Apigenin reverses behavioural impairments and cognitive decline in kindled mice via CREB-BDNF upregulation in the hippocampus.

Objectives: Apigenin is the most common bioflavonoid known to be biologically active after systemic administration and show multiple pharmacological effects. The present study was designed to explore the role of apigenin in pentylenetetrazole (PTZ) kindling associated cognitive and behavioural impairments in the mouse model. Methods: The animals were kindled by injecting a sub-convulsive dose of PTZ (35 mg/kg) on every alternate day, followed by 20 days treatment with apigenin at two different doses (10 and 20 mg/kg). Seizure severity was assessed on every 5th, 10th, 15th, and 20th day during apigenin treatment after a PTZ injection, followed by analysis of cognitive and behavioural functions. Results: Apigenin treatment displayed insignificant effect on seizure severity in kindled mice at both the tested doses, in comparison to control. However, the treatment showed marked increase in per cent spontaneous alterations and decline in the anxiety index in T-maze and elevated plus maze tests, respectively. Apigenin-treated groups showed significant decrease in immobility period in both forced swim and tail suspension tests, without any change in the total locomotor activity in the open field test. Furthermore, increase in the hippocampus protein expression of brain-derived neurotrophic factor (BDNF), cAMP response element binding protein (CREB), and phosphorylated CREB, with increased serotonin level were also observed in the treated animals. Discussion: The results of the present study showed that apigenin treatment prevents cognitive deficit and reverses behaviour impairments, without altering seizures severity in kindled mice. The observed effects can be attributed to CREB-BDNF upregulation in the hippocampus.

Enhanced bioluminescent sensor for longitudinal detection of CREB activation in living cells.

Cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) is associated with memory formation and controls cell survival and proliferation via regulation of downstream gene expression in tumorigenesis. As a transcription factor, CREB binds to cAMP response elements. Phosphorylation of CREB triggers transcriptional activation of CREB downstream genes following the interaction of the kinase-inducible domain (KID) of CREB with the KID interaction domain (KIX) of CREB-binding protein. Nevertheless, because of the lack of single-cell analytical techniques, little is known about spatiotemporal regulation of CREB phosphorylation. To analyze CREB activation in single living cells, we developed genetically encoded bioluminescent sensors using luciferase-fragment complementation: the sensors are designed based on KID-KIX interaction with a single-molecule format. The luminescence intensity of the sensor, designated as CREX (a sensor of CREB activation based on KID(CREB)-KIX interaction), increased by phosphorylation of CREB. Moreover, the luminescence intensity of CREX was sufficient to detect CREB activation in live-cell bioluminescence imaging for single-cell analysis because of the higher sensitivity. CREX sensor is expected to contribute to elucidation of the spatiotemporal regulation of CREB phosphorylation by applying single-cell analysis.

Challenges in the Structural-Functional Characterization of Multidomain, Partially Disordered Proteins CBP and p300: Preparing Native Proteins and Developing Nanobody Tools.

The structural and functional characterization of large multidomain signaling proteins containing long disordered linker regions represents special methodological and conceptual challenges. These proteins show extreme structural heterogeneity and have complex posttranslational modification patterns, due to which traditional structural biology techniques provide results that are often difficult to interpret. As demonstrated through the example of two such multidomain proteins, CREB-binding protein (CBP) and its paralogue, p300, even the expression and purification of such proteins are compromised by their extreme proteolytic sensitivity and structural heterogeneity. In this chapter, we describe the effective expression of CBP and p300 in a eukaryotic host, Sf9 insect cells, followed by their tandem affinity purification based on two terminal tags to ensure their structural integrity. The major focus of this chapter is on the development of novel accessory tools, single-domain camelid antibodies (nanobodies), for structural-functional characterization. Specific nanobodies against full-length CBP and p300 can specifically target their different regions and can be used for their marking, labeling, and structural stabilization in a broad range of in vitro and in vivo studies. Here, we describe four high-affinity nanobodies binding to the KIX and the HAT domains, either mimicking known interacting partners or revealing new functionally relevant conformations. As immunization of llamas results in nanobody libraries with a great sequence variation, deep sequencing and interaction analysis with different regions of the proteins provide a novel approach toward developing a panel of specific nanobodies.

