Seong-Gene Lee

Interaction of human Ku70 with TRF2

Ku, a heterodimer of 70‐ and 80‐kDa subunits, plays a general role in the metabolism of DNA ends in eukaryotic cells, including double‐strand DNA break repair, V(D)J recombination, and maintenance of telomeres. We have utilized the yeast two‐hybrid system to identify Ku70‐interacting proteins other than Ku80. Two reactive clones were found to encode the dimerization domain of TRF2, a mammalian telomeric protein that binds to duplex TTAGGG repeats at chromosome ends. This interaction was confirmed using bacterial fusion proteins and co‐immunoprecipitations from eukaryotic cells overexpressing TRF2. The transfected TFR2 colocalized with Ku70.

HPLC Analysis of Serotonin, Tryptamine, Tyramine, and the Hydroxycinnamic Acid Amides of Serotonin and Tyramine in Food Vegetables

Biogenic monoamines such as serotonin, tryptamine, and tyramine function as neurotransmitters and mitogenic factors in animals and are involved in flowering, morphogenesis, and protection from and adaptation to environmental changes in plants. In plants, serotonin and tyramine are conjugated to form phenolic compounds via thioester linkages during the synthesis of hydroxycinnamic acid amides, including p-coumaroylserotonin (CS), feruloylserotonin (FS), p-coumaroyltyramine (CT), and feruloyltyramine (FT). In this study, we determined the amounts of the biogenic monoamines CS, FS, CT, and FT in commonly consumed vegetables using high-performance liquid chromatography. Serotonin, tryptamine, and tyramine were detected in all vegetables tested. The serotonin levels ranged from 1.8 to 294 microg/g of dry weight, the tryptamine levels ranged from 0.8 to 372 microg/g of dry weight, and the tyramine levels ranged from 1.4 to 286 microg/g of dry weight. The highest serotonin and tryptamine contents were found in tomato and cherry tomato (140.3-222 microg/g of dry weight), while paprika and green pepper had higher tyramine contents than the other vegetables (286 and 141.5 microg/g of dry weight, respectively). Overall, the levels of CS, FS, CT, and FT ranged from 0.03 to 13.8 microg/g of dry weight, with green onion possessing the highest levels of CS (0.69 microg/g of dry weight), FT (1.99 microg/g of dry weight), and CT (13.85 microg/g of dry weight).

Anti-inflammatory activity of a water extract of Acorus calamus L. leaves on keratinocyte HaCaT cells.

ETHNOPHARMACOLOGICAL RELEVANCE Acorus calamus L., sweet flag, is a well-known medicinal plant that grows worldwide wildly along swamps, rivers, and lakes. AIM OF THE STUDY The aim of this study was to evaluate the anti-inflammatory activity of Acorus calamus leaf (ACL) extract and to explore its mechanism of action on human keratinocyte HaCaT cells. MATERIALS AND METHODS HaCaT cells treated with polyinosinic:polycytidylic acid (polyI:C) and peptidoglycan (PGN) induced the inflammatory reactions. The anti-inflammatory activities of ACL were investigated using RT-PCR, ELISA assay, immunoblotting, and immunofluorescence staining. RESULTS HaCaT cells induced the pro-inflammatory cytokines, interleukin-8 (IL-8) and/or interleukin-6 (IL-6) expressions after treatment with polyI:C or PGN. ACL inhibited the expression of IL-8 and IL-6 RNA and protein levels, and attenuated the activation of NF-kappaB and IRF3 after polyI:C treatment. ACL also inhibited expression of IL-8 and activation of NF-kappaB following PGN induction. CONCLUSIONS These results suggest that ACL inhibits the production of pro-inflammatory cytokines through multiple mechanisms and may be a novel and effective anti-inflammatory agent for the treatment of skin diseases.

