okadaic-acid and 4-hydroxy-2-nonenal

Osteoporosis is one of the most common bone diseases, which is characterized by a systemic impairment of bone mass and fragility fractures. Age-related oxidative stress is highly associated with impaired osteoblastic dysfunctions and subsequent osteoporosis. In osteoblasts (bone formation cells), reactive oxygen species (ROS) are continuously generated and further cause lipid peroxidation, protein damage, and DNA lesions, leading to osteoblastic dysfunctions, dysdifferentiations, and apoptosis. Although much progress has been made, the mechanism responsible for oxidative stress induced cellular alternations and osteoblastic toxicity is still not fully elucidated. Here, we demonstrate that protein phosphatase 2A (PP2A), a major protein phosphatase in mammalian cells, mediates oxidative stress induced apoptosis in osteoblasts. Our results showed that lipid peroxidation products (4-HNE) may induce dramatic oxidative stress, inflammatory reactions, and apoptosis in osteoblasts. These oxidative stress responses may ectopically activate PP2A phosphatase activity, which may be mediated by inactivation of AKT/mTOR pathway. Moreover, inhibition of PP2A activity by okadaic acid might partly prevent osteoblastic apoptosis under oxidative conditions. These findings may reveal a novel mechanism to clarify the role of oxidative stress for osteoblastic apoptosis and provide new possibilities for the treatment of related bone diseases, such as osteoporosis.

Topics: Aldehydes; Animals; Animals, Newborn; Apoptosis; Bone Remodeling; Inflammation; Lipid Peroxidation; Mice; Okadaic Acid; Osteoblasts; Osteoporosis; Oxidative Stress; Protein Phosphatase 2; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Signal Transduction

We have previously shown that monocyte conditioned medium (MCM) from patients with liver fibrosis stimulated proliferation of hepatic stellate cells (HSCs), the major cell involved in hepatic fibrosis. To investigate the potential role of fetuin and pentoxifylline in fibrosis we used MCM samples obtained from patients with biopsy proven hepatic fibrosis related to Hepatitis C (HCV). Our results indicate that the MCM obtained from patients with HCV-related liver fibrosis significantly stimulated collagen synthesis in HSCs as assessed by tritiated proline incorporation into a collagenase sensitive trichloroacetic acid (TCA) precipitate. Collagen synthesis was also stimulated in HSCs using transforming growth factor beta (TGFbeta) and this effect was neutralized using TGFbeta antibody. Incubation of HSCs with fetuin (but not TGFbeta antibody) significantly inhibited collagen synthesis in HSCs that were stimulated by HCV MCM samples. Patient MCM samples would also stimulate proliferation of HSCs as assessed by tritiated thymidine uptake but this effect was not attenuated by fetuin. Likewise the significant stimulatory effect of platelet derived growth factor (PDGF) on HSC proliferation and collagen synthesis was not inhibited by fetuin but could be significantly reduced by 70% and 40% respectively, when treated with pentoxifylline. We also investigated the ability of samples obtained from patients with hepatic fibrosis to inhibit HSC apoptosis, as determined by okadaic acid-induced 4-hydroxynonenal immunocytochemistry in HSCs. We have previously reported that okadaic acid induces apoptosis in HSCs as assessed by Hoescht and TUNEL. Okadaic acid treatment produced a positive 4-hydroxynonenal (4-HNE) immunoreactivity in HSCs and treatment with HCV patient MCM or TGFbeta decreased the 4-HNE positive immunoreactivity in HSCs treated with okadaic acid. Our results suggest that fetuin may be beneficial in hepatic fibrosis and suggest that combination of fetuin and pentoxifylline may target the two key events in hepatic fibrosis by modifying the effects of TGFbeta and PDGF, the two major growth factors in fibrosis.

