Table of contents

CREB targets

Several studies have characterized the upstream regulatory region of c-fos (see Drosophila Fos related antigen), and identified cis-acting elements, termed the cyclic AMP (cAMP) response elements (CREs), which are critical for c-fos transcription in response to a variety of extracellular stimuli. Although several transcription factors can bind to CREs in vitro, the identity of the transcription factor(s) that activates the c-fos promoter via the CRE in vivo remains unclear. To help identify the trans-acting factors that regulate stimulus-dependent transcription of c-fos via the CREs, there have been developed dominant-negative (D-N) inhibitor proteins that function by preventing DNA binding of B-ZIP proteins in a dimerization domain-dependent fashion. A D-N inhibitor of CREB, termed A-CREB, was constructed by fusing a designed acidic amphipathic extension onto the N terminus of the CREB leucine zipper domain. The acidic extension of A-CREB interacts with the basic region of CREB, forming a coiled-coil extension of the leucine zipper and thus preventing the basic region of wild-type CREB from binding to DNA. Other D-N inhibitors generated in a similar manner with the dimerization domains of Fos, Jun, C/EBP, ATF-2, or VBP do not block CREB DNA binding activity, nor do they inhibit transcriptional activation of a minimal promoter containing a single CRE in PC12 cells. A-CREB inhibits activation of CRE-mediated transcription evoked by three distinct stimuli: forskolin, which increases intracellular cAMP; membrane depolarization, which promotes Ca2+ influx, and nerve growth factor (NGF). A-CREB completely inhibits cAMP-mediated transcription of a reporter gene containing 750 bp of the native c-fos promoter, but A-CREB only partially inhibits Ca2+- and NGF-mediated transcription of the same reporter gene. Glutamate induction of c-fos expression in primary cortical neurons is dependent on CREB. In contrast, induction of c-fos transcription by UV light is not inhibited by A-CREB. A-CREB also attenuates NGF induction of morphological differentiation in PC12 cells. These results suggest that CREB or its closely related family members are general mediators of stimulus-dependent transcription of c-fos and are required for at least some of the long-term actions of NGF (Ahn, 1998).

Transcription of the neurotransmitter biosynthetic genes tyrosine hydroxylase and dopamine beta-hydroxylase (DBH) is regulated by cell type-specific transcription factors, including the homeoprotein Arix, and second messengers, including cyclic AMP. The cis-acting regulatory sites of the DBH gene which respond to Arix and cAMP lie adjacent to one another, between bases -180 and -150, in a regulatory element named DB1. Neither Arix nor cyclic AMP analogs alone effectively stimulate transcription from the DBH promoter in non-neuronal cell cultures. However, when Arix is present together with cAMP, transcription is substantially activated. Synergistic transcription from the DBH promoter can also be elicited by cotransfection of Arix with an expression vector encoding the catalytic subunit of protein kinase A. Nuclear extracts from PC12 cells display a cAMP-induced complex binding to the DB1 element. Antisera to transcription factors CREB, CREM, Fos, and Jun indicate that these proteins, or closely related family members, interact with DB1. A dominant negative construct of CREB inhibits the response of the DBH promoter to protein kinase A. These results demonstrate a synergistic interaction between a homeodomain protein and the cAMP signal transduction system and suggest that similar interactions may regulate the tissue-specific expression of neuroendocrine genes (Swanson, 1997).

Neurotrophins regulate neuronal survival, differentiation, and synaptic function. To understand how neurotrophins elicit such diverse responses, signaling pathways have been elucidated by which brain-derived neurotrophic factor (BDNF) activates gene expression in cultured neurons and hippocampal slices. The transcription factor cyclic AMP response element-binding protein (CREB) is an important regulator of BDNF-induced gene expression. Exposure of neurons to BDNF stimulates CREB phosphorylation and activation by at least two signaling pathways: (1) a calcium/calmodulin-dependent kinase IV (CaMKIV)-regulated pathway that is activated by the release of intracellular calcium and (2) a Ras-dependent pathway. These findings reveal a previously unrecognized, CaMK-dependent mechanism by which neurotrophins activate CREB and suggest that CREB plays a central role in mediating neurotrophin responses in neurons (Finkbeiner, 1997).

Cyclin A plays an essential role in the G1 to S phase transition of the cell cycle. The expression of cyclin A is restrained during G0 and G1, but steeply induced at the G1/S boundary. Analysis of the rat cyclin A promoter elements with the 5' sequential deletion derivatives of the promoter fused to the luciferase cDNA indicate that the ATF/CRE motif primary determines the of inducibility at G1/S. Gel shift analysis of the complex formed at the ATF/CRE site indicates that the complex was not formed with the G0/G1 cell extract, but maximally formed with the late-G1 cell extract. The complex is supershifted by anti-JunD antibody; Western blot analysis of the immune complexes prepared with anti-JunD antibody reveals the presence of ATF2, suggesting heterodimerization of JunD with ATF2. The cyclin A promoter in a reporter plasmid is activated nearly 10-fold in quiescent rat 3Y1 cells by cotransfection with the expression of plasmids encoding ATF2 and Jun family members. In contrast, cotransfection with the ATF4 expression plasmid suppresses the promoter activation mediated by ATF2 and Jun family members. The expression of Jun family members during G1 to S progression is induced biphasically in early and late G1 and the level of JunD increases markedly at the G1/S, while that of ATF family members is gradually increased along with the G1 to S progression. These results indicate that the cyclin A promoter activity is regulated, at least in part, by relative amounts of the ATF and Jun family members (Shimizu, 1998).

CREB is a transcription factor implicated in the control of adaptive neuronal responses. Although one function of CREB in neurons is believed to be the regulation of genes whose products control synaptic function, the targets of CREB that mediate synaptic function have not yet been identified. This report describes experiments demonstrating that CREB or a closely related protein mediates Ca2+-dependent regulation of BDNF, a neurotrophin that modulates synaptic activity. In cortical neurons, Ca2+ influx triggers phosphorylation of CREB, which by binding to a critical Ca2+ response element (CRE) within the BDNF gene activates BDNF transcription. Mutation of the BDNF CRE or an adjacent novel regulatory element as well as a blockade of CREB function results in a dramatic loss of BDNF transcription. These findings suggest that a CREB family member acts cooperatively with an additional transcription factor(s) to regulate BDNF transcription. It is concluded that the BDNF gene is a CREB family target whose protein product functions at synapses to control adaptive neuronal responses (Tao, 1998).

The effects of the pituitary adenylase cyclate-activating peptides (PACAP) 27 and 38 on proenkephalin (PENK) gene transcription were examined in PC12 (rat pheochromocytoma) cells using transient transfection assays. Both ligands stimulate PENK gene transcription in a dose-dependent manner, with an apparent ED50 close to 5 x 10(-11) M. Inactivation of cAMP dependent-protein kinase (PKA) with a dominant inhibitory mutant strongly reduces PACAP-stimulated PENK transcription. Using reporter genes driven by either the minimal TPA-responsive element (TRE: TGACTCA) or cAMP-responsive element (CRE: TGACGTCA), it has been shown that the two PACAPs activate transcription through both regulatory sequences. These effects could result from direct post-translational activation of Jun and CREB, as shown using GAL4-Jun or GAL4-CREB fusion proteins. Expression of a dominant inhibitory mutant of CREB decreases by 60% the response to PACAP, suggesting that CREB is implicated in PENK transactivation. Similarly, expression of c-fos antisense RNA reduces by 80% the stimulatory effects of PACAP. Taken together, these results indicate that PACAP stimulates PENK transcription by members of both the AP1 and the CREB families. However, AP1 by itself is not sufficient to increase PENK transcription, as insulin-like growth factor 1 (IGF1), which stimulates AP1 activity but not cAMP production, is unable to stimulate PENK transcription. These results indicate a cooperative effect of AP1 and CREB on PENK transcription (Monnier, 1998).

Proteins of the ATF/CREB class of transcription factors stimulate gene expression of several cell growth-related genes through protein kinase A-related cAMP response elements. The promoter activity of cell cycle regulated histone H4 genes is regulated by at least four principal cis-acting elements that mediate G1/S phase control and/or enhancement of transcription during the cell cycle. Using protein-DNA interaction assays it has been shown that the H4 promoter contains two ATF/CREB recognition motifs that interact with CREB, ATF1, and ATF2 but not with ATF4/CREB2. One ATF/CRE motif is located in the distal promoter at the nuclear matrix-associated Site IV, and the second motif is present in the proximal promoter at Site I. Both ATF/CRE motifs overlap binding sequences for the multifunctional YY1 transcription factor, which has previously been shown to be nuclear matrix associated. Subnuclear fractionation reveals that there are two ATF1 isoforms that appear to differ with respect to DNA binding activity and partition selectively between nuclear matrix and nonmatrix compartments, consistent with the role of the nuclear matrix in regulating gene expression. Site-directed mutational studies demonstrate that Site I and Site IV together support ATF1- and CREB-induced trans-activation of the H4 promoter. Thus, these data establish that ATF/CREB factors functionally modulate histone H4 gene transcription at distal and proximal promoter elements (Guo, 1997).

