Journal articles on the topic 'RhoA'
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1
Saracaloglu, Ahmet, Seniz Demiryürek, Sabit Kimyon, Alper Mete, Ebru Temiz, Gülper Nacarkahya, Oguzhan Saygili, Kıvanc Güngör, and Abdullah Tuncay Demiryürek. "RHO Gene Polymorphisms in Patients with Pterygium." Proceedings 2, no. 25 (December 6, 2018): 1572. http://dx.doi.org/10.3390/proceedings2251572.
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Pterygium is one of the most common ocular surface diseases, and characterized by inflammatory infiltrates, proliferation, fibrosis, angiogenesis, and extracellular matrix breakdown. We investigated the association of polymorphisms in the RHO genes RHOA, RHOB, RHOC, RHOD, and RND3 (RHOE). The results of this study demonstrate for the first time the association of RHO genes with the pterygium. We displayed that the RHO gene polymorphisms were significantly associated with pterygium in the Turkish population.
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Guasch, Rosa M., Peter Scambler, Gareth E. Jones, and Anne J. Ridley. "RhoE Regulates Actin Cytoskeleton Organization and Cell Migration." Molecular and Cellular Biology 18, no. 8 (August 1, 1998): 4761–71. http://dx.doi.org/10.1128/mcb.18.8.4761.
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ABSTRACT The actin cytoskeleton is regulated by Rho family proteins: in fibroblasts, Rho mediates the formation of actin stress fibers, whereas Rac regulates lamellipodium formation and Cdc42 controls filopodium formation. We have cloned the mouse RhoE gene, whose product is a member of the Rho family that shares (except in one amino acid) the conserved effector domain of RhoA, RhoB, and RhoC. RhoE is able to bind GTP but does not detectably bind GDP and has low intrinsic GTPase activity compared with Rac. The role of RhoE in regulating actin organization was investigated by microinjection in Bac1.2F5 macrophages and MDCK cells. In macrophages, RhoE induced actin reorganization, leading to the formation of extensions resembling filopodia and pseudopodia. In MDCK cells, RhoE induced the complete disappearance of stress fibers, together with cell spreading. However, RhoE did not detectably affect the actin bundles that run parallel to the outer membranes of cells at the periphery of colonies, which are known to be dependent on RhoA. In addition, RhoE induced an increase in the speed of migration of hepatocyte growth factor/scatter factor-stimulated MDCK cells, in contrast to the previously reported inhibition produced by activated RhoA. The subcellular localization of RhoE at the lateral membranes of MDCK cells suggests a role in cell-cell adhesion, as has been shown for RhoA. These results suggest that RhoE may act to inhibit signalling downstream of RhoA, altering some RhoA-regulated responses, such as stress fiber formation, but not affecting others, such as peripheral actin bundle formation.
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Königs, Volker, Richard Jennings, Thomas Vogl, Markus Horsthemke, Anne C. Bachg, Yan Xu, Kay Grobe, et al. "Mouse Macrophages Completely Lacking Rho Subfamily GTPases (RhoA, RhoB, and RhoC) Have Severe Lamellipodial Retraction Defects, but Robust Chemotactic Navigation and Altered Motility." Journal of Biological Chemistry 289, no. 44 (September 11, 2014): 30772–84. http://dx.doi.org/10.1074/jbc.m114.563270.
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RhoA is thought to be essential for coordination of the membrane protrusions and retractions required for immune cell motility and directed migration. Whether the subfamily of Rho (Ras homolog) GTPases (RhoA, RhoB, and RhoC) is actually required for the directed migration of primary cells is difficult to predict. Macrophages isolated from myeloid-restricted RhoA/RhoB (conditional) double knock-out (dKO) mice did not express RhoC and were essentially “pan-Rho”-deficient. Using real-time chemotaxis assays, we found that retraction of the trailing edge was dissociated from the advance of the cell body in dKO cells, which developed extremely elongated tails. Surprisingly, velocity (of the cell body) was increased, whereas chemotactic efficiency was preserved, when compared with WT macrophages. Randomly migrating RhoA/RhoB dKO macrophages exhibited multiple small protrusions and developed large “branches” due to impaired lamellipodial retraction. A mouse model of peritonitis indicated that monocyte/macrophage recruitment was, surprisingly, more rapid in RhoA/RhoB dKO mice than in WT mice. In comparison with dKO cells, the phenotypes of single RhoA- or RhoB-deficient macrophages were mild due to mutual compensation. Furthermore, genetic deletion of RhoB partially reversed the motility defect of macrophages lacking the RhoGAP (Rho GTPase-activating protein) myosin IXb (Myo9b). In conclusion, the Rho subfamily is not required for “front end” functions (motility and chemotaxis), although both RhoA and RhoB are involved in pulling up the “back end” and resorbing lamellipodial membrane protrusions. Macrophages lacking Rho proteins migrate faster in vitro, which, in the case of the peritoneum, translates to more rapid in vivo monocyte/macrophage recruitment.
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Han, Jian, Li Li, Jiongyu Hu, Lili Yu, Yingru Zheng, Jianxin Guo, Xiuhui Zheng, Ping Yi, and Yuanguo Zhou. "Epidermal Growth Factor Stimulates Human Trophoblast Cell Migration through Rho A and Rho C Activation." Endocrinology 151, no. 4 (February 11, 2010): 1732–42. http://dx.doi.org/10.1210/en.2009-0845.
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This study investigated the roles of Rho protein in epidermal growth factor (EGF)-induced trophoblast cell migration and its mechanism. Using choriocarcinoma cell lines JEG-3 and JAR and first-trimester human chorionic villus explant cultures on matrigel, we examined EGF-mediated stimulation of trophoblast migration. EGF is shown to have a dose-dependent effect on trophoblast migration. A low concentration of EGF (1 ng/ml) has a stimulatory effect on cell migration, whereas high concentrations of EGF (100 ng/ml) shows an inhibitory effect. EGF (1 ng/ml) activates RhoA and RhoC, but not RhoB, through elevated protein levels and activity. EGF-induced migration was shown to be inhibited by either cell-permeable C3 exoenzyme transferase or selective RhoA or RhoC small interfering RNAs. The inhibition was not mitigated by the addition of EGF, suggesting that RhoA and RhoC play an important role in trophoblast migration and are obligatory for EGF action. Treatment of JEG-3 and JAR cells with RhoA small interfering RNA induced F-actin cytoskeleton disruption and cell shrinkage, which is consistent with the effect of C3 exoenzyme transferase, and this action was not mitigated by EGF treatment. RhoC small interfering RNA had no apparent effect on the F-actin arrangement, suggesting that RhoA but not RhoC takes part in the EGF-induced migration through F-actin rearrangement. These results indicate that RhoA and RhoC play more important roles than RhoB in EGF-mediated migration of trophoblast cells, and RhoA but not RhoC regulates this migration through F-actin cytoskeleton reorganization.
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Tsubaki, Masanobu, Shuuji Genno, Tomoya Takeda, Takuya Matsuda, Naoto Kimura, Yuuma Yamashita, Yuusuke Morii, Kazunori Shimomura, and Shozo Nishida. "Rhosin Suppressed Tumor Cell Metastasis through Inhibition of Rho/YAP Pathway and Expression of RHAMM and CXCR4 in Melanoma and Breast Cancer Cells." Biomedicines 9, no. 1 (January 4, 2021): 35. http://dx.doi.org/10.3390/biomedicines9010035.
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The high mortality rate of cancer is strongly correlated with the development of distant metastases at secondary sites. Although Rho GTPases, such as RhoA, RhoB, RhoC, and RhoE, promote tumor metastasis, the main roles of Rho GTPases remain unidentified. It is also unclear whether rhosin, a Rho inhibitor, acts by suppressing metastasis by a downstream inhibition of Rho. In this study, we investigated this mechanism of metastasis in highly metastatic melanoma and breast cancer cells, and the mechanism of inhibition of metastasis by rhosin. We found that rhosin suppressed the RhoA and RhoC activation, the nuclear localization of YAP, but did not affect ERK1/2, Akt, or NF-κB activation in the highly metastatic cell lines B16BL6 and 4T1. High expression of YAP was associated with poor overall and recurrence-free survival in patients with breast cancer or melanoma. Treatment with rhosin inhibited lung metastasis in vivo. Moreover, rhosin inhibited tumor cell adhesion to the extracellular matrix via suppression of RHAMM expression, and inhibited SDF-1-induced cell migration and invasion by decreasing CXCR4 expression in B16BL6 and 4T1 cells. These results suggest that the inhibition of RhoA/C-YAP pathway by rhosin could be an extremely useful therapeutic approach in patients with melanoma and breast cancer.
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Tseliou, Melpomeni, Ahmed Al-Qahtani, Saud Alarifi, Saad H. Alkahtani, Christos Stournaras, and George Sourvinos. "The Role of RhoA, RhoB and RhoC GTPases in Cell Morphology, Proliferation and Migration in Human Cytomegalovirus (HCMV) Infected Glioblastoma Cells." Cellular Physiology and Biochemistry 38, no. 1 (2016): 94–109. http://dx.doi.org/10.1159/000438612.
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Background/Aims: Rho GTPases are crucial regulators of the actin cytoskeleton, membrane trafficking and cell signaling and their importance in cell migration and invasion is well- established. The human cytomegalovirus (HCMV) is a widespread pathogen responsible for generally asymptomatic and persistent infections in healthy people. Recent evidence indicates that HCMV gene products are expressed in over 90% of malignant type glioblastomas (GBM). In addition, the HCMV Immediate Early-1 protein (IE1) is expressed in >90% of tumors analyzed. Methods: RhoA, RhoB and RhoC were individually depleted in U373MG glioblastoma cells as well as U373MG cells stably expressing the HCMV IE1 protein (named U373MG-IE1 cells) shRNA lentivirus vectors. Cell proliferation assays, migration as well as wound-healing assays were performed in uninfected and HCMV-infected cells. Results: The depletion of RhoA, RhoB and RhoC protein resulted in significant alterations in the morphology of the uninfected cells, which were further enhanced by the cytopathic effect caused by HCMV. Furthermore, in the absence or presence of HCMV, the knockdown of RhoB and RhoC proteins decreased the proliferation rate of the parental and the IE1-expressing glioblastoma cells, whereas the knockdown of RhoA protein in the HCMV infected cell lines restored their proliferation rate. In addition, wound healing assays in U373MG cells revealed that depletion of RhoA, RhoB and RhoC differentially reduced their migration rate, even in the presence or the absence of HCMV. Conclusion: Collectively, these data show for the first time a differential implication of Rho GTPases in morphology, proliferation rate and motility of human glioblastoma cells during HCMV infection, further supporting an oncomodulatory role of HCMV depending on the Rho isoforms' state.
