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Dennis E Vance

Publications and source records attributed to Dennis E Vance.

At least 19 recordsLinked to original sources
PubMed2003

Enhanced expression and activation of CTP:phosphocholine cytidylyltransferase beta2 during neurite outgrowth.

During differentiation neurons increase phospholipid biosynthesis to provide new membrane for neurite growth. We studied the regulation of phosphatidylcholine (PC) biosynthesis during differentiation of two neuronal cell lines: PC12 cells and Neuro2a cells. We hypothesized that in PC12 cells nerve growth factor (NGF) would up-regulate the activity and expression of the rate-limiting enzyme in PC biosynthesis, CTP:phosphocholine cytidylyltransferase (CT). During neurite outgrowth, NGF doubled the amount of cellular PC and CT activity. CTbeta2 mRNA increased within 1 day of NGF application, prior to the formation of visible neurites, and continued to increase during neurite growth. When neurites retracted in response to NGF withdrawal, CTbeta2 mRNA, protein, and CT activity decreased. NGF specifically activated CTbeta2 by promoting its translocation from cytosol to membranes. In contrast, NGF did not alter CTalpha expression or translocation. The increase in both CTbeta2 mRNA and CT activity was inhibited by U0126, an inhibitor of mitogen-activated kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2). In Neuro2a cells, retinoic acid significantly increased CT activity (by 54%) and increased CTbeta2 protein, coincident with neurite outgrowth but did not change CTalpha expression. Together, these data suggest that the CTbeta2 isoform of CT is specifically up-regulated and activated during neuronal differentiation to increase PC biosynthesis for growing neurites.

Animals↗
PubMed2003

Inhibitors of hepatic microsomal triacylglycerol hydrolase decrease very low density lipoprotein secretion.

The presence of elevated circulating triacylglycerol (TG)-rich very low density lipoprotein (VLDL) and apolipoprotein B-100 (apoB-100) levels represents an independent risk factor for coronary artery disease. Triacylglycerol hydrolase catalyzes the mobilization of cytoplasmic TG stores. To test the hypothesis that the enzyme plays a role in the provision of core lipids for the assembly of VLDL, we inhibited the lipase activity in primary rat hepatocytes and analyzed lipid and apoB synthesis and secretion. Inhibition of lipolysis resulted in a dramatic decrease in secretion of TGs. In addition, secretion of cholesteryl ester and phosphatidylcholine was substantially decreased. Analysis of secreted apolipoproteins indicated that apoB-100 secretion was much more sensitive to lipase inhibition than was apoB-48 secretion, perhaps because of the ability of apoB-48 to be secreted as a relatively lipid-poor particle. The results agreed with those obtained with hepatoma cells transfected with triacylglycerol hydrolase cDNA, in which preferential lipidation of apoB-100 was observed. Together, our findings provide evidence that inhibition of intracellular TG hydrolysis significantly decreases apoB-100 secretion and suggest that triacylglycerol hydrolase may be a suitable pharmacological target in efforts to lower plasma lipid levels.

Animals↗
PubMed2003

Molecular dissection of the S-adenosylmethionine-binding site of phosphatidylethanolamine N-methyltransferase.

Phosphatidylethanolamine N-methyltransferase (PEMT) is a quatrotopic membrane protein that catalyzes the conversion of phosphatidylethanolamine to phosphatidylcholine through three sequential methylation reactions. Analysis of mice lacking a functional PEMT gene revealed a severe reduction in plasma homocysteine levels. Homocysteine is generated by the hydrolysis of S-adenosylhomocysteine, which is also a product of the PEMT reaction. To gain insight into the PEMT transmethylation reaction and the mechanism by which PEMT regulates homocysteine levels, we sought to define residues that are required for binding of the methyl group donor, S-adenosylmethionine (AdoMet). Bioinformatic analysis of the predicted amino acid sequence of human PEMT identified two putative AdoMet-binding motifs (98GXG100 and 180EE181). Site-directed mutagenesis experiments demonstrated the requirement for the conserved motifs in PEMT specific activity. Analysis of the AdoMet binding ability of mutant recombinant PEMT derivatives established that residues Gly100 and Glu180 are essential for binding of the AdoMet moiety. A significantly elevated KD with respect to AdoMet is observed following conservative mutagenesis of residues Gly98 (400 pmol) and Glu181 (666.7 pmol), relative to the unmodified enzyme (303.1 pmol), suggesting that these residues also participate in AdoMet binding. A model positions two separate AdoMet-binding motifs of PEMT in close proximity at the external leaflet of the endoplasmic reticulum membrane.

