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PubMed2003

Bile acid regulation of C/EBPbeta, CREB, and c-Jun function, via the extracellular signal-regulated kinase and c-Jun NH2-terminal kinase pathways, modulates the apoptotic response of hepatocytes.

Previously, we have demonstrated that deoxycholic acid (DCA)-induced signaling of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in primary hepatocytes is a protective response. In the present study, we examined the roles of the ERK and c-Jun NH(2)-terminal kinase (JNK) pathways, and downstream transcription factors, in the survival response of hepatocytes. DCA caused activation of the ERK1/2 and JNK1/2 pathways. Inhibition of either DCA-induced ERK1/2 or DCA-induced JNK1/2 signaling enhanced the apoptotic response of hepatocytes. Further analyses demonstrated that DCA-induced JNK2 signaling was cytoprotective whereas DCA-induced JNK1 signaling was cytotoxic. DCA-induced ERK1/2 activation was responsible for increased DNA binding of C/EBPbeta, CREB, and c-Jun/AP-1. Inhibition of C/EBPbeta, CREB, and c-Jun function promoted apoptosis following DCA treatment, and the level of apoptosis was further increased in the case of CREB and c-Jun, but not C/EBPbeta, by inhibition of MEK1/2. The combined loss of CREB and c-Jun function or of C/EBPbeta and c-Jun function enhanced DCA-induced apoptosis above the levels resulting from the loss of either factor individually; however, these effects were less than additive. Loss of c-Jun or CREB function correlated with increased expression of FAS death receptor and PUMA and decreased expression of c-FLIP-(L) and c-FLIP-(S), proteins previously implicated in the modulation of the cellular apoptotic response. Collectively, these data demonstrate that multiple DCA-induced signaling pathways and transcription factors control hepatocyte survival.

Amino Acid Sequence↗
PubMed2006

Transcriptional regulation of PEN-2, a key component of the gamma-secretase complex, by CREB.

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. Here, we characterized the upstream regions of the human PEN-2 gene and 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 showed 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 promoted the expression of PEN-2 mRNA and protein, whereas the other components of the gamma-secretase complex remained 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.

Amyloid Precursor Protein Secretases↗
PubMed2003

Activation of MAPK and CREB by GM1 induces survival of RGCs in the retina with axotomized nerve.

PURPOSE: Neuronal cells undergo apoptosis when the supply of neurotrophic factor is limited by injury, trauma, or neurodegenerative disease. Ganglioside has both neuritogenic and neurotropic functions. Exogenously administered monosialoganglioside (GM1) has been shown to have a stimulatory effect on neurite outgrowth and to prevent degeneration of neuronal cells in the central nervous system. Even though GM1 has been shown to mimic, or have synergy with, neurotrophic factors, the neuroprotective mechanism of GM1 has not been well understood. In this study, optic nerve transection, or axotomy, was used as an in vivo model system for injury, to examine the protective mechanism of GM1 in injured retinal ganglion cells. METHODS: GM1 was injected into the vitreous body before axotomy, and the protective effect of GM1 observed with regard to activation of mitogen-activated protein kinase (MAPK) and phosphorylation of cAMP-responsive element-binding (CREB) protein. Activation of MAPK and CREB were examined by Western blot analysis and immunohistochemistry, and the surviving retinal ganglion cells were counted after retrograde fluorescence labeling. RESULTS: GM1 inhibited the degeneration of axotomized retinal ganglion cells. In addition, GM1 enhanced the activation of MAPK and CREB with the treatment of GM1 in the retina with axotomized nerve. Treatment of MAPK inhibitor PD98059 with GM1 reduced the protective action of GM1 and prevented GM1-induced phosphorylation of CREB. CONCLUSIONS: GM1 protected the axotomized retinal ganglion cells (RGCs) from cell death after axotomy through the activation of MAPK and CREB.

Animals↗
PubMed2006

Induction of Fc gammaRIIA expression in myeloid PLB cells during differentiation depends on cytosolic phospholipase A2 activity and is regulated via activation of CREB by PGE2.

