CREB Function Research Articles

CREB Is Critically Implicated in Skin Mast Cell Degranulation Elicited via FcεRI and MRGPRX2.

Skin mast cells (MCs) mediate acute allergic reactions in the cutaneous environment and contribute to chronic dermatoses, including urticaria, and atopic or contact dermatitis. The cAMP response element binding protein (CREB), an evolutionarily well conserved transcription factor (TF) with over 4,000 binding sites in the genome, was recently found to form a feedforward loop with KIT, maintaining MC survival. The most selective MC function is degranulation with its acute release of prestored mediators. Herein, we asked whether CREB contributes to the expression and function of the degranulation-competent receptors FcεRI and MRGPRX2. Interference with CREB by pharmacological inhibition (CREBi, 666-15) or RNA interference only slightly affected the expression of these receptors, while KIT was strongly attenuated. Interestingly, MRGPRX2 surface expression moderately increased following CREB-knockdown, whereas MRGPRX2-dependent exocytosis simultaneously decreased. FcεRI expression and function were regulated consistently, although the effect was stronger at the functional level. Preformed MC mediators (tryptase, histamine, β-hexosaminidase) remained comparable following CREB attenuation, suggesting that granule synthesis did not rely on CREB function. Collectively, in contrast to KIT, FcεRI and MRGPRX2 moderately depend on unperturbed CREB function. Nevertheless, CREB is required to maintain MC releasability irrespective of stimulus, insinuating that CREB may operate by safeguarding the degranulation machinery. To our knowledge, CREB is the first factor identified to regulate MRGPRX2 expression and function in opposite direction. Overall, the ancient TF is an indispensable component of skin MCs, orchestrating not only survival and proliferation but also their secretory competence.

Inhibition of CREB Binding and Function with a Dual-Targeting Ligand.

CBP/p300 is a master transcriptional coactivator that regulates gene activation by interacting with multiple transcriptional activators. Dysregulation of protein-protein interactions (PPIs) between the CBP/p300 KIX domain and its activators is implicated in a number of cancers, including breast, leukemia, and colorectal cancer. However, KIX is typically considered "undruggable" because of its shallow binding surfaces lacking both significant topology and promiscuous binding profiles. We previously reported a dual-targeting peptide (MybLL-tide) that inhibits the KIX-Myb interaction with excellent specificity and potency. Here, we demonstrate a branched, second-generation analogue, CREBLL-tide, that inhibits the KIX-CREB PPI with higher potency and selectivity. Additionally, the best of these CREBLL-tide analogues shows excellent and selective antiproliferation activity in breast cancer cells. These results indicate that CREBLL-tide is an effective tool for assessing the role of KIX-activator interactions in breast cancer and expanding the dual-targeting strategy for inhibiting KIX and other coactivators that contain multiple binding surfaces.

CREB Is Activated by the SCF/KIT Axis in a Partially ERK-Dependent Manner and Orchestrates Survival and the Induction of Immediate Early Genes in Human Skin Mast Cells

cAMP response element binding protein (CREB) functions as a prototypical stimulus-inducible transcription factor (TF) that initiates multiple cellular changes in response to activation. Despite pronounced expression in mast cells (MCs), CREB function is surprisingly ill-defined in the lineage. Skin MCs (skMCs) are critical effector cells in acute allergic and pseudo-allergic settings, and they contribute to various chronic dermatoses such as urticaria, atopic dermatitis, allergic contact dermatitis, psoriasis, prurigo, rosacea and others. Using MCs of skin origin, we demonstrate herein that CREB is rapidly phosphorylated on serine-133 upon SCF-mediated KIT dimerization. Phosphorylation initiated by the SCF/KIT axis required intrinsic KIT kinase activity and partially depended on ERK1/2, but not on other kinases such as p38, JNK, PI3K or PKA. CREB was constitutively nuclear, where phosphorylation occurred. Interestingly, ERK did not translocate to the nucleus upon SCF activation of skMCs, but a fraction was present in the nucleus at baseline, and phosphorylation was prompted in the cytoplasm and nucleus in situ. CREB was required for SCF-facilitated survival, as demonstrated with the CREB-selective inhibitor 666-15. Knock-down of CREB by RNA interference duplicated CREB's anti-apoptotic function. On comparison with other modules (PI3K, p38 and MEK/ERK), CREB was equal or more potent at survival promotion. SCF efficiently induces immediate early genes (IEGs) in skMCs (FOS, JUNB and NR4A2). We now demonstrate that CREB is an essential partaker in this induction. Collectively, the ancient TF CREB is a crucial component of skMCs, where it operates as an effector of the SCF/KIT axis, orchestrating IEG induction and lifespan.

