Using U0126 to dissect the role of the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade in the regulation of gene expression by endothelin-1 in cardiac myocytes

The hypertrophic agonist endothelin-1 rapidly but transiently activates the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade (and other signalling pathways) in cardiac myocytes, but the events linking this to hypertrophy are not understood. Using Affymetrix rat U34A microarrays, we identified the short-term (2-4 h) changes in gene expression induced in neonatal myocytes by endothelin-1 alone or in combination with the ERK1/2 cascade inhibitor, U0126. Expression of 15 genes was significantly changed by U0126 alone, and expression of an additional 78 genes was significantly changed by endothelin-1. Of the genes upregulated by U0126, four are classically induced through the aryl hydrocarbon receptor (AhR) by dioxins suggesting that U0126 activates the xenobiotic response element in cardiac myocytes potentially independently of effects on ERK1/2 signalling. The 78 genes showing altered expression with endothelin-1 formed five clusters: (i) three clusters showing upregulation by endothelin-1 according to time course (4 h > 2 h; 2 h > 4 h; 2 h approximately 4 h) with at least partial inhibition by U0126; (ii) a cluster of 11 genes upregulated by endothelin-1 but unaffected by U0126 suggesting regulation through signalling pathways other than ERK1/2; (iii) a cluster of six genes downregulated by endothelin-1 with attenuation by U0126. Thus, U0126 apparently activates the AhR in cardiac myocytes (which must be taken into account in protracted studies), but careful analysis allows identification of genes potentially regulated acutely via the ERK1/2 cascade. Our data suggest that the majority of changes in gene expression induced by endothelin-1 are mediated by the ERK1/2 cascade.

Regulation of Ras.GTP loading and Ras-Raf association in neonatal rat ventricular myocytes by G protein-coupled receptor agonists and phorbol ester. Activation of the extracellular signal-regulated kinase cascade by phorbol ester is mediated by Ras.

The small G protein Ras has been implicated in hypertrophy of cardiac myocytes. We therefore examined the activation (GTP loading) of Ras by the following hypertrophic agonists: phorbol 12-myristate 13-acetate (PMA), endothelin-1 (ET-1), and phenylephrine (PE). All three increased Ras.GTP loading by 10-15-fold (maximal in 1-2 min), as did bradykinin. Other G protein-coupled receptor agonists (e.g. angiotensin II, carbachol, isoproterenol) were less effective. Activation of Ras by PMA, ET-1, or PE was reduced by inhibition of protein kinase C (PKC), and that induced by ET-1 or PE was partly sensitive to pertussis toxin. 8-(4-Chlorophenylthio)-cAMP (CPT-cAMP) did not inhibit Ras.GTP loading by PMA, ET-1, or PE. The association of Ras with c-Raf protein was increased by PMA, ET-1, or PE, and this was inhibited by CPT-cAMP. However, only PMA and ET-1 increased Ras-associated mitogen-activated protein kinase kinase 1-activating activity, and this was decreased by PKC inhibition, pertussis toxin, and CPT-cAMP. PMA caused the rapid appearance of phosphorylated (activated) extracellular signal-regulated kinase in the nucleus, which was inhibited by a microinjected neutralizing anti-Ras antibody. We conclude that PKC- and Gi-dependent mechanisms mediate the activation of Ras in myocytes and that Ras activation is required for stimulation of extracellular signal-regulated kinase by PMA.

Up-regulation of c-jun mRNA in cardiac myocytes requires the extracellular signal-regulated kinase cascade, but c-Jun N-terminal kinases are required for efficient up-regulation of c-Jun protein.

