• Journal of Microbiology and Biotechnology
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• 제18권1호
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• pp.95-103
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• 2008

Mushrooms are regarded as one of the well-known foods and biopharmaceutical materials with a great deal of interest. Polysaccharide ${\beta}$-glucan is the major component of mushrooms that displays various biological activities such as antidiabetic, anticancer, and antihyperlipidemic effects. In this study, we compared the immunostimulatory potency of polysaccharide fractions, prepared from liquid culture of pine-mushroom Tricholoma matsutake, with a potent immunogen lipopolysaccharide (LPS), and their molecular mechanisms on the functional activation of macrophages. We found that fraction II (TMF-II) was able to comparably upregulate or highly enhance the phenotypic functions of macrophages such NO production and cytokine (IL-$1{\beta}$, IL-6, IL-12, and TNF-${\alpha}$) expression, to LPS. TMF-II triggered the phosphorylation of $I{\kappa}B{\alpha}$, a critical step for NF-${\kappa}B$ activation and translocation. Of the upstream signaling enzymes tested, Src and Akt were thought to be the responsible upstream signaling components in induction of NO production, although TMF-II strongly upregulated the phosphorylation of all MAPK pathways. Therefore, our data suggest that T. matsutake-derived ${\beta}$-glucan may exert its immunostimulating activities with similar potency to LPS via activation of multiple signaling pathways linked to NF-${\kappa}B$ activation.

• Journal of Microbiology and Biotechnology
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• 제28권10호
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• pp.1635-1644
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• 2018

Asterias amurensis (starfish) is a marine organism that is harmful to the fishing industry, but is also a potential source of functional materials. The present study was conducted to analyze the profiles of fatty acids extracted from A. amurensis tissues and their anti-inflammatory effects on RAW264.7 macrophage cells. In different tissues, the component ratios of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids differed; particularly, polyunsaturated fatty acids such as dihomo-gamma-linolenic acid (20:3n-6) and eicosapentaenoic acid (20:5n-3) were considerably different. In lipopolysaccharide-stimulated RAW264.7 cells, fatty acids from A. amurensis skin, gonads, and digestive glands exhibited anti-inflammatory activities by reducing nitric oxide production and inducing nitric oxide synthase gene expression. Asterias amurensis fatty acids effectively suppressed the expression of inflammatory cytokines such as tumor necrosis $factor-{\alpha}$, interleukin-$1{\beta}$, and interleukin-6 in lipopolysaccharide-stimulated cells. Cyclooxygenase-2 and prostaglandin $E_2$, which are critical inflammation biomarkers, were also significantly suppressed. Furthermore, A. amurensis fatty acids reduced the phosphorylation of nuclear $factor-{\kappa}B$ p-65, p38, extracellular signal-related kinase 1/2, and c-Jun N-terminal kinase, indicating that these fatty acids ameliorated inflammation through the nuclear $factor-{\kappa}B$ and mitogen-activated protein kinase pathways. These results provide insight into the anti-inflammatory mechanism of A. amurensis fatty acids on immune cells and suggest that the species is a potential source of anti-inflammatory molecules.

• The Korean Journal of Physiology and Pharmacology
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• 제22권5호
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• pp.503-511
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• 2018

Lysophosphatidic acid (LPA) is known to play a critical role in breast cancer metastasis to bone. In this study, we tried to investigate any role of LPA in the regulation of osteoclastogenic cytokines from breast cancer cells and the possibility of these secretory factors in affecting osteoclastogenesis. Effect of secreted cytokines on osteoclastogenesis was analyzed by treating conditioned media from LPA-stimulated breast cancer cells to differentiating osteoclasts. Result demonstrated that IL-8 and IL-11 expression were upregulated in LPA-treated MDA-MB-231 cells. IL-8 was induced in both MDA-MB-231 and MDA-MB-468, however, IL-11 was induced only in MDA-MB-231, suggesting differential LPARs participation in the expression of these cytokines. Expression of IL-8 but not IL-11 was suppressed by inhibitors of PI3K, NF-kB, ROCK and PKC pathways. In the case of PKC activation, it was observed that $PKC{\delta}$ and $PKC{\mu}$ might regulate LPA-induced expression of IL-11 and IL-8, respectively, by using specific PKC subtype inhibitors. Finally, conditioned Medium from LPA-stimulated breast cancer cells induced osteoclastogenesis. In conclusion, LPA induced the expression of osteolytic cytokines (IL-8 and IL-11) in breast cancer cells by involving different LPA receptors. Enhanced expression of IL-8 by LPA may be via ROCK, PKCu, PI3K, and NFkB signaling pathways, while enhanced expression of IL-11 might involve $PKC{\delta}$ signaling pathway. LPA has the ability to enhance breast cancer cells-mediated osteoclastogenesis by inducing the secretion of cytokines such as IL-8 and IL-11.

