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PubMed1989

Transcription of Xenopus selenocysteine tRNA Ser (formerly designated opal suppressor phosphoserine tRNA) gene is directed by multiple 5'-extragenic regulatory elements.

A tRNA gene whose product is aminoacylated with serine and the serine moiety is then phosphorylated to form phosphoseryl-tRNA (see Hatfield, D. (1985) Trends Biochem. Sci. 10, 201-204 for review) has now been shown to form selenocysteyl-tRNA; hence the corresponding gene is designated as selenocysteine tRNA Ser (B. J. Lee, P. J. Worland, J. N. Davis, T. C. Stadtman, and D. Hatfield (1989) J. Biol. Chem. 264, in press). In the present study, we show that the expression of this unique tRNA gene is governed by at least three upstream regulatory elements. In initial studies, the relative efficiencies of transcription of the human, rabbit, chicken, and Xenopus selenocysteine tRNA genes were compared in vivo in Xenopus oocytes and in vitro in HeLa cell extracts. The Xenopus gene was severalfold more actively expressed, both in vivo and in vitro, than the human and rabbit genes, whereas the chicken gene was poorly expressed. Exchange of the 5'-flanking regions of the Xenopus and chicken genes, which have identical gene sequences, reversed their levels of transcription, demonstrating that a regulatory site or sites exist upstream of these genes. Deletion-substitution mutants in the Xenopus gene and its 5'-flanking sequence show in in vitro assays that 1) the level of transcription is reduced substantially when a GC-rich stretch that is immediately upstream of a TATA box in the -30 region is removed; 2) the level of transcription is virtually abolished when the TATA box is removed; and 3) deletions up to and further upstream of the GC-rich region do not affect the level of transcription. The same deletions, when used in in vivo assays, demonstrate a step-down in expression with the deletion removing the GC-rich region, a further step-down in expression with the deletion removing the TATA box, but the most pronounced reduction in expression was observed with a deletion removing an AT-rich region between nucleotides -62 and -76. Thus, a regulatory site was identified in vivo which was not detected in vitro, and transcription of the selenocysteine tRNA Ser gene is determined by multiple upstream regulatory elements.

Amino Acid Sequence↗

PubMed1994

SIS/PDGF-B promoter isolation and characterization of regulatory elements necessary for basal expression of the SIS/PDGF-B gene in U2-OS osteosarcoma cells.

Platelet-derived growth factor BB, encoded by the SIS/PDGF-B gene, is a potent mitogen for cells of mesenchymal origin, and the SIS/PDGF-B gene is expressed in a large percentage of human mesenchymal tumor cells establishing a growth-promoting, autocrine growth circuit. A 4-kb fragment, containing the SIS/PDGF-B promoter, was isolated from a human genomic library, and a series of 5'-nested deletions and linker-scanning mutants were used to identify positive regulatory elements that are necessary for the constitutive expression of this gene in human U2-OS osteosarcoma cells. A 250-bp fragment, lying immediately 5' to the SIS/PDGF-B mRNA initiation site (+1), retained full promoter activity, and positive regulatory elements at -228 to -219, -97 to -88 (SIS distal element) and -58 to -39 (SIS proximal element, SPE) were identified. Insertion of the 20-bp SPE into a heterologous, minimal promoter resulted in >5-fold transcriptional activation which was ablated by mutations to the SPE. High resolution mutagenesis within the 20-bp SPE, indicated the necessity of a CACCC motif for activity. Gel shift analysis of SPE-binding proteins in U2-OS nuclear extracts identified Sp1 and two additional binding factors that could be competed away from SPE binding by adding excess consensus Sp1 or CACC oligonucleotides. The individual and aggregate roles of the SPE and two weaker positive regulatory elements in regulating SIS/PDGF-B transcription in these tumor cells is considered.

Base Sequence↗

PubMed2004

Targeting promiscuous signaling pathways in cancer: another Notch in the bedpost.

