Induction Of Inducible cAMP Early Repressor Research Articles

Induction of ICER is superseded by smICER, challenging the impact of ICER under chronic beta-adrenergic stimulation.

ICER (inducible cAMP early repressor) isoforms are transcriptional repressors encoded by the Crem (cAMP responsive element modulator) gene. They were linked to the regulation of a multitude of cellular processes and pathophysiological mechanisms. Here, we show for the first time that two independent induction patterns for CREM repressor isoforms exist in the heart, namely for ICER and smICER (small ICER), which are induced in response to β-adrenergic stimulation in a transient- and saturation-like manner, respectively. This time-shifted induction pattern, driven by two internal promoters in the Crem gene, leads to the predominant transcription of smIcer after prolonged β-adrenergic stimulation. Using an ICER knockout mouse model with preserved smICER induction, we show that the transient-like induction of Icer itself has minor effects on gene regulation, cardiac hypertrophy or contractile function in the heart. We conclude that the functions previously linked to ICER may be rather attributed to smICER, also beyond the cardiac background.

GnRH Pulse Frequency Control of Fshb Gene Expression Is Mediated via ERK1/2 Regulation of ICER.

The pulsatile release of GnRH regulates the synthesis and secretion of pituitary FSH and LH. Two transcription factors, cAMP-response element-binding protein (CREB) and inducible cAMP early repressor (ICER), have been implicated in the regulation of rat Fshb gene expression. We previously showed that the protein kinase A pathway mediates GnRH-stimulated CREB activation. We hypothesized that CREB and ICER are activated by distinct signaling pathways in response to pulsatile GnRH to modulate Fshb gene expression, which is preferentially stimulated at low vs high pulse frequencies. In the LβT2 gonadotrope-derived cell line, GnRH stimulation increased ICER mRNA and protein. Blockade of ERK activation with mitogen-activated protein kinase kinase I/II (MEKI/II) inhibitors significantly attenuated GnRH induction of ICER mRNA and protein, whereas protein kinase C, calcium/calmodulin-dependent protein kinase II, and protein kinase A inhibitors had minimal effects. GnRH also stimulated ICER in primary mouse pituitary cultures, attenuated similarly by a MEKI/II inhibitor. In a perifusion paradigm, MEKI/II inhibition in LβT2 cells stimulated with pulsatile GnRH abrogated ICER induction at high GnRH pulse frequencies, with minimal effect at low frequencies. MEKI/II inhibition reduced GnRH stimulation of Fshb at high and low pulse frequencies, suggesting that the ERK pathway has additional effects on GnRH regulation of Fshb, beyond those mediated by ICER. Indeed, induction of the activating protein 1 proteins, cFos and cJun, positive modulators of Fshb transcription, by pulsatile GnRH was also abrogated by inhibition of the MEK/ERK signaling pathway. Collectively, these studies indicate that the signaling pathways mediating GnRH activation of CREB and ICER are distinct, contributing to the decoding of the pulsatile GnRH to regulate FSHβ expression.

Decompensation of β-cells in diabetes: when pancreatic β-cells are on ICE(R).

Insulin production and secretion are temporally regulated. Keeping insulin secretion at rest after a rise of glucose prevents exhaustion and ultimately failure of β-cells. Among the mechanisms that reduce β-cell activity is the inducible cAMP early repressor (ICER). ICER is an immediate early gene, which is rapidly induced by the cyclic AMP (cAMP) signaling cascade. The seminal function of ICER is to negatively regulate the production and secretion of insulin by repressing the genes expression. This is part of adaptive response required for proper β-cells function in response to environmental factors. Inappropriate induction of ICER accounts for pancreatic β-cells dysfunction and ultimately death elicited by chronic hyperglycemia, fatty acids, and oxidized LDL. This review underlines the importance of balancing the negative regulation achieved by ICER for preserving β-cell function and survival in diabetes.

