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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.


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The 25-OHC induces ER stress in HepG2 and Huh7 cells. A, B: HepG2 and Huh7 cells were treated with 10 μM 25-OHC for 24 h, and relative Bip and Chop mRNA levels were measured by qRT-PCR. C, D: The expression of ER stress markers, ATF4, Chop, phospho-PERK, and phospho-eIF2α, were determined by Western blotting. β-Actin was used as a loading control. The data represent mean ± SD from three individual experiments (n = 3, ***P < 0.001).
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f2: The 25-OHC induces ER stress in HepG2 and Huh7 cells. A, B: HepG2 and Huh7 cells were treated with 10 μM 25-OHC for 24 h, and relative Bip and Chop mRNA levels were measured by qRT-PCR. C, D: The expression of ER stress markers, ATF4, Chop, phospho-PERK, and phospho-eIF2α, were determined by Western blotting. β-Actin was used as a loading control. The data represent mean ± SD from three individual experiments (n = 3, ***P < 0.001).

Mentions: A previous report showed that 25-OHC could induce ER stress and apoptosis in macrophages (16). To determine whether ER stress was induced by 25-OHC in HepG2 and Huh7 cells, we first examined the expression of immunoglobulin heavy chain-binding protein (Bip) and Chop mRNAs, central components involved in ER stress responses (29). qRT-PCR analyses revealed that the Bip and Chop mRNAs were robustly induced after 24 h treatment of HepG2 and Huh7 cells with 10 μM 25-OHC, as compared with the control (Fig. 2A, B). We also examined the protein expression of ER stress markers, including ATF4, Chop, phospho-PERK, and phospho-eIF2α by Western blot analysis. All of these markers significantly increased after treatment for 24 h with 10 μM 25-OHC, whereas the expression did not change in cells treated with the vehicle (Fig. 2C, D).


Oxysterol binding protein-related protein 8 mediates the cytotoxicity of 25-hydroxycholesterol [S]
The 25-OHC induces ER stress in HepG2 and Huh7 cells. A, B: HepG2 and Huh7 cells were treated with 10 μM 25-OHC for 24 h, and relative Bip and Chop mRNA levels were measured by qRT-PCR. C, D: The expression of ER stress markers, ATF4, Chop, phospho-PERK, and phospho-eIF2α, were determined by Western blotting. β-Actin was used as a loading control. The data represent mean ± SD from three individual experiments (n = 3, ***P < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: The 25-OHC induces ER stress in HepG2 and Huh7 cells. A, B: HepG2 and Huh7 cells were treated with 10 μM 25-OHC for 24 h, and relative Bip and Chop mRNA levels were measured by qRT-PCR. C, D: The expression of ER stress markers, ATF4, Chop, phospho-PERK, and phospho-eIF2α, were determined by Western blotting. β-Actin was used as a loading control. The data represent mean ± SD from three individual experiments (n = 3, ***P < 0.001).
Mentions: A previous report showed that 25-OHC could induce ER stress and apoptosis in macrophages (16). To determine whether ER stress was induced by 25-OHC in HepG2 and Huh7 cells, we first examined the expression of immunoglobulin heavy chain-binding protein (Bip) and Chop mRNAs, central components involved in ER stress responses (29). qRT-PCR analyses revealed that the Bip and Chop mRNAs were robustly induced after 24 h treatment of HepG2 and Huh7 cells with 10 μM 25-OHC, as compared with the control (Fig. 2A, B). We also examined the protein expression of ER stress markers, including ATF4, Chop, phospho-PERK, and phospho-eIF2α by Western blot analysis. All of these markers significantly increased after treatment for 24 h with 10 μM 25-OHC, whereas the expression did not change in cells treated with the vehicle (Fig. 2C, D).

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