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Ap1-like Cis elements in the 5′-regulatory region of the human apolipoprotein A-I gene
Abstract
Ap1-like cis elements, interacting with transcription factors of the Ap1 and CREB/ATF families, were identified in the 5′-regulatory region
of the human apolipoprotein A-I gene (5′-apoA-I). The elements are beyond the hepatic enhancer (−220…−110) and region −595…−192, responsible for efficient transcription
in Caco2 cells. One of the elements (5′-TGAGGTCT-3′, Cre/jun2/apo) occurs in 5′-apoA-I in two copies, distal (−2176…−2165) and proximal (99…106). The electrophoretic mobility shift assay was used to characterize
this and two other 5′-apoA-I Ap1-like elements: 5′-TGACTCT-3′ (−1798…−1791, PF1) and 5′-TGACATCA-3′ (−1171…−1163, Cre/jun1). Experiments with specific
antibodies identified ATF2 as a component of the complexes formed by HepG2 nuclear proteins with Cre/jun2/apo and Cre/jun1.
Several 5′-apoA-I deletion derivatives were examined in HepG2 hepatoma cells (producing ApoA-I), Hutu80 duodenal adenocarcinoma cells (lacking
ApoA-I production), and SK-N-SH neuroblastoma cells (carrying defective apoA-I) by the luciferase reporter assay. Combined with cell cotransfection with c-jun and mekk1 expression vectors, the assays implicated the Ap1-like elements in the tissue-specific regulation of apoA-I expression, the proximal Cre/jun2/apo element playing the major role.
... PLASMIDS pCMVL, the expression vector for bacterial reporter gene lacZ under the control of the early human cytomegalovirus gene promoter (CMV), has been described previously [Lapikov et al., 2008]. pApoA-I(À2498/ þ72)-LUC and pAPOA-I(À256/þ72)-LUC, the plasmids containing the firefly luciferase reporter gene under control of deletion variants of the 5 0 -regulatory region of the human apoA-I gene (positions À2498 to þ72 and positions À256 to þ72, respectively) related to the transcription start point (TSP) of the human apoA-I gene, have been described earlier [Lapikov et al., 2008]. ...
... PLASMIDS pCMVL, the expression vector for bacterial reporter gene lacZ under the control of the early human cytomegalovirus gene promoter (CMV), has been described previously [Lapikov et al., 2008]. pApoA-I(À2498/ þ72)-LUC and pAPOA-I(À256/þ72)-LUC, the plasmids containing the firefly luciferase reporter gene under control of deletion variants of the 5 0 -regulatory region of the human apoA-I gene (positions À2498 to þ72 and positions À256 to þ72, respectively) related to the transcription start point (TSP) of the human apoA-I gene, have been described earlier [Lapikov et al., 2008]. The mutations of sites A and C within pAPOA-I (À256/þ72)-LUC were generated with QuikChange Site-Directed Mutagenesis Kit (Stratagen) using the following oligonucleotides: 5 0 -CCCGCCCCACTGAACGGTTATAAACTGCCCTGCAGCCCC-3 0 for site A and 5 0 -GCCCCAGGGACAGCGCTGAGATATCTACTCTTAAGGTTCC-AC-3 0 for site C and corresponding antisense oligonucleotides (the changed nucleotides are underlined). ...
Apolipoprotein A-I (ApoA-I) is the main anti-atherogenic component of human high-density lipoproteins (HDL). ApoA-I gene expression is regulated by several nuclear receptors, which are the sensors for metabolic changes during development of cardiovascular diseases. Activation of nuclear receptor PPARγ has been shown to impact lipid metabolism as well as inflammation. Here, we have shown that synthetic PPARγ agonist GW1929 decreases both ApoA-I mRNA and protein levels in HepG2 cells and the effect of GW1929 on apoA-I gene transcription depends on PPARγ. PPARγ binds to the sites A and C within the hepatic enhancer of apoA-I gene and the negative regulation of apoA-I gene transcription by PPARγ appears to be realized via the site C (-134 to -119). Ligand activation of PPARγ leads to an increase of LXRβ and a decrease of PPARα binding with the apoA-I gene hepatic enhancer in HepG2 cells. GW1929 abolishes the TNFα-mediated decrease of ApoA-I mRNA expression in both HepG2 and Caco-2 cells but does not block TNFα-mediated inhibition of ApoA-I protein secretion by HepG2 cells. These data demonstrate that complex of PPARγ with GW1929 is a negative regulator involved in the control of ApoA-I expression and secretion in human hepatocyte- and enterocyte-like cells. This article is protected by copyright. All rights reserved.
... The pCMV-LacZ plasmid containing the early human cytomegalovirus gene promoter and bacterial β-galactosidase reporter gene LacZ was described in [24]. The pA1(-256/+72)-Luc (pA1-Luc) plasmid containing the firefly luciferase reporter gene under control of the HE (positions -256 to +72 relative to the human APOA1 transcription initiation site) was described in [25]. The pA1(-256/+72) mutA(or mutC)-Luc plasmids with mutations in the sites A or C of the HE were obtained as described in [10]. ...
Adiponectin is an adipose tissue hormone, participating in energy metabolism and involved in atherogenesis. Previously, it was found that adiponectin increases expression of the APOA1 (apolipoprotein A-1) gene in hepatocytes, but the mechanisms of this effect remained unexplored. Our aim was to investigate the role of adiponectin receptors AdipoR1/R2, AMP-activated protein kinase (AMPK), nuclear peroxisome proliferator-activated receptor alpha (PPARα) and liver X receptors (LXRs) in mediating the action of adiponectin on hepatic APOA1 expression in human hepatoma HepG2 cells. The level of APOA1 expression was determined by RT-qPCR and ELISA. We showed that the siRNA-mediated knockdown of genes coding for AdipoR1, AdipoR2, AMPK, PPARα, and LXRα and β prevented adiponectin-induced APOA1 expression in HepG2 cells and demonstrated that interaction of PPARα and LXRs with the APOA1 gene hepatic enhancer is important for the adiponectin-dependent APOA1 transcription. The results of this study point out to the involvement of both types of adiponectin receptors, AMPK, PPARα, and LXRs in the adiponectin-dependent upregulation of the APOA1 expression.
... Plasmids The pCMVLacZ plasmid (Dizhe et al. 2006) and pA1(−256/+72) (Lapikov et al. 2008) were described elsewhere. The plasmids pA1(−256/+72) with disrupted A or C sites (Shavva et al. 2016b) and B site (Shavva et al. 2016a) were described in the previous works. ...
Reactive oxygen species damage various cell components including DNA, proteins, and lipids, and these impairments could be a reason for severe human diseases including atherosclerosis. Forkhead box O1 (FOXO1), an important metabolic transcription factor, upregulates antioxidant and proapoptotic genes during oxidative stress. Apolipoprotein A-I (ApoA-I) forms high density lipoprotein (HDL) particles that are responsible for cholesterol transfer from peripheral tissues to liver for removal in bile in vertebrates. The main sources for plasma ApoA-I in mammals are liver and jejunum. Hepatic apoA-I transcription depends on a multitude of metabolic transcription factors. We demonstrate that ApoA-I synthesis and secretion are decreased during H2O2-induced oxidative stress in human hepatoma cell line HepG2. Here, we first show that FOXO1 binds to site B of apoA-I hepatic enhancer and downregulates apoA-I gene activity in HepG2 cells. Moreover, FOXO1 and LXRα transcription factors participate in H2O2-triggered downregulation of apoA-I gene together with Src, JNK, p38, and AMPK kinase cascades. Mutations of sites B or C as well as the administration of siRNAs against FOXO1 or LXRα to HepG2 cells abolished the hydrogen peroxide-mediated suppression of apoA-I gene.
