2, and the least-characterized Rac3. We show here that Rac3 mRNA is widely and specifically expressed in the developing nervous system, with highest concentration at embryonic day 13 in the dorsal root ganglia and ventral spinal cord. At postnatal day 7 Rac3 appears particularly abundant in populations of projection neurons in several regions of the brain, including the fifth layer of the cortex and the CA1-CA3 region of the hippocampus. We generated mice deleted for the Rac3 gene with the aim of analyzing the function of this GTPase in vivo. Rac3 knockout animals survive embryogenesis and show no obvious developmental defects. Interestingly, specific behavioral differences were detected in the Rac3-deficient animals, since motor coordination and motor learning on the rotarod was superior to that of their wild-type littermates. No obvious histological or immunohistological differences were observed at major sites of Rac3 expression. Our results indicate that, in vivo, Rac3 activity is not strictly required for normal development in utero but may be relevant to later events in the development of a functional nervous system.
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Chae, Hee-Don, Katherine E. Lee, Aparna C. Jasti, David A. Williams, and Yi Gu. "Cross-Talk between Rho GTPases Regulates Actin Cytoskeleton and Chemotaxis of Hematopoietic Progenitor Cell." Blood 106, no. 11 (November 16, 2005): 266. http://dx.doi.org/10.1182/blood.v106.11.266.266.
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Abstract Movement of hematopoietic stem/progenitor cells into (engraftment) and out of (mobilization) the bone marrow involves actin cytoskeleton and chemotaxis. Members of the Rho GTPase family have been well known for their critical roles in morphogenesis and cell migration via regulating actin assembly. Loss of Rac1 and Rac2 alleles leads to defective engraftment and massive mobilization of hematopoietic progenitor cells (HPCs), which are associated with impaired chemotaxis and cortical filamentous (F)-actin polymerization (Gu et al., Science 302: 445–449). RhoH, a hematopoietic-specific member of the RhoE subfamily, negatively regulates HPC engraftment, chemotaxis, F-actin polymerization and Rac activities (Gu et al., Blood 105: 1467–1475). These findings suggest that RhoH may antagonize Rac function in regulating these cellular processes. However, molecular mechanism of the cross-talk between these Rho GTPases is not defined. In this study, we examined the role of RhoH in actin cytoskeleton organization, chemotaxis and Rac membrane translocation in response to stromal-derived factor 1α (SDF-1α) using RhoH-deficient HPCs and retrovirus-mediated expression of EGFP-fusion proteins. RhoH−/− HPCs exhibit increased migration in response to SDF-1α, especially at low concentration, as compared with wild-type (WT) cells [10ng/ml SDF-1α: 3.5 +/− 0.9 vs. 12.3 +/− 1.8; 100ng/ml SDF-1α: 21.4 +/− 1.7 vs. 32.3 +/− 3.4, migrated cells (%), WT vs. RhoH−/−, n=3, p< 0.01]. Migration without SDF-1α stimulation of RhoH−/− cells is also enhanced. RhoH−/− HPCs assemble cortical F-actin without SDF-1α stimulation, under conditions in which WT cells do not show F-actin polymerization [cells with F-actin (%): 8.9 +/− 0.9 vs. 72.8 +/− 4, WT vs. RhoH−/−, n=6, p<0.001]. Additionally, RhoH−/− HPCs exhibit increased active, GTP-bound Rac GTPases. PAK, a known downstream effector of Rac in regulating actin cytoskeleton, also shows hyperphosphorylation in RhoH-/− HPCs, suggesting that RhoH may regulate actin assembly and cell migration through Rac-mediated pathway. In support of this, expression of a dominant negative Rac1N17 mutant blocks cortical F-actin assembly in RhoH−/− cells [cells with F-actin (%): 60 +/− 1 vs. 19 +/− 7, EGFP-Rac1 vs. Rac1N17, n=2]. To further address the mechanism by which RhoH cross-talks to affect Rac signaling, we examine the role of RhoH in subcellular localization of EGFP-Rac proteins. SDF-1α induces activation of Rac, leading to translocation to the cell membrane where it co-localizes with lipid rafts and mediates cortical F-actin assembly in HPCs. In contrast, the dominant negative Rac1N17 does not localize to the cell membrane after SDF-1α stimulation. In RhoH−/− HPCs, EGFP-Rac protein presents at the cell membrane in the absence of SDF-1α [cells with membrane-localized EGFP-Rac1 (%): 7.5 +/− 3.9 vs. 44.5 +/− 6.4, WT vs. RhoH−/−, n=2]. In contrast, overexpression of RhoH in HPCs blocks translocation to the cell membrane after SDF-1α stimulation of Rac1, Rac2 and active Rac1V12. Finally, we found that RhoH, a constitutively active, GTP-bound protein, preferentially localizes to the cell membrane even in the absence of SDF-1α. This localization is dependent upon the prenylation site and the c-terminal domains of RhoH. Lack of membrane localization is associated with defective biological function. Together, our data suggest that RhoH is essential for proper cortical F-actin assembly and chemotaxis of HPCs via regulating Rac activation and membrane localization, and implicates a functional cross-talk between RhoH and Rac.
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Wunderlich, Mark, Kevin A. Link, Fu-Sheng Chou, and James C. Mulloy. "Constitutive Activation of Rac GTPases Is Induced by Cooperating Mutations in a Human Model of MLL-AF9 Leukemia." Blood 114, no. 22 (November 20, 2009): 2959. http://dx.doi.org/10.1182/blood.v114.22.2959.2959.
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Abstract Abstract 2959 Poster Board II-935 Retroviral transduction of primary human umbilical cord blood CD34+ cells with a gene encoding MLL-AF9 (MA9) efficiently promotes immortalization in vitro and development of leukemia in a xenotransplant model. MA9 cells remain cytokine dependent with a strict requirement for Flt3 signaling. Additionally, the cells are hypersensitive to inhibition of the RAC GTPases. In this current study, we have examined a potential link between these pathways. We found that MA9 cells proliferate in culture media containing Flt3 Ligand (FL) as the sole cytokine, indicating that Flt3 signaling is sufficient for growth and survival. Furthermore, introduction of a mutant Flt3-ITD gene in these cells (MA9-ITD cells) not only promotes cytokine independent growth, but also significantly decreases leukemia latency in xenotransplant experiments. Consistent with the persistent requirement for active Rac signaling, we found that MA9 cells stimulated with FL induce the activated form of Rac (Rac-GTP) while MA9-ITD cells contain constitutively high levels of Rac-GTP, even after serum starvation and in the absence of all cytokines. Interestingly, we found that expression of the activated mutant NRas-G12D in MA9 cells (MA9-NRas cells) also leads to constitutive Rac activation, cytokine independent growth, and decreased leukemia latency upon transplantation into immunodeficient mice. Taken together, these results indicate that RAC activation may be a common integrating pathway by which secondary mutations cooperate in the progression towards leukemia. Consistent with this notion, both MA9-ITD and MA9-NRas cells remain sensitive to Rac inhibition with a small molecule Rac inhibitor or shRNA knockdown of Rac1 or Rac2. These data indicate that Rac signaling remains critical for MA9 survival and proliferation even in the context of additional mutations and highlights the therapeutic potential for targeting Rac. Disclosures: No relevant conflicts of interest to declare.
