Immunoprecipitation of the low-density-lipoprotein (LDL) receptor and its precursor from human monocyte-derived macrophages.
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ABSTRACT:
SUBMITTER: Soutar AK
PROVIDER: S-EPMC1153086 | biostudies-other | 1986 Feb
REPOSITORIES: biostudies-other
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Project description:We have shown recently that lipoprotein-proteoglycan complexes isolated from human atherosclerotic lesions stimulated cholesteryl ester synthesis in human monocyte-derived macrophages [Vijayagopal, Srinivasan, Radhakrishnamurthy and Berenson (1992) Arterioscler. Thromb. 12, 237-249]. The present study was conducted to determine the mechanism of cellular uptake of the complexes. A chondroitin sulphate-dermatan sulphate proteoglycan was isolated from normal human aorta and complexed to 125I-labelled human low-density lipoprotein (LDL). The binding and degradation of 125I-LDL-proteoglycan complex were then studied in human monocyte-derived macrophages. The specific binding and degradation of the complex showed saturability and concentration-dependency. The Kd for binding was 1.5 x 10(-8) M, which was greater than that reported for LDL in monocyte-derived macrophages. Binding of the complex was not subject to down-regulation. Chloroquine inhibited degradation of the complex and the resultant stimulation of cholesteryl ester synthesis. Limited treatment of macrophages with proteolytic enzymes abolished binding and degradation of the complex significantly. Macrophages bound 125I-methyl-LDL-proteoglycan complex to the same extent as 125I-LDL-proteoglycan complex. Excess LDL and proteoglycan did not compete against the binding of the complex; however, excess acetyl-LDL competed for 61% of the binding. Likewise, excess LDL-proteoglycan complex inhibited the binding of 125I-acetyl-LDL by 64%. Polyinosinic acid and cytochalasin D inhibited the binding of 125I-LDL-proteoglycan complex by 60% and 36% respectively. Compared with that of acetyl-LDL, the degradation of LDL-proteoglycan complex was retarded in human macrophages. The results indicate that the uptake of LDL-proteoglycan complex in human monocyte-derived macrophages is not mediated through binding to the LDL receptor; but occurs predominantly via the scavenger receptor, with phagocytosis playing a minor role in the process.
| S-EPMC1132252 | biostudies-other
Project description:The importance of Chromatin Immunoprecipitation (ChIP) technology has grown exponentially along with an increased interest in epigenetic regulation. The correlation of transcription factors with histone marks is now well established as the center of epigenetic studies; therefore, precise knowledge about histone marks is critical to unravel their molecular function and to understand their role in biological systems. This knowledge constantly accumulates and is provided openly in the expanding hubs of information such as the USCS Genome Browser. Nevertheless, as we gain more knowledge, we realize that the DNA-protein interactions are not driven by a "one size fits all" rule. Also, the diversity of interactions between DNA, histones, and transcriptional regulators is much bigger than previously considered. Besides a detailed protocol of sample preparation for the ChIP assay from primary human monocyte-derived macrophages (MDM) [an acceptable in vitro model for primary, human macrophage cells], we show that differences between various types of cells exist. Furthermore, we can postulate that such variations exist between transformed macrophage-like cell lines and primary macrophages obtained from healthy volunteers. We found that the most efficient fixation time for MDM is 10min. Finally, to perform multiple analytical assays, we showed that even with thorough methodology, the yield of material obtained from primary cells is the major challenge.
| S-EPMC4268110 | biostudies-literature
Project description:On the basis of studies in vivo and in vitro that involved the use of pharmacological amounts of drugs and hormones or excess cholesterol supplementation, the expression of the low-density lipoprotein (LDL) receptor appears to be tightly coupled to the regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity and to extracellular levels of LDL. The present study was undertaken to see how these three entities are regulated under normal physiological conditions over a 24 h period. The results show that, in the rat, hepatic LDL-receptor expression and plasma LDL levels exhibit diurnal periodicity, with a 2-3-fold difference between the peak and trough of each rhythm. Both rhythms showed high inverse correlation (r = -0.86, P < 0.01), plasma LDL levels being lowest at the onset of darkness when LDL-receptor expression was at its peak. The results also showed that the LDL-receptor protein in rat liver has a shorter half-life than that reported for cultured fibroblasts or HepG2 cells. The maximal expression of the LDL receptor occurred several hours before the peak activity of HMG-CoA reductase and appeared not to be influenced by cellular or membrane cholesterol levels during the 24 h cycle. Treatment with dexamethasone increased the LDL-receptor activity significantly at both the lowest and highest points of the rhythm, but the receptor rhythm was still maintained, suggesting that the signal for the circadian variation of the receptor expression is not mediated by adrenal hormones.
