• BMB Reports
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• v.49 no.8
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• pp.437-442
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• 2016

We aimed to study the role of protein L-isoaspartyl methyltransferase (PIMT) in neuronal differentiation using basic fibroblast growth factor (bFGF)-induced neuronal differentiation, characterized by cell-body shrinkage, long neurite outgrowth, and expression of neuronal differentiation markers light and medium neurofilaments (NF). The bFGF-mediated neuronal differentiation of PC12 cells was induced through activation of mitogen-activated protein kinase (MAPK) signaling molecules [MAPK kinase 1/2 (MEK1/2), extracellular signal-regulated kinase 1/2 (ERK1/2), and p90RSK], and phosphatidylinositide 3-kinase (PI3K)/Akt signaling molecules PI3Kp110β, PI3Kp110γ, Akt, and mTOR. Inhibitors (adenosine dialdehyde and S-adenosylhomocysteine) of protein methylation suppressed bFGF-mediated neuronal differentiation of PC12 cells. PIMT-eficiency caused by PIMT-specific siRNA inhibited neuronal differentiation of PC12 cells by suppressing phosphorylation of MEK1/2 and ERK1/2 in the MAPK signaling pathway and Akt and mTOR in the PI3K/Akt signaling pathway. Therefore, these results suggested that PIMT was critical for bFGF-mediated neuronal differentiation of PC12 cells and regulated the MAPK and Akt signaling pathways.

• Journal of Microbiology and Biotechnology
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• v.16 no.3
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• pp.391-398
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• 2006

$NF-{\kappa}B$ is a transcription factor involved in the innate immunity against bacterial infection and inflammation. It is also known to render cells resistant to the apoptosis caused by some anticancer drugs. Such a chemoresistance of cancer cells may be related to the activation of $NF-{\kappa}B$ transcription factor; however, the mechanism of activation is not well understood. Here, we demonstrate that a chemotherapeutic agent, etoposide, independently stimulates the $I{\kappa}B{\alpha}$ degradation pathway and PI3-kinase/Akt signaling pathway: The classical $I{\kappa}B{\alpha}$ degradation pathway leads to the nuclear translocation and DNA binding of p65 subunit through $IKK{\beta}$ kinase, whereas the PI3-kinase/Akt pathway plays a distinct role in activating this transcription factor. The PI3-kinase/Akt pathway acts on the p50 subunit of the $NF-{\kappa}B$ transcription factor and enhances the DNA binding affinity of the p50 protein. It may also explain the role of the PI3-kinase/Akt pathway in the anti-apoptotic function of $NF-{\kappa}B$ during chemoresistance of cancer cells.

• Journal of Life Science
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• v.26 no.10
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• pp.1182-1188
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• 2016

Membrane-associated guanylate kinase inverted-3 (MAGI-3) is a member of the family of membrane-associated guanylate kinases (MAGUKs). MAGI-3 modulates the kinase activity of protein kinase B (PKB)/AKT through interactions with phosphatase and tensin homolog (PTEN)/MMAC. Coxsackievirus B3 (CVB3) is a common causative agent of acute myocarditis and chronic dilated cardiomyopathy. Activation of AKT and extracellular signal-regulated kinases 1/2 (ERK1/2) is essential for CVB3 replication, but the relation between MAGI-3 signaling and CVB3 replication is not well understood. This study investigated the role of MAGI-3 in CVB3 infection and replication. MAGI-3 was overexpressed in HeLa cells by polyethylenimine (PEI) transfection. To optimize the transfection conditions, different ratios of plasmid DNA to PEI concentrations were used. MAGI-3 and empty plasmid DNA were transfected into the HeLa cells. MAGI-3 overexpression alone was not sufficient to efficiently activate AKT. However, expression of the CVB3 capsid protein VP1 dramatically increased in the HeLa cells overexpressing MAGI-3 24 h after CVB3 infection. In addition, the activities of AKT and ERK were significantly induced in the CVB3-infected MAGI-3 cells overexpressing HeLa. These results demonstrate that MAGI-3 expression upregulates CVB3 replication through AKT and ERK signaling activation. MAGI-3 may be an important target to control CVB3 replication.

