Insights into apolipoprotein B biology from transgenic and gene-targeted mice.
(41/44874)
Over the past five years, several laboratories have used transgenic and gene-targeted mice to study apolipoprotein (apo) B biology. Genetically modified mice have proven useful for investigating the genetic and environmental factors affecting atherogenesis, for defining apoB structure/function relationships, for understanding the regulation of the apoB gene expression in the intestine, for defining the "physiologic rationale" for the existence of the two different forms of apoB (apoB48 and apoB100) in mammalian metabolism and for providing mechanistic insights into the human apoB deficiency syndrome, familial hypobetalipoproteinemia. This review will provide several examples of how genetically modified mice have contributed to our understanding of apoB biology, including our new discovery that human heart myocytes secrete nascent apoB-containing lipoproteins. (+info)
Paradoxical effect on atherosclerosis of hormone-sensitive lipase overexpression in macrophages.
(42/44874)
Foam cells formed from receptor-mediated uptake of lipoprotein cholesterol by macrophages in the arterial intima are critical in the initiation, progression, and stability of atherosclerotic lesions. Macrophages accumulate cholesterol when conditions favor esterification by acyl-CoA:cholesterol acyltransferase (ACAT) over cholesteryl-ester hydrolysis by a neutral cholesteryl-ester hydrolase, such as hormone-sensitive lipase (HSL), and subsequent cholesterol efflux mediated by extracellular acceptors. We recently made stable transfectants of a murine macrophage cell line, RAW 264.7, that overexpressed a rat HSL cDNA and had a 5-fold higher rate of cholesteryl-ester hydrolysis than control cells. The current study examined the effect of macrophage-specific HSL overexpression on susceptibility to diet-induced atherosclerosis in mice. A transgenic line overexpressing the rat HSL cDNA regulated with a macrophage-specific scavenger receptor promoter-enhancer was established by breeding with C57BL/6J mice. Transgenic peritoneal macrophages exhibited macrophage-specific 7-fold overexpression of HSL cholesterol esterase activity. Total plasma cholesterol levels in transgenic mice fed a chow diet were modestly elevated 16% compared to control littermates. After 14 weeks on a high-fat, high-cholesterol diet, total cholesterol increased 3-fold, with no difference between transgenics and controls. However, HSL overexpression resulted in thicker aortic fatty lesions that were 2.5-times larger in transgenic mice. HSL expression in the aortic lesions was shown by immunocytochemistry. Atherosclerosis was more advanced in transgenic mice exhibiting raised lesions involving the aortic wall, along with lipid accumulation in coronary arteries occurring only in transgenics. Thus, increasing cholesteryl-ester hydrolysis, without concomitantly decreasing ACAT activity or increasing cholesterol efflux, is not sufficient to protect against atherosclerosis. hormone-sensitive lipase overexpression in macrophages. (+info)
GABA- and glutamate-activated channels in green fluorescent protein-tagged gonadotropin-releasing hormone neurons in transgenic mice.
(43/44874)
Mice were generated expressing green fluorescent protein (GFP) under the control of the gonadotropin-releasing hormone (GnRH) promoter. Green fluorescence was observed in, and restricted to, GnRH-immunopositive neuronal somata in the olfactory bulb, ganglion terminale, septal nuclei, diagonal band of Broca (DBB), preoptic area (POA), and caudal hypothalamus, as well as GnRH neuronal dendrites and axons, including axon terminals in the median eminence and organum vasculosum of the lamina terminalis (OVLT). Whole-cell recordings from GFP-expressing GnRH neurons in the OVLT-POA-DBB region revealed a firing pattern among GFP-expressing GnRH neurons distinct from that of nonfluorescent neurons. Nucleated patches of GFP-expressing GnRH neurons exhibited pronounced responses to fast application of GABA and smaller responses to L-glutamate and AMPA. One-fifth of the nucleated patches responded to NMDA. The GABA-A, AMPA, and NMDA receptor channels on GnRH neurons mediating these responses may play a role in the modulation of GnRH secretory oscillations. (+info)
The 5'-flanking region of the mouse adenylyl cyclase type VIII gene imparts tissue-specific expression in transgenic mice.
(44/44874)
The calcium-stimulated adenylyl cyclases (ACs) play a central role in stimulus-dependent modification of synaptic function. The type VIII AC (AC8) is one of three mammalian calcium-stimulated isoforms, each of which is expressed in a region-specific manner in the CNS. To delineate the DNA sequences responsible for appropriate targeting of AC8 expression, we report here the complete structure of the AC8 gene and define the pattern of expression of the full-length cDNA and its splice variants. In addition to expression within the brain, robust expression of AC8 was also found in the lung. By in situ hybridization, we have found the highest expression of AC8 mRNA within the olfactory bulb, thalamus, habenula, cerebral cortex, and hypothalamic supraoptic and paraventricular nuclei. By generating transgenic mice whose expression of beta-galactosidase is controlled by the AC8 5'-flanking DNA sequences, we demonstrate that the DNA sequences within the 10 kb preceding exon 1 are critical for establishment of this region-specific pattern. This spectrum of sites of production is unique to AC8 among the calcium-stimulated adenylyl cyclases and suggests nonredundant functions with other adenylyl cyclases in neuroendocrine regulation and/or behavior. (+info)
Adult subventricular zone neuronal precursors continue to proliferate and migrate in the absence of the olfactory bulb.
