Cellular Cholesterol Homeostasis Research Articles

ORP1L, ORP1S, and ORP2: Lipid Sensors and Transporters

Lipid transfer proteins are crucial for intracellular cholesterol trafficking at sites of membrane contact. In the OSBP/ORPs (oxysterol binding protein and OSBP-related proteins) family of lipid transfer proteins, ORP1L, ORP1S and ORP2 play important roles in cholesterol transport. ORP1L is an endosome/lysosome-anchored cholesterol sensor which may also move cholesterol bidirectionally at the interface between the endoplasmic reticulum and the endosome/lysosome. ORP2 delivers cholesterol to the plasma membrane, driven by PI(4,5)P2hydrolysis. ORP1S may also transport cholesterol to the plasma membrane, although it is unclear if phosphoinositides are involved. The source of cholesterol delivered to the plasma membrane by ORP1S and ORP2 remains unclear. This review summarises the roles of these proteins in maintaining cellular cholesterol homeostasis and in human disease.

Enhanced acyl-CoA:cholesterol acyltransferase activity increases cholesterol levels on the lipid droplet surface and impairs adipocyte function

Cholesterol plays essential structural and signaling roles in mammalian cells, but too much cholesterol can cause cytotoxicity. Acyl-CoA:cholesterol acyltransferases 1 and 2 (ACAT1/2) convert cholesterol into its storage form, cholesteryl esters, regulating a key step in cellular cholesterol homeostasis. Adipose tissue can store >50% of whole-body cholesterol. Interestingly, however, almost no ACAT activity is present in adipose tissue, and most adipose cholesterol is stored in its free form. We therefore hypothesized that increased cholesterol esterification may have detrimental effects on adipose tissue function. Here, using several approaches, including protein overexpression, quantitative RT-PCR, immunofluorescence, and various biochemical assays, we found that ACAT1 expression is significantly increased in the adipose tissue of the ob/ob mice. We further demonstrated that ACAT1/2 overexpression partially inhibited the differentiation of 3T3-L1 preadipocytes. In mature adipocytes, increased ACAT activity reduced the size of lipid droplets (LDs) and inhibited lipolysis and insulin signaling. Paradoxically, the amount of free cholesterol increased on the surface of LDs in ACAT1/2-overexpressing adipocytes, accompanied by increased LD localization of caveolin-1. Moreover, cholesterol depletion in adipocytes by treating the cells with cholesterol-deficient media or β-cyclodextrins induced changes in cholesterol distribution that were similar to those caused by ACAT1/2 overexpression. Our results suggest that ACAT1/2 overexpression increases the level of free cholesterol on the LD surface, thereby impeding adipocyte function. These findings provide detailed insights into the role of free cholesterol in LD and adipocyte function and suggest that ACAT inhibitors have potential utility for managing disorders associated with extreme obesity.

Anti-ApoA-1 IgGs in Familial Hypercholesterolemia Display Paradoxical Associations with Lipid Profile and Promote Foam Cell Formation.

Aims: Anti-Apolipoprotein A-1 autoantibodies (anti-ApoA-1 IgG) promote atherogenesis via innate immune receptors, and may impair cellular cholesterol homeostasis (CH). We explored the presence of anti-ApoA-1 IgG in children (5–15 years old) with or without familial hypercholesterolemia (FH), analyzing their association with lipid profiles, and studied their in vitro effects on foam cell formation, gene regulation, and their functional impact on cholesterol passive diffusion (PD). Methods: Anti-ApoA-1 IgG and lipid profiles were measured on 29 FH and 25 healthy children. The impact of anti-ApoA-1 IgG on key CH regulators (SREBP2, HMGCR, LDL-R, ABCA1, and miR-33a) and foam cell formation detected by Oil Red O staining were assessed using human monocyte-derived macrophages. PD experiments were performed using a validated THP-1 macrophage model. Results: Prevalence of high anti-ApoA-1 IgG levels (seropositivity) was about 38% in both study groups. FH children seropositive for anti-ApoA-1 IgG had significant lower total cholesterol LDL and miR-33a levels than those who were seronegative. On macrophages, anti-ApoA-1 IgG induced foam cell formation in a toll-like receptor (TLR) 2/4-dependent manner, accompanied by NF-kB- and AP1-dependent increases of SREBP-2, LDL-R, and HMGCR. Despite increased ABCA1 and decreased mature miR-33a expression, the increased ACAT activity decreased membrane free cholesterol, functionally culminating to PD inhibition. Conclusions: Anti-ApoA-1 IgG seropositivity is frequent in children, unrelated to FH, and paradoxically associated with a favorable lipid profile. In vitro, anti-ApoA-1 IgG induced foam cell formation through a complex interplay between innate immune receptors and key cholesterol homeostasis regulators, functionally impairing the PD cholesterol efflux capacity of macrophages.

