Extracellular Signal-regulated Protein Kinase Activation Research Articles

Requirement for ERK activation in sinomenine-induced apoptosis of macrophages

Sinomenine (SN), an immunnosuppressive compound derived from the Chinese medicinal plant Sinomenium acutum, has been used to treat autoimmune diseases effectively. Previous studies show SN can inhibit lymphocytes proliferation and macrophage production of pro-inflammatory factors. However, little is known about the mechanisms by which SN inhibits macrophage functions. In this study, we demonstrated that SN could inhibit the proliferation of murine macrophages RAW264.7 by inducing apoptosis in a dose- and time-dependent manner. We found activation of extracellular signal-regulated protein kinase (ERK) in SN-treated macrophages, and requirement for ERK activation in SN-induced apoptosis of macrophages. Contemporarily, the expression of p27/KIP1, proapoptotic factor Bax increased, and expression of Bcl-2 decreased, which might cooperate to induce apoptosis. Inhibiting ERK activation reduced the increased expression of p27 and Bax, but had no effect on the decreased expression of Bcl-2, suggesting the involvement of ERK activation in the SN-induced increased expression of p27 and Bax. These results demonstrated that SN could induce apoptosis of macrophages through activation of ERK, and ERK activation might partially involve in the increased expression of p27 and Bax in apoptotic macrophages. Therefore, induction of macrophage apoptosis through ERK activation may be one of mechanisms by which SN exhibits its immunosuppressive function.

Role of Mitogen-Activated Protein Kinase Activation in Injured and Intact Primary Afferent Neurons for Mechanical and Heat Hypersensitivity after Spinal Nerve Ligation

To investigate whether activation of mitogen-activated protein kinase (MAPK) in damaged and/or undamaged primary afferents participates in neuropathic pain after partial nerve injury, we examined the phosphorylation of extracellular signal-regulated protein kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase (JNK) in the L4 and L5 dorsal root ganglion (DRG) in the L5 spinal nerve ligation (SNL) model. We first confirmed, using activating transcription factor 3 and neuropeptide Y immunoreactivity, that virtually all L4 DRG neurons are spared from axotomy in this model. In the injured L5 DRG, the L5 SNL induced the activation of ERK, p38, and JNK in different populations of DRG neurons. In contrast, in the uninjured L4 DRG, the L5 SNL induced only p38 activation in tyrosine kinase A-expressing small- to medium-diameter neurons. Intrathecal ERK, p38, and JNK inhibitor infusions reversed SNL-induced mechanical allodynia, whereas only p38 inhibitor application attenuated SNL-induced thermal hyperalgesia. Furthermore, the L5 dorsal rhizotomy did not prevent SNL-induced thermal hyperalgesia. We therefore hypothesized that p38 activation in the uninjured L4 DRG might be involved in the development of heat hypersensitivity in the L5 SNL model. In fact, the treatment of the p38 inhibitor and also anti-nerve growth factor reduced SNL-induced upregulation of brain-derived neurotrophic factor and transient receptor potential vanilloid type 1 expression in the L4 DRG. Together, our results demonstrate that the L5 SNL induces differential activation of MAPK in injured and uninjured DRG neurons and, furthermore, that MAPK activation in the primary afferents may participate in generating pain hypersensitivity after partial nerve injury.

Terrein: a new melanogenesis inhibitor and its mechanism

Terrein is a bioactive fungal metabolite whose effects are almost unknown. In this study, we found for the first time that terrein has a strong hypopigmentary effect in a spontaneously immortalized mouse melanocyte cell line, Mel-Ab. Treatment of Mel-Ab cells with terrein (10-100 microM) for 4 days significantly reduced melanin levels in a dose-dependent manner. In addition, terrein at the same concentration also reduced tyrosinase activity. We then investigated whether terrein influences the extracellular signal-regulated protein kinase (ERK) pathway and the expression of microphthalmia-associated transcription factor (MITF), which is required for tyrosinase expression. Terrein was found to induce sustained ERK activation and MITF down-regulation, and luciferase assays showed that terrein inhibits MITF promoter activity in a dose-dependent manner. To elucidate the correlation between ERK pathway activation and a decreased MITF transcriptional level, PD98059, a specific inhibitor of the ERK pathway, was applied before terrein treatment and found to abrogate the terrein-induced MITF attenuation. Terrein also reduced the tyrosinase protein level for at least 72 h. These results suggest that terrein reduces melanin synthesis by reducing tyrosinase production via ERK activation, and that this is followed by MITF down-regulation.

