蛋白质巯基亚硝基化对RSC-364细胞cAMP反应元件结合蛋白活性的影响

目的： 观察RSC-364细胞外源性亚硝基化对cAMP反应元件结合蛋白（CREB）活性的影响。方法：提取RSC-364细胞总蛋白,与100 μmol/L还原型谷胱甘肽(GSH)、100 μmol/L NO供体亚硝基化谷胱甘肽(GSNO)、10 mmol/L亚硝基化抑制剂二硫苏糖醇(DTT)及溶剂单独或联合应用孵育15 min完成外源性亚硝基化，采用生物素转化法与Western blotting技术检测亚硝基化蛋白表达水平；分别用溶剂或重组大鼠白细胞介素-1β(rIL-1β)10 μg/L孵育RSC-364细胞1 h，提取细胞核蛋白，经外源性亚硝基化后用电泳迁移率改变分析(electrophoresis mobility shift assay, EMSA)观察亚硝基化作用对CREB活性的影响。结果：RSC-364细胞总蛋白经GSNO孵育后亚硝基化蛋白表达水平明显增高，而应用DTT可抑制亚硝基化水平；GSNO与RSC-364细胞核蛋白孵育后，明显抑制了rIL-1β诱导的CREB活性(P<0.01)，GSNO的作用可被DTT所逆转(P<0.01) 。结论：NO可通过亚硝基化作用抑制RSC-364细胞CREB活性。     

Hippocalcin-deficient mice display a defect in cAMP response element-binding protein activation associated with impaired spatial and associative memory

Hippocalcin is a member of the neuronal calcium sensor (NCS) protein family that is highly expressed in hippocampal pyramidal cells and moderately expressed in the neurons of cerebral cortex, cerebellum and striatum. Here we examined the physiological roles of hippocalcin using targeted gene disruption. Hippocalcin-deficient (-/-) mice displayed no obvious structural abnormalities in the brain including hippocampal formation at the light microscopic level. Deletion of hippocalcin did not result in up-regulation of the hippocalcin-related proteins; neural visinin-like Ca(2+)-binding proteins (NVP) 1, 2, and 3. The synaptic excitability of hippocampal CA1 neurons appeared to be normal, as estimated by the shape of field excitatory postsynaptic potentials elicited by single- and paired-pulse stimuli, and by tetanic stimulation. However, N-methyl-d-aspartate stimulation- and depolarization-induced phosphorylation of cAMP-response element-binding protein (CREB) was significantly attenuated in -/- hippocampal neurons, suggesting an impairment in an activity-dependent gene expression cascade. In the Morris water maze test, the performance of -/- mice was comparable to that of wild-type littermates except in the probe test, where -/- mice crossed the previous location of the platform significantly less often than +/+ mice. Hippocalcin-deficient mice were also impaired on a discrimination learning task in which they needed to respond to a lamp illuminated on the left or right side to obtain food reinforcement. No abnormalities were observed in motor activity, anxiety behavior, or fear learning. These results suggest that hippocalcin plays a crucial role in the Ca(2+)-signaling pathway that underlies long-lasting neural plasticity and that leads to spatial and associative memory.     

Dexamethasone blocks the refeeding-induced phosphorylation of cAMP response element-binding protein in the rat hypothalamus

We previously reported that dexamethasone pretreatment abolishes the refeeding-induced neuronal nitric oxide synthase (nNOS) expression in the rat paraventricular nucleus (PVN). It was reported that nNOS upstream carries cAMP response element (CRE) and nNOS expression is mediated by a CRE-binding protein (CREB)-dependent mechanism. In this study, CREB phosphorylation was co-localized in the nNOS neurons of the rat PVN regardless of feeding conditions. The relative amount of phosphorylated CREB in the hypothalamic tissue lysates increased by 1 h of refeeding following 48 h of food deprivation, and interestingly, this increase was blocked by dexamethasone administration before the food onset. These results suggest that glucocorticoids exert an inhibitory role in CREB phosphorylation directed by nutritional stimuli in the rat hypothalamus, and this inhibition may be related to nNOS gene expression in this brain region.     