Bio-Inspired Functional Lipoprotein-like Nanoparticles (Flip-NPs) Cause Cholesterol Starvation and Ferroptosis in B-Cell Lymphomas: Studies in Cell Lines, Xenograft Models and Primary Patient Samples

Introduction: Hematologic malignancies, including B cell lymphomas such as diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL), have increased demands for cholesterol and cholesteryl esters to maintain membrane anchored pro-proliferative and pro-survival signaling pathways, including B cell receptor signaling. Recent evidence suggests that certain cancer cell lines, including several anaplastic large T cell lymphoma (ALCL) cell lines, are auxotrophic for cholesterol and are sensitive to cholesterol reduction-induced ferroptosis (Garcia-Bermudez, Nature 2019), an iron dependent form of programmed cell death characterized by accumulation of lipid peroxides. We have developed a cholesterol depleting functional lipoprotein-like nanoparticle (Flip-NP) that specifically targets the high-affinity HDL receptor, scavenger receptor type B1 (SCARB1), which maintains cellular and cell membrane cholesterol homeostasis. Our prior data demonstrated that Flip-NPs induce B cell lymphoma cell death in vitro and in in vivo xenograft models. Accordingly, we hypothesized that the mechanism of cell death by Flip-NPs in B cell lymphomas is ferroptosis, and that Flip-NPs would be potent therapy for an expanded number of cholesterol-addicted malignancies, including ALCL. Methods: After informed consent, primary B cell lymphoma cells were isolated from excisional biopsies from patients with FL or DLBCL. The SUDHL4 [germinal center (GC) DLBCL], Ramos [Burkitt's lymphoma], SUDHL1 [ALCL] and SR-786 [ALCL] cell lines were used for in vitro experiments. SCARB1 expression was quantified using flow cytometry and western blot analysis. Cell viability was quantified using the MTS assay and flow cytometry. Ferroptosis was measured using the lipophilic antioxidant ferrostatin-1 or the iron chelator deferoxamine. Gene expression changes were quantified using RT-qPCR. Lipid peroxidation was measured using C11-BODIPY and flow cytometry. SUDHL1 and SUDHL4 flank tumor xenografts were initiated in SCID-beige mice, with Flip-NPs administered 3 times per week IV. Results: Primary B cell lymphoma cells were isolated from patients with FL (n=4) or DLBCL (n=2), and all samples expressed some level of SCARB1 by flow cytometry. Flip-NPs increased cell death in 3 of the 4 FL samples and 1 of 2 DLBCL samples. In Ramos and SUDHL4 cells, RT-qPCR data showed that Flip-NP-mediated cholesterol reduction led to up-regulation of cholesterol biosynthesis genes and down-regulation of glutathione peroxidase-4 (GPX4), a critical protein responsible for degradation of lipid peroxides. Correspondingly, as shown with C11-BODIPY, Flip-NP treatment increased lipid peroxide accumulation in Ramos and SUDHL4 cells. Addition of ferrostatin-1 or deferoxamine reduced Flip-NP induced cell death, demonstrating that the mechanism-of-action of Flip-NPs involves, at least in part, ferroptosis. Given the sensitivity of cholesterol auxotrophic cell lines to cholesterol reduction-induced ferroptosis, we tested the efficacy of the Flip-NPs against cholesterol auxotrophic ALK+ ALCL cell lines SUDHL1 and SR-786. SCARB1 was expressed in both cell lines. Flip-NPs potently induced cell death in both SUDHL1 and SR-786 cells in vitro. In vivo, systemic administration of Flip-NPs reduced tumor volumes in both SUDHL4 and SUDHL1 tumor xenograft models. Conclusions: Our data show that Flip-NPs reduce GPX4 expression and increase lipid peroxide accumulation in B cell lymphoma cell lines, resulting in ferroptosis. Expanding on these results, Flip-NP efficacy was also demonstrated in cholesterol auxotrophic ALK+ ALCL cell lines and primary patient-derived B cell lymphoma cells. These in vitro results translated to in vivo murine models, as systemic administration of Flip-NPs potently reduced DLBCL and ALK+ ALCL tumor xenograft burden. Flip-NPs are a molecularly targeted, first-in-class therapy that may be effective for malignancies reliant upon cellular cholesterol. Disclosures Behdad: Pfizer: Other: Speaker; Thermo Fisher: Membership on an entity's Board of Directors or advisory committees; Loxo-Bayer: Membership on an entity's Board of Directors or advisory committees. Karmali:Astrazeneca: Speakers Bureau; Takeda, BMS: Other: Research Funding to Institution; Gilead/Kite; Juno/Celgene: Consultancy, Speakers Bureau. Thaxton:Zylem: Other: Co-founder of the biotech company Zylem. Gordon:Juno/Celgene: Other: Advisory Board, Research Funding; Gilead: Other: Advisory Board; Bayer: Other: Advisory Board; Zylem LLC: Other: co-founder; research in nanoparticles in cancer.

