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At least 739 records · Page 41Linked to original sources
PubMed1995

Activating transcription factor 1 and cyclic AMP response element modulator can modulate the activity of the immunoglobulin kappa 3' enhancer.

Previously we determined that the immunoglobulin kappa 3' enhancer (kappa E3') contains at least two functional DNA sequences (PU.1/NF-EM5 and E2A) within its 132-base pair active core. We have determined that the activities of these two sequences are insufficient to account for the entire activity of the 132-base pair core. Using site-directed linker scan mutagenesis across the core fragment we identified several additional functional sequences. We used one of these functional sequences to screen a lambda gt11 cDNA expression library resulting in the isolation of cDNA clones encoding the transcription factors ATF-1 (activating transcription factor) and CREM (cyclic AMP response element modulator). Because ATF-1 and CREM are known to bind to cAMP response elements (CRE), this functional sequence was named the kappa E3'-CRE. We show that dibutyryl cAMP can increase kappa E3' enhancer activity, and in transient expression assays ATF-1 caused a 4-5-fold increase in the activity of the core enhancer while CREM-alpha expression resulted in repression of enhancer activity. RNA analyses showed increased levels of ATF-1 mRNA during B cell development and some changes in CREM transcript processing. By joining various fragments of the kappa E3' enhancer to the kappa E3'-CRE, we observed that the kappa E3'-CRE can synergistically increase transcription in association with the PU.1/NF-EM5 binding sites, suggesting a functional interaction between the proteins that bind to these DNA sequences. Consistent with this possibility, we found that ATF-1 and CREM can physically interact with PU.1. The isolation of activator and repressor proteins that bind to the kappa E3'-CRE may relate to previous conflicting results concerning the role of the cAMP signal transduction pathway in kappa gene transcription.

Activating Transcription Factor 1↗
PubMed1998

The redox-sensitive human antioxidant responsive element induces gene expression under low oxygen conditions.

Transient transfection studies of human HepG2 and mouse Hepa hepatocarcinoma cells with a reporter gene construct regulated by a human antioxidant responsive element (ARE) from the NQO1 gene demonstrated that the element is responsive to low oxygen conditions. The antioxidant N-acetyl L-cysteine (NAC) strongly inhibited basal aerobic reporter gene activity in HepG2 cells without obviously affecting the hypoxic induction, as is consistent with ARE sensitivity to oxidative stress in aerobic cultures. Electrophoretic mobility shift (EMS) assays of nuclear extracts of HepG2 and Hepa cells lysed under aerobic or hypoxic conditions or after exposure to the phenolic compound 3-(2)-tert-butyl-4-hydroxyanisole (BHA), showed specific and constitutive protein binding to the ARE under all of these conditions. Taken together, these findings show that the ARE can mediate gene expression in response to low oxygen conditions. Co-ordinately regulated expression of ARE-dependent genes, such as phase II detoxification enzymes, may be an important phenotype of solid tumors containing significant regions of pathophysiological hypoxia.

Acetylcysteine↗
PubMed2000

The decorin high glucose response element and mechanism of its activation in human mesangial cells.

The decorin gene encodes a proteoglycan with putative structural and regulatory functions whose expression is markedly increased in human mesangial cells (HMC) exposed to high concentrations of glucose (15 to 30 mM). The gene has two promoters (P1 and P2) upstream of two alternative first exons. Transcripts driven by both promoters are present in HMC maintained in 4 mM D-glucose medium. After exposure to 30 mM D-glucose for 7 to 21 d, transcripts driven by P1 are markedly increased, whereas those driven by P2 decrease. Culture in 4 mM D-glucose medium containing transforming growth factor-beta1 (TGF-beta1) (1.25 ng/ml) has the same effect. However, addition of an excess of TGF-beta neutralizing antibody to the 30 mM D-glucose cultures only partly suppressed increased decorin transcription from P1. In transformed HMC transfected with a reporter (p-SAEP) driven by P1 or P2, P1 activity increased twofold on treatment with either 30 mM D-glucose or TGF-beta1 in 4 mM medium. P2 had little activity under any conditions. 5' deletion of P1 showed that basal transcriptional activity lies within the proximal 378 bp, while the major high glucose and TGF-beta response element is located in the -683 to -583-bp region. A putative cAMP response-like sequence (TGACGTTT) lies within this region. Electrophoretic mobility shift assays revealed the same pattern of multiple complexes between oligonucleotides containing this sequence and nuclear proteins extracted from HMC maintained in either 4 or 30 mM D-glucose conditions, but the latter were more prominent. cAMP response element binding protein (CREB) was identified as one transcription factor forming these complexes but other factors remain unidentified. Increased levels of phospho-(Ser 133) CREB were found in HMC exposed to 30 mM D-glucose. High glucose also activated and led to nuclear translocation of p42/44 mitogen-activated protein kinase and p38 mitogen-activated protein kinase, both of which can activate CREB by phosphorylation of serine 133.