Fc gammaRIIA expressed on neutrophils and monocytes has a fundamental role in combating bacterial infections. In the present study, the requirement of cytosolic phospholipase A2 (cPLA2) for induction of Fc gammaRIIA expression was studied in a model of cPLA2-deficient PLB-985 cells (PLB-D cells). Fc gammaRIIA was acquired only during differentiation of PLB but not of PLB-D cells induced by either 1,25-dihydroxyvitamin D3, retinoic acid, or interferon gamma. Addition of prostaglandin E2 (PGE2) to PLB-D cells undergoing differentiation restored the expression of Fc gammaRIIA protein, whereas addition of indomethacin to PLB cells during differentiation inhibited both the production of PGE2 and the expression of Fc gammaRIIA. Inhibition of PKA during PLB differentiation prevented Fc gammaRIIA expression, whereas dibutyryl cAMP (dbcAMP) induced its expression in both PLB and PLB-D cells. CREB phosphorylation and CREB-CRE interaction were detected only in differentiated PLB cells and not PLB-D cells and were inhibited by indomethacin. A reporter gene containing a Fc gammaRIIA gene promoter fragment with the CRE element was sufficient for CREB activation. Our results are the first to show that CREB activation is involved in up-regulation of Fc gammaRIIA expression in myeloid lineages. PGE2 formed via cPLA2 activates CREB through PKA and this process is dependent on development of PGE2 receptor 4.

Antigens, CD↗
PubMed2002

Interaction of early growth response protein 1 (Egr-1), specificity protein 1 (Sp1), and cyclic adenosine 3'5'-monophosphate response element binding protein (CREB) at a proximal response element is critical for gastrin-dependent activation of the chromogranin A promoter.

Recently, binding of specific protein 1 (Sp1) and cAMP response element binding protein (CREB) to a GC-rich element at -92/-62 has been identified as a critical step in gastrin-dependent regulation of the chromogranin A (CgA) gene in gastric epithelial cells. Here we demonstrate that binding of early growth response protein 1 (Egr-1) to the distal part of the -92/-62 site is also required for gastrin-dependent CgA transactivation. Gastrin elevated cellular and nuclear Egr-1 levels in a time-dependent manner and also increased Egr-1 binding to the CgA -92/-73 region. Disruption of this site reduced gastrin responsiveness without influencing basal promoter activity, while loss of Sp1 and/or CREB binding sites diminished basal and gastrin-stimulated CgA promoter activity. Ectopic Egr-1 overexpression potently stimulated the CgA promoter, whereas coexpression of Egr-1 with Sp1 and/or CREB resulted in additive effects. Functional analysis of Sp1-, Egr-1-, or CREB-specific promoter mutations in transfection studies confirmed the tripartite organization of the CgA -92/-62 element. Signaling studies revealed that MAPK kinase 1 (MEK1)/ERK1/2 cascades are critical for gastrin-dependent Egr-1 protein accumulation as well as Egr-1 binding to the CgA promoter. Our studies for the first time identify Egr-1 as a nuclear target of gastrin and show that functional interplay of Egr-1, Sp1, and CREB is indispensable for gastrin-dependent CgA transactivation in gastric epithelial cells.

Binding Sites↗
PubMed2005

LH/hCG-stimulated androgen production and selective HDL-cholesterol transport are inhibited by a dominant-negative CREB construct in primary cultures of rat theca-interstitial cells.

