• The Korean Journal of Physiology and Pharmacology
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• 제3권1호
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• pp.11-18
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• 1999

Nuclear factor $_{\kappa}B\;(NF-_{\kappa}B)$ activation is modulated by various protein kinases. Activation of $NF-_{\kappa}B$ is known to be important in the regulation of cell viability. The present study investigated the effect of inhibitors of protein tyrosine kinase (PTK), protein kinase C (PKC) and protein kinase A (PKA) on $NF-_{\kappa}B$ activity and the viability of bovine cerebral endothelial cells (BCECs). In serum-deprivation-induced BCEC death, low doses of $TNF{\alpha}$ showed a protective effect. $TNF{\alpha}$ induced $NF-_{\kappa}B$ activation within 4 h in serum-deprivation. PTK inhibitors (herbimycin A and genistein) and PKC inhibitor (calphostin C) prevented $NF-_{\kappa}B$ activation stimulated by $TNF{\alpha}.$ Likewise, these inhibitors prevented the protective effect of $TNF{\alpha}.$ In contrast to $TNF{\alpha}-stimulated\;NF-_{\kappa}B$ activity, basal $NF-_{\kappa}B$ activity of BCECs in media containing serum was suppressed only by calphostin C, but not by herbimycin A. As well BCEC death was also induced only by calphostin C in serum-condition. H 89, a PKA inhibitor, did not affect the basal and $TNF{\alpha}-stimulated\;NF-_{\kappa}B$ activities and the protective effect of $TNF{\alpha}$ on cell death. These data suggest that modulation of $NF-_{\kappa}B$ activation could be a possible mechanism for regulating cell viability by protein kinases in BCECs.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2003년도 Annual Meeting of KSAP : International Symposium on Pharmaceutical and Biomedical Sciences on Obesity
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• pp.78-78
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• 2003

Inulin, an active component of Chicorium intybus root, has been shown to stimulate the growth of bifidobacteria, and inhibit colon carcinogenesis. NO mediates a number of the host-defense functions of activated macrophages, including antimicrobial and tumoricidal activity. We examined the effect of inulin on the synthesis of NO in RAW 264.7 cells. Inulin alone had no effect, whereas inulin with IFN-ν synergistically increased the NO production and inducible NO synthase (iNOS) expression in RAW 264.7 cells. Synergy between IFN-ν and inulin was mainly dependent on inulin-induced TNF-${\alpha}$ secretion. Also, protein kinase C (PKC)-${\alpha}$ was involved in the inulin-induced NO production. Inulin-mediated NO production was inhibited by the protein tyrosine kinase (PTK) inhibitor, tyrphostin AG126. Since iNOS gene transcriptions have been shown to be under the control of the NF -$\kappa$B/Rel family of transcription factors, we assessed the effect of inulin on NF -$\kappa$B/Rel using an EMSA. Inulin produced strong induction of NF-$\kappa$B/Rel binding, whereas AP-l binding was slightly induced in RAW 264.7 cells. Inulin stimulated phosphorylation and degradation of I$\kappa$B-${\alpha}$. These results suggest that in IFN-ν-primed RAW 264.7 cells inulin might stimulate NO synthesis via activation of PKC-${\alpha}$ and PTK, resulting in the activation of NF-$\kappa$B.

• BMB Reports
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• 제35권4호
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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.

• Biomolecules & Therapeutics
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• 제21권6호
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• pp.435-441
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• 2013

Emodin, a naturally occurring anthraquinone derivative isolated from Polygoni cuspidati radix, has several beneficial pharmacologic effects, which include anti-cancer, anti-diabetic, and anti-inflammatory activities. In this study, the authors examined the effect of emodin on the production of proinflammatory cytokines, such as, tumor necrosis factor (TNF)-${\alpha}$ and interleukin (IL)-6, in mouse bone marrow-derived mast cells (BMMCs) stimulated with phorbol 12-myristate 13-acetate (PMA) plus the calcium ionophore A23187. To investigate the mechanism responsible for the regulation of pro-inflammatory cytokine production by emodin, the authors assessed its effects on the activations of transcriptional factor nuclear factor-${\kappa}B$ (NF-${\kappa}B$) and mitogen-activated protein kinases (MAPKs). Emodin attenuated the nuclear translocation of (NF)-${\kappa}B$ p65 and its DNA-binding activity by reducing the phosphorylation and degradation of $I{\kappa}B{\alpha}$ and the phosphorylation of $I{\kappa}B$ kinase B (IKK). Furthermore, emodin dose-dependently attenuated the phosphorylations of MAPKs, such as, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAP kinase, and the stress-activated protein kinases (SAPK)/c-Jun-N-terminal kinase (JNK). Taken together, the findings of this study suggest that the anti-inflammatory effects of emodin on PMA plus A23187-stimulated BMMCs are mediated via the inhibition of NF-${\kappa}B$ activation and of the MAPK pathway.