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, a key regulatory enzyme in the pathway for endogenous cholesterol synthesis, is a target for negative feedback regulation by cholesterol. When cellular sterol levels are low, the sterol regulatory element-binding proteins (SREBPs) are released from the endoplasmic reticulum membrane, allowing them to translocate to the nucleus and activate SREBP target genes. However, in all SREBP-regulated promoters studied to date, additional co-regulatory transcription factors are required for sterol-regulated activation of transcription. In addition to SREBPs, NF-Y/CBF is required for sterol-regulated transcription of HMG-CoA synthase. This heterotrimeric transcription factor functions as a co-regulator in several other SREBP-regulated promoters, as well. In addition to cis-acting sites for both SREBP and NF-Y/CBF, the sterol regulatory region of the synthase promoter also contains a consensus cAMP response element (CRE), an element that binds members of the CREB/ATF family of transcription factors. This consensus CRE is essential for sterol-regulated transcription of the synthase promoter. CREB is shown to bind to this CRE, and mutations within the CRE that result in a loss of CREB binding also result in a loss of sterol-regulated transcription. Efficient activation of the synthase promoter in Drosophila SL2 cells requires the simultaneous expression of all three factors: SREBPs, NF-Y/CBF, and CREB. To date this is the first promoter shown to require CREB for efficient sterol-regulated transcription, and to require two different co-regulatory factors in addition to SREBPs for maximal activation (Dooley, 1999).

Nerve growth factor (NGF) and other neurotrophins support survival of neurons through processes that are incompletely understood. The transcription factor CREB is a critical mediator of NGF-dependent gene expression, but whether CREB family transcription factors regulate expression of genes that contribute to NGF-dependent survival of sympathetic neurons is unknown. To determine whether CREB-mediated gene expression is necessary for NGF-dependent neuronal survival, this study monitored survival of sympathetic neurons after expression of either of two distinct inhibitors of CREB. One CREB inhibitor, A-CREB, is a potent and selective inhibitor of CREB DNA binding activity. The other, CREBm1, binds to CREB binding sites in DNA but is not activated because the transcriptional regulatory residue, serine 133, is mutated to alanine. CREB-mediated gene expression is both necessary for NGF-dependent survival and sufficient on its own to promote survival of sympathetic neurons. Moreover, expression of Bcl-2 is activated by NGF and other neurotrophins by a CREB-dependent transcriptional mechanism. A region of the bcl-2 gene between 1640 and 1337 relative to the translation start site is required for NGF-sensitive transcription. This region contains a near-perfect consensus CRE. Activated CREB can bind to this region of the bcl-2 promoter, and this interaction is critical for expression of Bcl-2 in a B lymphocyte cell line. Thus, a test was performed to see if the integrity of the bcl-2 CRE is necessary for the NGF-induced expression of bcl-2. A bcl-2 reporter construct harboring a two-base pair mutation of the CRE, rendering it unable to bind CREB, is impaired in its responsiveness to NGF. Overexpression of Bcl-2 reduces the death-promoting effects of CREB inhibition. Together, these data support a model in which neurotrophins promote survival of neurons, in part through a mechanism involving CREB family transcription factor-dependent expression of genes encoding prosurvival factors (Riccio, 1999).

The memory for sensitization of the gill withdrawal reflex in Aplysia is reflected in facilitation of the monosynaptic connection between the sensory and motor neurons of the reflex. The switch from short- to long-term facilitation requires activation of CREB1, derepression of ApCREB2, and induction of ApC/EBP. In search for genes that act downstream from CREB1, a transcription activator, ApAF, has been identified that is stimulated by protein kinase A and can dimerize with both ApC/EBP and ApCREB2. ApAF is necessary for long-term facilitation induced by five pulses of serotonin, by activation of CREB1, or by derepression of ApCREB2. Overexpression of ApAF enhances the long-term facilitation further. Thus, ApAF is a candidate memory enhancer gene downstream from both CREB1 and ApCREB2 (Bartsch, 2000).

The ApAF cDNA open reading frame encodes a putative novel basic-leucine zipper transcription factor that is 398 amino acids long. The C-terminal domain of the ApAF protein, which contains the bZIP, is homologous to a number of transcription factors involved in differentiation. The bZIP domain of ApAF is more than 60% homologous with the C-terminal domains of the mammalian PAR family of transcription factors: D-box binding protein (DBP), TEF, and HLF. ApAF also shares homology with the rodent, Xenopus, and human E4BP4, with the C. elegans transcription factor CES-2, and with the Drosophila gene giant (Bartsch, 2000 and references therein).

The experiments carried out with ApAF, both alone and in combination with other factors, suggest a model for the transition from short- to long-term facilitation in Aplysia sensory neurons. According to this model, the switch becomes activated when the transcription factor CREB1 becomes phosphorylated and the activity ratio between CREB1 and ApCREB2 is changed. Once activated, CREB1 regulates a cascade of downstream genes that are necessary for the induction of the long-term process. In addition to CREB1, two other downstream activators are involved in this switch: ApC/EBP and ApAF. Unlike ApC/EBP, which is present only at very low levels in the basal state and is induced by 5-HT, ApAF is constitutively expressed in the sensory neurons in the basal state, and its level of expression is not affected by the exposure of the sensory neurons to 5-HT. In addition to these three known activators, there are two known repressors: CREB1b and ApCREB2 (Bartsch, 2000).

Where in this cascade does ApAF act to enhance facilitation? ApAF forms poor homodimers but heterodimerizes well with both ApCREB2 and ApC/EBP. Although ApAF can dimerize with ApCREB2 in the basal state, evidence indicates that the function of ApAF necessary for long-term facilitation is downstream from ApCREB2. Injection of anti-ApCREB2 antibody, which facilitates the long-term facilitation by lowering the threshold of long-term facilitation to a single pulse of 5-HT, does so via ApAF. This long-term facilitation is blocked by coinjection of anti-ApAF antibody. The idea that ApAF is downstream from ApCREB2 is further supported by the converse finding that long-term facilitation, achieved by one pulse of 5-HT following removal of ApCREB2 with anti-ApCREB2 antibody, also is enhanced following the coinjection of ApAF protein (Bartsch, 2000).

The protein partner most likely to interact with ApAF is the other downstream activator, ApC/EBP. The data suggest that ApAF is recruited to act as an enhancer of long-term synaptic plasticity only after CREB1a is phosphorylated and the activity ratio between CREB1 and ApCREB2 is changed. The finding that ApAF only becomes critical for facilitating gene expression after the CREB1/ApCREB2 initiation complex is activated is further supported by the finding that the facilitation induced either by activation of CREB1a or by derepression of ApCREB2 can be blocked by both anti-ApAF antibodies or by a dominant negative inhibitor of ApAF. Once ApC/EBP and the downstream cascade of gene activation is induced, ApC/EBP can act in one of two ways: (1) it can act as a homodimer to activate downstream genes, and (2) ApC/EBP can recruit ApAF and ApCREB2 to form two new heterodimers, ApAF-ApC/EBP and ApCREB2-ApC/EBP. The specific roles of the individual complexes between ApAF, ApCREB2, and ApC/EBP in the formation and maintenance of long-term facilitation is not yet known. Since each dimer binds a different DNA motif, it is likely that this dimerization may target different DNA sequences and serve to broaden the number of targets that can be activated by the resulting complexes (Bartsch, 2000).

Even for a simple form of learning, the switch from short- to long-term is complex and involves a number of interrelated transcriptional activators and repressors. Because sensitization is a simple form of learning, it is thought that the several components encountered in Aplysia are likely to represent only the core conserved components of the switch. It is probable that with other, more complex learning processes, additional components will also be recruited (Bartsch, 2000).

In contrast to CREB1, phosphorylated ApAF alone is unable to induce long-term facilitation. However, ApAF is a powerful modulator of long-term facilitation. This is evident in several ways. (1) When paired with one pulse of 5-HT (which activates PKA and results in CREB1a phosphorylation), overexpression of ApAF converts short-term facilitation into long-term facilitation. (2) When paired with removal of ApCREB2, ApAF extends the long-term facilitation beyond limit set by five pulses of 5-HT. (3) ApAF also extends the long-term facilitation produced by five pulses of 5-HT (Bartsch, 2000).