| S-EPMC1137980 | biostudies-other
Project description:ObjectiveTMEM55B (transmembrane protein 55B) is a phosphatidylinositol-(4,5)-bisphosphate (PI[4,5]P2) phosphatase that regulates cellular cholesterol, modulates LDLR (low-density lipoprotein receptor) decay, and lysosome function. We tested the effects of Tmem55b knockdown on plasma lipids in mice and assessed the roles of LDLR lysosomal degradation and change in (PI[4,5]P2) in mediating these effects. Approach and Results: Western diet-fed C57BL/6J mice were treated with antisense oligonucleotides against Tmem55b or a nontargeting control for 3 to 4 weeks. Hepatic Tmem55b transcript and protein levels were reduced by ≈70%, and plasma non-HDL (high-density lipoprotein) cholesterol was increased ≈1.8-fold (P<0.0001). Immunoblot analysis of fast protein liquid chromatography (FPLC) fractions revealed enrichment of ApoE-containing particles in the LDL size range. In contrast, Tmem55b knockdown had no effect on plasma cholesterol in Ldlr-/- mice. In primary hepatocytes and liver tissues from Tmem55b knockdown mice, there was decreased LDLR protein. In the hepatocytes, there was increased lysosome staining and increased LDLR-lysosome colocalization. Impairment of lysosome function (incubation with NH4Cl or knockdown of the lysosomal proteins LAMP1 or RAB7) abolished the effect of TMEM55B knockdown on LDLR in HepG2 (human hepatoma) cells. Colocalization of the recycling endosome marker RAB11 (Ras-related protein 11) with LDLR in HepG2 cells was reduced by 50% upon TMEM55B knockdown. Finally, knockdown increased hepatic PI(4,5)P2 levels in vivo and in HepG2 cells, while TMEM55B overexpression in vitro decreased PI(4,5)P2. TMEM55B knockdown decreased, whereas overexpression increased, LDL uptake in HepG2 cells. Notably, the TMEM55B overexpression effect was reversed by incubation with PI(4,5)P2. Conclusions: These findings indicate a role for TMEM55B in regulating plasma cholesterol levels by affecting PI(4,5)P2-mediated LDLR lysosomal degradation.
| S-EPMC7197750 | biostudies-literature
Project description:ObjectiveThe role of hepatocyte Abca1 (ATP binding cassette transporter A1) in trafficking hepatic free cholesterol (FC) into plasma versus bile for reverse cholesterol transport (RCT) is poorly understood. We hypothesized that hepatocyte Abca1 recycles plasma HDL-C (high-density lipoprotein cholesterol) taken up by the liver back into plasma, maintaining the plasma HDL-C pool, and decreasing HDL-mediated RCT into feces. Approach and Results: Chow-fed hepatocyte-specific Abca1 knockout (HSKO) and control mice were injected with human HDL radiolabeled with 125I-tyramine cellobiose (125I-TC; protein) and 3H-cholesteryl oleate (3H-CO). 125I-TC and 3H-CO plasma decay, plasma HDL 3H-CO selective clearance (ie, 3H-125I fractional catabolic rate), liver radiolabel uptake, and fecal 3H-sterol were significantly greater in HSKO versus control mice, supporting increased plasma HDL RCT. Twenty-four hours after 3H-CO-HDL injection, HSKO mice had reduced total hepatic 3H-FC (ie, 3H-CO hydrolyzed to 3H-FC in liver) resecretion into plasma, demonstrating Abca1 recycled HDL-derived hepatic 3H-FC back into plasma. Despite similar liver LDLr (low-density lipoprotein receptor) expression between genotypes, HSKO mice treated with LDLr-targeting versus control antisense oligonucleotide had slower plasma 3H-CO-HDL decay, reduced selective 3H-CO clearance, and decreased fecal 3H-sterol excretion that was indistinguishable from control mice. Increased RCT in HSKO mice was selective for 3H-CO-HDL, since macrophage RCT was similar between genotypes.ConclusionsHepatocyte Abca1 deletion unmasks a novel and selective FC trafficking pathway that requires LDLr expression, accelerating plasma HDL-selective CE uptake by the liver and promoting HDL RCT into feces, consequently reducing HDL-derived hepatic FC recycling into plasma.
