Extracellular Signal-regulated Protein Kinase Activation Research Articles

Stimulation of fibroblast proliferation by the plant cysteine protease CMS2MS2 is independent of its proteolytic activity and requires ERK activation

The cysteine protease CMS2MS2 from Carica candamarcensis latex has been shown to enhance proliferation of L929 fibroblast and to activate the extracellular signal-regulated protein kinase (ERK). In experiments with CMS2MS2 irreversibly inhibited by E-64, the proliferative effect on fibroblasts remains unaffected. ERK phosphorylation mediated by CMS2MS2 was abolished in the presence of PD 98059 or U0126, both MAPK cascade inhibitors. In addition, these inhibitors suppress the mitogenic activity of intact CMS2MS2 or CMS2MS2-E-64. Furthermore, ERK phosphorylation and the mitogenic effect are partially suppressed by a phospholipase C (PLC) inhibitor. These data suggest that the mitogenic effect of CMS2MS2 on fibroblasts is independent of its proteolytic activity, requires ERK phosphorylation, and involves activation of PLC.

Multivalent ligand–receptor interactions elicit inverse agonist activity of AT1 receptor blockers against stretch-induced AT1 receptor activation

Type 1 angiotensin II (AT(1)) receptor has a critical role in the development of load-induced cardiac hypertrophy. Recently, we showed that mechanical stretching of cells activates the AT(1) receptor without the involvement of angiotensin II (AngII) and that this AngII-independent activation is inhibited by the inverse agonistic activity of the AT(1) receptor blocker (ARB), candesartan. Although the inverse agonist activity of ARBs has been studied in terms of their action on constitutively active AT(1) receptors, the structure-function relationship of the inverse agonism they exert against stretch-induced AT(1) receptor activation has not been fully elucidated. Assays evaluating c-fos gene expression and phosphorylated extracellular signal-regulated protein kinases (ERKs) have shown that olmesartan has strong inverse agonist activities against the constitutively active AT(1) receptor and the stretch-induced activation of AT(1) receptor, respectively. Ternary drug-receptor interactions, which occur between the hydroxyl group of olmesartan and Tyr(113) and between the carboxyl group of olmesartan and Lys(199) and His(256), were essential for the potent inverse agonist action olmesartan exerts against stretch-induced ERK activation and the constitutive activity of the AT(1)-N111G mutant receptor. Furthermore, the inverse agonist activity olmesartan exerts against stretch-induced ERK activation requires an additional drug-receptor interaction involving the tetrazole group of olmesartan and Gln(257) of the AT(1) receptor. These results suggest that multivalent interactions between an inverse agonist and the AT(1) receptor are required to stabilize the receptor in an inactive conformation in response to the distinct processes that lead to an AngII-independent activation of the AT(1) receptor.

Benzyl isothiocyanate-mediated generation of reactive oxygen species causes cell cycle arrest and induces apoptosis via activation of MAPK in human pancreatic cancer cells

In our previous studies, we have shown that benzyl isothiocyanate (BITC) inhibits the growth of human pancreatic cancer cells by inducing apoptosis. In the present study, we demonstrate the activation of all the three (MAPK) family members [extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK) and P38] in response to BITC treatment. Exposure of Capan-2 cells with varying concentrations of BITC for 24 h resulted in the phosphorylation (activation) of ERK at Thr202/Tyr204, JNK at Thr183/Tyr185 and P38 at Thr180/Tyr182, leading to the induction of apoptosis. Similar MAPK activation was also observed in MiaPaCa-2 cells in response to BITC treatment. However, normal human pancreatic ductal epithelial cells did not show the activation of MAPK's and remained unaffected by BITC treatment. To confirm the role of ERK, JNK and P38 in BITC-induced G(2)/M arrest and apoptosis, Capan-2 cells were pre-treated with MAPK-specific inhibitors or MAPK8-short hairpin RNA (shRNA) prior to BITC treatment. Significant protection from BITC-induced G(2)/M arrest was observed in the cells pre-treated with MAPK kinase (MEK-1) but not JNK or P38 inhibitors. On the other hand, BITC-induced apoptosis was almost completely abrogated in the cells pre-treated with MEK-1, JNK or P38 inhibitors. Similarly, MAPK8-shRNA also offered almost complete protection against BITC-induced G(2)/M arrest and apoptosis. Furthermore, we observed that BITC treatment leads to the generation of reactive oxygen species (ROS) in Capan-2 and MiaPaCa-2 cells, which in part was orchestrated by depletion of reduced glutathione (GSH) level. Blocking ROS generation with N-acetyl-L-cysteine (NAC) significantly prevented GSH depletion and activation of ERK and JNK but not P38. Further, NAC or tiron prevented G(2)/M arrest by blocking G(2)/M regulatory proteins and completely protected the cells from BITC-induced apoptosis. Taken together, our results suggest that BITC-mediated G(2)/M arrest is mediated through ERK activation, whereas apoptosis is via ERK, JNK and P38.