The chromosomal translocation t(7;9)(q34;q34.3) in human T-cell acute lymphoblastic leukemia results in the constitutive activation of Notch (Nic). Reported mutations in Ikaros cause the loss of DNA-binding, which in turn leads to a loss of repressive activity. Recently, these two mutations have been shown to cooperate in leukemogenesis. The current model proposes that the combination of the loss of Ikaros activity and the gain of constitutive Notch activity disrupts the normal balance between repression and activation at common regulatory elements. Furthermore, the model is extended to suggest that multiple transcription factors coordinate transcriptional repression and activation through these common regulatory elements. In leukemogenesis, the breakdown of this coordinate regulation underlies one of the pathophysiological mechanisms. Finally, using Notch as a template, potential points of interdiction by designer therapeutics are discussed.

DNA-Binding Proteins↗

PubMed1996

Angiotensin II induction of fibronectin biosynthesis in cultured human mesangial cells: association with CREB transcription factor activation.

We assessed the effect of angiotensin II on fibronectin biosynthesis a nd transcription factor activation in adult human mesangial cells in culture. We found that 10(-5) mol/L angiotensin II tended to increase fibronectin mRNA expression within 1 hour (1.2-fold +/- 0.3-fold of that in controls), with a significant increase after 4 hours (0.3-fold +/- 0.1-fold of that in controls, p < 0.05) and 24 hours (1.9-fold +/- 0.3-fold of that in controls, p < 0.02). In conjunction with increased fibronectin mRNA levels, angiotensin II exposure resulted in a significant elevation in immunoreactive fibronectin concentrations and the incorporation of (35S)-labeled methionine into fibronectin after 2 hours (224% +/- 23% of controls, p < 0.05). Angiotensin II also induced mesangial cell activation of the cyclic adenosine monophosphate response element binding protein (CREB) transcription factor, a DNA binding protein known to recognize specific regulatory elements present on the fibronectin gene promoter. Using the electrophoretic mobility shift assay, we showed that angiotensin II increased mesangial cell expression of the activated form of CREB after 4 hours (1.2-fold +/- 0.04-fold of that in controls, p < 0.05). To determine the importance of the CREB regulatory elements in mediating angiotensin II induction of fibronectin gene transcription, JEG-3 cells were transfected with plasmids containing fibronectin promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs with (FN510) or without (FN122) the CREB regulatory motifs. Angiotensin II resulted in a significant increase in CAT activity in FN510 transfectants (1.6-fold +/- 0.2-fold of that in controls, p < 0.05), but there was no effect of angiotensin II on FN122 transfected cells. These data demonstrate that angiotensin II stimulates fibronectin biosynthesis in adult human mesangial cells and suggest that the process may be regulated at the transcriptional level.

Angiotensin II↗

PubMed2003

Involvement of multiple transcription factors in the regulation of steroidogenic acute regulatory protein gene expression.

The rate-limiting, committed, and regulatable step in steroid hormone biosynthesis is the transport of cholesterol from the outer to the inner mitochondrial membrane, a process that is mediated by the steroidogenic acute regulatory (StAR) protein. In steroidogenic cells, the StAR protein is regulated by cAMP-dependent mechanisms. However, the StAR promoter lacks a consensus cAMP response-element (CRE), suggesting the involvement of alternate regulatory factor(s) in cAMP responsiveness. These regulatory elements are found to be located in a transcription factor-binding site-rich region (consisting of approximately 150 nucleotides upstream of the transcription start site) of the StAR promoter, and appears to be the most important region in regulating transcription of the StAR gene. The StAR promoter sequences in mouse, rat and human are highly homologous, and in the absence of a canonical CRE, multiple cis-elements have been shown to be instrumental in the regulation of StAR gene expression. Nevertheless, it has become apparent that functional cooperation, interaction, and alteration of different transcription factors are involved in the fine-tuning of the regulatory events associated with StAR gene transcription.

Animals↗

PubMed1992

Characterization of a minimal promoter element required for transcription of the mouse type II beta regulatory subunit (RII beta) of cAMP-dependent protein kinase.

