• Development and Reproduction
• /
• v.21 no.4
• /
• pp.407-415
• /
• 2017

In the present study, we investigated the role of binding immunoglobulin protein/glucose-regulated protein, 78-kDa (BIP/GRP78)-regulated endoplasmic reticulum (ER)-stress on meiotic maturation and cumulus cells expansion in porcine cumulus-oocyte complexes (COCs). Previously, it has been demonstrated that unfolded protein response (UPR)-related genes, such as molecules involved in ER-stress defense mechanisms, were expressed in matured oocytes and cumulus cells during in vitro maturation (IVM) of porcine oocytes. However, BIP/GRP78-mediated regulation of ER stress in porcine oocytes has not been reported. Firstly, we observed the effects of knockdown of BIP/GRP78 (an UPR initiation marker) using porcine-specific siRNAs (#909, #693, and #1570) on oocyte maturation. Among all siRNAs, siRNA #693 significantly reduced the protein levels of UPR marker proteins (BIP/GRP78, ATF4, and P90ATF6) in porcine COCs observed by Western blotting and immunofluorescence analysis. We also observed that the reduction of BIP/GRP78 levels by siRNA#693 significantly inhibited the meiotic maturation of oocytes (siRNA #693: $32.5{\pm}10.1%$ vs control: $77.8{\pm}5.3%$). In addition, we also checked the effect of ER-stress inhibitors, tauroursodeoxycholic acid (TUDCA, $200{\mu}M$) and melatonin ($0.1{\mu}M$), in BIP/GRP78-knockdown oocytes. TUDCA and melatonin treatment could restore the expression levels of ER-stress marker proteins (BIP/GRP78, $p-eIF2{\alpha}$, $eIF2{\alpha}$, ATF4, and P90ATF6) in siRNA #693-transfected matured COCs. In conclusion, these results demonstrated that BIP/GRP78-mediated regulation of UPR signaling and ER stress plays an important role in in vitro maturation of porcine oocytes.

• The Korean Journal of Zoology
• /
• v.38 no.2
• /
• pp.167-176
• /
• 1995

Glucose-regulated proteins (grp's), srp94 3nd grp78/BiP, are a group of stress proteins which are highly synthesized in cells exposed to a variety of stressful agents including tunicamycin 3nd Ca2+ ionophore. Grp78/BiP is hon to function as a molecular chaperone which regulates the folding and assembly of secretory or membrane proteins, but the biological function of grp941 remains to be elucidated. In this study, we have examined the intracellular distribution of grV's and the function of srp94. Grp's are resident in the endoplasmic reticulum (ERI 3nd a specific sequence (Lys-Asp-Glu-Leu) at their C-terminus is known to be responsible for their retention within the ER. However, it has been unclear whether upon disturbance of cellular Caa+ homeostasis by the Ca2+ ionophore, grp94 is retained within the ER or secreted into the medium. In this study, we showed that in the presence of C3a+ ionophore, grp94 and gif78/BiP are present in the cells, mainly within the ER. We have also investigated whether grp94 might function as a molecular chaperone. Here we showed that in the immunoglobulin (Ig)-secreting hvbridom3 cells, grp94 transientlY interacts with fully glycosylated Is heavy chain, suggesting that grpg94 may be involved in facilitating the folding and assembly of Ig heavy chains.

• The Korean Journal of Zoology
• /
• v.35 no.4
• /
• pp.456-465
• /
• 1992

The 94 KDa glucose-resulsted Protein (SH 94), one of stress Proteins, is a Ca2+-binding protein in the endoplasmic reticulum (ER). In this study, the possible effect of Ca2+ on the native conformation of grp 94 was examined. When the purified grp 94 was analyzed by Sel filtration in the presence of either EGTA or CaCl2, it was eluted with apparent molecular weight (MW) of 100 KDa in both cases. When similarly analyzed with microtome or cell Ivsate, however, srp 94 was eluted with apparent IW of 200 KDa in the presence of E6TA, while with apparent MW of 100 KDa in the presence of CaCl2, indicating possible association of grp 94 with one or more other proteins in the absence of CaCl2. Consequently, immunoprecipitation with anti-grp 94 was carried out to determine which proteins specifically interact with grp 94. It is sho%un that srp 94 may interact, in a Ca2+_dependent manner. with other proteins including BiP (grp 78) which is also a stress protein in the ER.

