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PubMed1997

Selective inhibition of oncogenic ras-p21 in vivo by agents that block its interaction with jun-N-kinase (JNK) and jun proteins. Implications for the design of selective chemotherapeutic agents.

We have obtained evidence that oncogenic and activated normal ras-p21 proteins utilize overlapping but distinct signal transduction pathways. Recently, we found that ras-p21 binds to both jun and its kinase, jun kinase (JNK). We now present evidence that suggests that oncogenic but not normal activated p21 depends strongly on early activation of JNK/jun. This early activation most likely involves direct interaction between oncogenic p21 and JNK/jun because p21 peptides that blocked the binding of p21 to JNK and jun strongly inhibited oncogenic p21-induced oocyte maturation while they did not inhibit insulin-activated normal cellular p21-induced maturation. Very similar results were also obtained for a newly characterized specific inhibitor of JNK which blocked oncogenic but not normal activated p21-induced oocyte maturation. We also found that both jun and JNK strongly enhanced oncogenic p21-induced oocyte maturation while they inhibited insulin-activated normal p21-induced oocyte maturation. These results suggest that the peptides and JNK inhibitor may be useful agents in selectively blocking the effects of oncogenic but not normal p21 in cells.

Animals↗
PubMed1992

Angiotensin-II-induced expression of proto-oncogene (c-fos, jun-B and c-jun) mRNA in bovine adrenocortical fasciculata cells (BAC) is mediated by AT-1 receptors.

We have shown previously that angiotensin-II (A-II) controls proto-oncogene (c-fos, jun-B and c-jun) mRNA accumulation in bovine adrenal fasciculata cells (BAC). Since BAC contain both subtypes (AT-1 and AT-2) of the A-II receptor, we have investigated which subtype was involved in the effect of A-II on proto-oncogene mRNA by using a selective antagonist for AT-1 (DUP 753) and for AT-2 (CGP 42112A). DUP 753, but not CGP 42112A, inhibited the stimulatory effect of A-II on proto-oncogene mRNA, with ID50s of 4 x 10(-7) M, 7 x 10(-7) M and 2 x 10(-6) M for c-fos, jun-B and c-jun, respectively. Neither of the two antagonists by themselves had a direct effect on proto-oncogene mRNA. As the A-II AT-1 receptors are coupled to the phospholipase C system in BAC, we have investigated whether the A-II effects on the proto-oncogenes were mediated by protein kinase C (PKC) or by Ca2+ calmodulin. First, activation of PKC by the phorbol ester, PMA, increased the level of three proto-oncogene mRNAs, whereas calcium ionophore had no effect. Second, staurosporine, a specific inhibitor of PKC, reduced the stimulatory action of A-II on proto-oncogene mRNA by 80-90%, whereas trifluoroperazine, an inhibitor of calmodulin, had no significant effect. These results demonstrate that the effects of A-II on proto-oncogene mRNA are mediated by AT1 receptor subtypes, mainly through activation of the PKC pathway.

Angiotensin II↗
PubMed1999

Induction of apoptosis by dexrazoxane (ICRF-187) through caspases in the absence of c-jun expression and c-Jun NH2-terminal kinase 1 (JNK1) activation in VM-26-resistant CEM cells.

Dexrazoxane (ICRF-187) is an inhibitor of the catalytic activity of DNA topoisomerase II (topo II) that does not stabilize DNA-topo II covalent complexes. Here, we examined cytotoxic signaling by ICRF-187 in human leukemic CEM cells and a teniposide (VM-26)-resistant subline, CEM/VM-1. Treatment of CEM and CEM/VM-1 cells with ICRF-187 induced apoptotic cell death characterized by internucleosomal DNA fragmentation, nuclear condensation, and induction of at least caspase-3- and -7-like protease activities (but not caspase 1). Treatment of these cells with Z-Asp-2,6-dichlorobenzoyloxymethyl-ketone, a potent inhibitor of apoptosis, inhibited ICRF-187-induced DEVD-specific caspase activity and apoptosis in a concentration-dependent manner. ICRF-187-induced apoptosis in CEM cells was associated with transient induction of c-jun and activation of c-Jun NH2-terminal kinase 1 (JNK1). However, CEM/VM-1 cells, which were 3-fold more sensitive than CEM cells to ICRF-187 due to a decrease in topo II activity, exhibited ICRF-187-induced apoptosis in the absence of c-jun induction and JNK1 activation. These results indicate that catalytic inhibition of topo II by ICRF-187 leads to apoptosis through at least a caspase-3- and -7-like protease-dependent mechanism and suggest that c-jun and JNK1 are not required in ICRF-187-induced apoptosis in CEM cells.

