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The lipid peroxidation product 4-hydroxy-2-nonenal induces tissue factor decryption via ROS generation and the thioredoxin system.

ABSTRACT: Many pathophysiologic agents transform cryptic tissue factor (TF) on cells to prothrombotic TF, and one such stimulus is 4-hydroxy-2-nonenal (HNE), the most abundant aldehyde produced by the oxidation of ?-6 polyunsaturated fatty acids. HNE was shown to induce reactive oxygen species (ROS) generation and p38 MAPK activation, but the link between them and their role in TF decryption are unclear. The present study was carried out to elucidate potential mechanisms involved in HNE-induced TF decryption in monocytic cells. The data presented herein show that mitochondria are the primary source for HNE-induced ROS generation. The inhibition of mitochondrial electron transport chain complex III and V blocked HNE-induced ROS generation, but not p38 MAPK activation. These inhibitors reduced phosphatidylserine (PS) externalization and TF decryption significantly, but not completely. HNE treatment inhibited the activities of thioredoxin reductase (TrxR) and thioredoxin (Trx), independent of ROS. Inhibition of the TrxR/Trx system by HNE or pharmacological inhibitors induced p38 MAPK activation, PS externalization, and TF decryption. Additional studies revealed that the inhibition of TrxR/Trx led to activation of apoptosis signal-regulating kinase (ASK-1) and mitogen-activated protein kinase kinase 3/6. Inhibition of ASK-1 expression by small interfering RNA or its activity by pharmacological inhibitors diminished HNE-induced TF decryption. Overall, our data suggest that HNE induces TF decryption by 2 distinctive pathways. One is ROS dependent but independent of p38 MAPK activation, and the other is via TrxR/Trx and is p38 MAPK activation dependent. However, both mechanisms result in the enhancement of PS at the outer leaflet that is responsible for TF decryption.

SUBMITTER: Ansari SA

PROVIDER: S-EPMC5729625 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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The lipid peroxidation product 4-hydroxy-2-nonenal induces tissue factor decryption via ROS generation and the thioredoxin system.

Ansari Shabbir A SA Pendurthi Usha R UR Rao L Vijaya Mohan LVM

Blood advances 20171127 25

Many pathophysiologic agents transform cryptic tissue factor (TF) on cells to prothrombotic TF, and one such stimulus is 4-hydroxy-2-nonenal (HNE), the most abundant aldehyde produced by the oxidation of ω-6 polyunsaturated fatty acids. HNE was shown to induce reactive oxygen species (ROS) generation and p38 MAPK activation, but the link between them and their role in TF decryption are unclear. The present study was carried out to elucidate potential mechanisms involved in HNE-induced TF decrypt ...[more]

PMID: 29296890

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Project description:Reactive oxygen species (ROS) are elevated in the heart in response to hemodynamic and metabolic stress and promote hypertrophic signaling. ROS also mediate the formation of lipid peroxidation-derived aldehydes that may promote myocardial hypertrophy. One lipid peroxidation by-product, 4-hydroxy-trans-2-nonenal (HNE), is a reactive aldehyde that covalently modifies proteins thereby altering their function. HNE adducts directly inhibit the activity of LKB1, a serine/threonine kinase involved in regulating cellular growth in part through its interaction with the AMP-activated protein kinase (AMPK), but whether this drives myocardial growth is unclear. We tested the hypothesis that HNE promotes myocardial protein synthesis and if this effect is associated with impaired LKB1-AMPK signaling. In adult rat ventricular cardiomyocytes, exposure to HNE (10 ?M for 1h) caused HNE-LKB1 adduct formation and inhibited LKB1 activity. HNE inhibited the downstream kinase AMPK, increased hypertrophic mTOR-p70S6K-RPS6 signaling, and stimulated protein synthesis by 27.1 ± 3.5%. HNE also stimulated Erk1/2 signaling, which contributed to RPS6 activation but was not required for HNE-stimulated protein synthesis. HNE-stimulated RPS6 phosphorylation was completely blocked using the mTOR inhibitor rapamycin. To evaluate if LKB1 inhibition by itself could promote the hypertrophic signaling changes observed with HNE, LKB1 was depleted in adult rat ventricular myocytes using siRNA. LKB1 knockdown did not replicate the effect of HNE on hypertrophic signaling or affect HNE-stimulated RPS6 phosphorylation. Thus, in adult cardiac myocytes HNE stimulates protein synthesis by activation of mTORC1-p70S6K-RPS6 signaling most likely mediated by direct inhibition of AMPK. Because HNE in the myocardium is commonly increased by stimuli that cause pathologic hypertrophy, these findings suggest that therapies that prevent activation of mTORC1-p70S6K-RPS6 signaling may be of therapeutic value.

Project description:Peroxidation of cellular membrane lipids, rich in polyunsaturated fatty acids, generates electrophilic, ?, ?-unsaturated aldehydes such as 4-hydroxy-2-nonenal (HNE). HNE is a highly reactive and cytotoxic molecule that can react with the nucleophilic sites in proteins causing posttranslational modification. The identification of protein targets is an important first step; however, quantitative profiling of site-specific modifications is necessary to understand the biological impact of HNE-induced carbonylation. We report a method that uses light (H(12)CHO) and heavy (D(13)CDO) isotopic variant of formaldehyde to differentially label primary amines (N-termini and ?-amino group of lysines) in peptides through reductive methylation and, combined with selective enrichment of modified peptides, permits comparison of the extent of carbonylation in two samples after mixing for simultaneous liquid chromatography-mass spectrometry. Specifically, dimethyl-labeled peptide carbonyls were fractionated from unmodified peptides using solid-phase hydrazide chemistry to immobilize them to porous glass beads and, after removing the unmodified peptides by thoroughly washing the beads, subsequently recover them by acid-catalyzed hydrolysis. The method was developed using HNE-modified synthetic peptides and also showing enrichment from a complex matrix of digested human plasma proteins. Applicability was confirmed using apomyoglobin as an analyte, implicating thereby its potential value to proteome-wide identification and relative quantification of posttranslational protein carbonylation with residue-specific information. Because HNE attachment may not necessarily cause change in protein abundance, this modification-focused quantification should facilitate the characterization of accompanied changes in protein function and, also, provide important insights into molecular signaling mechanisms and a better understanding of cellular processes associated with oxidative stress.

Dataset Information

The lipid peroxidation product 4-hydroxy-2-nonenal induces tissue factor decryption via ROS generation and the thioredoxin system.

Publications

The lipid peroxidation product 4-hydroxy-2-nonenal induces tissue factor decryption via ROS generation and the thioredoxin system.

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