• The Korean Journal of Physiology and Pharmacology
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• 제6권3호
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• pp.165-169
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• 2002

We have previously demonstrated that phytoestrogens isolated from safflower seeds significantly attenuated bone loss in ovariectomized rats, and directly stimulated proliferation and differentiation of cultured osteoblastic cells. In an attempt to elucidate underlying cellular mechanisms, in the present study we investigated effects of $17{\beta}-estradiol\;(E_2)$ and phytoestrogens such as matairesinol and acacetin, a type of lignan and flavonoid, respectively, on activation of mitogen activated protein (MAP) kinases, extracellular signal-regulated kinase 1 (ERK1) and ERK2, in cultured osteoblastic ROS 17/2.8 cells. Western blot analysis with anti-MAP kinase antibody showed that a wide range concentrations $(10^{-14}\;to\;10^{-6}\;M)\;of\;E_2$ as well as both phytoestrogens induced rapid and transient activation of ERK1/2 through phosphorylation within minutes. Maximum activation of MAP kinases by $E_2$ and phytoestrogens were observed at 10 and 15 min, respectively. $E_2-induced$ phosphorylation of ERK1/2 returned to the control level at 30 min, whereas phytoestrogen-induced phosphorylation was maintained at high level until 30 min. PD-98059, a highly selective inhibitor of MAP kinase, prevented phosphorylation of ERK1/2 in the cells treated either with $E_2$ or phytoestrogens. To examine a possible involvement of estrogen receptor in the activation process of MAP kinase, Western blot analysis was performed in the presence and absence of the estrogen receptor antagonists, ICI 182,780 and tamoxifen. These antagonists blocked MAP kinase phosphorylation induced not only by $E_2,$ but also by the phytoestrogens. To the best our knowledge, this study is the first to demonstrate that phytoestrogens such as flavonoid and lignan extracted from safflower seeds produce a rapid activation of MAP kinase, at least partially via membrane estrogen receptor of the cultured osteoblastic cells.

• Biomolecules & Therapeutics
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• 제32권1호
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• pp.56-64
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• 2024

Biased signaling or functional selectivity refers to the ability of an agonist or receptor to selectively activate a subset of transducers such as G protein and arrestin in the case of G protein-coupled receptors (GPCRs). Although signaling through arrestin has been reported from various GPCRs, only a few studies have examined side-by-side how it differs from signaling via G protein. In this study, two signaling pathways were compared using dopamine D₂ receptor (D₂R) mutants engineered via the evolutionary tracer method to selectively transduce signals through G protein or arrestin (D₂G and D₂Arr, respectively). D₂G mediated the inhibition of cAMP production and ERK activation in the cytoplasm. D₂Arr, in contrast, mediated receptor endocytosis accompanied by arrestin ubiquitination and ERK activation in the nucleus as well as in the cytoplasm. D₂Arr-mediated ERK activation occurred in a manner dependent on arrestin3 but not arrestin2, accompanied by the nuclear translocation of arrestin3 via importin1. D₂R-mediated ERK activation, which occurred in both the cytosol and nucleus, was limited to the cytosol when cellular arrestin3 was depleted. This finding supports the results obtained with D₂Arr and D₂G. Taken together, these observations indicate that biased signal transduction pathways activate distinct downstream mechanisms and that the subcellular regions in which they occur could be different when the same effectors are involved. These findings broaden our understanding on the relation between biased receptors and the corresponding downstream signaling, which is critical for elucidating the functional roles of biased pathways.

• 한국환경성돌연변이발암원학회지
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• 제25권2호
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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.

