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Results for “Cyclic AMP Response Element Modulator”

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PubMed2003

The rat pituitary promoter of the neuronal nitric oxide synthase gene contains an Sp1-, LIM homeodomain-dependent enhancer and a distinct bipartite gonadotropin-releasing hormone-responsive region.

The neuronal nitric oxide synthase (NOS I) is expressed and hormonally regulated in rat anterior pituitary gonadotropes. In the present study, we investigated the mechanisms that underlie the constitutive and GnRH up-regulated activity of the pituitary exon 1p promoter of the NOS I gene in these cells. Through the use of 5'-deletions and transient transfections in L beta T2, a gonadotrope-derived cell line, we delineated a NOS I cell-specific (NCS) enhancer region (-73/-59) that is required for constitutive activity. Independently of the NCS enhancer, GnRH responsiveness is supported by a bipartite regulatory domain referred to as the GnRH response element I and II located between -33/-10 and -4/+4, the latter consisting of a cAMP-like response element. By combining transient transfections, gel shift, and supershift assays, we demonstrate that Sp1 and LIM-homeodomain-related protein bind the NCS enhancer, whereas cAMP response element binding protein and cAMP regulatory element modulator-like factors bind the GnRH response element II motif. We further show that factors involved in GnRH regulation are also implicated in constitutive activity, suggesting intimate links between constitutive and regulated promoter activity. We speculate that specific expression of the NOS I gene in gonadotropes together with its regulation by GnRH is suggestive of a critical participation of NOS I in gonadotrope function.

Animals↗

PubMed2002

Synergistic effect of dimethyl sulfoxide on glucagon-like peptide 1 (GLP-1)-stimulated insulin secretion and gene transcription in INS-1 cells: characterization and implications.

Glucagon-like peptide 1 (GLP-1) is an incretin hormone that is secreted from the enteroendocrine L-cells of the gut in response to nutrient ingestion. GLP-1 enhances both insulin secretion and insulin gene expression in a glucose-dependent manner via activation of its putative G-protein-coupled receptor on pancreatic beta-cells. In the presence of DMSO (0.5-2.5%), these functional responses were enhanced significantly (2- to 2.5-fold) in a concentration-dependent manner in the beta-cell line INS-1, although basal levels were not affected. Rat insulin 1 (rINS1) promoter activity appeared to be augmented in a cAMP-response element (CRE)-dependent manner as the effect of DMSO was abolished following a mutation in the CRE of the rINS1 promoter. Also, expression of a generic cAMP-driven reporter gene was enhanced by 1.5% DMSO in response to GLP-1 (3.5-fold), forskolin (2-fold), and 3-isobutyl-1-methylxanthine (2-fold). Analysis of intracellular signaling components revealed that DMSO did not elevate cAMP levels, protein kinase A activity, or phosphorylated levels of CRE-binding protein (CREB), CRE-modulator (CREM), and activating transcription factor-1 (ATF-1). These data suggest that GLP-1 induces insulin gene transcription in a CREB, CREM, and ATF-1-independent manner in beta-cells. The mechanism by which DMSO imparts this amplifying action is unclear but may involve redistribution of intracellular compartments or a direct molecular interaction with a downstream target of the GLP-1 receptor signaling pathway in the beta-cell. These effects of DMSO on incretin action may provide novel applications with respect to further characterizing GLP-1 receptor signaling, identifying incretin-like compounds in screening assays, and as a therapeutic treatment in type 2 diabetes.

Activating Transcription Factor 1↗

PubMed1992

Transcriptional cross-talk: nuclear factors CREM and CREB bind to AP-1 sites and inhibit activation by Jun.

The proteins Fos and Jun dimerize to constitute the transcription factor AP-1 which is known to respond to treatment with phorbol esters. AP-1 binds to 12-O-tetradecanoylphorbol-13-acetate-responsive elements (TREs) palindromic sequences. cAMP-responsive elements (CREs) are very similar to TREs and CRE-binding proteins are similar in structure to Fos and Jun. Thus, the two main signal transduction pathways have closely related nuclear effectors which could possibly overlap and/or cross-talk. The gene CRE modulator (CREM) encodes both antagonists and an activator of the cAMP transcriptional response by alternative splicing. In this report we show that CREM antagonists are able to block the transcriptional activation elicited by c-Jun. The mechanism by which this repression is obtained does not require heterodimerization between CREM and the Fos and/or Jun proteins. In contrast, we show that both CREM and CRE-binding proteins (CREB) are able to bind TREs and therefore compete with c-Jun for this site. Removal of the phosphorylation domain in CREM does not affect the down-regulatory function. We also show that c-Fos does not affect the inhibitory function of CREM on c-Jun and that the transcriptional activation elicited by the other members of the jun family (JunB, JunD, and v-Jun) is also down-regulated by CREM.

