• Molecules and Cells
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• 제28권4호
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• pp.321-329
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• 2009

Rapid production of nitric oxide (NO) and reactive oxygen species (ROS) has been implicated in the regulation of innate immunity in plants. A potato calcium-dependent protein kinase (StCDPK5) activates an NADPH oxidase StRBOHA to D by direct phosphorylation of N-terminal regions, and heterologous expression of StCDPK5 and StRBOHs in Nicotiana benthamiana results in oxidative burst. The transgenic potato plants that carry a constitutively active StCDPK5 driven by a pathogen-inducible promoter of the potato showed high resistance to late blight pathogen Phytophthora infestans accompanied by HR-like cell death and $H_2O_2$ accumulation in the attacked cells. In contrast, these plants showed high susceptibility to early blight necrotrophic pathogen Alternaria solani, suggesting that oxidative burst confers high resistance to biotrophic pathogen, but high susceptibility to necrotrophic pathogen. NO and ROS synergistically function in defense responses. Two MAPK cascades, MEK2-SIPK and cytokinesis-related MEK1-NTF6, are involved in the induction of NbRBOHB gene in N. benthamiana. On the other hand, NO burst is regulated by the MEK2-SIPK cascade. Conditional activation of SIPK in potato plants induces oxidative and NO bursts, and confers resistance to both biotrophic and necrotrophic pathogens, indicating the plants may have obtained during evolution the signaling pathway which regulates both NO and ROS production to adapt to wide-spectrum pathogens.

• The Korean Journal of Physiology and Pharmacology
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• 제9권5호
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• pp.299-304
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• 2005

Cardiac hypertrophy contributes an increased risk to major cerebrovascular events. However, the molecular mechanisms underlying cerebrovascular dysfunction during cardiac hypertrophy have not yet been characterized. In the present study, we examined the molecular mechanism of isoproterenol (ISO)-evoked activation of Ras/Raf/MAPK pathways as well as PKA activity in cerebral artery of rabbits, and we also studied whether the activations of these signaling pathways were altered in cerebral artery, during ISO-induced cardiac hypertrophy compared to heart itself. The results show that the mRNA level of c-fos (not c-jun and c-myc) in heart and these genes in cerebral artery were considerably increased during cardiac hypertrophy. These results that the PKA activity and activations of Ras/Raf/ERK cascade as well as c-fos expression in rabbit heart during cardiac hypertrophy were consistent with previous reports. Interestingly, however, we also showed a novel finding that the decreased PKA activity might have differential effects on Ras and Raf expression in cerebral artery during cardiac hypertrophy. In conclusion, there are differences in molecular mechanisms between heart and cerebral artery during cardiac hypertrophy when stimulated with β2 adrenoreceptor (AR), suggesting a possible mechanism underlying cerebrovascular dysfunction during cardiac hypertrophy.

• BMB Reports
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• 제28권4호
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• pp.316-322
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• 1995

The GTPase activating protein (GAP) can function both as a negative regulator and an effector of $p21^{ras}$. Overexpression of GAP in NIH-3T3 cells has been shown to inhibit transformation by ms or src. To investigate the function of GAP in a differentiative system, we overexpressed this protein in the nerve growth factor (NGF)-responsive PC12 cell line. Two-fold overexpression of GAP caused a delay of several days in the onset of NGF- but not FGF-induced neuronal differentiation of PC12 cells. However, the NGF-induced activation or tyrosine phosphorylation of upstream (Trk, PLC-${\gamma}1$, SHC) and downstream (B-Raf and $p44^{mapk/erk1}$) components of $p21^{ras}$, signalling cascade was not altered by GAP overexpression. Therefore, the change of phenotype induced by GAP was probably not due to GAP functioning as a negative regulator of $p21^{ras}$. Rather, we found that NGF-induced tyrosine phosphorylation of SNT, a specific target of neurotrophin-induced tyrosine kinase activity, was inhibited by GAP overexpression. SNT is thought to function upstream or independent of $p21^{ras}$. Thus in PC12 cells, overexpressed GAP may control the rate of neuronal differentiation through a pathway involving SNT rather than the $p21^{ras}$ signalling pathway.

• 한국발생생물학회지:발생과생식
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• 제24권2호
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• pp.89-100
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• 2020

Clerodendrum trichotomum is a folk medicine exhibiting anti-hypertension, anti-arthritis, and anti-rheumatism properties. However, little is known about whether the material might prevent hyperuricemia and associated inflammation. In this study, we explored whether C. trichotomum leaf extract (CTE) prevented hyperuricemia induced by potassium oxonate (PO) in mice. CTE (400 mg/kg body weight) significantly reduced the serum uric acid (UA), blood urea nitrogen (BUN), and serum creatinine levels and increased urine UA and creatinine levels. CTE ameliorated PO-induced inflammation and apoptosis by reducing the levels of relevant proteins in kidney tissues. Also, CTE ameliorated both UA-induced inflammatory response in RAW 263.7 cells and UA-induced cytotoxicity in HK-2 cells. In addition, liver transcriptome analysis showed that CTE enriched mainly the genes for mediating positive regulation of MAPK cascade and apoptotic signaling pathways. Together, the results show that CTE effectively prevents hyperuricemia and associated inflammation in PO-induced mice.