Nitric oxide regulates pulmonary vascular smooth muscle cell expression of the inducible cAMP early repressor gene

Nitric oxide (NO) regulates vascular smooth muscle cell (VSMC) structure and function, in part by activating soluble guanylate cyclase (sGC) to synthesize cGMP. The objective of this study was to further characterize the signaling mechanisms by which NO regulates VSMC gene expression using transcription profiling. DNA microarrays were hybridized with RNA extracted from rat pulmonary artery smooth muscle cells (RPaSMC) exposed to the NO donor compound, S-nitroso-glutathione (GSNO). Many of the genes, whose expression was induced by GSNO, contain a cAMP-response element (CRE), of which one encoded the inducible cAMP early repressor (ICER). sGC and cAMP-dependent protein kinase, but not cGMP-dependent protein kinase, were required for NO-mediated phosphorylation of CRE-binding protein (CREB) and induction of ICER gene expression. Expression of a dominant-negative CREB in RPaSMC prevented the NO-mediated induction of CRE-dependent gene transcription and ICER gene expression. Pre-treatment of RPaSMC with the intracellular calcium (Ca2+) chelator, BAPTA-AM, blocked the induction of ICER gene expression by GSNO. The store-operated Ca2+ channel inhibitors, 2-ABP, and SKF-96365, reduced the GSNO-mediated increase in ICER mRNA levels, while 2-ABP did not inhibit GSNO-induced CREB phosphorylation. Our results suggest that induction of ICER gene expression by NO requires both CREB phosphorylation and Ca2+ signaling. Transcription profiling of RPaSMC exposed to GSNO revealed important roles for sGC, PKA, CREB, and Ca2+ in the regulation of gene expression by NO. The induction of ICER in GSNO-treated RPaSMC highlights a novel cross-talk mechanism between cGMP and cAMP signaling pathways.

Role for inducible cAMP early repressor in promoting pancreatic beta cell dysfunction evoked by oxidative stress in human and rat islets

Aims/hypothesisPro-atherogenic and pro-oxidant, oxidised LDL trigger adverse effects on pancreatic beta cells, possibly contributing to diabetes progression. Because oxidised LDL diminish the expression of genes regulated by the inducible cAMP early repressor (ICER), we investigated the involvement of this transcription factor and of oxidative stress in beta cell failure elicited by oxidised LDL.MethodsIsolated human and rat islets, and insulin-secreting cells were cultured with human native or oxidised LDL or with hydrogen peroxide. The expression of genes was determined by quantitative real-time PCR and western blotting. Insulin secretion was monitored by EIA kit. Cell apoptosis was determined by scoring cells displaying pycnotic nuclei.ResultsExposure of beta cell lines and islets to oxidised LDL, but not to native LDL raised the abundance of ICER. Induction of this repressor by the modified LDL compromised the expression of important beta cell genes, including insulin and anti-apoptotic islet brain 1, as well as of genes coding for key components of the secretory machinery. This led to hampering of insulin production and secretion, and of cell survival. Silencing of this transcription factor by RNA interference restored the expression of its target genes and alleviated beta cell dysfunction and death triggered by oxidised LDL. Induction of ICER was stimulated by oxidative stress, whereas antioxidant treatment with N-acetylcysteine or HDL prevented the rise of ICER elicited by oxidised LDL and restored beta cell functions.Conclusions/interpretationInduction of ICER links oxidative stress to beta cell failure caused by oxidised LDL and can be effectively abrogated by antioxidant treatment.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-011-2165-x) contains supplementary material, which is available to authorised users.

