Heparin-binding EGF-like growth factor (HB-EGF) antisense oligonucleotide protected against hyperlipidemia-associated atherosclerosis.

BACKGROUND AND AIMS: Heparin-binding EGF-like growth factor (HB-EGF) is a representative EGF family member that interacts with EGFR under diverse stress environment. Previously, we reported that the HB-EGF-targeting using antisense oligonucleotide (ASO) effectively suppressed an aortic aneurysm in the vessel wall and circulatory lipid levels. In this study, we further examined the effects of the HB-EGF ASO administration on the development of hyperlipidemia-associated atherosclerosis using an atherogenic mouse model. METHODS AND RESULTS: The male and female LDLR deficient mice under Western diet containing 21% fat and 0.2% cholesterol content were cotreated with control and HB-EGF ASOs for 12 weeks. We observed that the HB-EGF ASO administration effectively downregulated circulatory VLDL- and LDL-associated lipid levels in circulation; concordantly, the HB-EGF targeting effectively suppressed the development of atherosclerosis in the aorta. An EGFR blocker BIBX1382 administration suppressed the hepatic TG secretion rate, suggesting a positive role of the HB-EGF signaling for the hepatic VLDL production. We newly observed that there was a significant improvement of the insulin sensitivity by the HB-EGF ASO administration in a mouse model under the Western diet as demonstrated by the improvement of the glucose and insulin tolerances. CONCLUSION: The HB-EGF ASO administration effectively downregulated circulatory lipid levels by suppressing hepatic VLDL production rate, which leads to effective protection against atherosclerosis in the vascular wall.

Roles of scavenger receptor BI and APO A-I in selective uptake of HDL cholesterol by adrenal cells.

Adrenal cells obtain cholesterol for steroid production via the selective uptake of cholesteryl ester (CE) from HDL particles, a process in which CE is transferred to the plasma membrane without degradation of the HDL particle. Although this process has been studied for two decades, only recently have the receptor and the HDL ligand been identified. Scavenger class B, type I, (SR-BI) is regulated by ACTH in adrenocortical cells in parallel with steroid production. Antibody to SR-BI blocks the uptake and utilization of HDL CE for steroid production in Y1-BS1 adrenal cells. The adrenal glands of SR-BI knockout mice are depleted in cholesterol providing complementary evidence that SR-BI is responsible for HDL CE accumulation in adrenal cells. SR-BI-mediated HDL CE selective uptake is a two-step process in which SR-BI first interacts with multiple sites in apoA-I with the amphipathic inverted alpha-helical repeat units of apoA-I serving as recognition motifs. This is followed by efficient CE transfer down its concentration gradient to the plasma membrane, a process requiring the extracellular domain of SR-BI. Other scavenger receptors bind HDL but do not afford the CE transfer step. Adrenal glands from apoA-I knockout mice lack CE stores, indicating that apoAI is essential for HDL selective uptake in vivo. ApoA-I knockout HDL particles bind normally to SR-BI but do not permit efficient CE transfer to the cell. These findings suggest that apoA-I has an important role in the transfer of HDL CE that goes beyond its function as a ligand for interaction with SR-BI.

Scavenger receptor class B, type I, mediates selective uptake of low density lipoprotein cholesteryl ester.

