Lipid peroxidation and cell cycle signaling: 4-hydroxynonenal,a key molecule in stress mediated signaling

Role of lipid peroxidation products, particularly 4-hydroxynonenal (4-HNE) in cell cycle signaling is becoming increasingly clear. In this article, recent studies suggesting an important role of 4-HNE in stress mediated signaling for apoptosis are critically evaluated. Evidence demonstrating the modulation of UV, oxidative stress, and chemical stress mediated apoptosis by blocking lipid peroxidation by the alpha-class glutathione S-transferases (GSTs) is presented which suggest an important role of these enzymes in protection against oxidative stress and a role of lipid peroxidation products in stress mediated signaling. Overexpression of 4-HNE metabolizing GSTs (mGSTA4-4, hGSTA4-4, or hGST5.8) protects cells against 4-HNE, oxidative stress (H(2)O(2) or xanthine/xanthine oxidase), and UV-A mediated apoptosis by blocking JNK and caspase activation suggesting a role of 4-HNE in the mechanisms of apoptosis caused by these stress factors. The intracellular concentration of 4-HNE appears to be crucial for the nature of cell cycle signaling and may be a determinant for the signaling for differentiation, proliferation, transformation, or apoptosis. The intracellular concentrations of 4-HNE are regulated through a coordinated action of GSTs (GSTA4-4 and hGST5.8) which conjugate 4-HNE to GSH to form the conjugate (GS-HNE) and the transporter 76 kDa Ral-binding GTPase activating protein (RLIP76), which catalyze ATP-dependent transport of GS-HNE. A mild stress caused by heat, UV-A, or H(2)O(2)with no apparent effect on the cells in culture causes a rapid, transient induction of hGST5.8 and RLIP76. These stress preconditioned cells acquire ability to metabolize and exclude 4-HNE at an accelerated pace and acquire relative resistance to apoptosis by UV and oxidative stress as compared to unconditioned control cells. This resistance of stress preconditioned cells can be abrogated by coating the cells with anti-RLIP76 antibodies which block the transport of GS-HNE. These studies and previous reports discussed in this article strongly suggest a key role of 4-HNE in stress mediated signaling.     

Vascular smooth muscle cell activation and growth by 4-hydroxynonenal

The present study examines the signal transduction mechanism that is involved in the growth of vascular smooth muscle cells exposed to 4-hydroxynonenal (HNE) in vitro. This aldehyde component of oxidized low-density lipoprotein has been identified in atherosclerotic lesion. Exposure to HNE caused ERK, JNK, and p38 MAP kinase activation as well as the induction of c-fos and c-jun gene expression. AP-1 activity was also significantly induced by HNE treatment. These intracellular activities appear to be the mechanism of HNE-caused mitogenesis. Indeed, HNE induced vascular smooth muscle cell proliferation as determened by Alamar-Blue assay and stimulated DNA synthesis as determined by bromodeoxyuridine incorporation. These observations are consistent with a role of lipid peroxidation products in vascular smooth muscle cell growth in atherogenesis.     

4-hydroxynonenal and neurodegenerative diseases

The development of oxidative stress, in which production of highly reactive oxygen species (ROS) overwhelms antioxidant defenses, is a feature of many neurological diseases: ischemic, inflammatory, metabolic and degenerative. Oxidative stress is increasingly implicated in a number of neurodegenerative disorders characterized by abnormal filament accumulation or deposition of abnormal forms of specific proteins in affected neurons, like Alzheimer's disease (AD), Pick's disease, Lewy bodies related diseases, amyotrophic lateral sclerosis (ALS), and Huntington disease. Causes of neuronal death in neurodegenerative diseases are multifactorial. In some familiar cases of ALS mutation in the gene for Cu/Zn superoxide dismutase (SOD1) can be identified. In other neurodegenerative diseases ROS have some, usually not clear, role in early pathogenesis or implications on neuronal death in advanced stages of illness. The effects of oxidative stress on "post-mitotic cells", such as neurons may be cumulative, hence, it is often unclear whether oxidative damage is a cause or consequence of neurodegeneration. Peroxidation of cellular membrane lipids, or circulating lipoprotein molecules generates highly reactive aldehydes among which one of most important is 4-hydroxynonenal (HNE). The presence of HNE is increased in brain tissue and cerebrospinal fluid of AD patients, and in spinal cord of ALS patients. Immunohistochemical studies show presence of HNE in neurofibrilary tangles and in senile plaques in AD, in the cytoplasm of the residual motor neurons in sporadic ALS, in Lewy bodies in neocortical and brain stem neurons in Parkinson's disease (PD) and in diffuse Lewy bodies disease (DLBD). Thus, increased levels of HNE in neurodegenerative disorders and immunohistochemical distribution of HNE in brain tissue indicate pathophysiological role of oxidative stress in these diseases, and especially HNE in formation of abnormal filament deposites.     

