• Molecules and Cells
• /
• v.42 no.11
• /
• pp.783-793
• /
• 2019

When endoplasmic reticulum (ER) functions are perturbed, the ER induces several signaling pathways called unfolded protein response to reestablish ER homeostasis through three ER transmembrane proteins: inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Although it is important to measure the activity of ATF6 that can indicate the status of the ER, no specific cell-based reporter assay is currently available. Here, we report a new cell-based method for monitoring ER stress based on the cleavage of $ATF6{\alpha}$ by sequential actions of proteases at the Golgi apparatus during ER stress. A new expressing vector was constructed by using fusion gene of GAL4 DNA binding domain (GAL4DBD) and activation domain derived from herpes simplex virus VP16 protein (VP16AD) followed by a human $ATF6{\alpha}$ N-terminal deletion variant. During ER stress, the GAL4DBD-VP16AD(GV)-$hATF6{\alpha}$ deletion variant was cleaved to liberate active transcription activator encompassing GV-$hATF6{\alpha}$ fragment which could translocate into the nucleus. The translocated GV-$hATF6{\alpha}$ fragment strongly induced the expression of firefly luciferase in HeLa Luciferase Reporter cell line containing a stably integrated 5X GAL4 site-luciferase gene. The established double stable reporter cell line HLR-GV-$hATF6{\alpha}$(333) represents an innovative tool to investigate regulated intramembrane proteolysis of $ATF6{\alpha}$. It can substitute active pATF6(N) binding motif-based reporter cell lines.

• Journal of Life Science
• /
• v.15 no.6 s.73
• /
• pp.928-934
• /
• 2005

ATF2 is a cellular transcription factor which belongs to the CREB/ATF class and it is leucine zipper protein which generally binds to DNA as dimers. This paper presents the procedure for subcloning the ATF2 gene and the results of experiment used the expressed ATF2. The pET expression vector was used since it produced 6xHis fusion protein for easy purification using affinity column. The Nickel chelating chromatography was used for Purifying the expressed ATE2 from E- codi BL2l. Subsequen시y In vitro binding pull-down assay showed the binding specificity of ATF2 with AP-1 family factors such as BATF, c-Fos, c-Jun and ATF2 itselgf. ATF2 forms homodimer as well as strong heterodimer with BATF. It also forms stable dimer with c-Jun but barely binds with c-Fos.

• Clinical and Experimental Reproductive Medicine
• /
• v.25 no.3
• /
• pp.305-313
• /
• 1998

Steroid hormone is known to cause the dynamic changes of mammalian uterus during reproductive cycle. However there is little information about the effect of estrogen (E) and progesterone (P) on the expression of various transcription factors involved in gene expression. Thus the present study was designed to demonstrate E and/or P-induced expression of c-Fos, CREB, ATF and HSP70 in rat uterus. Rats, ovariectomized (OVX) for two weeks, were divided into 6 experimental groups, 1) OVX, 2) OVX+V, 3) OVX+E, 4) OVX+P, 5) OVX+E+V, 6) OVX+E+P, and western blotting assay for nuclear extract and immunohistochemical staining were carried out for each experimental group. Treatment of E $(10{\mu}g)$ showed to increase the expression of c-Fos, CREB, ATF, and HSP70, and maximal expression was occured at $3\sim6$ hr after E administration. P (1mg) also increased, but much less than E, the expression of c-Fos, ATF, and HSP70. However, P did not reveal any effect on the expression CREE. P treatment 4 hr after E injection decreased c-Fos, CREB, and ATF expression, but did not show any change in the E-induced HSP70 expression. In immunohistochemical study c-Fos-, CREB-, and ATF-immunoreactivities were confined to the cells of luminal epithelium of uterine endometrium. These results suggest that proliferation and differentiation of rat uterus during reproductive cycle may mediated via expression of transcription factors, such as c-Fos, CREB, ATF, and HSP70.

• The Korean Journal of Physiology and Pharmacology
• /
• v.20 no.4
• /
• pp.415-424
• /
• 2016

Berberine is an isoquinoline alkaloid found in Rhizoma coptidis, and elicits anti-inflammatory effects through diverse mechanisms. Based on previous reports that activating transcription factor-3 (ATF-3) acts as a negative regulator of LPS signaling, the authors investigated the possible involvement of ATF-3 in the anti-inflammatory effects of berberine. It was found berberine concentration-dependently induced the expressions of ATF-3 at the mRNA and protein levels and concomitantly suppressed the LPS-induced productions of proinflammatory cytokines ($TNF-{\alpha}$, IL-6, and $IL-1{\beta}$). In addition, ATF-3 knockdown abolished the inhibitory effects of berberine on LPS-induced proinflammatory cytokine production, and prevented the berberine-induced suppression of MAPK phosphorylation, but had little effect on AMPK phosphorylation. On the other hand, the effects of berberine, that is, ATF-3 induction, proinflammatory cytokine inhibition, and MAPK inactivation, were prevented by AMPK knockdown, suggesting ATF-3 induction occurs downstream of AMPK activation. The in vivo administration of berberine to mice with LPS-induced endotoxemia increased ATF-3 expression and AMPK phosphorylation in spleen and lung tissues, and concomitantly reduced the plasma and tissue levels of proinflammatory cytokines. These results suggest berberine has an anti-inflammatory effect on macrophages and that this effect is attributable, at least in part, to pathways involving AMPK activation and ATF-3 induction.

