Oxysterol 25-hydroxycholesterol as a metabolic pathophysiological factors of osteoarthritis induces apoptosis in primary rat chondrocytes.
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ABSTRACT:
SUBMITTER: Seo YS
PROVIDER: S-EPMC7193916 | biostudies-literature | 2020 May
REPOSITORIES: biostudies-literature
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Project description:Cholesterol, an essential component of mammalian cells, is also an important factor in the replicative-cycles of several human and animal viruses. The oxysterol, 25-hydroxycholesterol, is produced from cholesterol by the enzyme, cholesterol 25-hydroxylase. 25-hydroxycholesterol (25-HC) has been shown to have anti-viral activities against a wide range of viruses, including a range of positive-sense RNA viruses. In this study, we have investigated the role of 25-HC in norovirus replication using murine norovirus (MNV) as a model system. As a control, we employed herpes simplex virus-1 (HSV-1), a pathogen previously shown to be inhibited by 25-HC. Consistent with previous studies, 25-HC inhibited HSV-1 replication in the MNV-susceptible cell line, RAW264.7. Treating RAW264.7 cells with sub-cytotoxic concentrations of 25-HC reduced the MNV titers. However, other sterols such as cholesterol or the oxysterol, 22-S-hydroxycholesterol (22-S-HC), did not inhibit MNV replication. Moreover, treating MNV-infected RAW264.7 cells with 25-HC-stimulated caspase 3/7 activity, which leads to enhanced apoptosis and increased cell death. Our study adds noroviruses to the list of viruses inhibited by 25-HC and begins to offer insights into the mechanism behind this inhibition.
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Project description:Oxysterols are 27-carbon oxidized derivatives of cholesterol or by-products of cholesterol biosynthesis that can induce cell apoptosis in addition to a number of other bioactions. However, the mechanisms underlying this cytotoxicity are not completely understood. ORP8 is a member of the oxysterol binding protein-related protein (ORP) family, implicated in cellular lipid homeostasis, migration, and organization of the microtubule cytoskeleton. Here, we report that 25-hydroxycholesterol (OHC) induced apoptosis of the hepatoma cell lines, HepG2 and Huh7, via the endoplasmic reticulum (ER) stress response pathway, and ORP8 overexpression resulted in a similar cell response as 25-OHC, indicating a putative functional relationship between oxysterol cytotoxicity and ORP8. Further experiments demonstrated that ORP8 overexpression significantly enhanced the 25-OHC effect on ER stress and apoptosis in HepG2 cells. A truncated ORP8 construct lacking the ligand-binding domain or a closely related protein, ORP5, was devoid of this activity, evidencing for specificity of the observed effects. Importantly, ORP8 knockdown markedly dampened such responses to 25-OHC. Taken together, the present study suggests that ORP8 may mediate the cytotoxicity of 25-OHC.
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Project description:Neuroinflammation, a major hallmark of Alzheimer's disease and several other neurological and psychiatric disorders, is often associated with dysregulated cholesterol metabolism. Relative to homeostatic microglia, activated microglia express higher levels of Ch25h, an enzyme that hydroxylates cholesterol to produce 25-hydroxycholesterol (25HC). 25HC is an oxysterol with interesting immune roles stemming from its ability to regulate cholesterol metabolism. Since astrocytes synthesize cholesterol in the brain and transport it to other cells via ApoE-containing lipoproteins, we hypothesized that secreted 25HC from microglia may influence lipid metabolism as well as extracellular ApoE derived from astrocytes. Here, we show that astrocytes take up externally added 25HC and respond with altered lipid metabolism. Extracellular levels of ApoE lipoprotein particles increased after treatment of astrocytes with 25HC without an increase in Apoe mRNA expression. In mouse astrocytes-expressing human ApoE3 or ApoE4, 25HC promoted extracellular ApoE3 better than ApoE4. Increased extracellular ApoE was due to elevated efflux from increased Abca1 expression via LXRs as well as decreased lipoprotein reuptake from suppressed Ldlr expression via inhibition of SREBP. 25HC also suppressed expression of Srebf2, but not Srebf1, leading to reduced cholesterol synthesis in astrocytes without affecting fatty acid levels. We further show that 25HC promoted the activity of sterol-o-acyl transferase that led to a doubling of the amount of cholesteryl esters and their concomitant storage in lipid droplets. Our results demonstrate an important role for 25HC in regulating astrocyte lipid metabolism.