The mechanism of action of 4-hydroxynonenal in cell injury

The effect of the C9 ketoaldehyde, 4-hydroxynonenal (HNE), a cytotoxic product of lipid peroxidation, on DNA, RNA and protein synthesis has been investigated in cells in culture. Macromolecular synthesis is powerfully inhibited by this agent which readily enters the lipid-rich membranes and is considerably more toxic than the polar ketoaldehyde, methyl glyoxal (MG). The entry of HNE into membranes lowers their glutathione GSH content. This is associated with an increased lipid peroxidation measured in vitro which is blocked by added GSH or alpha-tocopherol. It is proposed that this latter sequence of events is the underlying cause of the cytopathic effect of HNE in cells in culture.     

Stimulation of HeLa cell growth by physiological concentrations of 4-hydroxynonenal

The aim of this study was to analyze the growth response of HeLa cells over a prolonged period of time to a single exposure of physiological and supraphysiological concentrations of 4-hydroxynonenal (HNE), a peroxidation product of omega-6-polyunsaturated fatty acids. Furthermore, the growth modulating effect of serum factors, particularly albumin, on the growth pattern was examined. The effects of HNE on the growth rate and viability of the cells, as well as on the incorporation of labelled amino acids were monitored daily over a period of four days. Fetal calf serum not only had a growth stimualting effect but also modulated the action of HNE. In neither respect was albumin able to substitute for serum indicating that the influence of serum was not exerted via an albumin–HNE conjugate. HNE had a clear dose-dependent effect and a distinction could be made between a supraphysiological concentration (100 μM), which was primarily cytotoxic and a physiological range (below 10 μM) which showed growth modulatory effects. These effects consisted of a transient inhibition in the initial phase of the cell growth, which under optimal conditions (in presence of serum) was followed by a period of increased proliferation, compared to untreated control cultures, until confluence was attained. It is suggested that HNE is not only a toxic product of lipid peroxidation, but a physiological growth regulating factor as well.     

Intracellular metabolism of 4-hydroxynonenal

4-hydroxynonenal (HNE) is a major aldehydic product of lipid peroxidation known to exert a multitude of biological, cytotoxic, and signal effects. Mammalian cells possess highly active pathways of HNE metabolism. The metabolic fate of HNE was investigated in various mammalian cells and organs such as hepatocytes, intestinal enterocytes, renal tubular cells, aortic and brain endothelial cells, synovial fibroblasts, neutrophils, thymocytes, heart, and tumor cells. The experiments were carried out at 37 degrees C at initial HNE concentrations between 1 microM--that means in the range of physiological and pathophysiologically relevant HNE levels--to 100 microM. In all cell types which were investigated, 90-95% of 100 microM HNE were degraded within 3 min of incubation. At 1 microM HNE the physiological blood serum level of about 0.1-0.2 microM was restored already after 10-30 s. As primary products of HNE in hepatocytes and other cell types the glutathione-HNE-1:1-conjugate, the hydroxynonenoic acid and the corresponding alcohol of HNE, the 1,4-dihydroxynonene, were identified. Furthermore, the beta-oxidation of hydroxynonenoic acid including the formation of water was demonstrated. The quantitative share of HNE binding to proteins was low with about 2-8% of total HNE consumption. The glycine-cysteine-HNE, cysteine-HNE adducts and the mercapturic acid from glutathione-HNE adduct were not formed in the most cell types, but in kidney cells and neutrophils. The rapid metabolism underlines the role of HNE degrading pathways in mammalian cells as important part of the secondary antioxidative defense mechanisms in order to protect proteins from modification by aldehydic lipid peroxidation products.     