Cardiac hypertrophy, an important adaptational response, is associated with up-regulation of the immediate early gene, c- jun, which encodes the c-Jun transcription factor. c-Jun may feed back to up-regulate its own transcription and, since the c-Jun N-terminal kinase (JNK) family of mitogen-activated protein kinases (MAPKs) phosphorylate c-Jun(Ser-63/73) to increase its transactivating activity, JNKs are thought to be the principal factors involved in c- jun up-regulation. Hypertrophy in primary cultures of cardiac myocytes is induced by endothelin-1, phenylephrine or PMA, probably through activation of one or more of the MAPK family. These three agonists increased c- jun mRNA with the rank order of potency of PMA approximately endothelin-1>phenylephrine. Up-regulation of c- jun mRNA by endothelin-1 was attenuated by inhibitors of protein kinase C (GF109203X) and the extracellular signal-regulated kinase (ERK) cascade (PD98059 or U0126), but not by inhibitors of the JNK (SP600125) or p38-MAPK (SB203580) cascades. Hyperosmotic shock (0.5 M sorbitol) powerfully activates JNKs, but did not increase c- jun mRNA. These data suggest that ERKs, rather than JNKs, are required for c- jun up-regulation. However, endothelin-1 and phenylephrine induced greater up-regulation of c-Jun protein than PMA and phosphorylation of c-Jun(Ser-63/73) correlated with the level of c-Jun protein. Up-regulation of c-Jun protein by endothelin-1 was attenuated by inhibitors of protein kinase C and the ERK cascade, probably correlating with a primary input of ERKs into transcription. In addition, SP600125 inhibited the phosphorylation of c-Jun(Ser-63/73), attenuated the increase in c-Jun protein induced by endothelin-1 and increased the rate of c-Jun degradation. Thus whereas ERKs are the principal MAPKs required for c- jun transcription, JNKs are necessary to stabilize c-Jun for efficient up-regulation of the protein.

Signaling through the extracellular signal-regulated kinase 1/2 cascade in cardiac myocytes.

The extracellular signal-regulated kinases 1/2 (ERK1/2) are particularly implicated in the growth response of cardiac myocytes. In these cells, the ERK1/2 pathway is potently activated by Gq protein-coupled receptor agonists (such as endothelin-1 or alpha-adrenergic agonists), which activate protein kinase C isoforms. Here, we review the mechanisms associated with the activation of the ERK1/2 pathway by these agonists with particular emphasis on signal integration into the pathway. Signaling to the nucleus and the regulation of transcription factor activity associated with ERK1/2 activation in cardiac myocytes are also discussed.

Peptide growth factors signal differentially through protein kinase C to extracellular signal-regulated kinases in neonatal cardiomyocytes

The extracellular signal-regulated kinases 1/2 (ERK1/2) are activated in cardiomyocytes by Gq protein-coupled receptors and are associated with induction of hypertrophy. Here, we demonstrate that, in primary cardiomyocyte cultures, ERK1/2 were also significantly activated by platelet-derived growth factor (PDGF), epidermal growth factor (EGF) or fibroblast growth factor (FGF), but insulin, insulin-like growth factor 1 (IGF-1) and nerve growth factor (NGF) had relatively minor effects. PDGF, EGF or FGF increased cardiomyocyte size via ERK1/2, whereas insulin, IGF-1 or NGF had no effect suggesting minimum thresholds/durations of ERK1/2 signaling are required for the morphological changes associated with hypertrophy. Peptide growth factors are widely accepted to activate phospholipase C gamma1 (PLCgamma1) and protein kinase C (PKC). In cardiomyocytes, only PDGF stimulated tyrosine phosphorylation of PLCgamma1 and nPKCdelta. Furthermore, activation of ERK1/2 by PDGF, but not EGF, required PKC activity. In contrast, EGF substantially increased Ras.GTP with rapid activation of c-Raf, whereas stimulation of Ras.GTP loading by PDGF was minimal and activation of c-Raf was delayed. Our data provide clear evidence for differential coupling of PDGF and EGF receptors to the ERK1/2 cascade, and indicate that a minimum threshold/duration of ERK1/2 signaling is required for the development of cardiomyocyte hypertrophy.

Monophosphothreonyl extracellular signal-regulated kinases 1 and 2 (ERK1/2) are formed endogenously in intact cardiac myocytes and are enzymically active

ERK1 and ERK2 (ERK1/2) are central to the regulation of cell division, growth and survival. They are activated by phosphorylation of the Thr- and the Tyr- residues in their Thr-Glu-Tyr activation loops. The dogma is that dually-phosphorylated ERK1/2 constitute the principal activities in intact cells. We previously showed that, in neonatal rat cardiac myocytes, endothelin-1 and phorbol 12-myristate 13-acetate (PMA) powerfully and rapidly (maximal at ~ 5 min) activate ERK1/2. Here, we show that dually-phosphorylated ERK1/2 rapidly (< 2 min) appear in the nucleus following stimulation with endothelin-1. We characterized the active ERK1/2 species in myocytes exposed to endothelin-1 or PMA using MonoQ FPLC. Unexpectedly, two peaks of ERK1 and two peaks of ERK2 activity were resolved using in vitro kinase assays. One of each of these represented the dually-phosphorylated species. The other two represented activities for ERK1 or ERK2 which were phosphorylated solely on the Thr- residue. Monophosphothreonyl ERK1/2 represented maximally ~ 30% of total ERK1/2 activity after stimulation with endothelin-1 or PMA, and their kcat values were estimated to be minimally ~ 30% of the dually-phosphorylated species. Appearance of monophosphothreonyl ERK1/2 was rapid but delayed in comparison with dually-phosphorylated ERK1/2. Of 10 agonists studied, endothelin-1 and PMA were most effective in terms of ERK1/2 activation and in stimulating the appearance of monophosphothreonyl and dually-phosphorylated ERK1/2. Thus, enzymically active monophosphothreonyl ERK1/2 are formed endogenously following activation of the ERK1/2 cascade and we suggest that monophosphothreonyl ERK1/2 arise by protein tyrosine phosphatase-mediated dephosphorylation of dually-phosphorylated ERK1/2.

