CREB Protein Levels Research Articles

Traditional Chinese medicine Bao Gan Ning increase phosphorylation of CREB in liver fibrosis in vivo and in vitro

Previous studies have demonstrated that traditional Chinese medicine Bao Gan Ning, which contains six different drugs: Trionyx sinensis Wiegmann shell, Prunus persica (L.) Batsch seed, Salvia miltiorrhiza Bge. root, Mallotus opelta (Lour.) Muell-Arg root, Astragalus membranaceus (Fisch.) Bge. var. mongho-licus (Bge.) Hsiao root and Scutellaria baicalensis Georgi root, was able to protect liver against fibrosis in CCL 4 models. In an effort to elucidate molecular mechanisms by which Bao Gan Ning exerts its anti-fibrosis activity, effects of Bao Gan Ning on liver fibrosis and cAMP response element binding protein (CREB), an important transcription factor involved in liver fibrosis, were evaluated in animal and cell models in this work. Results showed that Bao Gan Ning (2.16 or 4.32 g/kg/day) significantly decreased alanine aminotransferase (ALT) and hyaluronidase levels and reversed liver fibrosis in rat liver fibrosis models. The proliferation of HSC-T6, a hepatic stellate cell line, was also significantly inhibited by incubation with serums that were prepared from rats fed with Bao Gan Ning. Most interestingly, results from Western blot, immunohistochemistry and electrophoretic mobility shift assay (EMSA) showed that Bao Gan Ning up-regulated CREB phosphorylation both in rat liver fibrosis models and in HSC-T6 cells, but did not affect protein level of CREB and the DNA binding activity of CREB. These results suggested that up-regulation of CREB phosphorylation may be involved in anti-fibrosis activity of Chinese medicine Bao Gan Ning.

Short chain fatty acids regulate tyrosine hydroxylase gene expression through a cAMP-dependent signaling pathway

Multiple intracellular and extracellular regulatory factors affect transcription of the tyrosine hydroxylase (TH) gene encoding the rate-limiting enzyme in the biosynthesis of the neurotransmitters dopamine, norepinephrine and epinephrine. Short chain fatty acids like butyrate are known to alter TH gene expression, but the mechanism of action is unknown. In this report, transient transfection assays identified the proximal TH promoter to contain sufficient genetic information to confer butyrate responsiveness to a reporter gene. Deletion studies and gel shift analyses revealed that the promoter region spanning the cAMP response element is an absolute requirement for transcriptional activation by butyrate. The branched short chain fatty acid valproate is used for seizure control in humans. Significantly, it has a similar aliphatic structure to butyrate, and it was found to have similar effects on TH in PC12 cells. Site-directed mutagenesis indicated that the effects of both fatty acids were mediated through the canonical CRE. Butyrate treatment also resulted in CREB phosphorylation without changing CREB protein levels. The increased phosphorylation of CREB correlated with accumulation of TH mRNA. The adenylate cyclase inhibitor dideoxyadenosine blocked both CREB phosphorylation and accumulation of TH mRNA. The data are consistent with the conclusion that butyrate induces post-translational modifications of pre-existing CREB molecules in a cAMP/PKA-dependent manner to alter TH transcription. These results support the role of butyrate as a novel exogenous regulatory factor in TH gene expression. Our data delineate a molecular mechanism through which diet-derived environmental signals (e.g. butyrate) can modulate catecholaminergic systems by affecting TH gene transcription.

Dissociation of the effect of spatial behaviors on the phosphorylation of cAMP-response element binding protein (CREB) within the nucleus accumbens

