• Journal of Microbiology and Biotechnology
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• v.26 no.6
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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.

• Molecules and Cells
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• v.40 no.5
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• pp.371-377
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• 2017

Despite the importance of the receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-RANK signaling mechanisms on osteoclast differentiation, little has been studied on how RANK expression is regulated or what regulates its expression during osteoclastogenesis. We show here that insulin signaling increases RANK expression, thus enhancing osteoclast differentiation by RANKL. Insulin stimulation induced RANK gene expression in time- and dose-dependent manners and insulin receptor shRNA completely abolished RANK expression induced by insulin in bone marrow-derived monocyte/macrophage cells (BMMs). Moreover, the addition of insulin in the presence of RANKL promoted RANK expression. The ability of insulin to regulate RANK expression depends on extracellular signal-regulated kinase 1/2 (ERK1/2) since only PD98059, an ERK1/2 inhibitor, specifically inhibited its expression by insulin. However, the RANK expression by RANKL was blocked by all three mitogen-activated protein (MAP) kinases inhibitors. The activation of RANK increased differentiation of BMMs into tartrate-resistant acid phosphatase-positive ($TRAP^+$) osteoclasts as well as the expression of dendritic cell-specific transmembrane protein (DC-STAMP) and d2 isoform of vacuolar ($H^+$) ATPase (v-ATPase) Vo domain (Atp6v0d2), genes critical for osteoclastic cell-cell fusion. Collectively, these results suggest that insulin induces RANK expression via ERK1/2, which contributes to the enhancement of osteoclast differentiation.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2003.11a
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• pp.83-83
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• 2003

In our previous study, glucose deprivation was reported to induce the potentiated death and ATP loss in immunostimulated astroglia. And this vulnerability to glucose deprivation was due to overproduction of nitric oxide (NO) and hydrogen peroxide (H$_2$O$_2$). In the present study, the role of extracellular signal-regulated kinase 1/2 (ERK1/2) in the glucose deprivation-induced death of immunostimulated astroglia was examined. We showed that immunostimulation with LPS+IFN-ν activated the ERKl/2 signal pathway and produced a large amount of NO and H$_2$O$_2$. Generation of NO and H$_2$O$_2$ in immunostimulated astroglia was mediated via ERK1/2 signal pathways, since addition of the ERK kinase (MEKl) inhibitor PD98059 reduced NO and H$_2$O$_2$production. ERK1/2 activation-mediated NO and H$_2$O$_2$ production is due to an activation of iNOS and NADPH oxidase, respectively. Finally, we found that glucose deprivation caused ATP depletion and the augmented death in immunostimulated astroglia, which was also prevented by PD98059 treatment. These results demonstrate that the ERK1/2 signal pathways play an important role in glucose deprivation induced the death in immunostimulated astroglia by regulating the generation of NO and H$_2$O$_2$.

• Molecules and Cells
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• v.23 no.1
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• pp.30-38
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• 2007

Dipyrithione (2, 2'-dithiobispyridine-1, 1'-dioxide, PTS2), a pyrithione derivate, is highly bactericidal and fungicidal. In this study we examined its apoptotic effect on HeLa cells. PTS2 induced HeLa cell death in a dose and time dependent manner. ERK1/2 and p38 were markedly activated, but little JNK1/2 activation was detected. Suppression of p38 activation by SB203580 reduced the extent of apoptosis of the HeLa cells and also prevented induction of p21, release of cytochrome c, and cleavage of caspase-3 and PARP. Inhibition of ERK1/2 with PD98059 increased apoptosis, indicating that ERK1/2 activation has an anti-apoptotic effect on PTS2-induced HeLa cell apoptosis. PTS2 also inhibited murine sarcoma 180 and hepatoma 22 tumor growth in an animal tumor model. Our findings indicate that PTS2 possesses anti-tumor activity, that caspase-3 and poly (ADP-ribose) polymerase (PARP) are involved in PTS2-induced HeLa cell apoptosis and that ERK1/2 and p38 have opposing effects on this apoptosis.

• Molecules and Cells
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• v.40 no.7
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• pp.457-465
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• 2017

Streptozotocin (STZ)-induced murine models of type 1 diabetes have been used to examine ER stress during pancreatic ${\beta}$-cell apoptosis, as this ER stress plays important roles in the pathogenesis and development of the disease. However, the mechanisms linking type 1 diabetes to the ER stress-modulating anti-diabetic signaling pathway remain to be addressed, though it was recently established that ERK5 (Extracellular-signal-regulated kinase 5) contributes to the pathogeneses of diabetic complications. This study was undertaken to explore the mechanism whereby ERK5 inhibition instigates pancreatic ${\beta}$-cell apoptosis via an ER stress-dependent signaling pathway. STZ-induced diabetic WT and CHOP deficient mice were i.p. injected every 2 days for 6 days under BIX02189 (a specific ERK5 inhibitor) treatment in order to evaluate the role of ERK5. Hyperglycemia was exacerbated by co-treating C57BL/6J mice with STZ and BIX02189 as compared with mice administered with STZ alone. In addition, immunoblotting data revealed that ERK5 inhibition activated the unfolded protein response pathway accompanying apoptotic events, such as, PARP-1 and caspase-3 cleavage. Interestingly, ERK5 inhibition-induced exacerbation of pancreatic ${\beta}$-cell apoptosis was inhibited in CHOP deficient mice. Moreover, transduction of adenovirus encoding an active mutant form of $MEK5{\alpha}$, an upstream kinase of ERK5, inhibited STZ-induced unfolded protein responses and ${\beta}$-cell apoptosis. These results suggest that ERK5 protects against STZ-induced pancreatic ${\beta}$-cell apoptosis and hyperglycemia by interrupting the ER stress-mediated apoptotic pathway.