Gene–gene interaction between IL-13 and IL-13Rα1 is associated with total IgE in Korean children with atopic asthma

AbstractInterleukin (IL)-13, which is essential for IgE synthesis, mediates its effects by binding with a receptor composed of IL-4Rα and IL-13Rα1. We investigated the effects of IL-13 and IL-13Rα1 polymorphisms in Korean children with asthma, and whether these have been associated with IgE production. We enrolled 358 atopic asthmatic, 111 non-atopic asthmatic, and 146 non-atopic healthy children. IL-13 and IL-13Rα1 genotypes were identified using the PCR-RFLP method. There was an association between the asthma susceptibility and homozygosity for risk allele of IL-13 G+2044A. In children with atopic asthma, risk alleles in IL-13 (A−1512C and C−1112T) and IL-13Rα1 (A+1398G) showed increased total IgE (P=0.012, 0.015 and 0.017, respectively). Three-loci haplotype analysis for IL-13 showed that the haplotype composed of −1512C, −1112T and +2044A was associated with higher total IgE than other tested haplotypes in children with atopic asthma (P=0.003). The gene-gene interaction between risk alleles of each IL-13 promoter polymorphism and IL-13Rα1 polymorphism was associated with higher total IgE in children with atopic asthma (P=0.002, 0.010). These findings indicate that the IL-13 G+2044A is associated with asthma development and the IL-13 and IL-13Rα1 polymorphisms may interact to enhance IgE production.

Ginsenoside Rh1 suppresses matrix metalloproteinase-1 expression through inhibition of activator protein-1 and mitogen-activated protein kinase signaling pathway in human hepatocellular carcinoma cells

Invasion and metastasis are the major causes of treatment failure in patients with cancer. Here, we investigated the effects of ginsenoside Rh1 on tumor invasion and metastasis in human hepatocellular carcinoma HepG2 cells and its possible mechanism of action. Rh1 showed concentration- and time-dependent inhibition of HepG2 cell migration and invasion. Matrix metalloproteinase-1 (MMP-1) gene expression and its promoter activity were also concentration-dependently inhibited by Rh1 treatment. The inhibitory effect of Rh1 on MMP-1 expression was due to inactivation of the mitogen-activated protein kinases (MAPKs) ERK, JNK, and p38 MAPK. By transient transfection analysis with the MMP-1 promoter (-2846 to -29 nt) and AP-1 promoter, MMP-1 and AP-1 promoter activities were induced by phorbol myristate acetate (PMA) but were significantly inhibited by PD98059 (ERK1/2 inhibitor) or SP600125 (JNK inhibitor). The induction of MMP-1 and AP-1 promoters by PMA was attenuated by Rh1, and both promoter activities were synergistically inhibited by co-treatment with PD98059. To evaluate the effects of Rh1 on AP-1 dimers, expression analysis and electrophoretic mobility shift (EMSA) assay using radiolabeled AP-1-specific oligomers at proximal site (-73 nt) and distal site (-1600 nt) of the MMP-1 promoter were performed. The results showed that Rh1 inhibited the expression of c-Jun and c-Fos but did not affect the DNA binding ability of AP-1-specific oligomers. However, Rh1 attenuated the stability of c-Jun. Therefore, Rh1 has potential for development of novel chemotherapeutic agents for treatment of malignant cancers, including early hepatocellular carcinoma related to MMP-1 expression.

Anti-metastatic effects of ginsenoside Rd via inactivation of MAPK signaling and induction of focal adhesion formation.

