Peroxisome Proliferator-activated receptor γ activation by ligands and dephosphorylation induces proprotein convertase subtilisin kexin type 9 and low density lipoprotein receptor expression

Abstract

Proprotein convertase subtilisin kexin type 9 (PCSK9) plays an important role in cholesterol homeostasis by enhancing the degradation of LDL receptor (LDLR) protein. Peroxisome proliferator-activated receptor γ (PPARγ) has been shown to be atheroprotective. PPARγ can be activated by ligands and/or dephosphorylation with ERK1/2 inhibitors. The effect of PPARγ on PCSK9 and LDLR expression remains unknown. In this study, we investigated the effects of PPARγ on PCSK9 and LDLR expression. At the cellular levels, PPARγ ligands induced PCSK9 mRNA and protein expression in HepG2 cells. PCSK9 expression was induced by inhibition of ERK1/2 activity but inhibited by ERK1/2 activation. The mutagenic study and promoter activity assay suggested that the induction of PCSK9 expression by ERK1/2 inhibitors was tightly linked to PPARγ dephosphorylation. However, PPARγ activation by ligands or ERK1/2 inhibitors induced hepatic LDLR expression. The promoter assay indicated that the induction of LDLR expression by PPARγ was sterol regulatory element-dependent because PPARγ enhanced sterol regulatory element-binding protein 2 (SREBP2) processing. In vivo, administration of pioglitazone or U0126 alone increased PCSK9 expression in mouse liver but had little effect on PCSK9 secretion. However, the co-treatment of pioglitazone and U0126 enhanced both PCSK9 expression and secretion. Similar to in vitro, the increased PCSK9 expression by pioglitazone and/or U0126 did not result in decreased LDLR expression and function. In contrast, pioglitazone and/or U0126 increased LDLR protein expression and membrane translocation, SREBP2 processing, and CYP7A1 expression in the liver, which led to decreased total and LDL cholesterol levels in serum. Our results indicate that although PPARγ activation increased PCSK9 expression, PPARγ activation induced LDLR and CYP7A1 expression that enhanced LDL cholesterol metabolism.

FIGURE 1.

PPARγ ligands induce PCSK9 expression in HepG2 cells. A and B, confluent (∼95%) HepG2 cells in serum-free DMEM were treated with 15d-PGJ2 and pioglitazone at the indicated concentrations overnight (A) or treated with 15d-PGJ2 (2 μm) and pioglitazone (10 μm) for the indicated times (B). After treatment, the cellular proteins were extracted and used to determine both the precursor of PCSK9 (P) and mature PCSK9 (M) protein by Western blot as described under “Experimental Procedures.” The same membrane was reblotted with anti-β-actin antibody to verify equal loading. C, after extraction, the total cellular RNA was used to determine PCSK9 mRNA expression by real time PCR. *, significantly different from control (Ctrl) at p < 0.05 by the paired Student's t test (n = 3).

FIGURE 2.

Inhibition of ERK1/2 induces PCSK9 expression. A, HepG2 cells in serum-free medium were treated with PD98059 or U0126 at the indicated concentrations overnight. B, HepG2 cells were treated with EGF at the indicated concentrations overnight. C, HepG2 cells were treated with PD98059 (5 μm) or U0126 (0.5 μm) in the absence or presence of 15d-PGJ2 (0.5 μm) or pioglitazone (5 μm) overnight. Expression of PCSK9 was assessed by Western blot. Ctrl, control; P, precursor; M, mature.

FIGURE 3.

Induction of PCSK9 expression by PPARγ dephosphorylation. A, HepG2 cells were treated with PD98059 at the indicated concentrations overnight. Expression of total PPARγ or phospho-PPARγ (Pi-PPARγ) was determined by Western blot with anti-PPARγ or anti-phospho-PPARγ antibody. B, HepG2 cells were transfected with expression vector of wild type PPARγ (C2-PPARγ) or nonphosphorylated PPARγ mutant (γS112A) or constitutively phosphorylated PPARγ mutant (γS112D) at the indicated concentrations overnight. C, HepG2 cells were transfected with expression vector of C2-PPARγ or γS112A or γS112D (0.5 μg/well of each). After 12 h of transfection, the cells were treated with U0126 (1 μm) overnight. Expression of PCSK9 and endogenous (Endo) and exogenous (Exo) PPARγ was determined by Western blot. P, precursor; M, mature.

FIGURE 4.

