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.

Positive allosteric modulators of metabotropic glutamate receptor subtype 5 (mGlu5) have promising therapeutic potential. The effects of selective mGlu5 receptor positive allosteric modulators on signaling molecules in brain slices have not been previously reported. The current study demonstrated that the selective mGlu5 receptor positive allosteric modulator, N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2yl)-methyl]phenyl}-2-hydrobenzamide (CPPHA) potentiated the response to a subthreshold concentration of 3,5-dihydroxy-phenylglycine (DHPG) on extracellular signal-regulated protein kinase (ERK) and cyclic-AMP responsive element-binding protein (CREB) activity, as well as N-methyl d-aspartate (NMDA) receptor subunit NR1 phosphorylation in cortical and hippocampal slices. These results suggest that allosteric modulators of mGlu5 receptor could have physiologically significant effects by potentiating the actions of glutamate.

Activating transcription factor 1 (ATF1) and the closely related proteins CREB (cyclic AMP resonse element binding protein) and CREM (cyclic AMP response element modulator) constitute a subfamily of bZIP transcription factors that play critical roles in the regulation of cellular growth, metabolism, and survival. Previous studies demonstrated that CREB is phosphorylated on a cluster of conserved Ser residues, including Ser-111 and Ser-121, in response to DNA damage through the coordinated actions of the ataxia-telangiectasia-mutated (ATM) protein kinase and casein kinases 1 and 2 (CK1/2). Here, we show that DNA damage-induced phosphorylation by ATM is a general feature of CREB and ATF1. ATF1 harbors a conserved ATM/CK cluster that is constitutively and stoichiometrically phosphorylated by CK1 and CK2 in asynchronously growing cells. Exposure to DNA damage further induced ATF1 phosphorylation on Ser-51 by ATM in a manner that required prior phosphorylation of the upstream CK residues. Hyperphosphorylated ATF1 showed a 4-fold reduced affinity for CREB-binding protein. We further show that PP2A, in conjunction with its targeting subunit B56gamma, antagonized ATM and CK1/2-dependent phosphorylation of CREB and ATF1 in cellulo. Finally, we show that CK sites in CREB are phosphorylated during cellular growth and that phosphorylation of these residues reduces the threshold of DNA damage required for ATM-dependent phosphorylation of the inhibitory Ser-121 residue. These studies define overlapping and distinct modes of CREB and ATF1 regulation by phosphorylation that may ensure concerted changes in gene expression mediated by these factors.

Ion channels are essential for the regulation of neuronal functions. The significance of plasma membrane, mitochondrial, endoplasmic reticulum and lysosomal ion channels in the regulation of Ca(2+) is well established. In contrast, surprisingly little is known about the function of ion channels on the nuclear envelope (NE). Here we demonstrate the presence of functional large-conductance, calcium-activated potassium channels (BK channels) on the NE of rodent hippocampal neurons. Functionally, blockade of nuclear BK channels (nBK channels) induces NE-derived Ca(2+) release, nucleoplasmic Ca(2+) elevation and cyclic AMP response element binding protein (CREB)-dependent transcription. More importantly, blockade of nBK channels regulates nuclear Ca(2+)-sensitive gene expression and promotes dendritic arborization in a nuclear Ca(2+)-dependent manner. These results suggest that the nBK channel functions as a molecular link between neuronal activity and nuclear Ca(2+) to convey signals from synapse to nucleus and is a new modulator, operating at the NE, of synaptic activity-dependent neuronal functions.

Vasoactive intestinal peptide (VIP) is a modulator of inflammatory responses. VIP receptors are expressed in several tumor types, such as colorectal carcinoma. The study described herein was conducted to confirm the presence of VIP and its receptors (VPAC1 and VPAC2) in surgically resected hepatocellular carcinoma (HCC) tissues and in the HCC cell line Huh7. The mechanism responsible for apoptosis of HCC cells was then examined because VIP treatment (10-10  M) significantly suppressed proliferation of Huh7 cells. In examining apoptosis-related proteins, we found caspase-3 to be significantly increased and Bcl-xL and cyclic AMP (cAMP) response element-binding protein (CREB) to be significantly decreased in Huh7 cells cultured with VIP. Furthermore, the CREB level and phosphorylation were reduced. These effects were reversed by the addition of VIP receptor antagonist or cAMP antagonist Rp-cAMPS. Pretreatment with cAMP analogue blocked the increased apoptosis, suggesting that VIP induces apoptosis via a PKA-independent signaling mechanism. Our data indicate that VIP prevents the progression of HCC by apoptosis through the cAMP/Bcl-xL pathway.