Earlier work in Drosophila and Aplysia first indicated that long-term memory storage, and the synaptic plasticity that underlies it, can be both increased and decreased by modulating the activity at the level of CREB. These earlier studies indicated that long-term memory in the intact animal and long-term synaptic facilitation in the neural circuit storing a memory can either be depressed or enhanced by altering the ratio of CREB activators to CREB repressors. There is now evidence that positive and negative regulators of CRE and CREB are also part of the switch from short- to long-term memory in mice. In addition, there is now good pharmacological and genetic evidence in mice that synaptic plasticity and long-term memory can be enhanced by modulating the signaling pathways upstream from PKA and CREB. (1) Pharmacologically inhibiting type IV phosphodiesterases by rolipram increased cAMP response to forskolin in hippocampal slices and improved long-term memory retention in context conditioning. (2) Enhancement of memory has now also been shown in transgenic mice overexpressing in the forebrain NR2B, an NMDA receptor subunit with a long open time. These mice show both enhanced LTP in the hippocampus and improved memory formation (Bartsch, 2000 and references therein).

Evidence is provided for the enhancement of long-term synaptic plasticity at a functional step downstream from both CREB1a and ApCREB2. Similarly, ApAF and its potential mammalian homologs could also serve as modulators to enhance memory storage. Thus, analysis of the signaling cascades activated by learning processes reveals multiple sites of modulation. In addition to inhibitory constraints, (memory repressor genes), there seem also to be positive regulators of long-term synaptic strength that may serve as memory enhancer genes (Bartsch, 2000 and references therein).

Barx1 and Barx2 are homeodomain proteins originally identified using regulatory elements of genes encoding certain cell adhesion molecules (CAMs). In the present study, regions of Barx2 were characterized that bind to regulatory elements of genes encoding three CAMs, L1, neuron-glia CAM (Ng-CAM), and neural CAM (N-CAM); domains of Barx2 were identified that regulate N-CAM transcription. The homeodomain of Barx2 is sufficient for binding to homeodomain binding sites (HBS) from all three CAM genes. The presence of a 17-amino acid Barx basic region resulted in a 2-fold decrease in binding to HBS sequences from the Ng-CAM and L1 genes, whereas it led to a 6.5-fold increase in binding to the HBS from the N-CAM promoter. Thus, the Barx basic region influences the strength and specificity of Barx2 binding to DNA. In co-transfection experiments, Barx2 repressed N-CAM promoter activity. A 24-residue N-terminal region of Barx2 is essential for repression. When this region is absent, Barx2 activates the N-CAM promoter. A 63-residue C-terminal domain is required for this activation. In GST pull-down experiments, Barx2 binds to proteins of the CREB family, CREB1 and ATF2. Overall, these findings provide a framework for understanding developmental and physiological contexts that influence repressor or activator functions of Barx2 (Edelman, 2000).

A hidden Markov model (HMM) based on known cAMP responsive elements has been employed to search for putative CREB target genes. The best scoring sites are positionally conserved between mouse and human orthologs, suggesting that this parameter can be used to enrich for true CREB targets. Target validation experiments reveal a core promoter requirement for transcriptional induction via CREB; TATA-less promoters are unresponsive to cAMP, compared to TATA-containing genes, despite comparable binding of CREB to both sets of genes in vivo. Indeed, insertion of a TATA box motif rescues cAMP responsiveness on a TATA-less promoter. These results illustrate a mechanism by which subsets of target genes for a transcription factor are differentially regulated depending on core promoter configuration (Conkright, 2003).

The incretin hormone GLP1 promotes islet-cell survival via the second messenger cAMP. Mice deficient in the activity of CREB, caused by expression of a dominant-negative A-CREB transgene in pancreatic ß-cells, develop diabetes secondary to ß-cell apoptosis. Remarkably, A-CREB severely disrupts expression of IRS2, an insulin signaling pathway component that is shown in this study to be a direct target for CREB action in vivo. Since induction of IRS2 by cAMP enhances activation of the survival kinase Akt in response to insulin and IGF-1, these results demonstrate a novel mechanism by which opposing pathways cooperate in promoting cell survival (Jhala, 2003).

The CREB transcription factor regulates differentiation, survival, and synaptic plasticity. The complement of CREB targets responsible for these responses has not been identified, however. A novel approach was developed to identify CREB targets, termed serial analysis of chromatin occupancy (SACO), by combining chromatin immunoprecipitation (ChIP) with a modification of SAGE. Using a SACO library derived from rat PC12 cells, ~41,000 genomic signature tags (GSTs) were identified that map to unique genomic loci. CREB binding was confirmed for all loci supported by multiple GSTs. Of the 6302 loci identified by multiple GSTs, 40% were within 2 kb of the transcriptional start of an annotated gene, 49% were within 1 kb of a CpG island, and 72% were within 1 kb of a putative cAMP-response element (CRE). A large fraction of the SACO loci delineated bidirectional promoters and novel antisense transcripts. This study represents the most comprehensive definition of transcription factor binding sites in a metazoan species (Impey, 2004).

Gamma-secretase, which is responsible for the intramembranous cleavage of Alzheimer's beta-amyloid precursor protein (APP), the signaling receptor Notch, and many other substrates, is a multiprotein complex consisting of at least four components: presenilin (PS), nicastrin, APH-1, and PEN-2. Despite the fact that PEN-2 is known to mediate endoproteolytic cleavage of full-length PS and APH-1 and nicastrin are required for maintaining the stability of the complex, the detailed physiological function of each component remain elusive. Unlike that of PS, the transcriptional regulation of PEN-2, APH-1, and nicastrin has not been investigated. This study characterizes the upstream regions of the human PEN-2 gene and has identified a 238-bp fragment located 353 bp upstream of the translational start codon as the key region necessary for the promoter activity. Further analysis revealed a CREB binding site located in the 238-bp region that is essential for the transcriptional activity of the PEN-2 promoter. Mutation of the CREB site abolished the transcriptional activity of the PEN-2 promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis have shown the binding of CREB to the PEN-2 promoter region both in vitro and in vivo. Activation of the CREB transcriptional factor by forskolin dramatically promotes the expression of PEN-2 mRNA and protein, whereas the other components of the gamma-secretase complex remain unaffected. Forskolin treatment slightly increases the secretion of soluble APPalpha and Abeta without affecting Notch cleavage. These results demonstrate that expression of PEN-2 is regulated by CREB and suggest that the specific control of PEN-2 expression may imply additional physiological functions uniquely assigned to PEN-2 (Wang, 2006).

The mitogen-activated protein kinase p38 plays a critical role in inflammation, cell cycle progression, differentiation, and apoptosis. The activity of p38 is stimulated by a variety of extracellular stimuli, such as the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha), and subjected to regulation by other intracellular signaling pathways, including the cyclic AMP (cAMP) pathway. Yet the underlying mechanism by which cAMP inhibits p38 activation is unknown. This study shows that the induction of dynein light chain (DLC) by cAMP response element-binding protein (CREB) is required for cAMP-mediated inhibition of p38 activation. cAMP inhibits p38 activation via the protein kinase A-CREB pathway. The inhibition is mediated by the CREB target gene Dlc, whose protein product, DLC, interferes with the formation of the MKK3/6-p38 complex, thereby suppressing p38 phosphorylation activation by MKK3/6. The inhibition of p38 activation by cAMP leads to suppression of NF-kappaB activity and promotion of apoptosis in response to TNF-alpha. Thus, these results identify DLC as a novel inhibitor of the p38 pathway and provide a molecular mechanism by which cAMP suppresses p38 activation and promotes apoptosis (Zhang, 2006).

Endochondral ossification is the process of skeletal bone growth via the formation of a cartilage template that subsequently undergoes mineralization to form trabecular bone. Genetic mutations affecting the proliferation or differentiation of chondrocytes result in skeletal abnormalities. Activating transcription factor-2 (ATF-2) modulates expression of cell cycle regulatory genes in chondrocytes, and mutation of ATF-2 results in a dwarfed phenotype. This study investigated the regulatory role that ATF-2 plays in expression of the pocket proteins, cell cycle regulators important in cellular proliferation and differentiation. The spatial and temporal pattern of pocket protein expression was identified in wild type and mutant growth plates. Expression of retinoblastoma (pRb) mRNA and protein were decreased in ATF-2 mutant primary chondrocytes. pRb mRNA expression was coordinated with chondrogenic differentiation and cell cycle exit in ATDC5 cells. Type X collagen immunohistochemistry was performed to visualize a delay in differentiation in response to loss of ATF-2 signaling. Chondrocyte proliferation was also affected by loss of ATF-2. These studies suggest pRb plays a role in chondrocyte proliferation, differentiation and growth plate development by modulating cell cycle progression. ATF-2 regulates expression of pRb within the developing growth plate, contributing to the skeletal phenotype of ATF-2 mutant mice through the regulation of chondrocyte proliferation and differentiation (Vale-Cruz, 2008).