Modulating the masters: chemical tools to dissect CBP and p300 function.

Dysregulation of transcription is found in nearly every human disease, and as a result there has been intense interest in developing new therapeutics that target regulators of transcription. CREB binding protein (CBP) and its paralogue p300 are attractive targets due to their function as `master coactivators'. Although inhibitors of several CBP/p300 domains have been identified, the selectivity of many of these compounds has remained underexplored. Here, we review recent successes in the development of chemical tools targeting several CBP/p300 domains with selectivity acceptable for use as chemical probes. Additionally, we highlight recent studies which have used these probes to expand our understanding of interdomain interactions and differential coactivator usage.

GATA3 acetylation at K119 by CBP inhibits cell migration and invasion in lung adenocarcinoma.

GATA3 is a transcriptional factor involved in the development of multiple organs. Post translational modifications of GATA3 are critical to its function. Here, we report that GATA3 interacts with and is acetylated by the acetyltransferase CBP. Class I deacetylases HDAC1, HDAC2 and HDAC3 deacetylate GATA3. The major acetylated site of GATA3 in lung adenocarcinoma cells was determined at lysine 119 (AcK119). Functionally, GATA3-acetylation mimics K119Q mutant was found to inhibit lung adenocarcinoma cell migration and invasion with concomitant downregulation of EMT-controlling transcriptional factors Slug, Zeb1 and Zeb2. Taken together, we demonstrated that GATA3 acetylation at lysine 119 by CBP hinders the migration and invasion of lung adenocarcinoma cells.

Regulation of cyclic adenosine monophosphate response element binding protein on renin expression in kidney via complex cyclic adenosine monophosphate response element-binding-protein-binding protein/P300 recruitment.

INTRODUCTION: Renin synthesis and release is the rate-limiting step in the renin-angiotensin system, because cyclic adenosine monophosphate (cAMP) has been identified as dominant pathway for renin gene expression, and cAMP response element-binding protein (CREB) is found in the human and mouse renin promoter. This study aimed to evaluate the role of CREB in expression of the renin gene. MATERIALS AND METHODS: We created conditional deletion of CREB in mice with low-sodium diet, specifically in renin cells of the kidney. To assess the effect of CREB on renin expression, immunostaining of renin was used in samples from wild-type mice and mice with gene knock-down of CREB. Cyclic AMP response element-binding-protein-binding protein (CBP) and p300 were measured in cultured renin cells of the mice, and RNA detection was done with real-time polymerase chain reaction. RESULTS: With low-sodium diet, renin was expressed along the whole wall of the afferent glomerular arterioles in wild-type mice, while there was no increase or even decrease in renin expression in CREB-specific deletion mice; RNA level of renin in cultured cells decreased by 50% with single knock-down of CREB, CBP, or p300, and decreased 70% with triple knock-down of CREB, CBP, and p300. CONCLUSIONS: This study found that CREB was important for renin synthesis and the role of CREB can be achieved through the recruitment of co-activators CBP and p300.

Suppressing cAMP response element-binding protein transcription shortens the duration of status epilepticus and decreases the number of spontaneous seizures in the pilocarpine model of epilepsy.

