Basic Sciences

PCSK9 Induces CD36 Degradation and Affects Long-Chain Fatty Acid Uptake and Triglyceride Metabolism in Adipocytes and in Mouse LiverSignificance

Annie Demers, Samaneh Samami, Benjamin Lauzier, Christine Des Rosiers, Emilienne Tudor Ngo Sock, Huy Ong, Gaetan Mayer
https://doi.org/10.1161/ATVBAHA.115.306032
Arteriosclerosis, Thrombosis, and Vascular Biology. 2015;35:2517-2525
Originally published October 22, 2015

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Abstract

Objective—Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the degradation of the low-density lipoprotein receptor thereby elevating plasma low-density lipoprotein cholesterol levels and the risk of coronary heart disease. Thus, the use of PCSK9 inhibitors holds great promise to prevent heart disease. Previous work found that PCSK9 is involved in triglyceride metabolism, independently of its action on low-density lipoprotein receptor, and that other yet unidentified receptors could mediate this effect. Therefore, we assessed whether PCSK9 enhances the degradation of CD36, a major receptor involved in transport of long-chain fatty acids and triglyceride storage.

Approach and Results—Overexpressed or recombinant PCSK9 induced CD36 degradation in cell lines and primary adipocytes and reduced the uptake of the palmitate analog Bodipy FL C16 and oxidized low-density lipoprotein in 3T3-L1 adipocytes and hepatic HepG2 cells, respectively. Surface plasmon resonance, coimmunoprecipitation, confocal immunofluorescence microscopy, and protein degradation pathway inhibitors revealed that PCSK9 directly interacts with CD36 and targets the receptor to lysosomes through a mechanism involving the proteasome. Importantly, the level of CD36 protein was increased by >3-fold upon small interfering RNA knockdown of endogenous PCSK9 in hepatic cells and similarly increased in the liver and visceral adipose tissue of Pcsk9−/− mice. In Pcsk9−/− mice, increased hepatic CD36 was correlated with an amplified uptake of fatty acid and accumulation of triglycerides and lipid droplets.

Conclusions—Our results demonstrate an important role of PCSK9 in modulating the function of CD36 and triglyceride metabolism. PCSK9-mediated CD36 degradation may serve to limit fatty acid uptake and triglyceride accumulation in tissues, such as the liver.

Introduction

Maintenance of optimal blood lipid levels is central to vascular health. Dysregulation of circulating lipid homeostasis, such as elevated levels of low-density lipoprotein cholesterol (LDLc) and triglycerides, is associated with premature atherosclerosis and coronary heart disease.1 Naturally occurring genetic variations at the proprotein convertase subtilisin/kexin type 9 (PCSK9) locus are major determinant of plasma LDLc levels in humans.2 PCSK9 promotes the degradation of the hepatic LDL receptor (LDLR), the primary pathway of LDLc uptake from the circulation, thereby causing LDLc levels to rise.3 Gain-of-function mutations in PCSK9 can cause autosomal dominant hypercholesterolemia and premature death resulting from coronary atherosclerosis.4,5 Remarkably, PCSK9 loss-of-function mutations lower circulating LDLc and are associated with strikingly reduced incidence of coronary heart disease.6 As a result, inhibitors of PCSK9 are being developed and tested in clinical trials with the ultimate goal of lowering risk of heart diseases.7

PCSK9 is a secretory serine protease mainly expressed by the liver, intestine, and kidney.8,9 Unlike other proprotein convertases that lose their prosegment and are activated in the late secretory pathway,10 PCSK9 is secreted in complex with its prosegment trapped in the catalytic pocket.11 Hence, PCSK9 induces LDLR degradation independently of its catalytic activity.12 PCSK9 interacts with the LDLR epidermal growth factor–like repeat A (EGFA) domain at the cell surface13 and is internalized in endosomes, where its affinity for the receptor increases by several fold,11 thereby impeding LDLR recycling to the cell surface and promoting its degradation by lysosomal hydrolases.3,13 A previous study demonstrated that PCSK9 enhances the degradation of other receptors, such as the very low-density lipoprotein receptor and apolipoprotein E receptor 2, the closest structural members to LDLR, emphasizing its major role in cholesterol and lipid homeostasis.14 Furthermore, PCSK9 induces the degradation of CD81, a receptor for hepatitis C virus,15 the epithelial sodium channel (ENaC),16 and β-secretase 1 (BACE1).17 Hence, PCSK9 targets multiple proteins and receptors, of which some are not related to the LDLR family, suggesting that the convertase has other metabolic roles. Indeed, plasma PCSK9 levels correlate with multiple metabolic markers, such as fasting levels of glucose, insulin, triglycerides, and hepatic triglycerides content, indicating that it could target other receptors that remain to be uncovered.18,19