CREB and embryogenesis

The leucine zipper transcription factors cAMP response element binding protein (CREB), cAMP response element modulatory protein (CREM) and activating transcription factor 1 (ATF1) bind to the cAMP response element (CRE) with the palindromic consensus sequence TGACGTCA. Their transcriptional activities are dependent on serine phosphorylation induced by various extracellular signals such as hormones, growth factors and neurotransmitters. CREB is the predominant CRE-binding protein in Xenopus embryos and it plays an essential role during early development. The importance of CREB for morphogenetic processes was assessed by injection of RNA encoding a dominant-negative form of CREB; this form is fused to a truncated progesterone receptor ligand binding domain. In this fusion protein, a dominant-negative function can be induced by application of the synthetic steroid RU486 at given developmental stages. The inhibition of CREB at blastula and early gastrula stages leads to severe posterior defects of the embryos reflected by strong spina bifida, whereas the inhibition of CREB at the beginning of neurulation results in stunted embryos with microcephaly. In these embryos, initial induction of neural and mesodermal tissues is not dependent on CREB function, because genes such as Otx2, Krox20, Shh and MyoD are still expressed in injected embryos. But the expression domains of Otx2 and MyoD were found to be distorted reflecting abnormal development in both neural and somitic derivatives. In summary, these data show that CREB is essential during several developmental stages of Xenopus embryogenesis (Lutz, 1999).

The importance of the CREB family of transcriptional activators for endochondral bone formation was evaluated by expressing a potent dominant negative CREB inhibitor (A-CREB) in growth plate chondrocytes of transgenic mice. A-CREB transgenic mice exhibit short-limbed dwarfism and die minutes after birth, apparently due to respiratory failure from a diminished rib cage circumference. Consistent with the robust Ser133 phosphorylation and, hence, activation of CREB in chondrocytes within the proliferative zone of wild-type cartilage during development, chondrocytes in A-CREB mutant cartilage exhibit a profound decrease in proliferative index and a delay in hypertrophy. Correspondingly, the expression of certain signaling molecules in cartilage, most notably the Indian hedgehog (Ihh) receptor patched (Ptch), was lower in A-CREB expressing versus wild-type chondrocytes. CREB appears to promote Ptch expression in proliferating chondrocytes via an Ihh-independent pathway; phospho-CREB levels were comparable in cartilage from Ihh -/- and wild-type mice. These results demonstrate the presence of a distinct signaling pathway in developing bone that potentiates Ihh signaling and regulates chondrocyte proliferation, at least in part, via the CREB family of activators (Long, 2001).

Rho GTPases regulate several aspects of tissue morphogenesis during animal development. Mice lacking the Rho-inhibitory protein, p190-B RhoGAP, are 30% reduced in size and exhibit developmental defects strikingly similar to those seen in mice lacking the CREB transcription factor. In p190-B RhoGAP-deficient mice, CREB phosphorylation is substantially reduced in embryonic tissues. Embryo-derived cells contain abnormally high levels of active Rho protein, are reduced in size, and exhibit defects in CREB activation upon exposure to insulin or IGF-1. The cell size defect is rescued by expression of constitutively activated CREB, and in wild-type cells, expression of activated Rho or dominant-negative CREB results in reduced cell size. Together, these results suggest that activity of the Rho GTPase modulates a signal from insulin/IGFs to CREB that determines cell size and animal size during embryogenesis (Sordella, 2002).

These observations indicate that activity of the Rho GTPase and the CREB transcription factor are important determinates of cell and animal size during embryonic development, thereby defining a novel biological function for both of these widely expressed regulatory proteins. In fibroblasts derived from mice lacking p190-B RhoGAP, it appears that Rho is performing a largely cell-autonomous role that can influence responsiveness to insulin/IGFs through activation of the Rho target ROK. However, the fact that mutant fibroblasts are not completely defective in insulin/IGF-1 responsiveness suggests that at least some aspect of insulin-promoted growth is Rho insensitive. Moreover, the observation that mice lacking p190-B RhoGAP appear to be uniformly reduced in size, while phospho-CREB levels are not substantially reduced in every tissue, raises the possibility that there is additional complexity involved in determining organism size (Sordella, 2002).

It is possible, for example, that modulation of CREB is a more significant determinant of cell size at earlier stages of development in some tissues. In addition, there may be both cell-autonomous and -nonautonomous mechanisms involved in vivo. Indeed, PI-3 kinase, which appears to play a role in the pathway proposed here, has been previously implicated in cell size regulation, and has been reported to have both cell-autonomous and -nonautonomous functions in C. elegans. In addition, while IRS plays a well-documented cell-autonomous role in regulating cell size in Drosophila, studies in mice reveal an additional cell-nonautonomous role in controlling animal size. It is also worth noting that both insulin and IGF-1 gene expression have been found to be CREB responsive, raising the possibility that impaired CREB activity can also contribute in a cell-nonautonomous manner to growth regulation by affecting levels of secreted and circulating insulin/IGF. Such findings point to a complex regulatory system for the control of cell, organ, and animal size that involves both cell-autonomous and -nonautonomous mechanisms that may require an overlapping set of proteins. In fact, there may be additional compensatory mechanisms that serve to uniformly adjust the size of organs to accommodate changes that may be limited to a subset of organs in the mutant animals (Sordella, 2002).

In Xenopus embryos, body patterning and cell specification are initiated by transcription factors, which are themselves transcribed during oogenesis, and their mRNAs are stored for use after fertilization. The T-box transcription factor VegT is both necessary and sufficient to initiate transcription of all endoderm, and most mesoderm genes. In the absence of maternal VegT, no mesodermal organs (including the heart) or endodermal organs form. A second maternal transcription factor XTcf3 acts as a global repressor of transcription of dorsal genes, whose repression is inactivated on the dorsal side by a maternally encoded Wnt signaling pathway. In the absence of ß-catenin, no mesodermal or endodermal organs form. The maternally encoded transcription factor CREB is also essential for development. It is required for the initiation of expression of several mesodermal genes, including Xbra, Xcad2, and -3 and also regulates the cardiogenic gene Nkx 2-5. Maternal CREB-depleted embryos develop gastrulation defects that are rescued by the reintroduction of activated CREB mRNA. It is concluded that maternal CREB must be added to the list of essential maternal transcription factors regulating cell specification in the early embryo (Sundaram, 2003).

CREB and neural injury

Axonal injury increases intracellular Ca2+ and cAMP, and has been shown to induce gene expression, which is thought to be a key event for regeneration. Increases in intracellular Ca2+ and/or cAMP can alter gene expression via activation of a family of transcription factors that bind to and modulate the expression of CRE (Ca2+/cAMP response element) sequence-containing genes. Aplysia motor neurons were used to examine the role of CRE-binding proteins in axonal regeneration after injury. Axonal injury increases the binding of proteins to a CRE sequence-containing probe. Western blot analysis reveals that the level of ApCREB2, a CRE sequence-binding repressor, is enhanced as a result of axonal injury. The sequestration of CRE-binding proteins by microinjection of CRE sequence-containing plasmids enhances axon collateral formation (both number and length), as compared with control plasmid injections. These findings show that Ca2+/cAMP-mediated gene expression via CRE-binding transcription factors participates in the regeneration of motor neuron axons (Dash, 1998).

Inhibitors in myelin play a major role in preventing spontaneous axonal regeneration after CNS injury. Elevation of cAMP overcomes this inhibition, in a transcription-dependent manner, through the upregulation of Arginase I (Arg I) and increased synthesis of polyamines. Here, the cAMP effect requires activation of the transcription factor CREB to overcome myelin inhibitors; a dominant-negative CREB abolishes the effect, and neurons expressing a constitutively active form of CREB are not inhibited. Activation of CREB is also required for cAMP to upregulate Arg I, and the ability of constitutively active CREB to overcome inhibition is blocked by an inhibitor of polyamine synthesis. Finally, expression of constitutively active CREB in DRG neurons is sufficient to promote regeneration of subsequently lesioned dorsal column axons. These results indicate that CREB plays a central role in overcoming myelin inhibitors and so encourages regeneration in vivo (Gao, 2004).

CREB and Apoptosis

The mammalian ATF/CREB family of transcription factors comprises a large group of basic-region leucine zipper (bZIP) proteins whose members mediate diverse transcriptional regulatory functions. Expression of a specific mouse ATF gene, ATFx, is down-regulated in a variety of cells undergoing apoptosis following growth factor deprivation. When stably expressed in an interleukin 3 (IL-3)-dependent cell line, ATFx suppresses apoptosis resulting from cytokine deprivation. Conversely, a dominant-negative ATFx mutant induces apoptosis of cells cultured in the presence of growth factors. 24p3, a secreted lipocalin that induces apoptosis when added to hematopoietic cells, represses ATFx expression. However, constitutive expression of ATFx renders cells resistant to 24p3-mediated apoptosis. Collectively, these results indicate that ATFx is an anti-apoptotic factor, a novel role for an ATF protein (Persengiev, 2002).