The 5'-flanking DNA of the mouse RII beta subunit of the cAMP-dependent protein kinase gene was characterized by transient transfection of RII beta-CAT constructs into mouse neuroblastoma cells (NB2a) and Chinese hamster ovary (CHO) cells and by gel mobility shift and footprinting assays. The minimal promoter of the RII beta gene was composed of two adjacent functional elements. A 3'-element which supported enhanced CAT activity was located between base pairs (bp) -267/-168 from the translation initiation start site. CAT plasmids containing these RII beta sequences showed 12- and 16-fold increased CAT activity in the NB2a and CHO cells, respectively, compared to the basic CAT vector. Plasmids containing 20 additional bp 5' to the -267/-168 fragment showed 2-fold more CAT activity than the shorter fragment in NB2a cells, while CAT activity in CHO cells was nearly the same for both constructs. CAT plasmids containing only this 20-bp fragment showed 9- and 13-fold increased CAT activity in NB2a and CHO cells, respectively. The core promoter of the RII beta gene lacked classical TATA and CAT sequences, but contained 3 copies of the Sp1 core consensus sequence. Gel mobility shift assays using 32P-labeled 5'-flanking DNA containing bp -291/-49 and nuclear extracts from NB2a and CHO cells displayed several retarded bands in the gels suggesting complex formation with nuclear DNA-binding factors. Unlabeled DNA containing bp -291/-49 blocked the appearance of all retarded bands. Competition using an oligonucleotide corresponding to the Sp1 DNA-binding site effectively blocked the appearance of the two more slowly migrating bands but did not affect the major rapidly migrating bands. DNase I footprinting analysis using purified Sp1 protein confirmed that Sp1 could bind to the Sp1 sites. Methylation interference and mutational analysis showed that one of the faster migrating bands was the result of factor binding to the DNA sequence adjacent to the Sp1 sites. Additional tissue-specific nuclear-binding factor sequences were detected upstream of the core promoter. Our data suggest that the core promoter of the RII beta gene can initiate transcription from the DNA around the Sp1 sites but that there are tissue-specific nuclear factor-binding sites located distal to the Sp1 sites.

Animals↗

PubMed1993

Identification of positive and negative regulatory elements involved in the retinoic acid/cAMP induction of Fgf-3 transcription in F9 cells.

The proto-oncogene Fgf-3 has been implicated as an important signalling molecule in vertebrate development. In the mouse, it is expressed for a limited time at a multitude of sites from embryonic day 7 to birth. Transcription of Fgf-3 initiates at three promoter regions resulting in the generation of various mRNAs which nevertheless all encode the same protein products. A 1.7kb DNA fragment which encompasses these regions was joined to the CAT reporter gene and shown to function as a promoter in embryonal carcinoma cells. In stable transfectants the promoter retains its retinoic acid inducibility, initiating transcription at the same cap-sites as the endogenous gene. In differentiated F9 cells, transient transfection of progressive and targeted deletion mutants of the promoter region has revealed at least two positive and three negative regulatory elements. With one exception, loss of these elements was shown to dramatically affect promoter activity in stable transfectants of F9 cells. However the promoter remained inducible by retinoic acid to differing degrees, apart from deletions encompassing PS-4A which essentially abolished promoter activity in both undifferentiated and differentiated cells. The sequences of these potential regulatory regions were further defined using DNase-I footprinting, revealing some similarities to consensus binding sites for known transcription factors.

Animals↗

PubMed1999

The transcription factor Sp3 interacts with promoter elements of the lens specific MIP gene.