Cluster of differentiation 36 (CD36; scavenger receptor class B type 3 [SRB3] or fatty acid translocase) is a member of the scavenger receptor class B family that includes SRB1, the high-density lipoprotein receptor mediating cholesteryl ester uptake, and SRB2, a lysosomal integral membrane protein (LIMP2).20 CD36 has a hairpin-like structure with a large and extensively N-glycosylated extracellular loop, the C and N termini spanning the membrane and 2 cytosolic tails. It is a multiligand cell surface receptor expressed in several cells and tissues, such as macrophages, heart, adipose tissue, and liver.20 CD36 is a major receptor for oxidized LDL in macrophages and plays a critical role in the formation of lipid-laden foam cells and atherosclerosis. CD36 also contributes to muscle lipid utilization, adipose energy storage, and fat absorption in tissues with important lipid fluxes by binding long-chain fatty acids and facilitating their transport into cells.20 Recently, it has become apparent that CD36 also plays an important role in the liver by participating in the uptake of fatty acids and in triglyceride storage and secretion. Because of its function in lipid metabolism, CD36 has been linked to insulin resistance, obesity, and type 2 diabetes mellitus.21

PCSK9 and CD36 are both involved in the complex regulation of lipid and triglyceride metabolism, and therefore, we undertook to study their functional relationship. Herein, we show that PCSK9 induces the degradation of CD36 and regulates its function in mouse and human cell lines and in mice. Through direct protein–protein interaction, PCSK9 provokes CD36 degradation by a mechanism that depends on both lysosomal and proteasomal activity, which affects the uptake of CD36 ligands in adipocytes and hepatic cells. In Pcsk9−/− mice, CD36 protein levels were significantly increased in visceral adipose tissue and in the liver. Correspondingly, hepatic triglyceride content and uptake of long-chain fatty acids were found to be increased in the absence of PCSK9. Together, our data demonstrate that PCSK9 has an additional role in lipid homeostasis beyond its effect on LDLc through the regulation of CD36 levels and function.

Materials and Methods

Materials and Methods are available in the online-only Data Supplement.

Results

PCSK9 Induces the Degradation of CD36 Through a Proteasome-Sensitive Mechanism in a Post-Endoplasmic Reticulum, Acidic Compartment

To determine whether PCSK9 regulates the protein level of CD36 by enhancing its degradation, we first used HEK293 cells, which can be transfected with high efficiency. Cotransfection of hemagglutinin (HA)-tagged CD36 (CD36-HA) and increasing amount of PCSK9-V5 cDNA demonstrated that, similar to LDLR, PCSK9 dose dependently decreases CD36 protein levels by up to ≈70% (Figure 1A and 1B). Although CD36 protein levels were decreased only on its cotransfection with PCSK9 (Figure 1C), the level of its paralog LIMP2 was not modified by PCSK9, as previously reported for SRB1,22 or by any of the other proprotein convertases tested (proprotein convertase 5A [PC5A], subtilisin kexin isozyme-1 [SKI-1], or Furin; Figure 1D) attesting for the specificity of the PCSK9-induced CD36 degradation. The degradation of CD36 was also tested in the human hepatic cell line HepG2, which is commonly used to study PCSK9 function,12 stably expressing CD36-HA (Figure 1E). Transfection of these cells with PCSK9-V5 cDNA demonstrated a dose-dependent reduction of CD36 protein level (Figure 1F, first 4 lanes), which correlated with decreased internalization of CD36 ligand DiI-oxLDL (Figure 1G). Of note, Western blotting of CD36 yielded 2 bands in HepG2 cells, which are both reduced in the presence of PCSK9 (Figure 1F), likely reflecting differential glycosylation compared with HEK293 cells (Figure 1A and 1C). The PCSK9-induced degradation of CD36 was inhibited by the lysosomal function inhibitor ammonium chloride (NH4Cl) and the vesicle trafficking inhibitor brefeldin A (Figure 1F), suggesting that transport out of the endoplasmic reticulum and an acidic compartment are required, as previously shown for LDLR degradation.3 Interestingly, the effect of PCSK9 was also reversed by MG132 (Figure 1F), suggesting the involvement of the proteasome, which does not participate in the PCSK9-induced degradation of LDLR.3 Moreover, the use of a panel of proteasome inhibitors, ALLN (N-acetyl-L-leucyl-L-leucyl-L-norleucinal), lactacystin, proteasome inhibitor-1, or MG132, resulted in the inhibition of the PCSK9-induced CD36 degradation but not of LDLR degradation (Figure IA in the online-only Data Supplement). These results indicate that PCSK9 induces the degradation of CD36 by a mechanism that requires both proteasome and endosomes/lysosomes.