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Baumann, Kim. "RAB goes back to RAC." Nature Reviews Cancer 8, no. 9 (August 14, 2008): 663. http://dx.doi.org/10.1038/nrc2476.
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Scala, Marcello, Masashi Nishikawa, Koh-ichi Nagata, and Pasquale Striano. "Pathophysiological Mechanisms in Neurodevelopmental Disorders Caused by Rac GTPases Dysregulation: What’s behind Neuro-RACopathies." Cells 10, no. 12 (December 2, 2021): 3395. http://dx.doi.org/10.3390/cells10123395.
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Rho family guanosine triphosphatases (GTPases) regulate cellular signaling and cytoskeletal dynamics, playing a pivotal role in cell adhesion, migration, and cell cycle progression. The Rac subfamily of Rho GTPases consists of three highly homologous proteins, Rac 1–3. The proper function of Rac1 and Rac3, and their correct interaction with guanine nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs) are crucial for neural development. Pathogenic variants affecting these delicate biological processes are implicated in different medical conditions in humans, primarily neurodevelopmental disorders (NDDs). In addition to a direct deleterious effect produced by genetic variants in the RAC genes, a dysregulated GTPase activity resulting from an abnormal function of GEFs and GAPs has been involved in the pathogenesis of distinctive emerging conditions. In this study, we reviewed the current pertinent literature on Rac-related disorders with a primary neurological involvement, providing an overview of the current knowledge on the pathophysiological mechanisms involved in the neuro-RACopathies.
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Ming, Wenyu, Shijun Li, Daniel D. Billadeau, Lawrence A. Quilliam, and Mary C. Dinauer. "The Rac Effector p67phox Regulates Phagocyte NADPH Oxidase by Stimulating Vav1 Guanine Nucleotide Exchange Activity." Molecular and Cellular Biology 27, no. 1 (October 23, 2006): 312–23. http://dx.doi.org/10.1128/mcb.00985-06.
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ABSTRACT The phagocyte NADPH oxidase catalyzes the reduction of molecular oxygen to superoxide and is essential for microbial defense. Electron transport through the oxidase flavocytochrome is activated by the Rac effector p67 phox . Previous studies suggest that Vav1 regulates NADPH oxidase activity elicited by the chemoattractant formyl-Met-Leu-Phe (fMLP). We show that Vav1 associates with p67 phox and Rac2, but not Rac1, in fMLP-stimulated human neutrophils, correlating with superoxide production. The interaction of p67 phox with Vav1 is direct and activates nucleotide exchange on Rac, which enhances the interaction between p67 phox and Vav1. This provides new molecular insights into regulation of the neutrophil NADPH oxidase, suggesting that chemoattractant-stimulated superoxide production can be amplified by a positive feedback loop in which p67 phox targets Vav1-mediated Rac activation.
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Heyworth, P. G., U. G. Knaus, X. Xu, D. J. Uhlinger, L. Conroy, G. M. Bokoch, and J. T. Curnutte. "Requirement for posttranslational processing of Rac GTP-binding proteins for activation of human neutrophil NADPH oxidase." Molecular Biology of the Cell 4, no. 3 (March 1993): 261–69. http://dx.doi.org/10.1091/mbc.4.3.261.
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Rac1 and Rac2 are closely related, low molecular weight GTP-binding proteins that have both been implicated in regulation of phagocyte NADPH oxidase. This enzyme system is composed of multiple membrane-bound and cytosolic subunits and when activated catalyzes the one-electron reduction of oxygen to superoxide. Superoxide and its highly reactive derivatives are essential for killing microorganisms. Rac proteins undergo posttranslational processing, primarily the addition of an isoprenyl group to a carboxyl-terminal cysteine residue. We directly compared recombinant Rac1 and Rac2 in a human neutrophil cell-free NADPH oxidase system in which cytosol was replaced by purified recombinant cytosolic components (p47-phox and p67-phox). Processed Rac1 and Rac2 were both highly active in this system and supported comparable rates of superoxide production. Under different cell-free conditions, however, in which suboptimal amounts of cytosol were present in the assay mixture, processed Rac2 worked much better than Rac1 at all but the lowest concentrations. This suggests that a factor in the cytosol may suppress the activity of Rac1 but not of Rac2. Unprocessed Rac proteins were only weakly able to support superoxide generation in either system, but preloading of Rac1 or Rac2 with guanosine 5'-O-(3-thio-triphosphate) (GTP gamma S) restored activity. These results indicate that processing is required for nucleotide exchange but not for interaction with oxidase components.
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Arora, P. D., P. A. Marignani, and C. A. McCulloch. "Collagen phagocytosis is regulated by the guanine nucleotide exchange factor Vav2." American Journal of Physiology-Cell Physiology 295, no. 1 (July 2008): C130—C137. http://dx.doi.org/10.1152/ajpcell.00168.2008.
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Collagen phagocytosis is a crucial α2β1-integrin-dependent process that mediates extracellular matrix remodeling by fibroblasts. We showed previously that after initial contact with collagen, activated Rac1 accelerates collagen phagocytosis but the Rac guanine nucleotide exchange factors (GEFs) that regulate Rac are not defined. We examined here the GEFs that regulate collagen phagocytosis in mouse fibroblasts. Collagen binding enhanced Rac1 activity (5–20 min) but not Cdc42 or RhoA activity. Analysis of collagen bead-associated proteins showed enrichment with Vav2, which correlated temporally with increased Rac1 activity. Knockdown of Vav2 prevented Rac activation, recruitment of Rac1 to collagen bead binding sites, and collagen bead binding, but knockdown of Sos-1 or β-Pix had no effect on Rac activation or collagen binding. Vav2 was associated with the nucleotide-free Rac1 mutant (G15ARac1) after collagen binding. Collagen bead binding promoted phosphorylation of Vav2, which temporally correlated with Rac1 activation and which required Src kinase activity. Blockage of Src activity prevented collagen bead-induced Rac activation and collagen bead binding. Collectively these data indicate that Vav2 regulates the Rac1 activity associated with the binding step of collagen phagocytosis.