... The pCMVLacZ [Dizhe et al., 2006] and pA1(À256/þ72) [Lapikov et al., 2008] plasmids were described elsewhere. pCMVHNF4a, the expression vector of human transcription factor HNF4a, was a generous gift of Dr. Fukamizu (University of Tsukuba, Tsukuba, Japan). ...
Apolipoprotein A-I (ApoA-I) is a key component of High Density Lipoproteins which possess anti-atherosclerotic and anti-inflammatory properties. Insulin is a crucial mediator of the glucose and lipid metabolism that has been implicated in atherosclerotic and inflammatory processes. Important mediators of insulin signaling such as Liver X Receptors (LXRs) and Forkhead Box A2 (FOXA2) are known to regulate apoA-I expression in liver. Forkhead Box O1 (FOXO1) is a well-known target of insulin signaling and a key mediator of oxidative stress response. Low doses of insulin were shown to activate apoA-I expression in human hepatoma HepG2 cells. However, the detailed mechanisms for these processes are still unknown. We studied the possible involvement of FOXO1, FOXA2, LXRα and LXRβ transcription factors in the insulin-mediated regulation of apoA-I expression. Treatment of HepG2 cells with high doses of insulin (48 hours, 100 nM) suppresses apoA-I gene expression. siRNAs against FOXO1, FOXA2, LXRβ or LXRα abrogated this effect. FOXO1 forms a complex with LXRβ and insulin treatment impairs FOXO1/LXRβ complex binding to hepatic enhancer and triggers its nuclear export. Insulin as well as LXR ligand TO901317 enhance the interaction between FOXA2, LXRα and hepatic enhancer. These data suggest that high doses of insulin downregulate apoA-I gene expression in HepG2 cells through redistribution of FOXO1/LXRβ complex, FOXA2 and LXRα on hepatic enhancer of apoA-I gene. This article is protected by copyright. All rights reserved.
... pCMVHNF4D, the expression vector of the human HNF4R dominant-negative mutant, was kindly provided by T. Leff (Wayne State University School of Medicine, Detroit, MI). pAPOA-I(-2498/þ173)-Luc, pAPOA-I(-2498/þ72)-Luc, pAPOA-I(-256/þ173)-Luc, and pAPOA-I(-256/þ72)-Luc, the plasmids containing the firefly luciferase reporter gene under control of deletion variants of the 5 0 -regulatory region of the human apoA-I gene (positions -2498 to þ173, positions -2498 to þ72, positions -256 to þ173, and positions -256 to þ72, respectively) related to the transcription start point (TSP) of the human apoA-I gene, have been described previously (30). ...
The expression of the apolipoprotein A-I gene (apoA-I) in hepatocytes is repressed by pro-inflammatory cytokines such as IL-1beta and TNFalpha. In this work, we have demonstrated that treatment of HepG2 human hepatoma cells with chemical inhibitors for JNK, p38 protein kinases, and NFkappaB transcription factor abolishes the TNFalpha-mediated inhibition of human apoA-I gene expression in HepG2 cells. In addition, we have shown that TNFalpha decreases also the rate of secretion of apoA-I protein by HepG2 cells, and this effect depends on JNK and p38, but not on NFkappaB and MEK1/2 signaling pathways. The inhibitory effect of TNFalpha has been found to be mediated by the hepatic enhancer of the apoA-I gene. The decrease in the level of human apoA-I gene expression under the impact of TNFalpha appears to be partly mediated by the inhibition of HNF4alpha and PPARalpha gene expression. Treatment of HepG2 cells with PPARalpha antagonist (MK886) or LXR agonist (TO901317) abolishes the TNFalpha-mediated decrease in the level of apoA-I gene expression. PPARalpha agonist (WY-14643) abolishes the negative effect of TNFalpha on apoA-I gene expression in the case of simultaneous inhibition of MEK1/2, although neither inhibition of MEK1/2 nor addition of WY-14643 leads to the blocking of the TNFalpha-mediated decrease in the level of apoA-I gene expression individually. The ligand-dependent regulation of apoA-I gene expression by PPARalpha appears to be affected by the TNFalpha-mediated activation of MEK1/2 kinases, probably through PPARalpha phosphorylation. Treatment of HepG2 cells with PPARalpha and LXR synthetic agonists also blocks the inhibition of apoA-I protein secretion in HepG2 cells under the impact of TNFalpha. A chromatin immunoprecipitation assay demonstrates that TNFalpha leads to a 2-fold decrease in the level of PPARalpha binding with the apoA-I gene hepatic enhancer. At the same time, the level of LXRbeta binding with the apoA-I gene hepatic enhancer is increased 3-fold under the impact of TNFalpha. These results suggest that nuclear receptors HNF4alpha, PPARalpha, and LXRs are involved in the TNFalpha-mediated downregulation of human apoA-I gene expression and apoA-I protein secretion in HepG2 cells.
Human apolipoprotein A-I (ApoA-I) is a major structural and functional protein component of high-density lipoprotein (HDL). ApoA-I constitutes ~75% of the protein content of HDL. The main sites of ApoA-I synthesis in humans are the liver and the small intestine. The mechanisms that govern tissue-specific apoA-I transcription in tissues and organs other than the liver and the small intestine are poorly understood. It is known that the human apoA-I has two additional promoters, the proximal and the distal one. In this work these two alternative apoA-I promoters are characterized, their transcription start sites are mapped and their competition for apoA-I transcription is demonstrated; the role of the alternative promoters in apoA-I expression in human cells and tissues other than hepatocytes and enterocytes is discussed.
The genes coding for three of the proteins of the lipid transport system, apolipoproteins A-I (apoA-I), C-III (apoC-III), and A-IV (apoA-IV), are closely linked and tandemly organized as a multigene family in the human genome. The evolution of this multigene family was studied by cloning and extensive restriction mapping analysis of an approximately 20-kilobase genomic DNA fragment containing the rat apoA-I gene. Low stringency hybridization blotting analysis of this DNA fragment using human apoC-III and apoA-IV cDNA probes revealed that the apoA-I, apoC-III, and apoA-IV genes are also closely linked and tandemly organized in the rat genome. Complete characterization of the rat apoA-I, apoC-III, and apoA-IV genes showed that their relative location, size, direction of transcription, and intron-exon organization are remarkably similar to those of the corresponding human genes. The relative steady state apoA-I, apoC-III, and apoA-IV mRNA levels in various rat tissues were determined by quantitative dot blot hybridization of tissue total RNA using the corresponding gene probes. Adult liver and intestine, but not colon, brain, spleen, muscle, heart, lung, and kidney, contain apoA-I, apoC-III, and apoA-IV mRNAs. Fetal liver and intestine contain apoA-I but not apoC-III or apoA-IV mRNAs. During neonatal development the liver contains apoA-I and apoC-III but not apoA-IV while the intestine contains apoA-I, apoC-III, and substantial amounts of apoA-IV mRNAs. In adulthood and during aging both liver and intestine contain all three apoA-I, apoC-III, and apoA-IV mRNAs. These results indicate that the apolipoprotein A-I/C-III/A-IV multigene family was established before mammalian radiation and suggest that these genes are similarly organized in the genomes of all mammals. In addition, these results indicate that expression of the rat apoA-I, apoC-III, and apoA-IV genes is liver- and intestine-specific and regulated by fetal-, neonatal-, and aging-related factors.
