CREB Ser133 Research Articles

The Role of Basic Leucine Zipper Protein‐Mediated Transcription in Physiological and Pathological Myocardial Hypertrophy

Accumulating evidence suggests that nuclear transcription factors from the basic leucine zipper (bZIP) family play an important role in cardiac development and function. This class includes the CREB/ATF family of transcription factors, namely CREB, cAMP response element modulator (CREM), ATF, and the related AP-1 and C/EBP families. An effort has been made to elucidate the role of specific bZIP members in the heart. Unfortunately, little insight could be gained from knockout experiments, either due to embryonic lethal phenotypes or functional compensation by other bZIP family members. Surprisingly, cardiac overexpression of several inhibitory transcription factors from the bZIP family, such as a nonphosphorylatable form of CREB (CREB(ser133)), a nonfunctional isoform of CREM, or ATF3 resulted in massive atrial dilatation. In order to try and characterize this pathway we have expressed the potent bZIP inhibitory protein, Jun dimerization protein 2 (JDP2), specifically in the mouse heart in a temporally controlled manner. Expression of JDP2 resulted in massive biatrial dilatation; loss of connexin 40 (Cx40), connexin43 (Cx43), and myosin light chain 2 (MLC2a) expression; atrioventricular defects in conduction; and a lethal phenotype. All these effects were independent of any developmental events acquired during adulthood, and were totally reversible upon abolishing the bZIP inhibition. The results of this article suggest that bZIP inhibition is sufficient to cause atrial dilation, that this dilatation is acquired postnatally, and that it is reversible upon the relief of inhibition. Thus, bZIP repressors may serve as novel drug targets for the prevention of atrial dilatation a major risk of atrial fibrillation (AF).

Molecular Identification and Characterization of a Family of Kinases with Homology to Ca2+/Calmodulin-dependent Protein Kinases I/IV

Despite the critical importance of Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) II signaling in neuroplasticity, only a limited amount of work has so far been available regarding the presence and significance of another predominant CaMK subfamily, the CaMKI/CaMKIV family, in the central nervous system. We here searched for kinases with a core catalytic structure similar to CaMKI and CaMKIV. We isolated full-length cDNAs encoding three mouse CaMKI/CaMKIV-related kinases, CLICK-I (CL1)/doublecortin and CaM kinase-Like (DCAMKL)1, CLICK-II (CL2)/DCAMKL2, and CLICK-I,II-related (CLr)/DCAMKL3, the kinase domains of which had an intermediate homology not only to CaMKI/CaMKIV but also to CaMKII. Furthermore, CL1, CL2, and CLr were highly expressed in the central nervous system, in a neuron-specific fashion. CL1alpha and CL1beta were shorter isoforms of DCAMKL1, which lacked the doublecortin-like domain (Dx). In contrast, CL2alpha and CL2beta contained a full N-terminal Dx, whereas CLr only possessed a partial and dysfunctional Dx. Interestingly, despite a large similarity in the kinase domain, CL1/CL2/CLr had an impact on CRE-dependent gene expression distinct from that of the related CaMKI/CaMKIV and CaMKII. Although these were previously shown to activate Ca(2+)/cAMP-response element-binding protein (CREB)-dependent transcription, we here show that CL1 and CL2 were unable to significantly phosphorylate CREB Ser-133 and rather inhibited CRE-dependent gene expression by a dominant mechanism that bypassed CREB and was mediated by phosphorylated TORC2.

A Nitric Oxide Signaling Pathway Controls CREB-Mediated Gene Expression in Neurons

Prevailing views of neurotrophin action hold that the transcription factor CREB is constitutively bound to target genes with transcriptional activation occurring via CREB phosphorylation. However, we report that within several CRE-containing genes, CREB is not constitutively bound. Upon exposure of neurons to brain-derived neurotrophic factor (BDNF), CREB becomes rapidly bound to DNA coincident with phosphorylation at its transcriptional regulatory site, Ser133. This inducible CREB-DNA binding is independent of CREB Ser133 phosphorylation and is not affected by inhibition of the ERK or PI3K signaling pathways. Instead, BDNF regulates CREB binding by initiating a nitric oxide-dependent signaling pathway that leads to S-nitrosylation of nuclear proteins that associate with CREB target genes. Pharmacological manipulation of neurons in vitro and analysis of mice lacking neuronal nitric oxide synthase (nNOS) suggest that NO mediates BDNF and activity-dependent expression of CREB target genes. Thus, in conjunction with CREB phosphorylation, the NO pathway controls CREB-DNA binding and CRE-mediated gene expression.