Interactions between Extracellular Signal-regulated Protein Kinase 1, 14-3-3ϵ, and Heat Shock Factor 1 during Stress

Cytoprotection during the heat shock response is a complex phenomenon involving multiple inducible mechanisms. We have examined the interaction of two key molecular components in the response, heat shock transcription factor 1 (HSF1) and extracellular signal regulated protein kinase (ERK). Whereas both HSF1 and ERK are required to protect cells against apoptosis, ERK activation is paradoxically antagonistic to trans-activation of hsp promoters by HSF1 and HSP accumulation during heat shock. We have found that the two pathways interact directly and that heat shock causes the physical association of ERK1 with HSF1, an interaction that promotes the kinase activity of ERK in heat-shocked cells. ERK activation results in the recruitment of the phosphoserine binding protein 14-3-3epsilon in a manner dependent on previous HSF1 phosphorylation by ERK. The effects of 14-3-3epsilon binding on HSF1 were complex, however, depending on extracellular conditions, in that HSF1-14-3-3 binding at 37 degrees C led to the cytoplasmic sequestration and repression of HSF1, whereas heat shock overrode these effects and caused quantitative nuclear localization of HSF1. Although the effects of 14-3-3epsilon binding to HSF1 were overridden acutely by stress, during recovery from heat shock, 14-3-3epsilon association again led to enhanced cytoplasmic localization of HSF1, implicating a role for ERK/14-3-3epsilon in HSF1 deactivation in recovering cells. Association of HSF1 with ERK and 14-3-3epsilon during heat shock may thus modulate the amplitude of the response and lead to efficient termination of HSP expression on resumption of growth conditions.

Gap Junctions Regulate Extracellular Signal-regulated Kinase Signaling to Affect Gene Transcription

Osteoblasts are highly coupled by gap junctions formed by connexin43. Overexpression of connexin45 in osteoblasts results in decreased chemical and electrical coupling and reduces gene transcription from connexin response elements (CxREs) in the osteocalcin and collagen Ialpha1 promoters. Here, we demonstrate that transcription from the gap junction-dependent osteocalcin CxRE is regulated by extracellular signal-regulated protein kinase (ERK) and phosphatidylinositol 3-kinase (PI3K) cascades. Overexpression of a constitutively active mitogen-activated protein kinase kinase (MEK), Raf, or Ras can increase transcription more than twofold of the CxRE, whereas inhibition of MEK or PI3K can decrease transcription threefold from the osteocalcin CxRE. Importantly, disruption of gap junctional communication by overexpression of connexin45 or treatment with pharmacological inhibitors of gap junctions results in reduced Raf, ERK, and Akt activation. The consequence of attenuated gap junction-dependent signal cascade activation is a decrease in Sp1 phosphorylation by ERK, resulting in decreased Sp1 recruitment to the CxRE and inhibited gene transcription. These data establish that ERK/PI3K signaling is required for the optimal elaboration of transcription from the osteocalcin CxRE, and that disruption of gap junctional communication attenuates the ability of cells to respond to an extracellular cue, presumably by limiting the propagation of second messengers among adjacent cells by connexin43-gap junctions.

A Quantitative Cell-Based High-Content Screening Assay for the Epidermal Growth Factor Receptor-Specific Activation of Mitogen-Activated Protein Kinase