Phenylephrine promotes phosphorylation of Bad in cardiac myocytes through the extracellular signal-regulated kinases 1/2 and protein kinase A.

Studies in non-cardiomyocytic cells have shown that phosphorylation of the Bcl-2 family protein Bad on Ser-112, Ser-136 and Ser-155 decreases its pro-apoptotic activity. Both phenylephrine (100 microM) and the cell membrane-permeating cAMP analog, 8-(4-chlorophenylthio)-cAMP (100 microM), protected against 2-deoxy-D-glucose-induced apoptosis in neonatal rat cardiac myocytes as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). In cardiac myocytes, phenylephrine primarily stimulates the alpha-adrenoceptor, but, at high concentrations (100 microM), it also increases the activity of the cAMP-dependent protein kinase, protein kinase A (PKA) through the beta-adrenoceptor. Phenylephrine (100 microM) promoted rapid phosphorylation of Bad(Ser-112) and Bad(Ser-155), though we were unable to detect phosphorylation of Bad(Ser-136). Phosphorylation of Bad(Ser-112) was antagonized by either prazosin or propranolol, indicating that this phosphorylation required stimulation of both alpha(1)- and beta-adrenoceptors. Phosphorylation of Bad(Ser-155) was antagonized only by propranolol and was thus mediated through the beta-adrenoceptor. Inhibitor studies and partial purification of candidate kinases by fast protein liquid chromatography showed that the p90 ribosomal S6 kinases, p90RSK2/3 [which are activated by the extracellular signal-regulated kinases 1 and 2 (ERK1/2)] directly phosphorylated Bad(Ser-112), whereas the PKA catalytic subunit directly phosphorylated Bad(Ser-155). However, efficient phosphorylation of Bad(Ser-112) also required PKA activity. These data suggest that, although p90RSK2/3 phosphorylate Bad(Ser-112) directly, phosphorylation of this site is enhanced by phosphorylation of Bad(Ser-155). These phosphorylations potentially diminish the pro-apoptotic activity of Bad and contribute to the cytoprotective effects of phenylephrine and 8-(4-chlorophenylthio)-cAMP.

Activation of pro-survival Akt and ERK1/2 signalling pathways underlie the anti-apoptotic effects of flavanones in cortical neurons

There is growing interest in the potential beneficial effects of flavonoids in the aging and diseased brain. We have investigated the potential of the flavanone hesperetin and two of its metabolites, hesperetin-7-O-beta-D-glucuronide and 5-nitro-hesperetin, to inhibit oxidative stress-induced neuronal apoptosis. Exposure of cortical neurons to hydrogen peroxide led to the activation of apoptosis signal-regulating kinase 1 via its de-phosphorylation at Ser963, the phosphorylation of c-jun N-terminal kinase and c-Jun (Ser73) and the activation of caspase 3 and caspase 9. Whilst hesperetin glucuronide failed to exert protection, both hesperetin and 5-nitro-hesperetin were effective at preventing neuronal apoptosis via a mechanism involving the activation/phosphorylation of both Akt/protein kinase B and extracellular signal-regulated kinase 1 and 2 (ERK1/2). Protection against oxidative injury and the activation of Akt and ERK1/2 followed a bell-shaped response and was most apparent at 100 nmol/L concentrations. The activation of ERK1/2 and Akt by flavanones led to the inhibition of the pro-apoptotic proteins, apoptosis signal-regulating kinase 1, by phosphorylation at Ser83 and Bad, by phosphorylation at both Ser136 and Ser112 and to the inhibition of peroxide-induced caspase 9 and caspase 3 activation. Thus, flavanones may protect neurons against oxidative insults via the modulation of neuronal apoptotic machinery.

