CREB-binding Protein Knockdown Research Articles

PBA2, a novel inhibitor of the β-catenin/CBP pathway, eradicates chronic myeloid leukemia including BCR-ABL T315I mutation

BackgroundBCR-ABL is a constitutively active tyrosine kinase that stimulates multiple downstream signaling pathways to promote the survival and proliferation of chronic myeloid leukemia (CML) cells. The clinical application of specific BCR-ABL tyrosine kinase inhibitors (TKIs) has led to significantly improved prognosis and overall survival in CML patients compared to previous treatment regimens. However, direct targeting of BCR-ABL does not eradicate CML cells expressing T315I-mutated BCR-ABL. Our previous study revealed that inhibiting CREB binding protein (CBP) is efficacious in activating β-catenin/p300 signaling, promoting cell differentiation and inducing p53/p21-dependent senescence regardless of BCR-ABL mutation status. We hypothesize that the specific inhibition of CBP may represent a novel strategy to promote β-catenin/p300-mediated differentiation and suppress cancer cell proliferation for treating CML patients.MethodsThe anticancer efficacy of PBA2, a novel CBP inhibitor, in CML cells expressing wild-type or T315I-mutated BCR-ABL was investigated in vitro and in vivo. Cell differentiation was determined by the nitroblue tetrazolium (NBT) reduction assay. The extent of cellular senescence was assessed by senescence-associated β-galactosidase (SA-β-Gal) activity. Cytotoxicity was measured by MTS assay. RNA interference was performed to evaluate the cell proliferation effects of CBP knockdown. The interaction of β-catenin and CBP/p300 was examined by co-immunoprecipitation assay.ResultsPBA2 exhibited significantly higher anticancer effects than imatinib in CML cells harboring either wild-type or T315I-mutated BCR-ABL both in vitro and in vivo. Mechanistically, PBA2 reduced CBP expression and promoted β-catenin-p300 interaction to induce cell differentiation and senescence.ConclusionOur data supported the rational treatment of CML by inhibiting the β-catenin/CBP pathway regardless of BCR-ABL mutation status.

Liver X Receptor LXRα Promotes Grass Carp Reovirus Infection by Attenuating IRF3-CBP Interaction and Inhibiting RLR Antiviral Signaling.

Liver X receptors (LXRs) are nuclear receptors involved in metabolism and the immune response. Different from mammalian LXRs, which include two isoforms, LXRα and LXRβ, only a single LXRα gene exists in the piscine genomes. Although a study has suggested that piscine LXR inhibits intracellular bacterial survival, the functions of piscine LXRα in viral infection are unknown. In this study, we show that overexpression of LXRα from grass carp (Ctenopharyngodon idellus), which is named as gcLXRα, increases host susceptibility to grass carp reovirus (GCRV) infection, whereas gcLXRα knockdown in CIK (C. idellus kidney) cells inhibits GCRV infection. Consistent with these functional studies, gcLXRα knockdown promotes the transcription of antiviral genes involved in the RIG-I-like receptor (RLR) antiviral signaling pathway, including IFN regulatory factor (IRF3) and the type I IFN IFN1. Further results show that gcLXRα knockdown induces the expression of CREB-binding protein (CBP), a transcriptional coactivator. In the knockdown of CBP, the inhibitory effect of gcLXRα knockdown in limiting GCRV infection is completely abolished. gcLXRα also interacts with IRF3 and CBP, which impairs the formation of the IRF3/CBP transcription complex. Moreover, gcLXRα heterodimerizes with RXRg, which cooperatively impair the transcription of the RLR antiviral signaling pathway and promote GCRV infection. Taken together, to our knowledge, our findings provide new insight into the functional correlation between nuclear receptor LXRα and the RLR antiviral signaling pathway, and they demonstrate that gcLXRα can impair the RLR antiviral signaling pathway and the production of type I IFN via forming gcLXRα/RXRg complexes and attenuating IRF3/CBP complexes.

