MicroRNA-377 Inhibits Atherosclerosis by Regulating Triglyceride Metabolism Through the DNA Methyltransferase 1 in Apolipoprotein E-Knockout Mice.

BACKGROUND: Lipoprotein lipase (LPL) plays an important role in triglyceride metabolism. It is translocated across endothelial cells to reach the luminal surface of capillaries by glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1), where it hydrolyzes triglycerides in lipoproteins. MicroRNA 377 (miR-377) is highly associated with lipid levels. However, how miR-377 regulates triglyceride metabolism and whether it is involved in the development of atherosclerosis remain largely unexplored. Methods and Results: The clinical examination displayed that miR-377 expression was markedly lower in plasma from patients with hypertriglyceridemia compared with non-hypertriglyceridemic subjects. Bioinformatics analyses and a luciferase reporter assay showed that DNA methyltransferase 1 (DNMT1) was a target gene of miR-377. Moreover, miR-377 increased LPL binding to GPIHBP1 by directly targeting DNMT1 in human umbilical vein endothelial cells (HUVECs) and apolipoprotein E (ApoE)-knockout (KO) mice aorta endothelial cells (MAECs). In vivo, hematoxylin-eosin (H&E), Oil Red O and Masson's trichrome staining showed that ApoE-KO mice treated with miR-377 developed less atherosclerotic plaques, accompanied by reduced plasma triglyceride levels. CONCLUSIONS: It is concluded that miR-377 upregulates GPIHBP1 expression, increases the LPL binding to GPIHBP1, and reduces plasma triglyceride levels, likely through targeting DNMT1, inhibiting atherosclerosis in ApoE-KO mice.

Endothelial cells respond to hyperglycemia by increasing the LPL transporter GPIHBP1.

In diabetes, when glucose uptake and oxidation are impaired, the heart is compelled to use fatty acid (FA) almost exclusively for ATP. The vascular content of lipoprotein lipase (LPL), the rate-limiting enzyme that determines circulating triglyceride clearance, is largely responsible for this FA delivery and increases following diabetes. Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein [GPIHBP1; a protein expressed abundantly in the heart in endothelial cells (EC)] collects LPL from the interstitial space and transfers it across ECs onto the luminal binding sites of these cells, where the enzyme is functional. We tested whether ECs respond to hyperglycemia by increasing GPIHBP1. Streptozotocin diabetes increased cardiac LPL activity and GPIHBP1 gene and protein expression. The increased LPL and GPIHBP1 were located at the capillary lumen. In vitro, passaging EC caused a loss of GPIHBP1, which could be induced on exposure to increasing concentrations of glucose. The high-glucose-induced GPIHBP1 increased LPL shuttling across EC monolayers. GPIHBP1 expression was linked to the EC content of heparanase. Moreover, active heparanase increased GPIHBP1 gene and protein expression. Both ECs and myocyte heparan sulfate proteoglycan-bound platelet-derived growth factor (PDGF) released by heparanase caused augmentation of GPIHBP1. Overall, our data suggest that this protein "ensemble" (heparanase-PDGF-GPIHBP1) cooperates in the diabetic heart to regulate FA delivery and utilization by the cardiomyocytes. Interrupting this axis may be a novel therapeutic strategy to restore metabolic equilibrium, curb lipotoxicity, and help prevent or delay heart dysfunction that is characteristic of diabetes.

Interaction of RAS activation and lipid disorders accelerates the progression of glomerulosclerosis.

