Low-density Lipoprotein Receptor Abundance Research Articles

Hepatic inactivation of murine Surf4 results in marked reduction in plasma cholesterol.

PCSK9 negatively regulates low-density lipoprotein receptor (LDLR) abundance on the cell surface, leading to decreased hepatic clearance of LDL particles and increased levels of plasma cholesterol. We previously identified SURF4 as a cargo receptor that facilitates PCSK9 secretion in HEK293T cells (Emmer et al., 2018). Here, we generated hepatic SURF4-deficient mice (Surf4fl/fl Alb-Cre+) to investigate the physiologic role of SURF4 in vivo. Surf4fl/fl Alb-Cre+ mice exhibited normal viability, gross development, and fertility. Plasma PCSK9 levels were reduced by ~60% in Surf4fl/fl Alb-Cre+ mice, with a corresponding ~50% increase in steady state LDLR protein abundance in the liver, consistent with SURF4 functioning as a cargo receptor for PCSK9. Surprisingly, these mice exhibited a marked reduction in plasma cholesterol and triglyceride levels out of proportion to the partial increase in hepatic LDLR abundance. Detailed characterization of lipoprotein metabolism in these mice instead revealed a severe defect in hepatic lipoprotein secretion, consistent with prior reports of SURF4 also promoting the secretion of apolipoprotein B (APOB). Despite a small increase in liver mass and lipid content, histologic evaluation revealed no evidence of steatohepatitis or fibrosis in Surf4fl/fl Alb-Cre+ mice. Acute depletion of hepatic SURF4 by CRISPR/Cas9 or liver-targeted siRNA in adult mice confirms these findings. Together, these data support the physiologic significance of SURF4 in the hepatic secretion of PCSK9 and APOB-containing lipoproteins and its potential as a therapeutic target in atherosclerotic cardiovascular diseases.

N-Glycosylation Defects in Humans Lower Low-Density Lipoprotein Cholesterol Through Increased Low-Density Lipoprotein Receptor Expression.

The importance of protein glycosylation in regulating lipid metabolism is becoming increasingly apparent. We set out to further investigate this by studying patients with type I congenital disorders of glycosylation (CDGs) with defective N-glycosylation. We studied 29 patients with the 2 most prevalent types of type I CDG, ALG6 (asparagine-linked glycosylation protein 6)-deficiency CDG and PMM2 (phosphomannomutase 2)-deficiency CDG, and 23 first- and second-degree relatives with a heterozygous mutation and measured plasma cholesterol levels. Low-density lipoprotein (LDL) metabolism was studied in 3 cell models-gene silencing in HepG2 cells, patient fibroblasts, and patient hepatocyte-like cells derived from induced pluripotent stem cells-by measuring apolipoprotein B production and secretion, LDL receptor expression and membrane abundance, and LDL particle uptake. Furthermore, SREBP2 (sterol regulatory element-binding protein 2) protein expression and activation and endoplasmic reticulum stress markers were studied. We report hypobetalipoproteinemia (LDL cholesterol [LDL-C] and apolipoprotein B below the fifth percentile) in a large cohort of patients with type I CDG (mean age, 9 years), together with reduced LDL-C and apolipoprotein B in clinically unaffected heterozygous relatives (mean age, 46 years), compared with 2 separate sets of age- and sex-matched control subjects. ALG6 and PMM2 deficiency led to markedly increased LDL uptake as a result of increased cell surface LDL receptor abundance. Mechanistically, this outcome was driven by increased SREBP2 protein expression accompanied by amplified target gene expression, resulting in higher LDL receptor protein levels. Endoplasmic reticulum stress was not found to be a major mediator. Our study establishes N-glycosylation as an important regulator of LDL metabolism. Given that LDL-C was also reduced in a group of clinically unaffected heterozygotes, we propose that increasing LDL receptor-mediated cholesterol clearance by targeting N-glycosylation in the LDL pathway may represent a novel therapeutic strategy to reduce LDL-C and cardiovascular disease.