• Korean Journal of Plant Resources
• /
• v.30 no.5
• /
• pp.556-564
• /
• 2017

Appropriate soil temperature and early planting of potato is very important for the successful potato-soybean cropping system in central region of South Korea. This experiment was carried out to determine the effect of mulching materials on the growth and yield of potato (Solanum tuberosum L.). Five different mulch treatments were had been applied on an upland soil as follows ; no mulch (NM), transparent film (TF), transparent film + additional transparent film (TF + ATF), black film (BF), and black film + additional transparent film (ATF). In the period of sowing time to removing additional films, mean soil temperature of the treatments was in the order of TF+ATF > TF > BR+ATF > BF as $20.3^{\circ}C$ > $18.5^{\circ}C$ > $16.1^{\circ}C$ > $15.4^{\circ}C$, respectively and that of NM was $13.8^{\circ}C$. The accumulated soil temperature was TF > NM > BF during the removing additional films to earthing at inter-tillage. On the changes in the soil temperature during a whole day, the temperature in the BF was lower than NM during around 18:00 PM to 12:00 NM, while NM was higher than BF in the time period of 10:00AM to 21:00PM. The sequence of potato sprout emergence was 15 > 18 > 20 > 22 days of TF+ATF, TF, BF+ATF, and BF, respectively and that of NM was 24 days. Comparing to the NM, potato sprout emergence was observed on the TF+ATF treated plot as early as 9 days. At 10 days before harvest, the significant difference in the tuber dry weight had been observed and the sequence tuber weight was in the order of TF > TF+ATF > BF+ATF > BF > NM. The potato yields of TF, TF+ATF, and BF+ATF were increased of 40.7, 37.3, and 22% as compared to NM ($2,805kg\;10a^{-1}$), but almost same yield in the BF. The differences of tuber dry weight and potato yields was co-related with the temperature rise of soil by the application of mulching materials on soil. Based on these results, application of mulching film had been very effective to increase the tuber size and the yield of potato by the temperature rise during seedling stage of potato. Transparent mulching was better than black mulching especially for the emergence of sprout of potato in relation to minimizing cooling injury.

• Asian Pacific Journal of Cancer Prevention
• /
• v.17 no.2
• /
• pp.591-595
• /
• 2016

The liver is one of the most common sites of cancer in the world, hepatocellular carcinoma (HCC) predominating. Chronic hepatitis B virus infection (CHB) is considered as an important potential risk factors for HCC. Different people have diverse responses to HBV infection regarding the likelihood of HCC development, and host factors such as single nucleotide polymorphisms (SNPs) might account for this. The present study was conducted to evaluate any association between SNP frequencies in two genes, XRCC4 (rs1805377) and ATF6 (rs2070150), and the risk of CHB and HCC development in Thai patients. The study covered 369 subjects including 121 HCC patients, 141 with chronic hepatitis B virus infection (CHB) and 107 healthy controls. With TaqMan real-time PCR, the results showed that no significant association between XRCC4 (rs1805377) and ATF6 (rs2070150) and risk of HCC in the Thai population. From this first study of the 2 polymorphisms and HCC in Thailand it can concluded that rs1805377 and rs2070150 polymorphisms may not be applicable as genetic markers in the Thai population for HCC assessment.

• Korean Journal of Plant Resources
• /
• v.31 no.6
• /
• pp.612-621
• /
• 2018

Honey used as conventional medicine has various pharmacological properties. In the honey and anti-inflammatory effect, Gelam honey and Manuka honey has been reported to exert anti-inflammatory activity. However, the anti-inflammatory effect and potential mechanisms of acacia honey (AH) are not well understood. In this study, we investigated anti-inflammatory activity and mechanism of action of AH in LPS-stimulated RAW264.7 cells. AH attenuated NO production through inhibition of iNOS expression in LPS-stimulated RAW264.7 cells. AH also decreased the expressions of $IL-1{\beta}$, IL-6 and $TNF-{\alpha}$ as pro-inflammatory cytokines, and MCP-1 expression as a pro-inflammatory chemokine. In the elucidation of the molecular mechanisms, AH decreased LPS-mediated $I{\kappa}B$-${\alpha}$ degradation and subsequent nuclear accumulation of p65, which resulted in the inhibition of $NF-{\kappa}B$ activation in RAW264.7 cells. AH dose-dependently suppressed LPS-mediated phosphorylation of ERK1/2 and p38 in RAW264.7 cells. In addition, AH significantly inhibited ATF2 phosphorylation and nuclear accumulation of ATF2 in LPS-stimulated RAW264.7 cells. These results suggest that AH has an anti-inflammatory effect, inhibiting the production of pro-inflammatory mediators such as NO, iNOS, $TNF-{\alpha}$, IL-6, $IL-1{\beta}$ and MCP-1 via interruption of the $NF-{\kappa}B$ and MAPK/ATF2 signaling pathways.

