Response Element-binding Protein Activation Research Articles

Curcuminoids Promote Neurite Outgrowth in PC12 Cells through MAPK/ERK- and PKC-Dependent Pathways

Curcuminoids, the predominant polyphenolic compounds in the rhizome of Curcuma longa Linn., consist of curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC). They exhibit multiple desirable characteristics for a neuroprotective agent including antioxidant, anti-inflammatory, and antiamyloid activities. In this work, we report the first investigation of the neurotrophic action and mechanism of curcuminoids in PC12 cells, which respond to nerve growth factor (NGF) and therefore serve as a model system for primary neuronal cells. The percentages of neurite-bearing cells for those treated with 20 μM curcumin, DMC, and BDMC for 72 h reached 21.6 ± 2.0%, 16.3 ± 2.4%, and 19.9 ± 2.5%, respectively, and were significantly higher than that of the negative control (2.0 ± 0.3%, p < 0.05). In parallel, increased expression of the neuronal differentiation markers, growth-associated protein-43 (GAP-43), and neurofilament-L (NF-L) was found in curcuminoid-treated cells. All three curcuminoids (20 μM) activated extracellular signal-regulated protein kinase 1/2 (ERK1/2) and protein kinase C (PKC) signalings, and inhibition of these kinases with the respective pharmacological inhibitors effectively attenuated curcuminoid-induced neurite outgrowth. Furthermore, our results show that both curcumin and DMC, but not BDMC, induced phosphorylation of cAMP response element-binding protein (CREB) and CRE-reporter gene activity significantly (p < 0.05). These inductions were markedly attenuated by the addition of MEK/ERK or PKC inhibitor; as a consequence, ERK- and PKC-dependent pathways may be involved in curcuminoid-mediated neuritogenesis in PC12 cells. Moreover, activation of CREB coupling with CRE-dependent gene transcription may play a vital role for curcumin- or DMC-induced PC12 differentiation.

Anxiogenic-like effects of chronic cannabidiol administration in rats

Several pre-clinical and human-based studies have shown that acutely administered cannabidiol (CBD) can produce anxiolytic-like effects The present study investigated the effects of chronic administration of CBD on rat behaviour and on the expression of brain proteins. Male Lister-hooded rats (150-200 g, n = 8 per group) received daily injections of CBD (10 mg/kg, i.p.) for 14 days. The rats were subjected to two behavioural tests: locomotor activity and conditioned emotional response (CER). The expression of brain-derived neurotrophic factor (BDNF), its receptor tyrosine kinase B (Trk B), extracellular signal-regulated kinases (ERK1/2) and phospho-ERK1/2 and the transcription factor cyclic AMP response element binding protein activation (CREB) and phospho-CREB were determined in brain regions such as the frontal cortex and hippocampus using Western immunoblotting. CBD significantly increased the time spent freezing in the CER test with no effect on locomotor activity. CBD significantly reduced BDNF expression in the hippocampus and frontal cortex with no change in the striatum. In addition, CBD significantly reduced TrkB expression in the hippocampus with a strong trend towards reduction in the striatum but had no effect in the frontal cortex. In the hippocampus, CBD had no effect on ERK1/2 or phospho-ERK2, but in the frontal cortex, CBD significantly reduced phospho-ERK1/2 expression without affecting total ERK. Chronic administration of CBD produced an anxiogenic-like effect in clear opposition to the acute anxiolytic profile previously reported. In addition, CBD decreased the expression of proteins that have been shown to be enhanced by chronic treatment with antidepressant/anxiolytic drugs.

Age‐related decline in osteoblastogenesis and 1α‐hydroxylase/CYP27B1 in human mesenchymal stem cells: stimulation by parathyroid hormone

