The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Channing J Der - One of the best experts on this subject based on the ideXlab platform.
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dlc 1 suppresses non small cell lung cancer growth and invasion by RhoGap dependent and independent mechanisms
Molecular Carcinogenesis, 2008Co-Authors: Kevin D Healy, Louis Hodgson, Adam Shutes, Savitri Maddileti, Kendall T Harden, Tai Young Kim, Yungjue Bang, Klaus M. Hahn, Rudolph L. Juliano, Channing J DerAbstract:Expression of the tumor suppressor deleted in liver cancer-1 (DLC-1) is lost in non-small cell lung (NSCLC) and other human carcinomas, and ectopic DLC-1 expression dramatically reduces proliferation and tumorigenicity. DLC-1 is a multidomain protein that includes a Rho GTPase Activating Protein (RhoGAP) domain which has been hypothesized to be the basis of its tumor suppressive actions. To address the importance of the RhoGAP function of DLC-1 in tumor suppression, we performed biochemical and biological studies evaluating DLC-1 in NSCLC. Full length DLC-1 exhibited strong GAP activity for RhoA as well as RhoB and RhoC, but only very limited activity for Cdc42 in vitro. In contrast, the isolated RhoGAP domain showed 5- to 20-fold enhanced activity for RhoA, RhoB, RhoC and Cdc42. DLC-1 protein expression was absent in six of nine NSCLC cell lines. Restoration of DLC-1 expression in DLC-1-deficient NSCLC cell lines reduced RhoA activity, and experiments with a RhoA biosensor demonstrated that DLC-1 dramatically reduces RhoA activity at the leading edge of cellular protrusions. Furthermore, DLC-1 expression in NSCLC cell lines impaired both anchorage-dependent and -independent growth, as well as invasion in vitro. Surprisingly, we found that the anti-tumor activity of DLC-1 was due to both RhoGAP-dependent and -independent activities. Unlike the rat homologue p122RhoGAP, DLC-1 was not capable of activating the phospholipid hydrolysis activity of phospholipase C-δ1. Combined, these studies provide information on the mechanism of DLC-1 function and regulation, and further support the role of DLC-1 tumor suppression in NSCLC.
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dlc 1 suppresses non small cell lung cancer growth and invasion by RhoGap dependent and independent mechanisms
Molecular Carcinogenesis, 2008Co-Authors: Kevin D Healy, Louis Hodgson, Adam Shutes, Savitri Maddileti, Kendall T Harden, Tai Young Kim, Yungjue Bang, Klaus M. Hahn, Rudolph L. Juliano, Channing J DerAbstract:Expression of the tumor suppressor deleted in liver cancer-1 (DLC-1) is lost in non-small cell lung (NSCLC) and other human carcinomas, and ectopic DLC-1 expression dramatically reduces proliferation and tumorigenicity. DLC-1 is a multidomain protein that includes a Rho GTPase Activating Protein (RhoGAP) domain which has been hypothesized to be the basis of its tumor suppressive actions. To address the importance of the RhoGAP function of DLC-1 in tumor suppression, we performed biochemical and biological studies evaluating DLC-1 in NSCLC. Full length DLC-1 exhibited strong GAP activity for RhoA as well as RhoB and RhoC, but only very limited activity for Cdc42 in vitro. In contrast, the isolated RhoGAP domain showed 5- to 20-fold enhanced activity for RhoA, RhoB, RhoC and Cdc42. DLC-1 protein expression was absent in six of nine NSCLC cell lines. Restoration of DLC-1 expression in DLC-1-deficient NSCLC cell lines reduced RhoA activity, and experiments with a RhoA biosensor demonstrated that DLC-1 dramatically reduces RhoA activity at the leading edge of cellular protrusions. Furthermore, DLC-1 expression in NSCLC cell lines impaired both anchorage-dependent and -independent growth, as well as invasion in vitro. Surprisingly, we found that the anti-tumor activity of DLC-1 was due to both RhoGAP-dependent and -independent activities. Unlike the rat homologue p122RhoGAP, DLC-1 was not capable of activating the phospholipid hydrolysis activity of phospholipase C-δ1. Combined, these studies provide information on the mechanism of DLC-1 function and regulation, and further support the role of DLC-1 tumor suppression in NSCLC.
