Faculty Bibliography

Oxysterols constitute a class of cholesterol derivatives that exhibit broad biological effects ranging from cytotoxicity to regulation of nuclear receptors. The role of oxysterols such as 7-ketocholesterol (7-KC) in the development of retinal macular degeneration and atheromatous lesions is of particular interest, but little is known of their metabolic fate. We establish that the steroid/sterol sulfotransferase SULT2B1b, known to efficiently sulfonate cholesterol, also effectively sulfonates a variety of oxysterols, including 7-KC. The cytotoxic effect of 7-KC on 293T cells was attenuated when these cells, which do not express SULT2B1b, were transfected with SULT2B1b cDNA. Importantly, protection from 7-KC-induced loss of cell viability with transfection correlated with the synthesis of SULT2B1b protein and the production of the 7-KC sulfoconjugate (7-KCS). Moreover, when 7-KCS was added to the culture medium of 293T cells in amounts equimolar to 7-KC, no loss of cell viability occurred. Additionally, MCF-7 cells, which highly express SULT2B1b, were significantly more resistant to the cytotoxic effect of 7-KC. We extended the range of oxysterol substrates for SULT2B1b to include 7alpha/7beta-hydroxycholesterol and 5alpha,6alpha/5beta,6beta-epoxycholesterol as well as the 7alpha-hydroperoxide derivative of cholesterol. Thus, SULT2B1b, by acting on a variety of oxysterols, offers a potential pathway for modulating in vivo the injurious effects of these compounds

AIMS: To determine sensitivity and specificity of urine bile acid sulfate (UBAS) and non-sulfated urine bile acid (UBA) assays for detection of intrahepatic cholestasis of pregnancy (ICP). METHODS: Urine was collected prospectively from healthy and pruritic patients evaluated for ICP. Results were expressed as a ratio to urinary creatinine. RESULTS: Of 20 patients evaluated for ICP, 14 had confirmed ICP by serum testing and six had pruritus only. UBAS results were higher with ICP (P<0.001) and increased with gestational age in healthy controls. Adjusted for gestational age, the multiples of the median (MoM) were still higher (2.64+/-1.11, 1.05+/-0.54, P<0.001). The sensitivity was 100% and specificity 83% at 1.5 MoM in pruritic patients. UBA results were higher with ICP (23.0+/-9.8, 12.8+/-7.4, P<0.001), with sensitivity of 100% and specificity of 50% at 10.2 micromol/g cretinine. CONCLUSION: Urinary bile acids can detect or exclude ICP with serum abnormalities. Urine bile acid sulfates have higher specificity than non-sulfated urine bile acids at equivalent sensitivities, but co-detection of progestin sulfates is suspected

Sterol 27-hydroxylase (CYP27A1) is a mitochondrial P-450 enzyme with broad substrate specificity for C27 sterols including 7-ketocholesterol (7kCh). CYP27A1 is widely expressed in human tissues but has not been previously demonstrated in the retina. In this study, we examined the expression and localization of CYP27A1 in the monkey retina where it localized mainly to the photoreceptor inner segments. CYP27A1 was also observed in Muller cells with faint immuno staining detected in the RPE and choriocapillaris. We also determined that the 27-hydroxylation of 7-ketocholesterol (27OH7kCh) rendered it non-toxic to cultured RPE cells. Moreover, the 27OH7kCh when mixed with 7-ketocholesterol significantly reduced the toxicity of 7-ketocholesterol. These data, when taken in context of the known functions of CYP27A1 imply that expression in the retina serves to modify the biological activity of oxidized sterols that are either transported or generated locally by photo-oxidation

Phosphorylation of translation initiation factor 2alpha (eIF2alpha) coordinates a translational and transcriptional program known as the integrated stress response (ISR), which adapts cells to endoplasmic reticulum (ER) stress. A screen for small molecule activators of the ISR identified two related compounds that also activated sterol-regulated genes by blocking cholesterol biosynthesis at the level of CYP51. Ketoconazole, a known CYP51 inhibitor, had similar effects, establishing that perturbed flux of precursors to cholesterol activates the ISR. Surprisingly, compound-mediated activation of sterol-regulated genes was enhanced in cells with an ISR-blocking mutation in the regulatory phosphorylation site of eIF2alpha. Furthermore, induction of the ISR by an artificial drug-activated eIF2alpha kinase reduced the level of active sterol regulatory element binding protein (SREBP) and sterol-regulated mRNAs. These findings suggest a mechanism by which interactions between sterol metabolism, the ISR, and the SREBP pathway affect lipid metabolism during ER stress