We have isolated and characterized a 2.5-kilobase pairs genomic DNA fragment which includes the 5'-flanking region and the first and second exons of the human apolipoprotein (apo) A-I gene. The major transcriptional start site was determined by primer extension analysis and is 235 base pairs (bp) upstream from the AUG translational start codon in liver and 234 bp upstream in the intestine. TATA box-like and CAT box-like sequences and two GC box sequences are present in the intestine 30, 108, 220, and 440 bp upstream, respectively, from the transcriptional start site. Fragments of 570 bp (-487 to +71) and 2.15 kilobase pairs (-2067 to +99) containing the 5'-flanking region of the apoA-I gene were fused upstream to the bacterial chloramphenicol acetyltransferase (CAT) gene. These constructs, designated pA-I(0.6)CAT and pA-I(2.2)CAT, respectively, were introduced into human oral epithelial cells (KB), mouse NIH 3T3 cells, Chinese hamster ovary (CHO) cells, human hepatoma cells (Hep G2), human duodenal epithelial cells (Hutu 80), and human colonic epithelial cells (Caco-2) by calcium phosphate coprecipitation. When compared with control vectors, highly efficient CAT expression of both the pA-I(0.6)CAT and pA-I(2.2)CAT constructs were observed only in cells derived from the liver (Hep G2) and intestine (Caco-2), which is consistent with the tissue specificity of expression of the native gene. Analysis of deletion mutants of the human apoA-I 5'-flanking region revealed that: 1) the region from -250 to -199 bp, from -487 to -413 bp, and -1021 to -691 bp upstream from the transcriptional start site contain sequences required for maximum gene expression; and 2) the regions from -2067 to -1476 bp and -199 to -80 bp contain the sequences required for tissue-specific repression of apoA-I gene expression in non-apoA-I producing cells.
Studies using transgenic mice indicate that expression of the human apolipoprotein (apo) A-I gene in the liver and small intestine is controlled by spatially distinct cis-acting DNA elements; hepatic expression is controlled by a domain defined by nucleotides -256 to -1, while small intestinal expression requires elements positioned 9 kilobases 3' to the gene, between nucleotides -1300 and -200 of the convergently transcribed apoC-III gene. In this report we have mapped this enhancer to a 260-base pair (bp) region of the apoC-III promoter spanning nucleotides -780 to -520. The elements contained within this 260-bp apoC-III domain are sufficient to direct a pattern of expression in villus-associated enterocytes distributed along the duodenal-to-ileal axis that resembles that of mouse and human apoA-I. However, the elements produce inappropriate activation of apoA-I expression in proliferating and nonproliferating crypt epithelial cells, and in subpopulations of cholecystokinin- and serotonin-producing enteroendocrine cells. Cis-acting suppressors of these inappropriate patterns of expression are located outside of nucleotides -1300 to -200 of the human apoC-III gene. DNase I protection and gel mobility gel shift assays identified two 21-bp sequences, nucleotides -745 to -725 and -700 to -680 of human apoC-III, which bind nuclear proteins present in a human enterocyte-like cell line (Caco-2). These sequences are conserved in the orthologous mouse apoC-III gene. The 260-bp apoC-III element is the first intestinal enhancer that has been identified in an in vivo system and should provide insights about how cell lineage-specific, differentiation-dependent, and cephalocaudal patterns of gene expression are established and maintained in the perpetually renewing gut epithelium. In addition, novel intestinal transcription factors may bind to the enhancer and regulate its activity.
This study examines the transcriptional regulation of the bovine CYP11A (P450scc) gene by activators of protein kinase A and protein kinase C in bovine ovarian luteal cells. Cells were transfected with reporter gene constructs containing deletion mutations of the 5'-flanking region of the bovine CYP11A gene linked to the minimal beta-globin gene. A construct containing -118/-101 base pairs of CYP11A sequence retains the same degree of stimulation by forskolin and inhibition by co-treatment with phorbol 12-myristate 13-acetate as larger constructs. This sequence contains two putative binding sites for nuclear proteins, an AP1-like sequence and an overlapping GA box element. Gel shift analysis using nuclear extracts of bovine ovarian luteal cells demonstrated that both the wild-type -118/-101-base pair sequence and a consensus GC box bound Sp1 or Sp1-like proteins. Mutation of the GA box element completely suppressed stimulation by forskolin. Absence of binding using the same mutated sequence correlated with the reporter gene transcription results. Mutation of the AP1-like site had little effect on forskolin induction of phorbol 12-myristate 13-acetate inhibition. These results indicate that both stimulation by forskolin and inhibition by phorbol esters are mediated by the same GA box element, which binds Sp1 or an Sp1-like protein.
TRANSFAC is a database about eukaryotic transcription regulating DNA sequence elements and the transcription factors binding to and acting through them. This report summarizes the present status of this database and accompanying retrieval tools.
Three proximal regulatory elements, AIB, AIC, and AID, of the apoA-I gene are necessary and sufficient for its hepatic expression in vivo and in vitro. DNA binding and competition assays showed that elements AIB and AID contain hormone response elements composed of imperfect direct repeats that support the binding of the hepatic nuclear factor-4, other nuclear orphan receptors, and the ligand-dependent nuclear receptors retinoic X receptor (RXRalpha), RXRalpha/RARalpha, and RXRalpha/T3Rbeta. Substitution mutations on repeats 1 and 2 in the hormone response sites of elements AIB and AID, respectively, abolished the binding of all nuclear receptors and reduced promoter activity to background levels, indicating the importance of both hormone response elements for the hepatic expression of the apoA-I gene. Cotransfection experiments in HepG2 cells with normal and mutated promoter constructs and plasmids expressing nuclear hormone receptors showed that RXRalpha homodimers transactivated the wild type promoter 150% of control, in the presence of 9-cis-retinoic acid (RA), whereas RXR alpha/T3R beta heterodimers repressed transcription to 60% of control, in the presence of T3. RXR alpha/RAR alpha and hepatic nuclear factor-4 did not affect the transcription, driven by the proximal apoA-I promoter. Potassium permanganate and dimethyl sulfate interference experiments showed that RXRalpha homodimers, RXRalpha/RARalpha, and RXRalpha/T3Rbeta heterodimers participate in protein-DNA interactions with 12, 13, and 11 out of the 14 nucleotides, respectively, that span repeats 1 and 2 and the spacer region separating them on the hormone response element of element AID. The binding of RXRalpha homodimers and RXRalpha/T3Rbeta heterodimers is associated with ligand-dependent activation by 9-cis-RA or repression by T3. Upon deletion or mutation of repeat 1, homodimeric binding of RXRalpha is lost whereas heterodimeric binding is retained. This heterodimeric binding to the mutated element AID is mediated solely by interactions with repeat 2 and one adjacent nucleotide and is confined to a heptameric core recognition motif. The interactions of the RXRalpha heterodimers with repeat 2 are associated with low levels of ligand-independent transcriptional activity. The findings suggest that the specific types of homo- and heterodimers of nuclear hormone receptors occupying the hormone response elements of apoA-I and the availability of the ligand may play an important role in the transcriptional regulation of the human apoA-I gene.
