Biosynthesis of the regulatory oxysterol, 5-cholesten-3beta,25-diol 3-sulfate, in hepatocytes

Cellular cholesterol homeostasis is maintained through coordinated regulation of cholesterol synthesis, degradation, and secretion. Nuclear receptors for oxygenated cholesterol derivatives (oxysterols) are known to play key roles in the regulation of cholesterol homeostasis. We recently identified a sulfated oxysterol, 5-cholesten-3beta,25-diol 3-sulfate (25HC3S), that is localized to liver nuclei. The present study reports a biosynthetic pathway for 25HC3S in hepatocytes. Assays using mitochondria isolated from rats and sterol 27-hydroxylase (Cyp27A1) gene knockout mice indicated that 25-hydroxycholesterol (25HC) is synthesized by CYP27A1. Incubation of cholesterol or 25HC with mitochondrial and cytosolic fractions in the presence of 3'-phosphoadenosyl 5'-phosphosulfate resulted in the synthesis of 25HC3S. Real-time RT-PCR and Western blot analysis showed the presence of insulin-regulated hydroxycholesterol sulfotransferase 2B1b (SULT2B1b) in hepatocytes. 25HC3S, but not 25HC, decreased SULT2B1b mRNA and protein levels. Specific small interfering RNA decreased SULT2B1b mRNA, protein, and activity levels. These findings demonstrate that mitochondria synthesize 25HC, which is subsequently 3beta-sulfated to form 25HC3S.     

Cholesterol 25-hydroxylation activity of CYP3A

To date, many studies have been conducted using 25-hydroxycholesterol, which is a potent regulator of lipid metabolism. However, the origins of this oxysterol have not been entirely elucidated. Cholesterol 25-hydroxylase is one of the enzymes responsible for the metabolism of 25-hydroxycholesterol, but the expression of this enzyme is very low in humans. This oxysterol is also synthesized by sterol 27-hydroxylase (CYP27A1) and cholesterol 24-hydroxylase(CYP46A1), but it is only a minor product of these enzymes. We now report that CYP3A synthesizes a significant amount of 25-hydroxycholesterol and may participate in the regulation of lipid metabolism. Induction of CYP3A by pregnenolone-16α-carbonitrile caused the accumulation of 25-hydroxycholesterol in a cell line derived from mouse liver. Furthermore, treatment of the cells with troleandomycin, a specific inhibitor of CYP3A, significantly reduced cellular 25-hydroxycholesterol concentrations. In cells that overexpressed human recombinant CYP3A4, the activity of cholesterol 25-hydroxylation was found to be higher than that of cholesterol 4β-hydroxylation, a known marker activity of CYP3A4. In addition, 25-hydroxycholesterol concentrations in normal human sera correlated positively with the levels of 4β-hydroxycholesterol (r = 0.650, P < 0.0001, n = 78), but did not significantly correlate with the levels of 27-hydroxycholesterol or 24S-hydroxycholesterol. These results demonstrate the significance of CYP3A on the production of 25-hydroxycholesterol.     

Marked accumulation of 27-hydroxycholesterol in SPG5 patients with hereditary spastic paresis

Patients with a recessively inherited “pure” hereditary spastic paresis (SPG5) have mutations in the gene coding for the oxysterol 7 α hydroxylase (CYP7B1). One of the expected metabolic consequences of such mutations is accumulation of oxysterol substrates due to decreased enzyme activity. In accordance with this, we demonstrate here that four patients with the SPG5 disease have 6- to 9-fold increased plasma levels of 27-hydroxycholesterol. A much higher increase, 30- to 50-fold, was found in cerebrospinal fluid. The plasma levels of 25-hydroxycholesterol were increased about 100-fold. There were no measurable levels of this oxysterol in cerebrospinal fluid. The pattern of bile acids in serum was normal, suggesting a normal bile acid synthesis. The findings are discussed in relation to two transgenic mouse models with increased levels of 27-hydroxy cholesterol in the circulation but without neurological symptoms: the cyp27a1 transgenic mouse and the cyp7b1 knockout mouse. The absolute plasma levels of 27-hydroxycholesterol in the latter models are, however, only about 20% of those in the SPG5 patients. If the accumulation of 27-hydroxycholesterol is an important pathogenetic factor, a reduction of its levels may reduce or prevent the neurological symptoms. A possible strategy to achieve this is discussed.     