BACKGROUND: Bile acid synthesis accounts for more than 95% of total cholesterol catabolism per day. We have developed a minimally invasive technique in humans that quantifies the rates of plasma appearance of 7alpha- and 27-hydroxycholesterol, representing the first steps of the 'classical' and 'alternative' pathways of bile acid synthesis, respectively. METHODS: For this purpose, during the intravenous infusion of synthetic deuterated isotopomers of 7alpha-hydroxycholesterol and 27-hydroxycholesterol plasma samples are collected and analysed by a GC-MS based method that allows to quantify the exogenous/natural isotopomer ratio of the two sterols. From this data, the rates of plasma appearance of 7alpha- and 27-hydroxycholesterol are calculated. RESULTS: In a group of healthy individuals steady state kinetics are obtained during a 2 h period yielding mean values of 2.0+/-0.8 and 3.7+/-0.6 mg/h for 7alpha- and 27-hydroxycholesterol, respectively. The data are consistent with findings using older techniques that require studies over several days. CONCLUSION: Considering that at steady state of the exogenous/natural isotopomer ratio the plasma appearance of the two regulatory hydroxysterols are related to the rate of bile acid synthesis via the 'classical' and the 'alternative' pathways, respectively, the proposed method could be used to evaluate the immediate effects of different diets and drugs and other determinants on cholesterol catabolism

Feedback control of cholesterol synthesis is mediated in part by sterol-induced binding of HMG CoA reductase to Insig proteins in the endoplasmic reticulum (ER). Binding leads to ubiquitination and proteasomal degradation of reductase, a rate-controlling enzyme in cholesterol synthesis. Using in vitro and in vivo assays, we show that lanosterol, the first sterol intermediate in cholesterol synthesis, potently stimulates ubiquitination of reductase, whereas cholesterol has no effect at 10-fold higher concentrations. Lanosterol is not effective in mediating the other action of Insigs, namely to promote ER retention of SCAP-SREBP complexes, a reaction that is mediated directly by cholesterol. A pair of methyl groups located in the C4 position of lanosterol confers this differential response. These data indicate that buildup of cholesterol synthesis intermediates represses the pathway selectively at reductase and reveal a previously unappreciated link between feedback inhibition of reductase and carbon flow through the cholesterol synthetic pathway

Oxysteroids are a new classification for sterol intermediates in cholesterol synthesis that undergo enzyme-catalyzed stereo-specific 25R,26-hydroxylation and thus bypass cholesterol as the expected end-product. Recently, they were identified in micromolar amounts in the plasma of patients with Smith-Lemli-Opitz syndrome (SLOS). An additional three oxysteroids, the 25,26-hydroxy derivatives of lanosterol, zymosterol, and desmosterol, respectively, were generated in vitro by CYP27A1-transfected bacteria. As there are 19 steps between cholesterol and lanosterol, the first post-squalene sterol, a potentially large class of oxysteroids exists. Limited studies of 25r,26-7-dehydrocholesterol indicate a traditional role as a ligand for nuclear receptors, but complete evaluation of oxysteroids for novel biologic activities is lacking. Currently, the lack of authentic oxysteroid standards limits both their detection in biologic fluids and evaluation of their biologic effects

BACKGROUND: Cholesterol sulfate, the most important sterol sulfate in the human circulation, has emerged as a multifaceted molecule. Among its many demonstrated regulatory actions is its ability to influence blood clotting and fibrinolysis. Additionally, cholesterol sulfate is a constituent of human platelets, where it has been shown to support platelet aggregation. METHODS AND RESULTS: We have documented the presence of the enzyme (SULT2B1b) that sulfonates cholesterol in human platelets and examined the influence of plasma lipoproteins on the expression and activity of this enzyme. SULT2B1b mRNA was detected by reverse transcription-polymerase chain reaction and found to be the only steroid/sterol sulfotransferase expressed in these discoid anucleate particles. Using real-time polymerase chain reaction for quantification, we found that the level of SULT2B1b mRNA in platelets was maintained at 4 degrees C but substantially diminished over a period of 4 hours at 37 degrees C. The loss of SULT2B1b mRNA, however, was markedly reduced in the presence of HDL but not LDL. The stabilizing influence of HDL was attributable specifically to its apolipoprotein (apo) A-I component, whereas apoA-II and apoE were without effect. Importantly, there was a direct correlation between platelet SULT2B1b mRNA and protein levels in the presence or absence of lipoprotein that was reflected in enzymatic activity and cholesterol sulfate production. CONCLUSIONS: Human platelets selectively express SULT2B1b, the physiological cholesterol sulfotransferase. Furthermore, the stability of SULT2B1b mRNA and protein in platelets maintained at 37 degrees C is subject to regulation by the apoA-I component of HDL

A major biologic role of the ubiquitous mitochondrial P450 enzyme CYP27A1 is the generation of ligands such as 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid, which regulate the expression of nuclear receptors that govern many aspects of cholesterol homeostasis. We now report that sterol intermediates in cholesterol synthesis, beginning with the initial post-cyclization sterol, lanosterol, continuing with zymosterol, and ending with desmosterol are also substrates for the enzyme. Using the human enzyme expressed in Escherichia coli, we characterized the retention times and major mass fragments of these novel metabolites. Although sequestration of the enzyme in the inner mitochondrial membrane and normal subcellular organization probably greatly restrict the proportion of these and other intermediates in cholesterol synthesis that undergo side chain oxidation, disruption of compartmentalization can bypass cholesterol as the end product and give rise to potent ligands that further modify gene expression