• 동의생리병리학회지
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• 제28권2호
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• pp.195-199
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• 2014

Although the Herba Cirsii is known to posses beneficial health effects, the anti-diabetic effects and the mechanism of action have not been elucidated. In the present study we have shown that Herba Cirsii Extract (HCE) can stimulate glucose uptake in OP9 adipocytes. Unlike insulin, HCE did not stimulate the Ser473 phosphorylation and activation of Akt. The increasing effects of HCE on glucose uptake were inhibited by PD680509 and compound C pretreatment, which means that the glucose uptake effects by HCE were carried out by extracelluar signal-regulated kinase1/2(ERK1/2) and AMP-activated protein kinase (AMPK) activation. Further studies revealed that HCE stimulated glucose transport occurs through a mechanism involving ERK1/2 activation and AMPK activation.

• Journal of Photoscience
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• 제9권2호
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• pp.433-435
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• 2002

In the present study, we investigated the actions of sphingosine-I-phosphate (SPP) in Mel-Ab melanocytes. We observed the cytoprotective effect of SPP on UVB-induced cell death. Following exposure of cells to UVB, a significant protective effect was seen in cultures pretreated with SPP. Since SPP is well known as a mitogenic agent, it is possible that the mitogenic effect of SPP may contribute to cell survival. Surprisingly, we found that SPP inhibited DNA-synthesis significantly. We were next interested in the regulation of the extracellular signal-regulated protein kinase (ERK) and Akt pathways by SPP. We clearly observed that SPP potently stimulated the phosphorylation of both ERK and Akt against UVB-induced cell death. Based on these results, we conclude that SPP may show its cytoprotective effect through ERK and Akt activation.

• Journal of Microbiology and Biotechnology
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• 제26권6호
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• pp.1063-1066
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• 2016

Longan (Dimocarpus longan Lour.) has been used as a traditional oriental medicine and possesses a number of physiological activities. In this study, we used cell-based herbal extract screening to identify longan fruit extract (LFE) as an activator of osteoblast differentiation. LFE up-regulated alkaline phosphatase (ALP) activity, induced mineralization, and activated Runx2 gene expression in MC3T3-E1 cells. Furthermore, treatment of MC3T3-E1 cells with LFE promoted the phosphorylation of extracellular signal-regulated kinase1/2 (Erk1/2); however, abrogation of Erk1/2 activation with PD98059 resulted in down-regulation of the phospho-SMAD1/5/8 and Runx2 levels, which in turn reduced the ALP activity. Our findings suggest that LFE exerts its osteogenic activity through activation of the ERK signaling pathway and may have potential as an herbal therapeutic or a preventive agent for the treatment of osteoporosis.

• Journal of Microbiology and Biotechnology
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• 제15권4호
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• pp.756-766
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• 2005

The signaling mechanism underlying aburatubolactam C-induced FasL upregulation was investigated in human Jurkat T cells. After treatment with aburatubolactam C, the src-family PTKs $p56^{lck}\;and\;p59^{fyn}$, and MAP kinases ERK2 and JNK1, were activated prior to FasL upregulation; Both $p56^{lck}\;and\;p59^{fyn}$ were directly activated 2.4- and 2.2-fold, respectively, in vitro by aburatubolactam C. The aburatubolactam C-induced cellular changes, including the activation of ERK2 and INK1, and FasL upregulation, were completely prevented by the PTK inhibitor genistein. The activation of protein kinase C (PKC$\delta,\;\epsilon\;and\;\mu$ was also induced following aburatubolactam C treatment. Although the activation of $p56^{lck}$ and tyrosine phosphorylation of the cellular proteins were not blocked by the PKC inhibitor GFl09203X, the activation of ERK2 was completely abrogated, along with a detectably enhanced JNK1 activation; FasL upregulation, and apoptosis. However, the FasL upregulation and apoptosis were significantly inhibited by the PKC activator PMA, with a remarkable increase in the ERK2 activation. The cytotoxic effect of aburatubolactam C was reduced in the presence of the anti-Fas neutralizing antibody ZB-4. Although ectopic expression of Bcl-2 failed to completely block the cytotoxicity of aburatubolactam C, it was clearly suppressed. The c-Fos mRNA expression was upregulated in a biphasic manner, where the second phasic expression overlapped with the FasL upregulation. Accordingly, these results demonstrate that aburatubolactam C-induced apoptosis is exerted, at least in part, by FasL upregulation dictated by activation of the PTK ($p56^{lck}\;and\;p59^{fyn}$) /JNKI pathway, which is negatively affected by the concurrent activation of the PKC/ERK2 pathway proximal to PTK activation.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2023년도 임시총회 및 춘계학술대회
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• pp.47-47
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• 2023