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xpln a guanine nucleotide exchange factor for rhoa and rhob but not rhoc
Journal of Biological Chemistry, 2002Co-Authors: William T Arthur, Channing J Der, Shawn M Ellerbroek, Keith Burridge, Krister WennerbergAbstract:Rho proteins cycle between an inactive, GDP-bound state and an active, GTP-bound state. Activation of these GTPases is mediated by guanine nucleotide exchange factors (GEFs), which promote GDP to GTP exchange. In this study we have characterized XPLN, a Rho family GEF. Like other Rho GEFs, XPLN contains a tandem Dbl homology and pleckstrin homology domain topography, but lacks homology with other known functional domains or motifs. XPLN protein is expressed in the brain, skeletal muscle, heart, kidney, platelets, and macrophage and neuronal cell lines. In vitro, XPLN stimulates guanine nucleotide exchange on RhoA and RhoB, but not RhoC, RhoG, Rac1, or Cdc42. Consistent with these data, XPLN preferentially associates with RhoA and RhoB. The specificity of XPLN for RhoA and RhoB, but not RhoC, is surprising given that they share over 85% sequence identity. We determined that the inability of XPLN to exchange RhoC is mediated by isoleucine 43 in RhoC, a position occupied by valine in RhoA and RhoB. When expressed in cells, XPLN activates RhoA and RhoB, but not RhoC, and stimulates the assembly of stress fibers and focal adhesions in a Rho kinase-dependent manner. We also found that XPLN possesses transforming activity, as determined by focus formation assays. In conclusion, here we describe a Rho family GEF that can discriminate between the closely related RhoA, RhoB, and RhoC, possibly giving insight to the divergent functions of these three proteins.
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RhoG Signals in Parallel with Rac1 and Cdc42
The Journal of biological chemistry, 2002Co-Authors: Krister Wennerberg, Shawn M Ellerbroek, Keith Burridge, Rong Yu Liu, Antoine E. Karnoub, Channing J DerAbstract:RhoG is a member of the Rho family of small GTPases and shares high sequence identity with Rac1 and Cdc42. Previous studies suggested that RhoG mediates its effects through activation of Rac1 and Cdc42. To further understand the mechanism of RhoG signaling, we studied its potential activation pathways, downstream signaling properties, and functional relationship to Rac1 and Cdc42 in vivo. First, we determined that RhoG was regulated by guanine nucleotide exchange factors that also activate Rac and/or Cdc42. Vav2 (which activates RhoA, Rac1, and Cdc42) and to a lesser degree Dbs (which activates RhoA and Cdc42) activated RhoG in vitro. Thus, RhoG may be activated concurrently with Rac1 and Cdc42. Second, some effectors of Rac/Cdc42 (IQGAP2, MLK-3, PLD1), but not others (e.g. PAKs, POSH, WASP, Par-6, IRSp53), interacted with RhoG in a GTP-dependent manner. Third, consistent with this differential interaction with effectors, activated RhoG stimulated some (JNK and Akt) but not other (SRF and NF-kappaB) downstream signaling targets of activated Rac1 and Cdc42. Finally, transient transduction of a tat-tagged Rac1(17N) dominant-negative fusion protein inhibited the induction of lamellipodia by the Rac-specific activator, Tiam1, but not by activated RhoG. Together, these data argue that RhoG function is mediated by signals independent of Rac1 and Cdc42 activation and instead by direct utilization of a subset of common effectors.
Anne J Ridley - One of the best experts on this subject based on the ideXlab platform.
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effects of rhoa and rhoc upon the sensitivity of prostate cancer cells to glutamine deprivation
Small GTPases, 2021Co-Authors: Luciana Bueno De Paiva, Anne J Ridley, Vanessa Aline Bernusso, Joao Agostinho Machadoneto, Fabiola Traina, Sara T Olallasaad, Mariana LazariniAbstract:RhoA and RhoC contribute to the regulation of glutamine metabolism, which is a crucial determinant of cell growth in some types of cancer. Here we investigated the participation of RhoA and RhoC in the response of prostate cancer cells to glutamine deprivation. We found that RhoA and RhoC activities were up- or downregulated by glutamine reduction in PC3 and LNCaP cell lines, which was concomitant to a reduction in cell number and proliferation. Stable overexpression of wild type RhoA or RhoC did not alter the sensitivity to glutamine deprivation. However, PC3 cells expressing dominant negative RhoAN19 or RhoCN19 mutants were more resistant to glutamine deprivation. Our results indicate that RhoA and RhoC activities could affect cancer treatments targeting the glutamine pathway.