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Saracaloglu, Ahmet, Seniz Demiryürek, Sabit Kimyon, Alper Mete, Ebru Temiz, Gülper Nacarkahya, Betül Düzen, Oguzhan Saygili, Kıvanc Güngör, and Abdullah Tuncay Demiryürek. "Protein Expressions of the Small GTPase Rho Proteins in Pterygial Tissue and Leukocytes of Patients with Pterygium." Proceedings 2, no. 25 (December 6, 2018): 1571. http://dx.doi.org/10.3390/proceedings2251571.
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Pterygium is a benign fibrovascular proliferation that develops from the conjunctiva and invades the cornea. The etiology of this disorder remains unclear. Current treatment of pterygium is surgical. The postoperative recurrence rate of pterygium is reported be high. To the best of our knowledge, these results are the first to demonstrate the contribution of proteins expressions of the small GTPase Rho proteins in patients with pterygium. Our data showed that leukocyte RhoA, RhoB, RhoD, and RhoE protein expressions were markedly elevated in primary pterygium. However, no significant modifications were noted in pterygial tissues.
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Jackson, Ben, Karine Peyrollier, Esben Pedersen, Astrid Basse, Richard Karlsson, Zhipeng Wang, Tine Lefever, et al. "RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes." Molecular Biology of the Cell 22, no. 5 (March 2011): 593–605. http://dx.doi.org/10.1091/mbc.e09-10-0859.
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RhoA is a small guanosine-5’-triphosphatase (GTPase) suggested to be essential for cytokinesis, stress fiber formation, and epithelial cell–cell contacts. In skin, loss of RhoA was suggested to underlie pemphigus skin blistering. To analyze RhoA function in vivo, we generated mice with a keratinocyte-restricted deletion of the RhoA gene. Despite a severe reduction of cofilin and myosin light chain (MLC) phosphorylation, these mice showed normal skin development. Primary RhoA-null keratinocytes, however, displayed an increased percentage of multinucleated cells, defective maturation of cell–cell contacts. Furthermore we observed increased cell spreading due to impaired RhoA-ROCK (Rho-associated protein kinase)-MLC phosphatase-MLC–mediated cell contraction, independent of Rac1. Rho-inhibiting toxins further increased multinucleation of RhoA-null cells but had no significant effect on spreading, suggesting that RhoB and RhoC have partially overlapping functions with RhoA. Loss of RhoA decreased directed cell migration in vitro caused by reduced migration speed and directional persistence. These defects were not related to the decreased cell contraction and were independent of ROCK, as ROCK inhibition by Y27632 increased directed migration of both control and RhoA-null keratinocytes. Our data indicate a crucial role for RhoA and contraction in regulating cell spreading and a contraction-independent function of RhoA in keratinocyte migration. In addition, our data show that RhoA is dispensable for skin development.
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Riento, Kirsi, Rosa M. Guasch, Ritu Garg, Boquan Jin, and Anne J. Ridley. "RhoE Binds to ROCK I and Inhibits Downstream Signaling." Molecular and Cellular Biology 23, no. 12 (June 15, 2003): 4219–29. http://dx.doi.org/10.1128/mcb.23.12.4219-4229.2003.
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ABSTRACT RhoE belongs to the Rho GTPase family, the members of which control actin cytoskeletal dynamics. RhoE induces stress fiber disassembly in a variety of cell types, whereas RhoA stimulates stress fiber assembly. The similarity of RhoE and RhoA sequences suggested that RhoE might compete with RhoA for interaction with its targets. Here, we show that RhoE binds ROCK I but none of the other RhoA targets tested. The interaction of RhoE with ROCK I was confirmed by coimmunoprecipitation of the endogenous proteins, and the two proteins colocalized on the trans-Golgi network in COS-7 cells. Although RhoE and RhoA were not able to bind ROCK I simultaneously, RhoE bound to the amino-terminal region of ROCK I encompassing the kinase domain, at a site distant from the carboxy-terminal RhoA-binding site. Overexpression of RhoE inhibited ROCK I-induced stress fiber formation and phosphorylation of the ROCK I target myosin light chain phosphatase. These data suggest that RhoE induces stress fiber disassembly by directly binding ROCK I and inhibiting it from phosphorylating downstream targets.
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Pronk, Manon C. A., Jan S. M. van Bezu, Geerten P. van Nieuw Amerongen, Victor W. M. van Hinsbergh, and Peter L. Hordijk. "RhoA, RhoB and RhoC differentially regulate endothelial barrier function." Small GTPases 10, no. 6 (September 26, 2017): 466–84. http://dx.doi.org/10.1080/21541248.2017.1339767.
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Antoni, Angelika, Chelsea Ray, Ryan Kohn, Diana Andreyko, and Jerrold Levine. "Analysis of the misregulation of RhoA and RhoH in autoimmune mice. (HUM7P.302)." Journal of Immunology 192, no. 1_Supplement (May 1, 2014): 184.11. http://dx.doi.org/10.4049/jimmunol.192.supp.184.11.
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Abstract When cultured in the presence of apoptotic cells, cell bodies or lipids, RhoA is misregulated in macrophages from all 6 autoimmune mouse strains but none of the 7 normal strains tested. Misregulation of RhoA results in defective morphology, altered cytokine production, and increased adhesion. The same macrophages cultured without apoptotic cell lipids had properly regulated RhoA and behaved normally. Normal mouse macrophages cultured with the Rho inhibitor C3 toxin, had defective macrophage morphology and behavior. Therefore, we proposed that autoimmunity is due to an apoptotic cell recognition signaling defect and that normal macrophage function may be restored by a chemical reversal of the defect. Our new data demonstrate that lupus strain, MRL, macrophages cultured in the presence of C3 no longer had defects in morphology and function. The fact that a RhoA inhibitor restored RhoA function indicates that the misregulation in autoimmune macrophages involves a RhoA regulator also affected by C3 and not RhoA itself. RhoH is a known inhibitor of RhoA and it is only expressed in hematopoietic cells. Experiments comparing RhoH expression in BALB and MRL macrophages suggest at least four different sizes of RhoH protein are expressed in normal macrophages in response to lipid exposure and that lupus macrophages do not express the multiple forms of RhoH even in the presence of lipids. A lack of proper RhoH expression could be behind the autoimmune macrophage defect.
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Ocana-Morgner, Carlos, Christine Wahren, and Rolf Jessberger. "SWAP-70 regulates RhoA/RhoB-dependent MHCII surface localization in dendritic cells." Blood 113, no. 7 (February 12, 2009): 1474–82. http://dx.doi.org/10.1182/blood-2008-04-152587.
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Abstract Stimulated dendritic cells (DCs) mature and migrate to lymphoid organs to prime naive T cells. DC maturation augments antigen-presentation capacity of DCs by increasing peptide loading, half-life, and cell surface localization of MHC molecules. Activated SWAP-70−/− DCs fail to properly localize MHCII molecules in the plasma membrane, are strongly impaired in T-cell activation, and are altered in F-actin rearrangement. MHCII synthesis, invariant chain removal, and MHCII internalization, however, are unaffected. MHCII surface localization is known to require RhoGTPases. Surprisingly, SWAP70, hitherto known to bind F-actin and Rac, also binds RhoA-GTP. In SWAP-70−/− DCs, RhoA and RhoB are stimulus-independent and constitutively active. Surface localization of MHCII molecules and T-cell activation can be restored by blocking RhoA and RhoB before but not during DC activation. Thus, contrasting positive regulation of Rac, SWAP-70 negatively regulates RhoA and—indirectly—RhoB, preventing premature RhoA/RhoB activation. Through RhoA/RhoB regulation, SWAP-70 defines a new pathway to control surface localization of MHCII, a critical element in DC-dependent immune responses.
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Quinn, Kathrina, Melinda A. Brindley, Melodie L. Weller, Nikola Kaludov, Andrew Kondratowicz, Catherine L. Hunt, Patrick L. Sinn, et al. "Rho GTPases Modulate Entry of Ebola Virus and Vesicular Stomatitis Virus Pseudotyped Vectors." Journal of Virology 83, no. 19 (July 22, 2009): 10176–86. http://dx.doi.org/10.1128/jvi.00422-09.
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ABSTRACT To explore mechanisms of entry for Ebola virus (EBOV) glycoprotein (GP) pseudotyped virions, we used comparative gene analysis to identify genes whose expression correlated with viral transduction. Candidate genes were identified by using EBOV GP pseudotyped virions to transduce human tumor cell lines that had previously been characterized by cDNA microarray. Transduction profiles for each of these cell lines were generated, and a significant positive correlation was observed between RhoC expression and permissivity for EBOV vector transduction. This correlation was not specific for EBOV vector alone as RhoC also correlated highly with transduction of vesicular stomatitis virus GP (VSVG) pseudotyped vector. Levels of RhoC protein in EBOV and VSV permissive and nonpermissive cells were consistent with the cDNA gene array findings. Additionally, vector transduction was elevated in cells that expressed high levels of endogenous RhoC but not RhoA. RhoB and RhoC overexpression significantly increased EBOV GP and VSVG pseudotyped vector transduction but had minimal effect on human immunodeficiency virus (HIV) GP pseudotyped HIV or adeno-associated virus 2 vector entry, indicating that not all virus uptake was enhanced by expression of these molecules. RhoB and RhoC overexpression also significantly enhanced VSV infection. Similarly, overexpression of RhoC led to a significant increase in fusion of EBOV virus-like particles. Finally, ectopic expression of RhoC resulted in increased nonspecific endocytosis of fluorescent dextran and in formation of increased actin stress fibers compared to RhoA-transfected cells, suggesting that RhoC is enhancing macropinocytosis. In total, our studies implicate RhoB and RhoC in enhanced productive entry of some pseudovirions and suggest the involvement of actin-mediated macropinocytosis as a mechanism of uptake of EBOV GP and VSVG pseudotyped viral particles.
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Giang Ho, T. T., Audrey Stultiens, Johanne Dubail, Charles M. Lapière, Betty V. Nusgens, Alain C. Colige, and Christophe F. Deroanne. "RhoGDIα-dependent balance between RhoA and RhoC is a key regulator of cancer cell tumorigenesis." Molecular Biology of the Cell 22, no. 17 (September 2011): 3263–75. http://dx.doi.org/10.1091/mbc.e11-01-0020.
