• BMB Reports
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• 제49권4호
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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.

• Journal of Acupuncture Research
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• 제37권2호
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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.

• Tuberculosis and Respiratory Diseases
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• 제56권6호
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• pp.638-645
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• 2004

연구배경 : Uteroglobin은 정상 폐상피세포에서 발현되는 단백질로 비소세포암 조직이나 세포주에서는 발현이 저하되어있다. 항염증작용을 하며 암세포에 과발현 시키면 암의 형질이 소실됨이 밝혀지고 있다. 역시 염증작용과 관련이 있는 cPLA2와 COX-2도 암과의 관련성이 밝혀지고 있고, 암억제나 MMP의 억제 등의 공통점을 가지고 있으나 이들의 관련성에 대해서는 밝혀진 바가 없다. 또한, COX-2의 암과의 관련성을 설명하는 기전으로 ERK 활성화의 관련 가능성이 있으나, uteroglobin과 ERK의 관련성도 아직 밝혀지지 않고 있다. 비소세포폐암 세포주에 uteroglobin을 과발현시킨 후, cPLA2와 COX-2의 발현, 그리고 MMP-2, MMP-9, ERK의 활성화가 어떻게 변화하는지에 대해 실험하였다. 방 법 : 폐선암세포주인 A549와 NCI-H460 세포주에 adenovirus-uteroglobin, adenovirus-null을 각각 20,100,200 moi로 transduction 시킨 뒤, 48시간 배양한 후에 단백질을 추출하였다. Uteroglobin의 발현을 확인한 후, cPLA2, COX-2, pERK, total ERK에 대해 Western blot을 시행하였고, 배양액으로 zymography를 시행하였다. 결 과 : A549 세포주와 NCI-H460 세포주에서 uteroglobin의 발현을 확인한 세포주에서 cPLA2와 COX-2의 발현이 감소함을 Western blot으로 확인하였고, pERK가 증가함을 Western blot으로 보았고, ERK의 활성화가 증가함을 확인하였다. MMP-9은 활성이 저하되었고, MMP-2는 변화가 없었다. MEK inhibitor인 U0126을 이용하여 ERK의 활성화를 저해시킨 후, uteroglobin의 발현에는 영향이 없었고, MMP-9의 활성저하효과가 소실되었다. 결 론 : 폐암세포주에서 uteroglobin의 항암작용기전에 cPLA2 와 COX-2의 발현의 감소와 ERK의 활성화가 기여할 것으로 사료된다.

• BMB Reports
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• 제47권12호
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• pp.685-690
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• 2014

The Ras/Raf/MEK/Erk signaling pathway is important for regulation of cell growth, proliferation, differentiation, survival, and apoptosis in response to a variety of extracellular stimuli. Lack of Erk MAPK activation is observed in several cancer cells despite active activation of Ras. However, little is known about the modulation of Erk1/2 activity by active Ras. Here, we show that overexpression of active H-Ras (H-RasG12R) in NIH3T3 fibroblasts impaired FGF2-induced Erk1/2 phosphorylation, as compared to wild-type cells. Northern blot analysis revealed that prolonged expression of active Ras increased MAP kinase phosphatase 3 (MKP3) mRNA expression, a negative regulator of Erk MAPK. Inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway abrogated active Ras-induced up-regulation of MKP3 expression, leading to the rescue of Erk1/2 phosphorylation. Our results demonstrated that the Ras/Raf/MEK/Erk signaling cascade is negatively regulated by the PI3K/Aktdependent transcriptional activation of the MKP3 gene.

• The Korean Journal of Physiology and Pharmacology
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• 제8권4호
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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.

• Animal cells and systems
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• 제14권3호
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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.

• BMB Reports
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• 제40권6호
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• pp.899-910
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• 2007

Extracellular Regulated Kinases (ERK) and Protein Kinase B (Akt) are intermediaries in relaying extracellular growth signals to intracellular targets. Each pathway can become activated upon stimulation of G protein-coupled receptors mediated by $G_q$ and $G_{i/o}$ proteins subjected to regulation by RGS proteins. The goal of the study was to delineate the specificity in which cardiac RGS proteins modulate $G_{q^-}$ and $G_{i/o}$-induced ERK and Akt phosphorylation. To isolate $G_{q^-}$ and $G_{i/o}$-mediated effects, we exclusively expressed muscarinic $M_2$ or $M_3$ receptors in COS-7 cells. Western blot analyses demonstrated increase of phosphorylation of ERK 1.7-/3.3-fold and Akt 2.4-/6-fold in $M_{2^-}/M_{3^-}$ expressing cells through carbachol stimulation. In co-expressions, $M_3/G_q$-induced activation of Akt was exclusively blunted through RGS3s/RGS3, whereas activation of ERK was inhibited additionally through RGS2/RGS5. $M_2/G_{i/o}$ induced Akt activation was inhibited by all RGS proteins tested. RGS2 had no effect on $M_2/G_{i/o}$-induced ERK activation. The high degree of specificity in RGS proteins-depending modulation of $G_{q^-}$ and $G_{i/o}$-mediated ERK and Akt activation in the muscarinic network cannot merely be attributed exclusively to RGS protein selectivity towards $G_q$ or $G_{i/o}$ proteins. Counter-regulatory mechanisms and inter-signaling cross-talk may alter the sensitivity of GPCR-induced ERK and Akt activation to RGS protein regulation.

• Journal of Microbiology and Biotechnology
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• 제22권2호
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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.

• Molecules and Cells
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• 제25권4호
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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.

• 대한약침학회지
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• 제6권2호
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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.