Monohydroperoxidized fatty acids but not 4-hydroxynonenal induced acute cardiac cell damage

Unsaturated fatty acids constitutive of cardiac membranal lipid matrix are one of the primary targets for reactive oxygen species generated during ischemia-reperfusion cycle. Lipid peroxidation is a cascade of intricate reactions involving the successive formations of fatty acids hydroperoxides and aldehydic compounds such as alkenals derived from the oxidative fragmentation of these hydroperoxides. The potential deleterious effects of different classes of lipid peroxidation products on cardiac cells were compared using three in vitro approaches: (i) cardiomyocyte integrity, (ii) electromechanical activity of papillary muscle, and (iii) atrial contractility. The following products of lipid peroxidation were tested: (i) photoperoxidized arachidonic acid pooling hydroperoxidized derivatives and aldehydic compounds, (ii) fatty acids hydroperoxides, and (iii) 4-hydroxynonenal, a characteristic alkenal derived from the oxidative fragmentation of hydroperoxidized n-6 fatty acids. Only fatty acids hydroperoxides induced drasfic loss of cellular integrity and severe disturbances in electromechanical activity of cardiomyocytes. 4-hydroxynonenal induced only a slight leak of lactate dehydrogenase at high concentrations and did not modify the electromechanical behavior of cardiac preparations. Under our conditions, monohydroperoxidized fatty acids but not 4-hydroxynonenal induced acute cardiac cell damages. In conclusion, lipid hydroperoxides can be considered both as markers of oxidative injury and relay sources of oxidative stress.     

The chemistry of cell signaling by reactive oxygen and nitrogen species and 4-hydroxynonenal

During the past several years, major advances have been made in understanding how reactive oxygen species (ROS) and nitrogen species (RNS) participate in signal transduction. Identification of the specific targets and the chemical reactions involved still remains to be resolved with many of the signaling pathways in which the involvement of reactive species has been determined. Our understanding is that ROS and RNS have second messenger roles. While cysteine residues in the thiolate (ionized) form found in several classes of signaling proteins can be specific targets for reaction with H₂O₂ and RNS, better understanding of the chemistry, particularly kinetics, suggests that for many signaling events in which ROS and RNS participate, enzymatic catalysis is more likely to be involved than non-enzymatic reaction. Due to increased interest in how oxidation products, particularly lipid peroxidation products, also are involved with signaling, a review of signaling by 4-hydroxy-2-nonenal (HNE) is included. This article focuses on the chemistry of signaling by ROS, RNS, and HNE and will describe reactions with selected target proteins as representatives of the mechanisms rather attempt to comprehensively review the many signaling pathways in which the reactive species are involved.     

Self-regulatory role of 4-hydroxynonenal in signaling for stress-induced programmed cell death

Within the last two decades, 4-hydroxynonenal has emerged as an important second messenger involved in the regulation of various cellular processes. Our recent studies suggest that HNE can induce apoptosis in various cells through the death receptor Fas (CD95)-mediated extrinsic pathway as well as through the p53-dependent intrinsic pathway. Interestingly, through its interaction with the nuclear protein Daxx, HNE can self-limit its apoptotic role by translocating Daxx to cytoplasm where it binds to Fas and inhibits Fas-mediated apoptosis. In this paper, after briefly describing recent studies on various biological activities of HNE, based on its interactions with Fas, Daxx, and p53, we speculate on possible mechanisms through which HNE may affect a multitude of cellular processes and draw a parallel between signaling roles of H(2)O(2) and HNE.     

Signaling kinases modulated by 4-hydroxynonenal

The interaction of 4-hydroxynonenal (HNE) with a variety of kinases variously involved in cell signaling is now a matter of active investigation. In particular, findings with regard to the effect of HNE on different components of the protein kinase C family and the mitogen-activated protein kinase complex already provide reliable indications of a potential role of this aldehyde as a cell signal messenger. Such a role appears further supported by the clear-cut evidence of up-regulation of receptor tyrosine kinases and down-regulation of the nuclear factor kappa B system, produced by HNE concentrations actually detectable in pathophysiology.     