Cell Line↗
PubMed2005

The role of cyclic AMP response element binding protein in transactivation of scavenger receptor class B type I promoter in transfected cells and in primary cultures of rat theca-interstitial cells.

In the ovary, lutropin (LH) stimulates the selective uptake and transport of cholesterol for steroid biosynthesis from HDL particles via the scavenger receptor class B type I (SR-BI). Furthermore the expression of SR-BI mRNA in the ovary is stimulated by LH and cyclic AMP (cAMP). Since the promoter of the rat SR-BI gene is devoid of consensus cyclic AMP response element (CRE) sequences, this study examined if cAMP response element binding protein (CREB) plays a role in the transactivation of SR-BI promoter (SR-BIpr). The transactivation of SR-BIpr was examined in transfected 293T cells and human granulosa SVOG-4o cells, and in primary cultures of rat theca-interstitial cells infected with adenoviral constructs containing the SR-BIpr and a luciferase reporter gene. Dose-related increases in SR-BRpr activity ranging from 2- to 4-fold was induces by 293T cells co-transfected with the catalytic subunit of protein kinase A (cPKA). Co-transfections with CREB and cPKA produced a concentration-dependent increase ranging from 6- to 32-fold. The cAMP-mediated transactivation was significantly attenuated by co-transfection with CREB M1, a non-phosphorylatable, dominant-negative form of CREB. An increase in transactivation of SR-BIpr activity was also seen in SVOG-4o cells co-transfected with CREB. In primary cultures of rat theca-interstitial (T-I) cells infected with an adenoviral construct of SR-BIpr, forskolin produced a marked increase in promoter activity. These data indicate that stimulation of the cAMP-PKA-CREB pathway enhances rat SR-BIpr activity and substantiate the role of CREB as an intermediary in this process. The absence of canonical CRE sequences in the rat SR-BIpr suggests that the activation of SR-BI by CREB may occur either through non-canonical CRE sequences or through additional transcription factors that cooperate with CREB in the activation of SR-BI promoter activity.

Animals↗
PubMed1999

Regulation of cAMP responsive element binding modulator isoforms in cultured rat ovarian granulosa cells.

A pituitary glycoprotein hormone FSH stimulates ovarian granulosa cells to induce ovarian follicular development. In this study we identified rat ovarian genes that were rapidly induced by FSH in the cultured rat granulosa cells by means of subtraction cloning. Complementary DNA clones encoding cAMP responsive element binding modulator (CREM) were identified as one of the FSH inducible genes. Northern blotting and reverse transcription and polymerase chain reaction (RT-PCR) analyses revealed that only the repressor type of CREM gene products, ICER (inducible cAMP early repressor) isoforms, were induced by FSH treatment in cultured rat granulosa cells. The induction of ICER by FSH was mimicked by reagents known to increase intracellular cAMP levels, indicating that the induction is through cAMP and protein kinase A signal transduction system. Induction of ICER was also confirmed as the protein levels. Electrophoretic mobility shift assay of granulosa cell extracts with a radiolabeled double stranded oligonucleotide corresponding to somatostatin cAMP responsive element also revealed that only the ICER proteins were induced by FSH treatment, whereas levels of CREM proteins were nearly constant regardless of the FSH treatment. Our present study demonstrates that FSH-induced and cAMP-mediated induction and attenuation of transcriptional responses by CREM gene products may be a key mechanistic component for the granulosa cell differentiation and proliferation.

