Geranylgeranyl pyrophosphate amplifies Treg differentiation via increased IL-2 expression to ameliorate DSS-induced colitis

Blocking the mevalonate pathway for cholesterol reduction by using statin may have adverse effects including statin-induced colitis. Moreover, one of the predisposing factors for colitis is an imbalanced CD4⁺ T cell, which can be observed on the complete deletion of HMG-CoA reductase (HMGCR), a target of statins. In this study, we inquired geranylgeranyl pyrophosphate (GGPP) is responsible for maintaining the T-cell homeostasis. Following dextran sulfate sodium (DSS)-induced colitis, simvastatin increased the severity of disease, while cotreatment with GGPP, but not with cholesterol, reversed the disease magnitude. GGPP ameliorated DSS-induced colitis by increasing T_reg cells. GGPP amplified T_reg differentiation through increased IL-2/STAT 5 signaling. GGPP prenylated Ras protein, a prerequisite for extracellular signal-regulated kinase (ERK) pathway activation, leading to increased IL-2 production. Higher simvastatin dose increased the severity of colitis. GGPP ameliorated simvastatin-increased colitis by increasing T_reg cells. T_reg cells, which have the capacity to suppress inflammatory T cells and were generated through IL-2/STAT5 signaling, increased IL-2 production through prenylation and activation of the Ras/ERK pathway.     

Monomethylarsonous acid inhibited endogenous cholesterol biosynthesis in human skin fibroblasts

Human exposure to arsenic in drinking water is a widespread public health concern, and such exposure is known to be associated with many human diseases. The detailed molecular mechanisms about how arsenic species contribute to the adverse human health effects, however, remain incompletely understood. Monomethylarsonous acid [MMA(III)] is a highly toxic and stable metabolite of inorganic arsenic. To exploit the mechanisms through which MMA(III) exerts its cytotoxic effect, we adopted a quantitative proteomic approach, by coupling stable isotope labeling by amino acids in cell culture (SILAC) with LC-MS/MS analysis, to examine the variation in the entire proteome of GM00637 human skin fibroblasts following acute MMA(III) exposure. Among the ~ 6500 unique proteins quantified, ~ 300 displayed significant changes in expression after exposure with 2 μM MMA(III) for 24 h. Subsequent analysis revealed the perturbation of de novo cholesterol biosynthesis, selenoprotein synthesis and Nrf2 pathways evoked by MMA(III) exposure. Particularly, MMA(III) treatment resulted in considerable down-regulation of several enzymes involved in cholesterol biosynthesis. In addition, real-time PCR analysis showed reduced mRNA levels of select genes in this pathway. Furthermore, MMA(III) exposure contributed to a distinct decline in cellular cholesterol content and significant growth inhibition of multiple cell lines, both of which could be restored by supplementation of cholesterol to the culture media. Collectively, the present study demonstrated that the cytotoxicity of MMA(III) may arise, at least in part, from the down-regulation of cholesterol biosynthesis enzymes and the resultant decrease of cellular cholesterol content.     

Statins: Mechanisms of neuroprotection

Clinical trials report that the class of drugs known as statins may be neuroprotective in Alzheimer's and Parkinson's disease, and further trials are currently underway to test whether these drugs are also beneficial in multiple sclerosis and acute stroke treatment. Since statins are well tolerated and have relatively few side effects, they may be considered as viable drugs to ameliorate neurodegenerative diseases. However, the mechanism of their neuroprotective effects is only partly understood. In this article, we review the current data on the neuroprotective effects of statins and their underlying mechanisms.     

Simvastatin induces apoptosis by a Rho-dependent mechanism in cultured cardiac fibroblasts and myofibroblasts

Several clinical trials have shown the beneficial effects of statins in the prevention of coronary heart disease. Additionally, statins promote apoptosis in vascular smooth muscle cells, in renal tubular epithelial cells and also in a variety of cell lines; yet, the effects of statins on cardiac fibroblast and myofibroblast, primarily responsible for cardiac tissue healing are almost unknown. Here, we investigated the effects of simvastatin on cardiac fibroblast and myofibroblast viability and studied the molecular cell death mechanism triggered by simvastatin in both cell types.