Figure 1.
Figure 1.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) induces cluster of differentiation 36 (CD36) degradation. A, HEK293 cells transfected with CD36-HA cDNA (0.15 μg) and either with an empty pIRES-V5 vector (−) or with increasing amounts of PCSK9-V5 cDNA (lane 2, 0.05 μg; lane 3, 0.15 μg; and lane 4, 0.45 μg). Cell lysates were subjected to Western blotting (WB) with the anti-HA, anti-human (h)LDLR, anti-V5-horseradish peroxidase (HRP), and anti-β-actin antibodies. M indicates mature PCSK9; and P, pro-PCSK9. B, Densitometry calculations for WB data as shown in (A). The relative density of the bands was normalized to β-actin (n=10 per condition). *P<0.01 vs 3:0 ratio. C and D, WB analysis of CD36 and lysosomal integral membrane protein (LIMP2; anti-HA antibody) and proprotein convertases (anti-V5-HRP antibody) in lysates of HEK293 cells transfected with CD36-HA (C) or LIMP2-HA (D) and either with an empty pIRES-V5 vector (pIRES) or with V5-tagged PCSK9, PC5A, SKI-1, or Furin. β-actin was used as a control for loading. E, Immunolabeling of cell surface CD36 (arrow) in unpermeabilized stable HepG2-CD36-HA cells. Bar, 20 µm. F, Stable HepG2-CD36-HA cells transiently transfected with increasing amounts of PCSK9-V5 plasmid in the presence or absence of 1 μmol/L MG132, 10 mmol/L ammonium chloride (NH4Cl), or 5 μg/mL brefeldin A (BFA). CD36 (anti-HA), PCSK9 (anti-V5-HRP), and actin (anti-β-actin) were analyzed by WB. The asterisk denotes an additional band likely reflecting differential CD36 glycosylation in HepG2 cells compared with HEK293 cells (A and C). G, Stable HepG2-CD36-HA cells transiently transfected with PCSK9-V5 (transfected cells express EGFP [green nuclei]) were incubated with DiI-oxLDL (red) for 1 h at 37°C and fixed for fluorescence analysis. The dashed lines (right) outline PCSK9-expressing cells with low content of DiI-oxLDL compared with nontransfected cells (arrowheads). Bar, 20 µm. H and I, WB for CD36 (anti-HA), LDLR (anti-hLDLR), PCSK9 (anti-hPCSK9, which detects endogenous PCSK9 and rhPCSK9), and β-actin (anti-β-actin) in lysates of stable HepG2-CD36-HA cells transfected with an small interfering RNA (siRNA) targeting human PCSK9 or a control nontarget siRNA (H), or incubated with or without 2.5 µg/mL rhPCSK9 for 16 h (I). The asterisk indicates a nonspecific band. J, Stable HepG2-CD36-HA cells were incubated with conditioned media of HEK293 cells overexpressing pIRES-V5 (control) or V5-tagged PCSK9 (sPCSK9) for 16 h. Cell surface proteins were biotinylated, and cell lysates were either affinity precipitated with streptavidin agarose and analyzed by WB for CD36 (anti-HA) or analyzed by WB for LDLR (anti-hLDLR), PCSK9 (anti-V5-HRP), and β-actin (anti-β-actin). K, Quantification of band intensity for CD36 and LDLR from HepG2-CD36-HA cells treated with control medium (pIRES-V5, Ctl.) or V5-tagged PCSK9 (sPCSK9) or D374Y mutant, as described in (J). WB signals were normalized to β-actin. n=5 independent experiments for each condition. *P<0.01; **P<0.001 vs control. LDLR indicates low-density lipoprotein receptor.

We next evaluated the regulation of CD36 protein expression by endogenous PCSK9 in HepG2-CD36 cells. Transfection of a specific small interfering RNA against human PCSK9 in HepG2 cells resulted in a knockdown of >85% and caused a robust increased of CD36 protein level, as well as that of LDLR (Figure 1H). Then, to determine whether PCSK9 can induce CD36 degradation through an extracellular pathway,7 HepG2-CD36 cells were incubated with physiological levels of recombinant human PCSK918 (2.5 µg/mL rhPCSK9; Figure IB in the online-only Data Supplement). Western blotting results demonstrated that PCSK9 was capable of inducing the degradation of both CD36 and LDLR via an extracellular pathway (Figure 1I). In addition, secreted PCSK9 from conditioned media of HEK293 cells expressing PCSK9-V5 (2 µg/mL sPCSK9; Figure IB in the online-only Data Supplement) reduced the levels of cell surface CD36 in HepG2-CD36 cells (Figure 1J). Interestingly, the PCSK9-D374Y mutant did not result in a gain-of-function over CD36 as it does for LDLR5,7 (Figure 1K), suggesting that PCSK9 has different binding requirement for both receptors.

