guanosine-triphosphate and geranylgeranyl-pyrophosphate

Small GTPases (guanosine triphosphate, GTP) are involved in many critical cellular processes, including inflammation, proliferation, and migration. GTP loading and isoprenylation are two important post-translational modifications of small GTPases, and are critical for their normal function. In this study, we investigated the role of post-translational modifications of small GTPases in regulating endothelial cell inflammatory responses and junctional integrity.. Confluent human umbilical vein endothelial cell (HUVECs ) treated with atorvastatin demonstrated significantly decreased lipopolysaccharide (LPS)-mediated IL-6 and IL-8 generation. The inhibitory effect of atorvastatin (Atorva) was attenuated by co-treatment with 100 µM mevalonate (MVA) or 10 µM geranylgeranyl pyrophosphate (GGPP), but not by 10 µM farnesyl pyrophosphate (FPP). Atorvastatin treatment of HUVECs produced a time-dependent increase in GTP loading of all Rho GTPases, and induced the translocation of small Rho GTPases from the cellular membrane to the cytosol, which was reversed by 100 µM MVA and 10 µM GGPP, but not by 10 µM FPP. Atorvastatin significantly attenuated thrombin-induced HUVECs permeability, increased VE-cadherin targeting to cell junctions, and preserved junction integrity. These effects were partially reversed by GGPP but not by FPP, indicating that geranylgeranylation of small GTPases plays a major role in regulating endothelial junction integrity. Silencing of small GTPases showed that Rho and Rac, but not Cdc42, play central role in HUVECs junction integrity.. In conclusion, our studies show that post-translational modification of small GTPases plays a vital role in regulating endothelial inflammatory response and endothelial junction integrity. Atorvastatin increased GTP loading and inhibited isoprenylation of small GTPases, accompanied by reduced inflammatory response and preserved cellular junction integrity.

Topics: Antigens, CD; Atorvastatin; Cadherins; cdc42 GTP-Binding Protein; Guanosine Triphosphate; Heptanoic Acids; Human Umbilical Vein Endothelial Cells; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Intercellular Junctions; Interleukin-6; Interleukin-8; Lipopolysaccharides; Mevalonic Acid; Polyisoprenyl Phosphates; Prenylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Pyrroles; rho-Associated Kinases; Sesquiterpenes; Thrombin

The objective of this study was to determine whether geranylgeranyl diphosphate synthase inhibition, and therefore geranylgeranyl diphosphate depletion, interferes with breast cancer cell migration. Digeranyl bisphosphonate is a specific geranylgeranyl diphosphate synthase inhibitor. We demonstrate that digeranyl bisphosphonate depleted geranylgeranyl diphosphate and inhibited protein geranylgeranylation in MDA-MB-231 cells. Similar to GGTI-286, a GGTase I inhibitor, digeranyl bisphosphate significantly inhibited migration of MDA-MB-231 cells as measured by transwell assay. Similarly, digeranyl bisphosphonate reduced motility of MDA-MB-231 cells in a time-dependent manner as measured by large scale digital cell analysis system microscopy. Digeranyl bisphosphonate was mildly toxic and did not induce apoptosis. Treatment of MDA-MB-231 cells with digeranyl bisphosphonate decreased membrane while it increased cytosolic RhoA localization. In addition, digeranyl bisphosphonate increased RhoA GTP binding in MDA-MB-231 cells. The specificity of geranylgeranyl diphosphonate synthase inhibition by digeranyl bisphosphonate was confirmed by exogenous addition of geranylgeranyl diphosphate. Geranylgeranyl diphosphate addition prevented the effects of digeranyl bisphosphonate on migration, RhoA localization, and GTP binding to RhoA in MDA-MB-231 cells. These studies suggest that geranylgeranyl diphosphate synthase inhibitors are a novel approach to interfere with cancer cell migration.

Topics: Biosynthetic Pathways; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Diphosphonates; Female; Guanosine Triphosphate; Humans; Polyisoprenyl Phosphates; Protein Binding; Protein Transport; rhoA GTP-Binding Protein; Terpenes

Rho guanosine triphosphatases (GTPases) orchestrate signaling pathways leading to cell migration. They are typically responsible for the organization of actin filaments that support actomyosin contractility and cell-body translocation. The function of Rho GTPases depends on GTP-loading and isoprenylation by geranylgeranyl pyrophosphate (GGpp). The latter posttranslational modification may be manipulated by agents such as 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (HMGCRIs) that prevent de novo synthesis of isoprenoids such as GGpp. HMGCRIs have anti-inflammatory properties and substantially reduce infiltration of inflammatory immune cells into target tissues, including the central nervous system (CNS) during neuroinflammation. The depletion of the cellular isoprenoid pool is believed to result in the regulation of antigen-specific T cells outside the target organ and also to prevent migration of these cells into target organs, such as the CNS. In vivo treatment with HMGCRI in the experimental autoimmune encephalitis (EAE) rodent model of multiple sclerosis reduces the capacity of activated T cells to traffic to and within the brain. This presentation shows that geranylgeranylation is fundamental for RhoA-mediated downstream events such as influencing cytoskeletal organization and the migration of T cells. Tethering of RhoA to the membrane by GGpp is necessary for T cell migration and provides a mechanism by which HMGCRI may prevent T cell infiltration into inflamed compartments.

