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1.
Neuromolecular Med ; 24(4): 469-478, 2022 12.
Article in English | MEDLINE | ID: mdl-35482177

ABSTRACT

The orphan nuclear receptor Nurr1 is critical for the development, maintenance, and protection of midbrain dopaminergic neurons. Recently, we demonstrated that prostaglandins E1 (PGE1) and PGA1 directly bind to the ligand-binding domain (LBD) of Nurr1 and stimulate its transcriptional activation function. In this direction, here we report the transcriptional activation of Nurr1 by PGA2, a dehydrated metabolite of PGE2, through physical binding ably supported by NMR titration and crystal structure. The co-crystal structure of Nurr1-LBD bound to PGA2 revealed the covalent coupling of PGA2 with Nurr1-LBD through Cys566. PGA2 binding also induces a 21° shift of the activation function 2 (AF-2) helix H12 away from the protein core, similar to that observed in the Nurr1-LBD-PGA1 complex. We also show that PGA2 can rescue the locomotor deficits and neuronal degeneration in LRRK2 G2019S transgenic fly models.


Subject(s)
Nuclear Receptor Subfamily 4, Group A, Member 2 , Parkinson Disease , Prostaglandins A , Humans , Ligands , Nuclear Receptor Subfamily 4, Group A, Member 2/genetics , Nuclear Receptor Subfamily 4, Group A, Member 2/metabolism , Prostaglandins A/genetics , Prostaglandins A/metabolism , Animals, Genetically Modified , Drosophila , Disease Models, Animal
2.
Nat Chem Biol ; 16(8): 876-886, 2020 08.
Article in English | MEDLINE | ID: mdl-32451509

ABSTRACT

The orphan nuclear receptor Nurr1 is critical for the development, maintenance and protection of midbrain dopaminergic (mDA) neurons. Here we show that prostaglandin E1 (PGE1) and its dehydrated metabolite, PGA1, directly interact with the ligand-binding domain (LBD) of Nurr1 and stimulate its transcriptional function. We also report the crystallographic structure of Nurr1-LBD bound to PGA1 at 2.05 Å resolution. PGA1 couples covalently to Nurr1-LBD by forming a Michael adduct with Cys566, and induces notable conformational changes, including a 21° shift of the activation function-2 helix (H12) away from the protein core. Furthermore, PGE1/PGA1 exhibit neuroprotective effects in a Nurr1-dependent manner, prominently enhance expression of Nurr1 target genes in mDA neurons and improve motor deficits in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mouse models of Parkinson's disease. Based on these results, we propose that PGE1/PGA1 represent native ligands of Nurr1 and can exert neuroprotective effects on mDA neurons, via activation of Nurr1's transcriptional function.


Subject(s)
Alprostadil/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 2/metabolism , Prostaglandins A/metabolism , Animals , Cell Line, Tumor , Crystallography, X-Ray , Dopamine/metabolism , Humans , Ligands , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Neurons/metabolism , Neuroprotective Agents/pharmacology , Nuclear Receptor Subfamily 4, Group A, Member 2/chemistry , Nuclear Receptor Subfamily 4, Group A, Member 2/genetics , Protein Binding , Rats , Signal Transduction , Transcription, Genetic
3.
Biochem Biophys Res Commun ; 524(1): 231-235, 2020 03 26.
Article in English | MEDLINE | ID: mdl-31983432

ABSTRACT

Major depressive disorder (MDD) has become a potential cause of death and disability among young people worldwide. Numerous studies have indicated that the different cerebrospinal fluid (CSF) proteins may be used as mediums for MDD. Given the emergent interest of CSF proteins in MDD, we validated proteins expression in the choroid plexus (CP), the brain region that produces CSF in the lateral ventricle, the third ventricle, and the fourth ventricle of the central nervous system (CNS). The CSF constantly exchanges molecular substances with the brain tissue, which can dynamically reflect the metabolic microenvironment of the brain. In our previous study, Pepsin A (PGA) and periostin (POSTN) was associated with depressive-like behaviors of depressed macaca fascicularis models in CSF. Moreover, proteins that are expressed in the CP can be secreted into the CSF and may be associated MDD. This study sought to demonstrate the discrepancy of PGA and POSTN in the CP between Lipopolysaccharide (LPS)-induce depressed mice models and wild type (WT) mice. Our findings suggest that PGA and POSTN expression in CP of mice could be a possible candidate pathogenesis involved in MDD, which may contribute to a better understanding and treatment of MDD.


Subject(s)
Cell Adhesion Molecules/metabolism , Choroid Plexus/metabolism , Depression/metabolism , Prostaglandins A/metabolism , Animals , Behavior, Animal , Body Weight , Feeding Behavior , Hippocampus/metabolism , Immobilization , Male , Mice, Inbred C57BL , Sucrose , Time Factors
4.
Biochem J ; 476(19): 2757-2767, 2019 10 15.
Article in English | MEDLINE | ID: mdl-31492735

ABSTRACT

Nur77 is a transcription factor belonging to the NR4A subfamily of nuclear hormone receptors. Upon induction, Nur77 modulates the expression of its target genes and controls a variety of biological and pathophysiological processes. Prior research that revealed a structurally atypical ligand-binding domain (LBD) and failed to locate an endogenous ligand had led to a classification of Nur77 as an orphan receptor. However, several more recent studies indicate that small synthetic molecules and unsaturated fatty acids can bind to Nur77. Discovery of additional endogenous ligands will facilitate our understanding of the receptor's functions and regulatory mechanisms. Our data have identified prostaglandin A2 (PGA2), a cyclopentenone prostaglandin (PG), as such a ligand. Cyclopentenone PGs exert their biological effects primarily by forming protein adducts via the characteristic electrophilic ß-carbon(s) located in their cyclopentenone rings. Our data show that PGA2 induces Nur77 transcriptional activity by forming a covalent adduct between its endocyclic ß-carbon, C9, and Cys566 in the receptor's LBD. The importance of this endocyclic ß-carbon was substantiated by the failure of PGs without such electrophilic properties to react with Nur77. Calculated chemical properties and data from reactive molecular dynamic simulations, intrinsic reaction co-ordinate modeling, and covalent molecular docking also corroborate the selectivity of PGA2's C9 ß-carbon towards Nur77's Cys. In summary, our molecular, chemical, and structural characterization of the PGA2-Nur77 interaction provides the first evidence that PGA2 is an endogenous Nur77 agonist.