BACKGROUND: The activation of the renin-angiotensin system (RAS) and lipid disorders are major risk factors in progressive chronic kidney disease. This study aimed to investigate the potential synergistic mechanisms of RAS activation and lipid disorders that contribute to glomerulosclerosis. MATERIALS AND METHODS: Human renal mesangial cells (HMCs) were treated with 10(-7) mol/L angiotensin II (Ang II) or with 30 µg/ml cholesterol and 1 µg/ml 25-hydroxycholesterol (lipid loading) for 24 hours. Lipid accumulation in the cells was evaluated by Oil Red O staining and intracellular cholesterol quantitative assays. The gene and protein expression of molecules in the low-density lipoprotein receptor (LDLr) pathway, the RAS family, and the extracellular matrix were examined by real-time polymerase chain reaction and Western blotting. The translocation of sterol regulatory element-binding protein (SREBP) cleavage activating protein (SCAP), which escorts SREBP-2 from the endoplasmic reticulum (ER) to the Golgi, was examined by immunofluorescent staining. RESULTS: Ang II increased lipid droplet accumulation in HMCs. Further analysis revealed that Ang II increased the mRNA and protein expression of LDLr, SCAP, and SREBP-2. This increase was correlated with an enhanced translocation of the SCAP/SREBP-2 complex from the ER to the Golgi in HMCs that was induced by Ang II, thereby activating LDLr gene transcription. Interestingly, lipid loading increased the mRNA and protein expression of angiotensinogen, Ang II, renin, angiotensin-converting enzyme, angiotensin II type 1 receptor, and type 2 receptor in HMCs with increased mRNA and protein expression of collagen I, α-smooth muscle actin, and fibronectin. CONCLUSIONS: This study demonstrates that the interaction of RAS activation and lipid disorders accelerates the progression of glomerulosclerosis.

Reduced kidney lipoprotein lipase and renal tubule triglyceride accumulation in cisplatin-mediated acute kidney injury.

Peroxisome proliferator-activated receptor-α (PPARα) activation attenuates cisplatin (CP)-mediated acute kidney injury by increasing fatty acid oxidation, but mechanisms leading to reduced renal triglyceride (TG) accumulation could also contribute. Here, we investigated the effects of PPARα and CP on expression and enzyme activity of kidney lipoprotein lipase (LPL) as well as on expression of angiopoietin protein-like 4 (Angptl4), glycosylphosphatidylinositol-anchored-HDL-binding protein (GPIHBP1), and lipase maturation factor 1 (Lmf1), which are recognized as important proteins that modulate LPL activity. CP caused a 40% reduction in epididymal white adipose tissue (WAT) mass, with a reduction of LPL expression and activity. CP also reduced kidney LPL expression and activity. Angptl4 mRNA levels were increased by ninefold in liver and kidney tissue and by twofold in adipose tissue of CP-treated mice. Western blots of two-dimensional gel electrophoresis identified increased expression of a neutral pI Angptl4 protein in kidney tissue of CP-treated mice. Immunolocalization studies showed reduced staining of LPL and increased staining of Angptl4 primarily in proximal tubules of CP-treated mice. CP also increased TG accumulation in kidney tissue, which was ameliorated by PPARα ligand. In summary, a PPARα ligand ameliorates CP-mediated nephrotoxicity by increasing LPL activity via increased expression of GPHBP1 and Lmf1 and by reducing expression of Angptl4 protein in the proximal tubule.

Rat small intestine expresses the reelin-Disabled-1 signalling pathway.

Expression of reelin, reelin receptors [apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (VldlR)] and the Disabled-1 (Dab1) protein was investigated in rat intestinal mucosa. Intestinal reelin and Dab1 mRNA levels were maximal in the early stages of life, reaching adult levels in 1-month-old rats. Expression of reelin mRNA was restricted to fibroblasts, whereas mRNAs of Dab1, ApoER2, VldlR and integrins alpha3 and beta1 were observed in enterocytes, crypts and fibroblasts. Reelin protein was only observed in isolated intestinal fibroblasts and in a cell layer subjacent to the villus epithelium, which seems to be composed of myofibroblasts because it also reacted to alpha-smooth muscle actin. The Disabled-1 and VldlR protein signals were detected in the crypt and villus cells, and they were particularly abundant in the terminal web domain of the enterocytes. The ApoER2 protein signal was detected in the upper half of the villi but not in the crypts. This is the first report showing that rat intestinal mucosa expresses the reelin-Disabled-1 signalling system.

Apolipoprotein-D expression is increased during development and maturation of the human prefrontal cortex.