Amino Acid Sequence↗
PubMed2003

Insights into the requirement of phosphatidylcholine synthesis for liver function in mice.

Phosphatidylcholine (PC) is made in the liver by the CDP-choline pathway and via phosphatidylethanolamine N-methyltransferase (PEMT), which catalyzes the conversion of phosphatidylethanolamine to PC. Unexpectedly, hepatic apolipoprotein B-100 secretion is inhibited in male, but not female, Pemt-/- mice (Noga, A. A., Y. Zhao, and D. E. Vance. 2002. J. Biol. Chem. 277: 42358-42365; Noga, A. A., and D. E. Vance. 2003. J. Biol. Chem. 278: 21851-21859). To gain further insight into this process, we compared PC metabolism in male and female mice fed chow or a high-fat/high-cholesterol (HF/HC) diet. Immunoblot analyses demonstrated that twice as much PEMT2 was present in livers from female compared with male mice. In contrast, assays of CTP:phosphocholine cytidylyltransferase from livers of Pemt+/+ mice demonstrated more active cytidylyltransferase in male than in female mice. Secretion of PEMT-derived PC into lipoproteins was examined in vivo by injection of mice with [methyl-3H]methionine in the presence of Triton WR1339. The PEMT-derived PC shifts to smaller-sized particles in response to a HF/HC diet, but only in male mice. Secretion of PEMT-derived PC into bile was enhanced in mice fed a HF/HC diet. These results demonstrate that the synthesis and targeting of PC produced by the PEMT pathway in the livers of mice differs in a gender- and diet-specific manner.

Animals↗
PubMed2003

Localization of the PE methylation pathway and SR-BI to the canalicular membrane: evidence for apical PC biosynthesis that may promote biliary excretion of phospholipid and cholesterol.

To better understand the regulation of biliary phospholipid and cholesterol excretion, canalicular membranes were isolated from the livers of C57BL/6J mice and abundant proteins separated by SDS-PAGE and identified by matrix-assisted laser desorption/ionization mass spectrometry. A prominent protein revealed by this analysis was betaine homocysteine methyltransferase (BHMT). This enzyme catalyzes the first step in a three-enzyme pathway that promotes the methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC). Immunoblotting confirmed the presence of BHMT on the canalicular membrane, failed to reveal the presence of the second enzyme in this pathway, methionine adenosyltransferase, and localized the third enzyme of the pathway, PE N-methyltransferase (PEMT). Furthermore, immunfluorescence microscopy unambiguously confirmed the localization of PEMT to the canalicular membrane. These findings indicate that a local mechanism exists in or around hepatocyte canalicular membranes to promote phosphatidylethnolamine methylation and PC biosynthesis. Finally, immunoblotting revealed the presence and immunofluorescence microscopy unambiguously localized the scavenger receptor class B type I (SR-BI) to the canalicular membrane. Therefore, SR-BI, which is known to play a role in cholesterol uptake at the hepatocyte basolateral membrane, may also be involved in biliary cholesterol excretion. Based on these findings, a model is proposed in which local canalicular membrane PC biosynthesis in concert with the phospholipid transporter mdr2 and SR-BI, promotes the excretion of phospholipid and cholesterol into the bile.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2003

Activation of CTP:phosphocholine cytidylyltransferase alpha expression during the S phase of the cell cycle is mediated by the transcription factor Sp1.