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RhoGTPases are key signaling molecules regulating main cellular functions such as migration, proliferation, survival, and gene expression through interactions with various effectors. Within the RhoA-related subclass, RhoA and RhoC contribute to several steps of tumor growth, and the regulation of their expression affects cancer progression. Our aim is to investigate their respective contributions to the acquisition of an invasive phenotype by using models of reduced or forced expression. The silencing of RhoC, but not of RhoA, increased the expression of genes encoding tumor suppressors, such as nonsteroidal anti-inflammatory drug–activated gene 1 (NAG-1), and decreased migration and the anchorage-independent growth in vitro. In vivo, RhoC small interfering RNA (siRhoC) impaired tumor growth. Of interest, the simultaneous knockdown of RhoC and NAG-1 repressed most of the siRhoC-related effects, demonstrating the central role of NAG-1. In addition of being induced by RhoC silencing, NAG-1 was also largely up-regulated in cells overexpressing RhoA. The silencing of RhoGDP dissociation inhibitor α (RhoGDIα) and the overexpression of a RhoA mutant unable to bind RhoGDIα suggested that the effect of RhoC silencing is indirect and results from the up-regulation of the RhoA level through competition for RhoGDIα. This study demonstrates the dynamic balance inside the RhoGTPase network and illustrates its biological relevance in cancer progression.
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WHEELER, A., and A. RIDLEY. "Why three Rho proteins? RhoA, RhoB, RhoC, and cell motility." Experimental Cell Research 301, no. 1 (November 15, 2004): 43–49. http://dx.doi.org/10.1016/j.yexcr.2004.08.012.
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Pinzón-Daza, Martha L., Iris C. Salaroglio, Joanna Kopecka, Ruth Garzòn, Pierre-Olivier Couraud, Dario Ghigo, and Chiara Riganti. "The Cross-Talk between Canonical and Non-Canonical Wnt-Dependent Pathways Regulates P-Glycoprotein Expression in Human Blood–Brain Barrier Cells." Journal of Cerebral Blood Flow & Metabolism 34, no. 8 (June 4, 2014): 1258–69. http://dx.doi.org/10.1038/jcbfm.2014.100.
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In this work, we investigate if and how transducers of the ‘canonical’ Wnt pathway, i.e., Wnt/glycogen synthase kinase 3 (GSK3)/β-catenin, and transducers of the ‘non-canonical’ Wnt pathway, i.e., Wnt/RhoA/RhoA kinase (RhoAK), cooperate to control the expression of P-glycoprotein (Pgp) in blood–brain barrier (BBB) cells. By analyzing human primary brain microvascular endothelial cells constitutively activated for RhoA, silenced for RhoA or treated with the RhoAK inhibitor Y27632, we found that RhoAK phosphorylated and activated the protein tyrosine phosphatase 1B (PTP1B), which dephosphorylated tyrosine 216 of GSK3, decreasing the GSK3-mediated inhibition of β-catenin. By contrast, the inhibition of RhoA/RhoAK axis prevented the activation of PTP1B, enhanced the GSK3-induced phosphorylation and ubiquitination of β-catenin, and reduced the β-catenin-driven transcription of Pgp. The RhoAK inhibition increased the delivery of Pgp substrates like doxorubicin across the BBB and improved the doxorubicin efficacy against glioblastoma cells co-cultured under a BBB monolayer. Our data demonstrate that in human BBB cells the expression of Pgp is controlled by a cross-talk between canonical and non-canonical Wnt pathways. The disruption of this cross-talk, e.g., by inhibiting RhoAK, downregulates Pgp and increases the delivery of Pgp substrates across the BBB.
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Vega, Francisco M., Gilbert Fruhwirth, Tony Ng, and Anne J. Ridley. "RhoA and RhoC have distinct roles in migration and invasion by acting through different targets." Journal of Cell Biology 193, no. 4 (May 16, 2011): 655–65. http://dx.doi.org/10.1083/jcb.201011038.
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Several studies suggest that RhoA and RhoC, despite their sequence similarity, have different roles in cell migration and invasion, but the molecular basis for this is not known. Using RNAi, we show that RhoA-depleted cells became elongated and extended multiple Rac1-driven narrow protrusions in 2D and 3D environments, leading to increased invasion. These phenotypes were caused by combined but distinct effects of the Rho-regulated kinases ROCK1 and ROCK2. Depletion of ROCK2 induced multiple delocalized protrusions and reduced migratory polarity, whereas ROCK1 depletion selectively led to cell elongation and defective tail retraction. In contrast, RhoC depletion increased cell spreading and induced Rac1 activation around the periphery in broad lamellipodia, thereby inhibiting directed migration and invasion. These effects of RhoC depletion are mediated by the formin FMNL3, which we identify as a new target of RhoC but not RhoA. We propose that RhoA contributes to migratory cell polarity through ROCK2-mediated suppression of Rac1 activity in lamellipodia, whereas RhoC promotes polarized migration through FMNL3 by restricting lamellipodial broadening.
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Kovačević, Igor, Tomohisa Sakaue, Jisca Majoleé, Manon C. Pronk, Masashi Maekawa, Dirk Geerts, Mar Fernandez-Borja, Shigeki Higashiyama, and Peter L. Hordijk. "The Cullin-3–Rbx1–KCTD10 complex controls endothelial barrier function via K63 ubiquitination of RhoB." Journal of Cell Biology 217, no. 3 (January 22, 2018): 1015–32. http://dx.doi.org/10.1083/jcb.201606055.
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RhoGTPases control endothelial cell (EC) migration, adhesion, and barrier formation. Whereas the relevance of RhoA for endothelial barrier function is widely accepted, the role of the RhoA homologue RhoB is poorly defined. RhoB and RhoA are 85% identical, but RhoB’s subcellular localization and half-life are uniquely different. Here, we studied the role of ubiquitination for the function and stability of RhoB in primary human ECs. We show that the K63 polyubiquitination at lysine 162 and 181 of RhoB targets the protein to lysosomes. Moreover, we identified the RING E3 ligase complex Cullin-3–Rbx1–KCTD10 as key modulator of endothelial barrier integrity via its regulation of the ubiquitination, localization, and activity of RhoB. In conclusion, our data show that ubiquitination controls the subcellular localization and lysosomal degradation of RhoB and thereby regulates the stability of the endothelial barrier through control of RhoB-mediated EC contraction.
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RIDLEY, A. J. "RhoA, RhoB and RhoC have different roles in cancer cell migration." Journal of Microscopy 251, no. 3 (March 12, 2013): 242–49. http://dx.doi.org/10.1111/jmi.12025.
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Neisch, Amanda L., Olga Speck, Beth Stronach, and Richard G. Fehon. "Rho1 regulates apoptosis via activation of the JNK signaling pathway at the plasma membrane." Journal of Cell Biology 189, no. 2 (April 19, 2010): 311–23. http://dx.doi.org/10.1083/jcb.200912010.
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Precisely controlled growth and morphogenesis of developing epithelial tissues require coordination of multiple factors, including proliferation, adhesion, cell shape, and apoptosis. RhoA, a small GTPase, is known to control epithelial morphogenesis and integrity through its ability to regulate the cytoskeleton. In this study, we examine a less well-characterized RhoA function in cell survival. We demonstrate that the Drosophila melanogaster RhoA, Rho1, promotes apoptosis independently of Rho kinase through its effects on c-Jun NH2-terminal kinase (JNK) signaling. In addition, Rho1 forms a complex with Slipper (Slpr), an upstream activator of the JNK pathway. Loss of Moesin (Moe), an upstream regulator of Rho1 activity, results in increased levels of Rho1 at the plasma membrane and cortical accumulation of Slpr. Together, these results suggest that Rho1 functions at the cell cortex to regulate JNK activity and implicate Rho1 and Moe in epithelial cell survival.
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Konstantinidis, Diamantis G., Suvarnamala Pushkaran, Katie M. Giger, Ping Zhou, Paul R. Andreassen, Ursula Klingmuller, James Palis, Yi Zheng, and Theodosia A. Kalfa. "RhoA Gtpase Regulates Erythroblast Cytokinesis and Enucleation By Dynamic Coordination Of The Microtubule-Actomyosin Machineries For Successful Abscission." Blood 122, no. 21 (November 15, 2013): 311. http://dx.doi.org/10.1182/blood.v122.21.311.311.
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Abstract RhoA GTPase is known to regulate cell adhesion, actomyosin cytoskeleton, and cytokinesis. Specifically, it contributes to cytokinesis in sea urchin embryos, Xenopus and HeLa cells by associating with the microtubule ends at the abscission area marking the position for the daughter cell separation and by activating myosin through phosphorylation of the myosin regulatory light chain (MRLC) (Piekny et al, Trends in Cell Biology 2005). Erythroblast enucleation, the process through which orthochromatic erythroblasts expel their nucleus during the final stage of mammalian erythropoiesis, is a multi-step process resembling asymmetric cytokinesis (reviewed by Li, Developmental Cell 2013). It requires establishment of cell polarity through microtubule function, followed by formation of a contractile actomyosin ring between reticulocyte and pyrenocyte (Konstantinidis et al, Blood 2012). To define the mechanistic contribution of RhoA signaling in terminal erythroid maturation, we generated mice with erythroid specific RhoA deletion (EpoR-GFPcre-driven). Erythroid-specific deletion of RhoA, confirmed in the CD71-positive cells by PCR and immunoblotting, caused severe anemia, fatal in utero by E15.5. RhoA-deficient peripheral primitive blood cells were large with significant poikilocytosis and anisocytosis, frequently binuclear or multinuclear with polyploidy of the genetic material as demonstrated by flow cytometry with propidium iodide, and with incomplete clearance of the Golgi network and mitochondria. RhoAΔ/Δ fetal livers had progressively decreased cellularity. The erythroblast populations as determined by CD71-Ter119-FSC flow cytometry as well as by multispectral high-speed cell imaging in flow, demonstrated progressive decline and significantly decreased reticulocyte production (n=3, p<0.05), compatible with a fatal intrauterine anemia. RhoAΔ/Δ definitive erythroblasts also exhibited increased frequency of polyploidy. These polyploid cells were Ki67-negative by immunohistochemistry, indicating that they were not actively mitotic. Apoptosis was not significantly increased in the RhoA-deficient erythroblasts as determined by Annexin-V positivity. However, the late RhoAΔ/Δ erythroblasts demonstrated a significant increase in necrosis as determined by 7AAD and Annexin-V double-positivity (n=3, p<0.05). Increased phosphorylation of p53 was evident by immunoblotting, likely functioning as a polyploidy checkpoint after failure of abscission due to RhoA deficiency. Further evidence that RhoA is significant for microtubule organization in erythroblasts was attained by immuno-localization of RhoA at the microtubule ends and by decreased nuclear polarization in EpoR-Cre RhoAΔ/Δ orthochromatic erythroblasts, as shown by decreased delta centroid Ter119-Draq5 (the distance between the centers of the erythroblast and the nucleus) in multispectral high-speed cell imaging in flow. Phosphorylated myosin regulatory light chain (pMRLC) was found significantly decreased by immunoblotting of RhoAΔ/Δ fetal liver erythroblasts, indicating that RhoA controls phosphorylation of MRLC in erythropoiesis. Increased PAK phosphorylation was noted pointing towards compensatory upregulation of Rac GTPases in the RhoA-deficient cells, which was confirmed by quantitative RT-PCR. We further examined the erythroblastic islands in RhoAΔ/Δfetal liver by transmission electron microscopy and found that RhoA-deficient erythroblasts, frequently dysplastic and binucleated, were loosely associated with the central macrophage and there was a paucity of intracellular vacuoles in comparison to WT erythroblasts, indicating possible defects in vesicular transport, which has also been shown to play a role in erythroblast enucleation (Keerthivasan et al, Blood 2010). Thus, RhoA GTPase regulates erythroblast differentiation and enucleation by dynamic coordination of the microtubule-actomyosin machineries to achieve successful abscission. In addition, RhoA may also regulate erythroblast adhesive interactions with the central macrophage in erythroblastic islands and intracellular vesicular transport. Our results reveal novel molecular components and cellular processes in erythropoiesis that may be important for improving the efficiency of red blood cell production in vitro. Disclosures: No relevant conflicts of interest to declare.
