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Results for “Cyclic AMP Response Element Modulator”

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PubMed1999

Stress-induced stimulation of early growth response gene-1 by p38/stress-activated protein kinase 2 is mediated by a cAMP-responsive promoter element in a MAPKAP kinase 2-independent manner.

The p38/stress-activated protein kinase2 (p38/SAPK2) is activated by cellular stress and proinflammatory cytokines. Several transcription factors have been reported to be regulated by p38/SAPK2, and this kinase is involved in the control of expression of various genes. In human Jurkat T-cells, induction of the early growth response gene-1 (egr-1) by anisomycin is completely inhibited by SB203580, a specific inhibitor of p38/SAPK2a and -b. Northern blot and reporter gene experiments indicate that this block is at the level of mRNA biosynthesis. Using mutants of the egr-1 promoter, we demonstrate that a distal cAMP-responsive element (CRE; nucleotides -134 to -126) is necessary to control egr-1 induction by p38/SAPK2. Pull-down assays indicate that phospho-CRE binding protein (CREB) and phospho-activating transcription factor-1 (ATF1) bind to this element in a p38/SAPK2-dependent manner. In response to anisomycin, two known CREB kinases downstream to p38/SAPK2, MAPKAP kinase 2 (MK2) and mitogen- and stress-activated kinase 1 (MSK1), show increased activity. However, in MK2 -/- fibroblasts derived from mice carrying a disruption of the MK2 gene, the phosphorylation of CREB and ATF1 and the expression of egr-1 reach levels comparable with wild type cells. This finding excludes MK2 as an involved enzyme. We conclude that egr-1 induction by anisomycin is mediated by p38/SAPK2 and probably by MSK1. Phosphorylated CREB and ATF1 then bind to the CRE of the egr-1 promoter and cause a stress-dependent transcriptional activation of this gene.

Activating Transcription Factor 1↗
PubMed2000

Regulation of inducible cAMP early repressor expression by gastrin and cholecystokinin in the pancreatic cell line AR42J.

The CREM gene encodes both activators and repressors of cAMP-induced transcription. Inducible cAMP early repressor (ICER) isoforms are generated upon activation of an alternative, intronic promoter within the CREM gene. ICER is proposed to down-regulate both its own expression and the expression of other genes that contain cAMP-responsive elements such as a number of growth factors. Thus, ICER has been postulated to play a role in proliferation and differentiation. Here we show that ICER gene expression is induced by gastrin, cholecystokinin (CCK), and epidermal growth factor in AR42J cells. The time course of gastrin- and CCK-mediated ICER induction is rapid and transient, similar to forskolin- and phorbol 12-myristate 13-acetate-induced ICER expression. The specific CCK-B receptor antagonist L740,093 blocks the gastrin but not the CCK response, indicating that both the CCK-B and the CCK-A receptor can mediate ICER gene activation. Noteworthy, CREB is constitutively phosphorylated at Ser-133 in AR42J cells, and ICER induction proceeds in the absence of increased CREB Ser(P)-133. Gastrin-mediated ICER induction was not reduced in the presence of the protein kinase A inhibitor H-89, indicating a protein kinase A-independent mechanism. This is the first report on ICER inducibility via G(q)/G(11) protein-coupled receptors.

Animals↗
PubMed2003

Deacetylase activity is required for cAMP activation of a subset of CREB target genes.

Many hormones activate transcription by raising the level of cAMP within cells. In one well studied pathway, cAMP induces protein kinase A to phosphorylate the transcription factor CREB, which binds to a consensus sequence, the cAMP-regulated enhancer, found in many target genes. A generally accepted model suggests that phosphorylated CREB recruits the histone acetyltransferase CBP to activate transcription. In contrast, histone deacetylases have been linked to the cessation of CREB-dependent transcription. Here we tested this model in the regulation of endogenous CREB target genes. We used a constitutively active CREB mutant and microarray analysis to identify target genes in PC12 cells. We then tested the role of histone deacetylase activity in cAMP activation of four of these genes (c-FOS, ICER, NOR-1, and NUR77) by treating cells with the histone deacetylase inhibitor trichostatin A. Consistent with the generally accepted model, trichostatin A enhanced activation of c-FOS and NUR77 by cAMP. Surprisingly, trichostatin A blocked activation of ICER and NOR-1. The block of ICER and NOR-1 activation persisted in the presence of cycloheximide, indicating that the trichostatin A effect did not depend on new protein synthesis. This unexpected role of histone deacetylases in transcriptional activation of certain endogenous CREB target genes was not apparent in transfected reporter genes. Chromatin immunoprecipitation analysis indicated that the differential roles of histone deacetylases in activating or repressing CREB target genes was manifested at the level of preinitiation complex recruitment. These data indicate that histone deacetylases differentially regulate CREB target genes by contributing to either activation or cessation of transcription.

