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Signaling kinases modulated by 4-hydroxynonenal
Free Radical Biology & Medicine, Vol. 37, No. 11, pp. 1694–1702, 2004 Copyright D 2004 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/$-see front matter
doi:10.1016/j.freeradbiomed.2004.08.027
Serial Review: 4-Hydroxynonenal as a Signaling Molecule Serial Review Editor: Henry Jay Forman; Guest Co-Editor: Dale A. Dickinson SIGNALING KINASES MODULATED BY 4-HYDROXYNONENAL GABRIELLA LEONARDUZZI, FANNY ROBBESYN, and GIUSEPPE POLI Department of Clinical and Biological Sciences, University of Turin, San Luigi Hospital, 10043 Orbassano, Turin, Italy (Received 4 August 2004; Accepted 31 August 2004) Available online 25 September 2004
Abstract — 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. D 2004 Elsevier Inc. All rights reserved. Keywords —4-Hydroxynonenal, Cell signaling, Protein kinase C, MAP kinases, Tyrosine kinases, Free radicals
Contents Introduction . . . . . . . . . . . . Protein kinase C family . . . . . . Mitogen-activated protein kinases . The IkappaB kinase complex . . . Tyrosine kinase receptors . . . . . The serine/threonine kinase Akt. . Conclusions . . . . . . . . . . . . Acknowledgments . . . . . . . . . References. . . . . . . . . . . . . Abbreviations . . . . . . . . . . .
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This article is part of a series of reviews on b4-Hydroxynonenal as a Signaling Molecule.Q The full list of papers may be found on the home page of the journal. Gabriella Leonarduzzi, B.Sc. in Biological Sciences and Ph.D. in Experimental Molecular Pathology; Researcher at the Faculty of Medicine, University of Turin, Italy. Fanny Robbesyn, Ph.D. in The Biology of Aging, from the University of Toulouse; post-doctoral fellowship at the University of Turin. Giuseppe Poli, M.D., University of Turin, Ph.D. in Biochemistry, Brunel University of West London, Honorary Doctor Universitatis, University of Buenos Aires; Professor of General Pathology, Faculty of Medicine, University of Turin. Address correspondence to: Professor Giuseppe Poli, Department of Clinical and Biological Sciences, University of Turin, San Luigi Hospital, 10043 Orbassano, Turin, Italy; Fax: +39 011 2365401; E-mail: [email protected].
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INTRODUCTION
4-Hydroxynonenal (HNE) quantitatively represents a major aldehyde product of the nonenzymatic breakdown of two biologically important n-6 polyunsaturated fatty acids (PUFAs), namely linoleic and arachidonic acids [1,2]. In addition, it is of primary biochemical interest because the molecule contains three functional groups making it highly reactive: a CjC double bond, a carbonyl group, and a hydroxy group. HNE can react with the thiol and amino residues of a variety of biomolecules, such as proteins, peptides, lipids, and nucleic acids [3,4]. The reaction with SH residues on the double bond leads to the formation of addition products, 1694
HNE and signaling kinases
stabilized by the still reversible formation of acetals, while the reaction of the carbonyl group with amino acid residues (in particular cysteine, histidine, and lysine) generates relatively less stable Schiff’s bases. The biochemistry of HNE explains the ever-increasing literature on its potential involvement in cell signal transduction and gene expression [5–10]. Here we will focus on the most recent investigations of its interaction with a variety of kinases variously involved in cell signaling. PROTEIN KINASE C FAMILY
Protein kinase C (PKCs) is an isoenzyme family of at least 12 serine/threonine kinases primarily involved in intracellular transduction of proliferative, apoptotic, differentiating, and functional signals to the nucleus [11]. Of note, these signaling kinases contain several cysteine-rich regions, both in the zinc finger of the regulatory domain and at the catalytic site, which can be modified by various oxidants [12]. Thus, selective oxidation at the amino-terminal regulatory domain, destroying the zinc finger conformation, decreases autoinhibition by the regulatory domain itself and permits cofactor-independent catalytic activity. In contrast, oxidative modification at the carboxyl-terminal catalytic domain results in complete inactivation of the kinase due to loss of free sulfydrils [12]. Redox modulation of PKC activity may therefore play an important role in pathophysiology. Most cells express various PKC isoforms; moreover, differences among the isoenzyme activation or inactivation suggest that individual PKCs may mediate distinct biological processes [13]. Concentrations of HNE within the observed pathophysiological range (0.1–10 AM) have been shown to modulate various PKC isoforms. In several cell types where HNE exhibits intracellular migration, a consistent modulation of PKC isoenzymes has been demonstrated. In particular, it has been shown that treatment with low concentrations of HNE (0.1–1 AM) induces selective activation of PKC-h isoforms through direct interaction between the aldehyde and these isoenzymes. Using isolated rat hepatocytes, Chiarpotto and colleagues [14] showed that the types of PKC isoforms influenced by HNE varied with the concentration of the aldehyde: when the hepatocytes were incubated with nanomolar HNE (0.1 AM), strong activation of classic calcium-dependent PKC-hI and -hII isoforms was observed, whereas these kinases were inhibited in the presence of HNE in the low micromolar range (1–10 AM). The activity of the novel calcium-independent PKC-y isoform was the exact opposite, being inhibited by 0.1 AM HNE and markedly activated by 1–10 AM
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HNE [14]. All PKC isoforms, both Ca2+-dependent and Ca2+-independent, were consistently inactivated by very high aldehyde concentrations, i.e., between 10 and 100 AM. The up-regulation of the rat hepatocyte PKC-hI and -hII isoforms by the lowest concentrations of HNE was correlated to hepatic transport and secretion of glycoproteins. A selective inhibitor (Gf6976) of classic PKC isoforms was used to test the PKC-dependent transport of lysosomal cathepsin D from the trans-Golgi network to the endosomal–lysosomal compartment and the exocytosis of mature cathepsin D. This inhibitor fully prevented cathepsin D transport and secretion in hepatocytes incubated with HNE at the lowest concentration (0.1 AM) [14]. In moderate agreement with these data, Nitti and colleagues [15] observed that this aldehyde at 1 AM increased the release of monocyte chemotactic protein-1 (MCP-1) by murine J774.A1 macrophages, through PKC-hI and -hII activation. These isoenzymes showed biphasic behavior: their activity significantly increased when the cells were incubated with 1 AM HNE but progressively decreased in the presence of higher HNE concentrations. As in the case of the hepatocyte secretory pathway, the increased extracellular release of MCP-1 by macrophages treated with 1 AM HNE was also demonstrated to be a PKC-dependent event, since it was prevented by the hI and hII inhibitor Gf6976 [15]. Of interest, this finding supports the generally recognized role of PKCs in cell release of pro-inflammatory cytokines. Later studies on NT2 differentiated neuronal cells confirmed that the aldehyde selectively activated the PKC-hI and -hII isoforms when the cells were incubated with repeated doses of HNE, at final concentrations between 0.1 and 1 AM [16]. Since activation of certain PKC isoforms appears to be implicated in alteration of the amyloid precursor protein, HNE might affect neuronal amyloid h protein (Ah) production through PKC-h activation. Indeed, Paola and colleagues [16] showed that, at concentrations similar to those detected in brain tissue of patients with Alzheimer’s disease (0.1 to 1 AM), HNE induced activation of PKC-h isoforms and increased intracellular Ah production in NT2 cells. Thus, in the concentration range 0.1–1 AM, the aldehyde influences protein intracellular traffic and secretion in different cell types. These biochemical effects produced by very low levels of HNE appear essentially to be exerted through up-regulating classic PKC isoforms. On the contrary, relatively higher concentrations of the aldehyde appear to contribute to negative regulation of cell growth, in this case through up-regulation of novel Ca2+-independent PKC isoforms. In this connection, E. Chiarpotto and colleagues (unpublished) showed that, in the range 1–10 AM, HNE selectively activates PKC-y in isolated rat hepatocytes and eventually induces a
