Repressor Inducible cAMP Early Repressor Research Articles

The transcriptional repressor ICER binds to multiple loci throughout the genome

The events culminating in ovulation are controlled by the cyclical actions of hormones such as Follical Stimulating Hormone (FSH) and Luteinizing Hormone (LH). The secondary messenger, cyclic AMP (cAMP) conveys the intracellular activity of these hormones. It is well established that a family of transcription factors facilitate cAMP mediated gene expression, yet it remains unknown how these factors directly affect ovulation. One of these factors, Inducible cAMP Early Repressor (ICER) has been implicated in the transcriptional regulation of cAMP inducible genes during folliculogenesis and ovulation. In order to better determine the role of ICER in ovarian function we have identified novel targets using a genome-wide approach. Using a modification of the chromatin immunoprecipitation (ChIP) assay we directly cloned and sequenced the immunoprecipitated ICER-associated DNAs from an immortalized mouse granulose cell line (GRMO2). The analysis of the immunoprecipitated DNA fragments has revealed that ICER's binding to DNA has the following distribution; 16% within the promoter region, 31% within an intron, 14% were not within a gene, 6% were within 20 kb of a promoter and 3% were within the 3′ end of genes.

Inducible cAMP early repressor (ICER) is a novel regulator of RIG-I mediated IFN-β production

Antiviral responses can be triggered by the cytoplasmic RNA helicase RIG-I that binds to viral RNA. RIG-I-mediated signaling stimulates the transcription factors IRF3 and NF-κB and their activation mechanisms have been intensively studied. Here we examined Sendai virus (SV)-mediated activation of the transcription factor CREB and the role of its feedback repressor ICER in production of endogenous antiviral proteins. We show that SV infection and the mitochondrial adapter protein MAVS promote CREB phosphorylation that is dependent upon p38 MAPK and MK2. ICER is induced by CREB and acts as a feedback repressor of CRE-dependent transcription. We found that SV infection stimulated induction of ICER mRNA and protein expression. Surprisingly, ectopic expression and siRNA-mediated knockdown of ICER revealed that ICER is a positive regulator of the production of antiviral IFN-β and IP10 during SV infection. In contrast, ICER did not affect SV-elicited phosphorylation of IRF3, NF-κB or ATF2/c-Jun, transcription factors governing IFN-β and IP10 synthesis. However, expression of ICER increased total IRF3 protein levels during SV infection. These results point to a novel role of ICER in antiviral immune signaling acting to increase levels of antiviral effectors.

Mapping the ubiquitination sites of the transcriptional repressor ICER

Inducible cAMP Early Repressor (ICER) is a dominant negative transcriptional repressor that expresses tumor suppressor activity. In cancer cells, ICER is abnormally expressed and becomes modified by ubiquitination. Consequentially, ICER is degraded or re‐localized from the nucleus to the cytosol where it is non‐functional. When ICER is reintroduced into cancer cells, it inhibits the transformed phenotype of cancer cells. The goal of this project is to map the specific ubiquitinated lysines on ICER to define the functional consequences of ICER ubiquitination and cytosolic localization. Eleven mutant forms of ICER were constructed using site‐directed mutagenesis. All lysine residues were substituted to arginine to create an ICER molecule without lysines. Another 10 independent mutants were generated where all but one specific lysine was substituted by arginine. All clones were subcloned in a mammalian expression vector. By using these altered forms of ICER, we have found that ICER is ubiquitinated in several lysines. This data suggested alternative modes of regulating ICER degradation and subcellular localization. The long‐term goal of this project is to identify ways of inhibiting ICER degradation in cancer cells as a new mode for cancer treatment. This project was supported by a grant from the Sokol Institute of Pharmaceutical Sciences, College of Science and Mathematics, Montclair State University.