Scavenger receptor, class B, type I (SR-BI) is a cell-surface glycoprotein that mediates selective uptake of high density lipoprotein cholesteryl ester (CE) without the concomitant uptake and degradation of the particle. We have investigated the endocytic and selective uptake of low density lipoprotein (LDL)-CE by SR-BI using COS-7 cells transiently transfected with mouse SR-BI. Analysis of lipoprotein uptake data showed a concentration-dependent LDL-CE-selective uptake when doubly labeled LDL particles were incubated with SR-BI-expressing COS-7 cells. In contrast to vector-transfected cells, SR-BI-expressing COS-7 cells showed marked increases in LDL cell association and CE uptake by the selective uptake pathway, but only a modest increase in CE uptake by the endocytic pathway. SR-BI-mediated LDL-CE-selective uptake exceeded LDL endocytic uptake by 50-100-fold. SR-BI-mediated LDL-CE-selective uptake was not inhibited by the proteoglycan synthesis inhibitor, p-nitrophenyl-beta-D-xylopyranoside or by the sulfation inhibitor sodium chlorate, indicating that SR-BI-mediated LDL-CE uptake occurs independently of LDL interaction with cell-surface proteoglycan. Analyses with subclones of Y1 adrenocortical cells showed that LDL-CE-selective uptake was proportional to the level of SR-BI expression. Furthermore, antibody directed to the extracellular domain of SR-BI blocked LDL-CE-selective uptake in adrenocortical cells. Thus, in cells that normally express SR-BI and in transfected COS-7 cells SR-BI mediates the efficient uptake of LDL-CE via the selective uptake mechanism. These results suggest that SR-BI may influence the metabolism of apoB-containing lipoproteins in vivo by mediating LDL-CE uptake into SR-BI-expressing cells.

Scavenger receptor BI and cholesterol trafficking.

Scavenger receptor BI (SR-BI) mediates the selective uptake of HDL cholesteryl ester into steroidogenic cells and the liver and is a major determinant of the plasma HDL concentration in the mouse. Recent studies indicate that SR-BI also alters the metabolism of apolipoprotein B-containing particles and influences the development of atherosclerosis in several animal models. These results and the similar pattern of SR-BI expression in humans emphasize that it is important to learn how this receptor influences lipoprotein metabolism and atherosclerosis in people.

Mechanism of scavenger receptor class B type I-mediated selective uptake of cholesteryl esters from high density lipoprotein to adrenal cells.

Despite extensive studies and characterizations of the high density lipoprotein-cholesteryl ester (HDL-CE)-selective uptake pathway, the mechanisms by which the hydrophobic CE molecules are transferred from the HDL particle to the plasma membrane have remained elusive, until the discovery that scavenger receptor BI (SR-BI) plays an important role. To elucidate the molecular mechanism, we examined the quantitative relationships between the binding of HDL and the selective uptake of its CE in the murine adrenal Y1-BS1 cell line. A comparison of concentration dependences shows that half-maximal high affinity cell association of HDL occurs at 8.7 +/- 4.7 micrograms/ml and the Km of HDL-CE-selective uptake is 4.5 +/- 1.5 micrograms/ml. These values are similar, and there is a very high correlation between these two processes (r2 = 0.98), suggesting that they are linked. An examination of lipid uptake from reconstituted HDL particles of defined composition and size shows that there is a non-stoichiometric uptake of HDL lipid components, with CE being preferred over the major HDL phospholipids, phosphatidylcholine and sphingomyelin. Comparison of the rates of selective uptake of different classes of phospholipid in this system gives the ranking: phosphatidylserine > phosphatidylcholine approximately phosphatidylinositol > sphingomyelin. The rate of CE-selective uptake from donor particles is proportional to the amount of CE initially present in the particles, suggesting a mechanism in which CE moves down its concentration gradient from HDL particles docked on SR-BI into the cell plasma membrane. The activation energy for CE uptake from either HDL3 or reconstituted HDL is about 9 kcal/mol, indicating that HDL-CE uptake occurs via a non-aqueous pathway. HDL binding to SR-BI allows access of CE molecules to a "channel" formed by the receptor from which water is excluded and along which HDL-CE molecules move down their concentration gradient into the cell plasma membrane.

The selective pathway and a high-density lipoprotein receptor (SR-BI) in ovarian granulosa cells of the mouse.

