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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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 5

Association of PCSK9 and PCSK9(D374Y) with the LDLR.

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Association of PCSK9 and PCSK9(D374Y) with the LDLR. (A) Coimmunoprecipi...
(A) Coimmunoprecipitation of the LDLR and exogenously added wild-type PCSK9 or PCSK9(D374Y) protein. HepG2 cells were cultured for 18 hours in medium C prior to treatment for 1 hour in the presence of 0.1 mM chloroquine with 20 μg/ml or 2 μg/ml of purified human PCSK9 or PCSK9(D374Y), respectively. Cells were harvested and lysed and proteins immunoprecipitated with the indicated antibodies. Pellets of the immunoprecipitation were subjected to SDS-PAGE and immunoblot analysis. Polyclonal antiserum (Ab 3143) was used to detect LDLR, and IgG-15A6 was used to detect PCSK9. (B) Reduced (R) or nonreduced (NR) LDLR extracellular domain protein (2 μg) was resolved by SDS-PAGE. LDLR protein was detected with Coomassie brilliant blue R-250 stain. (C) Binding of PCSK9 to LDLR on ligand blots. LDLR protein (2 μg) was transferred to nitrocellulose and incubated with 5 μg/ml of purified PCSK9 or no addition (NA). Bound PCSK9 was detected by immunoblot analysis as described in Methods. (D) The indicated amounts of purified LDLR were subjected to nonreduced SDS-PAGE, transferred to nitrocellulose, and incubated with 5 μg/ml of purified wild-type PCSK9, PCSK9(D374Y), or buffer control (no addition). Bound PCSK9 was detected by immunoblot analysis as described in Methods. pep., FLAG octapeptide.