Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and inlivers of parabiotic mice
Thomas A. Lagace, … , Robert E. Hammer, Jay D. Horton
Thomas A. Lagace, … , Robert E. Hammer, Jay D. Horton
Published November 1, 2006
Citation Information: J Clin Invest. 2006;116(11):2995-3005. https://doi.org/10.1172/JCI29383.
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Research Article Cardiology

Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and inlivers of parabiotic mice

Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proteinase K subfamily of subtilases that reduces the number of LDL receptors (LDLRs) in liver through an undefined posttranscriptional mechanism. We show that purified PCSK9 added to the medium of HepG2 cells reduces the number of cell-surface LDLRs in a dose- and time-dependent manner. This activity was approximately 10-fold greater for a gain-of-function mutant, PCSK9(D374Y), that causes hypercholesterolemia. Binding and uptake of PCSK9 were largely dependent on the presence of LDLRs. Coimmunoprecipitation and ligand blotting studies indicated that PCSK9 and LDLR directly associate; both proteins colocalized to late endocytic compartments. Purified PCSK9 had no effect on cell-surface LDLRs in hepatocytes lacking autosomal recessive hypercholesterolemia (ARH), an adaptor protein required for endocytosis of the receptor. Transgenic mice overexpressing human PCSK9 in liver secreted large amounts of the protein into plasma, which increased plasma LDL cholesterol concentrations to levels similar to those of LDLR-knockout mice. To determine whether PCSK9 was active in plasma, transgenic PCSK9 mice were parabiosed with wild-type littermates. After parabiosis, secreted PCSK9 was transferred to the circulation of wild-type mice and reduced the number of hepatic LDLRs to nearly undetectable levels. We conclude that secreted PCSK9 associates with the LDLR and reduces hepatic LDLR protein levels.

Authors

Thomas A. Lagace, David E. Curtis, Rita Garuti, Markey C. McNutt, Sahng Wook Park, Heidi B. Prather, Norma N. Anderson, Y.K. Ho, Robert E. Hammer, Jay D. Horton

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Figure 4

LDLR-dependent endocytosis of PCSK9 in MEFs.

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LDLR-dependent endocytosis of PCSK9 in MEFs. (A) Immunoblot analysis of ...
(A) Immunoblot analysis of PCSK9 association with MEFs. Immortalized MEFs derived from wild-type, Ldlr–/–, Lrp–/–, and Ldlr–/–Lrp–/–mice were cultured for 18 hours in medium F prior to treatment with purified human PCSK9 for 4 hours. Cell lysates (30 μg) were subjected to SDS-PAGE and immunoblot analysis using IgG-15A6 to detect PCSK9 or a polyclonal antiserum (Ab 3143) that recognizes the LDLR, as described in Methods. Immunoblots of receptor-associated protein (RAP) were used as a loading control. (B) Indirect immunofluorescence localization of PCSK9 and the LDLR in wild-type MEFs. MEFs were incubated for 4 hours with 5 μg/ml purified human PCSK9 and processed for double immunofluorescence of PCSK9 (green) and the LDLR (red) as described in Methods. (C) Indirect immunofluorescence localization of PCSK9, LDLR, and the late-endosomal marker CI-MPR in MEFs cultured in the presence of chloroquine. Wild-type MEFs were incubated for 4 hours with 5 μg/ml purified human PCSK9 in the presence of 0.1 mM chloroquine and processed for double immunofluorescence of PCSK9 (green) and the LDLR (red) or CI-MPR (red) as described in Methods. The merged image shows areas of colocalization of PCSK9 with the LDLR and CI-MPR. Magnification, ×630. Similar results were obtained in 3 independent experiments.