The complexity of mitogen-activated protein kinase (MAPK) signaling pathways and their activation by different stimuli makes assaying the activation of particular MAPKs by specific receptors a challenging problem. The multiplexing capability of quantitative high-content screening (HCS) assays enables the simultaneous monitoring and correlation, in the same cell, of an MAPK's specific activation with a particular receptor's post-signaling behavior, such as its internalization. We demonstrate a cell-based HCS assay to quantify the epidermal growth factor (EGF) receptor-specific activation of the MAPK ERK. Activation was quantified by measuring immunofluorescently labeled phosphorylated extracellular signal-regulated protein kinases (ERK) in the nucleus. Specificity of ERK activation by the EGF receptor was simultaneously confirmed in the same cell by quantitatively monitoring fluorescent EGF's internalization and subsequent intracellular degradation. Quantitative analysis of the temporal behavior of these two activities showed that phosphorylated ERK's accumulation in the nucleus peaked at 5 min before falling to basal levels by 30 min. Cellular accumulation of fluorescent EGF was slower, peaking around 30 min, before being degraded. This assay strategy can serve as a paradigm to study other signaling pathways and their activation by specific receptors. The flexibility and multiplexing capability of HCS assays allow the use of additional targets to further qualify the specificity of response by including other MAPKs or receptors, to rule out cross-talk from competing signaling pathways, or to simultaneously monitor toxicity effects of compounds. This automated, non-subjective, easy-to-use assay procedure provides information rich, quantitative results, and demonstrates the potential of the HCS assay approach in deconvolving intracellular signaling pathways.

A Quantitative Cell-Based High-Content Screening Assay for the Epidermal Growth Factor Receptor-Specific Activation of Mitogen-Activated Protein Kinase

The complexity of mitogen-activated protein kinase (MAPK) signaling pathways and their activation by different stimuli makes assaying the activation of particular MAPKs by specific receptors a challenging problem. The multiplexing capability of quantitative high-content screening (HCS) assays enables the simultaneous monitoring and correlation, in the same cell, of an MAPK's specific activation with a particular receptor's post-signaling behavior, such as its internalization. We demonstrate a cell-based HCS assay to quantify the epidermal growth factor (EGF) receptor-specific activation of the MAPK ERK. Activation was quantified by measuring immunofluorescently labeled phosphorylated extracellular signal-regulated protein kinases (ERK) in the nucleus. Specificity of ERK activation by the EGF receptor was simultaneously confirmed in the same cell by quantitatively monitoring fluorescent EGF's internalization and subsequent intracellular degradation. Quantitative analysis of the temporal behavior of these two activities showed that phosphorylated ERK's accumulation in the nucleus peaked at 5 min before falling to basal levels by 30 min. Cellular accumulation of fluorescent EGF was slower, peaking around 30 min, before being degraded. This assay strategy can serve as a paradigm to study other signaling pathways and their activation by specific receptors. The flexibility and multiplexing capability of HCS assays allow the use of additional targets to further qualify the specificity of response by including other MAPKs or receptors, to rule out cross-talk from competing signaling pathways, or to simultaneously monitor toxicity effects of compounds. This automated, non-subjective, easy-to-use assay procedure provides information rich, quantitative results, and demonstrates the potential of the HCS assay approach in deconvolving intracellular signaling pathways.

Metalloproteinases and Transforming Growth Factor-α Mediate Substance P-induced Mitogen-activated Protein Kinase Activation and Proliferation in Human Colonocytes

Substance P (SP) participates in acute intestinal inflammation via binding to the G-protein-coupled neurokinin-1 receptor (NK-1R) and release of proinflammatory cytokines from colonic epithelial cells. SP also stimulates cell proliferation, a critical event in tissue healing during chronic colitis, via transactivation of the epidermal growth factor (EGF) receptor (EGFR) and activation of mitogen-activated protein kinase (MAPK). Here we examined the mechanism by which SP induces EGFR and MAPK activation. We used non-transformed human NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells) as well as untransfected U373 MG cells expressing high levels of endogenous NK-1R. Exposure of both cell lines to SP (10(-7) m) stimulated EGFR activation (1 min) followed by extracellular signal-regulated protein kinase (ERK1/2) activation (2-5 min). SP-induced ERK1/2 activation was blocked by pretreatment with the metalloproteinase inhibitor Batimastat/GM6001, the EGFR phosphorylation inhibitor AG1478, and the tumor necrosis factor-alpha-converting enzyme (TACE) inhibitor TAPI-1. Pretreatment with antibodies against potential EGFR ligands suggested that transforming growth factor-alpha (TGFalpha), but not the other EGFR ligands EGF, heparin-binding EGF, or amphiregulin, mediates SP-induced EGFR transactivation. SP stimulated TGFalpha release into the extracellular space that was measurable within 2 min, and this release was inhibited by metalloproteinase inhibitors and the TACE inhibitor TAPI-1. SP also induced MAPK-mediated cell proliferation that was inhibited by TACE, matrix metalloproteinase (MMP), EGFR, and MEK1 inhibitors. Thus, in human colonocytes, NK-1R-induced EGFR and MAPK activation and cell proliferation involve matrix metalloproteinases (most likely TACE) and the release of TGFalpha. These signaling mechanisms may be involved in the protective effects of NK-1R in chronic colitis.