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Project description:Interleukin 18 (IL-18) promotes inflammation and apoptosis in chondrocytes, thereby contributing to the development and progression of osteoarthritis (OA). Here, we investigated the effects of IL-18 treatment and inhibition in rat chondrocytes in vitro and in vivo. We used RT-PCR and Western blotting to measure the mRNA and protein levels of the chondrocyte-specific genes Collagen II and Aggrecan as well as the protein levels of apoptosis-related (Bax, Bcl2, Caspase3/9), autophagy-related (Atg5, Atg7, Beclin1, LC3), and mTOR pathway-related genes (PI3K, Akt, mTOR). We observed a decrease in Collagen II and Aggrecan mRNA and protein levels, upregulation of chondrocyte apoptosis, downregulation of chondrocyte autophagy, and activation of the PI3K/Akt/mTOR pathway upon IL-18 treatment. PI3K/Akt/mTOR pathway activation and inhibition tests using rat 740Y-P (PI3K activator), SC79 (AKT activator), 3BDO (mTOR activator), or LY294002 (PI3K inhibitor) revealed that activation of the PI3K/Akt/mTOR pathway enhances chondrocyte-specific gene degradation induced by IL-18, while its inhibition has protective effects on chondrocytes. We also found that treatment with rapamycin (a selective mTOR inhibitor) also exerts chondro-protective effects that ameliorate OA by promoting autophagy. These results suggest that inhibition of the mTOR pathway could be exploited for therapeutic benefits in the treatment of OA.
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Project description:OSBP (oxysterol-binding protein) homologues, ORPs (OSBP-related proteins), constitute a 12-member family in mammals. We employed an in vitro [3H]25OH (25-hydroxycholesterol)-binding assay with purified recombinant proteins as well as live cell photo-cross-linking with [3H]photo-25OH and [3H]photoCH (photo-cholesterol), to investigate sterol binding by the mammalian ORPs. ORP1 and ORP2 [a short ORP consisting of an ORD (OSBP-related ligand-binding domain) only] were in vitro shown to bind 25OH. GST (glutathione S-transferase) fusions of the ORP1L [long variant with an N-terminal extension that carries ankyrin repeats and a PH domain (pleckstrin homology domain)] and ORP1S (short variant consisting of an ORD only) variants bound 25OH with similar affinity (ORP1L, K(d)=9.7x10(-8) M; ORP1S, K(d)=8.4 x10(-8) M), while the affinity of GST-ORP2 for 25OH was lower (K(d)=3.9x10(-6) M). Molecular modelling suggested that ORP2 has a sterol-binding pocket similar to that of Saccharomyces cerevisiae Osh4p. This was confirmed by site-directed mutagenesis of residues in proximity of the bound sterol in the structural model. Substitution of Ile249 by tryptophan or Lys150 by alanine markedly inhibited 25OH binding by ORP2. In agreement with the in vitro data, ORP1L, ORP1S, and ORP2 were cross-linked with photo-25OH in live COS7 cells. Furthermore, in experiments with either truncated cDNAs encoding the OSBP-related ligand-binding domains of the ORPs or the full-length proteins, photo-25OH was bound to OSBP, ORP3, ORP4, ORP5, ORP6, ORP7, ORP8, ORP10 and ORP11. In addition, the ORP1L variant and ORP3, ORP5, and ORP8 were cross-linked with photoCH. The present study identifies ORP1 and ORP2 as OSBPs and suggests that most of the mammalian ORPs are able to bind sterols.
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Project description:This work investigates the relationship between high-glucose (HG) culture, CpG methylation of genes involved in cell signaling pathways, and the regulation of carbohydrate and lipid metabolism in hepatocytes. The results indicate that HG leads to an increase in nuclear 25-hydroxycholesterol (25HC), which specifically activates DNA methyltransferase-1 (DNMT1), and regulates gene expression involved in intracellular lipid metabolism. The results show significant increases in 5mCpG levels in at least 2,225 genes involved in 57 signaling pathways. The hypermethylated genes directly involved in carbohydrate and lipid metabolism are of PI3K, cAMP, insulin, insulin secretion, diabetic, and NAFLD signaling pathways. The studies indicate a close relationship between the increase in nuclear 25HC levels and activation of DNMT1, which may regulate lipid metabolism via DNA CpG methylation. Our results indicate an epigenetic regulation of hepatic cell metabolism that has relevance to some common diseases such as non-alcoholic fatty liver disease and metabolic syndrome.
