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PubMed2003

Overexpression and RNA interference of Ap-cyclic AMP-response element binding protein-2, a repressor of long-term facilitation, in Aplysia kurodai sensory-to-motor synapses.

cyclic AMP-response element binding protein-2 (CREB2) is a member of the CREB/transcription factor (CREB/ATF4) family. CREB2 is a transcription factor known to be involved in Aplysia long-term facilitation. To further examine the role of ApCREB2 on long-term synaptic facilitation, we isolated ApCREB2 from Aplysia kurodai in full-length cDNA library, and found that the overexpression of ApCREB2 blocked 5-hydroxytryptamine (5-HT)-induced long-term synaptic facilitation in Aplysia sensory-to-motor synapses. Furthermore, a single pulse of 5-HT, which normally induces only short-term facilitation, in the presence of ApCREB2 inhibition by RNA interference, induced long-term facilitation in Aplysia sensory-to-motor synapses. These results suggest that ApCREB2 is a functional repressor of long-term facilitation in Aplysia sensory-to-motor synapses.

Animals↗
PubMed1999

Functional consensus for mammalian osmotic response elements.

The molecular mechanisms underlying adaptation to hyperosmotic stress through the accumulation of organic osmolytes are largely unknown. Yet, among organisms, this is an almost universal phenomenon. In mammals, the cells of the renal medulla are uniquely exposed to high and variable salt concentrations; in response, renal cells accumulate the osmolyte sorbitol through increased transcription of the aldose reductase (AR) gene. In cloning the rabbit AR gene, we found the first evidence of an osmotic response region in a eukaryotic gene. More recently, we functionally defined a minimal essential osmotic response element (ORE) having the sequence CGGAAAATCAC(C) (bp -1105 to -1094). In the present study, we systematically replaced each base with every other possible nucleotide and tested the resulting sequences individually in reporter gene constructs. Additionally, we categorized hyperosmotic response by electrophoretic mobility shift assays of a 17-bp sequence (-1108 to -1092) containing the native ORE as a probe against which the test constructs would compete for binding. In this manner, binding activity was assessed for the full range of osmotic responses obtained. Thus we have arrived at a functional consensus for the mammalian ORE, NGGAAAWDHMC(N). This finding should accelerate the discovery of genes previously unrecognized as being osmotically regulated.

Aldehyde Reductase↗
PubMed2003

Involvement of Nrf2 and JNK1 in the activation of antioxidant responsive element (ARE) by chemopreventive agent phenethyl isothiocyanate (PEITC).

PURPOSE: Phenethyl isothiocyanate (PEITC) has been of great interest as a promising cancer chemopreventive agent. To better understand its chemopreventive activity, we examined the effect of PEITC on the antioxidant responsive element (ARE), which is an important gene regulatory element of many phase II drug-metabolizing/detoxification enzymes as well as cellular defensive enzymes. METHODS: HeLa cells were transiently transfected with different cDNA plasmids using calcium phosphate precipitation. Subsequently, the cells were maintained in fresh media, and various concentrations of PEITC were added to the transfected cells. After harvesting and lysing of the cells, ARE-luciferase reporter gene activity was measured and normalized against beta-galactosidase activity. RESULTS: Treatments of HeLa cells with PEITC transiently stimulated ARE-reporter gene expressions in a dose-dependent manner. Overexpression of wild-type NF-E2 related factor-2 (Nrf2) dramatically increased ARE-reporter gene expression in a dose-dependent manner. Similar effects were seen when wild-type c-Jun N-terminal kinase 1 (JNK1) was transfected, although the transactivating potential of JNK1 was much less than that of Nrf2. Cotransfection of Nrf2 and JNK1 showed additional enhancement of ARE reporter gene expression, implying that JNK1 might be an upstream activator of Nrf2. To support this, overexpression of dominant-negative JNK1 suppressed Nrf2-induced ARE reporter gene expression in a dose-dependent manner. When PEITC was added, slight enhancement of ARE reporter gene expression was observed in either Nrf2- or JNK1-transfected cells. Finally, ARE reporter activity induced by PEITC was substantially attenuated by transfection of either dominant-negative mutant of Nrf2 or dominant-negative mutant of JNK1. CONCLUSION: Taken together, these data suggest that JNK1 acts as an upstream activator of Nrf2 and that PEITC activates ARE-mediated phase II drug metabolism gene expressions via the JNK1- and Nrf2-dependent pathways.