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Rafa-Zabłocka, Katarzyna, Agnieszka Zelek-Molik, Beata Tepper, Piotr Chmielarz, Grzegorz Kreiner, Michał Wilczkowski, and Irena Nalepa. "Chronic restraint stress induces changes in the cerebral Galpha 12/13 and Rho-GTPase signaling network." Pharmacological Reports 73, no. 4 (June 11, 2021): 1179–87. http://dx.doi.org/10.1007/s43440-021-00294-4.
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Abstract Background Evidence indicates that Gα12, Gα13, and its downstream effectors, RhoA and Rac1, regulate neuronal morphology affected by stress. This study was aimed at investigating whether repeated stress influences the expression of proteins related to the Gα12/13 intracellular signaling pathway in selected brain regions sensitive to the effects of stress. Furthermore, the therapeutic impact of β(1)adrenergic receptors (β1AR) blockade was assessed. Methods Restraint stress (RS) model in mice (2 h/14 days) was used to assess prolonged stress effects on the mRNA expression of Gα12, Gα13, RhoA, Rac1 in the prefrontal cortex (PFC), hippocampus (HIP) and amygdala (AMY). In a separate study, applying RS model in rats (3–4 h/1 day or 14 days), we evaluated stress effects on the expression of Gα12, Gα11, Gαq, RhoA, RhoB, RhoC, Rac1/2/3 in the HIP. Betaxolol (BET), a selective β1AR antagonist, was introduced (5 mg/kg/p.o./8–14 days) in the rat RS model to assess the role of β1AR in stress effects. RT-qPCR and Western Blot were used for mRNA and protein assessments, respectively. Results Chronic RS decreased mRNA expression of Gα12 and increased mRNA for Rac1 in the PFC of mice. In the mice AMY, decreased mRNA expression of Gα12, Gα13 and RhoA was observed. Fourteen days of RS exposure increased RhoA protein level in the rats’ HIP in the manner dependent on β1AR activity. Conclusions Together, these results suggest that repeated RS affects the expression of genes and proteins known to be engaged in neural plasticity, providing potential targets for further studies aimed at unraveling the molecular mechanisms of stress-related neuropsychiatric diseases.
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Michaelson, David, Joseph Silletti, Gretchen Murphy, Peter D'Eustachio, Mark Rush, and Mark R. Philips. "Differential Localization of Rho Gtpases in Live Cells." Journal of Cell Biology 152, no. 1 (January 8, 2001): 111–26. http://dx.doi.org/10.1083/jcb.152.1.111.
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Determinants of membrane targeting of Rho proteins were investigated in live cells with green fluorescent fusion proteins expressed with or without Rho-guanine nucleotide dissociation inhibitor (GDI)α. The hypervariable region determined to which membrane compartment each protein was targeted. Targeting was regulated by binding to RhoGDIα in the case of RhoA, Rac1, Rac2, and Cdc42hs but not RhoB or TC10. Although RhoB localized to the plasma membrane (PM), Golgi, and motile peri-Golgi vesicles, TC10 localized to PMs and endosomes. Inhibition of palmitoylation mislocalized H-Ras, RhoB, and TC10 to the endoplasmic reticulum. Although overexpressed Cdc42hs and Rac2 were observed predominantly on endomembrane, Rac1 was predominantly at the PM. RhoA was cytosolic even when expressed at levels in vast excess of RhoGDIα. Oncogenic Dbl stimulated translocation of green fluorescent protein (GFP)-Rac1, GFP-Cdc42hs, and GFP-RhoA to lamellipodia. RhoGDI binding to GFP-Cdc42hs was not affected by substituting farnesylation for geranylgeranylation. A palmitoylation site inserted into RhoA blocked RhoGDIα binding. Mutations that render RhoA, Cdc42hs, or Rac1, either constitutively active or dominant negative abrogated binding to RhoGDIα and redirected expression to both PMs and internal membranes. Thus, despite the common essential feature of the CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif, the subcellular localizations of Rho GTPases, like their functions, are diverse and dynamic.
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ASPENSTRÖM, Pontus, Åsa FRANSSON, and Jan SARAS. "Rho GTPases have diverse effects on the organization of the actin filament system." Biochemical Journal 377, no. 2 (January 15, 2004): 327–37. http://dx.doi.org/10.1042/bj20031041.
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The Rho GTPases are related to the Ras proto-oncogenes and consist of 22 family members. These proteins have important roles in regulating the organization of the actin filament system, and thereby the morphogenesis of vertebrate cells as well as their ability to migrate. In an effort to compare the effects of all members of the Rho GTPase family, active Rho GTPases were transfected into porcine aortic endothelial cells and the effects on the actin filament system were monitored. Cdc42, TCL (TC10-like), Rac1–Rac3 and RhoG induced the formation of lamellipodia, whereas Cdc42, Rac1 and Rac2 also induced the formation of thick bundles of actin filaments. In contrast, transfection with TC10 or Chp resulted in the formation of focal adhesion-like structures, whereas Wrch-1 induced long and thin filopodia. Transfection with RhoA, RhoB or RhoC induced the assembly of stress fibres, whereas Rnd1–Rnd3 resulted in the loss of stress fibres, but this effect was associated with the formation of actin- and ezrin-containing dorsal microvilli. Cells expressing RhoD and Rif had extremely long and flexible filopodia. None of the RhoBTB or Miro GTPases had any major influence on the organization of the actin filament system; instead, RhoBTB1 and RhoBTB2 were present in vesicular structures, and Miro-1 and Miro-2 were present in mitochondria. Collectively, the data obtained in this study to some extent confirm earlier observations, but also allow the identification of previously undetected roles of the different members of the Rho GTPases.
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Konstantinidis, Diamantis, Suvarnamala Pushkaran, Ursula Klingmuller, James Palis, Yi Zheng, and Theodosia A. Kalfa. "Erythroid Specific RhoA Deficiency Causes Dysplastic and Inefficient Fetal Erythropoiesis with Lethal Embryonic Anemia Due to Defects in Erythroblast Cytokinesis and Enucleation." Blood 120, no. 21 (November 16, 2012): 82. http://dx.doi.org/10.1182/blood.v120.21.82.82.
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Abstract Abstract 82 RhoA GTPase is known to regulate cell adhesion, actomyosin cytoskeleton, and cytokinesis (Schwartz, J Cell Sci 2004). We recently demonstrated evidence that enucleation, the process through which orthochromatic erythroblasts expel their nucleus during the final stage of mammalian erythropoiesis, is a multi-step process resembling asymmetric cytokinesis. It requires establishment of cell polarity through microtubule function, followed by formation of a contractile actomyosin ring, and coalescence of lipid rafts between reticulocyte and pyrenocyte. We showed that RhoA-related Rac GTPases organize actin in the actomyosin ring and aggregate lipid rafts in the furrow between nascent reticulocyte and pyrenocyte during enucleation (Konstantinidis et al, Blood 2012). Based on the resemblances between erythroblast enucleation and cytokinesis we hypothesized that RhoA dynamically controls erythroblast enucleation by molecular pathways analogous to the ones involved in cytokinesis. To define the mechanistic contribution of RhoA signaling in erythropoiesis and enucleation, we generated mice with erythroid specific deletion (EpoRGFPcre/+ driven) of RhoA. Erythroid-specific deletion of RhoA caused severe anemia, which was fatal in utero by E14.5. These embryos as well as their yolk sacs had a pallid, anemic appearance and their peripheral primitive blood cells were large with significant poikilocytosis and anisocytosis, frequently binuclear or multinuclear, and with incomplete clearance of the Golgi network and mitochondria. RhoAΔ/Δ fetal livers at E12.5 had approximately 33% of the cellularity of WT littermates (n=9, p<0.001). Analysis of definitive erythropoiesis in the RhoAΔ/Δ fetal liver showed that despite decreased cellularity, the erythroid progenitors BFU-E and CFU-E were not different in total number per fetal liver. In contrast, the erythroblast populations, as determined by CD71-Ter119-FSC flow cytometry as well as by multispectral high-speed cell imaging in flow, demonstrated progressive decline and significantly decreased reticulocyte production (n=3, p<0.05), compatible with a fatal intrauterine anemia. RhoA-deficient fetal liver erythroblasts expressed significantly higher levels of the α4 and α5 integrin subunits, suggesting a compensatory increase of the adhesion receptors due to impaired downstream signaling. We further examined the erythroblastic islands in RhoAΔ/Δ fetal liver by transmission electron microscopy and found that RhoA-deficient erythroblasts, frequently dysplastic and binucleated, were more loosely associated with the central macrophage and there was a paucity of intracellular vacuoles in comparison to WT erythroblasts, indicating possible defects in vesicular transport, that has also been shown to play a role in erythroblast enucleation (Keerthivasan et al, Blood 2010). Upon evaluation of the RhoA protein level and phosphorylated myosin regulatory light chain (pMRLC) by immunoblotting of RhoAΔ/Δ fetal liver cells, pMRLC was found significantly decreased, indicating that one of the signaling pathways regulated by RhoA in erythropoiesis involves phosphorylation of MRLC. Moreover, treatment of wild type erythroblasts in in vitro erythropoiesis assays with the pharmacological inhibitor Y27632 of Rho-associated protein kinase (ROCK), a downstream target of RhoA participating in myosin regulation, resulted in a decrease of enucleation by up to 38% (n=3, p<0.05). Thus, RhoA GTPase regulates erythroblast differentiation and enucleation via regulation of the actomyosin cytoskeleton by ROCK-mediated phosphorylation of MRLC. In addition, RhoA may also regulate erythroblast adhesive interactions with the central macrophage in erythroblastic islands and intracellular vesicular transport. Further exploration of the role of RhoA in erythropoiesis has the potential to reveal targets for therapeutic interventions for anemias due to terminal erythroid maturation defects as well as for improving the efficiency of red blood cell production in vitro. Disclosures: No relevant conflicts of interest to declare.