Ginsenoside Rd is a protopanaxadiol-type ginsenoside found in ginseng and is the active ingredient in several Oriental herbal medicines. We investigated the effects of ginsenoside Rd on tumor invasion and metastasis in the human hepatocellular carcinoma HepG2 and its possible mechanism of action. HepG2 cells were treated with ginsenoside Rd at different concentrations. Scratch wound and Boyden chamber assays were used to determine the effects of ginsenoside Rd on the migration and invasiveness of HepG2 cells, respectively. The molecular mechanisms by which ginsenoside Rd inhibited the invasion and migration of HepG2 cells were investigated by RT-PCR, Western blotting, gelatin zymography, promoter assay, and treatment with inhibitors of MAPK signaling. Immunofluorescence analysis was conducted to evaluate the effect of ginsenoside Rd on focal adhesion formation in HepG2 cells. Treatment with ginsenoside Rd dose- and time-dependently inhibited the migration and invasion of HepG2 cells. It achieved this by reducing the expression of MMP-1, MMP-2, and MMP-7, by blocking MAPK signaling by inhibiting the phosphorylation of ERK and p38 MAPK, by inhibition of AP-1 activation, and by inducing focal adhesion formation and modulating vinculin localization and expression. Treatment of HepG2 cells with ginsenoside Rd significantly inhibited metastasis, most likely by blocking MMP activation and MAPK signaling pathways involved in cancer cell migration. These findings may be useful for the development of novel chemotherapeutic agents for the treatment of malignant cancers.

Caffeoylserotonin Protects Human Keratinocyte HaCaT Cells against H2O2-Induced Oxidative Stress and Apoptosis through Upregulation of HO-1 Expression via Activation of the PI3K/Akt/Nrf2 Pathway

Caffeoylserotonin (CaS) has strong radical scavenging activity as well as antioxidant activities, protecting cells from lipid peroxidation, intracellular reactive oxygen species generation, DNA damage, and cell death. The molecular mechanism by which CaS protects against oxidative stress is not well understood. Here, we analyzed the cytoprotective activity of CaS in hydrogen peroxide (H2O2)‐treated keratinocyte HaCaT cells. H2O2 induced apoptosis in the cells through activation of pro‐apoptotic p21, Bax, and caspase‐3. Pretreatment with CaS inhibited apoptotic gene expression and activated the anti‐apoptotic gene, Bcl‐xL. Although CaS did not directly affect heme oxygenase‐1 (HO‐1) expression, pretreatment with CaS augmented HO‐1 expression through an increase in NF‐E2‐related factor (Nrf2) stability and stimulation of Nrf2 translocation to the nucleus upon H2O2 exposure. H2O2 also induced the phosphorylation and subsequent activation of ERK, p38 MAPK, and Akt. Analysis using specific inhibitors of p38 MAPK and Akt demonstrated that only Akt activation was involved in HO‐1 and Nrf2 expressions. In addition, PI3K and PKC inhibitors suppressed HO‐1/Nrf2 expression and Akt phosphorylation. These results demonstrate that CaS protects against oxidative stress‐induced keratinocyte cell death in part through the activation of Nrf2‐mediated HO‐1 induction via the PI3K/Akt and/or PKC pathways, but not MAPK signaling. Copyright

The DEAD-box RNA helicase DDX3 interacts with DDX5, co-localizes with it in the cytoplasm during the G2/M phase of the cycle, and affects its shuttling during mRNP export.

DDX3 is involved in RNA transport, translational control, proliferation of RNA viruses, and cancer progression. From yeast two‐hybrid screening using the C‐terminal region of DDX3 as a bait, the DEAD‐box RNA helicase DDX5 was cloned. In immunofluorescence analysis, DDX3 and DDX5 were mainly co‐localized in the cytoplasm. Interestingly, cytoplasmic levels of DDX5 increased in the G2/M phase and consequently protein–protein interaction also increased in the cytoplasmic fraction. DDX3 was highly phosphorylated at its serine, threonine, and tyrosine residues in the steady state, but not phosphorylated at the serine residue(s) in the G2/M phase. DDX5 was less phosphorylated in the G1/S phase; however, it was highly phosphorylated at serine, threonine, and tyrosine residues in the G2/M phase. PP2A treatment of the cytoplasmic lysate from G2/M phase cells positively affected the interaction between DDX3 and DDX5, whereas, PTP1B treatment did not. In an analysis involving recombinant His‐DDX3 and His‐DDX5, PP2A pretreatment of His‐DDX5 increased the interaction with endogenous DDX3, and vice versa. Furthermore, the results of GST pull‐down experiments support the conclusion that dephosphorylation of serine and/or threonine residues in both proteins enhanced protein–protein interactions. UV cross‐linking experiments showed that DDX3 and DDX5 are involved in mRNP export. Additionally, DDX3 knockdown blocked the shuttling of DDX5 to the nucleus. These data demonstrate a novel interaction between DDX3 and DDX5 through the phosphorylation of both proteins, especially in the G2/M phase, and suggest a novel combined mechanism of action, involving RNP remodeling and splicing, for DEAD‐box RNA helicases involved in mRNP export. J. Cell. Biochem. 113: 985–996, 2012.