Kainate-induced neuronal injury leads to persistent phosphorylation of cAMP response element-binding protein in glial and endothelial cells in the hippocampus

Intracerebroventricular kainate treatment in rats induces neuronal cell death, followed by proliferation and hypertrophy of glial cells in the lesioned area. To further understand the activated signal transduction pathways and to get insights into potential target gene activation, the present study aims to elucidate long-term effects on the phosphorylation state of cAMP response element-binding protein (CREB) in the hippocampal formation. One to four weeks after kainate injection, we found high levels of phosphorylated and hence activated CREB (pCREB) in glial cells of the degenerating CA fields. As shown by electron microscopy, pCREB immunoreactivity was present in reactive astrocytes, oligodendrocyte precursor cells and endothelial cells of blood vessels. It is postulated that pCREB could drive the expression of downstream genes in these cells to promote cell proliferation and survival.     

Age-dependent changes in phosphorylated cAMP response element-binding protein immunoreactivity in motoneurons of the spinal nucleus of the bulbocavernosus of male rats

Phosphorylated cAMP response element-binding protein (CREB) immunoreactivity was examined in motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in young and old male rats by immunohistochemistry. In young animals, intense CREB immunoreactivity was confined to the cell nucleus, but not in the nucleolus of SNB motoneurons. In old animals, both the intensity of CREB immunoreactivity in the nuclei and number of CREB immunoreactive nuclei of the SNB motoneurons were significantly reduced. A marked decline in expression of CREB in the aged SNB motoneurons suggests alternation of cAMP-mediated regulation of gene expression in the SNB system with advancing age.     

Oxidant-mediated cAMP response element binding protein activation: calcium regulation and role in apoptosis of lung epithelial cells

Oxidant stress-mediated regulation of extracellular signal-regulated kinases (ERK1/2) is linked to pathologic outcomes in lung epithelium, yet a role for Ca2+ and Ca2+/cAMP-response element binding protein (CREB) in ERK1/2 signaling has not been defined. In this study, we tested the hypotheses that oxidants induce Ca2+-mediated phosphorylation of ERK and CREB, and that CREB is required for oxidant-induced proliferation and apoptosis. H2O2 initiated an influx of extracellular Ca2+ that was required for phosphorylation of both ERK and CREB in C10 lung epithelial cells. H2O2-mediated CREB phosphorylation was sensitive to MEK inhibition, suggesting that crosstalk between Ca2+, ERK, and CREB signaling pathways contributes to the oxidant-induced response. Reduction of CREB activity, using a dominant-negative CREB construct, inhibited c-fos steady-state mRNA levels, but unexpectedly enhanced bcl-2 steady-state mRNA levels after H2O2 exposure. Whereas inhibition of CREB activity had no detectable effect on H2O2 stimulation of cell cycle, loss of CREB activity significantly reduced the number of cells undergoing apoptosis. These data support a novel communication between Ca2+-ERK1/2 and CREB elicited by H2O2, and further provide evidence that CREB is an important regulator of apoptosis in oxidant-mediated responses of lung epithelial cells.     

Neonatal handling and the maternal odor preference in rat pups: Involvement of monoamines and cyclic AMP response element-binding protein pathway in the olfactory bulb

Early-life environmental events, such as the handling procedure, can induce long-lasting alterations upon several behavioral and neuroendocrine systems. However, the changes within the pups that could be causally related to the effects in adulthood are still poorly understood. In the present study, we analyzed the effects of neonatal handling on behavioral (maternal odor preference) and biochemical (cyclic AMP response element-binding protein (CREB) phosphorylation, noradrenaline (NA), and serotonin (5-HT) levels in the olfactory bulb (OB)) parameters in 7-day-old male and female rat pups. Repeated handling (RH) abolished preference for the maternal odor in female pups compared with nonhandled (NH) and the single-handled (SH) ones, while in RH males the preference was not different than NH and SH groups. In both male and female pups, RH decreased NA activity in the OB, but 5-HT activity increased only in males. Since preference for the maternal odor involves the synergic action of NA and 5-HT in the OB, the maintenance of the behavior in RH males could be related to the increased 5-HT activity, in spite of reduction in the NA activity in the OB. RH did not alter CREB phosphorylation in the OB of both male and females compared with NH pups. The repeated handling procedure can affect the behavior of rat pups in response to the maternal odor and biochemical parameters related to the olfactory learning mechanism. Sex differences were already detected in 7-day-old pups. Although the responsiveness of the hypothalamic–pituitary–adrenal axis to stressors is reduced in the neonatal period, environmental interventions may impact behavioral and biochemical mechanisms relevant to the animal at that early age.     