Ras‐induced melanoma transformation is associated with the proteasomal degradation of the transcriptional repressor ICER

Activation of the mitogen-activated protein kinase (MAPK) pathway targets the putative tumor suppressor protein inducible cAMP early repressor (ICER) to ubiquitin-mediated proteasomal degradation [Yehia et al. JBC 2001; 276: 35272-35279]. We demonstrate that ICER proteasomal degradation is implicated in Ras/MAPK-mediated melanoma tumorigenesis. In a system using Tyr/Tet-Ras INK4a-/- transgenic mice and melanoma cells in culture termed R545 cells isolated from Tyr/Tet-Ras INK4a-/- mice [Chin et al. Nature 1999; 400: 468-472], melanoma genesis and melanoma maintenance is strictly dependent upon expression of H-RasV12G. We found that ICER protein was not expressed during melanoma genesis but was strongly expressed in regressing melanomas. Similarly in R545 cells, ICER protein expression was negatively regulated by H-RasV12G. The expression of ICER mRNA was not affected by H-RasV12G expression, suggesting that ICER regulation was post-translational. Indeed, pharmacological inhibition of Ras activity or the proteasome abolished the degradation of ICER caused by H-RasV12G expression indicating that RAS oncogene regulates the expression of ICER protein by targeting ICER to proteasomal degradation. By engineering clones of R545 melanoma cells stably transfected with ICER we were able to determine the prerequisite for Ras-induced tumorigenesis. The reconstitution of physiological levels of ICER showed a significant decrease in cell growth, as well as inhibition of anchorage-independent cell growth and tumorigenicity in nude mice. ICER was found to efficiently repress the expression of cyclin D1 in R545 cells due to the binding of ICER to the CRE in the cyclin D1 promoter. Taken together, we postulate that ICER protein might be targeted to degradation in human tumors where Ras is mutated.

Determination of the DNA Binding Mode of the Transcriptional Repressor ICER

Inducible cAMP Early Repressor (ICER) is a leucine zipper protein, a product of the cAMP Responsive Element Modulator (CREM) gene. ICER functions as a transcriptional repressor and its expression is abnormal in certain types of cancer where ICER acts as a tumor repressor. ICER binds to cAMP Responsive Elements (CRE's) found in the promoter sequences of genes involved in cellular growth. It also binds to the four CRE sites on its own promoter, thereby regulating its own expression. These four sites located on the CREM gene promoter are designated CARE-1 through CARE-4, and each one consists of 8 to 9 bases. On the basis of the presence of four CRE sites, we hypothesize that ICER may autoregulate its expression by cooperative binding to its own promoter. To test this hypothesis, we are performing titrations of purified ICER with double stranded DNA that is labeled with a fluorophore-quencher pair and monitor the binding by measuring the change in fluorescence due to an increase in resonance energy transfer upon a decrease in fluorophore-quencher distance. The dsDNA contains one or more of the CARE sites. We are determining the dissociation constant of ICER for each of the four CARE sites as well as examine the potential for cooperative binding between multiple CARE sites. Experiments on dsDNA (35 bp) with CARE-1 and/or CARE-2 have each yielded a dissociation constants of kd = 0.16 ± 0.02 μM, and no obvious evidence for cooperative binding between CARE-1 and CARE-2 has yet been observed. Analysis of dsDNA with CARE-3 and CARE-4 as well as for the entire promoter region with all four CARE sites will be presented and a model for binding of ICER to its own promoter will be discussed.

Daytime Gating in the Syrian Hamster Pineal Gland

Melatonin synthesis in rodents is tightly regulated at the transcriptional level by stimulatory and inhibitory transcription factors. Among them, phosphorylated cAMP-related element binding protein (pCREB) and inducible cAMP early repressor (ICER), a strong inhibitor of cAMP-related element-driven genes, have an antagonistic action in activating/inhibiting the transcription of the Aa-nat gene, which is an important enzyme in melatonin synthesis. In the Syrian hamster, a rodent displaying a seasonal control of reproduction, melatonin synthesis is strongly gated to the second part of the night. Indeed, exogenous adrenergic stimulation is unable to stimulate Aa-nat gene transcription and melatonin synthesis during daytime. In the present study, we investigated whether ICER may be the cause of this daytime repression by comparing the dynamic of ICER and the adrenergic regulation of two genes whose expression is rapidly activated by cAMP-dependant mechanisms, c-fos and Icer. Adrenergic induction of c-fos and Icer expression was not possible during daytime, except at early day. ICER immunoreactivity was elevated throughout the daily cycle but reached the highest levels at early day, when gene expression can be induced by adrenergic agonists. Additionally, CREB phosphorylation was subjected to the same daily gating with an adrenergic induction occurring in the early but not in the late day. Taken together, our results indicate that the diurnal gating of pineal activity in the Syrian hamster is not caused by the repressor ICER and that it may occur at the level of noradrenergic receptor signalling.