PURPOSE: To characterize the cis regulatory elements and their interaction with transcription factors responsible for the lens specific expression of the MIP gene, which encodes the Major Intrinsic Protein of the lens fiber membranes. METHODS: Study interaction of factors present in newborn mouse lens nuclear extracts with DNA fragments corresponding to mouse MIP gene 5' flanking sequence by electrophoresis mobility shift assay (EMSA) and DNase I footprinting. RESULTS: We found a high degree of identity in the first 100 bp of 5' flanking sequence of mice and humans, however, a lower degree of conservation is observed further upstream. We have found by DNase I footprinting analysis that lens specific factors may interact with the first 100 bp of 5' flanking sequence. A domain containing an E box, conserved in mouse and human, may interact with a lens specific factor. However, general factors may interact with a NF-1 binding site. An overlapping GC and CT box is present in the mouse MIP gene. In the human MIP gene GC and CT boxes are found in different domains of the MIP gene promoter. Both CT boxes interact with factors present in lens nuclear extracts including Sp3. They are able to interact with purified Sp1but not with Sp1 present in mouse lens nuclear extracts. CONCLUSIONS: The transcription factor Sp3 may play an important role in regulating MIP gene expression in the lens.

Animals↗

PubMed2004

SREBF-1 gene polymorphisms are associated with obesity and type 2 diabetes in French obese and diabetic cohorts.

Sterol regulatory element-binding protein (SREBP)-1 transcription factors play a central role in energy homeostasis by promoting glycolysis, lipogenesis, and adipogenesis. The sterol regulatory element-binding protein gene (SREBF)-1 is a good candidate gene for obesity and obesity-related metabolic traits such as type 2 diabetes and dyslipidemia. The SREBF-1 molecular screening of 40 unrelated obese patients by PCR/single-strand conformation polymorphism revealed 19 single nucleotide polymorphisms (SNPs). Six SNPs were genotyped for an association study in large French obese and nonobese cohorts. Case-control studies using two independent nonobese cohorts indicated that SNP17 (54G/C, exon 18c) is associated with morbid obesity (odds ratio 1.5, P = 0.006 and P = 0.02, respectively). SNP3 (-150G/A, exon 1a), SNP5 (-36delG, exon 1a), and SNP17 are found in high linkage disequilibrium (D' > 0.8). The haplotype including wild-type alleles of these SNPs (C/G/G/T/C/G, HAP2) is identified as a risk factor for morbid obesity (P = 0.003). In the obese group, SNP3, SNP5, and SNP17 are associated with male-specific hypertriglyceridemia (P = 0.07, P = 0.01, and P = 0.05, respectively). SNP17 is also associated with type 2 diabetes (P = 0.03) and increased prevalence of nephropathy (P = 0.028) in a diabetic cohort. Our results indicate a role of the SREBF-1 gene in genetic predisposition of metabolic diseases such as obesity, type 2 diabetes, and dyslipidemia.

Adult↗

PubMed1997

The mouse prosaposin locus: promoter organization.

Prosaposin is a multifunctional protein that, when secreted, functions as a neurotrophic agent and, when retained in the lysosomes, is processed to essential glycosphingolipid hydrolase activator proteins. The prosaposin locus is temporarily and spatially regulated at the transcriptional and post-translational levels. The prosaposin gene has been partially characterized, but the 5' region has not. RACE, S1 nuclease protection, and sequence analysis were used to characterize the first intron and first exon as well as the 5'-flanking regions from murine P1 clones. The first intron is approximately 15 kb in length and the complete gene is approximately 25 kb. The transcriptional initiation sites are located 87 and 94 bp 5' to the ATG in exon 1. Using luciferase as a reporter gene and transfection into NS20Y, NIH-3T3, or SF-7 Sertoli cell cultures, deletion constructs from the 5' putative promoter region were shown to contain positive and negative regulatory elements within 2,400 bp 5' to the transcription start site. A negative regulatory element is located between 742 and 310 bp 5' to the transcription start site. These studies provide insight into the regulation of this unique "lysosomal" locus.

3T3 Cells↗

PubMed2006

A specific helical orientation underlies the functional contribution of the activin responsive unit to transcriptional activity of the murine gonadotropin-releasing hormone receptor gene promoter.