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Bayer, Cynthia A., Susan R. Halsell, James W. Fristrom, Daniel P. Kiehart, and Laurence von Kalm. "Genetic Interactions Between the RhoA and Stubble-stubbloid Loci Suggest a Role for a Type II Transmembrane Serine Protease in Intracellular Signaling During Drosophila Imaginal Disc Morphogenesis." Genetics 165, no. 3 (November 1, 2003): 1417–32. http://dx.doi.org/10.1093/genetics/165.3.1417.
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Abstract The Drosophila RhoA (Rho1) GTPase is essential for postembryonic morphogenesis of leg and wing imaginal discs. Mutations in RhoA enhance leg and wing defects associated with mutations in zipper, the gene encoding the heavy chain of nonmuscle myosin II. We demonstrate here that mutations affecting the RhoA signaling pathway also interact genetically with mutations in the Stubble-stubbloid (Sb-sbd) locus that encodes an unusual type II transmembrane serine protease required for normal leg and wing morphogenesis. In addition, a leg malformation phenotype associated with overexpression of Sb-sbd in prepupal leg discs is suppressed when RhoA gene dose is reduced, suggesting that RhoA and Sb-sbd act in a common pathway during leg morphogenesis. We also characterized six mutations identified as enhancers of zipper mutant leg defects. Three of these genes encode known members of the RhoA signaling pathway (RhoA, DRhoGEF2, and zipper). The remaining three enhancer of zipper mutations interact genetically with both RhoA and Sb-sbd mutations, suggesting that they encode additional components of the RhoA signaling pathway in imaginal discs. Our results provide evidence that the type II transmembrane serine proteases, a class of proteins linked to human developmental abnormalities and pathology, may be associated with intracellular signaling required for normal development.
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Bernusso, Vanessa Aline, Mariana Lazarini, João Agostinho Machado-Neto, Karin Spat Albino Barcellos, and Sara Teresinha Olalla Saad. "ARHGAP21 Is Upregulated and Triggers the Modulation of Rho Gtpase Signaling Pathways during Megakaryocytic Differentiation." Blood 126, no. 23 (December 3, 2015): 4760. http://dx.doi.org/10.1182/blood.v126.23.4760.4760.
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Abstract Introduction: During differentiation, the megakaryocyte goes through profound changes in the cytoskeleton of actin and tubulin through Rho GTPase activity. Microtubules provide the elongation of proplatelets, whereas actin microfilaments mediate force to increase branching and release of platelets. ARHGAP21 is a RhoGAP for RhoA, RhoC and Cdc42, which has been shown to interact with α-tubulin in cancer cells. Moreover, arhgap21+/- mice exhibit significant reduction in platelet number and increased platelet volume. Aim: To evaluate ARHGAP21 function in the activity of Rho GTPase and their effectors during megakaryocytic differentiation. Materials and Methods: Megakaryocyte differentiation was stimulated in HEL cells through treatment with 20 nM of phorbol myristate acetate -13 -12 (PMA) for 4 days and was confirmed by the expression of CD41a, CD42b and CD61 and polyploidy using flow cytometry. Morphological changes were observed by optical microscopy. The localization of ARHGAP21, F-Actin and α-Tubulin cytoskeletal proteins was assessed by confocal microscopy. The expression of ARHGAP21, and the Rho GTPases RhoA, RhoC, Cdc42 and downstream proteins Rock1 e2, phospho-MLC2, MLC2, phospho-cofilin, cofilin and mDia1 were analyzed by western blotting. Rho GTPases activity was determined through pull down assays using Rhotekin-GST (RhoA, RhoB and RhoC) and WASP-GST (Cdc42) constructions. Tubulin polymerization was evaluated by soluble and insoluble tubulin precipitation assay. ARHGAP21 silencing was performed by siRNA, after PMA treatment for 2 and 3 days and was followed by the analysis of the expression and activity of Rho GTPases and their effectors, ploidy and differentiation markers. Results: Megakaryocytic differentiation of HEL cells was accompanied by intense rearrangement of the cytoskeleton, increased cell size, polyploidy and increased expression of the membrane receptors CD61, CD41a and CD42b. Interestingly, a gradual upregulation of ARHGAP21 was observed during differentiation, especially on days 2 and 3 of treatment (both 9.33-fold increase) and mainly in extracts containing polymerized tubulin. ARHGAP21 upregulation was concomitant with the reduction of RhoA and Cdc42 activities (92% decreased and 52% decreased, respectively), but not in RhoC. Silencing of ARHGAP21 by siRNA was confirmed by western blot. Downregulation of ARHGAP21 in HEL cells trigged increased phosphorylation on serine 19 of myosin light chain2 (MLC2) on the day 2. Moreover, mDia1, a common effector of RhoA and Cdc42, was also increased at the same point. ARHGAP21 silencing induced an increase in CD42b on day 3 (5% increased, P<0.015). No difference was observed in the expression of CD61 and CD41a and in the ploidy of ARHGAP21 silenced cells compared to control. Conclusion: Our results suggest that the upregulation of ARHGAP21 during megakaryocytic differentiation is important to control the dynamics of the cytoskeleton through the regulation of RhoA and Cdc42. Silencing of ARHGAP21 induces increased phosphorylation of MLC2 and the expression of mDia1, which may impair megakaryocytic differentiation. Furthermore, ARHGAP21 appears to regulate the acquisition of CD42b receptor, participating in the final stages of megakaryopoiesis. Disclosures No relevant conflicts of interest to declare.
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von Kalm, Laurence, and Corey Seavey. "Abstract 2461: The cell migration inhibitor dihydromotuporamine C regulates actin-myosin contractility and actin polymerization." Cancer Research 83, no. 7_Supplement (April 4, 2023): 2461. http://dx.doi.org/10.1158/1538-7445.am2023-2461.
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Abstract The motuporamines are a promising class of anti-metastatic compounds. Dihydromotuporamine C (Motu33) has been shown to activate the small GTPase RhoA, however, little is known about subsequent downstream events leading to cell migration inhibition. In the present study, we investigated the mechanism of action of Motu33 and a synthetic derivative, Motu-(CH2)-33, in Drosophila by manipulating the gene dose of positive and negative regulators of actin dynamics. Consistent with previous findings, reduced gene dose of Rho1 (the Drosophila RhoA ortholog) attenuates motuporamine activity, confirming that RhoA/Rho1 is targeted by these compounds. Actin-myosin contraction is controlled by the RhoA-ROCK-myosin regulatory light chain (MRLC) pathway. Reduced gene dose of the myosin binding subunit of myosin phosphatase, a negative regulator of the RhoA-ROCK-MRLC pathway, enhances motuporamine activity indicating that the motuporamines stimulate actin-myosin contraction through activation of the myosin regulatory light chain. RhoA also activates diaphanous (dia) to control actin polymerization. Surprisingly, reduced gene dose of dia enhances motuporamine activity, suggesting that the motuporamines act on dia in a RhoA-independent manner. Reduction in gene dose of the Drosophila Rac orthologs also enhances motuporamine activity. In contrast, motuporamine activity is unaltered by reduced gene dose of slingshot (ssh) which acts to trigger actin severing and depolymerization. Since ssh is directly regulated by Rac, the enhanced activity of motuporamines observed when Rac gene dose is reduced may reflect an indirect mode of action on the Rac GTPases leading to increased RhoA activity. In summary, these findings demonstrate that motuporamines act through RhoA and diaphanous to regulate actin-myosin contractility and actin polymerization. Citation Format: Laurence von Kalm, Corey Seavey. The cell migration inhibitor dihydromotuporamine C regulates actin-myosin contractility and actin polymerization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2461.
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Riento, K., P. Villalonga, R. Garg, and A. Ridley. "Function and regulation of RhoE." Biochemical Society Transactions 33, no. 4 (August 1, 2005): 649–51. http://dx.doi.org/10.1042/bst0330649.
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The three Rnd proteins, Rnd1, Rnd2 and RhoE/Rnd3, are a subset of Rho family proteins that are unusual in that they bind but do not hydrolyse GTP, and are therefore not regulated by the classical GTP/GDP conformational switch of small GTPases. Increased expression of each Rnd protein induces loss of stress fibres in cultured fibroblasts and epithelial cells, acting antagonistically to RhoA, which stimulates stress fibre formation. RhoE is farnesylated and localizes partly on membranes, including the Golgi and plasma membrane, and in the cytosol. RhoE inhibits RhoA signalling in part by binding to the RhoA-activated serine/threonine kinase ROCK I (Rho-associated kinase I), thereby preventing it from phosphorylating its targets. RhoE activity is itself regulated by phosphorylation by ROCK I on multiple sites. RhoE phosphorylation enhances its stability, leading to an increase in RhoE levels. In addition, phosphorylation reduces its association with membranes and correlates with its ability to induce loss of stress fibres. RhoE also acts independently of ROCK to inhibit cell cycle progression, in part by preventing translation of cyclin D1, and to inhibit transformation of fibroblasts by oncogenic H-Ras. RhoE is therefore a multifunctional protein whose localization and actions are regulated by phosphorylation.
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Ho, T. T. Giang, Sofia D. Merajver, Charles M. Lapière, Betty V. Nusgens, and Christophe F. Deroanne. "RhoA-GDP Regulates RhoB Protein Stability." Journal of Biological Chemistry 283, no. 31 (June 4, 2008): 21588–98. http://dx.doi.org/10.1074/jbc.m710033200.