MicroRNAs as Potential Biomarkers in Atherosclerosis.

Atherosclerosis is a complex multifactorial disease that, despite advances in lifestyle management and drug therapy, remains to be the major cause of high morbidity and mortality rates from cardiovascular diseases (CVDs) in industrialized countries. Therefore, there is a great need in reliable diagnostic/prognostic biomarkers and effective treatment alternatives to reduce its burden. It was established that microRNAs (miRNAs/miRs), a class of non-coding single-stranded RNA molecules, can regulate the expression of genes at the post-transcriptional level and, accordingly, coordinate the cellular protein expression. Thus, they are involved not only in cell-specific physiological functions but also in the cellular and molecular mechanisms of human pathologies, including atherosclerosis. MiRNAs may be significant in the dysregulation that affects endothelial integrity, the function of vascular smooth muscle and inflammatory cells, and cellular cholesterol homeostasis that drives the initiation and growth of an atherosclerotic plaque. Besides, distinct expression patterns of several miRNAs are attributed to atherosclerotic and cardiovascular patients. In this article, the evidence indicating the multiple critical roles of miRNAs and their relevant molecular mechanisms related to atherosclerosis development and progression was reviewed. Moreover, the effects of miRNAs on atherosclerosis enabled to exploit them as novel diagnostic biomarkers and therapeutic targets that may lead to better management of atherosclerosis and CVDs.

Metabolism of cholesterol and progesterone is differentially regulated in primary trophoblastic subtypes and might be disturbed in recurrent miscarriages

During pregnancy, extravillous trophoblasts (EVTs) invade the maternal decidua and remodel the local vasculature to establish blood supply for the growing fetus. Compromised EVT function has been linked to aberrant pregnancy associated with maternal and fetal morbidity and mortality. However, metabolic features of this invasive trophoblast subtype are largely unknown. Using primary human trophoblasts isolated from first trimester placental tissues, we show that cellular cholesterol homeostasis is differentially regulated in EVTs compared with villous cytotrophoblasts. Utilizing RNA-sequencing, gene set-enrichment analysis, and functional validation, we provide evidence that EVTs display increased levels of free and esterified cholesterol. Accordingly, EVTs are characterized by increased expression of the HDL-receptor, scavenger receptor class B type I, and reduced expression of the LXR and its target genes. We further reveal that EVTs express elevated levels of hydroxy-delta-5-steroid dehydrogenase 3 beta- and steroid delta-isomerase 1 (HSD3B1) (a rate-limiting enzyme in progesterone synthesis) and are capable of secreting progesterone. Increasing cholesterol export by LXR activation reduced progesterone secretion in an ABCA1-dependent manner. Importantly, HSD3B1 expression was decreased in EVTs of idiopathic recurrent spontaneous abortions, pointing toward compromised progesterone metabolism in EVTs of early miscarriages. Here, we provide insights into the regulation of cholesterol and progesterone metabolism in trophoblastic subtypes and its putative relevance in human miscarriage.