PCSK9 Directly Interacts With CD36

We then sought to evaluate direct protein–protein interaction of PCSK9 and CD36. Using surface plasmon resonance, we found that purified rhPCSK9 bound to the immobilized extracellular domain of CD36 (hCD36ED) in a concentration-dependent manner with a dissociation constant (Kd) of 1.2 µmol/L at pH 7.4 (Figure 2A), which is similar to the reported Kd of CD36 ligands23 and similar to the Kd of PCSK9 binding to the LDLR EGFA domain (≈0.63–1 µmol/L at neutral pH).24,25 In addition, coimmunoprecipitation and confocal microscopy experiments showed that PCSK9 was specifically pulled down with CD36 and colocalized with the receptor in lysosomes in HEK293 cells (Figure 2B and 2C; Figure II in the online-only Data Supplement). This prompted us to test whether PCSK9 binding to LDLR was required for CD36 degradation. The PCSK9-F379A mutant, a PCSK9 loss-of-function mutation that does not interact with LDLR,26 was found to coimmunoprecipitate with CD36 and induced its degradation, with even more potency than wild-type (WT) PCSK9, but not that of LDLR (Figure IIIA and IIIB in the online-only Data Supplement). In addition, knockdown of LDLR (>90%) did not impeded PCSK9-induced CD36 degradation, suggesting that the PCSK9–LDLR interaction is not a requisite for CD36 degradation (Figure IIIC in the online-only Data Supplement). However, a PCSK9 neutralizing antibody that inhibits LDLR degradation also interfered with the extracellular pathway of CD36 degradation (Figure IIID in the online-only Data Supplement), suggesting that the catalytic domain of PCSK9, which is targeted by the neutralizing antibody, or a nearby region is involved in regulating CD36 protein levels.

Figure 2.
Figure 2.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) interacts and colocalizes with cluster of differentiation 36 (CD36). A, Purified recombinant PCSK9 (0.05–2 μmol/L) bound to the immobilized extracellular domain of CD36 with a Kd of 1.2 μmol/L at pH 7.4, as determined using surface plasmon resonance. B, Western blotting (WB) showing coimmunoprecipitation of CD36 with PCSK9 from HEK293 cells expressing an empty pIRES-V5 vector (−) or CD36-V5 and untagged PCSK9 in the presence or absence of 1 µmol/L MG132. Immunoprecipitated proteins (IP: V5) were detected by WB using the anti-V5-HRP and anti-hPCSK9 antibodies. Immunoblots of CD36-V5, PCSK9, and β-actin before IP (input) are shown. C, Immunofluorescence of HEK293 cells cotransfected with PCSK9-V5 and CD36-HA in the presence of 1 µmol/L MG132. Immunolabeling was performed under permeabilizing condition with the anti-V5 (blue) and anti-HA (red) antibodies. Colocalization (arrows) of PCSK9 with CD36 is exemplified by the purple color in the merge image. Nuclei were stained using DAPI (cyan). Bar, 10 μm. M indicates mature PCSK9; and P, pro-PCSK9.

PCSK9 Reduces Long-Chain Fatty Acid Uptake in Adipocytes and Regulates CD36 Protein Level in Visceral Adipose Tissue of Pcsk9−/− Mice

We next assessed the PCSK9-mediated regulation of endogenous CD36 in cell lines and tissues. Both PCSK9 and CD36 are well expressed in the small intestine.8,27 CD36 is highly expressed on the luminal surface of enterocytes in fasted mice and is degraded through a ubiquitin-dependent pathway in response to feeding.27 Therefore, we explored the effect of nutritional status on CD36 expression in the intestine of WT and Pcsk9−/− mice. Immunofluorescence of CD36 in the jejunum showed a weak staining in both genotypes under fed conditions and was remarkably increased at the apical surface of enterocytes under fasting conditions but without overt differences in intensity between WT and Pcsk9−/− mice (Figure IVA in the online-only Data Supplement). Similarly, CD36 levels were not significantly modified in the heart of Pcsk9−/− mice, whereas LDLR levels were slightly increased (Figure IVB in the online-only Data Supplement). Also, incubation of HL-1 cardiac muscle cells, with or without insulin stimulation,28 and PMA (phorbol 12-myristate 13-acetate)-induced THP-1 macrophages with sPCSK9 revealed no modification of CD36 protein level, whereas that of LDLR was significantly decreased in HL-1 cells (LDLR was barely detectable in THP-1 cells; Figure IVC and IVD in the online-only Data Supplement). However, incubation of 3T3-L1 adipocytes and mouse primary adipocytes with sPCSK9 demonstrated a significant reduction of endogenous CD36 and LDLR (Figure 3A; Figure VA and VB in the online-only Data Supplement). Moreover, as shown for HepG2 (Figure 1F) and HEK293 cells (Figure I in the online-only Data Supplement), incubation of 3T3-L1 adipocytes with inhibitors of the proteasome (MG132 or lactacystin) or 3 different lysosome inhibitors (NH4Cl, E64D (cysteine proteases inhibitor), or bafilomycin A1 (vacuolar type H+-ATPase [V-ATPase inhibitor]) inhibited the PCSK9-induced degradation of CD36 (Figure 3A; Figure VC in the online-only Data Supplement). MG132 had a slight effect on LDLR levels in adipocytes likely because of nonspecific inhibition of lysosomal enzymes.29