Topics: Animals; Brain; Cell Membrane; Cell Movement; Cytoskeleton; Encephalomyelitis, Autoimmune, Experimental; Guanosine Triphosphate; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Inflammation; Lymphocyte Activation; Mice; Multiple Sclerosis; Polyisoprenyl Phosphates; Protein Prenylation; rho GTP-Binding Proteins; rhoA GTP-Binding Protein; T-Lymphocytes

Rho GTPases orchestrate signaling pathways leading to cell migration. Their function depends on GTP loading and isoprenylation by geranylgeranyl pyrophosphate (GGpp). In this study, we show that in human T cells, geranylgeranylation-and not GTP loading-is necessary for RhoA-mediated downstream events. As a result of GGpp depletion with the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor atorvastatin, RhoA was sequestered from the membrane to the cytosol and, notwithstanding increased GTP loading, the constitutive activation of its substrate Rho-associated coiled-coil protein kinase-1 was blocked. In line with this, T cells expressing increased GTP-RhoA failed to form an intact cytoskeleton and to migrate toward a chemokine gradient. In vivo treatment with atorvastatin in the rodent model of multiple sclerosis markedly decreased the capacity of activated T cells to traffic within the brain, as demonstrated by multiphoton analysis. Thus, tethering of RhoA to the membrane by GGpp is determinant for T cell migration and provides a mechanism for preventing T cell infiltration into inflamed compartments by 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors.

Topics: Animals; Apoptosis; Brain; Caspase 3; Cell Line; Cell Movement; Cytoskeleton; Cytosol; Enzyme Activation; Guanosine Triphosphate; Humans; Mice; Phenalenes; Polyisoprenyl Phosphates; Prenylation; Protein Binding; rho-Associated Kinases; rhoA GTP-Binding Protein; T-Lymphocytes

A subset of nonsteroidal anti-inflammatory drugs (NSAIDs) has been shown to preferentially reduce the secretion of the highly amyloidogenic, 42-residue amyloid-beta peptide Abeta42. We found that Rho and its effector, Rho-associated kinase, preferentially regulated the amount of Abeta42 produced in vitro and that only those NSAIDs effective as Rho inhibitors lowered Abeta42. Administration of Y-27632, a selective Rock inhibitor, also preferentially lowered brain levels of Abeta42 in a transgenic mouse model of Alzheimer's disease. Thus, the Rho-Rock pathway may regulate amyloid precursor protein processing, and a subset of NSAIDs can reduce Abeta42 through inhibition of Rho activity.

Topics: Amides; Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspartic Acid Endopeptidases; Brain; Cell Line, Tumor; Endopeptidases; Enzyme Inhibitors; Guanosine Triphosphate; Humans; Ibuprofen; Intracellular Signaling Peptides and Proteins; Mice; Mice, Transgenic; Peptide Fragments; Polyisoprenyl Phosphates; Protein Serine-Threonine Kinases; Pyridines; rho GTP-Binding Proteins; rho-Associated Kinases; rhoA GTP-Binding Protein; Sesquiterpenes; Signal Transduction; Sulindac; Transfection

Lysophosphatidylcholine (LPC) is known to increase intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells. This study was conducted to investigate the effects of HMG-CoA reductase inhibitors (statins) on the increase in [Ca2+]i and membrane current induced by LPC.. [Ca2+]i was determined in cultured human aortic endothelial cells by fura-2 assay, and membrane current was measured by whole-cell patch clamp. The [Ca2+]i increase induced by LPC was abolished by inhibitors of phospholipase C (PLC). Statins markedly decreased the [Ca2+]i increase caused by LPC. This suppressive effect was quickly reversed by geranylgeranylpyrophosphate (GGPP) and was mimicked by inhibitors of Rho and Rho kinase. LPC induced the translocation of the GTP-bound active form of RhoA into membranes within 1 minute as determined by a pull-down assay and reduced the levels of RhoA in the cytoplasm, indicating that LPC quickly increases the GTP/GDP ratio of RhoA and induces membrane translocation. Statins prevented the GTP/GDP exchange of RhoA and its membrane translocation from the cytoplasm caused by LPC, and these effects of statins were reversed by GGPP. The responses of RhoA activation to statins and GGPP concurred with their effects on Ca2+ mobilization. LPC also induced a nonselective cation current after a lag. Statins prolonged the lag and decreased the current amplitude, and GGPP abolished the inhibitory effect on the current.. LPC induced Ca2+ mobilization and membrane current via a Rho activation-dependent PLC pathway in endothelial cells, and statins blocked these effects by preventing the GGPP-dependent lipid modification of Rho. The present study implicates Rho in LPC stimulation of Ca2+ movement.