Regulation of PCSK9 promoter activity by PPARγ activation. All of the cells were transfected with DNA of Renilla luciferase for internal normalization. After transfection and treatment, the cellular lysate was extracted and used to determine activity of firefly and Renilla luciferases. A, 293T cells were transfected with DNA for the PCSK9 promoter and the PPARγ2 expression vector (C2-γ2, 0.2 μg/well) as indicated followed by treatment with pioglitazone (Piog) overnight. § and ¶, significantly different from the control (pPCSK9+C2 vector) and the sample of (pPCSK9+C2-γ2), respectively, at p < 0.05 by the paired Student's t test (n = 3). B, cells were transfected with DNA for wild type PCSK9 promoter (pPCSK9) or PCSK9 promoter with DR1 mutation (pPCSK9-DR1mut) or deletion (pPCSK9-DR1del) followed by treatment with pioglitazone or 15d-PGJ2 at the indicated concentrations overnight. § and ¶, significantly different from control (promoter alone) and the corresponding samples in the group of wild type promoter, respectively, at p < 0.05 (n = 3). C, cells were transfected with DNA for the PCSK9 promoter followed by treatment with PD98059 and U0126 at the indicated concentrations overnight. §, significantly different from the control (pPCSK9) at p < 0.05 (n = 3). D, cells were transfected with DNA for PCSK9 promoter and expression vector of wild type PPARγ (C2-γ2) or nonphosphorylated PPARγ mutant (C2-γ2SA) or constitutively phosphorylated PPARγ mutant (C2-γ2SD) followed by treatment with pioglitazone at the indicated concentrations overnight. § and ¶, significantly different from the sample of (pPCSK9+C2 vector) and the corresponding control (pPCSK9+C2-γ2 or pPCSK9+C2-γ2SA), respectively, at p < 0.05 (n = 3). E, cells were transfected with DNA for the PCSK9 promoter and the PPARγ expression vector followed by treatment with pioglitazone or pioglitazone plus EGF at the indicated concentrations overnight. § and ¶, significantly different from the control (C2-γ2) and the pioglitazone-treated sample (C2-γ2 + pioglitazone (5 μm), respectively, at p < 0.05 (n = 3).

FIGURE 5.

PPARγ activation induces LDLR expression and binding of LDL to HepG2 cells. A, HepG2 cells in serum-free medium were treated with pioglitazone (Piog) or U0126 at the indicated concentrations overnight. Expression of LDLR protein was determined by Western blot. B and C, HepG2 cells were treated with U0126 (1 μm) or pioglitazone (5 μm) or their combination overnight. The cells were then used to determine LDLR protein expression or uptake of LDL as described under “Experimental Procedures.” D, 293T cells were transfected with DNA for wild type LDLR promoter (pLDLR) or promoter with SRE mutation (pLDLR-SREmut). After 4 h of transfection, the cells received the indicated treatment overnight. 25-Hydroxycholesterol or pitavastatin was used as a negative or positive control. * and #, significantly different from control in the corresponding group at p < 0.05 (n = 3). E, HepG2 cells were treated with pioglitazone or U0126 at the indicated concentrations overnight. Whole cellular protein was extracted and used to determine the precursor and mature forms of SREBP2 by Western blot. P, precursor; M, mature.

FIGURE 6.

PPARγ activation increases PCSK9 production in vivo. Male wild type C57 mice (n = 5) were fed normal chow or chow containing U0126 (5 mg/100 g of food) or pioglitazone (Piog, 30 mg/100 g of food) or U0126 plus pioglitazone for 10 days. After treatment, mouse liver and serum samples were prepared. A, expression of PCSK9 protein in mouse liver was determined by Western blot. B, serum PCSK9 was determined by ELISA. *, significantly different from control at p < 0.05 (n = 10, each serum sample was analyzed twice). P, precursor; M, mature.

FIGURE 7.

PPARγ activation induces expression of LDLR and 2CYP7A1 in mouse liver and reduces serum total and/or LDL cholesterol levels. The mice received the treatment as described in the legend to Fig. 6. Both liver and serum samples were collected and used for the following assays. A and D, expression of LDLR and SREBP2 protein in the liver was determined by Western blot. B, mouse liver sections were prepared and used to determine LDLR protein expression by immunohistochemical or immunofluorescent staining assay as described under “Experimental Procedures.” The arrows indicate the membrane location of LDLR protein. C and E, expression of LDLR and CYP7A1 mRNA in mouse liver was determined by real time PCR. *, significantly different from control (Ctrl) in the corresponding group at p < 0.05 (n = 5). F, serum lipid profiles were determined by enzymatic methods. * and #, significantly different from the corresponding control at p < 0.05 (n = 5). Piog, pioglitazone; P, precursor; M, mature.