Figure 3.
Figure 3.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates endogenous cluster of differentiation 36 (CD36) in 3T3-L1 adipocytes and in mouse adipose tissue. A, Western blotting (WB) of CD36 (anti-mCD36), low-density lipoprotein receptor (LDLR; anti-mLDLR), PCSK9 (anti-V5-HRP), and actin (anti-β-actin) in differentiated 3T3-L1 adipocytes incubated for 16 h with conditioned media obtained from HEK293 cells transfected with an empty pIRES-V5 vector (−) or V5-tagged PCSK9 (secreted PCSK9, sPCSK9) in the absence or presence of 2.5 μmol/L MG132 or 10 mmol/L NH4Cl. B, PCSK9-mediated degradation of CD36 reduces internalization of fatty acids. 3T3-L1 adipocytes were incubated for 16 h with control medium or with sPCSK9 as described in (A) and then incubated with Bodipy FL C16 for 30 min. Cell surface immunofluorescence of endogenous CD36 in nonpermeabilized cells (arrows) was performed using anti-mCD36. Confocal images of CD36 (red) and Bodipy FL C16 (green) were acquired at identical laser output, gain, and offset. Bar, 40 µm. C, Coimmunoprecipitation of endogenous CD36 with PCSK9 in 3T3-L1 adipocytes incubated for 16 h with DMEM (−) or with 2 µg/mL rhPCSK9 with or without 2.5 μmol/L MG132. Immunoprecipitation (IP) was performed using the anti-mCD36 antibody (IP: CD36). Immunoprecipitated proteins were analyzed by WB using anti-mCD36 and anti-His (to detect rhPCSK9) antibodies. Levels of CD36, rhPCSK9, and β-actin before IP (input) are shown. D, Confocal microscopy of 3T3-L1 adipocytes incubated with 2 µg/mL rhPCSK9 in the presence of 1 µmol/L MG132 for 16 h. Permeabilized cells (+Triton-X, TX) were immunolabeled with anti-hPCSK9 (green) and anti-mCD36 (red) antibodies. Arrows show colocalization of PCSK9 and CD36 in large vesicles in the perinuclear region. Bar, 20 µm. Insets show cell surface labeling and colocalization (arrow) of PCSK9 and CD36 in unpermeabilized (−TX) adipocytes. Bar, 20 µm. Magnified images of area within dashed square are shown (right and inset). Magnification: bars, 10 µm. E, WB of CD36 in perigonadal fat of 12-week-old C57BL/6 (+/+) and Pcsk9−/− mice. Quantification of the bands (right) was normalized to β-actin (n=4 mice/genotype). *P<0.05 vs +/+. F, Perigonadal fat CD36 mRNA levels as determined by quantitative polymerase chain reaction (n=5 mice/genotype). G, Representative immunofluorescence images of CD36 in perigonadal adipose tissue of 12-week-old C57BL/6 (+/+) and Pcsk9−/− mice. Bar, 50 µm. These data are representative of ≈3 independent experiments.

To determine whether PCSK9 reduces cell surface CD36 and affects its function, 3T3-L1 adipocytes were incubated for 16 hours with sPCSK9 and then with Bodipy FL C16 (Figure 3B). Compared with control medium, PCSK9-treated adipocytes showed a strong reduction of cell surface CD36 coinciding with significantly lower amount of internalized palmitate analog (Figure 3B). In addition, coimmunoprecipitation and confocal microscopy experiments from adipocytes incubated with 2 µg/mL purified rhPCSK9 revealed that endogenous CD36 interacts and colocalizes with PCSK9 at the cell surface and in vesicular endosome/lysosome-like compartments (Figure 3C and 3D; Figure VD in the online-only Data Supplement). Importantly, CD36 protein levels, but not its mRNA expression, were found significantly increased by ≈80% in perigonadal fat pads of Pcsk9−/− mice when compared with control C57BL/6 (+/+) mice (Figure 3E and 3F). In support of these results, confocal immunofluorescence microscopy of CD36 in mouse adipose tissue showed a strong increase of the immunolabeling at the surface of adipocytes in Pcsk9−/− mice (Figure 3G). Taken together, these results demonstrate that PCSK9 interacts with and decreases endogenous cell surface CD36 thereby reducing internalization of fatty acids in adipocytes and also suggest that circulating PCSK9 modifies CD36 protein levels and function in visceral adipose tissue.