Activin responsiveness of the murine GnRH receptor (GnRHR) gene promoter requires two spatially distinct regulatory elements termed the GnRH receptor activating sequence or GRAS and the downstream activin regulatory element or DARE. While GRAS interacts with multiple transcription factors, DARE activity requires tandem homeodomain binding motifs (TAAT) and displays specific binding to the LIM homeodomain protein LHX3. Herein, we find that both the murine GnRHR gene promoter and DARE fused to a minimal heterologous promoter are responsive to LHX3 overexpression. A dominant-repressor of LHX3 attenuates transcriptional activity of the murine GnRHR gene promoter but had no impact on activin responsiveness. Thus, LHX3 would not appear to be the protein mediating activin responsiveness of this promoter. Within DARE itself, the tandem TAAT motifs are separated by 4 bp. Although this arrangement differs from the prototypical P2 or P3 binding sites characterized for paired-like homeodomain proteins and from the directly abutting TAAT motifs found for LHX3, a LIM-class homeodomain protein, we find that separation of the TAAT sites by 5 and 10 bp decreases GnRHR promoter activity to a level similar to promoters containing loss of function mutations in either the proximal or distal TAAT motif. Thus, the juxtaposition of the TAAT sites is critical for the functional activity of DARE. That activin responsiveness of the GnRHR promoter requires both GRAS and DARE suggests that these elements may be both functionally and structurally coupled. As to the latter, GRAS and DARE are separated by 20 bp, thus placing the elements on the same side of the helical backbone. To determine if this spatial organization is functionally relevant, multiples of 5 bp were inserted or deleted between GRAS and DARE. Any insertion or deletion that resulted in a half-turn alteration in the helical positioning between the two elements reduced promoter activity. Thus, an important spatial relationship underlies functional cooperation between GRAS and DARE and the emergence of a complex activin responsive unit (ARU) within the mouse GnRHR promoter.

Activins↗

PubMed2005

Lymphoid cell growth and transformation are suppressed by a key regulatory element of the gene encoding PU.1.

Tight regulation of transcription factors, such as PU.1, is crucial for generation of all hematopoietic lineages. We previously reported that mice with a deletion of an upstream regulatory element (URE) of the gene encoding PU.1 (Sfpi1) developed acute myeloid leukemia. Here we show that the URE has an essential role in orchestrating the dynamic PU.1 expression pattern required for lymphoid development and tumor suppression. URE deletion ablated B2 cells but stimulated growth of B1 cells in mice. The URE was a PU.1 enhancer in B cells but a repressor in T cell precursors. TCF transcription factors coordinated this repressor function and linked PU.1 to Wnt signaling. Failure of appropriate PU.1 repression in T cell progenitors with URE deletion disrupted differentiation and induced thymic transformation. Genome-wide DNA methylation assessment showed that epigenetic silencing of selective tumor suppressor genes completed PU.1-initiated transformation of lymphoid progenitors with URE deletion. These results elucidate how a single transcription factor, PU.1, through the cell context-specific activity of a key cis-regulatory element, affects the development of multiple cell lineages and can induce cancer.

Animals↗

PubMed1998

Transcriptional regulation of the GluR2 gene: neural-specific expression, multiple promoters, and regulatory elements.

To understand how neurons control the expression of the AMPA receptor subunit GluR2, we cloned the 5' proximal region of the rat gene and investigated GluR2 promoter activity by transient transfection. RNase protection and primer extension of rat brain mRNA revealed multiple transcription initiation sites from -340 to -481 bases upstream of the GluR2 AUG codon. The relative use of 5' start sites was different in cortex and cerebellum, indicating complexity of GluR2 transcript expression among different sets of neurons. When GluR2 promoter activity was investigated by plasmid transfection into cultured cortical neurons, cortical glia, and C6 glioma cells, the promoter construct with the strongest activity, per transfected cell, was 29.4-fold (+/- 3.7) more active in neurons than in non-neural cells. Immunostaining of cortical cultures showed that >97% of the luciferase-positive cells also expressed the neuronal marker MAP-2. Evaluation of internal deletion and substitution mutations identified a functional repressor element I RE1-like silencer and functional Sp1 and nuclear respiratory factor-1 (NRF-1) elements within a GC-rich proximal GluR2 promoter region. The GluR2 silencer reduced promoter activity in glia and non-neuronal cell lines by two- to threefold, was without effect in cortical neurons, and could bind the RE1-silencing transcription factor (REST) because cotransfection of REST into neurons reduced GluR2 promoter activity in a silencer-dependent manner. Substitution of the GluR2 silencer by the homologous NaII RE1 silencer further reduced GluR2 promoter activity in non-neuronal cells by 30-47%. Maximal positive GluR2 promoter activity required both Sp1 and NRF-1 cis elements and an interelement nucleotide bridge sequence. These results indicate that GluR2 transcription initiates from multiple sites, is highly neuronal selective, and is regulated by three regulatory elements in the 5' proximal promoter region.