• Journal of Applied Biological Chemistry
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• v.64 no.4
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• pp.447-451
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• 2021

In this study, we discovered for the first time that linarin, a flavonoid compound, enhances melanin biosynthesis in B16F10 cells, and subsequently elucidated the underlying mechanism of linarin-induced melanogenesis. Linarin showed no cytotoxicity at a concentration of 42 μM and significantly increased intracellular tyrosinase activity and melanin content in B16F10 cells. Mechanistic analysis showed that linarin increased the expression of tyrosinase, tyrosinase-related protein 1 (TRP-1), and microphthalmia-associated transcription factor (MITF) that are related to melanogenesis. Moreover, linarin decreased the phosphorylation of extracellular signal-regulated kinase (ERK) and protein kinase B (AKT). Finally, we evaluated the effect of the structure-activity relationship of linarin and its aglycone on melanogenesis. The results indicated that linarin enhances the expression of melanogenic proteins by activating MITF expression via the modulation of mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and protein kinase B signaling pathways in B16F10 cells, thereby enhancing melanogenesis.

• Journal of Applied Biological Chemistry
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• v.48 no.4
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• pp.178-182
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• 2005

Nitric oxide (NO) produced from endothelial cells plays a critical role in vascular physiology. The regulation of endothelial NO synthase (eNOS) involves various mechanisms including multiple Ser/Thr phosphorylations. Recently, eNOS-Ser617 was newly recognized to be phosphorylated in response to humoral factors including vascular endothelial growth factor. However, it remains unknown whether and how eNOS-Ser617 phosphorylation is stimulated by shear stress, the primary stimulus of endothelial NO production. This issue was explored in the present study using cultured bovine aortic endothelial cells (BAECs). Over-expression of a constitutively active protein kinase B(Akt) mutant in BAECs increased Ser617 phosphorylation while constitutively active protein kinase A mutant had no effect. When BAECs were subjected to an arterial level of laminar shear stress, eNOS-Ser617 phosphorylation was clearly increased in a time-dependent manner. Shear stress also stimulated Akt phosphorylation at Thr308, one of the key regulatory sites. The time courses of eNOS-Ser617 and Akt-Thr308 phosphorylations appeared to be very similar. These results suggested that eNOS-Ser617 phosphorylation, mediated by Akt, is a physiological response to the mechanical shear stress, involved in the regulation of NO production in endothelial cells.

• Biomedical Science Letters
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• v.29 no.3
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• pp.190-200
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• 2023

Skeletal muscle, essential for metabolism, thermoregulation, and immunity, undergoes myogenic differentiation that results in myotube formation. Trans-anethole (TA), the major constituent in essential oil produced by anise, star anise, and fennel, whose function in skeletal muscle has not yet been elucidated. Therefore, we investigated whether TA influenced muscle differentiation in mouse C2C12 myoblasts. Cells were induced to differentiate using a differentiation medium with or without TA (50 or 200 mg/mL) daily for 5 days. We measured myotube length and diameter after differentiation days 1, 3, and 5 and analyzed the expression of myogenic markers (myoblast determination protein 1, myogenin, myocyte enhancer factor 2, muscle creatine kinase, and myosin heavy chain) and atrophy-related genes (atrogin-1 and muscle ring finger-1 [MuRF-1]) using quantitative real-time PCR. Additionally, we observed the expression of total protein kinase B (Akt) and phosphorylated Akt (p-Akt) using western blotting. Our data showed that TA significantly induced the formation of smaller and thinner myotubes and reduced the myogenic factor expression. Furthermore, the atrogin-1 and MuRF-1 expression markedly increased by TA. Consistent with these findings, TA significantly decreased the expression of total Akt and p-Akt. Taken together, these results indicate that TA inhibits myogenic differentiation of C2C12 cells via reduction of both total Akt and p-Akt. Our findings may provide valuable insights into the impact of PAA on individuals at risk of muscle atrophy.