The cyclic-AMP response element-binding (CREB) protein family of transcription factors plays a crucial role in supporting the survival of neurons. However, a cell-autonomous role has not been addressed in vivo. To investigate the cell-specific role of CREB, developing sympathetic neurons, whose survival in vitro is dependent on CREB activity, were used as a model. Mice were generated lacking CREB in noradrenergic (NA) and adrenergic neurons and compared with the phenotype of the germline CREB mutant. Whereas the germline CREB mutant revealed increased apoptosis of NA neurons and misplacement of sympathetic precursors, the NA neuron-specific mutation unexpectedly led to reduced levels of caspase-3-dependent apoptosis in sympathetic ganglia during the period of naturally occurring neuronal death. A reduced level of p75 neurotrophin receptor expression in the absence of CREB was shown to be responsible. Thus, this analysis indicates that the activity of cell-autonomous pro-survival signalling is operative in developing sympathetic neurons in the absence of CREB (Parlato, 2007).

The cyclic-AMP response element-binding (CREB) protein family of transcription factors plays a crucial role in supporting the survival of neurons. However, a cell-autonomous role has not been addressed in vivo. To investigate the cell-specific role of CREB, developing sympathetic neurons, whose survival in vitro is dependent on CREB activity, were used as a model. Mice were generated lacking CREB in noradrenergic (NA) and adrenergic neurons and compared with the phenotype of the germline CREB mutant. Whereas the germline CREB mutant revealed increased apoptosis of NA neurons and misplacement of sympathetic precursors, the NA neuron-specific mutation unexpectedly led to reduced levels of caspase-3-dependent apoptosis in sympathetic ganglia during the period of naturally occurring neuronal death. A reduced level of p75 neurotrophin receptor expression in the absence of CREB was shown to be responsible. Thus, this analysis indicates that the activity of cell-autonomous pro-survival signalling is operative in developing sympathetic neurons in the absence of CREB (Parlato, 2007).

CREB and longevity in C. elegans

Activating AMPK or inactivating calcineurin slows ageing in C. elegans and both have been implicated as therapeutic targets for age-related pathology in mammals. However, the direct targets that mediate their effects on longevity remain unclear. In mammals, CREB-regulated transcriptional coactivators (CRTCs; see Drosophila CRTC) are a family of cofactors involved in diverse physiological processes including energy homeostasis, cancer and endoplasmic reticulum stress. This study shows that both AMPK and calcineurin modulate longevity exclusively through post-translational modification of CRTC-1, the sole C. elegans CRTC. CRTC-1 is a direct AMPK target, and interacts with the CREB homologue-1 (CRH-1) transcription factor in vivo. The pro-longevity effects of activating AMPK or deactivating calcineurin decrease CRTC-1 and CRH-1 activity and induce transcriptional responses similar to those of CRH-1 null worms. Downregulation of crtc-1 increases lifespan in a crh-1-dependent manner and directly reducing crh-1 expression increases longevity, substantiating a role for CRTCs and CREB in ageing. Together, these findings indicate a novel role for CRTCs and CREB in determining lifespan downstream of AMPK and calcineurin, and illustrate the molecular mechanisms by which an evolutionarily conserved pathway responds to low energy to increase longevity (Mair, 2011).

These data indicate that CRTC-1 is the critical direct longevity target of both AMPK and calcineurin in C. elegans and identify a new role for CRTCs and CREB in modulating longevity. They also represent the first analysis of the transcriptional profiles of long-lived activated AMPK and deactivated calcineurin organisms and suggest the primary longevity-associated role of these perturbations is the modulation of CRTC-1 and CRH-1 transcriptional activity. Notably, both the FOXO transcription factor daf-16 and genes involved in autophagy have also been implicated in AMPK and calcineurin longevity, respectively. Further work to determine precisely where the AMPK-calcineurin-CRTC-1 pathway converges with FOXO and autophagy will be enlightening. It will also be interesting to determine if CRTC-1 mediates downstream effects of kinases other than AMPK. In mammals, CRTCs are regulated by multiple CAMKL kinase family members, and additive effects are seen of AMPK and related kinases on the localization of CRTC-1, in particular the MAP/microtubule affinity-regulating kinase (MARK) par-1, indicating that this kinase may also regulate CRTC-1 in vivo. At present, however, AMPK is the only CAMKL kinase shown to be a positive regulator of longevity (Mair, 2011).

Collectively, these data identify CRTC-1 as a central node linking the upstream lifespan modifiers AMPK and calcineurin to CREB activity via a shared signal-transduction pathway, and demonstrate that post-translational modification of CRTC-1 is required for their effects on longevity. Complementing the pro-longevity effects of inhibiting CRTC function in C. elegans, reducing components of the CRTC/CREB pathway has recently been shown to confer health benefits to mice. Given the evolutionary conservation of this pathway from C. elegans to mammals it will be fascinating to determine the role of CRTCs both as mammalian ageing modulators and as potential drug targets for patients with metabolic disorders and cancer (Mair, 2011).

A C. elegans thermosensory circuit regulates longevity through crh-1/CREB-dependent flp-6 neuropeptide signaling
Sensory perception, including thermosensation, shapes longevity in diverse organisms, but longevity-modulating signals from the sensory neurons are largely obscure. This study shows that CRH-1/CREB activation by CMK-1/CaMKI in the AFD thermosensory neuron is a key mechanism that maintains lifespan at warm temperatures in C. elegans. In response to temperature rise and crh-1 activation, the AFD neurons produce and secrete the FMRFamide neuropeptide FLP-6. Both CRH-1 and FLP-6 are necessary and sufficient for longevity at warm temperatures. These data suggest that FLP-6 targets the AIY interneurons and engages DAF-9 sterol hormone signaling. Moreover, it was shown that FLP-6 signaling downregulates ins-7/insulin-like peptide and several insulin pathway genes, whose activity compromises lifespan. This work illustrates how temperature experience is integrated by the thermosensory circuit to generate neuropeptide signals that remodel insulin and sterol hormone signaling and reveals a neuronal-endocrine circuit driven by thermosensation to promote temperature-specific longevity (Chen, 2016).

CREB mediates brain serotonin regulation of bone mass through its expression in ventromedial hypothalamic neurons

Serotonin is a bioamine regulating bone mass accrual differently depending on its site of synthesis. It decreases accrual when synthesized in the gut, and increases it when synthesized in the brain. The signal transduction events elicited by gut-derived serotonin once it binds to the Htr1b receptor present on osteoblasts have been identified and culminate in cAMP response element-binding protein (CREB) regulation of osteoblast proliferation. In contrast, it is not known how brain-derived serotonin favors bone mass accrual following its binding to the 5-hydroxytryptamine (serotonin) receptor 2C (Htr2c) receptor on neurons of the hypothalamic ventromedial nucleus (VMH). This study shows - through gene expression analysis, serotonin treatment of wild-type and Htr2c-/- hypothalamic explants, and cell-specific gene deletion in the mouse - that, following its binding to the Htr2c receptor on VMH neurons, serotonin uses a calmodulin kinase (CaMK)-dependent signaling cascade involving CaMKKβ and CaMKIV to decrease the sympathetic tone and increase bone mass accrual. It was further shown that the transcriptional mediator of these events is CREB, whose phosphorylation on Ser 133 is increased by CaMKIV following serotonin treatment of hypothalamic explants. A microarray experiment identified two genes necessary for optimum sympathetic activity whose expression is regulated by CREB. These results provide a molecular understanding of how serotonin signals in hypothalamic neurons to regulate bone mass accrual and identify CREB as a critical determinant of this function, although through different mechanisms depending on the cell type, neuron, or osteoblast in which it is expressed (Oury, 2010).

CREB function in axons and dendrites

In screening amplified poly(A) mRNA from hippocampal dendrites and growth cones in culture to determine candidates for local translation, select transcription factor mRNAs were found to be present. It is hypothesized that synthesis of transcription factor proteins within dendrites would provide a direct signaling pathway between the distal dendrite and the nucleus resulting in modulation of gene expression important for neuronal differentiation. To evaluate this possibility, radiolabeled amplified antisense RNA was used to probe slot blots of transcription factor cDNAs as well as arrayed blots of zinc finger transcription factors. The mRNAs encoding the cAMP response element binding protein (CREB), zif 268, and one putative transcription factor were detected. CREB protein is present in dendrites, translation of CREB mRNA in isolated dendrites is feasible and CREB protein found in dendrites can interact with the cis-acting cyclic AMP response element DNA sequence. Further, CREB protein in dendrites is not transported to this site from the cell body because fluorescently tagged CREB microperfused into the soma does not diffuse into the dendrites. In addition, CREB protein microperfused into dendrites is rapidly transported to the nucleus, its likely site of bioactivity. Using the isolated dendrite system, it was shown that phosphorylation of Ser-133 on CREB protein can occur in isolated dendrites independent of the nucleus. These data provide a regulatory pathway in which transcription factors synthesized and posttranslationally modified in dendrites directly alter gene expression bypassing the integration of signal transduction pathways that converge on the nucleus (Crino, 1998).