Small-Molecule Modulators of Sigma1 and Sigma2/TMEM97 in the Context of Cancer: Foundational Concepts and Emerging Themes.

There are two known subtypes of the so-called sigma receptors, Sigma1 and Sigma2. Sigma1 (encoded by the SIGMAR1 gene and also known as Sigma-1 receptor, S1R) is a unique pharmacologically regulated integral membrane chaperone or scaffolding protein that allosterically modulates the activity of its associated proteins. Sigma2, recently identified as transmembrane protein 97 (TMEM97), is an integral membrane protein implicated in cellular cholesterol homeostasis. A number of publications over the past two decades have suggested a role for both sigma proteins in tumor biology. Although there is currently no clinically used anti-cancer drug that targets Sigma1 or Sigma2/TMEM97, a growing body of evidence supports the potential of small-molecule compounds with affinity for these proteins, putative sigma ligands, as therapeutic agents to treat cancer. In preclinical models, these compounds have been reported to inhibit cancer cell proliferation, survival, adhesion, and migration; furthermore, they have been demonstrated to suppress tumor growth, to alleviate cancer-associated pain, and to exert immunomodulatory properties. Here, we will address the known knowns and the known unknowns of Sigma1 and Sigma2/TMEM97 ligand actions in the context of cancer. This review will highlight key discoveries and published evidence in support of a role for sigma proteins in cancer and will discuss several fundamental questions regarding the physiological roles of sigma proteins in cancer and sigma ligand mechanism of action.

Attenuated cholesterol metabolism pathway suppresses regulatory T cell development in prenatal nicotine exposed female mice

The recession of regulatory T cells (Tregs) contributes to development of autoimmune disease. Our previous study suggested that prenatal nicotine exposure (PNE) inhibited Tregs frequency in offspring, but the mechanisms are still uncertain. This study aimed to explore the molecular mechanisms of PNE-induced Tregs inhibition from the perspective of cellular cholesterol homeostasis both in vivo and in vitro. PNE mice model were established by 3 mg/kg/d nicotine administration in Balb/c strain from gestational day (GD) 9 to GD 18. The results showed that PNE significantly decreased thymic Tregs frequency in neonatal offspring. The activation of mTOR and downregulation of p-STAT5/Foxp3 pathway of Tregs were observed in PNE offspring. Mechanism study found that PNE elevated ATP-binding cassette transporter G1 (ABCG1) expression and decreased intracellular cholesterol content of Tregs in offspring, indicating impaired intracellular cholesterol homeostasis. Similar results were observed in 1 μM nicotine-treated primary thymocytes in vitro. Further, cholesterol-replenishment can abrogate nicotine-induced mTOR activation and the following suppression of p-STAT5/Foxp3 pathway and Tregs frequency. In addition, Abcg1 siRNA transfection can partly reverse the nicotine-decreased intracellular cholesterol content and cell frequency of Tregs. In conclusion, this study showed that PNE could suppress Tregs development in female mice by up-regulating ABCG1-dependent cholesterol efflux, and suggested that PNE-induced thymic Tregs recession of offspring at early life was the developmental origin mechanism of immune dysfunction in later life.

Mitochondria in alzheimer’s disease

The pathogenesis of Alzheimer disease (AD) is associated with aberrant proteolytic processing of the amyloid precursor protein (APP) - first by b-secretase to generate a 99-aa C-terminal fragment (C99) and then by g-secretase (containing presenilins-1 [PS1] or -2 [PS2]) that cleaves C99 to generate the APP intracellular domain and the b-amyloid (Ab) that is found in extraneuronal plaques - but the relationship of the former to the latter is unclear. Whereas plaques typically form late in disease progression, early events include aberrant lipid metabolism and mitochondrial dysfunction. We recently showed that PS1, PS2, and g-secretase activity itself are highly enriched in a subdomain of the endoplasmic reticulum (ER) that communicates with mitochondria, called mitochondria-associated ER membranes (MAM), and that ER-mitochondrial connectivity and MAM function are highly upregulated in presenilin-mutant cells and in cells from AD patients. However, the relationship of perturbed APP processing to MAM-related AD phenotypes has been unclear. MAM, which is a lipid raft rich in cholesterol and sphingomyelin, is a key regulator of lipid metabolism and of mitochondial dynamics, among other processes. We now show that C99 is highly enriched in the MAM, and that it plays a central role in regulating cellular cholesterol and sphingolipid homeostasis and mitochondrial bioenergetics. Furthermore, in cells from AD patients and in cell and animal models of AD, C99 levels are increased significantly, resulting in a disruption of lipid homeostasis that is triggered by the unregulated uptake of extracellular cholesterol, with downstream consequences that mimic the biochemical features of AD, including the bioenergetic deficits. We propose that MAM-localized C99 is a component of the cell's lipid sensing machinery and that its accumulation is a key and early event in AD pathogenesis that can explain many of the biochemical and morphological features of the disease.