Acetyltransferases↗
PubMed2004

PE-1/METS, an antiproliferative Ets repressor factor, is induced by CREB-1/CREM-1 during macrophage differentiation.

The molecular mechanisms involved in regulating the balance between cellular proliferation and differentiation remain poorly understood. Members of the Ets-domain family of transcription factors are candidates for proteins that might differentially regulate cell cycle control and cell type-specific genes during the differentiation of myeloid progenitor cells. The Ets repressor PE-1/METS has been suggested to contribute to growth arrest during terminal macrophage differentiation by repressing Ets target genes involved in Ras-dependent proliferation. An important feature of this regulatory model is that PE-1/METS is itself induced by the program of macrophage differentiation elicited by M-CSF. Here, we present evidence that the PE-1/METS gene is a transcriptional target of the cyclic AMP response element-binding protein-1 (CREB-1). CREB-1 expression is dramatically up-regulated during macrophage differentiation and phosphorylation of CREB-1 and the related factor CREM-1 are stimulated by M-CSF in a SAPK2/p38-dependent manner. Chromatin immunoprecipitation experiments demonstrate that CREB-1/CREM-1 are recruited to the PE-1/METS promoter as well as to the promoters of other genes that are up-regulated during terminal macrophage differentiation. Overexpression of CREB-1 stimulates the activities of the PE-1/METS, and macrosialin promoters, while expression of a dominant negative form of CREB-1 during macrophage differentiation inhibits expression of the PE-1/METS and macrosialin genes. Inhibition of CREB function also results in reduced expression of CD54 and impaired cell adhesion. Taken together, these findings reveal new roles of CREB-1/CREM-1 as regulators of macrophage differentiation.

Adenoviridae↗
PubMed2002

Ca2+-dependent block of CREB-CBP transcription by repressor DREAM.

The calcium-binding protein DREAM binds specifically to DRE sites in the DNA and represses transcription of target genes. Derepression at DRE sites following PKA activation depends on a specific interaction between alphaCREM and DREAM. Two leucine-charged residue-rich domains (LCD) located in the kinase-inducible domain (KID) and in the leucine zipper of alphaCREM and two LCDs in DREAM participate in a two-site interaction that results in the loss of DREAM binding to DRE sites and derepression. Since the LCD motif located within the KID in CREM is also present in CREB, and maps in a region critical for the recruitment of CBP, we investigated whether DREAM may affect CRE-dependent transcription. Here we show that in the absence of Ca2+ DREAM binds to the LCD in the KID of CREB. As a result, DREAM impairs recruitment of CBP by phospho CREB and blocks CBP-mediated transactivation at CRE sites in a Ca2+-dependent manner. Thus, Ca2+-dependent interactions between DREAM and CREB represent a novel point of cross-talk between cAMP and Ca2+ signalling pathways in the nucleus.

Brain↗
PubMed2000

Blockade of cyclic AMP-responsive element DNA binding in the brain of CREB delta/alpha mutant mice.

The cAMP-responsive element binding protein (CREB) gene transcription factor has been implicated in the synaptic plasticity and memory. Here, we investigated the mechanisms of CREB and/or cyclic AMP-responsive element modulatory protein (CREM) binding to CRE sites in brain tissues. CRE-DNA binding was determined in nuclear extracts obtained from the several brain structures of wild-type and CREB delta/alpha mutant mice. It was found that antibodies to CREB, phosphorylated CREB, and CREM supershifted the CRE-DNA binding complex in cortical nuclear extracts from wild-type mice, which suggests that the CRE-DNA binding complex contains both CREB and CREM proteins. In contrast, CRE-DNA binding is abolished in the cortex, hippocampus, cerebellum, and amygdala of CREB delta/alpha mutant mice. Because the CREB delta and alpha isoforms have been deleted in CREB mutant mice, consequently, other forms of CREB, such as CREB-beta and CREM, are up-regulated. These results suggest that the binding of CREM to CRE sites requires the presence of CREB delta/alpha, and that CREB-beta may be inefficient in binding to CRE-sites. Thus, these results indicate that CREB delta/alpha mutant mice are a useful animal model for studying the functional role of CREB-dependent gene expression.

