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Biomedical subjects

M Symons

Publications and source records attributed to M Symons.

At least 19 recordsLinked to original sources
PubMed2001

Rac1 protects epithelial cells against anoikis.

Rho family members play a critical role in malignant transformation. Anchorage-independent growth and the ability to avoid apoptosis caused by loss of anchorage (anoikis) are important features of transformed cells. Here we show that constitutive activation of Rac1 inhibits anoikis in Madin-Darby canine kidney (MDCK) epithelial cells. Constitutively active Rac1-V12 decreases DNA fragmentation and caspase activity by 50% in MDCK cells kept in suspension. In addition, expression of Rac1-V12 in MDCK cells in suspension conditions causes an increase in the number of surviving cells. We also investigated the signaling pathways that are activated by Rac1 to stimulate cell survival. We show that expression of Rac1-V12 in MDCK cells in suspension stimulates a number of signaling cascades that have been implicated in the control of cell survival, including the p42/44 ERK, p38, protein kinase B, and nuclear factor kappaB pathways. Using specific chemical or protein inhibitors of these respective pathways, we show that Rac1-mediated cell survival strongly depends on phosphatidylinositol 3-kinase activity and that activation of ERK, p38, and NF-kappaB are largely dispensable for Rac1 survival signaling. In conclusion, these studies demonstrate that Rac1 can suppress apoptosis in epithelial cells in anchorage-independent conditions and suggest a potential role for Rac1-mediated survival signaling in cell transformation.

Animals↗
PubMed2001

The thrombin receptor, PAR-1, causes transformation by activation of Rho-mediated signaling pathways.

We utilized a cDNA expression library derived from the B6SutA(1) mouse myeloid progenitor cell line to search for novel oncogenes that promote growth transformation of NIH3T3 cells. A 2.2 kb transforming cDNA was recovered that encodes the wild type thrombin-stimulated G protein-coupled receptor PAR-1. In addition to its potent focus forming activity, constitutive overexpression of PAR-1 in NIH3T3 cells promoted the loss of anchorage- and serum-dependent growth. Although inhibitors of thrombin failed to block PAR-1 transforming activity, a PAR-1 mutant that cannot be cleaved by thrombin was nontransforming. Since the foci of transformed cells induced by PAR-1 bear a striking resemblance to those induced by activated RhoA, we determined if PAR-1 transformation was due to the aberrant activation of a specific Rho family member. Like RhoA, PAR-1 cooperated with activated Raf-1 and caused synergistic enhancement of transforming activity, induced stress fibers when microinjected into porcine aortic endothelial cells, stimulated the activity of the serum response factor and NF-kappaB transcription factors, and PAR-1 transformation was blocked by co-expression of dominant negative RhoA. Finally, PAR-1 transforming activity was blocked by pertussis toxin and by co-expression of the RGS domain of Lsc, implicating Galpha(i) and Galpha(12)/Galpha(13) subunits, respectively, as mediators of PAR-1 transformation. Taken together, these observations suggest that PAR-1 growth transformation is mediated, in part, by activation of RhoA.

3T3 Cells↗
PubMed2001

Ras GTPases: singing in tune.

A review of the meeting "The Ras Superfamily of Small GTP-Binding Proteins," FASEB Summer Research Conference, Snowmass, Colorado, 15 through 20 July 2000 The molecular cloning of the human proto-oncogene encoding Ras was reported nearly 20 years ago. Since then, Ras has become the prototypical member of a superfamily of small guanosine triphosphatase proteins. Despite the maturity of this field of research, the discovery of new functions and interactions between the superfamily members continues unabated. Symons and Takai have written a meeting report on the latest findings on the Ras superfamily.

Animals↗
PubMed2001

The small GTPase Rac suppresses apoptosis caused by serum deprivation in fibroblasts.

BACKGROUND: The small GTPase Rac1 is a key signaling protein that mediates a number of important physiologic functions including the organization of the actin cytoskeleton, lipid metabolism, and gene transcription. Rac1 has also been implicated in oncogenic transformation. Expression of constitutively active Rac1 in Rat1 fibroblasts elicits serum- and anchorage-independent growth and causes tumorigenicity in nude mice. The signaling pathways that mediate the role of Rac in cell transformation remain to be identified. Here, we study the role of Rac in cell survival in the absence of serum. MATERIALS AND METHODS: The cell lines used in this study are Ratl fibroblasts that express constitutively active or dominant negative mutants of Rac1. We used long-term video time-lapse microscopy to analyze the effects of these Rac1 mutants on mitogenicity and apoptosis. RESULTS: We show that the increase in viability, which is stimulated by Rac1 in the absence of serum, is predominantly caused by an inhibition of apoptosis, with a minor increase in cell division. We also show that Rac1-stimulated cell viability in serum-starved cells is inhibited by chemical inhibition of phosphatidylinositol 3-kinase. CONCLUSIONS: Our observations indicate a role for Rac1 in survival signaling, possibly via activation of phosphatidylinositol 3-kinase. We propose that Rac1-stimulated cell survival may contribute to the role of Rac1 in serum-independent growth and cell transformation.