HIV protease inhibitor lopinavir-induced TNF-α and IL-6 expression is coupled to the unfolded protein response and ERK signaling pathways in macrophages

HIV protease inhibitor (PI)-associated cardiovascular risk, especially atherosclerosis, has become a major concern in the clinic. Macrophages are key players in the inflammatory response and atherosclerosis formation. We have previously shown that HIV PIs induce endoplasmic reticulum (ER) stress, activate the unfolded protein response (UPR), and increase the synthesis of the inflammatory cytokines, TNF-α and IL-6, by regulating the intracellular translocation of RNA binding protein HuR in macrophages. However, the underlying signaling mechanisms remain unclear. We show here that the HIV PI lopinavir significantly activated the extracellular-signal regulated protein kinase (ERK), but not c-Jun N-terminal kinase (JNK) and p38 MAPK. Lopinavir-induced cytosolic translocation of HuR and TNF-α and IL-6 synthesis was attenuated by specific chemical inhibitor of MEK (PD98058) or over-expression of dominant negative mutant of MEK1. In addition, we demonstrated that lopinavir-induced ERK activation and TNF-α and IL-6 expression were completely inhibited in macrophages from CHOP null mice. Taken together, these results indicate activation of the UPR plays an essential role in HIV PI-induced inflammatory cytokine synthesis and release by activating ERK, which increases the cytosolic translocation of HuR and subsequent binding to the 3′UTR of TNF-α and IL-6 mRNAs in macrophages.

Angiotensin II type 1 receptor blocker attenuates the activation of ERK and NADPH oxidase by mechanical strain in mesangial cells in the absence of angiotensin II

It has been reported that mechanical strain activates extracellular signal-regulated protein kinases (ERK) without the involvement of angiotensin II (Ang II) in cardiomyocytes. We examined the effects of mechanical strain on ERK phosphorylation levels in the absence of Ang II using rat mesangial cells. The ratio of phosphorylated ERK (p-ERK) to total ERK expression was increased by cyclic mechanical strain in a time- and elongation strength-dependent manner. With olmesartan [Ang II type 1 receptor (AT1R) antagonist] pretreatment, p-ERK plateau levels decreased in a dose-dependent manner (EC(50) = 1.3 x 10(-8) M, maximal inhibition 50.6 +/- 11.0% at 10(-5) M); a similar effect was observed with RNA interference against Ang II type 1A receptor (AT(1A)R) and Tempol, a superoxide dismutase mimetic. In addition to the inhibition of p-ERK levels, olmesartan blocked the increase in cell surface and phosphorylated p47(phox) induced by mechanical strain and also lowered the mRNA expression levels of NADPH oxidase subunits. These results demonstrate that mechanical strain stimulates AT1R to phosphorylate ERK in mesangial cells in the absence of Ang II. This mechanotransduction mechanism is involved in the oxidative stress caused by NADPH oxidase and is blocked by olmesartan. The inverse agonistic activity of this AT1R blocker may be useful for the prevention of mesangial proliferation and renal damage caused by mechanical strain/oxidative stress regardless of circulating or tissue Ang II levels.

