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PubMed2000

Response element and coactivator-mediated conformational change of the vitamin D(3) receptor permits sensitive interaction with agonists.

The vitamin D receptor (VDR) is the nuclear receptor for 1, 25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] that acts as a ligand-dependent transcription factor via combined contact with coactivator proteins (steroid receptor coactivator-1, transcriptional intermediary factor 2, and receptor associated coactivator 3) and specific DNA binding sites [vitamin D response elements (VDREs)]. Ligand-mediated conformational changes of the VDR contribute to the key mechanisms in this nuclear hormone signaling process. 1alpha,25(OH)(2)D(3), MC1288 [20-epi-1alpha,25(OH)(2)D(3)], ZK161422 [20-methyl-1alpha,25(OH)(2)D(3)], and Ro27-2310 (also called Gemini, having two side chains at carbon 20) were used as model VDR agonists. The analysis of agonist-induced VDR conformations and coactivator interactions were found to be insufficient for extrapolating in vivo activities. In DNA-independent assays, such as classical limited protease digestions and glutathione S-transferase pull downs, Gemini seemed to be up to 10,000-fold and the other VDR agonists 10- to 100-fold weaker than in functional in vivo assays. A more accurate description of the gene regulatory potential of VDR agonists was obtained with all tested VDR agonists by analyzing VDR conformations in the context of VDRE-bound VDR-retinoid X receptor heterodimers, in such assays as gel supershift, gel shift clipping, and limited protease digestion in the presence of DNA and cofactor. Coactivators were found to shift the ligand sensitivity (by a factor of 4 for Gemini) and the ratio of VDR conformations in the presence of DNA toward the high-affinity ligand binding conformation (c1(LPD)). In conclusion, the induction of response element- and coactivator-modulated VDR conformations appears to be a key step for the gene regulatory function of a VDR agonist. The quantification of these effects would be of central importance for the evaluation of the cell-specific efficacy of systemically applied 1alpha, 25(OH)(2)D(3) analogs.

Animals↗
PubMed2000

Cloning and expression analysis of testis-specific cyclic 3', 5'-adenosine monophosphate-responsive element modulator activators in the nonhuman primate (Macaca fascicularis): comparison with other primate and rodent species.

The cAMP-responsive element modulator (CREM) gene encodes a transcription factor that is essential for spermatogenesis. In mouse testis, several CREM repressors and activators have been identified. In contrast to the situation for the mouse, however, little is known about CREM isoforms in the primate testis. We analyzed CREM isoforms and mRNA expression in a clinically relevant primate model, the cynomolgus monkey (Macaca fascicularis). A cDNA library was generated from monkey testis; and two activator isoforms (tau2 with and without exon gamma) were identified, which displayed high sequence identity to mouse and human isoforms. The insertion of exon gamma was observed for the first time in the primate testis. CREM activator expression was confined to the testis, where it was seen in late pachytene spermatocytes and round spermatids in specific spermatogenic stages, as revealed by in situ hybridization. Comparison of the mRNA and the recently described protein expression indicated a lack of translational delay of CREM expression. Comparative analysis of testicular CREM expression by reverse transcription-polymerase chain reaction yielded several transcripts in the rat, mouse, hamster, and marmoset; two transcripts in cynomolgus and rhesus monkeys; and one transcript in men. These findings suggest an evolutionary trend from multiple activator isoforms to a single activator transcript in men.

Amino Acid Sequence↗
PubMed1993

Expression pattern of proteins that bind to the ultraviolet-responsive element (TGACAACA) in human keratinocytes.

We previously identified an ultraviolet (UV)-responsive element (URE; TGACAACA) that plays a role in both transcription and replication of polyoma sequences. Mouse polyclonal antibodies were raised against affinity-purified URE-bound proteins to characterize their expression patterns. These antibodies specifically recognized two of four URE-bound proteins, of 40 and 68 kDa. The 68-kDa protein was constitutively expressed in human keratinocytes, while the expression of the 40-kDa protein was induced by UV irradiation. Of the two, the 68-kDa protein bound to the URE with greater affinity than the 40-kDa protein, as determined by southwestern analysis. The expression of the 40-kDa protein increased as early as 1 h after UV irradiation of both rat fibroblasts and human keratinocytes and correlated with increased binding to the URE in an electrophoretic mobility shift assay. Other types of damage, as well as heat shock and serum stimulation, also induced the expression of this protein, suggesting that it may play a role in cellular response to stress or damage. The 40-kDa protein was expressed at the highest levels in the S phase of the cell cycle and was induced by aphidicolin, suggesting that it has a role in DNA replication. All together, these results suggest that exposure of human keratinocytes to damage- and stress-inducing agents modulates the expression of proteins that may play a role in regulating cellular response to DNA damage.