Subject(s)
Nuclear Receptor Subfamily 4, Group A, Member 1/agonists , Prostaglandins A/chemistry , Prostaglandins A/physiology , Cell Line , Humans , Nuclear Receptor Subfamily 4, Group A, Member 1/metabolism , Prostaglandins A/metabolism , Protein Binding , Protein Domains
5.
Biochem Biophys Res Commun ; 500(2): 204-210, 2018 06 02.
Article in English | MEDLINE | ID: mdl-29634926

ABSTRACT

Recently, the modulation of cellular inflammatory responses via endogenous regulators became a major focus of medically relevant investigations. Prostaglandins (PGs) are attractive regulatory molecules, but their synthesis and mechanisms of action in brain cells are still unclear. Astrocytes are involved in manifestation of neuropathology and their proliferation is an important part of astrogliosis, a cellular neuroinflammatory response. The aims of our study were to measure synthesis of PGs by astrocytes, and evaluate their influence on proliferation in combination with addition of inflammatory pathway inhibitors. With UPLC-MS/MS analysis we detected primary PGs (1410 ±â€¯36 pg/mg PGE2, 344 ±â€¯24 PGD2) and cyclopentenone PGs (cyPGs) (87 ±â€¯17 15d-PGJ2, 308 ±â€¯23 PGA2) in the extracellular medium after 24-h lipopolysaccharide (LPS) stimulation of astrocytes. PGs reduced astrocytic proliferation with the following order of potencies (measured as inhibition at 20 µM): most potent 15d-PGJ2 (90%) and PGA2 (80%), > PGD2 (40%) > 15d-PGA2 (20%) > PGE2 (5%), the least potent. However, PGF2α and 2-cyclopenten-1-one, and ciglitazone and rosiglitazone (synthetic agonists of PPARγ) had no effect. Combinations of cyPGs with SC-560 or NS-398 (specific anti-inflammatory inhibitors of cyclooxygenase-1 and -2, respectively) were not effective; while GW9662 (PPARγ antagonist) or MK-741 (inhibitor of multidrug resistance protein-1, MRP1, and CysLT1 receptors) amplified the inhibitory effect of PGA2 and 15d-PGJ2. Although concentrations of individual PGs and cyPGs are low, all of them, as well as primary PGs suppress proliferation. Thus, the effects are potentially additive, and activated PGs synthesis suppresses proliferation in astrocytes.


Subject(s)
Astrocytes/cytology , Astrocytes/metabolism , Cyclopentanes/metabolism , Prostaglandins/biosynthesis , Animals , Astrocytes/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Chromatography, Liquid , Lipopolysaccharides/pharmacology , PPAR gamma/agonists , PPAR gamma/metabolism , Prostaglandin D2/analogs & derivatives , Prostaglandin D2/chemistry , Prostaglandin D2/metabolism , Prostaglandins A/chemistry , Prostaglandins A/metabolism , Rats, Wistar , Tandem Mass Spectrometry
6.
Mol Biol Cell ; 28(12): 1622-1635, 2017 Jun 15.
Article in English | MEDLINE | ID: mdl-28428256

ABSTRACT

The role of prostaglandin A2 (PGA2) in modulation of vascular endothelial function is unknown. We investigated effects of PGA2 on pulmonary endothelial cell (EC) permeability and inflammatory activation and identified a receptor mediating these effects. PGA2 enhanced the EC barrier and protected against barrier dysfunction caused by vasoactive peptide thrombin and proinflammatory bacterial wall lipopolysaccharide (LPS). Receptor screening using pharmacological and molecular inhibitory approaches identified EP4 as a novel PGA2 receptor. EP4 mediated barrier-protective effects of PGA2 by activating Rap1/Rac1 GTPase and protein kinase A targets at cell adhesions and cytoskeleton: VE-cadherin, p120-catenin, ZO-1, cortactin, and VASP. PGA2 also suppressed LPS-induced inflammatory signaling by inhibiting the NFκB pathway and expression of EC adhesion molecules ICAM1 and VCAM1. These effects were abolished by pharmacological or molecular inhibition of EP4. In vivo, PGA2 was protective in two distinct models of acute lung injury (ALI): LPS-induced inflammatory injury and two-hit ALI caused by suboptimal mechanical ventilation and injection of thrombin receptor-activating peptide. These protective effects were abolished in mice with endothelial-specific EP4 knockout. The results suggest a novel role for the PGA2-EP4 axis in vascular EC protection that is critical for improvement of pathological states associated with increased vascular leakage and inflammation.