AdipoRon Attenuates Wnt Signaling by Reducing Cholesterol-Dependent Plasma Membrane Rigidity

The increasing prevalence of adult and adolescent obesity and its associated risk of colorectal cancer reinforces the urgent need to elucidate the underlying mechanisms contributing to the promotion of colon cancer in obese individuals. Adiponectin is an adipose tissue-derived adipokine, whose levels are reduced during obesity. Both epidemiological and preclinical data indicate that adiponectin suppresses colon tumorigenesis. We have previously demonstrated that both adiponectin and AdipoRon, a small-molecule adiponectin receptor agonist, suppress colon cancer risk in part by reducing the number of Lgr5+ stem cells in mouse colonic organoids. However, the mechanism by which the adiponectin signaling pathway attenuates colon cancer risk remains to be addressed. Here, we have hypothesized that adiponectin signaling supports colonic stem cell maintenance through modulation of the biophysical properties of the plasma membrane (PM). Specifically, we investigated the effects of adiponectin receptor activation by AdipoRon on the biophysical perturbations linked to the attenuation of Wnt-driven signaling and cell proliferation as determined by LEF luciferase reporter assay and colonic organoid proliferation, respectively. Using physicochemical sensitive dyes, Di-4-ANEPPDHQ and C-laurdan, we demonstrated that AdipoRon decreased the rigidity of the colonic cell PM. The decrease in membrane rigidity was associated with a reduction in PM free cholesterol levels and the intracellular accumulation of free cholesterol in lysosomes. These results suggest that adiponectin signaling plays a role in modulating cellular cholesterol homeostasis, PM biophysical properties, and Wnt-driven signaling. These findings are noteworthy because they may in part explain how obesity drives colon cancer progression.

Binding and intracellular transport of 25-hydroxycholesterol by Niemann-Pick C2 protein

Side-chain oxidized cholesterol derivatives, like 25-hydroxycholesterol (25-OH-Chol) are important regulators of cellular cholesterol homeostasis. How transport of oxysterols through the endo-lysosomal pathway contributes to their biological function is not clear. The Niemann-Pick C2 protein (NPC2) is a small lysosomal sterol transfer protein required for export of cholesterol from late endosomes and lysosomes (LE/LYSs). Here, we show that 25-hydroxy-cholestatrienol, (25-OH-CTL), an intrinsically fluorescent analogue of 25-OH-Chol, becomes trapped in LE/LYSs of NPC2-deficient fibroblasts, but can efflux from the cells even in the absence of NPC2 upon removal of the sterol source. Fluorescence recovery after photobleaching (FRAP) of 25-OH-CTL in endo-lysosomes was rapid and extensive and only partially dependent on NPC2 function. Using quenching of NPC2's intrinsic fluorescence, we show that 25-OH-Chol and 25-OH-CTL can bind to NPC2 though with lower affinity compared to cholesterol and its fluorescent analogues, cholestatrienol (CTL) and dehydroergosterol (DHE). This is confirmed by calculations of binding energies which additionally show that 25-OH-CTL can bind in two orientations to NPC2, in stark contrast to cholesterol and its analogues. We conclude that NPC2's affinity for all sterols is energetically favored over their self-aggregation in the lysosomal lumen. Lysosomal export of 25-OH-Chol is not strictly dependent on the NPC2 protein.

Translating the findings related to anti-apolipoprotein a-1 igg in familial hypercholesterolemia towards the identification of a new endogenous disruptor of cellular cholesterol homeostasis.

