geranylgeranyl-pyrophosphate and geranylgeraniol

The mevalonate synthesis inhibitors, statins, are mainstay therapeutics for cholesterol management and cardiovascular health. Thirty years of research have uncovered supportive roles for the mevalonate pathway in numerous cellular processes that support oncogenesis, most recently macropinocytosis. Central to the diverse mechanisms of statin sensitivity is an acquired dependence on one mevalonate pathway output, protein geranylgeranylation. New chemical prenylation probes and the discovery of a novel geranylgeranyl transferase hold promise to deepen our understanding of statin mechanisms of action. Further, insights into statin selection and the counterproductive role of dietary geranylgeraniol highlight how we should assess statins in the clinic. Lastly, rational combination strategies preview how statins will enter the oncology toolbox.

Topics: Alkyl and Aryl Transferases; Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Proliferation; Cell Survival; Disease Models, Animal; Diterpenes; Farnesyltranstransferase; Feeding Behavior; Food-Drug Interactions; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Metabolic Networks and Pathways; Mevalonic Acid; Mice; Neoplasms; Pinocytosis; Polyisoprenyl Phosphates; Prenylation

UbiA prenyltransferase domain-containing protein-1 (UBIAD1) utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K

Topics: Cell Line; Dimethylallyltranstransferase; Diterpenes; Endoplasmic Reticulum-Associated Degradation; Golgi Apparatus; Humans; Hydroxymethylglutaryl CoA Reductases; Phosphoric Monoester Hydrolases; Polyisoprenyl Phosphates

The mevalonate pathway (MVP) and the anti-angiogenic effect of bisphosphonates have been shown to play a role in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). This study determined the effect of the bisphosphonate, zoledronic acid and the replenishment of the MVP by geranylgeraniol on human gingival fibroblasts. Cell viability, apoptosis, morphological analysis using transmission electron microscopy, and gene expression for vascular endothelial growth factor A, bone morphogenic protein 2, ras homologue gene family member B, epiregulin and interferon-alpha were conducted. Results showed cellular viability was decreased in the presence of zoledronic acid and the co-addition of zoledronic acid with geranylgeraniol restored cell viability to control levels. Caspase 3/7 was detected in zoledronic-acid-treated cells indicating apoptosis. Transmission electron microscopy revealed dilation of the rough endoplasmic reticulum with zoledronic acid and the appearance of multiple lipid-like vesicles following the addition of geranylgeraniol. Zoledronic acid significantly (P < 0.05, FR > ± 2) up-regulated vascular endothelial growth factor A, bone morphogenic protein 2, ras homologue gene family member B and epiregulin at one or more time points but not interferon-alpha. Addition of geranylgeraniol resulted in a reduction in the expression of all five genes compared with zoledronic-acid-treated human gingival fibroblasts. The study concluded geranylgeraniol partially reversed the effects of zoledronic acid in human gingival fibroblasts both at the cellular and genetic levels, suggesting the regulation of these genes is mediated via the mevalonate pathway.

Topics: Adult; Apoptosis; Bone Density Conservation Agents; Bone Morphogenetic Protein 2; Cell Culture Techniques; Cell Survival; Cells, Cultured; Diphosphonates; Diterpenes; Epiregulin; Farnesol; Female; Fibroblasts; Gene Expression Regulation; Gingiva; Humans; Imidazoles; Interferon-alpha; Mevalonic Acid; Microscopy, Electron, Transmission; Middle Aged; Neovascularization, Physiologic; Polyisoprenyl Phosphates; rhoB GTP-Binding Protein; Sesquiterpenes; Signal Transduction; Vascular Endothelial Growth Factor A; Zoledronic Acid