Antineoplastic Agents↗
PubMed2002

An inhibitor of c-jun aminoterminal kinase (SP600125) represses c-Jun activation, DNA-binding and PMA-inducible 92-kDa type IV collagenase expression.

The 92-kDa type IV collagenase (MMP-9) contributes to tumor invasion and metastases and strategies to down-regulate its expression could ultimately be of clinical utility. Although the expression of this collagenase is regulated by numerous growth factors, the signaling pathways that transduce these signals are fewer in number and therefore represent pharmacological targets. In this regard, we previously reported that MMP-9 expression was regulated by the c-jun amino terminal kinase (JNK) signaling cascade. Therefore, we undertook a study to determine the efficacy of a novel compound (SP600125), which binds to the ATP binding site of all known JNKs, in repressing MMP-9 expression. In OVCAR-3 cells, SP600125 inhibited the PMA-dependent secretion of MMP-9 in a time-dependent manner and over a dose range that blocked c-Jun phosphorylation and AP-1 binding. SP600125 repressed the activity of a PMA-stimulated MMP-9 promoter-driven luciferase reporter, suggesting that diminished secretion of this collagenase reflected reduced transcription. Further, the activity of a GAL4-driven reporter in PMA-treated cells, co-transfected with an expression construct encoding the trans-activation domain of c-Jun fused to the DNA binding domain of GAL4, was repressed by SP600125. These findings indicate the efficacy of SP600125 in inhibiting c-Jun activation, DNA-binding and the PMA-dependent induction of MMP-9 expression.

Anthracenes↗
PubMed2004

Immunohistochemical analysis of tyrosine phosphorylation and AP-1 transcription factors c-Jun, Jun D, and Fos family during early ovarian follicle development in the mouse.

The growth control mechanism of early-stage ovarian follicles is unknown. Tyrosine phosphorylation of signaling molecules and changes in expression and activation of AP-1 transcription factors have been implicated in growth regulation of numerous cell types. In this study, we used immunohistochemistry to analyze tyrosine phosphorylation patterns and expression and activation of selected AP-1 transcription factors in mouse ovarian follicles. The ovaries were collected from B62F1/J mice in estrus. Representative sections were immunostained for phosphotyrosine, phospho-c-Jun, Jun D, and c-Fos. Phosphotyrosine staining was perioocytic from the transitional stage until approximately 5 to 7 layers of granulosa cells had formed. Perioocytic staining was then replaced by scattered stippled staining in granulosa cells of larger follicles. Phospho c-Jun was exclusively expressed in mitotic granulosa cells of follicles from transitional to antral stages. Jun D was expressed in the oocytes of primordial, primary, or transitional follicles and disappeared at the 2-layer preantral stage. Fos was present in corpora lutea and theca cells but not in granulosa cells. Collectively, these data indicate that phosphotyrosine signaling and AP-1 transcription factors are intimately involved in early stages of ovarian follicle growth.

Animals↗
PubMed2005

Induction of the RelB NF-kappaB subunit by the cytomegalovirus IE1 protein is mediated via Jun kinase and c-Jun/Fra-2 AP-1 complexes.

We recently demonstrated that the cytomegalovirus (CMV) immediate-early 1 (IE1) protein induces transcription of the gene encoding the RelB NF-kappaB subunit. The mechanism of this activation has been explored here. We report that the induction of the relB promoter by IE1 protein is mediated via activation of JNK and AP-1. The region controlling relB promoter induction was mapped to the upstream approximately 600-bp region between -1694 and -1096 bp. IE1 stimulated AP-1 activity in NIH 3T3 cells. Competition electrophoretic mobility shift assay (EMSA) confirmed the presence of one bona fide AP-1 element centered at -1503 bp. Introduction of a G-to-C mutation in the AP-1 binding site within the distal region of the relB promoter eliminated its activation by IE1 in both NIH 3T3 fibroblasts and vascular smooth muscle cells (SMCs). Supershift EMSA identified c-Jun, Fra-2, and c-Fos in AP-1 binding complexes in IE1 transfected NIH 3T3 cells. IE1 induced c-Jun phosphorylation, and treatment with SP600125, a selective JNK inhibitor, as well as overexpression of JNK-binding domain of JIP1, blocked IE1-mediated induction of AP-1 and relB promoter activity in NIH 3T3 cells and SMCs. Ectopic expression of c-Jun plus Fra-2, but not c-Fos, induced relB promoter activity. The relB promoter has two proximal NF-kappaB elements, and c-Jun/Fra-2 worked in synergy with p50/p65 NF-kappaB complexes. Overall, these findings demonstrate for the first time the role of AP-1 in transcriptional regulation of a gene encoding an NF-kappaB subunit, and its involvement in induction of RelB activity by the CMV IE1 protein.