Topics: Aldehydes; alpha-Fetoproteins; Animals; Apoptosis; Cell Differentiation; Cells, Cultured; Collagen; Culture Media, Conditioned; Cytokines; Hepatitis C; Humans; Liver; Liver Cirrhosis; Male; Monocytes; Okadaic Acid; Pentoxifylline; Platelet-Derived Growth Factor; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta

We studied the signal pathways for regulation of serine/threonine protein kinase Akt in Jurkat cells that had been treated with 4-hydroxynonenal (HNE) for caspase-dependent apoptosis induction. Treatment of cells with HNE led to a decrease in the level of Akt activity due to the dephosphorylation at Ser473, a major regulatory phosphorylation site. HNE-mediated dephosphorylation of Akt was prevented by a protein phosphatase 2A (PP2A) inhibitor, okadaic acid, and by a caspase-3 inhibitor, DEVD-CHO. HNE treatment resulted in an increase in the total level of PP2A activity, release of active tyrosine-dephosphorylated PP2A from the cytoskeleton and PP2A-Akt association, which were all dependent on caspase-3 activation. These results suggest that the level of PP2A activity is at least in part determined by its tyrosine phosphorylation, which is dually controlled by okadaic acid-sensitive phosphatases and protein-tyrosine kinases. Possibly underlying the mechanism of caspase-mediated activation of PP2A, HNE treatment resulted in downregulation of the activity of Src kinase, as a representative caspase-sensitive kinase to phosphorylate PP2A at tyrosine. In addition, activated caspase-3 partially cleaved Akt at a late stage of the apoptosis. These results indicate the existence of two distinct caspase-dependent signal pathways for downregulation of Akt that works as a mechanism of positive feedback regulation for HNE-triggered apoptotic signals.

Topics: Aldehydes; Apoptosis; Caspase 3; Caspase Inhibitors; Caspases; Down-Regulation; Enzyme Inhibitors; Feedback, Physiological; Humans; Jurkat Cells; Okadaic Acid; Oligopeptides; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 2; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Serine; Signal Transduction; src-Family Kinases

Here we show, for the first time, the in vitro formation of filamentous aggregates of phosphorylated tau protein in SH-SY5Y human neuroblastoma cells. The formation of such aberrant aggregates, similar to those occurring in vivo in Alzheimer's disease and other tauopathies, requires okadaic acid, a phosphatase inhibitor, to increase the level of phosphorylated tau, and hydroxynonenal, a product of oxidative stress that selectively adducts and modifies phosphorylated tau. Our findings suggest that both phosphorylation and oxidative modification are required for tau filament formation. Importantly, the in vitro formation of intracellular tau aggregates could be used as a model of tau polymerization and facilitate the development of novel therapeutic approaches.

Topics: Aldehydes; Humans; Neuroblastoma; Okadaic Acid; Phosphorylation; Polymers; tau Proteins; Tumor Cells, Cultured

Cyclin-dependent kinase 5 (cdk5) is believed to be involved in the phosphorylation of tau protein. We studied the expression of the protein levels of cdk5 and the neuron-specific cdk5 activator p35 as well as cdk5 activity and tau phosphorylation during apoptosis in rat hippocampal neuronal cultures. We observed that in cells treated with etoposide, cyclosporin A, 4-hydroxynonenal (HNE), or okadaic acid, there was an early reduction in the protein levels of p35, and later also in cdk5 with all treatments except etoposide. The level of p25, a calpain cleavage product of p35 suggested to have increased ability to activate cdk5, was reduced paralleling the amount of p35. The changes in the p35 and p25 protein levels coincided with decreases in cdk5 activity and tau phosphorylation after treatment with HNE and etoposide. However, the relationship between the p35 and p25 levels and cdk5 activity was complex. We conclude that neuronal apoptosis is accompanied with a decrease in the levels of p35, p25, and cdk5, and tau phosphorylation. These changes may reinforce the neuronal damage.

