Phosphorylated cAMP Response Element Binding Research Articles

Apurinic/apyrimidinic endonuclease APE1 is required for PACAP-induced neuroprotection against global cerebral ischemia

Inducible DNA repair via the base-excision repair pathway is an important prosurvival mechanism activated in response to oxidative DNA damage. Elevated levels of the essential base-excision repair enzyme apurinic/apyrimidinic endonuclease 1 (APE1)/redox effector factor-1 correlate closely with neuronal survival against ischemic insults, depending on the CNS region, protective treatments, and degree of insult. However, the precise mechanisms by which this multifunctional protein affords protection and is activated by upstream signaling pathways in postischemic neurons are not well delineated. Here we show that intracerebral administration of pituitary adenylate cyclase-activating polypeptide (PACAP), an endogenously occurring small neuropeptide, induces expression of APE1 in hippocampal neurons. Induction of APE1 expression requires PKA- and p38-dependent phosphorylation of cAMP response-element binding and activating transcription factor 2, which leads to transactivation of the APE1 promoter. We further show that PACAP markedly reduces oxidative DNA stress and hippocampal CA1 neuronal death following transient global ischemia. These effects occurred, at least in part, via enhanced APE1 expression. Furthermore, the DNA repair function of APE1 was required for PACAP-mediated neuroprotection. Thus, induction of DNA repair enzymes may be a unique strategy for neuroprotection against hippocampal injury.

Pincher-generated Nogo-A endosomes mediate growth cone collapse and retrograde signaling

Nogo-A is one of the most potent myelin-associated inhibitors for axonal growth, regeneration, and plasticity in the adult central nervous system. The Nogo-A-specific fragment NogoDelta20 induces growth cone collapse, and inhibits neurite outgrowth and cell spreading by activating RhoA. Here, we show that NogoDelta20 is internalized into neuronal cells by a Pincher- and rac-dependent, but clathrin- and dynamin-independent, mechanism. Pincher-mediated macroendocytosis results in the formation of NogoDelta20-containing signalosomes that direct RhoA activation and growth cone collapse. In compartmentalized chamber cultures, NogoDelta20 is endocytosed into neurites and retrogradely transported to the cell bodies of dorsal root ganglion neurons, triggering RhoA activation en route and decreasing phosphorylated cAMP response element binding levels in cell bodies. Thus, Pincher-dependent macroendocytosis leads to the formation of Nogo-A signaling endosomes, which act both within growth cones and after retrograde transport in the cell body to negatively regulate the neuronal growth program.

The memory-enhancing effects of Euphoria longan fruit extract in mice

Aim of the study The fruit of Euphoria longan (Lour.) Steud. (Sapindaceae) is sweet and edible. Dried Euphoria longan fruit is prescribed as a tonic and for the treatment of forgetfulness, insomnia, or palpitations caused by fright in traditional Chinese medicine. The effects of aqueous extract of Euphoria longan fruit (ELE) on learning and memory and their underlying mechanisms were investigated. Materials and methods Aqueous extract of Euphoria longan fruit (ELE) was administered to ICR mice for 14 days. Piracetam was used as a positive control for its known memory-enhancing effects. Memory performances were assessed using the passive avoidance task. The expressions of phosphorylated extracellular signal-regulated kinase (pERK) 1/2, phosphorylated cAMP response element binding protein (pCREB), brain-derived neurotrophic factor (BDNF), doublecortin (DCX) and the incorporation of 5-bromo-2-deoxyuridine (BrdU) in hippocampal dentate gyrus and CA1 regions were investigated using immunohistochemical methods. Results The step-through latency in the ELE-treated group was significantly increased compared with that in the vehicle-treated controls ( P < 0.05) in the passive avoidance task. Piracetam-treated group also showed enhanced cognitive performaces in the passive avoidance task. Immunohistochemical studies revealed that the number of cells immunopositive for BDNF, pCREB, or pERK 1/2 was significantly increased in the hippocampal dentate gyrus and CA1 regions after ELE treatment for 14 days ( P < 0.05). DCX and BrdU immunostaining also revealed that ELE significantly enhanced immature neuronal survival, but not neuronal cell proliferation in the subgranular zone of the dentate gyrus. Conclusions The present results suggest that subchronic administration of aqueous extract of Euphoria longan fruit enhances learning and memory, and that its beneficial effects are mediated, in part, by BDNF expression and immature neuronal survival.