• Molecules and Cells
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• 제28권1호
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• pp.13-17
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• 2009

Basic fibroblast growth factor (bFGF) plays an important role in angiogenesis. However, the underlying mechanisms are not clear. $Mg^{2+}$ is the most abundant intracellular divalent cation in the body and plays critical roles in many cell functions. We investigated the effect of bFGF on the intracellular $Mg^{2+}$ concentration ($[Mg^{2+}]_i$) in human umbilical vein endothelial cells (HUVECs). bFGF increased ($[Mg^{2+}]_i$) in a dose-dependent manner, independent of extracellular $Mg^{2+}$. This bFGF-induced $[Mg^{2+}]_i$ increase was blocked by tyrosine kinase inhibitors (tyrphostin A-23 and genistein), phosphatidylinositol 3-kinase (PI3K) inhibitors (wortmannin and LY294002) and a phospholipase $C{\gamma}$ ($PLC{\gamma}$) inhibitor (U73122). In contrast, mitogen-activated protein kinase inhibitors (SB202190 and PD98059) did not affect the bFGF-induced $[Mg^{2+}]_i$ increase. These results suggest that bFGF increases the $[Mg^{2+}]_i$ from the intracellular $Mg^{2+}$ stores through the tyrosine kinase/PI3K/$PLC{\gamma}$-dependent signaling pathways.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2002년도 창립10주년기념 및 국립독성연구원 의약품동등성평가부서 신설기념 국재학술대회:생물학적 동등성과 의약품 개발 전략을 위한 국제심포지움
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• pp.89-96
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• 2002

The importance of apoptosis in normal development and pathogenesis has been well recognized, and explosive progress towards dissecting its commitment step has been made during the past decade. Mitochondria, Apaf-1, caspase, and bcl-2 family members play central roles in the commitment step. However, it is still unclear how upstream cell survival pathways regulate apoptosis. It is also unknown whether the bcl-2 family members have any effect on the upstream survival pathways. We have demonstrated that the anti-apoptotic gene product bcl-2 greatly induces expression of the tissue inhibitor of metalloproteinase-1 (TIMP-1) in human breast epithelial cells. Surprisingly, we found that TIMP-1, like bcl-2, is a potent inhibitor of apoptosis induced by a variety of stimuli. Functional studies indicate that TIMP-1 inhibits a classical apoptotic pathway mediated by caspases, and that focal adhesion kinase (FAK)/Pl 3-kinase and mitogen activated protein kinase (MAPK) are critical for TIMP- 1 -mediated cell survival. We also showed specific association of TIMP-1 with the cell surface. Consistently, a 150-H)a surface protein was identified in MCF10A cells that specifically binds TIMP-1. Taken together, we hypothesize that TIMP-I binding on the cell surface induces a cell survival pathway that regulates the common apoptosis commitment step. The results of these studies will address a new paradigm in the regulation of apoptosis by an extracellular molecule TIMP-1, and also greatly enhance our understanding of TIMP-1's pleiotropic activity in many physiological and pathological processes. This information may also be useful in designing more rational therapeutic interventions aimed at modulating the anti-apoptotic activity of TIMP-1 .