Anticarcinogenic Agents↗
PubMed1991

Expression of the CRE-BP1 transcriptional regulator binding to the cyclic AMP response element in central nervous system, regenerating liver, and human tumors.

CRE-BP1 is a transcriptional regulator binding to the cyclic AMP response element (CRE). To understand the role of CRE-BP1 in vivo, we studied the expression of the CRE-BP1 gene in monkey tissues including the central nervous system, in rat regenerating liver, and in human cancer tissues compared with normal tissues. The CRE-BP1 mRNA was detected in all tissues examined, and was fairly abundant in brain. The CRE-BP1 mRNA was expressed in monkey brain tissues with different region specificities. In the hippocampus, frontal lobe, and parietal lobe, the CRE-BP1 mRNA was abundant and two mRNA species 4.0 kb and 3.7 kb in length were expressed. In rat liver, the expression of the CRE-BP1 gene was increased up to 4- to 5-fold of the normal level within 12-24 h after partial hepatectomy. Furthermore, the levels of CRE-BP1 mRNA in some clinical samples of human tumors were apparently higher than that in normal tissues. These results suggest that CRE-BP1 may be important for both the signal transduction in brain and cellular proliferation.

Animals↗
PubMedDate not supplied

Involvement of cAMP responsive element binding protein (CREB) in the synovial cell hyperfunction in patients with rheumatoid arthritis.

OBJECTIVE: To elucidate a possible role of cAMP responsive element binding protein (CREB) in rheumatoid arthritis (RA) synovial cell function, we have studied CREB expression of synovial cells and the effects of an inhibitor of the cAMP/CREB signal pathway on synovial cell function in patients with RA. METHODS: We examined CREB expression by immunohistochemical staining, immunocytochemical staining, and gel shift assays. Effects of cAMP/CREB inhibitor on the proliferation of RA synovial cells were assessed by [3H]-TdR incorporation, and those on proinflammatory cytokine and matrix metalloproteinase (MMP) production by reverse transcription PCR and ELISAs. RESULTS: Immunohistochemical staining of synovial tissue revealed that CREB is expressed mainly in the lining and sublining layers of synovium in patients with RA. DNA binding activity of CREB was ascertained by a gel shift assay. We also confirmed nuclear translocation and phosphorylation of CREB in TNF-alpha stimulated RA fibroblast-like synovial cells by immunocytochemical staining. Modulators of cAMP/CREB signaling pathway, such as Rp-cAMP, had an inhibitory potential on RA synovial cell proliferation in vitro. Rp-cAMP also inhibited the proinflammatory cytokine and MMP production. CONCLUSION: CREB is involved in the synovial cell activity in patients with RA. Inhibition of CREB activity by its inhibitor brings about the correction of aberrant synovial cell functions in patients with RA, thus suggesting a possible clinical application of cAMP/CREB inhibitors.

Aged↗
PubMed2002

Identification of a variant antioxidant response element in the promoter of the human glutamate-cysteine ligase modifier subunit gene. Revision of the ARE consensus sequence.