Animals↗
PubMed2006

The transcriptional repressor cAMP response element modulator alpha interacts with histone deacetylase 1 to repress promoter activity.

Transcriptional repression is a fundamental mechanism of gene regulation. cAMP response element (CRE) modulator (CREM)alpha is an ubiquitously expressed transcription factor and a counterpart of the activator CREB. In T cells, CREM is responsible for the termination of the IL-2 expression by a chromatin-dependent mechanism. We demonstrate in this study that CREMalpha associates with histone deacetylase (HDAC)1 through its H domain, which is located between the kinase inducible and DNA binding domains. The CREMalpha-mediated recruitment of HDAC1 to the CRE sites of the IL-2 and c-Fos promoter causes histone deacetylation and inaccessibility to restriction enzymes and limited transcriptional activity. Importantly, the CRE sites of these promoters are crucial for the activity and binding of HDAC1. Therefore, CREMalpha exerts its repressor activity by a mechanism that involves recruitment of HDAC1, increased deacetylation of histones, and repression of promoter activity.

Acetylation↗
PubMed1996

Human cholesteryl ester transfer protein gene proximal promoter contains dietary cholesterol positive responsive elements and mediates expression in small intestine and periphery while predominant liver and spleen expression is controlled by 5'-distal sequences. Cis-acting sequences mapped in transgenic mice.

The plasma cholesteryl ester transfer protein (CETP) facilitates the transfer of high density lipoprotein cholesteryl esters to other lipoproteins and appears to be a key regulated component of reverse cholesterol transport. Earlier studies showed that a CETP transgene containing natural flanking sequences (-3.4 kilobase pairs (kbp) upstream, +2.2 kbp downstream) was expressed in an authentic tissue distribution and induced in liver and other tissues in response to dietary or endogenous hypercholesterolemia. In order to localize the DNA elements responsible for these effects, we prepared transgenic mice expressing six new DNA constructs containing different amounts of natural flanking sequence of the CETP gene. Tissue-specific expression and dietary cholesterol response of CETP mRNA were determined. The native pattern of predominant expression in liver and spleen with cholesterol induction was shown by a -3.4 (5'), +0.2 (3') kbp transgene, indicating no major contribution of distal 3'-sequences. Serial 5'-deletions showed that a -570 base pairs (bp) transgene gave predominant expression in small intestine with cholesterol induction of CETP mRNA in that organ, and a -370 bp transgene gave highest expression in adrenal gland with partial dietary cholesterol induction of CETP mRNA and plasma activity. Further deletion to -138 bp 5'-flanking sequence resulted in a transgene that was not expressed in vivo. Both the -3.4 kbp and -138 bp transgenes were expressed when transfected into a cultured murine hepatocyte cell line, but only the former was induced by treating the cells with LDL. When linked to a human apoA-I transgene, the -570 to -138 segment of the CETP gene promoter gave rise to a relative positive response of hepatic apoA-I mRNA to the high cholesterol diet in two out of three transgenic lines. Thus, 5'-elements between -3,400 and -570 bp in the CETP promoter endow predominant expression in liver and spleen. Elements between -570 and -370 are required for expression in small intestine and some other tissues, and elements between -370 and -138 contribute to adrenal expression. The minimal CETP promoter element associated with a positive sterol response in vivo was found in the proximal CETP gene promoter between -370 and -138 bp. This region contains a tandem repeat of a sequence known to mediate sterol down-regulation of the HMG-CoA reductase gene, suggesting either the presence of separate positive and negative sterol response elements in this region or the use of a common DNA element for both positive and negative sterol responses.

Animals↗
PubMed2003

RNA-ligand interactions: affinity and specificity of aminoglycoside dimers and acridine conjugates to the HIV-1 Rev response element.