Enhanced phosphorylation of cyclic AMP response element binding protein in the brain of mice following repetitive hypoxic exposure

Cerebral ischemic/hypoxic preconditioning (I/HPC) is a phenomenon of endogenous protection that renders the brain tolerant to sustained ischemia/hypoxia. This profound protection induced by I/HPC makes it an attractive target for developing potential clinical therapeutic approaches. However, the molecular mechanism of I/HPC is unclear. Cyclic AMP (cAMP) response element binding protein (CREB), a selective nuclear transcriptional factor, plays a key role in the neuronal functions. Phosphorylation of CREB on Ser-133 may facilitate its transcriptional activity in response to various stresses. In the current study, we observed the changes in CREB phosphorylation (Ser-133) and protein expression in the brain of auto-hypoxia-induced HPC mice by using Western blot analysis. We found that the levels of phosphorylated CREB (Ser-133), but not protein expression of CREB, increased significantly ( p < 0.05) in the hippocampus and the frontal cortex of mice after repetitive hypoxic exposure (H2–H4, n = 6 for each group), when compared to that of the normoxic (H0, n = 6) or hypoxic exposure once group (H1, n = 6). In addition, a significant enhancement ( p < 0.05) of CREB phosphorylation (Ser-133) could also be found in the nuclear extracts from the whole hippocampus of hypoxic preconditioned mice (H2–H4, n = 6 for each group). These results suggest that the phosphorylation of CREB might be involved in the development of cerebral hypoxic preconditioning.

Inhibition of CREB transcriptional activity in human T lymphocytes by oxidative stress

Hydrogen peroxide (HP) induced the phosphorylation of cAMP response element binding protein (CREB) on Ser133 in Jurkat T lymphocytes via p38 and MSK1. Although CREB Ser133 was phosphorylated, increases in HP-stimulated CREB-mediated transcription were absent. T lymphocyte stimulation with anti-CD3 and anti-CD28 induced CREB Ser133 phosphorylation, as well as CREB-mediated transcriptional activity. When CD3/CD28-stimulated lymphocytes were treated with HP, Ser133 was phosphorylated, but TCR-induced CREB-mediated transcriptional activity was reduced. These data provide insight into a potential mechanism by which oxidative stress can alter T cell receptor-induced CREB activation and responsiveness.

Combinatorial control of the bradykinin B2 receptor promoter by p53, CREB, KLF-4, and CBP: implications for terminal nephron differentiation

Despite a wealth of knowledge regarding the early steps of epithelial differentiation, little is known about the mechanisms responsible for terminal nephron differentiation. The bradykinin B2 receptor (B2R) regulates renal function and integrity, and its expression is induced during terminal nephron differentiation. This study investigates the transcriptional regulation of the B2R during kidney development. The rat B2R 5'-flanking region has a highly conserved cis-acting enhancer in the proximal promoter consisting of contiguous binding sites for the transcription factors cAMP response element binding protein (CREB), p53, and Kruppel-like factor (KLF-4). The B2R enhancer drives reporter gene expression in inner medullary collecting duct-3 cells but is considerably weaker in other cell types. Site-directed mutagenesis and expression of dominant negative mutants demonstrated the requirement of CREB DNA binding and Ser-133 phosphorylation for optimal enhancer function. Moreover, helical phasing experiments showed that disruption of the spatial organization of the enhancer inhibits B2R promoter activity. Several lines of evidence indicate that cooperative interactions among the three transcription factors occur in vivo during terminal nephron differentiation: 1) CREB, p53, and KLF-4 are coexpressed in B2R-positive differentiating cells; 2) the maturational expression of B2R correlates with CREB/p53/KLF-4 DNA-binding activity; 3) assembly of CREB, p53, and KLF-4 on chromatin at the endogenous B2R promoter is developmentally regulated and is accompanied by CBP recruitment and histone hyperacetylation; and 4) CREB and p53 occupancy of the B2R enhancer is cooperative. These results demonstrate that combinatorial interactions among the transcription factors, CREB, p53, and KLF-4, and the coactivator CBP, may be critical for the regulation of B2R gene expression during terminal nephron differentiation.