Antisense-induced knockdown of cAMP response element-binding protein downregulates Per1 gene expression in the shell region of nucleus accumbens resulting in reduced alcohol consumption in mice.

We recently showed that circadian genes expressed in the shell region of nucleus accumbens (NAcSh) play a key role in alcohol consumption, though, the molecular mechanism of those effects is unclear. Because CREB-binding protein (CBP) promotes Per1 gene expression, we hypothesized that alcohol consumption would increase CBP expression in the NAcSh and antisense-induced knockdown of CBP would reduce Per1 expression and result in a reduction in alcohol consumption. To test our hypothesis, we performed two experiments. The Drinking-in-the-dark (DID) paradigm was used to evaluate alcohol consumption in male C57BL/6J mice. In Experiment 1 we examined the effects of alcohol consumption on CBP gene expression in the NAcSh. Control animals were exposed to, sucrose [10% (w/v) taste and calorie] and water (consummatory behavior). In Experiment 2 examined the effects of CBP gene silencing on the expression of the Per1 gene in the NAcSh and alcohol consumption in mice exposed to alcohol using the DID paradigm. CBP gene silencing was achieved by local infusion of two doses of either CBP antisense oligodeoxynucleotides (AS-ODNs; Antisense group) or nonsense ODNs (NS-ODNs; Nonsense group) bilaterally microinjected into the NAcSh within 24h before alcohol consumption on Day 4 of the DID paradigm. The microinfusion sites were verified by cresyl violet staining. Compared to sucrose, alcohol consumption, under the DID paradigm, significantly increased the expression of CBP in the NAcSh. Compared to Controls, bilateral infusion of CBP AS-ODNs significantly reduced the expression of Per1 in the NAcSh and alcohol consumption without affecting the amount of sucrose consumed. Our results suggest that CBP is an upstream regulator of Per1 expression in the NAcSh and may act via Per1 to modulate alcohol consumption.

CREB-binding protein regulates metamorphosis and compound eye development in the yellow fever mosquito, Aedes aegypti

Juvenile hormones (JH) and ecdysone coordinately regulate metamorphosis in Aedes aegypti. We studied the function of an epigenetic regulator and multifunctional transactivator, CREB binding protein (CBP) in A. aegypti. RNAi-mediated knockdown of CBP in Ae. aegypti larvae resulted in suppression of JH primary response gene, Krüppel-homolog 1 (Kr-h1), and induction of primary ecdysone response gene, E93, resulting in multiple effects including early metamorphosis, larval-pupal intermediate formation, mortality and inhibition of compound eye development. RNA sequencing identified hundreds of genes, including JH and ecdysone response genes regulated by CBP. In the presence of JH, CBP upregulates Kr-h1 by acetylating core histones at the Kr-h1 promoter and facilitating the recruitment of JH receptor and other proteins. CBP suppresses metamorphosis regulators, EcR-A, USP-A, BR-C, and E93 through the upregulation of Kr-h1 and E75A. CBP regulates the expression of core eye specification genes including those involved in TGF-β and EGFR signaling. These studies demonstrate that CBP is an essential player in JH and 20E action and regulates metamorphosis and compound eye development in Ae. aegypti.

Depletion of the transcriptional coactivators CREB-binding protein or EP300 downregulates CD20 in diffuse large B-cell lymphoma cells and impairs the cytotoxic effects of anti-CD20 antibodies