Coupling of binding and differential subdomain folding of the intrinsically disordered transcription factor CREB.

The cyclic AMP response element binding protein (CREB) contains a basic leucine zipper motif (bZIP) that forms a coiled coil structure upon dimerization and specific DNA binding. Although this state is well characterized, key features of CREB bZIP binding and folding are not well understood. We used single-molecule Förster resonance energy transfer (smFRET) to probe conformations of CREB bZIP subdomains. We found differential folding of the basic region and leucine zipper in response to different binding partners; a strong and previously unreported DNA-independent dimerization affinity; folding upon binding to nonspecific DNA; and evidence of long-range interdomain interactions in full-length CREB that modulate DNA binding. These studies provide new insights into DNA binding and dimerization and have implications for CREB function.

Distinct forms of regret linked to resilience versus susceptibility to stress are regulated by region-specific CREB function in mice.

Regret describes recognizing alternative actions could have led to better outcomes. It remains unclear whether regret derives from generalized mistake appraisal or instead comprises dissociable, action-specific processes. Using a neuroeconomic task, we found that mice were sensitive to fundamentally distinct types of regret following exposure to chronic social defeat stress or manipulations of CREB, a transcription factor implicated in stress action. Bias to make compensatory decisions after rejecting high-value offers (regret type I) was unique to stress-susceptible mice. Bias following the converse operation, accepting low-value offers (regret type II), was enhanced in stress-resilient mice and absent in stress-susceptible mice. CREB function in either the prefrontal cortex or nucleus accumbens was required to suppress regret type I but bidirectionally regulated regret type II. We provide insight into how maladaptive stress response traits relate to distinct forms of counterfactual thinking, which could steer therapy for mood disorders, such as depression, toward circuit-specific computations through a careful description of decision narrative.

Conserved age-related increases in hippocampal PDE11A4 cause unexpected proteinopathies and cognitive decline of social associative memories.

In humans, associative memories are more susceptible to age‐related cognitive decline (ARCD) than are recognition memories. Reduced cAMP/cGMP signaling in the hippocampus may contribute to ARCD. Here, we found that both aging and traumatic brain injury‐associated dementia increased the expression of the cAMP/cGMP‐degrading enzyme phosphodiesterase 11A (PDE11A) in the human hippocampus. Further, age‐related increases in hippocampal PDE11A4 mRNA and protein were conserved in mice, as was the increased vulnerability of associative versus recognition memories to ARCD. Interestingly, mouse PDE11A4 protein in the aged ventral hippocampus (VHIPP) ectopically accumulated in the membrane fraction and filamentous structures we term “ghost axons.” These age‐related increases in expression were driven by reduced exoribonuclease‐mediated degradation of PDE11A mRNA and increased PDE11A4‐pS117/pS124, the latter of which also drove the punctate accumulation of PDE11A4. In contrast, PDE11A4‐pS162 caused dispersal. Importantly, preventing age‐related increases in PDE11 expression via genetic deletion protected mice from ARCD of short‐term and remote long‐term associative memory (aLTM) in the social transmission of food preference assay, albeit at the expense of recent aLTM. Further, mimicking age‐related overexpression of PDE11A4 in CA1 of old KO mice caused aging‐like impairments in CREB function and remote social—but not non‐social—LTMs. RNA sequencing and phosphoproteomic analyses of VHIPP identified cGMP‐PKG—as opposed to cAMP‐PKA—as well as circadian entrainment, glutamatergic/cholinergic synapses, calcium signaling, oxytocin, and retrograde endocannabinoid signaling as mechanisms by which PDE11A deletion protects against ARCD. Together, these data suggest that PDE11A4 proteinopathies acutely impair signaling in the aged brain and contribute to ARCD of social memories.