Semisynthetic aminoglycoside derivatives may provide a means to selectively target viral RNA sites, including the HIV-1 Rev response element (RRE). The design, synthesis, and evaluation of derivatives based upon neomycin B, kanamycin A, and tobramycin conjugates of 9-aminoacridine are presented. To evaluate the importance of the acridine moiety, a series of dimeric aminoglycosides as well as unmodified "monomeric" aminoglycosides have also been evaluated for their nucleic acid affinity and specificity. Fluorescence-based binding assays that use ethidium bromide or Rev peptide displacement are used to quantify the affinities of these compounds to various nucleic acids, including the RRE, tRNA, and duplex DNA. All the modified aminoglycosides exhibit a high affinity for the Rev binding site on the RRE (K(d) <or= 10 nM), but few compounds have a high specificity for the RRE. Compared to the acridine conjugates, the dimeric and unmodified aminoglycosides exhibit good RNA over DNA selectivity, but show little differentiation between different RNA molecules. Neomycin-based derivatives consistently have the highest RNA and DNA affinities, but the lowest RRE specificity. To optimize these derivatives for RRE specificity, a series of neomycin-acridine conjugates with variable linker lengths were synthesized and evaluated. The neo-acridine conjugate with the shortest linker length has the optimal RRE specificity. Duplex DNA, on the other hand, prefers the acridine conjugate with the longest linker length, and duplex RNA (poly r[A]-r[U]) has the highest affinity for the conjugate with an intermediate linker length. Compared to neomycin B, the derivatives based upon tobramycin and kanamycin A have slightly lower RRE affinities, but better RRE specificities. These results illustrate how the binding affinity and specificity of aminoglycoside intercalator conjugates can be tuned by optimizing the linker length and by changing the identity of the aminoglycoside moiety. These results also indicate that many aminoglycoside-based ligands are capable of high-affinity binding of RNA, but achieving high site specificity remains a challenging objective.

Acridines↗
PubMed1998

Crosstalk between the interleukin-6 (IL-6)-JAK-STAT and the glucocorticoid-nuclear receptor pathway: synergistic activation of IL-6 response element by IL-6 and glucocorticoid.

Cytokines and steroid hormones use different sets of signal transduction pathways, which seem to be unrelated. Interleukin-6 (IL-6) uses JAK tyrosine kinase and STAT (signal transducer and activator of transcription) transcription factor. Glucocorticoid binds glucocorticoid receptor (GR), which is a member of the steroid receptor superfamily. We have studied the crosstalk between the IL-6-JAK-STAT and glucocorticoid-nuclear receptor pathways. IL-6 and glucocorticoid synergistically activated the IL-6 response element on the rat alpha2-macroglobulin promoter (APRE)-driven luciferase gene. The exogenous expression of GR enhanced the synergism. The exogenous expression of dominant negative STAT3 completely abolished the IL-6 plus glucocorticoid-induced activation of the APRE-luciferase gene. Tyrosine phosphorylation of STAT3 stimulated by IL-6 alone was not different from that by IL-6 plus glucocorticoid. The protein level of STAT3 was also not increased by glucocorticoid stimulation. The time course of STAT3 tyrosine phosphorylation by IL-6 plus glucocorticoid was not different from that by IL-6 alone. The synergism was studied on the two other IL-6 response elements, the junB promoter (JRE-IL-6) and the interferon regulatory factor-1 (IRF-1) promoter (IRF-GAS) which could be activated by STAT3. The synergistic activation by glucocorticoid on the IL-6-activated JRE-IL-6 and the IRF-GAS-driven luciferase gene was not detected. Glucocorticoid did not change the mobility of IL-6-induced APRE-binding proteins in a gel shift assay. These results suggest that the synergism was through the GR and STAT3, and the coactivation pathway which was specific for APRE was the target of glucocorticoid.

DNA-Binding Proteins↗
PubMed1996

Vitamin D receptor binding to the negative human parathyroid hormone vitamin D response element does not require the retinoid x receptor.