Animals↗

PubMed2003

Protein-tyrosine phosphatase 1B as new activator for hepatic lipogenesis via sterol regulatory element-binding protein-1 gene expression.

Like hyperglycemia, postprandial (diet-induced) hypertriglyceridemia is thought to play crucial roles in the pathogenesis of insulin resistant/metabolic syndrome. Sterol regulatory element-binding protein-1 (SREBP-1) is a key transcription factor to induce postprandial hypertriglyceridemia. We found that insulin-resistant rats fed a diet high in fructose showed an increased proteintyrosine phosphatase 1B (PTP1B) content with strong expression of SREBP-1 mRNA in the liver. To clarify the association of PTP1B with SREBP-1 gene expression, we overexpressed PTP1B in rat hepatocytes, which led to increased mRNA content and promoter activity of SREBP-1a and -1c, resulting in the increased mRNA expression of fatty-acid synthase, one of the SREBP-1-responsive lipogenic genes. Because PTP1B overexpression increased phosphatase 2A (PP2A) activity, we inhibited PP2A activity by expression of its selective inhibitor, SV40 small T antigen and found that this normalized the PTP1B-enhanced SREBP-1a and -1c mRNA expressions through activation of the Sp1 site. These results indicate that PTP1B may regulate gene expression of SREBP-1 via enhancement of PP2A activity, thus mediating hepatic lipogenesis and postprandial hypertriglyceridemia. We demonstrate here a unique serial activation of the PTP1B-PP2A axis as a novel mechanism for the regulation of gene expression in the biosynthesis of triglyceride.

Adenoviridae↗

PubMed2006

Sterol regulatory element-binding protein-2 negatively regulates low density lipoprotein receptor-related protein transcription.

Low density lipoprotein receptor-related protein (LRP1) binds aggregated LDL (agLDL) leading to a high intracellular cholesteryl ester (CE) accumulation. AgLDL up-regulates LRP1 expression concomitantly with an LDL receptor (LDLR) and sterol regulatory element binding protein (SREBP-2) down-regulation. The objectives were to investigate whether SREBP-2 regulates LRP1 transcription and determine the molecular mechanisms involved in the process. Down-regulation of active SREBP-2 by nLDL and agLDL led to LDLR down-regulation and LRP1 up-regulation. Enforced expression of an active form of SREBP-2 (SREBP-2-NT, amino acid residues 1-468) decreased LRP1 expression and LRP1 promoter (WT-LRP1) luciferase activity in a dose-dependent manner. LDL did not exert any significant effect on LRP1 promoter activity when a putative sterol regulatory element (SRE) (5-GTGGGGTGA-3'; +225 to +233) was mutated (SRE-MT-LRP1). SREBP-2 overexpression exerted stronger down-regulatory effects on WT-LRP1 than on SRE-MT-LRP1 promoter activity both in control, nLDL- and agLDL-exposed HeLa cells. Gel mobility shift assays showed that recombinant SREBP-2-NT protein (1-468) binds to a double-stranded LRP1 DNA fragment (215 to 245) containing a wild-type (wt) SRE sequence but not to a mutated SRE (mt) sequence (5-GAATTCGA-3'). Our results demonstrate that LDL stimulates LRP1 transcription and decreases SREBP-2 active form which negatively regulates LRP1 transcription. SRE sequence (+225 to +233) plays a pivotal role for the down-regulatory effect of SREBP-2 on LRP1 promoter activity.