With aging, there is a decline in bone mass and in osteoblast differentiation of human mesenchymal stem cells (hMSCs) in vitro. Osteoblastogenesis can be stimulated with 1,25-dihydroxyvitamin D(3) [1,25(OH)(2) D(3) ] and, in some hMSCs, by the precursor 25-hydroxyvitamin D(3) (25OHD(3) ). CYP27B1/1α-hydroxylase activates 25OHD(3) and, to a variable degree, hMSCs express CYP27B1. In this study, we tested the hypotheses (i) that age affects responsiveness to 25OHD(3) and expression/activity of CYP27B1 in hMSCs and (ii) that parathyroid hormone (PTH) upregulates CYP27B1 in hMSCs, as it does in renal cells. There were age-related declines in osteoblastogenesis (n=8, P=0.0286) and in CYP27B1 gene expression (n=27, r= -0.498; P=0.008) in hMSCs. Unlike hMSCs from young subjects (≤50 years), hMSCs from older subjects (≥55 years) were resistant to 25OHD(3) stimulation of osteoblastogenesis. PTH1-34 (100 nm) provided hMSCs with responsiveness to 25OHD(3) (P=0.0313, Wilcoxon matched pairs test) and with two episodes of increased 1,25(OH)(2) D(3) synthesis, of cAMP response element binding protein (CREB) activation, and of CYP27B1 upregulation. Both increases in CYP27B1 expression by PTH were obliterated by CREB-siRNA or KG-501 (which specifically inhibits the downstream binding of activated CREB). Only the second period of CREB signaling was diminished by AG1024, an inhibitor of insulin-like growth factor-I receptor kinase. Thus, PTH stimulated hMSCs from elders with responsiveness to 25OHD(3) by upregulating expression/activity of CYP27B1 and did so through CREB and IGF-I pathways.

Anti-inflammatory activity of ethanol extract derived from Phaseolus angularis beans

Ethnopharmacological significancePhaseolus angularis Wight (adzuki bean) is an ethnopharmacologically well-known folk medicine that is prescribed for infection, edema, and inflammation of the joints, appendix, kidney and bladder in Korea, China and Japan. Aim of studyThe anti-inflammatory effect of this plant and its associated molecular mechanisms will be investigated. Materials and methodsThe immunomodulatory activity of Phaseolus angularis ethanol extract (Pa-EE) in toll like receptor (TLR)-activated macrophages induced by ligands such as lipopolysaccharide (LPS), Poly (I:C), and pam3CSK was investigated by assessing nitric oxide (NO) and prostaglandin (PG)E2 levels. To identify which transcription factors such as nuclear factor (NF)-κB and their signaling enzymes can be targeted to Pa-EE, biochemical approaches including reporter gene assays, immunoprecipitation, kinase assays, and immunoblot analyses were also employed. Finally, whether Pa-EE was orally available, ethanol (EtOH)/hydrochloric acid (HCl)-induced gastritis model in mice was used. ResultsPa-EE dose-dependently suppressed the release of PGE2 and NO in LPS-, Poly(I:C)-, and pam3CSK-activated macrophages. Pa-EE strongly down-regulated LPS-induced mRNA expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2. Interestingly, Pa-EE markedly inhibited NF-κB, activator protein (AP)-1, and cAMP response element binding protein (CREB) activation; further, according to direct kinase assays and immunoblot analyses, Pa-EE blocked the activation of the upstream signaling molecules spleen tyrosine kinase (Syk), p38, and transforming growth factor β–activated kinase 1 (TAK1). Finally, orally administered Pa-EE clearly ameliorated EtOH/HCl-induced gastritis in mice. ConclusionOur results suggest that Pa-EE can be further developed as a promising anti-inflammatory remedy because it targets multiple inflammatory signaling enzymes and transcription factors.

Constitutive cAMP response element binding protein (CREB) activation by Alzheimer's disease presenilin-driven inositol trisphosphate receptor (InsP3R) Ca2+ signaling.

Mutations in presenilins (PS) account for most early-onset familial Alzheimer's disease (FAD). Accumulating evidence suggests that disrupted Ca(2+) signaling may play a proximal role in FAD specifically, and Alzheimer's disease (AD) more generally, but its links to the pathogenesis of AD are obscure. Here we demonstrate that expression of FAD mutant PS constitutively activates the transcription factor cAMP response element binding protein (CREB) and CREB target gene expression in cultured neuronal cells and AD mouse models. Constitutive CREB activation was associated with and dependent on constitutive activation of Ca(2+)/CaM kinase kinase β and CaM kinase IV (CaMKIV). Depletion of endoplasmic reticulum Ca(2+) stores or plasma membrane phosphatidylinositol-bisphosphate and pharmacologic inhibition or knockdown of the expression of the inositol trisphosphate receptor (InsP(3)R) Ca(2+) release channel each abolished FAD PS-associated constitutive CaMKIV and CREB phosphorylation. CREB and CaMKIV phosphorylation and CREB target gene expression, including nitric oxide synthase and c-fos, were enhanced in brains of M146V-KI and 3xTg-AD mice expressing FAD mutant PS1 knocked into the mouse locus. FAD mutant PS-expressing cells demonstrated enhanced cell death and sensitivity to Aβ toxicity, which were normalized by interfering with the InsP(3)R-CAMKIV-CREB pathway. Thus, constitutive CREB phosphorylation by exaggerated InsP(3)R Ca(2+) signaling in FAD PS-expressing cells may represent a signaling pathway involved in the pathogenesis of AD.

