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MG132, a proteasome inhibitor, enhances LDL uptake in HepG2 cells in vitro by regulating LDLR and PCSK9 expression.

Yan H, Ma YL, Gui YZ, Wang SM, Wang XB, Gao F, Wang YP - Acta Pharmacol. Sin. (2014)

Bottom Line: In contrast, a longer treatment with MG132 (0.3 μmol/L, 24 h) did not change LDLR mRNA, but markedly increased LDLR protein by reducing PCSK9-mediated lysosome LDLR degradation.Furthermore, MG132 time-dependently suppressed PCSK9 expression in the HepG2 cells through a SREBP-1c related pathway.Inhibition of proteasome by MG132 in HepG2 cells plays dual roles in LDLR and PCSK9 expression, and exerts a beneficial effect on cholesterol homeostasis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

ABSTRACT

Aim: Expression of liver low-density lipoprotein receptor (LDLR), a determinant regulator in cholesterol homeostasis, is tightly controlled at multiple levels. The aim of this study was to examine whether proteasome inhibition could affect LDLR expression and LDL uptake in liver cells in vitro.

Methods: HepG2 cells were examined. Real-time PCR and Western blot analysis were used to determine the mRNA and protein levels, respectively. DiI-LDL uptake assay was used to quantify the LDLR function. Luciferase assay system was used to detect the activity of proprotein convertase subtilisin/kexin type 9 (PCSK9, a major protein mediating LDLR degradation) promoter. Specific siRNAs were used to verify the involvement of PCSK9.

Results: Treatment of HepG2 cells with the specific proteasome inhibitor MG132 (0.03-3 μmol/L) dose-dependently increased LDLR mRNA and protein levels, as well as LDL uptake. Short-term treatment with MG132 (0.3 μmol/L, up to 8 h) significantly increased both LDLR mRNA and protein levels in HepG2 cells, which was blocked by the specific PKC inhibitors GF 109203X, Gö 6983 or staurosporine. In contrast, a longer treatment with MG132 (0.3 μmol/L, 24 h) did not change LDLR mRNA, but markedly increased LDLR protein by reducing PCSK9-mediated lysosome LDLR degradation. Furthermore, MG132 time-dependently suppressed PCSK9 expression in the HepG2 cells through a SREBP-1c related pathway. Combined treatment with MG132 (0.3 μmol/L) and pravastatin (5 μmol/L) strongly promoted LDLR expression and LDL uptake in HepG2 cells, and blocked the upregulation of PCSK9 caused by pravastatin alone.

Conclusion: Inhibition of proteasome by MG132 in HepG2 cells plays dual roles in LDLR and PCSK9 expression, and exerts a beneficial effect on cholesterol homeostasis.

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Related in: MedlinePlus

MG132 downregulates PCSK9 expression in HepG2 cells. (A, C) Real-time PCR and (B, D) Western blot analyses of the PCSK9 expression under MG132 treatment for the indicated time and dose. (E) Luciferase activities of the HepG2 cells transfected with PCSK9 promoter constructs containing wild-type, HNF mutant or SRE mutant sequences treated with MG132 (0.3 μmol/L, 24 h, normalized to β-galactosidase activity). Real-time PCR quantification of SREBP-1c (F, G), FAS, SCD, and SREBP-2 (H) mRNA levels. The data are representatives of three independent experiments. bP<0.05, cP<0.01.
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fig5: MG132 downregulates PCSK9 expression in HepG2 cells. (A, C) Real-time PCR and (B, D) Western blot analyses of the PCSK9 expression under MG132 treatment for the indicated time and dose. (E) Luciferase activities of the HepG2 cells transfected with PCSK9 promoter constructs containing wild-type, HNF mutant or SRE mutant sequences treated with MG132 (0.3 μmol/L, 24 h, normalized to β-galactosidase activity). Real-time PCR quantification of SREBP-1c (F, G), FAS, SCD, and SREBP-2 (H) mRNA levels. The data are representatives of three independent experiments. bP<0.05, cP<0.01.

Mentions: MG132 suppressed PCSK9 mRNA levels in a time- and dose-dependent manner (Figure 5A and 5C). The PCSK9 mRNA level was reduced by 70% compared with the control after 24 h treatment with 0.3 μmol/L MG132. The cellular and secreted PCSK9 protein levels were additionally diminished in a time- and dose-dependent manner in the presence of MG132 (Figures 5B, 5D).