Estrogen is an important modulator of reproductive activity through nuclear receptors and G protein-coupled estrogen receptor (GPER). Here, we observed that both estradiol and the GPER-specific agonist G1 rapidly induced cyclic adenosine monophosphate (cAMP) production in cumulus cells, leading to transient stimulation of phosphorylated cAMP response element binding protein (CREB), which was conducive to the transcription of epidermal growth factor (EGF)-like factors, amphiregulin, epiregulin, and betacellulin. Inhibition of GPER by G15 significantly reduced estradiol-induced CREB phosphorylation and EGF-like factor gene expression. Consistently, the silencing of GPER expression in cultured cumulus cells abrogated the estradiol-induced CREB phosphorylation and EGF-like factor transcription. In addition, the increase in EGF-like factor expression in the cumulus cells is associated with EGF receptor (EFGR) tyrosine kinase phosphorylation and extracellular signal-regulated kinase 1/2 (ERK1/2) activation. Furthermore, we demonstrated that GPER-mediated phosphorylation of EGFR and ERK1/2 was involved in reduced gap junction communication, cumulus expansion, increased oocyte mitochondrial activity and first polar body extrusion. Overall, our study identified a novel function for estrogen in regulating EGFR activation via GPER in cumulus cells during oocyte maturation.

The present study shows that the GABAB positive allosteric modulators (PAMs) CGP7930 and GS39783 stimulate extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) signalling in cells that do not express functional GABAB receptors. In human SH-SY5Y neuroblastoma cells, CGP7930 and GS39783 induced a time- and concentration-dependent increase in ERK1/2 phosphorylation with potencies similar to those displayed as GABAB PAMs. Conversely, γ-aminobutyric acid and the GABAB receptor agonists (-)baclofen and SKF97541 were completely inactive. CGP7930 and GS39783 enhanced the nuclear localization of phospho-ERK1/2 and CGP7930 promoted the phosphorylation of the transcription factors Elk-1 and CREB. CGP7930-induced ERK1/2 stimulation was insensitive to pertussis toxin, the Gq/11 antagonist YM254890 and the phospholipase C-β inhibitor U-73122, but was completely blocked by the MEK1/2 inhibitor PD98059. Inhibition of insulin-like growth factor-1, platelet--derived growth factor, phosphoinositide 3-kinase and Akt activities potentiated CGP7930-induced ERK1/2 phosphorylation. CGP7930 enhanced the phosphorylation of myristoylated alanine-rich protein kinase C (PKC) substrate and inhibition of PKC attenuated the ERK1/2 stimulation. Over-expression of N17Ras, a dominant negative mutant of c-Ras, or inhibition of c-Raf by GW5074 partially antagonized CGP7930-induced ERK1/2 activation. CGP7930 enhanced the phosphorylation of transforming growth factor-β-activated kinase 1 (TAK-1) and TAK-1 inhibition by 5Z-7-oxozeaenol reduced CGP7930-induced ERK1/2 phosphorylation. CGP7930 activated ERK1/2 in CHO-K1 fibroblasts, which lack endogenous GABAB receptors, but not in HEK-293 cells, indicating that the response displayed cell type specificity. These data demonstrate that CGP7930 and GS39783 can trigger ERK1/2 signalling, a critical modulator of mood and drug addiction, independently of an action on GABAB receptors.

