Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other

Abstract

Amyloid precursor-like protein 2 (APLP2) and sortilin were reported to individually bind the proprotein convertase subtilisin/kexin type 9 (PCSK9) and regulate its activity on the low-density lipoprotein receptor (LDLR). The data presented herein demonstrate that mRNA knockdowns of APLP2, sortilin, or both in the human hepatocyte cell lines HepG2 and Huh7 do not affect the ability of extracellular PCSK9 to enhance the degradation of the LDLR. Furthermore, mice deficient in APLP2 or sortilin do not exhibit significant changes in liver LDLR or plasma total cholesterol levels. Moreover, cellular overexpression of one or both proteins does not alter PCSK9 secretion, or its activity on the LDLR. We conclude that PCSK9 enhances the degradation of the LDLR independently of either APLP2 or sortilin both ex vivo and in mice. Interestingly, when co-expressed with PCSK9, both APLP2 and sortilin were targeted for lysosomal degradation. Using chemiluminescence proximity and co-immunoprecipitation assays, as well as biosynthetic analysis, we discovered that sortilin binds and stabilizes APLP2, and hence could regulate its intracellular functions on other targets.

Keywords: APLP2; Sortilin (SORT1); cardiovascular disease; dyslipidemia; lipoprotein metabolism; low-density lipoprotein (LDL); low-density lipoprotein receptor (LDLR) degradation; proprotein convertase subtilisin/kexin type 9 (PCSK9).

FIGURE 1.

Exogenous PCSK9 can induce degradation of the LDLR in the absence of APLP2. HepG2 (A) and Huh7 (B) cells were transfected with a control non-target siRNA (Ctrl) or 3 different siRNAs targeting APLP2. Cells were incubated overnight with serum-free conditioned media lacking or containing 1 μg/ml of PCSK9-V5. HepG2 and Huh7 cell lysates were then subjected to Western blotting using LDLR, APLP2, and β-actin antibodies. LDLR and APLP2 signals were normalized to that of β-actin. C, the input HEK293 conditioned medium was analyzed using mAb-V5 to detect PCSK9-V5. D, duplicate samples of Huh7 cells matching those in panel B were analyzed by FACS to assess the cell surface LDLR levels. Values were normalized to that of the first lane (control non-target siRNA in the absence of PCSK9). Error bars represent S.E. *, p < 0.05 (Student's t test). The data shown here are representative of two to three independent experiments.

FIGURE 2.

APLP2-deficient mice exhibit reduced levels of circulating PCSK9 but similar levels of total and cell surface LDLR in the liver. A, plasma levels of PCSK9 were measured by ELISA in Aplp2^+/+ (WT) and Aplp2^−/− mice. Error bars represent S.E. **, p < 0.01 (Student's t test). B, liver extracts of Aplp2^+/+ (n = 4), Aplp2^−/− (n = 3), and Pcsk9^−/− (n = 1) mice were analyzed by Western blot analysis. The latter showed a 2.5-fold higher level of LDLR. Note that liver APLP2 levels were not affected in Pcsk9^−/− mice (last lane). C, immunohistochemistry of surface LDLR (green) in the liver of Aplp2^+/+, Aplp2^−/−, Ldlr^−/−, and Pcsk9^−/− mice. Bar = 60 μm. D, LDLR levels were analyzed in primary hepatocytes isolated from Pcsk9^−/− (n = 1), Aplp2^+/+ and Aplp2^−/− (n = 3) mice. E, Aplp2^+/+ and Aplp2^−/− primary hepatocytes were incubated without or with purified human PCSK9 (10 μg/ml) for 2 and/or 16 h. LDLR relative intensities to β-actin obtained by Western blotting were normalized to that of the Aplp2^+/+ signal in the absence of PCSK9 (first lane in bold). These data are representative of at least two independent experiments.

FIGURE 3.

Sortilin depletion does not affect LDLR degradation by PCSK9 and has no cholesterol phenotype in mice. A, Huh7 cells were transfected with control non-target, APLP2-, and/or sortilin-specific siRNAs. After 48 h, cells were incubated in serum-free media for 24 h. Cell lysates were then subjected to Western blotting using LDLR, sortilin, APLP2, and β-actin antibodies. B, total liver extracts of Sort^+/+ (n = 3), Sort1^−/− (n = 3), Ldlr^−/− (n = 1), Pcsk9^+/+ (n = 1), and Pcsk9^−/− (n = 1) mice were analyzed by Western blot analysis. Accurate quantification of LDLR and sortilin signals and their normalization to that of β-actin was obtained by LI-COR analysis of a duplicate gel. Relative LDLR/β-actin signals were normalized to that of control (A and B in bold). C, immunohistochemistry of surface LDLR (green) in the liver of Sort^+/+, Sort1^−/−, Ldlr^−/−, and Pcsk9^−/− mice. Surface LDLR levels in the liver of Sort1^−/− and Sort^+/+ mice were similar. Bar = 60 μm. Plasma (D) total cholesterol and (E) PCSK9 measured by ELISA, in control Pcsk9^+/+ and Pcsk9^−/− mice, and in Sort^+/+ and Sort1^−/− mice. Error bars represent S.E. ***, p < 3 × 10⁻⁵ (Student's t test). F, Western blot analysis of total liver extracts of Pcsk9^+/+ (n = 3), Pcsk9^+/− (n = 3), and Pcsk9^−/− (n = 3) mice. Accurate quantification of LDLR and its normalization to that of β-actin was obtained by BioRad Image Lab 5.2 analysis of the same gel. Relative LDLR/β-actin signals were normalized to that of Pcsk9^+/+ (in bold). These data are representative of at least two independent experiments.

