Biomedical subjects

Y A Moon

Publications and source records attributed to Y A Moon.

10 recordsLinked to original sources

PubMed2001

Decreased lipid synthesis in livers of mice with disrupted Site-1 protease gene.

Site-1 protease (S1P) cleaves membrane-bound sterol regulatory element-binding proteins (SREBPs), allowing their transcription-stimulating domains to translocate to the nucleus where they activate genes governing lipid synthesis. S1P is a potential target for lipid-lowering drugs, but the effect of S1P blockade in animals is unknown. Here, we disrupt the S1P gene in mice. Homozygous germ-line disruptions of S1P were embryonically lethal. To disrupt the gene inducibly in liver, we generated mice homozygous for a floxed S1P allele and heterozygous for a transgene encoding Cre recombinase under control of the IFN-inducible MX1 promoter. When IFN was produced, 70-90% of S1P alleles in liver were inactivated, and S1P mRNA and protein were reduced. Nuclear SREBPs declined, as did mRNAs for SREBP target genes. Cholesterol and fatty acid biosynthesis in hepatocytes declined by 75%. Low density lipoprotein (LDL) receptor mRNA declined by 50%, as did the clearance of (125)I-labeled LDL from plasma, but plasma cholesterol fell, suggesting that LDL production was reduced. These data raise the possibility that S1P inhibitors may be effective lipid-lowering agents, but they suggest that nearly complete inhibition will be required.

Animals↗

PubMed2001

Identification of a mammalian long chain fatty acyl elongase regulated by sterol regulatory element-binding proteins.

Fatty acids are synthesized de novo from acetyl-CoA and malonyl-CoA through a series of reactions mediated by acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). In rodents, the principal fatty acid produced by FAS is palmitic acid (16:0). Sterol regulatory element-binding proteins (SREBPs) enhance the transcription of many genes responsible for fatty acid synthesis. In transgenic mice that overexpress SREBPs in liver, the rate of fatty acid synthesis is markedly increased, owing to the activation of these biosynthetic genes, which include ATP citrate lyase, ACC, FAS, and stearoyl-CoA desaturase. The fatty acids that accumulate in livers of SREBP transgenic mice are 18 carbons rather than 16 carbons in length, suggesting that the enzymes required for the elongation of palmitic to stearic acid may be induced. Here, we report the cDNA cloning of a murine long chain fatty acyl elongase (LCE) that was identified initially by oligonucleotide array analysis of mRNA from SREBP transgenic mouse livers. LCE mRNA is highly expressed in liver and adipose tissue. The cDNA encodes a protein of 267 amino acids that shares sequence identity with previously identified very long chain fatty acid elongases. Cells that overexpress LCE show enhanced addition of 2-carbon units to C12-C16 fatty acids. We provide evidence that LCE catalyzes the rate-limiting condensing step in this reaction. The current studies suggest that mouse LCE expression is increased by SREBPs and that the enzyme is a component of the elusive mammalian elongation system that converts palmitic to stearic acid.

Acetyltransferases↗

PubMed2001

SREBP cleavage-activating protein (SCAP) is required for increased lipid synthesis in liver induced by cholesterol deprivation and insulin elevation.

In liver, the synthesis of cholesterol and fatty acids increases in response to cholesterol deprivation and insulin elevation, respectively. This regulatory mechanism underlies the adaptation to cholesterol synthesis inhibitors (statins) and high calorie diets (insulin). In nonhepatic cells, lipid synthesis is controlled by sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors whose active domains are released proteolytically to enter the nucleus and activate genes involved in the synthesis and uptake of cholesterol and fatty acids. SCAP (SREBP cleavage-activating protein) is a sterol-regulated escort protein that transports SREBPs from their site of synthesis in the endoplasmic reticulum to their site of cleavage in the Golgi. Here, we produced a conditional deficiency of SCAP in mouse liver by genomic recombination mediated by inducible Cre recombinase. SCAP-deficient mice showed an 80% reduction in basal rates of cholesterol and fatty acid synthesis in liver, owing to decreases in mRNAs encoding multiple biosynthetic enzymes. Moreover, these mRNAs failed to increase normally in response to cholesterol deprivation produced by a cholesterol synthesis inhibitor and to insulin elevation produced by a fasting-refeeding protocol. These data provide in vivo evidence that SCAP and the SREBPs are required for hepatic lipid synthesis under basal and adaptive conditions.

Animals↗

PubMed2000

Cloning of human acetyl-CoA carboxylase beta promoter and its regulation by muscle regulatory factors.