| S-EPMC7225732 | biostudies-literature
Project description:Oxysterol-binding protein (OSBP) is the prototypical member of a class of phospholipid and oxysterol-binding proteins that interacts with the Golgi apparatus and regulates lipid and cholesterol metabolism. As a result of recent sequencing efforts, eleven other OSBP-related proteins (ORPs) have been identified in humans. We have investigated the structure, oxysterol-binding activity, cellular localization and function of ORP4 (also designated OSBP2 or HLM), a homologue that shares the highest degree of similarity with OSBP. Two ORP4 cDNAs were identified: a full-length ORP4 containing a pleckstrin homology (PH) domain and an oxysterol-binding region (designated ORP4-L), and a splice variant in which the PH domain and part of the oxysterol-binding domain were deleted (designated ORP4-S). ORP4 mRNA and protein expression overlapped partially with OSBP and were restricted to brain, heart, muscle and kidney. Like OSBP, ORP4-L bound [3H]25-hydroxycholesterol with high affinity and specificity. In contrast, ORP4-S did not bind [3H]25-hydroxycholesterol or [3H]7-ketocholesterol. Immunofluorescence localization in stably transfected Chinese hamster ovary cells showed that ORP4-S co-localized with vimentin and caused the intermediate filament network to bundle or aggregate. ORP4-L displayed a diffuse staining pattern that did not overlap with vimentin except when the microtubule network was disrupted with nocodazole. Oxysterols had no effect on the localization of either ORP4. Cells overexpressing ORP4-S had a 40% reduction in the esterification of low-density-lipoprotein-derived cholesterol, demonstrating that ORP4 interaction with intermediate filaments inhibits an intracellular cholesterol-transport pathway mediated by vimentin. These studies elucidate a hitherto unknown relationship between OSBPs and the intermediate filament network that influences cholesterol transport.
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Project description:Integrins are components of cell-matrix adhesions, and function as scaffolds for various signal transduction pathways. So far no lipid ligand for integrin has been reported. Here we show that a lipid, oxysterol 25-hydroxycholesterol (25HC), directly binds to α5β1 and αvβ3 integrins to activate integrin-focal adhesion kinase (FAK) signaling. Treatment of macrophages and epithelial cells with 25HC results in an increase in activated αvβ3 integrin in podosome and focal adhesion matrix adhesion sites. Moreover, activation of pattern recognition receptor on macrophages induces secretion of 25HC, triggering integrin signaling and the production of proinflammatory cytokines such as TNF and IL-6. Thus, the lipid molecule 25HC is a physiologically relevant activator of integrins and is involved in positively regulating proinflammatory responses. Our data suggest that extracellular 25HC links innate immune inflammatory response with integrin signaling.
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Project description:Osteoarthritis (OA) alters chondrocyte metabolism and mitochondrial biology. We explored whether OA and non-OA chondrocytes show persistent differences in metabolism and mitochondrial function and different responsiveness to cytokines and cAMP modulators. Hip chondrocytes from patients with OA or femoral neck fracture (non-OA) were stimulated with IL-1β, TNF, forskolin and opioid peptides. Mediators released from chondrocytes were measured, and mitochondrial functions and glycolysis were determined (Seahorse Analyzer). Unstimulated OA chondrocytes exhibited significantly higher release of IL-6, PGE2 and MMP1 and lower production of glycosaminoglycan than non-OA chondrocytes. Oxygen consumption rates (OCR) and mitochondrial ATP production were comparable in unstimulated non-OA and OA chondrocytes, although the non-mitochondrial OCR was higher in OA chondrocytes. Compared to OA chondrocytes, non-OA chondrocytes showed stronger responses to IL-1β/TNF stimulation, consisting of a larger decrease in mitochondrial ATP production and larger increases in non-mitochondrial OCR and NO production. Enhancement of cAMP by forskolin prevented IL-1β-induced mitochondrial dysfunction in OA chondrocytes but not in non-OA chondrocytes. Endogenous opioids, present in OA joints, influenced neither cytokine-induced mitochondrial dysfunction nor NO upregulation. Glycolysis was not different in non-OA and OA chondrocytes, independent of stimulation. OA induces persistent metabolic alterations, but the results suggest upregulation of cellular mechanisms protecting mitochondrial function in OA.
| S-EPMC9220245 | biostudies-literature
Project description:An excess of cholesterol and/or oxysterols induces apoptosis in macrophages, contributing to the development of advanced atherosclerotic lesions. In foam cells, these sterols are stored in esterified forms, which are hydrolyzed by two enzymes: neutral cholesterol ester hydrolase 1 (Nceh1) and hormone-sensitive lipase (Lipe). A deficiency in either enzyme leads to accelerated growth of atherosclerotic lesions in mice. However, it is poorly understood how the esterification and hydrolysis of sterols are linked to apoptosis. Remarkably, Nceh1-deficient thioglycollate-elicited peritoneal macrophages (TGEMs), but not Lipe-deficient TGEMs, were more susceptible to apoptosis induced by oxysterols, particularly 25-hydroxycholesterol (25-HC), and incubation with 25-HC caused massive accumulation of 25-HC ester in the endoplasmic reticulum (ER) due to its defective hydrolysis, thereby activating ER stress signaling such as induction of CCAAT/enhancer-binding protein-homologous protein (CHOP). These changes were nearly reversed by inhibition of ACAT1. In conclusion, deficiency of Nceh1 augments 25-HC-induced ER stress and subsequent apoptosis in TGEMs. In addition to reducing the cholesteryl ester content of foam cells, Nceh1 may protect against the pro-apoptotic effect of oxysterols and modulate the development of atherosclerosis.
| S-EPMC4174001 | biostudies-literature