An essential step during cell division is induction of phosphatidylcholine biosynthesis. In this pathway, CTP:phosphocholine cytidylyltransferase alpha (CT alpha) plays an important regulatory role. Previous studies (Golfman, L. S., Bakovic, M., and Vance, D. E. (2001) J. Biol. Chem. 276, 43688-43692) demonstrated that CT alpha mRNA accumulates during S phase in preparation for cellular mitosis. We now demonstrate that increased binding of the transcription factor Sp1 to the proximal promoter of CT alpha is responsible for increased transcription during the S phase. The Sp1 binding element present in position -67/-62 is essential for activation, and the Sp1 site in position -31/-9 is required to enhance transcription. Inhibition of Sp1 expression by RNA interference abolished the enhanced expression of CT alpha. Immunoprecipitation studies demonstrated that Sp1 interacts with cyclin E, cyclin A, and cyclin-dependent kinase 2 during the S phase. We conclude that Sp1 binding to the CT alpha proximal promoter is necessary to enhance transcription during the S phase. This is the first elucidation of a mechanism by which expression of a key enzyme in phospholipid biosynthesis is regulated during the cell cycle.

Animals↗
PubMed2003

A gender-specific role for phosphatidylethanolamine N-methyltransferase-derived phosphatidylcholine in the regulation of plasma high density and very low density lipoproteins in mice.

Phosphatidylethanolamine N-methyltransferase (PEMT)is involved in a secondary pathway for production of phosphatidylcholine (PC) in liver. We fed Pemt-/-mice a high fat/high cholesterol diet for 3 weeks to determine whether or not PC derived from PEMT is required for very low density lipoprotein secretion. Lipid analyses of plasma and liver indicated that male Pemt-/- mice accumulated triacylglycerols in their livers and were unable to secrete the same amount of triacylglycerols from the liver as did Pemt+/+ mice. Plasma levels of triacylglycerol and both apolipoproteins B100 and B48 were significantly decreased only in male Pemt-/- mice. Experiments in which mice were injected with Triton WR1339 showed that, whereas hepatic apoB100 secretion was decreased in male Pemt-/- mice, the decrease in plasma apoB48 in male Pemt-/- mice was not due to reduced secretion. Moreover, female and, to a lesser extent, male Pemt-/- mice showed a striking 40% decrease in plasma PC and cholesterol in high density lipoproteins. These results suggest that, even though the content of hepatic PC was normal in PEMT-deficient mice, plasma lipoprotein levels were profoundly altered in a gender-specific manner.

Animal Feed↗
PubMed2003

Identification of Ets-1 as an important transcriptional activator of CTP:phosphocholine cytidylyltransferase alpha in COS-7 cells and co-activation with transcriptional enhancer factor-4.

Phosphatidylcholine biosynthesis via the CDP-choline pathway is primarily regulated by CTP:phosphocholine cytidylyltransferase (CT). Transcriptional enhancer factor-4 (TEF-4) enhances the transcription of CTalpha in COS-7 cells by interactions with the basal transcription machinery (Sugimoto, H., Bakovic, M., Yamashita, S., and Vance, D.E. (2001) J. Biol. Chem. 276,12338-12344). To identify the most important transcription factor involved in basal CTalpha transcription, we made CTalpha promoter-deletion and -mutated constructs linked to a luciferase reporter and transfected them into COS-7 cells. The results indicate that an important site regulating basal CTalpha transcription is -53/-47 (GACTTCC), which is a putative consensus-binding site of Ets transcription factors (GGAA) in the opposite orientation. Gel shift analyses indicated the existence of a binding protein for -53/-47 (GACTTCC) in nuclear extracts of COS-7 cells. When anti-Ets-1 antibody was incubated with the probe in gel shift analyses, the intensity of the binding protein was decreased. The binding of endogenous Ets-1 to the promoter probe was increased when TEF-4 was expressed; however, the amount of Ets-1 detected by immunoblotting was unchanged. When cells were transfected with Ets-1 cDNA, the luciferase activity of CTalpha promoter constructs was greatly enhanced. Co-transfection experiments with Ets-1 and TEF-4 showed enhanced expression of reporter constructs as well as CTalpha mRNA. These results suggest that Ets-1 is an important transcriptional activator of the CTalpha gene and that Ets-1 activity is enhanced by TEF-4.