• International Journal of Oral Biology
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• v.45 no.4
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• pp.169-178
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• 2020

L-ascorbic acid (L-AA; vitamin C) induces apoptosis in cancer cells. This study aimed to elucidate the molecular mechanisms of L-AA-induced apoptosis in human laryngeal epidermoid carcinoma Hep-2 cells. L-AA suppressed the viability of Hep-2 cells and induced apoptosis, as shown by the cleavage and condensation of nuclear chromatin and increased number of Annexin V-positive cells. L-AA decreased Bcl-2 protein expression but upregulated Bax protein levels. In addition, cytochrome c release from the mitochondria into the cytosol and activation of caspase-9, -8, and -3 were enhanced by L-AA treatment. Furthermore, apoptosis-inducing factor (AIF) and endonuclease G (EndoG) were translocated into the nucleus during apoptosis of L-AA-treated Hep-2 cells. L-AA effectively inhibited the constitutive nuclear factor-κB (NF-κB) activation and attenuated the nuclear expression of the p65 subunit of NF-κB. Interestingly, L-AA treatment of Hep-2 cells markedly activated Akt and mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase [JNK]) and and LY294002 (Akt inhibitor), SB203580 (p38 inhibitor) or SP600125 (a JNK inhibitor) decreased the levels of Annexin V-positive cells. These results suggested that L-AA induces the apoptosis of Hep-2 cells via the nuclear translocation of AIF and EndoG by modulating the Bcl-2 family and MAPK/Akt signaling pathways.

• YAKHAK HOEJI
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• v.48 no.2
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• pp.105-110
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• 2004

Akt (or Protein Kinase B; PKB) is a serine/threonine kinase and is activated by phosphoinositide 3-kinase (PI3K) pathway. Recent evidence indicates that the abnormal activities or expression of Akt is closely associated with cancer, diabetes and neuro-degenerative diseases. These findings mean that Akt is likely to be a new therapeutic target for the treatment of disease. Here, we screened the effects of dithiolo-dithione derivatives such as SWU-20004, SWU-20009 and SWU-20025 on Akt activities. Among these compounds, only SWU-20009 (2-Thioxo-[1,3]dithiolo[4,5- $\beta$][1,4]dithiine-5,6-dicarboxylic acid dimethyl ester) inhibited the growth of KATOIII cell at micromolar range of concentration. Further investigation also revealed that SWU-20009 inhibited cellular Akt activity and induced apoptotic cell death.

• Journal of Applied Biological Chemistry
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• v.64 no.4
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• pp.323-331
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• 2021

Lincomycin is a lincosamide antibiotic isolated from the actinomycete Streptomyces lincolnensis. Moreover, it has been found to be effective against infections caused by Staphylococcus, Streptococcus, and Bacteroides fragillis. To identify the melanin-inducing properties of lincomycin, we used B16F10 melanoma cells in this study. The melanin content and intracellular tyrosinase activity in the cells were increased by lincomycin, without any cytotoxicity. Western blot analysis indicated that the protein expressions of tyrosinase, tyrosinase related protein 1 (TRP1) and TRP2 increased after lincomycin treatment. In addition, lincomycin enhanced the expression of master transcription regulator of melanogenesis, a microphthalmia-associated transcription factor (MITF). Lincomycin also increased the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and decreased the AKT phosphorylation. Moreover, the activation of tyrosinase activity by lincomycin was inhibited by the treatment with SB203580, which is p38 inhibitor. Furthermore, we also found that lincomycin-induced tyrosinase expression was reduced by H-89, a specific protein kinase A (PKA) inhibitor. These results indicate that lincomycin stimulate melanogenesis via MITF activation via p38 MAPK, AKT, and PKA signal pathways. Thus, lincomycin can potentially be used for treatment of hypopigmentation disorders.

• Molecules and Cells
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• v.25 no.3
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• pp.397-406
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• 2008

Computational modeling of signal transduction is currently attracting much attention as it can promote the understanding of complex signal transduction mechanisms. Although several mathematical models have been used to examine signaling pathways, little attention has been given to crosstalk mechanisms. In this study, an attempt was made to develop a computational model for the pathways involving growth-factor-mediated mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3'-kinase/protein kinase B (PI3K/Akt). In addition, the dynamics of the protein activities were analyzed based on a set of kinetic data. The simulation approach integrates the information on several levels and predicts systems behavior. The in-silico analysis conducted revealed that the Raf and Akt pathways act independently.