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rhoa rhob and rhoc have different roles in cancer cell migration
Journal of Microscopy, 2013Co-Authors: Anne J RidleyAbstract:Rho GTPases are well known to regulate cell motility through activation of a variety of downstream effector proteins, including enzymes, adaptor proteins and actin nucleators. The three closely related Rho GTPases RhoA, RhoB and RhoC all have the potential to interact with the same downstream effectors, yet they have substantially different effects on cell shape and migratory properties. Here I review the different ways in which RhoA, RhoB and RhoC expression is regulated in cancer and how they play distinct roles in cancer progression. I describe their main effectors known to contribute to cell motility. Recent results from our laboratory and others indicate that RhoA, RhoB and RhoC can be activated by specific stimuli and act through different effectors to control distinct aspects of cancer cell migration and invasion. This suggests that they each make unique contributions to cancer by participating in different protein complexes.
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rhoa and rhoc have distinct roles in migration and invasion by acting through different targets
Journal of Cell Biology, 2011Co-Authors: Francisco M Vega, Gilbert O Fruhwirth, Anne J RidleyAbstract: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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why three rho proteins rhoa rhob rhoc and cell motility
Experimental Cell Research, 2004Co-Authors: Ann P Wheeler, Anne J RidleyAbstract:Higher vertebrates have 3 Rho GTPases, RhoA, RhoB, and RhoC, which share 85% amino acid sequence identity. Here, we compare and contrast the roles of RhoA, B, and C in the regulation of the cytoskeleton and cell motility. Despite their similarity, some regulators and effectors show preferential interaction with RhoA, B, or C, and the three proteins show differences in function in cells. RhoA plays a key role in the regulation of actomyosin contractility. RhoB, which is localized primarily on endosomes, has been shown to regulate cytokine trafficking and cell survival, while RhoC may be more important in cell locomotion. In cancer cells, the expression and activity of RhoA, B, and C is altered in different ways. Together, this evidence suggests that although the 3 isoforms of Rho are structurally highly homologous, they have different cellular functions.
Klaus M. Hahn - One of the best experts on this subject based on the ideXlab platform.
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a rhoc biosensor reveals differences in the activation kinetics of rhoa and rhoc in migrating cells
PLOS ONE, 2013Co-Authors: Jon S Zawistowski, Gaudenz Danuser, Mohsen Sabourighomi, Klaus M. HahnAbstract:RhoA and RhoC GTPases share 92% amino acid sequence identity, yet play different roles in regulating cell motility and morphology. To understand these differences, we developed and validated a biosensor of RhoC activation (RhoC FLARE). This was used together with a RhoA biosensor to compare the spatio-temporal dynamics of RhoA and RhoC activity during cell protrusion/retraction and macropinocytosis. Both GTPases were activated similarly at the cell edge, but in regions more distal from the edge RhoC showed higher activation during protrusion. The two isoforms differed markedly in the kinetics of activation. RhoC was activated concomitantly with RhoA at the cell edge, but distally, RhoC activation preceded RhoA activation, occurring before edge protrusion. During macropinocytosis, differences were observed during vesicle closure and in the area surrounding vesicle formation.