Monoclonal antibodies targeting CD20 are central in the treatment of B-cell lymphomas. In diffuse large B-cell lymphoma (DLBCL), inactivating mutations of the histone acetyltransferases CREB-binding protein (CBP) and EP300 are common. Moreover, knockdown of CBP in DLBCL has been shown to result in aberrant transcriptional silencing. Expression of CD20 is sensitive to epigenetic manipulation, and histone deacetylase inhibitors have been found to potentiate treatment with anti-CD20 antibodies. Therefore, we studied the role of CBP and EP300 depletion on CD20 expression and effects of the anti-CD20 antibodies rituximab and obinutuzumab in DLBCL cells. Levels of CBP and EP300 were reduced by shRNA in the germinal centre-derived diffuse large B-cell lymphoma cell line SU-DHL4. The levels of CD20 mRNA and protein were determined by quantitative polymerase chain reaction, Western blot, and flow cytometry. Binding of the transcription factors PU.1 and FOXO1 to the CD20 promoter was determined by chromatin immunoprecipitation coupled with quantitative polymerase chain reaction. Response to the monoclonal anti-CD20 antibodies rituximab and obinutuzumab in CBP- or EP300-depleted cells was assessed by complement-dependent cell death, direct cell death, and antibody-dependent cellular cytotoxicity (ADCC). Our results suggest that depletion of CBP and EP300 levels leads to a strong reduction of CD20 expression, accompanied by reduced binding of PU.1 to the CD20 promoter. In CBP-depleted, but not EP300-depleted cells, increased binding of FOXO1 to the CD20 promoter was observed. Interestingly, CBP or EP300 depletion leads to decreased complement-dependent cell death and direct cell death in response to rituximab and obinutuzumab, which was most pronounced in response to rituximab in CBP-depleted cells. Our data suggest that inactivating mutations of CBP, and to a lesser extent EP300, may impair the response to anti-CD20 antibodies. However, these observations should be analyzed in future clinical trials.

Histone acetyltransferase CBP-related H3K23 acetylation contributes to courtship learning in Drosophila

BackgroundHistone modifications are critical in regulating neuronal processes. However, the impacts of individual histone modifications on learning and memory are elusive. Here, we investigated the contributions of histone H3 lysine modifications to learning and memory in Drosophila by using histone lysine-to-alanine mutants.ResultsBehavioural analysis indicated that compared to the H3WT group, mutants overexpressing H3K23A displayed impaired courtship learning. Chromatin immunoprecipitation analysis of H3K23A mutants showed that H3K23 acetylation (H3K23ac) levels were decreased on learning-related genes. Knockdown of CREB-binding protein (CBP) decreased H3K23ac levels, attenuated the expression of learning-related genes, led to a courtship learning defect and altered development of the mushroom bodies. A decline in courtship learning ability was observed in both larvae and adult treatments with ICG-001. Furthermore, treatment of Drosophila overexpressing mutated H3K23A with a CBP inhibitor did not aggravate the learning defect.ConclusionsH3K23ac, catalysed by the acetyltransferases dCBP, contributes to Drosophila learning, likely by controlling the expression of specific genes. This is a novel epigenetic regulatory mechanism underlying neuronal behaviours.

CREB-binding protein plays key roles in juvenile hormone action in the red flour beetle, Tribolium Castaneum

Juvenile hormones (JH) and ecdysteroids regulate many biological and metabolic processes. CREB-binding protein (CBP) is a transcriptional co-regulator with histone acetyltransferase (HAT) activity. Therefore, CBP is involved in activation of many transcription factors that regulate expression of genes associated with postembryonic development in insects. However, the function of CBP in JH action in insects is not well understood. Hence, we studied the role of CBP in JH action in the red flour beetle, Tribolium castaneum and the Tribolium cell line. CBP knockdown caused a decrease in JH induction of genes, Kr-h1, 4EBP and G13402 in T. castaneum larvae, adults and TcA cells whereas, Trichostatin A [TSA, a histone deacetylase (HDAC) inhibitor] induced the expression of these JH-response genes. Western blot analysis with specific antibodies revealed the requirement of CBP for the acetylation of H3K18 and H3K27 in both T. castaneum and TcA cells. Chromatin immunoprecipitation (Chip) assays showed the importance of CBP-mediated acetylation of H3K27 for JH induction of Kr-h1, 4EBP, and G13402 in TcA cells. These data suggest that CBP plays an important role in JH action in the model insect, T.castaneum.