Constitutive and inducible expression of the gene encoding the modulator subunit of human glutamate-cysteine ligase (GCLM) is regulated by either of two regions of the promoter; an antioxidant response element (ARE) at -302:-291 and a 44-bp fragment (-346:-303) upstream of the ARE. This second region includes a consensus AP-1 site previously considered responsible for the enhancer activity of the upstream fragment. Deletion of a 165-bp fragment (-348:-183) including the ARE and upstream 44-bp fragment totally ablated t-butyl hydroquinone (tBHQ) inducibility of a GCLM promoter/luciferase transgene. Mutation analyses confirmed that both the ARE and the -346:-303 fragment could support induction following tBHQ exposure but demonstrated that induction in the latter case did not involve the AP-1 site at -341:-335. A region sharing significant homology with the consensus ARE sequence except for a single nucleotide mismatch at -330 (5'-TTACnnnGCA-3' versus 5'-TGACnnnGCA-3') was identified at the 5'-end of the 44-bp fragment immediately adjacent to the AP-1 site. A G in this position has been considered an invariant requirement of functional ARE sequences. Mutation of T(-330) to A (a substitution known to ablate ARE function) or C eliminated basal and inducible expression. Substitution of a G at -330 enhanced basal expression relative to the wild-type sequence, but induction following tBHQ exposure was comparable, indicating that either sequence (5'-TTACnnnGCA-3' versus 5'-TGACnnnGCA-3') may function as an ARE, although the former sequence is less effective at directing basal expression. This possibility was confirmed by similar mutational analyses of the core sequence of hNQO1, a prototypic ARE. Electrophoretic mobility shift competition assays revealed that the 5'-TTACnnnGCA-3' sequence could compete with the hNQO1 ARE for protein binding but was less effective than a similar probe containing the 5'-TGACnnnGCA-3' motif. Probes including the T(-330)A or T(-330)C mutations were ineffective. These results reveal that the GCLM promoter includes two functional AREs, one having a variant sequence. The results indicate that the consensus ARE sequence should be revised to 5'-RTKAYnnnGCR-3'.

Amino Acid Sequence↗
PubMed2001

Phosphorylation of cyclic adenosine monophosphate response element binding protein in oligodendrocytes in the corpus callosum after focal cerebral ischemia in the rat.

Phosphorylation of cyclic adenosine monophosphate (AMP) response element binding protein (CREB) was examined immunohistochemically in the corpus callosum of the rat brain at various time points after 90-minute focal cerebral ischemia. Focal ischemia was induced by occlusion of the middle cerebral artery (MCA) using the intraluminal suture method. Sham animals showed that numerous oligodendrocytes (OLGs) constitutively express unphosphorylated CREB. Local cerebral blood flow (lCBF) measured by the 14C-iodoantipyrine method was reduced from 44.2 +/- 15.4 (mL 100 g(-1) min(-1)) to 18.4 +/- 3.8 and from 53.9 +/- 14.4 to 4.8 +/- 4.5 in the medial and the lateral regions of the corpus callosum, respectively, during MCA occlusion (MCAO). After release of the MCAO, lCBF recovered to the control level in each region. The medial region of the corpus callosum showed a marked increase in phosphorylated CREB-positive OLGs at 3.5 hours of recirculation, and it remained increased until 2 weeks of recirculation as it gradually declined. The activation of CREB phosphorylation in the OLGs was accompanied by expression of antiapoptotic protein bcl-2, normal staining with cresyl violet, and negative TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) staining. Myelination detected by immunostaining with anti-myelin basic protein (MBP) antibody and anti-myelin associated glycoprotein (MAG) antibody remained normal in the medial region of the corpus callosum. The lateral region of the corpus callosum showed a significant but only transient increase in phosphorylated CREB-positive OLGs at 3.5 hours of recirculation, which was followed by a rapid decrease during the subsequent recirculation period. Expression of bcl-2 was suppressed in this region, and demyelination became apparent. These findings suggest that signal transduction through CREB phosphorylation may be closely associated with survival of OLGs and maintenance of myelination in the corpus callosum after cerebral ischemia.

Adenomatous Polyposis Coli Protein↗
PubMed2001

Structure and characterization of the human insulin-like growth factor binding protein (IGFBP)-6 promoter: identification of a functional retinoid response element.

The 1.7 kb human insulin-like growth factor binding protein (IGFBP)-6 gene 5'-flanking region was sequenced and found to have promoter activity in human osteoblasts. The sequence contains four clustered transcription start sites and three retinoic acid response elements (RAREs) with widely spaced half-sites. Only the proximal DR15 RARE was functional. Retinoids increased IGFBP-6 promoter activity up to 3-fold.

