Wild-type PCSK9 inhibits LDL clearance but does not affect apoB-containing lipoprotein production in mouse and cultured cells

Lalanne, Florent; Lambert, Gilles; Amar, Marcelo; Chétiveaux, Maud; Zaïr, Yassine; Jarnoux, Anne-Laure; Ouguerram, Khadija; Friburg, Jose; Seidah, Nabil G.; Brewer, H. Bryan; Krempf, Michel; Costet, Philippe

doi:10.1194/jlr.m400396-jlr200

Cited by 95 publications

(86 citation statements)

References 35 publications

(34 reference statements)

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“…Our laboratory showed that PCSK9-S127R patients exhibit an overproduction of apoB, an effect confirmed on apoB100 in vitro by Sun et al (54) who showed the specificity of this trait for certain mutations and not others (3). In fed mice or primary mouse hepatocytes, wild type PCSK9 overexpression did not lead to apoB overproduction (7)(8)(9)(10). Our findings on PCSK9 insulin-dependent expression suggest that the nutritional status and especially insulin concentrations might be of importance when studying the link between PCSK9 and apoB production.…”

Section: Discussionmentioning

confidence: 78%

“…60 g of total or 40 g of cytoplasmic and 40 g of nuclear lysate proteins (Pierce Biotechnology), were resolved on NuPAGE 4 -12% BisTris gels in MES-SDS buffer (Invitrogen) under reducing conditions, as described elsewhere (8). Proteins were transferred onto a Protran nitrocellulose membrane (Schleicher & Shuell), probed with a polyclonal goat IgG directed against the extracellular domain of the mouse LDLr (R&D Systems, Minneapolis, MN) or polyclonal rabbit IgG directed against the CRSRHLAGASQELQ peptide (Neosystem, Strasbourg, France), an epitope of the C-terminal domain of human and mouse PCSK9, or polyclonal rabbit anti-SREBP-1 or anti-SREBP2 (Santa Cruz Biotechnology) or with the monoclonal anti ␤-actin AC-15 antibody (Sigma).…”

Section: Methodsmentioning

confidence: 99%

“…Studies on knock-out mice and plasma lipid profiles of patients with nonsense mutations, showed that PCSK9 deficiency results in low circulating LDL-cholesterol concentrations (4 -6). Although it is established that PCSK9 impairs the LDLr pathway by increasing the degradation of the receptor, its effect on apoB production is still controversial (7)(8)(9)(10).…”

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confidence: 99%

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Hepatic PCSK9 Expression Is Regulated by Nutritional Status via Insulin and Sterol Regulatory Element-binding Protein 1c