Stimulation of TRAF6/TAK1 degradation and inhibition of JNK/AP-1 signalling by ginsenoside Rg3 attenuates hepatitis B virus replication.

In present study, we investigated the effect of ginsenoside Rg3 on hepatitis B virus DNA replication and secretion of hepatitis B surface antigen and e antigen in HepG2.2.15 cells. Rg3 dose- and time-dependently inhibited hepatitis B surface antigen, e antigen, and hepatitis B viral particle secretion. To explore the effect of Rg3 on anti-hepatitis B activity, we analysed toll-like receptor-myeloid differentiation primary response gene 88 signalling. Rg3 did not affect the expression of toll-like receptors or myeloid differentiation primary response gene 88. However, it significantly inhibited the expression of TNF receptor-associated factor 6 and transforming growth factor β activated kinase-1, which are adaptor molecules that signal through a toll-like receptor-myeloid differentiation primary response gene 88-dependent pathway. The inhibitory effect of Rg3 on TNF receptor-associated factor 6/transforming growth factor β activated kinase-1 expression was caused by the downregulation of TNF receptor-associated factor 6 expression as well as the stimulation of ubiquitination and proteasomal degradation of TNF receptor-associated factor 6, followed by downregulation of transforming growth factor β activated kinase-1. Furthermore, Rg3 inhibited mitogen-activated protein kinase signalling by inhibiting c-Jun N-terminal kinase phosphorylation, reduced the expression of AP-1 transcription factors (especially c-Jun and JunB), and inhibited AP-1 promoter activity. The inhibitory effect of Rg3 on c-Jun N-terminal kinase/AP-1 signalling showed anti-inflammatory activity based on the reduction in the expression of pro-inflammatory cytokines, IL-8 and TNF-α, at both the transcriptional and translational levels. Therefore, Rg3 showed anti-hepatitis B activity via the degradation of TNF receptor-associated factor 6/transforming growth factor β activated kinase-1 and the inhibition of c-Jun N-terminal kinase/AP-1 signalling.

Induction of ATP synthase β by H2O2 induces melanogenesis by activating PAH and cAMP/CREB/MITF signaling in melanoma cells.

Hydrogen peroxide (H2O2) production due to oxidative stress is associated with apoptosis and melanogenesis in melanocytes. Here, we analyzed the effects of H2O2 on melanogenesis by measuring the melanin content and analyzing the expression of melanogenesis-related proteins, including cAMP-responsive element binding protein (CREB), microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), and phenylalanine hydroxylase (PAH). Treatment with 1mM H2O2 increased the cellular melanin content; the expression of PAH, TYR, and MITF; and the phosphorylation of CREB in B16F10 and SK-Mel-2 cells. In addition, H2O2 increased the expression of ATP synthase β (ATP5B), a mitochondrial F1 complex, and increased intracellular ATP levels. Studies using the ATP5B inhibitor oligomycin (OM) showed that the induction of cAMP resulted from an increase in ATP caused by the induction of ATP5B. OM treatment increased H2O2-mediated apoptosis via accelerated ATP depletion and apoptosis-related gene expressions. In summary, H2O2 may induce melanogenesis via the upregulation of PAH and activation of cAMP/p-CREB/MITF signaling by increasing intracellular cAMP levels through the induction of ATP5B.