Background and Aims: To explore i) the associations between anti-apolipoprotein (apo) A-1IgG ( an emergent cardiovascular disease biomarker), lipid profile, and microRNA-33 (mi-R33) in children with familial hypercholesterolemia (FH); ii) the in vitro effects of anti-apoA-1 IgG on macrophages cholesterol homeostasis (CH); and foam cell formation, the hallmark of atherogenesis.

Immunometabolic function of cholesterol in cardiovascular disease and beyond.

Inflammation represents the driving feature of many diseases, including atherosclerosis, cancer, autoimmunity and infections. It is now established that metabolic processes shape a proper immune response and within this context the alteration in cellular cholesterol homeostasis has emerged as a culprit of many metabolic abnormalities observed in chronic inflammatory diseases. Cholesterol accumulation supports the inflammatory response of myeloid cells (i.e. augmentation of toll-like receptor signalling, inflammasome activation, and production of monocytes and neutrophils) which is beneficial in the response to infections, but worsens diseases associated with chronic metabolic inflammation including atherosclerosis. In addition to the innate immune system, cells of adaptive immunity, upon activation, have also been shown to undergo a reprogramming of cellular cholesterol metabolism, which results in the amplification of inflammatory responses. Aim of this review is to discuss (i) the molecular mechanisms linking cellular cholesterol metabolism to specific immune functions; (ii) how cellular cholesterol accumulation sustains chronic inflammatory diseases such as atherosclerosis; (iii) the immunometabolic profile of patients with defects of genes affecting cholesterol metabolism including familial hypercholesterolaemia, cholesteryl ester storage disease, Niemann-Pick type C, and immunoglobulin D syndrome/mevalonate kinase deficiency. Available data indicate that cholesterol immunometabolism plays a key role in directing immune cells function and set the stage for investigating the repurposing of existing 'metabolic' drugs to modulate the immune response.

Acyl-CoA: cholesterol acyltransferases-2 gene polymorphism is associated with increased susceptibility to coronary artery disease in Uygur population in Xinjiang, China.

Background: Acyl-CoA: cholesterol acyltransferases (ACAT) is the only enzyme that catalyzes the synthesis of cholesterol esters (CE) from free cholesterol and long-chain fatty acyl-CoA and plays a critical role in cellular cholesterol homeostasis. In the present study, our primary objective was to explore whether the single-nucleotide polymorphisms (SNPs) in ACAT-2 gene were associated with coronary artery disease (CAD) in Uygur subjects, in Xinjiang, China.Methods: We designed a case–control study including 516 CAD patients and 318 age- and sex-matched control subjects. Using the improved multiplex ligation detection reaction (iMLDR) method, we genotyped two SNPs (rs28765985 and rs7308390) of ACAT-2 gene in all subjects.Results: We found that the genotypes, the dominant model (CC + CT vs TT) and over-dominant model (CT vs CC + TT) of rs28765985 were significantly different between CAD patients and the controls (P=0.027, P=0.012 and P=0.035, respectively). The rs28765985 C allele was associated with a significantly elevated CAD risk [CC/CT vs TT: odds ratio (OR) = 1.48, 95% confidence interval (CI) = 1.02–2.16, P=0.04] after adjustment for confounders. The TC and LDL-C levels were significantly higher in rs28765985 CC/CT genotypes than that in TT genotypes (P<0.05).Conclusions: Rs28765985 of ACAT-2 gene are associated with CAD in Uygur subjects. Subjects with CC/CT genotype or C allele of rs28765985 were associated with an increased risk of CAD.

Quantifying Cellular Cholesterol Efflux.

Measuring cholesterol efflux involves the tracking of cholesterol movement out of cells. Cholesterol efflux is an essential mechanism to maintain cellular cholesterol homeostasis, and this process is largely regulated via the LXR transcription factors and their regulated genes, the ATP-binding cassette (ABC) cholesterol transporters ABCA1 and ABCG1. Typically, efflux assays are performed utilizing radiolabeled cholesterol tracers to label intracellular cholesterol pools, and these assays may be tailored to quantify the efflux of exogenously delivered cholesterol or alternatively the efflux of newly synthesized (endogenous) cholesterol, in different cell types (macrophages, hepatocytes). Cholesterol efflux may also be customized to quantify cholesterol flux out of the cell to various exogenous cholesterol acceptors, such as apolipoprotein A-I, high-density lipoprotein, or methyl-beta-cyclodextrin, depending on the purpose of the experiment. Here, we provide comprehensive protocols to quantify the net flux of cholesterol out of cells and recommendations on how this assay may be tailored as a function of the experimental question at hand.