Covalent modifications of aminophospholipids by 4-hydroxynonenal

Lipid oxidation is implicated in a wide range of pathophysiological disorders, which leads to reactive compounds such as aldehydes. Among them 4-hydroxynonenal (4-HNE) reacts strongly with the NH₂ groups of amino acids and forms mainly Michael adducts and minor Schiff-base adducts. Such reactions occur also with compounds containing thiol groups. No data are available describing 4-HNE interactions with amino-phospholipids. To investigate such a possibility, 4-HNE was incubated with either phosphatidylethanolamine (PE) or phosphatidylserine (PS) in an aqueous-organic biphasic system and the resulting products were identified by liquid chromatography-mass spectrometry (LC-MS). Our study points out the potential capacity of 4-HNE to react with phospholipids containing amino groups and particularly PE. The main resulting compounds found were a Michael adduct plus a minor Schiff base adduct, which was partly cyclized as a pyrrole derivative via a loss of water. Its stabilization as a pyrrole derivative allows to differentiate 4-HNE from the other aldehydes generated via lipid oxidation (e.g., malondialdehyde, 2-nonenal) that lack the 4-hydroxyl group. Their formation seems not to be affected when the pH varies from 6.5 to 8.5. Surprisingly, PS reacted poorly producing only a small amount of Michael adduct, the Schiff-base adduct being nondetectable. We conclude that such adducts, if they are formed in cell membranes, could alter the phospholipase-dependent cell signaling.     

Synergistic effect of 4-hydroxynonenal and PPAR ligands in controlling human leukemic cell growth and differentiation

Peroxisome proliferator-activated receptors play an important role in the differentiation of different cell lines. In this study we demonstrate that PPAR-alpha ligands (clofibrate and ciprofibrate) and PPAR-gamma ligands (troglitazone and 15d-prostaglandin J2) inhibit growth and induce monocytic differentiation in HL-60 cells, whereas only PPAR-gamma ligands inhibit growth of U937 cells. Differentiation was demonstrated by the analysis of surface antigen expression CD11b and CD14, and by the characteristic morphological changes. PPAR-gamma ligands are more effective than PPAR-alpha ligands in the inhibition of cell growth and in the induction of differentiation. The physiological product of lipid peroxidation, 4-hydroxynonenal (HNE), which alone induces granulocytic-like differentiation of HL-60 cells, potentiates the monocytic differentiation induced by ciprofibrate, troglitazone, and 15d-prostaglandin J2. The same HNE treatment significantly inhibits U937 cell growth and potentiates the inhibition of cell growth in PPAR-gamma ligand-treated cells. However, HNE does not induce a significant number of CD14-positive U937 cells. HNE causes a great increase of PPAR-gamma expression in both HL-60 and U937 cells, whereas it does not modify the PPAR-alpha expression. This observation may account for the high synergistic effect displayed by HNE and PPAR-gamma ligands in the inhibition of cell growth and differentiation induction. These results represent the first evidence of the involvement of a product of lipid peroxidation in the modulation of PPAR ligand activity and suggest a relationship between HNE and PPAR ligand pathways in leukemic cell growth and differentiation.     

Stimulation of Epstein-Barr virus-infected human B cell growth by physiological concentrations of 4-hydroxynonenal

We had previously shown that cyclosporin A (CsA) directly promoted the immortalization of Epstein-Barr virus (EBV)-infected human B cells (EBV-B cells) via an oxidative stress mechanism. 4-Hydroxynonenal (HNE) is a reactive end-product of lipid peroxidation. We hypothesized that HNE may mediate a direct oxidative stress-promoting effect of CsA on EBV-B cells. HNE-protein adducts in CsA-treated EBV-B cell extracts were assayed immunochemically using a Slot-Blot method. Cell proliferation was assayed by [(3)H]-thymidine incorporation. EBV oncogene latent membrane protein-1 (LMP1) expression was assayed by using PE-conjugated anti-LMP1 antibody in flow cytometry. We found that CsA at 500 ng ml(-1) and 1000 ng ml(-1) significantly increased the level of HNE-protein adducts in EBV-B cells over the control (arbitrary units +/- SE) by 251.3 +/- 7.5 to 361.3 +/- 9.7 and 342.7 +/- 10.7, respectively (p < 0.05, n = 3). EBV-B cells treated with a physiological concentration of HNE (1 microM) for 0.5 and 1 h and cultured for 2 and 4 weeks showed significantly increased [(3)H]-thymidine incorporation. EBV-B cells treated with HNE (1 microM) for 1 h and subsequently cultured for 2 and 4 weeks had a significantly higher ( > 2.0 times) LMP1-positive cell population over the control. In conclusion, in accordance with our previous findings, we show that CsA treatment of EBV-B cells results in increased production of the lipid peroxidation reactive end-product HNE that directly promotes EBV-B cell proliferation and LMP1 expression. This observation provides evidence for further understanding the mechanism of CsA-induced oxidative stress on EBV-related post-transplant lymphoproliferative disorder (PTLD).     