• Molecules and Cells
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• v.41 no.5
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• pp.436-443
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• 2018

The actin cytoskeleton plays a key role in the entry of mitosis as well as in cytokinesis. In a previous study, we showed that actin disruption delays mitotic entry at G2/M by sustained activation of extracellular signal-related kinase 1/2 (ERK1/2) in primary cells but not in transformed cancer cell lines. Here, we examined the mechanism of cell cycle delay at G2/M by actin dysfunction in IMR-90 normal human fibroblasts. We observed that de-polymerization of actin with cytochalasin D (CD) constitutively activated ribosomal S6 kinase (RSK) and induced inhibitory phosphorylation of Cdc2 (Tyr 15) in IMR-90 cells. In the presence of an actin defect in IMR-90 cells, activating phosphorylation of Wee1 kinase (Ser 642) and inhibitory phosphorylation of Cdc25C (Ser 216) was also maintained. However, when kinase-dead RSK (DN-RSK) was overexpressed, we observed sustained activation of ERK1/2, but no delay in the G2/M transition, demonstrating that RSK functions downstream of ERK in cell cycle delay by actin dysfunction. In DN-RSK overexpressing IMR-90 cells treated with CD, phosphorylation of Cdc25C (Ser 216) was blocked and phosphorylation of Cdc2 (Tyr 15) was decreased, but the phosphorylation of Wee1 (Ser 642) was maintained, demonstrating that RSK directly controls phosphorylation of Cdc25C (Ser 216), but not the activity of Wee1. These results strongly suggest that actin dysfunction in primary cells activates ERK1/2 to inhibit Cdc2, delaying the cell cycle at G2/M by activating downstream RSK, which phosphorylates and blocks Cdc25C, and by directly activating Wee1.

• Biomolecules & Therapeutics
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• v.32 no.1
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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.

• Journal of Life Science
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• v.13 no.6
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• pp.849-855
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• 2003

Nitric oxide (NO) plays an important role as a signaling molecule in the proliferation of placenta trophoblasts. In this study, we investigated the effect of NO on the activation of phospholipase C (PLC) in BeWo cells, choriocar-cinoma cell line. Sodium nitroprusside (SNP), an agent to produce NO spontaneously in cells, alone increased $［^3H］$ thymidine incorporation of BeWo cells, indicating NO stimulates proliferation of the cells. NO-induced proliferation of BeWo cells was blocked by U73122, an inhibitor of PLC, suggesting that NO-induced PLC activation is involved in the cell proliferation. NO also stimulated extracellular signal-regulated kinase (ERK) in BeWo cells, indicated by increased phosphorylation of ERK1/2 in Western blotting using anti-phospho-ERK1/2 antibody. NO-induced phos-phorylation of ERK1/2 was not abrogated by U73122. $PLC\gamma_1$l but not$PLC\gamma_2$ was tyrosine phosphorylated by SNP in immunoprecipitation assay using anti-$PLC\gamma_1$/$PLC\gamma_2$ antibodies, and SNP-induced phosphorylation of $PLC\gamma_1$ was abrogated by pre-treatment of cells with genistein and PD98059, indicating that NO induced-phosphorylation of $PLC\gamma_1$ is mediated by ERK. These results suggest that NO stimulates the proliferation of BeWo cells through ERK and $PLC\gamma_1$.

• The Korean Journal of Physiology and Pharmacology
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• v.6 no.3
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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.

• BMB Reports
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• v.41 no.11
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• pp.803-807
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• 2008

We investigated the expression of muscarinic acetylcholine receptors (mAChRs) and their possible involvement in the regulation of cell proliferation in four colon cancer cell lines (SNU-61, SNU-81, SNU-407, and SNU-1033) derived from Korean colon carcinoma patients. A ligand binding assay showed that all four cell lines expressed mAChRs. Treatment of the four cell lines with the cholinergic agonist carbachol led to the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). In SNU-407 cells, carbachol significantly stimulated cell proliferation, which could be abolished by the muscarinic antagonist atropine and the ERK1/2 kinase inhibitor PD98059. These results indicate that mAChRs specifically mediate the proliferation of SNU-407 colon cancer cells via the ERK1/2 pathway.