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dlc 1 suppresses non small cell lung cancer growth and invasion by RhoGap dependent and independent mechanisms
Molecular Carcinogenesis, 2008Co-Authors: Kevin D Healy, Louis Hodgson, Adam Shutes, Savitri Maddileti, Kendall T Harden, Tai Young Kim, Yungjue Bang, Klaus M. Hahn, Rudolph L. Juliano, Channing J DerAbstract:Expression of the tumor suppressor deleted in liver cancer-1 (DLC-1) is lost in non-small cell lung (NSCLC) and other human carcinomas, and ectopic DLC-1 expression dramatically reduces proliferation and tumorigenicity. DLC-1 is a multidomain protein that includes a Rho GTPase Activating Protein (RhoGAP) domain which has been hypothesized to be the basis of its tumor suppressive actions. To address the importance of the RhoGAP function of DLC-1 in tumor suppression, we performed biochemical and biological studies evaluating DLC-1 in NSCLC. Full length DLC-1 exhibited strong GAP activity for RhoA as well as RhoB and RhoC, but only very limited activity for Cdc42 in vitro. In contrast, the isolated RhoGAP domain showed 5- to 20-fold enhanced activity for RhoA, RhoB, RhoC and Cdc42. DLC-1 protein expression was absent in six of nine NSCLC cell lines. Restoration of DLC-1 expression in DLC-1-deficient NSCLC cell lines reduced RhoA activity, and experiments with a RhoA biosensor demonstrated that DLC-1 dramatically reduces RhoA activity at the leading edge of cellular protrusions. Furthermore, DLC-1 expression in NSCLC cell lines impaired both anchorage-dependent and -independent growth, as well as invasion in vitro. Surprisingly, we found that the anti-tumor activity of DLC-1 was due to both RhoGAP-dependent and -independent activities. Unlike the rat homologue p122RhoGAP, DLC-1 was not capable of activating the phospholipid hydrolysis activity of phospholipase C-δ1. Combined, these studies provide information on the mechanism of DLC-1 function and regulation, and further support the role of DLC-1 tumor suppression in NSCLC.
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dlc 1 suppresses non small cell lung cancer growth and invasion by RhoGap dependent and independent mechanisms
Molecular Carcinogenesis, 2008Co-Authors: Kevin D Healy, Louis Hodgson, Adam Shutes, Savitri Maddileti, Kendall T Harden, Tai Young Kim, Yungjue Bang, Klaus M. Hahn, Rudolph L. Juliano, Channing J DerAbstract:Expression of the tumor suppressor deleted in liver cancer-1 (DLC-1) is lost in non-small cell lung (NSCLC) and other human carcinomas, and ectopic DLC-1 expression dramatically reduces proliferation and tumorigenicity. DLC-1 is a multidomain protein that includes a Rho GTPase Activating Protein (RhoGAP) domain which has been hypothesized to be the basis of its tumor suppressive actions. To address the importance of the RhoGAP function of DLC-1 in tumor suppression, we performed biochemical and biological studies evaluating DLC-1 in NSCLC. Full length DLC-1 exhibited strong GAP activity for RhoA as well as RhoB and RhoC, but only very limited activity for Cdc42 in vitro. In contrast, the isolated RhoGAP domain showed 5- to 20-fold enhanced activity for RhoA, RhoB, RhoC and Cdc42. DLC-1 protein expression was absent in six of nine NSCLC cell lines. Restoration of DLC-1 expression in DLC-1-deficient NSCLC cell lines reduced RhoA activity, and experiments with a RhoA biosensor demonstrated that DLC-1 dramatically reduces RhoA activity at the leading edge of cellular protrusions. Furthermore, DLC-1 expression in NSCLC cell lines impaired both anchorage-dependent and -independent growth, as well as invasion in vitro. Surprisingly, we found that the anti-tumor activity of DLC-1 was due to both RhoGAP-dependent and -independent activities. Unlike the rat homologue p122RhoGAP, DLC-1 was not capable of activating the phospholipid hydrolysis activity of phospholipase C-δ1. Combined, these studies provide information on the mechanism of DLC-1 function and regulation, and further support the role of DLC-1 tumor suppression in NSCLC.
Kevin D Healy - One of the best experts on this subject based on the ideXlab platform.