Animals↗
PubMed2002

Abscisic acid-induced transcription is mediated by phosphorylation of an abscisic acid response element binding factor, TRAB1.

The rice basic domain/Leu zipper factor TRAB1 binds to abscisic acid (ABA) response elements and mediates ABA signals to activate transcription. We show that TRAB1 is phosphorylated rapidly in an in vivo labeling experiment and by phosphatase-sensitive mobility shifts on SDS-polyacrylamide gels. We had shown previously that a chimeric promoter containing GAL4 binding sites became ABA inducible when a GAL4 binding domain-TRAB1 fusion protein was present. This expression system allowed us to assay the ABA response function of TRAB1. Using this system, we show that Ser-102 of TRAB1 is critical for this function. Because no ABA-induced mobility shift was observed when Ser-102 was replaced by Ala, we suggest that this Ser residue is phosphorylated in response to ABA. Cell fractionation experiments, as well as fluorescence microscopy observations of transiently expressed green fluorescent protein-TRAB1 fusion protein, indicated that TRAB1 was localized in the nucleus independently of ABA. Our results suggest that the terminal or nearly terminal event of the primary ABA signal transduction pathway is the phosphorylation in the nucleus of preexisting TRAB1.

Abscisic Acid↗
PubMed1995

Integration of MAP kinase signal transduction pathways at the serum response element.

The ternary complex factor (TCF) subfamily of ETS-domain transcription factors bind with serum response factor (SRF) to the serum response element (SRE) and mediate increased gene expression. The TCF protein Elk-1 is phosphorylated by the JNK and ERK groups of mitogen-activated protein (MAP) kinases causing increased DNA binding, ternary complex formation, and transcriptional activation. Activated SRE-dependent gene expression is induced by JNK in cells treated with interleukin-1 and by ERK after treatment with phorbol ester. The Elk-1 transcription factor therefore integrates MAP kinase signaling pathways in vivo to coordinate biological responses to different extracellular stimuli.

Animals↗
PubMed2006

Real-time monitoring of cAMP response element binding protein signaling in porcine granulosa cells modulated by ovarian factors.

The present study was performed to establish a real-time monitoring of the cAMP response element binding protein (CREB) signalling using granulosa cells, and to assess the modulation of CREB activity by potential ovarian autocrine/paracrine and oocyte-derived factors. Granulosa cells were isolated from porcine follicles and cultured for 2 days, and then transfected with CRE-containing pGL3. The cells were directly stimulated or cultured with FSH, LH, forskolin, or a permeable cAMP analog, and/or IGF-I, EGF, bFGF, TGF-beta2 or TNF-alpha, or cumulus-oocyte complex (COCs) for the real-time monitoring of CREB signaling. The activation pattern of CREB signaling consisted of three distinct phases, i.e., burst, attenuation and refractory. In contrast to FSH, LH, and forskolin, a cAMP analog induced the prolonged activation, although three distinct phases were observed at its high concentration. Of all the autocrine/paracrine factors, only IGF-I slightly induced CREB activity. On the other hand, TGF-beta2 and TNF-alpha significantly repressed FSH-stimulated transcriptional activation of CREB by 30% (P < 0.05) and 45% (P < 0.05), respectively. Additionally, coculture with COCs caused a significant suppression of transcriptional activation of CREB signaling stimulated by FSH. These results indicate that ovarian autocrine/paracrine factors such as IGF-I, TGF-beta2, TNF-alpha and oocyte-derived factors modulate the CREB signaling. The present study provides a new approach for direct signaling study on transcription factors under the influences of potential factors.

Animals↗
PubMed1999

Multiple androgen response elements and a Myc consensus site in the androgen receptor (AR) coding region are involved in androgen-mediated up-regulation of AR messenger RNA.