• BMB Reports
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• 제33권6호
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• pp.525-530
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• 2000

The nerve growth factor (NGF) induces neuronal differentiation and neurite outgrowth of PC12 cells, whereas epidermal growth factors (EGF) stimulate growth and proliferation of the cells. In spite of this difference, NGF-or EGF-treated PC12 cells share various properties in cellular-signaling pathways. These include the activation of the phosphoinositide (PI)-3 kinase, 70 kDa S6 kinase, and in the mitogen-activated protein (MAP) kinase pathway, following the binding of these growth factors to intrinsic receptor tyrosine kinases (RTKs). Therefore, many studies have been attempted to access the critical signaling events in determining the differentiation and proliferation of PC12 cells. In this study, we investigated the cytosolic phospholipase $A_2$ ($cPLA_2$) in neurite behavior in order to identify the differences of signaling pathways between the NGF-induced differentiation and the EGF-induced proliferation of PC12 cells. We have showed here that the $cPLA_2$ was translocated from cytosol to membrane only in NGF-treated cells. We also demonstrated that this translocation is associated with NGF-induced activation of phospholipase $C-{\gamma}(PLC-{\gamma})$, which elevates intracellular $Ca^{2+}$ concentration. These results reveal that the translocation of $cPLA_2$ may be a requisite event in the neuronal differentiation of PC12 cells. Various phospholipase inhibitors were used to confirm the importance of these enzymes in the differentiation of PC12 cells. Neomycin B, a PLC inhibitor, dramatically inhibited the neurite outgrowth, and two distinct $PLA_2$ inhibitors, 4-bromophenacyl bromide (BPB) and arachidonyltrifluoro-methyl ketone ($AACOCF_3$) also suppressed the neurite outgrowth of the cells, as well Taken together, these data indicated that $cPLA_2$ is involved in NGF-induced neuronal differentiation and neurite outgrowth of PC12 cells.

• Molecules and Cells
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• 제43권3호
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• pp.264-275
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• 2020

Reactive oxygen species (ROS) play a significant role in intracellular signaling and regulation, particularly when they are maintained at physiologic levels. However, excess ROS can cause cell damage and induce cell death. We recently reported that eIF2α phosphorylation protects hepatocytes from oxidative stress and liver fibrosis induced by fructose metabolism. Here, we found that hepatocyte-specific eIF2α phosphorylation-deficient mice have significantly reduced expression of the epidermal growth factor receptor (EGFR) and altered EGFR-mediated signaling pathways. EGFR-mediated signaling pathways are important for cell proliferation, differentiation, and survival in many tissues and cell types. Therefore, we studied whether the reduced amount of EGFR is responsible for the eIF2α phosphorylation-deficient hepatocytes' vulnerability to oxidative stress. ROS such as hydrogen peroxide and superoxides induce both EGFR tyrosine phosphorylation and eIF2α phosphorylation. eIF2α phosphorylation-deficient primary hepatocytes, or EGFR knockdown cells, have decreased ROS scavenging ability compared to normal cells. Therefore, these cells are particularly susceptible to oxidative stress. However, overexpression of EGFR in these eIF2α phosphorylation-deficient primary hepatocytes increased ROS scavenging ability and alleviated ROS-mediated cell death. Therefore, we hypothesize that the reduced EGFR level in eIF2α phosphorylation-deficient hepatocytes is one of critical factors responsible for their susceptibility to oxidative stress.

• Biomolecules & Therapeutics
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• 제20권3호
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• pp.299-305
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• 2012