Positive crosstalk between ERK and p38 in melanoma stimulates migration and in vivo proliferation

Melanoma is one of the most therapy-resistant cancers. Activating mutations in BRAF and NRAS are the source of extracellular signal regulated protein kinase (ERK) pathway activation. We show that melanoma cell lines, originating in different metastatic sites, with BRAF or NRAS mutations, in addition to active mitogen activated protein kinase (MAPK)-ERK, also have highly activated stress activated protein kinase (SAPK)-p38. This is in direct contrast to carcinoma cells in which the activity of the two kinases appears to be mutually exclusive; high level of p38 activity inhibits, through a negative feedback, ERK activity and prevents tumorigenesis. Melanomas are insensitive to ERK inhibition by p38 and utilize p38-signaling pathway for migration and growth in vivo. We found a positive functional loop linking the high ERK activity to surface expression of alphaVbeta3-integrin. This integrin, by interacting with vitronectin, induces p38 activity and increases IL-8 production, enhancing cell migration. Because the negative loop from p38 to ERK is lost, the two kinases can remain simultaneously activated. Inhibition of ERK and p38 activities is more effective in blocking in vivo growth than inhibition of each kinase individually. Future therapies might have to consider targeting of both pathways.

The Chondroprotective Agent ITZ-1 Inhibits Interleukin-1β–Induced Matrix Metalloproteinase–13 Production and Suppresses Nitric Oxide– Induced Chondrocyte Death

In a screening program aimed at discovering anti-osteoarthritis (OA) drugs, we identified an imidazo[5,1-c][1,4]thiazine derivative, ITZ-1, that suppressed both interleukin-1beta (IL-1beta)-induced proteoglycan and collagen release from bovine nasal cartilage in vitro and suppressed intra-articular infusion of IL-1beta-induced cartilage proteoglycan degradation in rat knee joints. ITZ-1 did not inhibit enzyme activities of various matrix metalloproteinases (MMPs), which have pivotal roles in cartilage degradation, while it selectively inhibited IL-1beta-induced production of MMP-13 in human articular chondrocytes (HAC). IL-1beta-induced MMP production has been shown to be mediated by extracellular signal-regulated protein kinase (ERK), p38 kinase, and c-Jun N-terminal kinase (JNK) of the mitogen-activated protein kinase (MAPK) family signal transduction molecules. An ERK-MAPK pathway inhibitor (U0126), but not a p38 kinase inhibitor (SB203580) or a JNK inhibitor (SP600125), also selectively inhibited IL-1beta-induced MMP-13 production in HAC. Furthermore, ITZ-1 selectively inhibited IL-1beta-induced ERK activation without affecting p38 kinase and JNK activation, which may account for its selective inhibition of MMP-13 production. Inhibition of nitric oxide (NO)-induced chondrocyte apoptosis has been another area of interest as a therapeutic strategy for OA, and ITZ-1 also suppressed NO-induced death in HAC. These results suggest that ITZ-1 is a promising lead compound for a disease modifying anti-OA drug program.

Thrombin induces Egr-1 expression in fibroblasts involving elevation of the intracellular Ca2+ concentration, phosphorylation of ERK and activation of ternary complex factor

