• Biomolecules & Therapeutics
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• 제17권3호
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• pp.276-281
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• 2009

Lactic acid bacteria (LAB) are considered as probiotics with immunostimulatory property. In this study, we investigated the molecular mechanism of its immunostimulating potency on macrophages using combined preparation of LAB (cpLAB). cpLAB is able to strongly stimulate nitric oxide (NO) production as well as inducible NO synthase (iNOS) expression from macrophage-like RAW264.7 cells. The cpLAB-induced NO release seemed to be mediated by extracellular signal-regulated kinase (ERK) but not p38 and C-Jun N-terminal kinase (JNK), since U0126, an ERK inhibitor, clearly suppressed NO production. cpLAB significantly diminished the binding of toll like receptor (TLR)-2 antibody up to 25%, implying that cpLAB-mediated activation of macrophages may be required for the functional activation of TLR-2, but not TLR-4. Therefore, our data suggest that cpLAB may directly allow macrophages to immunostimulating potency via activation of TLR-2 and ERK.

• Molecules and Cells
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• 제38권2호
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• pp.145-150
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• 2015

Continuous intra- and extracellular stresses induce disorder of $Ca^{2+}$ homeostasis and accumulation of unfolded protein in the endoplasmic reticulum (ER), which results in ER stress. Severe long-term ER stress triggers apoptosis signaling pathways, resulting in cell death. Neural epidermal growth factor-like like protein 2 (NELL2) has been reported to be important in protection of cells from cell death-inducing environments. In this study, we investigated the cytoprotective effect of NELL2 in the context of ER stress induced by thapsigargin, a strong ER stress inducer, in Cos7 cells. Overexpression of NELL2 prevented ER stress-mediated apoptosis by decreasing expression of ER stress-induced C/EBP homologous protein (CHOP) and increasing ER chaperones. In this context, expression of anti-apoptotic Bcl-xL was increased by NELL2, whereas NELL2 decreased expression of pro-apoptotic proteins, such as cleaved caspases 3 and 7. This anti-apoptotic effect of NELL2 is likely mediated by extracellular signal-regulated kinase (ERK) signaling, because its inhibitor, U0126, inhibited effects of NELL2 on the expression of anti- and pro-apoptotic proteins and on the protection from ER stress-induced cell death.

• Journal of Yeungnam Medical Science
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• 제30권1호
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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.

• Biomolecules & Therapeutics
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• 제25권6호
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• pp.593-598
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• 2017

The $Na^+/H^+$ exchanger-1 (NHE-1) is a ubiquitously expressed pH-regulatory membrane protein that functions in the brain, heart, and other organs. It is increased by intracellular acidosis through the interaction of intracellular $H^+$ with an allosteric modifier site in the transport domain. In the previous study, we reported that glutamate-induced NHE-1 phosphorylation mediated by activation of protein kinase C-${\beta}$ (PKC-${\beta}$) in cultured neuron cells via extracellular signal-regulated kinases (ERK)/p90 ribosomal s6 kinases (p90RSK) pathway results in NHE-1 activation. However, whether glutamate stimulates NHE-1 activity solely by the allosteric mechanism remains elusive. Cultured primary cortical neuronal cells were subjected to intracellular acidosis by exposure to $100{\mu}M$ glutamate or 20 mM $NH_4Cl$. After the desired duration of intracellular acidosis, the phosphorylation and activation of PKC-${\beta}$, ERK1/2 and p90RSK were determined by Western blotting. We investigated whether the duration of intracellular acidosis is controlled by glutamate exposure time. The NHE-1 activation increased while intracellular acidosis sustained for >3 min. To determine if sustained intracellular acidosis induced NHE-1 phosphorylation, we examined phosphorylation of NHE-1 induced by intracellular acidosis by transient exposure to $NH_4Cl$. Sustained intracellular acidosis led to activation and phosphorylation of NHE-1. In addition, sustained intracellular acidosis also activated the PKC-${\beta}$, ERK1/2, and p90RSK in neuronal cells. We conclude that glutamate stimulates NHE-1 activity through sustained intracellular acidosis, which mediates NHE-1 phosphorylation regulated by PKC-${\beta}$/ERK1/2/p90RSK pathway in neuronal cells.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
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• pp.79.1-79.1
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• 2003