Phosphatidylinositol 3-kinase activates ERK in primary sensory neurons and mediates inflammatory heat hyperalgesia through TRPV1 sensitization.

Although the PI3K (phosphatidylinositol 3-kinase) pathway typically regulates cell growth and survival, increasing evidence indicates the involvement of this pathway in neural plasticity. It is unknown whether the PI3K pathway can mediate pain hypersensitivity. Intradermal injection of capsaicin and NGF produce heat hyperalgesia by activating their respective TRPV1 (transient receptor potential vanilloid receptor-1) and TrkA receptors on nociceptor sensory nerve terminals. We examined the activation of PI3K in primary sensory DRG neurons by these inflammatory agents and the contribution of PI3K activation to inflammatory pain. We further investigated the correlation between the PI3K and the ERK (extracellular signal-regulated protein kinase) pathway. Capsaicin and NGF induce phosphorylation of the PI3K downstream target AKT (protein kinase B), which is blocked by the PI3K inhibitors LY294002 and wortmannin, indicative of the activation of PI3K by both agents. ERK activation by capsaicin and NGF was also blocked by PI3K inhibitors. Similarly, intradermal capsaicin in rats activated PI3K and ERK in C-fiber DRG neurons and epidermal nerve fibers. Injection of PI3K or MEK (ERK kinase) inhibitors into the hindpaw attenuated capsaicin- and NGF-evoked heat hyperalgesia but did not change basal heat sensitivity. Furthermore, PI3K, but not ERK, inhibition blocked early induction of hyperalgesia. In acutely dissociated DRG neurons, the capsaicin-induced TRPV1 current was strikingly potentiated by NGF, and this potentiation was completely blocked by PI3K inhibitors and primarily suppressed by MEK inhibitors. Therefore, PI3K induces heat hyperalgesia, possibly by regulating TRPV1 activity, in an ERK-dependent manner. The PI3K pathway also appears to play a role that is distinct from ERK by regulating the early onset of inflammatory pain.

Inhibition of activator protein 1 activation, vascular endothelial growth factor, and cyclooxygenase-2 expression by 15-deoxy-Delta12,14-prostaglandin J2 in colon carcinoma cells: evidence for a redox-sensitive peroxisome proliferator-activated receptor-gamma-independent mechanism.

Cyclooxygenase (COX)-2 and vascular endothelial growth factor (VEGF) are significantly associated with tumor growth and metastasis. Here we show that phorbol ester-mediated induction of VEGF and COX-2 expression in colon carcinoma cells is inhibited by 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)). This cyclopentenone was able to inhibit activator protein1 (AP-1)-dependent transcriptional induction of COX-2 and VEGF promoters induced by phorbol 12-myristate 13-acetate (PMA) or c-Jun overexpression. 15d-PGJ(2) interfered with at least two steps within the signaling pathway leading to AP-1 activation. First, 15d-PGJ(2) impaired AP-1 binding to a consensus DNA sequence. Second, 15d-PGJ(2) selectively inhibited c-Jun NH(2) terminal kinase (JNK) but not extracellular signal-regulated kinase or p38 mitogen-activated protein kinase activation induced by PMA. This led to a decreased ability of JNK to phosphorylate c-Jun and to activate its transactivating activity. Inhibition of AP-1 activation and COX-2 or VEGF transcriptional induction by this cyclopentenone was found to be independent of peroxisome proliferator-activated receptor-gamma (PPARgamma) because it was not affected by either expression of a dominant negative form of PPARgamma or the use of a PPARgamma antagonist. In contrast, we have found that the effects of 15d-PGJ(2) on AP-1 activation may occur through its ability to induce intracellular oxidative stress. The antioxidant N-acetylcysteine significantly reversed the inhibition by 15d-PGJ(2) of AP-1 activity and COX-2 or VEGF transcriptional induction. Together, these findings provide new insight into the antitumoral properties of 15d-PGJ(2) through the inhibition of the induction of AP-1-dependent genes involved in tumor progression, such as COX-2 and VEGF.