Multiple functions of CREB-binding protein during postembryonic development: identification of target genes

BackgroundJuvenile hormones (JH) and ecdysteroids control postembryonic development in insects. They serve as valuable targets for pest management. Hence, understanding the molecular mechanisms of their action is of crucial importance. CREB-binding protein (CBP) is a universal transcriptional co-regulator. It controls the expression of several genes including those from hormone signaling pathways through co-activation of many transcription factors. However, the role of CBP during postembryonic development in insects is not well understood. Therefore, we have studied the role of CBP in postembryonic development in Tribolium, a model coleopteran insect.ResultsCBP is ubiquitously expressed in the red flour beetle, Tribolium castaneum. RNA interference (RNAi) mediated knockdown of CBP resulted in a decrease in JH induction of Kr-h1 gene expression in Tribolium larvae and led to a block in their development. Moreover, the injection of CBP double-stranded RNA (dsRNA) showed lethal phenotypes within 8 days of injection. RNA-seq and subsequent differential gene expression analysis identified CBP target genes in Tribolium. Knockdown of CBP caused a decrease in the expression of 1306 genes coding for transcription factors and other proteins associated with growth and development. Depletion of CBP impaired the expression of several JH response genes (e.g., Kr-h1, Hairy, early trypsin) and ecdysone response genes (EcR, E74, E75, and broad complex). Further, GO enrichment analyses of the downregulated genes showed enrichment in different functions including developmental processes, pigmentation, anatomical structure development, regulation of biological and cellular processes, etc.ConclusionThese data suggest diverse but crucial roles for CBP during postembryonic development in the coleopteran model insect, Tribolium. It can serve as a target for RNAi mediated pest management of this stored product pest.

Sec8 modulates TGF-β induced EMT by controlling N-cadherin via regulation of Smad3/4

Sec8 is one of the subunits of the exocyst, which is an evolutionarily conserved complex of eight proteins, comprising Sec3 (EXOC1), Sec5 (EXOC2), Sec6 (EXOC3), Sec8 (EXOC4), Sec10 (EXOC5), Sec15 (EXOC6), Exo70 (EXOC7), and Exo84 (EXOC8) subunits. Sec8 knockout mice embryos initiate gastrulation but are unable to progress beyond the primitive streak stage and die shortly. During embryonic development, the first epithelial-mesenchymal transition (EMT) event occurs at gastrulation. Sec8 may be involved in the early embryonic development through EMT. However, the function of Sec8 in EMT remains unclear. In the present study, it was found that Sec8 regulates N-cadherin expression by controlling Smad3 and Smad4 expression at the basal transcriptional level, thereby modulating cell migration and adhesion. Furthermore, Sec8 knockdown decreased CREB binding protein (CBP) expression at mRNA and protein levels. However, CBP knockdown did not affect Sec8 expression. These results indicated that Sec8 regulates N-cadherin expression by controlling Smad3 and Smad4 expression through CBP, thereby mediating the EMT.

CREB-binding protein regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathway

CBP (CREB-binding protein) is a transcriptional co-activator which possesses HAT (histone acetyltransferases) activity and participates in many biological processes, including embryonic development, growth control and homeostasis. However, its roles and the underlying mechanisms in the regulation of carcinogenesis and tumor development remain largely unknown. Here we investigated the molecular mechanisms and potential targets of CBP involved in tumor growth and survival in lung cancer cells. Elevated expression of CBP was detected in lung cancer cells and tumor tissues compared to the normal lung cells and tissues. Knockdown of CBP by siRNA or inhibition of its HAT activity using specific chemical inhibitor effectively suppressed cell proliferation, migration and colony formation and induced apoptosis in lung cancer cells by inhibiting MAPK and activating cytochrome C/caspase-dependent signaling pathways. Co-immunoprecipitation and immunofluorescence analyses revealed the co-localization and interaction between CBP and CPSF4 (cleavage and polyadenylation specific factor 4) proteins in lung cancer cells. Knockdown of CPSF4 inhibited hTERT transcription and cell growth induced by CBP, and vice versa, demonstrating the synergetic effect of CBP and CPSF4 in the regulation of lung cancer cell growth and survival. Moreover, we found that high expression of both CBP and CPSF4 predicted a poor prognosis in the patients with lung adenocarcinomas. Collectively, our results indicate that CBP regulates lung cancer growth by targeting MAPK and CPSF4 signaling pathways.