Novel role of C terminus of Hsc70-interacting protein (CHIP) ubiquitin ligase on inhibiting cardiac apoptosis and dysfunction via regulating ERK5-mediated degradation of inducible cAMP early repressor

Growing evidence indicates a critical role of ubiquitin-proteosome system in apoptosis regulation. A cardioprotective effect of ubiquitin (Ub) ligase of the C terminus of Hsc70-interacting protein (CHIP) on myocytes has been reported. In the current study, we found that the cardioprotective effect of insulin growth factor-1 (IGF-1) was mediated by ERK5-CHIP signal module via inducible cAMP early repressor (ICER) destabilization. In vitro runoff assay and Ub assay showed ICER as a substrate of CHIP Ub ligase. Both disruption of ERK5-CHIP binding with inhibitory helical linker domain fragment (aa 101-200) of CHIP and the depletion of ERK5 by siRNA inhibited CHIP Ub ligase activity, which suggests an obligatory role of ERK5 on CHIP activation. Depletion of CHIP, using siRNA, inhibited IGF-1-mediated reduction of isoproterenol-mediated ICER induction and apoptosis. In diabetic mice subjected to myocardial infarction, the CHIP Ub ligase activity was decreased, with an increase in ICER expression. These changes were attenuated significantly in a cardiac-specific constitutively active form of MEK5α transgenic mice (CA-MEK5α-Tg) previously shown to have greater functional recovery. Furthermore, pressure overload-mediated ICER induction was enhanced in heterozygous CHIP(+/-) mice. We identified ICER as a novel CHIP substrate and that the ERK5-CHIP complex plays an obligatory role in inhibition of ICER expression, cardiomyocyte apoptosis, and cardiac dysfunction.

Cyclic AMP Inducible Early Repressor Mediates the Termination of Corticotropin Releasing Hormone Transcription in Hypothalamic Neurons

Elevations of inducible cAMP early repressor (ICER), the repressor isoform of the cAMP-responsive element modulator (CREM), are associated with protein binding to the corticotrophin releasing hormone (CRH) promoter and termination of CRH transcriptional responses to stress. To determine whether endogenous ICER production represses CRH transcription, we examined the effect of CREM siRNA on forskolin-stimulated ICER formation and CRH transcription in the hypothalamic cell line, 4B, and in primary cultures of hypothalamic neurons. Cotransfection of 4B cells with CREM siRNA and a CRH promoter-driven luciferase reporter gene markedly reduced the induction of ICER by forskolin and potentiated the stimulatory effect of forskolin on CRH promoter activity, compared with cells cotransfected with a nonspecific oligonucleotide. The role of ICER on endogenous CRH expression was studied in primary cultures of hypothalamic neurons by examining the effect of CREM siRNA on forskolin-induced primary transcript (CRH hnRNA) using intronic real-time PCR. As observed during stress in vivo, forskolin-stimulated CRH hnRNA was transient, increasing up to 60 min and declining to near basal values by 3 h. Transfection of CREM siRNA reduced forskolin-induced ICER by about 45% 48-h later and partially reversed the declining phase of CRH hnRNA production at 3 h. The data provide evidence that endogenous ICER formation is required for termination of CRH transcription and support the hypothesis that ICER is part of an intracellular feedback mechanism limiting the activation of CRH transcription during stress.

Surface Expression of GABAA Receptors Is Transcriptionally Controlled by the Interplay of cAMP-response Element-binding Protein and Its Binding Partner Inducible cAMP Early Repressor

The regulated expression of type A gamma-aminobutyric acid (GABA) receptor (GABA(A)R) subunit genes plays a critical role in neuronal maturation and synaptogenesis. It is also associated with a variety of neurological diseases. Changes in GABA(A) receptor alpha1 subunit gene (GABRA1) expression have been reported in animal models of epilepsy, alcohol abuse, withdrawal, and stress. Understanding the genetic mechanism behind such changes in alpha subunit expression will lead to a better understanding of the role that signal transduction plays in control over GABA(A)R function and brings with it the promise of providing new therapeutic tools for the prevention or cure of a variety of neurological disorders. Here we show that activation of protein kinase C increases alpha1 subunit levels via phosphorylation of CREB (pCREB) that is bound to the GABRA1 promoter (GABRA1p). In contrast, activation of protein kinase A decreases levels of alpha1 even in the presence of pCREB. Decrease of alpha1 is dependent upon the inducible cAMP early repressor (ICER) as directly demonstrated by ICER-induced down-regulation of endogenous alpha1-containing GABA(A)Rs at the cell surface of cortical neurons. Taken together with the fact that there are less alpha1gamma2-containing GABA(A)Rs in neurons after protein kinase A stimulation and that activation of endogenous dopamine receptors down-regulates alpha1 subunit mRNA levels subsequent to induction of ICER, our studies identify a transcriptional mechanism for regulating the cell surface expression of alpha1-containing GABA(A)Rs that is dependent upon the formation of CREB heterodimers.