Endothelin signalling in the cardiac myocyte and its pathophysiological relevance

Endothelin A (ET(A)) transmembrane receptors predominate in rat cardiac myocytes. These are G protein-coupled receptors whose actions are mediated by the G(q) heterotrimeric G proteins. Through these, ET-1 binding to ET(A)-receptors stimulates the hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate to diacylglycerol and inositol 1,4,5-trisphosphate. Diacylglycerol remains in the membrane whereas inositol 1,4,5-trisphosphate is soluble (though its importance in the cardiac myocyte is still debated). Isoforms of the phospholipid-dependent protein kinase, protein kinase C (PKC), are intracellular receptors for diacylglycerol. Cytoplasmic nPKCdelta and nPKCepsilon detect increases in membrane diacylglycerols and translocate to the membrane. This brings about PKC activation, though modifications additional to binding to phospholipids and diacylglycerol are involved. The next event (probably associated with PKC activation) is the activation of the membrane-bound small G protein Ras by exchange of GTP for GDP. Ras.GTP loading translocates Raf family mitogen-activated protein kinase (MAPK) kinase kinases to the membrane, initiates the activation of Raf, and thus activates the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade. Over longer times, two analogous protein kinase cascades, the c-Jun N-terminal kinase and p38-mitogen-activated protein kinase cascades, become activated. As the signals originating from the ET(A) receptor are transmitted through these protein kinase pathways, other signalling molecules become phosphorylated, thus changing their biological activities. For example, ET-1 increases the expression of the c-jun transcription factor gene, and increases abundance and phosphorylation of c-Jun protein. These changes in c-Jun expression and phosphorylation are likely to be important in the regulation of gene transcription.

The neurotoxicity of 5-S-cysteinyldopamine is mediated by the early activation of ERK1/2 followed by the subsequent activation of ASK1/JNK1/2 pro-apoptotic signalling

Parkinson's disease is characterized by the progressive and selective loss of dopaminergic neurons in the substantia nigra. It has been postulated that endogenously formed CysDA (5-S-cysteinyldopamine) and its metabolites may be, in part, responsible for this selective neuronal loss, although the mechanisms by which they contribute to such neurotoxicity are not understood. Exposure of neurons in culture to CysDA caused cell injury, apparent 12-48 h post-exposure. A portion of the neuronal death induced by CysDA was preceded by a rapid uptake and intracellular oxidation of CysDA, leading to an acute and transient activation of ERK2 (extracellular-signal-regulated kinase 2) and caspase 8. The oxidation of CysDA also induced the activation of apoptosis signal-regulating kinase 1 via its de-phosphorylation at Ser967, the phosphorylation of JNK (c-Jun N-terminal kinase) and c-Jun (Ser73) as well as the activation of p38, caspase 3, caspase 8, caspase 7 and caspase 9. Concurrently, the inhibition of complex I by the dihydrobenzothiazine DHBT-1 [7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid], formed from the intracellular oxidation of CysDA, induces complex I inhibition and the subsequent release of cytochrome c which further potentiates pro-apoptotic mechanisms. Our data suggest a novel comprehensive mechanism for CysDA that may hold relevance for the selective neuronal loss observed in Parkinson's disease.

Glial metabolism of quercetin reduces its neurotoxic potential

The neuroprotective effects of flavonoids will ultimately depend on their interaction with both neuronal and glial cells. in this study, we show that the potential neurotoxic effects of quercetin are modified by glial cell interactions. Specifically, quercetin is rapidly conjugated to glutathione within glial cells to yield 2 '-glutathionyl-quercetin, which is exported from cells but has significantly reduced neurotoxicity. In addion, quercetin underwent intracellular O-methylation to yield 3 '-O-methyl-quercetin and 4 '-O-methyl-quercetin, although these were not exported from glia at the same rate as the glutathionyl adduct. The neurotoxic potential of both quercetin and 2 '-glutathionyl-quercetin paralleled their ability to modulate the pro-survival Akt/PKB and extracellular signal-regulated kinase (ERK) signalling pathways. These data were supported by co-culture investigation, where the neurotoxic effects of quercetin were significantly reduced when they were cultured alongside glial cells. We propose that glial cells act to protect neurons against the neurotoxic effects of quercetin and that 2 '-glutathionyl-quercetin represents a novel quercetin metabolite. (c) 2008 Elsevier Inc. All rights reserved.