CD36 Protein Levels, Long-Chain Fatty Acid Uptake, and Triglyceride Content Are Increased in the Liver of Pcsk9−/− Mice

PCSK9 is predominantly expressed in the liver and preferentially reduces LDLR protein levels in hepatocytes.30 Thus, we tested whether hepatic CD36 was regulated in Pcsk9−/− mice. Our Western blotting results demonstrated that, compared with control +/+ mice and similar to LDLR, CD36 protein levels were strongly increased (≈3-fold, P<0.001) in membrane fractions of Pcsk9−/− liver, whereas SRB1 and pan-cadherin (herein used as membrane marker) levels were not modified (Figure 4A and 4B). Immunohistochemical analysis showed that CD36 was markedly increased at the basolateral plasma membrane of Pcsk9−/− hepatocytes, where it was colocalized with LDLR, a well-known basolateral membrane marker31 (Figure 4C). Importantly, quantitative polymerase chain reaction analyses showed no variation in liver CD36 and LDLR mRNA levels between +/+ and Pcsk9−/− mice, indicating a post-translational regulation of the receptors by PCSK9 (Figure 4D).3

Figure 4.
Figure 4.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates hepatic cluster of differentiation 36 (CD36) protein levels, long-chain fatty acid uptake, and triglyceride content. A, Representative Western blotting (WB) of CD36, low-density lipoprotein receptor [LDLR], SR-B1, and pan-cadherin in mouse liver total membrane fractions from C57BL/6 (+/+) and Pcsk9−/− mice. B, Densitometric analysis of CD36 and LDLR WB signals normalized over that of pan-cadherin. (+/+, n=10 mice; Pcsk9−/−, n=8 mice). *P<0.001 vs the +/+ group. C, Immunofluorescence of CD36 (left) and LDLR (middle) in liver cryosections of +/+ and Pcsk9−/− mice. Colocalization (arrows) of CD36 with LDLR at the basolateral membrane of hepatocytes is exemplified by the yellow color in the merge image. Nuclei were stained with DAPI (cyan). Bars, 40 µm. D, Hepatic CD36 and LDLR mRNA levels as determined by quantitative polymerase chain reaction (+/+, n=13 mice; Pcsk9−/−, n=10 mice). E, Confocal microscopy of cryosections of liver tissues from +/+ and Pcsk9−/− mice stained with Bodipy 493/503 (lipid droplets; green) and DAPI (nuclei; cyan). Bar, 20 µm. Inset bar, 10 µm. F, Hepatic triglyceride measurements from +/+ and Pcsk9−/− mice (+/+, n=12 mice; Pcsk9−/−, n=11 mice). *P<0.001 vs +/+. G, Liver fatty acid (FA) uptake measured after injection of Bodipy FL C16 in +/+ or Pcsk9−/− mice as indicated in experimental procedures (+/+, n=3 mice; Pcsk9−/−, n=3 mice). *P<0.05 vs +/+.

Accordingly, injection of rhPCSK9 to WT C57BL/6 mice, which reduces hepatic LDLR by ≈90% within 60 minutes (Figure VI in the online-only Data Supplement),30 reduced hepatic CD36 levels by 57% after 60 minutes and by 66% after 120 minutes, demonstrating that circulating PCSK9 can induce the degradation of CD36 (Figure VI in the online-only Data Supplement). CD36 protein levels are directly related to fatty acid uptake and triglyceride storage in the liver.21 Thus, we next compared hepatic lipid content and uptake in the absence or presence of PCSK9. Remarkably, neutral lipid staining of liver cryosections and quantification of triglycerides in liver extracts of Pcsk9−/− mice showed a significant accumulation of lipid droplets in hepatocytes and a ≈4-fold increase in triglyceride content (Figure 4E and 4F). In line with these results, acute injection of the fluorescent palmitate analog Bodipy FL C16 to control +/+ and Pcsk9−/− mice revealed a 2-fold increase of fatty acid uptake by the liver in Pcsk9−/− mice (Figure 4G). Altogether, these results indicate that Pcsk9−/− mice have an altered hepatic fatty acid metabolism.