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O'Connor, Kathleen L., Min Chen, and L. Nicole Towers. "Integrin α6β4 cooperates with LPA signaling to stimulate Rac through AKAP-Lbc-mediated RhoA activation." American Journal of Physiology-Cell Physiology 302, no. 3 (February 2012): C605—C614. http://dx.doi.org/10.1152/ajpcell.00095.2011.
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The α6β4 integrin promotes carcinoma invasion through its ability to promote directed migration and polarization of carcinoma cells. In this study, we explore how the α6β4 integrin cooperates with lysophosphatidic acid (LPA) to activate Rho and Rac small GTPases. Through the use of dominant negative Rho constructs, C3 exotransferase, and Rho kinase inhibitor, we find that Rho is critical for LPA-dependent chemotaxis and lamellae formation. However, utilization of specific Rho isoforms depends on integrin α6β4 expression status. Integrin α6β4-negative MDA-MB-435 cells utilize only RhoC for motility, whereas integrin α6β4-expressing cells utilize RhoC but additionally activate and utilize RhoA for LPA-dependent cell motility and lamellae formation. Notably, the activation of RhoA by cooperative LPA and integrin α6β4 signaling requires the Rho guanine nucleotide exchange factor AKAP-Lbc. We also determine that integrin α6β4 cannot activate Rac1 directly but promotes LPA-mediated Rac1 activation that is dependent on RhoA activity and de novo β1 integrin ligation. Finally, we find that the regulation of Rac1 and RhoA in response to LPA is differentially regulated by phosphodiesterases, PKA, and phosphatidylinositol 3-kinase, thus supporting their spatially distinct compartmentalization. In summary, signaling from integrin α6β4 facilitates LPA-stimulated chemotaxis through preferential activation of RhoA, which, in turn, facilitates activation of Rac1.
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Zaritsky, Assaf, Yun-Yu Tseng, M. Angeles Rabadán, Shefali Krishna, Michael Overholtzer, Gaudenz Danuser, and Alan Hall. "Diverse roles of guanine nucleotide exchange factors in regulating collective cell migration." Journal of Cell Biology 216, no. 6 (May 16, 2017): 1543–56. http://dx.doi.org/10.1083/jcb.201609095.
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Efficient collective migration depends on a balance between contractility and cytoskeletal rearrangements, adhesion, and mechanical cell–cell communication, all controlled by GTPases of the RHO family. By comprehensive screening of guanine nucleotide exchange factors (GEFs) in human bronchial epithelial cell monolayers, we identified GEFs that are required for collective migration at large, such as SOS1 and β-PIX, and RHOA GEFs that are implicated in intercellular communication. Down-regulation of the latter GEFs differentially enhanced front-to-back propagation of guidance cues through the monolayer and was mirrored by down-regulation of RHOA expression and myosin II activity. Phenotype-based clustering of knockdown behaviors identified RHOA-ARHGEF18 and ARHGEF3-ARHGEF28-ARHGEF11 clusters, indicating that the latter may signal through other RHO-family GTPases. Indeed, knockdown of RHOC produced an intermediate between the two phenotypes. We conclude that for effective collective migration, the RHOA-GEFs → RHOA/C → actomyosin pathways must be optimally tuned to compromise between generation of motility forces and restriction of intercellular communication.
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Shi, Geng-Xian, Won Seok Yang, Ling Jin, Michelle L. Matter, and Joe W. Ramos. "RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases." Proceedings of the National Academy of Sciences 115, no. 2 (December 26, 2017): E190—E199. http://dx.doi.org/10.1073/pnas.1708584115.
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Directed migration is essential for cell motility in many processes, including development and cancer cell invasion. RSKs (p90 ribosomal S6 kinases) have emerged as central regulators of cell migration; however, the mechanisms mediating RSK-dependent motility remain incompletely understood. We have identified a unique signaling mechanism by which RSK2 promotes cell motility through leukemia-associated RhoGEF (LARG)-dependent Rho GTPase activation. RSK2 directly interacts with LARG and nucleotide-bound Rho isoforms, but not Rac1 or Cdc42. We further show that epidermal growth factor or FBS stimulation induces association of endogenous RSK2 with LARG and LARG with RhoA. In response to these stimuli, RSK2 phosphorylates LARG at Ser1288 and thereby activates RhoA. Phosphorylation of RSK2 at threonine 577 is essential for activation of LARG-RhoA. Moreover, RSK2-mediated motility signaling depends on RhoA and -B, but not RhoC. These results establish a unique RSK2-dependent LARG-RhoA signaling module as a central organizer of directed cell migration and invasion.
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Leung, T., X. Q. Chen, E. Manser, and L. Lim. "The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton." Molecular and Cellular Biology 16, no. 10 (October 1996): 5313–27. http://dx.doi.org/10.1128/mcb.16.10.5313.
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The GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, motility, and cytokinesis. We recently reported on a p150 serine/threonine kinase (termed ROK alpha) binding RhoA only in its active GTP-bound state and on its cDNA; introduction of RhoA into HeLa cells resulted in translocation of the cytoplasmic kinase to plasma membranes, consistent with ROK alpha being a target for RhoA (T. Leung, E. Manser, L. Tan, and L. Lim, J. Biol. Chem. 256:29051-29054, 1995). Reanalysis of the cDNA revealed that ROK alpha contains an additional N-terminal region. We also isolated another cDNA which encoded a protein (ROK beta) with 90% identity to ROK alpha in the kinase domain. Both ROK alpha and ROK beta, which had a molecular mass of 160 kDa, contained a highly conserved cysteine/histidine-rich domain located within a putative pleckstrin homology domain. The kinases bound RhoA, RhoB, and RhoC but not Rac1 and Cdc42. The Rho-binding domain comprises about 30 amino acids. Mutations within this domain caused partial or complete loss of Rho binding. The morphological effects of ROK alpha were investigated by microinjecting HeLa cells with DNA constructs encoding various forms of ROK alpha. Full-length ROK alpha promoted formation of stress fibers and focal adhesion complexes, consistent with its being an effector of RhoA. ROK alpha truncated at the C terminus promoted this formation and also extensive condensation of actin microfilaments and nuclear disruption. The proteins exhibited protein kinase activity which was required for stress fiber formation; the kinase-dead ROK alpha K112A and N-terminally truncated mutants showed no such promotion. The latter mutant instead induced disassembly of stress fibers and focal adhesion complexes, accompanied by cell spreading. These effects were mediated by the C-terminal region containing Rho-binding, cysteine/histidine-rich, and pleckstrin homology domains. Thus, the multidomained ROK alpha appears to be involved in reorganization of the cytoskeleton, with the N and C termini acting as positive and negative regulators, respectively, of the kinase domain whose activity is crucial for formation of stress fibers and focal adhesion complexes.
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Marcos-Ramiro, Beatriz, Diego García-Weber, Susana Barroso, Jorge Feito, María C. Ortega, Eva Cernuda-Morollón, Natalia Reglero-Real, et al. "RhoB controls endothelial barrier recovery by inhibiting Rac1 trafficking to the cell border." Journal of Cell Biology 213, no. 3 (May 2, 2016): 385–402. http://dx.doi.org/10.1083/jcb.201504038.
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Endothelial barrier dysfunction underlies chronic inflammatory diseases. In searching for new proteins essential to the human endothelial inflammatory response, we have found that the endosomal GTPase RhoB is up-regulated in response to inflammatory cytokines and expressed in the endothelium of some chronically inflamed tissues. We show that although RhoB and the related RhoA and RhoC play additive and redundant roles in various aspects of endothelial barrier function, RhoB specifically inhibits barrier restoration after acute cell contraction by preventing plasma membrane extension. During barrier restoration, RhoB trafficking is induced between vesicles containing RhoB nanoclusters and plasma membrane protrusions. The Rho GTPase Rac1 controls membrane spreading and stabilizes endothelial barriers. We show that RhoB colocalizes with Rac1 in endosomes and inhibits Rac1 activity and trafficking to the cell border during barrier recovery. Inhibition of endosomal trafficking impairs barrier reformation, whereas induction of Rac1 translocation to the plasma membrane accelerates it. Therefore, RhoB-specific regulation of Rac1 trafficking controls endothelial barrier integrity during inflammation.
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Arthur, William T., Shawn M. Ellerbroek, Channing J. Der, Keith Burridge, and Krister Wennerberg. "XPLN, a Guanine Nucleotide Exchange Factor for RhoA and RhoB, But Not RhoC." Journal of Biological Chemistry 277, no. 45 (September 6, 2002): 42964–72. http://dx.doi.org/10.1074/jbc.m207401200.
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Vignal, E., A. Blangy, M. Martin, C. Gauthier-Rouvière, and P. Fort. "Kinectin Is a Key Effector of RhoG Microtubule-Dependent Cellular Activity." Molecular and Cellular Biology 21, no. 23 (December 1, 2001): 8022–34. http://dx.doi.org/10.1128/mcb.21.23.8022-8034.2001.
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ABSTRACT RhoG is a member of the Rho family of GTPases that activates Rac1 and Cdc42 through a microtubule-dependent pathway. To gain understanding of RhoG downstream signaling, we performed a yeast two-hybrid screen from which we identified kinectin, a 156-kDa protein that binds in vitro to conventional kinesin and enhances microtubule-dependent kinesin ATPase activity. We show that RhoGGTP specifically interacts with the central domain of kinectin, which also contains a RhoA binding domain in its C terminus. Interaction was confirmed by coprecipitation of kinectin with active RhoGG12V in COS-7 cells. RhoG, kinectin, and kinesin colocalize in REF-52 and COS-7 cells, mainly in the endoplasmic reticulum but also in lysosomes. Kinectin distribution in REF-52 cells is modulated according to endogenous RhoG activity. In addition, by using injection of anti-kinectin antibodies that challenge RhoG-kinectin interaction or by blocking anti-kinesin antibodies, we show that RhoG morphogenic activity relies on kinectin interaction and kinesin activity. Finally, kinectin overexpression elicits Rac1- and Cdc42-dependent cytoskeletal effects and switches cells to a RhoA phenotype when RhoG activity is inhibited or microtubules are disrupted. The functional links among RhoG, kinectin, and kinesin are further supported by time-lapse videomicroscopy of COS-7 cells, which showed that the microtubule-dependent lysosomal transport is facilitated by RhoG activation or kinectin overexpression and is severely stemmed upon RhoG inhibition. These data establish that kinectin is a key mediator of microtubule-dependent RhoG activity and suggest that kinectin also mediates RhoG- and RhoA-dependent antagonistic pathways.