Theca-interstitial (T-I) cells synthesize androgens that are converted to estrogen by the granulosa cells. In rat ovary, T-I cells primarily utilize HDL-derived cholesteryl esters (CE) as a precursor for androgen synthesis. The HDL-CE is delivered to steroidogenic cells by a process termed "selective" uptake in which CE is internalized without the simultaneous uptake of apolipoprotein(s). This process is mediated by an HDL receptor, scavenger receptor class B, type I (SR-BI) and is stimulated by trophic hormone (LH/hCG), which also activates the cAMP cascade. In this study, we tested whether the adenoviral (Ad)-mediated introduction of a dominant-negative analog of cyclic AMP response element binding protein (A-CREB) inhibits the stimulatory effect of LH/hCG on the selective uptake of high-density lipoprotein (HDL)-cholesterol and androgen production in primary cultures of rat T-I cells. Androstenedione production by cultured T-I cells was stimulated by hCG and by the adenoviral overexpression of wtCREB. Additionally, the stimulatory effect observed with hCG was amplified in the presence of HDL. Androgen synthesis was increased 17-fold in the presence of HDL and hCG but the stimulatory effect of hCG was inhibited by Ad A-CREB by approx 70%. In the selective up-take studies, cell-surface association of the labeled HDL was significantly enhanced by hCG treatment, and this effect was inhibited by Ad A-CREB. The selective uptake of HDL-cholesterol was also enhanced by hCG but exposure to Ad A-CREB also abrogated this effect. It is concluded that CREB plays an intermediary role in the stimulatory action of LH/hCG on androgen synthesis and the selective uptake of HDL-cholesterol in T-I cells.

Adenoviridae↗
PubMed2005

Identification of human myometrial target genes of the cAMP pathway: the role of cAMP-response element binding (CREB) and modulator (CREMalpha and CREMtau2alpha) proteins.

cAMP-response element (CRE) binding (CREB) and modulator (CREM) proteins, activated by protein kinase A-mediated phosphorylation, bind as homo- and heterodimers to promoters containing CRE and activator protein 1 (AP-1) sites to alter target-gene expression. We have previously reported differential expression of CREB and CREM splice variants CREMalpha and CREMtau2alpha in human myometrium during pregnancy and labour. Via microarray studies with cultured myometrial cells stably transfected with CREB, CREMalpha and CREMtau2alpha cDNAs, CREB affected the expression of 958 genes; 522 being up-regulated and 436 down-regulated. CREMalpha altered the expression of 118 genes; 71 were increased and 47 decreased. CREMtau2alpha affected 220 genes; 148 were activated and 72 repressed. Notably, genes affected by CREB, CREMalpha and CREMtau2alpha belong to largely discrete groups: less than 9% were affected by more than one factor. Genes involved in regulation of cell death and apoptosis, growth and maintenance, signal transduction, physiological and developmental processes, protein kinase cascades, extracellular matrix, cytoskeleton, cell-cycle regulation, transport, and a variety of enzymes, intracellular components and nucleic acid-binding proteins have been described, many of which are involved in the modulation of myometrial activity during pregnancy and parturition.

Cyclic AMP↗
PubMed2005

IGF-I and vasoactive intestinal peptide (VIP) regulate cAMP-response element-binding protein (CREB)-dependent transcription via the mitogen-activated protein kinase (MAPK) pathway in pituitary cells: requirement of Rap1.

In previous studies we demonstrated that vasoactive intestinal peptide (VIP) mediation, and interactions between mitogen-activated protein kinase (MAPK) and cAMP/protein kinase A (PKA) signaling pathways are implicated in insulin-like growth factor I (IGF-I)- and VIP-induced lactotroph proliferation. These facts led us to investigate the intracellular mechanisms involved in IGF-I- and VIP-induced lactotroph proliferation. Exposure of cultured male rat pituitary cells to IGF-I (10(-7) M) or VIP (10(-7) M) stimulated the MAPK cascade. Studies in GH4C1 cells, with an expression vector for Rap1 GTPase-activating protein (Rap1 GAP1), demonstrated reduced VIP-induced MAPK activation, indicating that VIP-dependent activation of the extracellular signal-regulated kinase (ERK) pathway requires PKA-Rap1 signaling. IGF-I induced cAMP-response element (CRE)-binding protein (CREB) phosphorylation through the Ras-MAPK pathway, whereas VIP phosphorylated CREB directly via PKA. The mechanisms that regulate IGF-I-and VIP-CREB-dependent gene transcription were examined using GH4C1 cells transiently transfected with a CRE reporter gene. IGF-I and VIP stimulation of CRE-mediated transcription required activation of both Ras-MAPK and cAMP/PKA signaling. This activation was blocked in the presence of Rap1 GAP1. In summary, we showed that IGF-I and VIP stimulated MAPK activity and the phosphorylation of CREB in pituitary cells. Furthermore, VIP-dependent activation of PKA-Rap1-ERK pathways mediated VIP and IGF-I effects on CREB-dependent transcription in GH4C1 cells. Thus, it is possible that VIP- and IGF-I-induced lactotroph proliferation may involve Rap1.

