• Title/Summary/Keyword: mitogen

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CLK3 is a Novel Negative Regulator of NF-κB Signaling (NF-κB 신호경로에서 CLK3의 새로운 음성 조절자로서의 기능)

  • Byeol-Eun, Jeon;Chan-Seong, Kwon;Ji-Eun, Lee;Ye-Lin, Woo;Sang-Woo, Kim
    • Journal of Life Science
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    • v.32 no.11
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    • pp.833-840
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    • 2022
  • Chronic inflammation has been shown to be closely associated with tumor development and progression. Nuclear factor kappa B (NF-κB) is composed of a family of five transcription factors. NF-κB signaling plays a crucial role in the inflammatory response and is often found to be dysregulated in various types of cancer, making it an attractive target in cancer therapeutics. In this study, CDC-like kinase 3 (CLK3) was identified as a novel kinase that regulates the NF-κB signaling pathway. Our data demonstrate that CLK3 inhibits the canonical and non-canonical NF-κB pathways. Luciferase assays following the transient or stable expression of CLK3 indicated that this kinase inhibited NF-κB activation mediated by Tumor necrosis factor-alpha (TNFα) and Phorbol 12-myristate 13-acetate (PMA), which are known to activate NF-κB signaling via the canonical pathway. Consistent with data on the ectopic expression of CLK3, CLK3 knockdown using shRNA constructs increased NF-κB activity 1.5-fold upon stimulation with TNFα in HEK293 cells compared with the control cells. Additionally, overexpression of CLK3 suppressed the activation of this signaling pathway induced by NF-κB-inducing kinase (NIK) or CD40, which are well-established activators of the non-canonical pathway. To further examine the negative impact of CLK3 on NF-κB signaling, we performed Western blotting following the TNFα treatment to directly identify the molecular components of the NF-κB pathway that are affected by this kinase. Our results revealed that CLK3 mitigated the phosphorylation/activation of transforming growth factor-α-activated kinase 1 (TAK1), inhibitor of NF-κB kinase alpha/beta (IKKα/α), NF-κB p65 (RelA), NF-κB inhibitor alpha (IκBα), and Extracellular signal-regulated kinase 1/2-Mitogen-activated protein kinase (ERK1/2-MAPK), suggesting that CLK3 inhibits both the NF-κB and MAPK signaling activated by TNFα exposure. Further studies are required to elucidate the mechanism by which CLK3 inhibits the canonical and non-canonical NF-κB pathways. Collectively, these findings reveal CLK3 as a novel negative regulator of NF-κB signaling.

Antioxidative Effects of Tenebrio molitor Larvae Extract Against Oxidative Stress in ARPE-19 Cells (ARPE-19 세포에서 산화적 스트레스에 대한 갈색거저리 추출물의 항산화 효과)

  • Bong Sun, Kim;Ra-Yeong, Choi;Eu-Jin, Ban;Joon Ha, Lee;In-Woo, Kim;Minchul, Seo
    • Journal of Life Science
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    • v.32 no.11
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    • pp.865-871
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    • 2022
  • Tenebrio molitor larvae is well known as edible insect. Then, although it has been widely studied that Tenebrio molitor larvae has various bioactive functions such as antioxidant, anti-wrinkle, and anticancer. Nevertheless, antioxidant effects of Tenebrio molitor larvae water extract (TMH) has not been well described in Adult Retina Pigment Epithelial cell line (ARPE-19). In this study, we demonstrated that antioxidant effects of TMH against H2O2-induced oxidative stress in ARPE-19. Thus, we selected for our studies and performed a series of dose-response assay to determine the working concentration that lead to a consistent and high degree of cytotoxicity, which we defined as the level of H2O2 that killed 40% of the ARPE-19 cells. ARPE-19 cells were pre-treated with various concentrations of TMH (0.1 up to 2 mg/ml) before exposure to 300 µM H2O2. As we expected, TMH effectively prevented ARPE-19 cells from 300 µM H2O2-induced cell death in a dose-dependent manner. Furthermore, TMH inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs) such as extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. Overall, the inhibitory effects of TMH on H2O2-induced apoptosis and oxidative stress were associated with the protection cleaved caspase-3, Bax, Bcl-2, and HO-1. The TMH suppressed H2O2-induced cell membrane leakage and oxidative stress in ARPE-19 cells. Thus, these results suggest that the TMH plays an important role in antioxidant effect in ARPE-19.

