Phosphorylated cyclic-AMP Response Element Binding Research Articles

Chaperone-mediated autophagy (CMA) alleviates cognitive impairment by reducing neuronal death in sepsis-associated encephalopathy (SAE)

Sepsis-associated encephalopathy (SAE) is a common and severe complication of sepsis, which causes long-term neurological deficits, such as cognitive impairment. Despite extensive research, there is still lack of specific treatments for SAE. Chaperone-mediated autophagy (CMA), a selective type of autophagy, has been reported to be related to cognitive dysfunctions in many neurodegenerative diseases. The aim of this study was to investigate the alteration of CMA activity in the hippocampus of SAE mice and explore the neuroprotective effect of enhanced CMA. Cecal ligation and puncture (CLP) was conducted to induce SAE. In the contextual fear conditioning test, the ratio of freezing time of CLP mice significantly decreased compared with that of the mice in the Sham group, indicating cognitive impairment in SAE mice. The expression of lysosome-associated membrane protein type 2A (Lamp2a) and chaperone heat shock cognate 71 kDa protein (Hsc70), positive markers for CMA activity, decreased in hippocampal neurons of SAE mice. Although overexpression of Lamp2a in neurons via adeno-associated virus injection in the hippocampus had little effect on the mortality of septic mice, this intervention significantly alleviated the memory impairments in contextual fear conditioning test, Y-maze test and novel objective recognition test, and attenuated the neural death observed in SAE mice. We further demonstrated that the overexpression of Lamp2a in the hippocampus increased the expression of phosphorylated cyclic-AMP response element binding protein (p-CREB), brain-derived neurotrophic factor (BDNF) and B-cell lymphoma-2 (Bcl-2), and suppressed the expression of cleaved caspase-3. Taken together, our study results suggested that the upregulation of CMA activity ameliorated cognitive impairments and neuron loss in SAE mice partially through the p-CREB-BDNF/Bcl-2 signaling pathways, providing a potential therapeutic target for SAE.

Effects of inhibiting astrocytes and BET/BRD4 chromatin reader on spatial memory and synaptic proteins in rats with Alzheimer's disease.

Communication between astrocytes and neurons has a profound effect on the pathophysiology of Alzheimer's disease (AD). Astrocytes regulate homeostasis and increase synaptic plasticity in physiological situations, however, they become activated during the progression of AD. Whether or not these reactions are supportive or detrimental for the central nervous system have not been understood yet. Considering epigenetic regulation of neuroinflammatory genes by chromatin readers, particularly bromodomain and extraterminal domain (BET) family, here we examined the effect of chronic co-inhibition of astrocytes metabolism (with fluorocitrate) and also BRD4 (with JQ1) on cognition deficit at early stages of AD. Forty adult male Wistar rats underwent stereotaxic cannulation for inducing AD by intrahippocampal injection of Aβ1-42 (4μg/8μl/rat). Then animals were divided into five groups of Saline+DMSO, Aβ + saline+DMSO, Aβ + JQ1, Aβ + FC (fluorocitrate), and Aβ + JQ1 + FC and received the related treatments. Two weeks later, spatial memory was recorded by Morris Water Maze (MWM), and the levels of phosphorylated cyclic-AMP response element binding protein (CREB), postsynaptic density 95 (PSD95), synaptophysin (SYP), and tumor necrosis factor-alpha (TNF-α) were measured in the hippocampus by western blotting and RT-qPCR. Administration of JQ1 significantly improved both acquisition and retrieval of spatial memory, which were evident by decreased escape latency and increased total time spent (TTS) in target quadrant, and significant rise in p-CREB, PSD95, and synaptophysin compared with Aβ + saline+DMSO group. In contrast, both groups receiving FC demonstrated memory decline, and reduction in p-CREB, PSD95 and synaptophysin in parallel with increase in TNF-α. Our data indicate that chronic inhibition of BRD4 significantly restores memory impaired by amyloid β partly via CREB signaling and upregulating synaptic proteins of PSD95 and synaptophysin. However, inhibition of astrocytes nullifies the memory-boosting effects of JQ1 and reduces CREB/PSD95/synaptophysin levels in hippocampus.

Regulation of basal expression of hepatic PEPCK and G6Pase by AKT2.