All-trans-retinoic acid (RA) is active in the treatment of Kaposi's sarcoma (KS), and retinoids inhibit KS cell growth in vitro. To understand the mechanism of retinoid action in KS, we studied the expression of autocrine growth factors of KS cells after RA treatment. We demonstrate that RA and its synthetic analogs inhibit the proliferation of KS cells by inhibiting the mRNA and protein levels of interleukin-6 (IL-6), an autocrine growth factor for KS cells. We further demonstrate that nuclear retinoid receptors (RA receptors [RARs] and retinoid X receptors [RXRs]) inhibit IL-6 promoter action by antagonizing the enhancer action of NF-IL6, a basic domain leucine zipper transcription factor belonging to the family of CAAT enhancer binding proteins. Furthermore, RARs and RXRs do not bind in vitro to an NF-IL6 binding site. However, the secondary folded structure of the DNA binding domain of RAR and RXR is obligatory for inhibiting NF-IL6 activity. Thus, NF-IL6 is a potential therapeutic target for the treatment of KS. Finally, using receptor-selective synthetic retinoids, we demonstrate that NF-IL6 antagonism and transactivation are separable functions of RAR alpha, thus indicating that synthetic retinoids with properties of NF-IL6 antagonism but lacking transactivation capabilities can be synthesized. Such retinoids might increase therapeutic potential in KS.
When deprived of nerve growth factor (NGF), developing sympathetic neurons die by apoptosis. This death is associated with an increase in the level of c-Jun protein and is blocked by expression of a c-Jun dominant negative mutant. Here we have investigated whether NGF withdrawal activates Jun kinases, a family of stress-activated protein kinases that can stimulate the transcriptional activity of c-Jun by phosphorylating serines 63 and 73 in the transactivation domain and which can activate c-jun gene expression. We found that sympathetic neurons contained high basal levels of Jun kinase activity that increased further after NGF deprivation. In contrast, p38 kinase, another stress-activated protein kinase that can also stimulate c-jun gene expression, was not activated after NGF withdrawal. Consistent with Jun kinase activation, we found using a phospho-c-Jun-specific antibody that c-Jun was phosphorylated on serine 63 after NGF withdrawal. Furthermore, expression of a constitutively active form of MEK kinase 1 (MEKK1), which strongly activates the Jun kinase pathway, increased c-Jun protein levels and c-Jun phosphorylation and induced apoptosis in the presence of NGF. This death could be prevented by co-expression of SEKAL, a dominant negative mutant of SAPK/ERK kinase 1 (SEK1), an activator of Jun kinase that is a target of MEKK1. In contrast, expression of SEKAL alone did not prevent c-Jun expression, increases in c-Jun phosphorylation, or cell death after NGF withdrawal. Thus, activation of Jun kinase and increases in c-Jun phosphorylation and c-Jun protein levels occur at the same time after NGF withdrawal, but c-Jun levels and phosphorylation are regulated by an SEK1-independent pathway.
Involucrin is a marker of keratinocyte terminal differentiation. Our previous studies show that involucrin mRNA levels are increased by the keratinocyte differentiating agent, 12-O-tetradecanoylphorbol-13-acetate (TPA) (Welter, J. F., Crish, J. F., Agarwal, C., and Eckert, R. L. (1995) J. Biol. Chem. 270, 12614-12622). We now study the signaling cascade responsible for this regulation. Protein kinase C and tyrosine kinase inhibitors inhibit both the TPA-dependent mRNA increase and the TPA-dependent increase in hINV promoter activity. The relevant response element is located within the promoter proximal regulatory region and includes an AP1 site, AP1-1. Co-transfection of the hINV promoter with dominant negative forms of Ras, MEKK1, MEK1, MEK7, MEK3, p38/RK, and c-Jun inhibit the TPA-dependent increase. Wild type MEKK1 enhances promoter activity and the activity can be inhibited by dominant negative MEKK1, MEK1, MEK7, MEK3, p38/RK, and c-Jun. In contrast, wild type Raf-1, ERK1, ERK2, MEK4, or JNK1 produced no change in activity and the dominant negative forms of these kinases failed to suppress TPA-dependent transcription. Treatment with an S6 kinase (S6K) inhibitor, or transfection with constitutively active S6K produced relatively minor changes in promoter activity, ruling out a regulatory role for S6K. These results suggest that activation of involucrin transcription involves a pathway that includes protein kinase C, Ras, MEKK1, MEK3, and p38/RK. Additional pathways that transfer MEKK1 activation via MEK1 and MEK7 also may function, but the downstream targets of these kinases need to be identified. AP1 transcription factors appear to be the ultimate target of this regulation.
The c-jun proto-oncogene encodes a component of the mitogen-inducible immediate-early transcription factor AP-1 and has been implicated as a positive regulator of cell proliferation and G1-to-S-phase progression. Here we report that fibroblasts derived from c-jun-/- mouse fetuses exhibit a severe proliferation defect and undergo a prolonged crisis before spontaneous immortalization. The cyclin D1- and cyclin E-dependent kinases (CDKs) and transcription factor E2F are poorly activated, resulting in inefficient G1-to-S-phase progression. Furthermore, the absence of c-Jun results in elevated expression of the tumor suppressor gene p53 and its target gene, the CDK inhibitor p21, whereas overexpression of c-Jun represses p53 and p21 expression and accelerates cell proliferation. Surprisingly, protein stabilization, the common mechanism of p53 regulation, is not involved in up-regulation of p53 in c-jun-/- fibroblasts. Rather, c-Jun regulates transcription of p53 negatively by direct binding to a variant AP-1 site in the p53 promoter. Importantly, deletion of p53 abrogates all defects of cells lacking c-Jun in cell cycle progression, proliferation, immortalization, and activation of G1 CDKs and E2F. These results demonstrate that an essential, rate-limiting function of c-Jun in fibroblast proliferation is negative regulation of p53 expression, and establish a mechanistic link between c-Jun-dependent mitogenic signaling and cell-cycle regulation.
The adenovirus E1A proteins activate the c-jun promoter through two Jun/ATF-binding sites, jun1 and jun2. P300, a transcriptional coactivator of several AP1 and ATF transcription factors has been postulated to play a role in this activation. Here, we present evidence that p300 can control c-jun transcription by acting as a cofactor for ATF2: (1) Over-expression of p300 was found to stimulate c-jun transcription both in the presence and absence of E1A. (2) Like E1A, p300 activates the c-jun promoter through the junl and jun2 elements and preferentially activates the N-terminal domain of ATF2. (3) Co-immunoprecipitation assays of crude cell extracts indicate that endogenous p300/CBP(-like) proteins and ATF2 proteins are present in a multiprotein complex that can bind specifically to the jun2 element. We further demonstrate that the Stress-Activated-Protein-Kinase (SAPK) target sites of ATF2, Thr69 and Thr71 are not required for the formation of the p300/CBP-ATF2 multiprotein complex. These data indicate that E1A does not inhibit all transcription activation functions of p300, and, in fact, cooperates with p300 in the activation of the ATF2 N-terminus.