Hypothalamic CREB Regulates the Expression of Pomc-Processing Enzyme Pcsk2

Background: The hypothalamic proopiomelanocortin (Pomc) neurons act as first-order sensors of systemic energy stores, providing signals that regulate caloric intake and energy expenditure. In experimental obesity, dietary saturated fatty acids affect Pomc endopeptidases (PCs), resulting in the abnormal production of the neurotransmitters α-melanocyte-stimulating hormone (α-MSH) and β-endorphin, thus impacting energy balance. The cAMP response element-binding protein (CREB) is one of the transcription factors that control the expression of Pomc endopeptidases; however, it was previously unknown if dietary fats could affect CREB and consequently the expression of Pomc endopeptidases. Methods: Here, we used single-cell RNA sequencing analysis, PCR, immunoblot, ELISA and immunofluorescence histological assays to determine the impact of a high-fat diet (HFD) on the expression and function of hypothalamic CREB and its impact on the melanocortinergic system. Results: The results indicate that CREB is expressed in arcuate nucleus Pomc neurons and is activated as early as nine hours after the introduction of a high-fat diet. The inhibition of hypothalamic CREB using a short-hairpin RNA lentiviral vector resulted in increased diet-induced body-mass gain and reduced energy expenditure. This was accompanied by reduced expression of the Pomc endopeptidases, protein convertase 2, which are encoded by Pcsk2, and by the loss of the high-fat-diet-induced effect to inhibit the production of α-MSH. Conclusions: This study provides the first evidence for the involvement of CREB in the abnormal regulation of the hypothalamic Pomc endopeptidase system in experimental obesity.

Purkinje cell-specific deletion of CREB worsens alcohol-induced cerebellar neuronal losses and motor deficits.

Exposure to alcohol during pregnancy can kill developing fetal neurons and lead to fetal alcohol spectrum disorder (FASD) in the offspring. However, not all fetuses are equally vulnerable to alcohol toxicity. These differences in vulnerability among individuals are likely due, at least in part, to genetic differences. Some genes encode neuroprotective molecules that act through signaling pathways to protect neurons against alcohol's toxic effects. One signaling pathway that can protect cultured neurons against alcohol-induced cell death invitro is the cAMP pathway. A goal of this study was to determine whether the cAMP pathway can exert a similar neuroprotective effect against alcohol invivo. A key molecule within the cAMP pathway is cAMP response element binding protein (CREB). In this study, CREB was specifically disrupted in cerebellar Purkinje cells to study its role in protection of cerebellar neurons against alcohol toxicity. Mice with Purkinje cell-specific knockout of CREB were generated with the Cre-lox system. A 2×2 design was used in which Cre-negative and Cre-positive mice received either 0.0 or 2.2mg/g ethanol by intraperitoneal (i.p.) injection daily over postnatal day (PD) 4-9. Stereological cell counts of cerebellar Purkinje cells and granule cells were performed on PD 10. Motor function was assessed on PD 40 using the rotarod. Purkinje cell-specific disruption of CREB alone (in the absence of alcohol) induced only a small reduction in Purkinje cell number. However, the loss of CREB function from Purkinje cells greatly increased the vulnerability of Purkinje cells to alcohol-induced cell death. While alcohol killed 20% of Purkinje cells in the Cre-negative (CREB-expressing) mice, alcohol killed 57% of Purkinje cells in the Cre-positive (CREB-nonexpressing) mice. This large loss of Purkinje cells did not lead to similar alcohol-induced losses of granule cells. In the absence of alcohol, lack of CREB function in Purkinje cells had no effect on rotarod performance. However, in the presence of alcohol, disruption of CREB in Purkinje cells substantially worsened rotarod performance. Disruption of a single gene (CREB) in a single neuronal population (Purkinje cells) greatly increases the vulnerability of that cell population to alcohol-induced cell death and worsens alcohol-induced brain dysfunction. The results suggest that the cAMP pathway can protect cells invivo against alcohol toxicity and underline the importance of genetics in determining the neuropathology and behavioral deficits of FASD.