African swine fever is one of the most devastating swine diseases caused by African swine fever virus (ASFV). Although ASFV encodes more than 160 viral proteins, the implication of a majority of ASFV proteins in regulating host immunity is yet to be explored, and the mechanisms of immune evasion by ASFV proteins are largely unknown. Here, we report that the I226R protein of ASFV significantly suppressed innate immune responses. The ectopic expression of ASFV I226R in 293T cells significantly inhibited the activation of interferon-stimulated response element promoters triggered by Sendai virus (SeV), poly(I:C), or cyclic GMP-AMP synthase (cGAS)/STING. The I226R protein caused a significant decrease in the expression of interferons and interferon-stimulating genes in cells infected with SeV. Similar results were obtained from experiments using I226R-overexpressed PK15 and 3D4/21 cells stimulated with vesicular stomatitis virus. We observed that I226R inhibited the activation of both nuclear factor-kappa B (NF-κB) and interferon regulatory factor 3 (IRF3). Furthermore, it was shown that overexpression of I226R suppressed IRF3 activation and caused the degradation of NF-κB essential modulator (NEMO) protein. The I226R-induced NEMO degradation could be prevented by treatment with MG132, a proteasome inhibitor. Together, these results reveal that the ASFV I226R protein impairs antiviral responses, likely through multiple mechanisms including the suppression of NF-κB and IRF3 activation, to counteract innate immune responses during the viral infection.

A diet high in total fat (HF) reduces hippocampal levels of brain-derived neurotrophic factor (BDNF), a crucial modulator of synaptic plasticity, and a predictor of learning efficacy. We have evaluated the capacity of voluntary exercise to interact with the effects of diet at the molecular level. Animal groups were exposed to the HF diet for 2 months with and without access to voluntary wheel running. Exercise reversed the decrease in BDNF and its downstream effectors on plasticity such as synapsin I, a molecule with a key role in the modulation of neurotransmitter release by BDNF, and the transcription factor cyclic AMP response element binding protein (CREB), important for learning and memory. Furthermore, we found that exercise influenced the activational state of synapsin as well as of CREB, by increasing the phosphorylation of these molecules. In addition, exercise prevented the deficit in spatial learning induced by the diet, tested in the Morris water maze. Furthermore, levels of reactive oxygen species increased by the effects of the diet were decreased by exercise. Results indicate that exercise interacts with the same molecular systems disrupted by the HF diet, reversing their effects on neural function. Reactive oxygen species, and BDNF in conjunction with its downstream effectors on synaptic and neuronal plasticity, are common molecular targets for the action of the diet and exercise. Results unveil a possible molecular mechanism by which lifestyle factors can interact at a molecular level, and provide information for potential therapeutic applications to decrease the risk imposed by certain lifestyles.

Synaptic plasticity and behaviors are likely dependent on the capacity of neurons to meet the energy demands imposed by neuronal activity. We used physical activity, a paradigm intrinsically associated with energy consumption/expenditure and cognitive enhancement, to study how energy metabolism interacts with the substrates for neuroplasticity. We found that in an area critical for learning and memory, the hippocampus, exercise modified aspects of energy metabolism by decreasing oxidative stress and increasing the levels of cytochrome c oxidase-II, a specific component of mitochondrial machinery. We infused 1,25-dihydroxyvitamin D3, a modulator of energy metabolism, directly into the hippocampus during 3 days of voluntary wheel running and measured its effects on brain-derived neurotrophic factor-mediated synaptic plasticity. Brain-derived neurotrophic factor is a central player for the effects of exercise on synaptic and cognitive plasticity. We found that 25-dihydroxyvitamin D3 decreased exercise-induced brain-derived neurotrophic factor but had no significant effect on neurotrophin-3 levels, thereby suggesting a level of specificity for brain-derived neurotrophic factor in the hippocampus. 25-Dihydroxyvitamin D3 injection also abolished the effects of exercise on the consummate end-products of brain-derived neurotrophic factor action, i.e. cyclic AMP response element-binding protein and synapsin I, and modulated phosphorylated calmodulin protein kinase II, a signal transduction cascade downstream to brain-derived neurotrophic factor action that is important for learning and memory. We also found that exercise significantly increased the expression of the mitochondrial uncoupling protein 2, an energy-balancing factor concerned with ATP production and free radical management. Our results reveal a fundamental mechanism by which key elements of energy metabolism may modulate the substrates of hippocampal synaptic plasticity.