32P-CTP radiolabeled aRNA was used to screen reverse Northern blots containing candidate transcription factor full-length cDNAs, including the [delta ] isoform of CREB, c-fos, c-jun, zif268, orthodenticle 1, brain factor 1/brain factor 2, and hairy enhancer of split 1 -- all of which are important in neuronal development. The alpha subunit of Ca2+/calmodulin-dependent kinase and microtubule-associated protein 2 cDNAs, previously identified in dendrites and dendritic growth cones, were detected and served as positive controls. CREB and zif 268 mRNAs are detected in growth cones but c-fos, c-jun, orthodenticle 1, hairy enhancer of split 1, brain factor 1, and brain factor 2 mRNAs are at or below pBS background. Comparison and expression of the relative levels of CREB as a percentage of calmodulin-dependent kinase mRNA from processes isolated from over 30 cells shows that CREB mRNA is present in processes at approximately 20% of the levels of calmodulin-dependent kinase mRNA. To gain a broader view of transcription factor mRNAs in the dendritic domain, arrayed blots of several hundred C2H2 zinc finger containing cDNAs were probed with radiolabeled aRNA from cell bodies or growth cones. Hybridization to several C2H2 cDNAs probed with cell body derived RNA (>15 positives) and growth cone derived RNA (five positives) was observed. Partial sequence of 2,400 bases of one of these cDNAs (C82) yielded a mouse zinc finger protein containing 12 zinc finger motifs with partial sequence homology to Krox20. The finding of only a few C2H2 transcription factor mRNAs and the data from the original reverse Northern analysis in dendrites highlights the selectivity of transcription factor mRNA localization in growth cones. Because of the potential importance of CREB in neuronal plasticity, the dendritic localization in cultured neurons was verified by single cell PCR, DNA sequencing of the PCR product, and in situ hybridization. In situ hybridization was performed on primary hippocampal cells by using a cRNA probe. Staining in the dendritic process confirms the subcellular localization of CREB. Additionally, to show that CREB mRNA is localized to dendrites in intact tissue and is not an artifact of dispersed cell culturing, as well as to show that dendritic CREB mRNA is present in other species, in situ hybridization was used to localize CREB mRNA in dendrites of human neocortical neurons in brain tissue sections. To determine whether CREB protein is present in neuronal processes, cultured hippocampal neurons were stained with polyclonal antibodies against either non- or phosphorylated CREB and the leucine zipper domain of CREB. Immuno-staining was dense in the nucleus and light in somatic, dendritic, and dendritic growth cone cytoplasm. Approximately 80% of the cultured neurons exhibit CREB immunoreactivity in dendritic processes. Growth cones with broadened lamellopodia were most heavily stained while many smaller growth cones were not, or were only lightly labeled. No CREB immunoreactivity is seen in morphologically identified axons (Crino, 1998).

CREB in dendrites may exist in either mono- or dimeric form. The majority of cytoplasmic CREB protein exists as nonphosphorylated monomers although a small fraction of cytoplasmic CREB (likely dimeric) is phosphorylated. The dimeric form of CREB is predominantly the form that interacts with the CRE, however, CREB monomers can bind the CRE in vitro. Thus, because both forms can bind to CRE, an attempt was made to determine whether dendritically localized CREB can bind to CRE concatemers. CRE concatemers bind to CREB protein in the nuclei, dendrites, and dendritic growth cones within neurons, corroborating the immunohistochemical identification of CREB in these domains. Indeed, the number of in situ Southwestern assay positive dendrites in a high power field matches the number that are CREB immunopositive. Labeling is dense within the nucleus and more punctate labeling is present within dendrites and dendritic growth cones. Axons showed no binding. Unligated 32P-CRE oligonucleotide does not bind to protein in the cells. Moreover, preincubation of the tissue section with excess (>5 mg) unlabeled ligated CRE effectively abolishes 32P-CRE concatemer binding. Finally, CRE concatemer binding does not reflect nonspecific interaction of double-stranded DNA with other transcription factors, because a nonsense palindrome probe (inverted CRE sequence, 5'-CAGTACTG-3') is ineffective. Experiments show that CREB mRNA is recognized by dendroplasmic translational machinery. The data show that the machinery necessary to transport CREB to the nucleus is present within the dendritic domain. Thus, CREB protein synthesized in dendrites can affect gene expression directly by its selective and rapid transport to the nucleus, likely via interaction of its nuclear localization signal with the translocation machinery. These CREB perfusion data also suggest that CREB does not act as an RNA-binding protein to chaperone RNA to the dendrite because microperfused fluorescent CREB does not move from the cell soma to the dendrites that would be expected for an RNA-transport protein. Transcriptional activation by CREB occurs only if Ser-133 is phosphorylated. To determine whether synaptic modulators could enhance phosphorylation of CREB in dendrites, DHPG, a mGluR1 agonist, which had previously been shown to stimulate protein synthesis in synaptodendrosome preparations was tested. Application of DHPG for 15 min induces phosphorylation of Ser-133 of CREB in processes severed from their somas as detected with a phospho-Ser-133 CREB specific antibody. There is little phospho-Ser133 CREB in nonstimulated neuronal cell nuclei or processes. These data show that Ser-133 phosphorylation can occur independent of the nucleus (Crino, 1998).

Many transcription factors such as CREB, regulate gene expression in response to membrane depolarization, Ca2+ influx, and cAMP-mediated second messenger systems. CREB phosphorylation is tightly coupled to synaptic plasticity and genesis of dendritic spines, suggesting that cellular events occurring at a distance from the nucleus can modulate the activation state of CREB within the nucleus. Because of this, most studies have focused on the regulation of CREB and other transcription factors within the nucleus. The hypothesis that transcription factor mRNAs present within dendrites are locally synthesized into proteins that are retrogradely transported into the nucleus is a view of how changes in gene expression during development may be regulated by distant cellular events. Indeed, the selective transport of microperfused CREB from the dendrite to the nucleus demonstrates that such a pathway exists in vivo and provides a mechanism by which trophic or activity-dependent cues induce local synthesis of transcription factors that are imported to the nucleus and modulate dendritic outgrowth and synaptic connectivity. Dendritic transcription factors may serve to imprint upon the nucleus an image of events occurring in the distal dendrite. Such a nuclear imprinting effect may play an important role in defining activity-dependent states and connectional status of dendritic synapses as dendritic arbors extend into cortex. Appearance of phospho-Ser-133 CREB in dendrites after mGluR1 stimulation provides compelling evidence for how specific stimuli can induce the formation of activated CREB. The possibility that the phosphorylation pattern of CREB differs in different cellular compartments is appealing. The composite of these phosphorylation events likely alters the transcriptional activity of CREB. Unfortunately, no reagents are currently available to assess the state of other putative phosphorylation sites on CREB. An important consideration, in the concept of nuclear imprinting is how the small amount of dendritically synthesized CREB contributes to the total functional nuclear pool. It is difficult to quantitatively compare basal CREB levels or increases in CREB levels in different subregions of neurons because of the large basal levels of CREB protein, and the harvesting of the large number of processes required for Western blot analysis is impractical. However, because basal levels of phospho-CREB are low the DHPG experiment permits an estimation of the relative contribution of dendritic phospho-CREB (dpCREB) to the functional pool of nuclear phospho-CREB (npCREB). Based upon the relative immunoreactivity, 5%-10% of the npCREB pool is present in an individual dendrite. Also, the dpCREB signal observed in a severed process is similar to that observed in the process that is still attached to the soma. This suggests that much of the phospho-CREB seen in the dendrite is phosphorylated in the dendrite and is not delivered by transport or diffusion from the nucleus to the process. It is reasonable to speculate that when a neuron is stimulated, in vivo, it receives a localized signal, which may differentially stimulate dpCREB production relative to CREB phosphorylation in the nucleus, such that there is dpCREB production with little to no stimulation of CREB phosphorylation in the nucleus. This dpCREB, synthesized in the absence of npCREB production, would likely be translocated to the nucleus where it would represent a much larger fraction of the npCREB pool. Hence, this 5%-10% estimate (based upon bath application of a receptor agonist) likely underestimates the relative contribution of dpCREB to the nuclear CREB pool. In toto, these data provide a mechanism by which spatially localized pharmacologic and electrophysiologic signals can effect changes in neuronal gene transcription. The phosphorylation and dephosphorylation of locally synthesized CREB and other transcription factors in the dendrite, which may be distinct from that in the nucleus, provides a compelling mechanism by which the spatial specificity of Ca2+ entry into the dendrite mediates regulation of gene expression (Crino, 1998).