Endochondral bone formation is the process by which mesenchymal cells condense to become chondrocytes, which ultimately form new bone. The process of chondrogenic differentiation and hypertrophy is critical for bone formation and as such is regulated by many factors. In this study, we aimed to indentify novel factors that regulate chondrogenesis. We investigated the possible role of isopsoralen in induction of chondrogenic differentiation in clonal mouse chondrogenic ATDC5 cells. Isopsoralen treatment stimulated the accumulation of cartilage nodules in a dose-dependent manner. Further, ATDC5 cells treated with isopsoralen were stained more intensely with Alcian blue than control cells, suggesting that isopsoralen increases the synthesis of matrix proteoglycans. Similarly, isopsoralen markedly induced the activation of alkaline phosphatase activity compared with control cells. Isopsoralen enhanced the expressions of chondrogenic marker genes such as collagen II, collagen X, OCN, Smad4 and Sox9 in a time-dependent manner. Furthermore, isopsoralen induced the activation of extracellular signal-regulated kinase (ERK) and p38 MAP kinase, but not that of c-jun N-terminal kinase (JNK). Isopsoralen significantly enhanced the protein expression of BMP-2 in a time-dependent manner. PD98059 and SB 203580, inhibitors of ERK and p38 MAPK, respectively, decreased the number of stained cells treated with isopsoralen. Taken together, these results suggest that isopsoralen mediates a chondromodulating effect by BMP-2 or MAPK signaling pathways, and is therefore a possible therapeutic agent for bone growth disorders.

• Molecules and Cells
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• 제38권1호
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• pp.20-25
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• 2015

TGF-${\beta}$ regulates pleiotropic cellular responses including cell growth, differentiation, migration, apoptosis, extracellular matrix production, and many other biological processes. Although non-Smad signaling pathways are being increasingly reported to play many roles in TGF-${\beta}$-mediated biological processes, Smads, especially receptor-regulated Smads (R-Smads), still play a central mediatory role in TGF-${\beta}$ signaling for epithelial-mesenchymal transition. Thus, the biological activities of R-Smads are tightly regulated at multiple points. Inhibitory Smad (I-Smad also called Smad7) acts as a critical endogenous negative feedback regulator of Smad-signaling pathways by inhibiting R-Smad phosphorylation and by inducing activated type I TGF-${\beta}$ receptor degradation. Roles played by Smad7 in health and disease are being increasingly reported, but the molecular mechanisms that regulate Smad7 are not well understood. In this study, we show that E3 ubiquitin ligase Itch acts as a positive regulator of TGF-${\beta}$ signaling and of subsequent EMT-related gene expression. Interestingly, the Itch-mediated positive regulation of TGF-${\beta}$ signaling was found to be dependent on Smad7 ubiquitination and its subsequent degradation. Further study revealed Itch acts as an E3 ubiquitin ligase for Smad7 polyubiquitination, and thus, that Itch is an important regulator of Smad7 activity and a positive regulator of TGF-${\beta}$ signaling and of TGF-${\beta}$-mediated biological processes. Accordingly, the study uncovers a novel regulatory mechanism whereby Smad7 is controlled by Itch.

• Journal of Veterinary Science
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• 제23권4호
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• pp.47.1-47.11
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• 2022

Background: In lipopolysaccharide-induced RAW264.7 cells, Aster tataricus (AT) inhibits the nuclear factor kappa-light-chain-enhancer of activated B cells and MAPKs pathways and critical pathways of osteoclast development and bone resorption. Objectives: This study examined how aster saponin A2 (AS-A2) isolated from AT affects the processes and function of osteoclastogenesis induced by receptor activator of nuclear factor kappa-B ligand (RANKL) in RAW264.7 cells and bone marrow macrophages (BMMs). Methods: The cell viability, tartrate-resistant acid phosphatase staining, pit formation assay, polymerase chain reaction, and western blot were carried out to determine the effects of AS-A2 on osteoclastogenesis. Results: In RAW264.7 and BMMs, AS-A2 decreased RANKL-initiated osteoclast differentiation in a concentration-dependent manner. In AS-A2-treated cells, the phosphorylation of ERK1/2, JNK, and p38 protein expression were reduced considerably compared to the control cells. In RAW264.7 cells, AS-A2 suppressed the RANKL-induced activation of osteoclast-related genes. During osteoclast differentiation, AS-A2 suppressed the transcriptional and translational expression of NFATc1 and c-Fos. AS-A2 inhibited osteoclast development, reducing the size of the bone resorption pit area. Conclusion: AS-A2 isolated from AT appears to be a viable therapeutic therapy for osteolytic illnesses, such as osteoporosis, Paget's disease, and osteogenesis imperfecta.