Topics: Aldehydes; Animals; Apoptosis; Blotting, Western; Caspase 3; Caspases; Cells, Cultured; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases; Cyclosporine; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Etoposide; Hippocampus; Nerve Tissue Proteins; Neurons; Nucleic Acid Synthesis Inhibitors; Okadaic Acid; Phosphorylation; Phosphotransferases; Rats; Rats, Wistar; tau Proteins; Time Factors

Increased oxyradical production and membrane lipid peroxidation occur in neurons under physiological conditions and in neurodegenerative disorders. Lipid peroxidation can alter synaptic plasticity and may increase the vulnerability of neurons to excitotoxicity, but the underlying mechanisms are unknown. We report that 4-hydroxy-2,3-nonenal (4HN), an aldehyde product of lipid peroxidation, exerts a biphasic effect on NMDA-induced current in cultured rat hippocampal neurons with current being increased during the first 2 h and decreased after 6 h. Similarly, 4HN causes an early increase and a delayed decrease in NMDA-induced elevation of intracellular Ca2+ levels. In contrast, 4HN affects neither the ion current nor the Ca2+ response to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA). The initial enhancement of NMDA-induced current is associated with increased phosphorylation of the NR1 receptor subunit, whereas the delayed suppression of current is associated with cellular ATP depletion and mitochondrial membrane depolarization. Cell death induced by 4HN is attenuated by an NMDA receptor antagonist, but not by an AMPA receptor antagonist. A secreted form of amyloid precursor protein, previously shown to protect neurons against oxidative and excitotoxic insults, prevented each of the effects of 4HN including the early and late changes in NMDA current, delayed ATP depletion, and cell death. These findings show that the membrane lipid peroxidation product 4HN can modulate NMDA channel activity, suggesting a role for this aldehyde in physiological and pathophysiological responses of neurons to oxidative stress.

Topics: Adenosine Triphosphate; Aldehydes; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Amyloid beta-Protein Precursor; Animals; Calcium; Cells, Cultured; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Hippocampus; Lipid Peroxidation; Membrane Lipids; Microscopy, Fluorescence; N-Methylaspartate; Neurons; Okadaic Acid; Patch-Clamp Techniques; Phosphorylation; Precipitin Tests; Rats; Receptors, N-Methyl-D-Aspartate; Rotenone; Time Factors; Uncoupling Agents

A correlation between hyperphosphorylation of tau protein and its aberrant assembly into paired helical filaments has lead to suggestions that phosphorylation controls assembly, but lacked a mechanistic basic. In this work, we have found that phosphorylated, but not native, tau protein is able to form polymers after the reaction with 4-hydroxy-2-nonenal, a highly toxic product of lipid peroxidation. Phosphorylation of tau by both proline or non-proline directed kinases, was able to assemble it into polymers.

Topics: Aldehydes; Animals; Calcium-Calmodulin-Dependent Protein Kinases; COS Cells; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Neurofibrillary Tangles; Okadaic Acid; Peptides; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Conformation; Protein Serine-Threonine Kinases; tau Proteins; Transfection

Peroxidation of membrane lipids occurs in many different neurodegenerative conditions including stroke, and Alzheimer's and Parkinson's diseases. Recent findings suggest that lipid peroxidation can promote neuronal death by a mechanism involving production of the toxic aldehyde 4-hydroxy-2,3-nonenal (HNE), which may act by covalently modifying proteins and impairing their function. The transcription factor NF-kappa B can prevent neuronal death in experimental models of neurodegenerative disorders by inducing the expression of anti-apoptotic proteins including Bcl-2 and manganese superoxide dismutase. We now report that HNE selectively suppresses basal and inducible NF-kappa B DNA binding activity in cultured rat cortical neurons. Immunoprecipitation-immunoblot analyses using antibodies against HNE-conjugated proteins and p50 and p65 NF-kappa B subunits indicate that HNE does not directly modify NF-kappa B proteins. Moreover, HNE did not affect NF-kappa B DNA-binding activity when added directly to cytosolic extracts, suggesting that HNE inhibits an upstream component of the NF-kappa B signaling pathway. Inhibition of the survival-promoting NF-kappa B signaling pathway by HNE may contribute to neuronal death under conditions in which membrane lipid peroxidation occurs.