Apolipoprotein D (apoD) is a lipid binding protein expressed in the brain where its function is largely unknown. Based on changes in lipid metabolism and deposition that occur in the human brain during postnatal development, we investigated changes in apoD expression in the prefrontal cortex in 69 normal cases ranging in age from 40 days to 49 years utilizing gene microarray, quantitative PCR and western blotting methods. In contrast to the high expression of apolipoprotein E (APOE), low-density lipoprotein receptor-related protein 8 (LRP8) and 3-hydroxy-3-methyl-glutaryl-CoA reducatase (HMGCR) (genes that play a role in lipid-related pathways in brain development) early in life, apoD expression was low in neonates and increased in expression throughout life resulting in six- to eight-fold higher levels at the mRNA and protein levels in adults. Recent studies suggest that apoD has a novel antioxidant function in the brain and we found that the increased apoD expression throughout development and into adulthood was correlated with the expression of antioxidant genes superoxide dismutase 1 (SOD1) and glutathione peroxidase 3 (GPX3) as well as proteins that were modified by the lipid peroxidation end-product 4-hydroxynonenal. These studies reveal that apoD expression is increased throughout life in the human prefrontal cortex and that this is correlated with genetic and biochemical markers of oxidative stress.

Selenium requirements are higher for glutathione peroxidase-1 mRNA than gpx1 activity in rat testis.

Selenium (Se) plays a critical role in testis, sperm, and reproduction, and testis Se levels are remarkably maintained in Se deficiency. In most other tissues, Se levels decrease dramatically as do levels of most selenoproteins and levels of a subset of Se-regulated selenoprotein mRNAs. Because of the recent identification of key molecules in the targeted trafficking of Se to the testis, we examined the hierarchy of Se regulation in testis by determining the dietary Se regulation of the full testis selenoproteome in rats fed graded levels of Se (0 to 0.8 microg Se/g) as Na2SeO3 for 28 d. Se status did not significantly affect testis weight or glutathione peroxidase 4 (Gpx4) activity (P>0.05). qRT-PCR analysis of selenoprotein mRNA expression revealed that 21 of the 24 selenoprotein mRNAs and ApoER2 mRNA (the selenoprotein P [Sepp1] receptor) were also not regulated significantly by dietary Se status. In contrast, Gpx1 activity decreased to 28% of Se-adequate levels, and mRNA levels for Gpx1, Sepp1, and Sepw1 (selenoprotein W) decreased significantly in Se-deficient rats to 45, 46, and 55%, respectively, of Se-adequate plateau levels. Overlap of hyperbolic Gpx4 activity and Sepw1 mRNA response curves with testis Se concentration, all with minimum dietary Se requirements<0.016 microg Se/g, showed the priority for synthesis of Gpx4. Higher minimum dietary Se requirements of 0.04 microg Se/g for Gpx1 activity and Sepp1 mRNA, and the even higher minimum dietary Se requirement of 0.08 microg Se/g for Gpx1 mRNA, suggest that the hierarchy of these biomarkers reflects distinct, lower priority pools, cell types, and roles for Se within the testis.

Olfactory bulb interneurons releasing NO exhibit the Reelin receptor ApoEr2 and part of those targeted by NO express Reelin.

Nitric oxide (NO) and Reelin both modulate neuronal plasticity in developing and mature synaptic networks. We recently showed a loss of neuronal nitric oxide synthase (nNOS) protein in the olfactory bulb of reeler mutants and advanced the hypothesis that the Reelin and NO signalling pathways may influence each other. We now studied the distribution of NO sensitive guanylyl cyclase (NOsGC), Reelin and its receptor Apolipoprotein E2 (ApoEr2) in the olfactory bulb by multiple fluorescence labelling and tested whether nNOS and ApoEr2 colocalize in this area. We also essayed the protein content of NOsGC in the reeler olfactory bulb and tested whether there are any changes in nNOS and NOsGC protein in other reeler brain areas. Olfactory bulb interneurons expressing ApoEr2 and nNOS are only few in the glomerular layer but represent the large majority of granule cell layer interneurons. Conversely, NOsGC interneurons are rare in the granule cell layer and abundant as periglomerular cells. Reelin containing periglomerular cells almost entirely belong to the NOsGC subset. These data further support the hypothesis of a reciprocal signalling between Reelin/NOsGC and ApoEr2/nNOS expressing neurons to affect olfactory bulb activity. We also show that a significant rise in NOsGC content accompanies the decrease of nNOS protein in the reeler olfactory bulb. The same reciprocal changes present in the cortex/striatum and the hippocampus of reeler mice. Thus, the influence that the deficit of extracellular Reelin seems to exert on nNOS and its receptor is not limited to the olfactory bulb but is a general feature of the reeler brain.

Prognostic value of LISCH7 mRNA in plasma and tumor of colon cancer patients.