Activation of glutamate transporters in the locus coeruleus paradoxically activates descending inhibition in rats

Descending noradrenergic inhibition is an important endogenous pain-relief mechanism which can be activated by local glutamate signaling. In the present study, we examined the effect of glutamate transporter activation by riluzole in the regulation of activity of locus coeruleus (LC) neurons, which provide the major inhibitory descending noradrenergic projection to the spinal cord. Local injection of riluzole into the LC dose-dependently reduced hypersensitivity in rats after L5-L6 spinal nerve ligation (SNL). This anti-hypersensitivity effect of LC-injected riluzole was blocked by intrathecal administration of the α2-adrenoceptor antagonist idazoxan and intra-LC co-injection of the AMPA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the gap-junction blockers, carbenoxolone (CBX) and meclofenamic acid (MEC). In brainstem slices from normal rats, riluzole increased phosphorylated cAMP response element binding protein (pCREB) expressing nuclei in dopamine-β-hydroxylase (DβH) containing cells in the LC. This riluzole-induced pCREB activation in LC neurons was also blocked by CNQX and CBX. In the primary astrocyte culture, riluzole enhanced glutamate-induced glutamate release. Contrary to expectations, these results suggest that activation of glutamate transporters in the LC results in increase of extracellular glutamate signaling, possibly via facilitation of glutamate release from astrocytes, and activation of LC neurons to induce descending inhibition, and that this paradoxical action of glutamate transporters in the LC requires gap-junction connections.

Activation of extracellular signal-regulated protein kinases 5 in the spinal cord contributes to the neuropathic pain behaviors induced by CCI in rats

Objective: To investigate whether activation and translocation of extracellular signal-regulated kinase 5 (ERK5) is involved in the induction and maintenance of neuropathic pain and observe the effects of activation and translocation of ERK5 on the expression of phosphorylated cAMP response element binding (pCREB) in the chronic neuropathic pain.Methods: Lumbar intrathecal catheters were chronically implanted in male Sprague–Dawley rats. The left sciatic nerve was loosely ligated proximal to the sciatica's trifurcation at ∼ 1.0 mm intervals with 4-0 silk sutures. The phosphorothioate-modified antisense oligonucleotides (AS-ODNs) were intrathecally administered every 12 hours, 1 day pre-chronic constriction injury (CCI) and 3 day post-CCI. Thermal and mechanical nociceptive thresholds were assessed with the paw withdrawal latency to a radiant heat and von Frey filaments. Expressions of phosphorylated ERK5 (pERK5), pCREB, were assessed by both Western blotting and immunohistochemical analysis.Results: Intrathecal injection of ERK5 AS-ODN significantly attenuated CCI-induced mechanical allodynia and thermal hyperalgesia. CCI significantly increased the expression of pERK5 neurons in the ipsilateral spinal dorsal horn to injury, not in the contralateral spinal dorsal horn. The time courses of pERK5 expression showed that the levels of both cytosol and nuclear pERK5 were increased at all points after CCI and reached a peak level on post-operative day 5. CCI significantly increased the expression of pERK5 neurons in the laminae I and II of ipsilateral spinal dorsal horn to injury, not in the contralateral spinal dorsal horn. Phospho-CREB-positive neurons were distributed in all laminae of the bilateral spinal cord. Intrathecal injection AS-ODN markedly suppressed the increase of CCI-induced pERK5, pCREB expression in the spinal cord.Conclusion: The activation of ERK5 pathways contributes to neuropathic pain in CCI rats, and the function of pERK5 may partly be accomplished via the CREB protein-dependent gene expression.

