ggti-286 and geranylgeranyl-pyrophosphate

Bronchial fibroblasts contribute to airway remodelling, including airway wall fibrosis. Transforming growth factor (TGF)-β1 plays a major role in this process. We previously revealed the importance of the mevalonate cascade in the fibrotic response of human airway smooth muscle cells. We now investigate mevalonate cascade-associated signaling in TGFβ1-induced fibronectin expression by bronchial fibroblasts from non-asthmatic and asthmatic subjects.. We used simvastatin (1-15 μM) to inhibit 3-hydroxy-3-methlyglutaryl-coenzyme A (HMG-CoA) reductase which converts HMG-CoA to mevalonate. Selective inhibitors of geranylgeranyl transferase-1 (GGT1; GGTI-286, 10 μM) and farnesyl transferase (FT; FTI-277, 10 μM) were used to determine whether GGT1 and FT contribute to TGFβ1-induced fibronectin expression. In addition, we studied the effects of co-incubation with simvastatin and mevalonate (1 mM), geranylgeranylpyrophosphate (30 μM) or farnesylpyrophosphate (30 μM).. Immunoblotting revealed concentration-dependent simvastatin inhibition of TGFβ1 (2.5 ng/ml, 48 h)-induced fibronectin. This was prevented by exogenous mevalonate, or isoprenoids (geranylgeranylpyrophosphate or farnesylpyrophosphate). The effects of simvastatin were mimicked by GGTI-286, but not FTI-277, suggesting fundamental involvement of GGT1 in TGFβ1-induced signaling. Asthmatic fibroblasts exhibited greater TGFβ1-induced fibronectin expression compared to non-asthmatic cells; this enhanced response was effectively reduced by simvastatin.. We conclude that TGFβ1-induced fibronectin expression in airway fibroblasts relies on activity of GGT1 and availability of isoprenoids. Our results suggest that targeting regulators of isoprenoid-dependent signaling holds promise for treating airway wall fibrosis.

Topics: Adult; Airway Remodeling; Alkyl and Aryl Transferases; Asthma; Bronchi; Case-Control Studies; Cells, Cultured; Dose-Response Relationship, Drug; Farnesyltranstransferase; Fibroblasts; Fibronectins; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Leucine; Methionine; Mevalonic Acid; Polyisoprenyl Phosphates; Sesquiterpenes; Simvastatin; Time Factors; Transforming Growth Factor beta1; Young Adult

We tested whether rosuvastatin (RST) protected against oxygen-glucose deprivation (OGD)-induced cell death in primary rat cortical neuronal cultures. OGD reduced neuronal viability (%naive controls, mean +/- SE, n = 24-96, P < 0.05) to 44 +/- 1%, but 3-day pretreatment with RST (5 microM) increased survival to 82 +/- 2% (P < 0.05). One-day RST treatment was not protective. RST-induced neuroprotection was abolished by mevalonate or geranylgeranyl pyrophosphate (GGPP), but not by cholesterol coapplication. Furthermore, RST-induced decreases in neuronal cholesterol levels were abolished by mevalonate but not by GGPP. Reactive oxygen species (ROS) levels were reduced in RST-preconditioned neurons after OGD, and this effect was also reversed by both mevalonate and GGPP. These data suggested that GGPP, but not cholesterol depletion, were responsible for the induction of neuroprotection. Therefore, we tested whether 3-day treatments with perillic acid, a nonspecific inhibitor of both geranylgeranyl transferase (GGT) GGT 1 and Rab GGT, and the GGT 1-specific inhibitor GGTI-286 would reproduce the effects of RST. Perillic acid, but not GGTI-286, elicited robust neuronal preconditioning against OGD. RST, GGTI-286, and perillic acid all decreased mitochondrial membrane potential and lactate dehydrogenase activity in the cultured neurons, but only RST and perillic acid reduced neuronal ATP and membrane Rab3a protein levels. In conclusion, RST preconditions cultured neurons against OGD via depletion of GGPP, leading to decreased geranylgeranylation of proteins that are probably not isoprenylated by GGT 1. Reduced neuronal ATP levels and ROS production after OGD may be directly involved in the mechanism of neuroprotection.