Animals↗
PubMed2006

PC12 cells regulate inducible cyclic AMP (cAMP) element repressor expression to differentially control cAMP response element-dependent transcription in response to nerve growth factor and cAMP.

Both cyclic AMP (cAMP) and nerve growth factor (NGF) have been shown to cause rapid activation of cAMP response element-binding protein (CREB) by phosphorylation of serine 133, but additional regulatory events contribute to CREB-targeted gene expression. Here, we have used stable transfection with a simple cAMP response element (CRE)-driven reporter to address the kinetics of CRE-dependent transcription during neuronal differentiation of PC12 cells. In naive cells, dibutyryl cAMP (dbcAMP) generated a rapid increase in CRE-driven luciferase activity by 5 h that returned to naive levels by 24 h. Luciferase induction after NGF treatment was delayed until 48 h when CRE-driven luciferase expression became TrkA dependent. Blocking histone deacetylase (HDAC) activity accelerated NGF-dependent CRE-driven luciferase expression by at least 24 h and resulted in a sustained cAMP-dependent expression of CRE-driven luciferase beyond 24 h. Inhibition of protein synthesis before stimulation with NGF or dbcAMP indicated that both stimuli induce expression of a transcriptional repressor that delays NGF-dependent and attenuates cAMP-dependent CRE-driven transcription. NGF caused a rapid but transient HDAC-dependent increase in inducible cAMP element repressor (ICER) expression, but ICER expression was sustained with increased cAMP. Depletion of ICER from PC12 cells indicated that HDAC-dependent ICER induction is responsible for the delay in CRE-dependent transcription after NGF treatment.

Animals↗
PubMed1994

A complex array of positive and negative elements regulates the chicken alpha A-crystallin gene: involvement of Pax-6, USF, CREB and/or CREM, and AP-1 proteins.

The abundance of crystallins (> 80% of the soluble protein) in the ocular lens provides advantageous markers for selective gene expression during cellular differentiation. Here we show by functional and protein-DNA binding experiments that the chicken alpha A-crystallin gene is regulated by at least five control elements located at sites A (-148 to -139), B (-138 to -132), C (-128 to -101), D (-102 to -93), and E (-56 to -41). Factors interacting with these sites were characterized immunologically and by gel mobility shift experiments. The results are interpreted with the following model. Site A binds USF and is part of a composite element with site B. Site B binds CREB and/or CREM to enhance expression in the lens and binds an AP-1 complex including CREB, Fra2 and/or JunD which interacts with USF on site A to repress expression in fibroblasts. Sites C and E (which is conserved across species) bind Pax-6 in the lens to stimulate alpha A-crystallin promoter activity. These experiments provide the first direct data that Pax-6 contributes to the lens-specific expression of a crystallin gene. Site D (-104 to -93) binds USF and is a negative element. Thus, the data indicate that USF, CREB and/or CREM (or AP-1 factors), and Pax-6 bind a complex array of positive and negative cis-acting elements of the chicken alpha A-crystallin gene to control high expression in the lens and repression in fibroblasts.

Animals↗
PubMed1995

Transcriptional regulation of the mouse alpha A-crystallin gene: activation dependent on a cyclic AMP-responsive element (DE1/CRE) and a Pax-6-binding site.