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significant increase in the number of apoptotic cells. This event was likely PKC-y dependent, since it was strongly reduced by cell cotreatment with rottlerin, a very specific inhibitor of novel PKCs. Finally, with regard to PKC down-regulation by relatively high doses of the aldehyde, the research performed by Arese’s group [17,18] on human monocytes and magrophages after phagocytosis of malarial pigment (hemozoin) is of particular interest. The latter event was followed by a net increase in lipid peroxides, and steady state HNE levels were found to be above 10 AM. Moreover, treatment of human monocytes with 10– 100 AM HNE greatly inhibited their total PKC activity but, more importantly, a marked accumulation of PKCHNE adducts occurred in these cells. Thus excessive alkylation of thiol groups due to PKC-HNE adduct formation may lead to PKC inhibition [17,18]. The biological outcome of such PKC inactivation, driven by aldehyde products of lipid peroxidation, is the functional blockage of phagocytes, once they have engulfed the malarial pigment. It is now generally accepted that modulation of PKC activity by 4-hydroxynonenal may have crucial consequences on downstream signaling pathways (which will be examined below), redox-sensitive transcription factors, and genes [19–21]. In this connection, the recent demonstration of a strong HNE-dependent activation of the transcription factor NF-E2-related factor 2 (Nrf2) in human smooth muscle cells is of particular interest [22]. This peptide regulates the cellular steady state levels of various antioxidant proteins, including heme oxygenase-1 (HO1); it is sequestered in the cytoplasm by the protein Keap1 under unstimulated conditions but, once activated upon exposure to oxidant stimuli, it translocates into the nucleus and transactivates the antioxidant-responsive element (ARE). Dissociation of the Nrf2/Keap1 complex was achieved by phosphorylation of Nrf2 on Ser40 and demonstrated to be PKC mediated [23]: nuclear translocation of Nrf2 and induction of HO-1 gene expression as induced by 10 AM HNE was inhibited by a specific PKC inhibitor (Ro-318220) [22]. HNE, in the concentration range 1–10 AM, has been shown to up-regulate the transcription factor activator protein 1 (AP-1) in all cytotypes tested thus far, including myofibroblasts, hepatocytes, neuronal cells, and cells of the macrophage lineage [10]. AP-1 regulates the expression of a variety of genes, through activation of PKC isoforms, in particular the novel PKC-y isoform: cotreatment of cells with rottlerin, a selective inhibitor of novel PKC isoforms, completely prevents HNE-induced AP-1 nuclear binding [10]. HNE-dependent up-regulation of AP-1 implies the PKC-mediated activation of the downstream c-Jun amino-terminal kinases (JNKs) rather
than extracellular-signal-regulated kinases (ERKs) [6,24]. MITOGEN-ACTIVATED PROTEIN KINASES
The serine/threonine kinases of the MAPKs family can also be regulated by oxidants [25]. MAPKs include extracellular-signal-regulated kinases, c-Jun N-terminal kinases, and p38 MAPK, which are involved in several cellular functions, ranging from proliferation to differentiation and apoptosis [26]. The mechanisms by which oxidative stimuli, such as reactive oxygen species (ROS) and lipid peroxidation products, activate MAPKs are unclear and the precise molecular targets are yet to be identified. Their activity is in part regulated by a family of phosphatases, MAPK phosphatases (MKPs) [27], which are dual phosphatases that specifically dephosphorylate both Thr and Tyr in MAPKs, such as ERK1 and ERK2, in a variety of cell types [28]. Several members of the family have now been identified and, like protein tyrosine phosphatases (PTPs), they contain a cysteine residue that is essential for their catalytic activity [29]. Another redox-sensitive upstream activator of MAPKs is Ras, a small G protein that transduces a signal from certain receptor tyrosine kinases to the MAPK cascade. Ras may also mediate activation of NADH/NADPH oxidase, with generation of intracellular ROS [30], and it is activated by oxidative stress [31]. It is now well recognized that HNE signaling to the nucleus involves the MAPK pathway. In particular, HNE appears to markedly up-regulate JNKs and p38 activities. 4-Hydroxynonenal-induced activation of JNKs was first demonstrated by Parola and colleagues [24] on primary cultures of human hepatic stellate cells (hHSC) treated with 1 AM aldehyde. Using monoclonal antibodies specific for HNE-histidine adducts, nuclear HNEprotein adducts of 46, 54, and 66 kDa were detected and p46 and p54 isoforms of JNKs were identified as HNE targets. A consequence of HNE’s direct interaction with critical histidine residues in JNKs is that it led not only to nuclear translocation of JNKs but also to their activation. This interpretation was suggested by the facts that upstream kinases in the JNK cascade were not involved and that JNK isoforms that had translocated into the nuclei of hHSC were not phosphorylated. Increased mRNA levels of c-jun associated with a biphasic increase in AP-1 DNA binding activity were also observed. In addition, at least in hepatic stellate cells, HNE did not affect the Ras/ERK pathway, c-fos expression, DNA synthesis, or NF-kB binding [24]. The preferential JNK/AP-1/c-jun signaling pathway elicited by HNE and the lack of significant effects on the ERK cascade were confirmed by Uchida’s group [6] in
HNE and signaling kinases
cultured rat liver epithelial RL34 cells; they also observed significant activation of p38 MAPK. It should be noted, however, that higher concentrations of the aldehyde (25 AM) were used in their work. Various other reports which stress the primary involvement of JNK activation in the proapoptotic action of HNE are now available [32]. Camandola and colleagues [33], in addition to confirming HNE-induced JNK activation by confocal microscopy, found a large increase in AP-1 DNA-binding activity in cultured rat cortical neurons exposed to a pro-apoptotic concentration of HNE (10 AM). Further, Song and colleagues [34] investigated the molecular mechanism of HNE-induced apoptosis in human PC12 neuroblastoma cells by measuring the activities of MAPKs. Again, JNKs were maximally activated within 15–30 min of 25 AM HNE treatment, while ERKs and p38 MAPK remained unchanged. Stress-activated protein kinase kinase 1 (SEK1 or SAPKK1), upstream of JNKs, was also activated. A net activation of JNKs through SEK1 was demonstrated by the transient transfection of COS-7 cells, a fibroblastlike cell line, with cDNA of wild-type SEK1 and JNK. On the contrary, a significant decrease in JNK activation and HNE-induced apoptosis occured when cells were cotransfected with JNK and the dominant negative mutant of SEK1. These data strongly suggest that cell death induced by HNE implicates activation of the SEK1/JNK pathway [34]. More recently, using murine sympathetic neurons deficient in JNK3 gene, Bruckner and Estus [35] demonstrated that JNK3 contributes to HNE-induced neuronal death; HNE (1 AM) induced c-Jun phosphorylation, c-jun induction, and caspase-dependent apoptosis in sympathetic neurons. All these events were significantly inhibited by JNK3 deficiency [35]. To mediate the sequence of specific pathological events, for example neuronal apoptosis induced by Ah peptides during the progression of Alzheimer’s disease, it may be necessary that HNE and other oxidative molecules be generated during oxidative processes. Indeed, involvement of HNE in Ah neurotoxicity has more than once been proposed [36,37]. Exposure of differentiated SK-N-BE neuronal cells to toxic Ah peptides induced apoptosis, through early production of HNE and hydrogen peroxide (H2O2). These oxidative stress-related molecules appeared to be responsible for JNK and p38 MAPK activation and for apoptosis, since cell cotreatment with the antioxidants a-tocopherol or N-acetylcysteine prevented their overproduction, kinase up-regulation, and at the same time apoptosis. Moreover, by using specific inhibitors of JNKs and p38 MAPK it was confirmed that HNE-induced activation of these kinases was required before cell death could be induced by Ah peptides [38].