Geranylgeranyl pyrophosphate (GGPP) and geranylgeraniol (GGOH) are used for the prenylation of GTP binding proteins and can reverse the antiresorptive action of nitrogen-containing bisphosphonates which inhibit farnesyl pyrophosphate synthase, an enzyme of the mevalonate pathway involved in the formation of GGPP. Previously, in cultures of fetal mouse long bones, we showed that GGOH stimulates osteoclastic bone resorption, but the cellular and molecular mode of action is not known. In cell homogenates, it has been found that GGOH can be metabolized to geranylgeranoic acid (GGA) which, like retinoic acid (RA), is a stimulator of retinoic acid receptor (RAR) expression. For this, we examined the involvement of the RAR in the action of GGOH on bone resorption. We show here that RA, GGOH, GGPP and GGA stimulate osteoclastic bone resorption and that this action is reversed by the RAR antagonist AGN-193109. These findings indicate the functional involvement of the RAR in the action of these polyisoprenoids. Moreover, RA, GGOH and GGA all stimulated RARbeta mRNA expression in bone explants. However, in contrast to GGOH and GGPP, GGA was not able to reverse the antiresorptive action of ibandronate, a nitrogen-containing bisphosphonate, suggesting that GGA is not involved in protein prenylation. In conclusion, our studies show that both GGOH and GGPP, independent of protein prenylation, stimulate osteoclastic bone resorption via RAR, probably via metabolism into GGA. Identification of such mechanism can help in the better understanding of the role of this metabolic pathway in the regulation of the activity and survival of osteoclasts.

Topics: Animals; Bone and Bones; Bone Density Conservation Agents; Bone Resorption; Calcium; Diphosphonates; Diterpenes; Dose-Response Relationship, Drug; Drug Synergism; Female; Ibandronic Acid; Mevalonic Acid; Mice; Osteoclasts; Polyisoprenyl Phosphates; Pregnancy; Protein Prenylation; Receptors, Retinoic Acid

Statins, competitive inhibitors of hydroxymethylglutaryl-CoA reductase, have recently been shown to have a therapeutic effect in rheumatoid arthritis (RA). In RA, synovial fibroblasts in the synovial lining, are believed to be particularly important in the pathogenesis of disease because they recruit leukocytes into the synovium and secrete angiogenesis-promoting molecules and proteases that degrade extracellular matrix. In this study, we show a marked reduction in RA synovial fibroblast survival through the induction of apoptosis when the cells were cultured with statins. Simvastatin was more effective in RA synovial fibroblasts than atorvastatin, and both statins were more potent on tumor necrosis factor-alpha-induced cells. In contrast, in osteoarthritis synovial fibroblasts, neither the statin nor the activation state of the cell contributed to the efficacy of apoptosis induction. Viability of statin-treated cells could be rescued by geranylgeraniol but not by farnesol, suggesting a requirement for a geranylgeranylated protein for synovial fibroblast survival. Phase partitioning experiments confirmed that in the presence of statin, geranylgeranylated proteins are redistributed to the cytoplasm. siRNA experiments demonstrated a role for Rac1 in synovial fibroblast survival. Western blotting showed that the activated phosphorylated form of Akt, a protein previously implicated in RA synovial fibroblast survival, was decreased by about 75%. The results presented in this study lend further support to the importance of elevated pAkt levels to RA synovial fibroblast survival and suggest that statins might have a beneficial role in reducing the aberrant pAkt levels in patients with RA. The results may also partly explain the therapeutic effect of atorvastatin in patients with RA.

Topics: Apoptosis; Arthritis, Rheumatoid; Biological Transport; Cell Survival; Cells, Cultured; Cytoplasm; Diterpenes; Fibroblasts; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Membrane Proteins; Osteoarthritis; Polyisoprenyl Phosphates; Proto-Oncogene Proteins c-akt; rac1 GTP-Binding Protein; RNA, Small Interfering; Synovial Membrane; Tissue Distribution; Tumor Necrosis Factor-alpha

Complement-mediated vascular injury is important in the pathophysiology of atherosclerosis and myocardial infarction. Because recent evidence shows that statins have beneficial effects on endothelial cell (EC) function independent of lipid lowering, we explored the hypothesis that statins modulate vascular EC resistance to complement through the upregulation of complement-inhibitory proteins. Human umbilical vein and aortic ECs were treated with atorvastatin or simvastatin, and decay-accelerating factor (DAF), membrane cofactor protein, and CD59 expression was measured by flow cytometry. A dose-dependent increase in DAF expression of up to 4-fold was seen 24 to 48 hours after treatment. Statin-induced upregulation of DAF required increased steady-state mRNA and de novo protein synthesis. L-Mevalonate and geranylgeranyl pyrophosphate reversed the effect, confirming the role of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibition and suggesting that constitutive DAF expression is negatively regulated by geranylgeranylation. Neither farnesyl pyrophosphate nor squalene inhibited statin-induced DAF expression, suggesting that the effect is independent of cholesterol lowering. Statin-induced DAF upregulation was mediated by the activation of protein kinase Calpha and inhibition of RhoA and was independent of phosphatidylinositol-3 kinase and NO activity. The increased DAF expression was functionally effective, resulting in significant reduction of C3 deposition and complement-mediated lysis of antibody-coated ECs. These observations provide evidence for a novel cytoprotective action of statins on vascular endothelium that is independent of the effect on lipids and results in enhanced protection against complement-mediated injury. Modulation of complement regulatory protein expression may contribute to the early beneficial effects of statins in reducing the morbidity and mortality associated with atherosclerosis.