CYP27A1 inhibits bladder cancer cells proliferation by regulating cholesterol homeostasis

ABSTRACTCYP27A1, an enzyme involved in regulating cellular cholesterol homeostasis, converts cholesterol into 27-hydroxycholesterol (27-HC). The relationship between CYP27A1 and cell proliferation was studied to determine the role of CYP27A1 in bladder cancer. The expression of CYP27A1 in three bladder cancer cell lines (T24, UM-UC-3 and 5637) were assessed by qRT-PCR and Western blotting, and cells with stable CYP27A1 expression were generated by lentiviral infection. Cell proliferation was detected by MTT assays, colony formation assays and a tumor xenograft model in vitro and in vivo, and the intracellular 27-HC and cholesterol secretion levels were detected by enzyme-linked immunosorbent assays (ELISA). The results revealed that CYP27A1 expression was downregulated in androgen receptor (AR)-positive T24/UM-UC-3 cells compared with AR-negative 5637 cell. After CYP27A1 expression was restored, cell proliferation was inhibited in vitro and in vivo because much more intracellular 27-HC was produced in the CYP27A1-overexpressing cells than in the control cells. Both T24 and UM-UC-3 cells treated with 27-HC showed similar results. In addition, CYP27A1/27HC could reduce the cellular cholesterol level in both T24 and UM-UC-3 cells by upregulating ATP-binding cassette transporters G1 and A1 (ABCG1 and ABCA1) through Liver X receptors (LXRs) pathway and downregulating low-density lipoprotein receptor (LDLR) expression. These findings all suggest that CYP27A1 is a critical cholesterol sensor in bladder cancer cells that may contribute significantly to bladder cancer proliferation.

Differential use of E2 ubiquitin conjugating enzymes for regulated degradation of the rate-limiting enzymes HMGCR and SQLE in cholesterol biosynthesis

Background and aimsCholesterol is an essential lipid for cellular function and membrane integrity, and hence its cellular levels and distribution must be tightly regulated. Biosynthesis of cholesterol is ramped when its cellular levels are low. Herein, the ER-resident and rate-limiting enzymes 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and squalene monooxygenase (SQLE) play a prominent role. We have recently reported that MARCH6, an E3 ubiquitin ligase, specifically promotes cholesterol-stimulated ubiquitylation and subsequent proteasomal degradation of SQLE, but not of HMGCR. To further delineate how post-translational regulation of SQLE and HMGCR is differentially achieved, we hypothesized that their sterol-dependent degradation machinery makes use of distinct E2 ubiquitin conjugating enzymes. MethodsTo study this possibility, we therefore used a CRISPR/Cas9-based approach to screen for ER-associated degradation (ERAD)-associated E2 enzymes that are essential for MARCH6-dependent degradation of SQLE. ResultsWe report here the identification of UBE2J2 as the primary E2 ubiquitin conjugating enzyme essential for this process in mammalian cells, in contrast to UBE2G2, which is essential for sterol-stimulated degradation of HMGCR. We demonstrate that ablating UBE2J2 disturbs cholesterol-accelerated SQLE degradation in multiple human cell types, including cells of hepatic origin, and that the ability of UBE2J2 to support SQLE degradation critically depends on its enzymatic activity. ConclusionsOur findings establish UBE2J2 as an important partner of MARCH6 in cholesterol-stimulated degradation of SQLE, thereby contributing to the complex regulation of cellular cholesterol homeostasis.

The long noncoding RNA CHROME regulates cholesterol homeostasis in primate.