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Uchida’s group [6], having shown marked JNK and p38 MAPK activation by HNE in rat liver epithelial RL34 cells, went on to produce clear evidence of the molecular mechanism underlying p38 kinase’s role in HNE-induced expression of cyclooxygenase-2 (COX-2), which plays a key role in converting free arachidonic acid to prostaglandins. Treatment of RL34 cells with 25 AM HNE rapidly (within 5 min) induced phosphorylation of p38 and of MAPK kinase 3/MAPK kinase 6 (MKK3/ MKK6), upstream of p38. The possibility of the p38 MAPK pathway being involved in HNE-induced COX-2 overexpression in this cell type was ruled out by its suppression in cells pretreated with a p38 MAPK selective inhibitor (SB203580) [39,40]. The conclusive demonstration, in many cell types, of a key role of JNKs in HNE signaling does not necessarily mean that other key elements of the MAPK pathway may not be involved: Ruef and colleagues [41] treated rat aortic smooth muscle cells with 1–2.5 AM HNE and observed strong but transient activation of ERK1/2, induction of c-fos and c-jun protein synthesis, and an increase in AP-1 DNA binding activity. In addition, HNE induced platelet-derived growth factor-AA protein synthesis. These data indicate that HNE may be involved in smooth muscle cell proliferation by inducing mitogenic signals, and this effect may be important in atherogenesis [41]. Later, again using vascular smooth muscle cells, Kakishita and Hattori [42] confirmed this observation, while in their hands 2.5 AM HNE activated not only ERK1/2 but also JNKs and p38 MAPK. Further, it has been observed that 25 AM HNE induced secretion of fibronectin by IMR-90 human lung fibroblasts, following marked activation of ERK1/2, with p38 MAPK being only slightly activated and JNKs being unchanged. The HNE-induced secretion of fibronectin was inhibited by selective ERKs and p38 inhibitors. These data suggest that lipid oxidation products such as HNE may stimulate airway remodeling by inducing lung fibroblasts to produce extracellular matrix proteins, such as fibronectin, via the ERK signal transduction pathway [43]. HNE-induced activation of ERKs, JNKs, and p38 MAPK in lung endothelial cells has also been reported. Activation of the MAPK pathway by 4-hydroxynonenal (1–100 AM) favored cytoskeletal remodeling, with regard to actin, and enhanced endothelial barrier function [44]. THE IKAPPAB KINASE COMPLEX
A major signaling pathway associated with oxidative stress and inflammation is mediated by the transcription factor nuclear factor kappa B (NF-nB). NF-nB is an ubiquitous redox-sensitive peptide that is activated by a large number of extracellular stimuli including pro-
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inflammatory cytokines, chemokines, growth factors, oxydative stress, bacterial and viral products [45–47]. The key step in activation of the NF-nB signaling pathway is phosphorylation of InB (inhibitory component of NF-nB) proteins, which is mediated by a highmolecular-weight InB kinase complex (IKK) (approximately 700 kDa). IKK is a multisubunit serine-specific kinase composed of at least two catalytic subunits, IKKa (IKK-1) and IKKh (IKK-2), plus a regulatory subunit, IKKg. In response to upstream stimuli, the IKK complex is activated through phosphorylation operated by a heterogenous group of tyrosine kinases named IKK kinases, namely PKC, protein kinase B/Akt (PKB/Akt), or MAPK/ERK kinase kinase-1, -2, and -3 [48–50]. With regard to the possible interaction of HNE with the IKK complex, the few reports available thus far all point to an inhibitory effect of the aldehyde. For example, NF-nB activation induced by the stimuli lipopolysaccharide, interleukin-1h, or phorbol ester in THP-1 monocytes [51] or by tetradecanoylphorbolacetate/ionomycin in human carcinoma cells [52] was prevented by cell pretreatment with HNE (25–50 AM). In agreement with these findings, activation of NF-nB induced by exposure of THP-1 cells to Chlamydia pneumoniae was significantly reduced by 50 AM HNE [53]; HNE (25–50 AM) appears to exert inhibitory action on the NF-nB system by preventing the phosphorylation and consequent degradation of the inhibitory proteins InBa, InBh, and InBq [51–53]. This inhibition appears to be caused by a direct reaction between HNE and certain components of the IKK complex, as suggested by the fact that covalent adducts can form between the unsaturated aldehyde and the IKK complex [52]. The possible impairment of NFnB transcription activity, by concentrations of HNE actually detectable in cells and tissues, may have a significant impact on pathophysiology; for instance, down-regulated expression of NF-nB-regulated genes, as inducible by HNE, may interfere with the immune response and thus indirectly sustain inflammation and degenerative processes that would otherwise be more easily terminated. TYROSINE KINASE RECEPTORS
Tyrosine kinase receptors (RTKs), such as the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (PDGFR), are transmembrane glycoproteins that display tyrosine kinase activity in their cytoplasmic region. Stimulation of RTKs by ligand-dependent or -independent mechanisms (radiation, metal ions, ROS) induced dimerization in the receptors and autophosphorylation of several tyrosine residues, followed by induction of the catalytic activity
of the kinase. Activation of RTKs and of the downstream signaling pathways are involved in various biological processes such as proliferation, differentiation, migration, apoptosis, and the genesis or progression of human diseases [54–56]. As was very recently reviewed by Negre-Salvayre and colleagues [57], oxidized LDL (oxLDL) and related lipid peroxidation products, including HNE, can interact with EGF and PDGF-h receptors: oxLDL added to vascular cells induced and sustained rapid tyrosine phosphorylation of EGFR and PDGFR-h; the receptor’s activation was associated with recruitment of SH2 domain (SH) proteins, such as SHPTP2, phospholipase Cg , Src, and phosphatidylinositol 3 kinase (PI3K), on the phosphorylated tyrosine, consequently triggering the downstream signaling pathway [58,59]. Further investigation by the same group [59,60] pointed to HNE as an early mediator of RTK activation by oxLDL. The aldehyde was demonstrated to be responsible for the early, antioxidant-insensitive phase of activation of these receptors. Late activation of RTKs was shown, on the contrary, to be antioxidant sensitive and is likely mediated by oxidative stress [59,60]: HNE added to the culture medium of human endothelial cell line (CRL-1998 EC) in the concentration range 0.1–1 AM mimicked the action of oxLDL in favoring the autophosphorylation and activation of the receptor intrinsic tyrosine kinase [58]. The aldehyde was demonstrated to directly react with RTKs, inducing formation of HNE-EGFR or HNEPDGFR adducts. The generation of such adducts was demonstrated by the