We recently reported that rat luteinized ovary tissue and primary cultures of rat ovarian granulosa cells reveal a remarkably tight functional correlation between expressed selective uptake of lipoprotein cholesteryl esters and the expression of an HDL receptor protein, scavenger receptor, class B, type I (SR-BI). In the current study, we examine these same processes in C57 mouse granulosa cells and report a different correlation. Unlike the rat cells, non-hormone stimulated mouse granulosa cells are able to effectively carry out their selective pathway functions and secrete HDL-derived progestins despite low levels of SR-BI and barely detectable levels of SR-BII (an isoform of SR-BI). Once stimulated with trophic hormones or Bt2cAMP, small (30-40%) increases are observed in selective pathway functions, but major (approximately 20-fold) increases are seen in SR-BI and SR-BII expression: thus, relatively little is gained in selective cholesteryl ester uptake by mouse granulosa cells even though SR-BI and SR-BII levels are greatly increased. The importance of the HDL receptor proteins to the selective pathway remains clear, however, since a significant portion of the selective process in both basal and stimulated granulosa cells is inhibitable by the use of blocking antibody. Another surface protein, caveolin, previously reported to co-localize with SR-BI in mouse cells shows no change in expression during periods when SR-BI/BII levels are undergoing major shifts.

Expression and microvillar localization of scavenger receptor, class B, type I (a high density lipoprotein receptor) in luteinized and hormone-desensitized rat ovarian models.

Steroidogenic cells in rats and mice obtain most of their cholesterol for steroid production and cholesteryl ester (CE) storage via the selective uptake pathway in which high density lipoprotein CE (HDL-CE) is taken into the cell without the uptake and degradation of the HDL particle. A number of recent studies show that the scavenger receptor, class B, type I (SR-BI) can mediate HDL-CE selective uptake in cultured cells and suggest that this receptor may be responsible for HDL-CE selective uptake in steroidogenic cells in vivo. In the current study we examine the relationship between SR-BI expression and HDL-CE selective uptake in the gonadotropin-primed, luteinized rat ovary and in the ovary that is desensitized by multiple gonadotropin treatments. Results from this study demonstrate a tight association between expression of SR-BI and measurements of HDL-CE selective uptake regardless of the steroidogenic state of the ovary. Thus, in the luteinized ovary (which is actively producing progestins), HDL-CE selective uptake is high, as is the expression of SR-BI. In the desensitized ovary (where CE content is reduced by 90% and progestin production is virtually absent), HDL-CE selective uptake and SR-BI are induced 2- to 3-fold compared with those in the luteinized ovary. These data argue that SR-BI can be regulated by the cholesterol status of the luteal cell independently of gonadotropic stimulation. Immunostaining at the light microscopic level showed strong expression of SR-BI specifically on the surface of luteal cells in the luteinized and desensitized ovary. Immunolocalization at the electron microscopic level showed that SR-BI was associated with microvilli and microvillar channels of the luteal cell surface. This result supports the hypothesis that microvilli and microvillar channels represent a cell surface compartment that is specialized for the selective uptake of lipoprotein cholesterol into steroidogenic cells.

Mutagenesis of phospholipase D defines a superfamily including a trans-Golgi viral protein required for poxvirus pathogenicity.

Phospholipase D (PLD) genes are members of a superfamily that is defined by several highly conserved motifs. PLD in mammals has been proposed to play a role in membrane vesicular trafficking and signal transduction. Using site-directed mutagenesis, 25 point mutants have been made in human PLD1 (hPLD1) and characterized. We find that a motif (HxKxxxxD) and a serine/threonine conserved in all members of the PLD superfamily are critical for PLD biochemical activity, suggesting a possible catalytic mechanism. Functional analysis of catalytically inactive point mutants for yeast PLD demonstrates that the meiotic phenotype ensuing from PLD deficiency in yeast derives from a loss of enzymatic activity. Finally, mutation of an HxKxxxxD motif found in a vaccinia viral protein expressed in the Golgi complex results in loss of efficient vaccinia virus cell-to-cell spreading, implicating the viral protein as a member of the superfamily and suggesting that it encodes a lipid modifying or binding activity. The results suggest that vaccinia virus and hPLD1 may act through analogous mechanisms to effect viral cellular egress and vesicular trafficking, respectively.

Improved parameters for the prediction of RNA hairpin stability.