Cyclic AMP response element-binding protein is required for normal maternal nurturing behavior

Analysis of mice with targeted disruptions of fosB or the gene encoding dopamine beta-hydroxylase suggests that FosB and adrenergic signaling play critical roles in maternal nurturing behavior. The majority of neonates born to null females from either mutation fail to thrive, and virgin mutant females of both lines exhibit impaired pup retrieval. Considering whether FosB and adrenergic signaling might share a signaling pathway important for maternal behavior, we examined the role of a potential intermediary, cyclic AMP response element-binding protein (CREB). Here we report that approximately 40% of neonates (all heterozygous) born to mice lacking the major isoforms of CREB (Creb-alphaDelta-/-) died within several days of birth. In contrast, heterozygotes born to Creb-alphaDelta+/- females thrived. Cross-fostering demonstrated that neonates born to Creb-alphaDelta(-/dagger/-) females thrived when reared by wild-type females, and that Creb-alphaDelta-/- females were capable of rearing neonates whose maternal care was initiated by wild-type females. Further, virgin Creb-alphaDelta-/- females were deficient in pup retrieval despite exhibiting normal investigation of pups and of novel objects. No maternal behavior phenotype was present in mice with a null mutation of the cyclic AMP response element modulator (Crem) gene. Interestingly, the number of cells immunostaining for phospho-CREB (on Ser(133)) in the medial preoptic area of the hypothalamus, a key region for the expression of maternal behavior, increased nearly three-fold in wild-type mice following exposure to pups but not to novel objects. On the other hand, basal expression and induction of FosB in response to pup exposure appeared to be independent of CREB because levels were equivalent between wild-type and Creb-alphaDelta-/- females. These results implicate CREB in maternal nurturing behavior and suggest that CREB is not critical for expression or induction of FosB in adult virgin female mice.     

Reengineering salivary gland cells to enhance protein secretion for use in developing artificial salivary gland device

Salivary glands (SGs) are considered exocrine glands, which mainly secrete water into the oral cavity. Nevertheless, they also exhibit a smaller endocrine secretory pathway toward the bloodstream. The concept of an artificial SG device for exocrine fluid secretion into the oral region in xerostomic patients has been previously studied. The purpose of the current study was to examine the potential of such a device for enhancing bioactive protein secretion. We engineered a plasmid encoding a SG-specific signal peptide sequence adjacent to a normally nonsecreted encoded reporter gene creating a chimera protein, and examined if this construct can enhance secretion from salivary epithelial cells. An N-terminal encoding epidermal growth factor (EGF) sequence was synthesized and inserted into a pGL3 control vector 5' of a firefly luciferase gene, creating a pGL3-EGF signal peptide (pGL3-EGFSP) fused vector. This vector was cotransfected with a pRL-CMV vector containing a Renilla luciferase gene, in 293 cells (serving as controls), and human submandibular gland ductal epithelial (HSG), rat submandibular gland acinar epithelial (SMIE), and rat submandibular gland ductal epithelial (A5) salivary cell lines. The transfected 293, SMIE, and HSG cells showed 8-, 18-, and 40-fold higher luciferase activity, respectively. These observations lead to the concept of an envisioned secretory device, which can serve as a potential biological pump for bioactive proteins.     

Lysophosphatidylcholine induces mast cell secretion and protein kinase C activation.

Lysophosphatidylcholine (lyso-PC), a natural product of phospholipase A2 activity, induced the secretion of both granule-associated beta-hexosaminidase and newly generated leukotriene C4 from mouse bone marrow-derived mast cells. Micromolar concentrations of lyso-PC potentiated the release of beta-hexosaminidase induced by specific antigen but not the calcium ionophore, A23187. Exogenous adenosine was relatively ineffective in enhancing beta-hexosaminidase release from cells challenged with lyso PC. Lyso-PC caused a marked increase in intracellular free-calcium levels and induced the activation of protein kinase C (PKC). These effects could not be abrogated by a prolonged preincubation with pertussis toxin. Staurosporine, an inhibitor of PKC, partially inhibited the abilities of antigen and A23187 to induce beta-hexosaminidase release but was ineffective when lyso-PC was the secretagogue. Lyso-PC appears to activate mast cell PKC, but its ability to stimulate mast cell mediator release appears to be related to its ability to elevate intracellular free calcium concentrations.     