Base Sequence↗

PubMed1993

Phosphorylation and negative regulation of the transcriptional activator CREM by p34cdc2.

Transcription factors that bind to cAMP-responsive elements (CREs) regulate the expression of target genes in response to activation of the adenylyl cyclase pathway. It is generally thought that activation is obtained through direct phosphorylation by the cAMP-dependent protein kinase-A. We have isolated the gene CRE modulator (CREM), which encodes multiple members of the CRE-binding protein family, by cell-specific alternative splicing. Various isoforms have been characterized, encoding both repressors (CREM alpha, -beta, and -gamma) as well as activators (CREM tau). Here we show that the function of the activator CREM tau is regulated by the p34cdc2 kinase. Multiple serine and threonine residues are phosphorylated in vivo as well as in vitro by p34cdc2. Although there is no effect of p34cdc2-mediated phosphorylation on CREM tau DNA binding, we observed a dramatic effect on the trans-regulatory function. Coexpression of a constitutively active p34cdc2 mutant shows that the trans-activation potential of CREM tau is strongly reduced by p34cdc2. This represents the first example of negative regulation of a transcription factor of this class by p34cdc2.

Amino Acid Sequence↗

PubMed2007

Transcriptional and epigenetic regulation of interleukin-2 gene in activated T cells by morphine.

Chronic morphine inhibits interleukin-2 (IL-2) at both the transcriptional and protein synthesis levels. The molecular mechanisms by which morphine decreases IL-2 are not fully understood. The production of IL-2 is tightly regulated by several transcription factors that bind to the IL-2 promoter. Herein, we show that chronic morphine treatment results in an increase in cAMP levels with a concurrent up-regulation of the cAMP inducible repressor inducible cAMP early repressor (ICER)/cAMP response element modulator (CREM) and down-regulation of p-cAMP-response element-binding protein (CREB) in activated T cells. Furthermore, ICER competes for p-CREB binding to the cAMP-responsive elements (CREs) site. This leads to the uncoupling of CBP/p300 thereby abrogating IL-2 transcription. Overexpression of either antisense CREM or CREB plasmid rescued morphine-induced inhibition of IL-2 promoter activity and protein production. In addition, we also found that chronic morphine treatment inhibited the acetylation and trimethylation of histones and decreased both DNA demethylation and accessibility of the IL-2 promoter. These findings suggest that chronic morphine treatment may function through both transcriptional and epigenetic mechanisms to inhibit IL-2 production.

Animals↗

PubMed1995

Modulation of Tax and PKA-mediated expression of HTLV-I promoter via cAMP response element binding and modulator proteins CREB and CREM.

Nuclear proteins of the human peripheral blood T lymphocytes that bind to the CREs located within three 21-bp repeat enhancers of the HTLV-I promoter belong to the CREB/CREM family of bZIP transcription factors. It has been shown previously that Tax enhances transactivation of these CREs by direct interactions with the bZIP domain of the transcription factors to stabilize DNA-binding. We show that CREB and CREM bind all three CRE sequences of the HTLV-I promoter which are important determinants in Tax-elicited transactivation as well as PKA-mediated activation of the HTLV-I promoter. Tax and PKA activate transcription from a HTLV-I-LTR CAT reporter plasmid transfected to NIH 3T3 cells, and CREM attenuates the activation. In the context of a GAL4 CREB fusion protein in which the DNA-binding bZIP domain of CREB is replaced by GAL4 binding domain, a single amino acid substitution of serine-133, phosphorylated by PKA and critical for the transactivation function of CREB, attenuates both Tax and PKA-mediated transcriptional responses. These observations suggest that Tax enhances CREB-mediated transactivation of the HTLV-I promoter by a mechanism apart from, and/or in addition to, the reported stabilization of DNA-binding by interaction with the bZIP domain of CREB.