The androgen receptor (AR) gene is transcriptionally regulated by AR (autoregulation); however, the androgen response elements (AREs) required for this process have not been found in the AR promoter or in the 5'-flanking region. We previously showed that the AR cDNA contains AREs involved in AR mRNA autoregulation and that auto(up)regulation is reproduced in PC3 cells (a human prostate cancer cell line) expressing the human AR cDNA driven by a heterologous promoter. A 350-bp fragment of the AR cDNA contains the requisite AREs (ARE-1 and ARE-2) and, when linked upstream of a reporter gene, confers androgen inducibility in a cell-specific manner. Here we report that, although an AR cDNA harboring silent mutations of ARE-1 and ARE-2 produces a transcriptionally active AR, AR mRNA encoded by this mutant cDNA is not up-regulated in androgen-treated PC3 cells. Thus, ARE-1 and ARE-2 are essential for androgen-mediated up-regulation of AR mRNA in this model. Since ARE-1 and ARE-2 are located on separate exons (exons D and E) in the AR gene, we evaluated these AREs in their native context, a 6.5-kb AR genomic fragment. Androgen regulated the 6.5-kb AR genomic fragment and the 350-bp region of the AR cDNA at comparable levels, suggesting that sequences in exons D and E are likely to be involved in androgen-mediated up-regulation of the native AR gene. Furthermore, androgen regulated both responsive regions in U2OS cells, a human osteoblastic cell line that exhibits androgen-mediated up-regulation of native AR mRNA. DNAse I footprinting of the 350-bp region with recombinant AR (DNA- and ligand-binding domains) suggested the presence of additional AREs. Gel shift analyses and mutational studies showed that maximal androgen regulation and AR binding were dependent on the integrity of four AREs (ARE-1, ARE-1A, IVSARE, and ARE-2). While the presence of multiple, nonconsensus AREs is common among other androgen-regulated enhancers, the androgen-responsive region of the AR gene is unique because it contains exonic AREs. DNA binding studies with nuclear extracts were performed to determine whether non-AR transcription factors contribute to androgen regulation of the 350-bp region. These studies, in conjunction with mutational analysis and reporter gene assays with dominant negative Myc and Max expression vectors, showed that Myc and Max interaction with a Myc consensus site is required for androgen regulation of the 350-bp fragment. These results represent a novel interaction between AR and the Myc family of proteins and support a model of androgenic control of AR mRNA via AR and Myc family interaction with a unique internal androgen-responsive region harboring multiple exonic regulatory sequences.

Androgens↗
PubMed2006

Bending of the estrogen response element by polyamines and estrogen receptors alpha and beta: a fluorescence resonance energy transfer study.

Estrogenic regulation of gene expression is mediated by the binding of the hormone to its receptors (ERalpha and ERbeta) followed by their binding to estrogen response element (ERE). Previous studies showed that natural polyamines -- putrescine, spermidine, and spermine -- facilitated ERalpha.ERE recognition. We determined the effects of natural and synthetic polyamines on the bending of a 27-mer oligonucleotide (ODN) harboring the ERE (ERE-ODN), using fluorescence resonance energy transfer (FRET) technique. Complementary strands of the ERE-ODN were labeled with fluorescein and tetramethylrhodamine, as donor and acceptor, respectively. The ERE-ODN was intrinsically bent with an end-to-end distance of 76 +/- 2 Angstrom, compared to a theoretical value of 98 Angstrom. The end-to-end distance of the ERE-ODN was reduced to 64 Angstrom in the presence of 250 microM spermine. A control ODN with scrambled sequence did not show intrinsic bending or spermine-induced bending. Alkyl substitution at the pendant amino groups reduced the ability of spermine to bend the ERE-ODN. Both ERalpha and ERbeta decreased the end-to-end distance of the ERE-ODN, although ERalpha was more efficient than ERbeta in inducing ERE bending. Spermine-induced bending of the ERE-ODN was significantly increased by ERalpha. Fluorescence anisotropy measurement showed that the equilibrium association constant of ERalpha-ERE binding increased by 12-fold in the presence of 250 microM spermine compared to control. The free energy change (Delta G) of ERalpha.ERE complex formation was -13.1 kcal/mol at 22 degrees C in the presence of spermine. Our results suggest that polyamine-induced bending of the ERE might be a mechanism for enhancing ERalpha-ERE binding affinity and thereby fine-tuning the transcriptional response of estrogen-responsive genes.

