Regulation Of CREB Phosphorylation Research Articles

Nuclear BK channels regulate CREB phosphorylation in RAW264.7 macrophages

BackgroundMacrophages are important cells of the innate immune system and contribute to a variety of physiological and pathophysiological responses. Monovalent and divalent ion channels have been studied in macrophage function, and while much research is still required, a role for these channels is beginning to emerge in macrophages. In addition to the plasma membrane, ion channels are also found in intracellular membranes including mitochondrial, lysosomal and nuclear membranes. While studying the function of plasma membrane located large conductance voltage- and calcium-activated potassium channels (BK channels) in a macrophage cell line RAW264.7, we became aware of the expression of these ion channels in other cellular locations.MethodsImmunofluorescence and Western blot analysis were used to identify the expression of BK channels. To demonstrate a functional role for the nuclear located channel, we investigated the effect of the lipid soluble BK channel inhibitor paxilline on CREB phosphorylation.ResultsTreatment of resting macrophages with paxilline resulted in increased CREB phosphorylation. To confirm a role for nuclear BK channels, these experiments were repeated in isolated nuclei and similar results were found. Ca2+ and calmodulin-dependent kinases (CaMK) have been demonstrated to regulate CREB phosphorylation. Inhibition of CaMKII and CaMKIV resulted in the reversal of paxilline-induced CREB phosphorylation.ConclusionsThese results suggest that nuclear BK channels regulate CREB phosphorylation in macrophages. Nuclear located ion channels may therefore be part of novel signalling pathways in macrophages and should be taken into account when studying the role of ion channels in these and other cells.Graphic abstract

Regulation of CREB Phosphorylation in Nucleus Accumbens after Relief Conditioning.

Relief learning is the association of environmental cues with the cessation of aversive events. While there is increasing knowledge about the neural circuitry mediating relief learning, the respective molecular pathways are not known. Therefore, the aim of the present study was to examine different putative molecular pathways underlying relief learning. To this purpose, male rats were subjected either to relief conditioning or to a pseudo conditioning procedure. Forty-five minutes or 6 h after conditioning, samples of five different brain regions, namely the prefrontal cortex, nucleus accumbens (NAC), dorsal striatum, dorsal hippocampus, and amygdala, were collected. Using quantitative Western blots, the expression level of CREB, pCREB, ERK1/2, pERK1/2, CaMKIIα, MAP2K, PKA, pPKA, Akt, pAkt, DARPP-32, pDARPP-32, 14-3-3, and neuroligin2 were studied. Our analyses revealed that relief conditioned rats had higher CREB phosphorylation in NAC 6 h after conditioning than pseudo conditioned rats. The data further revealed that this CREB phosphorylation was mainly induced by dopamine D1 receptor-mediated activation of PKA, however, other kinases, downstream of the NMDA receptor, may also contribute. Taken together, the present study suggests that CREB phosphorylation, induced by a combination of different molecular pathways downstream of dopamine D1 and NMDA receptors, is essential for the acquisition and consolidation of relief learning.

MiR-142-3p Regulates BDNF Expression in Activated Rodent Microglia Through Its Target CAMK2A.