BackgroundThe serine protease thrombin catalyzes fibrin clot formation by converting fibrinogen into fibrin. Additionally, thrombin stimulation leads to an activation of stimulus-responsive transcription factors in different cell types, indicating that the gene expression pattern is changed in thrombin-stimulated cells. The objective of this study was to analyze the signaling cascade leading to the expression of the zinc finger transcription factor Egr-1 in thrombin-stimulated lung fibroblasts.ResultsStimulation of 39M1-81 fibroblasts with thrombin induced a robust and transient biosynthesis of Egr-1. Reporter gene analysis revealed that the newly synthesized Egr-1 was biologically active. The signaling cascade connecting thrombin stimulation with Egr-1 gene expression required elevated levels of cytosolic Ca2+, the activation of diacylgycerol-dependent protein kinase C isoenzymes, and the activation of extracellular signal-regulated protein kinase (ERK). Stimulation of the cells with thrombin triggered the phosphorylation of the transcription factor Elk-1. Expression of a dominant-negative mutant of Elk-1 completely prevented Egr-1 expression in stimulated 39M1-81 cells, indicating that Elk-1 or related ternary complex factors connect the intracellular signaling cascade elicited by activation of protease-activated receptors with transcription of the Egr-1 gene. Lentiviral-mediated expression of MAP kinase phosphatase-1, a dual-specific phosphatase that dephosphorylates and inactivates ERK in the nucleus, prevented Elk-1 phosphorylation and Egr-1 biosynthesis in thrombin stimulated 39M1-81 cells, confirming the importance of nuclear ERK and Elk-1 for the upregulation of Egr-1 expression in thrombin-stimulated lung fibroblasts. 39M1-81 cells additionally express M1 muscarinic acetylcholine receptors. A comparison between the signaling cascades induced by thrombin or carbachol showed no differences, except that signal transduction via M1 muscarinic acetylcholine receptors required the transactivation of the EGF receptor, while thrombin signaling did not.ConclusionThis study shows that stimulus-transcription coupling in thrombin-treated lung fibroblasts relies on the elevation of the intracellular Ca2+-concentration and the activation of PKC and ERK. In the nucleus, ternary complex factors function as key proteins linking the intracellular signaling cascade with enhanced transcription of the Egr-1 gene. This study further shows that the dominant-negative Elk-1 mutant is a valuable tool to study Elk-1-mediated gene transcription.

ERK Regulates Renal Cell Proliferation and Renal Cyst Expansion in inv Mutant Mice

Nephronophthisis (NPHP) is the most frequent genetic cause of end-stage kidney disease in children and young adults. Inv mice are a model for human nephronophthisis type 2 (NPHP2) and characterized by multiple renal cysts and situs inversus. Renal epithelial cells in inv cystic kidneys show increased cell proliferation. We studied the ERK pathway to understand the mechanisms that induce cell proliferation and renal cyst progression in inv kidneys. We studied the effects of ERK suppression by administering PD184352, an oral mitogen-activated protein kinase kinase (MEK) inhibitor on renal cyst expansion, extracellular signal-regulated protein kinase (ERK) activity, bromo-deoxyuridine (BrdU) incorporation and expression of cell-cycle regulators in invΔC kidneys. Phosphorylated ERK (p-ERK) level increased along with renal cyst enlargement. Cell-cycle regulators showed a high level of expression in invΔC kidneys. PD184352 successfully decreased p-ERK level and inhibited renal cyst enlargement. The inhibitor also decreased expression of cell-cycle regulators and BrdU incorporation in renal epithelial cells. The present results showed that ERK regulated renal cell proliferation and cyst expansion in inv mutants.

(−)-Epigallocatechin-3-gallate induces up-regulation of Th1 and Th2 cytokine genes in Jurkat T cells

In the present study, we found that (−)-epigallocatechin-3-gallate (EGCG) significantly up-regulated the mRNA expression of the Th1/Th2 cytokines including IL-2, IFN-γ, IL-5 and IL-13 in Jurkat T cells. The EGCG-induced mRNA up-regulation of IL-2 and IL-5 was predominantly affected by the extracellular signal-regulated protein kinase (ERK) signalling, whereas IL-13 gene expression, the most responsive to the EGCG treatment, was dependent on neither ERK nor c-jun NH 2-terminal kinase (JNK) signalling. IFN-γ gene expression was partially mitigated by both inhibitors of the ERK and JNK pathways. Furthermore, catalase significantly attenuated the intracellular peroxide production, phosphorylation of ERK and JNK, and all cytokine gene expressions induced by EGCG. In addition, physiologically relevant concentrations of both EGCG and H 2O 2-induced up-regulation of IL-5 gene expression. Our findings provide biological evidence that EGCG induces Th1/Th2 cytokine mRNA expression via H 2O 2 production followed by activation of ERK or JNK in Jurkat T cells.