Allosteric Regulation↗
PubMed2007

An interferon-sensitive response element is involved in constitutive caspase-8 gene expression in neuroblastoma cells.

We previously identified a 1.2 Kb DNA element (P-1161/+16), 5' to caspase-8 exon-1, that acts as promoter in caspase-8-positive, but not in caspase-8-negative neuroblastoma (NB) cells. The P-1161/+16 DNA element regulates both constitutive and interferon IFN-gamma-inducible caspase-8 expression. Two GAS (IFN-activated sequence, STAT-1 binding site) and two ISRE (interferon sensitive response element, IRF binding site) were present in P-1161/+16. Deletion studies indicated that elements essential for promoter activity in NB cells were present in a 167 bp region 5' flanking exon-1 (P-151/+16), which contains an ISRE at position -32. The transcription initiation site was mapped by 5' rapid amplification of cDNA end (RACE) at position -20 from caspase-8 cDNA reference sequence. Disruption of the ISRE-32 indicated that it is required for both constitutive and IFN-gamma-inducible caspase-8 expression. IRF-1 and IRF-2 transcription factors bind to the (-151/+16) DNA fragment in vitro. Chromatin immunoprecipitation (ChIP) assays showed that IRF-1 and IRF-2 bind to the DNA region at the 5' of caspase-8 gene in NB cells, which show constitutive expression but not in caspase-8 negative cells. In these last cells, up-regulation of caspase-8 by IFN-gamma was associated to induction of IRF-1 and IRF-2 binding to caspase-8 promoter and increased histone acetylation. Moreover, RNA interference experiments also supported the involvement of IRF-1 and IRF-2 in constitutive caspase-8 expression in NB cells.

Antiviral Agents↗
PubMed1998

p38 Mitogen-activated protein kinase mediates the transcriptional induction of the atrial natriuretic factor gene through a serum response element. A potential role for the transcription factor ATF6.

In various cell types certain stresses can stimulate p38 mitogen-activated protein kinase (p38 MAPK), leading to the transcriptional activation of genes that contribute to appropriate compensatory responses. In this report the mechanism of p38-activated transcription was studied in cardiac myocytes where this MAPK is a key regulator of the cell growth and the cardiac-specific gene induction that occurs in response to potentially stressful stimuli. In the cardiac atrial natriuretic factor (ANF) gene, a promoter-proximal serum response element (SRE), which binds serum response factor (SRF), was shown to be critical for ANF induction in primary cardiac myocytes transfected with the selective p38 MAPK activator, MKK6 (Glu). This ANF SRE does not possess sequences typically required for the binding of the Ets-related ternary complex factors (TCFs), such as Elk-1, indicating that p38-mediated induction through this element may take place independently of such TCFs. Although p38 did not phosphorylate SRF in vitro, it efficiently phosphorylated ATF6, a newly discovered SRF-binding protein that is believed to serve as a co-activator of SRF-inducible transcription at SREs. Expression of an ATF6 antisense RNA blocked p38-mediated ANF induction through the ANF SRE. Moreover, when fused to the Gal4 DNA-binding domain, an N-terminal 273-amino acid fragment of ATF6 was sufficient to support trans-activation of Gal4/luciferase expression in response to p38 but not the other stress kinase, N-terminal Jun kinase (JNK); p38-activating cardiac growth promoters also stimulated ATF6 trans-activation. These results indicate that through ATF6, p38 can augment SRE-mediated transcription independently of Ets-related TCFs, representing a novel mechanism of SRF-dependent transcription by MAP kinases.

Activating Transcription Factor 6↗
PubMed2005

Group I metabotropic glutamate receptors, mGlu1a and mGlu5a, couple to cyclic AMP response element binding protein (CREB) through a common Ca2+ - and protein kinase C-dependent pathway.