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dlc 1 suppresses non small cell lung cancer growth and invasion by RhoGap dependent and independent mechanisms
Molecular Carcinogenesis, 2008Co-Authors: Kevin D Healy, Louis Hodgson, Adam Shutes, Savitri Maddileti, Kendall T Harden, Tai Young Kim, Yungjue Bang, Klaus M. Hahn, Rudolph L. Juliano, Channing J DerAbstract:Expression of the tumor suppressor deleted in liver cancer-1 (DLC-1) is lost in non-small cell lung (NSCLC) and other human carcinomas, and ectopic DLC-1 expression dramatically reduces proliferation and tumorigenicity. DLC-1 is a multidomain protein that includes a Rho GTPase Activating Protein (RhoGAP) domain which has been hypothesized to be the basis of its tumor suppressive actions. To address the importance of the RhoGAP function of DLC-1 in tumor suppression, we performed biochemical and biological studies evaluating DLC-1 in NSCLC. Full length DLC-1 exhibited strong GAP activity for RhoA as well as RhoB and RhoC, but only very limited activity for Cdc42 in vitro. In contrast, the isolated RhoGAP domain showed 5- to 20-fold enhanced activity for RhoA, RhoB, RhoC and Cdc42. DLC-1 protein expression was absent in six of nine NSCLC cell lines. Restoration of DLC-1 expression in DLC-1-deficient NSCLC cell lines reduced RhoA activity, and experiments with a RhoA biosensor demonstrated that DLC-1 dramatically reduces RhoA activity at the leading edge of cellular protrusions. Furthermore, DLC-1 expression in NSCLC cell lines impaired both anchorage-dependent and -independent growth, as well as invasion in vitro. Surprisingly, we found that the anti-tumor activity of DLC-1 was due to both RhoGAP-dependent and -independent activities. Unlike the rat homologue p122RhoGAP, DLC-1 was not capable of activating the phospholipid hydrolysis activity of phospholipase C-δ1. Combined, these studies provide information on the mechanism of DLC-1 function and regulation, and further support the role of DLC-1 tumor suppression in NSCLC.
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dlc 1 suppresses non small cell lung cancer growth and invasion by RhoGap dependent and independent mechanisms
Molecular Carcinogenesis, 2008Co-Authors: Kevin D Healy, Louis Hodgson, Adam Shutes, Savitri Maddileti, Kendall T Harden, Tai Young Kim, Yungjue Bang, Klaus M. Hahn, Rudolph L. Juliano, Channing J DerAbstract:Expression of the tumor suppressor deleted in liver cancer-1 (DLC-1) is lost in non-small cell lung (NSCLC) and other human carcinomas, and ectopic DLC-1 expression dramatically reduces proliferation and tumorigenicity. DLC-1 is a multidomain protein that includes a Rho GTPase Activating Protein (RhoGAP) domain which has been hypothesized to be the basis of its tumor suppressive actions. To address the importance of the RhoGAP function of DLC-1 in tumor suppression, we performed biochemical and biological studies evaluating DLC-1 in NSCLC. Full length DLC-1 exhibited strong GAP activity for RhoA as well as RhoB and RhoC, but only very limited activity for Cdc42 in vitro. In contrast, the isolated RhoGAP domain showed 5- to 20-fold enhanced activity for RhoA, RhoB, RhoC and Cdc42. DLC-1 protein expression was absent in six of nine NSCLC cell lines. Restoration of DLC-1 expression in DLC-1-deficient NSCLC cell lines reduced RhoA activity, and experiments with a RhoA biosensor demonstrated that DLC-1 dramatically reduces RhoA activity at the leading edge of cellular protrusions. Furthermore, DLC-1 expression in NSCLC cell lines impaired both anchorage-dependent and -independent growth, as well as invasion in vitro. Surprisingly, we found that the anti-tumor activity of DLC-1 was due to both RhoGAP-dependent and -independent activities. Unlike the rat homologue p122RhoGAP, DLC-1 was not capable of activating the phospholipid hydrolysis activity of phospholipase C-δ1. Combined, these studies provide information on the mechanism of DLC-1 function and regulation, and further support the role of DLC-1 tumor suppression in NSCLC.
Klaus Aktories - One of the best experts on this subject based on the ideXlab platform.
