Influence of PDZK1 on lipoprotein metabolism and atherosclerosis

Abstract

PDZK1 is a scaffold protein containing four PDZ protein interaction domains, which bind to the carboxy termini of a number of membrane transporter proteins, including ion channels (e.g., CFTR) and cell surface receptors. One of these, the HDL receptor, scavenger receptor class B type I (SR-BI), exhibits a striking, tissue-specific dependence on PDZK1 for its expression and activity. In PDZK1 knockout (KO) mice there is a marked reduction of SR-BI protein expression (approximately 95%) in the liver, but not in steroidogenic tissues or, as we show in this report, in bone marrow- or spleen-derived macrophages, or lung-derived endothelial cells. Because of hepatic SR-BI deficiency, PDZK1 KO mice exhibit dyslipidemia characterized by elevated plasma cholesterol carried in abnormally large HDL particles. Here, we show that inactivation of the PDZK1 gene promotes the development of aortic root atherosclerosis in apolipoprotein E (apoE) KO mice fed with a high fat/high cholesterol diet. However, unlike complete SR-BI-deficiency in SR-BI/apoE double KO mice, PDZK1 deficiency in PDZK1/apoE double knockout mice did not result in development of occlusive coronary artery disease or myocardial infarction, presumably because of their residual expression of SR-BI. These findings demonstrate that deficiency of an adaptor protein essential for normal expression of a lipoprotein receptor promotes atherosclerosis in a murine model. They also define PDZK1 as a member of the family of proteins that is instrumental in preventing cardiovascular disease by maintaining normal lipoprotein metabolism.

Figure 1. Influence of PDZK1 on SR-BI expression and function in macrophages and endothelial cells

SR-BI expression (shown as histograms of relative fluorescence intensity) in splenic macrophages (A) and lung endothelial cells (B) was examined by flow cytometric analysis using as a primary antibody either the anti-SR-BI antibody KKB-1 (‘αSR-BI’, thick lines) or non-immune control normal rabbit serum (‘control’, thin lines). SR-BI expression in cells from wild-type (WT) mice is represented by black lines, whereas that from SR-BI KO (left panels) or PDZK1 KO (right panels) mice are represented with gray lines. Results from two pooled mice per group are shown (Panels A, B) and are consistent with independent experiments using two individual mice. In panel A (left) the results from SR-BI KO with control serum (thin gray line) overlaps with SR-BI KO with anti-SR-BI antibody (thick gray line). C. SR-BI function was measured by flow cytometry as the uptake of the lipophilic dye DiI from DiI-labeled HDL by splenic macrophages. SR-BI lipid uptake activity (cellular DiI accumulation) in macrophages from wild-type mice is represented by black lines, whereas that from SR-BI KO (left panels) or PDZK1 KO (right panels) mice are represented with gray lines. Results shown are from representative individual mice (n=2). 100% cell number ~1–2×10³.

Figure 2. Immunoblot analysis of hepatic SR-BI expression and lipoprotein cholesterol profiles from apoE KO and PDZK1/apoE dKO mice

Mice were fed a standard chow (panel A) or a high fat/high cholesterol “Western” diet (panels A, B and C) for three months. A. Livers were harvested and subjected to immunoblotting using anti-SR-BI and anti-actin antibodies as described in Materials and methods. B. Plasma harvested from individual Western diet-fed animals was size fractionated using FPLC and the total cholesterol (note change of scale in inset) contents of the fractions (mg/dL plasma) were determined by enzymatic assay. Profiles averaged from six apoE KO (filled circles) and six PDZK1/apoE dKO (open circles) mice are shown. Approximate elution positions of human VLDL, IDL/LDL and HDL are indicated. C. Unesterified cholesterol (UC) and total cholesterol (TC), and the corresponding unesterified-to-total (UC:TC) cholesterol ratios, were determined for FPLC fractions 6–25 ( averages from five apoE KO (filled circles) and four PDZK1/apoE dKO (open circles) mice). UC values beyond fraction 25 were below the limits of detection. Error bars represent SEM. See also Materials and methods.

Figure 3. Aortic root atherosclerosis in Western diet-fed apoE KO and PDZK1/apoE KO mice

A–B. Representative cross-sections of Oil red O-stained aortic root lesions. Magnification, x 20. C. Average lesion areas (mm², horizontal lines) were: apoE KO, 0.314±0.024 (n=9); PDZK1/apoE dKO, 0.493±0.023 (n=8), *P<0.0001. D–E. Representative sections of hearts stained with Masson’s trichrome, showing no evidence of either fibrosis or myocardial infarction. Magnification, x 4.5.