Role of 4-hydroxynonenal and its metabolites in signaling

Available evidence from a multitude of studies on the effects of 4-hydroxynonenal (HNE) on cellular processes seem to converge on some common themes: (i) concentration-dependent opposing effects of HNE on key signaling components (e.g. protein kinase C, adenylate cyclase) predict that certain constitutive levels of HNE may be needed for normal cell functions - lowering of this constitutive HNE level in cells promotes proliferative machinery while an increase in this level promotes apoptotic signaling; (ii) HNE is a common denominator in stress-induced apoptosis caused by H(2)O(2), superoxide, UV, heat or oxidant chemicals such as doxorubicin; and (iii) HNE can modulate ligand-independent signaling by membrane receptors such as EGFR or Fas (CD95) and may act as a sensor of external stimuli for eliciting stress-response. Against a backdrop of various reported effects of HNE, in vitro and in vivo, we have critically evaluated the above mentioned hypotheses suggesting a key role of HNE in signaling.     

Metabolism of 4-hydroxynonenal by the rat hepatoma cell line MH1C1

The metabolism of the toxic lipid peroxidation product 4-hydroxynonenal was investigated in the well-differentiated rat heptoma cell line MH1C1. When exposed to 0.1 mM 4-hydroxynonenal (HNE), MH1C1 cells consumed it in a time-dependent manner. There was a linear relationship between the amount of aldehyde consumed and cell number in the range 0.5 - 4 X 10(6) cells ml-1. This process was unaffected by pyrazole, suggesting that alcohol dehydrogenase is not involved. The whole homogenate of MH1C1 cells consumed added HNE at a rate similar to that in intact cells. Fractionation of the homogenate showed that the highest HNE-metabolizing activity is in the cytosol. The dialysed cytosol had almost no capacity to metabolize HNE, but this was restored by supplementation with NAD, NADH, NADP and NADPH. The metabolism of HNE in MH1C1 cells is thus different from that in hepatocytes, which were shown to utilize cytosolic alcohol dehydrogenase for this process. Both reductive and oxidative pathways could be implicated in the metabolic activity of MH1C1 cells towards HNE as well as binding by glutathione.     

Glutathione transferase A4-4 resists adduction by 4-hydroxynonenal

4-Hydroxy-2-trans-nonenal (HNE) is a lipid peroxidation product that contributes to the pathophysiology of several diseases with components of oxidative stress. The electrophilic nature of HNE results in covalent adduct formation with proteins, fatty acids and DNA. However, it remains unclear whether enzymes that metabolize HNE avoid inactivation by it. Glutathione transferase A4-4 (GST A4-4) plays a significant role in the elimination of HNE by conjugating it with glutathione (GSH), with catalytic activity toward HNE that is dramatically higher than the homologous GST A1-1 or distantly related GSTs. To determine whether enzymes that metabolize HNE resist its covalent adduction, the rates of adduction of these GST isoforms were compared and the functional effects of adduction on catalytic properties were determined. Although GST A4-4 and GST A1-1 have striking structural similarity, GST A4-4 was insensitive to adduction by HNE under conditions that yield modest adduction of GST A1-1 and extensive adduction of GST P1-1. Furthermore, adduction of GST P1-1 by HNE eliminated its activity toward the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and toward HNE itself. HNE effects on GST A4-4 and A1-1 were less significant. The results indicate that enzymes that metabolize HNE may have evolved structurally to resist covalent adduction by it.     

Reactions of 4-hydroxynonenal with proteins and cellular targets

Peroxidative degradation of lipids yields the aldehyde 4-hydroxy-2-nonenal (4HNE) as a major product. The lipid aldehyde is an electrophile, and reactivity of 4HNE toward protein nucleophiles (i.e., Cys, His, and Lys) has been characterized. Through the use of purified enzymes and isolated cells, various pathways for biotransformation of the lipid aldehyde have been identified and include enzyme-mediated oxidation, reduction, and glutathione conjugation. Uncontrolled oxidative stress can yield excessive lipid peroxidation and 4HNE generation, however, and overwhelm these cellular defenses. Indeed, in vitro and in vivo production of 4HNE in response to pro-oxidant exposure has been demonstrated using antibodies to protein adducts of the lipid aldehyde. Recent evidence suggests a role for protein modification by 4HNE in the pathogenesis of several diseases (e.g., alcohol-induced liver disease); however, the precise mechanism(s) is currently unknown but likely results from adduction of proteins involved in cellular homeostasis or biological signaling.