Low Density Lipoprotein Receptor-Mediated Lipidome-Transcriptome Reprogramming Impulses to Cisplatin Insensitivity

Background: Platinum-based therapy remains the cornerstone for cancer therapy; however, efficacy varies. The role of tumor macroenvironmental lipid entry via lipoprotein receptors has been reported. Yet, the roles and mechanism of low-density lipoprotein receptor (LDLR) in chemo-sensitivities are in large. Method: Patient study: Single cohort or public database cDNA microarray to associate LDLR expressions of epithelial ovarian carcinomas (EOCs) with disease prognosis. Laboratory study: In vitro and in vivo add-in/silencing LDLR were introduced to determines cisplatin sensitivity. Transcriptome-Lipidome Trans-omics and bioinformatic analyses to model molecular mechanism. Translation study: LDLR-lipidome was implemented to formulate efficacy boosting liposome. Findings: The LDLR expression is associate with platin-based chemotherapy patients prognosis. The LDLR abundance in EOC subtypes is associated with cisplatin sensitivity. Knocked-down or overexpressed LDLR in EOC could reversed the chemosensitivity pattern in vitro and in vivo. Trans-omics analyses elucidated the LDLR→LPC (Lyso-PhosphotidylCholine)→FAM83B (phospholipase-related)→FGFRs (cisplatin sensitivity and phospholipase-related) regulatory axis in cisplatin insensitivity. Implementing LPC-liposome encapsulated cisplatin facilitated DNA-adduct formation via lipid droplets (LDs) delivery. Bioinformatics analyses found that the LDL/R-route alters LD homeostasis, which is critical for therapeutic prognosis. Lastly, using LPCliposome could improve multiple cancer types of cisplatin insensitive cells. Interpretation: The LDL/R-route as tumor macroenvironmental lipid entry which impulses platinum insensitivity and disease outcome. The drug specific lipidome for liposome manufacture might be an efficienct pharmaceutics strategy for chemoagents. Funding Statement: This study was supported by Taiwan Ministry of Science and Technology (MOST104-2628-B-039-001-MY4; MOST106-2221-E-039-011-MY3); National Health Research Institute (NHRI-EX107-10705BI); and China Medical University/Hospital (CMU107-S-05; CMU107-TC-02; DMR-108-080; DMR-CELL-17014; DMR-CELL-1806; DMR-108-079). Declaration of Interests: All the authors in this work claimed no interests conflict. Ethics Approval Statement: Access to the tissue samples was approved by the Internal Review Board of China Medical University Hospital (DMR101-IRB2-276; and CMU105-REC3-122(CR1)). All the animal studies were performed under the supervision and guidelines of the China Medical University Animal Care and Use Committee (#CMOIACUC-2018-089).

Chitosan oligosaccharides enhance lipid droplets via down-regulation of PCSK9 gene expression in HepG2 cells

Chitosan oligosaccharides (COS), linear polymers of N-acetyl-D-glucosamine and deacetylated glucosamine, exhibit diverse pharmacological effects such as antimicrobial, antitumor, antioxidant and anti-inflammatory activities. Here, we explored their hypocholesterolemic effects in vivo and the molecular mechanisms of COS in hepatic cells. Our in vivo study of dyslipidemic ApoE-/- male mice showed that COS treatment of 500 mg kg−1 d−1 for 4 weeks clearly reduced the lipid deposits in the aorta and significantly decreased the hepatic proprotein convertase subtilisin/kexin type 9 (PCSK9) protein levels versus HFD groups (p < 0.05). To elucidate the mechanisms behind these effects, the HepG2 cell line was treated with COS. We found that COS (200 μg/ml) increased the amount of cell-surface low-density lipoprotein receptor (LDLR) and enhanced the lipid droplets in HepG2 cells (p < 0.05). The mRNA levels of LDLR and HMG-CoA protein levels were not altered, and the mRNA levels of PCSK9 were down-regulated by COS treatment for 24 h. We also observed that the expression levels of SREBP-2 (125 kD) and HNF-1α were increased in total cell lysates, but nuclear SREBP-2 (nSREBP-2, 68 kD, the active subunit of SREBP-2) levels were decreased and FOXO3a levels increased in nuclear lysates after COS treatment for 24 h. We demonstrated that one of the reasons for regulation of lipid transfer with COS is that FOXO3a levels are up-regulated by COS, leading to a reduction in the PCSK9 promoter binding capacity of HNF-1α and thus suppressing PCSK9 gene expression, up-regulating LDLR levels, and enhancing the lipid droplets in HepG2 cells. In addition, decreased expression of the PCSK9 gene was also contributed to by down-regulation of SREBP-2 by COS. We further confirmed the effect of suppression of PCSK9 expression by COS by utilizing RNA interference to silence HNF-1α and SREBP-2. Finally, to the best of our knowledge, we are the first to demonstrate that PCSK9 expression and LDLR activity are synergistically changed by a combination of HNF-1α and SREBP-2 after COS treatment. Our findings indicate that COS may regulate PCSK9 to modulate hepatic LDLR abundance and activity.