MG132, a proteasome inhibitor, enhances LDL uptake in HepG2 cells in vitro by regulating LDLR and PCSK9 expression.

Yan H, Ma YL, Gui YZ, Wang SM, Wang XB, Gao F, Wang YP - Acta Pharmacol. Sin. (2014)

MG132 downregulates PCSK9 expression in HepG2 cells. (A, C) Real-time PCR and (B, D) Western blot analyses of the PCSK9 expression under MG132 treatment for the indicated time and dose. (E) Luciferase activities of the HepG2 cells transfected with PCSK9 promoter constructs containing wild-type, HNF mutant or SRE mutant sequences treated with MG132 (0.3 μmol/L, 24 h, normalized to β-galactosidase activity). Real-time PCR quantification of SREBP-1c (F, G), FAS, SCD, and SREBP-2 (H) mRNA levels. The data are representatives of three independent experiments. bP<0.05, cP<0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4125719&req=5

fig5: MG132 downregulates PCSK9 expression in HepG2 cells. (A, C) Real-time PCR and (B, D) Western blot analyses of the PCSK9 expression under MG132 treatment for the indicated time and dose. (E) Luciferase activities of the HepG2 cells transfected with PCSK9 promoter constructs containing wild-type, HNF mutant or SRE mutant sequences treated with MG132 (0.3 μmol/L, 24 h, normalized to β-galactosidase activity). Real-time PCR quantification of SREBP-1c (F, G), FAS, SCD, and SREBP-2 (H) mRNA levels. The data are representatives of three independent experiments. bP<0.05, cP<0.01.
Mentions: MG132 suppressed PCSK9 mRNA levels in a time- and dose-dependent manner (Figure 5A and 5C). The PCSK9 mRNA level was reduced by 70% compared with the control after 24 h treatment with 0.3 μmol/L MG132. The cellular and secreted PCSK9 protein levels were additionally diminished in a time- and dose-dependent manner in the presence of MG132 (Figures 5B, 5D).

Bottom Line: In contrast, a longer treatment with MG132 (0.3 μmol/L, 24 h) did not change LDLR mRNA, but markedly increased LDLR protein by reducing PCSK9-mediated lysosome LDLR degradation.Furthermore, MG132 time-dependently suppressed PCSK9 expression in the HepG2 cells through a SREBP-1c related pathway.Inhibition of proteasome by MG132 in HepG2 cells plays dual roles in LDLR and PCSK9 expression, and exerts a beneficial effect on cholesterol homeostasis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

ABSTRACT

Aim: Expression of liver low-density lipoprotein receptor (LDLR), a determinant regulator in cholesterol homeostasis, is tightly controlled at multiple levels. The aim of this study was to examine whether proteasome inhibition could affect LDLR expression and LDL uptake in liver cells in vitro.

Methods: HepG2 cells were examined. Real-time PCR and Western blot analysis were used to determine the mRNA and protein levels, respectively. DiI-LDL uptake assay was used to quantify the LDLR function. Luciferase assay system was used to detect the activity of proprotein convertase subtilisin/kexin type 9 (PCSK9, a major protein mediating LDLR degradation) promoter. Specific siRNAs were used to verify the involvement of PCSK9.

Results: Treatment of HepG2 cells with the specific proteasome inhibitor MG132 (0.03-3 μmol/L) dose-dependently increased LDLR mRNA and protein levels, as well as LDL uptake. Short-term treatment with MG132 (0.3 μmol/L, up to 8 h) significantly increased both LDLR mRNA and protein levels in HepG2 cells, which was blocked by the specific PKC inhibitors GF 109203X, Gö 6983 or staurosporine. In contrast, a longer treatment with MG132 (0.3 μmol/L, 24 h) did not change LDLR mRNA, but markedly increased LDLR protein by reducing PCSK9-mediated lysosome LDLR degradation. Furthermore, MG132 time-dependently suppressed PCSK9 expression in the HepG2 cells through a SREBP-1c related pathway. Combined treatment with MG132 (0.3 μmol/L) and pravastatin (5 μmol/L) strongly promoted LDLR expression and LDL uptake in HepG2 cells, and blocked the upregulation of PCSK9 caused by pravastatin alone.

Conclusion: Inhibition of proteasome by MG132 in HepG2 cells plays dual roles in LDLR and PCSK9 expression, and exerts a beneficial effect on cholesterol homeostasis.

Show MeSH
Related in: MedlinePlus