CaM kinase IV and CREB play a critical role in mediating calcium-induced dendritic growth in cortical neurons. Calcium-dependent dendritic growth is suppressed by CaM kinase inhibitors, a constitutively active form of CaM kinase IV induces dendritic growth in the absence of extracellular stimulation, and a kinase-dead form of CaM kinase IV suppresses dendritic growth induced by calcium influx. CaM kinase IV activates the transcription factor CREB, and expression of a dominant negative form of CREB blocks calcium- and CaM kinase IV-induced dendritic growth. In cortical slice cultures, dendritic growth is attenuated by inhibitors of voltage-sensitive calcium channels and by dominant negative CREB. These experiments indicate that calcium-induced dendritic growth is regulated by activation of a transcriptional program that involves CaM kinase IV and CREB-mediated signaling to the nucleus (Redmond, 2002).

While both CaM kinase IV and MAP kinase appear to be required for calcium-induced dendritic growth, it is intriguing that a constitutively active form of CaM kinase IV, but not of MEK, can induce dendritic growth. Although the basis of the difference in the effects of CaM kinase IV and MAP kinase is not yet known, one possibility is that it may be related to their ability to induce CREB/CBP-mediated transcription. Both CaM kinase IV and MAP kinase have been implicated in mediating calcium activation of CREB-dependent transcription, but they are thought to differ in their mechanism of action. While CaM kinase IV appears to be involved in the rapid phosphorylation of CREB, the MAP kinase pathway is activated with slower kinetics and appears to be important for the sustained phosphorylation of CREB. Both the rapid and sustained components of CREB phosphorylation are required for transcription. In addition, calcium signaling directly activates CBP, which is another important component of the CREB transcription complex. In these experiments, the fact that inhibitors of either CaM kinases or MAP kinase block calcium-dependent dendritic growth is consistent with the dual requirement of these kinases in activation of CREB. Constitutively active CaM kinase IV is probably effective in inducing dendritic growth because it can sustain CREB phosphorylation due to its continued presence in transfected neurons, and it is effective in activating CBP. Activation of MEK alone, in contrast, may not be effective in inducing dendritic growth because of its inability to activate CBP. Thus, the differential ability of CaM kinase IV and MEK to activate CBP may be the molecular distinction that underlies the distinct biological effects of these kinases (Redmond, 2002).

CRE-binding protein (CREB) belongs to a family of transcription factors that mediates stimulus-dependent gene expression in neuronal and non-neuronal cells. CREB is phosphorylated on its transcriptional regulatory site, Ser-133, in vivo in a neurotrophin-dependent manner. In mice harboring a null mutation in the Creb gene, sensory neurons exhibit excess apoptosis and degeneration, and display impaired axonal growth and projections. Interestingly, excess apoptosis is not observed in the central nervous system. CREB is required within sensory and sympathetic neurons for survival and axon extension since both of these neurotrophin-dependent processes are compromised in cultured neurons from CREB null mice. Thus, during their period of neurotrophin dependency, peripheral neurons require CREB-mediated gene expression for both survival and growth in vivo (Lonze, 2002).

Odorant stimulation enhances survival of olfactory sensory neurons via MAPK and CREB

Olfactory sensory neurons (OSNs) can be sensitized to odorants by repeated exposure, suggesting that an animal's responsiveness to olfactory cues can be enhanced at the initial stage of detection. However, because OSNs undergo a regular cycle of apoptosis and replacement by ostensibly naive, precursor-derived neurons, the advantage of sensitization would be lost in the absence of a mechanism for odorant-enhanced survival of OSNs. Using recombinant adenoviruses in conjunction with surgical and electrophysiological techniques, OSN survival and function were monitored in vivo; odorant exposure selectively rescues populations of OSNs from apoptosis. Odorant stimuli rescue OSNs in a cAMP-dependent manner by activating the MAPK/CREB-dependent transcriptional pathway, possibly as a result of expression of Bcl-2 (Watt, 2004).

Odorant activation of the MAPK pathway and CRE-mediated transcription as well as CREB phosphorylation in OSNs suggest that these neurons may possess some form of neuroplasticity akin to that observed in the central nervous system. Given the role of this pathway in the survival of certain neuronal tissues, the possibility that it could contribute to odorant-stimulated survival of peripheral OSNs was tested. Cotransduction of the olfactory turbinates with AdLacZ and Ad-HA dominant-negative MEK1 K97M (AdDNMEK) has no discernible effect on ß-gal expression, which was robust and uniform. However, inhibition of MAPK signaling by coexpression of this construct attenuates the ability of odorants to stimulate OSN survival in the Obx (olfactory bulbectomy) ipsilateral turbinates, demonstrating a requirement for MAPK activity in odorant-stimulated rescue. Cotransduction of the turbinates with AdLacZ and adenovirus carrying a constitutive-active FLAG-MEK1 R4F (AdCAMEK), without odorant exposure, produced the greatest survival of ß-gal-expressing OSNs, which was paralleled by persisting neuron-specific tubulin (NST) expression. The robust effect of the odorant citralva is likely due to its activation of numerous odorant receptor subtypes in these experiments (Watt, 2004).

In addition, electro-olfactogram analysis experiments have demonstrated that transduction with AdDNMEK blocks the ability of citralva to rescue odorant responsiveness after Obx, and AdCAMEK rescues responsiveness to all odorants tested without rescue odorant exposure. Thus, while in C. elegans the Ras/MAPK pathway appears to play a role in acute odorant detection, the current observations reveal effects that may be more likely to occur via downstream signaling events, perhaps resulting in gene transcription (Watt, 2004).

Activated MAPK has numerous targets in neurons including ion channels, cytoskeletal elements, and multiple transcription factors. To test the possibility that odorant-stimulated MAPK rescues OSNs from apoptosis by activation of its downstream target, the transcription factor CREB, the olfactory turbinates were cotransduced with AdLacZ and either the dominant-negative CREB-M1 (AdDNCREB) or constitutive-active FLAG-VP16-CREB (AdCACREB). Expression of DNCREB blocks the ability of odorants to rescue OSNs from apoptosis, while CACREB rescues a large number of OSNs in the absence of odorant stimuli, as well as robust NST expression. Furthermore, use of these viruses in EOG experiments has revealed that blockade of normal CREB function abolishes the ability of citralva to rescue OE responsiveness to odorants after Obx, while constitutive activation of CREB activity is sufficient to preserve responsiveness in the absence of exogenous rescue odorant (Watt, 2004).

One established mechanism for the survival of CNS neurons following apoptotic stimuli is the MAPK/CREB-regulated transcription of the protooncogene bcl-2. Importantly, OSNs in transgenic mice ectopically overexpressing Bcl-2 are refractory to Obx-induced apoptosis. Repeatedly exposing mice to odorants stimulates expression of Bcl-2 in OSNs; this stimulation was blocked by transduction with either AdDNMEK or AdDNCREB. Both AdCAMEK and AdCACREB induce expression of Bcl-2 in the absence of odorant stimuli. These results provide a mechanistic basis for the ability of odorant stimuli to rescue OSNs and indicate that activity-dependent regulation of Bcl-2 expression in neurons may have a more ubiquitous role in neuronal survival than previously thought (Watt, 2004).

PKA and CREB regulation of odorant receptor axonal projection

In mammals, odorant receptors (ORs) direct axons of olfactory sensory neurons (OSNs) toward targets in the olfactory bulb. G protein-mediated cAMP signals that regulate the expression of axon guidance molecules are essential for the OR-instructed axonal projection. Genetic manipulations of ORs, Gs, protein kinase A and a transcription factor, CREB, shifted the axonal projection sites along the anterior-posterior axis in the olfactory bulb. Thus it is the OR-derived cAMP signals, rather than direct action of OR molecules, that determines the target destinations of OSNs (Imai, 2006).