| S-EPMC6703909 | biostudies-literature
Project description:ObjectiveAtherosclerotic lesions are often characterized by accumulation of OxLDL (oxidized low-density lipoprotein), which is associated with vascular inflammation and lesion vulnerability to rupture. Extracellular AIBP (apolipoprotein A-I binding protein; encoded by APOA1BP gene), when secreted, promotes cholesterol efflux and regulates lipid rafts dynamics, but its role as an intracellular protein in mammalian cells remains unknown. The aim of this work was to determine the function of intracellular AIBP in macrophages exposed to OxLDL and in atherosclerotic lesions. Approach and Results: Using a novel monoclonal antibody against human and mouse AIBP, which are highly homologous, we demonstrated robust AIBP expression in human and mouse atherosclerotic lesions. We observed significantly reduced autophagy in bone marrow-derived macrophages, isolated from Apoa1bp-/- compared with wild-type mice, which were exposed to OxLDL. In atherosclerotic lesions from Apoa1bp-/- mice subjected to Ldlr knockdown and fed a Western diet, autophagy was reduced, whereas apoptosis was increased, when compared with that in wild-type mice. AIBP expression was necessary for efficient control of reactive oxygen species and cell death and for mitochondria quality control in macrophages exposed to OxLDL. Mitochondria-localized AIBP, via its N-terminal domain, associated with E3 ubiquitin-protein ligase PARK2 (Parkin), MFN (mitofusin)1, and MFN2, but not BNIP3 (Bcl2/adenovirus E1B 19-kDa-interacting protein-3), and regulated ubiquitination of MFN1 and MFN2, key components of mitophagy.ConclusionsThese data suggest that intracellular AIBP is a new regulator of autophagy in macrophages. Mitochondria-localized AIBP augments mitophagy and participates in mitochondria quality control, protecting macrophages against cell death in the context of atherosclerosis.
| S-EPMC8105271 | biostudies-literature
Project description:Lipoteichoic acid (LTA) and lipopolysaccharide (LPS) are bacterial lipids that stimulate pro-inflammatory cytokine production, thereby exacerbating sepsis pathophysiology. Proprotein convertase subtilisin/kexin type 9 (PCSK9) negatively regulates uptake of cholesterol by downregulating hepatic lipoprotein receptors, including low-density lipoprotein (LDL) receptor (LDLR) and possibly LDLR-related protein-1 (LRP1). PCSK9 also negatively regulates Gram-negative LPS uptake by hepatocytes, however this mechanism is not completely characterized and mechanisms of Gram-positive LTA uptake are unknown. Therefore, our objective was to elucidate the mechanisms through which PCSK9 regulates uptake of LTA and LPS by investigating the roles of lipoproteins and lipoprotein receptors. Here we show that plasma PCSK9 concentrations increase transiently over time in septic and non-septic critically ill patients, with highly similar profiles over 14 days. Using flow cytometry, we demonstrate that PCSK9 negatively regulates LDLR-mediated uptake of LTA and LPS by HepG2 hepatocytes through an LDL-dependent mechanism, whereas LRP1 and high-density lipoprotein do not contribute to this uptake pathway. Bacterial lipid uptake by hepatocytes was not associated with cytokine production or hepatocellular injury. In conclusion, our study characterizes an LDL-dependent and LDLR-mediated bacterial lipid uptake pathway regulated by PCSK9, and provides evidence in support of PCSK9 inhibition as a potential therapeutic strategy for sepsis.