Ethanol Attenuates the HFS‐Induced, ERK‐Mediated LTP in a Dose‐Dependent Manner in Rat Striatum

The striatum has been implicated to play a role in the control of voluntary behavior, and striatal synaptic plasticity is involved in instrumental learning. Ethanol is known to alter synaptic plasticity, in turn altering the behavior of human and animals. However, it remains unclear whether the striatum plays a role in the effects of ethanol on the central nervous system. The objective of this investigation was to study the effects of acute perfusion of ethanol on long-term potentiation (LTP) to elucidate the mechanisms of addictive drugs in the striatum. In addition, we investigated the contribution of intracellular extracellular signal regulated protein kinase (ERK) signaling pathway to corticostriatal LTP induction. The stimulation evoked population spikes (PS) were recorded from the dorsomedial striatum (DMS) slices of rat using the extracellular recording technique. The LTP in DMS slices was induced by high-frequency stimulation (HFS). The ERK level of the DMS was assessed with the Western blot technique. U0126, the inhibitor of ERK, eliminated or significantly attenuated the LTP induced by HFS of the PS in the DMS. MK801 and APV, N-methyl-d-aspartic acid receptor (NMDAR) antagonists, inhibited the induction of striatal LTP, and HFS-induced ERK activation decreased in the slices treated with MK801 in the DMS. Clinically relevant concentrations of ethanol (22 to 88 mM) dose-dependently attenuated the HFS-induced striatal LTP and ERK activation in this brain region. The LTP of the PS in the DMS is, at least partly, mediated by the ERK pathway coupling to NMDARs. Ethanol attenuated the HFS-induced, ERK-mediated LTP in a dose-dependent manner in this brain region. These results indicate that ethanol may change the synaptic plasticity of corticostriatal circuits underlying the learning of goal-directed instrumental actions, which is mediated by an intracellular ERK signaling pathway associated with NMDARs.

KRAS or BRAF mutation status is a useful predictor of sensitivity to MEK inhibition in ovarian cancer

This study examined the status of KRAS and BRAF mutations, in relation to extracellular signal-regulated protein kinase (ERK) activation in 58 ovarian carcinomas to clarify the clinicopathological and prognostic significance of KRAS/BRAF mutations. Somatic mutations of either KRAS or BRAF were identified in 12 (20.6%) out of 58 ovarian carcinomas. The frequency of KRAS/BRAF mutations in conventional serous high-grade carcinomas (4.0% : 1/25) was significantly lower than that in the other histological type (32.3% : 10/31). Phosphorylated ERK1/2 (p-ERK1/2) expression was identified in 18 (38.2%) out of 45 ovarian carcinomas. KRAS/BRAF mutation was significantly correlated with International Federation of Gynecology and Obstetrics (FIGO) stage I, II (P<0.001), and p-ERK1/2 (P<0.001). No significant correlations between KRAS/BRAF mutations or p-ERK1/2 expression and overall survival were found in patients with ovarian carcinoma treated with platinum and taxane chemotherapy (P=0.2460, P=0.9339, respectively). Next, to clarify the roles of ERK1/2 activation in ovarian cancers harbouring KRAS or BRAF mutations, we inactivated ERK1/2 in ovarian cancer cells using CI-1040. Cl-1040 is a compound that selectively inhibits MAP kinase kinase (MEK), an upstream regulator of ERK1/2, and thus prevents ERK1/2 activation. Profound growth inhibition and apoptosis were observed in CI-1040-treated cancer cells with mutations in either KRAS or BRAF in comparison with the ovarian cancer cells containing wild-type sequences. This was evident in both in vitro and in vivo studies. The findings in this study indicate that an activated ERK1/2 pathway is critical to tumour growth and survival of ovarian cancers with KRAS or BRAF mutations. Furthermore, they suggest that the CI-1040-induced phenotypes depend on the mutational status of KRAS and BRAF in ovarian cancers. Therefore, ovarian cancer patients with KRAS or BRAF mutations may benefit from CI-1040 treatment.