An important physiological control of PTH gene expression is its transcriptional repression by 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)]. The mechanism of this 1,25-(OH)(2)D(3)-mediated transcriptional repression is poorly understood. Previous investigations have identified a DNA sequence in the 5'-regulatory region of the human PTH (hPTH) gene that binds the vitamin D receptor (VDR) and mediates transcription repression in response to 1,25(OH)(2)D(3) in GH4CI cells. The hPTH gene sequence does not mediate transcriptional repression in ROS 17/2.8 cells, even though up-regulatory vitamin D response elements (VDREs) are active in these cells. The hPTH DNA sequence differs from the upregulatory VDREs in that it contains a single copy of a hexameric motif (AGGUC) homologous to those repeated in the up-regulatory VDREs. The protein-DNA interactions of this sequence were examined using nuclear extracts from bovine parathyroid, GH4CI, and ROS 17/2.8 cells. In bovine parathyroid nuclear extracts, the VDR binds the down-regulatory hPTH DNA sequence independently of the retinoid X receptor (RXR). In GH4C1 nuclear extracts, two VDR-containing complexes are observed: one lacking RXR and one containing RXR. In ROS 17/2.8 nuclear extracts, a single VDRdependent complex containing RXR is observed. When the up-regulatory rat osteocalcin VDRE is used as a probe, only VDR-RXR-containing complexes are generated using nuclear extracts from all three cell types. These results demonstrate that the sequence that mediates transcriptional repression in response to 1 ,25-(OH)(2)D(3) differs from the up-regulatory response elements both in sequence composition and in its ability to bind VDR independently of RXR.

Animals↗
PubMed1988

Receptors bound to antiprogestin from abortive complexes with hormone responsive elements.

The mechanism of action of antisteroids is not understood and explanations of their antagonistic activity have been sought at all levels of hormone action. It has been proposed that antisteroids, after binding to receptor, trap it into a non-activated (non DNA-binding) form possibly through interaction with a heat-shock protein of relative molecular mass (Mr) 90,000 (90 K), or that the antisteroids provoke binding of receptor to nonspecific DNA sites but not to hormone responsive elements (HREs), or that the antisteroid-receptor complexes can bind to HREs but form abortive complexes that fail to regulate transcription. We have constructed a deleted cDNA encoding a mutant form of rabbit progesterone receptor which exhibits constitutive activity, that is, binds to HREs in the absence of hormone and thus bypasses the first two steps discussed above. Co-transfection experiments allowed the expression of both constitutive and wild-type receptors in the same recipient cells. Antiprogestin RU486-wild-type receptor complexes completely suppressed the activity of the constitutive receptor on a reporter gene, showing that the inhibition is at the level of their common responsive elements.

Animals↗
PubMed1999

The human RC3 gene homolog, NRGN contains a thyroid hormone-responsive element located in the first intron.

NRGN is the human homolog of the neuron-specific rat RC3/neurogranin gene. This gene encodes a postsynaptic 78-amino acid protein kinase substrate that binds calmodulin in the absence of calcium, and that has been implicated in dendritic spine formation and synaptic plasticity. In the rat brain RC3 is under thyroid hormone control in specific neuronal subsets in both developing and adult animals. To evaluate whether the human gene is also a target of thyroid hormone we have searched for T3-responsive elements in NRGN cloned genomic fragments spanning the whole gene. Labeled DNA fragments were incubated with T3 receptors (T3R) and 9-cis-retinoic acid receptors and immunoprecipitated using an anti T3R antibody. A receptor-binding site was localized in the first intron, 3000 bp downstream from the origin of transcription. Footprinting analysis revealed the sequence GGATTAAATGAGGTAA, closely related to the consensus T3-responsive element of the direct repeat (DR4) type. This sequence binds the T3R-9-cis-retinoic acid receptors heterodimers, but not T3R monomers or homodimers, and is able to confer regulation by T3R and T3 when fused upstream of the NRGN or thymidine kinase promoters. The data reported in this work suggest that NRGN is a direct target of thyroid hormone in human brain, and that control of expression of this gene could underlay many of the consequences ofhypothyroidism on mental states during development as well as in adult subjects.

Animals↗
PubMed1989

The promoter-regulatory region of the major immediate-early gene of human cytomegalovirus responds to T-lymphocyte stimulation and contains functional cyclic AMP-response elements.

