Involvement of thyrotroph embryonic factor in calcium-mediated regulation of gene expression.
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In the present study, we used an expression cloning strategy to identify transcription factors that bind specifically to a limited region of the inducible cAMP early repressor (ICER) promoter and regulate transcription. Murine thyrotroph embryonic factor (mTEF) was isolated and was shown to bind to a site located at nucleotides -117 to -108 from the transcriptional start site. Transient expression of reporter constructs containing either a consensus TEFRE or the icerTEF binding site demonstrated that TEF-dependent transcription correlated with relative binding affinities, i.e. the consensus TEFRE bound TEF more tightly and was more responsive to TEF than the icerTEFRE. Because the icerTEFRE overlapped a cAMP response element, the responsiveness of these sequences to either cAMP or Ca(2+) was tested. Although TEF expression had no effect on the cAMP-regulated transcriptional response of the ICER promoter, TEF did confer calcium responsiveness to these sequences. Calcium also modestly increased the TEF-mediated transcription from a consensus TEFRE. Additional studies using Ca(2+)-activated kinases indicate that Ca(2+)/TEF/TEFRE-regulated transcription may be mediated through Ca(2+)/calmodulin-dependent kinase (CaMK) IV. Moreover, studies with the icerTEFRE in a CaMK IV-deficient cell line demonstrated that these cells were transcriptionally unresponsive to thapsigargin; however, responsiveness was restored by co-expression of the active CaMK IV. These studies are the first to demonstrate that TEF is a calcium-responsive transcription factor, and they suggest that there are two classes of TEF-regulated genes. One class, represented by a consensus TEFRE, is regulated by TEF in the resting cell; the second class, represented by icerTEFRE, is regulated by TEF in the calcium-activated cell. (+info)
Suppression of T cell function: a potential role for transcriptional repressor ICER.
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In this article, we review the inducible cAMP early repressor (ICER) and its possible critical involvement in modulation of T cell responsiveness by its capacity to transcriptionally attenuate interleukin-2 (IL-2) gene expression. It seems clear that the failure to produce the IL-2 is an important determinant of anergy induction. It is important that the CD28-responsive element (CD28RE), a composite DNA binding element consisting of NFAT and cyclic AMP-responsive (CRE)-like motifs in position of -160 of IL-2 promoter has the high affinity for ICER binding as well as NFAT/ICER complex formation. Moreover, CD28RE with adjacent DNA sequences was also shown to be essential for conferring anergy in T lymphocytes. Because ICER does not possess a transactivation domain required for the recruitment of CBP/p300, the binding of ICER to CD28RE and/or composite motifs containing CRE-like DNA motifs may lead to uncoupling of CBP/p300 thus extinguishing IL-2 expression as well as expression of numerous other cytokines and chemokines. (+info)
Induction of cAMP response element modulator (CREM) and inducible cAMP early repressor (ICER) expression in rat brain by uncompetitive N-methyl-D-aspartate receptor antagonists.
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The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor mediates fast excitatory neurotransmission, and agents that attenuate this function are neuroprotective, anesthetic, and psychotropic. To determine whether cAMP regulatable transcription factors play a role in the neurochemical actions of agents acting through NMDA receptors, the effects of the acute administration of uncompetitive and competitive antagonists on the expression of cAMP response element modulator (CREM) and inducible cAMP early repressor (ICER) transcription factors were examined. In situ hybridization to rat brain sections revealed that ICER mRNA expression was significantly increased by uncompetitive NMDA receptor antagonists (MK-801, phencyclidine, ketamine, memantine) but not by the competitive antagonist CPP [(+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid] or other psychotropic agents (clozapine, haloperidol, desipramine). Major brain regions where ICER transcripts were increased were the hippocampus, parietal cortex layers IV and VI, temporal cortex, cingulate cortex, thalamus, and granule cell layer of the olfactory bulb. Northern and Western blot analyses indicated that CREM mRNA and protein, respectively, were also increased after MK-801 treatment, but ICER isoforms predominate during both basal and induced conditions. MK-801 also transiently increased the binding of proteins to cAMP response element, which was supershifted by anti-CREM antibody, indicating that increased mRNA and protein levels have functional consequences on gene transcription. These results provide evidence for the involvement of CREM and ICER family transcription factors in the pharmacologic effects of uncompetitive NMDA receptor antagonists. (+info)
Expression of the cyclic AMP-dependent transcription factors, CREB, CREM and ATF2, in the human myometrium during pregnancy and labour.