Animals↗
PubMed1999

CCAAT/enhancer-binding protein beta is an accessory factor for the glucocorticoid response from the cAMP response element in the rat phosphoenolpyruvate carboxykinase gene promoter.

The cyclic AMP response element (CRE) of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene promoter is required for a complete glucocorticoid response. Proteins known to bind the PEPCK CRE include the CRE-binding protein (CREB) and members of the CCAAT/enhancer-binding protein (C/EBP) family. We took two different approaches to determine which of these proteins provides the accessory factor activity for the glucocorticoid response from the PEPCK CRE. The first strategy involved replacing the CRE of the PEPCK promoter/chloramphenicol acetyltransferase reporter plasmid (pPL32) with a consensus C/EBP-binding sequence. This construct, termed pDeltaCREC/EBP, binds C/EBPalpha and beta but not CREB, yet it confers a nearly complete glucocorticoid response when transiently transfected into H4IIE rat hepatoma cells. These results suggest that one of the C/EBP family members may be the accessory factor. The second strategy involved co-transfecting H4IIE cells with a pPL32 mutant, in which the CRE was replaced with a GAL4-binding sequence (pDeltaCREGAL4), and various GAL4 DNA-binding domain (DBD) fusion protein expression vectors. Although chimeric proteins consisting of the GAL4 DBD fused to either CREB or C/EBPalpha are able to confer an increase in basal transcription, they do not facilitate the glucocorticoid response. In contrast, a fusion protein consisting of the GAL4 DBD and amino acids 1-118 of C/EBPbeta provides a significant glucocorticoid response. Additional GAL4 fusion studies were done to map the minimal domain of C/EBPbeta needed for accessory factor activity to the glucocorticoid response. Chimeric proteins containing amino acid regions 1-84, 52-118, or 85-118 of C/EBPbeta fused to the GAL4 DBD do not mediate a glucocorticoid response. We conclude that the amino terminus of C/EBPbeta contains a multicomponent domain necessary to confer accessory factor activity to the glucocorticoid response from the CRE of the PEPCK gene promoter.

Animals↗
PubMed2004

Genomic mechanisms of p210BCR-ABL signaling: induction of heat shock protein 70 through the GATA response element confers resistance to paclitaxel-induced apoptosis.

Chronic myelogenous leukemia (CML) results from a t(9,22) translocation, producing the p210(BCR-ABL) oncoprotein, a tyrosine kinase that causes transformation and chemotherapy resistance. To further understand mechanisms mediating chemotherapy resistance, we identified 556 differentially regulated genes in HL-60 cells stably expressing p210(BCR-ABL) versus those expressing an empty vector using cDNA macro- and oligonucleotide microarrays. These BCR-ABL-regulated gene products play diverse roles in cellular function including apoptosis, cell cycle regulation, intracellular signaling, transcription, and cellular adhesion. In particular, we identified up-regulation of the inducible form of heat shock protein 70 (Hsp70), and further explored the mechanism for its up-regulation. In HL-60/BCR-ABL and K562 cells (expressing p210(BCR-ABL)), abundant cytoplasmic Hsp70 expression was detected by immunoblot analysis. Moreover, cells isolated from bone marrow aspirates of patients in different stages of CML (chronic, aggressive, and blast crisis) express Hsp70. Expression of p210(BCR-ABL) in BCR-ABL negative cells induced transcription of the proximal Hsp70 promoter. Mutational analysis mapped the major p210(BCR-ABL) responsive element to a high affinity 5'(A/T)GATA(A/G)-3' "GATA" response element (GATA-RE) that binds GATA-1 in CML cells. The GATA-RE was sufficient to confer p210(BCR-ABL)- and p185(BCR-ABL)-mediated trans-activation to an inert promoter. Short interfering RNA mediated "knockdown" of Hsp70 expression in K562 cells induced marked sensitivity to paclitaxel-induced apoptosis. Together these findings indicate that BCR-ABL confers chemotherapeutic resistance through intracellular signaling to the GATA-RE element found in the promoter region of the anti-apoptotic Hsp70 protein. We suggest that down-regulation of the GATA-Hsp70 pathway may be useful in the treatment of chemotherapy-resistant CML.