The Distribution of Messenger RNAs Encoding the Three Isoforms of the Transducer of Regulated cAMP Responsive Element Binding Protein Activity in the Rat Forebrain

Increasing evidence indicates that the cAMP responsive element binding protein (CREB)-dependent transcriptional activation of a number of genes requires the CREB co-activator: transducer of regulated CREB activity (TORC). Because of the central importance of CREB in many brain functions, we examined the topographic distribution of TORC1, 2, and 3 mRNAs in specific regions of the rat forebrain. In situ hybridisation analysis revealed that TORC1 is the most abundant isoform in most forebrain structures, followed by TORC2 and TORC3. All three TORC isoforms were found in a number of brain nuclei, the ventricular ependyma and pia mater. Although high levels of TORC1 were widely distributed in the forebrain, TORC2 was found in discrete nuclei and TORC3 mostly in the ependyma, and pia mater. The relative expression of TORC isoforms was confirmed by quantitative reverse transcriptase-polymerase chain reaction analysis in the hippocampus and hypothalamus. In the paraventricular nucleus of the hypothalamus, TORC1 and 2 mRNAs were abundant in the parvicellular and magnocellular neuroendocrine compartments, whereas TORC3 expression was low. All three isoform mRNAs were found elsewhere in the hypothalamus, with the most prominent expression of TORC1 in the ventromedial nucleus, TORC2 in the dorsomedial and arcuate nuclei, TORCs 1 and 2 in the supraoptic nucleus, and TORC2 in the suprachiasmatic nucleus. These differential distribution patterns are consistent with complex roles for all three TORC isoforms in diverse brain structures, and provide a foundation for further studies on the mechanisms of CREB/TORC signalling on brain function.

A phytoceramide analog stimulates the production of chemokines through CREB activation in human endothelial cells

Innate immunity is the front-line of self-defense against microbial infection. In mammals, innate immunity interacts with adaptive immunity and has a key role in the regulated immune response. From a pharmaceutical point of view, innate immunity is an ideal target for the development of immunoregulators. Therefore, we aimed to isolate and characterize a novel mammalian immunoregulator isolated from the thermophilic cellulotic fungus Talaromyces sp. 2′-(R)-hydroxy-C24 phytoceramide (C24(2′OH)Phy) was isolated from Talaromyces sp. using a Drosophila ex vivo culture system. C24(2′OH)Phy suppressed the immune deficiency (IMD) pathway-dependent expression of antibacterial peptides in Drosophila, whereas it stimulated the production of chemokines in human cells. Structure activity relationship studies of C24(2′OH)Phy analogs revealed that both the 2′-(R)-hydroxylignoceroyl group and phytoceramide backbone are essential for the biologic activity of C24(2′OH)Phy. Microarray analysis revealed that C24(2′OH)Phy selectively activates the transcription of inflammatory response genes, including chemokines. Furthermore, a reporter gene assay and small interfering RNA analysis demonstrated that C24(2′OH)Phy stimulates chemokine production through cAMP response element-binding protein activation in human cells. C24(2′OH)Phy may be a lead immunostimulating compound in humans.

Abstract 4785: Silver nanoparticles induce apoptosis in squamous lung cancer cell by regulation of cyclic AMP response element-binding protein: regulating its upstream oxidative stress-dependent Ras signaling pathways

Abstract Recently, much effort has been devoted to the development of biomedical applications (drug delivery system and as therapeutic drugs themselves) for nanoparticles (NPs). Silver NPs have been shown various therapeutic effects such as antimicrobial, antioxidant, and anti-inflammatory effects. Recently silver NPs have been shown to induce cytotoxicity in cancer cells. But exact mechanism is not yet established. We conducted this study to determine the mechanisms of silver NPs on cellular apoptosis in non-small cell lung cancer (NSCLC) cell lines using cyclic AMP response element-binding protein (CREB) activity which has been implicated to contribute an important pathobiologic role in lung carcinogenesis and is considered a potential therapeutic target for NSCLC. We hypothesized that constitutively increased CREB and its related signaling pathways can be blocked by silver NPs and it might be by regulation of oxidative stress in tumorigenesis. In H520 cell, human lung squamous cancer cell line, reactive oxygen species (ROS) were increased. In western blot analysis, Ras signaling molecule in which ROS is involved was constitutively overexpressed and the levels of pErk, pRsk, pAkt and pCREB were increased in H520 cell. Incubation of the H520 cells with silver NPs (at dose of 100 or 200 μM) substantially suppressed the ROS in H520 cell and Ras-Erk-Rsk-CREB and Ras-PI3K-Akt signaling pathway in a dose-dependent manner. We subsequently demonstrated that silver NPs inhibited the caspase-dependent pathway, determined by increased levels of PARP (poly (ADP-ribose) polymerase) cleavage. In this study, we suggest that silver NPs induce apoptosis in human lung cancer cells by attenuation of oxidative stress in lung cancer cell line and ROS-dependent Ras signaling which involves CREB signaling pathways and may be useful as a therapeutic strategy for cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4785. doi:10.1158/1538-7445.AM2011-4785

