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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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ORP8 knockdown partly abolishes the 25-OHC effect on ER stress and apoptosis. A: HepG2 cells were transfected with siORP8, siORP5, or siNT, and the knockdown efficiency was assessed by Western blot analysis. B, C: HepG2 cells were transfected with siORP8, siORP5, or ORP8 cDNA combined with siORP8, and then treated with or without 10 μM 25-OHC for 24 h, and 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. D: HepG2 cells were treated as indicated above, stained with annexin V-FITC and PI, and analyzed by flow cytometry for cell apoptosis. E: Cleaved caspase-9 and -3 protein levels were analyzed by Western blotting. The data represent mean ± SD from three individual experiments (n = 3, **P < 0.01, ***P < 0.001).
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f5: ORP8 knockdown partly abolishes the 25-OHC effect on ER stress and apoptosis. A: HepG2 cells were transfected with siORP8, siORP5, or siNT, and the knockdown efficiency was assessed by Western blot analysis. B, C: HepG2 cells were transfected with siORP8, siORP5, or ORP8 cDNA combined with siORP8, and then treated with or without 10 μM 25-OHC for 24 h, and 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. D: HepG2 cells were treated as indicated above, stained with annexin V-FITC and PI, and analyzed by flow cytometry for cell apoptosis. E: Cleaved caspase-9 and -3 protein levels were analyzed by Western blotting. The data represent mean ± SD from three individual experiments (n = 3, **P < 0.01, ***P < 0.001).

Mentions: We next assessed the role of endogenous ORP8 in the effect of 25-OHC-induced ER stress and cell apoptosis by employing a RNA interference approach. ORP8 knockdown (Fig. 5A) abolished the increase of Bip and Chop mRNA and ER stress protein markers’ expression induced by 25-OHC, while knockdown of ORP5 failed to do so (Fig. 5B, C). Consistently, annexin V-FITC/PI staining and Western blot analysis of cleaved caspase-9 and -3 showed a reduced degree of apoptosis in HepG2 cells subjected to ORP8 knockdown, but not ORP5 knockdown (Fig. 5D, E). In addition, ORP8 re-overexpression could rescue the decreased Bip and Chop mRNA expression (Fig. 5B) and cell apoptosis (Fig. 5D) upon ORP8 knockdown. These results provide mechanistic evidence supporting the view that ORP8 plays a distinct role in mediating the 25-OHC-induced ER stress and apoptosis in hepatic cells.


Oxysterol binding protein-related protein 8 mediates the cytotoxicity of 25-hydroxycholesterol [S]
ORP8 knockdown partly abolishes the 25-OHC effect on ER stress and apoptosis. A: HepG2 cells were transfected with siORP8, siORP5, or siNT, and the knockdown efficiency was assessed by Western blot analysis. B, C: HepG2 cells were transfected with siORP8, siORP5, or ORP8 cDNA combined with siORP8, and then treated with or without 10 μM 25-OHC for 24 h, and 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. D: HepG2 cells were treated as indicated above, stained with annexin V-FITC and PI, and analyzed by flow cytometry for cell apoptosis. E: Cleaved caspase-9 and -3 protein levels were analyzed by Western blotting. The data represent mean ± SD from three individual experiments (n = 3, **P < 0.01, ***P < 0.001).
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Related In: Results  -  Collection

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f5: ORP8 knockdown partly abolishes the 25-OHC effect on ER stress and apoptosis. A: HepG2 cells were transfected with siORP8, siORP5, or siNT, and the knockdown efficiency was assessed by Western blot analysis. B, C: HepG2 cells were transfected with siORP8, siORP5, or ORP8 cDNA combined with siORP8, and then treated with or without 10 μM 25-OHC for 24 h, and 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. D: HepG2 cells were treated as indicated above, stained with annexin V-FITC and PI, and analyzed by flow cytometry for cell apoptosis. E: Cleaved caspase-9 and -3 protein levels were analyzed by Western blotting. The data represent mean ± SD from three individual experiments (n = 3, **P < 0.01, ***P < 0.001).
Mentions: We next assessed the role of endogenous ORP8 in the effect of 25-OHC-induced ER stress and cell apoptosis by employing a RNA interference approach. ORP8 knockdown (Fig. 5A) abolished the increase of Bip and Chop mRNA and ER stress protein markers’ expression induced by 25-OHC, while knockdown of ORP5 failed to do so (Fig. 5B, C). Consistently, annexin V-FITC/PI staining and Western blot analysis of cleaved caspase-9 and -3 showed a reduced degree of apoptosis in HepG2 cells subjected to ORP8 knockdown, but not ORP5 knockdown (Fig. 5D, E). In addition, ORP8 re-overexpression could rescue the decreased Bip and Chop mRNA expression (Fig. 5B) and cell apoptosis (Fig. 5D) upon ORP8 knockdown. These results provide mechanistic evidence supporting the view that ORP8 plays a distinct role in mediating the 25-OHC-induced ER stress and apoptosis in hepatic cells.

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