KCl and forskolin synergistically up‐regulate cholecystokinin gene expression via coordinate activation of CREB and the co‐activator CBP

Cholecystokinin (CCK) is one of the most abundant peptide transmitters in the mammalian brain. Despite the physiological significance of CCK expression in long-term memory and psychiatric disorders, little is known about the factors that regulate the expression of CCK peptides. Here, we report that KCl and forskolin synergistically increase CCK gene transcription via protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) signalling pathways, activating cAMP response element-binding protein (CREB) associated with the CRE(- 80) element of the CCK promoter. Whereas, CREB Ser133 phosphorylation was essential for transcriptional activation, the synergistic stimulation was not correlated to the level of Ser133 phosphorylation, indicating that recruitment and/or activation of additional downstream factors were required for maximal stimulation. Transcriptional activation was reduced by co-expression of adenovirus 12S E1A, that inhibits binding of CREB-binding protein (CBP) to CREB. Moreover GAL4-CREB-DIEDML, which mediates the phosphorylation-independent binding of CBP, and the C-terminal domain of CBP was synergistically activated by forskolin and KCl. Taken together the results imply that neuronal CCK gene transcription is regulated by the cumulative action of calcium and cAMP via stimulation of the PKA and ERK signalling pathways and that synergy is accomplished by the coordinate activation of CREB and CBP.

Endothelin-1 promotes phosphorylation of CREB transcription factor in primary cultures of neonatal rat cardiac myocytes: implications for the regulation of c- jun expression

Cardiac myocyte hypertrophy is associated with an increase in expression of immediate early genes (e.g. c- jun) via activation of pre-existing transcription factors. The activity of CREB transcription factor is regulated through phosphorylation of Ser-133 by one of several protein kinases (e.g. protein kinase A (PKA), p90 ribosomal S6 kinases (RSKs) and the related kinase, MSK1). A cell-permeable form of cAMP, hypertrophic agonists (endothelin-1 (ET-1), phenylephrine (PE)) and hyperosmotic shock all promoted phosphorylation of CREB(Ser-133) in rat neonatal cardiac myocytes. The response to endothelin-1 required the extracellular signal-regulated kinase cascade which stimulates both RSKs and MSK1. Phosphorylation of CREB(Ser-133) in response to ET-1 was not associated with any increase in DNA binding to a consensus cAMP-response element (CRE). The rat c- jun promoter contains elements which may bind either c-Jun/ATF2 or CREB/ATF1 dimers. Using extracts from rat cardiac myocytes, we identified at least two complexes which bind to the most proximal of these elements, one of which contained CREB and the other c-Jun. Thus, phosphorylation and activation of CREB in cardiac myocytes may be effected by a range of different stimuli to influence the expression of immediate early genes such as c- jun.

Long-term effects of early-life malnutrition and status epilepticus: assessment by spatial navigation and CREB Serine-133 phosphorylation