Microglia, the innate immune effector cells of the mammalian central nervous system (CNS), are involved in the development, homeostasis, and pathology of CNS. Microglia become activated in response to various insults and injuries and protect the CNS by phagocytosing the invading pathogens, dead neurons, and other cellular debris. Recent studies have demonstrated that the epigenetic mechanisms ensure the coordinated regulation of genes involved in microglial activation. In this study, we performed a microRNA (miRNA) microarray in activated primary microglia derived from rat pup’s brain and identified differentially expressed miRNAs targeting key genes involved in cell survival, apoptosis, and inflammatory responses. Interestingly, miR-142-3p, one of the highly up-regulated miRNAs in microglia upon lipopolysaccharide (LPS)-mediated activation, compared to untreated primary microglia cells was predicted to target Ca2+/calmodulin dependent kinase 2a (CAMK2A). Further, luciferase reporter assay confirmed that miR-142-3p targets the 3′UTR of Camk2a. CAMK2A has been implicated in regulating the expression of brain-derived neurotrophic factor (BDNF) and long-term potentiation (LTP), a cellular mechanism underlying memory and learning. Given this, this study further focused on understanding the miR-142-3p mediated regulation of the CAMK2A-BDNF pathway via Cyclic AMP-responsive element-binding protein (CREB) in activated microglia. The results revealed that CAMK2A was downregulated in activated microglia, suggesting an inverse relationship between miR-142-3p and Camk2a in activated microglia. Overexpression of miR-142-3p in microglia was found to decrease the expression of CAMK2A and subsequently BDNF through regulation of CREB phosphorylation. Functional analysis through shRNA-mediated stable knockdown of CAMK2A in microglia confirmed that the regulation of BDNF by miR-142-3p is via CAMK2A. Overall, this study provides a database of differentially expressed miRNAs in activated primary microglia and reveals that microglial miR-142-3p regulates the CAMK2A-CREB-BDNF pathway which is involved in synaptic plasticity.

Sex Differences in the Rapid Cell Signaling Mechanisms Underlying the Memory-Enhancing Effects of 17β-Estradiol.

Little is known about how 17β-estradiol (E2) mediates memory formation in males. In ovariectomized (OVX) mice, bilateral dorsal hippocampal (DH) infusion of E2 enhances memory consolidation in object recognition (OR) and object placement (OP) tasks in a manner dependent on activation of extracellular signal-regulated kinase (ERK) and Akt signaling. Here, bilateral DH E2 infusion enhanced memory consolidation in both tasks among OVX female, gonadally-intact male, and castrated male mice, suggesting comparable facilitation of memory consolidation in both sexes, independent of testicular hormones in males. Contrary to previous reports in OVX mice, E2 did not increase DH ERK or Akt phosphorylation in males, nor did the ERK inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis (o-aminophenylmercapto) butadiene] prevent E2 from enhancing memory consolidation among intact and castrated males. These data suggest that ERK activation is not necessary for E2 to enhance memory consolidation in males, and compared with previous reports in females, reveal novel sex differences in the cell-signaling pathways through which E2 facilitates memory consolidation. To explore the mechanisms underlying E2-induced memory enhancements in males, phosphorylation of the transcription factor cAMP response element binding protein (CREB) in the DH was assessed. E2 increased phospho-CREB levels in both sexes, yet U0126 did not block these increases in castrated or intact males, indicating that E2 regulates CREB phosphorylation in males via an ERK-independent mechanism. Collectively, these findings suggest that the beneficial effects of hippocampal E2 on memory consolidation in males and females are mediated by different molecular mechanisms, which has important implications for the development of treatments to reduce memory dysfunction in men and women.

Phosphorylated delta sleep inducing peptide restores spatial memory and p-CREB expression by improving sleep architecture at high altitude

AimsSleep loss at high altitude (HA) play major role in worsening of neuropsychological functions, such as attention, memory and decision making. This study investigates the role of phosphorylated delta sleep inducing peptide (p-DSIP) in improving sleep architecture during chronic hypobaric hypoxia (HH) exposure and restoration of spatial navigational memory. MethodsMorris water maze (MWM) trained rats were exposed to HH at 7620 m. p-DSIP was injected intra-peritoneally (10 μg/Kg bw) during HH exposure as an intervention against sleep alteration. Sleep architecture was recorded telemetrically before and during HH exposure. Monoamines were estimated by high performance liquid chromatography from brain stem (BS) and hypothalamus. CREB and p-CREB level in hippocampus was studied by western blotting and expression of different monoamine regulatory enzymes in BS was measured by flow cytometry. Naloxone (1 mg/kg bw), a μ opioid receptor antagonist of sleep inducing effect of DSIP was also studied. Key findingsp-DSIP injection daily in circadian active period (18.30 h) during chronic HH enhanced non rapid eye movement sleep, rapid eye movement sleep as well as improved MWM performance of rats. p-DSIP treatment showed lower monoamine level and tyrosine hydroxylase (TH) expression and increased monoamine oxidase A (MAO A), glutamic acid decarboxylase (GAD) and Choline acetyltransferase (ChAT) expression. Further, naloxone altered navigational memory by decreasing the CREB and p-CREB level in hippocampus suggesting suppression of sleep inducing effect of p-DSIP. SignificanceOur study demonstrates that improvement of sleep quality by p-DSIP restores spatial memory by up regulating CREB phosphorylation during simulated high altitude hypoxia.