• Molecules and Cells
• /
• v.41 no.5
• /
• pp.401-412
• /
• 2018

Oxymatrine (OMT) often used in treatment for chronic hepatitis B virus infection in clinic. However, OMT-induced liver injury has been reported. In this study, we aim to investigate the possible mechanism of OMT-induced hepatotoxicity in human normal liver cells (L02). Exposed cells to OMT, the cell viability was decreased and apoptosis rate increased, the intracellular markers of oxidative stress were changed. Simultaneously, OMT altered apoptotic related proteins levels, including Bcl-2, Bax and pro-caspase-8/-9/-3. In addition, OMT enhanced the protein levels of endoplasmic reticulum (ER) stress makers (GRP78/Bip, CHOP, and cleaved-Caspase-4) and phosphorylation of c-Jun N-terminal kinase (p-JNK), as well as the mRNA levels of GRP78/Bip, CHOP, caspase-4, and ER stress sensors (IREI, ATF6, and PERK). Pre-treatment with Z-VAD-fmk, JNK inhibitor SP600125 and N-acetyl-l-cysteine (NAC), a ROS scavenger, partly improved the survival rates and restored OMT-induced cellular damage, and reduced caspase-3 cleavage. SP600125 or NAC reduced OMT-induced p-JNK and NAC significantly lowered caspase-4. Furthermore, 4-PBA, the ER stress inhibitor, weakened inhibitory effect of OMT on cells, on the contrary, TM worsen. 4-PBA also reduced the levels of p-JNK and cleaved-caspase-3 proteins. Therefore, OMT-induced injury in L02 cells was related to ROS mediated p-JNK and ER stress induction. Antioxidant, by inhibition of p-JNK or ER stress, may be a feasible method to alleviate OMT-induced liver injury.

• Journal of Life Science
• /
• v.28 no.10
• /
• pp.1186-1192
• /
• 2018

The purpose of this study was to compare the effects of either aerobic exercise or polyphenols supplementation on mRNA expression of endoplasmic reticulum stress in skeletal muscle of high fat diet-induced obese mice. In the study, mice were divided into five groups: (1) NC (normal diet for 16 weeks as a control, n=10), (2) HC (high fat diet for 16 weeks as a control, n=10), (3) H-Re (high fat diet with resveratrol 25 mg/kg supplementation for 16 weeks, n=10), (4) H-Ch (high fat diet with chrysin 50 mg/kg supplementation for 16 weeks, n=10), and (5) HE (high fat diet with aerobic exercise for 16 weeks, n=10). Aerobic exercise was performed on a treadmill for 40~60 min/day at 10~14 m/min, 0% grade, four days/week for 16 weeks. Endoplasmic reticulum stress related genes were measured by real-time polymerase chain reaction. ATF6, PERK, $IRE1{\alpha}$, and BIP/GRP78 mRNA were significantly decreased in HE compared with those in HC (p<0.05). Also, ATF6, $IRE1{\alpha}$, and BIP/GRP78 mRNA were significantly decreased in H-Re compared with those in HC (p<0.05). ATF6 mRNA was significantly decreased in H-Ch compared with that in HC (p<0.05). These findings suggest that aerobic exercise, resveratrol, and chrysin supplementation changed ER stress markers. However, aerobic exercise was most effective on ameliorating the high fat diet induced ER stress markers. Thus, it seems that aerobic exercise might have a more positive effect on skeletal muscle endoplasmic reticulum stress compared with polyphenol supplementation in high fat diet-induced obese mice.