Deficit in Long-Term Synaptic Plasticity Is Rescued by a Computationally Predicted Stimulus Protocol

Mutations in the gene encoding CREB-binding protein (CBP) cause deficits in long-term plasticity, learning, and memory. Here, long-term synaptic facilitation (LTF) at Aplysia sensorimotor synapses in cell culture was used as a model system to investigate methods for overcoming deficits in LTF produced by a CBP knockdown. Injecting CBP-siRNA into individual sensory neurons reduced CBP levels and impaired LTF produced by a standard protocol of five 5-min pulses of serotonin (5-HT) delivered at 20 min interstimulus intervals. A computational model, which simulated molecular processes underlying LTF induction, predicted a rescue protocol of five pulses of 5-HT at non-uniform interstimulus intervals that overcame the consequences of reduced CBP and restored LTF. These results suggest that complementary empirical and computational studies can identify methods for ameliorating impairments of learning attributable to molecular lesions.

CREB Binding Protein (CBP) Activation Is Required for Luteinizing Hormone Beta Expression and Normal Fertility in Mice

Normal function of the hypothalamic-pituitary-gonadal axis is dependent on gonadotropin-releasing hormone (GNRH)-stimulated synthesis and secretion of luteinizing hormone (LH) from the pituitary gonadotroph. While the transcriptional coactivator CREB binding protein (CBP) is known to interact with Egr-1, the major mediator of GNRH action on the Lhb gene, the role of CBP in Lhb gene expression has yet to be characterized. We show that in the LβT2 gonadotroph cell line, overexpression of CBP augmented the response to GNRH and that knockdown of CBP eliminated GNRH responsiveness. While GNRH-mediated phosphorylation of CBP at Ser436 increased the interaction with Egr-1 on the Lhb promoter, loss of this phosphorylation site eliminated GNRH-mediated Lhb expression in LβT2 cells. In vivo, loss of CBP phosphorylation at Ser436 rendered female mice subfertile. S436A knock-in mice had disrupted estrous cyclicity and reduced responsiveness to GNRH. Our results show that GNRH-mediated phosphorylation of CBP at Ser436 is required for Egr-1 to activate Lhb expression and is a requirement for normal fertility in female mice. As CBP can be phosphorylated by other factors, such as insulin, our studies suggest that CBP may act as a key regulator of Lhb expression in the gonadotroph by integrating homeostatic information with GNRH signaling.

Histone Deacetylase 6 (HDAC6) Deacetylates Survivin for Its Nuclear Export in Breast Cancer

Survivin is an oncogenic protein that is highly expressed in breast cancer and has a dual function that is dependent on its subcellular localization. In the cytosol, survivin blocks programmed cell death by inactivating caspase proteins; however, in the nucleus it facilitates cell division by regulating chromosomal movement and cytokinesis. In prior work, we showed that survivin is acetylated by CREB-binding protein (CBP), which restricts its localization to the nuclear compartment and thereby inhibits its anti-apoptotic function. Here, we identify histone deacetylase 6 (HDAC6) as responsible for abrogating CBP-mediated survivin acetylation in the estrogen receptor (ER)-positive breast cancer cell line, MCF-7. HDAC6 directly binds survivin, an interaction that is enhanced by CBP. In quiescent breast cancer cells in culture and in malignant tissue sections from ER+ breast tumors, HDAC6 localizes to a perinuclear region of the cell, undergoing transport to the nucleus following CBP activation where it then deacetylates survivin. Genetically modified mouse embryonic fibroblasts that lack mhdac6 localize survivin predominantly to the nuclear compartment, whereas wild-type mouse embryonic fibroblasts localize survivin to distinct cytoplasmic structures. Together, these data imply that HDAC6 deacetylates survivin to regulate its nuclear export, a feature that may provide a novel target for patients with ER+ breast cancer.