Several studies have reported a role for the nucleus accumbens (NAcc) in learning and synaptic plasticity. Many of them suggest that the NAcc is involved in translating cortico-limbic information to the motor system mediating spatial learning and memory processes. Previous studies from our laboratory have shown that protein kinase C is activated following training in a food search spatial learning task. The present study further characterizes the molecular substrates associated with NAcc-dependent spatial behavior. The cyclic AMP-response element binding protein (CREB), a transcription factor implicated in the formation of long-term memory, was studied in the NAcc following spatial training in a food search spatial learning task. Western blots were performed to detect phosphorylated (activated) and total CREB protein levels. Our results show that CREB is significantly phosphorylated in the NAcc 48 h after habituation and at 5 min and 1 h after the first spatial training session in comparison with the naive animals that remained in their home cages. Since published data show that NAcc plays a role in novelty detection and reactivity, we conducted further experiments in order to dissociate the effect on CREB phosphorylation and expression of spatial novelty (single exposure), exploration, and spatial learning in the food search apparatus. Results show that CREB phosphorylation is significantly increased 48 h after exposure to a novel environment. The present study suggests that CREB phosphorylation observed in the NAcc during habituation and spatial training may be mainly triggered by detection of spatial novelty.

CREB Is Amplified in AML Blasts and Is Associated with an Increased Risk of Relapse and Decreased Event-Free Survival.

The cAMP Responsive Element Binding Protein (CREB) is a basic leucine zipper transcription factor that regulates cell proliferation and survival. Greater than 60% of AML patients have elevated CREB protein levels in the bone marrow. Transgenic mice that overexpress CREB in myeloid cells have monocytosis and splenomegaly. Bone marrow progenitor cells from CREB transgenic mice exhibit properties of transformed cells including immortalization and growth factor-independence in colony assays. Our results suggest that CREB acts as a proto-oncogene in hematopoietic cells. We first sought to understand the mechanism of CREB overexpression in AML blasts. Since both CREB protein and mRNA levels were increased in primary AML cells, we hypothesized that CREB could be amplified. We performed fluorescence in situ hybridization (FISH) on blast cells from 4 CREB overexpressing patients using a CREB specific BAC clone. The CREB gene has been previously localized to chromosome 2q32.3–q34. In blast cells from 3 out of 4 AML patients that overexpressed CREB, we detected 3 to 4 signals from one homolog and 1 signal from the other in over 100 interphase nuclei analyzed. However, in normal control lymphocytes in metaphase and CREB nonamplified bone marrow cells in interphase, the CREB BAC showed two clear and unambiguous signals. Three out of the 4 AML patients whose bone marrow overexpressed CREB had a normal karyotype with no evidence of aneuploidy or hyperdiploidy. These results indicate that in certain AML patients there are more than two copies of the CREB gene. To evaluate whether increased CREB expression predicts outcome in leukemia patients, Kaplan-Meier analysis was performed on data from 45 AML patients (14 age 18 or under and 31 over age 18). Time to relapse or persistent disease was markedly shorter in the 31 CREB overexpressing, i.e. CREB positive, patients in comparison to the 14 not expressing CREB (proportion with persistent disease or relapsing by 1 year 0.68 ± 0.10 vs. 0.23 ± 0.14, p<0.001 both before and after stratification on cytogenetics). Likewise, event-free survival was noticeably worse in the CREB positive group (p<0.003). The seven patients who showed persistent disease after initial induction therapy were all CREB positive, but excluding them from the analysis still yielded noticeably shorter relapse times for the CREB positive group (p=0.01). No variable other than CREB overexpression had a significant association with adverse time to relapse or event-free survival. Initial treatment, cytogenetics, AML subtype, age, gender, initial white blood count, absolute blast count and follow-up times were similar between the two CREB groups. Percent blasts at diagnosis (mean ± SD) were higher in the CREB overexpressing group (69 ± 28 vs 51 ± 24, p=0.02). However, neither stratification on blast percent nor on any other variable altered the significance of CREB as a predictor of initial adverse outcome. In contrast, overall survival tended to differ in the expected way among the cytogenetic prognostic groups (p=0.03), but without strong evidence for an association with CREB expression (p=0.35). Our results suggest that CREB expression may be an important independent predictor of remission duration and event-free survival in AML patients.