• Molecules and Cells
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• 제20권2호
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• pp.280-287
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• 2005

The insulin-mediated Ras/mitogen-activated protein (MAP) kinase cascade was examined in SK-N-BE(2) and PC12 cells, which can and cannot produce reactive oxygen species (ROS), respectively. Tyrosine phosphorylation of the insulin receptor and insulin receptor substrate 1 (IRS-1) was much lower in SK-N-BE(2) cells than in PC12 cells when the cells were treated with insulin. The insulin-mediated interaction of IRS-1 with Grb2 was observed in PC12 but not in SK-N-BE(2) cells. Moreover, the activity of extracellular-signal-related kinase (ERK) was much lower in SK-N-BE(2) than in PC12 cells when the cells were treated with insulin. Application of exogenous $H_2O_2$ caused increased tyrosine phosphorylation and Grb2 binding to IRS-1 in SK-N-BE(2) cells, while exposure to an $H_2O_2$ scavenger (N-acetylcysteine) or to a phophatidylinositol-3 kinase inhibitor (wortmannin), and expression of a dominant negative Rac1, decreased the activation of ERK in insulin-stimulated PC12 cells. These results indicate that the transient increase of ROS is needed to activate ERK in insulin-mediated signaling and that an inability to generate ROS is the reason for the insulin insensitivity of SK-N-BE(2) cells.

• BMB Reports
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• 제38권6호
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• pp.639-645
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• 2005

Protein kinase C (PKC) isozymes, a family of serine-threonine kinases, are important regulators of cell proliferation and malignant transformation. Phorbol esters, the prototype PKC activators, cause PKC translocation to the plasma membrane in prostate cancer cells, and trigger an apoptotic response. Studies in recent years have determined that each member of the PKC family exerts different effects on apoptotic or survival pathways. $PKC{\delta}$, one of the novel PKCs, is a key player of the apoptotic response via the activation of the p38 MAPK pathway. Studies using RNAi revealed that depletion of $PKC{\delta}$ totally abolishes the apoptotic effect of the phorbol ester PMA. Activation of the classical $PKC{\alpha}$ promotes the dephosphorylation and inactivation of the survival kinase Akt. Studies have assigned a pro-survival role to $PKC{\varepsilon}$, but the function of this PKC isozyme remains controversial. Recently, it has been determined that the PKC apoptotic effect in androgen-dependent prostate cancer cells is mediated by the autocrine secretion of death factors. $PKC{\delta}$ stimulates the release of $TNF{\alpha}$ from the plasma membrane, and blockade of $TNF{\alpha}$ secretion or $TNF{\alpha}$ receptors abrogates the apoptotic response of PMA. Molecular analysis indicates the requirement of the extrinsic apoptotic cascade via the activation of death receptors and caspase-8. Dissecting the pathways downstream of PKC isozymes represents a major challenge to understanding the molecular basis of phorbol ester-induced apoptosis.

• Journal of Microbiology and Biotechnology
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• 제18권2호
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• pp.355-364
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• 2008

Mushrooms are regarded as one of the well-known foods and biopharmaceutical materials with a great deal of interest. ${\beta}$-Glucan is the major component of mushrooms that displays various biological activities such as antidiabetic, anticancer, and antihyperlipidemic effects. In this study, we explored the molecular mechanism of its immunostimulatory potency in immune responses of macrophages, using exopolysaccharides prepared from liquid culture of Lentinus edodes. We found that fraction II (F-II), with large molecular weight protein polysaccharides, is able to strongly upregulate the phenotypic functions of macrophages such as phagocytic uptake, ROS/NO production, cytokine expression, and morphological changes. F-II triggered the nuclear translocation of NF-${\kappa}B$ and activated its upstream signaling cascades such as PI3K/Akt and MAPK pathways, as assessed by their phosphorylation levels. The function-blocking antibodies to dectin-1 and TLR-2, but not CR3, markedly suppressed F-II-mediated NO production. Therefore, our data suggest that mushroom-derived ${\beta}$-glucan may exert its immunostimulating potency via activation of multiple signaling pathways.