Heme oxygenase-1 inhibition of MAP kinases, calcineurin/NFAT signaling, and hypertrophy in cardiac myocytes.

Heme oxygenases (HO) are the rate-limiting enzymes in heme degradation, catalyzing the breakdown of heme to equimolar quantities of biliverdin (BV), carbon monoxide (CO), and ferrous iron. The inducible HO isoform, HO-1, confers protection against ischemia/reperfusion (I/R)-injury in the heart. We hypothesized that HO-1 and its catalytic by-products constitute an antihypertrophic signaling module in cardiac myocytes. The G protein-coupled receptor (GPCR) agonist endothelin-1 (ET-1) (30 nmol/l) stimulated a robust hypertrophic response in cardiac myocytes isolated from 1- to 3-day-old Sprague-Dawley rats, with increases in cell surface area (planimetry), sarcomere assembly (confocal laser scanning microscopy), and prepro-atrial natriuretic peptide (ANP) mRNA expression. Adenoviral overexpression of HO-1, but not beta-galactosidase, significantly inhibited ET-1 induced cardiac myocyte hypertrophy. The antihypertrophic effects of HO-1 were mimicked by BV (10 micromol/l) and the CO-releasing molecule [Ru(CO)3Cl2]2 (10 micromol/l), strongly suggesting a critical involvement of BV and CO in the antihypertrophic effects of HO-1. Both BV and CO suppressed extracellular signal-regulated kinases (ERK1/ERK2) and p38 mitogen-activated protein kinase (MAPK) activation by ET-1 stimulation. Moreover, BV and CO inhibited the prohypertrophic calcineurin/NFAT pathway. This inhibition occurred upstream from calcineurin because BV and CO inhibited NFAT activation in response to ET-1 stimulation but not in response to adenoviral expression of a constitutively active calcineurin mutant. Upstream-inhibition of the calcineurin/NFAT pathway by CO occurred independent from cGMP and cGMP-dependent protein kinase type I (PKG I). Heme oxygenase-1 and its catalytic by-products, BV and CO, constitute a novel antihypertrophic signaling pathway in cardiac myocytes. Biliverdin and CO inhibition of MAPKs and calcineurin/NFAT signaling provides a mechanistic framework how heme degradation products may promote their antihypertrophic effects.

Role of ERK Activation in Cisplatin-Induced Apoptosis in A172 Human Glioma Cells

Cisplatin activates multiple signal transduction pathways associated with cell survival and apoptosis in various cell types. The present study was undertaken to determine the role of extracellular signal-regulated protein kinase (ERK), a member of the mitogen-activated protein kinase family, in cisplatin-induced apoptosis in human glioma cells. Cisplatin resulted in apoptosis in a dose- and time-dependent manner. Cisplatin-induced apoptosis was prevented by the hydrogen peroxide scavenger pyruvate and the antioxidant N-acetylcysteine, but not by the superoxide scavenger tiron. Western blot analysis demonstrated that cisplatin treatment induced time-dependent activation of ERK, which was inhibited by chemical inhibitors of the MEK signaling pathway (PD98059 and U0126) and N-acetylcysteine. These inhibitors prevented cisplatin-induced cell death. Transient transfection of constitutive active MEK1 increased cisplatin-induced apoptosis. Cisplatin resulted in a reduction in mitochondrial membrane potential and its effect was prevented by N-acetylcysteine and PD98059. Caspase inhibitors (Boc-D-FMK and zDEVD-FMK) protected against cisplatin-induced cell death. Cisplatin-induced activation of caspase-3 was inhibited by N-acetylcysteine and PD98059. Taken together, these findings suggest that the ERK activation plays an active role in mediating cisplatin-induced apoptosis of human glioma cells and functions upstream of mitochondrial dysfunction and caspase activation to the initiate the apoptotic signal.

Coordinate signaling by Src and p38 kinases in the induction of cortical cataracts.