Colocalization of phosphorylated CREB with calcium/calmodulin-dependent protein kinase IV in hippocampal neurons induced by ohmfentanyl stereoisomers

The transcription factor cAMP response element-binding protein (CREB) plays an important role in opioids dependence. To better understand the role of CREB in opioids dependence and underlying signal pathways, we compared the effects of three ohmfentanyl stereoisomers ((−)- cis-(3R,4S,2′R) OMF (F9202), (+)- cis-(3R,4S,2′S) OMF (F9204), (−)- cis-(3S,4S,2′R) OMF (F9203)) and morphine on CREB phosphorylation and the expression of Ca 2+/calmodulin-dependent protein kinase IV (CaMKIV) in hippocampus derived from mice which displayed conditioned place preference (CPP) behavior by Western blot, and immunohistochemistry analyses. Moreover, we studied the effects of OMF and morphine on CREB phosphorylation and colocalization of phosphorylated CREB (P-CREB) with CaMKIV in cultured rat hippocampal neurons by Western blot, and confocal fluorescence microscopy analyses. The results showed that F9202, F9204 or morphine, which could induce CPP, enhanced CREB phosphorylation and the expression of CaMKIV in hippocampus from CPP mice without affecting total CREB protein level. The CREB phosphorylation of cultured hippocampal neurons was also enhanced and reached its peak level at 30 min upon exposure to F9202 (100 nM), F9204 (100 nM) or morphine (1 μM), while the total CREB protein level was not altered. KN-62 (10 μM), an inhibitor of CaM kinases, prevented CREB phosphorylation induced by morphine, F9202, and F9204 without change of total CREB level. The results of confocal fluorescence microscopy further demonstrated that the activated CREB (P-CREB) was colocalized with CaMKIV in nucleus. F9203, which could not induce CPP, failed to increase the CREB phosphorylation and the colocalization of P-CREB with CaMKIV both in hippocampus from CPP mice and in cultured hippocampal neurons. This is the first evidence to suggest that the increased CREB phosphorylation via CaMKIV signal pathway in hippocampus is relevant to opioids psychological dependence.

Overexpression of CREB reduces CRE-mediated transcription: behavioral and cellular analyses in transgenic mice

The CREB transcription factor mediates neuronal plasticity in many systems, but the relationship between CREB levels and CRE-mediated transcription in individual neurons in vivo is unclear. In FVB/N nontransgenic mice, we observed that Purkinje cells showed low basal levels of Ser 133-phosphorylated CREB protein yet displayed strong CRE-directed transcription. Transgenic mice overexpressing CREB in Purkinje cells and dentate gyrus granule cells showed a decreased CRE-lacZ signal in the same cells, indicating repression of ATF/CREB family function. Dentate region long-term potentiation was not altered by these changes in CREB expression. CREB transgenic mice demonstrated an inability to perform the rotarod task, without signs of overt ataxia. Our results demonstrate that the level of phosphorylated CREB protein is not a reliable indicator of CRE-mediated function. Furthermore, we conclude that CRE-mediated transcription may be linked to only a subset of cerebellum-mediated motor behaviors and may not be universally required for long-lasting synaptic potentiation.

Dopamine-dependent increases in phosphorylation of cAMP response element binding protein (CREB) during precipitated morphine withdrawal in primary cultures of rat striatum.

Chronic morphine leads to compensatory up-regulation of cAMP signaling pathways in numerous brain regions. One potential consequence of up-regulated cAMP signaling is increased phosphorylation of cAMP response element binding protein (CREB), a transcription factor that may regulate neuroadaptations related to morphine dependence. Altered gene expression within the nucleus accumbens (NAc), a ventral component of the striatum that receives substantial dopaminergic input, may play a role in some of the motivational aspects of opiate withdrawal. To determine if morphine withdrawal leads to increased CREB phosphorylation in striatal tissues, we examined the effects of naloxone-precipitated morphine withdrawal on CREB phosphorylation in primary cultures of rat striatal neurons. Precipitated morphine withdrawal was associated with enhanced dopamine-, SKF 82958 (D1 receptor agonist)-, and forskolin-induced CREB phosphorylation. During precipitated withdrawal, D1 receptor-mediated CREB phosphorylation was dependent on cAMP-dependent protein kinase (PKA). Precipitated withdrawal also led to up-regulation of c-fos mRNA in response to SKF 82958. CREB protein levels were not altered by acute or chronic morphine. These results suggest that D1 receptor-mediated signal transduction is enhanced during morphine withdrawal. Furthermore, they are consistent with in vivo evidence suggesting that increased CREB activation in portions of the striatum (e.g. the NAc) is related to dysphoric states associated with drug withdrawal.