Inducible cAMP Early Repressor Inhibits Growth of Vascular Smooth Muscle Cell

The role of inducible cAMP early repressor (ICER), a transcriptional repressor, in the vascular remodeling process has not been determined. We examined whether ICER affects growth of vascular smooth muscle cells (VSMCs). Semi-quantitative RT-PCR and Western blot analysis showed that expression of ICER was increased in beraprost (a prostaglandin I2 analogue)-stimulated VSMCs in a time- and dose-dependent manner. The induction of ICER was inhibited by pretreatment with H89, a protein kinase A (PKA) inhibitor, suggesting that PKA mediates the induction of ICER expression. Beraprost suppressed platelet-derived growth factor-induced thymidine incorporation in VSMCs, which was reversed by transfection of short interfering RNA for ICER, not by scramble RNA. Overexpression of ICER by an adenovirus vector attenuated neointimal formation (intima/media ratio) by 50% compared with overexpression of LacZ. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive cells was increased and the number of Ki-67-positive cells was decreased in ICER-transduced artery. These results suggest that ICER induces apoptosis and inhibits proliferation of VSMCs, and plays a critical role in beraprost-mediated suppression of VSMC proliferation. ICER may be an important endogenous inhibitor of vascular proliferation.

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.

Activation of Extracellular Signal-Regulated Kinase 5 Reduces Cardiac Apoptosis and Dysfunction via Inhibition of a Phosphodiesterase 3A/Inducible cAMP Early Repressor Feedback Loop

Substantial evidence suggests that the progressive loss of cardiomyocytes caused by apoptosis significantly contributes to the development of heart failure. beta-Adrenergic receptor activation and subsequent persistent phosphodiesterase 3A (PDE3A) downregulation and concomitant inducible cAMP early repressor (ICER) upregulation (PDE3A/ICER feedback loop) has been proposed to play a key role in the pathogenesis of cardiomyocyte apoptosis. In contrast, insulin-like growth factor-1 can activate cell survival pathways, providing protection against cell death and restoring muscle function. In this study, we found that insulin-like growth factor-1 activates extracellular signal-regulated kinase 5 (ERK5) and inhibits PDE3A/ICER feedback loop. Insulin-like growth factor-1 normalized isoproterenol-mediated PDE3A downregulation and ICER upregulation via ERK5/MEF2 activation, and also inhibited isoproterenol-induced myocyte apoptosis. To determine the physiological relevance of ERK5 activation in regulating PDE3A/ICER feedback loop, we investigated the PDE3A/ICER expression and cardiomyocyte apoptosis in transgenic mice with cardiac specific expression of a constitutively active form of mitogen-activated protein (MAP)/extracellular signal-regulated protein kinase (ERK) kinase 5alpha (MEK5alpha) (CA-MEK5alpha-Tg). In wild-type mice, pressure overload- or doxorubicin-induced significant reduction of PDE3A expression and subsequent ICER induction. Cardiac specific expression of CA-MEK5alpha rescued pressure overload- or doxorubicin-mediated PDE3A downregulation and ICER upregulation and inhibited myocyte apoptosis as well as subsequent cardiac dysfunction in vivo. These data suggest that preventing the feedback loop of PDE3A/ICER by ERK5 activation could inhibit progression of myocyte apoptosis as well as cardiac dysfunction. These data suggest a new therapeutic paradigm for end stage of heart failure by inhibiting the PDE3A/ICER feedback loop via activating ERK5.