Methods

Rat neonatal cardiac fibroblasts and myofibroblasts were treated with simvastatin (0.1-10 μM) up to 72 h. Cell viability and apoptosis were evaluated by trypan blue exclusion method and by flow cytometry, respectively. Caspase-3 activation and Rho protein levels and activity were also determined by Western blot and pull-down assay, respectively.

Results

Simvastatin induces caspase-dependent apoptosis of cardiac fibroblasts and myofibroblasts in a concentration- and time-dependent manner, with greater effects on fibroblasts than myofibroblasts. These effects were prevented by mevalonate, farnesylpyrophosphate and geranylgeranylpyrophosphate, but not squalene. These last results suggest that apoptosis was dependent on small GTPases of the Rho family rather than Ras.

Conclusion

Simvastatin triggered apoptosis of cardiac fibroblasts and myofibroblasts by a mechanism independent of cholesterol synthesis, but dependent of isoprenilation of Rho protein. Additionally, cardiac fibroblasts were more susceptible to simvastatin-induced apoptosis than cardiac myofibroblasts. Thus simvastatin could avoid adverse cardiac remodeling leading to a less fibrotic repair of the damaged tissues.     

Simvastatin inhibits glucose uptake activity and GLUT4 translocation through suppression of the IR/IRS-1/Akt signaling in C2C12 myotubes

Simvastatin,a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor, is clinically used in the prevention and treatment of cardiovascular diseases. Numerous studies demonstrate that statins increase the risk of new-onset diabetes in long-term therapy, but mechanisms underpinning this effect are still unclear. Here, we investigated whether simvastatin inhibited the glucose uptake activity and the underlying mechanisms in C2C12 myotubes. Our studies showed that simvastatin significantly inhibited glucose uptake activity and GLUT4 translocation, whereas the effect was reversible with mevalonolactone (ML), which acts as an intermediate of cholesterol synthesis pathway. Mechanistically, the inhibition of glucose uptake and GLUT4 translocation elicited by simvastatin were associated with the suppression of the insulin receptor (IR)/IR substrate (IRS)/Akt signaling cascade. Simvastatin suppressed the phosphorylation of IR, IRS-1 and Akt, and total expression of IR or IRS-1, but did not affect Akt. Furthermore, simvastatin decreased Rac1 GTP binding. In conclusion, our findings indicate that simvastatin suppresses glucose uptake activity and GLUT4 translocation via IR-dependent IRS-1/PI3K/Akt pathway. These results provide an important new insight into the mechanism of statins on insulin sensitivity which may be associated with new-onset diabetes.     

Isoprenoid biosynthetic pathway inhibition disrupts monoclonal protein secretion and induces the unfolded protein response pathway in multiple myeloma cells

Myeloma is characterized by the overproduction and secretion of monoclonal protein. Inhibitors of the isoprenoid biosynthetic pathway (IBP) have pleiotropic effects in myeloma cells. To investigate whether IBP inhibition interferes with monoclonal protein secretion, human myeloma cells were treated with specific inhibitors of the IBP or prenyltransferases. These studies demonstrate that agents that inhibit Rab geranylgeranylation disrupt light chain trafficking, lead to accumulation of light chain in the endoplasmic reticulum, activate the unfolded protein response pathway and induce apoptosis. These studies provide a novel mechanism of action for IBP inhibitors and suggest that further exploration of Rab-targeted agents in myeloma is warranted.     