Modulation of Antiviral Immunity and Therapeutic Efficacy by 25-Hydroxycholesterol in Chronically SIV-Infected,ART-Treated Rhesus Macaques

Virologica Sinica - Cholesterol-25-hydroxylase (CH25H) and its enzymatic product 25-hydroxycholesterol (25HC) exert broadly antiviral activity including inhibiting HIV-1 infection. However, their...     

Synthesis of sterols oxygenated in the terminal fragment of their side chains

Methods of stereoselective synthesis of oxysterols are considered by the examples of (25R)-26-hydroxycholesterol, (24S)-24,25-epoxycholesterol, and (24S)-24-hydroxycholesterol containing functional groups in the terminal fragments of their side chain. Special attention is paid to the problems of construction of chiral centers C24 and C25.     

Direct control by calcium of 25-hydroxycholecalciferol-1-hydroxylase activity in chick kidney mitochondria

Kidney mitochondria were isolated from rachitic chicks and their activity in the metabolism of 25-OH-D₃ was studied in relation to the amount of calcium added $\text{in vitro}$. The addition of 0.050.2 mM calcium to a mitochondrial suspension caused a marked and dose-related stimulation of 1-hydroxylation. A sharp decline in the activity was induced by higher concentrations (0.3-0.5 mM) of calcium. The rate of 24-hydroxylation was not influenced by calcium. In these effects, calcium was relatively specific among various divalent cations. These data strongly suggest that calcium is directly involved in the regulation of the vitamin D activation in kidney mitochondria.     

Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS

We describe a highly sensitive and specific method for the quantification of key regulatory oxysterols in biological samples. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After alkaline hydrolysis of human serum (5 microl) or rat liver microsomes (1 mg protein), oxysterols were extracted, derivatized into picolinyl esters, and analyzed by LC-MS/MS using the electrospray ionization mode. The detection limits of the picolinyl esters of 4beta-hydroxycholesterol, 7alpha-hydroxycholesterol, 22R-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and 24S,25-epoxycholesterol were 2-10 fg (5-25 amol) on-column (signal-to-noise ratio = 3). Reproducibilities and recoveries of these oxysterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.8% to 12.7% and 2.9% to 11.9%, respectively. The recovery experiments were performed using rat liver microsomes spiked with 0.05 ng to 12 ng of oxysterols, and recoveries of the oxysterols ranged from 86.7% to 107.3%, with a mean recovery of 100.6%. This method provides reproducible and reliable results for the quantification of oxysterols in small amounts of biological samples.     

Oxysterols in the brain of the cholesterol 24-hydroxylase knockout mouse

Oxysterols are oxidised forms of cholesterol or its precursors. In this study we utilised the cholesterol 24-hydroxylase knockout mouse (Cyp46a1−/−) to study the sterol and oxysterol content of brain. Despite a great reduction in the abundance of 24S-hydroxycholesterol, the dominant metabolite of cholesterol in wild type brain, no other cholesterol metabolite was found to quantitatively replace this oxysterol in the Cyp46a1−/− mouse. Only minor amounts of other side-chain oxysterols including 22R-, 24R-, 25- and (25R),26-hydroxycholesterols were detected. In line with earlier studies, levels of cholesterol were similar in Cyp46a1−/− and wild type animals. However, the level of the cholesterol precursor, desomsterol, and its parallel metabolite formed via a shut of the mevalonate pathway, 24S,25-epoxycholesterol, were reduced in the Cyp46a1−/− mouse. The reduction in abundance of 24S,25-epoxycholesterol is interesting in light of a recent report indicating that this oxysterol promotes dopaminergic neurogenesis.     

Synthesis of deuterated isotopomers of 7alpha- and (25R,S)-26-hydroxycholesterol, internal standards for in vivo determination of the two biosynthetic pathways of bile acids

Deuterated isotopomers of 7alpha- and (25R,S)-26-hydroxycholesterol, internal standards for in vivo determination of the two biosynthetic pathways of bile acids formation from cholesterol, were prepared from [2,2,3,4,4,6-2H(6)]-cholesterol and (20S)-[7,7,21,21-2H(4)]-3beta-(tert-butyldimethylsilyl)oxy-20-methylpregna-5-en-21-ol, respectively.     