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Itoh, Reina E., Kazuo Kurokawa, Yusuke Ohba, Hisayoshi Yoshizaki, Naoki Mochizuki, and Michiyuki Matsuda. "Activation of Rac and Cdc42 Video Imaged by Fluorescent Resonance Energy Transfer-Based Single-Molecule Probes in the Membrane of Living Cells." Molecular and Cellular Biology 22, no. 18 (September 15, 2002): 6582–91. http://dx.doi.org/10.1128/mcb.22.18.6582-6591.2002.
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ABSTRACT Rho family G proteins, including Rac and Cdc42, regulate a variety of cellular functions such as morphology, motility, and gene expression. We developed fluorescent resonance energy transfer-based probes which monitored the local balance between the activities of guanine nucleotide exchange factors and GTPase-activating proteins for Rac1 and Cdc42 at the membrane. These probes, named Raichu-Rac and Raichu-Cdc42, consisted of a Cdc42- and Rac-binding domain of Pak, Rac1 or Cdc42, a pair of green fluorescent protein mutants, and a CAAX box of Ki-Ras. With these probes, we video imaged the Rac and Cdc42 activities. In motile HT1080 cells, activities of both Rac and Cdc42 gradually increased toward the leading edge and decreased rapidly when cells changed direction. Under a higher magnification, we observed that Rac activity was highest immediately behind the leading edge, whereas Cdc42 activity was most prominent at the tip of the leading edge. Raichu-Rac and Raichu-Cdc42 were also applied to a rapid and simple assay for the analysis of putative guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) in living cells. Among six putative GEFs and GAPs, we identified KIAA0362/DBS as a GEF for Rac and Cdc42, KIAA1256 as a GEF for Cdc42, KIAA0053 as a GAP for Rac and Cdc42, and KIAA1204 as a GAP for Cdc42. In conclusion, use of these single-molecule probes to determine Rac and Cdc42 activity will accelerate the analysis of the spatiotemporal regulation of Rac and Cdc42 in a living cell.
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Dumont, Celine, Agnieszka Corsoni-Tadrzak, Sandra Ruf, Jasper de Boer, Adam Williams, Martin Turner, Dimitris Kioussis, and Victor L. J. Tybulewicz. "Rac GTPases play critical roles in early T-cell development." Blood 113, no. 17 (April 23, 2009): 3990–98. http://dx.doi.org/10.1182/blood-2008-09-181180.
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Abstract The Rac1 and Rac2 GTPases play important roles in many processes including cytoskeletal reorganization, proliferation, and survival, and are required for B-cell development. Previous studies had shown that deficiency in Rac2 did not affect T-cell development, whereas the function of Rac1 in this process has not been investigated. We now show that simultaneous absence of both GTPases resulted in a very strong developmental block at the pre-TCR checkpoint and in defective positive selection. Unexpectedly, deficiency of Rac1 and Rac2 also resulted in the aberrant survival of thymocytes lacking expression of TCRβ, showing hallmarks of hyperactive Notch signaling. Furthermore, we found a similar novel phenotype in the absence of Vav1, Vav2, and Vav3, which function as guanine nucleotide exchange factors for Rac1 and Rac2. These results show that a pathway containing Vav and Rac proteins may negatively regulate Notch signaling during early thymic development.
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Sanchez-Aguilera, Abel, Ami tava Sengupta, Joseph P. Mastin, Kyung H. Chang, David A. Williams, and Jose A. Cancelas. "Rac2 GTPase Activation Is Necessary for Development of p190-BCR-ABL-Induced B-Cell Acute Lymphoblastic Leukemia." Blood 112, no. 11 (November 16, 2008): 3790. http://dx.doi.org/10.1182/blood.v112.11.3790.3790.
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Abstract 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-kB, 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.
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Dise, Rebecca S., Mark R. Frey, Robert H. Whitehead, and D. Brent Polk. "Epidermal growth factor stimulates Rac activation through Src and phosphatidylinositol 3-kinase to promote colonic epithelial cell migration." American Journal of Physiology-Gastrointestinal and Liver Physiology 294, no. 1 (January 2008): G276—G285. http://dx.doi.org/10.1152/ajpgi.00340.2007.
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Regulated intestinal epithelial cell migration plays a key role in wound healing and maintenance of a healthy gastrointestinal tract. Epidermal growth factor (EGF) stimulates cell migration and wound closure in intestinal epithelial cells through incompletely understood mechanisms. In this study we investigated the role of the small GTPase Rac in EGF-induced cell migration using an in vitro wound-healing assay. In mouse colonic epithelial (MCE) cell lines, EGF-stimulated wound closure was accompanied by a doubling of the number of cells containing lamellipodial extensions at the wound margin, increased Rac membrane translocation in cells at the wound margin, and rapid Rac activation. Either Rac1 small interfering (si)RNA or a Rac1 inhibitor completely blocked EGF-stimulated wound closure. Whereas EGF failed to activate Rac in colon cells from EGF receptor (EGFR) knockout mice, stable expression of wild-type EGFR restored EGF-stimulated Rac activation and migration. Pharmacological inhibition of either phosphatidylinositol 3-kinase (PI3K) or Src family kinases reduced EGF-stimulated Rac activation. Cotreatment of cells with both inhibitors completely blocked EGF-stimulated Rac activation and localization to the leading edge of cells and lamellipodial extension. Our results present a novel mechanism by which the PI3K and Src signaling cascades cooperate to activate Rac and promote intestinal epithelial cell migration downstream of EGFR.
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Schuringa, Jan Jacob, Marjan Rozenveld-Geugien, Inge Baas, Djoke van Gosliga, and Edo Vellenga. "Expansion of Normal and Leukemic Human Hematopoietic Stem/Progenitor Cells Requires Rac-Mediated Interaction with Stromal Cells." Blood 106, no. 11 (November 16, 2005): 1398. http://dx.doi.org/10.1182/blood.v106.11.1398.1398.