CREB Inactivation by HDAC1/PP1γ Contributes to Dopaminergic Neurodegeneration in Parkinson's Disease.

Understanding the pathogenesis of nigral dopaminergic neurodegeneration is critical for developing mechanism-based treatments for Parkinson's disease (PD). In the nigral dopaminergic neurons of postmortem human PD brains, we found that CREB, a well-recognized pro-survival transcription factor in neurons, was inactivated by dephosphorylation at Ser133. CREB dephosphorylation correlated with decreased expression of NURR1, one of its target genes crucial for dopaminergic neuron survival, confirming that CREB function was impaired in nigral dopaminergic neurons in PD. An MPTP mouse model was used to further elucidate the mechanism underlying CREB dephosphorylation. Protein phosphatase 1γ (PP1γ), which dephosphorylates CREB, was constitutively associated with histone deacetylase 1 (HDAC1). HDAC1 promotes CREB Ser133 dephosphorylation via a stable interaction with PP1γ. We found that CREB interacted with the HDAC1/PP1γ complex during dopaminergic neurodegeneration. Importantly, increased CREB/HDAC1 interaction occurred in the nigral dopaminergic neurons of PD patients as demonstrated using a proximity ligation assay. Disrupting CREB/HDAC1 interaction via either overexpression of GAL4 M1, a CREB mutant, or administration of trichostatin A, a pan-HDAC inhibitor, restored the expression levels of phospho-CREB (Ser133) and NURR1, and protected nigral dopaminergic neurons in the MPTP-treated mouse brain. Collectively, our results demonstrated that HDAC1/PP1γ-mediated CREB inactivation contributed to dopaminergic neuronal degeneration. Disruption of CREB/HDAC1 interaction has the potential to be a new approach for PD therapy.SIGNIFICANCE STATEMENT Parkinson's disease (PD) is the most common movement disorder attributed to the progressive loss of dopaminergic neurons in the substantia nigra. Understanding the pathogenesis of nigral dopaminergic neurodegeneration is critical for developing mechanism-based treatments for PD. We found in nigral dopaminergic neurons of postmortem human PD brains that CREB, a well-recognized pro-survival transcription factor in neurons, was inactivated by dephosphorylation at Ser133. HDAC1, constitutively associated with PP1γ, interacted with CREB to mediate its dephosphorylation during dopaminergic neurodegeneration. Disrupting CREB/HDAC1 interaction restored CREB activity and protected nigral dopaminergic neurons in the MPTP mouse brains. This work suggests that disruption of the CREB/HDAC1 interaction to restore CREB activity may be a potential therapeutic approach in PD.

PP-1β and PP-2Aα modulate cAMP response element-binding protein (CREB) functions in aging control and stress response through de-regulation of αB-crystallin gene and p300-p53 signaling axis.