Restoration of amphetamine-induced locomotor sensitization in dopamine D1 receptor-deficient mice

Amphetamine-induced sensitization is thought to involve dopamine D(1) receptors. Using mice lacking dopamine D(1) receptors (D (1) (-/-) ), we found that they exhibited blunted sensitization to low doses of amphetamine, while others using different treatment and testing regimens reported inconsistent results. We investigated whether experimental variables, alteration in gene expression or cholinergic input played a role in amphetamine-induced responses. D (1) (-/-) and wild-type (D (1) (+/+) ) mice pretreated with amphetamine (1 mg/kg, 3-7 days) or various doses of nicotine (chronically but intermittently) were challenged with amphetamine (0.7 and/or 1 mg/kg) after short and long abstinence periods. Expression of brain-derived neurotrophic factor (BDNF) and phosphorylated c-AMP response element binding protein (p-CREB) genes were measured under basal conditions and after acute or repeated amphetamine treatments. D (1) (-/-) mice failed to exhibit amphetamine-induced sensitization following short-term treatments and long abstinence periods, but expressed sensitization following prolonged amphetamine treatment or a shorter abstinence period. Basal expression of p-CREB (but not BDNF) was higher in D (1) (-/-) than D (1) (+/+) mice and was reduced after amphetamine treatment. Prolonged nicotine pretreatment augmented locomotor responses to amphetamine in both genotypes and restored sensitization in D (1) (-/-) mice. D(1) receptors were necessary for induction, but may not be necessary for expression of amphetamine-induced sensitization at low doses. The manifestation of amphetamine sensitization depended on the duration of treatment and length of the withdrawal period. Cholinergic-nicotinic stimulation restored amphetamine-induced sensitization in D (1) (-/-) mice. Enhanced basal expression of p-CREB in D (1) (-/-) mice may represent an adaptive mechanism related to lack of D(1) receptors.

Tanshinone I enhances learning and memory, and ameliorates memory impairment in mice via the extracellular signal‐regulated kinase signalling pathway

The intracellular signalling kinase, extracellular signal-regulated kinase 1/2 (ERK1/2) is required for new memory formation, suggesting that control of ERK signalling might be a target for the treatment of cognitive dysfunction. Previously, we reported that tanshinone congeners have ameliorating effects on drug-induced memory impairment in mice. Here, we have investigated possible modes of action of tanshinone I on learning and memory, associated with ERK phosphorylation. Using immunohistochemical, Western blot techniques, and behavioural testing, we studied the effect of tanshinone I on memory impairment induced by diazepam or dizocilpine (MK-801) in mice. Tanshinone I (2 or 4 mg.kg(-1), p.o.) increased latency times versus vehicle-treated control group in the passive avoidance task. Western blot analysis and immunohistochemical data showed that tanshinone I (4 mg.kg(-1)) increased levels of phosphorylated cAMP response element binding protein (pCREB) and phosphorylated ERK (pERK) in the hippocampus. These increases in pCREB and pERK were blocked by U0126 (inhibitor of ERK1/2), which also prevented the increase in passive avoidance task latency time after tanshinone I. In models of learning and memory impairment induced by diazepam and MK-801, tanshinone I (4 mg.kg(-1)) reversed learning and memory impairments detected by the passive avoidance test. Western blot analysis showed that tanshinone I reversed the diazepam- and MK-801-induced inhibitions of ERK and CREB activation in hippocampal tissues. These effects were also blocked by U0126. Tanshinone I ameliorates the learning and memory impairments induced by diazepam and MK-801 through activation of ERK signalling.

Effect of intrathecal administration of a mixture of butorphanol and ketamine on cAMP-PKA-CREB signal transduction pathway in spinal dorsal horn of rats with inflammatory pain