Malnutrition and/or seizure in the developing brain cause hippocampal damages. However, underlying mechanisms remain unclear. The malnutrition group (MN) subjected with malnutrition alone was culled to 20–22 rats per dam on postnatal day 1 (P1). The rats subjected to lithium–pilocarpine (Li/PC)-induced status epilepticus at P21 were grouped as the SE group. The rats subjected to malnutrition and subsequent status epilepticus were grouped as the MS group. Visual–spatial memory test using the Morris water maze task was performed at P80. Following behavioral tests, the hippocampus was evaluated for histological lesions and phosphorylated cAMP-responsive, element-binding protein at serine-133 ( pCREB Ser-133), an important transcription factor underlying learning and memory in the mammalian brain. Here, the MN group exhibited decreased body weight at P21. There was no significant difference in the seizure duration and mortality between the SE and MS groups. In adulthood (P80), both the SE and MS groups showed the spatial learning deficit, hippocampal cell loss and decreased pCREB Ser133 level within hippocampal CA1 region. Although the MN group demonstrated a decreased level of pCREB Ser133, no distinguishable changes in the cognitive deficit and hippocampal neuronal loss were detected. Collectively, the present results suggest that early-life malnutrition led to a reduced phosphorylation of CREB Ser133 in hippocampal CA1 in the absence of the long-term spatial learning deficit. This decreased phosphorylation of CREB Ser133 could suggest that cascades of signal transduction responsible for the phosphorylation of CREB Ser133 might be disturbed by early-life malnutrition. In addition, malnutrition caused no discernible synergistic effects on Li/PC-induced status epilepticus.

Phosphatase regulation of gene expression during development of the palate

In mammalian cells, including those of the embryonic palate, the level of phosphorylation of cellular proteins at any given time reflects the activities of protein kinases and protein phosphatases. Both protein phosphatase-1 (PP-1) and PP-2A inhibit cAMP-mediated increases in transcription by dephosphorylating CREB at ser-133. Western blot analysis indicated that protein phosphatase 1 (PP-1) was expressed constitutively in palatal tissue during its development. Expression of PP-2A was regulated developmentally with maximal expression on gestational day (gd) 14. Densitometric scanning revealed a 30% increase in expression from gd 13 to gd 14. Virtually all phosphatase activity in the tissue extracts could be inhibited by 5 μM okadaic acid, demonstrating that PP-1 and PP-2A account for all detectable ser/thr protein phosphatase activity present in the developing palate. Moreover, no significant differences in PP-1 and PP-2A activities were observed during the period of palate development. Treatment of primary cultures of murine embryonic palate mesenchymal (MEPM) cells with forskolin (20 μM) to elevate intracellular cAMP levels, resulted in a time-dependent increase in CREB ser-133 phosphorylation and a corresponding time dependent decrease in PP-1 and PP-2A levels. Moreover, treatment of MEPM cells with okadaic acid resulted in a dramatic increase in basal CREB ser-133 phosphorylation. This suggests that PP-1 activity may contribute to transcriptional regulation of CREB and that PP-1 and PP-2A are regulated differentially by cAMP. Treatment of MEPM cells with TGFβ1 (1 ng/ml) under conditions of TGFβ-induced CREB phosphorylation resulted in no effect on the expression of either PP-1 or PP-2A proteins and no significant alterations in total basal protein phosphatase activity. These results demonstrate that transcriptional regulation of CREB in embryonic palatal issue is dependent on the coordinate activity of specific kinases and phosphatases.

CREB Transcriptional Activity in Neurons Is Regulated by Multiple, Calcium-Specific Phosphorylation Events

The transcription factor CREB mediates diverse responses in the nervous system. It is not known how CREB induces specific patterns of gene expression in response to different extracellular stimuli. We find that Ca 2+ influx into neurons induces CREB phosphorylation at Ser133 and two additional sites, Ser142 and Ser143. While CREB Ser133 phosphorylation is induced by many stimuli, phosphorylation at Ser142 and Ser143 is selectively activated by Ca 2+ influx. The triple phosphorylation of CREB is required for effective Ca 2+ stimulation of CREB-dependent transcription, but the phosphorylation of Ser142 and Ser143, in addition to Ser133, disrupts the interaction of CREB with its cofactor CBP. These results suggest that Ca 2+ influx triggers a specific program of gene expression in neurons by selectively regulating CREB phosphorylation.

