• Molecular & Cellular Toxicology
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• v.4 no.3
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• pp.224-229
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• 2008

Eucommia ulmoides (Duchung) is commonly used for treatment of diabetes in Korean traditional medicine. However, the exact mechanism of its anti-diabetic effect has not yet been fully elucidated. In this study, the effect of E. ulmoides extract on glucose uptake was investigated in L6 rat skeletal muscle cells. E. ulmoides extract stimulated the activity of phosphatidylinositol (PI) 3-kinase that is a major regulatory molecule in glucose uptake pathway. Protein kinase B (PKB) and protein kinase C-${\xi}$ (PKC-${\xi}$), downstream mediators of PI 3-kinase, were also activated by E. ulmoides extract. We assessed the activity of AMP-activated protein kinase (AMPK), another regulatory molecule in glucose uptake pathway. Phosphorylation level of AMPK did not change with treatment of E. ulmoides extract. Phosphorylations of p38 mitogen activated protein kinase (p38 MAPK) and acetyl-CoA carboxylase (ACC), downstream mediators of AMPK, were not significantly different. Taken together, our results suggest that E. ulmoides may stimulate glucose uptake through PI 3-kinase but not AMPK in L6 skeletal muscle cells.

• Molecules and Cells
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• v.28 no.6
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• pp.589-593
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• 2009

In this study, the roles of the p38 MAPK, ERK1/2 and JNK signaling pathway in IGF-I-induced AR induction and activation were examined. C2C12 cells were treated with IGF-I in the absence or presence of various inhibitors of p38 MAPK (SB203580), ERK1/2 (PD98059), and JNK (SP600125). Inhibition of the MAPK pathway with SB203580, PD98059, or SP600125 significantly decreased IGF-I-induced AR phosphorylation and total AR protein expression. IGF-I-induced nuclear fraction of total AR and phosphorylated AR were significantly inhibited by SB203580, PD98059, or SP600125. Furthermore, IGF-I-induced AR mRNA and skeletal ${\alpha}-actin$ mRNA were blocked by those inhibitors in dose-dependent manner. Confocal images showed that IGF-I-induced AR nuclear translocation from cytosol was significantly blocked by SB203580, PD98059, or SP600125, suggesting that the MAPK pathway regulates IGF-I-induced AR nuclear localization in skeletal muscle cells. The present results suggest that the MAPK pathways are required for the ligand-independent activation of AR by IGF-I in C2C12 skeletal muscle cells.

• Animal Bioscience
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• v.36 no.6
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• pp.851-860
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• 2023

Objective: This study aims to evaluate the target genes of gga-miR-20a-5p and the regulated immune responses in the chicken macrophage cell line, HD11, by the exosome-mediated delivery of miR-20a-5p. Methods: Exosomes were purified from the chicken macrophage cell line HD11. Then, mimic gga-miR-20p or negative control miRNA were internalized into HD11 exosomes. HD11 cells were transfected with gga-miR-20a-5p or negative control miRNA containing exosomes. After 44 h of transfection, cells were incubated with or without 5 ㎍/mL poly(I:C) for 4 h. Then, expression of target genes and cytokines was evaluated by quantitative realtime polymerase chain reaction. Results: Using a luciferase reporter assay, we identified that gga-miR-20a-5p directly targeted interferon gamma receptor 2 (IFNGR2), mitogen-activated protein kinase 1 (MAPK1), mitogen-activated protein kinase kinase kinase 5 (MAP3K5), and mitogen-activated protein kinase kinase kinase 14 (MAP3K14). Moreover, the exosome-mediated delivery of gga-miR-20a-5p successfully repressed the expression of IFNGR2, MAPK1, MAP3K5, and MAP3K14 in HD11 cells. The expressions of interferon-stimulated genes (MX dynamin like GTPase 1 [MX1], eukaryotic translation initiation factor 2A [EIF2A], and oligoadenylate synthase-like [OASL]) and proinflammatory cytokines (interferon-gamma [IFNG], interleukin-1 beta [IL1B], and tumor necrosis factor-alpha [TNFA]) were also downregulated by exosomal miR-20a-5p. In addition, the proliferation of HD11 cells was increased by exosomal miR-20a-5p. Conclusion: The exosome-mediated delivery of gga-miR-20a-5p regulated immune responses by controlling the MAPK and apoptotic signaling pathways. Furthermore, we expected that exosomal miR-20a-5p could maintain immune homeostasis against highly pathogenic avian influenza virus H5N1 infection by regulating the expression of proinflammatory cytokines and cell death.