Negative Regulation of TLR Responses by the Neuropeptide CGRP Is Mediated by the Transcriptional Repressor ICER

Communication between the nervous and immune systems involves the release of neuropeptides, such as calcitonin gene-related peptide (CGRP), from sensory nerves during inflammation. CGRP may inhibit the activities of both innate and adaptive immune cells, but the molecular pathways underlying this function are largely unknown. In this study, we identify CGRP as a potent inhibitor of TLR-stimulated production of inflammatory mediators, such as TNF-alpha and CCL4, by murine dendritic cells. Inhibition of TLR responses was independent of IL-10 and did not involve perturbation of canonical TLR signaling, including activation of MAPK and NF-kappaB. Instead, the inhibitory activity of CGRP was mediated by the cAMP/protein kinase A pathway leading to rapid up-regulation of the transcriptional repressor, inducible cAMP early repressor (ICER). Ectopically expressed ICER directly repressed the LPS-stimulated activity of a synthetic Tnf promoter, as well as TNF-alpha protein production driven by the endogenous promoter. Inhibition of dendritic cell gene expression by CGRP was associated with the presence of a composite cAMP response element/kappaB promoter element. In a murine model of endotoxemia, CGRP markedly attenuated serum TNF-alpha levels, and this effect was associated with the up-regulation of ICER. Together, these results establish a novel pathway for the negative regulation of TLR responses through the nervous system that critically involves induction of the transcriptional repressor ICER by the neuropeptide CGRP.

Regulation of Phosphodiesterase 3 and Inducible cAMP Early Repressor in the Heart

Growing evidence suggests that multiple spatially, temporally, and functionally distinct pools of cyclic nucleotides exist and regulate cardiac performance, from acute myocardial contractility to chronic gene expression and cardiac structural remodeling. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing cAMP and cyclic GMP, regulate the amplitude, duration, and compartmentation of cyclic nucleotide-mediated signaling. In particular, PDE3 enzymes play a major role in regulating cAMP metabolism in the cardiovascular system. PDE3 inhibitors, by raising cAMP content, have acute inotropic and vasodilatory effects in treating congestive heart failure but have increased mortality in long-term therapy. PDE3A expression is downregulated in human and animal failing hearts. In vitro, inhibition of PDE3A function is associated with myocyte apoptosis through sustained induction of a transcriptional repressor ICER (inducible cAMP early repressor) and thereby inhibition of antiapoptotic molecule Bcl-2 expression. Sustained induction of ICER may also cause the change of other protein expression implicated in human and animal failing hearts. These data suggest that the downregulation of PDE3A observed in failing hearts may play a causative role in the progression of heart failure, in part, by inducing ICER and promoting cardiac myocyte dysfunction. Hence, strategies that maintain PDE3A function may represent an attractive approach to circumvent myocyte apoptosis and cardiac dysfunction.