Hepatic glucose metabolism signaling downstream of insulin can diverge to multiple pathways including AKT. Genetic studies suggest that AKT is necessary for insulin to suppress gluconeogenesis. To specifically address the role of AKT2, the dominant liver isoform of AKT in the regulation of gluconeogenesis genes, we generated hepatocytes lacking AKT2 (Akt2-/-). We found that, in the absence of insulin signal, AKT2 is required for maintaining the basal level expression of phosphoenolpyruvate carboxyl kinase (PEPCK) and to a lesser extent G6Pase, two key rate-limiting enzymes for gluconeogenesis that support glucose excursion due to pyruvate loading. We further showed that this function of AKT2 is mediated by the phosphorylation of cyclic AMP response element binding (CREB). Phosphorylation of CREB by AKT2 is needed for CREB to induce the expression of PEPCK and likely represents a priming event for unstimulated cells to poise to receive glucagon and other signals. The inhibition of gluconeogenesis by insulin is also dependent on the reduced FOXO1 transcriptional activity at the promoter of PEPCK. When insulin signal is absent, this activity appears to be inhibited by AKT2 in manner that is independent of its phosphorylation by AKT. Together, this action of AKT2 on FOXO1 and CREB to maintain basal gluconeogenesis activity may provide fine-tuning for insulin and glucocorticoid/glucagon to regulate gluconeogenesis in a timely manner to meet metabolic needs.

Therapeutic Effect of a Novel Chimeric Molecule Targeting Both Somatostatin and Dopamine Receptors on Growth Hormone-Secreting Pituitary Adenomas.

BackgroundAcromegaly is a rare disease primarily caused by growth hormone (GH)-secreting pituitary adenomas, and its treatment is costly. Moreover, some patients are unresponsive to treatment. Hence, there are increasing efforts to develop new drugs with improved effectiveness for this disease. BIM23B065 is a novel chimeric molecule that acts on both somatostatin and dopamine receptors. This study aimed to investigate the effects of BIM23B065 compared with those of a somatostatin receptor analog and a dopamine agonist.MethodsThe effects of BIM23B065 on the proliferation, GH and insulin-like growth factor-1 (IGF-1) levels, and extracellular signal-regulated kinase (ERK) 1/2 and cyclic AMP response element binding (CREB) phosphorylation of GH3 cells were investigated with MTS assay, enzyme-linked immunosorbent assay, and Western blotting, respectively. The dosage and treatment duration of BIM23B065 were tested in animal models of GH-secreting pituitary adenoma. The effect of BIM23B065 (3 mg/kg/day) on changes in IGF-1 levels before and after treatment was further investigated.ResultsIn vitro, BIM23B065 treatment decreased GH release in the culture media and downregulated ERK 1/2 and CREB phosphorylation to 22% and 26%, respectively. In vivo, IGF-1 expression decreased to 50 % after 4 weeks of treatment with BIM23B065 using an osmotic pump implant. Moreover, magnetic resonance imaging results showed that the tumor size decreased significantly following treatment with BIM23B065 for 4 weeks.ConclusionThe novel chimeric molecule was effective in decreasing IGF-1 and GH levels and may serve as an effective therapeutic agent for acromegaly.

Effect of dapagliflozin, a sodium-glucose co-transporter-2 inhibitor, on gluconeogenesis in proximal renal tubules.

To investigate the effect of dapagliflozin, a sodium-glucose co-transporter-2 (SGLT2) inhibitor, on renal gluconeogenesis in vitro, ex vivo and in vivo. We treated HK-2 cells (human renal proximal tubule cells) and mouse primary renal proximal tubule cells with dapagliflozin, and evaluated the process of renal gluconeogenesis. We also examined the effect of dapagliflozin on renal gluconeogenesis in normoglycaemic and hyperglycaemic mice. Dapagliflozin enhanced renal gluconeogenesis in vitro, ex vivo and in vivo. It increased phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), peroxisome proliferative activated receptor-gamma co-activator 1α (PGC-1α) and phosphorylated cyclic-AMP response element binding protein (CREB) expression and decreased phosphorylated Forkhead Box O1 (FOXO1) expression in HK-2 cells, mouse primary renal proximal tubule cells, and the mouse renal cortex. Glutamine enhanced the gluconeogenic effect of dapagliflozin in HK-2 cells. Also, dapagliflozin increased 14 C-glutamine utilization in HK-2 cells. Glucagon did not affect dapagliflozin-induced enhancement in renal gluconeogenesis in HK-2 cells. SGLT2 gene knockdown with siRNA resulted in an increase of gluconeogenic gene expression and associated transcription factors in HK-2 cells. Dapagliflozin reduced fasting plasma glucose levels and improved oral glucose tolerance and insulin tolerance in high-fat diet-fed hyperglycaemic mice, although renal gluconeogenesis was enhanced. Dapagliflozin increased levels of gluconeogenic enzyme in the renal cortex and consequently increased renal gluconeogenesis, which is mediated by SGLT2 inhibition.

Contextual Fear Conditioning Alter Microglia Number and Morphology in the Rat Dorsal Hippocampus.