183 Novel glycomimetics inhibit gly-ldl induced smooth muscle cell calcification via creb

Advanced glycated end-products (AGEs) are known drivers of cardiovascular complications such as vascular calcification, which is currently untreatable, and are increased in diabetic subjects in part due to oxidative stress...

Glucocorticoid‐induced inhibition of AKT leads to CREB phosphorylation and increased myostatin expression via a PDE/cAMP/PKA pathway in skeletal muscle

Muscle atrophy in diseases like chronic kidney disease and diabetes is associated with dysfunctional regulation of insulin/Akt/FoxO3 and myostatin‐SMAD3 signaling pathways. We recently found CREB, a transcription factor that regulates maintenance and regeneration of skeletal muscle, is phosphorylated in wasting conditions. In silico analysis identifies a CREB binding site in the myostatin promoter. This study investigated the mechanism by which atrophy‐inducing conditions regulate CREB phosphorylation and its impact on myostatin expression. Differentiated C2C12 myotubes were incubated with combinations of dexamethasone (Dex), LY294002 (PI‐3 kinase inhibitor), H89 (protein kinase A inhibitor), isobutylmethyl xanthine (IBMX, a general phosphodiesterase (PDE) inhibitor), milrinone (PDE3 inhibitor), or rolipram (PDE4 inhibitor) for up to 24 h. Cells were harvested for analysis of proteins or mRNAs. Treatment of myotubes with Dex for 6–24h increased CREB phosphorylation (pCREB) within 3 h and sustained the increase over 24h. Dex also inhibited Akt phosphorylation (pAkt) over a similar time frame. Inhibition of pAkt by LY294002 mimicked Dex by increasing pCREB; the actions of LY294002 and Dex on pCREB were not additive. Since Akt can activate PDE3/PDE4 and reduce cAMP/PKA signaling in non‐muscle cells, we tested whether Dex‐induced inhibition of PDE3/4 increases pCREB. Without Dex, IBMX, milrinone, and rolipram increased pCREB. The Dex‐induced increase in pCREB was attenuated by the PKA inhibitor H89. When added individually, Dex, milrinone, rolipram and IBMX increase myostatin mRNA and protein significantly. Our study identifies a new mechanism by which atrophy‐inducing conditions activate CREB and contribute to myostatin expression. Signals that attenuate insulin/Akt signaling reduce the PDE3/4 activity. This leads to an increase in cAMP, PKA activity, CREB phosphorylation and myostatin expression.Support or Funding InformationFunded by NIH RO1 DK95610 and VA Merit X01BX001456

Period1 gates the circadian modulation of memory-relevant signaling in mouse hippocampus by regulating the nuclear shuttling of the CREB kinase pP90RSK.

