Effects of currently prescribed LDL-C-lowering drugs on PCSK9 and implications for the next generation of LDL-C-lowering agents

Abstract

Background: During the past decade, proprotein convertase subtilisin kexin type 9 (PCSK9) has been identified as a key regulator of serum LDL-cholesterol (LDL-C) levels. PCSK9 is secreted by the liver into the plasma and binds the hepatic LDL receptor, causing its subsequent degradation. In humans, gain-of-function mutations in PCSK9 cause a form of familial hypercholesterolemia that manifests with dramatically increased serum levels of LDL-C, while loss-of-function mutations in PCSK9 are associated with significantly decreased LDL-C and cardiovascular risk.

Results: Initial studies in animals and cultured cells demonstrated that statins increased PCSK9 mRNA expression, resulting in many research groups exploring the effect of statins on PCSK9 levels in humans. We first reported that statins increased human PCSK9 circulating protein levels. Additional researchers subsequently confirmed these observations, further prompting many laboratories including our own to examine the effect of other lipid lowering medications on PCSK9 levels. Our observation that fenofibrate (200 mg/day) significantly increased PCSK9 levels was confirmed by another laboratory, and an additional group demonstrated that ezetimibe also increased PCSK9 levels.

Conclusions: It has become clear that the major classes of commonly prescribed lipid-lowering medications increase serum PCSK9 levels. These observations almost certainly explain why these agents are not more effective in lowering LDL-C and suggest that efforts should be made toward the development of new LDL-C lowering medications that either do not increase circulating PCSK9 levels or work through decreasing or inhibiting PCSK9.

Figure 1

PCSK9 protein structure and selected mutations. PCSK9 protein includes a pro-domain (Pro) that is self-cleaved during secretion and remains tightly associated, a catalytic domain, and a C-terminal domain. Activating mutations such as D374Y cause increased affinity for LDLR. Inactivating mutations such as Y142X and C679X block self-cleavage and secretion of the protein. The R46L mutation results in decreased circulating levels of PCSK9 protein, although the mechanism is not completely understood.

Figure 2

Effect of atorvastatin on hepatocyte LDLR and PCSK9. Atorvastatin increases the activity/nuclear translocation of sterol regulatory element-binding protein-2 (SREBP-2), which is a transcription factor that activates both the LDLR and PCSK9 genes. This results in increased expression and secretion of PCSK9 protein, which binds the LDLR and targets it for lysosomal degradation. This likely prevents atorvastatin from causing as much increased LDLR protein from being present on the hepatocyte plasma membrane as might otherwise be expected.

Figure 3

Characterization of PCSK9 protein in human serum. When immunoprecipitated from human serum and analyzed in our laboratory by subsequent western blotting under reducing and denaturing conditions, PCSK9 migrates as an intact band (top arrow) that co-migrates with recombinant PCSK9 protein (rPCSK9) [51,60]. In addition, a band of approximately 55 kD is also present (middle arrow), and this band had been described as representing a furin cleavage product of the intact protein [49]. The band at the very bottom of the blot represents the propeptide which remains tightly but non-covalently associated during the immunoprecipitation step, but is subsequently separated by denaturation prior to western blotting (lower arrow).