Cue-elicited drug craving represses ERK activation in mice prefrontal association cortex

Morphine addiction is characterized by compulsive drug-taking behavior and high rates of relapse that reflect reward-controlled learning, consolidation and reconsolidation of drug cues. Extracellular signal-regulated protein kinase (ERK) is one of the cellular molecules that have been highly implicated in the synaptic plasticity processes of learning and memory in cocaine addiction. However, the roles of ERK in the morphine-paired conditioned place preference (CPP) are not clear. In the present study, we found that compared to the morphine-unpaired and saline-paired and saline-unpaired groups, morphine-paired mice showed depressed ERK2 activity in the Frontal Association Cortex (FrA), whereas ERK1 activity was not changed in the same region. In the Accumbens Nucleus (Acb) and Caudate Putamen (CPu) that are associated with cocaine addiction, the activities of ERK1 and ERK2 among four groups showed no difference. These results suggest that the FrA plays an important role in morphine craving and that ERK2 is involved in eliciting the environment-related morphine craving, which is totally different from those induced by morphine itself.

TRAIL induces MMP-9 expression via ERK activation in human astrocytoma cells

Matrix metalloproteinase-9 (MMP-9) is an important angiogenic and prognostic factor in malignant tumors. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known as the death ligand, which induces preferential apoptosis of transformed tumor cells. In this study, we investigated the biological functions of TRAIL, other than its role in induction of apoptosis. We demonstrated that TRAIL induces MMP-9 expression in human astrocytoma cells, which is preceded by activation of extracellular signal-regulated protein kinase (ERK). In addition, TRAIL induces the DNA-binding activity of NF-κB, an important transcription factor for MMP-9 induction. The specific MEK inhibitor, U0126, significantly blocks TRAIL-mediated NF-κB activation and subsequent MMP-9 induction. These findings indicate that TRAIL treatment in human astrocytoma cells leads to the activation of NF-κB and subsequent expression of MMP-9, which are dependent on ERK activation. Collectively, these results suggest that TRAIL has alternative biological functions in addition to its role in inducing apoptosis in human malignant astrocytoma cells.

Calcium influx into MIN6 insulinoma cells induces expression of Egr-1 involving extracellular signal-regulated protein kinase and the transcription factors Elk-1 and CREB

Glucose induces many changes in the transcriptional pattern of β-cells derived from the endocrine pancreas. The zinc finger protein Egr-1 belongs to the transcription factors that are activated in glucose-treated β-cells. Egr-1 expression is additionally induced by treatment of MIN6 pancreatic β-cells with tolbutamide, a compound that triggers a closure of ATP-dependent potassium channels, K ATP, in the plasma membrane or by KCl that depolarizes the cell membrane. Stimulation with glucose, tolbutamide or KCl induces a Ca 2+ influx into the β-cells via L-type Ca 2+ channels. Accordingly, incubation of the cells with the L-type Ca 2+ channel blocker nifedipine or the acetoxymethylester of the cytosolic Ca 2+ chelator BAPTA prevented Egr-1 expression. Moreover, diacylgycerol-dependent protein kinase C isoenzymes and activation of extracellular signal-regulated protein kinase (ERK) are required for glucose-, tolbutamide- and KCl-induced Egr-1 expression. The signaling cascade was blocked by MAP kinase phosphatase-1 (MKP-1) overexpression that dephosphorylated ERK in the nucleus. Stimulation of β-cells by glucose, tolbutamide and KCl induced the phosphorylation of the transcription factors Elk-1 and CREB. ChIP experiments revealed that phosphorylated Elk-1 and CREB bound under physiological conditions to the Egr-1 gene. Lentiviral-mediated expression of dominant-negative mutants of Elk-1 or CREB interfered with glucose-, tolbutamide- and KCl-induced upregulation of Egr-1 biosynthesis. Together, these data indicate that stimulus-induced transcription of the Egr-1 gene in β-cells requires combinatorial regulation by Elk-1 and CREB following activation of ERK. The newly synthesized Egr-1 is biologically active and binds under physiological conditions to the genes encoding basic fibroblast growth factor, tumor necrosis factor α, transforming growth factor β and PTEN.