Memory performance varies over a 24-h day/night cycle. While the detailed underlying mechanisms are yet unknown, recent evidence suggests that in the mouse hippocampus, rhythmic phosphorylation of mitogen-activated protein kinase (MAPK) and cyclic adenosine monophosphate response element-binding protein (CREB) are central to the circadian (~24h) regulation of learning and memory. We recently identified the clock protein PERIOD1 (PER1) as a vehicle that translates information encoding time of day to hippocampal plasticity. We here elaborate how PER1 may gate the sensitivity of memory-relevant hippocampal signaling pathways. We found that in wild-type mice (WT), spatial learning triggers CREB phosphorylation only during the daytime, and that this effect depends on the presence of PER1. The time-of-day-dependent induction of CREB phosphorylation can be reproduced pharmacologically in acute hippocampal slices prepared from WT mice, but is absent in preparations made from Per1-knockout (Per1(-/-) ) mice. We showed that the PER1-dependent CREB phosphorylation is regulated downstream of MAPK. Stimulation of WT hippocampal neurons triggered the co-translocation of PER1 and the CREB kinase pP90RSK (pMAPK-activated ribosomal S6kinase) into the nucleus. In hippocampal neurons from Per1(-/-) mice, however, pP90RSK remained perinuclear. A co-immunoprecipitation assay confirmed a high-affinity interaction between PER1 and pP90RSK. Knocking down endogenous PER1 in hippocampal cells inhibited adenylyl cyclase-dependent CREB activation. Taken together, the PER1-dependent modulation of cytoplasmic-to-nuclear signaling in the murine hippocampus provides a molecular explanation for how the circadian system potentially shapes a temporal framework for daytime-dependent memory performance, and adds a novel facet to the versatility of the clock gene protein PER1. We provide evidence that the circadian clock gene Period1 (Per1) regulates CREB phosphorylation in the mouse hippocampus, sculpturing time-of-day-dependent memory formation. This molecular mechanism constitutes the functional link between circadian rhythms and learning efficiency. In hippocampal neurons of wild-type mice, pP90RSK translocates into the nucleus upon stimulation with forskolin (left), whereas in Period1-knockout (Per1(-/-) ) mice (right) the kinase is trapped at the nuclear periphery, unable to efficiently phosphorylate nuclear CREB. Consequently, the presence of PER1 in hippocampal neurons is a prerequisite for the time-of-day-dependent phosphorylation of CREB, as it regulates the shuttling of pP90RSK into the nucleus. Representative immunofluorescence images show a temporal difference in phosphorylated cAMP response element-binding protein (pCREB; green color) levels in all regions of the dorsal hippocampus between a wild-type C3H mouse (WT; left) and a Period1-knockout (Per1(-/-) ; right) mouse. Images were taken 2h after lights on, thus, when fluctuating levels of pCREB peak in WT mouse hippocampus. Insets show a representative hippocampal neuron, in response to activating cAMP signaling, stained for the neuronal marker NeuN (red), the nuclear marker DAPI (blue) and the activated CREB kinase pP90RSK (green). The image was taken 2h after light onset (at the peak of the endogenous CREB phosphorylation that fluctuates with time of day). Magnification: 100X, inset 400X. Read the Editorial Highlight for this article on page 650. Cover image for this issue: doi: 10.1111/jnc.13332.

Calpain-mediated cleavage of DARPP-32 in Alzheimer's disease.

Toxicity induced by aberrant protein aggregates in Alzheimer’s disease (AD) causes synaptic disconnection and concomitant progressive neurodegeneration that eventually impair cognitive function. cAMP-response element-binding protein (CREB) is a transcription factor involved in the molecular switch that converts short-term to long-term memory. Although disturbances in CREB function have been suggested to cause memory deficits in both AD and AD animal models, the mechanism of CREB dysfunction is still unclear. Here, we show that the dopamine- and cAMP-regulated phosphoprotein 32 kDa (DARPP-32), a key inhibitor of protein phosphate-1 (PP-1) that regulates CREB phosphorylation, is cleaved by activated calpain in both AD brains and neuronal cells treated with amyloid-β or okadaic acid, a protein phosphatase-2A inhibitor that induces tau hyperphosphorylation and neuronal death. We found that DARPP-32 is mainly cleaved at Thr153 by calpain and that this cleavage of DARPP-32 reduces CREB phosphorylation via loss of its inhibitory function on PP1. Our results suggest a novel mechanism of DARPP-32–CREB signalling dysregulation in AD.

Appetitive cue-evoked ERK signaling in the nucleus accumbens requires NMDA and D1 dopamine receptor activation and regulates CREB phosphorylation.