PURPOSE: LISCH7 is a gene potentially regulated by p53 that is up-regulated in metastasis development. Our hypothesis was that the expression of LISCH7 in primary colorectal tumors determined certain characteristics of the tumors, as well as their behavior, and that its identification in plasma could serve as a prognostic marker. EXPERIMENTAL DESIGN: We tested this hypothesis in a series of 115 tumors and normal tissues and in 83 plasmas from patients with sporadic colorectal carcinomas, as well as in 20 healthy control plasmas in which the expression levels of the gene were measured by real-time PCR. The expression data were contrasted with clinicopathologic variables. RESULTS: Although LISCH7 expression was not detected in any control plasma samples, it was positive in 25 (30.1%) plasmas from patients (P = 0.002). LISCH7 mRNA in plasma was significantly associated with the pathologic stage (P = 0.019), with lymph node metastasis (P = 0.008) and with vascular invasion (P = 0.005). Expression was not detected in any normal tissues but was detected in 80 tumor tissues, with a clear association found with vascular invasion (P = 0.027). Moreover, we show that LISCH7 was specifically expressed by the epithelial tumor cells. The adjusted overall survival study showed independent prognostic values for LISCH7 expression levels in tumor tissues (hazard ratio, 3.45; 95% confidence interval, 1.19-9.98). CONCLUSIONS: Our results suggest that LISCH7 is a good tumor marker whose expression levels could be considered as a poor prognosis factor in human colon cancer. Furthermore, plasma is suggested as a feasible source of nucleic acids for subsequent noninvasive prognostic studies.

Peroxisome proliferator-activated receptor γ activation favours selective subcutaneous lipid deposition by coordinately regulating lipoprotein lipase modulators, fatty acid transporters and lipogenic enzymes.

AIM: Peroxisome proliferator-activated receptor (PPAR) γ activation is associated with preferential lipoprotein lipase (LPL)-mediated fatty acid storage in peripheral subcutaneous fat depots. How PPARγ agonism acts upon the multi-level modulation of depot-specific lipid storage remains incompletely understood. METHODS: We evaluated herein triglyceride-derived lipid incorporation into adipose tissue depots, LPL mass and activity, mRNA levels and content of proteins involved in the modulation of LPL activity and fatty acid transport, and the expression/activity of enzymes defining adipose tissue lipogenic potential in rats treated with the PPARγ ligand rosiglitazone (30 mg kg(-1)  day(-1) , 23 days) after either a 10-h fasting period or a 17-h fast followed by 6 h of ad libitum refeeding. RESULTS: Rosiglitazone stimulated lipid accretion in subcutaneous fat (SF) ~twofold and significantly reduced that of visceral fat (VF) to nearly half. PPARγ activation selectively increased LPL mass, activity and the expression of its chaperone LMF1 in SF. In VF, rosiglitazone had no effect on LPL activity and downregulated the mRNA levels of the transendothelial transporter GPIHBP1. Overexpression of lipid uptake and fatty acid transport proteins (FAT/CD36, FATP1 and FABP4) and stimulation of lipogenic enzyme activities (GPAT, AGPAT and DGAT) upon rosiglitazone treatment were of higher magnitude in SF. CONCLUSIONS: Together these findings demonstrate that the depot-specific transcriptional control of LPL induced by PPARγ activation extends to its key interacting proteins and post-translational modulators to favour subcutaneous lipid storage.

Role for reelin in the development of granule cell dispersion in temporal lobe epilepsy.

The reelin signaling pathway plays a crucial role during the development of laminated structures in the mammalian brain. Reelin, which is synthesized and secreted by Cajal-Retzius cells in the marginal zone of the neocortex and hippocampus, is proposed to act as a stop signal for migrating neurons. Here we show that a decreased expression of reelin mRNA by hippocampal Cajal-Retzius cells correlates with the extent of migration defects in the dentate gyrus of patients with temporal lobe epilepsy. These results suggest that reelin is required for normal neuronal lamination in humans, and that deficient reelin expression may be involved in migration defects associated with temporal lobe epilepsy.

Susceptibility to murine cholesterol gallstone formation is not affected by partial disruption of the HDL receptor SR-BI.