Two cis-acting promoter elements (-108 to -100 and -49 to -33) of the mouse alpha A-crystallin gene, which is highly expressed in the ocular lens, were studied. Here we show that DE1 (-108 to -100; 5'TGACGGTG3'), which resembles the consensus cyclic AMP (cAMP)-responsive element sequence (CRE; 5'TGACGT[A/C][A/G]3'), behaves like a functional CRE site. Transfection experiments and electrophoretic mobility shift assays (EMSAs) using site-specific mutations correlated a loss of function with deviations from the CRE consensus sequence. Results of EMSAs in the presence of antisera against CREB, delta CREB, and CREM were consistent with the binding of CREB-like proteins to the DE1 sequence. Stimulation of alpha A-crystallin promoter activity via 8-bromo-cAMP, forskolin, or human T-cell leukemia virus type I Tax1 in transfections and reduction of activity of this site in cell-free transcription tests by competition with the somatostatin CRE supported the idea that DE1 is a functional CRE. Finally, Pax-6, a member of the paired-box family of transcription factors, activated the mouse alpha A-crystallin promoter in cotransfected COP-8 fibroblasts and bound to the -59 to -29 promoter sequence in EMSAs. These data provide evidence for a synergistic role of Pax-6 and CREB-like proteins for high expression of the mouse alpha A-crystallin gene in the lens.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

A Drosophila CREB/CREM homolog encodes multiple isoforms, including a cyclic AMP-dependent protein kinase-responsive transcriptional activator and antagonist.

We have characterized a Drosophila gene that is a highly conserved homolog of the mammalian cyclic AMP (cAMP)-responsive transcription factors CREB and CREM. Uniquely among Drosophila genes characterized to date, it codes for a cAMP-responsive transcriptional activator. An alternatively spliced product of the same gene is a specific antagonist of cAMP-inducible transcription. Analysis of the splicing pattern of the gene suggests that the gene may be the predecessor of the mammalian CREB and CREM genes.

Alternative Splicing↗
PubMed2000

A family of LIM-only transcriptional coactivators: tissue-specific expression and selective activation of CREB and CREM.

Transcription factors of the CREB family control the expression of a large number of genes in response to various signaling pathways. Regulation mediated by members of the CREB family has been linked to various physiological functions. Classically, activation by CREB is known to occur upon phosphorylation at an essential regulatory site (Ser133 in CREB) and the subsequent interaction with the ubiquitous coactivator CREB-binding protein (CBP). However, the mechanism by which selectivity is achieved in the identification of target genes, as well as the routes adopted to ensure tissue-specific activation, remains unrecognized. We have recently described the first tissue-specific coactivator of CREB family transcription factors, ACT (activator of CREM in testis). ACT is a LIM-only protein which associates with CREM in male germ cells and provides an activation function which is independent of phosphorylation and CBP. Here we characterize a family of LIM-only proteins which share common structural organization with ACT. These are referred to as four-and-a-half-LIM-domain (FHL) proteins and display tissue-specific and developmentally regulated expression. FHL proteins display different degrees of intrinsic activation potential. They provide powerful activation function to both CREB and CREM when coexpressed either in yeast or in mammalian cells, specific combinations eliciting selective activation. Deletion analysis of the ACT protein shows that the activation function depends on specific arrangements of the LIM domains, which are essential for both transactivation and interaction properties. This study uncovers the existence of a family of tissue-specific coactivators that operate through novel, CBP-independent routes to elicit transcriptional activation by CREB and CREM. The future identification of additional partners of FHL proteins is likely to reveal unappreciated aspects of tissue-specific transcriptional regulation.

Amino Acid Motifs↗
PubMed2000

DREAM-alphaCREM interaction via leucine-charged domains derepresses downstream regulatory element-dependent transcription.

Protein kinase A-dependent derepression of the human prodynorphin gene is regulated by the differential occupancy of the Dyn downstream regulatory element (DRE) site. Here, we show that a direct protein-protein interaction between DREAM and the CREM repressor isoform, alphaCREM, prevents binding of DREAM to the DRE and suggests a mechanism for cyclic AMP-dependent derepression of the prodynorphin gene in human neuroblastoma cells. Phosphorylation in the kinase-inducible domain of alphaCREM is not required for the interaction, but phospho-alphaCREM shows higher affinity for DREAM. The interaction with alphaCREM is independent of the Ca(2+)-binding properties of DREAM and is governed by leucine-charged residue-rich domains located in both alphaCREM and DREAM. Thus, our results propose a new mechanism for DREAM-mediated derepression that can operate independently of changes in nuclear Ca(2+).

Amino Acid Motifs↗
PubMed1999

A semiquantitative image-analytical method for the recording of dose-response curves in immunocytochemical preparations.