Paeonia lactiflora roots (PLR) are a medicinal plant widely used for treating inflammatory diseases. However, PLR has been recently reported to increase the production of proinflammatory mediators and activates phagocytosis in macrophages. Thus, in this study, we tried to verify the macrophage activation of PLR and elucidate its mechanism of action. PLR upregulated the production of proinflammatory mediators and activated phagocytosis in RAW264.7 cells. However, these effects were reversed by inhibition of TLR2/4. In addition, the inhibition of p38, JNK, and ERK1/2 reduced the PLR-mediated production of proinflammatory mediators, and the PLR-mediated activation of p38, JNK, and ERK1/2 was blocked by the TLR4 inhibition. These findings indicate that PLR may activate macrophages through TLR4-dependent activation of p38, JNK, and ERK1/2. These indicate that PLR has immunostimulatory activity. Thus, it is believed that PLR can be used as a functional food agent that enhances the immune system.

• 한국발생생물학회지:발생과생식
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• 제24권2호
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• pp.113-123
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• 2020

Metformin has been widely used as an antidiabetic drug, and reported to inhibit cell proliferation in many cancers including non-small cell lung cancer (NSCLC). In NSCLC cells, metformin suppresses PI3K/AKT/mTOR signaling pathway, but effect of metformin on RAS/RAF/MEK/ERK signaling pathway is controversial; several studies showed the inhibition of ERK activity, while others demonstrated the activation of ERK in response to metformin exposure. Metformin-induced activation of ERK is therapeutically important, since metformin could enhance cell proliferation through RAS/RAF/MEK/ERK pathway and lead to impairment of its anticancer activity suppressing PI3K/AKT/mTOR pathway, requiring blockade of both signaling pathways for more efficient antitumor effect. The present study tested the combination therapy of metformin and trametinib by monitoring the alterations of regulatory effector proteins of cell signaling pathways and the effect of the combination on cell viability in NCI-H2087 NSCLC cells with NRAS and BRAF mutations. We show that metformin alone blocks PI3K/AKT/mTOR signaling pathway but induces the activation and phosphorylation of ERK. The combination therapy synergistically decreased cell viability in treatment with low doses of two drugs, while it gave antagonistic effect with high doses. These findings suggest that the efficacy of metformin and trametinib combination therapy may depend on the alteration of ERK activity induced by metformin and specific cellular context of cancer cells.

• IMMUNE NETWORK
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• 제7권1호
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• pp.26-30
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• 2007

Background: Mycobacterial antigens released as PIM, LM, LAM, lipoproteins and other cellular factors may contribute to macrophage and dendritic cell activation through pattern recognition receptors such as TLRs. In this study, we assessed cytokine production and ERK activation with stimulation of several major mycobacterial antigens. Methods: Purified mycobacterial antigens (10, 22, 30, 38kDa) and recombinant antigens (6, 16, 19, 38kDa, Ag85A antigen) were studied. The production of cytokines (TNF-${\alpha}$, IL-12, IL-6) was measured by ELISA. The ERK activation was detected by western blotting. The expression of TLR2 or TLR4 was measured by flow cytometry. Results: Among purified antigens only 30kDa antigen induced production of IL-6 or TNF-${\alpha}$ in THP-1 macrophage cells. When THP-1 macrophage cells were treated with 30kDa antigen, phosphorylation of ERK was detected. ERK activation also occurred in TLR2 transfectant HEK293 cells with 30kDa antigen stimulation. Conclusion: 30kDa antigen is one of the major mycobacterial antigens inducing cytokine production and MAP kinases phosphorylation in macrophages.