High density lipoprotein (HDL) promotes reverse cholesterol transport from peripheral tissues to the liver where its cholesterol is secreted preferentially into bile. The scavenger receptor class B type I (SR-BI) is believed to play a pivotal role in unloading HDL cholesterol and its ester to hepatocytes. Here, using male SR-BI "att" mice with a dysfunctional mutation in the Sr-b1 promoter, we studied whether approximately 50% of normal SR-BI expression influences gallstone susceptibility in these mice fed a lithogenic diet containing 1% cholesterol, 0.5% cholic acid and 15% butterfat. Our results showed that the disruption of SR-BI expression reduced cholesterol secretion by 37% in the chow-fed state and 10% on the lithogenic diet, and while delaying incidence slightly, did not influence cumulative susceptibility to cholesterol gallstones. The lithogenic diet induced marked increases in biliary cholesterol and phospholipid secretion rates but not of bile salts. Basal expression of hepatic SR-BI protein was dissimilar in both wild-type and SR-BI mice, and remained unaltered in response to the lithogenic diet. By two independent dual isotope methods, intestinal cholesterol absorption was unimpaired by attenuation of the SR-BI which also displays low-density expression on small intestinal enterocytes. We conclude that although HDL cholesterol is a principal source of biliary cholesterol in the basal state, uptake of cholesterol from chylomicron remnants appears to be the major contributor to biliary cholesterol hypersecretion during diet-induced cholelithogenesis in the mouse.

Functional interplay between the macrophage scavenger receptor class B type I and pitavastatin (NK-104).

BACKGROUND: Scavenger receptor class B type I (SR-BI), a receptor for high-density lipoprotein (HDL), plays an important role in the bidirectional cholesterol exchange between cells and HDL particles and the atherosclerotic lesion development. Enhancement of SR-BI expression significantly reduces, whereas lack of SR-BI expression accelerates, the atherosclerotic lesion development in proatherogenic mice. Statins, a class of inhibitors for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, significantly suppress de novo cholesterol synthesis and reduce the incidence of coronary heart disease. Statins also display multiple pleiotropic effects independently of cholesterol synthesis in the vascular cells. Here, we investigated the effects of pitavastatin (NK-104), a newly synthesized statin, on macrophage SR-BI expression. METHODS AND RESULTS: We found that pitavastatin significantly increased SR-BI mRNA and protein expression in a macrophage cell line in a concentration- and time-dependent manner. It also increased SR-BI expression in both mouse peritoneal and human monocyte-derived macrophages. Associated with increased SR-BI expression, pitavastatin enhanced macrophage HDL binding, uptake of [14C]cholesteryl oleate/HDL, and efflux of [3H]cholesterol to HDL. Pitavastatin abolished the inhibition of macrophage SR-BI expression by cholesterol biosynthetic intermediates. It also restored SR-BI expression inhibited by lipopolysaccharide and tumor necrosis factor-alpha through its inactivation of the transcription factor nuclear factor-kappaB. CONCLUSIONS: Our data demonstrate that pitavastatin can stimulate macrophage SR-BI expression by reduction of cholesterol biosynthetic intermediates and antiinflammatory action and suggest additional pleiotropic effects of statins by which they may reduce the incidence of coronary heart disease.

Scavenger receptor class B type I-mediated protection against atherosclerosis in LDL receptor-negative mice involves its expression in bone marrow-derived cells.

OBJECTIVE: Scavenger receptor class B type I (SR-BI) is a cell-surface HDL receptor that is implicated in reverse cholesterol transport and protection against atherosclerosis. We have previously demonstrated that SR-BI/apolipoprotein E double-knockout mice develop severe occlusive coronary artery disease and myocardial infarction and die at approximately 6 weeks of age. To determine if this is a general effect of a lack of SR-BI, we generated mice deficient in both SR-BI and the LDL receptor. METHODS AND RESULTS: Complete ablation of SR-BI expression in LDL receptor knockout mice resulted in increased plasma cholesterol associated with HDL particles of abnormally large size and a 6-fold increase in diet-induced aortic atherosclerosis but no macroscopic evidence of early-onset coronary artery disease, cardiac pathology, or early death. Furthermore, selective elimination of SR-BI expression in bone marrow-derived cells resulted in increased diet-induced atherosclerosis in LDL receptor knockout mice without concomitant alterations in the distributions of plasma lipoprotein cholesterol. CONCLUSIONS: SR-BI expression protects against atherosclerosis in LDL receptor-deficient as well as apolipoprotein E-deficient mice, and its expression in bone marrow-derived cells contributes to this protection.

