Cellular and secreted pro-protein convertase subtilisin/kexin type 9 catalytic activity in hepatocytes.

OBJECTIVES: Pro-protein convertase subtilisin/kexin type 9 (PCSK9) impairs the low density lipoprotein receptor (LDLr) recyling. To reach the LDLr, the pro-protein must cleave itself in the endoplasmic reticulum. Using a fluorogenic peptide corresponding to the cleavage site, we directly monitored for the first time the cleavage activity of purified human PCSK9 and that of endogenous human wild-type PCSK9 and naturally occurring variants in hepatocytes. METHODS: Validation of the assay was performed with wild type or PCSK9 deficient primary mouse hepatocytes and immortalized human hepatocytes transfected with antiPCSK9 siRNA. An analysis of the cleaved peptide was performed using mass spectrometry. Pharmacological regulation of the enzyme was studied in human hepatocytes. Expression vectors coding for the variants S127R, D374Y, F216L, S386A were transfected in primary hepatocytes from PCSK9 deficient mice. RESULTS: PCSK9 activity was measured in cell lysates and media, at levels 100 times higher than with the human purified recombinant protein. The assay is highly specific for PCSK9 in cell lysate and cell culture media but not in plasma. Pharmacological up- or down-regulation of PCSK9 expression produced paralleled effects on the activity. The catalytic activity of gain-of-function variants S127R, D374Y recapitulated roughly the maturation efficiency estimated by western blots, in contrast with the F216L variant that presented with a 54% lower catalytic activity than the wild-type protein, despite similar proPCSK9 to PCSK9 ratios. Thus, other factors might be involved in the maturation of PCSK9. CONCLUSION: All together, these results shed a new light on PCSK9 enzymatic activity and could help identifying proPCSK9 inhibitors.

Dual mechanisms for the fibrate-mediated repression of proprotein convertase subtilisin/kexin type 9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with familial autosomal dominant hypercholesterolemia and is a natural inhibitor of the LDL receptor (LDLr). PCSK9 is degraded by other proprotein convertases: PC5/6A and furin. Both PCSK9 and the LDLr are up-regulated by the hypocholesterolemic statins. Thus, inhibitors or repressors of PCSK9 should amplify their beneficial effects. In the present study, we showed that PPARalpha activation counteracts PCSK9 induction by statins by repressing PCSK9 promoter activity and by increasing PC5/6A and furin expression. Quantification of mRNA and protein levels showed that various fibrates decreased PCSK9 and increased PC5/6A and furin expression. Fenofibric acid (FA) reduced PCSK9 protein content in immortalized human hepatocytes (IHH) as well as its cellular secretion. FA suppressed PCSK9 induction by statins or by the liver X receptor agonist TO901317. PCSK9 repression is occurring at the promoter level. We showed that PC5/6A and furin fibrate-mediated up-regulation is PPARalpha-dependent. As a functional test, we observed that FA increased by 30% the effect of pravastatin on the LDLr activity in vitro. In conclusion, fibrates simultaneously decreased PCSK9 expression while increasing PC5/6A and furin expression, indicating a broad action of PPARalpha activation in proprotein convertase-mediated lipid homeostasis. Moreover, this study validates the functional relevance of a combined therapy associating PCSK9 repressors and statins.

Fasting induces hyperlipidemia in mice overexpressing proprotein convertase subtilisin kexin type 9: lack of modulation of very-low-density lipoprotein hepatic output by the low-density lipoprotein receptor.