Research progress on neurobiology of neuronal nitric oxide synthase

Neuronal nitric oxide synthase (nNOS) is mainly expressed in neurons, to some extent in astrocytes and neuronal stem cells. The alternative splicing of nNOS mRNA generates 5 isoforms of nNOS, including nNOS-α, nNOS-β, nNOS-µ, nNOS-γ and nNOS-2. Monomer of nNOS is inactive, and dimer is the active form. Dimerization requires tetrahydrobiopterin (BH4), heme and L-arginine binding. Regulation of nNOS expression relies largely on cAMP response element-binding protein (CREB) activity, and nNOS activity is regulated by heat shock protein 90 (HSP90)/HSP70, calmodulin (CaM), phosphorylation and dephosphorylation at Ser847 and Ser1412, and the protein inhibitor of nNOS (PIN). There are primarily 9 nNOS-interacting proteins, including post-synaptic density protein 95 (PSD95), clathrin assembly lymphoid leukemia (CALM), calcium/calmodulin-dependent protein kinase II alpha (CAMKIIA), Disks large homolog 4 (DLG4), DLG2, 6-phosphofructokinase, muscle type (PFK-M), carboxy-terminal PDZ ligand of nNOS (CAPON) protein, syntrophin and dynein light chain (LC). Among them, PSD95, CAPON and PFK-M are important nNOS adapter proteins in neurons. The interaction of PSD95 with nNOS controls synapse formation and is implicated in N-methyl-D-aspartic acid-induced neuronal death. nNOS-derived NO is implicated in synapse loss-mediated early cognitive/motor deficits in several neuropathological states, and negatively regulates neurogenesis under physiological and pathological conditions.

Abstract A18: Silver nanoparticles induce apoptosis by regulation of cyclic AMP response element-binding protein in lung cancer cells

Abstract Recently, much effort has been devoted to the development of biomedical applications for nanoparticles (NPs). Silver NPs have been shown various therapeutic effects such as antimicrobial, antioxidant, and anti-inflammatory effects. Recently silver NPs have been shown to induce the apoptotic pathway in vitro. But exact mechanism is not yet established. We conducted this study to determine the mechanisms of silver NPs on cellular apoptosis in non-small cell lung cancer (NSCLC) cell lines using cyclic AMP response element-binding protein (CREB) activity which has been implicated to contribute an important pathobiologic role in lung carcinogenesis and is considered a potential therapeutic target for NSCLC. We hypothesized that constitutively increased CREB and its related signaling pathways can be blocked by silver NPs. To determine cytotoxic effect of silver NPs, several NSCLC cell lines were treated with various concentrations for different times. Among these lung cancer cell lines, H520, human lung squamous carcinoma cell lines, was the most sensitive. FACS analysis also showed that silver NPs induced cell death of H520 cells. Treating H520 cells with silver NPs (100, 200, or 500 M) inhibited CREB activation by blocking the activity of extracellular signal kinase/ribosomal s6 kinase and also by blocking the activity of phosphatidylinositol 3-kinase/Akt. We subsequently confirmed the expression of Bax and Bcl-2 in H520 cells was substantially regulated by silver NPs. We also demonstrated that silver NPs induced the cleavage of apoptosis-related proteins, poly (ADP-ribose) polymerase and caspase-3. In this study, we suggest that silver NPs induce apoptosis in H520 cells by regulation of CREB signaling pathways and may be useful as a therapeutic strategy for cancer. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A18.