• The Korean Journal of Physiology and Pharmacology
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• 제4권5호
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• pp.339-346
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• 2000

Using murine immortalized microglial cells (BV-2), we examined the regulation of RANTES production stimulated by lipopolysaccharide (LPS), focusing on the role of mitogen-activated protein kinase (MAPK) and nuclear factor $(NF)-{\kappa}B.$ The result showed that RANTES (regulated upon activation of normal T cell expressed and secreted) was induced at the mRNA and protein levels in a dose- and time-dependent manner in response to LPS. From investigations of second messenger pathways involved in regulating the secretion of RANTES, we found that LPS induced phosphorylation of extracellular signal-regulated kinase (Erk), p38 MAPK and c-Jun-N-terminal kinase (JNK), and activated $(NF)-{\kappa}B.$ To determine whether this MAPK phosphorylation is involved in LPS-stimulated RANTES production, we used specific inhibitors for p38 MAPK and Erk, SB 203580 and PD 98059, respectively. LPS-induced RANTES production was reduced approximately 80% at $25\;{\mu}M$ of SB 203580 treatment. But PD 98059 did not affect RANTES production. Pyrrolidine-dithiocarbamate (PDTC), $(NF)-{\kappa}B$ inhibitor, reduced RANTES secretion. These results suggest that LPS-induced RANTES production in microglial cells (BV-2) is mainly mediated by the coordination of p38 MAPK and $(NF)-{\kappa}B$ cascade.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제34권1호
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• pp.28-35
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• 2008

Purpose: The nitric oxide (NO) release by inducible nitric oxide synthase (iNOS) is the key events in macrophage response to lipopolysaccharide (LPS) which is suggested to be a crucial mediator for inflammatory and innate immune responses. NO is an important mediator involved in many host defense action and may also lead to a harmful host response to bacterial infection. However, given the importance of iNOS in a variety of pathophysiological conditions, control of its expression and signaling events in response to LPS has been the subject of considerable investigation. Materials and Methods: The Raw264.7 macrophage cell line was used to observe LPS-stimulated iNOS expression. The expression of iNOS is observed by Western blot analysis and real-time RT-PCR. Protein kinase C $(PKC)-{\alpha}$ overexpressing Raw264.7 cells are established to determine the involvement of $PKC-{\alpha}$ in LPS-mediated iNOS expression. $NF-{\kappa}B$ activity is measured by $I{\kappa}B{\alpha}$ degradation and $NF-{\kappa}B$ luciferase activity assay. Results: We found that various PKC isozymes regulate LPS-induced iNOS expression at the transcriptional and translational levels. The involvement of $PKC-{\alpha}$ in LPS-mediated iNOS induction was further confirmed by increased iNOS expression in $PKC-{\alpha}$ overexpressing cells. $NF-{\kappa}B$ dependent transactivation by LPS was observed and $PKC-{\alpha}$ specific inhibitory peptide abolished this activation, indicating that $NF-{\kappa}B$ activation is dependent on $PKC-{\alpha}$. Conclusion: Our data suggests that $PKC-{\alpha}$ is involved in LPS-mediated iNOS expression and that its downstream target is $NF-{\kappa}B$. Although $PKC-{\alpha}$ is a crucial mediator in the iNOS regulation, other PKC isozymes may contribute LPS-stimulated iNOS expression. This finding is needed to be elucidated in further study.

• BMB Reports
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• 제42권3호
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• pp.148-153
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• 2009

Pyrrolidine dithiocarbamate (PDTC) is a stable anti-oxidant or pro-oxidant, depending on the situation, and it is widely used to inhibit the activation of NF-${\kappa}B$. We recently reported that PDTC activates the MIP-2 gene in a NF-${\kappa}B$-independent and c-Jun-dependent manner in macrophage cells. In this work, we found that PDTC activates signal transduction pathways in mouse ES cells. Among the three different mitogen-activated protein kinase (MAPK) pathways, including the extracellular-signal-regulated kinase (ERK), p38 MAP kinase, and stress-activated protein kinase (SAPK)/Jun N-terminal kinase (JNK) pathways, only the ERK pathway was significantly activated in mouse ES cells after stimulation with PDTC. Additionally, we observed a synergistic activation of ERK and induction of c-Fos after stimulation with PDTC in the presence of mouse embryonic fibroblast (MEF) conditioned medium. In contrast, another NF-${\kappa}B$ inhibitor, BMS-345541, did not activate the MAP kinase pathways or induce expression of c-Fos. These results suggest that changes in the presence of the NF-${\kappa}B$ inhibitor PDTC should be carefully considered when it used with mouse ES cells.