Conditioned stimuli (CS) can modulate reward-seeking behavior. This modulatory effect can be maladaptive and has been implicated in excessive reward seeking and relapse to drug addiction. We previously demonstrated that exposure to an appetitive CS causes an increase in the activation of extracellular signal-regulated kinase (ERK) and cyclic-AMP response-element binding protein (CREB) in the nucleus accumbens (NAc) of rats, and that CS-evoked ERK activation is critical for CS control over reward seeking. To elucidate the mechanism that mediates CS-driven ERK activation in the NAc, we selectively blocked NMDA glutamate or D1 dopamine receptors in the NAc. To determine whether CS-driven ERK and CREB activation are linked, we selectively blocked ERK signaling in the NAc. We found that both NMDA and D1 receptors are critical for CS-driven ERK signaling in the NAc, and that this recruitment of the ERK cascade is responsible for increased CREB activation in the presence of the CS. Our findings suggest that activation of the NMDAR-D1R/ERK/CREB signal transduction pathway plays a critical role in the control of reward-seeking behavior by reward-predictive cues.

Spinal synaptic scaffolding protein Homer 1b/c regulates CREB phosphorylation and c-fos activation induced by inflammatory pain in rats

Previous studies have shown that spinal Homer 1b/c plays an important role in the maintenance of chronic inflammatory pain induced by complete Freund's adjuvant (CFA). This study investigated the possible mechanism underlying Homer 1b/c mediating CFA-induced inflammatory pain. Chronic inflammation was induced by CFA injection into the left hind ankle of the rat. Homer 1b/c antisense or missense oligonucleotides were administered intrathecally (10μg/10μl) from 5 to 8 days following the onset of inflammation. Immunohistochemistry was conducted to detect the expression of phosphorylated cAMP response element binding protein (pCREB) and Fos protein in the spinal dorsal horn. Intrathecal administration of Homer 1b/c antisense oligonucleotides not only markedly reduced the expression of Homer 1b/c protein, but also attenuated CFA-induced inflammation, spinal CREB phosphorylation, and Fos expression. These results demonstrate for the first time that Homer 1b/c regulates CREB phosphorylation and c-fos activation in the spinal dorsal horn during the maintenance of chronic inflammatory pain, suggesting that Homer 1b/c may be involved in the development of CFA-induced inflammation.

Regulation Of DKK-1 Expression By p38 MAPK Signaling Via CREB Myeloma Cells

Bone destruction is a hallmark of multiple myeloma and severely compromises a patient's quality of life. Recently, we have demonstrated that p38 mitogen-activated protein kinase (MAPK), which is constitutively activated in myeloma cells, is a master regulator of myeloma-mediated bone destruction. We have observed that myeloma cell p38 MAPK upregulated the production of dickkopf-1 (DKK-1), of which inhibits osteoblast differentiation and bone formation by inhibiting Wnt signaling and enhances osteoclast differentiation and bone resorption by upregulating RANKL production. Our results have shown that treatment of SB203580 (SB20), a p38 MAPK inhibitor, downregulated, while treatment of anisomycin, a p38 MAPK activator, upregulated DKK-1 expression. However, the mechanism underlying the regulation of DKK-1 expression by p38 MAPK in myeloma cells remains poorly elucidated. Previous studies have suggested that CREB is a p38 MAPK-targeted transcriptional factor. We therefore hypothesized that myeloma cell p38 MAPK may upregulate the transcriptional levels of DKK-1 through CREB. To verify whether p38 MAPK regulates CREB phosphorylation in myeloma cells, ARP-1 and U266 cells were treated with SB20 or anisomycin. Western blotting results showed that SB20 treatment significantly reduced, while anisomycin treatment enhanced phosphorylation of CREB in both cell lines. Database analysis of DKK-1 promoter regions predicted a couple of potential CREB-binding sites localized around 1.3 kb upstream from the starting codons. CHIP assay further indicated that CREB specifically bound to the one of the putative binding sites (-1,354 bp). To validate the CHIP assay results, we designed three constructs: construct 1 containing the whole promoter (-2,542 bp to -220 bp), which has previously been shown to have strong transcriptional activity; construct 2, as a truncated portion of the promoter containing the putative CREB-binding sites (-1,392 bp to -220 bp); and construct 3, as a truncated portion of the promoter containing neither CREB-binding sites (-1073 bp to -220 bp). These constructs were amplified by PCR and cloned into a luciferase reporter gene vector (pGL2) respectively. To examine the effect of CREB on the transcription activities of DKK1 in myeloma cells, reporter constructs were transfected into ARP-1 and U266 cells. In line with our findings in CHIP assay, we observed that in both myeloma cells, the construct 2 had similar strong transcriptional activity as the construct 1 did, the construct 3 had little activity. These results demonstrated that there was a CREB-binding site in DKK-1 promoter, and is required for p38 MAPK-upregulated DKK-1 expression in myeloma cells. In conclusion, our results uncover a mechanism of myeloma cell p38 MAPK in osteoblast inhibition and osteoclast activation by which p38 MAPK transcriptionally upregulates DKK-1 expression via CREB. Disclosures:Orlowski:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array Biopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees.