Pairing of forskolin and KCl increases differentiation of immortalized hippocampal neurons in a CREB Serine 133 phosphorylation-dependent and extracellular-regulated protein kinase-independent manner

Although cAMP response element binding protein (CREB)- and extracellular-regulated protein kinase (ERK)-mediated pathways have been linked to each other in neuronal differentiation, involvement of these in hippocampal neuronal cell line has not been defined. Using an immortalized hippocampal cell line, HiB5, we have tried a pairing of forskolin with KCl depolarization, which acts as an ERK and CREB kinase activator in hippocampal neurons, to investigate if an activation of ERK and phosphorylation of CREB at the critical regulatory site, serine 133 might be coupled in differentiation. Differentiation toward a neuronal phenotype was synergistically and markedly increased by the pairing of forskolin and KCl depolarization. The synergistic effect was accompanied by an increase in phosphorylation of CREB Ser-133, but not phosphorylation of ERK, and was not inhibited by MEK inhibitor, PD98059. These findings indicate that phosphorylation of the transcriptional factor CREB may function to facilitate differentiation of HiB5 cells.

HIV-1 Tat protein down-regulates CREB transcription factor expression in PC12 neuronal cells through a phosphatidylinositol 3-kinase/AKT/cyclic nucleoside phosphodiesterase pathway.

The addition of low concentrations (0.1-1 nM) of extracellular HIV-1 Tat protein to PC12 neuronal cells stimulated a rapid (peak at 5 min) elevation of the cAMP intracellular levels, which in turn induced the phosphorylation of CREB transcription factor (peak at 15 min) on serine-133 (Ser-133). On the contrary, at later time points (60-120 min) Tat induced a significant decline of intracellular cAMP with respect to the basal levels observed in control cells treated with bovine serum albumin. In blocking experiments performed with pharmacological inhibitors, Tat decreased the intracellular levels of cAMP and CREB Ser-133 phosphorylation through a signal transduction pathway involving the sequential activation of phosphatidylinositol 3-kinase, AKT, and cyclic nucleoside phosphodiesterases. Moreover, in transient transfection experiments, Tat inhibited transcription of CREB promoter in a manner strictly dependent on the presence of the cAMP-responsive elements (CRE) in the CREB promoter. Consistently, the expression of endogenous CREB protein was significantly reduced in PC12 cells by prolonged (24-48 h) treatment with Tat. This decline in the expression of CREB, which plays an essential role in the survival and function of neuronal cells, anticipated a progressive increase of apoptosis in Tat-treated cells. Although obtained in a neuronal cell line, our findings might help to explain some aspects of the pathogenesis of HIV-1-associated dementia.

Multiple signals required for cyclic AMP-responsive element binding protein (CREB) binding protein interaction induced by CD3/CD28 costimulation.

The optimal activation of cAMP-responsive element binding protein (CREB), similar to the full activation of T lymphocytes, requires the stimulation of both CD3 and CD28. Using a reporter system to detect interaction of CREB and CREB-binding protein (CBP), in this study we found that CREB binds to CBP only by engagement of both CD3 and CD28. CD3/CD28-promoted CREB-CBP interaction was dependent on p38 mitogen-activated protein kinase (MAPK) and calcium/calmodulin-dependent protein kinase (CaMK) IV in addition to the previously identified extracellular signal-regulated kinase pathway. Extracellular signal-regulated kinase, CaMKIV, and p38 MAPK were also the kinases involved in CREB Ser(133) phosphorylation induced by CD3/CD28. A reconstitution experiment illustrated that optimum CREB-CBP interaction and CREB trans-activation were attained when these three kinase pathways were simultaneously activated in T cells. Our results demonstrate that coordinated activation of different kinases leads to full activation of CREB. Notably, CD28 ligation activated p38 MAPK and CaMKIV, the kinases stimulated by CD3 engagement, suggesting that CD28 acts by increasing the activation extent of p38 MAPK and CaMKIV. These results support the model of a minimum activation threshold for CREB-CBP interaction that can be reached only when both CD3 and CD28 are stimulated.

