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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

A combination of MG132 and pravastatin enhances LDLR while suppressing PCSK9 expression in HepG2 cells. Real-time PCR quantification (A, B) and Western blot analysis (C) of LDLR and PCSK9 expression levels and DiI-LDL uptake (D) in cells treated with MG132 (0.3 μmol/L), pravastatin (5 μmol/L), or both for the indicated times. The data are presented as the mean±SEM of three or more independent experiments. bP<0.05, cP<0.01 vs the vehicle-treated groups.
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fig6: A combination of MG132 and pravastatin enhances LDLR while suppressing PCSK9 expression in HepG2 cells. Real-time PCR quantification (A, B) and Western blot analysis (C) of LDLR and PCSK9 expression levels and DiI-LDL uptake (D) in cells treated with MG132 (0.3 μmol/L), pravastatin (5 μmol/L), or both for the indicated times. The data are presented as the mean±SEM of three or more independent experiments. bP<0.05, cP<0.01 vs the vehicle-treated groups.

Mentions: In view of the finding that MG132 upregulates LDLR but suppresses PCSK9 expression, we investigated whether a combination of MG132 and statins has a concomitant stimulatory effect on LDLR expression while suppressing the PCSK9 expression induced by statins alone. The HepG2 cells were treated with MG132 (0.3 μmol/L) and pravastatin (5 μmol/L) either alone or in combination. The combination treatment led to an increase in the LDLR mRNA level by 2.3-fold after 8 h, while pravastatin alone enhanced the transcript level by ∼1.4-fold. The combination additionally suppressed the increase in the PCSK9 mRNA induced by pravastatin by 63% at 24 h (P<0.01, Figure 6A, 6B). Western blot analysis revealed a robust increase in the LDLR expression in cells receiving both MG132 and pravastatin treatment for 8 or 24 h and a significant decrease in PCSK9 expression after 24 h (Figure 6C). The combined MG132 and pravastatin treatment additionally augmented the LDL uptake by 43% and 39%, while pravastatin alone increased LDL uptake by 20% and 17% after 8 and 24 h, respectively (Figure 6D).


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)

A combination of MG132 and pravastatin enhances LDLR while suppressing PCSK9 expression in HepG2 cells. Real-time PCR quantification (A, B) and Western blot analysis (C) of LDLR and PCSK9 expression levels and DiI-LDL uptake (D) in cells treated with MG132 (0.3 μmol/L), pravastatin (5 μmol/L), or both for the indicated times. The data are presented as the mean±SEM of three or more independent experiments. bP<0.05, cP<0.01 vs the vehicle-treated groups.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: A combination of MG132 and pravastatin enhances LDLR while suppressing PCSK9 expression in HepG2 cells. Real-time PCR quantification (A, B) and Western blot analysis (C) of LDLR and PCSK9 expression levels and DiI-LDL uptake (D) in cells treated with MG132 (0.3 μmol/L), pravastatin (5 μmol/L), or both for the indicated times. The data are presented as the mean±SEM of three or more independent experiments. bP<0.05, cP<0.01 vs the vehicle-treated groups.
Mentions: In view of the finding that MG132 upregulates LDLR but suppresses PCSK9 expression, we investigated whether a combination of MG132 and statins has a concomitant stimulatory effect on LDLR expression while suppressing the PCSK9 expression induced by statins alone. The HepG2 cells were treated with MG132 (0.3 μmol/L) and pravastatin (5 μmol/L) either alone or in combination. The combination treatment led to an increase in the LDLR mRNA level by 2.3-fold after 8 h, while pravastatin alone enhanced the transcript level by ∼1.4-fold. The combination additionally suppressed the increase in the PCSK9 mRNA induced by pravastatin by 63% at 24 h (P<0.01, Figure 6A, 6B). Western blot analysis revealed a robust increase in the LDLR expression in cells receiving both MG132 and pravastatin treatment for 8 or 24 h and a significant decrease in PCSK9 expression after 24 h (Figure 6C). The combined MG132 and pravastatin treatment additionally augmented the LDL uptake by 43% and 39%, while pravastatin alone increased LDL uptake by 20% and 17% after 8 and 24 h, respectively (Figure 6D).

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