Lysophosphatidic Acid Receptors 1 and 2 Play Roles in Regulation of Vascular Injury Responses but Not Blood Pressure

Phenotypic modulation of vascular smooth muscle cells (SMCs) is essential for the development of intimal hyperplasia. Lysophosphatidic acid (LPA) is a serum component that can promote phenotypic modulation of cultured SMCs, but an endogenous role for this bioactive lipid as a regulator of SMC function in vivo has not been established. Ligation injury of the carotid artery in mice increased levels in the vessel of both autotaxin, the lysophospholipase D enzyme responsible for generation of extracellular LPA, and 2 LPA responsive G protein-coupled receptors 1 (LPA1) and 2 (LPA2). LPA1(-/-)2(-/-) mice were partially protected from the development of injury-induced neointimal hyperplasia, whereas LPA1(-/-) mice developed larger neointimal lesions after injury. Growth in serum, LPA-induced extracellular signal-regulated protein kinase activation, and migration to LPA and serum were all attenuated in SMCs isolated from LPA1(-/-)2(-/-) mice. In contrast, LPA1(-/-) SMCs exhibited enhanced migration resulting from an upregulation of LPA3. However, despite their involvement in intimal hyperplasia, neither LPA1 nor LPA2 was required for dedifferentiation of SMCs following vascular injury or dedifferentiation of isolated SMCs in response to LPA or serum in vitro. Similarly, neither LPA1 nor LPA2 was required for LPA to elicit a transient increase in blood pressure following intravenous administration of LPA to mice. These results identify a role for LPA1 and LPA2 in regulating SMC migratory responses in the context of vascular injury but suggest that additional LPA receptor subtypes are required for other LPA-mediated effects in the vasculature.

Activation of ERK-FAK Signaling Pathway and Enhancement of Cell Migration Involved in the Early Interaction Between Oral Keratinocytes and Candida albicans

To investigate the early molecular events in oral keratinocytes induced by Candida albicans challenge. The oral keratinocyte cell line, Tca8113, was used to study the molecular events induced by C. albicans challenge in oral keratinocytes. The phosphorylation statuses of extracellular signal-regulated protein kinase (ERK) and focal adhesion kinase (FAK) upon C. albicans challenge were assessed using specific antibodies and western blotting. Specific inhibitors for ERK and FAK were used to validate the involvement of ERK-FAK signaling cascade. A Transwell insert system-based migration study was performed to evaluate the involvement of the C. albicans-dependent ERK-FAK activation with cell migration. Following the stimulation with C. albicans, a transient activation of ERK was observed, which reached a peak at 10 min post stimulation. Similarly, a transient activation of FAK, the downstream substrate of ERK, was also observed upon C. albicans challenges, which reach the maximum at 20 min. Specific inhibitors for ERK and FAK abolished the C. albicans-induced ERK and FAK activations. The elevated migratory ability of oral keratinocyte was observed upon stimulation with C. albicans, and was synchronous with the ERK-FAK activation. ERK-FAK signaling cascades are involved in the early interaction between the oral keratinocytes and C. albicans, which appears to be linked with the enhanced cell migration.

Induction of hepatocyte growth factor expression by maleic acid in human fibroblasts through MAPK activation