Serum amyloid A (SAA) is an acute phase protein of unknown function that is involved in systemic amyloidosis and may also be involved in atherogenesis. The precise role of SAA in these processes has not been established. SAA circulates in plasma bound to high density lipoprotein-3 (HDL3). The pathway for the production of SAA-containing HDL is not known. To test whether apolipoprotein (apo)A-I-HDL is required in the production of SAA-HDL, we analyzed the lipopolysaccharide (LPS)-induced changes in apoA-I+/+ and apoA-I-/- mice. In apoA-I+/+ mice, after injection of LPS, remodeling of HDL occurred: total cholesterol increased and apoA-I decreased slightly and shifted to lighter density. Dense (density of HDL3) but large (size of HDL2 ) SAA-containing particles were formed. Upon fast phase liquid chromatography fractionation of plasma, >90% of SAA eluted with HDL that was enriched in cholesterol and phospholipid and shifted "leftward" to larger particles. Non-denaturing immunoprecipitation with anti-mouse apoA-I precipitated all of the apoA-I but not all of the SAA, confirming the presence of SAA-HDL devoid of apoA-I. In the apoA-I-/- mice, which normally have very low plasma lipid levels, LPS injection resulted in significantly increased total and HDL cholesterol. Greater than 90% of the SAA was lipid associated and was found on dense but large, spherical HDL particles essentially devoid of other apolipoproteins.We conclude that serum amyloid A (SAA) is able to sequester lipid, forming dense but large HDL particles with or without apoA-I or other apolipoproteins. The capacity to isolate lipoprotein particles containing SAA as the predominant or only apolipoprotein provides an important system to further explore the biological function of SAA.
The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) gene family and are essential for cell proliferation, differentiation, and apoptosis. Previously we found that activation of JNK in T-cells required costimulation of both T-cell receptor and auxiliary receptors such as CD28. In this study, we cloned a full-length human MEK kinase (MEKK) 2 cDNA from Jurkat T-cells and demonstrated that it was a major upstream MAPK kinase kinase for the JNK cascade in T-cells. The human MEKK2 cDNA encoded a polypeptide of 619 amino acids and was the human counterpart of the reported murine MEKK2. It was 94% homologous with human and murine MEKK3 at the catalytic domains and 60% homologous at the N-terminal noncatalytic region. Northern blot analysis showed that MEKK2 was ubiquitously expressed, with the highest level in peripheral blood leukocytes. In T cells, MEKK2 was found to be a strong activator of JNK but not of extracellular signal-regulated kinase MAPKs and to activate JNK-dependent AP-1 reporter gene expression. MEKK2 also synergized with anti-CD3 antibody to activate JNK in T cells, and stimulation of T cells led to induction of MEKK2 tyrosine phosphorylation. Significantly, the JNK activation induced by anti-CD3 and anti-CD28 antibodies, but not by 12-O-tetradecanoylphorbol-13-acetate and Ca(2+) ionophore A23187, was inhibited by dominant negative MEKK2 mutants. AP-1 and interleukin-2 reporter gene induction in T-cells was also inhibited by dominant negative MEKK2 mutants. Taken together, our results showed that human MEKK2 is a key signaling molecule for T-cell receptor/CD3-mediated JNK MAPK activation and interleukin-2 gene expression.
Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), essential components in the pathogenesis of immunoinflammatory diseases, are strongly induced in monocytes by direct contact with stimulated T lymphocytes. This study demonstrates that adult human serum (HS) but not fetal calf or cord blood serum displays inhibitory activity toward the contact-mediated activation of monocytes by stimulated T cells, decreasing the production of both TNF-alpha and IL-1beta. Fractionation of HS and N-terminal microsequencing as well as electroelution of material subjected to preparative electrophoresis revealed that apolipoprotein A-I (apo A-I), a "negative" acute-phase protein, was the inhibitory factor. Functional assays and flow cytometry analyses show that high-density lipoprotein (HDL)-associated apo A-I inhibits contact-mediated activation of monocytes by binding to stimulated T cells, thus inhibiting TNF-alpha and IL-1beta production at both protein and messenger RNA levels. Furthermore, apo A-I inhibits monocyte inflammatory functions in peripheral blood mononuclear cells activated by either specific antigens or lectins without affecting cell proliferation. These results demonstrate a new anti-inflammatory activity of HDL-associated apo A-I that might have modulating functions in nonseptic conditions. Therefore, because HDL has been shown to bind and neutralize lipopolysaccharide, HDL appears to play an important part in modulating both acute and chronic inflammation. The novel anti-inflammatory function of apo A-I reported here might lead to new therapeutic approaches in inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, and atherosclerosis.
The etiology of chronic immuno-inflammatory diseases including rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus (SLE), and atherosclerosis is far from being elucidated. It is generally accepted that multiple factors are involved in the development of such pathologies, including factors of genetic susceptibility that interact in complex ways with diverse environmental factors, i.e. gender, nutrition, environment, etc. Furthermore, infection has often been pinpointed as playing a causal role. However, no distinctive pattern has yet emerged from the tremendous number of compiled results that would provide a generally acceptable hypothesis of the etiology of immuno-inflammatory diseases, and the possibility of a persistent antigenic stimulus arising from an infection cannot be confirmed or refuted. At the cellular level, chronic inflammation is characterized by the infiltration of immuno-inflammatory cells into the target tissue, which mostly precedes tissue damage. At the inflammatory site, monocytes and T lymphocytes are in close proximity. We have demonstrated that contact-mediated activation of monocytes by stimulated T lymphocytes is a major stimulus triggering the production of large amounts of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) whose importance in chronic inflammation is well known. We recently established that high-density lipolipoprotein (HDL)-associated apolipoprotein (apo) A-I is a specific inhibitor of cytokine production in monocyte-macrophages upon contact with stimulated T cells. HDL-associated apo A-I is a negative acute-phase protein, i.e. a protein whose level is lowered by more than 25% during the acute phase. This review aims at highlighting the fact that HDL-associated apo A-I might play the role of a constitutive anti-inflammatory factor. The decrease of plasma levels of HDL-associated apo A-I upon acute inflammation may be a sign of the possible development of chronic inflammation, i.e. individuals presenting with risk factors might develop chronic inflammatory diseases after infection. We thus hypothesize that HDL-associated apo A-I might be the missing link between infection and chronic inflammation.