Coupling of the group I metabotropic glutamate receptors, mGlu1a and mGlu5a, to the cAMP response element binding protein (CREB) has been studied in Chinese hamster ovary cell lines where receptor expression is under the control of an inducible promoter. Both receptors stimulate CREB phosphorylation with similar time courses, and agonist potency was also comparable between the two receptors. Stimulation of cells in Ca(2+)-free medium containing EGTA (100 microm), with or without the additional depletion of intracellular stores, caused marked decreases in agonist-mediated responses in both cell lines. Down-regulation of protein kinase C (PKC) activity by phorbol ester treatment, or treatment with the broad spectrum PKC inhibitor Ro 31-8220, partially attenuated both mGlu1a and mGlu5a receptor-mediated responses. Furthermore, stimulation of cells in the absence of extracellular Ca(2+) following prior PKC down-regulation resulted in additive inhibitory effects. The involvement of extracellular signal-regulated kinases (ERK1/2), Ca(2+)/calmodulin or Ca(2+)/calmodulin-dependent protein kinases was assessed using pharmacological inhibitors. Results indicated that coupling of the group I mGlu receptors to CREB phosphorylation occurs independently of these pathways. Thus, although the [Ca(2+)](i) signatures activated by these mGlu receptors differ, they couple to CREB with comparable potency and recruit similar downstream components to execute CREB phosphorylation.

Analysis of Variance↗
PubMed1994

Identification of 3',5'-cyclic adenosine monophosphate response element and other cis-acting elements in the human androgen receptor gene promoter.

Androgen and androgen receptor (AR) play an important role in sexual differentiation and prostate proliferation. To investigate AR gene transcriptional regulation, a 2.3-kilobase AR gene promoter region was isolated, sequenced, and characterized. Chloramphenicol acetyltransferase (CAT) assay and sequence homology search of AR gene promoter among human, rat, and mouse revealed some potential cis-acting elements, including a GC box, a suppressor region, and a purine-rich element. Deletion analysis and gel retardation assay using a 50-base pair (bp) double-strand purine-rich element showed that this purine-rich element can bind to specific proteins in nuclear extract of LNCaP and HeLa cells and may be essential for AR gene transcription. Furthermore, to investigate the effect of cAMP on AR gene transcription, we treated LNCaP and HeLa cells with 10 mM (Bu)2cAMP after transfection with CAT gene reporter plasmids linked to the AR gene promoter. This treatment induced several folds of CAT activity in LNCaP cells only, and the induction was further confirmed at AR mRNA level by Northern blot analysis and reverse transcription-polymerase chain reaction assay. Deletion analysis of the AR gene promoter showed that a region between 530 bp and 380 bp upstream of AR gene transcription initiation site, which includes one potential cAMP response element (CRE), is responsible for cAMP induction. Gel retardation analysis using this CRE (AR/CRE1) showed that AR/CRE1 can bind to specific proteins in nuclear extract of LNCaP cells, which appears to form a different binding complex compared to somatostatin/CRE.

Animals↗
PubMed1994

Antiestrogens activate an estrogen receptor mutant exhibiting enhanced binding to the estrogen response element.

The appearance of constitutively active and antiestrogen resistant estrogen receptor variants has been proposed as one of several factors leading to the development of antiestrogen resistant breast cancers. We recently described the ER2, estrogen receptor mutant, which exhibits enhanced binding to the estrogen response element on DNA, and partial constitutive estrogen-independent) ability to activate transcription. In this work we used transient transfections to show that the antiestrogens trans hydroxytamoxifen and ICI 164,384 are unable to suppress the constitutive activity of the ER2 mutant. Instead, both antiestrogens were concentration-dependent activators of the ER2 mutant. The ER2 mutant appears to be the first estrogen receptor mutant to show activation of a simple estrogen response element-containing promoter using the "pure" antiestrogen ICI 164,384.

Animals↗