Animals↗
PubMed1999

L-Type Ca(2+) channels are essential for glutamate-mediated CREB phosphorylation and c-fos gene expression in striatal neurons.

The second messenger pathways linking receptor activation at the membrane to changes in the nucleus are just beginning to be unraveled in neurons. The work presented here attempts to identify in striatal neurons the pathways that mediate cAMP response element-binding protein (CREB) phosphorylation and gene expression in response to NMDA receptor activation. We investigated the phosphorylation of the transcription factor CREB, the expression of the immediate early gene c-fos, and the induction of a transfected reporter gene under the transcriptional control of CREB after stimulation of ionotropic glutamate receptors. We found that neither AMPA/kainate receptors nor NMDA receptors were able to stimulate independently a second messenger pathway that led to CREB phosphorylation or c-fos gene expression. Instead, we saw a consecutive pathway from AMPA/kainate receptors to NMDA receptors and from NMDA receptors to L-type Ca(2+) channels. AMPA/kainate receptors were involved in relieving the Mg(2+) block of NMDA receptors, and NMDA receptors triggered the opening of L-type Ca(2+) channels. The second messenger pathway that activates CREB phosphorylation and c-fos gene expression is likely activated by Ca(2+) entry through L-type Ca(2+) channels. We conclude that in primary striatal neurons glutamate-mediated signal transduction is dependent on functional L-type Ca(2+) channels.

Animals↗
PubMed1999

CREB and COUP-TF mediate transcriptional activation of the human immunodeficiency virus type 1 genome in Jurkat T cells in response to cyclic AMP and dopamine.

Infection of lymphocytes by the human immunodeficiency virus type 1 (HIV-1) is associated with an increase in intracellular cAMP levels. Recent studies have shown that lymphocytes are able to synthesize and bind the dopamine, known to affect multiple cellular pathways, such as the cAMP pathway. Here we have investigated the molecular mechanisms by which cAMP and dopamine regulate HIV-1 gene transcription in Jurkat T cells. Transient expression experiments revealed that dopamine and forskolin lead to a synergistic stimulation of long terminal repeat (LTR)-driven transcription. This action is mediated through the cAMP response element binding (CREB) protein and chicken ovalbumin upstream promoter transcription factor (COUP-TF). CREB and COUP-TF act indirectly through the minimal -40/+80 and -68/+80 LTR region, respectively. We have previously demonstrated that COUP-TF stimulates HIV-1 transcription via the -68/+29 LTR region without direct DNA binding. Here, gel supershift experiments show that CREB does not directly bind to the -45/+85 proximal LTR sequences. Moreover, our data reveal novel functional interactions between COUP-TF and CREB, which lead to synergistic cAMP- and dopamine-induced transactivation of the HIV-1 LTR. These findings reveal that dopamine-induced signals and the cAMP pathway stimulate HIV-1 gene transcription in lymphocytes by converging to the minimal -68/+80 LTR region, through the transcription factors CREB and COUP-TF.

Binding Sites↗
PubMed2000

CREB (cAMP response element binding protein) and C/EBPalpha (CCAAT/enhancer binding protein) are required for the superstimulation of phosphoenolpyruvate carboxykinase gene transcription by adenoviral E1a and cAMP.

