• Development and Reproduction
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• v.13 no.4
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• pp.291-296
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• 2009

In most larvae of ascidian, two sensory pigment cells, otolith and ocellus, lie in their brain vesicle. They also have a third type of sensory cells: hydrostatic pressure receptor (Hpr) cells. The Hpr cells were presumed to be hydrostatic pressure-detection cells, but their precise functions is still disputed. In this study, we investigated whether an FGF signaling is involved in formation of Hpr cells. When fertilized eggs were injected with Hr-FGF9/16/20 antisense MO, the resulting larvae showed severe abnormalities with no expression of the Hpr cell-specific Hpr-1 antigen. Similar results were obtained using an FGF receptor inhibitor, SU5402, and an MEK inhibitor, U0126. Embryos treated with SU5402 or U0126 during the 32-cell and hatching stages did not express the Hpr-1 antigen. To elucidate the temporal requirement for the FGF signaling in formation of Hpr cells, embryos were treated with SU5402 for 2 h, or U0126 for 20 min during various embryonic stages. Larvae treated with SU5402 from the 16-cell stage to the 64-cell stage did not express the Hpr-1 antigen, whereas those treated at the early gastrula stage expressed the Hpr-1 antigen. When U0126 treatment was carried out at various stages between the 8-cell and late gastrula stages, larvae scarcely formed the Hpr cells. They showed expression of the Hpr-1 antigen when embryos were placed in U0126 just before the neural plate stage. These results suggest that FGF9/16/20 signaling is involved in formation of Hpr cells from the primary neural induction stage to the late gastrula stage.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.11 no.6
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• pp.2124-2128
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• 2010

In this study, we determined effects of the mitogen-activated protein kinase (MAPK) inhibitor, U0126 on meiotic maturation, microtubule organization and actin filament assembly in the porcine oocyte. The phosphorylated MAPK was first detected at 12 h after the initiation of maturation cultures, fully activated at 24h, and remained until metaphase II. Treatment of germinal vesicle (GV) stage oocytes with $20{\mu}M$ U0126 completely blocked MAPK phosphorylation, but germinal vesicle breakdown (GVBD) was normally proceeded. However, the oocytes didn‘t progress to the metaphase I. The inhibition of MAPK resulted in abnormal spindles. In oocytes treated with U0126 after GVBD, polar body extrusion was normal, but the organization of the metaphase plate and chromosome segregation were abnormal. In conclusion, MAPK activity plays an important regulatory role in GV chromatin configuration and meiotic progress in porcine oocyte maturation.

• Animal cells and systems
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• v.7 no.3
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• pp.221-225
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• 2003

The tadpole larva of the ascidian Halocynthia roretzi has two sensory pigment cells in its brain vesicle. To elucidate the temporal requirement for FGF signaling in formation of the pigment cells, embryos were treated with an FGF receptor 1 inhibitor, SU5402, or an MEK inhibitor, U0126 during various embryonic stages. In the present study, it is shown that the embryos treated with SU5402 from the 16-cell stage to the early gastrula stage do not form pigment cells, whereas those treated after the early gastrula stage form pigment cells. In pigment cell formation, embryos suddenly exhibited the sensitivity to SU5402 only for 1 h at the neural plate stage(-4 h after the beginning of gastrulation). When U0126 treatment was carried out at various stages between the 8-cell and late neurula stages, the embryos scarcely formed pigment cells. Pigment cell formation occurred when the embryos were placed in U0126 at early tail bud stage. These results indicate that FGF signaling is involved in pigment cell formation at two separate processes during ascidian embryogenesis, whereas more prolonged period is required for MEK signaling.

• Molecules and Cells
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• v.23 no.2
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• pp.138-144
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• 2007

Previously we showed that the human GM3 synthase gene was expressed during the induction of megakaryocytic differentiation in human leukemia K562 cells by phorbol 12-myristate 13-acetate (PMA). In this study we found that treatment of PMA-induced K562 cells with $G{\ddot{o}}6976$, a specific inhibitor of PKC, and U0126, an inhibitor of the extracellular signal-regulated kinase (ERK) reduced expression of GM3 synthase, whereas wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K) did not. Moreover, activation of ERK and cAMP response element binding protein (CREB) was prevented by pretreatment with $G{\ddot{o}}6976$ and U0126. PMA stimulated the promoter activity of the 5'-flanking region from -177 to -83 region of the GM3 synthase gene, and mutation or deletion of a CREB site located around -143 of the promoter reduced PMA-stimulated promoter activity, as did the inhibitors $G{\ddot{o}}6976$ and U0126. Our results demonstrate that induction of GM3 synthase during megakaryocytic differentiation in PMA-stimulated human leukemia K562 cells depends upon the PKC/ERK/CREB pathway.