Topics: Adenosine Triphosphate; Alkyl and Aryl Transferases; Animals; Cell Death; Cell Hypoxia; Cell Survival; Cells, Cultured; Cerebral Cortex; Cholesterol; Cyclohexenes; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fluorobenzenes; Glucose; Glutathione; L-Lactate Dehydrogenase; Leucine; Membrane Potential, Mitochondrial; Mevalonic Acid; Mitogen-Activated Protein Kinases; Monoterpenes; Neurons; Neuroprotective Agents; Phosphatidylinositol 3-Kinases; Polyisoprenyl Phosphates; Pyrimidines; rab3A GTP-Binding Protein; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Rosuvastatin Calcium; Sulfonamides; Time Factors

We evaluated the pharmacological effect of statins (3-hydroxy-3-methylglutaryl-CoA reductase inhibitors) on mast cell degranulation in RBL-2H3 cells. A hydrophilic statin (pravastatin) did not inhibit degranulation induced by dinitrophenol-human serum albumin (DNP-HSA); in contrast, lipophilic statins (simvastatin, fluvastatin and atorvastatin) inhibited DNP-HSA-induced degranulation in that order. The inhibitory effects were completely attenuated by simultaneous treatment with 100-1000 microM mevalonic acid for 4 h. We used fluvastatin to clarify the mechanism of the statin-mediated inhibitory action of mast cell degranulation. Fluvastatin (3 microM) had no effect on Ca(2+) release from the endoplasmic reticulum or Ca(2+) influx in the DNP-HSA- or thapsigargin-stimulated cells. Fluvastatin treatment also had no effect on the total granule content of the cell or sensitivity to DNP-HSA and IgE. Fluvastatin (3 microM, 24 h treatment) also failed to affect the morphology, proliferation, and viability of RBL-2H3 cells. Geranylgeranyl transferase inhibitor, GGTI-286 (20 microM), but not farnesyl transferase inhibitor, FPTIII (20 microM), inhibited the DNP-HSA-induced degranulation. The GGTI-286-induced inhibitory action was not associated with a decrease in the cytoplasmic Ca(2+) level. In conclusion, fluvastatin at a lower concentration range inhibited DNP-HSA-induced degranulation without affecting the cytoplasmic Ca(2+) response and also without changing the amount of granule content and proliferation of the mast cells. The statin-induced inhibitory action may be mediated by the suppression of geranylgeranyl transferase via the depletion of intracellular mevalonic acid.

Topics: Alkyl and Aryl Transferases; Calcium; Cell Degranulation; Cell Proliferation; Cell Survival; Cholesterol; Cytoplasm; Cytoplasmic Granules; Dinitrophenols; Enzyme Inhibitors; Fatty Acids, Monounsaturated; Fluvastatin; Humans; Indoles; Intracellular Space; Leucine; Mast Cells; Mevalonic Acid; Organophosphonates; Polyisoprenyl Phosphates; Serum Albumin; Thapsigargin

Erythropoietin (Epo) is a cytokine that is required for the survival of erythroid progenitors through interaction with its receptor on the surface of these cells. Recent studies showed that erythropoietin receptor (EpoR) is expressed on many cancer cells. The factors that govern EpoR expression on the cell surface are poorly understood. Using both biotinlyation and radiolabeled Epo binding experiments, we show here that Epo starvation of the Epo-dependent erythroleukemia cell line, ASE2, leads to a time-dependent increase in both forms of EpoR, the maturing 64 kDa and the mature 66 kDa proteins. Mevalonate depletion inhibits the formation of the highly glycosylated mature form of EpoR without affecting the other form. Treatment of cells with lovastatin, a selective inhibitor of the rate-limiting enzyme in the mevalonate pathway leads to inhibition of cell surface EpoR that is induced by Epo starvation. The effect of lovastatin appears to be the consequence of inhibition of two processes, glycosylation and geranylgeranylation. Adding back geranylgeranyl pyrophosphate to lovastatin-treated cells completely prevents the lovastatin effect on EpoR expression. Dolichol, the sugar carrier in N-linked glycosylation that is derived from the mevalonate pathway, partially reverses lovastatin's effect. The glycosylation inhibitor tunicamycin also partially suppresses EpoR surface expression. Inhibiting protein geranylgeranylation mimics the effect of lovastatin and inhibits EpoR surface expression in a concentration-dependent manner. Finally, lovastatin inhibits Epo's stimulatory effects on cell proliferation. These results indicate that mevalonate derivatives are required for normal EpoR expression on the cell surface through two pathways, glycosylation and geranylgeranylation.