Developmentally Regulated NMDA Receptor-Dependent Dephosphorylation of cAMP Response Element-Binding Protein (CREB) in Hippocampal Neurons

Developmental changes in the signaling properties of NMDA receptors have been proposed to underlie the loss of plasticity that accompanies brain maturation. Calcium influx through postsynaptic NMDA receptors can stimulate neuronal gene expression via signaling pathways such as the Ras-MAP kinase (MAPK) pathway and the transcription factor cAMP response element-binding protein (CREB). We analyzed MAPK (Erk1/2) and CREB activation in response to NMDA receptor stimulation during the development of hippocampal neurons in culture. At all stages of development NMDA stimulation induced a rapid phosphorylation of CREB on Ser-133 (phospho-CREB). However, the time course of decline in phospho-CREB changed dramatically with neuronal maturation. At 7 d in vitro (7 DIV) phospho-CREB remained elevated 2 hr after strong NMDA stimulation, whereas at 14 DIV phospho-CREB rose only transiently and fell back to below basal levels within 30 min. Moreover, at 14 DIV, but not at 7 DIV, NMDA receptor stimulation induced a dephosphorylation of CREB that previously had been phosphorylated by KCl depolarization or forskolin, suggesting an NMDA receptor-dependent activation of a CREB phosphatase. There was no developmental change in the time course of phospho-CREB induction that followed KCl depolarization or PKA activation, nor was there a developmental change in the time course of phospho-Erk1/2 induced by NMDA receptor activation. We suggest that, during neuronal maturation, NMDA receptor activation becomes linked specifically to protein phosphatases that act on Ser-133 of CREB. Such a developmentally regulated switch in the mode of NMDA receptor coupling to intracellular signaling pathways may contribute to the changes in neural plasticity observed during brain development.

Sp1 and CREB Mediate Gastrin-dependent Regulation of Chromogranin A Promoter Activity in Gastric Carcinoma Cells

Chromogranin A (CgA) is a multifunctional acidic protein that in the stomach is expressed predominantly in enterochromaffin-like cells (ECL cells) where it is regulated by gastrin. In order to investigate the transcriptional response of the mouse CgA (mCgA) promoter to gastrin stimulation, we studied a 4.8-kilobase mCgA promoter-luciferase reporter gene construct in transiently transfected AGS-B cells. 5'-Deletion analysis and scanning mutagenesis of mCgA 5'-flanking DNA showed that a Sp1/Egr-1 site spanning -88 to -77 base pairs (bp) and a cyclic AMP-responsive element (CRE) at -71 to -64 bp are essential for gastrin-dependent mCgA transactivation. Gastrin stimulation increased cellular Sp1 protein levels and Sp1-binding to the mCgA -88 to -77 bp element, as well as binding of CREB to its consensus motif at -71 to -64 bp. Gastrin also stimulated CREB Ser-133 phosphorylation, and abundance of cellular CREB protein levels. Overexpression of either Sp1 or phosphorylated CREB transactivated the mCgA promoter dose dependently, while coexpression of both transcription factors resulted in an additive mCgA promoter response. mCgA -92 to -64 bp, comprising the Sp1/Egr-1 site and the CRE motif, conferred gastrin responsiveness to a heterologous thymidine kinase promoter system, and therefore functions as a "true" enhancer element. This report demonstrates that Sp1 and CREB mediate CCK-B/gastrin receptor-dependent gene regulation, and that the effect of gastrin on the CgA gene is brought about by cooperative action of both transcription factors.

Nerve growth factor activates extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways to stimulate CREB serine 133 phosphorylation.

The mechanisms by which growth factor-induced signals are propagated to the nucleus, leading to the activation of the transcription factor CREB, have been characterized. Nerve growth factor (NGF) was found to activate multiple signaling pathways that mediate the phosphorylation of CREB at the critical regulatory site, serine 133 (Ser-133). NGF activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs), which in turn activate the pp90 ribosomal S6 kinase (RSK) family of Ser/Thr kinases, all three members of which were found to catalyze CREB Ser-133 phosphorylation in vitro and in vivo. In addition to the ERK/RSK pathway, we found that NGF activated the p38 MAPK and its downstream effector, MAPK-activated protein kinase 2 (MAPKAP kinase 2), resulting in phosphorylation of CREB at Ser-133. Inhibition of either the ERK/RSK or the p38/MAPKAP kinase 2 pathway only partially blocked NGF-induced CREB Ser-133 phosphorylation, suggesting that either pathway alone is sufficient for coupling the NGF signal to CREB activation. However, inhibition of both the ERK/RSK and the p38/MAPKAP kinase 2 pathways completely abolished NGF-induced CREB Ser-133 phosphorylation. These findings indicate that NGF activates two distinct MAPK pathways, both of which contribute to the phosphorylation of the transcription factor CREB and the activation of immediate-early genes.