Contextual fear conditioning is a Pavlovian conditioning paradigm capable of rapidly creating fear memories to contexts, such as rooms or chambers. Contextual fear conditioning protocols have long been utilized to evaluate how fear memories are consolidated, maintained, expressed, recalled, and extinguished within the brain. These studies have identified the lateral portion of the amygdala and the dorsal portion of the hippocampus as essential for contextual fear memory consolidation. The current study was designed to evaluate how two different contextual fear memories alter amygdala and hippocampus microglia, brain derived neurotrophic factor (BDNF), and phosphorylated cyclic-AMP response element binding (pCREB). We find rats provided with standard contextual fear conditioning to have more microglia and more cells expressing BDNF in the dentate gyrus as compared to a context only control group. Additionally, standard contextual fear conditioning altered microglia morphology to become amoeboid in shape – a common response to central nervous system insult, such as traumatic brain injury, infection, ischemia, and more. The unpaired fear conditioning procedure (whereby non-reinforced and non-overlapping auditory tones were provided at random intervals during conditioning), despite producing equivalent levels of fear as the standard procedure, did not alter microglia, BDNF or pCREB number in any dorsal hippocampus or lateral amygdala brain regions. Despite this, the unpaired fear conditioning protocol produced some alterations in microglia morphology, but less compared to rats provided with standard contextual fear conditioning. Results from this study demonstrate that contextual fear conditioning is capable of producing large alterations to dentate gyrus plasticity and microglia, whereas unpaired fear conditioning only produces minor changes to microglia morphology. These data show, for the first time, that Pavlovian fear conditioning protocols can induce similar responses as trauma, infection or other insults within the central nervous system.

Involvement of the Akt-dependent CREB signaling pathway in hydrogen-peroxide-induced early growth response protein-1 expression in rat vascular smooth muscle cells 1.

Increased generation of reactive oxygen species is believed to play a key role in the pathophysiology of cardiovascular diseases. Excessive growth and proliferation of vascular smooth muscle cells (VSMCs) have been suggested to be major contributors to vascular dysfunction. Potential involvement of early growth response protein-1 (Egr-1), a zinc finger transcription factor, in the development of vascular diseases has been suggested. Recent studies have shown that the reactive oxygen species hydrogen peroxide (H2O2) increases Egr-1 expression in VSMCs; however, signaling events leading to H2O2-induced Egr-1 expression are not fully understood. Therefore, we aimed to determine the signaling pathways implicated in H2O2-induced Egr-1 expression in rat VSMCs. Pharmacological blockade of the phosphatidylinositol 3-kinase/Akt pathway by wortmannin or SC66 significantly inhibited the protein and mRNA levels of Egr-1 induced by H2O2. H2O2-induced Egr-1 expression was associated with increased phosphorylation of cyclic AMP response element-binding (CREB) protein, and pharmacological inhibition or silencing of Akt attenuated both H2O2-induced CREB phosphorylation and Egr-1 expression. Moreover, RNA interference-mediated depletion of CREB almost completely suppressed the stimulatory effect of H2O2 on Egr-1 expression. Pharmacological blockade or silencing of c-Src resulted in significant suppression of H2O2-induced Egr-1 expression as well as Akt and CREB phosphorylation. These data show that H2O2 enhances the expression of Egr-1, which was associated with increased phosphorylation of Akt, and H2O2 triggers its effects on Egr-1 expression through c-Src-mediated Akt and CREB-dependent signaling events in VSMCs.

Upregulation of P21-Activated Kinase 1 (PAK1)/CREB Axis in Squamous Non-Small Cell Lung Carcinoma

Background/Aims: p21-activated Ser/Thr kinase 1 (PAK1) is essential for the genesis and development of many cancers. The purpose of this study was to investigate the role of the PAK1–cyclic AMP response element-binding (CREB) axis in non-small cell lung cancer (NSCLC) tumorigenesis and its related mechanisms. Methods: Western blot assay and immunohistochemical staining were employed to investigate the PAK1 and CREB expression in the tissue microarray of human squamous NSCLC. Co-immunoprecipitation and immunofluorescence confocal assays were performed to determine the link between PAK1 and CREB. NSCLC xenograft models were used to study oncogenic function of PAK1 in vivo. Results: We observed that PAK1 and CREB expression levels were significantly elevated in human squamous NSCLC-tissue specimens, compared with those in adjacent normal bronchial or bronchiolar epithelial-tissue specimens, as well as their phosphorylated forms, based on western blotting. We showed in vitro that PAK1 knockdown by small-interfering RNA (siRNA) blocked CREB phosphorylation, whereas plasmid-based PAK1 overexpression resulted in CREB phosphorylation at Ser133, based on western blotting. In addition, PAK1 interacted with CREB in co-immunoprecipitation assays. Additionally, our in vitro findings detected by flow cytometry revealed that PAK1 silencing attenuated cell cycle progression, inducing apoptosis. Inhibition of PAK1 expression reduced tumor sizes and masses by modulating CREB expression and activation in xenograft models. Conclusion: These results suggest a novel mechanism whereby the PAK1–CREB axis drives carcinogenesis of squamous-cell carcinomas, and have important implications in the development of targeted therapeutics for squamous-cell lung cancer.