Enzymatically degraded LDL preferentially binds to CD14(high) CD16(+) monocytes and induces foam cell formation mediated only in part by the class B scavenger-receptor CD36.

Heterogeneity of peripheral blood monocytes is characterized by specific patterns in the membrane expression of Fc gamma-receptor III (FcgammaRIII/CD16) and the lipopolysaccharide receptor (LPS receptor CD14), allowing discrimination of distinct subpopulations. The aim was to analyze the correlation of these phenotypic differences to the early interaction of freshly isolated monocytes with modified lipoproteins by the use of either enzymatically degraded low density lipoprotein (E-LDL), acetylated low density lipoprotein (ac-LDL), oxidized low density lipoprotein (ox-LDL), or native low density lipoprotein. Highest E-LDL binding was observed on CD14(high) CD16(+) monocytes as determined by flow cytometry, suggesting a selective interaction of E-LDL with distinct subpopulations of monocytes. E-LDL induced rapid foam cell formation both in predifferentiated monocyte-derived macrophages and, in contrast to ac-LDL or ox-LDL, also in freshly isolated peripheral blood monocytes. This was accompanied by upregulation of the 2 class B scavenger receptors CLA-1/SR-BI (CD36 and LIMPII Analogous-1/scavenger receptor type B class I) and CD36. Cellular binding and uptake of E-LDL was neither competed by ac-LDL nor the class A scavenger-receptor inhibitor polyinosinic acid but was partially inhibited by an excess of ox-LDL. In predifferentiated monocyte-derived macrophages, an anti-CD36 antibody inhibited cellular binding and uptake of E-LDL by approximately 20%, suggesting that recognition of these hydrolase-modified low density lipoprotein particles is mediated only in part by the class B scavenger receptor CD36.

Prolonged stimulation of the adrenals by corticotropin suppresses hepatic low-density lipoprotein and high-density lipoprotein receptors and increases plasma cholesterol.

Pituitary ACTH has been shown to strongly stimulate adrenal receptors for low-density lipoprotein (LDL) and high-density lipoprotein (HDL) scavenger receptor class B type 1(SR-BI) to provide precursor cholesterol for glucocorticoid synthesis. The present study aimed to determine the effects of ACTH on hepatic cholesterol metabolism and plasma lipoproteins. Treatment of Sprague Dawley rats or normal C57BL/6J mice with ACTH for 3.5 d reduced hepatic SR-BI and LDL receptors. Simultaneously, cholesterol in plasma LDL and HDL was increased. None of these effects could be reproduced using glucocorticoids instead of ACTH, and they were abolished in adrenalectomized rats, indicating an obligate role of the adrenals for the effects of ACTH observed in the liver. When ACTH was given to LDL receptor-deficient mice, plasma LDL did not increase and the increase in HDL cholesterol remained, as did the suppression of hepatic SR-BI. Our data show that prolonged ACTH treatment suppresses hepatic SR-BI and LDL receptors in vivo in rodents, resulting in elevated plasma HDL and LDL. The adrenals are obligate for these effects, suggesting that ACTH releases some factor(s) that suppresses hepatic LDL and SR-BI receptors. Hypothetically, this novel mechanism would further promote channeling of cholesterol to the adrenals in situations of prolonged stress.

Identification and expression of scavenger receptor SR-BI in endothelial cells and smooth muscle cells of rat aorta in vitro and in vivo.

In this study, we used immunoelectron microscopy to investigate the subcellular localization of scavenger receptor class B type I (SR-BI) in the arterial walls of rats. The expression of SR-BI in cultured endothelial and smooth muscle cells of rat aorta after exposure to high-density lipoprotein (HDL) was also investigated by immunofluorescence microscopy and immunoblotting analysis. A peptide containing residues 495-509 from mouse SR-BI (mSR-BI) plus an NH2-terminal cysteine was coupled to hemocyanin to generate mSR-BI antiserum in rabbits. Reactivity of antiserum against the synthetic peptides was confirmed with an enzyme-linked immunosorbent assay (ELISA). The results showed that SR-BI was specifically localized on the surface of the endothelial cells and smooth muscle cells. SR-BI was also observed in the cytoplasm of smooth muscle cells. Immunoblotting analysis indicated that SR-BI was expressed in the cell membrane. The levels of SR-BI increased gradually from 1 to 3 h and decreased at 24 and 48 h after cholesterol-loaded cells were incubated in the culture medium containing HDL. We conclude that SR-BI, a functional receptor for HDL, is expressed in the aortic endothelial cells as well as in smooth muscle cells. This receptor also responds to the presence of HDL in the culture medium.