Acetoacetyl-CoA ligase activity in the isolated rat hepatocyte: Effects of 25-hydroxycholesterol and high density lipoprotein

The activity of acetoacetyl-CoA (AcAc-CoA) ligase (E.C.6.2.1.16) in hepatocytes from rats was shown to be the same as the activity in homogenates of their livers. In hepatocytes treated with 25-hydroxycholesterol, AcAc-CoA ligase, 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase and rates of sterol synthesis were substantially decreased. Hepatocytes treated with high density lipoprotein (HDL) exhibited a 2 to 4 fold induction of HMG-CoA reductase activity; however an accompanying increase in AcAc-CoA ligase activity and the rate of cholesterol synthesis was not observed. We conclude (a) that increases in the activity of HMG-CoA reductase when mediated by HDL in hepatocytes do not result in a corresponding change in the capacity for sterol synthesis and (b) that changes in the activity state of HMG-CoA reductase can be dissociated from that of AcAc-CoA ligase.     

Transcriptional regulation of cholesterol 24-hydroxylase by histone deacetylase inhibitors

The mechanistic basis for the tissue specific expression of cholesterol elimination pathways is poorly understood. To gain additional insight into this phenomenon we considered it of interest to investigate if epigenetic mechanisms are involved in the regulation of the brain-specific enzyme cholesterol 24-hydroxylase (CYP46A1), a key regulator of brain cholesterol elimination. We demonstrated a marked time-dependent derepression of the expression of CYP46A1, in response to treatment with the potent histone deacetylase (HDAC) inhibitor Trichostatin A. The pattern of expression of the genes in the genomic region surrounding CYP46A1 was found to be diametrically opposite in brain and liver. Intraperitoneal injection of HDAC inhibitors in mice led to a significant derepression of hepatic Cyp46a1 mRNA expression and tissue specific changes in Hmgcr and Cyp39a1 mRNA expression. These results are discussed in the context of the phenomenology of tissue specific cholesterol balance.     

TBC1D25 alleviates nonalcoholic steatohepatitis by inhibiting abnormal lipid accumulation and inflammation

Nonalcoholic fatty liver disease (NAFLD) is a strong stimulant of cardiovascular diseases, affecting one-quarter of the world's population. TBC1 domain family member 25 (TBC1D25) regulates the development of myocardial hypertrophy and cerebral ischemia–reperfusion injury; however, its effect on NAFLD/nonalcoholic steatohepatitis (NASH) has not been reported. In this study, we demonstrated that TBC1D25 expression is upregulated in NASH. TBC1D25 deficiency aggravated hepatic steatosis, inflammation, and fibrosis in NASH. In vitro tests revealed that TBC1D25 overexpression restrained NASH responses. Subsequent mechanistic validation experiments demonstrated that TBC1D25 interfered with NASH progression by inhibiting abnormal lipid accumulation and inflammation. TBC1D25 deficiency significantly promoted NASH occurrence and development. Therefore, TBC1D25 may potentially be used as a clinical therapeutic target for NASH treatment.     

Nicotine induces the expression of C-reactive protein via MAPK-dependent signal pathway in U937 macrophages

Atherosclerosis is an inflammatory disease in the vessel wall. Nicotine, a major component of cigarette smoke, is an independent risk factor for cardiovascular diseases including atherosclerosis. As an inflammatory molecule, C- reactive protein (CRP) participates in atherogenesis. Although it has been confirmed that CRP level in smoking patient is significantly higher than non-smokers and cigarette withdrawal, it is unknown whether nicotine induces CRP expression in macrophages. The present study was to observe effect of nicotine on CRP production and the related signal pathway in U937 macrophages. The results showed that nicotine significantly increased mRNA and protein expression of CRP in U937 macrophages in time- and concentration-dependent ways. Nicotinic acetylcholine receptor (nAChR) blocker hexamethonium, MEK1/2 inhibitor PD98059, p38 MAPK inhibitor SB203580 and NF-κB inhibitor PDTC almost completely abolished nicotineinduced CRP expression in mRNA and protein levels in U937 macrophages. The further study indicated that hexamethonium, PD98059, and SB203580 significantly inhibited ERK1/2 and p38 MAPK phosphorylation. These demonstrate that nicotine has ability to induce CRP expression in macrophages through nAChR-ERK1/2/p38 MAPK-NF-κB signal pathway, which contributes to better understanding of the pro-inflammatory and pro-atherosclerotic effects of nicotine in cigarette smokers.     