• BMB Reports
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• 제46권12호
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• pp.594-599
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• 2013

The anti-inflammatory activity of eriodictyol and its mode of action were investigated. Eriodictyol suppressed tumor necrosis factor (mTNF)-${\alpha}$, inducible nitric oxide synthase (miNOS), interleukin (mIL)-6, macrophage inflammatory protein (mMIP)-1, and mMIP-2 cytokine release in LPS-stimulated macrophages. We found that the anti-inflammatory cascade of eriodictyol is mediated through the Toll-like Receptor (TLR)4/CD14, p38 mitogen-activated protein kinases (MAPK), extracellular-signal-regulated kinase (ERK), Jun-N terminal kinase (JNK), and cyclooxygenase (COX)-2 pathway. Fluorescence quenching and saturation-transfer difference (STD) NMR experiments showed that eriodictyol exhibits good binding affinity to JNK, $8.79{\times}10^5M^{-1}$. Based on a docking study, we propose a model of eriodictyol and JNK binding, in which eriodictyol forms 3 hydrogen bonds with the side chains of Lys55, Met111, and Asp169 in JNK, and in which the hydroxyl groups of the B ring play key roles in binding interactions with JNK. Therefore, eriodictyol may be a potent anti-inflammatory inhibitor of JNK.

• Molecules and Cells
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• 제24권1호
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• pp.95-104
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• 2007

The mechanism of acacetin-induced apoptosis of human breast cancer MCF-7 cells was investigated. Acacetin caused 50% growth inhibition ($IC_{50}$) of MCF-7 cells at $26.4{\pm}0.7{\mu}M$ over 24 h in the MTT assay. Apoptosis was characterized by DNA fragmentation and an increase of sub-G1 cells and involved activation of caspase-7 and PARP (poly-ADP-ribose polymerase). Maximum caspase 7 activity was observed with $100{\mu}M$ acacetin for 24 h. Caspase 8 and 9 activation cascades mediated the activation of caspase 7. Acacetin caused a reduction of Bcl-2 expression leading to an increase of the Bax:Bcl-2 ratio. It also caused a loss of mitochondrial membrane potential that induced release of cytochrome c and apoptosis inducing factor (AIF) into the cytoplasm, enhancing ROS generation and subsequently resulting in apoptosis. Pretreatment of cells with N-acetylcysteine (NAC) reduced ROS generation and cell growth inhibition, and pretreatment with NAC or a caspase 8 inhibitor (Z-IETD-FMK) inhibited the acacetin-induced loss of mitochondrial membrane potential and release of cytochrome c and AIF. Stress-activated protein kinase/c-Jun $NH_4$-terminal kinase 1/2 (SAPK/JNK1/2) and c-Jun were activated by acacetin but extracellular-regulated kinase 1/2 (Erk1/2) nor p38 mitogen-activated protein kinase (MAPK) were not. Our results show that acacetin-induced apoptosis of MCF-7 cells is mediated by caspase activation cascades, ROS generation, mitochondria-mediated cell death signaling and the SAPK/JNK1/2-c-Jun signaling pathway, activated by acacetin-induced ROS generation.

• Asian Pacific Journal of Cancer Prevention
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• 제15권13호
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• pp.5271-5276
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• 2014

Mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 3 (MEKK3) is an important protein kinase and a member of the MAPK family, which regulates cellular responses to environmental stress and serves as key integration points along the signal transduction cascade that not only link diverse extracellular stimuli to subsequent signaling molecules but also amplify the initiating signals to ultimately activate effector molecules and induce cell proliferation, differentiation and survival. To explore the relationship between MEKK3 and cell apoptosis, clinicopathology and prognosis, we characterize the expression of MEKK3 and survivin in cervical cancer. MEKK3 and survivin expression was measured by RT-PCR and Western blotting of fresh surgical resections from 30 cases of cervical cancer and 25 cases of chronic cervicitis. Protein expression was detected by tissue microarray and immunochemistry (En Vision) in 107 cases of cervical cancer, 86 cases of cervical intraepithelial neoplasia (CIN), and 35 cases of chronic cervicitis. Expression patterns were analyzed for their association with clinicopathological factors and prognosis in cervical cancer. Expression of MEKK3 and survivin mRNA was significantly higher in cervical cancer than in the controls (p<0.05). MEKK3 and survivin expression differed significantly between cervical carcinoma, CIN, and cervicitis (p<0.05) and correlated with clinical stage, infiltration depth, and lymph node metastasis (p<0.05). MEKK3 expression was positively correlated with survivin (p<0.05). Kaplan-Meier survival analysis showed that MEKK3 and survivin expression, lymph node metastasis, depth of invasion, and FIGO stage reduce cumulative survival. Cox multivariate regression analysis showed that MEKK3, survivin, and clinical staging are independent prognostic factors in cervical cancer (p<0.05). Expression of MEKK3 and survivin are significantly increased in cervical cancer, their overexpression participating in the occurrence and development of cervical cancer, with protein expression and clinical staging acting as independent prognostic factors for patients with cervical cancer.