Antineoplastic Agents, Phytogenic↗
PubMed2002

A transcription factor response element for gene expression during circadian night.

Mammalian circadian clocks consist of complex integrated feedback loops that cannot be elucidated without comprehensive measurement of system dynamics and determination of network structures. To dissect such a complicated system, we took a systems-biological approach based on genomic, molecular and cell biological techniques. We profiled suprachiasmatic nuclei and liver genome-wide expression patterns under light/dark cycles and constant darkness. We determined transcription start sites of human orthologues for newly identified cycling genes and then performed bioinformatical searches for relationships between time-of-day specific expression and transcription factor response elements around transcription start sites. Here we demonstrate the role of the Rev-ErbA/ROR response element in gene expression during circadian night, which is in phase with Bmal1 and in antiphase to Per2 oscillations. This role was verified using an in vitro validation system, in which cultured fibroblasts transiently transfected with clock-controlled reporter vectors exhibited robust circadian bioluminescence.

Animals↗
PubMed1997

The orphan nuclear receptor TR2 suppresses a DR4 hormone response element of the mouse CRABP-I gene promoter.

The mouse orphan nuclear receptor TR2-11-f suppressed the expression of reporters fused to a hormone response element of the mouse cellular retinoic acid-binding protein I gene promoter. TR2-11-f was able to bind to a direct repeat with four nucleotides in the spacer (5'TGACCTTTGGGGACCT3') located within this hormone response element as homodimers. The specificity of protein-DNA interactions was demonstrated by competition in gel retardation and antibody-mediated supershift reactions. The residues critical for TR2-11-f binding were mapped to both repeated sequences, whereas the spacer and the flanking sequences were less important. The Kd and Bmax of TR2-11-f homodimer binding to this direct repeat were determined to be 2.6 nM and 0.012 nM, respectively. By using a yeast two-hybrid system, it was demonstrated that dimerization of TR2-11-f was mediated by its ligand-binding domain. The actions of TR2-11-f in regulating cellular retinoic acid-binding protein I gene will likely influence retinoic action and availability within the cells.

Animals↗
PubMed1992

An iron-sulfur cluster plays a novel regulatory role in the iron-responsive element binding protein.

Post-transcriptional regulation of genes important in iron metabolism, ferritin and the transferrin receptor (TfR), is achieved through regulated binding of a cytosolic protein, the iron-responsive element binding protein (IRE-BP), to RNA stem-loop motifs known as iron-responsive elements (IREs). Binding of the IRE-BP represses ferritin translation and represses degradation of the TfR mRNA. The IRE-BP senses iron levels and accordingly modifies binding to IREs through a novel sensing mechanism. An iron-sulfur cluster of the IRE-BP reversibly binds iron; when cytosolic iron levels are depleted, the cluster becomes depleted of iron and the IRE-BP acquires the capacity to bind IREs. When cytosolic iron levels are replete, the IRE-BP loses RNA binding capacity, but acquires enzymatic activity as a functional aconitase. RNA binding and aconitase activity are mutually exclusive activities of the IRE-BP, and the state of the iron-sulfur cluster determines how the IRE-BP will function.

Aconitate Hydratase↗
PubMed1999

Coordinate regulation of NAD(P)H:quinone oxidoreductase and glutathione-S-transferases in primary cultures of rat neurons and glia: role of the antioxidant/electrophile responsive element.

NAD(P)H:quinone oxidoreductase (QR) and glutathione-S-transferases (GSTs) are among the enzymes believed to protect an organism against oxidative stress. To test if redox-cycling compounds regulate the expression of these enzymes in cells of neural origin, primary cultures of rat cerebellar neurons and glia were treated with tert-butylhydroquinone (tBHQ) and hydroquinone (HQ). Basal levels of endogenous QR and GST activity were significantly greater in glia than neurons; and QR, GSTP1, and A3 were increased in glial but not neuronal cultures by treatment with tBHQ and HQ. A possible role for protein kinase C (PKC) in the tBHQ-mediated increase in QR and GST was evaluated by activating PKC with phorbol 12-myristate 13-acetate or inhibiting PKC with bisindolylmaleimide I. PKC was not involved in maintaining basal expression or mediating the increased expression of GST or QR by tBHQ. Transcriptional activation of QR and rGSTP1 by tBHQ could be mediated through a common responsive element present in the 5'-flanking region of both genes, the antioxidant/electrophile responsive element (ARE/EpRE). Transient transfection of the glial cultures with rGSTP1- or rQR1-ARE/EpRE-luciferase reporter constructs demonstrated that tBHQ transcriptionally activates the ARE/EpRE. Thus, the increased expression of genes regulated by the ARE/EpRE in cells of the central nervous system may provide protection against oxidative stress.