The impact of flavonoids on memory: physiological and molecular considerations

Emerging evidence suggests that a group of dietary-derived phytochemicals known as flavonoids are able to induce improvements in memory acquisition, consolidation, storage and retrieval. These low molecular weight polyphenols are widespread in the human diet, are absorbed to only a limited degree and localise in the brain at low concentration. However, they have been found to be highly effective in reversing age-related declines in memory via their ability to interact with the cellular and molecular architecture of the brain responsible for memory. These interactions include an ability to activate signalling pathways, critical in controlling synaptic plasticity, and a potential to induce vascular effects capable of causing new nerve cell growth in the hippocampus. Their ability to activate the extracellular signal-regulated kinase (ERK1/2) and the protein kinase B (PKB/Akt) signalling pathways, leading to the activation of the cAMP response element-binding protein (CREB), a transcription factor responsible for increasing the expression of a number of neurotrophins important in de. ning memory, will be discussed. How these effects lead to improvements in memory through induction of synapse growth and connectivity, increases in dendritic spine density and the functional integration of old and new neurons will be illustrated. The overall goal of this critical review is to emphasize future areas of investigation as well as to highlight these dietary agents as promising candidates for the design of memory-enhancing drugs with relevance to normal and pathological brain ageing (161 references).

Food for thought: the role of dietary flavonoids in enhancing human memory, learning and neuro-cognitive performance

Emerging evidence suggests that dietary-derived flavonoids have the potential to improve human memory and neuro-cognitive performance via their ability to protect vulnerable neurons, enhance existing neuronal function and stimulate neuronal regeneration. Long-term potentiation (LTP) is widely considered to be one of the major mechanisms underlying memory acquisition, consolidation and storage in the brain and is known to be controlled at the molecular level by the activation of a number of neuronal signalling pathways. These pathways include the phosphatidylinositol-3 kinase/protein kinase B/Akt (Akt), protein kinase C, protein kinase A, Ca-calmodulin kinase and mitogen-activated protein kinase pathways. Growing evidence suggests that flavonoids exert effects on LTP, and consequently memory and cognitive performance, through their interactions with these signalling pathways. Of particular interest is the ability of flavonoids to activate the extracellular signal-regulated kinase and the Akt signalling pathways leading to the activation of the cAMP-response element-binding protein, a transcription factor responsible for increasing the expression of a number of neurotrophins important in LTP and long-term memory. One such neurotrophin is brain-derived neurotrophic factor, which is known to be crucial in controlling synapse growth, in promoting an increase in dendritic spine density and in enhancing synaptic receptor density. The present review explores the potential of flavonoids and their metabolite forms to promote memory and learning through their interactions with neuronal signalling pathways pivotal in controlling LTP and memory in human subjects.

(-)Epicatechin stimulates ERK-dependent cyclic AMP response element activity and up-regulates GluR2 in cortical neurons

Emerging evidence suggests that the cellular actions of flavonoids relate not simply to their antioxidant potential but also to the modulation of protein kinase signalling pathways. We investigated in primary cortical neurons, the ability of the flavan-3-ol, (-)epicatechin, and its human metabolites at physiologically relevant concentrations, to stimulate phosphorylation of the transcription factor cAMP-response element binding protein (CREB), a regulator of neuronal viability and synaptic plasticity. (-)Epicatechin at 100-300 nmol/L stimulated a rapid, extracellular signal-regulated kinase (ERK)- and PI3K-dependent, increase in CREB phosphorylation. At micromolar concentrations, stimulation was no longer apparent and at the highest concentration tested (30 mu mol/L) (-)epicatechin was inhibitory. (-)Epicatechin also stimulated ERK and Akt phosphorylation with similar bell-shaped concentration-response characteristics. The human metabolite 3 '-O-methyl-(-)epicatechin was as effective as (-)epicatechin at stimulating ERK phosphorylation, but (-)epicatechin glucuronide was inactive. (-)Epicatechin and 3 '-O-methyl-(-)epicatechin treatments (100 nmol/L) increased CRE-luciferase activity in cortical neurons in a partially ERK-dependent manner, suggesting the potential to increase CREB-mediated gene expression. mRNA levels of the glutamate receptor subunit GluR2 increased by 60%, measured 18 h after a 15 min exposure to (-)epicatechin and this translated into an increase in GluR2 protein. Thus, (-)epicatechin has the potential to increase CREB-regulated gene expression and increase GluR2 levels and thus modulate neurotransmission, plasticity and synaptogenesis.