Adiponectin down-regulates CREB and inhibits proliferation of A549 lung cancer cells

IntroductionAdipokines are known to play a relevant role in a number of cancer related molecular pathways. Adiponectin is a major adipokine with anti-inflammatory and beneficial metabolic actions. Furthermore, it has been shown to exert anti-carcinogenic effects in various tumor models and some clinical studies suggested an inverse relationship between circulating levels of adiponectin and an increased risk for development of malignancies. On the other hand, the cyclic AMP response element binding (CREB) transcription factor has been clearly linked to lung cancer. Methodswe analyzed cell proliferation, cell cycle of A549 cells treated with adiponectin as well as CREB activation status in human lung adenocarcinoma A549 cells and in non-small cell lung cancer (NSCLC) samples. Resultsadiponectin treatment, at concentrations ranging between 5 and 50 μg/ml mimicking human serum levels, has a significant effect on reducing tumor cell proliferation of A549 cells, mainly by altering cell cycle progression. Importantly, we provide evidence that adiponectin clearly inhibits in a dose- and time-dependent manner CREB phosphorylation (activation) and, at least in part, also the level of CREB protein itself, preceding and accompanying the anti-proliferative effects in response to adiponectin. Moreover, in agreement with previous studies demonstrating that CREB over-expression occurs in many tumors, we also show by western-blotting from lung specimen that CREB is significantly up-regulated in NSCLC samples compared to adjacent normal tissues from six patients. ConclusionsOverall, our results represent the first evidence of CREB inhibition by adiponectin and may provide new insight into therapeutic strategies for lung cancer.

BE360, a new selective estrogen receptor modulator, produces antidepressant and antidementia effects through the enhancement of hippocampal cell proliferation in olfactory bulbectomized mice

We have reported that the carborane compound BE360 is a novel selective estrogen receptor modulator and new therapy option for osteoporosis. The aim of this study was to explore the effects and underlying mechanisms of BE360 on depressive-like behavior and memory impairment in the olfactory bulbectomized (OBX) mice, an experimental animal model of depression and dementia. BE360 was administered subcutaneously to mice using a mini-osmotic pump for 2 weeks. Depressive-like behavior was measured as the reduced intake of a sweet solution in the sucrose preference test. Short-term memory was assessed using the Y-maze test. Cell proliferation was assessed by the analysis of cells expressing 5-bromo-2′-deoxyuridine (BrdU) uptake. The expression of phosphorylated cyclic-AMP response element binding protein (pCREB) and brain-derived neurotrophic factor (BDNF) were measured by immunoblot. The depressive-like behavior and memory impairment in OBX mice were improved by the chronic treatment with BE360. Immunohistochemical analysis showed that the number of BrdU-positive cells in the dentate gyrus of the hippocampus significantly decreased in OBX mice whereas they increased after the chronic treatment with BE360. Immunoblotting studies revealed that pCREB and BDNF were significantly increased in the hippocampus of OBX mice treated with BE360. The present study has shown that BE360 has antidepressant and antidementia effects characterized by hippocampal cell proliferation potentially activated via CREB/BDNF signaling pathways. These results indicate that BE360 may have valuable therapeutic potential against depression and neurodegenerative diseases.

Abstract 924: GM-CSF induces CREB signaling pathways and modulates tobacco carcinogen-induced pancreatic tumorigenesis