Subject(s)
Prostaglandins A/metabolism , Receptors, Prostaglandin E, EP4 Subtype/metabolism , Animals , Antigens, CD/metabolism , Cadherins/metabolism , Capillary Permeability , Cell Adhesion , Cell Line , Cell Membrane Permeability/physiology , Cyclic AMP-Dependent Protein Kinases/metabolism , Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Humans , Inflammation/metabolism , Intercellular Adhesion Molecule-1/metabolism , Lipopolysaccharides/pharmacology , Lung/metabolism , Mice , NF-kappa B/metabolism , Permeability , Signal Transduction/drug effects
7.
J Neuroinflammation ; 13(1): 148, 2016 06 14.
Article in English | MEDLINE | ID: mdl-27301644

ABSTRACT

BACKGROUND: Hyperactivation of microglia is considered to be a key hallmark of brain inflammation and plays a critical role in regulating neuroinflammatory events. Neuroinflammatory responses in microglia represent one of the major risk factors for various neurodegenerative diseases. One of the strategies to protect the brain and slow down the progression of these neurodegenerative diseases is by consuming diet enriched in anti-oxidants and polyphenols. Therefore, the present study aimed to evaluate the anti-inflammatory effects of rice bran extract (RBE), one of the rich sources of vitamin E forms (tocopherols and tocotrienols) and gamma-oryzanols, in primary rat microglia. METHODS: The vitamin E profile of the RBE was quantified by high-performance liquid chromatography (HPLC). Microglia were stimulated with lipopolysaccharide (LPS) in the presence or absence of RBE. Release of prostaglandins (prostaglandin (PG) E2, 8-iso-prostaglandin F2α (8-iso-PGF2α)) were determined with enzyme immunoassay (EIA). Protein levels and genes related to PGE2 synthesis (Cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1)) and various pro- and anti-inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-10), were assessed by western blot, ELISA, and quantitative real-time PCR. Furthermore, to elucidate the molecular targets of RBE, the phosphorylated state of various mitogen-activated protein kinase (MAPK) signaling molecules (p38 MAPK, ERK 1/2, and JNK) and activation of NF-kB pathway was studied. RESULTS: RBE significantly inhibited the release of PGE2 and free radical formation (8-iso-PGF2α) in LPS-activated primary microglia. Inhibition of PGE2 by RBE was dependent on reduced COX-2 and mPGES-1 immunoreactivity in microglia. Interestingly, treatment of activated microglia with RBE further enhanced the gene expression of the microglial M2 marker IL-10 and reduced the expression of pro-inflammatory M1 markers (TNF-α, IL-1ß). Further mechanistic studies showed that RBE inhibits microglial activation by interfering with important steps of MAPK signaling pathway. Additionally, microglia activation with LPS leads to IkB-α degradation which was not affected by the pre-treatment of RBE. CONCLUSIONS: Taken together, our data demonstrate that RBE is able to affect microglial activation by interfering in important inflammatory pathway. These in vitro findings further demonstrate the potential value of RBE as a nutraceutical for the prevention of microglial dysfunction related to neuroinflammatory diseases, including Alzheimer's disease.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Microglia/drug effects , Mitogen-Activated Protein Kinase Kinases/metabolism , Oryza/chemistry , Signal Transduction/drug effects , Animals , Animals, Newborn , Cell Survival/drug effects , Cells, Cultured , Cerebral Cortex/cytology , Cyclooxygenase 2/metabolism , Cytokines/metabolism , Dinoprostone/metabolism , Lipopolysaccharides/pharmacology , Prostaglandins A/metabolism , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Time Factors
8.
Mol Pharmacol ; 89(1): 42-52, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26487510

ABSTRACT

Aldose reductase (AKR1B1) is a critical drug target because of its involvement in diabetic complications, inflammation, and tumorigenesis. However, to date, development of clinically useful inhibitors has been largely unsuccessful. Cyclopentenone prostaglandins (cyPGs) are reactive lipid mediators that bind covalently to proteins and exert anti-inflammatory and antiproliferative effects in numerous settings. By pursuing targets for modification by cyPGs we have found that the cyPG PGA1 binds to and inactivates AKR1B1. A PGA1-AKR1B1 adduct was observed, both by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and by SDS-PAGE using biotinylated PGA1 (PGA1-B). Insight into the molecular interactions between AKR1B1 and PGA1 was advanced by molecular modeling. This anticipated the addition of PGA1 to active site Cys298 and the potential reversibility of the adduct, which was supported experimentally. Indeed, loss of biotin label from the AKR1B1-PGA1-B adduct was favored by glutathione, indicating a retro-Michael reaction, which unveils new implications of cyPG-protein interaction. PGA1 elicited only marginal inhibition of aldehyde reductase (AKR1A1), considered responsible for the severe adverse effects of many AKR1B1 inhibitors. Interestingly, other prostaglandins (PGs) inhibited the enzyme, including non-electrophilic PGE1 and PGE2, currently used in clinical practice. Moreover, both PGA1 and PGE1 reduced the formation of sorbitol in an ex-vivo model of diabetic cataract to an extent comparable to that attained by the known AKR inhibitor epalrestat. Taken together, these results highlight the role of PGs as AKR1B1 inhibitors and the interest in PG-related molecules as leads for the development of novel pharmacological tools.


Subject(s)
Aldehyde Reductase/antagonists & inhibitors , Aldehyde Reductase/metabolism , Prostaglandins A/metabolism , Prostaglandins A/pharmacology , Animals , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Humans , Male , Prostaglandins/metabolism , Prostaglandins/pharmacology , Protein Binding/physiology , Rats , Rats, Wistar
9.
Antiviral Res ; 117: 1-9, 2015 May.
Article in English | MEDLINE | ID: mdl-25666760

ABSTRACT

Previous studies have demonstrated that cyclopentenone prostaglandins (cyPGs) inhibit the replication of a wide variety of DNA and RNA viruses in different mammalian cell types. We investigated a new role for prostaglandin A1 (PGA1) in the inhibition of hepatitis C virus (HCV)-IRES-mediated translation. PGA1 exhibited dose-dependent inhibitory effects on HCV translation in HCV replicon cells. Furthermore, repetitive PGA1 treatment demonstrated the potential to safely induce the suppression of HCV translation. We also validated a new role for PGA1 in the inhibition of HCV-IRES-mediated translation by targeting cellular translation factors, including the small ribosomal subunit (40S) and eukaryotic initiation factors (eIFs). In pull-down assays, biotinylated PGA1 co-precipitated with the entire HCV IRES RNA/eIF3-40S subunit complex. Moreover, the interactions between PGA1 and the elongation factors and ribosomal subunit were dependent upon HCV IRES RNA binding, and the PGA1/HCV IRES RNA/eIF3-40S subunit complex inhibited HCV-IRES-mediated translation. The novel mechanism revealed in this study may aid in the search for more effective anti-HCV drugs.