In the present study, we observed superstimulated levels of cAMP-stimulated transcription from the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter in cells infected with wild-type adenovirus expressing 12 S and 13 S E1a proteins, or in cells expressing 13 S E1a alone. cAMP-stimulated transcription was inhibited in cells expressing only 12 S E1a, but slightly elevated in cells expressing E1a proteins with mutations in conserved regions 1 or 2, leading us to conclude that the superstimulation was mediated by conserved region 3 of 13 S E1a. E1a failed to enhance cAMP-stimulated transcription from promoters containing mutations that abolish binding by cAMP response element binding protein (CREB) or CCAAT/enhancer binding proteins (C/EBPs). This result was supported by experiments in which expression of dominant-negative CREB and/or C/EBP proteins repressed E1a- and cAMP-stimulated transcription from the PEPCK gene promoter. In reconstitution experiments using a Gal4-responsive promoter, E1a enhanced cAMP-stimulated transcription when chimaeric Gal4-CREB and Gal4-C/EBPalpha were co-expressed. Phosphorylation of CREB on serine-133 was stimulated in cells treated with dibutyryl cAMP, whereas phosphorylation of C/EBPalpha was increased by E1a expression. Our data support a model in which cAMP agonists increase CREB activity and stimulate PEPCK gene transcription, a process that is enhanced by E1a through the phosphorylation of C/EBPalpha.

Adenovirus E1A Proteins↗
PubMed1990

Heterodimer formation between CREB and JUN proteins.

DNA binding protein families have been identified that contain a leucine zipper dimerization motif preceded by a conserved, highly basic domain involved in direct specific interaction with DNA. Members of two of these families, the Jun and Fos related proteins, have been shown to directly interact and form heterodimeric complexes. A third such family known as the CREB or ATF proteins, bind to a sequence element present in promoters from a number of viral and cellular genes; this element can confer cAMP-inducibility and E1A-inducibility of transcription. In this report we show that one member of the CREB family can efficiently form a heterodimeric complex with the cJun protein. The DNA binding specificity of the heterodimer was indistinguishable from CREB alone. Transfection studies in undifferentiated F9 cells suggest that the CREB/cJun heterodimer can form in vivo, but that the complex does not activate transcription. The heterodimer formation between CREB and Jun proteins is highly specific; only one of the two CREB proteins would heterodimerize with cJun and it would not form dimers with JunB or cFos. The interaction of members of these two families of proteins increases the repertoire of possible regulatory complexes that could play an important role in the regulation of transcription of specific cellular genes.

Base Sequence↗
PubMed1994

Three distinct regions within the constitutive activation domain of cAMP regulatory element-binding protein (CREB) are required for transcription activation.

The transcription factor cAMP regulatory element-binding protein (CREB) mediates both constitutive and cAMP-induced gene expression through distinct, independently acting domains. The constitutive activation domain (CAD) (amino acids (aa) 165-252) encompasses and overlaps exon 9 of the CREB gene (E9, aa 180-243). In the present study, deletion of either the CAD or exon 9 from CREB-GAL4 (CRG) reduced constitutive activity to less than 2-fold, without affecting kinase inducible activity. However, fusion of the CAD to the GAL4 DNA binding domain (CAD-G4) stimulated transcription, whereas fusion of exon 9 sequences did not. Deletion of the amino-terminal flanking region of exon 9 (aa 165-180), but not COOH-terminal flanking sequences (aa 243-252), decreased constitutive activation in either the CAD-G4 or CRG background. Deletion of the previously characterized glutamine-rich region (Q3, aa 218-252) or of a region containing a hydrophobic cluster of amino acids (HC, aa 180-218) also reduced constitutive activation by either CAD-G4 or CRG. No single mutation of hydrophobic residues within HC impaired activity of the CAD, but double and triple mutations did, suggesting that multiple weak interactions are involved in function of the HC region. Thus, exon 9 of the CREB gene is necessary but not sufficient for constitutive activation. The CAD requires three distinct regions for function, suggesting that CREB may interact with multiple targets in the RNA polymerase II complex.

Amino Acid Sequence↗
PubMed1994

Amphetamine regulates gene expression in rat striatum via transcription factor CREB.