Regulation of HIV-1 and IL-2 transcription by inducible CAMP early repressor (ICER)

HIV-1 infection of human monocytes induces production of prostaglandin E2 (PGE2) in vitro and in vivo, which has capacity to make the bystander T cells unresponsive (anergic). ICER, a potent transcriptional repressor induced by PGE2 leads to transcriptional attenuation of interleukin-2 (IL-2) and an induction of T cell suppression. We propose that this HIV-1 triggered mechanism may subvert physiologically relevant suppression of IL-2 by regulatory CD4+CD25+ T (Treg) cells. Our preliminary studies suggest that the suppressive mechanism conveyed by Treg cells stems from coexpression of ICER and Foxp3, which inhibits IL-2 expression. Moreover, naive CD4+CD25- responder T cells retrovirally transduced with Foxp3 can induce the accumulation of ICER and replace natural Treg cells. Importantly, ICER expression is induced in activated responder T cells early and correlates with a sharp decrease of IL-2 expression. Our preliminary studies indicate that ICER inhibits HIV-1 LTR expression and binds the cyclic responsive element (CRE)-like motifs termed DSE positioned downstream of transcription initiation site of HIV-1 LTR. Moreover, in monocytes PGE2 induced transcriptional attenuation of CCR5 expression tightly correlates with upregulation of ICER. We conclude that ICER is a critical component of Treg-mediated inhibitory function that affects transcriptional attenuation of IL-2 production in T cells and that HIV-1 subverts this mechanism via PGE2 and/or Tat-mediated induction of ICER.

Induction of Inducible cAMP Early Repressor Expression in Nucleus Accumbens by Stress or Amphetamine Increases Behavioral Responses to Emotional Stimuli

Previous research has shown that cAMP response element (CRE)-mediated transcription is activated in the nucleus accumbens, a major brain reward region, by a variety of environmental stimuli and contributes to neuroadaptations to these stimuli. CRE-binding protein (CREB) is the most studied activator of CRE transcription and has been implicated in this brain region as a gating mechanism for behavioral responses to emotional stimuli. Little attention, however, has been given to naturally occurring inhibitors of CRE-mediated transcription, such as the inducible cAMP early repressor (ICER), an inhibitory product of the CRE modulator gene. In the present study, we investigated the extent to which ICER is induced in the nucleus accumbens by two types of environmental stimuli, stress and amphetamine, and characterized how induction of ICER in this region affects complex behavior. We show that stress and amphetamine each induces ICER expression and that overexpression of ICER in the nucleus accumbens, using viral-mediated gene transfer, increases behavioral responses to both rewarding and aversive emotional stimuli. For example, ICER overexpression increases sensitivity to amphetamine-stimulated locomotor activity as well as to natural rewards such as sucrose and social grooming. However, ICER overexpression also increases measures of anxiety in the elevated plus maze and neophobia to novel tastes. Finally, ICER produces an antidepressant-like effect in the forced swim test, further indication of an enhanced active response to stress. These results suggest that ICER is an important mechanism for modulating CRE-mediated transcription in the nucleus accumbens.

Suppression of MIP-1β transcription in human T cells is regulated by inducible cAMP early repressor (ICER)