Subject(s)
Hepacivirus/growth & development , Hepacivirus/genetics , Hepacivirus/metabolism , Prostaglandins A/metabolism , Prostaglandins A/pharmacology , Replicon/drug effects , Ribosome Subunits, Small/drug effects , Cell Line, Tumor , Eukaryotic Initiation Factor-3/metabolism , Humans , Internal Ribosome Entry Sites , Protein Biosynthesis/drug effects , RNA, Viral/genetics , Replicon/physiology , Ribosome Subunits, Small/metabolism
10.
Chin Med J (Engl) ; 127(9): 1608-12, 2014.
Article in English | MEDLINE | ID: mdl-24791862

ABSTRACT

BACKGROUND: Pulmonary hypertension (PH) is a common complication of chronic obstructive pulmonary disease (COPD). Although alveolar hypoxia is considered as a main cause of PH in COPD, structural and functional changes of pulmonary circulation are apparent at the initial stage of COPD. We hypothesized that an inflammatory response and oxidative stress might contribute to the formation of PH in COPD. METHODS: We measured the levels of interleukin-6 (IL-6) and 8-iso-prostaglandin (8-iso-PSG) in exhaled breath condensate (EBC) and serum in 40 patients with COPD only or in 45 patients with COPD combined with PH. Pulmonary arterial systolic pressure (PASP) was assessed by Doppler echocardiography and defined as PH when the value of systolic pressure was greater than 40 mmHg. RESULTS: Compared with the COPD only group, the level of IL-6 in EBC was significantly increased in all 45 patients with COPD combined with PH ((8.27±2.14) ng/L vs. (4.95±1.19) ng/L, P < 0.01). The level of IL-6 in serum was also elevated in patients with COPD combined with PH compared with the COPD only group ((72.8±21.6) ng/L vs. (43.58±13.38) ng/L, P < 0.01). Similarly, we also observed a significant increase in the level of 8-iso-PSG in both EBC and serum in the COPD with PH group, compared with the COPD only group (EBC: (9.00±2.49) ng/L vs. (5.96±2.31) ng/L, P < 0.01 and serum: (41.87±9.75) ng/L vs. (27.79±11.09) ng/L, P < 0.01). Additionally, the value of PASP in the PH group was confirmed to be positively correlated with the increase in the levels of IL-6 and 8-iso-PSG in both EBC and serum (r = 0.477-0.589, P < 0.05). CONCLUSION: The increase in the levels of IL-6 and 8-iso-PSG in EBC and serum correlates with the pathogenesis of PH in COPD.


Subject(s)
Hypertension, Pulmonary/blood , Hypertension, Pulmonary/metabolism , Interleukin-6/blood , Interleukin-6/metabolism , Prostaglandins A/blood , Prostaglandins A/metabolism , Pulmonary Disease, Chronic Obstructive/blood , Pulmonary Disease, Chronic Obstructive/metabolism , Aged , Breath Tests , Female , Humans , Male , Middle Aged
11.
Atherosclerosis ; 214(1): 122-8, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21056418

ABSTRACT

OBJECTIVES: We examined the time-dependent effects of atorvastatin and rosuvastatin on in vivo oxidative stress and platelet activation, to assess whether these phenomena are related to any pleiotropic effect of any statin or to their LDL-lowering effect. We also asked whether the presence of specific allele frequencies in carriers of the 3'UTR/lectin-like oxidized LDL receptor-1 (LOX-1) polymorphism may influence the effect of either statin. METHODS: We included 60 hypercholesterolemic subjects, previously screened for LOX-1 3'UTR polymorphism, randomized, according to genetic profile (15 T and 15 C carriers for each arm), to atorvastatin 20mg/day or rosuvastatin 10mg/day. RESULTS: After 8 weeks, atorvastatin and rosuvastatin were associated with comparable, significant reductions in LDL cholesterol (40.8% and 43.6%, respectively), plasma hs-CRP (9.5% vs. 13.8%), urinary 11-dehydro-thromboxane (TX) B(2) (38.9% vs. 27.1%) and 8-iso-prostaglandin (PG) F(2α) (39.4% vs. 19.4%). The impact of rosuvastatin or atorvastatin on CRP, 8-iso-PGF(2α), and 11-dehydro-TXB(2) did not differ according to the LOX-1 haplotype. On multiple regression analyses, only CRP and LDL were independent predictors of 11-dehydro-TXB(2), and only LDL was a significant predictor of 8-iso-PGF(2α). CONCLUSIONS: Both atorvastatin and rosuvastatin cause comparable reductions of thromboxane-dependent platelet activation, lipid peroxidation and inflammation. The presence of 3'UTR/LOX-1 polymorphism does not affect the changes induced by either statin.