detection of HNE-protein epitopes on immunoprecipitated RTKs using anti-HNE-histidine antibodies and by the loss of free amino group content in the receptor molecules. The derivatization of RTKs was first confirmed in vitro by incubating rabbit smooth muscle cells (SMCs) with [H3]HNE-labeled oxLDL. A small but significant part of the added [H3]HNE was recovered bound to PDGFR-h [59]. The detection of PDGFR-h-HNE adducts in atherosclerotic aorta of cholesterol-fed rabbits provided the conclusive demonstration that HNE actually reacts and activates membrane RTKs [59]. Analysis of the biological responses triggered by HNE-dependent activation of tyrosine kinase receptors has shown that the aldehyde may up-regulate different signaling pathways depending upon incubation time and concentration: short incubation of vascular cells with a relatively low concentration of HNE (0.1–1 AM) induced the derivatization and autophosphorylation of RTKs, the subsequent activation of a signaling cascade that involved the PI3K/Akt survival pathway, and consequently the mitogenic response of SMCs [61]. On the contrary, incubation of human epidermoid carcinoma A431 cells with a relatively high concentration of
HNE and signaling kinases
exogenous HNE (500 AM) activated EGFR, ERK1/2, and JNK1/2 but inhibited cell growth [62]. In line with this finding, HNE concentrations in the medium-high pathophysiological range have been observed also to exert growth inhibition in other cell types, by inducing cell cycle arrest or even apoptosis [63,64]. Of note, interaction of HNE with RTKs apparently does not always result in their activation. At least in hHSC in culture, a low concentration of HNE (1 AM) inhibited tyrosine phosphorylation of PDGFR-h by its own ligand PDGFR-BB. This event was accompanied by inhibition of MAPKs and PI3K and, as a consequence, by a decrease in the PDGF-dependent DNA synthesis [65]. THE SERINE/THREONINE KINASE AKT
The serine/threonine kinase Akt, also called protein kinase B (PKB) because of the high homology of its kinase domain with those of protein kinases A and C, is a critical component of the signaling pathway triggered by PI3K. Following activation of PI3K by ligand-stimulated growth factor receptors and generation of phosphatidylinositol-3,4,5-triphosphate by the activated enzyme, PKB/Akt is recruited from the cytosol to the plasma membrane. It is then activated by phosphorylation on Thr308 and Ser473, by kinases such as phosphoinositidedependent kinase 1 (PDK1). Activated PKB/Akt leads to the phosphorylation of a large number of other proteins involved in the regulation of glucose metabolism, cell proliferation, apoptosis, cell migration, and gene expression. Termination of Akt signaling is mediated by the action of phosphatases such as protein phosphatase 2A
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(PP2A), which dephosphorylates Thr308 and Ser473 and brings PKB/Akt back to an inactive state in the cytosol [66–68]. PKB/Akt activity may also be affected by HNE. However, the only two reports on this matter available thus far provide opposing evidence: Liu and colleagues [69] showed that HNE (20 AM) induces apoptosis in human T-cell leukemia Jurkat cells through impairment of the Akt-mediated cell survival pathway. The aldehyde activated protein phosphatase 2A (PP2A), which in turn dephosphorylated Akt on Ser473. HNE-mediated dephosphorylation and inactivation of Akt were prevented by okadaic acid, a selective PP2A inhibitor. On the contrary, using a very different type of cells, namely human neuroblastoma IRM-32 cells, Dozza and colleagues [70] observed that challenge with 10 AM HNE provoked significant inhibition of the glycogen synthase kinase 3h, likely through the phosphorylation and activation of both Akt and ERK2: this HNE-dependent effect was also abolished by cell cotreatment with LY294002 or U0126, inhibitors of the PI3K and ERK pathways, respectively. The mechanism by which HNE up-regulates Akt has yet to be clarified; the aldehyde might act through activation of protein tyrosine kinases, such as membrane growth factor receptors [70]. CONCLUSIONS
Due to its peculiar biochemical structure and its relatively high diffusibility from the site of origin, 4hydroxynonenal is a good candidate molecule for integrating/modulating physiological cell signaling. This probability is strengthened by the fact that HNE
Fig. 1. Potential signaling by HNE in pathophysiology.
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concentrations that have been found to be active in signal transduction are within the range detectable in a variety of cells and tissues, under a number of different pathophysiological conditions. With regard to HNEinduced modulation of signaling kinases, experimental findings obtained thus far for PKCs and MAPKs appear to indicate that (i) in the lower concentration range the aldehyde influences PKC-dependent physiological events such as protein trafficking and secretion, while at relatively higher concentrations it tends to favor apoptotic death through involvement of novel rather than classic PKC isoforms and (ii) the latter effect is likely connected to HNE’s up-regulation of JNKs, while up-regulation of ERKs is more probably related to functional aspects such as cytoskeletal remodeling, the effect of growth factors, and so on. It is also now clear that 4-hydroxynonenal activates signaling pathways regulated by RTKs, a biochemical effect that would clearly qualify HNE as a signal transducing molecule. Further, the net demonstration of IKK inhibition by pathophysiological amounts of the aldehyde undoubtedly explains the HNE-induced downregulation of the NF-kB pathway that has been widely reported in different model systems. In most cases, HNE appears to react chemically with the various kinases to form more or less stable adducts. In addition to the influence on any effect exerted by the cell type involved and the concentration and cellular compartmentalization of the aldehyde, the location of HNE-adduct formation, whether in the regulatory or in the catalytic domain of the kinase, appears to be crucial. The former event would better allow enzyme upregulation by the aldehyde, while the latter appears more likely to lead to inactivation of the kinase, especially if there are numerous SH residues at that site of the molecule. Finally, Fig. 1 summarizes the pathophysiological processes to whose modulation 4-hydroxynonenal may, at least in principle, contribute. It reports the various signaling kinases possibly regulated by HNE in the different pathways. Acknowledgments — The research was supported by grants from the Italian Ministry of the University, PRIN 2001, 2002, the Monzino Foundation, Milan, Italy, the Piedmont Region, the University of Turin, and the Compagnia di San Paolo, Turin, Italy. Fanny Robbesyn was a recipient of a fellowship from Fondation pour la Recherche Me´dicale, Paris, France.