抑郁症与cAMP反应元件结合蛋白基因的关联研究

目的 探讨cAMP反应元件结合蛋白(cyclic adenosine monophosphate response elementbinding protein,CREB1)基因与抑郁症的关联关系.方法 采用聚合酶链反应-限制性片段长度多态性方法检测105个抑郁症核心家系CREB1基因上单核苷酸多态性(single nucleotide polymorphisms,SNP)rs10932201和rs6740584的等位基因与基因型分布情况.进行单位点及单倍型的传递/不平衡检验(transmission disequilibrium test,TDT).结果 CREB1基因上SNP位点rs10932201和rs6740584与抑郁症均无显著性关联,TDT χ2 值分别为2.700(P=0.1004)和0.458(P=0.4986),差异均无统计学意义.单倍型TDT分析结果显示由rs10932201和rs6740584构成的单倍型与抑郁症存在显著性关联,差异有统计学意义(总χ2=23.458,df=3,P=0.00003241).单个单倍型A-C和A-T与抑郁症也均有显著性关联,差异有统计学意义(χ2 值分别为5.405和13.623,P值分别为0.020和0.00022).结论 CREB1基因上SNP位点rs10932201和rs6740584与抑郁症均无显著性关联,但由这2个SNP位点构成的单倍型与抑郁症存在显著性关联,提示CREB1基因rs10932201-rs6740584单倍型可能在抑郁症的遗传学发病机制中具有重要作用.     

Involvement of GANP in B cell activation in T cell-dependent antigen response

Adaptive immunity is dependent on proliferation of antigen-driven B cells for clonal expansion in germinal centers (GCs) against T cell-dependent antigens (TD-Ag), accompanied with somatic hypermutation of variable-region gene and class switching of B cell antigen receptors. To study molecular mechanisms for B cell differentiation in GCs, we have identified and studied a 210kDa GANP protein expressed in GC-B cells. GANP has domains for MCM3-binding and RNA-primase activities and is selectively up-regulated in centrocytes surrounded with follicular dendritic cells (FDCs) upon immunization with TD-Ag in vivo and in B cells stimulated with anti-CD40 monoclonal antibody in vitro, which suggested that GANP plays a certain important role in the maturation of immunoglobulin or selection of B cells in GC during the immune response to TD-Ag. Since this up-regulation has not been detected in T cells in GCs and in Concanavalin A-stimulated T cells in vitro, selective function of GANP molecule on B cell proliferation and differentiation might exist.     

Involvement of thrombin in activation of heparin secretion by mast cells in immobilization stress in rats

Activation of heparin secretion by connective tissue mast cells under conditions of immobilization stress is determined by activation of the sympathoadrenal system, secretion of adrenocorticotropic hormone, and possible generation of thrombin. Generation of thrombin in the blood under these conditions is confirmed by a significant drop in the proenzyme protein C concentration by 23%, a decrease in the activity of factor V (substrate of protein C) by 36%, and prolongation of activated partial thromboplastin time by 40%. It is shown that 30-min immobilization leads to a 3-fold depletion of the heparin pool in mast cells. Intravenous injection of hirudin, a specific thrombin inhibitor, before immobilization slightly diminishes the stimulating effect of stress on heparin secretion. These data suggest that apart from catecholamines and adrenocorticotropic hormone, thrombin generated in the bloodstream during stress also markedly contributes to activation of heparin secretion by mast cells. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 123, No. 2, pp. 143–145, February, 1997     

Involvement of cAMP in neuronal survival and axonal regeneration

In vitro, cAMP elevation alters neuronal responsiveness to diffusible growth factors and overcomes myelin-associated inhibitory molecules. Significant advances have been made recently in understanding the role of increases in cAMP in promoting axonal growth. Importantly, it has now been shown that cAMP elevation can promote axonal regeneration and functional recovery after central nervous system injury. Elevation of cAMP can be achieved via either direct application of cAMP analogs or an inhibitor of the enzyme phosphodiesterase that degrades cAMP in vivo. Current information points to a number of protein kinase A-mediated pathways (mitogen-activated protein kinase/extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/akt pathway activation and Rho inactivation) underlying cAMP elevation-induced neuronal survival and axonal regeneration.