The human genome encodes thousands of long non-coding RNAs (lncRNAs), the majority of which are poorly conserved and uncharacterized. Here we identify a primate-specific lncRNA (CHROME), elevated in the plasma and atherosclerotic plaques of individuals with coronary artery disease, that regulates cellular and systemic cholesterol homeostasis. LncRNA CHROME expression is influenced by dietary and cellular cholesterol via the sterol-activated liver X receptor transcription factors, which control genes mediating responses to cholesterol overload. Using gain- and loss-of-function approaches, we show that CHROME promotes cholesterol efflux and HDL biogenesis by curbing the actions of a set of functionally related microRNAs that repress genes in those pathways. CHROME knockdown in human hepatocytes and macrophages increases levels of miR-27b, miR-33a, miR-33b and miR-128, thereby reducing expression of their overlapping target gene networks and associated biologic functions. In particular, cells lacking CHROME show reduced expression of ABCA1, which regulates cholesterol efflux and nascent HDL particle formation. Collectively, our findings identify CHROME as a central component of the non-coding RNA circuitry controlling cholesterol homeostasis in humans.

Nutritionally Derived Metabolic Cues Typical of the Obese Microenvironment Increase Cholesterol Efflux Capacity of Adipose Tissue Macrophages.

BackgroundCholesterol retention within plasma membranes of macrophages is associated with increased inflammatory signaling. Cholesterol efflux via the transporters ABCA1, ABCG1, and SR‐BI to high‐density lipoprotein (HDL) particles is a critical mechanism to maintain cellular cholesterol homeostasis. Little is known about the impact of the obese microenvironment on cholesterol efflux capacity (CEC) of macrophages. In this study, the CEC of obese‐derived primary adipose‐tissue macrophages (ATM) is evaluated and the in vivo microenvironment is modeled in vitro to determine mechanisms underlying modulated CEC.Materials and MethodsF4/80+ ATM are labeled with 3H‐cholesterol ex vivo, and CEC and ABCA1/ABCG1 protein levels are determined. Total, ABCA1‐dependent, and ABCA1‐independent CECs are determined in J774 macrophages polarized to M1 (LPS&IFNγ), M2 (IL‐4&IL‐13), or metabolic phenotypes (glucose, insulin, and palmitic acid).ResultsObese ATM exhibit enhanced CEC and ABCA1 and ABCG1 expression compared to lean ATM. In contrast, ABCA1‐CEC is suppressed from M1 polarized macrophages compared to untreated in vitro, by activation of the JAK/STAT pathway. Incubation of macrophages in vitro in high glucose augments cAMP‐induced ABCA1 protein expression and ABCA1‐CEC.ConclusionsThese novel findings demonstrate remarkable plasticity of macrophages to respond to their environment with specific modulation of ABCA1 depending on whether classical pro‐inflammatory or metabolic cues predominate.

79. LXR and ABC-transporter-expression patterns in the placenta in IUGR

Introduction Atherogenic lipid oxidation and cholesterol accumulation is increased in the placenta in IUGR. Both, hypoxic conditions and the formation of small molecules like oxysterols however are involved in cellular cholesterol homeostasis by regulating the cholesterol efflux ABC-transporter expression ABCA1 and ABCG1 via activating liver-X-receptors (LXR). Objectives/hypothesis Since it is assumed that hypoxia and oxysterol production or accumulation is increased in the IUGR placenta we questioned whether the LXR-ABCA1 pathway is activated to counteract cholesterol excess. Methodes Placentas of 40 IUGR and 40 controls (CTRL) arranged in tissue micro arrays (TMA) were analyzed immunohistochemically for LXRa, LXRb, ABCA1, and ABCG1. The expression was assessed in trophoblasts and endothelium cells semi-quantitatively using Immunoreactivity-Score (IRS). Expression values were related to maternal and fetal lipid profiles. Expression patterns were statistically accomplished via the Mann–Whitney test. Results The LXRa und ABCA1-expression was increased in trophoblasts as compared to controls (LXRa Median IRS (95%CI) IUGR = 6.3 (5.59–7.06), CTRL = 4.8 (4.51–5.85), p = 0.0207; ABCA1 Median IRS (95%CI) IUGR = 7.0 (6.33–7.87), CTRL = 3.9 (3.87–5.67), p = 0.0001). No differences were found in LXRb and ABCG1 expression patterns. Discussion An increased expression of cholesterol transport molecules in trophoblasts may be a compensatory mechanism against toxic effects of cholesterol excess and oxidation within the placenta.