• BMB Reports
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• v.49 no.4
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• pp.220-225
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• 2016

Cancer cells have different characteristics due to the genetic differences where these unique features may strongly influence the effectiveness of therapeutic interventions. Here, we show that the spontaneous reactivation of extracellular signalregulated kinase (ERK), distinct from conventional ERK activation, represents a potent mechanism for cancer cell survival. We studied ERK1/2 activation in vitro in SW480 colorectal cancer cells. Although ERK signaling tends to be transiently activated, we observed the delayed reactivation of ERK1/2 in epidermal growth factor (EGF)-stimulated SW480 cells. This effect was observed even after EGF withdrawal. While phosphorylated ERK1/2 translocated into the nucleus following its primary activation, it remained in the cytoplasm during late-phase activation. The inhibition of primary ERK1/2 activation or protein trafficking, blocked reactivation and concurrently increased caspase 3 activity. Our results suggest that the biphasic activation of ERK1/2 plays a role in cancer cell survival; thus, regulation of ERK1/2 activation may improve the efficacy of cancer therapies that target ERK signaling.

• Animal cells and systems
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• v.14 no.3
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• pp.155-160
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• 2010

Extracellular signal-regulated kinases 1/2 (ERK1/2) play important roles in a variety of biological processes including cell growth and differentiation. We have previously reported that GAR-3 activates ERK1/2 via phospholipase C and protein kinase C, presumably through pertussis toxin (PTX)-insensitive Gq proteins, in Chinese hamster ovary (CHO) cells. Here we provide evidence that GAR-3 also activates ERK1/2 through PTX-sensitive G proteins, phosphatidylinositol 3-kinase (PI 3-kinase), and Src family kinases in CHO cells. We further show that in human embryonic kidney (HEK293) cells, epidermal growth factor receptor and Ras are required for efficient ERK1/2 activation by GAR-3. Taken together, our data indicate that GAR-3 evokes ERK1/2 activation through multiple signaling pathways in cultured mammalian cells.

• The Korean Journal of Physiology and Pharmacology
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• v.8 no.4
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• pp.181-185
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• 2004

Extracellular regulated protein kinase1/2 (pERK1/2) is one of the major regulatory factors for transcription of the c-fos oncogene in neurons. The purpose of this study was to evaluate the expression of phosphorylated ERK1/2 within the vestibular nuclei (VN) of rats following acute arterial hypotension. Following the acute arterial hypotension induced by rapid hemorrhage, a significant number of pERK1/2-immunoreactive neurons appeared bilaterally in the caudal aspect of the medial and inferior VN. No labeling of pERK1/2 was observed in the lateral VN. The peak expression of pERK1/2 in these nuclei occurred within 5 min after hemorrhage. However, in bilaterally labyrinthectomized rats, the appearance of pERK1/2-immunoreactive neurons was eliminated in the VN. Western blot confirmed the effect of bilateral labyrinthectomy on pERK1/2 protein expression in the medial vestibular nucleus 5 min after hemorrhage. These results suggest that, following acute hypotension, afferent signals from the peripheral vestibular receptors are required for activation of ERK 1/2 in the VN.

• Molecules and Cells
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• v.25 no.4
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• pp.504-509
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• 2008

Three G-protein-linked acetylcholine receptors (GARs) exist in the nematode C. elegans. GAR-3 is pharmacologically most similar to mammalian muscarinic acetylcholine receptors (mAChRs). We observed that carbachol stimulated ERK1/2 activation in Chinese hamster ovary (CHO) cells stably expressing GAR-3b, the predominant alternatively spliced isoform of GAR-3. This effect was substantially reduced by the phospholipase C (PLC) inhibitor U73122 and the protein kinase C (PKC) inhibitor GF109203X, implying that PLC and PKC are involved in this process. On the other hand, GAR-3b-mediated ERK1/2 activation was inhibited by treatment with forskolin, an adenylate cyclase (AC) activator. This inhibitory effect was blocked by H89, an inhibitor of cAMP-dependent protein kinase A (PKA). These results suggest that GAR-3b-mediated ERK1/2 activation is negatively regulated by cAMP through PKA. Together our data show that GAR-3b mediates ERK1/2 activation in CHO cells and that GAR-3b can couple to both stimulatory and inhibitory pathways to modulate ERK1/2.