Animals↗
PubMed2004

c-Jun N-terminal protein kinase 1 (JNK1), but not JNK2, is essential for tumor necrosis factor alpha-induced c-Jun kinase activation and apoptosis.

Two ubiquitously expressed isoforms of c-Jun N-terminal protein kinase (JNK), JNK1 and JNK2, have shared functions and different functions. However, the molecular mechanism is unknown. Here we report that JNK1, but not JNK2, is essential for tumor necrosis factor alpha (TNF-alpha)-induced c-Jun kinase activation, c-Jun expression, and apoptosis. Using mouse fibroblasts deficient in either Jnk1 or Jnk2, we found that JNK1 was activated by TNF-alpha, whereas JNK2 activation was negligible. In addition, JNK2 interfered with JNK1 activation via its "futile" phosphorylation by upstream kinases. Consequently, expression and activation of c-Jun, which depends on JNK activity, were impaired in Jnk1 null cells but enhanced in Jnk2 null cells. TNF-alpha-induced apoptosis was also suppressed in Jnk1 null fibroblasts but increased in Jnk2 null cells. Thus, our results provide a molecular mechanism underlying the different biological functions of JNK isoforms.

Animals↗
PubMed1995

The leucine zipper of c-Jun binds to ribosomal protein L18a: a role in Jun protein regulation?

Recently we developed a method called direct interaction rescue (DIRE) for selective cloning in filamentous phage. The rescue is effected by the interaction of two heterologous proteins, one fused to the N-terminus of gene 3 adhesion protein, the other fused to the C-terminus. When heterologous fusion proteins interact with each other, gene 3 protein activity in restored thereby rescuing phage infectivity. We have used the leucine zipper of c-Jun protein as a 'bait' to select for interacting proteins from a human cDNA library. Two interacting clones were isolated, one coding for ribosomal protein L18a, a component of the large ribosomal subunit, and the other for tropomyosin, a component of the cytoskeleton. L18a contains two zipper-like domains which probably interact with c-Jun. We consider it possible that L18a (and tropomyosin) are involved in the cellular regulation of Jun protein levels.

Amino Acid Sequence↗
PubMed1999

A comparative study of fibrous dysplasia and osteofibrous dysplasia with regard to expressions of c-fos and c-jun products and bone matrix proteins: a clinicopathologic review and immunohistochemical study of c-fos, c-jun, type I collagen, osteonectin, osteopontin, and osteocalcin.

Fibrous dysplasia and osteofibrous dysplasia are both benign fibro-osseous lesions of the bone and are generally seen during childhood or adolescence. Histologically, the features of these bone lesions sometimes look quite similar, but their precise nature remains controversial. We retrospectively studied clinicopathologic findings in 62 cases of fibrous dysplasia and 20 cases of osteofibrous dysplasia with regard to their anatomic location and histological appearance. From among these cases, the immunohistochemical expressions of c-fos and c-jun proto-oncogene products and bone matrix proteins of type I collagen, osteonectin, osteopontin, and osteocalcin were evaluated in 20 typical fibrous dysplasias and 17 osteofibrous dysplasias using paraffin sections, and these expressions were then assessed semiquantitatively. Microscopically, fibrous dysplasia showed various secondary changes, such as hyalinization, hemorrhage, xanthomatous reaction, and cystic change in 22 of the 62 cases (35%). This was a higher incidence than in osteofibrous dysplasia, in which only 2 of the 20 cases (10%) showed such changes. In the elderly fibrous dysplasia cases, the cellularity of fibroblast-like cells was rather low, and those cases were hyalinized. Almost all of the cases of fibrous dysplasia and osteofibrous dysplasia showed positive expressions of c-fos and c-jun products. The expressions of type I collagen and osteopontin showed no difference between fibrous dysplasia and osteofibrous dysplasia. Immunoreactivity for osteonectin in bone matrix was detected in only 1 case of fibrous dysplasia (1 of 20), whereas it was recognized in 14 of the 17 cases of osteofibrous dysplasia. Furthermore, the immunoreactivity for osteocalcin in bone matrix and fibroblast-like cells was higher in fibrous dysplasia than it was in osteofibrous dysplasia, semiquantitatively. Our immunohistochemical results regarding osteonectin and osteocalcin suggest that the bone matrix of fibrous dysplasia is somewhat more mature than that of osteofibrous dysplasia, and that the fibroblast-like cells in fibrous dysplasia share some phenotypic features with osteoprogenitor cells of normal osteogenic tissues. Fibrous dysplasia and osteofibrous dysplasia share some similar histological features, including c-fos and c-jun expressions, although different clinicohistologic features and immunohistochemical expressions of osteonectin and osteocalcin were observed. These features suggest that the mechanisms behind the development of fibrous dysplasia and osteofibrous dysplasia are similar, but this is not necessarily indicative of a closer relationship between the 2 diseases.