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Shields, Sarah-Kim, Catalin Nicola, and Chandan Chakraborty. "Rho Guanosine 5′-Triphosphatases Differentially Regulate Insulin-Like Growth Factor I (IGF-I) Receptor-Dependent and -Independent Actions of IGF-II on Human Trophoblast Migration." Endocrinology 148, no. 10 (October 1, 2007): 4906–17. http://dx.doi.org/10.1210/en.2007-0476.
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Both IGF-I and IGF-II stimulate migration of human extravillous trophoblast (EVT) cells. Although IGF-I is known to signal through IGF type 1 receptor (IGF1R), IGF-II signals through IGF1R as well as in an IGF1R-independent manner. The purpose of this study was to investigate the roles of Rho GTPases in IGF1R-independent and -dependent actions of IGF-II on EVT cell migration. To distinguish IGF1R-dependent and -independent actions, we used picropodophyllin, a selective inhibitor of IGF1R tyrosine kinase, and IGF analogs with differential affinities for IGF1R, IGF-II/cation-independent mannose 6-phosphate receptor, and IGF-binding proteins. IGF1R-dependent actions of IGF-II were confirmed by showing the effects of IGF1R-selective agonist Des1–3 IGF-I. We used pharmacological inhibitors or selective small interfering RNAs to investigate the roles of RhoA, RhoC, Rac1, Cdc42, and Rho effector kinases called ROCK-I and -II in IGF-induced EVT cell migration. Although basal migration of EVT cells required each member of the Rho GTPase family studied, IGF1R-dependent and -independent EVT cell migration exhibited differential requirements for these enzymes. IGF1R-mediated EVT cell migration was found to depend on RhoA and RhoC but not on Rac1 or Cdc42. However, IGF1R-independent effect of IGF-II on EVT cell migration required ROCKs but not RhoA, RhoC, Rac1, or Cdc42. Most importantly, IGF1R-independent action of IGF-II was found to be exaggerated when RhoA or RhoC was down-regulated. Thus, different members of the Rho GTPase family regulate IGF-II-mediated EVT cell migration differentially, depending upon whether it signals through IGF1R or in an IGF1R-independent manner.
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Kilic, Ertugrul, Ayman ElAli, Ülkan Kilic, Zeyun Guo, Milas Ugur, Unal Uslu, Claudio L. Bassetti, Martin E. Schwab, and Dirk M. Hermann. "Role of Nogo-A in Neuronal Survival in the Reperfused Ischemic Brain." Journal of Cerebral Blood Flow & Metabolism 30, no. 5 (January 20, 2010): 969–84. http://dx.doi.org/10.1038/jcbfm.2009.268.
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Nogo-A is an oligodendroglial neurite outgrowth inhibitor, the deactivation of which enhances brain plasticity and functional recovery in animal models of stroke. Nogo-A's role in the reperfused brain tissue was still unknown. By using Nogo-A−/− mice and mice in which Nogo-A was blocked with a neutralizing antibody (11C7) that was infused into the lateral ventricle or striatum, we show that Nogo-A inhibition goes along with decreased neuronal survival and more protracted neurologic recovery, when deactivation is constitutive or induced 24 h before, but not after focal cerebral ischemia. We show that in the presence of Nogo-A, RhoA is activated and Rac1 and RhoB are deactivated, maintaining stress kinases p38/MAPK, SAPK/JNK1/2 and phosphatase-and-tensin homolog (PTEN) activities low. Nogo-A blockade leads to RhoA deactivation, thus overactivating Rac1 and RhoB, the former of which activates p38/MAPK and SAPK/JNK1/2 via direct interaction. RhoA and its effector Rho-associated coiled-coil protein kinase2 deactivation in turn stimulates PTEN, thus inhibiting Akt and ERK1/2, and initiating p53-dependent cell death. Our data suggest a novel role of Nogo-A in promoting neuronal survival by controlling Rac1/RhoA balance. Clinical trials should be aware of injurious effects of axonal growth-promoting therapies. Thus, Nogo-A antibodies should not be used in the very acute stroke phase.
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Onishi, Masayuki, Nolan Ko, Ryuichi Nishihama, and John R. Pringle. "Distinct roles of Rho1, Cdc42, and Cyk3 in septum formation and abscission during yeast cytokinesis." Journal of Cell Biology 202, no. 2 (July 22, 2013): 311–29. http://dx.doi.org/10.1083/jcb.201302001.
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In yeast and animal cytokinesis, the small guanosine triphosphatase (GTPase) Rho1/RhoA has an established role in formation of the contractile actomyosin ring, but its role, if any, during cleavage-furrow ingression and abscission is poorly understood. Through genetic screens in yeast, we found that either activation of Rho1 or inactivation of another small GTPase, Cdc42, promoted secondary septum (SS) formation, which appeared to be responsible for abscission. Consistent with this hypothesis, a dominant-negative Rho1 inhibited SS formation but not cleavage-furrow ingression or the concomitant actomyosin ring constriction. Moreover, Rho1 is temporarily inactivated during cleavage-furrow ingression; this inactivation requires the protein Cyk3, which binds Rho1-guanosine diphosphate via its catalytically inactive transglutaminase-like domain. Thus, unlike the active transglutaminases that activate RhoA, the multidomain protein Cyk3 appears to inhibit activation of Rho1 (and thus SS formation), while simultaneously promoting cleavage-furrow ingression through primary septum formation. This work suggests a general role for the catalytically inactive transglutaminases of fungi and animals, some of which have previously been implicated in cytokinesis.
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Nishizuka, Makoto, Rina Komada, and Masayoshi Imagawa. "Knockdown of RhoE Expression Enhances TGF-β-Induced EMT (epithelial-to-mesenchymal transition) in Cervical Cancer HeLa Cells." International Journal of Molecular Sciences 20, no. 19 (September 22, 2019): 4697. http://dx.doi.org/10.3390/ijms20194697.
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Cervical cancer with early metastasis of the primary tumor is associated with poor prognosis and poor therapeutic outcomes. Since epithelial-to-mesenchymal transition (EMT) plays a role in acquisition of the ability to invade the pelvic lymph nodes and surrounding tissue, it is important to clarify the molecular mechanism underlying EMT in cervical cancer. RhoE, also known as Rnd3, is a member of the Rnd subfamily of Rho GTPases. While previous reports have suggested that RhoE may act as either a positive or a negative regulator of cancer metastasis and EMT, the role of RhoE during EMT in cervical cancer cells remains unclear. The present study revealed that RhoE expression was upregulated during transforming growth factor-β (TGF-β)-mediated EMT in human cervical cancer HeLa cells. Furthermore, reduced RhoE expression enhanced TGF-β-mediated EMT and migration of HeLa cells. In addition, we demonstrated that RhoE knockdown elevated RhoA activity and a ROCK inhibitor partially suppressed the acceleration of TGF-β-mediated EMT by RhoE knockdown. These results indicate that RhoE suppresses TGF-β-mediated EMT, partially via RhoA/ROCK signaling in cervical cancer HeLa cells.
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Garg, Ritu, Kirsi Riento, Nicholas Keep, Jonathan D. H. Morris, and Anne J. Ridley. "N-terminus-mediated dimerization of ROCK-I is required for RhoE binding and actin reorganization." Biochemical Journal 411, no. 2 (March 27, 2008): 407–14. http://dx.doi.org/10.1042/bj20071342.
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ROCK-I (Rho-associated kinase 1) is a serine/threonine kinase that can be activated by RhoA and inhibited by RhoE. ROCK-I has an N-terminal kinase domain, a central coiled-coil region and a RhoA-binding domain near the C-terminus. We have previously shown that RhoE binds to the N-terminal 420 amino acids of ROCK-I, which includes the kinase domain as well as N-terminal and C-terminal extensions. In the present study, we show that N-terminus-mediated dimerization of ROCK-I is required for RhoE binding. The central coiled-coil domain can also dimerize ROCK-I in cells, but this is insufficient in the absence of the N-terminus to allow RhoE binding. The kinase activity of ROCK-I1–420 is required for dimerization and RhoE binding; however, inclusion of part of the coiled-coil domain compensates for lack of kinase activity, allowing RhoE to bind. N-terminus-mediated dimerization is also required for ROCK-I to induce the formation of stellate actin stress fibres in cells. These results indicate that dimerization via the N-terminus is critical for ROCK-I function in cells and for its regulation by RhoE.
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Bilodeau, Diane, Sylvie Lamy, Richard R. Desrosiers, Denis Gingras, and Richard Béliveau. "Regulation of Rho protein binding to membranes by rhoGDI: inhibition of releasing activity by physiological ionic conditions." Biochemistry and Cell Biology 77, no. 1 (March 1, 1999): 59–69. http://dx.doi.org/10.1139/o99-004.
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The Rho GDP dissociation inhibitor (GDI) is an ubiquitously expressed regulatory protein involved in the cycling of Rho proteins between membrane-bound and soluble forms. Here, we characterized the Rho solubilization activity of a glutathione S-transferase (GST) - GDI fusion protein in a cell-free system derived from rat kidney. Addition of GST-GDI to kidney brush border membranes resulted in the specific release of Cdc42 and RhoA from the membranes, while RhoB and Ras were not extracted. The release of Cdc42 and RhoA by GST-GDI was dose dependent and saturable with about 50% of both RhoA and Cdc42 extracted. The unextracted Rho proteins were tightly bound to membranes and could not be solubilized by repeated GST-GDI treatment. These results demonstrated that kidney brush border membranes contained two populations of RhoA and Cdc42. Furthermore, the GST-GDI solubilizing activity on membrane-bound Cdc42 and RhoA was abolished at physiological conditions of salt and temperature in all tissues examined. When using bead-immobilized GST-GDI, KCl did not reduced the binding of Rho proteins. However, washing brush border membranes with KCl prior treatment by GST-GDI inhibited the extraction of Rho proteins. Taken together, these results suggest that the binding of GDI to membrane-bound Cdc42 and RhoA occurs easily under physiological ionic strength conditions, but a complementary factor is required to extract these proteins from membranes. These observations suggest that the shuttling activity of GDI upon Rho proteins could be normally downregulated under physiological conditions.Key words: rhoGDI, rho proteins, ionic strength, kidney.
