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Combined effects of multiple endoplasmic reticulum stresses on cytokine secretion in macrophage.

Kim HM, Do CH, Lee DH - Biomol Ther (Seoul) (2012)

Bottom Line: However, secretion of cytokines was ceased upon dual treatments with BFA and TG.This result strongly implies that cells may differently deal with various polypeptides depending on the urgency in cellular function under ER stress.Considering IL-6 is one of the most important signal molecules in macrophage, the molecule might be able to circumvent ER stress and UPR to reach its targeting site.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, University of Seoul, Seoul 130-743.

ABSTRACT
Cells show various stress signs when they are challenged with severe physiological problems. Majority of such cellular stresses are conveyed to endoplasmic reticulum (ER) and unfolded protein response (UPR) serves as typical defense mechanism against ER stress. This study investigated an interaction between ER stress agents using macropage cell line Raw 264.7. When activated by lipopolysaccharide (LPS), the cell lines showed typical indicators of ER stress. Along with molecular chaperones, the activation process leads to the production of additional infl ammatory mediators. Following activation, the macrophage cell line was further treated with TUN and characterized in terms of chaperone expression and cytokine secretion. When treated with TUN, the activated macrophage cell leads to increased secretion of IL-6 although expression of ER stress markers, GRP94 and GRP78 increased. The secretion of cytokines continued until the addition of BFA which inhibits protein targeting from ER to Golgi. However, secretion of cytokines was ceased upon dual treatments with BFA and TG. This result strongly implies that cells may differently deal with various polypeptides depending on the urgency in cellular function under ER stress. Considering IL-6 is one of the most important signal molecules in macrophage, the molecule might be able to circumvent ER stress and UPR to reach its targeting site.

No MeSH data available.


Expression of molecular chaperones in macrophages treated with tunicamycin or LPS. Raw 264.7 cells were treated with tunicamycin for 0-24 h. Cell lysates were analyzed for the expression of GRP94 and GRP78 by western blotting (A). The expression of molecular chaperones was quantitated in (B). Relative intensity refers to the converted value of protein expression assuming the band intensity at time 0 as 1.0. (C) Raw 264.7 cells treated with LPS (100 ng/ml) for 0-12 h. The level of molecular chaperones was analyzed as in Fig. 1-(B).
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Figure 001: Expression of molecular chaperones in macrophages treated with tunicamycin or LPS. Raw 264.7 cells were treated with tunicamycin for 0-24 h. Cell lysates were analyzed for the expression of GRP94 and GRP78 by western blotting (A). The expression of molecular chaperones was quantitated in (B). Relative intensity refers to the converted value of protein expression assuming the band intensity at time 0 as 1.0. (C) Raw 264.7 cells treated with LPS (100 ng/ml) for 0-12 h. The level of molecular chaperones was analyzed as in Fig. 1-(B).

Mentions: After each sample was fixed in the plate, mouse IL-6 ELISAset (eBioscience) and anti-mouse IL-6 was sequentially added up to 100 μl/well, followed by incubation overnight at 4℃. Plates were then washed three times with PBST before addition of 1X Assay diluents and the diluted samples were further incubated for 1 h. At the termination of incubation, plates were washed three times with PBST and incubated with streptavidin-HRP diluted in 1X Assay diluents. When the plates were thoroughly washed again, TMB peroxidase sub-strate and 50 μl of 2N sulfuric acid were added to the wells to stop the enzyme reaction. The enzyme activity was detected


Combined effects of multiple endoplasmic reticulum stresses on cytokine secretion in macrophage.

Kim HM, Do CH, Lee DH - Biomol Ther (Seoul) (2012)

Expression of molecular chaperones in macrophages treated with tunicamycin or LPS. Raw 264.7 cells were treated with tunicamycin for 0-24 h. Cell lysates were analyzed for the expression of GRP94 and GRP78 by western blotting (A). The expression of molecular chaperones was quantitated in (B). Relative intensity refers to the converted value of protein expression assuming the band intensity at time 0 as 1.0. (C) Raw 264.7 cells treated with LPS (100 ng/ml) for 0-12 h. The level of molecular chaperones was analyzed as in Fig. 1-(B).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3794534&req=5

Figure 001: Expression of molecular chaperones in macrophages treated with tunicamycin or LPS. Raw 264.7 cells were treated with tunicamycin for 0-24 h. Cell lysates were analyzed for the expression of GRP94 and GRP78 by western blotting (A). The expression of molecular chaperones was quantitated in (B). Relative intensity refers to the converted value of protein expression assuming the band intensity at time 0 as 1.0. (C) Raw 264.7 cells treated with LPS (100 ng/ml) for 0-12 h. The level of molecular chaperones was analyzed as in Fig. 1-(B).
Mentions: After each sample was fixed in the plate, mouse IL-6 ELISAset (eBioscience) and anti-mouse IL-6 was sequentially added up to 100 μl/well, followed by incubation overnight at 4℃. Plates were then washed three times with PBST before addition of 1X Assay diluents and the diluted samples were further incubated for 1 h. At the termination of incubation, plates were washed three times with PBST and incubated with streptavidin-HRP diluted in 1X Assay diluents. When the plates were thoroughly washed again, TMB peroxidase sub-strate and 50 μl of 2N sulfuric acid were added to the wells to stop the enzyme reaction. The enzyme activity was detected

Bottom Line: However, secretion of cytokines was ceased upon dual treatments with BFA and TG.This result strongly implies that cells may differently deal with various polypeptides depending on the urgency in cellular function under ER stress.Considering IL-6 is one of the most important signal molecules in macrophage, the molecule might be able to circumvent ER stress and UPR to reach its targeting site.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, University of Seoul, Seoul 130-743.

ABSTRACT
Cells show various stress signs when they are challenged with severe physiological problems. Majority of such cellular stresses are conveyed to endoplasmic reticulum (ER) and unfolded protein response (UPR) serves as typical defense mechanism against ER stress. This study investigated an interaction between ER stress agents using macropage cell line Raw 264.7. When activated by lipopolysaccharide (LPS), the cell lines showed typical indicators of ER stress. Along with molecular chaperones, the activation process leads to the production of additional infl ammatory mediators. Following activation, the macrophage cell line was further treated with TUN and characterized in terms of chaperone expression and cytokine secretion. When treated with TUN, the activated macrophage cell leads to increased secretion of IL-6 although expression of ER stress markers, GRP94 and GRP78 increased. The secretion of cytokines continued until the addition of BFA which inhibits protein targeting from ER to Golgi. However, secretion of cytokines was ceased upon dual treatments with BFA and TG. This result strongly implies that cells may differently deal with various polypeptides depending on the urgency in cellular function under ER stress. Considering IL-6 is one of the most important signal molecules in macrophage, the molecule might be able to circumvent ER stress and UPR to reach its targeting site.

No MeSH data available.