Several proprotein convertase subtilisin kexin type 9 (PCSK9) mutations lead to familial hypercholesterolemia by virtue of its role as a negative modulator of the low-density lipoprotein receptor (LDLr). Here, we uncover that upon dietary challenge, the down-regulation of the LDLr is also a key mechanism by which PCSK9 modulates the hepatic production of apolipoprotein-B-containing lipoproteins. Thus, adenoviral-mediated overexpression of PCSK9 in 24-h fasted mice results in massive hyperlipidemia, due to a striking increase in very-low-density lipoprotein (VLDL) triglycerides and apolipoprotein B100 hepatic output. Similar studies in LDLr (-/-) mice demonstrate that PCSK9-mediated alteration of VLDL output in the fasted state requires the LDLr. This increased production of VLDL was associated with a concomitant reduction of intrahepatic lipid stores as well as a lack of down-regulation of peroxisome proliferator-activated receptor-alpha activity and target genes expression. Finally, we show that PCSK9 hepatic expression is inhibited by the hypotriglyceridemic peroxisome proliferator-activated receptor-alpha agonist fenofibrate. In summary, the negative modulation of LDLr expression by PCSK9, which decreases plasma LDL clearance, also promotes an overproduction of nascent VLDL in vivo upon fasting.

Hepatic PCSK9 expression is regulated by nutritional status via insulin and sterol regulatory element-binding protein 1c.

Familial autosomal dominant hypercholesterolemia is associated with high risk for cardiovascular accidents and is related to mutations in the low density lipoprotein receptor or its ligand apolipoprotein B (apoB). Mutations in a third gene, proprotein convertase subtilisin kexin 9 (PCSK9), were recently associated to this disease. PCSK9 acts as a natural inhibitor of the low density lipoprotein receptor pathway, and both genes are regulated by depletion of cholesterol cell content and statins, via sterol regulatory element-binding protein (SREBP). Here we investigated the regulation of PCSK9 gene expression during nutritional changes. We showed that PCSK9 mRNA quantity is decreased by 73% in mice after 24 h of fasting, leading to a 2-fold decrease in protein level. In contrast PCSK9 expression was restored upon high carbohydrate refeeding. PCSK9 mRNA increased by 4-5-fold in presence of insulin in rodent primary hepatocytes, whereas glucose had no effect. Moreover, insulin up-regulated hepatic PCSK9 expression in vivo during a hyperinsulinemic-euglycemic clamp in mice. Adenoviral mediated overexpression of a dominant or negative form of SREBP-1c confirmed the implication of this transcription factor in insulin-mediated stimulation of PCSK9 expression. Liver X receptor agonist T0901317 also regulated PCSK9 expression via this same pathway (a 2-fold increase in PCSK9 mRNA of primary hepatocytes cultured for 24 h in presence of 1 microm T0901317). As our last investigation, we isolated PCSK9 proximal promoter and verified the functionality of a SREBP-1c responsive element located from 335 bp to 355 bp upstream of the ATG. Together, these results show that PCSK9 expression is regulated by nutritional status and insulinemia.

Wild-type PCSK9 inhibits LDL clearance but does not affect apoB-containing lipoprotein production in mouse and cultured cells.

Mutations in Proprotein Convertase Subtilisin Kexin 9 (PCSK9) have been associated with autosomal dominant hypercholesterolemia. In vivo kinetic studies indicate that LDL catabolism was impaired and apolipoprotein B (apoB)-containing lipoprotein synthesis was enhanced in two patients presenting with the S127R mutation on PCSK9. To understand the physiological role of PCSK9, we overexpressed human PCSK9 in mouse and cellular models as well as attenuated the endogenous expression of PCSK9 in HuH7 hepatoma cells using RNA interference. Here, we show that PCSK9 dramatically impairs the expression of the low density lipoprotein receptor (LDLr) and, in turn, LDL cellular binding as well as LDL clearance from the plasma compartment in C57BL6/J mice but not in LDLr-deficient mice, establishing a definitive role for PCSK9 in the modulation of the LDLr metabolic pathway. In contrast to data obtained in S127R-PCSK9 patients presenting with increased apoB production, our study indicates that wild-type PCSK9 does not significantly alter the production and/or secretion of VLDL apoB in either cultured cells or mice. Finally, we show that unlike PCSK9 overexpression in mice, the S127R mutation in patients led to increased VLDL apoB levels, suggesting a potential gain of function for S127R-PCSK9 in humans.