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ABBREVIATIONS
Ah — amyloid h protein AP-1 — activator protein 1 ARE — antioxidant-responsive element
COX-2 — cyclooxygenase-2 DAG — diacylglycerol EGFR — epidermal growth factor receptor ERKs — extracellular-signal-regulated kinases HNE — 4-hydroxynonenal HO-1 — heme oxygenase-1 H2O2 — hydrogen peroxide InB — inhibitory component of NF-nB IKK — IkappaB kinase IP3 — inositol triphosphate JNK — c-Jun N-terminal kinases MAPKs — mitogen-activated protein kinases MCP-1 — monocyte chemotactic protein-1 MKK — MAPK kinase MKPs — MAPK phosphatases NF-nB — nuclear factor kappa B Nrf2 — NF-E2-related factor 2 OxLDL — oxidized LDL PDGFR — platelet-derived growth factor receptor PDK1 — phosphoinositide-dependent kinase 1 PI3K — phosphatidylinositol 3 kinase PKB — protein kinase B PKC — protein kinase C PP2A — protein phosphatase 2A PTPs — protein tyrosine phosphatases PUFAs — polyunsaturated fatty acids ROS — reactive oxygen species RTKs — tyrosine kinase receptor SAPKK1 (or SEK1) — stress-activated protein kinase kinase 1 SH — SH2 domain
doi:10.1016/j.freeradbiomed.2004.08.027
Serial Review: 4-Hydroxynonenal as a Signaling Molecule Serial Review Editor: Henry Jay Forman; Guest Co-Editor: Dale A. Dickinson SIGNALING KINASES MODULATED BY 4-HYDROXYNONENAL GABRIELLA LEONARDUZZI, FANNY ROBBESYN, and GIUSEPPE POLI Department of Clinical and Biological Sciences, University of Turin, San Luigi Hospital, 10043 Orbassano, Turin, Italy (Received 4 August 2004; Accepted 31 August 2004) Available online 25 September 2004
Abstract — 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. D 2004 Elsevier Inc. All rights reserved. Keywords —4-Hydroxynonenal, Cell signaling, Protein kinase C, MAP kinases, Tyrosine kinases, Free radicals
Contents Introduction . . . . . . . . . . . . Protein kinase C family . . . . . . Mitogen-activated protein kinases . The IkappaB kinase complex . . . Tyrosine kinase receptors . . . . . The serine/threonine kinase Akt. . Conclusions . . . . . . . . . . . . Acknowledgments . . . . . . . . . References. . . . . . . . . . . . . Abbreviations . . . . . . . . . . .
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This article is part of a series of reviews on b4-Hydroxynonenal as a Signaling Molecule.Q The full list of papers may be found on the home page of the journal. Gabriella Leonarduzzi, B.Sc. in Biological Sciences and Ph.D. in Experimental Molecular Pathology; Researcher at the Faculty of Medicine, University of Turin, Italy. Fanny Robbesyn, Ph.D. in The Biology of Aging, from the University of Toulouse; post-doctoral fellowship at the University of Turin. Giuseppe Poli, M.D., University of Turin, Ph.D. in Biochemistry, Brunel University of West London, Honorary Doctor Universitatis, University of Buenos Aires; Professor of General Pathology, Faculty of Medicine, University of Turin. Address correspondence to: Professor Giuseppe Poli, Department of Clinical and Biological Sciences, University of Turin, San Luigi Hospital, 10043 Orbassano, Turin, Italy; Fax: +39 011 2365401; E-mail: [email protected].
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INTRODUCTION
4-Hydroxynonenal (HNE) quantitatively represents a major aldehyde product of the nonenzymatic breakdown of two biologically important n-6 polyunsaturated fatty acids (PUFAs), namely linoleic and arachidonic acids [1,2]. In addition, it is of primary biochemical interest because the molecule contains three functional groups making it highly reactive: a CjC double bond, a carbonyl group, and a hydroxy group. HNE can react with the thiol and amino residues of a variety of biomolecules, such as proteins, peptides, lipids, and nucleic acids [3,4]. The reaction with SH residues on the double bond leads to the formation of addition products, 1694
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stabilized by the still reversible formation of acetals, while the reaction of the carbonyl group with amino acid residues (in particular cysteine, histidine, and lysine) generates relatively less stable Schiff’s bases. The biochemistry of HNE explains the ever-increasing literature on its potential involvement in cell signal transduction and gene expression [5–10]. Here we will focus on the most recent investigations of its interaction with a variety of kinases variously involved in cell signaling. PROTEIN KINASE C FAMILY
Protein kinase C (PKCs) is an isoenzyme family of at least 12 serine/threonine kinases primarily involved in intracellular transduction of proliferative, apoptotic, differentiating, and functional signals to the nucleus [11]. Of note, these signaling kinases contain several cysteine-rich regions, both in the zinc finger of the regulatory domain and at the catalytic site, which can be modified by various oxidants [12]. Thus, selective oxidation at the amino-terminal regulatory domain, destroying the zinc finger conformation, decreases autoinhibition by the regulatory domain itself and permits cofactor-independent catalytic activity. In contrast, oxidative modification at the carboxyl-terminal catalytic domain results in complete inactivation of the kinase due to loss of free sulfydrils [12]. Redox modulation of PKC activity may therefore play an important role in pathophysiology. Most cells express various PKC isoforms; moreover, differences among the isoenzyme activation or inactivation suggest that individual PKCs may mediate distinct biological processes [13]. Concentrations of HNE within the observed pathophysiological range (0.1–10 AM) have been shown to modulate various PKC isoforms. In several cell types where HNE exhibits intracellular migration, a consistent modulation of PKC isoenzymes has been demonstrated. In particular, it has been shown that treatment with low concentrations of HNE (0.1–1 AM) induces selective activation of PKC-h isoforms through direct interaction between the aldehyde and these isoenzymes. Using isolated rat hepatocytes, Chiarpotto and colleagues [14] showed that the types of PKC isoforms influenced by HNE varied with the concentration of the aldehyde: when the hepatocytes were incubated with nanomolar HNE (0.1 AM), strong activation of classic calcium-dependent PKC-hI and -hII isoforms was observed, whereas these kinases were inhibited in the presence of HNE in the low micromolar range (1–10 AM). The activity of the novel calcium-independent PKC-y isoform was the exact opposite, being inhibited by 0.1 AM HNE and markedly activated by 1–10 AM
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HNE [14]. All PKC isoforms, both Ca2+-dependent and Ca2+-independent, were consistently inactivated by very high aldehyde concentrations, i.e., between 10 and 100 AM. The up-regulation of the rat hepatocyte PKC-hI and -hII isoforms by the lowest concentrations of HNE was correlated to hepatic transport and secretion of glycoproteins. A selective inhibitor (Gf6976) of classic PKC isoforms was used to test the PKC-dependent transport of lysosomal cathepsin D from the trans-Golgi network to the endosomal–lysosomal compartment and the exocytosis of mature cathepsin D. This inhibitor fully prevented cathepsin D transport and secretion in hepatocytes incubated with HNE at the lowest concentration (0.1 AM) [14]. In moderate agreement with these data, Nitti and colleagues [15] observed that this aldehyde at 1 AM increased the release of monocyte chemotactic protein-1 (MCP-1) by murine J774.A1 macrophages, through PKC-hI and -hII activation. These isoenzymes showed biphasic behavior: their activity significantly increased when the cells were incubated with 1 AM HNE but progressively decreased in the presence of higher HNE concentrations. As in the case of the hepatocyte secretory pathway, the increased extracellular release of MCP-1 by macrophages treated with 1 AM HNE was also demonstrated to be a PKC-dependent event, since it was prevented by the hI and hII inhibitor Gf6976 [15]. Of interest, this finding supports the generally recognized role of PKCs in cell release of pro-inflammatory cytokines. Later studies on NT2 differentiated neuronal cells confirmed that the aldehyde selectively activated the PKC-hI and -hII isoforms when the cells were incubated with repeated doses of HNE, at final concentrations between 0.1 and 1 AM [16]. Since activation of certain PKC isoforms appears to be implicated in alteration of the amyloid precursor protein, HNE might affect neuronal amyloid h protein (Ah) production through PKC-h activation. Indeed, Paola and colleagues [16] showed that, at concentrations similar to those detected in brain tissue of patients with Alzheimer’s disease (0.1 to 1 AM), HNE induced activation of PKC-h isoforms and increased intracellular Ah production in NT2 cells. Thus, in the concentration range 0.1–1 AM, the aldehyde influences protein intracellular traffic and secretion in different cell types. These biochemical effects produced by very low levels of HNE appear essentially to be exerted through up-regulating classic PKC isoforms. On the contrary, relatively higher concentrations of the aldehyde appear to contribute to negative regulation of cell growth, in this case through up-regulation of novel Ca2+-independent PKC isoforms. In this connection, E. Chiarpotto and colleagues (unpublished) showed that, in the range 1–10 AM, HNE selectively activates PKC-y in isolated rat hepatocytes and eventually induces a