Transcription factor 3',5'-cyclic adenosine 5'-monophosphate-responsive element-binding protein (CREB) is decreased during human adrenal cortex tumorigenesis and fetal development.

Various cellular and molecular alterations of the cAMP pathway have been observed in adrenal Cushing syndrome. We recently reported the loss of cAMP-responsive element-binding protein (CREB) expression in the adrenocortical cancer cell line H295R. CREB is the major nuclear target of the cAMP pathway. This study therefore aimed to analyze the status of the CREB protein in various types of human adrenocortical tumors and normal fetal adrenal cortex. CREB protein status was studied by Western blotting in adrenocortical adenomas (AAs, n = 27) and adrenocortical carcinomas (ACs, n = 24). A decrease of CREB protein was noticed in the majority of the adrenocortical tumors. The dramatic decrease in CREB protein levels was more pronounced in ACs than in AAs. Levels of the phosphorylated form of CREB were also low in adrenocortical tumors, with a greater decrease in ACs than in AAs. EMSAs also showed decreases in the amounts of CREB- containing complexes in nuclear extracts from adrenocortical tumors. The secretory status of adenomas was strongly correlated with CREB levels, significantly lower in nonfunctioning AAs (n = 9) than in functioning AAs (n = 9). CREB levels, determined by Western blotting and immunohistochemistry, were very low in the fetal zone of human fetal adrenal cortex, whereas they were normal in the definitive zone. In tumors, adrenocortical cells in several zones were weakly immunohistochemically stained for CREB, whereas CREB was uniformly detected in nonendocrine cell nuclei (e.g. vascular cells, fibroblasts). These results suggest that the absence of CREB may be linked to the development of a highly aggressive tumor with a dedifferentiated benign (nonfunctioning AA) or malignant (AC) phenotype. These findings highlight the similarities between the normal human fetal adrenal gland and adrenal cancers previously observed in terms of parallelism in IGF-II production.

Expression of cAMP response element-binding protein and sodium iodide symporter in benign non-functioning and malignant thyroid tumours.

Reduced expression or defective targeting of the sodium/iodide symporter (NIS) to the cell membrane in thyroid tumours has been reported. The expression of the NIS gene is up-regulated by TSH through the cAMP pathway and the characterization of the promoter region of the rat NIS gene revealed the existence of a degenerate cAMP response element (CRE) sequence. The cAMP-dependent transcription factor cAMP response element-binding protein (CREB) binds to CRE acting, upon phosphorylation, as a transcriptional activator. In this study we evaluated the expression of CREB and NIS gene in thyroid non-functioning adenomas (n=18) and carcinomas (n=20), as well as in the corresponding normal tissue. The levels of CREB and NIS mRNA were determined by quantitative real-time RT-PCR, whereas CREB protein (total and phosphorylated) was analyzed by Western blot. The levels of CREB mRNA in thyroid carcinomas, but not in adenomas, were significantly lower than in the corresponding normal tissue (4.63+/-0.89 vs 9.51+/-2.01 pg/microg total RNA, means+/-s.e., P=0.025). CREB protein levels, which were determined in a subset of samples, were in quite good agreement with mRNA data. NIS mRNA levels did not differ in adenomas or carcinomas, compared with the corresponding normal tissue and no significant relationship with the levels of CREB mRNA was observed. Our results have indicated for the first time that reduced levels of CREB expression are a feature of thyroid carcinomas, and confirm that different factors are likely to modulate NIS expression.

Molecular profiling of midbrain dopamine regions in cocaine overdose victims.