Effect of cytokines and lipopolysaccharide on CD14 antigen expression in human monocytes and macrophages.

The 52 kD myeloid membrane glycoprotein CD14 represents the receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein (LBP); it is involved in LPS induced tumor necrosis factor-alpha production. Expression of CD14 increases in monocytes differentiating into macrophages, and it is reduced by rIFNg in monocytes in vitro. In the present study CD14 membrane antigen expression was investigated in cultures of human mononuclear leucocytes (PBL), in elutriated, purified monocytes, and in blood monocyte derived Teflon cultured macrophages. Cells were incubated for 15 or 45 h with rIL-1, rIL-2, rIL-3, rIL-5, rIL-6, rTNFa, rGM-CSF, rM-CSF, rTGFb1, rIFNa, lipopolysaccharide (LPS), and, as a control, rIFNg. The monoclonal antibodies Leu-M3 and MEM 18 were used for labelling of CD14 antigen by indirect immunofluorescence and FACS analysis of scatter gated monocytes or macrophages. IFNg concentrations were determined in PBL culture supernatants by ELISA. rIFNa and rIL-2 reduced CD14 in 15 and 45 h PBL cultures, an effect mediated by endogenous IFNg, since it was abolished by simultaneous addition of an anti-IFNg antibody. rIFNa and rIL-2 were ineffective in purified monocytes or macrophages. rIL-4 strongly reduced CD14 in PBL and purified monocytes after 45 h, whereas in macrophages the decrease was weak, although measurable after 15 h. The other cytokines investigated did not change CD14 antigen expression. Cycloheximide alone reduced CD14, but when added in combination with rIFNg the effect on CD14 downregulation was more pronounced. The effect of rIFNg on CD14 in PBL cultures was dose-dependently inhibited by rIL-4 and this inhibition is probably due to an IL-4 mediated blockade of IFNg secretion. LPS at a low dose increased CD14, at a high dose it produced a variable decrease of CD14 in PBL, which was probably due to LPS induced IFNg secretion. LPS strongly enhanced CD14 in 45 h cultures of purified monocytes. The results, showing that CD14 antigen expression is upregulated by LPS and downregulated by rIFNg and rIL-4, suggest that the LPS-LBP receptor is involved in the feedback response of IFNg and IL-4 to LPS stimulation.     

Altered expression of microRNA-365 is related to the occurrence and development of non-small-cell lung cancer by inhibiting TRIM25 expression

The purpose of this current study is to elucidate whether altered microRNA-365 (miR-365) has an association with the initiation and development of non-small-cell lung cancer (NSCLC) by targeting TRIM25 expression. The expression of miR-365 and TRIM25 in NSCLC tissues, adjacent normal tissues, and NSCLC cell lines were detected. The relationship between miR-365 expression and TRIM25 with the clinicopathological characteristics of NSCLC was analyzed. The putative binding site between miR-365 and TRIM25 was determined by luciferase activity assay. miR-365 inhibitors and miR-365 mimics were transfected to human NSCLC A549 cells, and the cell viability was detected by cell counting kit-8 assay; flow cytometry was carried out to determine cell cycle and apoptosis rate. Poorly expressed miR-365 and overexpressed TRIM25 was found in NSCLC tissues. TRIM25 was determined as a target gene of miR-365. The miR-365 and TRIM25 expression were related to the clinicopathological features of NSCLC, such as pathological classification, differentiation degree, TNM stage as well as lymph node metastasis. miR-365 suppressed the expression of TRIM25 and elevated the expression of the proapoptotic protein in NSCLC cells. Our study demonstrates that altered expression of miR-365 has a close association with the occurrence and development of NSCLC by inhibiting TRIM25 expression.