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Abstract We have studied Rac signal transduction in human cord-blood (CB) and acute myeloid leukemia (AML) CD34+ cells and determined that Rac proteins are critically involved in the interaction between human stem/progenitor cells and stroma. Constitutive activation of Rac signaling was achieved by retroviral introduction of Rac1-V12 into CB-derived CD34+ cells, while inhibition of Rac signaling was established by retroviral introduction of dominant negative Rac1-N17 or by utilizing the Rac inhibitor NSC23766. Inhibition of Rac signaling resulted in a proliferative disadvantage of CB-CD34+ cells when cultured on MS5 stromal cells. Cells were severely disturbed in their migration towards and direct association with MS5 stroma when Rac signaling was inhibited. The Long Term Culture-Initiating Cells (LTC-ICs) migrated underneath the stromal MS5 layer within 24 hrs after plating, and similar results were obtained for about 50% of the Colony Forming Cells (CFCs). However, transient inhibition of Rac signaling for 24–72 hrs resulted in a shift of LTC-ICs and CFCs to the suspension phase as determined by colony assays and CAFC week 5 enumeration. When Rac signaling signal transduction was inhibited during the 5 week coculture period a dramatic decrease in LTC-IC frequency from 0.6% to 0.15% was observed. Many of these phenotypes were reversed in the presence of activated Rac1-V12, including improved migration towards and association with MS5 cells and elevated LTC-IC frequencies. Importantly, in CAFC assays using AML cells (n=8) that were enriched for leukemic stem cells on the basis of a CD34+/CD38low phenotype we observed a dramatic decrease in leukemic CAFC formation as well as strongly diminished clonal expansion in the presence of the Rac inhibitor NSC23766. Taken together, our data indicate that Rac signal transduction is required for the maintenance and expansion of both normal as well as leukemic stem/progenitor cells by mediating their interaction with stromal cells.
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Yamazaki, Daisuke, Toshiki Itoh, Hiroaki Miki, and Tadaomi Takenawa. "srGAP1 regulates lamellipodial dynamics and cell migratory behavior by modulating Rac1 activity." Molecular Biology of the Cell 24, no. 21 (November 2013): 3393–405. http://dx.doi.org/10.1091/mbc.e13-04-0178.
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The distinct levels of Rac activity differentially regulate the pattern of intrinsic cell migration. However, it remains unknown how Rac activity is modulated and how the level of Rac activity controls cell migratory behavior. Here we show that Slit-Robo GAP 1 (srGAP1) is a modulator of Rac activity in locomotive cells. srGAP1 possesses a GAP activity specific to Rac1 and is recruited to lamellipodia in a Rac1-dependent manner. srGAP1 limits Rac1 activity and allows concomitant activation of Rac1 and RhoA, which are mutually inhibitory. When both GTPases are activated, the protrusive structures caused by Rac1-dependent actin reorganization are spatially restricted and periodically destabilized, causing ruffling by RhoA-induced actomyosin contractility. Depletion of srGAP1 overactivates Rac1 and inactivates RhoA, resulting in continuous spatiotemporal spreading of lamellipodia and a modal shift of intrinsic cell motility from random to directionally persistent. Thus srGAP1 is a key determinant of lamellipodial dynamics and cell migratory behavior.
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Zhang, Baolin, Yaqin Zhang, and Emily Shacter. "Caspase 3-Mediated Inactivation of Rac GTPases Promotes Drug-Induced Apoptosis in Human Lymphoma Cells." Molecular and Cellular Biology 23, no. 16 (August 15, 2003): 5716–25. http://dx.doi.org/10.1128/mcb.23.16.5716-5725.2003.
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ABSTRACT The Rac members of the Rho family GTPases control signaling pathways that regulate diverse cellular activities, including cytoskeletal organization, gene transcription, and cell transformation. Rac is implicated in apoptosis, but little is known about the mechanism by which it responds to apoptotic stimuli. Here we demonstrate that endogenous Rac GTPases are caspase 3 substrates that are cleaved in human lymphoma cells during drug-induced apoptosis. Cleavage of Rac1 occurs at two unconventional caspase 3 sites, VVGD11/G and VMVD47/G, and results in inactivation of the GTPase and effector functions of the protein (binding to the p21-activated protein kinase PAK1). Expression of caspase 3-resistant Rac1 mutants in the cells suppresses drug-induced apoptosis. Thus, proteolytic inactivation of Rac GTPases represents a novel, irreversible mechanism of Rac downregulation that allows maximal cell death following drug treatment.
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Chae, Young Chan, Jung Hwan Kim, Kyung Lock Kim, Hyun Wook Kim, Hye Young Lee, Won Do Heo, Tobias Meyer, Pann-Ghill Suh, and Sung Ho Ryu. "Phospholipase D Activity Regulates Integrin-mediated Cell Spreading and Migration by Inducing GTP-Rac Translocation to the Plasma Membrane." Molecular Biology of the Cell 19, no. 7 (July 2008): 3111–23. http://dx.doi.org/10.1091/mbc.e07-04-0337.
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Small GTPase Rac is a crucial regulator of actin cytoskeletal rearrangement, and it plays an important role in cell spreading, migration, mitogenesis, phagocytosis, superoxide generation, and axonal growth. It is generally accepted that Rac activity is regulated by the guanosine triphosphate (GTP)/guanosine diphosphate (GDP) cycle. But, it is suggested that in addition to Rac-GTP loading, membrane localization is required for the initiation of downstream effector signaling. However, the molecular mechanisms that control the targeting of GTP-Rac to the plasma membrane remain largely unknown. Here, we have uncovered a signaling pathway linking phospholipase D (PLD) to the localized functions of Rac1. We show that PLD product phosphatidic acid (PA) acts as a membrane anchor of Rac1. The C-terminal polybasic motif of Rac1 is responsible for direct interaction with PA, and Rac1 mutated in this region is incapable of translocating to the plasma membrane and of activating downstream target p21-activated kinase upon integrin activation. Finally, we show that PA induces dissociation of Rho-guanine nucleotide dissociation inhibitor from Rac1 and that PA-mediated Rac1 localization is important for integrin-mediated lamellipodia formation, cell spreading, and migration. These results provide a novel molecular mechanism for the GTP-Rac1 localization through the elevating PLD activity, and they suggest a general mechanism for diverse cellular functions that is required localized Rac activation.
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KITAMURA, Yukari, Tadahiro KITAMURA, Hiroshi SAKAUE, Tetsuo MAEDA, Hikaru UENO, Shoko NISHIO, Shigeo OHNO, et al. "Interaction of Nck-associated protein 1 with activated GTP-binding protein Rac." Biochemical Journal 322, no. 3 (March 15, 1997): 873–78. http://dx.doi.org/10.1042/bj3220873.