Immunostimulatory and Anti-Obesity Activity of Lonicera insularis Nakai Extracts in Mouse Macrophages RAW264.7 Cells and Mouse Adipocytes 3T3-L1 Cells (섬괴불나무(Lonicera insularis Nakai) 추출물의 면역자극 및 항비만 활성)

  • Yu, Ju Hyeong;Yeo, Joo Ho;Choi, Min Yeong;Lee, Jae Won;Geum, Na Gyeong;An, Mi-Yun;Jeong, Jin Boo
    • Korean Journal of Plant Resources
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    • v.35 no.4
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    • pp.417-427
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    • 2022
  • In this study, we investigated in vitro immuno-stimulatory and anti-obesity activity of fruit (LIF), leaves (LIL) and stems (LIS) from Lonicera insularis Nakai in mouse macrophages RAW264.7 cells and mouse pre-adipocytes 3T3-L1 cells. LIF, LIL and LIS increased the production of immunostimulatory factors such as nitric oxide (NO), inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α) and activated phagocytosis in RAW264.7 cells. Inhibition of toll-like receptor 2/4 (TLR2/4) partly blocked LIF, LIL and LIS mediated production of immunostimulatory factors. In addition, inhibition of mitogen-activated protein kinases (MAPK) signaling attenuated the production of immunostimulatory factors induced by LIF, LIL and LIS. Based on these results of this study, LIF, LIL and LIS is thought to activate macrophages the production of immunostimulatory factors and phagocytosis through toll-like receptor 2/4 (TLR2/4) and MAPKs signaling pathway. In anti-obesity study, LIF reduced the lipid accumulation in 3T3-L1 cells. LIF increased the protein phosphorylation expressions such as AMP-activated protein kinase (AMPK), hormone sensitive lipase (HSL), adipose triglyceride lipase (ATGL) related to the lipolysis of the adipocytes. In addition, LIF increased the expression of proteins involved in energy metabolism and brown adipose tissues differentiation such as peroxisome proliferator-activated receptor gamma coativator 1α (PGC-1α) and PR domain-containing16 (PRDM16). These results suggest that LIF is involved in lipid accumulation inhibition through expressing the proteins such as lipolysis and differentiation of white adipocytes to brown adipocytes.

Protective Effects of Trifolium pratense L. Extract against H2O2-induced Oxidative Stress in HaCaT Keratinocytes (인간 피부각질세포에서 Hydrogen peroxide로 유도된 산화적 스트레스에 대한 붉은 토끼풀 추출물의 세포 보호 효과)

  • Mi Song Shin;You Kyeong Lee;Seo Young Choi;Ji Sun Hwang;Parkyong Song;Hyeon Cheal Park;Keun Ki Kim;Hong-Joo Son;Yu-Jin Kim;Kwang Min Lee
    • Journal of the Korean Applied Science and Technology
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    • v.40 no.2
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    • pp.223-232
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    • 2023
  • Oxidative stress plays a significant role in the pathogenesis of various skin conditions, resulting in cellular and tissue damage that can contribute to the development of skin tone unevenness, roughness and wrinkles. In this study, we found that Trifolium pratense L. extract (TE) attenuated oxidative-induced damage in HaCaT cells and elucidated the underlying molecular mechanism. Our finding demonstrated that TE effectively protected HaCaT cells against H2O2-induced cell death by inhibiting caspase-3 activation, downregulating Bax and upregulating Bcl-2, and attenuating the activation of three mitogen-activated protein kinases (MAPKs). Our results suggest that TE has remarkable cytoprotective properties against oxidative damage in HaCaT cells and could serve as a complementary or alternative approach to prevent and treat skin damage.