Prior studies have demonstrated that a small proportion of blood lymphocytes from patients with human cytomegalovirus (HCMV) infection express only the viral immediate-early (IE) genes (L. Einhorn and A. Ost, J. Infect. Dis. 149:207-214, 1984; G. P. A. Rice, R. D. Schrier, and M. B. A. Oldstone, Proc. Natl. Acad. Sci. USA 81:6134-6138, 1984). The present studies demonstrate that the IE genes of HCMV are transcribed in Jurkat cells (T lymphocytes) only after activation of the cells with mitogens. Transcription of the IE genes is from an upstream enhancer promoter-regulatory region containing several different repeated sequence motifs. Chimeric plasmids were constructed with just a single copy or three copies of a synthetic oligonucleotide sequence of either the 16-, 18-, 19-, or 21-base-pair (bp) repeat elements upstream of the minimal wild-type promoter sequence to drive expression of the indicator gene, chloramphenicol acetyltransferase (CAT). The 18- or 19-bp motifs in the enhancer region were found to be important in mediating the effect of the mitogens. However, the CAT activity detected with the 19-bp repeat was always significantly higher than that found with the 18-bp repeat. There was an additive effect by multiple copies of the 18- or 19-bp repeat sequences on gene expression. The 19-bp repeat contains a sequence identical to that described for a cyclic AMP (cAMP) response element, and plasmids containing only this sequence and the minimal promoter sequences upstream of the CAT gene respond to agents which increase intracellular cAMP. Functional cAMP response elements are present in the wild-type promoter-regulatory region and are associated with the 19-bp repeat sequences. It is proposed that activation of lymphocytes results in expression of the IE genes of HCMV, in part via the activation of cellular trans-acting factors which interact with the 18- and 19-bp motifs in the HCMV IE promoter-regulatory region. The 19-bp repeat is the major contributor to the strength of this enhancer-containing promoter-regulatory region.

Base Sequence↗
PubMed1993

PPAR-RXR heterodimer activates a peroxisome proliferator response element upstream of the bifunctional enzyme gene.

A DNA sequence that confers a response to a class of rodent hepatocarcinogens termed peroxisome proliferators has been identified 2947bp upstream of the rat peroxisomal bifunctional enzyme gene. Two members of the steroid hormone receptor family, termed the peroxisome proliferator activated receptor (PPAR alpha) and the retinoid X receptor (RXR alpha), co-operate to bind specifically to this sequence. Importantly, this response element (PPRE) is similar to that identified upstream of other peroxisome proliferator responsive genes such as those encoding acyl CoA oxidase and cytochrome P450 IVA6. These data therefore provide further evidence that PPAR alpha plays an important role in mediating the action of peroxisome proliferators.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1996

Transactivation of a cellular promoter by the NS1 protein of the parvovirus minute virus of mice through a putative hormone-responsive element.

The promoter of the thyroid hormone receptor alpha gene (c-erbA-1) is activated by the nonstructural protein 1 (NS1) of parvovirus minute virus of mice (prototype strain [MVMp]) in ras-transformed FREJ4 cells that are permissive for lytic MVMp replication. This stimulation may be related to the sensitivity of host cells to MVMp, as it does not take place in parental FR3T3 cells, which are resistant to the parvovirus killing effect. The analysis of a series of deletion and point mutants of the c-erbA-1 promoter led to the identification of an upstream region that is necessary for NS1-driven transactivation. This sequence harbors a putative hormone-responsive element and is sufficient to render a minimal promoter NS1 inducible in FREJ4 but not in FR3T3 cells, and it is involved in distinct interactions with proteins from the respective cell lines. The NS1-responsive element of the c-erbA-1 promoter bears no homology with sequences that were previously reported to be necessary for NS1 DNA binding and transactivation. Altogether, our data point to a novel, cell-specific mechanism of promoter activation by NS1.

3T3 Cells↗
PubMed2004

Identification of a prostaglandin-responsive element in the Na,K-ATPase beta 1 promoter that is regulated by cAMP and Ca2+. Evidence for an interactive role of cAMP regulatory element-binding protein and Sp1.