Subject(s)
Blood Platelets/drug effects , Fluorobenzenes/pharmacology , Heptanoic Acids/pharmacology , Hypercholesterolemia/metabolism , Pyrimidines/pharmacology , Pyrroles/pharmacology , Sulfonamides/pharmacology , Thromboxane B2/metabolism , 3' Untranslated Regions , Anticholesteremic Agents/pharmacology , Atorvastatin , Double-Blind Method , Female , Humans , Lipids/chemistry , Male , Middle Aged , Oxidative Stress , Platelet Activation , Polymorphism, Genetic , Prospective Studies , Prostaglandins A/metabolism , Regression Analysis , Rosuvastatin Calcium
12.
Prostaglandins Other Lipid Mediat ; 91(1-2): 30-7, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20044024

ABSTRACT

HL-60 cells treated by prostaglandin (PG) A(2) showed characteristics of apoptosis such as accumulation of hypodiploid and annexin V positive cells, condensed and fragmented nuclei, cytochrome c (Cyt C) release from mitochondria and activation of caspase-1, -2, -3, -7 and -9. PGA(2)-induced cell death was rescued by inhibitors of caspase-9 and -3, but PGA(2)-induced Cyt C release was not prevented by caspase inhibitors. During Cyt C release by PGA(2), mitochondrial transmembrane potential was maintained and mitochondrial permeability transition pore was not formed. In addition, anti-apoptotic BCL-2 family proteins like BCL-2 and BCL-XL, and ROS scavengers including ascorbic acid and 2,2,6,6-tetramethyl-1-piperidinyloxy were not able to inhibit Cyt C release as well as apoptosis by PGA(2). Finally, it was shown that PGA(2)-induced Cyt C release in vitro from purified mitochondria in the absence of cytosolic components. Furthermore, thiol-containing compounds such as N-acetylcysteine, l-cysteine and monothioglycerol prevented Cyt C release, and hence induction of apoptosis. Taken together, these results suggest that PGA(2) activates intrinsic apoptotic pathway by directly stimulating mitochondrial outer membrane permeabilization to release Cyt C, in which thiol-reactivity of PGA(2) plays a pivotal role.


Subject(s)
Apoptosis/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , Prostaglandins A/metabolism , Prostaglandins A/pharmacology , Signal Transduction/drug effects , Animals , Caspase 3/metabolism , Cell Membrane Permeability/drug effects , Cytochromes c/metabolism , Enzyme Activation/drug effects , Free Radical Scavengers/metabolism , HL-60 Cells , Humans , Membrane Potential, Mitochondrial/drug effects , Mitochondrial Membranes/drug effects , Mitochondrial Membranes/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Reactive Oxygen Species/metabolism , Sulfhydryl Compounds/metabolism
13.
Free Radic Biol Med ; 46(6): 799-809, 2009 Mar 15.
Article in English | MEDLINE | ID: mdl-19159678

ABSTRACT

Resveratrol (RSV), a naturally occurring phytoalexin that can be found in red wine, berries, and peanuts, has been shown to extend both mean and maximum life span in model organisms. RSV has also been reported to shift the physiology of middle-aged mice on a high-calorie diet toward that of mice on a standard diet. These beneficial effects of RSV have been suggested to resemble caloric restriction. Our study in F2 four-way cross-hybrid mice was the first to evaluate the effects of aging and long-term RSV treatment (14.09+/-3.4 mg/L in drinking water for 6 or 12 months) on biomarkers of oxidative damage to DNA, 8-hydroxy-2'-deoxyguanosine (8OHdG); lipid, 8-iso-prostaglandin(2 alpha) (8-iso-PGF(2 alpha)); and protein, protein carbonyl content (PCC). There was a significant age-dependent accumulation of oxidative damage to DNA, lipid, and protein as well as a clear increase in urine 8-iso-PGF(2 alpha) levels in the majority of mouse tissues. Rates of age-dependent increases in damage biomarkers varied between tissues. Chronic RSV treatment elevated total RSV plasma levels and reduced the observed age-dependent accumulation of (1) 8OHdG in liver and heart, (2) 8-iso-PGF(2 alpha) in heart and urine, and (3) PCC in liver and kidney. However, a 12-month RSV intake resulted in significant elevation of 8-iso-PGF(2 alpha) and PCC in kidney. Our studies demonstrate that RSV treatment consistently attenuated oxidative damage in tissues where age-related oxidative damage accumulation was prominent, but also suggested that chronic RSV treatment may induce nephrotoxicity.


Subject(s)
Biomarkers/metabolism , Deoxyguanosine/analogs & derivatives , Kidney/metabolism , Prostaglandins A/metabolism , Stilbenes/administration & dosage , 8-Hydroxy-2'-Deoxyguanosine , Administration, Oral , Aging/immunology , Animals , Breeding , DNA Damage , Deoxyguanosine/genetics , Deoxyguanosine/metabolism , Kidney/immunology , Kidney/pathology , Mice , Myocardium/metabolism , Myocardium/pathology , Organ Specificity , Oxidative Stress/immunology , Prostaglandins A/genetics , Protein Carbonylation/immunology , Resveratrol , Stilbenes/adverse effects
14.
Circ Res ; 103(9): 1037-46, 2008 Oct 24.
Article in English | MEDLINE | ID: mdl-18802021

ABSTRACT

Isoprostanes are endogenously formed end products of lipid peroxidation. Furthermore, they are markers of oxidative stress and independent risk markers of coronary heart disease. In patients experiencing coronary heart disease, impaired angiogenesis may exacerbate insufficient blood supply of ischemic myocardium. We therefore hypothesized that isoprostanes may exert detrimental cardiovascular effects by inhibiting angiogenesis. We studied the effect of isoprostanes on vascular endothelial growth factor (VEGF)-induced migration and tube formation of human endothelial cells (ECs), and cardiac angiogenesis in vitro as well as on VEGF-induced angiogenesis in the chorioallantoic membrane assay in vivo. The isoprostanes 8-iso-PGF(2alpha), 8-iso-PGE(2), and 8-iso-PGA(2) inhibited VEGF-induced migration, tube formation of ECs, and cardiac angiogenesis in vitro, as well as VEGF-induced angiogenesis in vivo via activation of the thromboxane A(2) receptor (TBXA2R): the specific TBXA2R antagonists SQ-29548, BM 567, and ICI 192,605 but not the thromboxane A(2) synthase inhibitor ozagrel blocked the effect of isoprostanes. The isoprostane 8-iso-PGA(2) degraded into 2 biologically active derivatives in vitro, which also inhibited EC tube formation via the TBXA2R. Moreover, short hairpin RNA-mediated knockdown of the TBXA2R antagonized isoprostane-induced effects. In addition, Rho kinase inhibitor Y-27632 reversed the inhibitory effect of isoprostanes and the thromboxane A(2) mimetic U-46619 on EC migration and tube formation. Finally, the various isoprostanes exerted a synergistic inhibitory effect on EC tube formation. We demonstrate for the first time that isoprostanes inhibit angiogenesis via activation of the TBXA2R. By this mechanism, isoprostanes may contribute directly to exacerbation of coronary heart disease and to capillary rarefaction in disease states of increased oxidative stress.