Animals↗
PubMed2003

Oncogenic Ha-Ras transformation modulates the transcription of the CTP:phosphocholine cytidylyltransferase alpha gene via p42/44MAPK and transcription factor Sp3.

We have shown previously that expression of the murine CTP:phosphocholine cytidylyltransferase (CT) alpha gene is regulated during cell proliferation (Golfman, L. S., Bakovic, M., and Vance, D. E. (2001) J. Biol. Chem. 276, 43688-43692). We have now characterized the role of Ha-Ras in the transcriptional regulation of the CTalpha gene. The expression of CTalpha and CTbeta2 proteins and mRNAs was stimulated in C3H10T1/2 murine fibroblasts expressing oncogenic Ha-Ras. Incubation of cells with the specific inhibitor (PD98059) of p42/44(MAPK) decreased the expression of both CT isoforms. Transfection of fibroblasts with CTalpha promoter-luciferase constructs resulted in an approximately 2-fold enhanced luciferase expression in Ha-Ras-transformed, compared with nontransformed, fibroblasts. Electromobility shift assays indicated enhanced binding of the Sp3 transcription factor to the CTalpha promoter in Ha-Ras-transformed cells. Expression of several forms of Sp3 was increased in nuclear extracts of Ha-Ras-transformed fibroblasts compared with nontransformed cells. Tyrosine phosphorylation of one Sp3 form was decreased, whereas phosphorylation of two other forms of Sp3 was increased in nuclear extracts of Ha-Ras-transformed cells. When control fibroblasts were transfected with a Sp3-expressing plasmid, an enhanced expression of CTalpha and CTbeta was observed. However, the expression of CTalpha or CTbeta was not increased in Ha-Ras-transformed cells transfected with a Sp3 plasmid presumably because expression was already maximally enhanced. The results suggest that Sp3 is a downstream effector of a Ras/p42/44(MAPK) signaling pathway which increases CTalpha gene transcription.

3T3 Cells↗
PubMed2003

Expression of phosphatidylethanolamine N-methyltransferase in human hepatocellular carcinomas.

OBJECTIVE: Hepatic phosphatidylethanolamine is converted into phosphatidylcholine by the enzyme phosphatidylethanolamine N-methyltransferase (PEMT) when the dietary choline supply is inadequate. Our previous reports implicated PEMT in the regulation of hepatocyte growth and transformation. In the present study, we analyzed PEMT activity, PEMPT gene status and its mRNA expression in 29 human hepatocellular carcinomas (HCC). METHODS: The status of the PEMPT gene and PEMT2 mRNA expression were evaluated with Southern and Northern blotting, respectively, in HCC and the noninvolved liver. PEMT activity was assessed by biochemical assay. Cell proliferation markers were defined by immunohistochemical or histoautoradiographic methods. RESULTS: PEMT activity was lower in HCC than in the noninvolved liver and it was negligible in 62% of the tumors. No deletions or mutations of the PEMPT gene were found and PEMT2 mRNA expression was absent or reduced in HCC compared with peritumoral liver tissue. PEMT2 mRNA expression was inversely related to tumor proliferation and to histologic grade. Patients whose HCC did not express PEMT2 mRNA showed poorer outcomes for cancer-related survival than those with PEMT2-positive HCC. CONCLUSIONS: The present findings suggest that (1). clones lacking PEMT2 expression may have been selected during liver tumorigenesis and progression, and (2). PEMT2 expression seems to be associated with clinical progression.

Aged↗
PubMed2002

Plasma homocysteine is regulated by phospholipid methylation.