3T3 Cells↗

PubMed1994

Targeted mutation of the CREB gene: compensation within the CREB/ATF family of transcription factors.

The cAMP response element binding protein (CREB) has been implicated as a key regulator in the transcriptional control of many genes. To assess the functional importance of CREB in vivo and its role in development, we used gene targeting to generate mice with a disruption of the CREB gene. Homozygous mutant mice appeared healthy and exhibited no impairment of growth or development. In this report we demonstrate that CREB and two other members of the CREB/ATF family, cAMP response element modulation protein (CREM) and activating transcription factor 1 (ATF1), appear to form a unique subgroup within this extensive class of transcription factors. Examination of CREM mRNA and protein levels in CREB mutant mice demonstrated overexpression of CREM in all tissues examined, but no change in ATF1 levels. These data demonstrate that CREB is not the sole mediator of cAMP-dependent transcriptional regulation and probably acts in concert with a specific subset of cAMP response element-binding proteins to transduce the cAMP signal and, in its absence, these same proteins can compensate for CREB function in vivo.

Activating Transcription Factor 1↗

PubMed2000

The inducible cAMP early repressor ICERIIgamma inhibits CREB and AP-1 transcription but not AT1 receptor gene expression in vascular smooth muscle cells.

Growth factors and agonists of both G alpha s and G alpha q-coupled receptor agonists each down regulate angiotensin AT1 receptor gene expression in vascular smooth muscle cells. To test whether the transcription factor CREB is in the pathway involved in modulation of AT1-receptor gene expression, AT1 receptor mRNA levels were measured after stimulation with forskolin, angiotensin II and PDGF-BB in VSMC expressing the inducible cAMP early repressor protein, ICERIIgamma, fused to the green fluorescent protein (eGFP). This fusion protein inhibits luciferase induction from promoters driven by either CREB response elements (CRE) or, unexpectedly, by tetradecanoylphorbol acetate response elements (TRE), which bind activator protein (AP-1). EGFP-ICERIIgamma expression inhibits occupancy of CRE and TRE elements by endogenous VSMC nuclear proteins. Nevertheless, agonist-induced AT1-receptor mRNA down regulation is unaffected by eGFP-ICERIIgamma expression. Although CREB transcription is activated by multiple classes of agonists in VSMC, and ICER can inhibit agonist-induced transcription mediated by both CREB and AP-1 enhancers, these transcription factors are not obligate components of the pathways regulating AT1-R gene expression in rat VSMC.

Angiotensin II↗

PubMed1993

Transcriptional response to cAMP in brain: specific distribution and induction of CREM antagonists.

Changes in cAMP levels are often associated with the modulation of neuronal function. The CREM gene encodes both antagonists and activators of the cAMP-dependent transcriptional response by alternative splicing. CREM transcripts in rat brain show a characteristic pattern of expression, being specific for the inner layer of the cerebral cortex, anterior thalamus, hippocampus, and hypothalamus. Strikingly, the CREM transcripts correspond to the antagonist isoforms in these areas, suggesting a down-regulatory role for CREM in brain; in contrast, the expression of CREM tau and CREB activators is more diffuse and generalized. In the supraoptic nucleus, CREM expression is induced after osmotic stimulus. Importantly, this demonstrates physiological inducibility of CREM, which is novel within the CRE/ATF family.

Animals↗

PubMed1997

The dynamics of the transcriptional response to cyclic adenosine 3',5'-monophosphate: recurrent inducibility and refractory phase.

Activation of the cAMP signal transduction pathway results in the transcriptional induction of many genes. Several of them are induced with kinetics characteristic of the early response. One of these, the cAMP response element modulator (CREM) gene, is cAMP-inducible by virtue of an intronic promoter that directs the synthesis of the dominant negative inducible cAMP early repressor (ICER). ICER is involved in the down-regulation of its own promoter via an autoregulatory loop. Thus, while phosphorylation of cAMP response element binding protein (CREB) by the cAMP-dependent protein kinase A is the prerequisite for induction, it has been proposed that the following attenuation involves both CREB dephosphorylation and repression by the inducible repressor ICER. Here we show that ectopic expression of sense or antisense ICER in corticotroph AtT20 cells dramatically modifies the normal CREM inducibility profile. We have investigated the kinetics of CREM inducibility by recurrent stimulation of the cAMP-signaling pathway. We define the presence of a refractory phase that follows the first induction cycle. Accumulation of cAMP, protein kinase A activity, CREB/CREM phosphorylation, and ICER levels contribute to the refractory period. Strikingly, the length of the refractory period is determined by the length of the stimulation by cAMP responsible for the first cycle of induction.

