Dissection of the Endogenous Cellular Pathways of PCSK9-induced Low Density Lipoprotein Receptor Degradation: EVIDENCE FOR AN INTRACELLULAR ROUTE

Steve Poirier,Gaetan Mayer,Viviane Poupon,Peter S Mcpherson,Roxane Desjardins,Kevin Ly,Marie-Claude Asselin,Robert Day,Franck J Duclos,Mark Witmer,Rex Parker,Annik Prat,Nabil G Seidah

doi:10.1074/jbc.m109.037085

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.

Highlights

High levels of circulating low-density lipoprotein (LDL)3cholesterol represent a major risk factor that leads to coronary
Enously expresses both proteins. (i) Upon 6-h incubation of (ii) siRNA-mediated knockdown of ARH (KDARH) in naïve HepG2 cells with Dynasore, a cell-permeable inhibitor of HepG2 cells resulted in Ͼ80% reduction of its protein levels and as expected enriched cell surface LDL receptor (LDLR) levels (Fig. 1c), it did not affect total LDLR levels (Fig. 1d), in agreement with the reported unchanged levels of LDLR in the liver of ArhϪ/Ϫ mice
We showed that LDLR and proprotein convertases subtilisin kexin 9 (PCSK9) interact early in the secretory pathway [10] and questioned whether some PCSK91⁄7LDLR complex or PCSK9 alone can be targeted directly from the transGolgi network to late endosomes/lysosomes (LE/L), without cycling through the cell surface

Summary

Intracellular Pathway of LDLR Degradation

ARH did not affect hepatic LDLR subcellular localization in LE/L or protein levels [17]. This is not the expected result if PCSK9 mostly targets LDLR by the extracellular pathway [18], as one would have expected that in ArhϪ/Ϫ mice total LDLR levels should have been more elevated. We focused on the relative contribution of the intra- versus extracellular pathways of endogenous PCSK9-induced LDLR degradation. This information should guide the choice of therapeutic approaches that will best target the site of PCSK9-LDLR interaction to control hypercholesterolemia and coronary heart disease

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION