cAMP反应元件结合蛋白在药物依赖形成中的作用

反复应用成瘾性药物引起中枢神经系统特定部位cAMP信号系统出现适应性改变 ,cAMP反应元件结合蛋白(cyclicAMPresponseelementbindingprotein ,CREB)是受cAMP调节的主要转录因子 ,在动物产生药物依赖时脑内CREB含量及磷酸化程度发生变化 ,与动物躯体依赖和精神依赖有关。确定CERB在药物依赖中的作用有益于阐明药物依赖分子机制及采取相应的防治措施     

Differential regulation of phosphorylation of the cAMP response element-binding protein after activation of EP2 and EP4 prostanoid receptors by prostaglandin E2

The EP2 and EP4 prostanoid receptors are G-protein-coupled receptors whose activation by their endogenous ligand, prostaglandin (PG) E2, stimulates the formation of intracellular cAMP. We have previously reported that the stimulation of cAMP formation in EP4-expressing cells is significantly less than in EP2-expressing cells, despite nearly identical levels of receptor expression (J Biol Chem 277:2614-2619, 2002). In addition, a component of EP4 receptor signaling, but not of EP2 receptor signaling, was found to involve the activation of phosphatidylinositol 3-kinase (PI3K). In this study, we report that PGE2 stimulation of cells expressing either the EP2 or EP4 receptor results in the phosphorylation of the cAMP response element binding protein (CREB) at serine-133. Pretreatment of cells with N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H-89), an inhibitor of protein kinase A (PKA), attenuated the PGE2-mediated phosphorylation of CREB in EP2-expressing cells, but not in EP4-expressing cells. Pretreatment of cells with wortmannin, an inhibitor of PI3K, had no effects on the PGE2-mediated phosphorylation of CREB in either EP2- or EP4-expressing cells, although it significantly increased the PGE2-mediated activation of PKA in EP4-expressing cells. However, combined pretreatment with H-89 and wortmannin blocked PGE2-mediated phosphorylation in EP2-expressing cells as well as in EP2-expressing cells. PGE2-mediated intracellular cAMP formation was not affected by pretreatment with wortmannin, or combined treatment with wortmannin and H-89, in either the EP2- or EP4-expressing cells. These findings suggest that PGE2 stimulation of EP4 receptors, but not EP2 receptors, results in the activation of a PI3K signaling pathway that inhibits the activity of PKA and that the PGE2-mediated phosphorylation of CREB by these receptors occurs through different signaling pathways     

Protein O-N-acetylglucosaminylation modulates promoter activities of cyclic AMP response element and activator protein 1 and enhances E-selectin expression on HuH-7 human hepatoma cells

High glucose accelerates O-N-acetylglucosaminylation (O-GlcNAcylation) of proteins and causes diabetic complications. In the present study, we found that treatment of HuH-7 human hepatoma cells with high glucose or the protein O-N-acetylglucosaminidase (O-GlcNAcase) inhibitor O-(2-acetoamide-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) increased the cell surface expression of E-selectin. A dual luciferase reporter assay indicated that high glucose and PUGNAc suppressed promoter activities of the cyclic AMP response element (CRE) and enhanced those of activator protein 1 (AP-1). Enhanced CRE promoter activities in HuH-7 cells treated with dibutyryl cAMP or co-transfected with a protein kinase A expression vector pFC-PKA that enhances the phosphorylation of CRE binding protein (CREB) were suppressed by PUGNAc. In contrast, PUGNAc further increased the enhanced AP-1 promoter activity in cells transfected with a mitogen-activated protein kinase kinase kinase expression vector pFC-MEKK that enhances c-Jun phosphorylation. Immuno-blotting using an anti-O-GlcNAc antibody revealed that high glucose and PUGNAc accelerated protein O-GlcNAcylation and that there were substantial differences in the O-GlcNAcylated proteins in the cytoplasmic and nuclear fractions. In addition, PUGNAc increased the nuclear import of O-GlcNAcylated CREB. These results suggest that protein O-GlcNAcylation modulates the promoter activities of E-selectin gene, suppression of CRE and enhancement of AP-1, and enhances E-selectin protein expression on hepatocytes.     