Animals↗
PubMed2000

Synaptojanin 2, a novel Rac1 effector that regulates clathrin-mediated endocytosis.

The small GTPase Rac has been implicated in a wide range of cellular processes, including the organization of the actin cytoskeleton, transcriptional control and endocytic vesicle trafficking [1-3]. The signaling components that mediate these functions downstream of Rac largely remain to be identified. In this study, we have identified synaptojanin 2, a polyphosphoinositide phosphatase as a novel Rac1 effector. Synaptojanin 2 directly and specifically interacts with Rac1 in a GTP-dependent manner. Expression of constitutively active Rac1 caused the translocation of synaptojanin 2 from the cytoplasm to the plasma membrane. Both activated Rac1 and a membrane-targeted version of synaptojanin 2 inhibited endocytosis of the epidermal growth factor (EGF) and transferrin receptors, a process that is known to be dependent on polyphosphoinositide lipids. Endocytosis of growth factor receptors is thought to play an important role in the regulation of cell proliferation. Thus, these results suggest that synaptojanin 2 may mediate the inhibitory effect of Rac1 on endocytosis and could contribute to Rac1-mediated control of cell growth.

Actins↗
PubMed2000

Regulation of Tiam1 nucleotide exchange activity by pleckstrin domain binding ligands.

Rho family GTPases play roles in cytoskeletal organization and cellular transformation. Tiam1 is a member of the Dbl family of guanine nucleotide exchange factors that activate Rho family GTPases. These exchange factors have in common a catalytic Dbl homology and adjacent pleckstrin homology domain. Previous structural studies suggest that the pleckstrin domain, a putative phosphoinositide-binding site, may serve a regulatory function. We identified ascorbyl stearate as a compound that binds to the pleckstrin domain of p120 Ras GTPase-activating protein. Furthermore, ascorbyl stearate appears to be a general pleckstrin domain ligand, perhaps by mimicking an endogenous amphiphilic ligand. Tiam1 nucleotide exchange activity was greatly stimulated by ascorbyl stearate. Certain phosphoinositides also stimulated Tiam1 activity but were less potent than ascorbyl stearate. Tiam1 contains an additional N-terminal pleckstrin domain, but only the C-terminal pleckstrin domain was required for activation. Our results suggest that the pleckstrin domains of Dbl-type proteins may not only be involved in subcellular localization but may also directly regulate the nucleotide exchange activity of an associated Dbl homology domain. In addition, this paper introduces ascorbyl stearate as a pleckstrin domain ligand that can modulate the activity of certain pleckstrin domain-containing proteins.

Animals↗
PubMed2000

G2A is an oncogenic G protein-coupled receptor.

G2A is a heptahelical cell surface protein that has recently been described as a potential tumor suppressor, based on its ability to counteract transformation of pre-B cells and fibroblasts by Bcr-Abl, an oncogenic tyrosine kinase. We have isolated cDNAs encoding G2A in the course of screening libraries for clones that cause oncogenic transformation of NIH3T3 fibroblasts. When expressed at high levels in NIH3T3 cells by retroviral transduction, G2A induced a full range of phenotypes characteristic of oncogenic transformation, including loss of contact inhibition, anchorage-independent survival and proliferation, reduced dependence on serum, and tumorigenicity in mice. When expressed by transfection, G2A greatly enhanced the ability of a weakly oncogenic form of Raf-1 to transform NIH3T3 cells. These results demonstrate that G2A is potently oncogenic both on its own and in cooperation with another oncogene. Expression of G2A in fibroblasts and endothelial cells resulted in changes in cell morphology and cytoskeleton structure that were equivalent to those induced by the G protein subunit Galpha13. Transformation of NIH3T3 cells via G2A expression was completely suppressed by co-expression of LscRGS, a GTPase activating protein that suppresses signaling by Galpha12 and Galpha13. Hyperactivity of Galpha12 or Galpha13 has previously been shown to result in activation of Rho GTPases. G2A expression resulted in activation of Rho, and transformation via G2A was suppressed by a dominant negative form of RhoA. These results indicate that G2A may be directly coupled to Galpha13, and that it is the activation of this Rho-activating Galpha protein which is responsible for the ability of G2A to transform fibroblasts.

3T3 Cells↗
PubMed2000

Adhesion signaling: PAK meets Rac on solid ground.