Objective To investigate the effect of intrathecal administration of a mixture of butorphanol and ketamine on cAMP-PKA-CREB signal transductian pathway in the spinal dorsal ham of the rats with inflammatory pain. Methods Twenty-four male SD rats, weighing 240-280 g,in which intrathecal catheters were successfully placed, were divided into 4 groups randomly (n = 6 each): inflammatory pain group (group IP), butorphanol group (group B), ketamine group (group K), and butorphanol + ketamine group (group BK). The inflammatory pain was induced by injection of 5% formalin 50 μl into the plantar surface of left hind paw. Normal saline 10 μl, butorphannl 12.5 μg, ketamine 50 μg, and a mixture of butorphanol 12.5 μg and ketamine 50 μg was injected intrathecally 30 min before subcutaneous injection of formalin in group IP, B, K and BK respectively.Pain intensity score (PIS) was used to assess pain behavior every 5 min within an hour after subcutaneous injection of formalin. The animals were killed at 2 h after subcutaneous injection of formalin, and the L5 segment of the spinal cord was removed for determination of protein kinase A (PKA) and phosphorylated cAMP response element binding protein (p-CREB) expression using immunohistochemistry. Results Fonnahn administration induced pain behaviour expressed as two phases. PIS scores, PKA and p-CBEB expression, and staining scores were significantly lower during the fast and second phases in group BK than in group IP (P 0.05). Conclusion lntrathecal injection of a mixture of butorphanol and ketamine can reduce inflammatory pain in rats, and the mechanism may be related to the cAMP-PKA-CREB signal transduction pathway. Key words: Butorphanol; Ketamine; Cyclic AMP; Cyclic AMP-dependent protein kinnses; Cyclic AMP response element-binding protein; Pain; Injections, spinal

Regulation of CREB Activation by P38 Mitogen Activated Protein Kinase during Human Primary Erythroblast Differentiation

Among the molecular events underlying erythroid differentiation, we analyzed the signalling pathway leading to cAMP response element binding (CREB) nuclear transcription factor activation. Normal donor blood light density cells differentiated to pro-erythroblasts during the proliferative phase (10 days) of the human erythroblast massive amplification (HEMA) culture, and to orthochromatic erythroblasts, during the differentiation phase (4 additional days) of the culture. Since erythropoietin was present all over the culture, also pro-erythroblasts left in proliferative medium for 14 days continued their maturation without reaching the final steps of differentiation. p38 mitogen activated protein kinase (p38 MAPK) and CREB maximal activation occurred upon 4 days of differentiation induction, whereas a lower activation was detectable in the cells maintained in parallel in proliferative medium (14 days). Interestingly, when SB203580, a specific p38 MAPK inhibitor, was added to the culture the percentage of differentiated cells decreased along with p38 MAPK and CREB phosphorylation. All in all, our results evidence a role for p38 MAPK in activating CREB metabolic pathway in the events leading to erythroid differentiation.

α-Tocopherol decreases the somatostatin receptor-effector system and increases the cyclic AMP/cyclic AMP response element binding protein pathway in the rat dentate gyrus

Neuronal survival has been shown to be enhanced by α-tocopherol and modulated by cyclic AMP (cAMP). Somatostatin (SST) receptors couple negatively to adenylyl cyclase (AC), thus leading to decreased cAMP levels. Whether α-tocopherol can stimulate neuronal survival via regulation of the somatostatinergic system, however, is unknown. The aim of this study was to investigate the effects of α-tocopherol on the SST signaling pathway in the rat dentate gyrus. To that end, 15-week-old male Sprague–Dawley rats were treated daily for 1 week with (+)-α-tocopherol or vehicle and sacrificed on the day following the last administration. No changes in either SST-like immunoreactivity (SST-LI) content or SST mRNA levels were detected in the dentate gyrus as a result of α-tocopherol treatment. A significant decrease in the density of the SST binding sites and an increase in the dissociation constant, however, were detected. The lower SST receptor density in the α-tocopherol-treated rats correlated with a significant decrease in the protein levels of the SST receptor subtypes SSTR1-SSTR4, whereas the corresponding mRNA levels were unaltered. G-protein-coupled-receptor kinase 2 expression was decreased by α-tocopherol treatment. This vitamin induced a significant increase in both basal and forskolin-stimulated AC activity, as well as a decrease in the inhibitory effect of SST on AC. Whereas the protein levels of AC type V/VI were not modified by α-tocopherol administration, ACVIII expression was significantly enhanced, suggesting it might account for the increase in AC activity. In addition, this treatment led to a reduction in Giα1-3 protein levels and in Gi functionality. α-Tocopherol did not affect the expression of the regulator of G-protein signaling 6/7 (RGS6/7). Finally, α-tocopherol induced an increase in the levels of phosphorylated cAMP response element binding protein (p-CREB) and total CREB in the dentate gyrus. Since CREB synthesis and phosphorylation promote the survival of many cells, including neurons, whereas SST inhibits the cAMP-PKA pathway, which is known to be involved in CREB phosphorylation, the α-tocopherol-induced reduction of SSTR observed here might possibly contribute, via increased cAMP levels and CREB activity, to the mechanism by which this vitamin promotes the survival of newborn neurons in the dentate gyrus.