Chronic cocaine use in humans and animal models is known to lead to pronounced alterations in neuronal function in brain regions associated with drug reinforcement. To evaluate whether the alterations in gene expression in cocaine overdose victims are associated with specific dopamine populations in the midbrain, cDNA arrays and western blotting were used to compare gene and protein expression patterns between cocaine overdose victims and age-matched controls in the ventral tegmental area (VTA) and lateral substantia nigra (l-SN). Array analysis revealed significant up-regulation of numerous transcripts in the VTA, but not in the l-SN, of cocaine overdose victims including NMDAR1, GluR2, GluR5 and KA2 receptor mRNA (p < 0.05). No significant alterations between overdose victims and controls were observed for GluR1, R3 or R4 mRNA levels. Correspondingly, western blot analysis revealed VTA-selective up-regulation of CREB (p < 0.01), NMDAR1 (p < 0.01), GluR2 (p < 0.05), GluR5 (p < 0.01) and KA2 (p < 0.05) protein levels of cocaine overdose victims. The present results indicate that selective alterations of CREB and certain ionotropic glutamate receptor (iGluR) subtypes appear to be associated with chronic cocaine use in humans in a region-specific manner. Moreover, as subunit composition determines the functional properties of iGluRs, the observed changes may indicate alterations in the excitability of dopamine transmission underlying long-term biochemical and behavioral effects of cocaine in humans.

MyoD stimulates RB promoter activity via the CREB/p300 nuclear transduction pathway.

The induction of RB gene transcription by MyoD is a key event in the process of skeletal muscle differentiation, because elevated levels of the retinoblastoma protein are essential for myoblast cell cycle arrest as well as for the terminal differentiation and survival of postmitotic myocytes. We previously showed that MyoD stimulates transcription from the RB promoter independently of direct binding to promoter sequences. Here we demonstrate that stimulation by MyoD requires a cyclic AMP-responsive element (CRE) in the RB promoter, bound by the transcription factor CREB in differentiating myoblasts. We also show that both the CREB protein level and the level of phosphorylation of the CREB protein at Ser-133 rapidly increase at the onset of muscle differentiation and that both remain high throughout the myogenic process. Biochemical and functional evidence indicates that in differentiating myoblasts, MyoD becomes associated with CREB and is targeted to the RB promoter CRE in a complex also containing the p300 transcriptional coactivator. The resulting multiprotein complex stimulates transcription from the RB promoter. These and other observations strongly suggest that MyoD functions by promoting the efficient recruitment of p300 by promoter-bound, phosphorylated CREB.

Abnormal Expression and Functional Characteristics of Cyclic Adenosine Monophosphate Response Element Binding Protein in Postmortem Brain of Suicide Subjects

Cyclic adenosine monophosphate response element binding protein (CREB) is a transcription factor that, on phosphorylation by protein kinases, is activated, and in response, regulates the transcription of many neuronally expressed genes. In view of the recent observations that catalytic properties and/or expression of many kinases that mediate their physiological responses through the activation of CREB are altered in the postmortem brain of subjects who commit suicide (hereafter referred to as suicide subjects), we examined the status of CREB in suicidal behavior. These studies were performed in Brodmann area (BA) 9 and hippocampus obtained from 26 suicide subjects and 20 nonpsychiatric healthy control subjects. Messenger RNA levels of CREB and neuron-specific enolase were determined in total RNA by means of quantitative reverse transcriptase-polymerase chain reaction. Protein levels and the functional characteristics of CREB were determined in nuclear fractions by means of Western blot and cyclic adenosine monophosphate response element (CRE)-DNA binding activity, respectively. In the same nuclear fraction, we determined the catalytic activity of cyclic adenosine monophosphate-stimulated protein kinase A by means of enzymatic assay. We observed a significant reduction in messenger RNA and protein levels of CREB, CRE-DNA binding activity, and basal and cyclic adenosine monophosphate-stimulated protein kinase A activity in BA 9 and hippocampus of suicide subjects, without any change in messenger RNA levels of neuron-specific enolase in BA 9. Except for protein kinase A activity, changes in CREB expression and CRE-DNA binding activity were present in all suicide subjects, irrespective of diagnosis. These changes were unrelated to postmortem intervals, age, sex, or antidepressant treatment. Given the significance of CREB in mediating various physiological functions through gene transcription, our results of decreased expression and functional characteristics of CREB in postmortem brain of suicide subjects suggest that CREB may play an important role in suicidal behavior.

