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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 modulates LDLR mRNA and protein levels through a PKC-dependent pathway. Western blot analysis of LDLR expression in HepG2 cells incubated with MG132 (0.3 μmol/L) under cholesterol addition (A, +cho, 2 mg/L 25-OH cholesterol and 10 mg/L cholesterol) or cholesterol depletion (B, -cho, 10 μmol/L pravastatin and 100 μmol/L mevalonlactone) conditions for the indicated times, (dep-, control of cholesterol depletion group; cho-, control of cholesterol addition group; great increase of LDLR expression in the dep group and decrease in the cho group indicating increased or decreased SREBP-2 activity). Western blot analysis of (C) mature SREBP-2 and (D) phosphorylated ERK (Pra-, pravastatin 5 μmol/L; Ber-, berberine 15 μg/L as positive controls, respectively). (E) LDLR mRNA was measured via real-time PCR, and the decay curves were plotted versus the time (MG132 0.3 μmol/L, 6 h). (F) Real-time PCR analysis of LDLR transcription in HepG2 cells incubated with GF 109203X (10 μmol/L), Gö 6983 (10 μmol/L) and staurosporine (400 nmol/L) in the presence or absence of MG132 0.3 μmol/L for 6 h, respectively. All of the values are presented as the mean±SEM of three or more independent experiments. bP<0.05, cP<0.01 vs the control groups.
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fig3: MG132 modulates LDLR mRNA and protein levels through a PKC-dependent pathway. Western blot analysis of LDLR expression in HepG2 cells incubated with MG132 (0.3 μmol/L) under cholesterol addition (A, +cho, 2 mg/L 25-OH cholesterol and 10 mg/L cholesterol) or cholesterol depletion (B, -cho, 10 μmol/L pravastatin and 100 μmol/L mevalonlactone) conditions for the indicated times, (dep-, control of cholesterol depletion group; cho-, control of cholesterol addition group; great increase of LDLR expression in the dep group and decrease in the cho group indicating increased or decreased SREBP-2 activity). Western blot analysis of (C) mature SREBP-2 and (D) phosphorylated ERK (Pra-, pravastatin 5 μmol/L; Ber-, berberine 15 μg/L as positive controls, respectively). (E) LDLR mRNA was measured via real-time PCR, and the decay curves were plotted versus the time (MG132 0.3 μmol/L, 6 h). (F) Real-time PCR analysis of LDLR transcription in HepG2 cells incubated with GF 109203X (10 μmol/L), Gö 6983 (10 μmol/L) and staurosporine (400 nmol/L) in the presence or absence of MG132 0.3 μmol/L for 6 h, respectively. All of the values are presented as the mean±SEM of three or more independent experiments. bP<0.05, cP<0.01 vs the control groups.

Mentions: Next, we explored the mechanisms involved in MG132-mediated LDLR mRNA enhancement in HepG2 cells. High cellular cholesterol levels inhibit SREBP-2 maturation, thus decreasing LDLR transcription, and vice versa. To determine whether these processes are related to LDLR transcription upregulation by MG132, high levels of sterols (10 mg/L cholesterol and 2 mg/L 25-OH cholesterol) were added to the culture media. The suppressive sterol levels almost abolished the LDLR expression but did not block the effect of MG132 (0.3 μmol/L) in increasing the LDLR expression (Figure 3A). Moreover, depleting the cellular cholesterol levels by adding 10 μmol/L pravastatin and 100 μmol/L mevalonlactone in the medium also had no effect on the induction of LDLR by MG132 (Figure 3B). We also detected the mature form of SREBP-2 by immunoblotting (Figure 3C), and no significant increase was observed, although SREBP is reported to be degraded by the proteasome14. This lack of a significant increase may be attributed to the low doses of MG132 used in this experiment, leading to only partial proteasome inhibition, while the majority of activity was preserved, as noted above in Figure 2. Our data confirmed that the MG132-mediated augmentation of the LDLR mRNA level is independent of the SREBPs.