Simvastatin disrupts cytoskeleton and decreases cardiac fibroblast adhesion, migration and viability

Statins reduce the isoprenoids farnesyl and geranylgeranyl pyrophosphate, essential intermediates, which control a diversity of cellular events such as cytoskeleton integrity, adhesion, migration and viability. Cardiac fibroblasts are the major non-myocyte cell constituent in the normal heart, and play a key role in the maintenance of extracellular matrix. The effects of simvastatin on cardiac fibroblast processes previously mentioned remain unknown. Our aims were to investigate the effects of simvastatin on cytoskeleton structure and focal adhesion complex assembly and their relationships with cell adhesion, migration and viability in cultured cardiac fibroblasts. To this end, cells were treated with simvastatin for 24 h and changes in actin cytoskeleton, levels of vimentin and paxillin as well as their subcellular localization were analyzed by Western blot and immunocytochemistry, respectively. Cell adhesion to plastic or collagen coated dishes, migration in Transwell chambers, and cell viability were analyzed after simvastatin treatment. Our results show that simvastatin disrupts actin cytoskeleton and focal adhesion complex evaluated by phalloidin stain and immunocytochemistry for paxillin and vinculin. All these effects occurred by a cholesterol synthesis-independent mechanism. Simvastatin decreased cell adhesion, migration and viability in a concentration-dependent manner. Finally, simvastatin decreased angiotensin II-induced phospho-paxillin levels and cell adhesion. We concluded that simvastatin disrupts cytoskeleton integrity and focal adhesion complex assembly in cultured cardiac fibroblasts by a cholesterol-independent mechanism and consequently decreases cell migration, adhesion and viability.     

Do tocotrienols have potential as neuroprotective dietary factors?

Tocotrienols (T₃) belong to the family of vitamin E compounds (α-, β-, γ-, δ-tocopherols and -tocotrienols) and have unique biological properties that make them potential neuroprotective dietary factors. In addition to their antioxidant activity, T₃ at micromolar concentrations exert cholesterol-lowering activities in cells, animal models and some, but not all, human studies by means of inhibition of the activity of the rate-limiting enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase. At lower concentrations (∼10 nmol/L), T₃ modulate signalling pathways involved in neuronal cell death in cell culture experiments. Targets of T₃ include prenyl transferases, non-receptor tyrosine kinase, phospholipase A₂, 12-lipoxygenase, cyclooxygenase-2, and nuclear factor κB. The low bioavailability and rapid excretion of T₃ represents a major hurdle in their preventive use. Fasting plasma concentrations, even after supplementation with high doses, are below 1 μmol/L. T₃ bioavailability may be enhanced by ingestion with a high-fat meal, self-emulsifying drug delivery systems, or phytochemicals that inhibit T₃ metabolism and excretion. T₃ have no known adverse effects when consumed as part of a normal diet and the studies reviewed here support the notion that they may have potential as neuroprotective agents. However, experiments in relevant animal models and randomised human intervention trials addressing the neuroprotection mediated by T₃ are scarce and, thus, highly warranted.     

Combining metabolic and protein engineering of a terpenoid biosynthetic pathway for overproduction and selectivity control

A common strategy of metabolic engineering is to increase the endogenous supply of precursor metabolites to improve pathway productivity. The ability to further enhance heterologous production of a desired compound may be limited by the inherent capacity of the imported pathway to accommodate high precursor supply. Here, we present engineered diterpenoid biosynthesis as a case where insufficient downstream pathway capacity limits high-level levopimaradiene production in Escherichia coli. To increase levopimaradiene synthesis, we amplified the flux toward isopentenyl diphosphate and dimethylallyl diphosphate precursors and reprogrammed the rate-limiting downstream pathway by generating combinatorial mutations in geranylgeranyl diphosphate synthase and levopimaradiene synthase. The mutant library contained pathway variants that not only increased diterpenoid production but also tuned the selectivity toward levopimaradiene. The most productive pathway, combining precursor flux amplification and mutant synthases, conferred approximately 2,600-fold increase in levopimaradiene levels. A maximum titer of approximately 700 mg/L was subsequently obtained by cultivation in a bench-scale bioreactor. The present study highlights the importance of engineering proteins along with pathways as a key strategy in achieving microbial biosynthesis and overproduction of pharmaceutical and chemical products.