Anti-inflammatory Effects of Rumohra adiantiformis Extracts Fermented with Bovista plumbea Mycelium in LPS-stimulated RAW 264.7 Cells (LPS로 자극된 RAW 264.7 세포에서 찹쌀떡버섯 균사체로 생물전환된 루모라고사리 추출물의 항염증 효과)

  • Ji-Hye Hong;Eun-Seo Jang;Myung-Chul Gil;Gye Won Lee;Young Ho Cho
    • Journal of Life Science
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    • v.33 no.6
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    • pp.471-480
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    • 2023
  • This study was designed to evaluate the anti-inflammatory effects of Rumohra adiantiformis extracts fermented with Bovista plumbea mycelium (B-RAE) in LPS-stimulated RAW 264.7 cells. The total polyphenol and total flavonoid content of B-RAE were 379.26±7.77 mg/g and 50.85±3.08 mg/g, respectively. The results of measuring the antioxidant activity of B-RAE showed that it scavenges 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and superoxide anion radical in a dose-dependent manner. B-RAE inhibited nitric oxide (NO) production in a dose-dependent manner without affecting cell viability. The gene expression of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-lβ (IL-1β), and IL-6 was measured using real time quantitative reverse transcription PCR (qRT-PCR). We found that, compared to the LPS-treated group, B-RAE significantly reduced the mRNA levels of the pro-inflammatory cytokines in a concentration-dependent manner. The expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), the phosphorylation of transcription factors such as nuclear factor-κB (NF-κB), and the mitogen-activated protein kinase (MAPK) signaling pathway proteins were assessed using Western blot analysis. We found that B-RAE significantly suppressed the expression of iNOS and COX-2, but their expression was increased by LPS treatment. In addition, the phosphorylation of NF-κB and IκB, which was increased by LPS treatment, was reduced with B-RAE treatment. The effect of B-RAE on the phosphorylation of the MAPK signaling pathway proteins was measured, and the phosphorylation of extracellular signal-regulated kinase (ERK) and the p38 MAPK proteins decreased in a dose-dependent manner, while the phosphorylation of c-Jun N-terminal kinase (JNK) increased. These anti-inflammatory effects of B-RAE may thus have been achieved through the high antioxidant activity, the inhibition of NO production through the suppression of iNOS and COX-2 expression, the inhibition of the NF-κB pathway, and the suppression of pro-inflammatory cytokine expression.

The Role of c-Jun N-terminal Kinase in the Radiation-Induced Lung Fibrosis (방사선에 의한 폐 섬유화증에서 c-Jun N-terminal Kinase(JNK)의 역할)