Abstract Introduction: Nicotine and nitrosamine exposure from smoking causes pancreatic cell injury and contributes to a cascade of oncogenic events that may be contributing to the rising rate of pancreatic cancer (PDAC). Cytokines activate kinases and transcription factors including cyclic AMP response element binding (CREB) protein. CREB activation through phosphorylation regulates diverse cellular responses. We studied whether granulocyte-macrophage colony stimulating factor (GM-CSF)-dependent phosphorylated CREB plays a role in smoking-induced pathogenesis of PDAC. Experimental procedure: Human tissue microarray analysis was performed to determine the significance of pCREB expression amongst smokers and non-smokers. Total RNA extracted from immortalized human pancreatic ductal cell (H6c7) and pancreatic intraepithelial neoplasia (PanIN) mouse cell lines (LSL-KrasG12D/+; Pdx1Cre/+) were treated with tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and RNA sequencing (RNA-seq) was performed. Network, pathway and functional analyses of the transcriptome were conducted. To analyze the signaling pathway involved in NNK-induced tumorigenicity, we performed a phospho-kinase antibody array and cytokine antibody array on NNK treated H6c7 and PanIN cells or conditioned media (CM) protein. Western blot and ELISA were used to validate the array data findings. NNK-induced activation of GM-CSF, G-CSF and IL-6 were blocked using monoclonal antibodies. PKT (Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox) mice and nude mice with MiaPaCa2 flank xenografts were treated with MEK inhibition (AZD6244) with or without NNK/in vivo smoking. Tumors from these mice were immunoblotted for phosphorylation of MEK1/2, MSK1/2, RSK and CREB. PDAC cells with CREB siRNA or human rGM-CSF were treated with NNK and analyzed for in vitro functional assays and EMT characteristics. Results: Expression of pCREB was significantly higher (p<0.001) in smokers when compared to non-smokers. Overall survival of smokers with high pCREB expression in their primary tumor was associated with significantly decreased median survival when compared to non-smokers with low pCREB expression. Exposure of cells to NNK resulted in phosphorylation of CREB, c-Jun and β-catenin, and release of GM-CSF, G-CSF and IL-6. RNA-seq analysis confirmed activation of MEK/ERK signaling. Studies were performed to elucidate the possible regulatory mechanism behind NNK mediated induction of GM-CSF and its downstream signaling. Importantly, blocking GM-CSF inhibited NNK-induced phosphorylation of CREB and it was mediated through MEK signaling. The in vivo and in vitro tumorigenicity assays showed an increase in the tumorigenic potential and EMT of NNK treated PDAC cells. Conclusions: Our study demonstrates that NNK induces pancreatic tumorigenesis through GM-CSF mediated activation of CREB. Citation Format: Jason Castellanos, Kumaraswamy Honnenahally, Chanjuan Shi, Nipun Merchant, Nagaraj Nagathihalli. GM-CSF induces CREB signaling pathways and modulates tobacco carcinogen-induced pancreatic tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 924. doi:10.1158/1538-7445.AM2015-924

Abstract T P226: Cilostazol Can Attenuate Oxidative Stress by Suppressing Nox2 on Neurons in Mice Permanent Focal Ischemia

It has been reported that phosphodiesterase (PDE) 3 inhibitor cilostazol, which is used as an antiplatelet drug, could ameliorate ischemic brain injury. We aimed to investigate how cilostazol affected permanent focal ischemia. Male Balb/c mice were subjected to permanent middle cerebral artery occlusion. They were treated with cilostazol (10- mg/kg or 20-mg/kg) or vehicle 30 min and 24 h post-ischemia, and the infarct volume was assessed 48 h post-ischemia. The animals treated with 20-mg/kg cilostazol or vehicle were sacrificed 6 h or 24 h post-ischemia, and then the immunohistochemistry was performed to the brain sections. We demonstrated that the treatment with 20- mg/kg cilostazol significantly reduced infarct volume (70.1% vs. vehicle group). Immunohistochemistry against 8-hydroxydeoxyguanosine (OHdG) showed that some neurons suffered from oxidative stress in peri-infarct 6 h post-ischemia. The treatment with cilostazol reduced the number of 8-OHdG-positive neurons significantly (65.8 ± 33.5% in vehicle group and 21.3 ± 9.9% in cilostazol group). Moreover, the number of NADPH oxidase (NOX) 2-positive neurons was also reduced significantly (57.0 ± 13.6% in vehicle group and 26.5 ± 16.7% in cilostazol group). On the other hand, immunohistochemistry against phosphorylated cyclic-AMP response element binding protein (pCREB) showed that the number of the pCREB-positive neurons in peri-infarct of the cilostazol-treated animals was significantly higher than in vehicle-treated ones 24 h post-ischemia. These findings suggested that cilostazol could have multi-mechanisms to ameliorate the tissue damage, one was to attenuate oxidative stress by suppressing NOX2 on ischemic neurons, and another was anti-apoptotic effect through the pathway of Akt/pCREB.