抑郁症信号转导通路研究

长期给予抗抑郁剂可以增加脑内cAMP依赖性PKA的表达水平,继而激活cAMP反应元件结合蛋白。CREB可以调节脑源性神经生长因子的表达。大量的研究表明cAMP和BDNF是多种抗抑郁剂的共同通路,现就此进行综述,探讨其与抗抑郁剂之间的关系,为精神药理和新药研发提供依据。     

cAMP反应元件结合蛋白与神经退行性疾病

cAMP反应元件结合蛋白(cAMP response elem ent b ind-ing prote in,CREB)是位于细胞核内的转录因子。CREB的活性受到信号通路中许多因子的调控。CREB活化后与真核生物靶基因CRE序列结合并调节其转录,发挥多种生物学效应。在中枢神经系统,CREB调节着神经细胞生长发育,参与神经细胞突触可塑性、长时程记忆的形成过程。CREB参与阿尔采末病、血管性痴呆、亨廷顿舞蹈病及H IV-相关痴呆等神经退行性疾病的病理生理机制研究也取得了进展,成为以CREB作为靶点控制神经退行性疾病病程的理论基础。     

Neurobiology and treatment of anxiety: signal transduction and neural plasticity

The stress-dependence and chronic nature of anxiety disorders along with the anxiolytic effectiveness of antidepressant drugs suggests that neuronal plasticity may play a role in the pathophysiology of anxiety. Intracellular signaling pathways are known in many systems to be critical links in the cascades from surface signals to the molecular alterations that result in functional plasticity. Chronic antidepressant treatments can regulate intracellular signaling pathways and can induce molecular, cellular, and structural changes over time. These changes may be important to the anxiolytic effectiveness of these drugs. In addition, the signaling proteins implicated in the actions of chronic antidepressant action, such as cAMP response element binding protein (CREB), have also been implicated in conditioned fear and in anxiety. The cellular mechanisms underlying conditioned fear indicate roles for additional signaling pathways; however, less is known about such mechanisms in anxiety. The challenge to identify intracellular signaling pathways and related molecular and structural changes that are critical to the etiology and treatment of anxiety will further establish the importance of mechanisms of neuronal plasticity in functional outcome and improve treatment strategies.     

羟甲芬太尼立体异构体对培养的海马神经元cAMP-反应元件结合蛋白磷酸化的影响

AIM: To define the effects and signal pathways of ohmefentanyl stereoisomers [(-)-cis-(3R,4S,2'R) OMF (F9202),( )-cis-(3R,4S,2'S) OMF (F9204), and (-)-cis-(3S,4S,2'R) OMF (F9203)] on the phosphorylation of cAMP-re-sponse element binding protein (CREB) in cultured rat hippocampal neurons. METHODS: The effects of the three OMF stereoisomers and morphine (Mor) on cAMP accumulation and CREB phosphorylation were monitored by radioimmunoassay and Western blot analysis, respectively. RESULTS: The three OMF stereoisomers and Mor could all partially inhibit forskolin-stimulated (25μmol/L, 15min) cAMP accumulation in a dose-dependent manner and this effect could be reversed by naloxone. F9202, F9204, and Mor could significantly increase CREB phosphorylation from 2.88 to 3.59 folds over control levels after 30-min exposure. This effect was reversed by naloxone,but F9203 failed to increase CREB phosphorylation. KN-62 and staurosporine significantly blocked the opioidsinduced CREB phosphorylation, while H-89 and PD 98059 had no effect on the actions. CONCLUSION: Mor,F9202, and F9204, which could induce psychological dependence affected via the μ-opioid receptor, stimulated intracellular signal pathways involving Ca^2 /calmodulin-dependent protein kinases (CCDPK) and protein kinase C(PKC) pathways, which in turn initiated CREB phosphorylation. F9203, which could not induce dependence, had no effect on CREB phosphorylation in hippocampal neurons. The increased CREB phosphorylation in hippocampal neurons may play a role in opioids dependence.     