Abstract 17031: Epigenetic Modifications at VEGFR1 Locus in Response to Angiogenic Signal

Vascular endothelial growth factor (VEGF) and its receptors (VEGFR1/VEGFR2) are primary regulators of angiogenesis. Among them, VEGFR1 has a dual role of either stimulating or suppressing angiogenesis. So, it is highly important to understand the molecular mechanism underlying its regulation. Earlier we found that angiogenic signal-ETS1-HIF2α regulatory axis is involved in the activation of Vegfr1 chromatin domain in endothelial cells. However, the dynamic nucleoprotein structure that assembles at the Vegfr1 chromatin locus is poorly understood. Here, we report that dynamic alteration in Vegfr1 transcription is associated with epigenetic marks that facilitate or repress transcriptional activation. The rapid induction of Vegfr1 transcription in response to angiogenic signal FGF2/EGF in yolk sac endothelial cells (YSECs) is associated with rapid increase in acetylated histone H3 at lysine 9 (H3acK9), 56 (H3acK56) and 27 (H3acK27), levels at the Vegfr1 chromatin domain. A time course analysis revealed that these acetylation levels are gradually reduced with time and is correlated with transcriptional repression of Vegfr1 . The increase in acetylation is associated with the induction in expression of histone acetyl transferase, Creb Binding Protein (CBP) in YSECs. Knockdown of CBP by RNA interference inhibited FGF2/EGF induced VEGFR1 expression with loss in histone acetylation marks. Level of repressive modification of histone H3 methylated at lysine27 (H3me3K27) at the Vegfr1 locus correlated with transcriptional repression of Vegfr1 . Member of the polycomb group family might regulate the transcriptional repression of Vegfr1 in endothelial cells as well as during differentiation of embryonic stem cells towards endothelial progenitors. This approach will provide important insights about molecular mechanisms of gene regulation in angiogenic signal-activated vs. repressed endothelial cells and will lead to therapeutic modalities for controlling angiogenesis.

Dysregulation of upstream binding factor-1 acetylation at K352 is linked to impaired ribosomal DNA transcription in Huntington's disease

Huntington's disease (HD) is an autosomal-dominant neurological disorder caused by expanded CAG repeats in the Huntingtin (Htt) gene, but it is not known how this mutation causes neurodegeneration. Herein, we found that dysfunction of upstream binding factor-1 (UBF-1) is linked to reduced ribosomal DNA (rDNA) transcription in HD. We identified that UBF1 acetylation at Lys (K) 352 by CREB binding protein (CBP) is crucial for the transcriptional activity of rDNA. UBF1 mutation (K352A, K352Q, and K352R) decreased rDNA transcriptional activity. Moreover, both CBP-dHAT mutant and knockdown of CBP by siRNA reduced acetylation of UBF1 and resulted in the decreased transcription of rDNA into rRNA. ChIP analysis showed a significant reduction of UBF1 occupancy in the promoter of rDNA in STHdh(Q111) cell line model of HD. These results demonstrate that abnormal activity of UBF1 and its acetylation by CBP are linked to impaired rDNA transcription in HD. This novel mechanism suggests that modulation of UBF-mediated rDNA synthesis by CBP may be a therapeutic target for improving neuronal rDNA transcription in HD.