The function of the transcription factor, cAMP response element‐binding protein (CREB), is activated through S133 phosphorylation by PKA and others. Regarding its inactivation, it is not well defined. cAMP response element‐binding protein plays an essential role in promoting cell proliferation, neuronal survival and the synaptic plasticity associated with long‐term memory. Our recent studies have shown that CREB is an important player in mediating stress response. Here, we have demonstrated that CREB regulates aging process through suppression of αB‐crystallin and activation of the p300‐p53‐Bak/Bax signaling axis. First, we determined that two specific protein phosphatases, PP‐1β and PP‐2Aα, can inactivate CREB through S133 dephosphorylation. Subsequently, we demonstrated that cells expressing the S133A‐CREB, a mutant mimicking constant dephosphorylation at S133, suppress CREB functions in aging control and stress response. Mechanistically, S133A‐CREB not only significantly suppresses CREB control of αB‐crystallin gene, but also represses CREB‐mediated activation of p53 acetylation and downstream Bak/Bax genes. cAMP response element‐binding protein suppression of αB‐crystallin and its activation of p53 acetylation are major molecular events observed in human cataractous lenses of different age groups. Together, our results demonstrate that PP‐1β and PP‐2Aα modulate CREB functions in aging control and stress response through de‐regulation of αB‐crystallin gene and p300‐p53‐Bax/Bak signaling axis, which regulates human cataractogenesis in the aging lens.

Roflumilast Ameliorates Isoflurane-Induced Inflammation in Astrocytes via the CREB/BDNF Signaling Pathway.

Background and purpose: Astrocyte-mediated neuroinflammation plays an important role in anesthetic isoflurane-induced cognitive impairment. Roflumilast, a selective inhibitor of phosphodiesterase-4 (PDE-4) used for the treatment of chronic obstructive pulmonary disease (COPD), has displayed a wide range of anti-inflammatory capacity in different types of cells and tissues. In the current study, we aimed to investigate whether roflumilast possesses a protective effect against isoflurane-induced insults in mouse primary astrocytes. Methods: Primary astrocytes were isolated from the cerebral cortices of immature rats. The production of NO was determined using DAF-FM DA staining assay. QRT-PCR and western blot were used to evaluate the expression levels of iNOS, COX-2, and BDNF in the astrocytes treated with different therapies. The gene expressions and concentrations of IL-6 and MCP-1 released by the astrocytes were detected using qRT-PCR and ELISA, respectively. The expression levels of phosphorylated CREB and PGE2 were determined using western blot and ELISA, respectively. H89 was introduced to evaluate the function of CREB. Recombinant human BDNF and ANA-12 were used to verify the role of BDNF. Results: The upregulated iNOS, excessive production of NO, IL-6, and MCP-1, and activated COX-2/PGE2 signaling pathways in the astrocytes induced by isoflurane were significantly reversed by the introduction of roflumilast, in a dose-dependent manner. Subsequently, we found that BDNF could be upregulated by roflumilast, which was verified to be related to the activation of CREB and blocked by H89 (a CREB inhibitor). In addition, the COX-2/PGE2 signaling pathway activated by isoflurane can be inactivated by recombinant human BDNF. Finally, the regulatory effect of roflumilast against the isoflurane-activated COX-2/PGE2 signaling pathway was significantly blocked by ANA-12, which is a BDNF inhibitor. Conclusion: Roflumilast might ameliorate isoflurane-induced inflammation in astrocytes via the CREB/BDNF signaling pathway.

Osmolytes dynamically regulate mutant Huntingtin aggregation and CREB function in Huntington\u2019s disease cell models

Osmolytes are organic solutes that change the protein folding landscape shifting the equilibrium towards the folded state. Herein, we use osmolytes to probe the structuring and aggregation of the intrinsically disordered mutant Huntingtin (mHtt) vis-a-vis the pathogenicity of mHtt on transcription factor function and cell survival. Using an inducible PC12 cell model of Huntington’s disease (HD), we show that stabilizing polyol osmolytes drive the aggregation of Htt103QExon1-EGFP from a diffuse ensemble into inclusion bodies (IBs), whereas the destabilizing osmolyte urea does not. This effect of stabilizing osmolytes is innate, generic, countered by urea, and unaffected by HSP70 and HSC70 knockdown. A qualitatively similar result of osmolyte-induced mHtt IB formation is observed in a conditionally immortalized striatal neuron model of HD, and IB formation correlates with improved survival under stress. Increased expression of diffuse mHtt sequesters the CREB transcription factor to repress CREB-reporter gene activity. This repression is mitigated either by stabilizing osmolytes, which deplete diffuse mHtt or by urea, which negates protein–protein interaction. Our results show that stabilizing polyol osmolytes promote mHtt aggregation, alleviate CREB dysfunction, and promote survival under stress to support the hypothesis that lower molecular weight entities of disease protein are relevant pathogenic species in neurodegeneration.