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Bacterially expressed glutathione S-transferase fusion proteins containing Rac1 were used to identify binding proteins of this Rho family GTPase present in a bovine brain extract. Five proteins of 85, 110, 125, 140 and 170 kDa were detected, all of which were associated exclusively with guanosine 5´-[γ-thio]triphosphate-bound Rac1, not with GDP-bound Rac1. The 85 and 110 kDa proteins were identified as the regulatory and catalytic subunits respectively of phosphatidylinositol 3-kinase. Several lines of evidence suggested that the 125 kDa protein is identical with Nck-associated protein 1 (Nap1). The mobilities of the 125 kDa protein and Nap1 on SDS/PAGE were indistinguishable, and the 125 kDa protein was depleted from brain extract by preincubation with the Src homology 3 domain of Nck to which Nap1 binds. Furthermore, antibodies to Nap1 reacted with the 125 kDa protein. Nap1 was co-immunoprecipitated with a constitutively active form of Rac expressed in Chinese hamster ovary cells. The observation that complex formation between activated Rac and PAK, but not that between Rac and Nap1, could be reproduced in vitro with recombinant proteins indicates that the interaction of Nap1 with Rac is indirect. The 140 kDa Rac-binding protein is a potential candidate for a link that connects Nap1 to Rac. The multimolecular complex comprising Rac, Nap1 and probably the 140 kDa protein might mediate some of the biological effects transmitted by the multipotent GTPase.
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Kalfa, Theodosia A., Suvarnamala Pushkaran, Narla Mohandas, John H. Hartwig, Velia M. Fowler, James F. Johnson, Clinton H. Joiner, David A. Williams, and Yi Zheng. "Rac GTPases regulate the morphology and deformability of the erythrocyte cytoskeleton." Blood 108, no. 12 (December 1, 2006): 3637–45. http://dx.doi.org/10.1182/blood-2006-03-005942.
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Abstract Actin oligomers are a significant structural component of the erythrocyte cytoskeleton. Rac1 and Rac2 GTPases regulate actin structures and have multiple overlapping as well as distinct roles in hematopoietic cells; therefore, we studied their role in red blood cells (RBCs). Conditional gene targeting with a loxP-flanked Rac1 gene allowed Crerecombinase–induced deletion of Rac1 on a Rac2 null genetic background. The Rac1–/–;Rac2–/– mice developed microcytic anemia with a hemoglobin drop of about 20% and significant anisocytosis and poikilocytosis. Reticulocytes increased more than 2-fold. Rac1–/–;Rac2–/– RBCs stained with rhodamine-phalloidin demonstrated F-actin meshwork gaps and aggregates under confocal microscopy. Transmission electron microscopy of the cytoskeleton demonstrated junctional aggregates and pronounced irregularity of the hexagonal spectrin scaffold. Ektacytometry confirmed that these cytoskeletal changes in Rac1–/–;Rac2–/– erythrocytes were associated with significantly decreased cellular deformability. The composition of the cytoskeletal proteins was altered with an increased actin-to-spectrin ratio and increased phosphorylation (Ser724) of adducin, an F-actin capping protein. Actin and phosphorylated adducin of Rac1–/–;Rac2–/– erythrocytes were more easily extractable by Triton X-100, indicating weaker association to the cytoskeleton. Thus, deficiency of Rac1 and Rac2 GTPases in mice alters actin assembly in RBCs and causes microcytic anemia with reticulocytosis, implicating Rac GTPases as dynamic regulators of the erythrocyte cytoskeleton organization.
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Kunisaki, Yuya, Akihiko Nishikimi, Yoshihiko Tanaka, Ryosuke Takii, Mayuko Noda, Ayumi Inayoshi, Ken-ichi Watanabe, et al. "DOCK2 is a Rac activator that regulates motility and polarity during neutrophil chemotaxis." Journal of Cell Biology 174, no. 5 (August 28, 2006): 647–52. http://dx.doi.org/10.1083/jcb.200602142.
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Neutrophils are highly motile leukocytes, and they play important roles in the innate immune response to invading pathogens. Neutrophil chemotaxis requires Rac activation, yet the Rac activators functioning downstream of chemoattractant receptors remain to be determined. We show that DOCK2, which is a mammalian homologue of Caenorhabditis elegans CED-5 and Drosophila melanogaster Myoblast City, regulates motility and polarity during neutrophil chemotaxis. Although DOCK2-deficient neutrophils moved toward the chemoattractant source, they exhibited abnormal migratory behavior with a marked reduction in translocation speed. In DOCK2-deficient neutrophils, chemoattractant-induced activation of both Rac1 and Rac2 were severely impaired, resulting in the loss of polarized accumulation of F-actin and phosphatidylinositol 3,4,5-triphosphate (PIP3) at the leading edge. On the other hand, we found that DOCK2 associates with PIP3 and translocates to the leading edge of chemotaxing neutrophils in a phosphatidylinositol 3-kinase (PI3K)–dependent manner. These results indicate that during neutrophil chemotaxis DOCK2 regulates leading edge formation through PIP3-dependent membrane translocation and Rac activation.
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Makino, Ayako, Michael Glogauer, Gary M. Bokoch, Shu Chien, and Geert W. Schmid-Schönbein. "Control of neutrophil pseudopods by fluid shear: role of Rho family GTPases." American Journal of Physiology-Cell Physiology 288, no. 4 (April 2005): C863—C871. http://dx.doi.org/10.1152/ajpcell.00358.2004.
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Blood vessels and blood cells are under continuous fluid shear. Studies on vascular endothelium and smooth muscle cells have shown the importance of this mechanical stress in cell signal transduction, gene expression, vascular remodeling, and cell survival. However, in circulating leukocytes, shear-induced signal transduction has not been investigated. Here we examine in vivo and in vitro the control of pseudopods in leukocytes under the influence of fluid shear stress and the role of the Rho family small GTPases. We used a combination of HL-60 cells differentiated into neutrophils (1.4% dimethyl sulfoxide for 5 days) and fresh leukocytes from Rac knockout mice. The cells responded to shear stress (5 dyn/cm2) with retraction of pseudopods and reduction of their projected cell area. The Rac1 and Rac2 activities were decreased by fluid shear in a time- and magnitude-dependent manner, whereas the Cdc42 activity remained unchanged (up to 5 dyn/cm2). The Rho activity was transiently increased and recovered to static levels after 10 min of shear exposure (5 dyn/cm2). Inhibition of either Rac1 or Rac2 slightly but significantly diminished the fluid shear response. Transfection with Rac1-positive mutant enhanced the pseudopod formation during shear. Leukocytes from Rac1-null and Rac2-null mice had an ability to form pseudopods in response to platelet-activating factor but did not respond to fluid shear in vitro. Leukocytes in wild-type mice retracted pseudopods after physiological shear exposure, whereas cells in Rac1-null mice showed no retraction during equal shear. On leukocytes from Rac2-null mice, however, fluid shear exerted a biphasic effect. Leukocytes with extended pseudopods slightly decreased in length, whereas initially round cells increased in length after shear application. The disruption of Rac activity made leukocytes nonresponsive to fluid shear, induced cell adhesion and microvascular stasis, and decreased microvascular density. These results suggest that deactivation of Rac activity by fluid shear plays an important role in stable circulation of leukocytes.