• Journal of Acupuncture Research
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• v.37 no.2
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• pp.123-127
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• 2020

Background: This study was designed using a mouse model of atopic dermatitis [phthalic anhydride (PA)-treated mice], to investigate the anti-inflammatory effect of bee venom pharmacopuncture (BVP) in keratinocytes. Methods: Western blot analysis was performed to investigate inflammation related protein expression of iNOS, COX-2, phospho-ERK (p-ERK), and ERK, in LPS (1 ㎍/mL)-activated keratinocytes, following BVP treatment, and in PA-treated mice, after BVP treatment. Griess reaction was performed to investigate NO concentration. Enzyme-linked immunosorbent assays were used to determine the concentrations of interleukin (IL)-4+, IL-17A+, IL-13 and IL-4 in PA-treated mice after BVP treatment. In addition, monocyte, macrophage, neutrophil, and eosinophil counts were measured to observe the changes in white blood cell infiltration. Results: The keratinocytes of the BVP-treated group showed a decreased expression of iNOS, COX-2, ERK at 5 OX-2, ERK E, and p-ERK at 1, 2 and 5 RKRK ERK ERK, and a dose-dependent decrease in NO concentration at 2 and 5 ntrationof s. In the BVP-treated groups (0.1 μ.1-trea μ.1-treated gr), PA-treated mice showed recovery after 4 weeks which was dose-dependent, showing a significant decrease in clinical scores for AD, and a decreased concentration of IL-13 and IL-4 with BV treatment. There was a dose-dependent decrease in the infiltration of eosinophils, neutrophils, monocytes, macrophages, and a decreased thickness of the epidermis due to inflammation, and decreased expressions of iNOS, COX-2, p-ERK, ERK, especially in the 0.1 μ0/mL BVP-treated group, Conclusion: These results suggest that BVP may be an effective alternative treatment for atopic dermatitis.

• Natural Product Sciences
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• v.15 no.1
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• pp.32-36
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• 2009

Glutamate causes neurotoxicity through formation of reactive oxygen species and activation of mitogen-activated protein kinase (MAPK) pathways. MAPK phosphatase-1 (MKP-1) is one of the phosphatases responsible for dephosphorylation/deactivation of three MAPK families: the extracellular signal-regulated kinase-1/2 (ERK-1/2), the c-Jun N-terminal kinase-1/2 (JNK-1/2), and the p38 MAPK. In this report, the potential involvement of MKP-1 in neuroprotective effects of curcumin, the active ingredient of turmeric (Curcuma longa), was examined using HT22 cells. Glutamate caused cell death and activation of ERK-1/2 but not p38 MAPK or JNK-1/2. Blockage of ERK-1/2 by its inhibitor protected HT22 cells against glutamate-induced toxicity. Curcumin attenuated glutamate-induced cell death and ERK-1/2 activation. Interestingly, curcumin induced MKP-1 activation. In HT22 cells transiently transfected with small interfering RNA against MKP-1, curcumin failed to inhibit glutamate-induced ERK-1/2 activation and to protect HT22 cells from glutamate-induced toxicity. These results suggest that curcumin can attenuate glutamate-induced neurotoxicity by activating MKP-1 which acts as the negative regulator of ERK-1/2. This novel pathway may contribute to and explain at least one of the neuroprotective actions of curcumin.

• Journal of Microbiology and Biotechnology
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• v.22 no.2
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• pp.170-175
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• 2012

Clostridium difficile toxin A glucosylates Rho family proteins, resulting in actin filament disaggregation and cell rounding in cultured colonocytes. Given that the cellular toxicity of toxin A is dependent on its receptor binding and subsequent entry into the cell, we herein sought to identify additional colonocyte proteins that might bind to toxin A following its internalization. Our results revealed that toxin A interacted with ERK1 and ERK2 in two human colonocyte cell lines (NCM460 and HT29). A GST-pulldown assay also showed that toxin A can directly bind to ERK1 and ERK2. In NCM460 cells exposed to PMA (an ERK1/2 activator), the phosphorylation of ERK1/2 did not affect the interaction between toxin A and ERK1/2. However, an in vitro kinase assay showed that the direct binding of toxin A to ERK1 or ERK2 inhibited their kinase activities. These results suggest a new molecular mechanism for the cellular toxicity seen in cells exposed to toxin A.

