Dissection of the endogenous cellular pathways of PCSK9-induced low density lipoprotein receptor degradation: evidence for an intracellular route

Abstract

Elevated levels of plasma low density lipoprotein (LDL)-cholesterol, leading to familial hypercholesterolemia, are enhanced by mutations in at least three major genes, the LDL receptor (LDLR), its ligand apolipoprotein B, and the proprotein convertase PCSK9. Single point mutations in PCSK9 are associated with either hyper- or hypocholesterolemia. Accordingly, PCSK9 is an attractive target for treatment of dyslipidemia. PCSK9 binds the epidermal growth factor domain A (EGF-A) of the LDLR and directs it to endosomes/lysosomes for destruction. Although the mechanism by which PCSK9 regulates LDLR degradation is not fully resolved, it seems to involve both intracellular and extracellular pathways. Here, we show that clathrin light chain small interfering RNAs that block intracellular trafficking from the trans-Golgi network to lysosomes rapidly increased LDLR levels within HepG2 cells in a PCSK9-dependent fashion without affecting the ability of exogenous PCSK9 to enhance LDLR degradation. In contrast, blocking the extracellular LDLR endocytosis/degradation pathway by a 4-, 6-, or 24-h incubation of cells with Dynasore or an EGF-AB peptide or by knockdown of endogenous autosomal recessive hypercholesterolemia did not significantly affect LDLR levels. The present data from HepG2 cells and mouse primary hepatocytes favor a model whereby depending on the dose and/or incubation period, endogenous PCSK9 enhances the degradation of the LDLR both extra- and intracellularly. Therefore, targeting either pathway, or both, would be an effective method to reduce PCSK9 activity in the treatment of hypercholesterolemia and coronary heart disease.

FIGURE 1.

Minor contribution of endogenous secreted PCSK9 to LDLR intracellular degradation. a, HepG2 cells were incubated for 4 h with 0.3% v/v of dimethyl sulfoxide (DMSO) (vehicle) or 80 μm Dynasore monohydrate, a cell-permeable inhibitor of dynamin together with 4 μg/ml diI-LDL (red) and analyzed by immunocytochemistry. b, after a 6-h incubation with dimethyl sulfoxide or 80 μm Dynasore, protein extracts were analyzed by Western blotting. c, immunocytochemistry of nonpermeabilized HepG2 cells that were transfected with either a nonsilencing siRNA (siCtl) or an ARH siRNA at 72 h after transfection is shown. Arrows emphasize the increased cell surface LDLR. (d) At 72 h after transfection, protein extracts were analyzed by Western blotting. These data are representative of three to six separate experiments. Scale bars, 20 μm.

FIGURE 2.

Extracellular EGF-AB does not affect endogenous PCSK9 effect on LDLR. a and b, HepG2 cells were preincubated for either 4 h (a) or 24 h (b) with 0.01, 0.1, 0.3, 1, 3, and 10 μm EGF-AB peptide together with 0, 67, 135 or 200 nm pure human PCSK9. Then, 5 μg/ml diI-LDL was added, and the incubation continued for another 2 h. Cells were washed and analyzed for fluorescence incorporation. Normalized data are presented. d, Western blot analyses of HepG2 cells incubated for 24 h with 0, 1, 5, and 12.5 μm EGF-AB are shown. These data are representative of three independent experiments.

FIGURE 3.

Secreted PCSK9 does not reduce LDLR levels in PCSK9 KO hepatocytes. Primary hepatocytes of Pcsk9^−/− (−/−) mice were incubated for 24 h in duplicate with either 100 nm PCSK9 or with 48-h conditioned media from PCSK9 KO or WT hepatocytes (from two mice, WT₁ and WT₂). Note the lower LDLR levels in Pcsk9^+/+ (+/+) hepatocytes, the source of WT media, versus (−/−) hepatocytes. Western blot analyses of LDLR, human PCSK9, and β-actin are shown. Note the level of cell-associated human PCSK9 (hPCSK9) after the 100 nm incubation using a human PCSK9 antibody that does not recognize mouse PCSK9 on Western blots (20). Similar data were obtained in two separate experiments.

FIGURE 4.

The gain-of-function mutant PCSK9-D374Y, but not WT PCSK9, decreases cell surface LDLR on adjacent cells. a, immunocytochemistry of cell surface LDLR (blue) in HepG2 transiently overexpressing a bicistronic vector (pIRES) encoding WT PCSK9 and EGFP (green) is shown. b–d, at 24 h after transfection, cells overexpressing a nonsecreted catalytically inactive PCSK9 (H226A), WT, or the gain-of-function mutant (D374Y) were sorted for EGFP and LDLR. Graphs were derived from FACS analyses (supplemental Fig. S1), and values were normalized to those of the H226A-expressing cells. Secreted human PCSK9 levels were measured using an ELISA (b). Error bars represent three separate experiments. Scale bar, 20 μm.

FIGURE 5.

KD of CLCs increases endogenous LDLR levels only in PCSK9-expressing cells. HepG2 or HEK293 cells were transiently transfected with either a nonsilencing siRNA (siCtl) or with siRNAs against both CLC a and b isoforms (siCLCa+b). a, immunocytochemistry analyses confirmed that KD_CLCs (green) results in the clustering of the cation-independent mannose 6-phosphate receptor, characteristic of disruption of the intracellular Golgi-lysosomal pathway (blue; see arrows). b and c, at 72 h after transfection, HepG2 and HEK293 cells were analyzed by Western blotting. d, immunocytochemistry under nonpermeabilizing conditions of cell surface LDLR (green) upon KD_CLCs (siCLCa+b) compared with nonsilencing siRNA transfected cells (siCtl) is shown. e, at 48 h after transfection, addition of 40 nm exogenous PCSK9 enhances the degradation of LDLR irrespective of KD_CLCs. (f) Effect of KD_CLCs on LDLR protein levels in HepG2 cells with (shNT) or without (shPCSK9*) endogenous PCSK9 is shown. The 160 kDa band representing mature LDLR was quantified and normalized with those of actin. These data are representative of 3–6 independent experiments. Scale bars, 20 μm.

FIGURE 6.

Synergic effect of KD_CLCs with LPDS treatment. HepG2 cells were transiently transfected with either a nonsilencing siRNA or with siRNAs against both CLC a and b isoforms (siCLCa+b). At 48 h after transfection, cells were washed twice with DMEM and incubated with either complete (10% FBS) or with 5% LPDS medium. After a 24-h incubation, cells were washed and incubated for 6 h in DMEM, and protein extracts and media were analyzed by Western blotting. These data are representative of three independent experiments.