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Oxysterol binding protein-related protein 8 mediates the cytotoxicity of 25-hydroxycholesterol [S]

View Article: PubMed Central - PubMed

ABSTRACT

Oxysterols are 27-carbon oxidized derivatives of cholesterol or by-products of cholesterol biosynthesis that can induce cell apoptosis in addition to a number of other bioactions. However, the mechanisms underlying this cytotoxicity are not completely understood. ORP8 is a member of the oxysterol binding protein-related protein (ORP) family, implicated in cellular lipid homeostasis, migration, and organization of the microtubule cytoskeleton. Here, we report that 25-hydroxycholesterol (OHC) induced apoptosis of the hepatoma cell lines, HepG2 and Huh7, via the endoplasmic reticulum (ER) stress response pathway, and ORP8 overexpression resulted in a similar cell response as 25-OHC, indicating a putative functional relationship between oxysterol cytotoxicity and ORP8. Further experiments demonstrated that ORP8 overexpression significantly enhanced the 25-OHC effect on ER stress and apoptosis in HepG2 cells. A truncated ORP8 construct lacking the ligand-binding domain or a closely related protein, ORP5, was devoid of this activity, evidencing for specificity of the observed effects. Importantly, ORP8 knockdown markedly dampened such responses to 25-OHC. Taken together, the present study suggests that ORP8 may mediate the cytotoxicity of 25-OHC.

No MeSH data available.


Related in: MedlinePlus

ER stress is involved in 25-OHC-induced apoptosis. A, B: HepG2 cells were treated with or without 2 mM 4-PBA for 1 h, and then incubated with 10 μM 25-OHC for 24 h, relative Bip and Chop mRNA levels were measured by qRT-PCR. ATF4, Chop, phospho-PERK, and phospho-eIF2α protein levels were analyzed by Western blotting. C: HepG2 cells were treated with or without 4-PBA and 25-OHC as indicated above, stained with annexin V-FITC and PI, and analyzed by FCM for cell apoptosis. D: Cleaved caspase-9 and -3 protein levels were analyzed by Western blotting in the 25-OHC-treated HepG2 cells. The data represent mean ± SD from three individual experiments (n = 3, **P < 0.01, ***P < 0.001).
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f3: ER stress is involved in 25-OHC-induced apoptosis. A, B: HepG2 cells were treated with or without 2 mM 4-PBA for 1 h, and then incubated with 10 μM 25-OHC for 24 h, relative Bip and Chop mRNA levels were measured by qRT-PCR. ATF4, Chop, phospho-PERK, and phospho-eIF2α protein levels were analyzed by Western blotting. C: HepG2 cells were treated with or without 4-PBA and 25-OHC as indicated above, stained with annexin V-FITC and PI, and analyzed by FCM for cell apoptosis. D: Cleaved caspase-9 and -3 protein levels were analyzed by Western blotting in the 25-OHC-treated HepG2 cells. The data represent mean ± SD from three individual experiments (n = 3, **P < 0.01, ***P < 0.001).

Mentions: To further confirm the role of 25-OHC in ER stress, HepG2 cells were pretreated with 4-PBA, a chemical molecular chaperone, which has been used to improve the misfolding and mislocalization of proteins and to reduce ER stress-mediated cell damage in vivo and in vitro (30–32). The results showed that, when treated with 10 μM 25-OHC for 24 h, the increase of Bip and Chop mRNAs induced by 25-OHC was significantly reversed (Fig. 3A) in the presence of 4-PBA. Expectedly, the ATF4, Chop, phospho-PERK, and phospho-eIF2α protein levels also showed a parallel reduction (Fig. 3B). In order to confirm whether the ER stress induced by 25-OHC contributes to cellular apoptosis, HepG2 cells treated with 25-OHC were subjected to annexin V-FITC/PI double-staining followed by flow cytometry. The results showed an increase in apoptotic cells upon treatment with 25-OHC as compared with the control (Fig. 3C), but the apoptotic cell rate was significantly reduced when the 25-OHC treatment was combined with the addition of 4-PBA (Fig. 3C). We also analyzed the cleaved caspase-9 and -3 levels by Western blotting. Cleavage of the caspases was clearly enhanced in 25-OHC-treated cells, but this response was abolished by addition of 4-PBA, suggesting that apoptosis of the 25-OHC-treated cells is induced by ER stress (Fig. 3D).