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Teng, Guigen, Yun Liu, Ting Wu, Weihong Wang, Huahong Wang, and Fulian Hu. "Efficacy of Sucralfate-Combined Quadruple Therapy on Gastric Mucosal Injury Induced by Helicobacter pylori and Its Effect on Gastrointestinal Flora." BioMed Research International 2020 (August 31, 2020): 1–14. http://dx.doi.org/10.1155/2020/4936318.
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Background. This study explored the therapeutic efficacy of standard triple therapy combined with sucralfate suspension gel as well as the mechanisms of action in mouse models of H. pylori infection. Materials and Methods. C57BL/6J mice were randomly divided into 5 groups: NC (natural control), HP (H. pylori infection), RAC (rabeprazole, amoxicillin, and clarithromycin), RACS (RAC and sucralfate suspension gel), and RACB (RAC and bismuth potassium citrate). HE staining and electron microscopy were performed to estimate histological and ultrastructural damages. The IL-8, IL-10, and TNF-α of gastric antrum tissues were measured by immunohistochemistry and qRT-PCR. ZO-1 and Occludin were also detected with immunohistochemistry. The genomes of gastric and fecal microbiota were sequenced. Results. The eradication rate of H. pylori in the RACS group was higher than the RAC group. RACS therapy had protective effects on H. pylori-induced histological and ultrastructural damages, which were superior to the RAC group. RACS therapy reduced the protein and mRNA levels of IL-8 compared with the RAC group. The expression of Occludin in the RACS group was significantly higher than that of the RAC group. The composition of gastric and fecal microbiota for RACS was similar to the RACB group according to PCA. Conclusions. The RACS regimen eradicated H. pylori infection effectively and showed RACS had protective effects against H. pylori-induced histological and ultrastructural damage. The mechanisms of RACS effects included decreasing IL-8, enhancing Occludin, and transforming gastric microbiota. Moreover, RACS and RACB have a similar effect on gastrointestinal flora.
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Casado-Medrano, Victoria, Martin J. Baker, Cynthia Lopez-Haber, Mariana Cooke, Shaofei Wang, Maria J. Caloca, and Marcelo G. Kazanietz. "The role of Rac in tumor susceptibility and disease progression: from biochemistry to the clinic." Biochemical Society Transactions 46, no. 4 (July 31, 2018): 1003–12. http://dx.doi.org/10.1042/bst20170519.
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The family of Rho GTPases are involved in the dynamic control of cytoskeleton reorganization and other fundamental cellular functions, including growth, motility, and survival. Rac1, one of the best characterized Rho GTPases, is an established effector of receptors and an important node in signaling networks crucial for tumorigenesis and metastasis. Rac1 hyperactivation is common in human cancer and could be the consequence of overexpression, abnormal upstream inputs, deregulated degradation, and/or anomalous intracellular localization. More recently, cancer-associated gain-of-function mutations in Rac1 have been identified which contribute to tumor phenotypes and confer resistance to targeted therapies. Deregulated expression/activity of Rac guanine nucleotide exchange factors responsible for Rac activation has been largely associated with a metastatic phenotype and drug resistance. Translating our extensive knowledge in Rac pathway biochemistry into a clinical setting still remains a major challenge; nonetheless, remarkable opportunities for cancer therapeutics arise from promising lead compounds targeting Rac and its effectors.
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Santos, Iwldson Guilherme da Silva, José Francisco de Oliveira Júnior, Isnaldo Isaac Barbosa, Luis Felipe Francisco Ferreira da Silva, William Max de Oliveira Romão, Vitória Rejane Marques dos Santos, Kelvy Rosalvo Alencar Cardoso, and Caroline Cristina da Silva de Andrade. "Rede neural artificial aplicada aos casos notificados de dengue cases em Maceió – Alagoas." Research, Society and Development 11, no. 14 (October 31, 2022): e406111436382. http://dx.doi.org/10.33448/rsd-v11i14.36382.
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A dengue é um dos graves problemas de saúde pública mundial. O Nordeste do Brasil (NEB) possui um clima e ambiente urbano ideal para a proliferação do mosquito Aedes (aegypti e albopictus), vetor da doença. O Estado de Alagoas, principalmente a sua capital, tem epidemias da doença de forma frequente. Portanto, o objetivo deste estudo é avaliar a aplicação de Rede Neural Artificial (RNA) nos casos notificados de dengue (CND) nas regiões administrativas (RA) de Maceió. As RAs são divididas em: RA1, RA2, RA3, RA4, RA5, RA6, RA7 e RA8. Os CND foram submetidos a RNA não linear autorregressiva (NAR) – (RNA-NAR). O período de estudo foi de 2011 a 2020. Os resultados obtidos de CND se destacaram em anos específicos (2012, 2013, 2017, 2018 e 2020), por outro lado houve superestimativas das previsões via RNA. Em algumas RAs houve subnotificações e, por isso interferiu nos resultados das previsões. A RNA-NAR foi validada, visto que a maioria das previsões apresentou correlação positiva e com resposta aos dados observados, exceto as RAs com subnotificações. O uso da RNA é adequado no alerta e previsão da donça, onde tal instrumento pode ser usado em ações preventivas de controle da doença.
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Lagresle-Peyrou, Chantal, Aurélien Olichon, Hanem Sadek, Philippe Roche, Claudine Tardy, Natael Sorel, Boris Bessot, et al. "An Autosomal Dominant Form of Ras-Related C3 Botulinum Toxin Substrate 2 (RAC2) Is Associated with Haematopoiesis Failure." Blood 138, Supplement 1 (November 5, 2021): 4306. http://dx.doi.org/10.1182/blood-2021-150114.