• The Korea Journal of Herbology
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• v.22 no.2
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• pp.147-153
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• 2007

Objectives : Sophora flavescens (SF) is widely used in traditional herbal medicine in Korea and is well recognized for its anti-inflammatory effect. However, its effect on Tumornecrosis factor alpha (Tnf) production in BV2 microglial cell is not yet known. Methods : We investigated the effect of SF on the production and expression of Tnf, a well known inflammatory mediator, in lipopolysaccaride (LPS)-activated BV2 microglial cells. Results : The LPS-induced Tnf production was markedly reduced by treatment with SF (50 ${\mu}g/ml$). In reverse transcription polymerase chain reaction (RT-PCR) analysis, SF suppressed the LPS activated expression of Tnf mRNA. In addition, Western blot analysis confirmed that SF suppressed the expression of Tnf. Sophora flavescens also inhibited the LPS-induced phosphylation of extracellular signal-regulated kinases (ERK), which mediate the Tnfproduction signaling pathway whereas LPS-induced phosphylation of p38 mitogen activated protein kinase (p38 MAPK), and c-Jun NH2-terminal kinases (JNK) was not inhibited by SF, which implies that SF suppresses LPS-induced Tnf production via the ERK mediated pathway. Conclusion : Taken together, these findings indicated that SF inhibits LPS-induce Tnf production, and that this inhibitory effect is mediated via the ERK pathway.

• Journal of the korean academy of Pediatric Dentistry
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• v.29 no.3
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• pp.337-344
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• 2002

Runx2, a Runt-related osteoblast-specific transcription factor, is essential for osteoblast differentiation and function. Runx2 was identified as a key regulator of osteoblast-specific gene expression through its binding to the OSE2 element present in these genes. However, little is known about the signaling mechanism regulating Runx2 activity. This study examines the role of protein kinase C (PKC) pathway and mitogen-activated protein kinase (MAPK) pathway in regulating Runx2 and bone marker genes (osteopontin; OP, osteocalcin; OC). Luciferase assay and Northern blot analysis suggested that the stimulation of PKC by PMA increased transcription activity of Runx2 and bone marker genes (OP and OC) and also increased expression of Runx2. The stimulation of MAPK by okadaic acid increased transcription activity of Runx2 and bone marker genes (OP and OC). Pretreatment with PD98059 (Erk pathway inhibitor) and SB203580 (P38 pathway inhibitor) prior to PMA treatment decreased PMA stimulated Runx2 activity. Together these results indicate that both PKC and MAPKs are involved in the regulation of Runx2 activity and also the stimulation of Runx2 transcriptional activity by the PKC pathway is through activation of MAPK pathway.

• Journal of Photoscience
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• v.9 no.2
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• pp.25-28
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• 2002

The chicken pineal gland has been used for studies on the circadian clock, because it retains an intracellular phototransduction pathway regulating the phase of the intrinsic clock oscillator. Previously, we identified chicken clock genes expressed in the gland (cPer2, cPer3, cBmal1, cBmal2, cCry1, cCry2, and cClock), and showed that a cBMALl/2-cCLOCK heteromer acts as a regulator transactivating cPer2 gene through the CACGTG E-box element found in its promoter. Notably, mRNA expression of cPer2 gene is up-regulated by light as well as is driven by the circadian clock, implying that light-dependent clock resetting may involve the up-regulation of cPer2 gene. To explore the mechanism of light-dependent gene expression unidentified in animals, we first focused on pinopsin gene whose mRNA level is also up-regulated by light. A pinopsin promoter was isolated and analyzed by transcriptional assays using cultured chicken pineal cells, resulting in identification of an 18-bp light-responsive element that includes a CACGTG E-box sequence. We also investigated a role of mitogen-activated protein kinase (MAPK) in the clock resetting, especially in the E-box-dependent transcriptional regulation, because MAPK is phospholylated (activated) in a circadian manner and is rapidly dephosphorylated by light in the gland. Both pulldown analysis and kinase assay revealed that MAPK directly associates with BMAL1 to phosphorylate it at several Ser/Thr residues. Transcriptional analyses implied that the MAPK-mediated phosphorylation may negatively regulate the BMAL-CLOCK-dependent transactivation through the E-box. These results suggest that the CACGTG E-box serves not only as a clock-controlled element but also as a light-responsive element.

• BMB Reports
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• v.35 no.4
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• pp.371-376
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• 2002

The receptor activator of nuclear factor kappa B (RANK) is a member of the tumor necrosis factor (TNF) receptor superfamily. It plays a critical role in osteoclast differentiaion, lymph node organogenesis, and mammary gland development. The stimulation of RANK causes the activation of transcription factors NF-${\kappa}B$ and activator protein 1 (AP1), and the mitogen activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK). In the signal transduction of RANK, the recruitment of the adaptor molecules, TNF receptor-associated factors (TRAFs), is and initial cytoplasmic event. Recently, the association of the MAPK kinase kinase, transforming growth factor-$\beta$-activated kinase 1 (TAK1), with TRAF6 was shown to mediate the IL-1 signaling to NF-${\kappa}B$ and JNK. We investigated whether or not TAK1 plays a role in RANK signaling. A dominant-negative form of TAK1 was discovered to abolish the RANK-induced activation of AP1 and JNK. The AP1 activation by TRAF2, TRAF5, and TRAF6 was also greatly suppressed by the dominant-negative TAK1. the inhibitory effect of the TAK1 mutant on RANK-and TRAF-induced NF-${\kappa}B$ activation was also observed, but less efficiently. Our findings indicate that TAK1 is involved in the MAPK cascade and NF-${\kappa}B$ pathway that is activated by RANK.