5' Untranslated Regions↗

PubMed1987

Different transcript abundance of two divergent ATP synthase subunit 9 genes in the mitochondrial genome of Petunia hybrida.

The mitochondrial genome of Petunia hybrida line 3704 has been found to contain two transcribed genes for the proteolipid subunit of the ATP synthase complex (atp 9). The 5' and 3' flanking sequences of the second atp 9 gene differ from those of another atp 9 gene previously sequenced by Young et al. (1986), while the coding region exhibits only one silent base change. The transcript termini of the two divergent atp 9 genes map to different locations, most of which are surrounded by sequences homologous to putative mitochondrial transcription signals (Hiesel and Brennicke 1985; Schuster et al. 1986; Young et al. (1986). Because transcripts from the atp 9 genes differ in abundance, divergence in the flanking regions of these two genes must affect regulatory elements which control either transcription rate or transcript stability.

Amino Acid Sequence↗

PubMed2001

Induction of nuclear transcription factors, cytochrome P450 monooxygenases, and glutathione S-transferase alpha gene expression in Aroclor 1254-treated rat hepatocyte cultures.

Aroclor 1254 is a complex mixture of polychlorinated biphenyls and is well known for its potency to induce drug-metabolising enzymes, but little is known about its ability to modulate gene expression of transcription factors, which code for proteins that bind to the regulatory elements of DNA and facilitate transcriptional activation. We therefore investigated the gene expression of the liver-specific transcription factors CCAAT/enhancer-binding protein alpha (c/EBPalpha), hepatic nuclear factor (HNF) 1 and 4, and major cytochrome P450 (CYP) isozymes in addition to glutathione S-transferase alpha 2 (GSTA-2) in cultures of primary rat hepatocytes. We found highly significant and dose-dependent increases of c/EBPalpha (up to 62-fold), HNF-1 (up to 7-fold), HNF-4 (up to 8-fold), and 50- and 4-fold inductions of GSTA-2 and CYP monooxygenases, respectively. Based on the ethoxyresorufin-O-deethylase assay, the gene expression and enzyme activity for CYP1A1 were in good agreement, but for other CYP isozymes similar correlations could not be obtained. In conclusion, the simultaneous induction of liver-specific TFs and of several detoxifying enzymes may point to a coordinate genomic response in cultures of rat hepatocytes upon treatment with Aroclor 1254.

Animals↗

PubMed1998

Blunted feedback suppression of SREBP processing by dietary cholesterol in transgenic mice expressing sterol-resistant SCAP(D443N).

Feedback regulation of cholesterol biosynthesis is mediated by membrane-bound transcription factors designated sterol regulatory element-binding proteins (SREBP)-1 and -2. In sterol-deprived cultured cells, SREBPs are released from membranes by a proteolytic process that is stimulated by SREBP cleavage-activating protein (SCAP), a membrane protein containing a sterol-sensing domain. Sterols suppress SREBP cleavage by blocking the action of SCAP, thereby decreasing cholesterol synthesis. A point mutation in SCAP(D443N) causes resistance to sterol suppression. In this article, we produced transgenic mice that express mutant SCAP(D443N) in liver. In these livers the nuclear content of SREBP-1 and -2 was increased, mRNAs encoding proteins involved in uptake and synthesis of cholesterol and fatty acids were elevated, and the livers were engorged with cholesteryl esters and triglycerides enriched in monounsaturated fatty acids. When the mice were challenged with a high cholesterol diet, cleavage of SREBP-1 and -2 was reduced in wild-type livers and less so in transgenic livers. We conclude that SCAP(D443N) stimulates proteolytic processing of native SREBPs in liver and decreases the normal sterol-mediated feedback regulation of SREBP cleavage, suggesting a central role for SCAP as a sterol sensor in liver.

Animals↗

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