Proprotein convertase subtilisin/kexin type 9 (PCSK9): Hepatocyte-specific low-density lipoprotein receptor degradation and critical role in mouse liver regeneration

Abstract

The gene encoding the proprotein convertase subtilisin/kexin type 9 (PCSK9) is linked to familial hypercholesterolemia, as are those of the low-density lipoprotein receptor (LDLR) and apolipoprotein B. PCSK9 enhances LDLR degradation, resulting in low-density lipoprotein accumulation in plasma. To analyze the role of hepatic PCSK9, total and hepatocyte-specific knockout mice were generated. They exhibit 42% and 27% less circulating cholesterol, respectively, showing that liver PCSK9 was responsible for two thirds of the phenotype. We also demonstrated that, in liver, PCSK9 is exclusively expressed in hepatocytes, representing the main source of circulating PCSK9. The data suggest that local but not circulating PCSK9 regulates cholesterol levels. Although transgenic mice overexpressing high levels of liver and circulating PCSK9 led to the almost complete disappearance of the hepatic LDLR, they did not recapitulate the plasma cholesterol levels observed in LDLRdeficient mice. Single LDLR or double LDLR/PCSK9 knockout mice exhibited similar cholesterol profiles, indicating that PCSK9 regulates cholesterol homeostasis exclusively through the LDLR. Finally, the regenerating liver of PCSK9-deficient mice exhibited necrotic lesions, which were prevented by a high-cholesterol diet. However, lipid accumulation in hepatocytes of these mice was markedly reduced under both chow and high-cholesterol diets, revealing that PCSK9 deficiency confers resistance to liver steatosis. Conclusion: Although PCSK9 is a target for controlling hypercholesterolemia, our data indicate that upon hepatic damage, patients lacking PCSK9 could be at risk. (HEPATOLOGY 2008;48:646-654.) P roprotein convertase subtilisin/kexin type 9 (PCSK9) 1 is the ninth member of the proprotein convertase family. 2 The first seven members, including furin, cleave protein precursors of hormones, growth factors, receptors, or surface glycoproteins at basic sites (after Arg or Lys residues). The eighth member, SKI-1 3 or S1P, 4 is known to cleave membrane-bound transcription factors such as the SREBPs 5 in their luminal domains, resulting in the release of their DNA-binding domain. Proprotein convertases can also inactivate secreted substrates, such as endothelial lipase 6 and PCSK9. 7 PCSK9 is synthesized as a precursor that undergoes autocatalytic cleavage of its N-terminal prosegment in the ER, 1 a step required for its exit from this compartment and its efficient secretion. Secreted PCSK9 remains associated with its prosegment. 1 Different from the other proprotein convertases, this serine protease has no known substrate other than itself. In addition, the tight association of the prosegment with the active site 8 raises the question of the existence of an in trans PCSK9 protease

Dissection of the Endogenous Cellular Pathways of PCSK9-induced Low Density Lipoprotein Receptor Degradation

“…Incubations-In agreement with whole liver data (20,25), LDLR levels increased by ϳ2.5-fold in primary hepatocytes from Pcsk9 (Fig. 3).…”

“…Western blot analyses of LDLR, human PCSK9, and ␤-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.…”

“…Immunoprecipitation of PCSK9-Secreted mouse PCSK9 derived from isolated primary hepatocytes was detected, as described (20). Briefly, 1 ϫ 10 6 hepatocytes were plated on 6-well plates in serum-free hepatoZYME medium and incubated at 37°C for 48 h. Using the rabbit IgG TrueBlot kit (eBioscience), 500 l of conditioned medium (1:4), 500 l of complete radioimmune precipitation assay buffer (0.5ϫ), 50 l of anti-rabbit IgG beads, and 3 l of primary mouse PCSK9 antibody were incubated overnight on a rocking platform at 4°C.…”

PCSK9 knock-out mice are protected from neointimal formation in response to perivascular carotid collar placement

“…The positive effect of PCSK9 on cell proliferation has been previously observed after partial hepatectomy in PCSK9 -/-mice 28 and in hepatocyte cell line HepG2 following PCSK9 downregulation by shRNA 29 . Moreover, since in tumor cells the positive effect of PCSK9 on cell number is also associated with a reduction of apoptosis 29 .…”

Differences in allele frequencies of autosomal dominant hypercholesterolemia SNPs in the Malaysian population

“…[10][11][12] Conversely, the depletion or removal of PCSK9 protein can substantially increase the level of LDLR and decrease the plasma cholesterol. [13][14][15][16] It is very important that haplotype signatures of prominent SNPs of these three genes are studied in all populations rather than inferring or assuming frequencies from the HAPMAP data, where only a few populations are tested. LDLR is a highly polymorphic gene but how relevant is the CHB (Han Chinese in Beijing) frequencies to other similar races in other locations?…”