Receptor-Ck-dependent regulation of genes in HL-60 cell line.

The selective and conspicuous absence of a novel receptor-Ck (having affinity for cholesterol moiety in lipoproteins and intrinsic tyrosine kinase activity) in various leukemic cell lines/patients, prompted us to explore the nature of interrelationship between receptor-Ck deficiency and over-expression of genes coding for bcl-2, cyclin 'D', chimeric bcr-abl, c-myc and LDL-receptor in the human promyelocytic leukemic cell line (HL-60). This study revealed unambiguously that deregulated expression of these genes is primarily due to the inability of these cells to express the receptor-Ck gene. Based upon the observations we propose that 'receptor-Ck' deficiency may be responsible for the initiation of chronic myeloid leukemia.

Pharmacological characterization of lysophospholipid receptor signal transduction pathways in rat cerebrocortical astrocytes.

Lysophosphatidic acid (1-acyl-2-lyso-sn-glycero-3-phosphate; LPA) and sphingosine-1-phosphate (S1P) are bioactive phospholipids which respectively act as agonists for the G-protein-coupled lpA receptors (LPA1, LPA2, and LPA3) and s1p receptors (S1P1, S1P2, S1P3, S1P4, and S1P5), collectively referred to as lysophospholipid receptors (lpR). Since astrocytes are responsive to LPA and S1P, we examined mechanisms of lpR signaling in rat cortical secondary astrocytes. Rat cortical astrocyte mRNA expression by quantitative TaqMan polymerase chain reaction (PCR) analysis revealed the following order of relative expression of lpR mRNAs: s1p3>s1p1>lpa1>s1p2=lpa3>>s1p5. Activation of lpRs by LPA or S1P led to multiple pharmacological effects, including the influx of calcium, phosphoinositide (PI) hydrolysis, phosphorylation of extracellular receptor regulated kinase (ERK) and release of [3H]-arachidonic acid (AA). These signalling events downstream of lpR activation were inhibited to varying degrees by pertussis toxin (PTX) pretreatment or by the inhibition of sphingosine kinase (SK), a rate-limiting enzyme in the biosynthesis of S1P from sphingosine. These results suggest that astrocyte lpR signalling mechanisms likely involve both Gi- and Gq-coupled GPCRs and that receptor-mediated activation of SK leads to intracellular generation of S1P, which in turn amplifies the lpR signalling in a paracrine/autocrine manner.

Microglial activation state and lysophospholipid acid receptor expression.

We used a simple commercial magnetic immunobead method for the preparation of acutely isolated microglial cells from postnatal days 1-3 rat brain. With the exception of a 15 min enzyme incubation, all stages are carried out at 4 degrees C, minimizing the opportunity for changes in gene expression during the isolation to be reflected in changes in accumulated mRNA. The composition of the isolated cells was compared with that of microglial cultures prepared by conventional tissue culture methods, and the purity of microglia was comparable between the two preparations. RT-PCR analysis of several genes related to inflammatory products indicated that the acutely prepared cells were in a less activated condition than the conventionally tissue cultured cells. We examined the pattern of expression of receptors for lysophosphatidic acid (lpa) and sphingosine-1-phosphate (S1P) using quantitative real-time PCR (TaqMan PCR) techniques. mRNA for LPA1, S1P1, S1P2, S1P3 and S1P5 was detected in these preparations, but the levels of the different receptor mRNAs varied according to the state of activation of the cells. mRNA for LPA3 was only detected significantly in cultured cell after lipopolysaccharide (LPS) stimulation, being almost absent in cultured microglia and undetectable in the acutely isolated preparations. The levels of mRNA of LPA1 and S1P receptors was reduced by overnight exposure to S1P, while the same treatment significantly up-regulated the level of LPA3 mRNA.