OBJECTIVE: Current epilepsy therapies directed at altering the function of neurotransmitter receptors or ion channels, or release of synaptic vesicles fail to prevent seizures in approximately 30% of patients. A better understanding of the molecular mechanism underlying epilepsy is needed to provide new therapeutic targets. The activity of cyclic AMP (cAMP) response element-binding protein (CREB), a major transcription factor promoting CRE-mediated transcription, increases following a prolonged seizure called status epilepticus. It is also increased in the seizure focus of patients with medically intractable focal epilepsy. Herein we explored the effect of acute suppression of CREB activity on status epilepticus and spontaneous seizures in a chronic epilepsy model. METHODS: Pilocarpine chemoconvulsant was used to induce status epilepticus. To suppress CREB activity, a transgenic mouse line expressing an inducible dominant negative mutant of CREB (CREB(IR) ) with a serine to alanine 133 substitution was used. Status epilepticus and spontaneous seizures of transgenic and wild-type mice were analyzed using video-electroencephalography (EEG) to assess the effect of CREB suppression on seizures. RESULTS: Our findings indicate that activation of CREB(IR) shortens the duration of status epilepticus. The frequency of spontaneous seizures decreased in mice with chronic epilepsy during CREB(IR) induction; however, the duration of the spontaneous seizures was unchanged. Of interest, we found significantly reduced levels of phospho-CREB Ser133 upon activation of CREB(IR) , supporting prior work suggesting that binding to the CRE site is important for CREB phosphorylation. SIGNIFICANCE: Our results suggest that CRE transcription supports seizure activity both during status epilepticus and in spontaneous seizures. Thus, blocking of CRE transcription is a novel target for the treatment of epilepsy.

Expression of p300 and CBP is associated with poor prognosis in small cell lung cancer.

PURPOSE: To investigate the correlation among p300, CBP and MLL expression and the clinicopathological characteristics in resected SCLC patients. METHODS: Two hundred and twenty-two resected SCLC patients were included in this study. We evaluated p300, CBP and MLL expression by immunohistochemistry. RESULTS: Patients with high p300 expression had shorter OS and DFS than those with low p300 expression (p = 0.01; p = 0.009, respectively). The patients with CBP-positive tumors had significantly lower OS and DFS than those with CBP-negative tumors (p = 0.005 and p = 0.007, respectively). Moreover, the p300- and CBP-positive (+) group had a significantly poor OS and DFS. The multivariate Cox regression analysis showed that high p300 and CBP expression are independent markers of poor overall survival (p = 0.006; p = 0.017, respectively) in operable SCLC patients. CONCLUSIONS: High p300 and CBP expression are independent prognostic markers of poor overall survival for resected SCLC patients. The combination of p300 and CBP expression may be useful in identifying patients with increased risks of cancer recurrence of SCLC.

Magnolol protects osteoblastic MC3T3-E1 cells against antimycin A-induced cytotoxicity through activation of mitochondrial function.

Antimycin A treatment of cells blocks the mitochondrial electron transport chain and leads to elevated ROS generation. In the present study, we investigated the protective effects of magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, on antimycin A-induced toxicity in osteoblastic MC3T3-E1 cells. Osteoblastic MC3T3-E1 cells were pre-incubated with magnolol before treatment with antimycin A. Cell viability and mineralization of osteoblasts were assessed by MTT assay and Alizarin Red staining, respectively. Mitochondrial dysfunction in cells was measured by mitochondrial membrane potential (MMP), complex IV activity, and ATP level. The cellular antioxidant effect of magnolol in osteoblastic MC3T3-E1 cells was assessed by measuring cardiolipin oxidation, mitochondrial superoxide levels, and nitrotyrosine content. Phosphorylated cAMP-response element-binding protein (CREB ) was evaluated using ELISA assay. Pretreatment with magnolol prior to antimycin A exposure significantly reduced antimycin A-induced osteoblast dysfunction by preventing MMP dissipation, ATP loss, and CREB inactivation. Magnolol also reduced cardiolipin peroxidation, mitochondrial superoxide, and nitrotyrosine production induced by antimycin A. These results suggest that magnolol has a protective effect against antimycin A-induced cell damage by its antioxidant effects and the attenuation of mitochondrial dysfunction. All these data indicate that magnolol may reduce or prevent osteoblast degeneration in osteoporosis or other degenerative disorders.

The nucleosome map of the mammalian liver.

Binding to nucleosomal DNA is critical for 'pioneer' transcription factors such as the winged-helix transcription factors Foxa1 and Foxa2 to regulate chromatin structure and gene activation. Here we report the genome-wide map of nucleosome positions in the mouse liver, with emphasis on transcriptional start sites, CpG islands, Foxa2 binding sites and their correlation with gene expression. Despite the heterogeneity of liver tissue, we could clearly discern the nucleosome pattern of the predominant liver cell, the hepatocyte. By analyzing nucleosome occupancy and the distributions of heterochromatin protein 1 (Hp1), CBP (also known as Crebbp) and p300 (Ep300) in Foxa1- and Foxa2-deficient livers, we find that the maintenance of nucleosome position and chromatin structure surrounding Foxa2 binding sites is independent of Foxa1 and Foxa2.