Inducible cAMP early repressor suppresses gastrin-mediated activation of cyclin D1 and c-fosgene expression

The gastric hormone gastrin and its precursors promote proliferation in several gastrointestinal cell types. Here we show that gastrin induces transcription of cell cycle gene cyclin D1 and protooncogene c-fos in the neuroendocrine pancreatic cell line AR42J and that this gastrin response is inhibited by endogenous inducible cAMP early repressor (ICER). The transcriptional repressor ICER is known to downregulate both its own expression and the expression of other genes containing cAMP-responsive elements (CREs). Using siRNA, we also show that CRE promoter elements are the targets of endogenous ICER in AR42J cells as well as in the neuroendocrine cell line RIN5F. Our results suggest that ICER plays an important role in molecular mechanisms governing gastrin-mediated growth by modulating gastrin's transcriptional activation of growth-related genes. Our finding that ICER modulates pituitary adenylate cyclase-activating polypeptide-activated gene expression also indicates a regulatory effect of ICER in the responses of neuroendocrine cells to peptides other than gastrin.

Coupling cAMP Signaling to Transcription in the Liver: Pivotal Role of CREB and CREM

Transcriptional factors binding to cAMP-responsive elements (CREs) in the promoters of various genes belong to the basic domain-leucine zipper superfamily and are composed of three genes in mammals, CREB, CREM, and ATF-1. A large number of CREB, CREM, and ATF-1 proteins are generated by posttranscriptional events, mostly alternative splicing, and regulate gene expression by acting as activators or repressors. Activation is classically brought about by signaling-dependent phosphorylation of a key acceptor site (Ser133 in CREB) by a number of possible kinases, including PKA, CamKIV, and Rsk-2. Phosphorylation is the prerequisite for the interaction of CBP (CREB-binding protein), a co-activator that has also histone acetyltransferase activity. Repression may involve dynamic dephosphorylation of the activators and thus decreased association with CBP. Another pathway of transcriptional repression on CRE sites implicates the inducible repressor ICER (inducible cAMP early repressor), a product of the CREM gene. Being an inducible repressor, ICER is involved in autoregulatory feedback loops of transcription that govern the down-regulation of early response genes, such as the proto-oncogene c-fos. The liver represents a remarkable physiological setting where cAMP-responsive signaling plays a major role. Indeed, a finely tuned program of gene expression is triggered by partial hepatectomy, so that through specific checkpoints a coordinated regeneration of the tissue is obtained. Temporal kinetics of transcriptional activation after hepatectomy reveals a pattern of early induction for several genes, some of them controlled by the CREB/CREM transcription factors. An important role of CREM in liver physiology was suggested by the robust induction of ICER after partial hepatectomy. The delay in tissue regeneration in CREM-deficient mice confirmed the important function of this factor in regulating hepatocyte proliferation. As gene induction is accompanied by critical changes in chromatin organization, the deciphering of the specific modification codes that histones display during liver regeneration and physiology will provide exciting new insights into the dynamics of chromatin architecture.

Differential inducibility of the transcriptional repressor ICER and its role in modulation of Fas ligand expression in T and NK lymphocytes

The engagement of antigen receptor can initiate apoptosis of T lymphocytes through the induced expression of Fas ligand (FasL). Forskolin, an activator of the cAMP/PKA pathway, results in antagonism of Fas-dependent, activation-induced cell death (AICD) by suppressed expression of the FasL. We report that forskolin-mediated induction of inducible cAMP early repressor (ICER) correlates with transcriptional attenuation of FasL expression in the AICD model 2B4 T cell hybridoma. ICER is inducible in human peripheral blood CD3(+) T cells, but in CD19(+) B cells, its induction is less responsive to forskolin treatment. Increased expression of ICER correlates with decreased FasL expression in both T and NK cells. ICER binds specifically to the proximal DNA binding site of the nuclear factor of activated T cells (NFAT) in the FasL promoter and in the presence of the minimal NFAT DNA-binding domain, the proximal NFAT motif allows ICER and NFAT to form an NFAT/ICER ternary complex in vitro. Moreover, in the activated 2B4 T cell hybridoma, the proximal NFAT motif participates in the down-regulation of the FasL promoter mediated by ICER. These findings provide further insight into the mechanism involved in cAMP-mediated transcriptional attenuation of FasL expression in T and NK lymphocytes.

Glucagon stimulates expression of the inducible cAMP early repressor and suppresses insulin gene expression in pancreatic beta-cells.

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.