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Abstract Severe combined immunodeficiencies (SCIDs) constitute a heterogeneous group of life-threatening genetic disorders that typically present in the first year of life. Reticular dysgenesis (RD) is an autosomal recessive form of human Severe Combined Immunodeficiency (SCIDs) characterized by the absence of blood neutrophils and T lymphocytes. This pathology is due to biallelic mutations in the adenylate kinase 2 (AK2) gene, encoding for a mitochondrial protein which regulates the homeostasis of adenine nucleotides. In three newborns presenting a RD-like clinical phenotype as frequent infections and a profound leukopenia, we identified an heterozygous dominant missense mutation in the gene coding for Rac Family Small GTPase 2 (RAC2) protein (p.G12R). Hematopoietic stem cell transplantation (HSCT) performed soon after birth was successful in two out of the three patients attesting that the inherited defect was intrinsic and not micro-environmental. RAC2 protein belongs to the Rac subfamily of RHO small GTPases. In the inactive GDP-bound state, RAC2 is located in the cytosol and upon stimulation, the active RAC2-GTP-bound form translocates to the plasma membrane. Unlike the other members of the Rac subfamily (RAC1 and RAC3), RAC2 is mostly expressed on hematopoietic cells and during T cell differentiation. To gain insight into the disease, we transduced human hematopoietic stem and progenitor cells (HSPCs) with a lentiviral construct containing the RAC2 mutated form. The mutation inhibits HSPCs proliferation and differentiation toward the myeloid and lymphoid lineages reproducing the patients' clinical phenotype. In this condition, we also observed high apoptosis level and an alteration of mitochondrial activity and Ros production. In a biochemical model, we demonstrated that the substitution of the glycine (G) amino-acid by a bulky flexible arginine (R) may prevent GTP hydrolysis. Lastly, our findings suggest that RAC2 gene sequencing must be considered in newborn screening programs for SCID detection. To decipher the mechanisms regulating RAC2 functions, we studied the impact of the p.G12R mutation on a human AML cell line expressing RAC2 protein. Our preliminary data highlight that the cell cycle and mitochondrial activity are disrupted by G12R mutation. By holotomography, we also observed morphological changes and accumulation of lipid droplets into the cells. All these data suggest that RAC2 defective signalling pathway is linked to cell metabolism imbalance and further investigations are ongoing to better understand how RAC2 controls cell energy demand, especially during differentiation. Disclosures Cavazzana: Smart Immune: Other: co-founder.
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Bosco, E. E., J. C. Mulloy, and Y. Zheng. "Rac1 GTPase: A “Rac” of All Trades." Cellular and Molecular Life Sciences 66, no. 3 (December 30, 2008): 370–74. http://dx.doi.org/10.1007/s00018-008-8552-x.
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Tuma, Rabiya S. "Activating Rac." Journal of Cell Biology 174, no. 5 (August 28, 2006): 609a. http://dx.doi.org/10.1083/jcb.1745iti5.
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Konstantinidis, Diamantis G., Suvarnamala Pushkaran, James F. Johnson, Jose A. Cancelas, Stefanos Manganaris, Chad E. Harris, David A. Williams, Yi Zheng, and Theodosia A. Kalfa. "Signaling and cytoskeletal requirements in erythroblast enucleation." Blood 119, no. 25 (June 21, 2012): 6118–27. http://dx.doi.org/10.1182/blood-2011-09-379263.
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Abstract To understand the role of cytoskeleton and membrane signaling molecules in erythroblast enucleation, we developed a novel analysis protocol of multiparameter high-speed cell imaging in flow. This protocol enabled us to observe F-actin and phosphorylated myosin regulatory light chain (pMRLC) assembled into a contractile actomyosin ring (CAR) between nascent reticulocyte and nucleus, in a population of enucleating erythroblasts. CAR formation and subsequent enucleation were not affected in murine erythroblasts with genetic deletion of Rac1 and Rac2 GTPases because of compensation by Rac3. Pharmacologic inhibition or genetic deletion of all Rac GTPases altered the distribution of F-actin and pMRLC and inhibited enucleation. Erythroblasts treated with NSC23766, cytochalasin-D, colchicine, ML7, or filipin that inhibited Rac activity, actin or tubulin polymerization, MRLC phosphorylation, or lipid raft assembly, respectively, exhibited decreased enucleation efficiency, as quantified by flow cytometry. As assessed by high-speed flow-imaging analysis, colchicine inhibited erythroblast polarization, implicating microtubules during the preparatory stage of enucleation, whereas NSC23766 led to absence of lipid raft assembly in the reticulocyte-pyrenocyte border. In conclusion, enucleation is a multistep process that resembles cytokinesis, requiring establishment of cell polarity through microtubule function, followed by formation of a contractile actomyosin ring, and coalescence of lipid rafts between reticulocyte and pyrenocyte.
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Tolias, Kimberley F., Anthony D. Couvillon, Lewis C. Cantley, and Christopher L. Carpenter. "Characterization of a Rac1- and RhoGDI-Associated Lipid Kinase Signaling Complex." Molecular and Cellular Biology 18, no. 2 (February 1, 1998): 762–70. http://dx.doi.org/10.1128/mcb.18.2.762.
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ABSTRACT Rho family GTPases regulate a number of cellular processes, including actin cytoskeletal organization, cellular proliferation, and NADPH oxidase activation. The mechanisms by which these G proteins mediate their effects are unclear, although a number of downstream targets have been identified. The interaction of most of these target proteins with Rho GTPases is GTP dependent and requires the effector domain. The activation of the NADPH oxidase also depends on the C terminus of Rac, but no effector molecules that bind to this region have yet been identified. We previously showed that Rac interacts with a type I phosphatidylinositol-4-phosphate (PtdInsP) 5-kinase, independent of GTP. Here we report the identification of a diacylglycerol kinase (DGK) which also associates with both GTP- and GDP-bound Rac1. In vitro binding analysis using chimeric proteins, peptides, and a truncation mutant demonstrated that the C terminus of Rac is necessary and sufficient for binding to both lipid kinases. The Rac-associated PtdInsP 5-kinase and DGK copurify by liquid chromatography, suggesting that they bind as a complex to Rac. RhoGDI also associates with this lipid kinase complex both in vivo and in vitro, primarily via its interaction with Rac. The interaction between Rac and the lipid kinases was enhanced by specific phospholipids, indicating a possible mechanism of regulation in vivo. Given that the products of the PtdInsP 5-kinase and the DGK have been implicated in several Rac-regulated processes, and they bind to the Rac C terminus, these lipid kinases may play important roles in Rac activation of the NADPH oxidase, actin polymerization, and other signaling pathways.