• Nutrition Research and Practice
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• v.4 no.4
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• pp.276-282
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• 2010

The principal objective of this study was to evaluate the chemopreventive and therapeutic effects of a combination of all-trans-retinoic acid (RA) and knockdown of delta-like 1 homologue (Drosophila) (DLK1) on neuroblastoma, the most common malignant disease in children. As unfavorable neuroblastoma is poorly differentiated, neuroblastoma cell was induced differentiation by RA or DLK1 knockdown. Neuroblastoma cells showed elongated neurite growth, a hallmark of neuronal differentiation at various doses of RA, as well as by DLK1 knockdown. In order to determine whether or not a combination of RA and DLK1 knockdown exerts a greater chemotherapeutic effect on neuroblastoma, cells were incubated at 10 nM RA after being transfected with SiRNA-DLK1. Neuronal differentiation was increased more by a combination of RA and DLK1 knockdown than by single treatment. Additionally, in order to assess the signal pathway of neuroblastoma differentiation induced by RA and DLK1 knockdown, treatment with the specific MEK/ERK inhibitors, U0126 and PD 98059, was applied to differentiated neuroblastoma cells. Differentiation induced by RA and DLK1 knockdown increased ERK phosphorylation. The MEK/ERK inhibitor U0126 completely inhibited neuronal differentiation induced by both RA and DLK1 knockdown, whereas PD98059 partially blocked neuronal differentiation. After the withdrawal of inhibitors, cellular differentiation was fully recovered. This study is, to the best of our knowledge, the first to demonstrate that the specific inhibitors of the MEK/ERK pathway, U0126 and PD98059, exert differential effects on the ERK phosphorylation induced by RA or DLK1 knockdown. Based on the observations of this study, it can be concluded that a combination of RA and DLK1 knockdown increases neuronal differentiation for the control of the malignant growth of human neuroblastomas, and also that both MEK1 and MEK2 are required for the differentiation induced by RA and DLK1 knockdown.

• The Korean Journal of Physiology and Pharmacology
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• v.21 no.3
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• pp.301-308
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• 2017

27-Hydroxycholesterol induces differentiation of monocytic cells into mature dendritic cells, mDCs. In the current study we sought to determine roles of the PI3K and the ERK pathways in the 27OHChol-induced differentiation. Up-regulation of mDC-specific markers like CD80, CD83 and CD88 induced by stimulation with 27OHChol was significantly reduced in the presence of LY294002, an inhibitor of PI3K, and U0126, an inhibitor of ERK. Surface expression of MHC class I and II molecules elevated by 27OHChol was decreased to basal levels in the presence of the inhibitors. Treatment with LY294002 or U0126 resulted in recovery of endocytic activity which was reduced by 27OHChol. CD197 expression and cell adherence enhanced by 27OHChol were attenuated in the presence of the inhibitors. Transcription and surface expression of CD molecules involved in atherosclerosis such as CD105, CD137 and CD166 were also significantly decreased by treatment with LY294002 and U0126. These results mean that the PI3K and the ERK signaling pathways are necessary for differentiation of monocytic cells into mDCs and involved in over-expression of atherosclerosis-associated molecules in response to 27OHChol.

• The Journal of Internal Korean Medicine
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• v.25 no.4
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• pp.75-85
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• 2004

목적 : 黃芩(황금)과 黃芩(황금)의 주요 flavonoid 성분인 baicalein이 신장세뇨관 상피세포에서 산화제에 의한 apoptosis에 미치는 효과를 살펴보고자 한다. 방법 : 신장세뇨관 상피세포주인 opossum kidney (OK) 세포를 유기산화제인 t-butylhydroperoxide (tBHP)에 노출시켜 apoptosis를 일으킨 후 관련된 변화를 관찰하였다. 결과 : tBHP는 농도에 의존하여 apoptosis를 유발시켰는데, 이러한 효과는 黃芩(황금)과 baicalein에 의해 농도 의존적으로 방지 되었다. tBHP에 의한 OK 세포사는 항산화제인 Trolox와 N-acetylcysteine에 의해 방지 되었다. tBHP는 mitogen-activated protein kinase의 subfamily인 extracellular signal-regulated kinase (ERK)를 활성화시켰다. ERK 억제제인 PD98059와 U0126은 tBHP에 의한 세포 사망을 방지하였다. tBHP에 의한 ERK 활성화는 U0126에 의해 억제되었으나 黃芩(황금)과 baicalein에 의해서는 영향을 받지 않았다. 철착염제인 deferoxamine은 tBHP에 의한 세포 사망과 ERK 활성화를 방지하였다. tBHP에 의한 세포 사망은 casopase 억제제인 BOD-U-FMK와 zDEVD-FMK에 의해 방지되었다. 결론 : 黃芩(황금)은 산화제에 의한 세포 사망을 방지하는데, 이는 kinase 억제, 항산화제 역할 및 철착염제의 작용에 기인하지 않았다. 黃芩(황금)의 이러한 효과는 산화제에 의관 신부전 예방 및 치료제로 개발하는데 이용될 수 있는 가능성을 보였다.