Adolescent↗
PubMed1995

The Bcr-Abl leukemia oncogene activates Jun kinase and requires Jun for transformation.

The leukemogenic tyrosine kinase fusion protein Bcr-Abl activates a Ras-dependent pathway required for transformation. To examine subsequent signal transduction events we measured the effect of Bcr-Abl on two mitogen-activated protein kinase (MAPK) cascades--the extracellular signal-regulated kinase (ERK) pathway and the Jun N-terminal kinase (JNK) pathway. We find that Bcr-Abl primarily activates JNK in fibroblasts and hematopoietic cells. Bcr-Abl enhances JNK function as measured by transcription from Jun responsive promoters and requires Ras, MEK kinase (MAPK/ERK kinase kinase), and JNK to do so. Dominant-negative mutants of c-Jun, which inhibit the endpoint of the JNK pathway, impair Bcr-Abl transforming activity. These findings implicate the JNK pathway in transformation by a human leukemia oncogene.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

Hypoglycemia-induced c-Jun phosphorylation is mediated by c-Jun N-terminal kinase 1 and Lyn kinase in drug-resistant human breast carcinoma MCF-7/ADR cells.

We studied the signal transduction mechanism that is involved in c-Jun phosphorylation evident after glucose deprivation in MCF-7/ADR cells. Glucose deprivation caused an immediate increase in tyrosine phosphorylation in MCF-7/ADR cells and specifically activated Lyn kinase, a src family tyrosine kinase. In addition, hypoglycemic treatment strongly activated c-Jun N-terminal kinase 1 (JNK1), leading to the phosphorylation and activation of c-Jun. Experiments with Lyn antisense oligonucleotides demonstrated that Lyn kinase activation was responsible for the activation of JNK1 but not extracellular signal-regulated kinase. We also observed glucose deprivation-induced Ras activation in MCF-7/ADR cells. These results indicate a possible Ras-dependent signaling pathway involving Lyn kinase and JNK1, which leads to the glucose deprivation-induced responses in MCF-7/ADR cells.

Breast Neoplasms↗
PubMed1997

Signal transduction in fibroblasts stably transformed by [Val12]Ras--the activities of extracellular-signal-regulated kinase and Jun N-terminal kinase are only moderately increased, and the activity of the delta-inhibitor of c-Jun is not alleviated.

Ras-transformed cells often show high levels of expression of activating protein-1 and Ets and of genes regulated by these transcription factors. In analogy with the effects of transient stimulation of Ras, it is assumed that the increase in transcription-factor transactivation in stably transformed cells is due to Ras-induced constitutive activation of mitogen-activated protein kinases. However, this has not been extensively studied. Using specific substrate peptides, we have examined here the activities of two types of mitogen-activated protein kinase, extracellular-signal-regulated kinase (ERK) and Jun N-terminal kinase (JNK), in [Val12]Ras-transformed rat embryo fibroblast cell lines. These activities were elevated 2-3-fold in Ras-transformed cells compared with non-transformed cells with a similar growth rate. Increased ERK activity was not necessarily accompanied by a similar increase in JNK activity. In transformed cells, ERK and JNK activities could be stimulated fourfold and ninefold by phorbol ester and ultraviolet-light treatment, respectively, indicating that only a fraction of these enzymes were constitutively activated in these cells. It has been suggested that inactive JNK downregulates c-Jun transcriptional activity by binding to the c-Jun delta-domain. No decrease in delta-inhibitor activity could be demonstrated in Ras-transformed cells compared with control cells, consistent with the presence of mainly inactive JNK in transformed cells. Treatment of transformed cells wih benzodiazepine 5B, an inhibitor of Ras farnesylation, decreased ERK and JNK activities, and concomitantly caused morphological reversion, reduced growth rate, and normalization of transformation-related gene expression. We conclude that in stably Ras-transformed cells the moderately increased ERK/JNK activities are not coregulated, and that ERK rather than JNK activity correlated with transformation-related gene expression.