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significant increase in the number of apoptotic cells. This event was likely PKC-y dependent, since it was strongly reduced by cell cotreatment with rottlerin, a very specific inhibitor of novel PKCs. Finally, with regard to PKC down-regulation by relatively high doses of the aldehyde, the research performed by Arese’s group [17,18] on human monocytes and magrophages after phagocytosis of malarial pigment (hemozoin) is of particular interest. The latter event was followed by a net increase in lipid peroxides, and steady state HNE levels were found to be above 10 AM. Moreover, treatment of human monocytes with 10– 100 AM HNE greatly inhibited their total PKC activity but, more importantly, a marked accumulation of PKCHNE adducts occurred in these cells. Thus excessive alkylation of thiol groups due to PKC-HNE adduct formation may lead to PKC inhibition [17,18]. The biological outcome of such PKC inactivation, driven by aldehyde products of lipid peroxidation, is the functional blockage of phagocytes, once they have engulfed the malarial pigment. It is now generally accepted that modulation of PKC activity by 4-hydroxynonenal may have crucial consequences on downstream signaling pathways (which will be examined below), redox-sensitive transcription factors, and genes [19–21]. In this connection, the recent demonstration of a strong HNE-dependent activation of the transcription factor NF-E2-related factor 2 (Nrf2) in human smooth muscle cells is of particular interest [22]. This peptide regulates the cellular steady state levels of various antioxidant proteins, including heme oxygenase-1 (HO1); it is sequestered in the cytoplasm by the protein Keap1 under unstimulated conditions but, once activated upon exposure to oxidant stimuli, it translocates into the nucleus and transactivates the antioxidant-responsive element (ARE). Dissociation of the Nrf2/Keap1 complex was achieved by phosphorylation of Nrf2 on Ser40 and demonstrated to be PKC mediated [23]: nuclear translocation of Nrf2 and induction of HO-1 gene expression as induced by 10 AM HNE was inhibited by a specific PKC inhibitor (Ro-318220) [22]. HNE, in the concentration range 1–10 AM, has been shown to up-regulate the transcription factor activator protein 1 (AP-1) in all cytotypes tested thus far, including myofibroblasts, hepatocytes, neuronal cells, and cells of the macrophage lineage [10]. AP-1 regulates the expression of a variety of genes, through activation of PKC isoforms, in particular the novel PKC-y isoform: cotreatment of cells with rottlerin, a selective inhibitor of novel PKC isoforms, completely prevents HNE-induced AP-1 nuclear binding [10]. HNE-dependent up-regulation of AP-1 implies the PKC-mediated activation of the downstream c-Jun amino-terminal kinases (JNKs) rather
than extracellular-signal-regulated kinases (ERKs) [6,24]. MITOGEN-ACTIVATED PROTEIN KINASES
The serine/threonine kinases of the MAPKs family can also be regulated by oxidants [25]. MAPKs include extracellular-signal-regulated kinases, c-Jun N-terminal kinases, and p38 MAPK, which are involved in several cellular functions, ranging from proliferation to differentiation and apoptosis [26]. The mechanisms by which oxidative stimuli, such as reactive oxygen species (ROS) and lipid peroxidation products, activate MAPKs are unclear and the precise molecular targets are yet to be identified. Their activity is in part regulated by a family of phosphatases, MAPK phosphatases (MKPs) [27], which are dual phosphatases that specifically dephosphorylate both Thr and Tyr in MAPKs, such as ERK1 and ERK2, in a variety of cell types [28]. Several members of the family have now been identified and, like protein tyrosine phosphatases (PTPs), they contain a cysteine residue that is essential for their catalytic activity [29]. Another redox-sensitive upstream activator of MAPKs is Ras, a small G protein that transduces a signal from certain receptor tyrosine kinases to the MAPK cascade. Ras may also mediate activation of NADH/NADPH oxidase, with generation of intracellular ROS [30], and it is activated by oxidative stress [31]. It is now well recognized that HNE signaling to the nucleus involves the MAPK pathway. In particular, HNE appears to markedly up-regulate JNKs and p38 activities. 4-Hydroxynonenal-induced activation of JNKs was first demonstrated by Parola and colleagues [24] on primary cultures of human hepatic stellate cells (hHSC) treated with 1 AM aldehyde. Using monoclonal antibodies specific for HNE-histidine adducts, nuclear HNEprotein adducts of 46, 54, and 66 kDa were detected and p46 and p54 isoforms of JNKs were identified as HNE targets. A consequence of HNE’s direct interaction with critical histidine residues in JNKs is that it led not only to nuclear translocation of JNKs but also to their activation. This interpretation was suggested by the facts that upstream kinases in the JNK cascade were not involved and that JNK isoforms that had translocated into the nuclei of hHSC were not phosphorylated. Increased mRNA levels of c-jun associated with a biphasic increase in AP-1 DNA binding activity were also observed. In addition, at least in hepatic stellate cells, HNE did not affect the Ras/ERK pathway, c-fos expression, DNA synthesis, or NF-kB binding [24]. The preferential JNK/AP-1/c-jun signaling pathway elicited by HNE and the lack of significant effects on the ERK cascade were confirmed by Uchida’s group [6] in
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cultured rat liver epithelial RL34 cells; they also observed significant activation of p38 MAPK. It should be noted, however, that higher concentrations of the aldehyde (25 AM) were used in their work. Various other reports which stress the primary involvement of JNK activation in the proapoptotic action of HNE are now available [32]. Camandola and colleagues [33], in addition to confirming HNE-induced JNK activation by confocal microscopy, found a large increase in AP-1 DNA-binding activity in cultured rat cortical neurons exposed to a pro-apoptotic concentration of HNE (10 AM). Further, Song and colleagues [34] investigated the molecular mechanism of HNE-induced apoptosis in human PC12 neuroblastoma cells by measuring the activities of MAPKs. Again, JNKs were maximally activated within 15–30 min of 25 AM HNE treatment, while ERKs and p38 MAPK remained unchanged. Stress-activated protein kinase kinase 1 (SEK1 or SAPKK1), upstream of JNKs, was also activated. A net activation of JNKs through SEK1 was demonstrated by the transient transfection of COS-7 cells, a fibroblastlike cell line, with cDNA of wild-type SEK1 and JNK. On the contrary, a significant decrease in JNK activation and HNE-induced apoptosis occured when cells were cotransfected with JNK and the dominant negative mutant of SEK1. These data strongly suggest that cell death induced by HNE implicates activation of the SEK1/JNK pathway [34]. More recently, using murine sympathetic neurons deficient in JNK3 gene, Bruckner and Estus [35] demonstrated that JNK3 contributes to HNE-induced neuronal death; HNE (1 AM) induced c-Jun phosphorylation, c-jun induction, and caspase-dependent apoptosis in sympathetic neurons. All these events were significantly inhibited by JNK3 deficiency [35]. To mediate the sequence of specific pathological events, for example neuronal apoptosis induced by Ah peptides during the progression of Alzheimer’s disease, it may be necessary that HNE and other oxidative molecules be generated during oxidative processes. Indeed, involvement of HNE in Ah neurotoxicity has more than once been proposed [36,37]. Exposure of differentiated SK-N-BE neuronal cells to toxic Ah peptides induced apoptosis, through early production of HNE and hydrogen peroxide (H2O2). These oxidative stress-related molecules appeared to be responsible for JNK and p38 MAPK activation and for apoptosis, since cell cotreatment with the antioxidants a-tocopherol or N-acetylcysteine prevented their overproduction, kinase up-regulation, and at the same time apoptosis. Moreover, by using specific inhibitors of JNKs and p38 MAPK it was confirmed that HNE-induced activation of these kinases was required before cell death could be induced by Ah peptides [38].