Discussion

PCSK9 was shown to cause the post-translational degradation of LDLR and familial hypercholesterolemia >10 years ago, but its role in enhancing the degradation of other cell surface receptors is still emerging. In this work, we characterized a novel function of PCSK9 causing the degradation of CD36, a major receptor involved in fatty acid and triglyceride metabolism. Our results demonstrate that PCSK9 directly interacts with CD36 with a Kd of 1.2 µmol/L, which is similar to its binding with the LDLR EGFA domain at neutral pH24,25 and within the range of micromolar-binding affinity of other CD36 ligands.23 Accordingly, we found that PCSK9 and CD36 coimmunoprecipitate and colocalize at the cell surface and in lysosomes either under overexpression or physiological conditions in hepatic and adipocyte cell lines. PCSK9 elicited the degradation of CD36 both through an intracellular or extracellular pathway and reduced the level of CD36 at the cell surface, likely by impeding the recycling or translocation of CD36 to the plasma membrane and directing the receptor to the lysosomes. Functionally, reduction of CD36 levels significantly decreased internalization of ligands, such as oxidized-LDL in hepatic cells and a palmitate analog in adipocytes. Conversely, CD36 protein levels were robustly increased, as well as that of LDLR, on small interfering RNA knockdown of PCSK9 gene expression in HepG2-CD36 cells. In vivo, injection of recombinant PCSK9 to WT C57BL/6 mice induced hepatic CD36 degradation. Inversely, the absence of PCSK9 resulted in the strong upregulation of CD36 protein levels in the liver and adipose tissue and was associated with an increase in hepatic fatty acid uptake and triglyceride content.

The mechanism by which PCSK9 promotes CD36 degradation was evaluated and compared with that of LDLR. Our results show that inhibitors of proteasome (MG132, lactacystin, ALLN, and proteasome inhibitor-1) or lysosomes (NH4Cl, E64D, and bafilomycin A1) prevented the PCSK9-induced destruction of CD36, which contrast with that of LDLR that is solely inhibited by lysosome inhibitors.3,32 The inhibition of CD36 degradation by proteasome and lysosome inhibitors, added to the fact that degradation was prevented in brefeldin A–treated cells, might suggest the involvement of ubiquitin-mediated targeting of CD36 to lysosomes and the crosstalk between the ubiquitin–proteasome and autophagy–lysosome systems.33 Indeed, the addition of ubiquitin tags to many cell surface receptors serve as a sorting signal to lysosomes.34 Deubiquitination of endosomal CD36, which is required for trafficking to multivesicular bodies and lysosomes, may be mediated by the proteasome.35 However, ubiquitin is essential for LDLR degradation by the inducible degrader of the LDLR, but not by PCSK9,32 and if PCSK9 promotes CD36 ubiquitination remains to be demonstrated.

Binding of PCSK9 to LDLR requires key residues found on the surface of its catalytic domain.26 Our results indicate that the same residues are not involved for PCSK9–CD36 complex formation. The PCSK9-D374Y mutant, which increases the affinity and degradation potency of PCSK9 toward LDLR,11 did not increase CD36 degradation compared with WT PCSK9. In addition, the PCSK9-F379A mutant, which greatly diminishes binding to and degradation of LDLR,26 coimmunoprecipitated with CD36 and induced its degradation with more potency than WT PCSK9. Although it is not excluded that LDLR may be involved in the trafficking of the CD36/PCSK9 complex, our data suggest that PCSK9-mediated CD36 degradation could occur in the absence of LDLR, as demonstrated previously for very low-density lipoprotein receptor, APOER2 (apolipoprotein E receptor 2), and CD81.14,15 These results suggest that critical PCSK9 residues differ for LDLR and CD36 but that the catalytic domain (aa 153–421) affects binding to CD36. Indeed, we showed that a PCSK9 neutralizing antibody against the catalytic domain prevented CD36 degradation and LDLR. Similar to PCSK9-mediated LDLR degradation,14,30 CD36 degradation is cell type and tissue dependent. Although the mechanism is still unclear, it has been proposed that inhibition of PCSK9 binding to LDLR by annexin A2 in extrahepatic tissues36 or that PCSK9 dissociates from the LDLR within early endosomes37 contribute to PCSK9 resistance. Whether this applies to CD36 remains to be demonstrated and, as for LDLR, the complete mechanism remains to be resolved. CD36 endocytosis can be mediated through clathrin-dependent or lipid raft pathways,38 whereas LDLR endocytosis and degradation are clathrin dependent.7,32 Therefore, PCSK9 could be internalized with CD36 through different endocytotic pathways and, in some cell types or tissues, this might not always result in degradation of the receptor. CD36 was resistant to PCSK9 in insulin-stimulated HL-1 cardiac muscle cells and in PMA-induced THP-1 macrophages, in conditions favoring CD36 translocation at the cell surface, as well as not regulated by PCSK9 in mouse heart and jejunum. In line with these results, it was shown that PCSK9 failed to induce the degradation of CD36 in polarized intestinal Caco-2 cells.39 Moreover, through a yet undefined mechanism, the addition of PCSK9 to the basolateral medium of Caco-2 cells grown on filter inserts, which separates the basolateral and apical domain, increased the expression of CD36 and NPC1L1 at the apical surface resulting in enhanced cholesterol uptake and chylomicron secretion.39 However, our results demonstrated that, independently of the nutritional status, apical CD36 levels were not overtly modified in the jejunum of Pcsk9−/− mice, suggesting that intestinal CD36 may not participate to the reduced postprandial hypertriglyceridemia phenotype of Pcsk9−/− mice.19