Costet

Cariou

Lambert

et al. 2006

Journal of Biological Chemistry

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271

224

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Familial autosomal dominant hypercholesterolemia is associated with high risk for cardiovascular accidents and is related to mutations in the low density lipoprotein receptor or its ligand apolipoprotein B (apoB). Mutations in a third gene, proprotein convertase subtilisin kexin 9 (PCSK9), were recently associated to this disease. PCSK9 acts as a natural inhibitor of the low density lipoprotein receptor pathway, and both genes are regulated by depletion of cholesterol cell content and statins, via sterol regulatory elementbinding protein (SREBP). Here we investigated the regulation of PCSK9 gene expression during nutritional changes. We showed that PCSK9 mRNA quantity is decreased by 73% in mice after 24 h of fasting, leading to a 2-fold decrease in protein level. In contrast PCSK9 expression was restored upon high carbohydrate refeeding. PCSK9 mRNA increased by 4 -5-fold in presence of insulin in rodent primary hepatocytes, whereas glucose had no effect. Moreover, insulin up-regulated hepatic PCSK9 expression in vivo during a hyperinsulinemic-euglycemic clamp in mice. Adenoviral mediated overexpression of a dominant or negative form of SREBP-1c confirmed the implication of this transcription factor in insulinmediated stimulation of PCSK9 expression. Liver X receptor agonist T0901317 also regulated PCSK9 expression via this same pathway (a 2-fold increase in PCSK9 mRNA of primary hepatocytes cultured for 24 h in presence of 1 M T0901317). As our last investigation, we isolated PCSK9 proximal promoter and verified the functionality of a SREBP-1c responsive element located from 335 bp to 355 bp upstream of the ATG. Together, these results show that PCSK9 expression is regulated by nutritional status and insulinemia.Autosomal dominant hypercholesterolemia is associated with mutations in genes involved in the regulation of LDL 2 homeostasis. The most common and severe form of monogenic hypercholesterolemia is familial hypercholesterolemia, caused by mutations in the LDL receptor (LDLr) (1). Familial hypercholesterolemia is characterized by elevated plasma LDL-cholesterol levels and premature cardiovascular disease.Another form of this disease, familial defective apolipoprotein (apo)B100, is caused by mutations in the LDLr binding domain of ApoB100 (1). ApoB100 is synthesized by the liver and is the major protein component of very low density lipoprotein and LDL. Recently, proprotein convertase subtilisin kexin 9 (PCSK9) has been identified as the third gene involved in autosomal dominant hypercholesterolemia (5). Proprotein convertases are proteolytic enzymes that cleave their substrate, producing a biologically active molecule (2). We showed that patients mutated in PCSK9 prodomain (mutation S127R) have decreased LDL catabolism and increased very low density lipoprotein, intermediary density lipoprotein, and LDL production (3). Studies on knock-out mice and plasma lipid profiles of patients with nonsense mutations, showed that PCSK9 deficiency results in low circulating LDL-cholesterol concentrations (4 -6). Althou...

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Section: Discussionmentioning

confidence: 78%

Section: Methodsmentioning

confidence: 99%

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Hepatic PCSK9 Expression Is Regulated by Nutritional Status via Insulin and Sterol Regulatory Element-binding Protein 1c

Costet

Cariou

Lambert

et al. 2006

Journal of Biological Chemistry

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271

224

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“…Huh7 cells are derived from human hepatoma and retain the ability to secrete VLDL (Higashi et al, 2002(Higashi et al, , 2003Lalanne et al, 2005). At least 25% of ApoB in the conditioned medium was recovered as lipoproteins of Ͻ1.063 g/cm 3 (our unpublished data).…”

Section: Apob Around Clds Is Increased By Proteasome Inhibitorsmentioning

confidence: 99%

Cytoplasmic Lipid Droplets Are Sites of Convergence of Proteasomal and Autophagic Degradation of Apolipoprotein B

Ohsaki

Cheng

Fujita

et al. 2006

MBoC

195

186

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Lipid esters stored in cytoplasmic lipid droplets (CLDs) of hepatocytes are used to synthesize very low-density lipoproteins (VLDLs), into which apolipoprotein B (ApoB) is integrated cotranslationally. In the present study, by using Huh7 cells, derived from human hepatoma and competent for VLDL secretion, we found that ApoB is highly concentrated around CLDs to make "ApoB-crescents." ApoB-crescents were seen in <10% of Huh7 cells under normal conditions, but the ratio increased to nearly 50% after 12 h of proteasomal inhibition by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal. Electron microscopy showed ApoB to be localized to a cluster of electron-lucent particles 50 -100 nm in diameter adhering to CLDs. ApoB, proteasome subunits, and ubiquitinated proteins were detected in the CLD fraction, and this ApoB was ubiquitinated. Interestingly, proteasome inhibition also caused increases in autophagic vacuoles and ApoB in lysosomes. ApoB-crescents began to decrease after 12-24 h of proteasomal inhibition, but the decrease was blocked by an autophagy inhibitor, 3-methyladenine. Inhibition of autophagy alone caused an increase in ApoB-crescents. These observations indicate that both proteasomal and autophagy/lysosomal degradation of ApoB occur around CLDs and that the CLD surface functions as a unique platform for convergence of the two pathways. INTRODUCTIONLipid droplets (CLDs) consist of a neutral lipid core with a surrounding phospholipid monolayer (Murphy and Vance, 1999;Tauchi-Sato et al., 2002). CLDs are prominent in adipose cells and steroidogenic cells, but they also exist in other cell types. With the exception of a few cell types, CLDs have been considered as inert excess lipid deposits. However, recent studies have shown that a variety of proteins are localized in CLDs, suggesting that they may play more active functional roles than previously thought. In addition to PAT family proteins, enzymes involved in eicosanoid formation, enzymes for cholesterol synthesis, signaling proteins, caveolins, and Rab proteins have been reported in CLDs (Ozeki et al., 2005, and references therein). Proteomic studies identified many more proteins of both known and unknown functions in the CLD-rich fraction (Brasaemle et al., 2004;Fujimoto et al., 2004;Liu et al., 2004;Umlauf et al., 2004). The presence of functional proteins implies that CLDs are not mere lipid storage vessels but may be involved in various cellular activities.In hepatocytes, the triglycerides in CLDs are thought to be used for the synthesis of very low-density lipoprotein (VLDL) (Gibbons et al., 2000). Enzyme activities that hydrolyze and reesterify neutral lipids are found in CLDs and/or endoplasmic reticulum (ER), but the detailed mechanism by which the CLD content is mobilized and incorporated into lipoprotein particles is still unclear (Murphy, 2001). Apolipoprotein B (ApoB)-100, the primary protein component of VLDL, is assembled with lipids in the ER lumen, and further lipidation and maturation of the lipoprotein particles occur in the ER or the pre-Go...