The levels of plasma HDL cholesterol and apoA-I in NFκB p50 subunit-deficient mice were significantly higher than those in
wild-type mice under regular and high fat diets, without any significant difference in the level of total cholesterol. To
examine the role of NFκBin lipid metabolism, we studied its effect on the regulation of apoA-I secretion from human hepatoma
HepG2 cells. Lipopolysaccharide-induced activation of NFκB reduced the expression of apoA-I mRNA and protein, whereas adenovirus-mediated
expression of IκBα super-repressor ameliorated the reduction. This IκBα-induced apoA-I increase was blocked by preincubation
with MK886, a selective inhibitor of peroxisome proliferator-activated receptor α (PPARα), suggesting that NFκB inactivation
induces apoA-I through activation of PPARα. To further support this idea, the expression of IκBα increased apoA-I promoter
activity, and this increase was blocked by preincubation with MK886. Mutations in the putative PPARα-binding site in the apoA-I
promoter or lack of the site abrogated these changes. Taking these results together, inhibition of NFκB increases apoA-I and
HDL cholesterol through activation of PPARα in vivo and in vitro. Our data suggest a new aspect of lipid metabolism and may lead to a new paradigm for prevention and treatment of atherosclerotic
disease.
Cyclooxygenase-2 (COX-2) overexpression has been linked to cell survival, transformation, and hyperproliferation. We examined the regulation of the tumor suppressor gene p53 and p53 target genes by prostaglandin E(2) (PGE(2)) in human synovial fibroblasts (HSF). PGE(2) induced a time-dependent increase in p53 Ser(15) phosphorylation, with no discernible change in overall p53 levels. PGE(2)-dependent Ser(15) phosphorylation was apparently mediated by activated p38 MAP kinase as SB202190, a p38 kinase inhibitor, blocked the response. Overexpression of a MKK3 construct, but not MKK1, stimulated SB202190-sensitive p53 Ser(15) phosphorylation. PGE(2)-stimulated [phospho-Ser(15)]p53 transactivated a p53 response element (GADD45)-luciferase reporter in transiently transfected HSF (SN7); the effect was compromised by overexpression of a dominant-negative mutant (dnm) of p53 or excess p53S15A expression plasmid but mimicked by a constitutively active p53S15E expression construct. PGE(2), wtp53 expression in the presence of PGE(2), and p53S15E suppressed steady-state levels of MEKK1-induced MMP-1 mRNA, effects nullified with co-transfection of p53 dnm or p53S15A. MEKK1-induced MMP-1 promoter-driven luciferase activity was largely dependent on a c/EBPbeta-NF-kappaB-like enhancer site at -2008 to -1972 bp, as judged by deletion and point mutation analyses. PGE(2), overexpression of p53wt with PGE(2), or p53S15E abolished the MEKK1-induced MMP-1 promoter luciferase activity. Gel-shift/super gel-shift analyses identified c/EBPbeta dimers and c/EBPbeta/NF-kappaB p65 heterodimers as binding species at the apparent site of MEKK1-dependent transactivation. PGE(2)-stimulated [phospho-Ser(15)]p53 abrogated the DNA binding of c/EBPbeta dimers and c/EBPbeta/NF-kappaB p65 heterodimers. Our data suggest that COX-2 prostaglandins may be implicated in p53 function and p53 target gene expression.
TRANSFAC is a database about eukaryotic transcription regulating DNA sequence elements and the transcription factors binding
to and acting through them. This report summarizes the present status of this database and accompanying retrieval tools.
Bile acids regulate the cholesterol 7α-hydroxylase gene (CYP7A1), which encodes the rate-limiting enzyme in the classical pathway of bile acid synthesis. Here we report a novel mechanism
whereby bile acid feedback regulatesCYP7A1 transcription through the nuclear receptorhepatocyte nuclear factor-4 (HNF-4), which binds to the bile acid response element (BARE) at nt −149/−118 relative to the transcription start site. Using
transient transfection assays of HepG2 cells with Gal4-HNF-4 fusion proteins, we show that chenodeoxycholic acid (CDCA) dampened
the transactivation potential of HNF-4. Overexpression of a constitutive active form of MEKK1, an upstream mitogen-activated
protein kinase (MAPK) module triggered by stress signals, strongly repressed the promoter activity ofCYP7A1 via the consensus sequence for HNF-4 embedded in the BARE. Similarly, MEKK1 inhibited the activity of HNF-4 in the Gal4-based
assay. The involvement of the MEKK1-dependent pathway in the bile acid-mediated repression of CYP7A1 was confirmed by co-transfecting a dominant negative form of the stress-activated protein kinase kinase, SEK, which abolished
the effect of CDCA upon CYP7A1transcription. Treatment of transfected HepG2 cells with tumor necrosis factor α (TNF-α), an activator of the MEKK1 pathway,
led to the repression of CYP7A1 via the HNF-4 site in the BARE. TNF-α also inhibited the transactivation potential of HNF-4. Collectively, our results demonstrate
for the first time that HNF-4, in combination with a MAPK signaling pathway, acts as a bile acid sensor in the liver. Furthermore,
the effects of CDCA and TNF-α converge to HNF-4, which binds to the BARE of CYP7A1, suggesting a link between the cascades elicited by bile acids and pro-inflammatory stimuli in the liver.
Human apolipoprotein A-I gene (apoA-I) inserted into a plasmid expression vector was transferred in vivo into C57Bl/6 mice using hydrodynamic injections into the tail vein. Two types of plasmid expression vectors were used: (1) pCMVcapoAI which contained cDNA of apoA-I driven by the human cytomegalovirus (CMV) early gene promoter and (2) pAlg, which contained a genomic locus of intron-containing apoA-I driven by its own extended 5-regulatory region (APOAI). Hydrodynamic intravenous injections of both expression vectors led to the appearance of human apoA-I mRNA in the liver and human ApoA-I protein in the serum of injected mice. The dynamics of human ApoA-I content in the sera of mice injected with pCMVcapoAI and pAlg were different. When pCMVcapoAI was used, the concentration of human ApoA-I in mouse serum was maximal one day after injection and decreased to zero within the next two weeks. In the case of pAlg, the content of human ApoA-I in serum was maximal (up to 20 g/ml) on days 5–7 after injection and then gradually decreased for several months (six months after injection, for example, it decreased to 25% of the maximal value). Experiments on saved pAlg plasmid isolated from the nuclei of hepatocytes 50 days after injection showed that the plasmid was retained for a long time in the form of an episome. A significant content of human ApoA-I in serum and its long-term persistence after injecting mice with pAlg may be accounted for by the properties of APOAI and/or the exon–intron structure of the apoA-I gene. Injecting mice with different variants of APOAI coupled with the luciferase gene did not lead to long-term expression of luciferase in the liver. It is concluded that the presence of introns in the apoA-I gene is required for its efficient and long-term expression after transfer to mice by means of hydrodynamic injections.