PCSK9-deficient mice have visceral fat accumulation (our unpublished observations).40 This phenotype was shown to be LDLR independent and may be in part mediated by very low-density lipoprotein receptor. CD36 is a major regulator of adipose tissue growth and function; CD36 null mice are significantly leaner than WT mice and display increased plasma levels of triglyceride and fatty acids.41 Our results demonstrate that CD36 level is regulated by PCSK9 in mouse primary adipocytes and 3T3-L1 adipocytes. In addition, CD36 protein level is increased by ≈80% in perigonadal fat pads of Pcsk9−/− mice, suggesting that it represents an important factor causing the increase in adiposity in Pcsk9−/− mice. Through an LDLR-independent mechanism, PCSK9 was shown to modulate hepatic and intestinal secretion and uptake of triglyceride-rich lipoproteins and postprandial triglyceride levels.19,42 Herein, we show that CD36 levels were increased by ≈3-fold in the liver of Pcsk9−/− mice and, in accordance with the function of CD36 in fatty acid uptake, long-chain fatty acid analog uptake, triglycerides, and lipid droplets were significantly increased in the liver of Pcsk9−/− mice. Unlike young ≈2- to 3-month-old Pcsk9−/− mice,22 our experiments, conducted with ≈6- to 7-month-old mice, showed an increase in hepatic triglyceride content, suggesting that the long-term effect of elevated CD36 in the liver may result in accumulation of lipids.21 Indeed, enhanced CD36-mediated hepatic fat uptake is associated with greater susceptibility to nonalcoholic fatty liver disease in mice and humans.43 In addition, PCSK9 loss-of-function mutations, which can result in familial hypobetalipoproteinemia, could be a cause of mild fatty liver disease.44 Whether this might occur after long-term inhibition of PCSK9 in humans remains to be evaluated.

In conclusion, we have demonstrated for the first time that PCSK9 induces the degradation of CD36 via a proteasome-sensitive mechanism in a post-endoplasmic reticulum, acidic compartment. PCSK9-mediated modulation of CD36 levels in tissues with important lipid fluxes, such as in the liver and visceral adipose tissue, may restrict fatty acid internalization and triglyceride storage and provide additional mechanistic support to the role of PCSK9 in triglyceride metabolism.

Acknowledgments

We thank Nicolas Bousette and John D. Rioux at the Montreal Heart Institute for their generous gift of cell lines. We are also grateful to Steve Bourgault (Université du Québec à Montréal) for expert help with SPR analyses and to Catherine Martel (Montreal Heart Institute) for critical reading of the article.

Sources of Funding

This work was supported by research grants from the Canadian Institutes of Health Research (CIHR—Institute of Nutrition, Metabolism and Diabetes; MOP133598), the Heart and Stroke Foundation of Canada, the Fonds de la Recherche en Santé du Québec, and the Montreal Heart Institute Foundation to Dr Mayer.

Disclosures

None.

Footnotes

  • Received June 9, 2015.
  • Accepted October 12, 2015.

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Significance

Proprotein convertase subtilisin/kexin type 9 (PCSK9) reduces the protein levels of low-density lipoprotein receptor that is essential for clearance of atherogenic low-density lipoprotein cholesterol from the bloodstream. Accordingly, the use of PCSK9 inhibitors aimed at reducing hypercholesterolemia could prevent major cardiovascular events. However, the mechanism by which PCSK9 regulates triglyceride metabolism is not well understood. Herein, we provide evidence that PCSK9 induces the degradation of CD36, a major receptor for long-chain fatty acids. Our results show that PCSK9 regulates CD36 protein levels and function in vitro in hepatic and adipose cell lines, ex vivo in primary adipocytes, and in vivo in visceral adipose tissue and liver of mice. Importantly, our data show that the absence of PCSK9 causes increased hepatic fatty acid uptake and triglyceride storage, likely through CD36 upregulation. These data support that PCSK9-induced CD36 degradation may serve to limit the entry of fatty acids and triglyceride accumulation in tissues, such as the liver.

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  • PCSK9 Induces CD36 Degradation and Affects Long-Chain Fatty Acid Uptake and Triglyceride Metabolism in Adipocytes and in Mouse LiverSignificance
    Annie Demers, Samaneh Samami, Benjamin Lauzier, Christine Des Rosiers, Emilienne Tudor Ngo Sock, Huy Ong and Gaetan Mayer
    Arteriosclerosis, Thrombosis, and Vascular Biology. 2015;35:2517-2525, originally published October 22, 2015
    https://doi.org/10.1161/ATVBAHA.115.306032
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