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Cruz Morales, Elisa, Yan Song, Romela Marin, Bernard Khor, and Deanna Nguyen. "Rac GTPases have opposite roles in T cells and dendritic cells in mucosal immune function (MUC8P.730)." Journal of Immunology 194, no. 1_Supplement (May 1, 2015): 204.10. http://dx.doi.org/10.4049/jimmunol.194.supp.204.10.
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Abstract Variants in RAC1 and RAC2, which encode small Rho-family GTPases, are associated with risk for inflammatory bowel disease. We have shown reduced regulatory T-cell (Treg) proportions in Rac2 knockout (KO) mice with additional dendritic cells (DC)-specific deletion of Rac1 compared to WT. Since Rac is crucial to TCR signaling, we hypothesized that a T-cell specific deletion of Rac1 would also result in defects in Treg development. Treg proportions and susceptibility to dextran sulfate sodium(DSS)-induced intestinal inflammation were assessed in mice deficient in Rac2 in addition to either T cell- or DC-specific deletion of Rac1 (cDKO). Surprisingly, elevated Treg proportions were observed in T-cell specific cDKO mice compared to WT as well as higher Treg proportions within the cDKO compared to the WT compartment in mixed bone marrow chimera. Furthermore, cDKO naïve CD4+ T cells were able to differentiate into Tregs, unlike cDKO DCs, which led to defective Treg generation of WT naïve CD4+ T cells in in vitro Treg generation assays. In vivo Treg generation was reduced in DC-specific cDKO mice compared to WT controls with ovalbumin (OVA) feeding following transfer of CD4+ T cells transgenic for OVA. Lastly, exacerbated DSS colitis was observed in DC-specific, but not T-cell specific cDKO mice, compared to WT. In conclusion, deficiency of Rac proteins result in contrasting effects on mucosal immune function in DCs compared to T cells. The precise mechanism is under investigation.
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Faroudi, Mustapha, Miroslav Hons, Agnieszka Zachacz, Celine Dumont, Ruth Lyck, Jens V. Stein, and Victor L. J. Tybulewicz. "Critical roles for Rac GTPases in T-cell migration to and within lymph nodes." Blood 116, no. 25 (December 16, 2010): 5536–47. http://dx.doi.org/10.1182/blood-2010-08-299438.
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Abstract Naive T cells continuously recirculate between secondary lymphoid tissue via the blood and lymphatic systems, a process that maximizes the chances of an encounter between a T cell and its cognate antigen. This recirculation depends on signals from chemokine receptors, integrins, and the sphingosine-1-phosphate receptor. The authors of previous studies in other cell types have shown that Rac GTPases transduce signals leading to cell migration and adhesion; however, their roles in T cells are unknown. By using both 3-dimensional intravital and in vitro approaches, we show that Rac1- and Rac2-deficient T cells have multiple defects in this recirculation process. Rac-deficient T cells home very inefficiently to lymph nodes and the white pulp of the spleen, show reduced interstitial migration within lymph node parenchyma, and are defective in egress from lymph nodes. These mutant T cells show defective chemokine-induced chemotaxis, chemokinesis, and adhesion to integrin ligands. They have reduced lateral motility on endothelial cells and transmigrate in-efficiently. These multiple defects stem from critical roles for Rac1 and Rac2 in transducing chemokine and sphingosine-1-phosphate receptor 1 signals leading to motility and adhesion.
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Azim, Anser C., Hongmei Cao, Xiaopei Gao, Myungsoo Joo, Asrar B. Malik, Richard B. van Breemen, Ruxana T. Sadikot, GyeYoung Park, and John W. Christman. "Regulation of cyclooxygenase-2 expression by small GTPase Rac2 in bone marrow macrophages." American Journal of Physiology-Lung Cellular and Molecular Physiology 293, no. 3 (September 2007): L668—L673. http://dx.doi.org/10.1152/ajplung.00043.2007.
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Cyclooxygenase 2 (COX-2) is induced by microbial products, proinflammatory cytokines, growth factors, and oncogenes. The Rho family includes RhoA, Rac1, Rac2, Rac3, and Cdc42 and is involved in regulation of the actin cytoskeleton organization, cell growth, vesicular cell trafficking, and transcriptional regulation. Rac2 binds to NADPH oxidase protein complex, and Rac2 null neutrophils are known to have poor phagocytic activity. We examined whether Rac2, the predominant small GTPase in hematopoietic cells, influences COX-2 expression in bone marrow-derived macrophages (BMDM). We showed that BMDM from Rac2−/−null mice have reduced COX-2 expression in response to treatment with endotoxin. Despite a compensatory increase in Rac1, BMDM from Rac2−/−null mice have less biologically active GTP-bound Rac in response to LPS stimulation. Signaling molecules (downstream of Rac2 and Toll-like receptor 4) such as p42/44, p38, and pAKT were also affected in BMDM from Rac2−/−null mouse macrophages. We also observed that BMDM from Rac2−/−null failed to degrade IκBα significantly and had less immunoreactive PU.1. We show that both NF-κB pathway and PU.1 are involved in normal macrophage function and play a role in macrophage COX-2 expression. In summary, these data indicate that Rac2 regulates COX-2 expression in BMDM.
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Chen, Feng, Kai Wu, Lijian Ren, Jianan Xu, and Huiming Zheng. "Internal Curing Effect and Compressive Strength Calculation of Recycled Clay Brick Aggregate Concrete." Materials 12, no. 11 (June 4, 2019): 1815. http://dx.doi.org/10.3390/ma12111815.
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In order to investigate the internal curing effect of recycled brick aggregate (RBA) in recycled aggregate concrete (RAC) and calculate its contribution to the final compressive strength, two RAC groups with different recycled aggregates and 6 replacement ratios (r) under 4 curing ages were tested. Results show that the compressive strengths of RACI and RACII decrease steadily with the increase of r when below 40%, and that there is a significant drop once the r is higher than 60%. The internal curing effect for RAC with a low RBA ratio is mainly reflected during the curing age of 14–21 days, while for RAC with a high RBA ratio, this internal curing effect appears earlier, during 7–14 days, and becomes very obvious after 14 days. In addition, the actual tested compressive strength of RAC replaced by 100% RBA exceeds around 40% of the expected compressive strength at the age of 28 days. When the age of RAC entirely with RBA is 28 days, the compressive strength caused by the internal curing effect accounts for around 28% of the actual tested compressive strength. The most appropriate r of RBA for RAC production is between 40% to 60%. Finally, the equations for calculating the compressive strength of RAC are presented considering the curing ages, the replacement ratios and the internal curing effect of RBA. Further, a unified equation is suggested for convenience in calculation.