Aster scaber T. (Asteraceae) has been used in traditional Korean and Chinese medicine to treat bruises, snakebites, headaches and dizziness. (-) 3,5-Dicaffeoyl-muco-quinic acid (DQ) isolated from Aster scaber induced neurite outgrowth in PC12 cells. It has been reported that the activation of the extracellular signal regulated kinase1/2 (Erk 1/2) and phosphoinositide 3 (P13) kinase plays a crucial role in the NGF-induced differentiation of PC12 cells. This study showed that the effect of DQ on neurite outgrowth is mediated via the Erk 1/2 and PI3 kinase-dependent pathways like NGF. (omitted)

• Journal of Applied Biological Chemistry
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• 제63권1호
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• pp.103-110
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• 2020

Objective: The objective of our study was to investigate anti-cancer effects of Danggui Liuhuang Decoction extract bioconverted by protease liquid coenzyme of Aspergillus kawachii (DLD-BE), compared to a non-bioconverted DLD extract (DLD-E) and determine the underlying mechanisms. Methods: DLD-E and DLD-BE were evaluated for their ability to modulate these signaling pathways and suppress the proliferation of human colorectal cancer (CRC) cells, HCT-116, LoVo, and HT-29. The anti-cancer effects of DLD-E and DLD-BE were measured by using proliferation and migration assays, cell cycle analysis, Western blots, and real-time PCR. Results: In this study, treatment with DLD-E and DLD-BE at concentrations of 25-100 ㎍/mL inhibited proliferation and migration in human CRC cells. DLD-BE induced apoptotic cell death and decreased COX-2 expression in HT-29 cells. The mechanisms of action included modulation of the AKT and extracellular-signal-regulated kinase signaling cascades along with inhibition of COX-2 expression. The results demonstrate novel anti-cancer mechanisms of DLD-BE against the growth of human CRC cells. Thus, we propose that DLD-BE can be developed as a more potent supplement to inhibit colorectal tumor growth and intestinal inflammation than DLD-E.

• BMB Reports
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• 제36권5호
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• pp.468-474
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• 2003

The molecular mechanism of hypoxia-induced apoptosis has not been clearly elucidated. In this study, we investigated the involvement of extracellular signal-regulated protein kinase (ERK 1/2) in hypoxia-induced apoptosis using cobalt chloride in HeLa human cervical cancer cells. The cobalt chloride was used for the induction of hypoxia, and its $IC_{50}$ was $471.4\;{\mu}M$. We demonstrated the DNA fragmentation after incubation with concentrations more than $50\;{\mu}M$ cobalt chloride for 24 h, and also evidenced the morphological changes of the cells undergoing apoptosis with electron microscopy. Next, we examined the signaling pathway of cobalt chloride-induced apoptosis in HeLa cells. ERK1/2 activation occurred 6 and 9 h after treatment with $600\;{\mu}M$ cobalt chloride. Meanwhile, the pretreatment of the MEK 1 inhibitor (PD98059) completely blocked the cobalt chloride-induced ERK 1/2 activation. At the same time, the activated ERK 1/2 translocated into the nucleus and phosphorylated its transcriptional factor, c-Jun. In addition, the pretreatment of PD98059 inhibited the cobalt chloride-induced DNA fragmentation and apoptotic cell death. These results suggest that cobalt chloride is able to induce apoptotic activity in HeLa cells, and its apoptotic mechanism may be associated with signal transduction via ERK 1/2.

• 생명과학회지
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• 제28권1호
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• pp.43-49
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• 2018