Animals↗
PubMed1996

Synergistic activation of cAMP and calcium on cAMP-response-element-mediated gene expression in GH3 pituitary tumor cells.

Signals responsible for expression of the vasoactive intestinal peptide (VIP)-stimulated prolactin gene in GH3 pituitary tumor cells were examined. Transfection with a deoxyribonucleic acid (DNA) construct containing the chloramphenicol acetyltransferase (CAT) gene fused to the 2.5-kb prolactin 5'-upstream regulatory sequence indicated that VIP stimulated CAT expression. However, this effect could not be mimicked by 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), and was inhibited by the L-type Ca(2+)-channel blocker verapamil. While KCl had little effect on CAT activity, combined treatment with KCl and 8-Br-cAMP synergistically activated CAT expression. Potentiation between KCl and 8-Br-c-AMP was also seen with c-fos messenger ribonucleic acid (mRNA) expression. In addition, KCl and 8-Br-cAMP synergistically activated cAMP response element (CRE)-mediated CAT expression, and the synergism was abolished by verapamil. In the presence of okadaic acid, cAMP had no significant activation on CRE-driven CAT expression, whereas KCl-stimulated CAT expression was greatly potentiated. These results indicate that cAMP and Ca2+ synergistically activated CRE-driven gene expression through non-overlapping phosphorylation events in GH3 cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2000

Defective Th function induced by a dominant-negative cAMP response element binding protein mutation is reversed by Bcl-2.

cAMP response element binding protein (CREB) is a critical regulator of diverse stimulus-dependent transcriptional events. Following TCR stimulation, CREB is rapidly induced in CD4+ Th cell precursors, but not in effector Th cells. However, its role in mature T cell function is incompletely defined. Transgenic mice expressing a CREB dominant-negative (dn) mutation in the T cell lineage exhibited normal T cell development in the thymus, normal T cell homeostasis in the periphery, and normal T cell clonal expansion following Ag challenge. However, this mutation caused selective inhibition of Th cell function in vitro and in vivo, and increased susceptibility of Th cells to activation-induced cell death. Th cells expressing the CREB-dn mutation contained reduced levels of the inhibitor of programmed cell death, BCL-2; overexpression of BCL-2 in transgenic mice reversed both susceptibility to activation-induced cell death in CREB-dn T cells and the defect in effector cytokine production. Thus, CREB plays a critical role in Th cell function and development of Th cell-mediated adaptive immune responses, at least in part, by inhibiting stimulus-dependent cell death.

Alanine↗
PubMed1996

Identification and charaterization of the second retinoic acid response element in the phosphoenolpyruvate carboxykinase gene promoter.

A previously characterized retinoic acid response element (RARE1) in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter confers approximately 50% of the response of this gene to retinoic acid (RA). Transient transfection experiments were performed using constructs containing progressive 5' deletions of the PEPCK promoter to locate other elements that contribute to the RA response. A second RARE (RARE2) was located between -402 and -306. Methylation interference and mobility gel shift assays indicated that RAR/RXR bound specifically to a segment of DNA located between -337 and -321. This region contains consensus and degenerate half-sites for receptor binding separated by 5 bp. Mutations in either half-site selectively decreased the RA response and diminished RAR/RXR binding in mobility gel shift assays. When both RARE1 and RARE2 were mutated, 80% of the RA response was lost. Finally, RARE2 conferred a RA response in a heterologous promoter context. We conclude that RAR/RXR binds to RARE2, and that this DR5-type element is a major contributor to the response of the PEPCK gene to RA.