Animals↗
PubMed1999

Extracellular-regulated kinase 1/2, Jun N-terminal kinase, and c-Jun are involved in NF-kappa B-dependent IL-6 expression in human monocytes.

In the present study we investigated the possible involvement of the mitogen-activated protein kinase family members extracellular-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) in mediating IL-6 gene expression in human monocytes, in particular their role in enhancing NF-kappa B activity. Freshly isolated monocytes treated with the protein phosphatase inhibitor okadaic acid secreted high levels of IL-6 protein, which coincided with enhanced binding activity of NF-kappa B as well as with phosphorylation and activation of the ERK1/2 and JNK proteins. The ERK pathway-specific inhibitor PD98059 inhibited IL-6 secretion from monocytes. Transient overexpression of inactive mutants of either Raf-1 or JNK1 showed that both pathways were involved in kappa B-dependent IL-6 promoter activity. By using PD98059, we demonstrated that the Raf1/MEK1/ERK1/2 pathway did not affect the DNA binding of NF-kappa B but, rather, acted at the level of transcriptional activity of NF-kappa B. Interestingly, it was shown that NF-kappa B-mediated gene transcription, both in the context of the IL-6 promoter as well as on its own, was dependent on both serine kinase activity and interaction with c-Jun protein. We conclude that okadaic acid-induced IL-6 gene expression is at least partly mediated through the ERK1/2 and JNK pathway-dependent activation of NF-kappa B transcriptional capacity. Our results suggest that the JNK pathway may regulate NF-kappa B-mediated gene transcription through its phosphorylation and activation of c-Jun.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1999

The JNKK2-JNK1 fusion protein acts as a constitutively active c-Jun kinase that stimulates c-Jun transcription activity.

c-Jun N-terminal protein kinase (JNK), a member of the mitogen-activated protein (MAP) kinase family, regulates gene expression in response to various extracellular stimuli. JNK is activated by JNK-activating kinase (JNKK1 and JNKK2), a subfamily of the dual specificity MAP kinase kinase (MEK) family, through phosphorylation on threonine (Thr) 183 and tyrosine (Tyr) 185 residues. The physiological functions of the JNK pathway, however, are not completely understood. A major obstacle is the lack of specific and activated kinase components that can stimulate the JNK pathway in the absence of any stimulus. Here we show that fusion of JNK1 to its upstream activator JNKK2 resulted in its constitutive activation. In HeLa cells, the JNKK2-JNK1 fusion protein showed significant JNK activity, which was comparable with that of JNK1 activated by many stimuli and activators, including EGF, TNF-alpha, anisomycin, UV irradiation, MEKK1, and small GTP binding proteins Rac1 and Cdc42Hs. Immunoblotting analysis indicated that JNK1 was phosphorylated by JNKK2 in the fusion protein on both Thr(183) and Tyr(185) residues. Like JNKK2, the JNKK2-JNK1 fusion protein was highly specific for the JNK pathway and did not activate either p38 or ERK2. Transient transfection assays demonstrated that the JNKK2-JNK1 fusion protein was sufficient to stimulate c-Jun transcriptional activity in the absence of any stimulus. Immunofluorescence analysis revealed that the JNKK2-JNK1 fusion protein was predominantly located in the nucleus of transfected HeLa cells. These results indicate that the JNKK2-JNK1 fusion protein is a constitutively active Jun kinase, which will facilitate the investigation of the physiological roles of the JNK pathway.

Fluorescent Antibody Technique↗
PubMed2002

Methamphetamine causes coordinate regulation of Src, Cas, Crk, and the Jun N-terminal kinase-Jun pathway.