A time-dependent role for the transcription factor CREB in neuronal allocation to an engram underlying a fear memory revealed using a novel in vivo optogenetic tool to modulate CREB function.

The internal representation of an experience is thought to be encoded by long-lasting physical changes to the brain ("engrams") . Previously, we and others showed within the lateral amygdala (LA), a region critical for auditory conditioned fear, eligible neurons compete against one other for allocation to an engram. Neurons with relatively higher function of the transcription factor CREB were more likely to be allocated to the engram. In these studies, though, CREB function was artificially increased for several days before training. Precisely when increased CREB function is important for allocation remains an unanswered question. Here, we took advantage of a novel optogenetic tool (opto-DN-CREB) to gain spatial and temporal control of CREB function in freely behaving mice. We found increasing CREB function in a small, random population of LA principal neurons in the minutes, but not 24 h, before training was sufficient to enhance memory, likely because these neurons were preferentially allocated to the underlying engram. However, similarly increasing CREB activity in a small population of random LA neurons immediately after training disrupted subsequent memory retrieval, likely by disrupting the precise spatial and temporal patterns of offline post-training neuronal activity and/or function required for consolidation. These findings reveal the importance of the timing of CREB activity in regulating allocation and subsequent memory retrieval, and further, highlight the potential of optogenetic approaches to control protein function with temporal specificity in behaving animals.

Memory strength gates the involvement of a CREB-dependent cortical fear engram in remote memory

Encoding and retrieval of contextual memories is initially mediated by sparsely activated neurons, so-called engram cells, in the hippocampus. Subsequent memory persistence is thought to depend on network-wide changes involving progressive contribution of cortical regions, a process referred to as systems consolidation. Using a viral-based TRAP (targeted recombination in activated populations) approach, we studied whether consolidation of contextual fear memory by neurons in the medial prefrontal cortex (mPFC) is modulated by memory strength and CREB function. We demonstrate that activity of a small subset of mPFC neurons is sufficient and necessary for remote memory expression, but their involvement depends on the strength of conditioning. Furthermore, selective disruption of CREB function in mPFC engram cells after mild conditioning impairs remote memory expression. Together, our data demonstrate that memory consolidation by mPFC engram cells requires CREB-mediated transcription, with the functionality of this network hub being gated by memory strength.

Engram-specific transcriptome profiling of contextual memory consolidation

Sparse populations of neurons in the dentate gyrus (DG) of the hippocampus are causally implicated in the encoding of contextual fear memories. However, engram-specific molecular mechanisms underlying memory consolidation remain largely unknown. Here we perform unbiased RNA sequencing of DG engram neurons 24 h after contextual fear conditioning to identify transcriptome changes specific to memory consolidation. DG engram neurons exhibit a highly distinct pattern of gene expression, in which CREB-dependent transcription features prominently (P = 6.2 × 10−13), including Atf3 (P = 2.4 × 10−41), Penk (P = 1.3 × 10−15), and Kcnq3 (P = 3.1 × 10−12). Moreover, we validate the functional relevance of the RNAseq findings by establishing the causal requirement of intact CREB function specifically within the DG engram during memory consolidation, and identify a novel group of CREB target genes involved in the encoding of long-term memory.