The 280-kDa beta-isoform of acetyl-CoA carboxylase (ACCbeta) is predominantly expressed in heart and skeletal muscle, whereas the 265-kDa alpha-isoform (ACCalpha) is the major ACC in lipogenic tissues. The ACCbeta promoter showed myoblast-specific promoter activity and was strongly induced by MyoD in NIH3T3 cells. Serial deletions of the promoter revealed that MyoD acts on the E-boxes located at positions -498 to -403 and on the proximal region including the 5'-untranslated region. Destruction of the E-boxes at positions -498 to -403 by site-directed mutagenesis resulted in a significant decrease of MyoD responsiveness. The "TGAAA" at -32 to -28 and the region around the transcription start site play important roles in basal transcription, probably as a TATA box and an Inr element, respectively. Mutations of another E-box at -14 to -9 and a "GCCTGTCA" sequence at +17 to +24 drastically decreased the MyoD responsiveness. The novel cis-element GCCTGTCA was preferentially bound by MyoD homodimer in EMSA and conferred MyoD responsiveness to a luciferase reporter, which was repressed by the overexpression of E12. This finding is unique since activation via E-boxes is mediated by heterodimers of MyoD and E-proteins. We screened a human skeletal muscle cDNA library to isolate clones expressing proteins that bind to the region around the GCCTGTCA (+8 to +27) sequence, and isolated Myf4 and Myf6 cDNAs. Electrophoretic mobility shift assay showed that recombinant Myf4 and Myf6 bind to this novel cis-element. Moreover, transient expression of Myf6 induced significant activation on the ACCbeta promoter or an artificial promoter harboring this novel cis-element. These findings suggest that muscle regulatory factors, such as MyoD, Myf4, and Myf6, contribute to the muscle-specific expression of ACCbeta via E-boxes and the novel cis-element GCCTGTCA.

Acetyl-CoA Carboxylase↗

PubMed2000

The roles of sterol regulatory element-binding proteins in the transactivation of the rat ATP citrate-lyase promoter.

ATP citrate-lyase (ACL) is a key enzyme supplying acetyl-CoA for fatty acid and cholesterol synthesis. Its expression is drastically up-regulated when an animal is fed a low fat, high carbohydrate diet after prolonged fasting. In this report, we describe the role of sterol regulatory element-binding proteins (SREBPs) in the transactivation of the rat ACL promoter. ACL promoter activity was markedly stimulated by the overexpression of SREBP-1a and, to a lesser extent, by SREBP-2 in Alexander human hepatoma cells. The promoter elements responsive to SREBPs were located within the 55-base pair sequences from -114 to -60. The gel mobility shift assay revealed four SREBP-1a binding sites in this region. Of these four elements, the -102/-94 region, immediately upstream of the inverted Y-box, and the -70/-61 region, just adjacent to Sp1 binding site, played critical roles in SREBPs-mediated stimulation. The mutation in the inverted Y-box and the coexpression of dominant negative nuclear factor-Y (NF-Y) significantly attenuated the transactivation by SREBP-1a, suggesting that NF-Y binding is a prerequisite for SREBPs to activate the ACL promoter. However, the multiple Sp1 binding sites did not affect the transactivation of the ACL promoter by SREBPs. The binding affinity of SREBP-1a to SREs of the ACL promoter also was much higher than that of SREBP-2. The transactivation potencies of the chimeric SREBPs, of which the activation domains (70 amino acids of the amino terminus) were derived from the different species of their carboxyl-terminal region, were similar to those of SREBPs corresponding to their carboxyl termini. Therefore, it is suggested that the carboxyl-terminal portions of SREBPs containing DNA binding domains are important in determining their transactivation potencies to a certain promoter.

ATP Citrate (pro-S)-Lyase↗

PubMed1999

Characterization of regulatory elements on the promoter region of human ATP-citrate lyase.

ATP-citrate lyase (ACL), an enzyme catalyzing the first step in biosynthesis of fatty acids, is induced during the lipogenesis and cholesterologenesis. We demonstrate that the region -213 to -128 of human ACL promoter is responsible for conferring glucose-mediated transcription. This region in the ACL promoter contains Sp1 binding sites determined by DNase I foot-printing assay. Gel retardation assay using oligonucleotides from -179 to -141 and -140 to -110 showed two specific DNA-protein complexes postulated to be formed by transcription factor Sp1. Competition gel shift and supershift assays have confirmed that these DNA-protein complexes were the result of induced Sp1 as well as another Sp1-related proteins. Western blot analysis also demonstrated that transcription factor Sp1 was slightly increased in the nuclear proteins extracted from Alexander cells following supplementation of glucose. In addition, expression of 110 kDa protein reacting with antibody against Sp3 was dramatically increased by glucose supplementation, while isoforms of Sp3, about 80 kDa in size was decreased in its amounts. Our results suggest that changes in the expression of Sp1 family proteins play an important role in activation of the ACL promoter by glucose.