Phosphorylation of Transcription Factor CREB in Rat Spinal Cord after Formalin-Induced Hyperalgesia: Relationship toc-fosInduction

The involvement of cAMP-responsive element-binding protein (CREB) signaling in tissue injury-induced inflammation and hyperalgesia has been characterized by measuring phosphorylation of CREB at serine-133 (CREB Ser133) using a specific antibody. In the unstimulated state, unphosphorylated CREB was observed in most nuclei of spinal neurons except for motor neurons, where only a small portion of neurons were stained. A few dorsal root ganglion (DRG) neurons were also CREB-positive. After a unilateral injection of formalin into the hindpaw, a strong and bilateral phosphorylation of CREB Ser133 was induced, as assessed by both immunohistochemistry and Western blot. PhosphoCREB (pCREB)-positive neurons were found in laminae I, II, V, and X of spinal cord on both sides. CREB phosphorylation was very rapid and reached peak levels within 10 min of formalin treatment, whereas few pCREB-positive neurons were seen in unstimulated spinal cord. The induction of pCREB was predominantly postsynaptic, because only 5% of DRG neurons were labeled after inflammation. In contrast to CREB phosphorylation, the induction of c-Fos expression reached peak levels 2 hr after formalin treatment and c-Fos induction was mainly ipsilateral. Both formalin-evoked CREB phosphorylation and c-Fos expression in the spinal cord were suppressed by pretreatment with the NMDA receptor antagonist MK-801 (3.5 mg/kg, i.p.) or halothane anesthesia. These results suggest that CREB signaling may play a role in the long-term facilitation of spinal cord neurons after hyperalgesia. Furthermore, our results indicate that CREB phosphorylation may be necessary but not sufficient for c-fos induction.

TGF-β Signaling in Murine Embryonic Palate Cells Involves Phosphorylation of the CREB Transcription Factor

A number of studies over the last several years have demonstrated a crucial role for TGF-β in epithelial and mesenchymal differentiation during development of the embryonic palate. Molecular mechanism(s) of signal transduction responsible for eliciting these responses remain unresolved. Since cAMP signaling also modulates the same tissue differentiation in the developing palate and palate-derived cells, we hypothesized that TGF-β activity may be mediated through cAMP-inducible pathways. We thus examined the effects of TGF-β on activation of the cAMP regulatory element binding protein CREB, a nuclear transcription factor which mediates transcription of genes containing CRE recognition sequences in their promoters. We examined the ability of TGF-β-treated murine embryonic palate mesenchymal (MEPM) cells to phosphorylate CREB on the amino acid residue serine 133, phosphorylation of which is indispensable for transcriptional activation. TGF-β treatment led to increasedphosphorylation of CREB ser-133 in a time- and dose-dependent manner. Inhibition of serine-threonine phosphatases by okadaic acid enhanced but did not prolong this response. TGF-β failed to induce the activity of protein kinase A (PKA), a known CREB kinase. Inhibition of either PKA or calcium/calmodulin kinase II (CaMK II) did not abrogate phosphorylation of CREB by TGF-β. TGF-β treatment also did not induce phosphorylation of mitogen-activated protein kinases, erk-1 and erk-2, on tyrosine 185, suggesting that these kinases do not mediate CREB phosphorylation by TGF-β. Additionally, TGF-β had no effect on CREB binding to known CREB DNA consensus recognition sequences, CRE and TRE. Together, these data suggest an alternative or novel CREB kinase in MEPM cells through which TGF-β acts to induce CREB ser-133 phosphorylation and subsequent activation of CRE-containing genes.

Calcium regulation of gene expression: isn't that spatial?

Calcium regulation of gene expression: isn't that spatial?