• BMB Reports
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• v.57 no.5
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• pp.238-243
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• 2024

Bone marrow-derived mesenchymal stem cells (BM-MSCs) can differentiate into endothelial cells in an inflammatory microenvironment. However, the regulatory mechanisms underlying this process are not entirely understood. Here, we found that TIE2 in BM-MSCs was upregulated at the transcriptional level after stimulation with tumor necrosis factor-alpha (TNFα), a major pro-inflammatory cytokine. Additionally, the STAT-binding sequence within the proximal region of TIE2 was necessary for TNFα-induced TIE2 promoter activation. TIE2 and STAT3 knockdown reduced TNFα-induced endothelial tube formation in BM-MSCs. Among the major TNFα-activated MAP kinases (ERK1/2, JNK1/2, and p38 MAPK) in BM-MSCs, only inhibition of the p38 kinase abrogated TNFα-induced TIE2 upregulation by inhibiting the JAK-STAT signaling pathway. These findings suggest that p38 MAP contributes to the endothelial differentiation of BM-MSCs by activating the JAK-STAT-TIE2 signaling axis in the inflammatory microenvironment.

• Journal of Acupuncture Research
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• v.18 no.4
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• pp.101-115
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• 2001

Objective : To characterize the antitumorigenic potential of Apamin, one of the major components of bee venom, its effects on cell proliferation and the mitogen-activated protein kinase (MAPK) signal transduction pathway were characterized using the human melanoma cell line SK-MEL-2. Methods & Results : Cell counting analysis for cell death demonstrated that consistent with a previous results, SK-MEL-2 cells treated with $0.5-2.0{\mu}g/ml$ of Apamin showed no recognizable cytotoxic effect whereas detectable induction of cell death was identified at concentrations over $5.0{\mu}g/ml$. [3H]thymidine incorporation assay for cell proliferation demonstrated that DNA replication of SK-MEL-2 cells is inhibited by Apamin in a dose- and time-dependent manner. To explore whether Apamin-induced growth suppression is associated with the MAPK signaling pathway, phosphorylation of Erk, a function mediator of MAPK growth-stimulating signal, was examined Western blot assay using a phospho-specific Erkl/2 antibody. A significant increase of Erkl/2 phosphorylation level was observed in Apamin-treated cells compared with untreated control cells. Qantitative RT-PCR analysis revealed that Apamin inhibit expression of MAPK downstream genes such as c-Jun, c-Fos, and cyclin D1 but not expression of MAPK pathway component genes including Ha-Ras, c-Raf-1, MEK1, and Erk. Conclusion : It is strongly suggested that the antitumorigenic activity of Apamin might result in part from its inhibitory effect on the MAPK signaling pathway in human melanoma cells SK-MEL-2.

• Journal of Nutrition and Health
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• v.53 no.5
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• pp.452-463
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• 2020

Purpose: Aster tataricus (AT) is one of the Asteraceae perennial herbs used in traditional Chinese medicine. The herb contains various bioactive substances, such as flavonoids, isoflavonoids, and phenolic compounds in the roots, and exhibits a range of effects including anti-bacterial, anti-oxidant, and anti-inflammatory activities. This study compared the immunomodulatory effects of ethanol and water extracts of whole AT, except the roots, and analyzed the molecular mechanisms for the regulatory effects on cytokine secretion from THP-1 cells. Methods: The effects of AT extract on the cell viability and proliferation of THP-1 cells were analyzed using the Cell Counting Kit-8 method. The concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the cell culture supernatant of the AT-treated THP-1 cells were measured using an enzyme-linked immunosorbent assay. The protein levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), inhibitor of nuclear factor kappa B (IκBα), and mitogen-activated protein kinase (MAPK) phosphorylation in the cell lysates were determined by western blotting. Results: The water extract and the ethanol extract of AT did not affect the cell viability, and increased the proliferation of THP-1 cells significantly compared to the vehicle. The water extract increased the secretion of IL-1β from THP-1 cells in a dose-dependent manner, but the ethanol extract had no effect. The expression of COX-2 and iNOS protein and the phosphorylation of MAPK and Akt were induced in AT-treated cells. In addition, IκBα was degraded by AT in a concentration-dependent manner. IL-1β secretion by AT was reduced by extracellular-signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) inhibitors, while TNF-α secretion was decreased by inhibitors of ERK, p38 MAPK, and JNK. Interestingly, the p38 MAPK inhibitor increased the production of IL-1β by AT further. Conclusion: The water extract of the above-ground parts of AT contains immunomodulatory bioactive substances that stimulate immune cells through the MAPK signaling pathway.