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequences of the type GGXANmAYCC, where XY is the set of four Watson-Crick base pairs and the underlined loop sequences are three to nine nucleotides. A nearest neighbor analysis of the data indicates the free energy of loop formation at 37 degrees C is dependent upon loop size and closing base pair. The model previously developed to predict the stability for RNA hairpin loops (n > 3) includes contributions from the size of the loop, the identity of the closing base pair, the free energy increment (deltaGo(37mm)) for the interaction of the closing base pair with the first mismatch and an additional stabilization term for GA and UU first mismatches [Serra, M. J., Axenson, T. J., & Turner, D. H. (1994) Biochemistry 33, 14289]. The results presented here allow improvements in the parameters used to predict RNA hairpin stability. For hairpin loops of n = 4-9, deltaGo(37iL)(n) is 4.9, 5.0, 5.0, 5.0, 4.9, and 5.5 kcal/mol, respectively, and the penalty for hairpin closure by AU or UA is +0.6 kcal/mol. deltaGo(37iL)(n) is the free energy for initiating a loop of n nucleotides. The model for predicting hairpin loop stability for loops larger than three becomes deltaGo(37L)(n) = deltaGo(37iL)(n) + deltaGo(37mm) + 0.6(if closed by AU or UA) - 0.7(if first mismatch is GA or UU). Hairpin loops of three are modeled as independent of loop sequence with deltaGo(37iL)(3) = 4.8 and the penalty for AU closure of +0.6 kcal/mol. Thermodynamic parameters for hairpin formation in 1 M NaCl for 11 naturally occurring RNA hairpin sequences are reported. The model provides good agreement with the measured values for both T(M) (within 10 degrees C of the measured value) and deltaGo(37) (within 0.8 kcal/mol of the measured value) for hairpin formation. In general, the nearest neighbor model allows prediction of RNA hairpin stability to within 5-10% of the experimentally measured values.

Scavenger receptor class B, type I (SR-BI) is the major route for the delivery of high density lipoprotein cholesterol to the steroidogenic pathway in cultured mouse adrenocortical cells.

The class B, type I scavenger receptor, SR-BI, binds high density lipoprotein (HDL) and mediates the selective uptake of HDL cholesteryl ester (CE) by cultured transfected cells. The high levels of SR-BI expression in steroidogenic cells in vivo and its regulation by tropic hormones provides support for the hypothesis that SR-BI is a physiologically relevant HDL receptor that supplies substrate cholesterol for steroid hormone synthesis. This hypothesis was tested by determining the ability of antibody directed against murine (m) SR-BI to inhibit the selective uptake of HDL CE in Y1-BS1 adrenocortical cells. Anti-mSR-BI IgG inhibited HDL CE-selective uptake by 70% and cell association of HDL particles by 50% in a dose-dependent manner. The secretion of [3H]steroids derived from HDL containing [3H]CE was inhibited by 78% by anti-mSR-BI IgG. These results establish mSR-BI as the major route for the selective uptake of HDL CE and the delivery of HDL cholesterol to the steroidogenic pathway in cultured mouse adrenal cells.

Regulation by adrenocorticotropic hormone of the in vivo expression of scavenger receptor class B type I (SR-BI), a high density lipoprotein receptor, in steroidogenic cells of the murine adrenal gland.

The class B, type I scavenger receptor, SR-BI, binds high density lipoprotein (HDL) and can mediate selective uptake of HDL cholesteryl esters by cultured cells. The high levels of expression of SR-BI in steroidogenic tissues and the importance of selective uptake from HDL as a source of cholesterol for steroidogenesis raised the possibility that SR-BI may participate in cholesterol delivery to steroidogenic tissues in vivo. We have used immunoblotting and immunohistochemical methods to show that SR-BI is specifically expressed in a distinctive pattern on the surfaces of steroid-producing cells in the murine adrenal gland's cortex and that its expression in vivo is induced by adrenocorticotropic hormone and suppressed by glucocorticoids. Thus, expression of SR-BI protein is coordinately regulated with adrenal steroidogenesis. These data provide strong support for the hypothesis that SR-BI is a physiologically relevant HDL receptor that provides substrate cholesterol for steroid hormone synthesis.