| S-EPMC6043579 | biostudies-literature
Project description:Background & aimsDespite high-risk behaviour, 10%-20% of HCV multiple exposed individuals remain uninfected (MEU), whilst the remainder become infected (MEI). We hypothesize that host factors play a role in HCV susceptibility. We aimed to identify polymorphisms in host genes that encode for proteins involved in viral entry: CD81, Scavenger receptor 1 (SR-1), Low-density lipoprotein receptor (LDL-R), Claudin-1 (CLDN1), Occludin (OCLN) and Niemann-Pick C1-like 1 (NPC1L1).MethodsMultiple exposed infected and MEU from two observational cohorts were selected. From the MSM study of acute infection with HCV (MOSAIC), HIV-1 infected MEU cases (n = 30) and HIV-1 infected MEI controls (n = 32) were selected based on reported high-risk behaviour. From the Amsterdam Cohorts Studies (ACS) injecting drug users (IDU) cohort, MEU cases (n = 40) and MEI controls (n = 22) were selected who injected drugs for ≥2 years, in the nineties, when HCV incidence was high. Selected single nucleotide polymorphisms (SNPs) were determined by sequencing or SNP assays.ResultsNo associations were found for SNPs within genes coding for CD81, SR-1, Claudin-1 or Occludin between the MEU and MEI individuals from either cohort. We did observe a significant association for rs688 within the LDL-R gene with HCV infection (OR: 0.41 P = 0.001), however, LDL cholesterol levels did not vary between individuals carrying the differential SNPs. Additionally, a marginal significant effect was found for rs217434 and rs2072183 (OR: 2.07 P = 0.032 and OR: 1.76 P = 0.039, respectively) within NPC1L1.ConclusionsOur results demonstrate that the rs688 SNP within the LDL-R gene associates with HCV susceptibility through mucosal as well as intravenous exposure.
| S-EPMC6588020 | biostudies-literature
Project description:Low-density lipoprotein receptor-related protein 6 (LRP6), a member of the low-density lipoprotein receptor (LDLR) family, displays a unique structure and ligand-binding function. As a co-receptor of the Wnt/β-catenin signaling pathway, LRP6 is a novel therapeutic target that plays an important role in the regulation of cardiovascular disease, lipid metabolism, tumorigenesis, and some classical signals. By using capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX), with recombinant human LRP-6 as the target, four candidate aptamers with a stem-loop structure were selected from an ssDNA library-AptLRP6-A1, AptLRP6-A2, AptLRP6-A3, and AptLRP6-A4. The equilibrium dissociation constant KD values between these aptamers and the LRP6 protein were in the range of 0.105 to 1.279 μmol/L, as determined by CE-LIF analysis. Their affinities and specificities were further determined by the gold nanoparticle (AuNP) colorimetric method. Among them, AptLRP6-A3 showed the highest affinity with LRP6-overexpressed human breast cancer cells. Therefore, the LRP6 aptamer identified in this study constitutes a promising modality for the rapid diagnosis and treatment of LRP6-related diseases.
| S-EPMC10180154 | biostudies-literature
Project description:ObjectiveAdrenal gland secretes stress-induced glucocorticoids (iGCs) to coping with stress. Previous study showed that SR-BI (scavenger receptor BI) null (SR-BI-/-) mice failed to generate iGC in stress conditions, suggesting that SR-BI-mediated cholesterol uptake from HDL (high-density lipoprotein) is a key regulator for iGC production. However, the LDL (low-density lipoprotein)/LDLr (LDL receptor) pathway can also provide cholesterol for iGC synthesis, but rodents have limited LDL levels in circulation. Here, we generated SR-BI-/-ApoBtg (apolipoprotein B transgenic) mice with normal LDL levels in circulation to determine the relative contribution of the HDL/SR-BI and LDL/LDLr pathways to iGC production in stress conditions. Approach and Results: To obtain mouse models with normal LDL levels, SR-BI-/- mice were bred to ApoBtg mice. Then, the F1 SR-BI±ApoBtg mice were backcrossed to SR-BI-/- to obtain SR-BI-/-ApoBtg, SR-BI-/-ApoBwt (apolipoprotein B wild type), and SR-BI+/+ApoBtg mice. We first examined the lipoprotein profile, which shows a 6.5-fold increase in LDL levels in SR-BI-/-ApoBtg mice compared with SR-BI-/-ApoBwt mice. Then, we induced stress with adrenocorticotropic hormone and cecal ligation and puncture. One hour after adrenocorticotropic hormone stimulation, SR-BI+/+ApoBtg control mice produced iGC (14.9-fold), but both SR-BI-/-ApoBwt and SR-BI-/-ApoBtg showed no iGC production (P<0.001). Three hours after cecal ligation and puncture treatment, SR-BI+/+ApoBtg control mice showed iGC production (6.4-fold), but both SR-BI-/-ApoBwt and SR-BI-/-ApoBtg mice showed no iGC production (P<0.001).ConclusionsSR-BI-/-ApoBtg mice fail to produce iGC in stress conditions even though with restored LDL levels in circulation. These findings clarify that the HDL/SR-BI, not LDL/LDLr, pathway is responsible for iGC production in stress conditions.
| S-EPMC7526689 | biostudies-literature