CaMKIIδ-dependent Inhibition of cAMP-response Element-binding Protein Activity in Vascular Smooth Muscle

One transcription factor mediator of Ca(2+)-signals is cAMP response element-binding protein (CREB). CREB expression and/or activity negatively correlates with vascular smooth muscle (VSM) cell proliferation and migration. Multifunctional Ca(2+)/calmodulin-dependent protein kinases, including CaMKII, have been demonstrated to regulate CREB activity through both positive and negative phosphorylation events in vitro, but the function of CaMKII as a proximal regulator of CREB in intact cell systems, including VSM, is not clear. In this study, we used gain- and loss-of-function approaches to determine the function of CaMKIIδ in regulating CREB phosphorylation, localization, and activity in VSM. Overexpression of constitutively active CaMKIIδ specifically increased CREB phosphorylation on Ser(142) and silencing CaMKIIδ expression by siRNA or blocking endogenous CaMKII activity with KN93 abolished thrombin- or ionomycin-induced CREB phosphorylation on Ser(142) without affecting Ser(133) phosphorylation. CREB-Ser(142) phosphorylation correlated with transient nucleocytoplasmic translocation of CREB. Thrombin-induced CREB promoter activity, CREB binding to Sik1 and Rgs2 promoters, and Sik1/Rgs2 transcription were enhanced by a kinase-negative CaMKIIδ2 (K43A) mutant and inhibited by a constitutively active (T287D) mutant. Taken together, these studies establish negative regulation of CREB activity by endogenous CaMKIIδ-dependent CREB-Ser(142) phosphorylation and suggest a potential mechanism for CaMKIIδ/CREB signaling in modulating proliferation and migration in VSM cells.

Glycogen synthase kinase-3 (GSK-3) regulates TGF-β₁-induced differentiation of pulmonary fibroblasts.

Chronic lung diseases such as asthma, COPD and pulmonary fibrosis are characterized by abnormal extracellular matrix (ECM) turnover. TGF-β is a key mediator stimulating ECM production by recruiting and activating lung fibroblasts and initiating their differentiation process into more active myofibroblasts. Glycogen synthase kinase-3 (GSK-3) regulates various intracellular signalling pathways; its role in TGF-β₁-induced myofibroblast differentiation is currently largely unknown. To determine the contribution of GSK-3 signalling in TGF-β₁-induced myofibroblast differentiation. We used MRC5 human lung fibroblasts and primary pulmonary fibroblasts of individuals with and without COPD. Protein and mRNA expression were determined by immunoblotting and RT-PCR analysis respectively. Stimulation of MRC5 and primary human lung fibroblasts with TGF-β₁ resulted in time- and dose-dependent increases of α-sm-actin and fibronectin expression, indicative of myofibroblast differentiation. Pharmacological inhibition of GSK-3 by SB216763 dose-dependently attenuated TGF-β₁-induced expression of these myofibroblasts markers. Moreover, silencing of GSK-3 by siRNA or pharmacological inhibition by CT/CHIR99021 fully inhibited the TGF-β₁-induced expression of α-sm-actin and fibronectin. The effect of GSK-3 inhibition on α-sm-actin expression was similar in fibroblasts from individuals with and without COPD. Neither smad, NF-κB nor ERK1/2 were involved in the inhibitory actions of GSK-3 inhibition by SB126763 on myofibroblast differentiation. Rather, SB216763 increased the phosphorylation of CREB, which in its phosphorylated form acts as a functional antagonist of TGF-β/smad signalling. We demonstrate that GSK-3 signalling regulates TGF-β₁-induced myofibroblast differentiation by regulating CREB phosphorylation. GSK-3 may constitute a useful target for treatment of chronic lung diseases.