Subject(s)
Angiogenesis Inhibitors/metabolism , Cell Movement , Coronary Vessels/metabolism , Endothelial Cells/metabolism , Isoprostanes/metabolism , Neovascularization, Physiologic , Oxidative Stress , Receptors, Thromboxane A2, Prostaglandin H2/metabolism , Vascular Endothelial Growth Factor A/metabolism , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology , Actins/metabolism , Angiogenesis Inhibitors/pharmacology , Animals , Apoptosis , Bridged Bicyclo Compounds, Heterocyclic , Cell Movement/drug effects , Cells, Cultured , Chick Embryo , Chorioallantoic Membrane/blood supply , Coronary Vessels/drug effects , Dinoprost/analogs & derivatives , Dinoprost/metabolism , Dinoprostone/analogs & derivatives , Dinoprostone/metabolism , Dioxanes/pharmacology , Endothelial Cells/drug effects , Endothelial Cells/enzymology , Endothelial Cells/pathology , Extracellular Signal-Regulated MAP Kinases/metabolism , Fatty Acids, Unsaturated , Focal Adhesion Kinase 1/metabolism , Humans , Hydrazines/pharmacology , Mice , Neovascularization, Physiologic/drug effects , Phosphorylation , Prostaglandins A/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Receptors, Thromboxane A2, Prostaglandin H2/drug effects , Receptors, Thromboxane A2, Prostaglandin H2/genetics , Stress Fibers/metabolism , Sulfonylurea Compounds/pharmacology , Tissue Culture Techniques
15.
J Neurosci Res ; 86(5): 1132-41, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18074385

ABSTRACT

We have previously reported that prostaglandin A(1) (PGA(1)) reduces infarct size in rodent models of focal ischemia. This study seeks to elucidate the possible molecular mechanisms underlying PGA(1)'s neuroprotective effects against ischemic injury. Rats were subjected to permanent middle cerebral artery occlusion (pMCAO) by intraluminal suture blockade. PGA(1) was injected intracerebroventricularly (icv) immediately after ischemic onset. Western blot analysis was employed to determine alterations in IkappaBalpha, pIKKalpha, and peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Immunohistochemistry was used to confirm the nuclear translocation of nuclear factor-kappaB (NF-kappaB) p65 and the expression of PPAR-gamma. RT-PCR was used to detect levels of c-Myc mRNA. The contribution of PPAR-gamma to PGA(1)'s neuroprotection was evaluated by pretreatment with the PPAR-gamma irreversible antagonist GW9662. A brief increase in pIKKalpha levels and rapid reduction in IkappaBalpha were observed after ischemia. PGA(1) blocked ischemia-induced increases in pIKKalpha levels and reversed the decline in IkappaBalpha levels. Ischemia-induced nuclear translocation of NF-kappaB p65 was attenuated by PGA(1). PGA(1) also repressed the ischemia-induced increase in expression of NF-kappaB target gene c-Myc mRNA. Immunohistochemistry demonstrated an increase in PPAR-gamma immunoreactivity in the nucleus of striatal cells at 3 hr after pMCAO. Western blot analysis revealed that the expression of PPAR-gamma protein significantly increased at 12 hr and peaked at 24 hr. PGA(1) enhanced the ischemia-triggered induction of PPAR-gamma protein. Pretreatment with the irreversible PPAR-gamma antagonist GW9662 attenuated PGA(1)'s neuroprotection against ischemia. These findings suggest that PGA(1)-mediated neuroprotective effect against ischemia appears to be associated with blocking NF-kappaB activation and likely with up-regulating PPAR-gamma expression.


Subject(s)
Brain Infarction/drug therapy , Brain Ischemia/drug therapy , NF-kappa B/antagonists & inhibitors , Nerve Degeneration/drug therapy , PPAR gamma/agonists , Prostaglandins A/pharmacology , Active Transport, Cell Nucleus/drug effects , Active Transport, Cell Nucleus/genetics , Anilides/pharmacology , Animals , Brain Infarction/metabolism , Brain Ischemia/metabolism , Corpus Striatum/drug effects , Corpus Striatum/metabolism , Cytoprotection/drug effects , Disease Models, Animal , I-kappa B Proteins/drug effects , I-kappa B Proteins/metabolism , Infarction, Middle Cerebral Artery/drug therapy , Infarction, Middle Cerebral Artery/metabolism , Male , NF-kappa B/metabolism , Nerve Degeneration/metabolism , Neuroprotective Agents/metabolism , Neuroprotective Agents/pharmacology , PPAR gamma/antagonists & inhibitors , PPAR gamma/metabolism , Prostaglandins A/metabolism , Proto-Oncogene Proteins c-myc/genetics , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Transcription Factor RelA/antagonists & inhibitors , Transcription Factor RelA/metabolism , Up-Regulation/drug effects
16.
EMBO J ; 26(24): 5020-32, 2007 Dec 12.
Article in English | MEDLINE | ID: mdl-18034160