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Uchida’s group [6], having shown marked JNK and p38 MAPK activation by HNE in rat liver epithelial RL34 cells, went on to produce clear evidence of the molecular mechanism underlying p38 kinase’s role in HNE-induced expression of cyclooxygenase-2 (COX-2), which plays a key role in converting free arachidonic acid to prostaglandins. Treatment of RL34 cells with 25 AM HNE rapidly (within 5 min) induced phosphorylation of p38 and of MAPK kinase 3/MAPK kinase 6 (MKK3/ MKK6), upstream of p38. The possibility of the p38 MAPK pathway being involved in HNE-induced COX-2 overexpression in this cell type was ruled out by its suppression in cells pretreated with a p38 MAPK selective inhibitor (SB203580) [39,40]. The conclusive demonstration, in many cell types, of a key role of JNKs in HNE signaling does not necessarily mean that other key elements of the MAPK pathway may not be involved: Ruef and colleagues [41] treated rat aortic smooth muscle cells with 1–2.5 AM HNE and observed strong but transient activation of ERK1/2, induction of c-fos and c-jun protein synthesis, and an increase in AP-1 DNA binding activity. In addition, HNE induced platelet-derived growth factor-AA protein synthesis. These data indicate that HNE may be involved in smooth muscle cell proliferation by inducing mitogenic signals, and this effect may be important in atherogenesis [41]. Later, again using vascular smooth muscle cells, Kakishita and Hattori [42] confirmed this observation, while in their hands 2.5 AM HNE activated not only ERK1/2 but also JNKs and p38 MAPK. Further, it has been observed that 25 AM HNE induced secretion of fibronectin by IMR-90 human lung fibroblasts, following marked activation of ERK1/2, with p38 MAPK being only slightly activated and JNKs being unchanged. The HNE-induced secretion of fibronectin was inhibited by selective ERKs and p38 inhibitors. These data suggest that lipid oxidation products such as HNE may stimulate airway remodeling by inducing lung fibroblasts to produce extracellular matrix proteins, such as fibronectin, via the ERK signal transduction pathway [43]. HNE-induced activation of ERKs, JNKs, and p38 MAPK in lung endothelial cells has also been reported. Activation of the MAPK pathway by 4-hydroxynonenal (1–100 AM) favored cytoskeletal remodeling, with regard to actin, and enhanced endothelial barrier function [44]. THE IKAPPAB KINASE COMPLEX
A major signaling pathway associated with oxidative stress and inflammation is mediated by the transcription factor nuclear factor kappa B (NF-nB). NF-nB is an ubiquitous redox-sensitive peptide that is activated by a large number of extracellular stimuli including pro-
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inflammatory cytokines, chemokines, growth factors, oxydative stress, bacterial and viral products [45–47]. The key step in activation of the NF-nB signaling pathway is phosphorylation of InB (inhibitory component of NF-nB) proteins, which is mediated by a highmolecular-weight InB kinase complex (IKK) (approximately 700 kDa). IKK is a multisubunit serine-specific kinase composed of at least two catalytic subunits, IKKa (IKK-1) and IKKh (IKK-2), plus a regulatory subunit, IKKg. In response to upstream stimuli, the IKK complex is activated through phosphorylation operated by a heterogenous group of tyrosine kinases named IKK kinases, namely PKC, protein kinase B/Akt (PKB/Akt), or MAPK/ERK kinase kinase-1, -2, and -3 [48–50]. With regard to the possible interaction of HNE with the IKK complex, the few reports available thus far all point to an inhibitory effect of the aldehyde. For example, NF-nB activation induced by the stimuli lipopolysaccharide, interleukin-1h, or phorbol ester in THP-1 monocytes [51] or by tetradecanoylphorbolacetate/ionomycin in human carcinoma cells [52] was prevented by cell pretreatment with HNE (25–50 AM). In agreement with these findings, activation of NF-nB induced by exposure of THP-1 cells to Chlamydia pneumoniae was significantly reduced by 50 AM HNE [53]; HNE (25–50 AM) appears to exert inhibitory action on the NF-nB system by preventing the phosphorylation and consequent degradation of the inhibitory proteins InBa, InBh, and InBq [51–53]. This inhibition appears to be caused by a direct reaction between HNE and certain components of the IKK complex, as suggested by the fact that covalent adducts can form between the unsaturated aldehyde and the IKK complex [52]. The possible impairment of NFnB transcription activity, by concentrations of HNE actually detectable in cells and tissues, may have a significant impact on pathophysiology; for instance, down-regulated expression of NF-nB-regulated genes, as inducible by HNE, may interfere with the immune response and thus indirectly sustain inflammation and degenerative processes that would otherwise be more easily terminated. TYROSINE KINASE RECEPTORS
Tyrosine kinase receptors (RTKs), such as the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (PDGFR), are transmembrane glycoproteins that display tyrosine kinase activity in their cytoplasmic region. Stimulation of RTKs by ligand-dependent or -independent mechanisms (radiation, metal ions, ROS) induced dimerization in the receptors and autophosphorylation of several tyrosine residues, followed by induction of the catalytic activity
of the kinase. Activation of RTKs and of the downstream signaling pathways are involved in various biological processes such as proliferation, differentiation, migration, apoptosis, and the genesis or progression of human diseases [54–56]. As was very recently reviewed by Negre-Salvayre and colleagues [57], oxidized LDL (oxLDL) and related lipid peroxidation products, including HNE, can interact with EGF and PDGF-h receptors: oxLDL added to vascular cells induced and sustained rapid tyrosine phosphorylation of EGFR and PDGFR-h; the receptor’s activation was associated with recruitment of SH2 domain (SH) proteins, such as SHPTP2, phospholipase Cg , Src, and phosphatidylinositol 3 kinase (PI3K), on the phosphorylated tyrosine, consequently triggering the downstream signaling pathway [58,59]. Further investigation by the same group [59,60] pointed to HNE as an early mediator of RTK activation by oxLDL. The aldehyde was demonstrated to be responsible for the early, antioxidant-insensitive phase of activation of these receptors. Late activation of RTKs was shown, on the contrary, to be antioxidant sensitive and is likely mediated by oxidative stress [59,60]: HNE added to the culture medium of human endothelial cell line (CRL-1998 EC) in the concentration range 0.1–1 AM mimicked the action of oxLDL in favoring the autophosphorylation and activation of the receptor intrinsic tyrosine kinase [58]. The aldehyde was demonstrated to directly react with RTKs, inducing formation of HNE-EGFR or HNEPDGFR adducts. The generation of such adducts was demonstrated by the detection of HNE-protein epitopes on immunoprecipitated RTKs using anti-HNE-histidine antibodies and by the loss of free amino group content in the receptor molecules. The derivatization of RTKs was first confirmed in vitro by incubating rabbit smooth muscle cells (SMCs) with [H3]HNE-labeled oxLDL. A small but significant part of the added [H3]HNE was recovered bound to PDGFR-h [59]. The detection of PDGFR-h-HNE adducts in atherosclerotic aorta of cholesterol-fed rabbits provided the conclusive demonstration that HNE actually reacts and activates membrane RTKs [59]. Analysis of the biological responses triggered by HNE-dependent activation of tyrosine kinase receptors has shown that the aldehyde may up-regulate different signaling pathways depending upon incubation time and concentration: short incubation of vascular cells with a relatively low concentration of HNE (0.1–1 AM) induced the derivatization and autophosphorylation of RTKs, the subsequent activation of a signaling cascade that involved the PI3K/Akt survival pathway, and consequently the mitogenic response of SMCs [61]. On the contrary, incubation of human epidermoid carcinoma A431 cells with a relatively high concentration of