Topics: Anti-Bacterial Agents; Anticholesteremic Agents; Antiviral Agents; Biotinylation; Blotting, Western; Cell Line, Tumor; Cell Survival; Dolichols; Dose-Response Relationship, Drug; Erythropoietin; Glycosylation; Humans; Iodine Radioisotopes; Janus Kinase 2; Leucine; Lovastatin; Mevalonic Acid; Phosphorylation; Polyisoprenyl Phosphates; Protein Prenylation; Receptors, Erythropoietin; STAT5 Transcription Factor; Tunicamycin

3-hydroxyl-3-methyl coenzyme A reductase inhibitors (statins) can function to protect the vasculature in a manner that is independent of their lipid-lowering activity. The main feature of the antithrombotic properties of endothelial cells is an increase in the expression of thrombomodulin (TM) without induction of tissue factor (TF) expression. We investigated the effect of statins on the expression of TM and TF by endothelial cells.. The incubation of endothelial cells with pitavastatin led to a concentration- and time-dependent increase in cellular TM antigen and mRNA levels. In contrast, the expression of TF mRNA was not induced under the same conditions. A nuclear run-on study revealed that pitavastatin accelerates TM transcription rate. The stimulation of TM expression by pitavastatin was prevented by either mevalonate or geranylgeranylpyrophosphate. Specific inhibition of geranylgeranyltransferase-I and Rac/Cdc42 by GGTI-286 and Clostridium sordellii lethal toxin, respectively, enhanced TM expression, whereas inactivation of Rho by Clostridium botulinum C3 exoenzyme was ineffective.. Statins regulate TM expression via inhibition of small G proteins of the Rho family; Rac/Cdc42. A statin-mediated increase in TM expression by endothelial cells may contribute to the beneficial effects of statins on endothelial function.

Topics: Bacterial Proteins; Bacterial Toxins; Cells, Cultured; Endothelium, Vascular; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Leucine; Mevalonic Acid; Monomeric GTP-Binding Proteins; Polyisoprenyl Phosphates; Quinolines; RNA, Messenger; Thrombomodulin; Thromboplastin; Umbilical Veins; Up-Regulation

Mevalonate depletion by inhibition of hydroxymethylglutaryl coenzyme A reductase impairs post-translational processing of Ras and Ras-related proteins. We have previously shown that this mevalonate depletion also leads to the upregulation of Ras, Rap1a, RhoA, and RhoB. This upregulation may result from global inhibition of isoprenylation or depletion of key regulatory isoprenoid species. Studies utilizing specific isoprenoid pyrophosphates in mevalonate-depleted cells reveal that farnesyl pyrophosphate (FPP) restores Ras processing and prevents RhoB upregulation while geranylgeranyl pyrophosphate (GGPP) restores Rap1a processing and prevents RhoA and RhoB upregulation. Either FPP or GGPP completely prevents lovastatin-induced upregulation of RhoB mRNA. Inhibition of FPP or squalene synthase allowed for the further identification of the putative regulatory species. Studies involving the specific isoprenyl transferase inhibitors FTI-277 and GGTI-286 demonstrate that selective inhibition of protein isoprenylation does not mimic lovastatin's ability to increase Ras and RhoA synthesis, decrease Ras and RhoA degradation, increase RhoB mRNA, or increase total levels of Ras, Rap1a, RhoA, and RhoB. In aggregate, these findings reveal a novel role and mechanism for isoprenoids to influence levels of Ras and Ras-related proteins.

Topics: Antineoplastic Agents; Blotting, Northern; Blotting, Western; Cycloheximide; Enzyme Inhibitors; Farnesyl-Diphosphate Farnesyltransferase; Humans; K562 Cells; Leucine; Lovastatin; Methionine; Mevalonic Acid; Polyisoprenyl Phosphates; Protein Biosynthesis; Protein Processing, Post-Translational; Protein Synthesis Inhibitors; rap1 GTP-Binding Proteins; ras Proteins; rhoA GTP-Binding Protein; rhoB GTP-Binding Protein; RNA, Messenger; Sesquiterpenes; Time Factors; Transcription, Genetic; Up-Regulation