The clinical abuse of methamphetamine (METH) is a major concern because it can cause long-lasting neurodegenerative effects in humans. Current concepts of the molecular mechanisms underlying these complications have centered on the formation of reactive oxygen species. Herein, we provide cDNA microarray evidence that METH administration caused the induction of c-Jun and of other members involved in the pathway leading to c-Jun activation [stress-activated protein kinase/Jun N-terminal kinase (JNK3), Crk-associated substrate-Cas and c-Src] after environmental stresses or cytokine stimulation. Reverse transcription-polymerase chain reaction analysis confirmed these increases and also showed that the expression of JNK1 and JNK3 but not JNK2 was also increased in the METH-treated mice. Western blot analysis showed that METH increased the expression of c-Jun phosphorylated at serine-63 and serine-73 residues. Other upstream members of the JNK pathway, including phosphorylated JNKs, mitogen-activated protein kinase kinase 4, mitogen-activated protein kinase kinase 7, Crk II, Cas, and c-Src were also increased at the protein level. These values returned to baseline by 1 week after drug treatment. These results are discussed in terms of their support for a possible role of the activation of the JNK/Jun pathway in the pathophysiological effects of METH.

Animals↗
PubMed1993

Temporal and spatial patterns of expression of c-fos, zif/268, c-jun and jun-B mRNAs in rat brain following seizures evoked focally from the deep prepiriform cortex.

Using in situ hybridization, we previously investigated (17) the regional pattern of c-fos mRNA increases in the brain following convulsive seizures elicited from a highly circumscribed epileptogenic site located in the deep prepiriform cortex. In this paper, we focus on the hippocampus and examine mRNAs encoding other immediate early genes (IEGs), namely c-jun, jun-B, and zif/268, for changes following the focally evoked seizures. Although the anatomic distribution of increases in each IEG mRNA was qualitatively comparable, the temporal analysis indicated that increases in zif/268 mRNA appeared prior to the other genes studied. Each of the mRNAs reached a maximum increase by 30 min and declined to basal levels within 3 h following seizure initiation. The data indicate that these four IEGs respond in a coordinated fashion to propagated seizure activity with increases in mRNA and, furthermore, that increased expression of all four genes appears to occur in the same cell types in the hippocampus.

Animals↗
PubMed1992

Expression of c-fos, c-jun, jun-B, metallothionein and metalloproteinase genes in human chondrocyte.

Normal and osteoarthritic (OA) human articular cartilage chondrocytes, released enzymatically in the presence of 0.5% fetal calf serum, display constitutive expression of early response activating protein (AP-1) genes; c-fos, c-jun and jun-B. Among the late AP-1 responsive genes, total metallothionein (MT) and stromelysin mRNAs were expressed at high levels in both normal and OA chondrocytes, while collagenase and hMT-IIA mRNA levels were elevated only in OA individuals. Despite the common AP-1 sequences present in their promoter regions, the three late genes were differentially expressed.

Aged↗
PubMed2001

Flavonoids protect neurons from oxidized low-density-lipoprotein-induced apoptosis involving c-Jun N-terminal kinase (JNK), c-Jun and caspase-3.

Oxidative stress has been associated with neuronal loss in neurodegenerative diseases and during age-associated cognitive decline. Flavonoids have been proposed to play a useful role in protecting the central nervous system against oxidative and excitotoxic stress, although the mechanism of action is unknown. Using oxidized low-density lipoprotein (oxLDL) as the oxidative insult we investigated the mechanism of neurotoxicity and attempted to identify possible sites of action of two of the most potent protective flavonoids, epicatechin and kaempferol, in cultured primary neurons. Using cultured striatal neurons and selective phosphospecific antibodies we addressed the potential role of extracellular signal-regulated kinases 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). OxLDL stimulated a Ca(2+)-dependent activation of both ERK1/2 and JNK that was strongly inhibited by pre-treatment with low micromolar concentrations of epicatechin. Neurotoxicity induced by oxLDL, however, was neither reduced nor enhanced by inhibiting ERK1/2 activation with mitogen-activated protein kinase kinase (MEK) inhibitors, suggesting that this cascade is unlikely to be involved in either oxLDL toxicity or the protective effects of flavonoids. oxLDL caused a sustained activation of JNK that resulted in the phosphorylation of the transcription factor c-Jun, which was abolished in neurons pre-treated with flavonoids. Furthermore, oxLDL induced the cleavage of procaspase-3 and increased caspase-3-like protease activity in neurons, an effect which was strongly inhibited by pre-exposure to either epicatechin or kaempferol. In addition, a caspase-3 inhibitor reduced oxLDL-induced neuronal death, implicating an apoptotic mechanism. A major in vivo metabolite of epicatechin, 3'-O-methyl-epicatechin was as effective as epicatechin in protecting neurons. Thus dietary flavonoids might have potential as protective agents against neuronal apoptosis through selective actions within stress-activated cellular responses, including protein kinase signalling cascades.

Animals↗