Cathepsin D, a Lysosomal Protease, Regulates ABCA1-mediated Lipid Efflux

doi:10.1074/jbc.M605095200

Cathepsin D, a Lysosomal Protease, Regulates ABCA1-mediated Lipid Efflux*

^‡Lipoprotein and Atherosclerosis Research Group, University of Ottawa Heart Institute, and Departments of Biochemistry, Microbiology, and Immunology, and Pathology and Laboratory Medicine, and Medicine (Cardiology), University of Ottawa, Ottawa, Ontario, K1Y 4W7, Canada and ^§GlaxoSmithKline, King of Prussia, Pennsylvania 19406

2 To whom correspondence should be addressed: University of Ottawa Heart Institute, 40 Ruskin St., Ottawa, Ontario K1Y 4W7, Canada. Tel.: 613-761-5254; Fax: 613-761-5281; E-mail: ylmarcel{at}ottawaheart.ca.

Abstract

To identify genes involved in the regulation of plasma high density lipoprotein (HDL) cholesterol (HDL-C) levels, patients with low HDL-C and age- and sex-matched controls (normal HDL-C) were extensively characterized. Comparative transcriptome analysis was carried out in cholesterol-loaded monocyte-derived macrophages from low HDL subjects segregated into groups with or without cholesterol efflux defects or ABCA1 mutations. Clusters of differentially regulated genes were evident in the low HDL groups as compared with controls. Of particular note, expression of cathepsin D (CTSD), a lysosomal proteinase, was reduced by ∼50% in monocyte-derived macrophages of low HDL-C subjects, most significantly those with cholesterol efflux defects but without mutations in ABCA1 (p < 0.01). These results were verified by reverse transcription-PCR and replicated in a second cohort. We show here that blocking the activity or expression of CTSD, by pepstatin or CTSD small interfering RNA, respectively, reduced ABCA1 expression and protein abundance in both macrophages and CHO cells and apolipoprotein A-I-mediated lipid efflux by more than 70%. Conversely, expression of CTSD increased both ABCA1 mRNA expression and cellular ABCA1 protein. Consistent with its role in the proteolytic processing of prosaposin, inactivation of CTSD function resulted in the accumulation of glycosphingo-lipid and free cholesterol in late endosomes/lysosomes, a phenotype similar to NPC1 deficiency. Inhibition of CTSD also caused retention of ABCA1 in lysosomal compartments, reducing its trafficking to the plasma membrane. These studies demonstrate a novel and potentially important role for CTSD in intracellular cholesterol trafficking and ABCA1-mediated efflux. Therefore, decreased CTSD expression may contribute to low plasma HDL-C levels.

Footnotes

↵3 The abbreviations used are: HDL-C, high density lipoprotein cholesterol; HDL, high density lipoprotein; LDL, low density lipoprotein; MDM, monocyte-derived macrophage(s); CTSD, cathepsin D; GFP, green fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; RT, reverse transcription; FBS, fetal bovine serum; siRNA, small interfering RNA; dsRNA, double-stranded RNA; GSL, glycosphingolipid; CHO, Chinese hamster ovary; BODIPY, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene.
↵4 L. Sarov-Blat, R. S. Kiss, B. Haidar, N. Kavaslar, M. Jaye, M. Bertiaux, K. Steplewski, D. Sprecher, R. McPherson, and Y. L. Marcel, manuscript submitted for publication.
↵5 R. S. Kiss, N. Kavaslar, K. Okuhira, M. W. Freeman, S. Walter, R. W. Milne, R. McPherson, and Y. L. Marcel, manuscript submitted for publication.
↵* This work was supported in part by grants from the Ontario Heart and Stroke Foundation (to Y. L. M.) and from the Canadian Institutes of Health Research (Grant 44359 (to Y. L. M.) and group Grant 64519). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1.
↵1 Supported by a postdoctoral fellowship from the Heart and Stroke Foundation of Canada.
- Received May 30, 2006.
- Revision received October 5, 2006.
The American Society for Biochemistry and Molecular Biology, Inc.