Expression of phosphorylated CREB in the fasted rat hippocampus

Fasting facilitates synaptic plasticity and promotes learning consolidation. However, cellular signaling mechanisms underlying the effect of fasting on memory consolidation are not completely understood. We investigated the effect of fasting on the hippocampal neuron by testing the hypothesis of whether fasting promoted phosphorylation of CREB (c‐AMP response element binding protein), an important nuclear protein to consolidate memories by inducing gene expression. We used in vivo rat model of fasting and applied anti‐phospho‐CREB (Ser133) raised in the rabbit to cardially‐perfused rat brain sections (40 μm thick). The cell layer of CA1 hippocampal subfield in fasted animals exhibited strong expression of phosphorylated CREB (pCREB). As reported previously, the hypothalamus was also strongly immunoreactive and provided a positive control in the present study. Our finding suggests fasting may stimulate cAMP‐dependent signaling cascades in the hippocampal CA1 neuron. A molecule involved in and responsible for the activation of this signaling process is currently under the investigation. Supported by NIH grants R15DA021683 and SC1GM081179 (to MI). NME is an American Physiological Society Undergraduate Summer Research Fellow in 2008.

The neuroprotective effects of the seeds of Cassia obtusifolia on transient cerebral global ischemia in mice

The aim of this study was to determine the mechanism underlying the neuroprotective effects of the ethanolic extract of the seeds of Cassia obtusifolia (COE) (10 or 50 mg/kg/day, p.o) on transient cerebral global ischemia induced by bilateral common carotid artery occlusion (2VO) in mice. Immunohistochemical and western blot studies showed that levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the hippocampal CA1 region at 1 day post-2VO were attenuated by COE (50 mg/kg/day, p.o), which was administered immediately after 2VO. Furthermore, OX-42 – and glial fibrillary acidic protein (GFAP)-positive cell numbers at 4 days post-2VO were markedly attenuated by COE (50 mg/kg/day, p.o) treatment for 4 days in CA1. Viable neurons detected by Nissl at 7 days post-2VO were increased by administering COE (50 mg/kg/day, p.o) for 7 days. In addition, COE increased the expressions of phosphorylated cAMP response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF) in CA1 in naïve-control within 1 and 6 h, respectively, and these expressions were also profoundly increased in 2VO-treated mice by COE at immediately post-2VO. These results suggest that the neuroprotective effects of COE are due to its anti-inflammatory effects and to its upregulation of BDNF expression and CREB phosphorylation.

Transgenic overexpression of aldehyde dehydrogenase-2 rescues chronic alcohol intake-induced myocardial hypertrophy and contractile dysfunction.

Chronic alcoholism leads to the onset and progression of alcoholic cardiomyopathy through toxic mechanisms of ethanol and its metabolite, acetaldehyde. This study examined the impact of altered acetaldehyde metabolism through systemic transgenic overexpression of aldehyde dehydrogenase-2 (ALDH2) on chronic alcohol ingestion-induced myocardial damage. ALDH2 transgenic mice were produced with the chicken beta-actin promoter. Wild-type FVB and ALDH2 mice were placed on a 4% alcohol diet or a control diet for 14 weeks. Myocardial and cardiomyocyte contraction, intracellular Ca(2+) handling, histology (hematoxylin and eosin, Masson trichrome), protein damage, and apoptosis were determined. Western blot was used to monitor the expression of NADPH oxidase, calcineurin, apoptosis-stimulated kinase (ASK-1), glycogen synthase kinase-3beta (GSK-3beta), GATA4, and cAMP-response element binding (CREB) protein. ALDH2 reduced the chronic alcohol ingestion-induced elevation in plasma and tissue acetaldehyde levels. Chronic alcohol consumption led to cardiac hypertrophy, reduced fractional shortening, cell shortening, and impaired intracellular Ca(2+) homeostasis, the effect of which was alleviated by ALDH2. In addition, the ALDH2 transgene significantly attenuated chronic alcohol intake-induced myocardial fibrosis, protein carbonyl formation, apoptosis, enhanced NADPH oxidase p47(phox) and calcineurin expression, as well as phosphorylation of ASK-1, GSK-3beta, GATA4, and CREB. The present results suggest that transgenic overexpression of ALDH2 effectively antagonizes chronic alcohol intake-elicited myocardial hypertrophy and contractile defect through a mechanism that is associated, at least in part, with phosphorylation of ASK-1, GSK-3beta, GATA4, and CREB. These data strongly support the notion that acetaldehyde may be an essential contributor to the chronic development of alcoholic cardiomyopathy.

