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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

PCSK9 is involved in MG132-mediated LDLR protein upregulation. (A) Western blot analysis of LDLR expression in HepG2 cells treated with MG132 (0.3 μmol/L, 24 h) in the presence or absence of NH4Cl (a lysosome pH desruptor). (B) Western blot analysis of the LDLR expression in HepG2 cells transfected with siRNA-PCSK9 and then treated with MG132 (0.3 μmol/L, 24 h). (C) Western blot analysis of the LDLR protein level following the PCSK9-Flag addition (conditioned medium, 6 h). DiI-LDL uptake of the HepG2 cells with or without the exogenous addition of PCSK9 using the conditioned medium (6 h) of HEK293 cells transfected with PCSK9-Flag constructs. (D) Representative fluorescence microscopy images of cell-associated DiI-LDL (Red) and Hoechst-stained nuclei (Blue). Scale bar 50 μm. (E) Fluorescence of isopropanol-extracted DiI (520–570 nm, normalized to the cell protein). The data are the representative results of three independent experiments. Mean±SEM.
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fig4: PCSK9 is involved in MG132-mediated LDLR protein upregulation. (A) Western blot analysis of LDLR expression in HepG2 cells treated with MG132 (0.3 μmol/L, 24 h) in the presence or absence of NH4Cl (a lysosome pH desruptor). (B) Western blot analysis of the LDLR expression in HepG2 cells transfected with siRNA-PCSK9 and then treated with MG132 (0.3 μmol/L, 24 h). (C) Western blot analysis of the LDLR protein level following the PCSK9-Flag addition (conditioned medium, 6 h). DiI-LDL uptake of the HepG2 cells with or without the exogenous addition of PCSK9 using the conditioned medium (6 h) of HEK293 cells transfected with PCSK9-Flag constructs. (D) Representative fluorescence microscopy images of cell-associated DiI-LDL (Red) and Hoechst-stained nuclei (Blue). Scale bar 50 μm. (E) Fluorescence of isopropanol-extracted DiI (520–570 nm, normalized to the cell protein). The data are the representative results of three independent experiments. Mean±SEM.

Mentions: LDLR is synthesized in a premature form by ribosomes bound to the endoplasmic reticulum (ER) and then translocated into the ER lumen and subsequently processed to its mature form in the Golgi apparatus. Mature LDLR is guided to the cell surface, where it functions as an LDL-clearing mediator. After LDL binding and endocytosis, LDLR releases its ligand in endosomes, followed by recycling to the cell surface or degradation in the lysosome16. PCSK9 affects LDLR recycling by directly binding to LDLR and increasing its degradation in the lysosome in a pH-dependent manner3. NH4Cl, a lysosome pH disruptor, abolished MG132-induced LDLR protein augmentation at 24 h, suggesting that MG132 influences LDLR degradation in the lysosome (Figure 4A). Moreover, the disruption of endogenous PCSK9 expression by the targeted siRNA abrogated the elevation of the mature LDLR by MG132 at 24 h (Figure 4B). Our data support the involvement of endogenous PCSK9 in stabilizing the LDLR protein during long-term MG132 treatment.


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)

PCSK9 is involved in MG132-mediated LDLR protein upregulation. (A) Western blot analysis of LDLR expression in HepG2 cells treated with MG132 (0.3 μmol/L, 24 h) in the presence or absence of NH4Cl (a lysosome pH desruptor). (B) Western blot analysis of the LDLR expression in HepG2 cells transfected with siRNA-PCSK9 and then treated with MG132 (0.3 μmol/L, 24 h). (C) Western blot analysis of the LDLR protein level following the PCSK9-Flag addition (conditioned medium, 6 h). DiI-LDL uptake of the HepG2 cells with or without the exogenous addition of PCSK9 using the conditioned medium (6 h) of HEK293 cells transfected with PCSK9-Flag constructs. (D) Representative fluorescence microscopy images of cell-associated DiI-LDL (Red) and Hoechst-stained nuclei (Blue). Scale bar 50 μm. (E) Fluorescence of isopropanol-extracted DiI (520–570 nm, normalized to the cell protein). The data are the representative results of three independent experiments. Mean±SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4125719&req=5

fig4: PCSK9 is involved in MG132-mediated LDLR protein upregulation. (A) Western blot analysis of LDLR expression in HepG2 cells treated with MG132 (0.3 μmol/L, 24 h) in the presence or absence of NH4Cl (a lysosome pH desruptor). (B) Western blot analysis of the LDLR expression in HepG2 cells transfected with siRNA-PCSK9 and then treated with MG132 (0.3 μmol/L, 24 h). (C) Western blot analysis of the LDLR protein level following the PCSK9-Flag addition (conditioned medium, 6 h). DiI-LDL uptake of the HepG2 cells with or without the exogenous addition of PCSK9 using the conditioned medium (6 h) of HEK293 cells transfected with PCSK9-Flag constructs. (D) Representative fluorescence microscopy images of cell-associated DiI-LDL (Red) and Hoechst-stained nuclei (Blue). Scale bar 50 μm. (E) Fluorescence of isopropanol-extracted DiI (520–570 nm, normalized to the cell protein). The data are the representative results of three independent experiments. Mean±SEM.
Mentions: LDLR is synthesized in a premature form by ribosomes bound to the endoplasmic reticulum (ER) and then translocated into the ER lumen and subsequently processed to its mature form in the Golgi apparatus. Mature LDLR is guided to the cell surface, where it functions as an LDL-clearing mediator. After LDL binding and endocytosis, LDLR releases its ligand in endosomes, followed by recycling to the cell surface or degradation in the lysosome16. PCSK9 affects LDLR recycling by directly binding to LDLR and increasing its degradation in the lysosome in a pH-dependent manner3. NH4Cl, a lysosome pH disruptor, abolished MG132-induced LDLR protein augmentation at 24 h, suggesting that MG132 influences LDLR degradation in the lysosome (Figure 4A). Moreover, the disruption of endogenous PCSK9 expression by the targeted siRNA abrogated the elevation of the mature LDLR by MG132 at 24 h (Figure 4B). Our data support the involvement of endogenous PCSK9 in stabilizing the LDLR protein during long-term MG132 treatment.

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