Xanthohumol Suppresses Mylip/Idol Gene Expression and Modulates LDLR Abundance and Activity in HepG2 Cells.

Xanthohumol, a prenylated flavonoid found in hops (Humulus lupulus L.), exhibits multiple biological activities such as antiatherosclerosis and hypolipidemic activities. In this study, we aim to investigate the hypocholesterolemic effects and molecular mechanisms of xanthohumol in hepatic cells. We found that xanthohumol (10 and 20 μM) increased the amount of cell-surface low-density lipoprotein receptor (LDLR) from 100.0 ± 2.1% to 115.0 ± 1.3% and 135.2 ± 2.7%, and enhanced the LDL uptake activity from 100.0 ± 0.9% to 139.1 ± 13.2% in HepG2 cells (p < 0.01). The mRNA levels of LDLR, HMGCR, and PCSK9 were not altered. Xanthohumol (20 μM) reduced the expression of inducible degrader of the LDL receptor (Mylip/Idol) mRNA and protein by approximately 45% (p < 0.01), which was reported to be associated with increases of LDLR level. We demonstrated that xanthohumol suppressed hepatic Mylip/Idol expression via counteracting liver X receptor (LXR) activation. The molecular docking results predicted that xanthohumol has a high binding affinity to interact with the LXRα ligand-binding domain, which may result in attenuation of LXRα-induced Mylip/Idol expression. Finally, we demonstrated that the Mylip/Idol expression and LDLR activity were synergistically changed by a combination of xanthohumol and simvastatin treatment. Our findings indicated that xanthohumol may regulate the LXR-Mylip/Idol axis to modulate hepatic LDLR abundance and activity.

The Deubiquitylase USP2 Regulates the LDLR Pathway by Counteracting the E3-Ubiquitin Ligase IDOL.

The low-density lipoprotein (LDL) receptor (LDLR) is a central determinant of circulating LDL-cholesterol and as such subject to tight regulation. Recent studies and genetic evidence implicate the inducible degrader of the LDLR (IDOL) as a regulator of LDLR abundance and of circulating levels of LDL-cholesterol in humans. Acting as an E3-ubiquitin ligase, IDOL promotes ubiquitylation and subsequent lysosomal degradation of the LDLR. Consequently, inhibition of IDOL-mediated degradation of the LDLR represents a potential strategy to increase hepatic LDL-cholesterol clearance. To establish whether deubiquitylases counteract IDOL-mediated ubiquitylation and degradation of the LDLR. Using a genetic screening approach, we identify the ubiquitin-specific protease 2 (USP2) as a post-transcriptional regulator of IDOL-mediated LDLR degradation. We demonstrate that both USP2 isoforms, USP2-69 and USP2-45, interact with IDOL and promote its deubiquitylation. IDOL deubiquitylation requires USP2 enzymatic activity and leads to a marked stabilization of IDOL protein. Paradoxically, this also markedly attenuates IDOL-mediated degradation of the LDLR and the ability of IDOL to limit LDL uptake into cells. Conversely, loss of USP2 reduces LDLR protein in an IDOL-dependent manner and limits LDL uptake. We identify a tri-partite complex encompassing IDOL, USP2, and LDLR and demonstrate that in this context USP2 promotes deubiquitylation of the LDLR and prevents its degradation. Our findings identify USP2 as a novel regulator of lipoprotein clearance owing to its ability to control ubiquitylation-dependent degradation of the LDLR by IDOL.

Elevated Plasma PCSK9 Level Is Equally Detrimental for Patients With Nonfamilial Hypercholesterolemia and Heterozygous Familial Hypercholesterolemia, Irrespective of Low-Density Lipoprotein Receptor Defects