Animals↗

PubMed1999

Requirement for cAMP-response element (CRE) binding protein/CRE modulator transcription factors in thyrotropin-induced proliferation of dog thyroid cells in primary culture.

In several cell types, mostly of epithelial origin, activation of the cAMP pathway triggers DNA synthesis and cell division. Regulation of gene expression by cAMP involves phosphorylation by pyruvate kinase A and activation of cAMP-response element binding protein (CREB)/CRE modulator (CREM) transcription factors which bind DNA to CRE sites. On the other hand, several CREM isoforms are transcriptional repressors, such as the inducible cAMP early repressor (ICER) transcription factors, which are synthesized from an intronic promoter of the CREM gene. This study investigated the potential role of CREB/CREM transcription factors in the cAMP mitogenic pathway, using an experimental model of epithelial cells in primary culture, i.e. dog thyroid cells stimulated by thyroid-stimulating hormone (TSH). In response to TSH, CREB/CREM transcription factors were phosphorylated on the serine residue of the pyruvate kinase A consensus site. In addition, the synthesis of ICER mRNAs was strongly induced by TSH. This transient upregulation of ICER expression correlated with increased protein levels. It was restricted to the cAMP pathway, as neither epidermal growth factor nor phorbol myristate acetate, which are potent mitogens for dog thyroid cells, induced ICER expression. On the other hand, increased expression of ICER mRNAs was not detected in dog thyroids chronically stimulated by TSH in vivo. The requirement for CREB/CREM transcription factors in the mitogenic effect of TSH was assessed by transfecting expression vectors encoding CREM repressors into dog thyrocytes in order to interfere with CRE-mediated gene transcription. The ectopic expression of ICER Igamma or CREM alpha isoforms inhibited DNA replication in dog thyrocytes stimulated by TSH. This inhibitory effect was dependent on the ability of CREM repressors to form dimers but did not involve their DNA-binding capacity. Together these results show that CREB/CREM transcription factors are tightly regulated, at the transcriptional and post-translational levels, by TSH in dog thyroid cells, and provide clear evidence that their activity is required for the cAMP-dependent proliferation of cells in primary culture. Moreover, the transient induction of ICER transcription factors during mitogenic stimulation by TSH raises questions about the role of these potent repressors of CRE-dependent transcription as timers of cellular proliferation.

Animals↗

PubMed2006

Transcription factors, cAMP-responsive element modulator (CREM) and Tisp40, act in concert in postmeiotic transcriptional regulation.

We previously isolated 80 TISP (transcript induced in spermiogenesis) genes whose transcription is dramatically induced during spermiogenesis. Our analysis here of the expression of these genes in the testis of the cAMP-responsive element modulator (CREM)-null mouse revealed that 54 TISP genes are under the transcriptional regulation of CREM. One CREM-regulated gene is TISP40, which encodes a basic leucine zipper (bZip)-type transcription factor bearing a transmembrane domain that generates the two proteins Tisp40alpha and Tisp40beta. Both of these proteins function by binding to UPRE (unfolded protein-response element) but do not recognize CRE motifs. We show here that Tisp40alpha mRNA is generated under the direct transcriptional regulation of CREM. CREMtau and Tisp40 form a heterodimer, which functions through CRE but not through UPRE. Furthermore, binding ability of CREM to CRE is dramatically up-regulated by forming a heterodimer with Tisp40alphaDeltaTM, a truncated form of Tisp40alpha that lacks the transmembrane domain. We confirmed that Tisp40 and CREM actually bind to the Tisp40 promoter in vivo by chromatin immunoprecipitation assay. Finally, we demonstrate that the Tisp40DeltaTM-CREMtau heterodimer acts as a recruiter of HIRA, a histone chaperone, to CRE. Taken together, we propose that Tisp40 is an important transcriptional regulator during spermiogenesis.