4-Methylnitrosamino-1-3-pyridyl-1-butanone (NNK) promotes lung cancer cell survival by stimulating thromboxane A2 and its receptor

The role of thromboxane A(2) (TxA(2)) in smoking-associated lung cancer is poorly understood. This study was conducted to study the role of TxA(2) in smoking carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-promoted cell survival and growth in human lung cancer cells. We found that NNK increased TxA(2) synthase (TxAS) expression and thromboxane B(2) (TxB(2)) generation in cultured lung cancer cells, the result of which was supported by the increased level of TxAS in lung cancer tissues of smokers. Both TxAS-specific inhibitor furegrelate and TxA(2) receptor antagonist SQ29548 completely blocked NNK-mediated cell survival and growth via inducting apoptosis. TxA(2) receptor agonist U46619 reconstituted a near-full survival and growth response to NNK when TxAS was inhibited, affirming the role of TxA(2) receptor in NNK-mediated cell survival and growth. Suppression of cyclic adenosine monophosphate response element binding protein (CREB) activity by its small interference RNA blocked the effect of NNK. Phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) also had a positive role. Altogether, our results have revealed that NNK stimulates TxA(2) synthesis and activates its receptor in lung cancer cells. The increased TxA(2) may then activate CREB through PI3K/Akt and extracellular ERK pathways, thereby contributing to the NNK-promoted survival and growth of lung cancer cells.

Promotion of cAMP Responsive Element-Binding Protein Activity Ameliorates Radiation-Induced Suppression of Hippocampal Neurogenesis in Adult Mice

This study was performed to examine whether elevated activity of cAMP responsive element-binding protein (CREB) attenuates the detrimental effects of acute gamma (γ) -irradiation on hippocampal neurogenesis and related functions. C57BL/6 male mice were treated with rolipram (1.25 mg/kg, i.p., twice a day for 5 consecutive days) to activate the cAMP/CREB pathway against cranial irradiation (2 Gy) , and were euthanized at 24 h post-irradiation. Exposure to γ-rays decreased both CREB phosphorylation and immunohistochemical markers for neurogenesis, including Ki-67 and doublecortin (DCX) , in the hippocampal dentate gyrus (DG) . However, the rolipram treatment protected from γ-irradiation-induced decreases of CREB phosphorylation, and Ki-67 and DCX immunoreactivity in the hippocampal DG. In an object recognition memory test, mice trained 24 h after acute γ-irradiation (2 Gy) showed significant memory impairment, which was attenuated by rolipram treatment. The results suggest that activation of CREB signaling ameliorates the detrimental effects of acute γ-irradiation on hippocampal neurogenesis and related functions in adult mice.

Src-dependent TrkA transactivation is required for pituitary adenylate cyclase-activating polypeptide 38-mediated Rit activation and neuronal differentiation.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent neuropeptide that possesses both neurotrophic and neurodevelopmental effects. Recently, the Rit GTPase was found to be activated by a novel Galpha/cAMP/exchange protein activated by cyclic AMP (Epac)-dependent signaling pathway and required for PACAP-dependent cAMP response element-binding protein activation and neuronal differentiation. However, Epac did not function as a Rit guanine nucleotide exchange factor (GEF), and the nature of the PACAP regulatory cascade remained unclear. Here, we show that PACAP-mediated Rit activation involves Src family kinase-dependent TrkA receptor transactivation. PACAP receptor (PACR1) stimulation triggered both G(i)alpha and G(s)alpha/cAMP/Epac regulatory cascades resulting in Src kinase activity, which in turn induced TrkA kinase tyrosine phosphorylation. Importantly, Src inhibition, or the lack of functional Trk receptors, was found to inhibit PACAP-mediated Rit activation, whereas constitutively active Src alone was sufficient to stimulate Rit-guanosine triphosphate levels. A single tyrosine (Y(499)) phosphorylation event was identified as critical to both PACAP-mediated transactivation and TrkA-dependent Rit activation. Accordingly, PACAP stimulation resulted in TrkA-dependent phosphorylation of both the Shc adaptor and son of sevenless (SOS)1/2 GEFs, and Rit activation was inhibited by RNA interference silencing of SOS1/2, implicating a TrkA/Shc/SOS signaling complex in Rit regulation. Together, these observations expand upon the nature of PACR1-mediated transactivation and identify TrkA-Rit signaling as a key contributor to PACAP-dependent neuronal differentiation.