Coactivation of the CLOCK–BMAL1 complex by CBP mediates resetting of the circadian clock

The transcription factor CLOCK-BMAL1 is a core component of the molecular clock machinery that drives circadian gene expression and physiology in mammals. Recently, we reported that this heterodimeric transcription factor functions as a signaling molecule in response to the resetting stimuli via the Ca²+-dependent protein kinase C pathway. Here, we demonstrate that the CREB-binding protein (CBP) plays a key role in rapid activation of the CLOCK-BMAL1 heterodimer that leads to phase resetting of the circadian clock. Under physiological conditions, a bimolecular fluorescence complementation (BiFC) assay revealed that CLOCK and BMAL1 dimerize in the cytoplasm and subsequently translocate into the nucleus in response to serum stimuli (mean time duration was 29.2 minutes and mean velocity 0.7 μm/minute). Concomitantly, BMAL1 rapidly recruited CBP on Per1 promoter E-box, but not p300 (a functional analog of CBP), in the discrete nuclear foci. However, recruitment of CBP by cAMP/Ca²+ response element-binding (CREB) protein on CRE was not markedly increased upon delivery of the resetting stimuli. Furthermore, overexpression of CBP greatly potentiated the CLOCK-BMAL1-mediated Per1 transcription, and this effect was completely abolished by site-directed mutation of E-box elements, but not by the mutation of CRE in the Per1 promoter. Furthermore, molecular knockdown of CBP severely dampened circadian oscillation of clock gene expression triggered by the resetting stimuli. These findings suggest that CBP recruitment by BMAL1 mediates acute transactivation of CLOCK-BMAL1, thereby inducing immediate-early Per1 transcription and phase resetting of the circadian clock.

CBP knockdown inhibits angiotensin II-induced vascular smooth muscle cells proliferation through downregulating NF-kB transcriptional activity

CREB binding protein (CBP), a powerful transcriptional co-activator for various transcriptional factors, regulates cell behavior in many cell types. Angiotensin II (Ang II) contributes to vascular lesion by promoting vascular smooth muscle cells (VSMCs) proliferation and migration. Therefore, we examined whether CBP knockdown could suppress Ang II-induced VSMCs proliferation, and elucidated its underlying molecular mechanism. We constructed lentiviral vector expressing CBP-specific short hairpin RNAs (shRNAs) that efficiently silenced CBP. VSMCs proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation assay. Protein and mRNA expression of CBP and relevant cytokines were examined by Western blot, ELISA, and real-time PCR, respectively. We also used luciferase reporter gene and electrophoretic mobility shift assay (EMSA) to detect Nuclear factor kappaB (NF-kB) transcriptional activity and DNA binding. Meanwhile, NF-kB p65 subunit nuclear translocation was confirmed by immunoblotting. Lentiviral-mediated CBP-shRNAs at different multiplicities of infection (MOI = 100, 150) both significantly suppressed Ang II-induced CBP expression. Knockdown of CBP markedly inhibited Ang II-stimulated VSMCs proliferation and cytokines (TNF-alpha and IL-6) production. However, this inhibitory effect was not enhanced at MOI of 150 compared with MOI of 100 (P > 0.05). CBP siRNA showed the potent inhibition on Ang II-induced NF-kB transcriptional activity. Similarly, no significant difference was found between CBP siRNA lentivirus treatment groups. Furthermore, CBP gene silencing had no effect on NF-kB nuclear translocation and DNA binding. These findings suggest that CBP knockdown inhibits Ang II-induced VSMCs proliferation and the mechanism is involved with downregulation of NF-kB transcriptional activity, not through reduction in NF-kB nuclear translocation or DNA binding. Maintaining proper CBP level may be a potential therapeutic target for Ang II-induced cardiovascular disorders.

Lentivirus-mediated RNAi Targeting CREB Binding Protein Attenuates Neointimal Formation and Promotes Re-endothelialization in Balloon Injured Rat Carotid Artery