The Na,K-ATPase is a transmembrane protein responsible for maintaining electrochemical gradients across the plasma membrane in all mammalian cells, a process that is subject to regulation at the transcriptional as well as post-transcriptional level. Included among physiologic regulators in the kidney are prostaglandins. Previously, we demonstrated that prostaglandin E(1) (PGE(1)) increases the activity and expression of the Na,K-ATPase in Madin-Darby canine kidney cells (Taub, M., Borsick, M., Geisel, J., Matlhagela, K., Rajkhowa, T., and Allen, C. (2004) Exp. Cell Res. 299, 1-14; Taub, M. L., Wang, Y., Yang, I. S., Fiorella, P., and Lee, S. M. (1992) J. Cell. Physiol. 151, 337-346). In this work, we present evidence that transcription of the Na,K-ATPase beta(1) subunit is stimulated by PGE(1), an effect that may be mediated through the cAMP and Ca(2+) pathways. Transient transfection studies using 5'-deletion mutants of the human beta(1) subunit promoter indicated that region -100 to -92 containing the sequence AGTCCCTGC (a prostaglandin-responsive element (PGRE)) is required to elicit the stimulatory effects of PGE(1), 8-bromo-cAMP, phorbol 12-myristate 13-acetate, and okadaic acid. Electrophoretic mobility shift assays indicated that both the cAMP regulatory element-binding protein (CREB) and Sp1 bind to the PGRE present within this region of the beta(1) subunit promoter. The involvement of the PGRE and Sp1 sites in regulation by PGE(1) was further confirmed by the increased PGE(1) stimulation that was observed following insertion of the PGRE into a promoter/luciferase construct containing a portion of a heterologous promoter and the fibronectin promoter with four GC boxes. Further evidence suggesting an interaction between Sp1 and CREB was obtained from experiments conducted with pLuc-MCS-beta 72-167, which contains region -167 to -72 in the human beta(1) subunit promoter. The PGE(1) stimulation observed in Madin-Darby canine kidney cells transiently transfected with pLuc-MCS-beta 72-167 was reduced when the two GC boxes immediately upstream from the PGRE were translocated farther upstream. Also consistent with an interaction between CREB and Sp1 are the results of our immunoprecipitation studies indicating that CREB co-immunoprecipitated with Sp1 when an antibody against CREB, Sp1, or the CREB-binding protein was used.

Alprostadil↗
PubMed2005

Calcium-dependent dephosphorylation of brain mitochondrial calcium/cAMP response element binding protein (CREB).

Calcium-mediated signaling regulates nuclear gene transcription by calcium/cAMP response element binding protein (CREB) via calcium-dependent kinases and phosphatases. This study tested the hypothesis that CREB is also present in mitochondria and subject to dynamic calcium-dependent modulation of its phosphorylation state. Antibodies to CREB and phosphorylated CREB (pCREB) were used to demonstrate the presence of both forms in isolated mitochondria and mitoplasts from rat brain. When energized mitochondria were exposed to increasing concentrations of Ca2+ in the physiological range, pCREB was lost while total CREB remained constant. In the presence of Ru360, an inhibitor of the mitochondrial Ca2+ uptake uniporter, calcium-dependent loss of pCREB levels was attenuated, suggesting that intramitochondrial calcium plays an important role in pCREB dephosphorylation. pCREB dephosphorylation was not, however, inhibited by the phosphatase inhibitors okadaic acid and Tacrolimus. In the absence of Ca2+, CREB phosphorylation was elevated by the addition of ATP to the mitochondrial suspension. Exposure of mitochondria to the pore-forming molecule alamethicin that causes osmotic swelling and release of intermembrane proteins enriched mitochondrial pCREB immunoreactivity. These results further suggest that mitochondrial CREB is located in the matrix or inner membrane and that a kinase and a calcium-dependent phosphatase regulate its phosphorylation state.

Adenosine Triphosphate↗
PubMed1999

Immunohistochemical analysis of cyclic AMP response element binding protein phosphorylation in focal cerebral ischemia in rats.