Neuroprotective effects of cilostazol are mediated by multiple mechanisms in a mouse model of permanent focal ischemia

The phosphodiesterase (PDE) 3 inhibitor cilostazol, used as an anti-platelet drug, reportedly can also ameliorate ischemic brain injury. Here, we investigated the effects of cilostazol in a permanent focal ischemia mice model. Male Balb/c mice were subjected to permanent middle cerebral artery occlusion. Mice were then treated with either cilostazol (10 or 20mg/kg) or vehicle administered at 30min and 24h post-ischemia, and infarct volumes were assessed at 48h post-ischemia. Mice treated with 20mg/kg of cilostazol or vehicle were sacrificed at 6h or 24h post-ischemia and immunohistochemistry was used for brain sections. Treatment with 20mg/kg of cilostazol significantly reduced infarct volumes to 70.1% of those with vehicle treatment. Immunohistochemistry results for 8-hydroxydeoxyguanosine (OHdG) expression showed that some neurons underwent oxidative stress around the ischemic boundary zone at 6h post-ischemia. Cilostazol treatment significantly reduced the percentage of 8-OHdG-positive neurons (65.8±33.5% with vehicle and 21.3±9.9% with cilostazol). Moreover, NADPH oxidase (NOX) 2-positive neurons were significantly reduced with cilostazol treatment. In contrast, immunohistochemistry results for phosphorylated cyclic-AMP response element binding protein (pCREB) showed that there were significantly more pCREB-positive neurons around the ischemic boundary zone of cilostazol-treated mice than in those of vehicle-treated mice at 24h post-ischemia. These results suggested that cilostazol might have multiple mechanisms of action to ameliorate ischemic tissue damage, by attenuating oxidative stress mediated by suppressing NOX2 expression by ischemic neurons and an anti-apoptotic effect mediated through the pCREB pathway.

Pain behavior and the expression of phosphorylated cAMP response element binding protein and brain-derived neurotrophic factor in dorsal horn of spinal cord in a mouse model of bone cancer pain

Objective To investigate the role of phosphorylated cyclic-AMP response element binding protein (pCREB) and brain-derived neurotrophic factor (BDNF) in bone cancer pain,via observing the change of pain behavior and the expression of pCREB and BDNF in dorsal horn of spinal cord.Methods A total of 52 male mice (C3H/HeJ) were randomly divided into tumor group (group T,n=26)and sham group (group S,n=26).In group T,bone cancer pain was induced by implantation 20 μl of α-minimal essence medium (α-MEM) containing osteosarcoma cells into the intramedullary space of right femur.In group S,20 μl culture medium α-MEM containing no cancer cells was injected instead.Pain behavior of animals in both groups was assessed by the spontaneous lifting times (SLTs) and paw withdrawal mechanical threshold (PWMT) measured at 1 day before and at 4,7,10,14 d,and 21 d after inoculation.Another 3 mice were sacrificed at the corresponding time point and the L3-5 spinal cords were obtained for determining the expressions of pCREB and BDNF by western blotting.Results Compared with preoperative base level,the levels of SLTs in group S were significantly increased at 7,10,14,21 d after inoculation,the obvious increasing of SLTs [(4.43±0.91),(7.10±1.03),(11.27 ±1.35),(13.03 ±0.58),respectively] (P＜0.05),at 10,14,21 d after inoculation,the significant decreasing of PWMT[(0.63±0.20),(0.32±0.12),(0.24±0.12) g] were observed in group T (P＜0.05) and the expressions of pCREB[(3.78±0.58),(3.92±0.46),(4.92±0.37)] and BDNF [(2.13±0.31),(2.88±0.15),(2.58±0.41)] in group T were obviously increased (P＜0.05).Conclusions The expressions of pCREB and BDNF in dorsal horns of spinal cords were increased in a mouse model of bone cancer pain,accompanied by the pain behavior,suggesting spinal cord pCREB and BDNF may be involved in the development and maintenance of bone cancer pain in mice. Key words: Bone cancer pain; Mice; Phosphorylated cAMP response element binding protein; Brain-derived neurotrophic factor; Dorsal horn of spinal cord

Morphine treatment selectively regulates expression of rat pituitary POMC and the prohormone convertases PC1/3 and PC2

The prohormone convertases, PC1/3 and PC2 are thought to be responsible for the activation of many prohormones through processing including the endogenous opioid peptides. We propose that maintenance of hormonal homeostasis can be achieved, in part, via alterations in levels of these enzymes that control the ratio of active hormone to prohormone. In order to test the hypothesis that exogenous opioids regulate the endogenous opioid system and the enzymes responsible for their biosynthesis, we studied the effect of short-term morphine or naltrexone treatment on pituitary PC1/3 and PC2 as well as on the level of pro-opiomelanocortin (POMC), the precursor gene for the biosynthesis of the endogenous opioid peptide, β-endorphin. Using ribonuclease protection assays, we observed that morphine down-regulated and naltrexone up-regulated rat pituitary PC1/3 and PC2 mRNA. Immunofluorescence and Western blot analysis confirmed that the protein levels changed in parallel with the changes in mRNA levels and were accompanied by changes in the levels of phosphorylated cyclic-AMP response element binding protein. We propose that the alterations of the prohormone processing system may be a compensatory mechanism in response to an exogenous opioid ligand whereby the organism tries to restore its homeostatic hormonal milieu following exposure to the opioid, possibly by regulating the levels of multiple endogenous opioid peptides and other neuropeptides in concert.

