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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

Effects of MG132 on LDLR expression in and LDL uptake by HepG2 cells. (A) Real-time quantification of the LDLR mRNA level and (B) Western blot analysis of LDLR protein in HepG2 cells treated with MG132 (0.03, 0.1, 0.3, 1, and 3 μmol/L) for 6 h. Time-course of the LDLR mRNA (C) and protein (D) levels in HepG2 cells exposed to MG132 (0.3 μmol/L) for 24 h. DiI-LDL uptake was assessed in cells treated with the indicated concentrations of MG132 for 24 h; (E) Representative images of cells associated with DiI-LDL (Red) and Hoechst-stained nuclei (Blue). Scale bar 50 μm; (F) The normalized fluorescence of isopropanol-extracted DiI (520 and 570 nm). Data are presented as the mean±SEM of at least three independent experiments. bP<0.05, cP<0.01 vs the vehicle-treated groups.
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fig1: Effects of MG132 on LDLR expression in and LDL uptake by HepG2 cells. (A) Real-time quantification of the LDLR mRNA level and (B) Western blot analysis of LDLR protein in HepG2 cells treated with MG132 (0.03, 0.1, 0.3, 1, and 3 μmol/L) for 6 h. Time-course of the LDLR mRNA (C) and protein (D) levels in HepG2 cells exposed to MG132 (0.3 μmol/L) for 24 h. DiI-LDL uptake was assessed in cells treated with the indicated concentrations of MG132 for 24 h; (E) Representative images of cells associated with DiI-LDL (Red) and Hoechst-stained nuclei (Blue). Scale bar 50 μm; (F) The normalized fluorescence of isopropanol-extracted DiI (520 and 570 nm). Data are presented as the mean±SEM of at least three independent experiments. bP<0.05, cP<0.01 vs the vehicle-treated groups.

Mentions: MG132 promoted a remarkable increase in both the LDLR mRNA and protein levels (Figure 1A–1D). LDLR mRNA was upregulated transiently and the levels peaked at 4 h (2.2-fold that of the control, P<0.01) and then declined to background levels after 12 h (MG132 0.3 μmol/L). However, the LDLR protein level continued to show a robust elevation throughout the 4 to 24 h treatment (Figure 1D), indicating the involvement of a post-translation pathway.


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)

Effects of MG132 on LDLR expression in and LDL uptake by HepG2 cells. (A) Real-time quantification of the LDLR mRNA level and (B) Western blot analysis of LDLR protein in HepG2 cells treated with MG132 (0.03, 0.1, 0.3, 1, and 3 μmol/L) for 6 h. Time-course of the LDLR mRNA (C) and protein (D) levels in HepG2 cells exposed to MG132 (0.3 μmol/L) for 24 h. DiI-LDL uptake was assessed in cells treated with the indicated concentrations of MG132 for 24 h; (E) Representative images of cells associated with DiI-LDL (Red) and Hoechst-stained nuclei (Blue). Scale bar 50 μm; (F) The normalized fluorescence of isopropanol-extracted DiI (520 and 570 nm). Data are presented as the mean±SEM of at least three independent experiments. bP<0.05, cP<0.01 vs the vehicle-treated groups.
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Related In: Results  -  Collection

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

fig1: Effects of MG132 on LDLR expression in and LDL uptake by HepG2 cells. (A) Real-time quantification of the LDLR mRNA level and (B) Western blot analysis of LDLR protein in HepG2 cells treated with MG132 (0.03, 0.1, 0.3, 1, and 3 μmol/L) for 6 h. Time-course of the LDLR mRNA (C) and protein (D) levels in HepG2 cells exposed to MG132 (0.3 μmol/L) for 24 h. DiI-LDL uptake was assessed in cells treated with the indicated concentrations of MG132 for 24 h; (E) Representative images of cells associated with DiI-LDL (Red) and Hoechst-stained nuclei (Blue). Scale bar 50 μm; (F) The normalized fluorescence of isopropanol-extracted DiI (520 and 570 nm). Data are presented as the mean±SEM of at least three independent experiments. bP<0.05, cP<0.01 vs the vehicle-treated groups.
Mentions: MG132 promoted a remarkable increase in both the LDLR mRNA and protein levels (Figure 1A–1D). LDLR mRNA was upregulated transiently and the levels peaked at 4 h (2.2-fold that of the control, P<0.01) and then declined to background levels after 12 h (MG132 0.3 μmol/L). However, the LDLR protein level continued to show a robust elevation throughout the 4 to 24 h treatment (Figure 1D), indicating the involvement of a post-translation pathway.

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