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Villalonga, Priam, Rosa M. Guasch, Kirsi Riento, and Anne J. Ridley. "RhoE Inhibits Cell Cycle Progression and Ras-Induced Transformation." Molecular and Cellular Biology 24, no. 18 (September 15, 2004): 7829–40. http://dx.doi.org/10.1128/mcb.24.18.7829-7840.2004.
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ABSTRACT Rho GTPases are major regulators of cytoskeletal dynamics, but they also affect cell proliferation, transformation, and oncogenesis. RhoE, a member of the Rnd subfamily that does not detectably hydrolyze GTP, inhibits RhoA/ROCK signaling to promote actin stress fiber and focal adhesion disassembly. We have generated fibroblasts with inducible RhoE expression to investigate the role of RhoE in cell proliferation. RhoE expression induced a loss of stress fibers and cell rounding, but these effects were only transient. RhoE induction inhibited cell proliferation and serum-induced S-phase entry. Neither ROCK nor RhoA inhibition accounted for this response. Consistent with its inhibitory effect on cell cycle progression, RhoE expression was induced by cisplatin, a DNA damage-inducing agent. RhoE-expressing cells failed to accumulate cyclin D1 or p21cip1 protein or to activate E2F-regulated genes in response to serum, although ERK, PI3-K/Akt, FAK, Rac, and cyclin D1 transcription was activated normally. The expression of proteins that bypass the retinoblastoma (pRb) family cell cycle checkpoint, including human papillomavirus E7, adenovirus E1A, and cyclin E, rescued cell cycle progression in RhoE-expressing cells. RhoE also inhibited Ras- and Raf-induced fibroblast transformation. These results indicate that RhoE inhibits cell cycle progression upstream of the pRb checkpoint.
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Oinuma, Izumi, Kana Kawada, Kiyoka Tsukagoshi, and Manabu Negishi. "Rnd1 and Rnd3 targeting to lipid raft is required for p190 RhoGAP activation." Molecular Biology of the Cell 23, no. 8 (April 15, 2012): 1593–604. http://dx.doi.org/10.1091/mbc.e11-11-0900.
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The Rnd proteins Rnd1, Rnd2, and Rnd3/RhoE are well known as key regulators of the actin cytoskeleton in various cell types, but they comprise a distinct subgroup of the Rho family in that they are GTP bound and constitutively active. Functional differences of the Rnd proteins in RhoA inhibition signaling have been reported in various cell types. Rnd1 and Rnd3 antagonize RhoA signaling by activating p190 RhoGAP, whereas Rnd2 does not. However, all the members of the Rnd family have been reported to bind directly to p190 RhoGAP and equally induce activation of p190 RhoGAP in vitro, and there is no evidence that accounts for the functional difference of the Rnd proteins in RhoA inhibition signaling. Here we report the role of the N-terminal region in signaling. Rnd1 and Rnd3, but not Rnd2, have a KERRA (Lys-Glu-Arg-Arg-Ala) sequence of amino acids in their N-terminus, which functions as the lipid raft-targeting determinant. The sequence mediates the lipid raft targeting of p190 RhoGAP correlated with its activation. Overall, our results demonstrate a novel regulatory mechanism by which differential membrane targeting governs activities of Rnd proteins to function as RhoA antagonists.
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Troeger, Anja, Amy J. Johnson, Jenna Wood, William G. Blum, Leslie A. Andritsos, John C. Byrd, and David A. Williams. "RhoH is critical for cell-microenvironment interactions in chronic lymphocytic leukemia in mice and humans." Blood 119, no. 20 (May 17, 2012): 4708–18. http://dx.doi.org/10.1182/blood-2011-12-395939.
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Abstract Trafficking of B-cell chronic lymphocytic leukemia (CLL) cells to the bone marrow and interaction with supporting stromal cells mediates important survival and proliferation signals. Previous studies have demonstrated that deletion of Rhoh led to a delayed disease onset in a murine model of CLL. Here we assessed the impact of RhoH on homing, migration, and cell-contact dependent interactions of CLL cells. Rhoh−/− CLL cells exhibited reduced marrow homing and subsequent engraftment. In vitro migration toward the chemokines CXCL12 and CXCL13 and cell-cell interactions between Rhoh−/− CLL cells and the supporting microenvironment was reduced. In the absence of RhoH the distribution of phosphorylated focal adhesion kinase, a protein known to coordinate activation of the Rho GTPases RhoA and Rac, appeared less polarized in chemokine-stimulated Rhoh−/− CLL cells, and activation and localization of RhoA and Rac was dysregulated leading to defective integrin function. These findings in the Rhoh−/− CLL cells were subsequently demonstrated to closely resemble changes in GTPase activation observed in human CLL samples after in vitro and in vivo treatment with lenalidomide, an agent with known influence on microenvironment protection, and suggest that RhoH plays a critical role in prosurvival CLL cell-cell and cell-microenvironment interactions with this agent.
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Meriane, Mayya, Pierre Roux, Michael Primig, Philippe Fort, and Cécile Gauthier-Rouvière. "Critical Activities of Rac1 and Cdc42Hs in Skeletal Myogenesis: Antagonistic Effects of JNK and p38 Pathways." Molecular Biology of the Cell 11, no. 8 (August 2000): 2513–28. http://dx.doi.org/10.1091/mbc.11.8.2513.
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The Rho family of GTP-binding proteins plays a critical role in a variety of cellular processes, including cytoskeletal reorganization and activation of kinases such as p38 and C-jun N-terminal kinase (JNK) MAPKs. We report here that dominant negative forms of Rac1 and Cdc42Hs inhibit the expression of the muscle-specific genes myogenin, troponin T, and myosin heavy chain in L6 and C2 myoblasts. Such inhibition correlates with decreased p38 activity. Active RhoA, RhoG, Rac1, and Cdc42Hs also prevent myoblast-to-myotube transition but affect distinct stages: RhoG, Rac1, and Cdc42Hs inhibit the expression of all muscle-specific genes analyzed, whereas active RhoA potentiates their expression but prevents the myoblast fusion process. We further show by two different approaches that the inhibitory effects of active Rac1 and Cdc42Hs are independent of their morphogenic activities. Rather, myogenesis inhibition is mediated by the JNK pathway, which also leads to a cytoplasmic redistribution of Myf5. We propose that although Rho proteins are required for the commitment of myogenesis, they differentially influence this process, positively for RhoA and Rac1/Cdc42Hs through the activation of the SRF and p38 pathways, respectively, and negatively for Rac1/Cdc42Hs through the activation of the JNK pathway.
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Huang, Qingqing, Jiarong Xie, and Jayaraman Seetharaman. "Crystal Structure of Schizosaccharomyces pombe Rho1 Reveals Its Evolutionary Relationship with Other Rho GTPases." Biology 11, no. 11 (November 7, 2022): 1627. http://dx.doi.org/10.3390/biology11111627.
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The Rho protein, a homolog of Ras, is a member of the Ras superfamily of small GTPases. Rho family proteins are involved in cytoskeletal organization, cell mobility, and polarity, and are implicated in cancer morphogenesis. Although Rho homologs from higher-order mammalian organisms are well studied, there are few studies examining Rho proteins in lower-level single-celled organisms. Here, we report on the crystal structure of Rho1 from Schizosaccharomyces pombe (SpRho1) in complex with GDP in the presence of Mg2+ at a 2.78 Å resolution. The overall structure is similar to that of known Rho homologs, including human RhoA, human RhoC, and Aspergillus fumigatus Rho1 (AfRho1), with some exceptions. We observed subtle differences at the Switch I and II regions, in β2 and β3, and in the Rho insert domain and loop from Phe107 to Pro112. Our analysis suggests that SpRho is evolutionarily closer to HsRhoC than HsRhoA, as previously believed.
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Seccia, Teresa M., Matteo Rigato, Verdiana Ravarotto, and Lorenzo A. Calò. "ROCK (RhoA/Rho Kinase) in Cardiovascular–Renal Pathophysiology: A Review of New Advancements." Journal of Clinical Medicine 9, no. 5 (May 2, 2020): 1328. http://dx.doi.org/10.3390/jcm9051328.
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Rho-associated, coiled-coil containing kinases (ROCK) were originally identified as effectors of the RhoA small GTPase and found to belong to the AGC family of serine/threonine kinases. They were shown to be downstream effectors of RhoA and RhoC activation. They signal via phosphorylation of proteins such as MYPT-1, thereby regulating many key cellular functions including proliferation, motility and viability and the RhoA/ROCK signaling has been shown to be deeply involved in arterial hypertension, cardiovascular–renal remodeling, hypertensive nephropathy and posttransplant hypertension. Given the deep involvement of ROCK in cardiovascular–renal pathophysiology and the interaction of ROCK signaling with other signaling pathways, the reports of trials on the clinical beneficial effects of ROCK’s pharmacologic targeting are growing. In this current review, we provide a brief survey of the current understanding of ROCK-signaling pathways, also integrating with the more novel data that overall support a relevant role of ROCK for the cardiovascular–renal physiology and pathophysiology.
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Xing, Lei, Xiaodi Yao, Kathryn R. Williams, and Gary J. Bassell. "Negative regulation of RhoA translation and signaling by hnRNP-Q1 affects cellular morphogenesis." Molecular Biology of the Cell 23, no. 8 (April 15, 2012): 1500–1509. http://dx.doi.org/10.1091/mbc.e11-10-0867.
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The small GTPase RhoA has critical functions in regulating actin dynamics affecting cellular morphogenesis through the RhoA/Rho kinase (ROCK) signaling cascade. RhoA signaling controls stress fiber and focal adhesion formation and cell motility in fibroblasts. RhoA signaling is involved in several aspects of neuronal development, including neuronal migration, growth cone collapse, dendrite branching, and spine growth. Altered RhoA signaling is implicated in cancer and neurodegenerative disease and is linked to inherited intellectual disabilities. Although much is known about factors regulating RhoA activity and/or degradation, little is known about molecular mechanisms regulating RhoA expression and the subsequent effects on RhoA signaling. We hypothesized that posttranscriptional control of RhoA expression may provide a mechanism to regulate RhoA signaling and downstream effects on cell morphology. Here we uncover a cellular function for the mRNA-binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) Q1 in the control of dendritic development and focal adhesion formation that involves the negative regulation of RhoA synthesis and signaling. We show that hnRNP-Q1 represses RhoA translation and knockdown of hnRNP-Q1 induced phenotypes associated with elevated RhoA protein levels and RhoA/ROCK signaling. These morphological changes were rescued by ROCK inhibition and/or RhoA knockdown. These findings further suggest that negative modulation of RhoA mRNA translation can provide control over downstream signaling and cellular morphogenesis.