ATP Citrate (pro-S)-Lyase↗

PubMed1996

Cloning and identification of exon-intron organization of the rat ATP-citrate lyase gene.

The rat ATP-citrate lyase gene was cloned and the complete exon-intron organization of the gene has been identified. The ATP-citrate lyase gene, spanning about 55 kb, is divided into 29 exons that range in size from 30 to 986 base pairs. The sequences bordering the splice site junctions universally follow the GT/AG rule. Reverse transcription-polymerase chain reaction showed two forms of ACL mRNA; the one containing complete exons and the other lacking exon 14 were found in the brain, kidney, mammary gland, lung and liver. Also, the restriction fragment length polymorphisms were observed at intron 10 and intron 11 regions.

ATP Citrate (pro-S)-Lyase↗

PubMed1996

Regulation of ATP-citrate lyase gene transcription.

It has been suggested that glucose metabolites and insulin are the most important factors inducing ATP-citrate lyase (ACL) by a high carbohydrate diet. We have used a primary culture of rat hepatocytes to confirm the role of glucose and insulin in terms of ACL gene expression. The results showed that glucose displayed a direct effect on ACL gene expression and the insulin helps the glucose effect. The nucleotide sequences from -512 to -485 of the ACL promoter are highly homologous (70%) to the sequences surrounding the carbohydrate response element (ChoRE) of the S14 gene. The gel retardation analysis using ChoRE of the S14 gene showed that the ACL promoter which contains the ChoRE-like sequence specifically inhibited the formation of the complex by the nuclear proteins isolated from rat liver. To localize the regions which are involved in the regulation of ACL gene expression, transient expression assay using ACL promoter-CAT (chloramphenicol acetyltransferase) constructs containing various lengths of a 5' flanking region of the ACL gene were carried out. The proximal promoter region -419 to -1 containing several potential Sp1 binding sites showed the strong enhancing effect, which increases the transcription of CAT genes in the various cell lines, such as the CHO (Chinese hamster ovary) cell, the HepG2 cell, and primary cultured rat hepatocytes. In response to glucose, among the ACL promoter-CAT constructs, only pNP33-CAT (-1342 to -1) showed a 2.64 fold increase in CAT activity by a high concentration of glucose. The activation of ACL gene expression by glucose seems to be regulated in a complicated manner involving interactions between the contexts of the several sequence elements and various transacting factors, which is not a simple mechanism directed only by a short sequence element.

ATP Citrate (pro-S)-Lyase↗

PubMed1994

Organization of the 5' region of the rat ATP citrate lyase gene.

A genomic clone, encompassing the 5' flanking region and the first seven exons of rat ATP citrate lyase gene, was isolated from a rat genomic library and sequenced. Primer-extension analysis showed that mRNA is transcribed at 4407 nucleotides upstream from the translation start site. Primer-extension analysis and sequencing of ATP citrate lyase cDNA amplified by PCR showed that the promoter used for transcription is identical in mammary gland, lung, liver, brain and kidney. Southern-blot analysis showed that the ATP citrate lyase gene exists as a single copy. The 5' flanking region contains several consensus sequences defined as promoter elements. These include a CAAT box and Sp1-binding sites. However, a TATA box lacks this promoter. The expression of the chloramphenicol acetyltransferase gene was induced by the 5' flanking region (-2370 to -1) in the CHO cell line. The 5' flanking region also contains several sequence elements that may be involved in the transcriptional regulation of the gene.

ATP Citrate (pro-S)-Lyase↗

PubMed1994

Expression and localization of human papillomavirus type 16 E6 and E7 open reading frame proteins in human epidermal keratinocyte.

Over 60 different types of human papillomavirus (HPV) have been identified, and they are classified into high and low risk groups based on the risk for malignant progression of HPV associated lesions. HPVs belonging to a high risk group have been shown to express two major transforming proteins, E6 and E7. With respect to the transforming activity of these proteins, many investigators have reported the location of these proteins in the cell, but their results are still controversial. In the present study, HPV type 16 E6 or E7 open reading frame (ORF) proteins were expressed and localized in human epidermal keratinocytes (RHEK-1) using the vaccinia virus as an expression vector. Immunofluorescence detection using monoclonal antibodies against E6 or E7 ORF proteins revealed that E6 or E7 proteins of HPV type 16 were located in the cytoplasm of RHEK-1 cells. These results suggest that E6 and E7 proteins bind to the tumor suppressor counterparts, thereby preventing transport of these proteins into the nucleus. These antioncogene products that fail to be rapidly transported out of the cytosol may be degraded by certain proteases such as the ubiquitin dependent system. In this way, the precise function of antioncogene products in the regulation of cell growth could be destroyed, and abnormal cell growth could occur.

Animals↗