The effect of tyrosine nitration of L‐type Ca2+ channels on excitation–transcription coupling in colonic inflammation

Excitation-transcriptional coupling involves communication between plasma membrane ion channels and gene expression in the nucleus. Calcium influx through L-type Ca(2+) channels induces phosphorylation of the transcription factor, cyclic-AMP response element binding protein (CREB) and downstream activation of the cyclic-AMP response element (CRE) promoter regions. Tyrosine nitration of Ca(2+) channels attenuates interactions with c-Src kinase, decreasing Ca(2+) channel currents and smooth muscle contraction during colonic inflammation. In this study we examined the effect of tyrosine nitration and colonic inflammation on Ca(2+) channel mediated phosphorylation of CREB and CRE activation. CREB and phospho-CREB were detected by Western blots and CRE activation measured by dual luciferase assay. Chinese hamster ovary (CHO) cells were transfected with hCa(v)1.2b and hCa(v)1.2b c-terminal mutants. Colonic inflammation was induced by intracolonic instillation of 2,4,6 trinitrobenzene sulphonic acid in mouse colon. In hCa(v)1.2b transfected CHO cells and in native colonic smooth muscle, depolarization with 80 mM KCl induced CREB phosphorylation (pCREB). Treatment with peroxynitrite inhibited KCl-induced pCREB. Following experimental colitis, KCl-induced CREB phosphorylation was abolished in smooth muscle, concomitant with tyrosine nitration of Ca(2+) channels. Depolarization increased CRE activation in hCa(v)1.2b CHO cells by 2.35 fold which was blocked by nifedipine and by protein nitration of Ca(2+) channels with peroxynitrite. The Src-kinase inhibitor, PP2, blocked depolarization-induced CRE activation. Mutation of the C-terminus tyrosine residue, Y2134F, but not Y1861F, blocked CRE activation. Post-translational modification of Ca(2+) channels due to tyrosine nitration modified excitation-transcriptional coupling in colonic inflammation.

Involvement of Transducer of Regulated cAMP Response Element-Binding Protein Activity on Corticotropin Releasing Hormone Transcription

We have recently shown that phospho-cAMP response element-binding protein (CREB) is essential but not sufficient for activation of CRH transcription, suggesting the requirement of a coactivator. Here, we test the hypothesis that the CREB coactivator, transducer of regulated CREB activity (TORC), is required for activation of CRH transcription, using the cell line 4B and primary cultures of hypothalamic neurons. Immunohistochemistry and Western blot experiments in 4B cells revealed time-dependent nuclear translocation of TORC1,TORC 2, and TORC3 by forskolin [but not by the phorbol ester, phorbol 12-myristate 13-acetate (PMA)] in a concentration-dependent manner. In reporter gene assays, cotransfection of TORC1 or TORC2 potentiated the stimulatory effect of forskolin on CRH promoter activity but had no effect in cells treated with PMA. Knockout of endogenous TORC using silencing RNA markedly inhibited forskolin-activated CRH promoter activity in 4B cells, as well as the induction of endogenous CRH primary transcript by forskolin in primary neuronal cultures. Coimmunoprecipitation and chromatin immunoprecipitation experiments in 4B cells revealed association of CREB and TORC in the nucleus, and recruitment of TORC2 by the CRH promoter, after 20-min incubation with forskolin. These studies demonstrate a correlation between nuclear translocation of TORC with association to the CRH promoter and activation of CRH transcription. The data suggest that TORC is required for transcriptional activation of the CRH promoter by acting as a CREB coactivator. In addition, cytoplasmic retention of TORC during PMA treatment is likely to explain the failure of phorbolesters to activate CRH transcription in spite of efficiently phosphorylating CREB.

Exploring the molecular basis of addiction: drug-induced neuroadaptations.