Clinicopathological significance of cell cycle regulatory factors and differentiation-related factors in pancreatic neoplasms.

OBJECTIVES: The aim of the present study was to compare the expression levels of the cyclins and the differentiation-related factors in pancreatic neoplasms. METHODS: The expression levels of cyclins A and B1, E1A-like inhibitor of differentiation 1 (EID-1), p300, 3'-5'-cyclic sdenosine monophosphate response element binding protein (CREB) binding protein (CBP), and acetylated histone H3 (AcH3) in ordinary ductal carcinoma (ODC) and intraductal papillary mucinous neoplasms (IPMNs) of the pancreas were investigated. RESULTS: More cells positive for cyclin A and EID-1 were present in the ODC than in the IPMNs. Cells positive for both cyclins and EID-1 were observed more frequently in invasive carcinoma derived from the IPMN than from the IP mucinous carcinoma. Multivariate regression analysis revealed that EID-1 and cyclin A overexpressions were independent factors associated with poor prognosis. Overall survival was significantly lower in ODC patients with overexpressions of cyclin A, EID-1, and AcH3 than in those without such overexpressions. There were significant differences in the survival curves between patients with ODC and invasive carcinoma derived from IPMN, regarding high frequency for cyclin A or B1. CONCLUSIONS: These results indicated that the expressions of cyclins A and B1, EID-1, and AcH3 may be correlated with a malignant potential in IPMNs. Invasive carcinoma derived from IPMN may be slow growing as compared with ODC.

Environmental neurotoxic pesticide increases histone acetylation to promote apoptosis in dopaminergic neuronal cells: relevance to epigenetic mechanisms of neurodegeneration.

Pesticide exposure has been implicated in the etiopathogenesis of Parkinson's disease (PD); in particular, the organochlorine insecticide dieldrin is believed to be associated with PD. Emerging evidence indicates that histone modifications play a critical role in cell death. In this study, we examined the effects of dieldrin treatment on histone acetylation and its role in dieldrin-induced apoptotic cell death in dopaminergic neuronal cells. In mesencephalic dopaminergic neuronal cells, dieldrin induced a time-dependent increase in the acetylation of core histones H3 and H4. Histone acetylation occurred within 10 min of dieldrin exposure indicating that acetylation is an early event in dieldrin neurotoxicity. The hyperacetylation was attributed to dieldrin-induced proteasomal dysfunction, resulting in accumulation of a key histone acetyltransferase (HAT), cAMP response element-binding protein. The novel HAT inhibitor anacardic acid significantly attenuated dieldrin-induced histone acetylation, Protein kinase C delta proteolytic activation and DNA fragmentation in dopaminergic cells protected against dopaminergic neuronal degeneration in primary mesencephalic neuronal cultures. Furthermore, 30-day exposure of dieldrin in mouse models induced histone hyperacetylation in the striatum and substantia nigra. For the first time, our results collectively demonstrate that exposure to the neurotoxic pesticide dieldrin induces acetylation of core histones because of proteasomal dysfunction and that hyperacetylation plays a key role in dopaminergic neuronal degeneration after exposure of dieldrin.

Somatic and germ-line mosaicism in Rubinstein-Taybi syndrome.