ABSTRACT

The signaling lipid molecule 15-deoxy-delta 12,14-prostaglandin J2 (15d-PGJ2) has multiple cellular functions, including anti-inflammatory and antineoplastic activities. Here, we report that 15d-PGJ2 blocks translation through inactivation of translational initiation factor eIF4A. Binding of 15d-PGJ2 to eIF4A blocks the interaction between eIF4A and eIF4G that is essential for translation of many mRNAs. Cysteine 264 in eIF4A is the target site of 15d-PGJ2. The antineoplastic activity of 15d-PGJ2 is likely attributed to inhibition of translation. Moreover, inhibition of translation by 15d-PGJ2 results in stress granule (SG) formation, into which TRAF2 is sequestered. The sequestration of TRAF2 contributes to the anti-inflammatory activity of 15d-PGJ2. These findings reveal a novel cross-talk between translation and inflammatory response, and offer new approaches to develop anticancer and anti-inflammatory drugs that target translation factors including eIF4A.


Subject(s)
Anti-Inflammatory Agents/metabolism , Eukaryotic Initiation Factor-4A/metabolism , Prostaglandin D2/analogs & derivatives , Protein Biosynthesis , Arachidonic Acid/metabolism , Arsenites/metabolism , Chromans/metabolism , Cyclopentanes/metabolism , Cytoplasmic Granules/chemistry , Cytoplasmic Granules/metabolism , Dinoprostone/metabolism , Emetine/metabolism , Enzyme Inhibitors/metabolism , Eukaryotic Initiation Factor-2/genetics , Eukaryotic Initiation Factor-2/metabolism , Eukaryotic Initiation Factor-4A/genetics , Gene Expression Regulation , HeLa Cells , Humans , Hypoglycemic Agents/metabolism , Inflammation/genetics , PPAR gamma/metabolism , Poly(A)-Binding Proteins/genetics , Poly(A)-Binding Proteins/metabolism , Prostaglandin D2/metabolism , Prostaglandins A/metabolism , Protein Synthesis Inhibitors/metabolism , Rosiglitazone , Signal Transduction/physiology , Sodium Compounds/metabolism , T-Cell Intracellular Antigen-1 , TNF Receptor-Associated Factor 2/genetics , TNF Receptor-Associated Factor 2/metabolism , Thiazolidinediones/metabolism , Troglitazone , Tumor Necrosis Factor-alpha/metabolism
17.
J Mass Spectrom ; 42(11): 1474-84, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17960581

ABSTRACT

Prostaglandins with cyclopentenone structure (cyPG) display potent antiproliferative actions that have elicited their study as potential anticancer agents. Several natural and synthetic analogs of the cyPG prostaglandin A(1) (PGA(1)) have proven antitumoral efficacy in cancer cell lines and animal models. In addition, PGA(1) has been used as an inhibitor of transcription factor NF-kappaB-mediated processes, including inflammatory gene expression and viral replication. An important determinant for these effects is the ability of cyPG to form Michael adducts with free thiol groups. The chemical nature of this interaction implies that PGA(1) could covalently modify cysteine residues in a large number of cellular proteins potentially involved in its beneficial effects. However, only a few targets of PGA(1) have been identified. In previous work, we have observed that a biotinylated analog of PGA(1) that retains the cyclopentenone moiety (PGA(1)-B) binds to multiple targets in fibroblasts. Here, we have addressed the identification of these targets through a proteomic approach. Cell fractionation followed by avidin affinity chromatography yielded a fraction enriched in proteins modified by PGA(1)-B. Analysis of this fraction by SDS-PAGE and LC-MS/MS allowed the identification of the chaperone Hsp90, elongation and initiation factors for protein synthesis and cytoskeletal proteins including actin, tubulin and vimentin. Furthermore, we have characterized the modification of vimentin both in vitro and in intact cells. Our observations indicate that cysteine 328 is the main site for PGA(1) addition. These results may contribute to a better understanding of the mechanism of action of PGA(1) and the potential of cyPG-based therapeutic strategies.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/metabolism , Antineoplastic Agents/metabolism , Prostaglandins A/metabolism , Vimentin/metabolism , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Avidin/chemistry , Biotinylation , COS Cells , Chlorocebus aethiops , Cysteine/chemistry , Cysteine/genetics , Cysteine/metabolism , Eukaryotic Initiation Factor-4A/chemistry , Eukaryotic Initiation Factor-4A/metabolism , Glial Fibrillary Acidic Protein/chemistry , Glial Fibrillary Acidic Protein/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HSP90 Heat-Shock Proteins/chemistry , HSP90 Heat-Shock Proteins/metabolism , Humans , Mice , Mutation , NIH 3T3 Cells , Peptide Elongation Factor 1/chemistry , Peptide Elongation Factor 1/metabolism , Prostaglandins A/chemistry , Prostaglandins A/pharmacology , Protein Processing, Post-Translational/drug effects , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tandem Mass Spectrometry , Transfection , Tubulin/chemistry , Tubulin/metabolism , Vimentin/chemistry , Vimentin/genetics
18.
Biochemistry ; 46(22): 6607-16, 2007 Jun 05.
Article in English | MEDLINE | ID: mdl-17489560

ABSTRACT

Cyclopentenone prostanoids (cyP) arise as important modulators of inflammation and cell proliferation. Although their physiological significance has not been fully elucidated, their potent biological effects have spurred their study as leads for the development of therapeutic agents. A key determinant of cyP action is their ability to bind to thiol groups in proteins or in glutathione through Michael addition. Even though several protein targets for cyP addition have been identified, little is known about the structural determinants from the protein or the cyP that drive this modification. The results herein presented provide the first evidence that cyP with different structures target distinct thiol sites in a protein molecule, namely, H-Ras. Whereas 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) and Delta12-PGJ2 preferentially target the C-terminal region containing cysteines 181 and 184, PGA1 and 8-iso-PGA1 bind mainly to cysteine 118, located in the GTP-binding motif. The biological counterparts of this specificity are the site-selective modification and activation of H-Ras in cells and the differential interaction of cyP with H, N, and K-Ras proteins. Cysteine 184 is unique to H-Ras, whereas cysteine 118 is present in the three Ras homologues. Consistent with this, PGA1 binds to and activates H-, N-, and K-Ras, thus differing from the preferential interaction of 15d-PGJ2 with H-Ras. These results put forward the possibility of influencing the selectivity of cyP-protein addition by modifying cyP structure. Furthermore, they may open new avenues for the development of cyP-based drugs.