Topics: Atorvastatin; CD55 Antigens; Cell Line; Cells, Cultured; Complement Activation; Complement C3; Cytoprotection; Diterpenes; Endothelium, Vascular; Heptanoic Acids; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Mevalonic Acid; Nitric Oxide; Phosphatidylinositol 3-Kinases; Polyisoprenyl Phosphates; Protein Kinase C; Pyrroles; rho GTP-Binding Proteins; RNA, Messenger; Simvastatin; Up-Regulation

Geranylgeranylation of RhoA small G-protein is essential for its localization to cell membranes and for its biological functions. Many RhoA effects are mediated by its downstream effector RhoA kinase. The role of protein geranylgeranylation and the RhoA pathway in the regulation of endothelial cell survival has not been elucidated. The hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor lovastatin depletes cellular pools of geranylgeranyl pyrophosphate and farnesol pyrophosphate and thereby inhibits both geranylgeranylation and farnesylation. Human umbilical vein endothelial cells (HUVECs) were exposed to lovastatin (3 microm-30 microm) for 48 h, and cell death was quantitatively determined by cytoplasmic histone-associated DNA fragments as well as caspase-3 activity. The assays showed that lovastatin caused a dose-dependent endothelial cell death. The addition of geranylgeraniol, which restores geranylgeranylation, rescued HUVEC from apoptosis. The geranylgeranyltransferase inhibitor GGTI-298, but not the farnesyltransferase inhibitor FTI-277, induced apoptosis in HUVEC. Cell death was also induced by a blockade of RhoA function by exoenzyme C3. In addition, treatment of HUVEC with the RhoA kinase inhibitors Y-27632 and HA-1077 caused dose-dependent cell death. Y-27632 did not inhibit other well known survival pathways, such as NF-kappa B, ERK, and phosphatidylinositol 3-kinase/Akt. However, there was an increase in p53 protein level concomitant with Y-27632-induced cell death. Unlike the apoptosis induced by TNF-alpha, which occurs only with inhibition of new protein synthesis, apoptosis induced by inhibitors of HMG-CoA reductase, geranylgeranyltransferase, or RhoA kinase was blocked by cycloheximide. Our data indicate that inhibition of protein geranylgeranylation and RhoA pathways induce apoptosis in HUVEC and that induction of p53 or other proapoptotic proteins is required for this process.

Topics: Apoptosis; Benzamides; Caspase 3; Caspases; Cell Death; Cells, Cultured; Diterpenes; Endothelium, Vascular; Enzyme Activation; Enzyme Inhibitors; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Intracellular Signaling Peptides and Proteins; Lovastatin; Methionine; Polyisoprenyl Phosphates; Protein Prenylation; Protein Serine-Threonine Kinases; rho-Associated Kinases; rhoA GTP-Binding Protein; Umbilical Veins