Animals↗

PubMed1994

An isoform of transcription factor CREM expressed during spermatogenesis lacks the phosphorylation domain and represses cAMP-induced transcription.

cAMP response element-binding protein (CREB) and modulator protein (CREM) regulate the transcription of cAMP-responsive genes via phosphorylation by cAMP-dependent protein kinase A. Reverse transcription and polymerase chain amplification of RNA from male germ cells identify an alternatively spliced CREM isoform, CREM delta C-G, lacking four exons including those encoding the protein kinase A-regulated phosphorylation domain and the flanking glutamine-rich transcriptional activation domains. CREM delta C-G retains exons that encode the basic-leucine zipper (bZIP) DNA-binding domain, binds to cAMP response elements (CREs), and competitively inhibits binding of CREB and CREM to CREs. Expression of CREM delta C-G inhibits transcription of a CRE-containing chloramphenicol acetyltransferase reporter plasmid induced by endogenous CREB. Antiserum to CREM detects CREM delta C-G in elongated spermatids from rat testis. These observations indicate that CREM delta C-G is a unique form of a competitive negative regulator of CREB-mediated gene transcription expressed in a maturation-dependent manner in haploid germ cells. The developmental specificity of CREM delta C-G suggests that it may play a role in transcriptional regulation during spermatogenesis.

Alternative Splicing↗

PubMed2006

Tumor necrosis factor-alpha activates transcription of inducible repressor form of 3',5'-cyclic adenosine 5'-monophosphate-responsive element binding modulator and represses P450 aromatase and inhibin alpha-subunit expression in rat ovarian granulosa cells by a p44/42 mitogen-activated protein kinase-dependent mechanism.

The proinflammatory cytokine TNFalpha has important actions at the level of the ovary, including inhibition of P450 aromatase (P450AROM) activity and the secretion of inhibin, two proteins that are markers of the granulosa cell's differentiated status. Because the transcription of both P450AROM and inhibin alpha-subunit can be suppressed in the ovary by the inducible repressor isoform of cAMP-responsive element binding modulator (ICER), we have investigated whether TNFalpha and its intracellular messenger ceramide can induce ICER expression and the mechanisms whereby the induction is accomplished. ICER mRNA levels were assessed by RT-PCR in granulosa cells treated with TNFalpha, the ceramide-mobilizing enzyme sphingomyelinase (SMase), or C6-cer, a cell-permeant ceramide analog. Rapid (3 h) yet transient increases in the four isoforms of ICER were observed in response to all treatments. Likewise, ICER protein measured by immunoprecipitation with a specific antibody increases after TNFalpha, SMase, or C6-cer treatment. The mandatory phosphorylation of cAMP-responsive element binding was also observed in response to TNFalpha, SMase, or C6-cer and shown to be prevented by the p44/42 MAPK-specific inhibitor PD098059 but no other kinase blockers. Activation of p44/42 MAPK by the cytokine and its messenger was subsequently demonstrated as well as the inhibition of ICER expression by PD098059. Finally, the blocking of p44/42 MAPK activation prevented TNFalpha inhibition of FSH-dependent increases in P450AROM and inhibin alpha-subunit mRNA levels, thus indicating that p44/42 MAPK-mediated ICER expression may be accountable for the effects of TNFalpha on the expression of both proteins.

Animals↗

PubMed2002

The expanding family of CREB/CREM transcription factors that are involved with spermatogenesis.