Regulation of cocaine- and amphetamine-regulated transcript mRNA expression by calcium-mediated signaling in GH3 cells

Cocaine- and amphetamine-regulated-transcript (CART) peptides are associated with multiple physiological processes, including, feeding, body weight, and the response to drugs of abuse. CART mRNA and peptide levels and the expression of the CART gene appears to be under the control of a number of extra- and intra-cellular factors including the transcription factor, cAMP response element binding protein (CREB). Similar to the effects of CART, Ca 2+ signaling leads to the phosphorylation of CREB and has been associated with both feeding and the actions of psychostimulants; therefore, we hypothesized that Ca 2+ may play a role in CART gene regulation. We used real-time PCR (rtPCR) and GH3 cells to examine the effect of ionomycin, which increases intracellular Ca 2+, on CART mRNA levels. Ionomycin increased CART mRNA in a dose- and time-dependent manner. The effect of ionomycin appeared transient as CART mRNA had returned to control levels 3 h following treatment. Calmidazolium and KN93, inhibitors of calmodulin and Ca 2+-modulated protein (CaM) kinases respectively, attenuated the effect of ionomycin (10 μM) on CART mRNA levels suggesting a calmodulin-dependent mechanism. Western immunoblotting indicated that ionomycin increased phosphorylated cAMP response element binding protein (pCREB) levels and electrophoretic mobility shift assay/supershift assay using antibodies against pCREB demonstrated increased levels of a CART oligo/pCREB protein complex. Finally, we showed that injection of ionomycin into the rat nucleus accumbens increases CART mRNA levels. To our knowledge, this is the first study providing evidence that the CART gene is, in part, regulated by Ca 2+/CaM/CREB-dependent cell signaling.

Dopamine D1 receptor, but not dopamine D2 receptor, is a critical regulator for acute cocaine-enhanced gene expression

Objective: The aim of present study is to investigate the roles of dopamine receptor subfamilies and their subsequent molecular events in cocaine-enhanced gene expression in striatum.Methods: Acute cocaine-treated mice models were build to address this issue. Specific antagonists for dopamine D1 and D2 receptors (SCH 23390 and raclopride, respectively) and specific inhibitor for extracellular signal-regulated protein kinase 1/2 (ERK1/2) kinase were pretreated. Immunofluorescence was used to detect the expressions of c-Fos, phosphorylated cAMP response element binding (p-CREB) and phosphorylated Elk-1 (p-Elk-1) in striatum.Results: Acute cocaine injection significantly enhanced expressions of c-Fos, p-CREB and p-Elk-1 in the striatum. Notably, these enhancements were totally blocked to normal level by SCH 23390 pre-treatment, while no changes occurred in the presence of raclopride. Moreover, we found that dopamine D1 receptor was involved in acute cocaine-induced activation of ERK1/2 in the striatum. Blockade of this dopamine D1 receptor-dependent ERK1/2 activation by SL 327 could reduce cocaine-enhanced expressions of c-Fos, p-CREB and p-Elk-1 in the striatum.Discussion: These results suggest that dopamine D1 receptor, but not dopamine D2 receptor, plays a critical role in regulating acute cocaine-enhanced gene expression in the striatum, and ERK1/2 pathway may contribute to this regulation.