Abuse of a number of psychoactive substances can eventually control an individual's behavior by producing dependence and/or addiction. Recent surveys estimate that there are about 200 million users of illegal drugs worldwide, which represent 3.4% of the world population. An ever-increasing number of neuroscientists are searching for clues regarding the molecular determinants of addictive behavior. The low-hanging fruit would be to study dopamine receptors and transporters in the nucleus accumbens (NAc); however, scientists are now exploring mechanisms far beyond dopaminergic targets. For example, some scientists have chosen to target molecular mechanisms within the hippocampus because of its role in encoding and retrieving information in the central nervous system. In addition, this brain region sends and receives projections from the mesolimbic dopamine system so often implicated in addiction (Bannerman et al, 2004). Further, the hippocampus has been directly implicated in addiction behavior (Vorel et al, 2001), likely because of the fact that there is increasing evidence suggesting that drug addiction represents a conditioning phenomenon that is largely dependent on associations between drug effects and environmental cues (Berke and Hyman, 2000). Coordinated hippocampal/accumbal regulation is likely at the heart of the protective addiction phenotype produced by environmental enrichment. We find that rats reared in an enriched condition (large cages with cohorts and novel objects) self-administer cocaine or amphetamine less readily than rats in the isolated control group (Green et al, 2009). This protective phenotype is likely mediated by a coordinated decrease in cAMP response element binding protein (CREB) activity in the accumbens coupled with an increase in the hippocampus. Further, CREB has been linked to neuronal excitability (Dong et al, 2006), suggesting that some yet to be identified CREB target gene may produce this protective phenotype by decreasing excitability in the accumbens and increasing excitability in the hippocampus. Addiction-related molecular targets in the hippocampus are also being interrogated using proteomics approaches. It is known that exposure to drugs of abuse alters the expression of certain synaptic proteins. Indeed, a recent study uses this state-of-the-art technology to examine the effects of morphine administration on the protein expression profile at hippocampal synapses (Moron et al, 2007). This study finds that repeated morphine administration alters the synaptic distribution of endocytic proteins. This finding has functional implications, as receptor trafficking largely depends on endocytosis, and therefore morphine may alter receptor localization by affecting synaptic redistribution of the endocytic machinery. The idea that endocytic proteins, such as clathrin, may be involved in morphine-induced changes at hippocampal synapses is quite innovative and opens up a new direction for the study of the mechanisms underlying morphine-induced neuroadaptations. In addition, these findings suggest that the study of hippocampal neuroadaptations induced by repeated morphine treatment has great potential to reveal the mechanisms contributing to the development of opiate addiction. Addiction is a complex polygenic psychiatric condition involving many brain regions, proteins and physiological effects, not to mention varied etiologies. Understanding the molecular mechanisms underlying addictive behavior will someday allow for therapeutic intervention that will be both efficacious and safe.

Calmodulin activation is required for the enhancement of hippocampal neurogenesis following environmental enrichment

Objective: An enriched environmental exposure has been shown to promote hippocampal neurogenesis, which is an important mechanism underlying enrichment-induced memory improvement. The cAMP response element-binding protein is an important stimulus-inducible factor in the brain and is crucial for neurogenesis and memory ability. Calmodulin activation is important for activity-induced cAMP response element-binding protein phosphorylation. The aim of this study was to explore the effects of calmodulin activation on hippocampal neurogenesis and spatial memory improvement in rats exposed to enriched environment.Methods: Ninety-six neonatal pups were assigned to four groups: control group, enriched environment group, control + trifluoperazine (trifluoperazine, 5 mg/kg) group and enriched environment + trifluoperazine group. Pups in the enriched environment and enriched environment + trifluoperazine groups were exposed to enriched environments for 25 min from P10 to P24. Half of pups in each group were intraperitoneally injected with 50 mg/kg BrdU every other day from P10 to P24. Pups without BrdU injection were euthanized at P24, and hippocampal nuclear Ca2+-calmodulin and phosphorylated cAMP response element-binding protein were detected. Morris water maze acquisition and subsequent probe trial retention were performed in remained rats after P50 and immunohistochemistry was used to identify proliferating cells in the dentate gyrus 1 day after the probe trial.Results: The trifluoperazine injection could completely inhibit the environmental enrichment-induced activations of calmodulin and cAMP response element-binding protein. Enriched environmental exposure could improve Morris water maze performance and increase hippocampal BrdU-labeled cells, but the effects could be completely blocked by trifluoperazine injection.Conclusion: This study proves that the activation of calmodulin is essential for an enriched environmental exposure-induced cAMP response element-binding protein activation, hippocampal neurogenesis and spatial memory improvement.

2, 3, 5, 4′-tetrahydroxystilbene-2-O-β- d-glucoside (THSG) induces melanogenesis in B16 cells by MAP kinase activation and tyrosinase upregulation

Aims The 2, 3, 5, 4′-tetrahydroxystilbene-2-O-β- d-glucoside (THSG), a water-soluble active component extracted from dried tuber root of Polygonum multiflorum, has been found to induce pigmentation in B16 cells, but the details of the underlying mechanism remain unknown. The present study was conducted to investigate the mechanism of stimulatory effect of THSG on melanogenesis using B16F1 melanoma cells. Main methods Several experiments were performed in B16F1 melanoma cells. We studied melanin content, tyrosinase activity, cell viability, and performed reverse transcription polymerase chain reaction and Western blots for proteins involved in melanogenesis. Key findings THSG increased the melanin content and tyrosinase activity in a concentration-dependent manner and treatment with 10 µg/ml THSG enhanced the expression of tyrosinase time-dependently in B16 cells. We then investigated whether THSG influences the expression of microphthalmia-associated transcription factor (MITF), which is required for tyrosinase expression. THSG was found to induce sustained MITF up-regulation and cAMP response element (CRE) binding protein (CREB) activation, suggesting that THSG-mediated MITF activation may be cAMP dependent. Furthermore, Western blot analysis revealed that THSG elevated the level of phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) significantly at 1–6 h; a p38 MAPK inhibitor, SB203580, almost completely attenuated the THSG-mediated up-regulation of melanin synthesis and induction of MITF and tyrosinase expression. Significance THSG exerts its stimulatory effect on melanogenesis by MAP kinase activation and MITF-induction of tyrosinase.