  • Uh, Soo-Taek;Hong, Ki-Young;Lee, Young-Mok;Kim, Ki-Up;Kim, Do-Jin;Moon, Seung-Hyuk;Kim, Yong-Hoon;Park, Choon-Sik;Yeom, Uk;Kim, Eun-Suk;Choi, Doo-Ho
    • Tuberculosis and Respiratory Diseases
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    • v.50 no.4
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    • pp.450-461
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    • 2001
  • Background : The underlying pathogenesis of radiation-induced lung fibrosis (RTLF) has not been very well defined. However, the role of TGF-$\beta$ in the generation of RTLF has been a major focus because there is an increase in the expression of both the TGF-${\beta}m$-RNA and its protein preceding RTLF lesions. The down stream signal after a TGF-$\beta$ stimulated lung fibrosis includes the activation of many mediators such as Smad and c-Jun N-terminal kinase (JNK) through TAK1. It is we hypothesized that JNK activation may play a pivotal role in RTLF pathogenesis through increased transcription of the fibrogenic cytokines. The present study evaluates JNK activity in alveolar macrophages after irradiation and the relationship between JNK activity and the amount of collagen in the lung tissues. Methods : C57BL/6 mice(20-25 gr, males) received chlorotetracycline(2g/L) in their drinking water 1 week prior to irradiation and continuously there after. The mice were irradiated once with 1400 cGy of $60CO{\gamma}$-ray over the whole chest. The cellular composition of the whole lung bronchoalveoalr lavage fluids(BALF), elastin expression in the lung tissues, the level of hydroxyproline in lung tissues, and an in vitro JNK assay was measured before irradiation and one, four, and eight weeks after irradiation (RT). Results : The volumes of BALF retrieved from instilled 4 mL of saline with 2% heparin were 3.7-3.8 mL for each group. The cell numbers were similar before($4.1{\times}10^4{\pm}0.5{\times}10^4/mL$) and 1 week($3.1{\times}10^4{\pm}0.5{\times}10^4/mL$) after RT. At four and eight weeks after RT, the cell number reached to $14.0{\times}10^4{\pm}1.5{\times}10^4mL$ and $10.0{\times}10^4{\pm}1.3{\times}10^4/mL$, respectively. There we no changes in the lymphocytes and neutrophils population observed in the BALF after RT. The H-E stain of the lung tissues did not show any structural and fibrotic change in the lung tissues at 4 and 8 weeks after RT. In addition, the amount of elastin and collagen were not different on Verhoeff staining of the lung tissues before RT to eight weeks after RT. The hydroxyproine content was measured with the left lung dissected from the left main bronchus. The lung were homogenized and hydrolyzed with 6 N Hel for 12 hours at $110^{\circ}C$ then measured as previously described. The content of hydroxyproline, standardized with a lung protein concentration, reached a peak 4 weeks after RT, and thereafter showed a plateau. AnIn vitro JNK assay using c-$Jun_{1-79}$-GST sepharose beads were performed with the alveolar macrophages obtained from the BAL. JNK activity was not detected prior to RT, However, the JNK activity increased from one week after RT and reached a peak four weeks after RT. Conclusion : JNK may be involved in the pathogenesis because the JNK activity showed similar pattern observed with the hydroxyproine content. However, it is necessary to clarify that the JNK increases the transcription of fibrogenic cyiokines through the transcription factor.

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Sesquiterpenoids Bioconversion Analysis by Wood Rot Fungi