Mild hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Homocysteine, a non-protein amino acid, is formed from S-adenosylhomocysteine and partially secreted into plasma. A potential source for homocysteine is methylation of the lipid phosphatidylethanolamine to phosphatidylcholine by phosphatidylethanolamine N-methyltransferase in the liver. We show that mice that lack phosphatidylethanolamine N-methyltransferase have plasma levels of homocysteine that are approximately 50% of those in wild-type mice. Hepatocytes isolated from methyltransferase-deficient mice secrete approximately 50% less homocysteine. Rat hepatoma cells transfected with phosphatidylethanolamine N-methyltransferase secrete more homocysteine than wild-type cells. Thus, phosphatidylethanolamine N-methyltransferase is an important source of plasma homocysteine and a potential therapeutic target for hyperhomocysteinemia.

Animals↗
PubMed2002

Trafficking of cholesterol from cell bodies to distal axons in Niemann Pick C1-deficient neurons.

Niemann Pick type C (NPC) disease is a progressive neurodegenerative disorder. In cells lacking functional NPC1 protein, endocytosed cholesterol accumulates in late endosomes/lysosomes. We utilized primary neuronal cultures in which cell bodies and distal axons reside in separate compartments to investigate the requirement of NPC1 protein for transport of cholesterol from cell bodies to distal axons. We have recently observed that in NPC1-deficient neurons compared with wild-type neurons, cholesterol accumulates in cell bodies but is reduced in distal axons (Karten, B., Vance, D. E., Campenot, R. B., and Vance, J. E. (2002) J. Neurochem. 83, 1154-1163). We now show that NPC1 protein is expressed in both cell bodies and distal axons. In NPC1-deficient neurons, cholesterol delivered to cell bodies from low density lipoproteins (LDLs), high density lipoproteins, or cyclodextrin complexes was transported into axons in normal amounts, whereas transport of endogenously synthesized cholesterol was impaired. Inhibition of cholesterol synthesis with pravastatin in wild-type and NPC1-deficient neurons reduced axonal growth. However, LDLs restored a normal rate of growth to wild-type but not NPC1-deficient neurons treated with pravastatin. Thus, although LDL cholesterol is transported into axons of NPC1-deficient neurons, this source of cholesterol does not sustain normal axonal growth. Over the lifespan of NPC1-deficient neurons, these defects in cholesterol transport might be responsible for the observed altered distribution of cholesterol between cell bodies and axons and, consequently, might contribute to the neurological dysfunction in NPC disease.

Androstenes↗
PubMed2002

Membrane topography of human phosphatidylethanolamine N-methyltransferase.

In liver, phosphatidylethanolamine is converted to phosphatidylcholine through a series of three sequential methylation reactions. Phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes each transmethylation reaction, and S-adenosylmethionine is the methyl group donor. Biochemical analysis of human liver revealed that the methyltransferase activity is primarily localized to the endoplasmic reticulum and mitochondria-associated membranes. Bioinformatic analysis of the predicted amino acid sequence suggested that the enzyme adopts a polytopic conformation in those membranes. To elucidate the precise membrane topography of PEMT and thereby provide the basis for in-depth functional characterization of the enzyme, we performed endoproteinase-protection analysis of epitope-tagged, recombinant protein. Our data suggest a topographical model of PEMT in which four transmembrane regions span the membrane such that both the N and C termini of the enzyme are localized external to the ER. Two hydrophilic connecting loops protrude into the luminal space of the microsomes whereas a corresponding loop on the cytosolic side remains proximate to the membrane. Further support for this model was obtained following endoproteinase-protection analysis of mutant recombinant PEMT derivatives in which specific protease cleavage sites had been genetically engineered or ablated.

Adult↗
PubMed2002

An unexpected requirement for phosphatidylethanolamine N-methyltransferase in the secretion of very low density lipoproteins.

Phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes the conversion of phosphatidylethanolamine to phosphatidylcholine (PC). We investigated whether there was diminished secretion of lipoproteins from hepatocytes derived from mice that lacked PEMT (Pemt(-/-)) compared with Pemt(+/+) mice. Hepatocytes were incubated with 0.75 mm oleate, the media were harvested, and triacylglycerol (TG), PC, apolipoprotein (apo) B100, and apoB48 were isolated and quantified. Compared with hepatocytes from Pemt(+/+) mice, hepatocytes from Pemt(-/-) mice secreted 50% less TG, whereas secretion of PC was unaffected. Fractionation of the secreted lipoproteins on density gradients demonstrated that the decrease in TG was in the very low density lipoprotein (VLDL)/low density lipoprotein fractions. The secretion of apoB100 was decreased by approximately 70% in VLDLs/low density lipoproteins, whereas there was no significant decrease in apoB48 secretion in any fraction. Transfection of McArdle hepatoma cells (that lack PEMT) with PEMT cDNA enhanced secretion of TG in the VLDLs. Because the levels of PC in the hepatocytes and hepatoma cells were unaffected by the lack of PEMT expression, there appears to be an unexpected requirement for PEMT in the secretion of apoB100-containing VLDLs.

Animals↗
PubMed2002

Structure-function analysis of human triacylglycerol hydrolase by site-directed mutagenesis: identification of the catalytic triad and a glycosylation site.

Triacylglycerol hydrolase is a microsomal enzyme that hydrolyzes stored cytoplasmic triacylglycerol in the liver and participates in the lipolysis/re-esterification cycle during the assembly of very-low-density lipoproteins. The structure-activity relationship of the enzyme was investigated by site-directed mutagenesis and heterologous expression. Expression of human TGH in Escherichia coli yields a protein without enzymatic activity, which suggests that posttranslational processing is necessary for the catalytic activity. Expression in baculovirus-infected Sf-9 cells resulted in correct processing of the N-terminal signal sequence and yielded a catalytically active enzyme. A putative catalytic triad consisting of a nucleophilic serine (S221), glutamic acid (E354), and histidine (H468) was identified. Site-directed mutagenesis of the residues (S221A, E354A, and H468A) yielded a catalytically inactive enzyme. CD spectra of purified mutant proteins were very similar to that of the wild-type enzyme, which suggests that the mutations did not affect folding. Human TGH was glycosylated in the insect cells. Mutagenesis of the putative N-glycosylation site (N79A) yielded an active nonglycosylated enzyme. Deletion of the putative C-terminal endoplasmic reticulum retrieval signal (HIEL) did not result in secretion of the mutant protein. A model of human TGH structure suggested a lipase alpha/beta hydrolase fold with a buried active site and two disulfide bridges (C87-C116 and C274-C285).

Animals↗
PubMed2002

Heterologous expression, purification, and characterization of human triacylglycerol hydrolase.

Triacylglycerol hydrolase mobilizes stored triacylglycerol some of which is used for very-low-density lipoprotein assembly in the liver. A full-length cDNA coding for a human triacylglycerol hydrolase (hTGH) was isolated from a human liver cDNA library. The cDNA has an open reading frame of 576 amino acids with a cleavable 18-amino-acid signal sequence. The deduced amino acid sequence shows that the protein belongs to the carboxylesterase family. The hTGH was highly expressed in Escherichia coli as a 6xHis-tagged fusion protein, with the tag at the N-terminus in place of the signal peptide. However, the expressed protein was insoluble and inactive. Expression was confirmed by immunoblotting and N-terminal amino acid sequencing of the purified protein. Expression of hTGH with its native signal sequence and a C-terminal 6xHis-tag in Sf9 cells using the baculovirus expression system yielded active enzyme. N-terminal amino acid sequencing of the purified expressed protein showed correct processing of the signal peptide. The enzyme also undergoes glycosylation within the endoplasmic reticulum lumen. The results suggest that hTGH expressed in insect cells is properly folded. Therefore, baculovirus expression of hTGH and facile purification of the His-tagged enzyme will allow detailed characterization of the structure/activity relationship.

Amino Acid Sequence↗