One of the molecular mechanisms shown to have played a major role in orchestrating the expression of the many genes with unique cellular and temporal specificity in spermatogenesis, is the cAMP-dependent signaling pathway. In this pathway, gene expression is mediated primarily by two members of the bZIP transcription factors-cAMP-response element binding protein (CREB) and cAMP-responsive element modulator (CREM). Both bind a specific cis element, cAMP-response element (CRE), in the promoter of target genes, both are activated by protein kinase A (PKA) phosphorylation that enables binding of CREB binding protein (CBP) and recruitment of the basal transcription machinery, and both are characterized by multiple alternatively spliced forms. Some of these alternatively spliced forms lack the transactivation domains and hence function as transcription suppressors. In Sertoli cells, CREB levels fluctuate in a cyclical manner that depends on the specific cell associations along the spermatogenic wave. Follicle stimulating hormone (FSH) activates the cAMP signaling pathway and consequently, CREB positively auto-regulates its own expression (by binding to a CRE like element in its promoter). Subsequently, activated CREB activates transcription of genes essential for proper germ cell differentiation. In addition, TNFalpha secreted by round spermatids, activates NF-kappaB dependent CREB expression in Sertoli cells and thus, contributes to the elevated CREB levels as long as these cells are intimately associated. Inducible cAMP early repressor (ICER), a suppressor isoform of CREM, also activated by CREB, down regulates CREB expression together with its own expression, resetting CREB to basal level that enables a new spermatogenic wave. In germ cells, antagonist forms of CREM (alpha, beta and gamma) are present in premeiotic cells and early prophase spermatocytes. A prominent switch to the CREMtau and CREMtheta; activating forms starts in pachytene spermatocytes leading to the activation of haploid genes important for spermiogenesis in round spermatids. Interestingly, in germ cells, CREM exerts activation of haploid genes independent of its phosphorylation state. It associates with activator of CREM in testis (ACT), that has an intrinsic transcriptional activity, rather than with CBP. These and other findings suggest that the expanding CREB/CREM proteins and potentially other members of the CREB family are key molecular regulators at all stages of spermatogenesis.

Animals↗

PubMed2002

Regulation of steroidogenesis and the steroidogenic acute regulatory protein by a member of the cAMP response-element binding protein family.

The mitochondrial phosphoprotein, the steroidogenic acute regulatory (StAR) protein, is an essential component in the regulation of steroid biosynthesis in adrenal and gonadal cells through cAMP-dependent pathways. In many cases transcriptional induction by cAMP is mediated through the interaction of a cAMP response-element binding protein (CREB) family member with a consensus cAMP response element (CRE; 5'-TGACGTCA-3') found in the promoter of target genes. The present investigation was carried out to determine whether a CRE-binding protein (CREB) family member [CREB/CRE modulator (CREM) family] was involved in the regulation of steroidogenesis and StAR protein expression. Transient expression of wild- type CREB in MA-10 mouse Leydig tumor cells further increased the levels of (Bu)2cAMP-induced progesterone synthesis, StAR promoter activity, StAR mRNA, and StAR protein. These responses were significantly inhibited by transfection with a dominant-negative CREB (A-CREB), or with a CREB mutant that cannot be phosphorylated (CREB-M1), the latter observation indicating the importance of phosphorylation of a CREB/CREM family member in steroidogenesis and StAR expression. The CREB/CREM-responsive region in the mouse StAR gene was located between -110 and -67 bp upstream of the transcriptional start site. An oligonucleotide probe (-96/-67 bp) containing three putative half-sites for 5'-canonical CRE sequences (TGAC) demonstrated the formation of protein-DNA complexes in EMSAs with recombinant CREB protein as well as with nuclear extracts from MA-10 or Y-1 mouse adrenal tumor cells. The predominant binding factor observed with EMSA was found to be the CREM protein as demonstrated using specific antibodies and RT-PCR analyses. The CRE elements identified within the -96/-67 bp region were tested for cAMP responsiveness by generating mutations in each of the CRE half-sites either alone or in combination. Although each of the CRE sites contribute in part to the CREM response, the CRE2 appears to be the most important site as determined by EMSA and by reporter gene analyses. Binding specificity was further assessed using specific antibodies to CREB/CREM family members, cold competitors, and mutations in the target sites that resulted in either supershift and/or inhibition of these complexes. We also demonstrate that the inducible cAMP early repressor markedly diminished the endogenous effects of CREM on cAMP-induced StAR promoter activity and on StAR mRNA expression. These are the first observations to provide evidence for the functional involvement of a CREB/CREM family member in the acute regulation of trophic hormone-stimulated steroidogenesis and StAR gene expression.

5' Flanking Region↗

PubMed1997

Coupling signalling pathways to transcriptional control: nuclear factors responsive to cAMP.