Suppression of T cell function: a potential role for transcriptional repressor ICER.

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.

Stress-induced expression of transcriptional repressor ICER in the adrenal gland

Second messenger cyclic AMP plays a central role in signalling within the hypothalamo-pituitary-adrenal (HPA) axis. Changes in gene expression are central to long-term adaptations made in response to stress in the adrenal gland. Here we demonstrate that expression of the cAMP inducible transcriptional repressor, ICER (Inducible cAMP Early Repressor), is rapidly and powerfully induced in response to surgical stress in the rat adrenal gland. Hypophysectomisation blocks stress-induced ICER expression. Finally we demonstrate that injection of the pituitary hormone ACTH (Adrenocorticotropin Hormone) induces robust ICER expression in the adrenal cortex. Thus, induction of the transcriptional repressor ICER is coupled to the HPA axis response to stress.

Inducible cAMP early repressor ICER down-regulation of CREB gene expression in Sertoli cells

The cAMP response element binding protein (CREB) and the cAMP-responsive element modulator (CREM) are cyclically expressed in the seminiferous tubules during spermatogenesis. In the somatic Sertoli cells, which are the major supporters of germ cell development in the seminiferous tubules, the expression of CREB is cyclical and appears to be regulated by the levels of cAMP produced in response to the pituitary derived follicle-stimulating hormone FSH. Cyclic AMP response elements (CREs) located in the promoter of the CREB gene were shown earlier to be implicated in an autopositive feedback loop that up-regulates the expression of CREB. Here we show that in Sertoli cells FSH-mediated induction of the CREM repressor isoform, ICER (inducible cAMP early repressor) is correlated with the inhibition and delay of CREB gene expression in the seminiferous tubules. ICER binds to the two CREs located in the promoter of the CREB gene and in transient transfection assays of Sertoli cells, ICER expression vectors down-regulate transcription of a reporter gene driven by the CREB gene promoter. In addition, analyses of ICER and CREB gene expression in isolated segments of rat seminiferous tubules reveals stage-specific and cycle-dependent expression of ICER. The periods of enhanced expression of ICER correspond to the stages of spermatogenesis with the lowest levels of CREB expression. We suggest that the expression of ICER in Sertoli cells may contribute to the periodic repression of CREB gene expression during the repeated 12-day cycles of spermatogenesis, and may be required to reset the levels of activator CREB prior to the initiation of each new cycle of spermatogenesis.

Role of Transcriptional Repressor ICER in Cyclic AMP-mediated Attenuation of Cytokine Gene Expression in Human Thymocytes

Proliferating human medullary thymocytes can exhibit characteristic T helper cell type 1 cytokine responses exemplified by the immediate early expression of interleukin-2, interferon-gamma, tumor necrosis factor-alpha, and lymphotoxin-beta. Here we report that cAMP-mediated attenuation of the transcription of T helper-1-specific cytokine genes in human medullary thymocytes correlates with the induction of the cAMP-mediated transcriptional repressor ICER (inducible cAMP early repressor). We show that ICER binds specifically to several NFAT/AP-1 (nuclear factor of activated T cells/activating protein-1) composite DNA sites essential for the activation of the interleukin (IL)-2 promoter as well as to a homologous DNA motif present in the proximal segment of the interferon-gamma promoter. In the presence of the minimal NFAT DNA-binding domain, which is sufficient for both DNA binding and AP-1 complex formation, ICER and NFAT form NFAT/ICER ternary complexes on several NFAT/AP-1 DNA composite sites previously identified as essential for the expression of the immunoregulatory cytokines such as IL-2, IL-4, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha. In extracts prepared from human medullary thymocytes treated with forskolin and ionomycin, these composite sites bind endogenously expressed ICER either singly or in complexes. Moreover, in Jurkat cells, ectopically expressed ICER represses transcription from NFAT-mediated, phorbol ester/ionophore-activated IL-2, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha promoters. We present evidence that ICER interactions with NFAT/AP-1 composite DNA sites correlate with its ability to repress transcription. These findings provide further insight into the mechanisms involved in cAMP-mediated transcriptional attenuation of cytokine expression.