The goals of this study were to determine whether MAP kinase signaling pathways play a role in the formation of lens cataracts and to examine the potential signaling relationship between Src and MAP kinases in signaling the induction of lens opacities. Embryonic day (E)10 chick lenses were cultured in Medium 199 containing 10% fetal bovine serum. The activation state of Src kinases and the MAP kinases extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK), and p38 in the lens epithelium was determined over a time course from 10 minutes to 10 days in culture by immunoblot analysis. Src kinase activation was suppressed by exposure to the Src family kinase-specific inhibitor PP1. To examine the role of specific MAP kinases in the development of lens opacities, lenses were grown for 10 days in the presence or absence of inhibitors of ERK (U0126), JNK (SP600125), and p38 (SB203580). Lenses were observed and photographed daily, and the degree of opacification was quantified by using image-analysis software. Within a short time after placing embryonic lenses in culture conditions that induce the formation of cataracts, there occurred a great increase in the activation state of the MAP kinase ERK. Activation of ERK was both rapid and transient. No activation of the MAP kinase JNK was observed in the cataract cultures beyond that which occurred in normal lens epithelium, even though JNK activation is often linked to the cellular response to stress. In contrast, although p38 activation was barely detected in the normal embryonic lens, this stress-activated protein kinase exhibited a robust activation in cataract cultures that was sustained throughout the culture period. Studies conducted to map the cataract signaling pathways indicate that the p38 MAP kinase functions upstream of the Src kinase. To analyze the potential role of ERK, JNK, and p38 in cataract induction, lenses were cultured in the presence of specific MAP kinase inhibitors. Although the inhibitors of ERK and JNK did not interfere with the formation of cataract, p38 inhibitors blocked the development of lens opacities with an efficacy similar to that of the Src kinase inhibitor PP1. Activation of both Src and p38 kinases lead to the induction of cataract.

Differential effects of peroxisome proliferator activated receptor-γ (PPARγ) ligands in proximal tubular cells: Thiazolidinediones are partial PPARγ agonists

Background. Peroxisome proliferator activated receptors (PPARs) are ligand-activated transcription factors with multiple effects on target cell function. PPAR gamma activity is regulated by extracellular signal-regulated protein kinase (ERK), mitogen-activated protein (MAP) kinase, and PPAR gamma ligands have varying effects on activity of ERK. Different PPAR gamma ligands have been shown to have both protective and detrimental effects in the kidney. Since transcriptional activation by different PPAR agonists is ligand- and depot-specific PPAR gamma, we have examined the effects of different agonists on PPAR activity in the proximal tubule. Opossum kidney cells were used in all experiments, transiently transfected with a PPAR response element luciferase reporter and subject to stimulation with various PPAR ligands. The role of ERK and phosphorylation in PPAR gamma activation were studied, as were the effects of PPAR agonists on ERK activation and cell proliferation. Transcriptional activity of PPAR was not stimulated by PPAR alpha agonists, and only very modestly stimulated by a PPAR beta agonist. The PPAR gamma agonists 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), ciglitazone, and troglitazone stimulated significant transcriptional activation and phosphorylation of PPAR gamma. These effects were more marked with 15d-PGJ(2). Thiazolidinediones attenuated 15d-PGJ(2) evoked PPAR gamma activation and phosphorylation. ERK activity positively regulated PPAR activation. Only 15d-PGJ(2) stimulated ERK activity and cell proliferation, and these effects were also inhibited by thiazolidinediones. PPAR gamma agonists exert differential effects in proximal tubule cells with thiazolidinediones behaving as partial agonists.

Mitogen-induced Rapid Phosphorylation of Serine 795 of the Retinoblastoma Gene Product in Vascular Smooth Muscle Cells Involves ERK Activation