Local production of macrophage inflammatory protein-1beta (MIP-1beta), a beta-chemokine that blocks human immunodeficiency virus type 1 (HIV-1) entry into CD4+ CC chemokine receptor 5+ target cells, may be a significant factor in resistance to HIV-1 infection and control of local viral spread. The mechanisms governing MIP-1beta expression in T cells, however, are not well understood. Our results suggest that MIP-1beta RNA expression in T cells is dynamically regulated by transcriptional factors of the cyclic adenosine monophosphate (cAMP) responsive element (CRE)-binding (CREB)/modulator family. Transient transfection of primary human T cells with 5' deletion and site-specific mutants of the human MIP-1beta promoter identified an activated protein-1 (AP-1)/CRE-like motif at position -74 to -65 base pairs, relative to the TATA box as a vital cis-acting element and a binding site for inducible cAMP early repressor (ICER). Ectopic expression of ICER or induction of endogenous ICER with the cAMP agonists forskolin and prostaglandin E2 resulted in the formation of ICER-containing complexes, including an ICER:CREB heterodimer to the AP-1/CRE-like site and inhibition of MIP-1beta promoter activity. Our data characterize an important binding site for the dominant-negative regulator ICER in the MIP-1beta promoter and suggest that dynamic changes in the relative levels of ICER and CREB play a crucial role in cAMP-mediated attenuation of MIP-1beta transcription in human T cells.

A positive feedback loop of phosphodiesterase 3 (PDE3) and inducible cAMP early repressor (ICER) leads to cardiomyocyte apoptosis

cAMP plays crucial roles in cardiac remodeling and the progression of heart failure. Recently, we found that expression of cAMP hydrolyzing phosphodiesterase 3A (PDE3A) was significantly reduced in human failing hearts, accompanied by up-regulation of inducible cAMP early repressor (ICER) expression. Angiotensin II (Ang II) and the beta-adrenergic receptor agonist isoproterenol (ISO) also induced persistent PDE3A down-regulation and concomitant ICER up-regulation in vitro, which is important in Ang II- and ISO-induced cardiomyocyte apoptosis. We hypothesized that interactions between PDE3A and ICER may constitute an autoregulatory positive feedback loop (PDE3A-ICER feedback loop), and this loop would cause persistent PDE3A down-regulation and ICER up-regulation. Here, we demonstrate that ICER induction repressed PDE3A gene transcription. PDE3A down-regulation activated cAMP/PKA signaling, leading to ICER up-regulation via PKA-dependent stabilization of ICER. With respect to Ang II, the initiation of the PDE3A-ICER feedback loop depends on activation of Ang II type 1 receptor (AT1R), classical PKC(s), and CREB (cAMP response element binding protein). We further show that the PDE3A-ICER feedback loop is essential for Ang II-induced cardiomyocyte apoptosis. ISO and PDE3 inhibitors also induced the PDE3A-ICER feedback loop and subsequent cardiomyocyte apoptosis, highlighting the importance of this PDE3A-ICER feedback loop and cAMP signaling in cardiomyocyte apoptosis. Our findings may provide a therapeutic paradigm to prevent cardiomyocyte apoptosis and the progression of heart failure by inhibiting the PDE3A-ICER feedback loop.

Lithium‐induced gene expression of inducible cyclic adenosine monophosphate early repressor in the rat adrenal gland

Lithium has acute and chronic effects on the hypothalamic-pituitary-adrenal gland (HPA) axis that are important for both therapeutic (e.g., treatment of mood disorders) and experimental (e.g., as the toxin in conditioned taste aversion studies) applications. We visualized lithium-induced activation of the HPA axis in rats by the adrenal expression of inducible cAMP early repressor (ICER), which is activated by elevated intracellular cAMP. We have shown that 1) intraperitoneal lithium chloride (LiCl) induces transient expression of ICER and c-fos mRNAs in the rat adrenal cortex and increases plasma level of corticosterone; 2) the cortical expression of ICER mRNA by LiCl occurs in a dose-dependent manner; 3) adrenal induction of ICER expression is delayed compared with c-fos expression; 4) dexamethasone pretreatment (4 mg/kg) blocks corticosterone release and adrenocortical ICER induction either by systemic LiCl (76 mg/kg) or by restraint stress; and 5) intracerebroventricular LiCl (127 microg/5 microl) is sufficient for adrenocortical, but not medullary, ICER induction. These results suggest that adrenocortical ICER expression could serve as a reliable marker for lithium-induced activation of the HPA axis. Understanding the activation of immediate-early genes such as c-fos or ICER in response to a single LiCl injection is an important first step in understanding the long-term changes in gene expression elicited by lithium that are involved in its therapeutic and toxic effect. The pattern and mechanism by which lithium stimulates ICER transcription in the adrenal gland would serve as a useful model system in future studies of lithium.