Metabolic demand stimulates CREB signaling in the limbic cortex: Implication for the induction of hippocampal synaptic plasticity by intrinsic stimulus for survival

Caloric restriction by fasting has been implicated to facilitate synaptic plasticity and promote contextual learning. However, cellular and molecular mechanisms underlying the effect of fasting on memory consolidation are not completely understood. We hypothesized that fasting-induced enhancement of synaptic plasticity was mediated by the increased signaling mediated by CREB (cAMP response element binding protein), an important nuclear protein and the transcription factor that is involved in the consolidation of memories in the hippocampus. In the in vivo rat model of 18 h fasting, the expression of phosphorylated CREB (pCREB) was examined using anti-phospho-CREB (Ser133) in cardially-perfused and cryo-sectioned rat brain specimens. When compared with control animals, the hippocampus exhibited up to a twofold of increase in pCREB expression in fasted animals. The piriform cortex, the entorhinal cortex, and the cortico-amygdala transitional zone also significantly increased immunoreactivities to pCREB. In contrast, the amygdala did not show any change in the magnitude of pCREB expression in response to fasting. The arcuate nucleus in the medial hypothalamus, which was previously reported to up-regulate CREB phosphorylation during fasting of up to 48 h, was also strongly immunoreactive and provided a positive control in the present study. Our findings demonstrate a metabolic demand not only stimulates cAMP-dependent signaling cascades in the hypothalamus, but also signals to various limbic brain regions including the hippocampus by activating the CREB signaling mechanism. The hippocampus is a primary brain structure for learning and memory. It receives hypothalamic and arcuate projections directly from the fornix. The hippocampus is also situated centrally for functional interactions with other limbic cortexes by establishing reciprocal synaptic connections. We suggest that hippocampal neurons and those in the surrounding limbic cortexes are intimately involved in the metabolism-dependent plasticity, which may be essential and necessary for successful achievement of adaptive appetitive behavior.

Trichosanthin suppresses HeLa cell proliferation through inhibition of the PKC/MAPK signaling pathway

Trichosanthin (TCS) possesses a broad spectrum of biological and pharmacological activities, including anti-tumor activities. Our previous studies have shown that TCS inhibits HeLa cell proliferation by activating the apoptotic pathway. In particular, the transcriptional factor cAMP response element binding (CREB) protein plays a pivotal role in apoptotic HeLa cells. However, no information, to date, is available about the signaling pathways involved in the inhibition of cell proliferation induced by TCS. The present study showed that PKA and PKC activities were significantly inhibited by TCS treatment. However, specific inhibitor of PKA activity failed to affect the inhibition of HeLa cell proliferation induced by TCS, even in the presence of cAMP agonists. In contrast, PKC activator/inhibitor significantly attenuated/enhanced the inhibitory effect of TCS on cell proliferation. In particular, the reversed effect of cAMP agonist on cell proliferation was partly prevented by PKC, ERK1/2, and p38 MAPK blockade. Consistent with these results, the reversed effect of cAMP agonists on CREB phosphorylation was significantly decreased by inhibitors of these kinases, but not PKA inhibitor. Therefore, our results suggested that HeLa cell proliferation was inhibited by TCS via suppression of PKC/MAPK signaling pathway.

The coactivators CBP/p300 and the histone chaperone NAP1 promote transcription-independent nucleosome eviction at the HTLV-1 promoter

The human T cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T cell leukemia/lymphoma. The multifunctional virally encoded oncoprotein Tax is responsible for malignant transformation and potent activation of HTLV-1 transcription. Tax, in complex with phosphorylated cAMP response element binding protein (pCREB), strongly recruits the cellular coactivators CREB binding protein (CBP)/p300 to the viral promoter concomitant with transcriptional activation. Although the mechanism of activator/coactivator-mediated transcriptional activation is poorly understood, the recruitment of CBP/p300 by regulatory factors appears to function, in part, by promoting changes in chromatin architecture that are permissive to transcriptional activation. Here, we show that CBP/p300 recruitment promotes histone acetylation and eviction of the histone octamer from the chromatin-assembled HTLV-1 promoter template in vitro. Nucleosome disassembly is strictly acetyl-CoA dependent and is not linked to ATP utilization. We find that the histone chaperone, nucleosome assembly protein 1 (NAP1), cooperates with CBP/p300 in eviction of the acetylated histones from the chromatin template. These findings reveal a unique mechanism in which the DNA-bound Tax/pCREB complex recruits CBP/p300, and together with NAP1, the coactivators cooperate to dramatically reduce nucleosome occupancy at the viral promoter in an acetylation-dependent and transcription-independent fashion.