• 대한약학회:학술대회논문집
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• 대한약학회 2001년도 Proceedings of International Convention of the Pharmaceutical Society of Korea
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• pp.109-113
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• 2001

Oxidized low-density lipoprotein (oxLDL) has been shown to modulate transactivations by the peroxisome proliferator activated receptor (PPAR)$\gamma$ and nuclear factor-kappa B (NF$\kappa$B). In this study, the oxLDL signaling pathways involved with the NF$\kappa$B transactivation were investigated by utilizing a reporter construct driven by three upstream NF$\kappa$B binding sites, and various pharmacological inhibitors. OxLDL and its constituent lysophophatidylcholine (lysoPC) induced a rapid and transient increase of intracellular calcium and stimulated the NF-KB transactivation in resting RAW264.7 macrophage cells in an oxidation-dependent manner. The NF$\kappa$B activation by oxLDL or lysoPC was inhibited by protein kinase C inhibitors or an intracellular calcium chelator. Tyrosine kinase or PI3 kinase inhibitors did not block the NF$\kappa$B transactivation. Furthermore, the oxLDL-induced NF$\kappa$B activity was abolished by the PPAR$\gamma$ ligands. When the endocytosis of oxLDL was blocked by cytochalasin B, the NF$\kappa$B transactivation by oxLDL was synergistically increased, while PPAR transactivation was blocked. These results suggest that oxLDL activates NF-$\kappa$B in resting macrophages via protein kinase C- and/or calcium-dependent pathways, which does not involve the endocytic processing of oxLDL. The endocytosis-dependent PPAR$\gamma$ activation by oxLDL may function as an inactivation route of the oxLDL induced NF$\kappa$B signal. Short heterodimer partner (SHP), specifically expressed in liver and a limited number of other tissues, is an unusual orphan nuclear receptor that lacks the conventional DNA-binding domain. In this work, we found that SHP expression is abundant in murine macrophage cell line RAW 264.7 but suppressed by oxLDL and its constituent I3-HODE, a ligand for peroxisome proliferator-activated receptor y. Furthermore, SHP acted as a transcription coactivator of nuclear factor-$\kappa$B (NF$\kappa$B) and was essential for the previously described NF$\kappa$B transactivation by lysoPC, one of the oxLDL constituents. Accordingly, NF$\kappa$B, transcriptionally active in the beginning, became progressively inert in oxLDL-treated RAW 264.7 cells, as oxLDL decreased the SHP expression. Thus, SHP appears to be an important modulatory component to regulate the transcriptional activities of NF$\kappa$B in oxLDL-treated, resting macrophage cells.

• BMB Reports
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• 제50권11호
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• pp.584-589
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• 2017

Intercellular adhesion molecule-1 (ICAM-1), which is induced by tumor necrosis factor (TNF)-${\alpha}$, contributes to the entry of immune cells into the site of inflammation in the skin. Here, we show that protein tyrosine phosphatase non-receptor type 21 (PTPN21) negatively regulates ICAM-1 expression in human keratinocytes. PTPN21 expression was transiently induced after stimulation with TNF-${\alpha}$. When overexpressed, PTPN21 inhibited the expression of ICAM-1 in HaCaT cells but PTPN21 C1108S, a phosphatase activity-inactive mutant, failed to inhibit ICAM-1 expression. Nuclear factor-${\kappa}B$ (NF-${\kappa}B$), a key transcription factor of ICAM-1 gene expression, was inhibited by PTPN21, but not by PTPN21 C1108S. PTPN21 directly dephosphorylated phospho-inhibitor of ${\kappa}B$ ($I{\kappa}B$)-kinase ${\beta}$ ($IKK{\beta}$) at Ser177/181. This dephosphorylation led to the stabilization of $I{\kappa}B{\alpha}$ and inhibition of NF-${\kappa}B$ activity. Taken together, our results suggest that PTPN21 could be a valuable molecular target for regulation of inflammation in the skin by dephosphorylating p-$IKK{\beta}$ and inhibiting NF-${\kappa}B$ signaling.

• The Korean Journal of Physiology and Pharmacology
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• 제5권1호
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• pp.55-63
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• 2001

In macrophages, lipopolysaccharide (LPS) alone or in combination with $interferon-{\gamma}\;(IFN-{\gamma})$ has been shown to release a nitric oxide (NO) through the increase of the transcription of the inducible nitric oxide synthase (iNOS) gene. To investigate the exact intracellular signaling pathway of the regulation of iNOS gene transcription by LPS plus $IFN-{\gamma},$ the effects of protein tyrosine kinase (PTK) inhibitor and protein kinase C (PKC) inhibitors on NO production, iNOS mRNA expression, nuclear $factor-_{\kappa}B\;(NF-_{\kappa}B)$ binding activity and the promoter activity of iNOS gene containing two $NF-_{\kappa}B$ sites have been examined in a mouse macrophage RAW 264.7 cells. LPS or $IFN-{\gamma}$ stimulated NO production, and their effect was enhanced synergistically by mixture of LPS and $IFN-{\gamma}.$ The PTK inhibitor such as tyrphostin reduced LPS plus $IFN-{\gamma}-induced$ NO production, iNOS mRNA expression and $NF-_{\kappa}B$ binding activity. In contrast, PKC inhibitors such as H-7, Ro-318220 and staurosporine did not show any effect on them. In addition, transfection of RAW 264.7 cells with iNOS promoter linked to a CAT reporter gene revealed that tyrphostin inhibited the iNOS promoter activity through the $NF-_{\kappa}B$ binding site, whereas PKC inhibitors did not. Taken together, these suggest that PTK, but not PKC pathway, is involved in the regulation of the iNOS gene transcription through the $NF-_{\kappa}B$ sites of iNOS promoter in RAW 264.7 macrophages by LPS plus $IFN-{\gamma}$.