Subject(s)
Prostaglandins/pharmacology , ras Proteins/drug effects , ras Proteins/metabolism , Animals , Anti-Inflammatory Agents/metabolism , Anti-Inflammatory Agents/pharmacology , Binding Sites/drug effects , Cysteine/chemistry , Cysteine/drug effects , Gene Expression Regulation , HeLa Cells , Humans , Mice , Prostaglandin D2/analogs & derivatives , Prostaglandin D2/metabolism , Prostaglandin D2/pharmacology , Prostaglandins/chemistry , Prostaglandins/metabolism , Prostaglandins A/metabolism , Prostaglandins A/pharmacology , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/classification , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tandem Mass Spectrometry , Tumor Cells, Cultured , ras Proteins/chemistry , ras Proteins/classification
19.
Biochem Biophys Res Commun ; 349(4): 1387-93, 2006 Nov 03.
Article in English | MEDLINE | ID: mdl-16979589

ABSTRACT

Pathophysiological processes associated with oxidative stress lead to the generation of reactive lipid species. Among them, lipids bearing unsaturated aldehyde or ketone moieties can form covalent adducts with cysteine residues and modulate protein function. Through proteomic techniques we have identified actin as a target for the addition of biotinylated analogs of the cyclopentenone prostaglandins 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) and PGA(1) in NIH-3T3 fibroblasts. This modification could take place in vitro and mapped to the protein C-terminal end. Other electrophilic lipids, like the isoprostane 8-iso-PGA(1) and 4-hydroxy-2-nonenal, also bound to actin. The C-terminal region of actin is important for monomer-monomer interactions and polymerization. Electron microscopy showed that actin treated with 15d-PGJ(2) or 4-hydroxy-2-nonenal formed filaments which were less abundant and displayed shorter length and altered structure. Streptavidin-gold staining allowed mapping of biotinylated 15d-PGJ(2) at sites of filament disruption. These results shed light on the structural implications of actin modification by lipid electrophiles.


Subject(s)
Actins/metabolism , Actins/ultrastructure , Muscle, Skeletal/metabolism , Prostaglandin D2/analogs & derivatives , Prostaglandins A/metabolism , Actins/chemistry , Animals , Binding Sites , Mice , NIH 3T3 Cells , Prostaglandin D2/chemistry , Prostaglandin D2/metabolism , Prostaglandins A/chemistry , Protein Binding , Protein Conformation , Rabbits
20.
J Neurochem ; 97(5): 1301-13, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16638022

ABSTRACT

Oxidative stress and subsequent lipid peroxidation are involved in the pathogenesis of numerous neurodegenerative conditions, including stroke. Cyclopentenone isoprostanes (IsoPs) are novel electrophilic lipid peroxidation products formed under conditions of oxidative stress via the isoprostane pathway. These cyclopentenone IsoPs are isomeric to highly bioactive cyclopentenone prostaglandins, yet it has not been determined if these products are biologically active or are formed in the brain. Here we demonstrate that the major cyclopentenone IsoP isomer 15-A2t-IsoP potently induces apoptosis in neuronal cultures at submicromolar concentrations. We present a model in which 15-A2t-IsoP induced neuronal apoptosis involves initial depletion of glutathione and enhanced production of reactive oxygen species, followed by 12-lipoxygenase activation and phosphorylation of extracellular signal-regulated kinase 1/2 and the redox sensitive adaptor protein p66shc, which results in caspase-3 cleavage. 15-A2t-IsoP application also dramatically potentiates oxidative glutamate toxicity at concentrations as low as 100 nm, demonstrating the functional importance of these molecules in neurodegeneration. Finally, we employ novel mass spectrometric methods to show that cyclopentenone IsoPs are formed abundantly in brain tissue under conditions of oxidative stress. Together these findings suggest that cyclopentenone IsoPs may contribute to neuronal death caused by oxidative insults, and that their activity should perhaps be addressed when designing neuroprotective therapies.


Subject(s)
Cyclopentanes/chemistry , Isoprostanes/toxicity , Lipid Peroxidation/physiology , Nerve Degeneration/chemically induced , Nerve Degeneration/metabolism , Oxidative Stress/drug effects , Adaptor Proteins, Signal Transducing/metabolism , Animals , Arachidonate 12-Lipoxygenase/metabolism , Cells, Cultured , Dose-Response Relationship, Drug , Excitatory Amino Acid Agonists/pharmacology , Glutamic Acid/toxicity , Glutathione/deficiency , Isoprostanes/chemistry , Isoprostanes/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Models, Neurological , Molecular Structure , Nerve Degeneration/physiopathology , Neurons/drug effects , Neurons/metabolism , Neurons/pathology , Oxidative Stress/physiology , Phosphorylation/drug effects , Prostaglandins A/chemistry , Prostaglandins A/metabolism , Prostaglandins A/toxicity , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Shc Signaling Adaptor Proteins , Src Homology 2 Domain-Containing, Transforming Protein 1
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