Different signal transduction pathways elicited by basic fibroblast growth factor and interleukin 1β regulate CREB phosphorylation in Sertoli cells.

Basic fibroblast growth factor (bFGF) and interleukin 1β (IL1β) belong to the set of intratesticular regulators that provide for the fine-tuning of processes implicated in the maintenance of spermatogenesis. The aim of this study was to investigate if bFGF and IL1β activate CREB, what signaling pathways may be participating and the possible relationship between CREB activation and the regulation of Sertoli cell function. Twenty-day-old rat Sertoli cell cultures were used. Cultures stimulated with bFGF and IL1β produced a time-dependent increment in phosphorylated CREB levels that reached maximal values in 5- and 15-minute incubations respectively. MEK inhibitors--PD98059 and U0126--blocked the effect of bFGF on phosphorylated CREB while a p38-MAPK inhibitor--SB203580--blocked the effect of IL1β on phosphorylated CREB. A possible correlation between CREB regulation and two Sertoli cell-differentiated functions, Ldh A and transferrin expression, was explored. PD98059 blocked the ability of bFGF to stimulate Ldh A expression and SB203580 blocked the ability of IL1β to stimulate Ldh A expression and LDH activity. Concerning transferrin, PD98059 and U0126 were able to inhibit the ability of bFGF to stimulate its secre tion. On the contrary, SB203580 was unable to block IL1β induced increase in transferrin secretion suggesting that the p38-MAPK pathway does not participate in the mechanism of action of the cytokine to regulate transferrin. The results presented herein suggest that CREB is stimulated in response to bFGF and IL1β through p42/p44-MAPK and p38-MAPK pathways and that this transcription factor may be partially responsible for the regulation of Sertoli cell function.

Membrane estrogen receptors activate the metabotropic glutamate receptors mGluR5 and mGluR3 to bidirectionally regulate CREB phosphorylation in female rat striatal neurons

Along with its ability to directly regulate gene expression, estradiol influences cell signaling and brain functions via rapid, membrane-initiated events. In the female rat striatum, estradiol activates membrane-localized estrogen receptors to influence synaptic neurotransmission, calcium channel activity, and behaviors related to motor control. Yet, the mechanism by which estradiol acts to rapidly affect striatal physiology has remained elusive. Here we find that membrane estrogen receptors (ERs) couple to the metabotropic glutamate receptors mGluR5 and mGluR3, providing the framework to understand how membrane estrogen receptors affect striatal function. Using CREB phosphorylation as a downstream measure of ER/mGluR activation, membrane-localized estrogen receptor α (ERα) activates mGluR5 signaling to mediate mitogen-activated protein kinase (MAPK)-dependent CREB phosphorylation. Further, ERα and estrogen receptor β (ERβ) activate mGluR3 to attenuate L-type calcium channel-dependent CREB signaling. Interestingly, while this fundamental mechanism of ER/mGluR signaling was initially characterized in hippocampal neurons, estrogen receptors in striatal neurons are paired with a different set of mGluRs, resulting in the potential to functionally isolate membrane-initiated estrogen signaling across brain regions via use of specific mGluR modulators. These results provide both a mechanism for the rapid actions of estrogens within the female striatum, as well as demonstrate that estrogen receptors can interact with a more diverse set of surface membrane receptors than previously recognized.

Impairment of memory consolidation by galanin correlates with in vivo inhibition of both LTP and CREB phosphorylation

Changes in the state of CREB phosphorylation and in LTP in the hippocampus have been associated with learning and memory. Here we show that galanin, the neuropeptide released in the hippocampal formation from cholinergic and noradrenergic fibers, that has been shown to produce impairments in memory consolidation in the Morris water maze task inhibits both LTP and CREB phosphorylation in the rat hippocampus in vivo. While there are many transmitters regulating CREB phosphorylation none has been shown to suppress behaviorally-induced hippocampal CREB phosphorylation as potently as galanin. The in vivo inhibition of dentate gyrus-LTP and of CREB phosphorylation by the agonist occupancy of GalR1 and GalR2-type galanin receptors provides strong in vivo cellular and molecular correlates to galanin-induced learning deficits and designates galanin as a major regulator of the memory consolidation process.