Bilirubin as an endogenous modulator of neurotrophin redox signaling

Bilirubin is neurotoxic upon excess accumulation in the brain, but it also plays important physiological roles related to its antioxidant properties. Here we report that exposure of PC12 and primary rat cerebellar granule neurons to bilirubin (0.5-10 microM) drastically decreases nerve growth factor (NGF)/brain-derived neurotrophic factor signaling to Akt and extracellular signal-regulated kinases (ERKs), indicating a direct interference of the molecule with crucial prosurvival signaling pathways. This effect likely involves the scavenging capacity of bilirubin, the latter being able to inhibit, in PC12 cells, accumulation of intracellular reactive oxygen species and phosphorylation of Akt and ERKs in response to extracellular hydrogen peroxide. Interestingly, in the absence of exogenous growth factor, bilirubin elicited the phosphorylation of ERKs and of the cAMP responsive element binding (CREB) transcription factor, a signature of NGF-dependent survival signaling. These growth factor-like signaling effects were paralleled by the induction of the neuronal nitric oxide synthase (nNOS) and generation of nitric oxide (NO). Pharmacological dissection of the signaling cascade triggered by bilirubin revealed that phosphorylation of ERKs requires NO signaling through soluble guanylyl cyclase, and, further upstream, influx of extracellular calcium is necessary for nNOS induction and NO release, likely through calcium-dependent phosphorylation of CREB. Importantly, the cascade elicited by bilirubin through NO and ERK is cytoprotective, as revealed by exacerbated bilirubin toxicity in cultures treated by either NOS or MEK inhibitors. Taken together, these observations indicate an important action of bilirubin on redox signaling by neurotrophins, with either inhibitory or agonistic effects based on growth factor availability.

Bone morphogenetic protein-4 interacts with activin and GnRH to modulate gonadotrophin secretion in LβT2 gonadotrophs

We have shown previously that, in sheep primary pituitary cells, bone morphogenetic proteins (BMP)-4 inhibits FSHβ mRNA expression and FSH release. In contrast, in mouse LβT2 gonadotrophs, others have shown a stimulatory effect of BMPs on basal or activin-stimulated FSHβ promoter-driven transcription. As a species comparison with our previous results, we used LβT2 cells to investigate the effects of BMP-4 on gonadotrophin mRNA and secretion modulated by activin and GnRH. BMP-4 alone had no effect on FSH production, but enhanced the activin+GnRH-induced stimulation of FSHβ mRNA and FSH secretion, without any effect on follistatin mRNA. BMP-4 reduced LHβ mRNA up-regulation in response to GnRH (±activin) and decreased GnRH receptor expression, which would favour FSH, rather than LH, synthesis and secretion. In contrast to sheep pituitary gonadotrophs, which express only BMP receptor types IA (BMPRIA) and II (BMPRII), LβT2 cells also express BMPRIB. Smad1/5 phosphorylation induced by BMP-4, indicating activation of BMP signalling, was the same whether BMP-4 was used alone or combined with activin±GnRH. We hypothesized that activin and/or GnRH pathways may be modulated by BMP-4, but neither the activin-stimulated phosphorylation of Smad2/3 nor the GnRH-induced ERK1/2 or cAMP response element-binding phosphorylation were modified. However, the GnRH-induced activation of p38 MAPK was decreased by BMP-4. This was associated with increased FSHβ mRNA levels and FSH secretion, but decreased LHβ mRNA levels. These results confirm 1. BMPs as important modulators of activin and/or GnRH-stimulated gonadotrophin synthesis and release and 2. important species differences in these effects, which could relate to differences in BMP receptor expression in gonadotrophs.