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

It is well documented that 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors prevent cultured mammalian cells from progressing through the cell cycle, suggesting a critical role for a mevalonate-derived product. Recently, it has been shown that free geranylgeraniol (GG-OH) and farnesol (F-OH) can be utilized by C6 glioma cells for protein isoprenylation. The ability of GG-OH and F-OH to restore protein geranylgeranylation or farnesylation selectively has enabled us to examine the possibility that mevalonate is essential for cell proliferation because it is a precursor of farnesyl pyrophosphate or geranylgeranyl pyrophosphate, the isoprenyl donors involved in the posttranslational modification of key regulatory proteins. In this study we report that GG-OH, as well as mevalonate, overcomes the arrest of cell proliferation of C6 glioma cells treated with lovastatin, as assessed by increased cell numbers and a stimulation in [3H]thymidine incorporation. The increase in cell number and [3H]thymidine incorporation were significantly lower when F-OH was added. Under these conditions [3H]mevalonate and [3H]GG-OH are actively incorporated into a set of isoprenylated proteins in the size range of small, GTP-binding proteins (19-27 kDa) and a polypeptide with the molecular size (46 kDa) of the smaller isoform of 2 ',3'-cyclic nucleotide 3'-phosphodiesterase. Analysis of the proteins metabolically labeled by [3H]mevalonate and [3H]GG-OH reveals the presence of labeled proteins containing geranylgeranylated cysteinyl residues. Consistent with geranylgeranylated proteins playing a critical role in the entry of C6 cells into the cell cycle, a (phosphonoacetamido)oxy derivative of GG-OH, a drug previously shown to interfere with protein geranylgeranylation, prevented the increase in cell number when mevalonate or GG-OH was added to lovastatin-treated cells. These results strongly suggest that geranylgeranylated proteins are essential for progression of C6 cells into the S phase of the cell cycle and provide the first evidence that the "salvage" pathway for the utilization of the free isoprenols is physiologically significant in the CNS.

Topics: Animals; Cell Division; Diterpenes; Electrophoresis, Polyacrylamide Gel; Farnesol; Glioma; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin; Mevalonic Acid; Molecular Weight; Nerve Tissue Proteins; Polyisoprenyl Phosphates; Protein Prenylation; Rats; Thymidine; Tumor Cells, Cultured

[14C]Geranylgeranyldiphosphate was produced by a prenyl transferase system extracted from the mutant strain SG4 of Gibberella fujikuroi (wild-type strain IMI 58289), which is blocked in carotenoid biosynthesis. The fungus was grown in liquid medium containing only potato-dextrose broth. Crude extracts of 12- to 14-day-old mycelia in Tris buffer, pH 8.5, were centrifuged at 100,000g. The supernatant was desalted and used to produce [4,8,12,16-14C]geranylgeranyldiphosphate from R,S-[2-14C]mevalonic acid. Maximum yield of [14C]geranylgeranyldiphosphate was obtained using mycelia from the stationary growth phase with incubation of the enzyme preparation at pH 8.5 for 5 h. Routinely, 65% of the biogenetic available R-mevalonic acid was converted to [14C]geranylgeranyldiphosphate, which was identified by cochromatography with authentic [1-3H]geranylgeranyldiphosphate by means of radio-TLC, radio-HPLC, and hydrolysis to geranylgeraniol.

Topics: Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Dimethylallyltranstransferase; Diterpenes; Gibberella; Hydrolysis; Mevalonic Acid; Mutagenesis; Oxidoreductases; Polyisoprenyl Phosphates

Topics: Chlorophyllides; Chloroplasts; Diterpenes; Phytol; Polyisoprenyl Phosphates

The esterification of chlorophyllide a was investigated an irradiated etioplast-membrane fractions ('broken etioplasts') from oat seedlings (Avena sativa L.). As a substrate, [1(-3)H]geranylgeraniol and its monophosphate and diphosphate derivatives were prepared by chemical synthesis. Geranylgeraniol and its monphosphate derivative are incorporated into chlorophyll only in the presence of ATP whereas the diphosphate derivative is incorporated also without ATP. The yield of esterified chlorophyll is 80-90% of chlorophyllide with saturating substrate concentrations. The term 'chlorophyll synthetase' is used to describe the enzyme activity which is different from chlorophyllase. Other substrates are phytol and farnesol either with ATP or as the diphosphate derivatives. The relative specificity of 'chlorophyll synthetase' for thse substrates is geranylgeraniol:phytol:farnesol = 6:3:1. In these experiments in vitro, a new chlorphyll esterified with farnesol was detected which does not occur in intact plants. Geraniol and n-pentadecanol are no substrates for the enzyme. Protochlorphyllide which is present in non-irradiated etioplast membrane fractions is not esterified under the same conditions.

Topics: Carbon-Oxygen Ligases; Chlorophyllides; Chromatography, High Pressure Liquid; Diterpenes; Edible Grain; Kinetics; Ligases; Plants; Polyisoprenyl Phosphates; Substrate Specificity