Oxidative stress‐mediated down‐regulation of bcl‐2 promoter in hippocampal neurons

Generation of oxidative stress/reactive oxygen species (ROS) is one of the causes of neuronal apoptosis. We have examined the effects of ROS at the transcriptional level in an immortalized hippocampal neuronal cell line (H19-7) and in rat primary hippocampal neurons. Treatment of H19-7 cells with hydrogen peroxide (150 micro m) resulted in a 40% decrease in Bcl-2 protein and a parallel decrease in bcl-2 mRNA levels. H19-7 cells overexpressing bcl-2 were found to be resistant to ROS-induced apoptosis. We had previously shown that bcl-2 promoter activity is positively regulated by the transcription factor cyclic AMP response element binding protein (CREB) in neurons. In the present study, we demonstrate that ROS decreases the activity of luciferase reporter gene driven by a cyclic AMP response element site containing bcl-2 promoter. Exposure of neurons to ROS for 6 h resulted in basal and fibroblast growth factor-2-stimulated phosphorylation/activation of CREB. Chronic 24 h treatment with ROS led to a significant (p < 0.01) decrease in CREB protein and CREB mRNA levels. Adenoviral overexpression of wild type CREB in H19-7 cells resulted in significant (p < 0.01) protection against ROS-induced apoptosis through up-regulation of Bcl-2 expression whereas dominant negative CREB exaggerated the injury. These findings demonstrate that loss of CREB function contributes to oxidative stress-induced neuronal dysfunction.

Ohmefentanyl stereoisomers induce changes of CREB phosphorylation in hippocampus of mice in conditioned place preference paradigm.

The present study was designed to determine the changes of phosphorylation of cAMP- response element binding protein (CREB) in hippocampus induced by ohmefentanyl stereoisomers (F9202 and F9204) in conditioned place preference (CPP) paradigm. The results showed that mice receiving F9202 and F9204 displayed obvious CPP. They could all significantly stimulate CREB phosphorylation and maintained for a long time without affecting total CREB protein levels. The effect of F9204 was similar to morphine which effect was more potent and longer than F9202. We also examined the effects of ketamine, a noncompetitive N-mthyl-D-aspartate receptor (NR) antagonist, on morphine-, F9202- and F9204- induced CPP and phosphorylation of CREB in hippocampus. Ketamine could suppress not only the place preference but also the phosphorylation of CREB produced by morphine, F9202 and F9204. These findings suggest that alterations in the phosphorylation of CREB be relevant to opiates signaling and the development of opiates dependence. NR antagonists may interfere with opiates dependence and may have potential therapeutic implications.

Expression of cAMP-responsive element binding protein and inducible cAMP early repressor in hyperfunctioning thyroid adenomas.

The pathogenesis of thyroid hyperfunctioning adenomas is still only partially understood and controversy exists about the frequency of gain-of-function mutations of the TSH receptor or G(s)alpha gene, which activate the cAMP pathway. The nuclear transcription factors cAMP-responsive element binding protein (CREB) and inducible cAMP early repressor (ICER) are among the final targets of this signalling cascade. In our study we focused on the expression of CREB and ICER genes in the nodular as well as in the extranodular tissue of hyperfunctioning tumours of the thyroid. RT-PCR and Western blot analysis were performed in a series of 14 patients. The presence of an activating mutation of the TSH receptor or of the G(s)alpha gene was ascertained by direct sequencing. The levels of CREB transcripts did not significantly differ in the adenomas and in the normal tissues (CREB/GAPDH, mean optical density+/-s.e.: 0.98+/-0.18 vs 0.88+/-0.27 respectively, P = not significant (N.S.)), although case-to-case variability was observed. The absence of a significant difference between the adenoma and the surrounding normal tissue was maintained after dividing the patients into two groups, according to TSH receptor status. Accordingly, no significant difference in the levels of CREB protein (total and Ser(133)-phosphorylated) was observed between the nodular and the extranodular tissue. In addition, no difference was found in the levels of ICER transcripts (ICER/GAPDH, mean optical density+/-s.e.: 0.52+/-0.11, nodule vs 0.36+/-0.11, normal thyroid, P=N.S.), independently of the TSH receptor gene status (i.e. wild-type or mutated). Our results support the recent hypothesis that the activation of the cAMP pathway in hyperfunctioning adenomas of the thyroid might be counteracted by opposite events and suggest that complex molecular mechanisms might take part in the pathogenesis of hyperfunctioning tumours.

