Plasma PCSK9 preferentially reduces liver LDL receptors in mice

Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that regulates the expression of LDL receptor (LDLR) protein. Gain-of-function mutations in PCSK9 cause hypercholesterolemia, and loss-offunction mutations result in lower plasma LDL-cholesterol. Here, we investigate the kinetics and metabolism of circulating PCSK9 relative to tissue levels of LDLRs. The administration of recombinant human PCSK9 (32 mg) to mice by a single injection reduced hepatic LDLRs by ?90% within 60 min, and the receptor levels returned to normal within 6 h. The half-life of the PCSK9 was estimated to be ?5 min. Continuous infusion of PCSK9 (32 mg/h) into wild-type mice caused a ?90% reduction in hepatic LDLRs within 2 h and no associated change in the level of LDLR in the adrenals. Parallel studies were performed using a catalytically inactive form of PCSK9, PCSK9(S386A), and similar results were obtained. Infusion of PCSK9(D374Y), a gain-of-function mutation, resulted in accelerated clearance of the mutant PCSK9 and a greater reduction in hepatic LDLRs. Combined, these data suggest that exogenously administrated PCSK9 in plasma preferentially reduces LDLR protein levels in liver at concentrations found in human plasma and that PCSK9's action on the LDLR is not dependent on catalytic activity in vivo. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proteinase K subfamily of enzymes (1-4). In mice, overexpression of human PCSK9 results in decreased hepatic LDL receptors (LDLRs) and hypercholesterolemia (2-5), whereas Pcsk9 2/2 mice have increased hepatic LDLRs and reduced plasma cholesterol concentrations (1). Similarly, humans carrying gain-of-function alleles for PCSK9 have increased plasma LDL-cholesterol (LDL-C) levels and premature coronary artery disease (6), whereas individuals with loss-of-function alleles have significantly reduced plasma LDL-C and are protected from atherosclerosis (7-9).PCSK9 is rapidly secreted from cultured hepatocytes and is found in the plasma of humans (10-12). The addition of recombinant human PCSK9 to the medium of HepG2 cells leads to the degradation of LDLRs in a timeand concentration-dependent manner (10). PCSK9 directly binds selectively to the epidermal growth factor-like repeat A domain of the LDLR (13, 14), and this binding and subsequent internalization are required for exogenous PCSK9 to reduce LDLR levels (10, 13). Several studies suggest that PCSK9 also functions intracellularly to degrade LDLRs (2, 15, 16). Currently, the relative contribution of the intracellular pathway versus the exogenous pathway of PCSK9-mediated LDLR degradation is not known.In parabiosis experiments, human PCSK9 secreted from livers of transgenic mice decreased hepatic LDLR protein levels when it was transferred via shared circulation to recipient wild-type mice (10). These studies demonstrated that human PCSK9 in plasma is capable of reducing hepatic LDLRs, although the plasma levels of PCSK9 (17-246 mg/ml) in the transgenic mice were ?100-to 1,000-fold higher ...

Endoplasmic Reticulum Stress and Ca2+ Depletion Differentially Modulate the Sterol Regulatory Protein PCSK9 to Control Lipid Metabolism

“…Veh, vehicle; NS, not significant. expression (37,54,55). Although it has been reported that TM induces the VLDLR via the PERK-ATF4 pathway (13), the potential mechanisms by which TM affects LDLR expression are numerous.…”

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

“…Although hepatocyte-specific transgenic mice weakly overexpressing PCSK9 (3-fold) exhibit no significant change in circulating LDL-cholesterol, a ϳ30-fold overexpression increased it by ϳ5-fold (20). Data from transgenic mice expressing very high levels of human PCSK9 in mouse kidney (35) or liver (16), or continuous infusions of recipient WT mice with recombinant PCSK9 (36) indicated that Ͼ100 nM amounts of circulating PCSK9 are required to affect liver LDLR protein levels significantly, without affecting extrahepatic LDLR. In that context, we recently showed that annexin A2 inhibits the effect of extracellular PCSK9 on LDLR, and in view of its absence from hepatocytes in vivo, it may exert its inhibitory effect in extrahepatic tissues (14).…”

Mutations and polymorphisms in the proprotein convertase subtilisin kexin 9 (PCSK9) gene in cholesterol metabolism and disease

“…Finally, the low plasma LDL-C levels and the reduction of CHD displayed in PCSK9 loss-of-function mutation carriers, which seem furthermore healthy, indicate that the inhibition of PCSK9 should be an effective target in the treatment of hypercholesterolemia with no known adverse side effects. The recent elucidation of the crystal structure of PCSK9 [Cunningham et al, 2007;Piper et al, 2007], especially in complex with the LDL receptor [Kwon et al, 2008], should provide essential clues for the identification of small pharmacological molecules or antibodies that might block the interaction of PCSK9 and the LDLR and thus inhibit PCSK9 function and constitute new cholesterol-lowering drugs [Kwon et al, 2008;Grefhorst et al, 2008].…”