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exogenous HNE (500 AM) activated EGFR, ERK1/2, and JNK1/2 but inhibited cell growth [62]. In line with this finding, HNE concentrations in the medium-high pathophysiological range have been observed also to exert growth inhibition in other cell types, by inducing cell cycle arrest or even apoptosis [63,64]. Of note, interaction of HNE with RTKs apparently does not always result in their activation. At least in hHSC in culture, a low concentration of HNE (1 AM) inhibited tyrosine phosphorylation of PDGFR-h by its own ligand PDGFR-BB. This event was accompanied by inhibition of MAPKs and PI3K and, as a consequence, by a decrease in the PDGF-dependent DNA synthesis [65]. THE SERINE/THREONINE KINASE AKT
The serine/threonine kinase Akt, also called protein kinase B (PKB) because of the high homology of its kinase domain with those of protein kinases A and C, is a critical component of the signaling pathway triggered by PI3K. Following activation of PI3K by ligand-stimulated growth factor receptors and generation of phosphatidylinositol-3,4,5-triphosphate by the activated enzyme, PKB/Akt is recruited from the cytosol to the plasma membrane. It is then activated by phosphorylation on Thr308 and Ser473, by kinases such as phosphoinositidedependent kinase 1 (PDK1). Activated PKB/Akt leads to the phosphorylation of a large number of other proteins involved in the regulation of glucose metabolism, cell proliferation, apoptosis, cell migration, and gene expression. Termination of Akt signaling is mediated by the action of phosphatases such as protein phosphatase 2A
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(PP2A), which dephosphorylates Thr308 and Ser473 and brings PKB/Akt back to an inactive state in the cytosol [66–68]. PKB/Akt activity may also be affected by HNE. However, the only two reports on this matter available thus far provide opposing evidence: Liu and colleagues [69] showed that HNE (20 AM) induces apoptosis in human T-cell leukemia Jurkat cells through impairment of the Akt-mediated cell survival pathway. The aldehyde activated protein phosphatase 2A (PP2A), which in turn dephosphorylated Akt on Ser473. HNE-mediated dephosphorylation and inactivation of Akt were prevented by okadaic acid, a selective PP2A inhibitor. On the contrary, using a very different type of cells, namely human neuroblastoma IRM-32 cells, Dozza and colleagues [70] observed that challenge with 10 AM HNE provoked significant inhibition of the glycogen synthase kinase 3h, likely through the phosphorylation and activation of both Akt and ERK2: this HNE-dependent effect was also abolished by cell cotreatment with LY294002 or U0126, inhibitors of the PI3K and ERK pathways, respectively. The mechanism by which HNE up-regulates Akt has yet to be clarified; the aldehyde might act through activation of protein tyrosine kinases, such as membrane growth factor receptors [70]. CONCLUSIONS
Due to its peculiar biochemical structure and its relatively high diffusibility from the site of origin, 4hydroxynonenal is a good candidate molecule for integrating/modulating physiological cell signaling. This probability is strengthened by the fact that HNE
Fig. 1. Potential signaling by HNE in pathophysiology.
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concentrations that have been found to be active in signal transduction are within the range detectable in a variety of cells and tissues, under a number of different pathophysiological conditions. With regard to HNEinduced modulation of signaling kinases, experimental findings obtained thus far for PKCs and MAPKs appear to indicate that (i) in the lower concentration range the aldehyde influences PKC-dependent physiological events such as protein trafficking and secretion, while at relatively higher concentrations it tends to favor apoptotic death through involvement of novel rather than classic PKC isoforms and (ii) the latter effect is likely connected to HNE’s up-regulation of JNKs, while up-regulation of ERKs is more probably related to functional aspects such as cytoskeletal remodeling, the effect of growth factors, and so on. It is also now clear that 4-hydroxynonenal activates signaling pathways regulated by RTKs, a biochemical effect that would clearly qualify HNE as a signal transducing molecule. Further, the net demonstration of IKK inhibition by pathophysiological amounts of the aldehyde undoubtedly explains the HNE-induced downregulation of the NF-kB pathway that has been widely reported in different model systems. In most cases, HNE appears to react chemically with the various kinases to form more or less stable adducts. In addition to the influence on any effect exerted by the cell type involved and the concentration and cellular compartmentalization of the aldehyde, the location of HNE-adduct formation, whether in the regulatory or in the catalytic domain of the kinase, appears to be crucial. The former event would better allow enzyme upregulation by the aldehyde, while the latter appears more likely to lead to inactivation of the kinase, especially if there are numerous SH residues at that site of the molecule. Finally, Fig. 1 summarizes the pathophysiological processes to whose modulation 4-hydroxynonenal may, at least in principle, contribute. It reports the various signaling kinases possibly regulated by HNE in the different pathways. Acknowledgments — The research was supported by grants from the Italian Ministry of the University, PRIN 2001, 2002, the Monzino Foundation, Milan, Italy, the Piedmont Region, the University of Turin, and the Compagnia di San Paolo, Turin, Italy. Fanny Robbesyn was a recipient of a fellowship from Fondation pour la Recherche Me´dicale, Paris, France.
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ABBREVIATIONS
Ah — amyloid h protein AP-1 — activator protein 1 ARE — antioxidant-responsive element
COX-2 — cyclooxygenase-2 DAG — diacylglycerol EGFR — epidermal growth factor receptor ERKs — extracellular-signal-regulated kinases HNE — 4-hydroxynonenal HO-1 — heme oxygenase-1 H2O2 — hydrogen peroxide InB — inhibitory component of NF-nB IKK — IkappaB kinase IP3 — inositol triphosphate JNK — c-Jun N-terminal kinases MAPKs — mitogen-activated protein kinases MCP-1 — monocyte chemotactic protein-1 MKK — MAPK kinase MKPs — MAPK phosphatases NF-nB — nuclear factor kappa B Nrf2 — NF-E2-related factor 2 OxLDL — oxidized LDL PDGFR — platelet-derived growth factor receptor PDK1 — phosphoinositide-dependent kinase 1 PI3K — phosphatidylinositol 3 kinase PKB — protein kinase B PKC — protein kinase C PP2A — protein phosphatase 2A PTPs — protein tyrosine phosphatases PUFAs — polyunsaturated fatty acids ROS — reactive oxygen species RTKs — tyrosine kinase receptor SAPKK1 (or SEK1) — stress-activated protein kinase kinase 1 SH — SH2 domain