Secreted PCSK 9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentrationand time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization inHepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR.—Qian, Y-W., R. J. Schmidt, Y. Zhang, S. Chu, A. Lin, H. Wang, X. Wang, T. P. Beyer, W. R. Bensch, W. Li, M. E. Ehsani, D. Lu, R. J. Konrad, P. I. Eacho, D. E. Moller, S. K. Karathanasis, and G. Cao. Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis. J. Lipid Res. 2007. 48: 1488–1498. Supplementary key words proprotein convertase subtilisin/kexin type 9 Increased plasma LDL cholesterol is a major risk factor for atherosclerotic cardiovascular disease. Importantly, recent studies have suggested further benefits of very aggressive LDL cholesterol lowering compared with the typical clinical targets in place today (1, 2). Plasma LDL cholesterol is controlled primarily by hepatic cholesterol biosynthesis and hepatic low density lipoprotein receptor (LDLR) levels (3). The master transcription factor controlling mRNA levels that encode key enzymes involved in cholesterol biosynthesis and LDLR is sterol-responsive element binding protein 2 (SREBP2). When cellular cholesterol levels are low, SREBP2 is activated through sequential proteolytic cleavage by two proteases. The activated N terminus of the protein then enters the cell nucleus to mediate the transcription of genes that contain sterol response element(s) in their promoter or enhancer region (4). Cellular cholesterol levels are thus tightly regulated through this feedback mechanism. Recent human genetic studies have revealed that proprotein convertase subtilisin/kexin type 9 (PCSK9) is a critically important additional mechanism that regulates cellular LDLR levels. Although the molecular basis has yet to be determined, multiple mutations in the PCSK9 gene have been described to result in reduced cellular LDLR levels and thus significantly increased plasma LDL cholesterol (5–8). More importantly, opposite to these “gainof-function” mutations, Cohen et al. (9–13) found apparent loss-of-function mutations that are presumed to lead to increased cellular LDLR protein levels. In humans carrying these mutations, plasma LDL cholesterol is reduced by 30–40% compared with controls. More strikingly, an apparently healthy human subject with mutations affecting both alleles of the PCSK9 gene was described with an LDL cholesterol level of 14 mg/dl. This finding further indicates a critical role for PCSK9 in modulating LDLR Manuscript received 8 February 2007 and in revised form 21 March 2007. Published, JLR Papers in Press, April 20, 2007. DOI 10.1194/jlr.M700071-JLR200 1 Y-W. Qian, R. J. Schmidt, and Y. Zhang contributed equally to this work. 2 To whom correspondence should be addressed. e-mail: guoqing_cao@lilly.com Copyright D 2007 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org 1488 Journal of Lipid Research Volume 48, 2007 by gest, on A uust 8, 2017 w w w .j.org D ow nladed fom and plasma LDL cholesterol levels and provides evidence that loss of PCSK9 function in humans is not associated with apparent deleterious effects (12). PCSK9 belongs to the proprotein convertase family and was only recently cloned from brain tissue as a secreted protein (14). The closest homolog is site 1 protease, another protein intimately involved in cholesterol homeostasis (15). Beyond the function implied by human genetic studies, the physiological function of PCSK9 was not clear until the recent demonstration that hepatic overexpression in mice greatly reduced hepatic LDLR levels and led directly to increased plasma LDL cholesterol (16, 17). Consistent with these observations, PCSK9 deficiency in mice resulted in significantly increased hepatic LDLR levels (18). Furthermore, PCSK9 deficiency augmented statin-induced increase of LDLR protein levels and thus strongly suggested the value of PCSK9 as a pharmacological target for LDL cholesterol lowering (18). Although the function of PCSK9 in reducing hepatic LDLR protein levels is firmly established, the precise molecular basis for this effect has remained elusive. To date, the direct substrates and the active form of the enzyme are not known. In this report, we have investigated the potential molecular mechanisms of PCSK9 activity.