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specific role of rhoc in tumor invasion and metastasis
Oncotarget, 2017Co-Authors: Sarah Lang, Klaus Aktories, Hauke Busch, Melanie Boerries, Tilman Brummer, Sylvia Timme, Silke Lassmann, Gudula SchmidtAbstract:// Sarah Lang 1 , Hauke Busch 2, 3 , Melanie Boerries 3, 4 , Tilman Brummer 3, 4, 5 , Sylvia Timme 6 , Silke Lassmann 4, 5, 6 , Klaus Aktories 1 and Gudula Schmidt 1 1 Institute for Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, Albert-Ludwigs-University, Freiburg, Germany 2 Lubeck Institute of Experimental Dermatology, Institute for Cardiogenetics, University of Lubeck, Lubeck, Germany 3 Institute of Molecular Medicine and Cell Research, Faculty of Medicine, University of Freiburg, Freiburg, Germany 4 German Cancer Consortium (DKTK), Freiburg, Germany, German Cancer Research Center (DKFZ), Heidelberg, Germany 5 Center for Biological Signalling Studies (BIOSS), University of Freiburg, Freiburg, Germany 6 Institute for Surgical Pathology, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany Correspondence to: Gudula Schmidt, email: Gudula.Schmidt@pharmakol.uni-freiburg.de Keywords: Rho GTPase, invasion, cyclooxygenase, breast cancer Received: June 14, 2017 Accepted: August 26, 2017 Published: September 16, 2017 ABSTRACT Rho GTPases are regulators of many cellular functions and are often dysregulated in cancer. However, the precise role of Rho proteins for tumor development is not well understood. In breast cancer, overexpression of RhoC is linked with poor prognosis. Here, we aim to compare the function of RhoC and its homolog family member RhoA in breast cancer progression. We established stable breast epithelial cell lines with inducible expression of RhoA and RhoC, respectively. Moreover, we made use of Rho-activating bacterial toxins (Cytotoxic Necrotizing Factors) to stimulate the endogenous pool of Rho GTPases in benign breast epithelial cells and simultaneously knocked down specific Rho proteins. Whereas activation of Rho GTPases was sufficient to induce an invasive phenotype in three-dimensional culture systems, overexpression of RhoA or RhoC were not. However, RhoC but not RhoA was required for invasion, whereas RhoA and RhoC equally regulated proliferation. We further identified downstream target genes of RhoC involved in invasion and identified PTGS2 (COX-2) being preferentially upregulated by RhoC. Consistently, the COX-2 inhibitor Celecoxib blocked the invasive phenotype induced by the Rho-activating toxins.
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RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes
Molecular biology of the cell, 2011Co-Authors: Ben Jackson, Gudula Schmidt, Karine Peyrollier, Esben Pedersen, Astrid L. Basse, Richard Karlsson, Zhipeng Wang, Tine Lefever, Alexandra M. Ochsenbein, Klaus AktoriesAbstract: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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role of rho and rho kinase in the activation of volume regulated anion channels in bovine endothelial cells
The Journal of Physiology, 1999Co-Authors: Bernd Nilius, Klaus Aktories, Thomas Voets, Jean Prenen, Holger Barth, Kozo Kaibuchi, Guy Droogmans, Jan EggermontAbstract:In most mammalian cells, cell swelling activates a Cl− current (ICl,swell) through volume-regulated anion channels (VRACs). These channels are involved in cell volume regulation, electrogenesis, control of electrochemical gradients for ion channels and transporters and possibly in cell proliferation and differentiation (Strange et al. 1996; Nilius et al. 1996a, 1997a;Okada, 1997; Kirk, 1997; Kirk & Strange, 1998). The nature of the cellular volume sensor, as well as the transduction apparatus that controls the gating of VRACs, is unknown. The cell swelling-induced activation of VRACs requires one or more tyrosine phosphorylation steps, since inhibitors of protein tyrosine kinases inhibit ICl,swell whereas inhibitors of protein tyrosine phosphatases potentiate ICl,swell (Tilly et al. 1993; Voets et al. 1998). It was recently shown that p56lck, a member of the src protein tyrosine kinase family, mediates activation of ICl,swell in lymphocytes (Lepple-Wienhues et al. 1998). We have previously shown that ICl,swell is also activated by reducing the intracellular ionic strength (Γi) (Nilius et al. 1998) or by intracellular perfusion with GTPγS (Voets et al. 1998). How reduced Γi or intracellular GTPγS activate VRACs is unknown, but similar to the swelling-induced