Several endocrine and neuronal functions are governed by the cAMP-dependent signalling pathway. In eukaryotes, transcriptional regulation upon stimulation of the adenylyl cyclase signalling pathway is mediated by a family of cAMP-responsive nuclear factors. This family consists of a large number of members that may act as activators or repressors. These factors contain the basic domain/ leucine zipper motifs and bind as dimers to cAMP-response elements (CRE). The function of CRE-binding proteins (CREBs) is modulated by phosphorylation by several kinases. Direct activation of gene expression by CREB requires phosphorylation by the cAMP-dependent protein kinase A to the serine-133 residue. Among the repressors, ICER (Inducible cAMP Early Repressor) deserves special mention. ICER is generated from an alternative CREM promoter and constitutes the only inducible cAMP-responsive element binding protein. Furthermore, ICER negatively autoregulates the alternative promoter, thus generating a feedback loop. In contrast to the other members of the CRE-binding protein family, ICER expression is tissue specific and developmentally regulated. The kinetics of ICER expression are characteristic of an early response gene. Our results indicate that CREM plays a key physiological and developmental role within the hypothalamic-pituitary-gonadal axis. We have previously shown that the transcriptional activator CREM is highly expressed in postmeiotic cells. Spermiogenesis is a complex process by which postmeiotic male germ cells differentiate into mature spermatozoa. This process involves remarkable structural and biochemical changes that are under the hormonal control of the hypothalamic-pituitary axis. We have addressed the specific role of CREM in spermiogenesis using CREM-mutant mice generated by homologous recombination. Analysis of the seminiferous epithelium from mutant male mice reveals that spermatogenesis stops at the first step of spermiogenesis. Late spermatids are completely absent, while there is a significant increase in apoptotic germ cells. A series of postmeiotic germ cell-specific genes are not expressed. Mutant male mice completely lack spermatozoa. This phenotype is reminiscent of cases of human infertility. We have shown that ICER is regulated in a circadian manner in the pineal gland, the site of the hormone melatonin production. This night-day oscillation is driven by the endogenous clock (located in the suprachiasmatic nucleus, SCN). The synthesis of melatonin is regulated by a rate-limiting enzyme, the serotonin N-acetyltransferase (NAT). By using the CREM-deficient mice and by analysis of the regulatory region of the gene encoding the serotonin NAT, we have established that ICER is responsible for the amplitude and rhythmicity of NAT and thus for the oscillation in the hormonal synthesis of melatonin.

Adenylyl Cyclases↗

PubMed1997

Human endometrial stromal cells express novel isoforms of the transcriptional modulator CREM and up-regulate ICER in the course of decidualization.

Decidualization of human endometrial stromal (ES) cells in culture can be triggered by a sustained elevation of intracellular cAMP for several days and is characterized by activation of the cAMP-responsive decidual PRL (dPRL) gene promoter. We investigated the expression of the cAMP response element (CRE) binding protein CREB, and the modulators CREM (cAMP response element modulator) and ICER (inducible cAMP early repressor), in relation to decidualization of ES cells. We isolated all four known ICER isoforms from ES cells, which differ by the presence or absence of the small exon gamma and the presence of either DNA-binding domain (DBD) I or II. Of the various CREM isoforms, we cloned six transcript species, all containing DBD I. These were the known repressor CREM-alpha, the potential activator CREM-tau 2 alpha, and four novel forms whose reading frames were blocked upstream of the DBD. Two of these forms contained a novel exon psi, which is 100 bp in length, resides downstream of the first protein-coding exon of the CREM gene, and introduces an early in-frame stop codon. Surprisingly, in cotransfection assays, all four novel CREM isoforms were potent inhibitors of protein kinase A-stimulated transcription of a reporter gene construct driven by a CRE. By in vitro transcription/translation of all six CREM cDNAs, we demonstrated internal translation initiation at three different methionine residues, giving rise to novel short and very short C-terminal proteins comprising DBD I. These proteins bound to a cAMP response element as homodimers or as heterodimers with each other or with CREB. Immunofluorescence showed nuclear localization of C-terminal CREM proteins expressed from all six CREM cDNAs. Comparison of undifferentiated and decidualized ES cells showed no difference in the level of expression of any of the CREM transcript species. Likewise, CREB was evenly expressed between the two populations. In contrast, ICER transcripts were strongly up-regulated in decidualized ES cells in parallel with the induction of dPRL expression. It appears paradoxical that in vivo, in response to a permanent cAMP stimulus, ICER is up-regulated without displaying negative autoregulation of its own gene or suppression of the dPRL promoter. Elevated ICER levels in decidualized ES cells may be indicative of the presence of overriding amounts of transcriptional activators such as full length CREM-tau or CREB which, in turn, upon cAMP-induced phosphorylation, contribute to the induction of the dPRL gene.

Animals↗