Phosphorylation of cyclic AMP response element binding protein (CREB) is one of the most important mechanisms controlling various gene transcriptions. In the present study, the phosphorylation of CREB was examined immunohistochemically at 24 h of recirculation following 1.5 h of middle cerebral artery occlusion (MCAO) in rats. MCAO was induced by the intraluminal suture method. The infarct core revealed a significant reduction in the number of immunoreactive cells with the anti-phosphorylated CREB and with the anti-CREB antibody, which binds to both unphosphorylated and phosphorylated CREB. In contrast, the peri-infarct area exhibited a marked increase in the number of immunopositive cells as well as in the intensity of nuclear staining with each antibody, so that almost all of the cells expressing CREB demonstrated phosphorylation of CREB. On the other hand, about half of the CREB immunopositive cells reacted weakly with the anti-phosphorylated CREB antibody in the sham group. These findings indicated that the expression as well as phosphorylation of CREB protein was significantly activated in the regions surrounding the infarct area. Since phosphorylation of CREB has recently been implicated in signal transductions that promote the survival and differentiation of neurons, the present data suggest that tissue repair mechanisms may be markedly activated in the peri-infarct area.

Animals↗
PubMed2003

Finding of TRE (TPA responsive element) in the sequence of human taurine transporter promoter.

Activity of the taurine transporter (TAUT) is regulated by signal transduction in response to diverse stimuli including tumor promoters such as phobol ester. Regulation of the transcription rate of TAUT appears to play an important role in exerting biological roles of taurine in mammalian tissues in adverse environments. Although cDNA of human TAUT has been cloned and sequenced in placenta, thyroid cells, and retinal pigment epithelial cells, the promoter region of TAUT has never been reported. In order to clone the upstream region of the human TAUT promoter, we have compared TAUT cDNA sequences with the entire human genome sequence. Polymerase chain reaction (PCR) was performed from genomic DNA prepared from a SK-Hep-1 cell line for the amplification of the TAUT promoter region including the partial exon (150 bp) and the 5' untranslated region (UTR, 380 bp). The PCR product of the promoter region, which was 1800 bp long, was ligated into the pGEM-T vector, and sequenced. The 5' flanking region of the TAUT promoter was analysed for the identification of enhancer and regulation motifs. Surprisingly we found the consensus TPA responsive element (TGAGTCAG) which is responsible for gene regulation by the protein kinase C (PKC)-mediated signal transduction pathway. The well known fact that proto-oncogene AP1 (cFos/cJun heterodimer or cJun/cJun homodimer) binds to TRE implies that TAUT expression might be closely linked to tumor promotion. Since AP1 activity is also tightly regulated in nerve cells, AP1-regulated TAUT transcription might be an important step in nerve cell function. Furthermore, the TFIID binding site, cap signal for transcription initiation, PEA3 motif, heat shock factor binding motif, and many other motifs were found in the TAUT promoter region, and require characterization.

Base Sequence↗
PubMed2001

Ectopic expression of the cAMP-responsive element binding protein inhibits phorbol ester-mediated induction of tissue-type plasminogen activator gene expression.

The human tissue-type plasminogen activator (t-PA) gene is regulated in a cell-type dependent manner. The t-PA gene is transcriptionally induced by the phorbol ester PMA in HeLa cells, but suppressed by PMA in HT-1080 cells. A cAMP responsive element (tPACRE) and a Sp-1 site located within the proximal t-PA gene promoter are functionally important in both cell systems. HeLa and HT-1080 cells contain a different repertoire of factors that associate with the tPACRE. In HT-1080 cells, CREB and c-Jun are the two major t-PACRE binding proteins identified, while activating transcription factor 2 (ATF-2) is a predominant t-PACRE binding protein in HeLa cells. To determine whether alteration in the distribution of tPACRE binding proteins would influence the differential regulation of the t-PA gene in these cells, the tPACRE binding profiles in these two cell systems were manipulated by over expressing ATF-2 in HT-1080 cells and CREB in HeLa cells. Supershift experiments confirmed that the overexpression of these factors resulted in binding to the tPACRE site. However, the presence of ATF-2 in HT-1080 cells did not affect either constitutive or PMA-mediated suppression of the endogenous t-PA gene. In contrast, enforced tPACRE-binding activity of CREB in HeLa cells significantly reduced the magnitude of PMA-mediated induction of t-PA mRNA in HeLa cells. These results indicate that the introduction of CREB into HeLa cells disrupts the regulation of the t-PA gene.

Activating Transcription Factor 2↗