COGNITIVE IMPAIRMENT IS A PREDICTOR OF ONE-YEAR MORTALITY IN PATIENTS REFERRED FOR CARDIAC CATHETERIZATION

Cognitive dysfunction and memory loss are common features of Alzheimer9s disease (AD). Abnormalities in the expression profile of immediate early genes that play a critical role in memory formation, such as the cAMP-response element binding protein (CREB), have been reported in the brains of AD patients. Here we show that amyloid-β (Aβ) accumulation, which plays a primary role in the cognitive deficits of AD, interferes with CREB activity. We further show that restoring CREB function via brain viral delivery of the CREB-binding protein (CBP) improves learning and memory deficits in an animal model of AD. Notably, such improvements occur without changes in Aβ and tau pathology, and instead are linked to an increased level of brain-derived neurotrophic factor. The resulting data suggest that Aβ-induced learning and memory deficits are mediated by alterations in CREB function, based on the finding that restoring CREB activity by directly modulating CBP levels in the brains of adult mice is sufficient to ameliorate learning and memory. Therefore, increasing CBP expression in adult brains may be a valid therapeutic approach not only for AD, but also for various brain disorders characterized by alterations in immediate early genes, further supporting the concept that viral vector delivery may be a viable therapeutic approach in neurodegenerative diseases.

CAMP/Protein Kinase A Signaling Inhibits Dlx5 Expression via Activation of CREB and Subsequent C/EBPβ Induction in 3T3-L1 Preadipocytes.

Distal-less homeobox 5 (Dlx5) is a negative regulator of adipogenesis. Dlx5 expression is decreased by adipogenic stimuli, but the mechanisms of Dlx5 downregulation by adipogenic stimuli have not yet been determined. Here, we tested the impact of cAMP/PKA (protein kinase A) signaling induced by 3-isobutyl-1 methyl xanthine (IBMX), forskolin, and 8-CPT-cAMP on the expression of Dlx5 in 3T3-L1 preadipocytes. Significant downregulation of Dlx5 mRNA expression and protein production levels were observed via cAMP/PKA-dependent signaling. Forced expression of cAMP-responsive element-binding protein (CREB) and CCAAT/enhancer-binding protein β (C/EBPβ) was sufficient for downregulation of Dlx5 expression and revealed that CREB functions upstream of C/EBPβ. In addition, C/EBPβ knockdown by siRNA rescued Dlx5 expression in IBMX-treated 3T3-L1 preadipocytes. Luciferase assays using a Dlx5-luc-2935 reporter construct demonstrated the requirement of the Dlx5 promoter region, ranging from −774 to −95 bp that contains two putative C/EBPβ binding elements (site-1: −517 to −510 bp and site-2: −164 to −157 bp), in the suppression of Dlx5 transcription. Consequently, chromatin immunoprecipitation analysis confirmed the importance of site-1, but not site-2, in C/EBPβ binding and transcriptional suppression of Dlx5. In conclusion, we elucidated the underling mechanism of Dlx5 downregulation in IBMX-induced adipogenesis. IBMX activated cAMP/PKA/CREB signaling and subsequently upregulated C/EBPβ, which binds to the Dlx5 promoter to suppress Dlx5 transcription.

CAMP Response Element-Binding Protein (CREB): A Possible Signaling Molecule Link in the Pathophysiology of Schizophrenia.