Interaction of cells with the extracellular matrix influences various aspects of cellular behavior. A recent study shows that cell-substrate adhesion is necessary for effective coupling of the small GTPase Rac to its effector PAK.

Animals↗
PubMed2000

Motility and invasion are differentially modulated by Rho family GTPases.

Cell migration in vivo often requires invasion through tissue matrices. Currently little is known regarding the signaling pathways that regulate cell invasion through three-dimensional matrices. The small GTPases Cdc42, Rac and Rho are key regulators of actin cytoskeletal and adhesive structures. We now show that expression of dominant negative forms of either Cdc42, Rac or Rho inhibited PDGF-BB-stimulated Rat1 fibroblast invasion into 3D collagen matrices, indicating that the activity of each of these GTPases is necessary for cell invasion. In contrast, only Rac activation was required for PDGF-BB-stimulated locomotion across a planar substrate in the Boyden chamber. Interestingly, PDGF-induced invasion was also strongly inhibited by expression of constitutively active forms of Cdc42 or Rho, and to a lesser extent by constitutively active Rac. We also show that constitutively active V12-Rac significantly stimulated basal Rat1 fibroblast invasion, independent of PI-3-kinase activity, and that this effect was suppressed by the effector mutant V12/H40-Rac. These results suggest that cellular invasion may require an optimal level of activation of Cdc42, Rho and Rac, and that migration and invasion are differentially modulated by Rho family GTPases.

Animals↗
PubMed2000

Rho family GTPases: more than simple switches.

Rho family GTPases control a large variety of biological processes. Cycling of Rho proteins between the GDP-bound and the GTP-bound state is controlled by several classes of regulatory proteins. In this review, we discuss the signal-transduction mechanisms that control these regulators. We will emphasize the subcellular localization of Rho GTPases and their regulatory proteins and the role of GTP hydrolysis in signal transmission.

Guanosine Diphosphate↗
PubMed1999

Fruit and vegetable consumption and prevention of cancer: the Black Churches United for Better Health project.

OBJECTIVES: This study assessed the effects of the Black Churches United for Better Health project on increasing fruit and vegetable consumption among rural African American church members in North Carolina. METHODS: Ten counties comprising 50 churches were pair matched and randomly assigned to either intervention or delayed intervention (no program until after the follow-up survey) conditions. A multicomponent intervention was conducted over approximately 20 months. A total of 2519 adults (77.3% response rate) completed both the baseline and 2-year follow-up interviews. RESULTS: The 2 study groups consumed similar amounts of fruits and vegetables at baseline. AT the 2-year follow-up, the intervention group consumed 0.85 (SE = 0.12) servings more than the delayed intervention group (P < .0001). The largest increases were observed among people 66 years or older (1 serving), those with education beyond high school (0.92 servings), those widowed or divorced (0.96 servings), and those attending church frequently (1.3 servings). The last improvement occurred among those aged 18 to 37 years and those who were single. CONCLUSIONS: The project was a successful model for achieving dietary change among rural African Americans.

Adolescent↗
PubMed1998

The transcription factor AP-1 is required for EGF-induced activation of rho-like GTPases, cytoskeletal rearrangements, motility, and in vitro invasion of A431 cells.

Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun, TAM67, suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, we establish that sustained TAM67 expression inhibits growth factor-induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion.

Actins↗
PubMed1998

Integrin-mediated signals regulated by members of the rho family of GTPases.

The organization of the actin cytoskeleton can be regulated by soluble factors that trigger signal transduction events involving the Rho family of GTPases. Since adhesive interactions are also capable of organizing the actin-based cytoskeleton, we examined the role of Cdc42-, Rac-, and Rho-dependent signaling pathways in regulating the cytoskeleton during integrin-mediated adhesion and cell spreading using dominant-inhibitory mutants of these GTPases. When Rat1 cells initially adhere to the extracellular matrix protein fibronectin, punctate focal complexes form at the cell periphery. Concomitant with focal complex formation, we observed some phosphorylation of the focal adhesion kinase (FAK) and Src, which occurred independently of Rho family GTPases. However, subsequent phosphorylation of FAK and paxillin occurs in a Rho-dependent manner. Moreover, we found Rho dependence of the assembly of large focal adhesions from which actin stress fibers radiate. Initial adhesion to fibronectin also stimulates membrane ruffling; we show that this ruffling is independent of Rho but is dependent on both Cdc42 and Rac. Furthermore, we observed that Cdc42 controls the integrin-dependent activation of extracellular signal-regulated kinase 2 and of Akt, a kinase whose activity has been demonstrated to be dependent on phosphatidylinositol (PI) 3-kinase. Since Rac-dependent membrane ruffling can be stimulated by PI 3-kinase, it appears that Cdc42, PI 3-kinase, and Rac lie on a distinct pathway that regulates adhesion-induced membrane ruffling. In contrast to the differential regulation of integrin-mediated signaling by Cdc42, Rac, and Rho, we observed that all three GTPases regulate cell spreading, an event that may indirectly control cellular architecture. Therefore, several separable signaling pathways regulated by different members of the Rho family of GTPases converge to control adhesion-dependent changes in the organization of the cytoskeleton, changes that regulate cell morphology and behavior.