Animals↗
PubMed2002

Expression of cyclic adenosine monophosphate response-element binding protein in acute leukemia.

Cyclic adenosine monophosphate response-element binding protein (CREB) is a nuclear protein that regulates expression of genes that control cell proliferation, differentiation, and survival. To analyze CREB expression in leukemia cells, we conducted Western blot analysis of bone marrow cells obtained from patients with acute lymphoblastic leukemia, patients with acute myeloid leukemia, and patients without active leukemia. CREB was expressed at a higher frequency in bone marrow cells from patients with acute lymphoid or myeloid leukemia than in patients with leukemia remission or without leukemia. Our results indicate that CREB expression could be a useful marker for leukemia in patients with acute disease and suggest a role for CREB in leukemogenesis.

Adolescent↗
PubMed1999

Trithorax- and Polycomb-group response elements within an Ultrabithorax transcription maintenance unit consist of closely situated but separable sequences.

In Drosophila, two classes of genes, the trithorax group and the Polycomb group, are required in concert to maintain gene expression by regulating chromatin structure. We have identified Trithorax protein (TRX) binding elements within the bithorax complex and have found that within the bxd/pbx regulatory region these elements are functionally relevant for normal expression patterns in embryos and confer TRX binding in vivo. TRX was localized to three closely situated sites within a 3-kb chromatin maintenance unit with a modular structure. Results of an in vivo analysis showed that these DNA fragments (each approximately 400 bp) contain both TRX- and Polycomb-group response elements (TREs and PREs) and that in the context of the endogenous Ultrabithorax gene, all of these elements are essential for proper maintenance of expression in embryos. Dissection of one of these maintenance modules showed that TRX- and Polycomb-group responsiveness is conferred by neighboring but separable DNA sequences, suggesting that independent protein complexes are formed at their respective response elements. Furthermore, we have found that the activity of this TRE requires a sequence (approximately 90 bp) which maps to within several tens of base pairs from the closest neighboring PRE and that the PRE activity in one of the elements may require a binding site for PHO, the protein product of the Polycomb-group gene pleiohomeotic. Our results show that long-range maintenance of Ultrabithorax expression requires a complex element composed of cooperating modules, each capable of interacting with both positive and negative chromatin regulators.

Animals↗
PubMed2005

Role of cAMP response element binding protein in cardiovascular remodeling: good, bad, or both?

The cAMP response element binding protein (CREB) is a ubiquitously expressed nuclear transcription factor that is activated by various extracellular stimuli. CREB is known to regulate the expression of genes important to cell proliferation, differentiation, adaptation, and survival in many cell types. Loss of CREB function by transgenic overexpression of dominant negative CREB or targeted deletion of the CREB gene revealed that CREB is involved in the differentiation of T lymphocytes, production of growth hormone, and the long-term potentiation of neuronal memory. The role of CREB in cardiovascular system is incompletely characterized and several controversies remain. A growing body of recent evidence, however, has suggested that CREB plays an important role in the cardiovascular remodeling process, including inflammation, cell migration, and apoptosis. Thus, CREB may be a possible target for the treatment of cardiovascular diseases such as atherosclerosis, restenosis, and reperfusion injury.

Animals↗
PubMed1989

Fine mapping of a mouse metallothionein gene metal response element.

Metal-regulated transcription of metallothionein (MT) genes in higher eucaryotes involves multiple copies of a highly conserved 17-base-pair metal-regulatory element (MRE). We have assayed by transient transfection the ability of mouse MT-I element d (MREd) to confer metal responsivity to constructs containing the mouse MT-I TATA box and the bacterial chloramphenicol acetyltransferase indicator gene. A single copy of MREd works bidirectionally to afford a three- to fourfold induction, and dual copies act cooperatively to yield a 10- to 20-fold response. Element d responds to the same spectrum of heavy metals as doses the complete MT gene promoter. The sequences involved in induction by metals were delineated by analyzing point mutations in MREd. While nucleotides of the highly conserved core sequence TGCPuCXC are critical, substitutions in the less conserved regions affect the induction response only marginally. These sequences include residues of a potential Sp1-binding site, suggesting that if Sp1 binds to MREd, it has little if any role in induction by metals.

Animals↗