Dopamine is a brain neurotransmitter involved in the pathology of schizophrenia. The dopamine hypothesis states that, in schizophrenia, dopaminergic signal transduction is hyperactive. The cAMP-response element binding protein (CREB) is an intracellular protein that regulates the expression of genes that are important in dopaminergic neurons. Dopamine affects the phosphorylation of CREB via G protein-coupled receptors. Neurotrophins, such as brain derived growth factor (BDNF), are critical regulators during neurodevelopment and synaptic plasticity. The CREB is one of the major regulators of neurotrophin responses since phosphorylated CREB binds to a specific sequence in the promoter of BDNF and regulates its transcription. Moreover, susceptibility genes associated with schizophrenia also target and stimulate the activity of CREB. Abnormalities of CREB expression is observed in the brain of individuals suffering from schizophrenia, and two variants (-933T to C and -413G to A) were found only in schizophrenic patients. The CREB was also involved in the therapy of animal models of schizophrenia. Collectively, these findings suggest a link between CREB and the pathophysiology of schizophrenia. This review provides an overview of CREB structure, expression, and biological functions in the brain and its interaction with dopamine signaling, neurotrophins, and susceptibility genes for schizophrenia. Animal models in which CREB function is modulated, by either overexpression of the protein or knocked down through gene deletion/mutation, implicating CREB in schizophrenia and antipsychotic drugs efficacy are also discussed. Targeting research and drug development on CREB could potentially accelerate the development of novel medications against schizophrenia.

Abstract 692: Development of the Gene Therapy With CRE Decoy ODN to Prevent Vascular Intimal Hyperplasia

Background: Intimal hyperplasia (IH) is the main cause of vein graft stenosis or failure after bypass surgery. However, in the previous study, no therapeutic targets for the treatment of IH have been identified. Our recent research have been reported that the inhibition of Cyclic adenosine monophosphate response-element (CRE) binding protein (CREB) activation is a key role for vein graft IH. Objective: In consideration of future clinical application, we focused on decoy oligodeoxynucleotide (ODN) transfection as gene therapy strategy for suppressing IH. The goal of the present study is to identify whether the CRE decoy ODN had the therapeutic efficacy for suppressing IH. Methods: We developed phosphorothioate CRE decoy ODN and checked binding ability to a CRE sequence using CREB transcription assay . We chose a decoy ODN having high first-binding ability and transfected it to vascular smooth muscle cell(VSMC) in vitro. Proliferation was assessed using MTS assay and migration was assessed using a modified Boyden-chamber assay. We examined CRE activity using Luciferase reporter gene assay. We checked expression of mRNAs using qRT-PCR. In wire-injury mouse model(C57BL6,n=6), CRE decoy ODN was transfected to injured vessel wall using ultrasound-sonoporation method in vivo. Results: CREB transcription factor assay showed CRE decoy had about 80 folds binding ability of control decoy in IC50 value (vs.control,n=5). Transfer of CRE decoy ODN resulted in significantly downregulated CRE activity(vs.control,P<.01,n=12) and inhibited VSMC proliferation (vs.control,P<.01,n=12) and migration (vs.control,P<.01,n=5). In a wire-injury mouse model, histopathological analysis revealed that transfer of the CRE decoy ODN significantly repressed IH whereas intensive IH was observed in the control group. In this vessel tissue, Levels of mRNA CCNA2 and Bcl2 were significantly lower in CRE decoy group than in control group(n=6,P<.01,respectively). CRE-activated gene expression was strongly repressed. Conclusions: The present result suggested that CRE decoy ODN inhibiting CREB function provide an effective therapeutic approach to suppressing IH.

Targeting the overexpressed CREB inhibits esophageal squamous cell carcinoma cell growth.

Although several studies highlight the important role of cAMP-responsive element binding protein(CREB) in tumor progression, little is known concerning the expression and function of CREB in esophageal cancer. In the present study, the expression of CREB was evaluated using a human esophageal squamous cell carcinoma tissue array by immunohistochemical analysis, which was confirmed by western blot analysis of tissues from esophageal cancer, and adjacent esophageal tissue. The role of CREB on esophageal cancer cell growth was analyzed invitro and invivo. Results showed that CREB was overexpressed in esophageal squamous cell carcinomas tissues, which was positively correlated with lymph node metastasis and tumor-node-metastasis(TNM) stage of esophageal cancer patients. Downregulating the expression of CREB effectively reduced esophageal cell growth invitro and invivo, induced Sphase cell cycle arrest, triggered apoptosis and inhibited cell migration and invasion. These findings suggested CREB as an attractive drug target for esophageal cancer.