Knowledge about intracellular signal transduction cascades is largely based on investigations of cultured cells whose responses to different stimuli are typically quantified via RIA, ELISA, or immunoblots. These techniques, which require relatively large amounts of biological material, are performed with homogenized cells and therefore do not allow localization of the molecules under investigation. We describe a protocol for recording dose-response curves directly from immunocytochemical preparations using rat pinealocytes as a model system. The cells were exposed to beta-adrenergic stimuli inducing the phosphorylation of the transcription factor CREB (mediated by PKA), an increase in ICER protein levels, and synthesis and release of melatonin. Melatonin concentrations were determined by ELISA. cPKA, phosphorylated CREB, and ICER were demonstrated by immunocytochemistry and immunoblots. Dose-response curves were recorded by measuring the integrated density of the immunoreactive sites with an image analysis program. Dose-response curves from immunoblots and immunocytochemical preparations showed almost identical dynamics, validating the immunocytochemical approach, which minimizes the amount of biological material needed for such studies, allows combined quantification and localization of biomolecules, and may even be more sensitive than immunoblotting.

Animals↗
PubMed2005

Repression of the inhibin alpha-subunit gene by the transcription factor CCAAT/enhancer-binding protein-beta.

Inhibin is a dimeric peptide hormone produced in ovarian granulosa cells that suppresses FSH synthesis and secretion in the pituitary. Expression of inhibin alpha- and beta-subunit genes in the rodent ovary is positively regulated by FSH and negatively regulated after the preovulatory LH surge. We have investigated the role of the transcription factor CCAAT/enhancer-binding protein-beta (C/EBPbeta) in repressing the inhibin alpha-subunit gene. C/EBPbeta knockout mice fail to appropriately down-regulate inhibin alpha-subunit mRNA levels after treatment with human chorionic gonadotropin, indicating that C/EBPbeta may function to repress inhibin gene expression. The expression and regulation of C/EBPbeta were examined in rodent ovary, and these studies show that C/EBPbeta is expressed in ovary and granulosa cells and is induced in response to human chorionic gonadotropin. Transient cotransfections with an inhibin promoter-luciferase reporter in a mouse granulosa cell line, GRMO2 cells, show that C/EBPbeta is capable of repressing both basal and forskolin-stimulated inhibin gene promoter activities. An upstream binding site for C/EBPbeta in the inhibin alpha-subunit promoter was identified by electrophoretic mobility shift assays, which, when mutated, results in elevated inhibin promoter activity. However, C/EBPbeta also represses shorter promoter constructs lacking this site, and this component of repression is dependent on the more proximal promoter cAMP response element (CRE). Electrophoretic mobility shift assays show that C/EBPbeta effectively competes with CRE-binding protein for binding to this atypical CRE. Thus, there are two distinct mechanisms by which C/EBPbeta represses inhibin alpha-subunit gene expression in ovarian granulosa cells.

Animals↗
PubMed2005

Mechanism of repression of the inhibin alpha-subunit gene by inducible 3',5'-cyclic adenosine monophosphate early repressor.

The rodent ovary is regulated throughout the reproductive cycle to maintain normal cyclicity. Ovarian follicular development is controlled by changes in gene expression in response to the gonadotropins FSH and LH. The inhibin alpha-subunit gene belongs to a group of genes that is positively regulated by FSH and negatively regulated by LH. Previous studies established an important role for inducible cAMP early repressor (ICER) in repression of alpha-inhibin. These current studies investigate the mechanisms of repression by ICER. It is not clear whether all four ICER isoforms expressed in the ovary can act as repressors of the inhibin alpha-subunit gene. EMSAs demonstrate binding of all isoforms to the inhibin alpha-subunit CRE (cAMP response element), and transfection studies demonstrate that all isoforms can repress the inhibin alpha-subunit gene. Repression by ICER is dependent on its binding to DNA as demonstrated by mutations to ICER's DNA-binding domain. These mutational studies also demonstrate that repression by ICER is not dependent on heterodimerization with CREB (CRE-binding protein). Competitive EMSAs show that ICER effectively competes with CREB for binding to the inhibin alpha CRE in vitro. Chromatin immunoprecipitation assays demonstrate a replacement of CREB dimers bound to the inhibin alpha CRE by ICER dimers in ovarian granulosa cells in response to LH signaling. Thus, there is a temporal association of transcription factors bound to the inhibin alpha-CRE controlling inhibin alpha-subunit gene expression.

Animals↗