• Tuberculosis and Respiratory Diseases
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• v.65 no.4
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• pp.343-350
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• 2008

PD-L1 is expressed in a variety of antigen-presenting cells and provides T cell tolerance via ligation with its receptor PD-1 and B7-1 on T cells. Stimulation with lipopolysaccharide (LPS) can increase the level of PD-L1 expression in B cells and macrophages, which suggests that this molecule plays a role in the immunosuppression observed in severe sepsis. The aim of this study was to identify which of the downstream pathways of TLR4 are involved in the up-regulation of PD-L1 by LPS in macrophages. Flow cytometry was used to examine the expression of PD-L1 in RAW 264.7 macrophages stimulated with LPS. The following chemical inhibitors were used to evaluate the role of each pathway: LY294002 for PI3K/Akt, SB202190 for p38 MAPK, and U0126 for MEK. LPS induced the expression of PD-L1 in a time- and dose-dependent manner. Transfection of siRNA for TLR4 suppressed the induction of PD-L1. Pretreatment with LY294002 and SB202190 decreased the level of PD-L1 expression but U0126 did not. Overall, the PI3K/Akt and p38 MAPK pathways are involved in the up-regulation of PD-L1 expression in RAW 264.7 macrophages stimulated with LPS.

• Journal of Yeungnam Medical Science
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• v.30 no.1
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• pp.10-16
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• 2013

Background: Toll-like receptors (TLRs) are well-known pattern recognition receptors. Among the 13 TLRs, TLR2 is the most known receptor for immune response. It activates mitogen-activated protein kinases (MAPKs), which are counterbalanced by MAPK phosphatases [MKPs or dual-specificity phosphatases (DUSPs)]. However, the regulatory mechanism of DUSPs is still unclear. In this study, the effect of a TLR2 ligand (TLR2L, Pam3CSK4) on DUSP4 expression in Raw264.7 cells was demonstrated. Methods: A Raw264.7 mouse macrophage cell line was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% antibiotics (100 U/mL penicillin and 100 g/mL streptomycin) at $37^{\circ}C$ in 5% $CO_2$. TLR2L (Pam3CSK4)-mediated DUSP4 expressions were confirmed with RT-PCR and western blot analysis. In addition, the detection of reactive oxygen species (ROS) was measured with lucigenin assay. Results: Pam3CSK4 induced the expression of DUSP1, 2, 4, 5 and 16. The DUSP4 expression was also increased by TLR4 and 9 agonists (lipopolysaccharide and CpG ODN, respectively). Pam3CSK4 also induced ERK1/2 phosphorylation and ROS production, and the Pam3CSK4-induced DUSP4 expression was decreased by ERK1/2 (U0126) and ROS (DPI) inhibitors. U0126 suppressed the ROS production by Pam3CSK4. Conclusion: Pam3CSK4-mediated DUSP4 expression is regulated by ERK1/2 and ROS. This finding suggests the physiological importance of DUSP4 in TLR2-mediated immune response.

• The Korean Journal of Physiology and Pharmacology
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• v.8 no.4
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• pp.227-235
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• 2004

The aim of the present study was to examine the effect of leptin on CA release from the isolated perfused model of the rat adrenal gland, and to establish its mechanism of action. Leptin $(1{\sim}100\;ng/ml)$, when perfused into an adrenal vein of the rat adrenal gland for 60 min, enhanced a dose-dependently the secretory responses of CA evoked by ACh $(5.32{\times}10^{-3}\;M)$, DMPP $(10^{-4}\;M)$ and McN-A-343 $(10^{-4}\;M)$, although it alone has weak effect on CA secretion. However, it did not affect the CA secretion evoked by excess $K^+\;(5.6{\times}10^{-2}\;M)$. Leptin alone produced a weak secretory response of the CA. Moreover, leptin (10 ng/ml) in to an adrenal vein for 60 min also augmented the CA release evoked by BAY-K-8644, an activator of the dihydropyridine L-type $Ca^{2+}$ channels, and cyclopiazonic acid, an inhibitor of cytoplasmic $Ca^{2+}$ ATPase. However, in the presence of U0126 $(1\;{\mu}M)$, an inhibitor of mitogen-activated protein kinase (MAPK), leptin no longer enhanced the CA secretion evoked by ACh and DMPP. Furthermore, in the presence of anti-leptin (10 ng/ml), an antagonist of Ob receptor, leptin (10 ng/ml) also no longer potentiated the CA secretory responses evoked by DMPP and Bay-K-8644. Collectively, these experimental results suggest that leptin enhances the CA secretion from the rat adrenal medulla evoked by cholinergic stimulation (both nicotininc and muscarinic receptors), but does not that by membrane depolarization. It seems that this enhanced effect of leptin may be mediated by activation of U0126-sensitive MAPK through the leptin receptors, which is probably relevant to the activation of the dihydropyridine L-type $Ca^{2+}$ channels located on the rat adrenomedullary chromaffin cells.