Each olfactory sensory neuron (OSN) in the mouse expresses only one functional odorant receptor (OR) gene out of ~1,000 members. Axons from OSNs expressing a given OR converge onto a specific site, glomerulus, in the olfactory bulb. It has been proposed that OR molecules at axon termini may directly recognize guidance cues on the olfactory bulb and mediate homophilic interactions of like-axons. OR molecules are G protein-coupled receptors (GPCRs) that transduce the odorant-binding signals by activating the olfactory-specific heterotrimeric G protein (Golf) expressed in mature OSNs. Activation of Golf stimulates adenylyl cyclase type III, generating cAMP, which opens cyclic nucleotide-gated (CNG) channels. Mice deficient for Golf and CNGA2 are anosmic, but form a normal glomerular map, suggesting that a G protein, other than Golf, may aid targeting OSNs independent of CNG channels (Imai, 2006).

OR molecules are rhodopsin-like type A GPCRs that contain a conserved tripeptide motif, Asp-Arg-Tyr (DRY), at the cytoplasmic end of transmembrane domain III, which is required for coupling of GPCRs to the partner G proteins. To examine whether the G protein signaling is involved in guidance of OSN axons, a DRYmotif mutant (D126R/R127D) was generated for the rat OR gene, I7, and it was expressed using a transgenic system. Axons from OSNs expressing the wild-type I7, I7(WT), converged to a specific site in the olfactory bulb, while those expressing the DRY-motif mutant, I7(RDY), remained in the anterior region of the olfactory bulb, failing to converge onto a specific glomerulus. The I7(RDY)-expressing axons never penetrated the glomerular layer, but stayed within the olfactory nerve layer. These axon termini were devoid of synaptotagmin (presynaptic marker) and MAP2 (dendritic marker) immunoreactivities, and thus likely did not form synapses. OSNs expressing a nonfunctional OR gene can activate other OR genes and will fail to converge onto a single glomerulus. However, the inability of I7(RDY) axons to converge on a specific glomerulus was not due to the co-expression of other OR genes; OSNs expressing the I7(RDY) transgene expressed no other OR genes. OSNs expressing I7(WT) all showed Ca2+ signals in response to octanal (agonist of I7 receptor), whereas those expressing I7(RDY) did not. Thus the I7(RDY) mutant is deficient in both axon targeting and G protein coupling (Imai, 2006).

Both Go and Gs genes are expressed in immature mouse OSNs. Although the Gs knock-out mutation is embryonic lethal, the Go deficient mouse shows no obvious anatomical defect in the olfactory system. Since the DRY-motif mutant was assumed to be incapable of coupling with G proteins, whether the constitutively-active Gs mutant (caGs) would rescue the defective phenotype of I7(RDY) in axonal projection was examined. The caGs gene was inserted into the I7(RDY) construct with an internal ribosome entry site (IRES), generating the I7(RDY)-caGs. In OSNs expressing this construct, cAMP signals should be generated constitutively by caGs in a receptor-independent manner. Axons expressing I7(RDY)-caGs (cyan) converged to a specific site in the olfactory bulb, whereas axons expressing I7(RDY) did not. YFP-positive and CFP-positive axons did not intermingle or co-converge, suggesting that homophilic interaction of OR molecules is unlikely. Axons expressing I7(RDY)-caGs were found within a glomerular structure, and were immunoreactive for synaptotagmin. Gs stimulates adenylyl cyclase to produce cAMP, which in turn activates protein kinase A (PKA). A constitutively-active (ca) PKA rescued the defective phenotype of I7(RDY) in OSN projection and glomerular formation, although a few projection sites were found in the posterior region in the olfactory bulb. When the I7(RDY) was coexpressed with a constitutively-active variant of CREB, a PKA-regulated transcription factor, axon termini were found within glomerular structures although with incomplete convergence. These results confirm the role of G proteins in OSN axon targeting, and suggest involvement of cAMP in transcriptional regulation of axon guidance molecules (Imai, 2006).

To study cAMP signaling in OSN projection, the effect of caGs on OSNs expressing the wild-type OR was examined. Two transgenic constructs, I7(WT)-Cre and I7(WT)-caGs were analyzed. The Cre recombinase gene was assumed not to affect the Gs-mediated signaling. Axons from OSNs expressing I7(WT)-Cre or I7(WT) converged in similar regions, whereas those expressing I7(WT)-caGs projected to more posterior regions. Note that additional cAMP signals are generated by caGs. In OSNs expressing I7(WT)-caGs, cAMP signals are generated by both the transgenic caGs and endogenous Gs, whereas in OSNs expressing I7(RDY)-caGs, generation of cAMP signals by endogenous Gs is blocked. The glomerulus for I7(WT)-caGs showed a smaller posterior shift from that for I7(RDY)-caGs. Thus, the signaling level of the endogenous Gs appears to be relatively low, when coupled with the wild-type OR. Whether decreased levels of cAMP signals would affect the OSN projection was also tested. Axons expressing a dominant-negative (dn) PKA with the wild-type OR converged to the anterior part of the olfactory bulb. Unlike axons carrying I7(RDY), axons expressing the I7(WT)-dnPKA generated glomerular structures. These transgenic experiments indicate that increased or decreased levels of cAMP signals shift the glomerular target of OSNs posteriorly or anteriorly, respectively (Imai, 2006).

To examine the effect of excessive cAMP signals on OSN projection, the transgenic construct, caGshi, where the OR coding sequence has been replaced with the caGs, was generated. More caGs was translated from the cap-dependent caGshi than from the IRES-mediated I7(RDY)-caGs. Althougha posteriorly shifted, but scattered pattern of projection with caGshi was expected, only one or a few glomeruli were detected. Projection sites driven by caGshi were located posterior to the I7(RDY)-caGs glomeruli. In situ hybridization and single-cell RT-PCR indicate that OSNs expressing the caGshi express multiple OR species. In the double transgenic mouse carrying CFP-tagged I7(WT) and YFP-tagged caGshi, a few I7(WT)- expressing axons that probably also expressed caGshi projected to the caGshi glomerulus. Thus the caGshi glomerulus represent a heterogeneous population of axons expressing different ORs. It is possible that caGshi produces saturated levels of cAMP signals and generates a distinct glomerular structure regardless of the OR species (Imai, 2006).

In contrast to Golf, Gs is expressed early in OSN differentiation. These experiments suggest involvement of a PKA-regulated transcription factor, CREB, in OSN projection. Microarray and RT-PCR analyses was used to screen for genes with expression levels correlated with cAMP signals. cDNA libraries were prepared from single OSNs from four different transgenic mice, and gene expression profiles were compared between caGshi and I7(RDY), and between I7(WT) and I7(WT)-dnPKA. Among the genes differentially expressed were some encoding axon guidance molecules, e.g., Neuropilin-1 (Nrp1). Nrp1 was expressed in the caGshi OSNs (where cAMP signals might be high), but not in the I7(RDY)-expressing OSNs (where cAMP signaling is blocked). Immunostaining demonstrated a gradient of Nrp1 expression, with low expression in the anterior and high expression in the posterior of the olfactory bulb. In the I7(WT) / I7(WT)-dnPKA mouse, the I7(WT) glomerulus was Nrp1-positive, and the I7(WT)-dnPKA glomerulus was Nrp1-negative. Nrp1 has been implicated in guidance of OSN axons because disruption of the Sema3A gene, which encodes a repulsive ligand for Nrp1, alters glomerular arrangements along the anterior-posterior axis. It is suggested that Gs-mediated cAMP signals regulate transcription of genes encoding axon guidance molecules, which in turn guide positioning of glomeruli (Imai, 2006).

These results explain some puzzling observations about OSN targeting. The α2-adrenergic receptor (α2-AR) but not a vomeronasal receptor (V1rb2), can substitute for an OR in OR-instructed axonal outgrowth and glomerular formation. The explanation may be that the α2-AR can couple to Gs, but the V1rb2 can not. This is consistent with the idea that the Gs-mediated cAMP levels set by the receptors determine the target sites of OSN axons. Another puzzling observation is that alterations in OR expression levels can affect OSN projection. The level of cAMP signals may be affected by both OR identity and amount of OR protein, which would be a factor of transcription and translation parameters. OR-instructed Gs signals are not dependent on odorants, and disruption of Golf or CNGA2 genes did not affect positioning of glomeruli, which suggests that Gs-mediated cAMP signaling is distinct from that mediated by odor-evoked neuronal activity. It has been thought that ORs at axon termini may recognize guidance cues on the olfactory bulb and mediate the homophilic interactions of like-axons. However, the results favor a model in which cAMP signals posterior axis. These results complement previous studies indicating that the dorsal-ventral arrangement of glomeruli is determined by the locations of OSNs within the olfactory epithelium. It is proposed that a combination of dorsal-ventral patterning based on anatomical locations of OSNs and anterior-posterior patterning based on OR-derived cAMP signals establish olfactory bulb topography. After OSN axons reach their approximate destinations in the olfactory bulb, further refinement of the glomerular map may occur through fasciculation and segregation of axon termini in an activity-dependent manner (Imai, 2006).

Table of contents

CrebB-17A: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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