Chrysoeriol은 alfalfa에서 주로 발견되는, 식물계에 많이 분포하고 있는 flavone으로 전통의학에서 소화불량, 천식, 비뇨기계 이상의 치료에 사용되어 왔다. 최근의 연구에서는 항염증 효과가 있는 것으로 밝혀졌으나 항산화 효과에 대한 분석은 없었다. 본 연구에서는 chrysoeriol의 항산화 효과와 그 분자적 기전을 RAW 264.7 cell에서 세포생존율, reactive oxygen species (ROS)와 Western blot분석을 통해 알아보고자 하였다. Chrysoeriol은 lipopolysaccharide(LPS)에 의해 발생한 ROS를 세포독성없이 농도의존적으로 제거하였다. 그리고 항산화효과를 보이는 2상 효소 중 하나인 heme oxygenase (HO)-1의 발현을 강하게 유도하였고, 그와 동시에 전사인자인 Nrf2의 핵내 이동도 촉진하는 것으로 밝혀졌다. 특히, 산화스트레스에 대한 세포내 산화환원항상성 유지에 중요한 역할을 하고 있는 것으로 알려진 mitogen activated protein kinase (MAPK)와 phosphoinositide 3-kinase (PI3K)의 분석결과, chrysoeriol은 extracellular signal regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK)와 p38의 인산화를 통해 HO-1의 발현을 유도하는 것으로 나타났다. HO-1에 의한 항산화 효과를 확인하기 위하여 chrysoeriol을 전처리한 후 t-BHP에 의한 산화 스트레스에 세포를 노출시킨 결과, chrysoeriol 처리에 의해 세포사멸이 줄어드는 것을 확인하였고, HO-1의 유도제와 억제제의 처리에 따라 세포생존율 또한 조절되는 것을 확인할 수 있었다. 따라서, chrysoeriol은 HO-1의 발현을 유도하여 항산화 효과를 높이고 이것은 Nrf2/MAPK 신호전달 체계에 의한다는 것을 알 수 있었다.

• Molecules and Cells
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• 제44권8호
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• pp.569-579
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• 2021

Cyclase-associated protein 2 (CAP2) has been addressed as a candidate biomarker in various cancer types. Previously, we have shown that CAP2 is expressed during multi-step hepatocarcinogenesis; however, its underlying mechanisms in liver cancer cells are not fully elucidated yet. Here, we demonstrated that endoplasmic reticulum (ER) stress induced CAP2 expression, and which promoted migration and invasion of liver cancer cells. We also found that the ER stress-induced CAP2 expression is mediated through activation of protein kinase C epsilon (PKCε) and the promotor binding of activating transcription factor 2 (ATF2). In addition, we further demonstrated that CAP2 expression promoted epithelial-mesenchymal transition (EMT) through activation of Rac1 and ERK. In conclusion, we suggest that ER stress induces CAP2 expression promoting EMT in liver cancer cells. Our results shed light on the novel functions of CAP2 in the metastatic process of liver cancer cells.

• 생명과학회지
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• 제33권6호
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• pp.463-470
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• 2023

Desmarestia tabacoides Okamura는 전 세계적으로 널리 분포하는 갈조류 중 하나이다. 몇몇 산말류의 항종양, 멜라닌 생성 억제 및 광보호 활성에 대한 연구는 있었으나 D. tabacoides Okamura의 항염증 기전에 대해서는 보고되지 않아 본 연구에서는 LPS (lipopolysaccharide)로 자극된 RAW 264.7 세포에서 D. tabacoides Okamura 에탄올 추출물(DTEE)의 항염증 기전을 inducible nitric oxide synthase (iNOS)와 cyclooxygenase (COX)-2의 발현 및 이들의 상위신호전달물질인 nuclear factor (NF)-κB, mitogen-activated protein kinase (MAPK) 그리고 phosphoinositide-3-kinase (PI3K)/Akt의 인산화 조절 정도를 통해 분석하였다. DTEE의 처리는 세포 독성 없이 LPS로 유도된 NO와 prostaglandin (PG) E₂의 생성과 이들의 생성 효소인 iNOS 및 COX-2의 발현을 유의하게 억제하였다. 그리고 LPS에 의해 활성화된 NF-κB 및 상위 신호 전달 물질인 extracellular signal-regulated kinase (ERK), c-Jun NH₂-terminal kinase (JNK) 및 p38은 DTEE 처리에 의해 유의적으로 억제되었다. DTEE의 처리는 RAW 264.7 세포에서 LPS에 의해 활성화되는 adaptor molecule인 Toll-like receptor (TLR) 4 및 myeloid differentiation primary response (MyD) 88 또한 유의적으로 억제하였다. 이 결과를 통해 DTEE는 LPS에 의해 유도된 TLR4와 NF-κB 및 MAPK의 활성을 억제함으로써 염증 매개인자의 발현을 조절하였고, 이는 DTEE가 염증을 완화할 수 있는 기능성 식품의 소재로써 유용하게 사용될 수 있음을 시사한다.