Copper induces oxidative stress and apoptosis of hippocampal neuron via pCREB/BDNF/ and Nrf2/HO-1/NQO1 pathway

Disordered copper metabolism has been suggested to occur to several neurological conditions, including Alzheimer's disease and Parkinson's disease. However, the underlying mechanism was still unclear. This might link to copper-induced hippocampal neuronal apoptosis and decrease in neurons viability. Our vitro experiment showed copper exposure induced oxidative stress and promoted apoptosis of HT22 murine hippocampal neuronal cell. Mechanistically, we found copper, on the one hand, prevented phosphorylation of cAMP response element binding protein (CREB) to decrease expression its downstream target protein Brain-derived neurotrophic factor (BDNF), and to decrease mitochondrial membrane potential and Bcl-2/Bax ratio; on the other hand, copper-induced reactive oxygen species (ROS), promoted lipid peroxidation, reduced antioxidant enzyme activity of GSH-Px. Copper-induced oxidative damage further decreased the phosphorylation of CREB, decreased expression of Bcl-2, enhanced expression of Bax, and accelerated the dissociation of keap1-Nrf2 complex, promoted the nuclear translocation of Nrf2, stimulate the expression of antioxidant molecules HO-1 and NQO1. In conclusion, we found copper inhibited pCREB/BDNF signaling pathway by prevent CREB from phosphorylation, further found that oxidative damage not only inhibited neuroprotective signaling pathways and induced apoptosis, but activated antioxidant protection signals Nrf2/HO-1/NQO1 signaling pathway.     

Learning/memory and drug dependence

We investigated the possible mechanisms of development of latent learning and morphine dependence by the methods of behavioral pharmacology and confirmed them by using mutant mice. The heterozygous mice for the tyrosine hydroxylase (TH) gene and for the cyclic AMP (cAMP) response element binding protein (CREB) binding protein (CBP) gene showed the impairment of latent learning in the water finding task, and these mice did not develop morphine dependence. The spatial learning and hippocampal long-term potentiation (LTP) were normal in the both mutants. TH heterozygous mice showed a reduction of high K(+)-evoked noradrenaline release in the frontal cortex measured by the microdialysis technique and of cAMP content in the brain. In conclusion, the results of mutant mice suggest that the alternation of catecholamine biosynthesis and cAMP signal pathways may play a key role in development of latent learning and morphine dependence, and they furthermore show that the expression of genes mediated by phosphorylated CREB may be involved in the development of latent learning and morphine dependence.     

Induction of cyclooxygenase-2 by bovine type I collagen in macrophages via C/EBP and CREB activation by multiple cell signaling pathways

Bovine type I collagen (Col-I) is utilized for medical purposes such as cosmetic surgery and wrinkle removal. Cyclooxygenase-2 (COX-2) plays roles in pathophysiological processes including inflammation and tumorigenesis. This study examines the effects of Col-I on the COX-2 expression and the signaling pathways in macrophages. Col-I increased the levels of COX-2 protein and mRNA in serum-stimulated Raw264.7 cells in a time- and concentration-dependent manner. Treatment of cells with Col-I increased CCAAT/enhancer binding protein (C/EBP) DNA binding. Antibody supershift experiments revealed that C/EBP DNA binding activity induced by Col-I depended largely on C/EBPbeta and C/EBPdelta. Immunocytochemistry showed that Col-I induced nuclear translocation of C/EBPbeta and C/EBPdelta, whose activation contributes to COX-2 induction. Overexpression of the dominant-negative mutant form of C/EBP abolished COX-2 induction by Col-I. Col-I also increased cyclic-AMP response element binding protein (CREB) binding to DNA. Inhibition of focal adhesion kinase (FAK) or downstream phosphoinositide 3-kinase and p70S6 kinase by specific chemical inhibitors prevented COX-2 induction by Col-I, and C/EBP and CREB from binding to their consensus DNA oligonucleotides. Experiments using chemical inhibitors or dominant-negative mutant vectors showed that the mitogen-activated protein (MAP) kinase pathways including p38-kinase and extracellular signal-regulated kinase (ERK1/2), but not c-Jun N-terminal kinase (JNK1), simultaneously regulated COX-2 induction by Col-I. This was in agreement with inhibition of Col-I-inducible C/EBP and CREB DNA binding by concomitant treatment with SB203580 and PD98059. These results provide evidence that Col-I induces COX-2 in serum-stimulated macrophages and that the multiple cell signaling pathways involving Src-focal adhesion kinase, phosphoinositide 3-kinase, and MAP kinases regulate COX-2 induction by Col-I via C/EBP and CREB activation.     