Topics: Aldehydes; Alzheimer Disease; Animals; Apoptosis; Cell Survival; Cells, Cultured; Cerebral Cortex; Cycloheximide; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Lipid Peroxidation; Nerve Degeneration; Neurons; NF-kappa B; Okadaic Acid; Protein Synthesis Inhibitors; Rats; Stroke; Transcription Factor AP-1; Vanadates

Extensively oxidized low density lipoprotein (ox-LDL), a modulator of atherogenesis, down-regulates the lipopolysaccharide (LPS)-induced activation of transcription factor NF-kappaB. We investigated whether 4-hydroxynonenal (HNE), a prominent aldehyde component of ox-LDL, represents one of the inhibitory substances. NF-kappaB activation by stimuli such as LPS, interleukin (IL)-1beta, and phorbol ester, but not tumor necrosis factor (TNF), was reversibly inhibited by HNE in a dose-dependent manner in human monocytic cells, whereas AP-1 binding was unaffected. Using similar HNE concentrations, LPS-induced kappaB- and TNF or IL-8 promoter-dependent transcription was prevented. Furthermore, pretreatment with HNE suppressed TNF production but not lactate dehydrogenase levels. Under these conditions the binding of LPS to monocytic cells was not significantly affected. However, induced proteolysis of the inhibitory proteins IkappaB-alpha, IkappaB-beta, and, at a later time point, IkappaB-epsilon was prevented. This is not due to inhibition of the proteasome, the major proteolytic activities of which remain unaffected, but rather to a specific prevention of the activation-dependent phosphorylation of IkappaB-alpha. This is the first report which demonstrates that HNE specifically inhibits the NF-kappaB/Rel system. Down-modulation of NF-kappaB-regulated gene expression may contribute at certain stages of atherosclerosis to low levels of chronic inflammation and may also be involved in other inflammatory/degenerative diseases.

Topics: Aldehydes; Cell Line; Cysteine Endopeptidases; DNA-Binding Proteins; Enzyme Activation; Humans; Hydrolysis; I-kappa B Kinase; I-kappa B Proteins; Leukocyte Elastase; Multienzyme Complexes; NF-kappa B; Okadaic Acid; Phosphorylation; Proteasome Endopeptidase Complex; Protein Serine-Threonine Kinases; Tumor Necrosis Factor-alpha

In Alzheimer's disease (AD) the microtubule-associated protein tau is excessively phosphorylated in degenerating neurons, but the mechanisms underlying the increased phosphorylation are unknown. Recent findings suggest that oxidative stress, and membrane lipid peroxidation in particular, contributes to the neurodegenerative process in AD. We now report that following exposure of cultured rat hippocampal neurons to 4-hydroxynonenal (HNE), an aldehydic product of membrane lipid peroxidation, tau is resistant to dephosphorylation. Immunocytochemical and Western blot analyses using phosphorylation-sensitive tau antibodies showed that HNE treatment causes a moderate increase in basal levels of tau phosphorylation, and prevents tau dephosphorylation by alkaline phosphatase in neurons pretreated with the phosphatase inhibitor okadaic acid. Studies with anti-HNE antibodies showed that HNE binds directly to tau, and that HNE immunoreactivity localizes to cell bodies and axons, cell compartments that contain tau. These data suggest a role for HNE in altered tau phosphorylation and neurofibrillary degeneration in AD.

Topics: Aldehydes; Animals; Blotting, Western; Cells, Cultured; Cysteine Proteinase Inhibitors; Hippocampus; Lipid Peroxidation; Microscopy, Confocal; Okadaic Acid; Phosphorylation; Rats; tau Proteins