Activation and inactivation of cAMP-response element-mediated gene transcription in cardiac myocytes.

Chronic beta-adrenergic stimulation of the cAMP-dependent signalling pathway is implicated in functionally relevant expressional changes in congestive heart failure. We studied activation and inactivation of the cardiac gene transcription mediated by the cAMP-response element (CRE) and the CRE-binding protein (CREB) as an important mechanism of a cAMP-dependent gene regulation. We investigated the transcriptional activation by forskolin, an activator of the adenylyl cyclase, in chick embryonic cardiomyocytes transfected with a CRE-controlled luciferase construct in comparison to the phosphorylation and expression of CREB determined on immunoblots. Forskolin (10 micromol/l; 8 h) increased CRE-mediated transcription and phosphorylation of CREB 13- and 1.5-fold, respectively. The phosphorylation was further elevated in combination with cantharidin, an inhibitor of type 1+2A protein phosphatases. The transcriptional response to forskolin was desensitized by pretreatment with forskolin (1 micromol/l; 24 h) while CREB phosphorylation was increased. In forskolin-pretreated cells, total CREB protein levels were decreased. Cantharidin did not restore the attenuated transcriptional response. In cardiomyocytes, there is an activation of the CRE-mediated gene transcription by forskolin that is attenuated after prolonged stimulation, and this attenuation is not dependent from a dephosphorylation of CREB. We suggest that attenuation of the CRE-mediated transcription through chronic stimulation of the cAMP-pathway, e.g. by elevated catecholamines, contributes to the altered expressional regulation in congestive heart failure.

Effects of protracted nicotine exposure and withdrawal on the expression and phosphorylation of the CREB gene transcription factor in rat brain.

Addiction to nicotine may result in molecular adaptations in the neurocircuitry of specific brain structures via changes in the cyclic AMP-responsive element binding protein (CREB)-dependent gene transcription program. We therefore investigated the effects of chronic nicotine exposure and its withdrawal on CREB and phosphorylated CREB (p-CREB) protein levels in the rat brain. We report here that chronic nicotine exposure (1-h withdrawal) had no effect on the expression of CREB and p-CREB in the rat cortex and amygdala. On the other hand, decreases in the expression of CREB protein and phosphorylation of CREB occur in the cingulate gyrus, and in the parietal and the piriform but not in the frontal cortex during nicotine withdrawal (18 h) after nicotine exposure. It was also observed that CREB and p-CREB protein levels were significantly decreased in the medial and basolateral, but not in the central amygdala during nicotine withdrawal (18 h) after chronic nicotine exposure. Furthermore, it was found that nicotine withdrawal (18 h) after chronic nicotine exposure leads to decreased CRE-DNA binding without modulating cAMP-dependent protein kinase A activity in the cortex and the amygdala of rats. In addition, chronic nicotine treatment produced anxiolytic effects whereas nicotine withdrawal (18 h) produced anxiety in rats as measured by the elevated plus-maze test. These results provide the first evidence that decreased CREB activity and/or expression in specific cortical and amygdaloid brain structures may be involved in the underlying molecular mechanisms of nicotine dependence.

Increased levels of transcription factors Elk-1, cyclic adenosine monophosphate response element-binding protein, and activating transcription factor 2 in the cerebellar vermis of schizophrenic patients.