Possible mechanisms in latent learning formation investigated by using mutant mice

We examined possible mechanisms in the development of latent learning by methods of behavioral pharmacology and confirmed them by using mutant mice. Mice that received dopamine agonists, a noradrenergic neurotoxin or a traumatic brain injury showed impairment of latent learning. This impairment was suggested to be mediated by imbalance of dopaminergic and noradrenergic systems since the impairment was attenuated by a noradrenaline uptake inhibitor or a dopamine-D2 antagonist. The heterozygous mice for the tyrosine hydroxylase (TH) gene and for the cyclic AMP (cAMP) response element binding protein (CREB) binding protein (CBP) gene showed impairment of latent learning in the water finding task. The spatial learning and hippocampal long-term potentiation (LTP) were normal in both the mutants. TH heterozygous mice showed a reduction of high K(+)-evoked noradrenaline release in the frontal cortex by the microdialysis technique and a reduction of cAMP of the brain cAMP content. The central noradrenergic systems and/or cAMP signal pathways play an important role in latent learning, but not spatial memory. In contrast with TH and CBP mutant mice, nociceptin-knockout mice showed an enhanced retention of latent learning in the water finding task, greater learning ability in the water maze task and larger LTP than wild-type mice. Such mice showed hyperfunction of dopaminergic systems in the cortex. Nociceptin itself induced latent learning impairment in wild-type mice. These results suggest that the nociceptin system seems to play negative roles in learning and memory. In conclusion, the results of mutant mice further supported our previous results of behavioral pharmacology and suggest that the alternation of catecholamine biosynthesis and cAMP signal pathways may play a key role in development of latent learning. They further suggest that the expression of genes mediated by phosphorylated CREB may be involved in the development of latent learning.     

Upregulation of cyclooxygenase-2 by 4-nonylphenol is mediated through the cyclic amp response element activation pathway

The organic compound nonylphenol (NP) belongs to the family of alkylphenols and is a product of industrial synthesis formed during phenol alkylation. Nonylphenol is considered to be an endocrine disruptor due to weak ability to mimic estrogen and subsequently to disrupt the natural balance of hormones in a given organism. Since the endocrine and immune systems share portions of common signaling pathways, it is conceivable that NP may also affect immune system functions. However, the influence of NP on inflammation and macrophages responsiveness to NP is unclear. Thus, the effects of NP were investigated on cyclooxygenase (COX)-2 expression in cultured macrophages. NP induced COX-2 protein and gene expression in murine macrophage RAW264.7 cells and enhanced COX-2 promoter activity and prostaglandin E(2) production. Transfection of RAW264.7 cells with hCOX-2 or various deletion and mutation promoter constructs revealed that the cyclic AMP response element (CRE) was the predominant mediator responsive to NP-induced effects. Moreover, transfection with pCRE-Luc plasmid followed by immunoblotting demonstrated that NP activated CRE sites and CRE binding protein (CREB) phosphorylation. NP also increased nuclear CREB accumulation and CREB binding to the COX-2 promoter. Phosphatidylinositol 3 (PI3)-kinase, Akt, and the mitogen-activated protein kinases (MAP kinases) p38 and JNK were also significantly activated by NP. Our data demonstrate that NP induces COX-2 expression through the PI3-kinase/Akt/MAP kinases/CRE pathway. These findings provide insight into the signal transduction pathways involved in the inflammatory responses induced by NP in macrophages.     

Scopoletin suppresses IL-6 production from fibroblast-like synoviocytes of adjuvant arthritis rats induced by IL-1β stimulation

Scopoletin, a coumarin compound naturally occurring in many medicinal plants, has previously been demonstrated to ameliorate synovial inflammation and destruction of cartilage and bone in adjuvant arthritis (AA) rats. As interleukin (IL)-6 is critically involved in the initiation and development of rheumatoid arthritis (RA), the present study was performed to investigate the effect of scopoletin on IL-6 production from fibroblast-like synoviocytes (FLS) to get insight into its anti-RA mechanisms. FLS were isolated from synovial membrane tissues of AA rats, and stimulated with IL-1β (10 ng/mL). Scopoletin, at concentrations of 15, 30, and 60 μM, was shown to only moderately inhibit FLS proliferation, but dramatically reduce IL-6 production at both mRNA and protein levels. It also inhibited the phosphorylation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK), protein kinase C (PKC) and cAMP response element binding protein (CREB). These findings suggest that scopoletin exerts anti-RA action probably through suppressing IL-6 production from FLS via MAPK/PKC/CREB pathways.