Carboxylic acids have various biological activities and play critical roles in cellular metabolic pathways such as the tricarboxylic acid (TCA) cycle. It has been shown that some carboxylic acids induce cell proliferation and production of cytokines or growth factors. However, there have been no reports on effects of carboxylic acids on hepatocyte growth factor (HGF) expression. In this study, we found that only maleic acid among various carboxylic acids examined markedly induced HGF production from human dermal fibroblasts. Maleic acid also induced HGF production from human lung fibroblasts and neuroblastoma cells. The stimulatory effect was accompanied by upregulation of HGF gene expression. Increase in phosphorylation of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) but not in phosphorylation of p38 was observed from 6 h and up to 24 h after maleic acid addition. The ERK kinase inhibitor PD98059 and the JNK inhibitor SP600125 potently inhibited maleic acid-induced HGF production, while the p38 inhibitor SB203580 did not significantly inhibit the production. The protein synthesis inhibitor cycloheximide completely inhibited upregulation of HGF mRNA induced by maleic acid but superinduced HGF mRNA expression upregulated by 12-O-tetradecanoylphorbol 13-acetate (TPA). These results suggest that maleic acid indirectly induced HGF expression from human dermal fibroblasts through activation of ERK and JNK and that de novo protein synthesis is required for maleic acid-induced upregulation of HGF mRNA.

Coactivation of M1Muscarinic and α1 Adrenergic Receptors Stimulates Extracellular Signal-Regulated Protein Kinase and Induces Long-Term Depression at CA3–CA1 Synapses in Rat Hippocampus

Intact cholinergic innervation from the medial septum and noradrenergic innervation from the locus ceruleus are required for hippocampal-dependent learning and memory. However, much remains unclear about the precise roles of acetylcholine (ACh) and norepinephrine (NE) in hippocampal function, particularly in terms of how interactions between these two transmitter systems might play an important role in synaptic plasticity. Previously, we reported that activation of either muscarinic M(1) or adrenergic alpha1 receptors induces activity- and NMDA receptor-dependent long-term depression (LTD) at CA3-CA1 synapses in acute hippocampal slices, referred to as muscarinic LTD (mLTD) and norepinephrine LTD (NE LTD), respectively. In this study, we tested the hypothesis that mLTD and NE LTD are independent forms of LTD, yet require activation of a common Galphaq-coupled signaling pathway for their induction, and investigated the net effect of coactivation of M(1) and alpha1 receptors on the magnitude of LTD induced. We find that neither mLTD nor NE LTD requires phospholipase C activation, but both plasticities are prevented by inhibiting the Src kinase family and extracellular signal-regulated protein kinase (ERK) activation. Interestingly, LTD can be induced when M(1) and alpha1 agonists are coapplied at concentrations too low to induce LTD when applied separately, via a summed increase in ERK activation. Thus, because ACh and NE levels in vivo covary, especially during periods of memory encoding and consolidation, cooperative signaling through M(1) and alpha1 receptors could function to induce long-term changes in synaptic function important for cognition.

BDNF induces late-phase LTP of C-fiber evoked field potentials in rat spinal dorsal horn

Several lines of evidence have shown that in some brain regions brain-derived neurotrophic factor (BDNF) is important for long-term potentiation (LTP), a synaptic model of memory storage. In the present work we evaluate the role of BDNF in LTP of C-fiber evoked field potentials in spinal dorsal horn, a synaptic model of pain memory. We found that spinal application of BDNF-induced LTP of C-fiber evoked field potentials with a long latency, lasting for > 8 h, and the effect was blocked by either tyrosine kinase inhibitor (K252a) or BNDF scavenger (TrkB-Fc). The potentiation produced by BDNF was occluded by late-phase LTP (L-LTP) but not by early-phase LTP (E-LTP) induced by electrical stimulation. Pretreatment of K252a or TrkB-Fc selectively blocked spinal L-LTP induced by low-frequency stimulation (LFS) but not E-LTP. BDNF-induced LTP was completely abolished by the protein synthesis inhibitor (anisomycin), by N-methyl-D-aspartate (NMDA) receptor blocker (MK-801), by extracellular signal-regulated protein kinase (ERK) inhibitor (PD98059) or by p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580) but not by c-Jun N-terminal kinase (JNK) inhibitor (SP600125). Nuclear factor-kappaB (NF-κB) inhibitor (PDTC) also suppressed spinal BDNF-LTP. The results suggest that BDNF play a crucial role in protein synthesis-dependent L-LTP in spinal dorsal horn via activation of ERK, p38 MAPK and NF-κB signal pathways.