• Journal of Pharmacopuncture
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• v.6 no.2
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• pp.77-90
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• 2003

The purpose of this study was to investigate the effect of Bee Venom on the lipopolysaccharide(LPS), sodium nitroprusside(SNP), hydrogen peroxide($H_2O_2$)-induced expressions of cyclooxygenase-2(COX-2), p38, jun N-terminal Kinase(JNK) and extra-signal response kinase(ERK) in RAW 264.7 cells, a murine macrophage cell line. Method : The expressions of COX-2, p38, JNK and ERK were determined by western blotting with corresponding antibodies. Results : 1. The 0.5, 1 and $5\;{\mu}g/ml$ of bee venom inhibited significantly LPS and SNP-induced expression of COX-2 compared with control, respectively. The 0.5, 1 and $5\;{\mu}g/ml$ of bee venom inhibited insignificantly $H_2O_2$-induced expression of COX-2 compared with control, respectively. 2. The 0.5, 1 and $5\;{\mu}g/ml$ of bee venom inhibited significantly LPS, SNP and $H_2O_2$-induced expression of p38 compared with control, respectively. 3. The 1 and $5\;{\mu}g/ml$ of bee venom inhibited significantly SNP-induced expression of JNK compared with control, respectively. All of bee venom inhibited insignificantly LPS and $H_2O_2$-induced expression of JNK compared with control, respectively. 4. The $5\;{\mu}g/ml$ of bee venom inhibited significantly SNP-induced expression of ERK, the $0.5\;{\mu}g/ml$ of bee venom increased significantly $H_2O_2$-induced expression of ERK compared with control. The 0.5, 1 and $5\;{\mu}g/ml$ of bee venom inhibited insignificantly LPS-induced expression of ERK compared with control, respectively.

• Environmental Mutagens and Carcinogens
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• v.25 no.2
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• pp.51-59
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• 2005

Overexpression of HSP25 delayed cell growth, increased the level of $p21^{waf}$, reduced the levels of cyclin D1, cylcin A and cdc2, and induced radioresistance in L929 cells. We demonstrated that extracellular regulated kinase (ERK) and MAP kinase/ERK kinase (MEK) expressions as well as their activation (phospho-forms) were inhibited by hsp25 overexpression. To confirm the relationship between ERK1/2 and hsp25-mediated radioresistance, ERK1 or ERK2 cDNA was transiently transfected into the hsp25 overexpressed cells and their radioresistance was examined. HSP25-mediated radioresistance was abolished by overexpression of ERK2, but not by overexpression of ERK1. Alteration of cell cycle distribution and cell cycle related protein expressions (cyclin D, cyclin A and cdc2) by hsp25 overexpression were also recovered by ERK2 cDNA transfection. Increase in Bc1-2 protein by hsp25 gene transfection was also reduced by subsequent ERK2 cDNA-transfection. In addition, HSP25 overexpression reduced reactive oxygen species (ROS) and increased expression of manganese superoxide dismutase (MnSOD) gene. Increased activation of NF-kB (IkB degradation) was also found in hsp25-overexpressed cells. Moreover, transfection of hsp25 antisense gene abrogated all the HSP25-mediated phenomena. To further elucidate the exact relationship between MnSOD induction and NF-kB activation, dominant negative $I-kB\alpha(I-kB\alpha-DN)$ construction was transfected to HSP25 overexpressed cells. $I-kB\alpha-DN$ inhibited HSP25 mediated MnSOD gene expression. In addition, HSP25 mediated radioresistance was blocked by $I-kB\alpha-DN$ transfection. Blockage of MnSOD with antisense oligonucleotides in HSP25 overexpressed cells, prevented apoptosis and returned the ERK1/2 activation to the control level. From the above results, we suggest for the first time that reduced oxidative damage by HSP25 was due to MnSOD-mediated down regulation of ERK1/2.

• Korean Journal of Biological Psychiatry
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• v.20 no.4
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• pp.159-165
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• 2013

Objectives Mechanisms of clinical synergistic effects, induced by co-treatments of lithium and valproate, are unclear. Extracellular signal-regulated kinase (ERK) has been suggested to play important roles in mechanisms of the action of mood stabilizers. In this study, effects of co-treatments of lithium and valproate on the ERK1/2 signal pathway and its down-stream transcription factors, ELK1 and C-FOS, were investigated in vitro. Methods PC12 cells, human pheochromocytoma cells, were treated with lithium chloride (30 mM), valproate (1 mM) or lithium chloride + valproate. The phosphorylation of ERK1/2 was analyzed with immunoblot analysis. Transcriptional activities of ELK1 and C-FOS were analyzed with reporter gene assay. Results Single treatment of lithium and valproate increased the phosphorylation of ERK and transcriptional activities of ELK1 and C-FOS, respectively. Combined treatments of lithium and valproate induced more robust increase in the phosphorylation of ERK1/2 and transcriptional activities of ELK1 and C-FOS, compared to those in response to single treatment of lithium or valproate. Conclusions Co-treatments of lithium and valproate induced synergistic increase in the phosphorylation of ERK1/2 and transcriptional activities of its down-stream transcription factors, ELK1 and C-FOS, compared to effects of single treatment. The findings might suggest potentiating effects of lithium and valproate augmentation treatment strategy.