  • Lee, Su-Yeon;Ryu, Sun-Hwa;Choi, In-Gyu;Kim, Myungkil
    • 한국균학회소식:학술대회논문집
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    • 2016.05a
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    • pp.19-20
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    • 2016
  • Sesquiterpenoids are defined as $C_{15}$ compounds derived from farnesyl pyrophosphate (FPP), and their complex structures are found in the tissue of many diverse plants (Degenhardt et al. 2009). FPP's long chain length and additional double bond enables its conversion to a huge range of mono-, di-, and tri-cyclic structures. A number of cyclic sesquiterpenes with alcohol, aldehyde, and ketone derivatives have key biological and medicinal properties (Fraga 1999). Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Fungal suspensions of 11 white rot species were inoculated in modified medium containing $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ for 20 days. Cultivation was stopped by solvent extraction via separation of the mycelium. The metabolites were identified as follows: propionic acid (1), mevalonic acid lactone (2), ${\beta}$-eudesmane (3), and ${\beta}$-eudesmol (4), respectively (Figure 1). The main peaks of ${\beta}$-eudesmane and ${\beta}$-eudesmol, which were indicative of sesquiterpene structures, were consistently detected for 5, 7, 12, and 15 days These results demonstrated the existence of terpene metabolism in the mycelium of P. brumalis. Polyporus spp. are known to generate flavor components such as methyl 2,4-dihydroxy-3,6-dimethyl benzoate; 2-hydroxy-4-methoxy-6-methyl benzoic acid; 3-hydroxy-5-methyl phenol; and 3-methoxy-2,5-dimethyl phenol in submerged cultures (Hoffmann and Esser 1978). Drimanes of sesquiterpenes were reported as metabolites from P. arcularius and shown to exhibit antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus (Fleck et al. 1996). The main metabolites of P. brumalis, ${\beta}$-Eudesmol and ${\beta}$-eudesmane, were categorized as eudesmane-type sesquiterpene structures. The eudesmane skeleton could be biosynthesized from FPP-derived IPP, and approximately 1,000 structures have been identified in plants as essential oils. The biosynthesis of eudesmol from P. brumalis may thus be an important tool for the production of useful natural compounds as presumed from its identified potent bioactivity in plants. Essential oils comprising eudesmane-type sesquiterpenoids have been previously and extensively researched (Wu et al. 2006). ${\beta}$-Eudesmol is a well-known and important eudesmane alcohol with an anticholinergic effect in the vascular endothelium (Tsuneki et al. 2005). Additionally, recent studies demonstrated that ${\beta}$-eudesmol acts as a channel blocker for nicotinic acetylcholine receptors at the neuromuscular junction, and it can inhibit angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase (MAPK) signaling pathway (Seo et al. 2011). Variation of nutrients was conducted to determine an optimum condition for the biosynthesis of sesquiterpenes by P. brumalis. Genes encoding terpene synthases, which are crucial to the terpene synthesis pathway, generally respond to environmental factors such as pH, temperature, and available nutrients (Hoffmeister and Keller 2007, Yu and Keller 2005). Calvo et al. described the effect of major nutrients, carbon and nitrogen, on the synthesis of secondary metabolites (Calvo et al. 2002). P. brumalis did not prefer to synthesize sesquiterpenes under all growth conditions. Results of differences in metabolites observed in P. brumalis grown in PDB and modified medium highlighted the potential effect inorganic sources such as $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ on sesquiterpene synthesis. ${\beta}$-eudesmol was apparent during cultivation except for when P. brumalis was grown on $MgSO_4$-free medium. These results demonstrated that $MgSO_4$ can specifically control the biosynthesis of ${\beta}$-eudesmol. Magnesium has been reported as a cofactor that binds to sesquiterpene synthase (Agger et al. 2008). Specifically, the $Mg^{2+}$ ions bind to two conserved metal-binding motifs. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving groups of FPP and resulting in the generation of a highly reactive allylic cation. Effect of magnesium source on the sesquiterpene biosynthesis was also identified via analysis of the concentration of total carbohydrates. Our current study offered further insight that fungal sesquiterpene biosynthesis can be controlled by nutrients. To profile the metabolites of P. brumalis, the cultures were extracted based on the growth curve. Despite metabolites produced during mycelia growth, there was difficulty in detecting significant changes in metabolite production, especially those at low concentrations. These compounds may be of interest in understanding their synthetic mechanisms in P. brumalis. The synthesis of terpene compounds began during the growth phase at day 9. Sesquiterpene synthesis occurred after growth was complete. At day 9, drimenol, farnesol, and mevalonic lactone (or mevalonic acid lactone) were identified. Mevalonic acid lactone is the precursor of the mevalonic pathway, and particularly, it is a precursor for a number of biologically important lipids, including cholesterol hormones (Buckley et al. 2002). Farnesol is the precursor of sesquiterpenoids. Drimenol compounds, bi-cyclic-sesquiterpene alcohols, can be synthesized from trans-trans farnesol via cyclization and rearrangement (Polovinka et al. 1994). They have also been identified in the basidiomycota Lentinus lepideus as secondary metabolites. After 12 days in the growth phase, ${\beta}$-elemene caryophyllene, ${\delta}$-cadiene, and eudesmane were detected with ${\beta}$-eudesmol. The data showed the synthesis of sesquiterpene hydrocarbons with bi-cyclic structures. These compounds can be synthesized from FPP by cyclization. Cyclic terpenoids are synthesized through the formation of a carbon skeleton from linear precursors by terpene cyclase, which is followed by chemical modification by oxidation, reduction, methylation, etc. Sesquiterpene cyclase is a key branch-point enzyme that catalyzes the complex intermolecular cyclization of the linear prenyl diphosphate into cyclic hydrocarbons (Toyomasu et al. 2007). After 20 days in stationary phase, the oxygenated structures eudesmol, elemol, and caryophyllene oxide were detected. Thus, after growth, sesquiterpenes were identified. Per these results, we showed that terpene metabolism in wood-rotting fungi occurs in the stationary phase. We also showed that such metabolism can be controlled by magnesium supplementation in the growth medium. In conclusion, we identified P. brumalis as a wood-rotting fungus that can produce sesquiterpenes. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.

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