Mitigation of augmented extrasynaptic NMDAR signaling and apoptosis in cortico-striatal co-cultures from Huntington's disease mice

We recently reported evidence for disturbed synaptic versus extrasynaptic NMDAR transmission in the early pathogenesis of Huntington's disease (HD), a late-onset neurodegenerative disorder caused by CAG repeat expansion in the gene encoding huntingtin. Studies in glutamatergic cells indicate that synaptic NMDAR transmission increases phosphorylated cyclic-AMP response element binding protein (pCREB) levels and drives neuroprotective gene transcription, whereas extrasynaptic NMDAR activation reduces pCREB and promotes cell death. By generating striatal and cortical neuronal co-cultures to investigate the glutamatergic innervation of striatal neurons, we demonstrate that dichotomous synaptic and extrasynaptic NMDAR signaling also occurs in GABAergic striatal medium-sized spiny neurons (MSNs), which are acutely vulnerable in HD. Further, we show that wild-type (WT) and HD transgenic YAC128 MSNs co-cultured with cortical cells have similar levels of glutamatergic synapses, synaptic NMDAR currents and synaptic GluN2B and GluN2A subunit-containing NMDARs. However, NMDAR whole-cell, and especially extrasynaptic, current is elevated in YAC128 MSNs. Moreover, GluN2B subunit-containing NMDAR surface expression is markedly increased, irrespective of whether or not the co-cultured cortical cells express mutant huntingtin. The data suggest that MSN cell-autonomous increases in extrasynaptic NMDARs are driven by the HD mutation. Consistent with these results, we find that extrasynaptic NMDAR-induced pCREB reductions and apoptosis are also augmented in YAC128 MSNs. Moreover, both NMDAR-mediated apoptosis and CREB-off signaling are blocked by co-application of either memantine or the GluN2B subunit-selective antagonist ifenprodil in YAC128 MSNs. GluN2A-subunit-selective concentrations of the antagonist NVP-AAM077 did not reduce cell death in either genotype. Cortico-striatal co-cultures provide an in vitro model system in which to better investigate striatal neuronal dysfunction in disease than mono-cultured striatal cells. Results from the use of this system, which partially recapitulates the cortico-striatal circuit and is amenable to acute genetic and pharmacological manipulations, suggest that pathophysiological NMDAR signaling is an intrinsic frailty in HD MSNs that can be successfully targeted by pharmacological interventions.

Dysregulated phosphorylation and nuclear translocation of cyclic AMP response element binding protein (CREB) in rat liver after chronic ethanol binge

Binge ethanol during chronic ethanol abuse augments liver injury but the underlying mechanism remains unknown. CREB (cyclic AMP response element binding protein) is implicated as a key transcription factor in liver regeneration and hepatic glucose and lipid metabolism. We examined the effects of ethanol on the phosphorylation of CREB in hepatocytes, and in vivo in rat liver after chronic ethanol binge. For in vivo studies, rats were fed ethanol in liquid diet for 4weeks followed by single binge administration of ethanol (intragastric, 5g/kg body weight). Four hours after binge administration, liver samples were collected and analyzed. Treatment of hepatocytes with ethanol caused increased phosphorylation of p38 MAPK (mitogen activated protein kinase), MSK-1 (mitogen and stress activated kinase) and CREB in the nuclear compartment without activation of ERK1/2 (extracellular regulated kinase); whereas angiotensin II induced activation of CREB was accompanied by activation of ERK1/2. In chronic ethanol-binge studies, analysis of the whole cell extracts showed increased phosphorylation of CREB, with no effect on CREB protein levels; increased phospho-ERK1/2, and decreased phospho-p38 MAPK. In contrast, the nuclear levels of phospho-CREB and CREB protein were reduced. Reduction in phospho-CREB and CREB proteins in the nuclear extracts was accompanied by suppression of mRNA levels for CPT-1 (carnitine palmitoyl transferase-1) and increase in hepatic steatosis after binge. It is concluded that binge ethanol causes defect in the nuclear accumulation of CREB protein, phospho-CREB, and an exaggerated hepatic steatosis. These in vivo effects are distinct from the effects of ethanol on hepatocytes in vitro.