Do elevated proprotein convertase subtilisin/kexin type 9 (PCSK9) levels constitute an even greater risk for patients who already have reduced low-density lipoprotein receptor (LDLR) levels, such as those with heterozygous familial hypercholesterolemia (HeFH)? As a circulating inhibitor of LDLR, PCSK9 is an attractive target for lowering LDL-cholesterol (LDL-C) levels. Circulating PCSK9 levels were measured by enzyme-linked immunosorbent assay in nontreated patients with HeFH carrying a D206E (n = 237), V408M (n = 117), or D154N (n = 38) LDLR missense mutation and in normolipidemic controls (n = 152). Skin fibroblasts and lymphocytes were isolated from a subset of patients and grown in 0.5% serum and mevastatin with increasing amounts of recombinant PCSK9. LDLR abundance at the cell surface was determined by flow cytometry. PCSK9 reduced LDLR expression in a dose-dependent manner in control and FH fibroblasts to similar extents, by up to 77 ± 8% and 82 ± 7%, respectively. Likewise, PCSK9 reduced LDLR abundance by 39 ± 8% in nonfamilial hypercholesterolemia (non-FH) and by 45 ± 10% in HeFH lymphocytes, irrespective of their LDLR mutation status. We found positive correlations of the same magnitude between PCSK9 and LDL-C levels in controls (beta = 0.22; p = 0.0003), D206E (beta = 0.20; p = 0.0002), V408M (beta = 0.24; p = 0.0002), and D154N (beta = 0.25; p = 0.048) patients with HeFH. The strengths of these associations were all similar. Elevated PCSK9 levels are equally detrimental for patients with HeFH or non-FH: a 100-ng/ml increase in PCSK9 will lead to an increase in LDL-C of 0.20 to 0.25 mmol/l in controls and HeFH alike, irrespective of their LDLR mutation. This explains why patients with non-FH or HeFH respond equally well to monoclonal antibodies targeting PCSK9.

Post-transcriptional regulation of lipoprotein receptors by the E3-ubiquitin ligase inducible degrader of the low-density lipoprotein receptor

The hepatic low-density lipoprotein receptor (LDLR) pathway is essential for clearing circulating LDL and is an important therapeutic target for treating cardiovascular disease. Abundance of the LDLR is subject to both transcriptional and nontranscriptional control. Here, we highlight a new post-transcriptional mechanism for controlling LDLR function via ubiquitination of the receptor by the E3-ubiquitin ligase inducible degrader of the LDLR (IDOL). IDOL is a recently identified transcriptional target of the liver X receptors. Acting as an E3-ubiquitin ligase IDOL promotes ubiquitination of the LDLR, thereby marking it for lysosomal degradation. The determinants required for degradation of the LDLR by IDOL have been largely identified. IDOL also targets two related lipoprotein receptors, the very low-density lipoprotein receptor and apolipoprotein E receptor 2. Despite several similarities, the IDOL, and PCSK9 pathways for controlling LDLR abundance seem independent of each other. Genome-wide association studies have recently identified IDOL as a locus influencing variability in circulating levels of LDL, thereby highlighting the possible role of IDOL in human lipoprotein metabolism. Transcriptional induction of IDOL by liver X receptor defines a new post-transcriptional pathway for controlling LDLR abundance and LDL uptake independent of sterol regulatory element binding proteins. Targeting IDOL activity may offer a novel therapeutic approach complementary to statins for treating cardiovascular disease.

Distinct Functional Domains Contribute to Degradation of the Low Density Lipoprotein Receptor (LDLR) by the E3 Ubiquitin Ligase Inducible Degrader of the LDLR (IDOL)

We recently identified the liver X receptor-regulated E3 ubiquitin ligase inducible degrader of the LDL receptor (IDOL) as a modulator of lipoprotein metabolism. Acting as an E3 ubiquitin ligase, IDOL triggers ubiquitination and subsequent degradation of the low density lipoprotein receptor (LDLR). We demonstrate here that this outcome requires the conserved FERM and RING domains present in IDOL. The RING domain promotes ubiquitination in vitro and Lys-63-specific ubiquitination of the LDLR in vivo in response to IDOL or liver X receptor activation. We further identify RING residues that differentially influence ubiquitination of the LDLR or stability of IDOL. The FERM domain interacts with the LDLR and in living cells co-localizes with the receptor at the plasma membrane. Homology modeling revealed a phosphotyrosine-binding element embedded in the FERM domain. Mutating residues within this region or residues in the LDLR preceding the NPVY endocytosis motif abrogate LDLR degradation by IDOL. Collectively, our results indicate that both the FERM and RING domains are required for promoting lysosomal degradation of the LDLR by IDOL. Our findings may facilitate development of structure-based IDOL inhibitors aimed at increasing LDLR abundance in therapeutic strategies to treat cardiovascular disease.