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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

Proteasome activity. (A) Chymotrypsin-like protease activities of HepG2 cells treated with vehicle, 20 μmol/L MG132 (positive control) or 0.3 μmol/L MG132 for indicated times. (B) Western blot analysis of ubiquitinated proteins in HepG2 cells. The data are representatives of three independent experiments. cP<0.01 vs control.
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fig2: Proteasome activity. (A) Chymotrypsin-like protease activities of HepG2 cells treated with vehicle, 20 μmol/L MG132 (positive control) or 0.3 μmol/L MG132 for indicated times. (B) Western blot analysis of ubiquitinated proteins in HepG2 cells. The data are representatives of three independent experiments. cP<0.01 vs control.

Mentions: To determine the proteasome inhibition ability of MG132 under currently used doses, we examined the chymotrypsin-like proteasome activity in the HepG2 cells treated with MG132. The proteasome activity was greatly inhibited by a high concentration of MG132 (20 μmol/L), but it was only partially inhibited by 37.8%±1.5% and 35.6%±13.4% after 4 and 8 h of treatment with 0.3 μmol/L MG132, respectively, and the activity returned to normal levels after 24 h of incubation (Figure 2A). In addition, a modest increase in the ubiquitinated proteins was observed after 4 and 8 h of treatment, but no significant increase was observed at 24 h, which was comparable to the results obtained from the chymotrypsin-like proteasome activity (Figure 2B). Proteasome inhibition is reported to induce an Nrf-1 protein-dependent compensatory increase in the proteasome subunit gene expression and to restore proteasome function13. MG132 consistently induced an increase in the expression of specific proteasome subunit genes (Supplementary Figure 2), indicating that the restoration of proteasome function at 24 h may be caused by the compensatory expression of proteasome subunit genes.


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)

Proteasome activity. (A) Chymotrypsin-like protease activities of HepG2 cells treated with vehicle, 20 μmol/L MG132 (positive control) or 0.3 μmol/L MG132 for indicated times. (B) Western blot analysis of ubiquitinated proteins in HepG2 cells. The data are representatives of three independent experiments. cP<0.01 vs control.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Proteasome activity. (A) Chymotrypsin-like protease activities of HepG2 cells treated with vehicle, 20 μmol/L MG132 (positive control) or 0.3 μmol/L MG132 for indicated times. (B) Western blot analysis of ubiquitinated proteins in HepG2 cells. The data are representatives of three independent experiments. cP<0.01 vs control.
Mentions: To determine the proteasome inhibition ability of MG132 under currently used doses, we examined the chymotrypsin-like proteasome activity in the HepG2 cells treated with MG132. The proteasome activity was greatly inhibited by a high concentration of MG132 (20 μmol/L), but it was only partially inhibited by 37.8%±1.5% and 35.6%±13.4% after 4 and 8 h of treatment with 0.3 μmol/L MG132, respectively, and the activity returned to normal levels after 24 h of incubation (Figure 2A). In addition, a modest increase in the ubiquitinated proteins was observed after 4 and 8 h of treatment, but no significant increase was observed at 24 h, which was comparable to the results obtained from the chymotrypsin-like proteasome activity (Figure 2B). Proteasome inhibition is reported to induce an Nrf-1 protein-dependent compensatory increase in the proteasome subunit gene expression and to restore proteasome function13. MG132 consistently induced an increase in the expression of specific proteasome subunit genes (Supplementary Figure 2), indicating that the restoration of proteasome function at 24 h may be caused by the compensatory expression of proteasome subunit genes.

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