Plasma inflammatory cytokines are elevated in obese subjects as well as in those with type 2 diabetes. This presumably results in systemic insulin resistance, characterized by a pro-atherogenic plasma lipid profile and reduced apolipoprotein AI (apoAI) protein levels. To determine how cytokine-mediated insulin resistance suppresses apoAI gene expression, we investigated the effect of tumor necrosis factor α (TNF α) and interleukin-1β (IL-1β) on apoAI protein, mRNA, and transcriptional activity in the human hepatoma cell line HepG2. ApoAI secretion was suppressed in a dose-dependent manner in HepG2 cells treated with both cytokines. ApoAI protein levels were 2892±22.0, 2263±117, 2458±25.0, 3401±152, 2333±248, 1520±41.5 and 956.0±11.0 arbitrary units (AU) in cells treated with 0, 0.3, 1.0, 3.0, 10, 30, and 100 ng/ml TNF α, achieving statistical significance in the 30 and 100 ng/ml range (P<0.0009). ApoAI protein levels were 4055±360, 3697±101, 3347±327, 1561±33.0, 1581±182, 810.0±59.5, and 1766±717 AU in cells treated with similar doses of IL-1β, achieving statistical significance within the range of 3–100 ng/ml (P<0.02). ApoAI mRNA levels were suppressed 50.8% in HepG2 cells treated with 30 ng/ml TNF α for 24 h (P<0.05), and remained suppressed for up to 96 h. Similarly, treatment of cells with 30 ng/ml IL-1β for 24 h, resulted in 42.9% reduction in apoAI mRNA levels (P<0.05) and remained suppressed for up to 96 h.
During continuous stimulation by agonist, beta 1- and beta 2-adrenergic receptors (ARs) undergo processes that lead to decreases in receptor expression. This receptor down-regulation serves to limit the cellular cAMP response during chronic agonist exposure. In the recently described third subtype of the beta AR, denoted beta 3AR, we found four potential cAMP response elements in the 5' flanking region, suggesting that expression of this receptor might be positively regulated by agonists. These elements were cloned into the vector pA10CAT2, which contains a chloramphenicol acetyltransferase reporter gene, and transiently expressed in VERO cells. Three of these elements, TGACTCCA, TGAGGTCT, and CGAGGTCA (located 518, 622, and 1125 bases upstream of the beta 3AR coding block, respectively) were found to increase transcription of the chloramphenicol acetyltransferase gene in response to cAMP analogues and agents that increase intracellular cAMP. 3T3-F442A cells, when differentiated into the adipocyte phenotype by insulin, expressed beta 3AR, and nuclear runoff studies from such cells confirmed cAMP enhancement of beta 3AR mRNA transcription. In these cells, beta 3AR mRNA increased in response to exposure to the beta 3AR agonist isoproterenol and remained elevated during exposures of up to 24-30 hr. During prolonged exposure to agonist, no downregulation of beta 3AR expression in 3T3-F442A cells occurred. Indeed, beta 3AR expression increased during agonist exposure to approximately 165% of basal expression. In marked contrast, beta 1AR expression declined by approximately 70% in response to chronic agonist exposure. These studies reveal a subtype-specific prolonged transcriptional regulation of a beta AR gene by the end product of its signal transduction pathway. Thus, the beta 3AR undergoes a paradoxical increase in receptor expression during chronic agonist exposure.
We have shown that the transcriptional activity of the protooncogene jun (c-jun) promoter is repressed by a transcription factor, the cAMP response element-binding protein (CREB). This repression can be alleviated when CREB is phosphorylated by the catalytic subunit of protein kinase A. Repression cannot be alleviated by a mutant CREB deficient in the protein kinase A phosphorylation site (M1 CREB Ser-133----Ala), suggesting that phosphorylation of CREB at this site is essential for the relief of repression. Repression by CREB requires its binding to the c-jun promoter. In NIH 3T3 cells stably expressing CREB, c-jun is no longer induced by serum, but this repression can be relieved by treatment of the cells with forskolin, an agonist of the adenylate cyclase pathway. Thus, CREB has a dual function, that of a repressor in the absence of phosphorylation and an activator when phosphorylated by protein kinase A.
High-density lipoprotein (HDL) cholesterol (HDL-C) concentrations are inversely associated with coronary heart disease (CHD)
in humans [1,2]. The initial descriptions of this inverse relationship in the early 1950s [3–5] were “rediscovered” in the
1970s [6–9]. The major mechanism proposed for the protective effect of HDL is reverse cholesterol transport, a process in
which excess cholesterol from peripheral cells is transported back to the liver for removal from the body [10]. The higher
the plasma levels of HDL, the more efficient is the transport to the liver of excess cholesterol from peripheral cells.
We have used apolipoprotein genes to investigate the signal transduction mechanisms involved in the control of intestinal specific gene expression. The human apoAI, apoCIII, and apoAIV genes are tandemly organized within a 15-kb DNA segment and are expressed predominantly in the liver and intestine. Transient transfection of various human apoAI gene plasmid constructs into human hepatoma (HepG2) and colon carcinoma (Caco-2) cells showed that apoAI gene transcription is under the control of two separate and distinct cell-specific promoters. The region between nucleotides -192 and -41 is essential for expression in HepG2 cells, whereas the region from -595 to -192 is essential for expression in Caco-2 cells. A third 0.6 kb DNA fragment in the apoCIII gene promoter region, approximately 5 kb down-stream from the human apoAI gene, enhances transcription mediated by either of these two tissue-specific apoAI promoters. In Caco-2 cells, expression of the apoAI gene and activation by the distal enhancer required the presence of a nuclear hormone receptor response element (NHRRE) located in the -214 to -192 apoAI promoter region. Overexpression of the orphan receptor hepatocyte nuclear factor 4 (HNF-4), which binds to the NHRRE, dramatically stimulates apoAI gene expression in Caco-2 cells but not in HepG2 cells. Maximal stimulation of transcription by HNF-4 in Caco-2 cells required the presence of both the intestinal specific promoter, the NHRRE, and distal enhancer elements. Transactivation by HNF-4 thus appears to result from functional synergy between the NHRRE binding HNF-4 and distal DNA elements containing intestinal-specific DNA binding activities. The apoAI gene provides a model system to define the mechanism(s) governing intestinal cell specific gene regulation and the role of nuclear hormone receptors in the establishment and regulation of enterocytic gene transcription.
The adenovirus E1A proteins differentially regulate AP-1-responsive genes. Collagenase and stromelysin are repressed by E1A, whereas the expression of c-jun is elevated. Inhibition of collagenase has been found to be exerted through the consensus AP-1 binding site TGAGTCA. Here we show that the distal AP-1 binding site in the c-jun promoter, the jun2TRE (TTACCTCA), is the decisive element of this promoter in mediating the positive response to the 243 amino acid E1A product. In vitro binding studies revealed that, in contrast to the consensus AP-1 site which is preferentially targeted by dimers composed of the Jun and Fos families, the jun2TRE binds heterodimers composed of cJun and ATF-2(-like) proteins. Since stimulation of c-jun transcription is a function of the transforming domain of E1A encoded by conserved region 1, cJun--ATF-2 may be one of the effector factors involved in transformation. The data further suggest that E1A can distinguish between cJun--cJun and cJun--ATF-2 in imposing opposite states of activity.
The transcription of eukaryotic genes is a complex biological event involving numerous proteins—including RNA polymerase II, the proteins of the basal transcription initiation complex, and a variety of promoter- and enhancer-specific transcription factors—and requiring an ATP-dependent activation step.1 2 3 4 5 6 7 8 9 10 11 12 The regulation of transcription is responsible for the tissue-specific gene expression as well as gene expression during differentiation and development and in response to intracellular and extracellular stimuli such as hormones and metabolites.