• Journal of Life Science
• /
• v.22 no.12
• /
• pp.1672-1679
• /
• 2012

The effect of high stocking density (HSD) on the expression of stress and lipid metabolism associated genes in the liver of broiler chickens was examined by chicken genome array analysis. The chickens in a control group were randomly assigned to a $495cm^2/bird$ stocking density, whereas the chickens in a HSD group were arranged in a $245cm^2/bird$ stocking density with feeding ad libitum for 35 days. The chickens assigned to the HSD group had a significantly lower body weight, weight gain, and feed intake compared with those of the control group (p<0.05). The mortality of chickens was higher in the HSD group than in the control group. The microarray analysis indicated up-regulation of stress associated genes such as HMGCR, $HSP90{\alpha}$, HSPA5 (GRP78/Bip), DNAJC3 and ATF4, and down-regulation of interferon-${\gamma}$ and PDCD4 genes. The endoplasmic reticulum stress associated genes, HSPA5 (GRP78/Bip), DNAJC3 and ATF4, were highly expressed in the HSD group. The genes, ACSL5, TMEM195 and ELOVL6, involved in fatty acid synthesis, were elevated in the HSD group. The genes, ACAA1, ACOX1, EHHADH, LOC423347 and CPT1A, related to fatty acid oxidation, were also activated in the HSD group. These results suggest that a HSD rearing system stimulates the genes associated with fatty acid synthesis as well as fatty acid oxidation in the liver of broiler chickens.

• BMB Reports
• /
• v.45 no.8
• /
• pp.482-487
• /
• 2012

To identify the novel inhibitors of endoplasmic reticulum stress-induced cell death, we performed a high throughput assay with a chemical library containing a total of 3,280 bioactive small molecules. Cyclosporine A and bromocriptine were identified as potent inhibitors of thapsigargiin-induced cell death (cut-off at $4{\sigma}$ standard score). However, U74389G, the potent inhibitor of lipid peroxidation had lower activity in inhibiting cell death. The inhibition effect of cyclosporine A and bromocriptine was specific for only thapsigargin-induced cell death. The mechanism of inhibition by these compounds was identified as modification of the expression of glucose regulated protein-78 (GRP-78/Bip) and inhibition of phosphorylation of p38 mitogen activated protein kinase (MAPK). However, these compounds did not inhibit the same events triggered by tunicamycin, which was in agreement with the cell survival data. We suggest that the induction of protective unfolded protein response by these compounds confers resistance to cell death. In summary, we identified compounds that may provide insights on cell death mechanisms stimulated by ER stress.

• The Korean Journal of Physiology and Pharmacology
• /
• v.11 no.6
• /
• pp.239-246
• /
• 2007

Expressions of endoplasmic reticulum stress response (ERSR) genes were examined during the neuronal differentiation of rat fetal cortical precursor cells (rCPC) and rat pheochromocytoma PC12 cells. When rCPC were differentiated into neuronal cells for 7 days, early stem cell marker, nest in, expression was decreased from day 4, and neuronal markers such as neurofilament-L, -M and Tuj1 were increased after day 4. In this condition, expressions of BIP, ATF6, and phosphorylated PERK as well as their down stream signaling molecules such as CHOP, ATF4, XBP1, GADD34, Nrf2 and $p58^{IPK}$ were significantly increased, suggesting the induction of ERSR during neuronal differentiation of rCPC. ERSR was also induced during the differentiation of PC12 cells for 9 days with NGF. Neurofilament-L transcript was time-dependently increased. Both mRNA and protein levels of Tuj1 were increased after the induction, and the significant increase in NeuN was observed at day 9. Similar to the expression patterns of neuronal markers, BIP/GRP78 and CHOP mRNAs were highly increased at day 9, and ATF4 mRNA was also increased from day 7. These results strongly suggest the induction and possible role of ERSR in neuronal differentiation process. Further study to identify targets responsible for neuronal induction will be necessary.