Different effects of corticotropin-releasing factor and urocortin 2 on apoptosis of prostate cancer cells in vitro

Urocortin (Ucn), a corticotropin-releasing factor (CRF)-related neuropeptide binding both CRF type 1 receptor (CRFR1) and CRFR2, has recently been found in prostate cancer. However, no report has yet been known to elucidate the roles of Ucn in prostate cancer via the two receptors. In this study, the expression of both CRFR1 and CRFR2 in the mouse prostate cancer cell line RM-1 were detected and cellular apoptosis was monitored in the presence of CRF or Ucn2, the CRFR1- and CRFR2-selective agonist respectively. CRF promoted apoptosis while Ucn2 exerted the opposite effect. CRF reduced Bcl-2 expression, induced Bax expression, and hyperpolarized the mitochondrial membrane potential to activate caspase-9. On the contrary, Ucn2 increased Bcl-2 expression and decreased Bax expression, in which phosphorylation of Akt and cyclic AMP response element-binding (CREB) was involved. Pretreatment with phosphatidylinositide 3-kinase/Akt inhibitor (LY-294002) prior to Ucn2 led to downregulation of CREB phosphorylation and hence reduced Bcl-2 expression. These effects of CRF and Ucn2 were abolished by antalarmin (Anta) and antisauvagine-30, the CRFR1- and CRFR2-selective antagonist respectively. In LNCaP cell line, similar effects on cell apoptosis by CRF and Ucn2 were observed. In summary, our results demonstrated CRFR1 and CRFR2 expression in prostate cancer and indicated the opposite apoptotic roles of the two different CRFRs. These data may contribute to uncovering the pathophysiological function of endogenous Ucn in prostate tumorigenesis and progression.

Dose-related effects of venlafaxine on pCREB and brain-derived neurotrophic factor (BDNF) in the hippocampus of the rat by chronic unpredictable stress.

Background: The molecular pathogenesis of depression and psychopharmacology of antidepressants remain elusive. Recent hypotheses suggest that changes in neurogenesis and plasticity may underlie the aetiology of depression. The hippocampus is affected by depression and shows neuronal remodelling during adulthood.Objective: The present study on the adult rat hippocampus, was to evaluate the dose-related effects of chronic venlafaxine on the expression of brain-derived neurotrophic factor (BDNF) and phosphorylated cyclic-AMP response element binding protein (pCREB).Methods: Sprague-Dawley rats were exposed to a variety of chronic unpredictable stressors (CUSs) to establish a depression model. Rats were treated for either 14 or 28 days with venlafaxine (5 and 10 mg/kg, respectively). The hippocampal expression of pCREB and BDNF mRNA and protein was assessed by using immunohistochemistry, western blotting and reverse transcription polymerase chain reaction (RT-PCR).Results: Rats subjected to CUS procedure consumed less sucrose solution compared with non-stressed rats. The CUS influenced exploratory activity resulting in a reduction of the motility counts. Chronic low dose (5 mg/kg, 14 and 28 days), but not high dose (10 mg/kg, 14 and 28 days) of venlafaxine treatment increased the expression of pCREB and BDNF mRNA and protein in the CUS rat hippocampus.Conclusion: Neuronal plasticity-associated proteins such as pCREB and BDNF play an important role both in stress-related depression and in antidepressant effect.

Cognitive dysfunction induced by chronic cerebral hypoperfusion in a rat model associated with arteriovenous malformations

The relationship between chronic cerebral hypoperfusion and cognitive function has not been completely delineated. In the present studies, we developed an experimental model associated with arteriovenous malformation to investigate the effects of chronic cerebral hypoperfusion on cognitive function and neuropathological changes. The rat model was established by creating a fistula through an end-to-side anastomosis between the right distal external jugular vein and the ipsilateral common carotid artery, followed by ligation of the left vein draining the transverse sinus and bilateral external carotid arteries. Age-matched rats comprised a control group. Three months after surgery, cognitive functions were evaluated by the Morris water maze and hippocampal long-term potentiation (LTP). Neuropathological changes were examined using light and electron microscopic techniques. We found that both learning capacity and spatial memory were significantly impaired in rats with chronic cerebral hypoperfusion concomitant with LTP inhibition and neurodegeneration in the hippocampal CA1 region of model rats compared with control rats. In addition, model rats showed a decrease at the protein level of cyclic AMP response element binding (CREB) phosphorylation in hippocampal tissues. Therefore, cognitive impairment caused by chronic cerebral hypoperfusion associated with arteriovenous malformations may be partially explained by the neurodegeneration and reduction of CREB phosphorylation in rat hippocampus.