Actins↗
PubMed1998

Transforming potential of Dbl family proteins correlates with transcription from the cyclin D1 promoter but not with activation of Jun NH2-terminal kinase, p38/Mpk2, serum response factor, or c-Jun.

The dbl family of oncogenes encodes a large, structurally related, family of growth-regulatory molecules that possess guanine nucleotide exchange factor activity for specific members of the Rho family of Ras-related GTPases. We have evaluated matched sets of weakly and strongly transforming versions of five Dbl family proteins (Lfc, Lsc, Ect2, Dbl, and Dbs) to determine their ability to stimulate signaling pathways that are activated by Rho family proteins. We found that the transforming potential of this panel did not correlate directly with their ability to activate Jun NH2-terminal kinase, p38/Mpk2, serum response factor, or c-Jun. In contrast, transient stimulation of transcription from the cyclin D1 promoter provided a strong correlation with transforming potential, and we found constitutive up-regulation of cyclin D1 protein in Dbl family protein-transformed cells. In addition, we observed that at least two Dbl family members (Lfc and Ect2) induced changes in the actin cytoskeleton and exhibited nuclear signaling profiles that are consistent with a broader range of in vivo substrate utilization than is predicted from their in vitro exchange specificities. In summary, although Dbl family proteins exhibit signaling profiles that are consistent with their in vivo activation of Rho proteins, stimulation of cyclin D1 transcription is the only activity that correlates with transforming potential, thus suggesting that deregulated cell cycle progression may be important for Dbl family protein transformation.

3T3 Cells↗
PubMed1998

Role of Rac1 and oxygen radicals in collagenase-1 expression induced by cell shape change.

Integrin-mediated reorganization of cell shape leads to an altered cellular phenotype. Disruption of the actin cytoskeleton, initiated by binding of soluble antibody to alpha5beta1 integrin, led to increased expression of the collagenase-1 gene in rabbit synovial fibroblasts. Activation of the guanosine triphosphate-binding protein Rac1, which was downstream of the integrin, was necessary for this process, and expression of activated Rac1 was sufficient to increase expression of collagenase-1. Rac1 activation generated reactive oxygen species that were essential for nuclear factor kappa B-dependent transcriptional regulation of interleukin-1alpha, which, in an autocrine manner, induced collagenase-1 gene expression. Remodeling of the extracellular matrix and consequent alterations of integrin-mediated adhesion and cytoarchitecture are central to development, wound healing, inflammation, and malignant disease. The resulting activation of Rac1 may lead to altered gene regulation and alterations in cellular morphogenesis, migration, and invasion.

Animals↗
PubMed1998

Mas oncogene signaling and transformation require the small GTP-binding protein Rac.

The Mas oncogene encodes a novel G-protein-coupled receptor that was identified originally as a transforming protein when overexpressed in NIH 3T3 cells. The mechanism and signaling pathways that mediate Mas transformation have not been determined. We observed that the foci of transformed NIH 3T3 cells caused by Mas were similar to those caused by activated Rho and Rac proteins. Therefore, we determined if Mas signaling and transformation are mediated through activation of a specific Rho family protein. First, we observed that, like activated Rac1, Mas cooperated with activated Raf and caused synergistic transformation of NIH 3T3 cells. Second, both Mas- and Rac1-transformed NIH 3T3 cells retained actin stress fibers and showed enhanced membrane ruffling. Third, like Rac, Mas induced lamellipodium formation in porcine aortic endothelial cells. Fourth, Mas and Rac1 strongly activated the JNK and p38, but not ERK, mitogen-activated protein kinases. Fifth, Mas and Rac1 stimulated transcription from common DNA promoter elements: NF-kappaB, serum response factor (SRF), Jun/ATF-2, and the cyclin D1 promoter. Finally, Mas transformation and some of Mas signaling (SRF and cyclin D1 but not NF-kappaB activation) were blocked by dominant negative Rac1. Taken together, these observations suggest that Mas transformation is mediated in part by activation of Rac-dependent signaling pathways. Thus, Rho family proteins are common mediators of transformation by a diverse variety of oncogene proteins that include Ras, Dbl family, and G-protein-coupled oncogene proteins.

3T3 Cells↗