(45/44874)
Neurons continue to be born in the subventricular zone (SVZ) of the lateral ventricles of adult mice. These cells migrate as a network of chains through the SVZ and the rostral migratory stream (RMS) into the olfactory bulb (OB), where they differentiate into mature neurons. The OB is the only known target for these neuronal precursors. Here, we show that, after elimination of the OB, the SVZ and RMS persist and become dramatically larger. The proportion of dividing [bromodeoxyuridine (BrdU)-labeled] or dying (pyknotic or terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeled) cells in the RMS was not significantly affected at 3 d or 3 weeks after bulbectomy (OBX). However, by 3 months after OBX, the percentage of BrdU-labeled cells in the RMS decreased by half and that of dying cells doubled. Surprisingly, the rostral migration of precursors continued along the RMS after OBX. This was demonstrated by focal microinjections of BrdU and grafts of SVZ cells carrying LacZ under the control of a neuron-specific promoter gene. Results indicate that the OB is not essential for proliferation and the directional migration of SVZ precursors. (+info)
Molecular basis of the interaction between plasma platelet-activating factor acetylhydrolase and low density lipoprotein.
(46/44874)
The platelet-activating factor acetylhydrolases are enzymes that were initially characterized by their ability to hydrolyze platelet-activating factor (PAF). In human plasma, PAF acetylhydrolase (EC 3.1.1.47) circulates in a complex with low density lipoproteins (LDL) and high density lipoproteins (HDL). This association defines the physical state of PAF acetylhydrolase, confers a long half-life, and is a major determinant of its catalytic efficiency in vivo. The lipoprotein-associated enzyme accounts for all of the PAF hydrolysis in plasma but only two-thirds of the protein mass. To characterize the enzyme-lipoprotein interaction, we employed site-directed mutagenesis techniques. Two domains within the primary sequence of human PAF acetylhydrolase, tyrosine 205 and residues 115 and 116, were important for its binding to LDL. Mutation or deletion of those sequences prevented the association of the enzyme with lipoproteins. When residues 115 and 116 from human PAF acetylhydrolase were introduced into mouse PAF acetylhydrolase (which normally does not associate with LDL), the mutant mouse PAF acetylhydrolase associated with lipoproteins. To analyze the role of apolipoprotein (apo) B100 in the formation of the PAF acetylhydrolase-LDL complex, we tested the ability of PAF acetylhydrolase to bind to lipoproteins containing truncated forms of apoB. These studies indicated that the carboxyl terminus of apoB plays a key role in the association of PAF acetylhydrolase with LDL. These data on the molecular basis of the PAF acetylhydrolase-LDL association provide a new level of understanding regarding the pathway for the catabolism of PAF in human blood. (+info)
Lower plasma levels and accelerated clearance of high density lipoprotein (HDL) and non-HDL cholesterol in scavenger receptor class B type I transgenic mice.
(47/44874)
Recent studies have indicated that the scavenger receptor class B type I (SR-BI) may play an important role in the uptake of high density lipoprotein (HDL) cholesteryl ester in liver and steroidogenic tissues. To investigate the in vivo effects of liver-specific SR-BI overexpression on lipid metabolism, we created several lines of SR-BI transgenic mice with an SR-BI genomic construct where the SR-BI promoter region had been replaced by the apolipoprotein (apo)A-I promoter. The effect of constitutively increased SR-BI expression on plasma HDL and non-HDL lipoproteins and apolipoproteins was characterized. There was an inverse correlation between SR-BI expression and apoA-I and HDL cholesterol levels in transgenic mice fed either mouse chow or a diet high in fat and cholesterol. An unexpected finding in the SR-BI transgenic mice was the dramatic impact of the SR-BI transgene on non-HDL cholesterol and apoB whose levels were also inversely correlated with SR-BI expression. Consistent with the decrease in plasma HDL and non-HDL cholesterol was an accelerated clearance of HDL, non-HDL, and their major associated apolipoproteins in the transgenics compared with control animals. These in vivo studies of the effect of SR-BI overexpression on plasma lipoproteins support the previously proposed hypothesis that SR-BI accelerates the metabolism of HDL and also highlight the capacity of this receptor to participate in the metabolism of non-HDL lipoproteins. (+info)
pp60(v-src) induction of cyclin D1 requires collaborative interactions between the extracellular signal-regulated kinase, p38, and Jun kinase pathways. A role for cAMP response element-binding protein and activating transcription factor-2 in pp60(v-src) signaling in breast cancer cells.
(48/44874)
The cyclin D1 gene is overexpressed in breast tumors and encodes a regulatory subunit of cyclin-dependent kinases that phosphorylate the retinoblastoma protein. pp60(c-src) activity is frequently increased in breast tumors; however, the mechanisms governing pp60(c-src) regulation of the cell cycle in breast epithelium are poorly understood. In these studies, pp60(v-src) induced cyclin D1 protein levels and promoter activity (48-fold) in MCF7 cells. Cyclin D1-associated kinase activity and protein levels were increased in mammary tumors from murine mammary tumor virus-pp60(c-src527F) transgenic mice. Optimal induction of cyclin D1 by pp60(v-src) involved the extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase members of the mitogen-activated protein kinase family. Cyclin D1 promoter activation by pp60(v-src) involved a cAMP response element-binding protein (CREB)/activating transcription factor 2 (ATF-2) binding site. Dominant negative mutants of CREB and ATF-2 but not c-Jun inhibited pp60(v-src) induction of cyclin D1. pp60(v-src) induction of CREB was blocked by the p38 inhibitor SB203580 or by mutation of CREB at Ser133. pp60(v-src) induction of ATF-2 was abolished by the c-Jun N-terminal kinase inhibitor JNK-interacting protein-1 or by mutation of ATF-2 at Thr69 and Thr71. CREB and ATF-2, which bind to a common pp60(v-src) response element, are transcriptionally activated by distinct mitogen-activated protein kinases. Induction of cyclin D1 activity by pp60(v-src) may contribute to breast tumorigenesis through phosphorylation and inactivation of the retinoblastoma protein. (+info)