Background/Aims: Lentiviral vectors provide a promising strategy for the treatment of cardiovascular diseases, owing to their ability to govern efficient and durable gene transfer. However, relatively few studies have been addressed on restenosis after balloon or stent associated arterial injury. We previously found that CREB binding protein (CBP), a powerful transcriptional coactivator, regulated cell proliferation and apoptosis in vascular endothelial and smooth muscle cells. Therefore, we investigated whether inhibition of CBP by lentivirus-mediated small interfering RNA can reduce neointimal formation after arterial injury. Methods: The carotid arteries from Sprague-Dawley rats were injured by balloon catheter, followed by incubating with 100 µl lentivirus expressing CBP or negative control (NC)-specific short hairpin RNAs (shRNAs) or PBS solution for 30 minutes. The rats were euthanized for real-time PCR, Western blot, immunohistochemical staining, and morphometric analysis at 4 weeks after balloon injury and in vivo gene transfer. Results: Lentiviral shRNA targeting CBP markedly reduced CBP expression. Moreover, CBP siRNA showed potent inhibition on balloon injury-induced Nuclear factor kappaB (NF-ĸB) acetylation. Compared with controls, the significant decrease of neointimal formation by CBP siRNA was accompanied by reduced cell proliferation in the neointima of injured arteries. However, no changes in medial area were observed among these different groups. Interestingly, endothelial cell marker CD31 immunostaining and morphometric analysis both showed that CBP knockdown significantly accelerated re-endothelialization. Conclusions: These findings suggest that CBP is involved in the control of neointimal formation and re-endothelialization via regulating NF-ĸB acetylation. Lentivirus-mediated CBP silencing may represent a novel therapeutic approach for the prevention of restenosis after vascular interventions.

TNF-α Preconditioning Protects Neurons via Neuron-Specific Up-Regulation of CREB-Binding Protein

Despite being a proinflammatory cytokine, TNF-alpha preconditions neurons against various toxic insults. However, underlying molecular mechanisms are poorly understood. The present study identifies the importance of CREB-binding protein (CBP) in facilitating TNF-alpha-mediated preconditioning in neurons. Treatment of rat primary neurons with fibrillar amyloid beta1-42 (Abeta) resulted in the loss of CBP protein. However, this loss was compensated by TNF-alpha preconditioning as the expression of neuronal CBP was up-regulated in response to TNF-alpha treatment. The induction of CBP by TNF-alpha was observed only in neurons, but not in astroglia and microglia, and it was contingent on the activation of transcription factor NF-kappaB. Interestingly, antisense knockdown of CBP abrogated the TNF-alpha-mediated preconditioning of neurons against Abeta and glutamate toxicity. Similarly in vivo, preadministration of TNF-alpha in mouse neocortex prevented Abeta-induced apoptosis and loss of choline acetyltransferase-positive cholinergic neurons. However, coadministration of cbp antisense, but not scrambled oligonucleotides, negated the protective effect of TNF-alpha against Abeta neurotoxicity. This study illustrates a novel biological role of TNF-alpha in increasing neuron-specific expression of CBP for preconditioning that may have therapeutic potential against neurodegenerative disorders.

Functional Role of CREB-Binding Protein in the Circadian Clock System of Drosophila melanogaster

Rhythmic histone acetylation underlies the oscillating expression of clock genes in the mammalian circadian clock system. Cellular factors that contain histone acetyltransferase and histone deacetylase activity have been implicated in these processes by direct interactions with clock genes, but their functional relevance remains to be assessed by use of appropriate animal models. Here, using transgenic fly models, we show that CREB-binding protein (CBP) participates in the transcriptional regulation of the Drosophila CLOCK/CYCLE (dCLK/CYC) heterodimer. CBP knockdown in pigment dispersing factor-expressing cells lengthens the period of adult locomotor rhythm with the prolonged expression of period and timeless genes, while CBP overexpression in timeless-expressing cells causes arrhythmic circadian behaviors with the impaired expression of these dCLK/CYC-induced clock genes. In contrast to the mammalian circadian clock system, CBP overexpression attenuates the transcriptional activity of the dCLK/CYC heterodimer in cultured cells, possibly by targeting the PER-ARNT-SIM domain of dCLK. Our data suggest that the Drosophila circadian clock system has evolved a distinct mechanism to tightly regulate the robust transcriptional potency of the dCLK/CYC heterodimer.