FIGURE 4.

Sortilin and APLP2 are novel cellular targets of PCSK9. A, overexpressed PCSK9 induces sortilin and APLP2 degradation in HEK293 cells. Triplicate Western blot analyses revealing that both sortilin-Myc and APLP2-V5 expression levels in HEK293 cells were reduced by 90 and 40%, respectively, upon transfection with a PCSK9 plasmid, as compared with a control empty pIRES vector (V). Quantification of sortilin and APLP2 band intensities were normalized against those of β-actin. B, HEK293 cells transfected with a cDNA coding for an empty vector control (pIRES; V) or individually with human sortilin or APLP2 tagged at the C terminus with a Myc or V5 epitope, respectively, or together in the absence or presence of a cDNA coding for untagged PCSK9. The following day the cells were washed and then pulsed for 4 h with [³⁵S]Met + Cys in the presence or absence of 5 mm NH₄Cl. The cells were then extracted and their lysates immunoprecipitated (IP) with a mAb-V5 or mAb-Myc or a polyclonal antibody for PCSK9. The precipitates were separated on an 8% SDS-PAGE. The dried gel was then autoradiographed. Notice the co-precipitation of sortilin and APLP2 in the presence of NH₄Cl. These data are representative of at least three independent experiments.

FIGURE 5.

Sortilin, APLP2, and soluble APLP2 are degraded by both PCSK9 and ER-localized PCSK9-KDEL isoforms. HEK293 cells were transfected with indicated DNA amounts of vectors encoding a control protein 7B2, sortilin (no tag), APLP2-V5, soluble APLP2-V5 (sAPLP2-V5), PCSK9-V5, or PCSK9-V5-KDEL, as indicated. After 48 h, lysates and media were analyzed by Western blotting for the indicated proteins. The data show that overexpressed PCSK9 or PCSK9-KDEL induces degradation of sortilin (A), APLP2 (B), and sAPLP2 (C) in HEK293 cells. Quantification of sortilin and APLP2 band intensities were normalized against those of β-actin or GAPDH. These data are representative of two independent experiments.

FIGURE 6.

Co-expression of sortilin, APLP2, or both with PCSK9 has no major effect on LDLR degradation. Huh7 cells were transfected with a total of 3 μg using 1 μg of each vector encoding for either a control protein 7B2 (−), sortilin (+), APLP2 (+), or PCSK9 (+), as indicated. After 48 h, lysates were analyzed by Western blotting for expression of the LDLR, sortilin-Myc, APLP2-V5, intracellular pro- and mature-PCSK9-V5, and β-actin. Media were analyzed for secreted endogenous and overexpressed PCSK9-V5 using a rabbit polyclonal human PCSK9 antibody. Quantification of LDLR expression was normalized against that of β-actin. These data are representative of at least 3 different experiments showing similar results.

FIGURE 7.

Sortilin binds APLP2. A, schematic diagram of sortilin and APLP2 fused to the G. princeps luciferase half-domains, Gluc-1 and Gluc-2, respectively. B, heat map generated by G. princeps luciferase complementation assay showing the interaction profile of 36 protein pairs. Normalized luminescence ratio ranging from strong to null interactions is displayed on a light blue to black scale. C, validation of sortilin-Myc and APLP2-V5 interaction was confirmed by co-expression in HEK293 cells and immunoprecipitation with mAb-Myc followed by Western blotting (WB) using a mAb-Myc or mAb-V5.

FIGURE 8.

Comparative in situ hybridization histochemistry of APLP2, PCSK9, and sortilin mRNA expression. Comparative in situ hybridization localization of APLP2, PCSK9, and sortilin mRNA in cryostat sagittal sections of whole body postnatal mouse at post-partum day 10 (P10) mice (left panel, magnification ×2.4) and in the liver of adult mice (right panel, magnification ×4). A, x-ray film autoradiography showing widespread APLP2 expression pattern with high mRNA concentration in the eye retina and lens, brain, cerebellum, brown fat, liver, spleen, skin, small intestine, colon, and kidney. B, tissue-specific PCSK9 expression pattern with moderate to high level mRNA concentration in the eye retina inner nuclear layer, brain olfactory tract and cerebellum, spleen, liver, small intestine, and colon. C, tissue-specific sortilin expression pattern with moderate to high level mRNA concentration in the eye retina and lens, brain, cerebellum, skin, kidney medulla, colon, and small intestine. Abbreviations: BF, brown fat; BM, bone marrow; Br, brain; Cb, cerebellum; Co, colon; Ki, kidney; LG, lacrimal gland; Le, eye lens; Li, liver; Lu, lung; Mo, molars; OT, olfactory tract; Re, retina; SIn, small intestine; Sk, skin; Spl, spleen; St, stomach; Str, brain striatum; Th, thymus. *, indicates nonspecific staining of the aorta.