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 modulates LDLR mRNA and protein levels through a PKC-dependent pathway. Western blot analysis of LDLR expression in HepG2 cells incubated with MG132 (0.3 μmol/L) under cholesterol addition (A, +cho, 2 mg/L 25-OH cholesterol and 10 mg/L cholesterol) or cholesterol depletion (B, -cho, 10 μmol/L pravastatin and 100 μmol/L mevalonlactone) conditions for the indicated times, (dep-, control of cholesterol depletion group; cho-, control of cholesterol addition group; great increase of LDLR expression in the dep group and decrease in the cho group indicating increased or decreased SREBP-2 activity). Western blot analysis of (C) mature SREBP-2 and (D) phosphorylated ERK (Pra-, pravastatin 5 μmol/L; Ber-, berberine 15 μg/L as positive controls, respectively). (E) LDLR mRNA was measured via real-time PCR, and the decay curves were plotted versus the time (MG132 0.3 μmol/L, 6 h). (F) Real-time PCR analysis of LDLR transcription in HepG2 cells incubated with GF 109203X (10 μmol/L), Gö 6983 (10 μmol/L) and staurosporine (400 nmol/L) in the presence or absence of MG132 0.3 μmol/L for 6 h, respectively. All of the values are presented as the mean±SEM of three or more independent experiments. bP<0.05, cP<0.01 vs the control groups.
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fig3: MG132 modulates LDLR mRNA and protein levels through a PKC-dependent pathway. Western blot analysis of LDLR expression in HepG2 cells incubated with MG132 (0.3 μmol/L) under cholesterol addition (A, +cho, 2 mg/L 25-OH cholesterol and 10 mg/L cholesterol) or cholesterol depletion (B, -cho, 10 μmol/L pravastatin and 100 μmol/L mevalonlactone) conditions for the indicated times, (dep-, control of cholesterol depletion group; cho-, control of cholesterol addition group; great increase of LDLR expression in the dep group and decrease in the cho group indicating increased or decreased SREBP-2 activity). Western blot analysis of (C) mature SREBP-2 and (D) phosphorylated ERK (Pra-, pravastatin 5 μmol/L; Ber-, berberine 15 μg/L as positive controls, respectively). (E) LDLR mRNA was measured via real-time PCR, and the decay curves were plotted versus the time (MG132 0.3 μmol/L, 6 h). (F) Real-time PCR analysis of LDLR transcription in HepG2 cells incubated with GF 109203X (10 μmol/L), Gö 6983 (10 μmol/L) and staurosporine (400 nmol/L) in the presence or absence of MG132 0.3 μmol/L for 6 h, respectively. All of the values are presented as the mean±SEM of three or more independent experiments. bP<0.05, cP<0.01 vs the control groups.
Mentions: Next, we explored the mechanisms involved in MG132-mediated LDLR mRNA enhancement in HepG2 cells. High cellular cholesterol levels inhibit SREBP-2 maturation, thus decreasing LDLR transcription, and vice versa. To determine whether these processes are related to LDLR transcription upregulation by MG132, high levels of sterols (10 mg/L cholesterol and 2 mg/L 25-OH cholesterol) were added to the culture media. The suppressive sterol levels almost abolished the LDLR expression but did not block the effect of MG132 (0.3 μmol/L) in increasing the LDLR expression (Figure 3A). Moreover, depleting the cellular cholesterol levels by adding 10 μmol/L pravastatin and 100 μmol/L mevalonlactone in the medium also had no effect on the induction of LDLR by MG132 (Figure 3B). We also detected the mature form of SREBP-2 by immunoblotting (Figure 3C), and no significant increase was observed, although SREBP is reported to be degraded by the proteasome14. This lack of a significant increase may be attributed to the low doses of MG132 used in this experiment, leading to only partial proteasome inhibition, while the majority of activity was preserved, as noted above in Figure 2. Our data confirmed that the MG132-mediated augmentation of the LDLR mRNA level is independent of the SREBPs.

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