Numerous studies have established that a precise array of regulatory elements exists in each promoter/enhancer and these elements are occupied by transcription factors. It has been proposed that this promoter/enhancer-specific arrangement of factors permits the formation of stereospecific DNA-protein complexes. These complexes may directly or indirectly interact with the basal transcription system, thus leading to the transcriptional activation of the target gene.8 13
Several experimental advances have facilitated the study of eukaryotic promoters and have led to the identification and characterization of several eukaryotic transcription factors. These include the following: (1) Definition of the long-range regulatory elements that confer tissue specificity or developmentally regulated expression. This analysis utilizes transgenic mouse technologies.14 15 (2) Definition of the promoter region a few kilobases upstream of the transcription initiation site necessary for gene transcription. This analysis monitors the expression of a reporter gene under the control of normal and mutated promoters after transfection of cell cultures. (3) Identification of the different factors that bind to a specific promoter region and definition of their binding sites on the DNA. For this purpose, several techniques are used, including DNase I footprinting, in vivo footprinting,16 17 18 gel electrophoretic mobility shift assays,19 supershift assays, and DNA binding interference assays that involve modification of T residues by KMnO4 and …
With the goal of developing non-viral techniques for exogenous gene delivery into mammalian cells, we have studied receptor-mediated gene transfer using complexes of plasmid DNA and galactosylated poly-L-lysine, poly(L-Lys)Gal. To evaluate the optimal parameters for efficient gene transfer into human hepatoma HepG2 cells by the DNA-poly(L-Lys)Gal complexes, the bacterial reporter genes lacZ and cat were used. Examination of the reporter gene expression level showed that the efficiency of DNA delivery into the cells depends on the structure of DNA--poly(L-Lys)Gal complexes formed at various ionic strength values. The efficiency of DNA transfer into the cells also depends on DNA/poly(L-Lys)Gal molar ratio in the complexes. Plasmid vector carrying human apolipoprotein A-I (apoA-I) gene was injected as its complex with poly(L-Lys)Gal into rat tail vein. Some level of ApoA-I was detected in the serum of the injected rats. Also, the human apoA-I-containing plasmid was found to be captured specifically by the rat liver cells and transported into the cell nuclei, where it can persist as an episome-like structure for at least a week. After repeated injections of DNA--poly(L-Lys)Gal complexes, the level of human ApoA-I in rat serum increases, probably, due to accumulation of functional human apoA-I gene in the liver cell nuclei. The data seem to be useful for the development of non-viral approaches to gene therapy of cardiovascular diseases.
Bile acids regulate the cholesterol 7alpha-hydroxylase gene (CYP7A1), which encodes the rate-limiting enzyme in the classical pathway of bile acid synthesis. Here we report a novel mechanism whereby bile acid feedback regulates CYP7A1 transcription through the nuclear receptor hepatocyte nuclear factor-4 (HNF-4), which binds to the bile acid response element (BARE) at nt -149/-118 relative to the transcription start site. Using transient transfection assays of HepG2 cells with Gal4-HNF-4 fusion proteins, we show that chenodeoxycholic acid (CDCA) dampened the transactivation potential of HNF-4. Overexpression of a constitutive active form of MEKK1, an upstream mitogen-activated protein kinase (MAPK) module triggered by stress signals, strongly repressed the promoter activity of CYP7A1 via the consensus sequence for HNF-4 embedded in the BARE. Similarly, MEKK1 inhibited the activity of HNF-4 in the Gal4-based assay. The involvement of the MEKK1-dependent pathway in the bile acid-mediated repression of CYP7A1 was confirmed by co-transfecting a dominant negative form of the stress-activated protein kinase kinase, SEK, which abolished the effect of CDCA upon CYP7A1 transcription. Treatment of transfected HepG2 cells with tumor necrosis factor alpha (TNF-alpha), an activator of the MEKK1 pathway, led to the repression of CYP7A1 via the HNF-4 site in the BARE. TNF-alpha also inhibited the transactivation potential of HNF-4. Collectively, our results demonstrate for the first time that HNF-4, in combination with a MAPK signaling pathway, acts as a bile acid sensor in the liver. Furthermore, the effects of CDCA and TNF-alpha converge to HNF-4, which binds to the BARE of CYP7A1, suggesting a link between the cascades elicited by bile acids and pro-inflammatory stimuli in the liver.
Human apolipoprotein A-I gene (apoA-I) plasmid expression vectors were transferred into mice by hydrodynamic injections into tail vein. Two types of expression vectors were used. First one -pCMVcapoAI contains cDNA of apo A-I driven by human cytomegalovirus early gene promoter (CMV). Second one--pAlg contains genomic locus of intron-containing apo A-I under control of own extended 5'-regulatory region (APOAI). Hydrodynamic intravenous injections of both expression vectors led to appearance of human apo A-I mRNA in the liver and human Apo A-I protein in the serum of injected mice. Dynamics of human Apo A-I content in the serum of mice injected by pCMVcapoAI and pAlg were different. When pCMVcapoAI was used, maximal concentration of human Apo A-I protein in the mouse serum was detected one day after injection with following decline to zero level during next two weeks. Under the same conditions injections of pAlg led to maximal level of human Apo A-I concentration in the mouse serum (up to 20 mkg/ml in some animals) on the 5th-7th day of experiment with following graduate decline during several months (human Apo A-I concentration in the serum of oldest analyzed mouse (6 months after injection) was about 25% of its maximal level in the same animal). Levels of human Apo A-I concentration in the mouse serum were compatible after injections of both expression vectors, in spite of much more strong activity of CMV promoter in comparison with APOAI in cultured human hepatoma cells HepG2. We ascribe the revealed difference in dynamics of human Apo A-I expression to delay of apo A-I transcription from pAlg vector, that was confirmed by nested RT-PCR. Significant level and long-term persistence of human Apo A-I in the serum of mice injected by pAlg could be explained by properties of APOAI or (and) exon-intron structure of genomic apo A-I gene. To test the role of APOAI in long-term expression of human Apo A-I in the mice we performed hydrodynamic injections of plasmid vectors containing cDNA of reporter gene encoding luciferase driven by variants of APOAI. No long-term expression of luciferase was found in the livers of injected mice. Therefore, our data suggest the role of exon-intron structure in maintaining of efficient and long-term expression of transferred human apo A-I.
In recent years, a link between the transcriptional regulators of lineage-specific gene expression and progenitor proliferation control has emerged. A main exponent of this phenomenon is the CCAAT/enhancer binding protein (C/EBP) family of basic region-leucine zipper proteins. These transcription factors control the differentiation of a range of cell types, and have key roles in regulating cellular proliferation through interaction with cell cycle proteins. More recently, their position at the crossroads between proliferation and differentiation has made them strong candidate regulators of tumorigenesis, and C/EBPs have been described as both tumor promoters and tumor suppressors.
Molecular Cloning: A Laboratory Manual. Cold Spring Harbor
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Klimov A.N., Nikul'cheva N.G. 1995. Lipidy, lipopro-teidy i ateroskleroz (Lipids, Lipoproteins, and Athero-sclerosis). St. Petersburg: Piter Press.
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Klimov A.N., Nikul'cheva N.G. 1999. Obmen lipidob i lipoproteidov i ego narusheniya (Lipid and Lipoprotein Metabolism and Its Disturbances). St. Petersburg: Piter.
1989 Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells
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1989 Rapid detection of octamer binding proteins with
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