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“…9 Studies demonstrated that the overexpression of PCSK9 results in depletion of LDLR, and thus a dramatic increase in plasma level of cholesterol. [10][11][12] Conversely, the depletion or removal of PCSK9 protein can substantially increase the level of LDLR and decrease the plasma cholesterol. [13][14][15][16] It is very important that haplotype signatures of prominent SNPs of these three genes are studied in all populations rather than inferring or assuming frequencies from the HAPMAP data, where only a few populations are tested.…”

Section: Introductionmentioning

confidence: 99%

Differences in allele frequencies of autosomal dominant hypercholesterolemia SNPs in the Malaysian population

Alex¹,

Chahil²,

Lye³

et al. 2012

J Hum Genet

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Hypercholesterolemia is caused by different interactions of lifestyle and genetic determinants. At the genetic level, it can be attributed to the interactions of multiple polymorphisms, or as in the example of familial hypercholesterolemia (FH), it can be the result of a single mutation. A large number of genetic markers, mostly single nucleotide polymorphisms (SNP) or mutations in three genes, implicated in autosomal dominant hypercholesterolemia (ADH), viz APOB (apolipoprotein B), LDLR (low density lipoprotein receptor) and PCSK9 (proprotein convertase subtilisin/kexin type-9), have been identified and characterized. However, such studies have been insufficiently undertaken specifically in Malaysia and Southeast Asia in general. The main objective of this study was to identify ADH variants, specifically ADH-causing mutations and hypercholesterolemia-associated polymorphisms in multiethnic Malaysian population. We aimed to evaluate published SNPs in ADH causing genes, in this population and to report any unusual trends. We examined a large number of selected SNPs from previous studies of APOB, LDLR, PCSK9 and other genes, in clinically diagnosed ADH patients (n ¼ 141) and healthy control subjects (n ¼ 111). Selection of SNPs was initiated by searching within genes reported to be associated with ADH from known databases. The important finding was 137 mono-allelic markers (44.1%) and 173 polymorphic markers (55.8%) in both subject groups.

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Wild-type PCSK9 inhibits LDL clearance but does not affect apoB-containing lipoprotein production in mouse and cultured cells

Cited by 95 publications

References 35 publications

Hepatic PCSK9 Expression Is Regulated by Nutritional Status via Insulin and Sterol Regulatory Element-binding Protein 1c

Hepatic PCSK9 Expression Is Regulated by Nutritional Status via Insulin and Sterol Regulatory Element-binding Protein 1c

Cytoplasmic Lipid Droplets Are Sites of Convergence of Proteasomal and Autophagic Degradation of Apolipoprotein B

Differences in allele frequencies of autosomal dominant hypercholesterolemia SNPs in the Malaysian population

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