Role of cyclic‐AMP responsive element binding (CREB) proteins in cell proliferation in a rat model of hepatocellular carcinoma

The role of cyclic adenosine monophosphate (cAMP) is poorly understood in the regulation of normal and abnormal hepatic cell growth. In this study, we examined the regulation of intracellular cAMP levels and its effect on nuclear cAMP responsive elements (CREs) in a rat model of hepatocellular carcinoma (HCC). Tumorigenic liver cells were cultured from an in vivo model of HCC and the role of cAMP in cell mitogenesis determined. These data demonstrated agents that elevate intracellular cAMP ([cAMP]i) levels caused significant dose-dependent inhibition of serum-stimulated mitogenesis in HCC cells. Cells were next analyzed for transcription factor expression and activity following increased [cAMP]i. These data demonstrated time- and dose-dependent increases in CRE binding protein (pCREB) activity, a maximal response occurring after 10-20 min before returning to basal levels within 60 min. In contrast, increased [cAMP]i levels led to sustained inducible cAMP early repressor (ICER) II/IIgamma mRNA and protein induction. To understand these data in relation to the in vivo setting, HCC tumors were analyzed and compared to pair-matched normal liver (NL) samples. These studies demonstrated significantly elevated Gsalpha-protein expression in HCC versus NL in the absence of significant changes in basal cAMP levels. Analysis of total and active CREB demonstrated significantly increased total CREB/pCREB in HCC versus NL. Further analysis of CRE expression demonstrated significantly increased expression of ICER mRNA and protein in HCC versus sham operated (Sh). These data demonstrate cAMP, while capable of stimulating promitogenic CREB activation inhibits cell mitogenesis in HCC possibly via ICER induction.

Functional Role of Phosphodiesterase 3 in Cardiomyocyte Apoptosis

Myocyte apoptosis plays an important role in pathological cardiac remodeling and the progression of heart failure. cAMP signaling is crucial in the regulation of myocyte apoptosis and cardiac remodeling. Multiple cAMP-hydrolyzing phosphodiesterases (PDEs), such as PDE3 and PDE4, coexist in cardiomyocytes and elicit differential temporal/spatial regulation of cAMP signaling. However, the role of PDE3 and PDE4 in the regulation of cardiomyocyte apoptosis remains unclear. Although chronic treatment with PDE3 inhibitors increases mortality in patients with heart failure, the contribution of PDE3 expression/activity in heart failure is not well known. In this study we report that PDE3A expression and activity were significantly reduced in human failing hearts as well as mouse hearts with chronic pressure overload. In primary cultured cardiomyocytes, chronic inhibition of PDE3 but not PDE4 activity by pharmacological agents or adenovirus-delivered antisense PDE3A promoted cardiomyocyte apoptosis. Both angiotensin II (Ang II) and the beta-adrenergic receptor agonist isoproterenol selectively induced a sustained downregulation of PDE3A expression and induced cardiomyocyte apoptosis. Restoring PDE3A via adenovirus-delivered expression of wild-type PDE3A1 completely blocked Ang II- and isoproterenol-induced cardiomyocyte apoptosis, suggesting the critical role of PDE3A reduction in cardiomyocyte apoptosis. Moreover, we defined a crucial role for inducible cAMP early repressor expression in PDE3A reduction-mediated cardiomyocyte apoptosis. Our results suggest that PDE3A reduction and consequent inducible cAMP early repressor induction are critical events in Ang II- and isoproterenol-induced cardiomyocyte apoptosis and may contribute to the development of heart failure. Drugs that maintain PDE3A function may represent an attractive therapeutic approach to treat heart failure.

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.

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.