Chronic morphine administration induces over-expression of aldolase C with reduction of CREB phosphorylation in the mouse hippocampus

In recent studies, alterations in the activity and expression of metabolic enzymes, such as those involved in glycolysis, have been detected in morphine-dependent patients and animals. Increasing evidence demonstrates that the hippocampus is an important brain region associated with morphine dependence, but the molecular events occurring in the hippocampus following chronic exposure to morphine are poorly understood. Aldolase C is the brain-specific isoform of fructose-1, 6-bisphosphate aldolase which is a glycolytic enzyme catalyzing reactions in the glycolytic, gluconeogenic, and fructose metabolic pathways. Using Western blot and immunofluorescence assays, we found the expression of aldolase C was markedly increased in the mouse hippocampus following chronic morphine treatment. Naloxone pretreatment before morphine administration suppressed withdrawal jumping, weight loss, and overexpression of aldolase C. CREB is a transcription factor regulated through phosphorylation on Ser133, which is known to play a key role in the mechanism of morphine dependence. When detecting the expression of phosphorylated CREB (p-CREB) in the mouse hippocampus using Western blot and immunohistochemistry, we found CREB phosphorylation was clearly decreased following chronic morphine treatment. Interestingly, laser-confocal microscopy showed that overexpression of aldolase C in mouse hippocampal neurons was concomitant with the decreased immunoreactivity of p-CREB. The results suggest potential links between the morphine-induced alteration of aldolase C and the regulation of CREB phosphorylation, a possible mechanism of morphine dependence.

Calcium/calmodulin-dependent protein kinase II regulates the phosphorylation of CREB in NMDA-induced retinal neurotoxicity

We examined the role of the phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII) and cyclic AMP-response element binding protein (CREB) in N-methyl-d-aspartate (NMDA)-induced neurotoxicity in the rat retina. Western blot analysis showed early elevation of phosphorylated CaMKII (p-CaMKII) protein levels and subsequential elevation of phosphorylated CREB (p-CREB) protein after NMDA injection. Immunohistochemistry showed that p-CaMKII was colocalized with Thy-1-positive retinal ganglion cells (RGCs) after NMDA injection. The increase in the p-CaMKII protein level was significantly inhibited by the preinjection of CaMKII small interfering RNA (siRNA), whereas negative control siRNA did not affect. Moreover, the increase in the p-CREB protein level after NMDA injection was also prevented by preinjection of CaMKII siRNA. In addition, our morphometric study of neurotracer retrograde labeling and Thy-1-positive cells showed that CaMKII siRNA significantly accelerated NMDA-induced RGC loss. Furthermore, the prevention of CREB binding by CRE decoy oligonucleotide also exacerbated RGC loss. These results suggest that the activation of CaMKII may regulate CREB phosphorylation and that the transient phosphorylation of CaMKII and CREB may be a neuroprotective response against NMDA-induced neurotoxicity.

D-Serine regulates CREB phosphorylation induced by NMDA receptor activation in Müller glia from the retina

D-serine is an N-methyl-D-aspartate (NMDA) receptor coagonist predominantly produced by glial cells in the brain and the retina. Whereas a role for D-serine as a modulator of NMDA receptors in neurons has been suggested, its function in glial cells has not been analyzed. We here show that D-serine modulates gene expression in Müller glial cells from the retina through the induction of transcription factor CREB phosphorylation and the expression of the immediate-early gene c-fos. Pharmacological analysis indicates that D-serine effect involves NMDA receptor activation. Comparison of the effect of D-serine in Müller cells, hippocampal astrocytes and hippocampal neurons suggests that D-serine could function as a retinal NMDA receptor coagonist activating functionally relevant transcription factor pathways in glial cells.