Oxysterol binding protein-related protein 8 mediates the cytotoxicity of 25-hydroxycholesterol [S]
ER stress is involved in 25-OHC-induced apoptosis. A, B: HepG2 cells were treated with or without 2 mM 4-PBA for 1 h, and then incubated with 10 μM 25-OHC for 24 h, relative Bip and Chop mRNA levels were measured by qRT-PCR. ATF4, Chop, phospho-PERK, and phospho-eIF2α protein levels were analyzed by Western blotting. C: HepG2 cells were treated with or without 4-PBA and 25-OHC as indicated above, stained with annexin V-FITC and PI, and analyzed by FCM for cell apoptosis. D: Cleaved caspase-9 and -3 protein levels were analyzed by Western blotting in the 25-OHC-treated HepG2 cells. The data represent mean ± SD from three individual experiments (n = 3, **P < 0.01, ***P < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5036365&req=5

f3: ER stress is involved in 25-OHC-induced apoptosis. A, B: HepG2 cells were treated with or without 2 mM 4-PBA for 1 h, and then incubated with 10 μM 25-OHC for 24 h, relative Bip and Chop mRNA levels were measured by qRT-PCR. ATF4, Chop, phospho-PERK, and phospho-eIF2α protein levels were analyzed by Western blotting. C: HepG2 cells were treated with or without 4-PBA and 25-OHC as indicated above, stained with annexin V-FITC and PI, and analyzed by FCM for cell apoptosis. D: Cleaved caspase-9 and -3 protein levels were analyzed by Western blotting in the 25-OHC-treated HepG2 cells. The data represent mean ± SD from three individual experiments (n = 3, **P < 0.01, ***P < 0.001).
Mentions: To further confirm the role of 25-OHC in ER stress, HepG2 cells were pretreated with 4-PBA, a chemical molecular chaperone, which has been used to improve the misfolding and mislocalization of proteins and to reduce ER stress-mediated cell damage in vivo and in vitro (30–32). The results showed that, when treated with 10 μM 25-OHC for 24 h, the increase of Bip and Chop mRNAs induced by 25-OHC was significantly reversed (Fig. 3A) in the presence of 4-PBA. Expectedly, the ATF4, Chop, phospho-PERK, and phospho-eIF2α protein levels also showed a parallel reduction (Fig. 3B). In order to confirm whether the ER stress induced by 25-OHC contributes to cellular apoptosis, HepG2 cells treated with 25-OHC were subjected to annexin V-FITC/PI double-staining followed by flow cytometry. The results showed an increase in apoptotic cells upon treatment with 25-OHC as compared with the control (Fig. 3C), but the apoptotic cell rate was significantly reduced when the 25-OHC treatment was combined with the addition of 4-PBA (Fig. 3C). We also analyzed the cleaved caspase-9 and -3 levels by Western blotting. Cleavage of the caspases was clearly enhanced in 25-OHC-treated cells, but this response was abolished by addition of 4-PBA, suggesting that apoptosis of the 25-OHC-treated cells is induced by ER stress (Fig. 3D).

View Article: PubMed Central - PubMed

ABSTRACT

Oxysterols are 27-carbon oxidized derivatives of cholesterol or by-products of cholesterol biosynthesis that can induce cell apoptosis in addition to a number of other bioactions. However, the mechanisms underlying this cytotoxicity are not completely understood. ORP8 is a member of the oxysterol binding protein-related protein (ORP) family, implicated in cellular lipid homeostasis, migration, and organization of the microtubule cytoskeleton. Here, we report that 25-hydroxycholesterol (OHC) induced apoptosis of the hepatoma cell lines, HepG2 and Huh7, via the endoplasmic reticulum (ER) stress response pathway, and ORP8 overexpression resulted in a similar cell response as 25-OHC, indicating a putative functional relationship between oxysterol cytotoxicity and ORP8. Further experiments demonstrated that ORP8 overexpression significantly enhanced the 25-OHC effect on ER stress and apoptosis in HepG2 cells. A truncated ORP8 construct lacking the ligand-binding domain or a closely related protein, ORP5, was devoid of this activity, evidencing for specificity of the observed effects. Importantly, ORP8 knockdown markedly dampened such responses to 25-OHC. Taken together, the present study suggests that ORP8 may mediate the cytotoxicity of 25-OHC.

No MeSH data available.


Related in: MedlinePlus