• Molecules and Cells
• /
• v.41 no.11
• /
• pp.964-970
• /
• 2018

Atherosclerosis preferentially involves in prone area of low and disturbed blood flow while steady and high levels of laminar blood flow are relatively protected from atherosclerosis. Disturbed flow induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). ER stress is caused under stress that disturbs the processing and folding of proteins resulting in the accumulation of misfolded proteins in the ER and activation of the UPR. Prolonged or severe UPR leads to activate apoptotic signaling. Recent studies have indicated that disturbed flow significantly up-regulated $p-ATF6{\alpha}$, $p-IRE1{\alpha}$, and its target spliced XBP-1. However, the role of laminar flow in ER stress-mediated endothelial apoptosis has not been reported yet. The present study thus investigated the role of laminar flow in ER stress-dependent endothelial cell death. The results demonstrated that laminar flow protects ER stress-induced cleavage forms of PARP-1 and caspase-3. Also, laminar flow inhibits ER stress-induced $p-eIF2{\alpha}$, ATF4, CHOP, spliced XBP-1, ATF6 and JNK pathway; these effects are abrogated by pharmacological inhibition of PI3K with wortmannin. Finally, nitric oxide affects thapsigargin-induced cell death in response to laminar flow but not UPR. Taken together, these findings indicate that laminar flow inhibits UPR and ER stress-induced endothelial cell death via PI3K/Akt pathway.

• Proceedings of the Plant Resources Society of Korea Conference
• /
• 2018.10a
• /
• pp.97-97
• /
• 2018

Honey used as conventional medicine has various pharmacological properties. In the honey and anti-inflammatory effect, Gelam honey and Manuka honey has been reported to exert anti-inflammatory activity. However, the anti-inflammatory effect and potential mechanisms of acacia honey (AH) are not well understood. In this study, we investigated anti-inflammatory activity and mechanism of action of AH in LPS-stimulated RAW264.7 cells. AH attenuated NO production through inhibition of iNOS expression in LPS-stimulated RAW264.7 cells. AH also decreased the expressions of $IL-1{\beta}$, IL-6 and $TNF-{\alpha}$ as pro-inflammatory cytokines, and MCP-1 expression as a pro-inflammatory chemokine. In the elucidation of the molecular mechanisms, AH decreased LPS-mediated $I{\kappa}B-{\alpha}$ degradation and subsequent nuclear accumulation of p65, which resulted in the inhibition of $NF-{\kappa}B$ activation in RAW264.7 cells. AH dose-dependently suppressed LPS-mediated phosphorylation of ERK1/2 and p38 in RAW264.7 cells. In addition, AH significantly inhibited ATF2 phosphorylation and nuclear accumulation of ATF2 in LPS-stimulated RAW264.7 cells. These results suggest that AH has an anti-inflammatory effect, inhibiting the production of pro-inflammatory mediators such as NO, iNOS, $TNF-{\alpha}$, IL-6, $IL-1{\beta}$ and MCP-1 via interruption of the $NF-{\kappa}B$ and MAPK/ATF2 signaling pathways.

• Tribology and Lubricants
• /
• v.33 no.6
• /
• pp.269-274
• /
• 2017

Currently, fuel efficiency becomes one of critical issues for automotive industries as concerns about environmental and energy problems grow. In an automatic transmission of an automobile, a drag torque due to a viscous drag of a fluid between friction and clutch plates is one of factors that degrade fuel economy. In this work, the drag torque characteristics of a wet clutch was experimentally investigated with respect to rotational speed, temperature of automatic transmission fluid (ATF), and gap between friction and clutch plates. The experimental results showed that drag torque increases to a certain level, and then decrease to the steady state value with increasing rotational speed. This behavior may be associated with two-phase flow of air and ATF at gap between friction and clutch plates. Also, it was found that the maximum drag torque value decreased as ATF viscosity decreases with increasing temperature. However, it was shown that the point at which the maximum drag torque occurs was not significantly affected by the ATF temperature. In addition, maximum drag torque was found to decrease as the gap between friction and clutch plates increased from 0.1 mm to 0.2 mm. Furthermore, it was observed that the generation of maximum drag torque was delayed as the gap increased. The outcomes of this work are expected to be helpful to gain a better understanding of drag torque characteristic of a wet clutch, and may therefore be useful in the design of wet clutch systems with improved performance.