Rubinstein-Taybi syndrome (RSTS) is a rare autosomal dominant genetic disease and is characterized by mental retardation, distinctive facial features, broad and often angulated thumbs and great toes, short stature, and growth retardation. CREBBP and EP300 are the only genes currently known to be associated with RSTS. Mutations in CREBBP and EP300 were identified in approximately 50% and 3% of RSTS patients, respectively. To date, most of CREBBP mutations were de novo mutations and the recurrence rate in a family was low. Families with more than one affected child are extremely rare. In this study, we have shown a family with two affected siblings; the same mutation was found in both siblings. However, the mutation was not found in the blood or saliva DNA samples from the parents, suggesting the mechanism of germ-line mosaicism. In addition, we identified low-level mosaicism of a CREBBP mutation in the father from a second family with one affected child. Among the three analyzed tissue samples from the father, low-level mosaicism is present only significantly in the blood sample. We hypothesize mutations in CREBBP in these two families occur in the postzygotic stage in one of the parents (one generation ahead) of the affected individual. Additional family studies are required to determine how common somatic and/or gonadal mosaicism is present in RSTS patients.

ERK1/2 control phosphorylation and protein level of cAMP-responsive element-binding protein: a key role in glucose-mediated pancreatic beta-cell survival.

cAMP-responsive element-binding protein (CREB) is required for beta-cell survival by regulating expression of crucial genes such as bcl-2 and IRS-2. Using MIN6 cells and isolated rat pancreatic islets, we investigated the signaling pathway that controls phosphorylation and protein level of CREB. We observed that 10 mmol/l glucose-induced CREB phosphorylation was totally inhibited by the protein kinase A (PKA) inhibitor H89 (2 micromol/l) and reduced by 50% with the extracellular signal-regulated kinase (ERK)1/2 inhibitor PD98059 (20 micromol/l). This indicates that ERK1/2, reported to be located downstream of PKA, participates in the PKA-mediated CREB phosphorylation elicited by glucose. In ERK1/2-downregulated MIN6 cells by siRNA, glucose-stimulated CREB phosphorylation was highly reduced and CREB protein content was decreased by 60%. In MIN6 cells and islets cultured for 24-48 h in optimal glucose concentration (10 mmol/l), which promotes survival, blockade of ERK1/2 activity with PD98059 caused a significant decrease in CREB protein level, whereas CREB mRNA remained unaffected (measured by real-time quantitative PCR). This was associated with loss of bcl-2 mRNA and protein contents, caspase-3 activation, and emergence of ultrastructural apoptotic features detected by electron microscopy. Our results indicate that ERK1 and -2 control the phosphorylation and protein level of CREB and play a key role in glucose-mediated pancreatic beta-cell survival.

Nuclear receptor coactivators function in estrogen receptor- and progestin receptor-dependent aspects of sexual behavior in female rats.

The ovarian hormones, estradiol (E) and progesterone (P) facilitate the expression of sexual behavior in female rats. E and P mediate many of these behavioral effects by binding to their respective intracellular receptors in specific brain regions. Nuclear receptor coactivators, including Steroid Receptor Coactivator-1 (SRC-1) and CREB Binding Protein (CBP), dramatically enhance ligand-dependent steroid receptor transcriptional activity in vitro. Previously, our lab has shown that SRC-1 and CBP modulate estrogen receptor (ER)-mediated induction of progestin receptor (PR) gene expression in the ventromedial nucleus of the hypothalamus (VMN) and hormone-dependent sexual receptivity in female rats. Female sexual behaviors can be activated by high doses of E alone in ovariectomized rats, and thus are believed to be ER-dependent. However, the full repertoire of female sexual behavior, in particular, proceptive behaviors such as hopping, darting and ear wiggling, are considered to be PR-dependent. In the present experiments, the function of SRC-1 and CBP in distinct ER- (Exp. 1) and PR- (Exp. 2) dependent aspects of female sexual behavior was investigated. In Exp. 1, infusion of antisense oligodeoxynucleotides to SRC-1 and CBP mRNA into the VMN decreased lordosis intensity in rats treated with E alone, suggesting that these coactivators modulate ER-mediated female sexual behavior. In Exp. 2, antisense to SRC-1 and CBP mRNA around the time of P administration reduced PR-dependent ear wiggling and hopping and darting. Taken together, these data suggest that SRC-1 and CBP modulate ER and PR action in brain and influence distinct aspects of hormone-dependent sexual behaviors. These findings support our previous studies and provide further evidence that SRC-1 and CBP function together to regulate ovarian hormone action in behaviorally-relevant brain regions.