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Elevated concentrations of cyclic AMP (cAMP) in the human myometrium may promote uterine quiescence during pregnancy by protein kinase A (PKA)-mediated phosphorylation and subsequent inactivation of myosin light-chain kinase, as well as by the phosphorylation and activation of cAMP-dependent transcription factors. In this context, we show that the altered expression of cAMP response-element binding protein (CREB), cAMP response-element modulator protein (CREM) and activating transcription factor 2 (ATF2) are implicated in the maintenance of myometrial quiescence during fetal maturation and the switch to uterine activation at term. Using electrophoretic mobility shift and super shift assays, as well as immunoblotting of paired myometrial tissue samples from non-pregnant, pregnant non-labouring and spontaneous labouring women, we defined the patterns of expression of various isoforms of these proteins in the human uterus. Here, we report spatio-temporal changes in the expression of a 43 kDa form of CREB, a 28 kDa CREM-like protein, and a novel 28 kDa ATF2-like protein which are differentially expressed, depending on the gestational state of the uterus. Changes in the pattern of expression of these potent transcription factors may have an important role in the control of uterine activity throughout pregnancy. (+info)
Glucagon stimulates expression of the inducible cAMP early repressor and suppresses insulin gene expression in pancreatic beta-cells.
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The hormone glucagon is secreted by the alpha-cells of the endocrine pancreas (islets of Langerhans) during fasting and is essential for the maintenance of blood glucose levels by stimulation of hepatic glucose output. Excessive production and secretion of glucagon by the alpha-cells of the islets is a common accompaniment to diabetes. The resulting hyperglucagonemia stimulates hepatic glucose production, thereby contributing to hyperglycemia of diabetes. The reduced insulin secretion in diabetes and resultant failure to suppress glucagon secretion by intra-islet paracrine mechanisms is believed to cause the hypersecretion of glucagon. Here, we report the discovery of a new mechanism by which glucagon suppresses insulin secretion. We show that glucagon, but not glucagon-like peptide 1 (GLP-1), or pituitary adenylyl cyclase-activating peptide (PACAP) specifically induces the expression of the transcriptional repressor inducible cAMP early repressor (ICER) in pancreatic beta-cells, resulting in a repression of the transcriptional expression of the insulin gene. Remarkably, glucagon, GLP-1, and PACAP all stimulate the formation of cAMP to a comparable extent in rat pancreatic islets, but only glucagon activates the expression of ICER and represses insulin gene transcription in beta-cells. These findings lead us to propose that hyperglucagonemia may additionally aggravate the diabetic phenotype via a suppression of insulin gene expression mediated by the transcriptional repressor ICER. (+info)
Overexpression of a dominant negative CREB protein in HT-1080 cells selectively disrupts plasminogen activator inhibitor type 2 but not tissue-type plasminogen activator gene expression.
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The tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 2 (PAI-2) genes are differentially regulated by 12-phorbol 13-myristate acetate (PMA) in HT-1080 fibrosarcoma cells. PMA transcriptionally down-regulates the t-PA gene in HT-1080 cells, while the PAI-2 gene is simultaneously induced by this agonist. The t-PA and PAI-2 gene promoters harbour a cAMP-response element (CRE) which influences the expression of both genes. We have compared the binding activity of nuclear factors that recognise these CRE sites. We show that CREB (CRE binding protein) recognises each CRE and that the degree of constitutive Ser119-phosphorylated t-PA CRE-bound CREB was greater than for PAI-2 CRE bound CREB. Stable transfection of HT-1080 cells with a plasmid containing a CREB that could not be phosphorylated on Ser119 (pCI-CREB(ala119)) did not influence PMA-mediated suppression of t-PA mRNA, but markedly impaired PMA-mediated induction of PAI-2 mRNA. Our results demonstrate that the Ser119 residue of CREB plays a crucial role in PMA-mediated induction of PAI-2 gene expression, whereas PMA-mediated suppression of t-PA in HT-1080 cells requires a different process. (+info)
CREM activator and repressor isoforms in human testis: sequence variations and inaccurate splicing during impaired spermatogenesis.
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cAMP responsive element modulator (CREM) activators are specifically expressed in haploid germ cells prior to cell elongation and are essential for spermatid development in mice. Recent studies indicate that CREM activators are also involved in the process of spermatid maturation in men. Unlike the activators, CREM repressors were suggested to be absent from adult mouse and dog testes. The present work investigates CREM transcripts in human testis with normal (n = 4) and impaired spermatogenesis (n = 2). Two activator transcripts could be identified corresponding to the tau2 isoform with and without exon gamma. Interestingly, four CREM repressors could be isolated from specimens with complete spermatogenesis. These were gamma-repressor (exons B, E, F, H, I(b)), CREM DeltaC-F, beta (exons B, G, H, I(b)), CREM DeltaC-G, beta (exons B, H, I(b)), and CREM DeltaC-G, alpha (exons B, H, I(a), I(b)). These isoforms were also present in cynomolgus monkey testes. A novel CREM splice variant (CREM DeltaC-H) was detected in a specimen with spermatid maturation defect. Beyond that, inaccurate CREM splicing, giving rise to inactive transcripts, was encountered in a specimen with impaired spermatogenesis. In conclusion, several CREM repressor transcripts are present in adult human testes, and altered transcript splicing is associated with impaired spermatogenesis. (+info)
A family of LIM-only transcriptional coactivators: tissue-specific expression and selective activation of CREB and CREM.
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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. (+info)