We examined the relationship between mitogen-activated MEK (mitogen and extracellular signal-regulated protein kinase kinase) and phosphorylation of the gene product encoded by retinoblastoma (hereafter referred to as Rb) in vascular smooth muscle cells. Brief treatment of the cells with 100 nm angiotensin II or 1 microm serotonin resulted in serine phosphorylation of Rb that was equal in magnitude to that induced by treating cells for 20 h with 10% fetal bovine serum ( approximately 3 x basal). There was no detectable rapid phosphorylation of two close cousins of Rb, p107 and p130. Phosphorylation state-specific antisera demonstrated that the rapid phosphorylation occurred on Ser(795), but not on Ser(249), Thr(252), Thr(373), Ser(780), Ser(807), or Ser(811). Phosphorylation of Rb Ser(795) peaked at 10 min, lagging behind phosphorylation of MEK and ERK (extracellular signal-regulated protein kinase). Rb Ser(795) phosphorylation could be blocked by PD98059, a MEK inhibitor, and greatly attenuated by apigenin, an inhibitor of the Ras --> Raf --> MEK --> ERK pathway. The effect also appears to be mediated by CDK4. Immunoprecipitation/immunoblot studies revealed that serotonin and angiotensin II induced complex formation between CDK4, cyclin D1, and phosphorylated ERK. These studies show a rapid, novel, and selective phosphorylation of Rb Ser(795) by mitogens and demonstrate an unexpected rapid linkage between the actions of the Ras --> Raf --> MEK --> ERK pathway and the phosphorylation state of Rb.

Extracellular Signal-Regulated Protein Kinase Activation Is Required for Metabotropic Glutamate Receptor-Dependent Long-Term Depression in Hippocampal Area CA1

Activation of group 1 metabotropic glutamate receptors (mGluRs) induces long-term depression (LTD) of synaptic transmission that relies on dendritic protein synthesis. We investigated the signal transduction pathways required for mGluR-LTD to identify candidate mechanisms for mGluR regulation of synaptic protein synthesis. Our results demonstrate a role for extracellular signal-regulated protein kinase (ERK), a subclass of the mitogen-activated protein kinases (MAPKs), in mGluR-LTD in area CA1 of the rat hippocampus. Inhibitors of the upstream kinase of ERK, MAP/ERK kinase significantly reduce mGluR-LTD induced by the group 1 agonist dihydroxyphenylglycine (DHPG) and synaptic stimulation but do not affect NMDA receptor-dependent LTD. In contrast, inhibitors of p38 MAPK were ineffective against DHPG-induced LTD. Consistent with the role of ERK in mGluR-LTD, we observed that DHPG treatment of hippocampal slices (isolated CA1), at concentrations that induce LTD, results in a robust phosphorylation of ERK but not of p38 MAPK. These results point to ERK as an important regulator of mGluR-LTD and a potential mechanism for mGluR regulation of synaptic protein synthesis.

Activation of extracellular signal-regulated protein kinase in dorsal horn neurons in the rat neuropathic intermittent claudication model

Extracellular signal-regulated protein kinase (ERK) is a mitogen-activated protein kinase (MAPK) that mediates several cellular responses to mitogenic and differentiation signals, and activation of ERK in dorsal horn neurons by noxious stimulation is known to contribute to pain hypersensitivity. In order to elucidate the pathophysiological mechanisms of the cauda equina syndrome, secondary to spinal canal stenosis, we evaluated walking dysfunction triggered by forced exercise and activation of ERK in the dorsal horn using a rat model of neuropathic intermittent claudication. Rats in the lumbar canal stenosis (LCS) group showed a shorter running distance from 1 to 14 days after surgery. Two minutes after running on the treadmill apparatus, phosphorylation of ERK was induced in neurons in the superficial laminae in the LCS group but not in the sham group, whereas there was no change in the deeper laminae. Intrathecal administration of the MAPK kinase inhibitor, U0126, 30 min before running, clearly increased the running distance, whereas there was no significant change in the vehicle control group 3 days after surgery. In addition, a prostaglandin E1 analog, OP-1206 α-CD, administered orally, improved the walking dysfunction, and further, inhibited activation of ERK following running 7 days after surgery. These findings suggest that intermittent claudication triggered by forced walking might affect the phosphorylation of ERK in the superficial laminae, possibly via transient (partial) ischemia of the spinal cord. ERK activation in the dorsal horn neurons may be involved in the transient pain in the neuropathic intermittent claudication model.