activation one or more tyrosine phosphorylation events are involved. Furthermore, the Γi- and GTPγS-activated Cl− currents are still sensitive to changes in cell volume, as indicated by the blocking effect of cell shrinkage induced by extracellular hypertonicity (Voets et al. 1998; Nilius et al. 1998). The GTPγS effect suggests a crucial role of GTP-binding proteins in the activation of VRACs. Rho (or p21rho) is a monomeric 21 kDa GTPase of which there are three mammalian isoforms (RhoA, RhoB and RhoC) and which, together with Rac, Cdc42, RhoD, RhoE, RhoG and TC10, form the Rho GTPase family (Mackay & Hall, 1998). Rho proteins are involved in the organization of the actin cytoskeleton, in the formation of stress fibres, in exo/endocytosis and in formation of focal adhesions (Ridley, 1996; Symons, 1996). It has recently been shown that inactivation of Rho by pretreatment with the Clostridium botulinum C3 exoenzyme greatly reduced the swelling-induced efflux of iodide in human Intestine 407 cells (Tilly et al. 1996). A signalling cascade whereby activation of Rho results in the activation of p125FAK (a protein tyrosine kinase localized to focal adhesion contacts) and phosphatidylinositol-3-kinase was suggested to be critically involved in the activation of VRACs (Tilly et al. 1996). However, several alternative signalling pathways downstream of Rho have been described, among which are protein kinases that are activated by binding to GTP-bound Rho (Ridley, 1996; Tapon & Hall, 1997). A subclass of these Rho-activated protein kinases is formed by Rho kinase which is a serine/threonine protein kinase with structural homology to the myotonic dystrophy kinase (Matsui et al. 1996). Two distinct isoforms (Rho Kinase/ROKα/ROCK-II and p160ROCK/ROKβ/ROCK-I) with approximately 90 % identity in their kinase domain have been identified (Leung et al. 1996; Narumiya et al. 1997). Rho-dependent activation of Rho kinase promotes the formation of focal adhesions and actin stress fibres in fibroblasts and HeLa cells and increases the Ca2+ sensitivity of the contractile apparatus in smooth muscle (Narumiya et al. 1997). In this study we have investigated the role of the Rho-Rho kinase signalling cascade in the activation of ICl,swell in calf pulmonary artery endothelial (CPAE) cells. We show that inhibiting Rho or Rho kinase results in an impaired activation of ICl,swell by either cell swelling or GTPγS. We therefore conclude that in CPAE cells the Rho-Rho kinase pathway modulates the gating process of VRACs.
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The Ras-related small GTP-binding protein RhoB is immediate-early inducible by DNA damaging treatments
Journal of Biological Chemistry, 1995Co-Authors: G Fritz, Bernd Kaina, Klaus AktoriesAbstract:The low molecular weight GTP-binding proteins RhoA, RhoB, and RhoC are characterized as specific substrates for the ADP-ribosyltransferase C3 from Clostridium botulinum and are supposed to he involved in the organization of the microfilamental network and transformation. rhoB is known to be immediate-early inducible by growth factors and protein-tyrosine kinases. Since increasing evidence indicates overlapping of growth factor- and UV- induced signal pathways, we studied the effect of UV light and other genotoxic agents on early rhoB transcription. Within 30 min after UV irradiation of NIH3T3 cells, the amount of rhoB mRNA increased 3-4-fold. Elevated rhoB mRNA was accompanied by an increase in RhoB protein, as detected by C3-mediated [32P]ADP-ribosylation. The transcription inhibitor actinomycin D prevented the UV-induced increase in rhoB mRNA and proved rhoB mRNA to be unstable with a half-life of 20 min. Transcriptional activation of rhoB by UV light was confirmed by run-on analysis. The increase in rhoB mRNA after UV irradiation was prevented by inhibitors of protein kinase A (H9) and C (H7, Go18). The tyrosine kinase inhibitor genistein did not affect UV induction of rhoB. In addition to UV, N-methyl-N-nitrosourea and the cytostatic drug cisplatin evoked rhoB response. Cycloheximide was likewise effective in increasing the amount of rhoB mRNA, whereas Bt2cAMP, 12-O- tetradecanoylphorbol-13-acetate, and retinoic acid were without effect. Prior down-regulation of signaling by 12-O-tetradecanoyl-phorhol-13-acetate and serum pretreatment reduced UV-stimulated rhoB expression. The data indicate that rhoB represents a novel DNA damage-inducible function involved in early steps of signal transduction upon genotoxic stress.