We investigated the levels of transcription factors associated with activation of the mitogen-activated protein (MAP) kinase pathway in schizophrenics using postmortem brain samples. These studies were done to determine whether our previous findings of abnormal levels of the MAP kinases in the cerebellar vermis were linked to additional downstream targets of this signal transduction pathway. We measured the protein levels of 3 transcription factors in nuclear fractions of postmortem samples from cerebellar vermis of 10 patients with schizophrenia and 13 control subjects: Elk-1, cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), and activating transcription factor 2 (ATF-2). Studies in rats examined the postmortem stability and effect of haloperidol and risperidone on levels of Elk-1, cAMP, and ATF-2 proteins. We found a significant increase in the protein levels of Elk-1 (mean+SD, 4489+/-659 vs 2915+/-583 arbitrary densitometric units [P<.001]), CREB (mean +/- SD, 2149 1061 vs 904+/-711 arbitrary densitometric units [P=.003]) and ATF-2 (mean+/-SD, 1421 854 vs 512+/-394 arbitrary densitometric units [P=.003]) in the cerebellar vermis of schizophrenic subjects. Complementary studies in rats indicate that these findings can not be attributed to subacute treatment with antipsychotic medications. Taken together with the alterations of MAP kinases previously reported, and the findings of elevations of downstream transcription targets, we suggest that the MAP kinase signal transduction pathway contributes to the cerebellar abnormalities in schizophrenia.

CREB mediates the cAMP-responsiveness of the tyrosine hydroxylase gene: use of an antisense RNA strategy to produce CREB-deficient PC12 cell lines

cAMP initiates the PKA signaling cascade in rat pheochromocytoma PC12 cells, resulting in transcriptional activation of the tyrosine hydroxylase (TH) gene. This effect is mediated primarily through the cAMP responsive element (CRE), located at position −45 to −38 within the TH gene promoter. In this study, we applied an antisense RNA strategy to evaluate the role of the cAMP responsive element binding protein (CREB) in regulating TH gene expression. CREB antisense RNA expression vectors were stably introduced into PC12 cells to generate cell lines deficient in CREB. CREB protein and mRNA levels were diminished up to 90% in the stably transfected cell lines. Promoter analysis experiments demonstrated that cAMP-mediated inducibility of either TH gene proximal promoter activity or the activity of the TH CRE by itself fused upstream of a basal promoter was diminished in CREB-deficient cell lines. PKA activity in the CREB-deficient cell lines was comparable to the activity in control cell lines. In addition, neither ATF1, nor CREM proteins were significantly down-regulated in the CREB-deficient cells. Most significantly, the cAMP-inducibility of endogenous TH mRNA was completely blocked in the CREB-deficient cells, indicating that the response of the endogenous gene to cAMP was dependent on CREB. These results support the hypothesis that CREB (not other CRE-binding proteins) is the key transcription factor that is required for regulating TH gene expression in response to cAMP. Furthermore, our studies indicate that these CREB-deficient PC12 cells are excellent tools to study the participation of CREB in gene regulation.

サイクリックＡＭＰ応答エレメント結合蛋白（ＣＲＥＢ）による細胞増殖およびアポトーシスの制御

In this review, we will discuss cyclic AMP response element binding protein (CREB), a cAMP-and Ca2+-dependent transcription factor originally isolated from rat brain, and its roles in diverse cellular activities. In the introduction, we briefly describe the molecular mechanism for transcriptional enhancement by CREB and CREB/ATF family proteins. We also review the biological importance of CREB in long-term memory and T lymphocyte activation established by knockout and transgenic mice, respectively. The involvement of CREB in inflammatory responses through an induction of immunoregulatory and preinflammatoy cytokine genes is also discussed. Then we focus on the possible involvement of CREB in the regulation of cell cycle progression and apoptosis induction, presenting our published data along with our preliminary observations. We show that the expression level of CREB protein highly correlates with the growth activity of the cells and an over-expression of CREB enhances cyclin A expression. Surprisingly, an enforced expression of CREB induces apoptosis. Deletion analysis indicates that neither the transactivation domain nor kinase inducible domain is required but the C-terminal b-Zip structure, consisting of DNA-binding and dimerization domain, and the juxta four amino acids to its N-terminus are important for apoptosis induction, suggesting that the two functions of CREB, transcriptional enhancement and apoptosis induction, are accomplished by distinct mechanisms. The physiological relevance of CREB induced apoptosis and the possible therapeutic applications for diseases with malignant cell proliferation will be discussed.