Suppression of cadmium-induced JNK/p38 activation and HSP70 family gene expression by LL-Z1640-2 in NIH3T3 cells

When NIH3T3 cells were exposed to CdCl 2, the three major mitogen-activated protein kinases (MAPKs), extracellular signal-regulated protein kinase (ERK), c-Jun NH 2-terminal kinase (JNK), and p38, were phosphorylated in a time (1–9 h)- and dose (1–20 μM)-dependent manner. Treatment with a macrocyclic nonaketide compound, LL-Z1640-2 (10–100 ng/ml), suppressed the phosphorylation of MAPKs without affecting the total protein level in cells exposed to 10 μM CdCl 2 for 6 h. CdCl 2-induced phosphorylation of c-Jun on Ser63 and that on Ser73, and resultant accumulation of total c-Jun protein were also suppressed by LL-Z1640-2 treatment. The in vitro kinase assays also showed significant inhibitory effects of LL-Z1640-2 (at 10 or 25 ng/ml) on JNK and p38 but less markedly. In contrast to JNK and p38, ERK activity was inhibited moderately only at 50 or 100 ng/ml LL-Z1640-2. On the other hand, other JNK inhibitors, SP600125 and L-JNKI1, failed to suppress CdCl 2-induced activation of the JNK pathway. Among the mouse stress response genes upregulated in response to CdCl 2 exposure, the expressions of hsp68 (encoding for heat shock 70 kDa protein 1; Hsp70-1) and grp78 (encoding for 78 kDa glucose-regulated protein; Grp78) genes were suppressed by treatment with 25 ng/ml LL-Z1640-2. Thus, LL-Z1640-2 could suppress CdCl 2-induced activation of JNK/p38 pathways and expression of HSP70 family genes in NIH3T3 cells. LL-Z1640-2 seems to be useful to analyze functions of toxic metal-induced JNK/p38 activation.

Mitogen-activated protein kinase feedback phosphorylation regulates MEK1 complex formation and activation during cellular adhesion.

Cell adhesion and spreading depend on activation of mitogen-activated kinase, which in turn is regulated both by growth factor and integrin signaling. Growth factors, such as epidermal growth factor, are capable of activating Ras and Raf, but integrin signaling is required to couple Raf to MEK and MEK to extracellular signal-regulated protein kinase (ERK). It was previously shown that Rac-p21-activated kinase (PAK) signaling regulated the physical association of MEK1 with ERK2 through phosphorylation sites in the proline-rich sequence (PRS) of MEK1. It was also shown that activation of MEK1 and ERK by integrins depends on PAK phosphorylation of S298 in the PRS. Here we report a novel MEK1-specific regulatory feedback mechanism that provides a means by which activated ERK can terminate continued PAK phosphorylation of MEK1. Activated ERK can phosphorylate T292 in the PRS, and this blocks the ability of PAK to phosphorylate S298 and of Rac-PAK signaling to enhance MEK1-ERK complex formation. Preventing ERK feedback phosphorylation on T292 during cellular adhesion prolonged phosphorylation of S298 by PAK and phosphorylation of S218 and S222, the MEK1 activating sites. We propose that activation of ERK during adhesion creates a feedback system in which ERK phosphorylates MEK1 on T292, and this in turn blocks additional S298 phosphorylation in response to integrin signaling.

MAPK activation in nociceptive neurons and pain hypersensitivity

Extracellular signal-regulated protein kinase (ERK) is a mitogen-activated protein kinase (MAPK) that mediates intracellular signal transduction in response to a variety of stimuli. ERK is involved in cell proliferation and differentiation and in neuronal plasticity, including long-term potentiation, learning, and memory. Here, we present recently accumulating data about the roles of MAPK pathways in mediating the neuronal plasticity that contributes to pain hypersensitivity. The phosphorylation of ERK in the dorsal root ganglion (DRG) and dorsal horn neurons occurs in response to noxious stimulation of the peripheral tissue or electrical stimulation to the peripheral nerve, i.e., activity-dependent activation of ERK in nociceptive neurons. In addition, the activation of ERK occurs in these nociceptive neurons after peripheral inflammation and axotomy and contributes to persistent inflammatory and neuropathic pain, via transcriptional regulation of key gene products. On the other hand, peripheral inflammation and axotomy also induces p38 MAPK activation in DRG neurons. Taken together, these findings indicate that activation of MAPK in nociceptive neurons may participate in generating pain hypersensitivity through transcription-dependent and -independent means. Thus, inhibition of MAPK signaling in the primary afferents, as well as in the spinal cord, may provide a fruitful strategy for the development of novel analgesics.