The inducible cyclic adenosine 3’,5’-monophosphate early repressor (ICER) enhances drug sensitivity in acute myeloid leukemia

e22045 Background: The Inducible Cyclic Adenosine 3’,5’-monophosphate early repressor (ICER) is a transcription factor that principally counteracts the cAMP response element binding protein (CREB) activity. CREB was previously demonstrated to be overexpressed in acute leukemia, whereas ICER was found rapidly degradated being unable to control gene transcription. ICER exogenous expression was demonstrated to repress CREB targets preventing leukemia progression. We hypothesized that ICER restoration deserves a special consideration for playing a role in CREB oncogenic feature and in modeling leukemic cell phenotype. Methods: We constructed an expression vector for ICER and induced its exogenous expression in HL60 cells (HL60+ICER). We monitored transcription and translation of a series of genes involved in different pathways by quantitative gene expression and western blot analysis. We investigate ICER's role in cell death after treatment with chemotherapic drugs. Results: We revealed that ICER was able to control gene expression in leukemia, principally of genes involved in cell death and survival. In particular, we focused on DUSP1 and DUSP4 phosphatases which were found significantly repressed. HL60+ICER after being treated with drugs increased apoptosis. Cell cycle analyses revealed a block in G2 phase, a lowered cell proliferation and clonogenic potential with respect to HL60 treated at the same conditions. DUSPs downstream targets (IL6, RB, RAS) were lowered expressed and p38 constitutively phosphorylated confirming their role in mediating cellular stress. ICER driven apoptosis was found to be caspases mediated. An enhanced activity of p38 was observed in treated HL60+ICER, and by the use of specific p38 inhibitors apoptosis decreased. Other possible apoptotic pathways (AKT, ERK, JNK) were excluded because found severely disrupted. Conclusions: Finally, we described an enhanced sensibility to drugs in HL60 induced by ICER exogenous expression and that p38 stress signaling pathway is the direct target of this phenomenon. The ability of ICER to modify leukemic cells phenotype respect to chemotherapeutic treatment gave an important information for targets and therapeutic possibilities. No significant financial relationships to disclose.

AMP-activated protein kinase and carbohydrate response element binding protein: A study of two potential regulatory factors in the hepatic lipogenic program of broiler chickens

This study investigated the effects of fasting and refeeding on AMP-activated protein kinase (AMPK) and carbohydrate response element binding protein (ChREBP) mRNA, protein and activity levels; as well as the expression of lipogenic genes involved in regulating lipid synthesis in broiler chicken (Gallus gallus) liver. Fasting for 24 or 48 h produced significant declines in plasma glucose (at 24 h), insulin and thyroid hormone (T3) levels that were accompanied by changes in mRNA expression levels of hepatic lipogenic genes. The mRNA levels of malic enzyme (ME), ATP-citrate lyase (ACL), acetyl-CoA carboxylase alpha (ACCalpha), fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD-1) and thyroid hormone responsive Spot 14 (Spot 14) declined in response to fasting. Refeeding for 24 h increased mRNA levels for each of these genes, characterized by a significant increase ('overshoot') above fed control values. No change in mRNA expression of the two AMPK alpha subunit genes was observed in response to fasting or refeeding. In contrast, ChREBP and sterol regulatory element binding protein-1 (SREBP-1) mRNA levels decreased during fasting and increased with refeeding. Phosphorylation of AMPK alpha subunits increased modestly after a 48 h fast. However, there was no corresponding change in the phosphorylation of ACC, a major downstream target of AMPK. Protein level and DNA-binding activity of ChREBP increased during fasting and declined upon refeeding as measured in whole liver tissue extracts. In general, evidence was found for coordinate transcriptional regulation of lipogenic program genes in broiler chicken liver, but specific regulatory roles for AMPK and ChREBP in that process remain to be further characterized.