Cross-talk in signal transduction: Ras-dependent induction of cAMP-responsive transcriptional repressor ICER by nerve growth factor.

The CREM gene encodes both activators and repressors of cAMP-induced transcription. By virtue of an alternative, intronic promoter within the gene, the ICER (Inducible cAMP Early Repressor) isoform is generated. ICER acts as a dominant negative regulator and is cAMP-inducible in various neuroendocrine cells and tissues. ICER negatively autoregulates its own expression, and appears to participate in the molecular events governing oscillatory hormonal regulations. Here we report that ICER is inducible with nerve growth factor (NGF). This is the first example of cAMP-independent induction of ICER expression. Importantly, induction by NGF occurs via a subset of the CREs present in the ICER promoter which were previously shown to direct cAMP-inducibility. ICER induction correlates with a NGF-mediated phosphorylation of CREB. Both CREB phosphorylation and ICER inducibility require an intact Ras-dependent signalling pathway. We show that increased ICER levels result in the attenuation of c-fos expression. The activation of a powerful repressor of cAMP-responsive transcription by NGF, whose transduction signalling is cAMP-independent, constitutes a notable example of nuclear cross-talk and thus is likely to have relevant physiological implications.

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.

The transcriptional repressor ICER and cAMP-induced programmed cell death.

The cAMP pathway plays a central role in the response to hormonal signals for cell proliferation, differentiation and apoptosis. In IPC-81 leukaemia cells, activation of the cAMP pathway by prostaglandin E1 treatment, or other cAMP-elevating agents, induces apoptosis within 4-6 h. Inhibition of mRNA or protein synthesis during the first 2 h of cAMP induction protects cells from apoptosis, suggesting a requirement for early gene expression. cAMP-dependent protein kinase phosphorylates a class of nuclear factors and thereby regulates the transcription of a specific set of genes. Here we show that CREM (cAMP Responsive Element Modulator) expression is induced rapidly upon prostaglandin E1 treatment of IPC-81 cells. The induced transcripts correspond to the early product ICER (Inducible cAMP Early Repressor). ICER expression remains elevated until the burst of cell death. Protein synthesis inhibitors which prevent cAMP-induced apoptosis also block de novo ICER synthesis. Transfected IPC-81 cell lines, constitutively expressing high level of ICER are resistant to cAMP-induced cell death. In these transfected cells, cAMP fails to upregulate the ICER transcripts demonstrating that ICER exerts strongly its repressor function on CRE-containing genes. That an early expression of ICER blocks apoptosis, suggests that gene repression by endogenous ICER in IPC-81 is insufficient or occurs too late to protect cells against death. ICER transfected cells rescued from cAMP-induced apoptosis are growth arrested. It shows for the first time that CREM activation directly participates to the decision of the cell to die. ICER, by sequentially repressing distinct sets of CRE-containing genes could modulate cell fate.

Cyclic AMP signalling pathway and cellular proliferation: induction of CREM during liver regeneration

The CREM gene encodes both activators and repressors of cAMP-induced gene expression. An isoform of CREM encodes the powerful transcriptional repressor ICER (Inducible cAMP Early Repressor), which has been shown to be inducible by virtue of an alternative, intronic promoter. The CREM gene belongs to the early response class and displays a characteristic neuroendocrine cell- and tissue-specific expression. To date ICER inducibility has been described in non-replicating, terminally differentiated tissues. In this paper we document a robust induction of CREM expression in the regenerating rat liver after partial hepatectomy. This represents the first link of inducible CREM expression to the phenomenon of cellular proliferation. Furthermore, it represents the first example of transcriptional activation of a cAMP-responsive factor in the regenerating liver. This has significant physiological relevance since the adenylate cyclase signalling pathway is strongly implicated in liver regeneration. Finally, we show that the repressor ICER is inducible in the hepatoma cell line H35 upon activation of the adenylate cyclase and phosphorylation of the activator CREB.