Topics: ADP Ribose Transferases; Amides; Aorta; Botulinum Toxins; Calcium; Cells, Cultured; Endothelium, Vascular; Enzyme Inhibitors; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Intracellular Fluid; Intracellular Signaling Peptides and Proteins; Lysophosphatidylcholines; Membrane Potentials; Mevalonic Acid; Patch-Clamp Techniques; Polyisoprenyl Phosphates; Protein Serine-Threonine Kinases; Pyridines; rho-Associated Kinases; rhoA GTP-Binding Protein; Signal Transduction; Type C Phospholipases

Prostate cancer cells derived from transgenic mice with adenocarcinoma of the prostate (TRAMP cells) were treated with the HMG-CoA reductase inhibitor, lovastatin. This caused inactivation of the small GTPase RhoA, actin stress fiber disassembly, cell rounding, growth arrest in the G1 phase of the cell cycle, cell detachment and apoptosis. Addition of geranylgeraniol (GGOL) in the presence of lovastatin, to stimulate protein geranylgeranylation, prevented lovastatin's effects. That is, RhoA was activated, actin stress fibers were assembled, the cells assumed a flat morphology and cell growth resumed. The following observations support an essential role for RhoA in TRAMP cell growth: (1) TRAMP cells expressing dominant-negative RhoA (T19N) mutant protein displayed few actin stress fibers and grew at a slower rate than controls (35 h doubling time for cells expressing RhoA (T19N) vs 20 h for untransfected cells); (2) TRAMP cells expressing constitutively active RhoA (Q63L) mutant protein displayed a contractile phenotype and grew faster than controls (13 h doubling time). Interestingly, addition of farnesol (FOL) with lovastatin, to stimulate protein farnesylation, prevented lovastatin-induced cell rounding, cell detachment and apoptosis, and stimulated cell spreading to a spindle shaped morphology. However, RhoA remained inactive and growth arrest persisted. The morphological effects of FOL addition were prevented in TRAMP cells expressing dominant-negative H-Ras (T17N) mutant protein. Thus, it appears that H-Ras is capable of inducing cell spreading, but incapable of supporting cell proliferation, in the absence of geranylgeranylated proteins like RhoA.

Topics: Actin Cytoskeleton; Adenocarcinoma; Alkyl and Aryl Transferases; Animals; Antineoplastic Agents; Apoptosis; Cell Adhesion; Cell Division; Cell Size; Diterpenes; Drug Interactions; Enzyme Activation; Farnesol; G1 Phase; Genes, ras; GTP-Binding Proteins; Guanosine Triphosphate; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin; Male; Mevalonic Acid; Mice; Mice, Transgenic; Polyisoprenyl Phosphates; Prostatic Neoplasms; Protein Prenylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins p21(ras); rac GTP-Binding Proteins; rhoA GTP-Binding Protein; Sesquiterpenes; Tumor Cells, Cultured

The mechanism by which 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors increase endothelial nitric oxide synthase (eNOS) expression is unknown. To determine whether changes in isoprenoid synthesis affects eNOS expression, human endothelial cells were treated with the HMG-CoA reductase inhibitor, mevastatin (1-10 microM), in the presence of L-mevalonate (200 microM), geranylgeranylpyrophosphate (GGPP, 1-10 microM), farnesylpyrophosphate (FPP, 5-10 microM), or low density lipoprotein (LDL, 1 mg/ml). Mevastatin increased eNOS mRNA and protein levels by 305 +/- 15% and 180 +/- 11%, respectively. Co-treatment with L-mevalonate or GGPP, but not FPP or LDL, reversed mevastatin's effects. Because Rho GTPases undergo geranylgeranyl modification, we investigated whether Rho regulates eNOS expression. Immunoblot analyses and [35S]GTPgammaS-binding assays revealed that mevastatin inhibited Rho membrane translocation and GTP binding activity by 60 +/- 5% and 78 +/- 6%, both of which were reversed by co-treatment with GGPP but not FPP. Furthermore, inhibition of Rho by Clostridium botulinum C3 transferase (50 microg/ml) or by overexpression of a dominant-negative N19RhoA mutant increased eNOS expression. In contrast, activation of Rho by Escherichia coli cytotoxic necrotizing factor-1 (200 ng/ml) decreased eNOS expression. These findings indicate that Rho negatively regulates eNOS expression and that HMG-CoA reductase inhibitors up-regulate eNOS expression by blocking Rho geranylgeranylation, which is necessary for its membrane-associated activity.

Topics: ADP Ribose Transferases; Bacterial Toxins; Botulinum Toxins; Cells, Cultured; Cytosol; Cytotoxins; Endothelium, Vascular; Escherichia coli Proteins; Gene Expression Regulation, Enzymologic; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Kinetics; Lipoproteins, LDL; Lovastatin; Membrane Proteins; Mevalonic Acid; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Polyisoprenyl Phosphates; ras Proteins; rhoA GTP-Binding Protein; rhoB GTP-Binding Protein; RNA Processing, Post-Transcriptional; RNA, Messenger; Sesquiterpenes