Amphetamine is a psychostimulant drug of abuse that can produce long-lived changes in behavior including sensitization and dependence. The neural substrates of these drug effects remain unknown, but based on their prolonged time course, we hypothesize that they involve drug-induced alterations in gene expression. It has recently been demonstrated that amphetamine regulates the expression of several genes, including c-fos, via dopamine D1 receptors in rat striatum. Here we report that amphetamine induces phosphorylation of transcription factor cAMP response element binding protein (CREB) in rat striatum in vivo and that dopamine D1 receptor stimulation induces phosphorylation of CREB within specific complexes bound to cAMP regulatory elements. In addition, we show by antisense injection that CREB is necessary for c-fos induction by amphetamine in vivo. Since CREB has been implicated in the activation of a number of immediate-early genes as well as several neuropeptide genes, CREB phosphorylation may be an important early nuclear event mediating long-term consequences of amphetamine administration.

Activating Transcription Factor 2↗
PubMed1996

Hormonal regulation of CREB phosphorylation in the anteroventral periventricular nucleus.

The anteroventral periventricular nucleus (AVPV) is a nodal point in neural circuits regulating secretion of gonadotropin and contains sexually dimorphic populations of hormonally regulated dopamine-, dynorphin-, and enkephalin-containing neurons. Because the tyrosine hydroxylase (TH), prodynorphin (PDYN), and proenkephalin (PENK) genes contain cAMP response elements that control their expression in their promoters, we used histochemical methods to determine whether ovarian steroids alter expression of the cAMP response element-binding protein (CREB) in the AVPV. Because the ability of CREB to activate transcription depends on phosphorylation at Ser133, we also evaluated the effects of acute steroid treatment on levels of phosphorylated CREB (pCREB) in AVPV neurons by using an antibody that differentiates between CREB and pCREB. Treatment of ovariectomized rats with estradiol treatments caused a significant induction in the number of pCREB-immunoreactive nuclei within 30 min that was maintained for at least 4 hr, but did not alter CREB immunostaining in the AVPV. Pretreatment with the estrogen antagonist Nafoxidine blocked this induction. In contrast, acute administration of progesterone to estrogen-primed animals suppressed and then increased pCREB staining in the ASVPV at 30 and 60 min, respectively; no significant differences between experimental and control animals were apparent by 2 hr after progesterone treatment. Double-labeling experiments showed that pCREB was colocalized with PDYN, PENK, or TH mRNA in the AVPV, suggesting that pCREB may mediate the effect of steroid hormones on gene expression in these neurons.

Animals↗
PubMed1996

Phosphorylation of CREB in ovine pars tuberalis is regulated both by cyclic AMP-dependent and cyclic AMP-independent mechanisms.

This study used a combination of Western blotting and immunocytochemistry to test whether signalling pathways independent of cyclic AMP have the potential to induce phospho-CREB (pCREB)-like immunoreactivity (-ir) in the oPT. Western blot analysis of extracts of primary cultures of oPT using an antiserum against CREB, revealed a major band of CREB-ir at 44 KDa. The intensity of this band did not vary systematically with treatment. In extracts from untreated cells, Western blot analysis revealed a major band of pCREB-ir at 42 KDa which was not sensitive to agonist treatment. Treatment of cells with forskolin (10(-6) M) increased the intensity of a number of other pCREB-ir bands at between ca. 38 and 44 KDa. The band at 44 KDa probably represented native pCREB whilst the other bands induced by forskolin probably represented pCREB-like proteins. Melatonin (10(-6) M) alone had no effect on pCREB-ir, but it did inhibit the effect of forskolin on the ca. 38 and 44 KDa pCREB-ir bands. Treatment with lamb serum (1%) consistently increased the intensity of the ca. 38 and 44 KDa pCREB-ir bands relative to control cells, as assessed by Western blot. However, Western blot analysis did not reveal a consistent effect of melatonin on the pCREB-ir response to serum. The effect of serum on pCREB-ir in oPT cells was characterized further by immunocytochemical analysis. In contrast to experiments utilizing Western blotting, untreated cells did not possess detectable pCREB-ir. In serum-starved oPT and oPD cultures, treatment with serum induced exclusively nuclear pCREB-ir. A large majority of oPT cells (> or = 90%) were sensitive to serum (1%), and serum caused a time- and dose-dependent increase of nuclear pCREB-ir. Melatonin attenuated the response to serum in the oPT. This inhibition of the response to serum was not apparent in the oPD, demonstrating that the effect of melatonin was specific for a tissue known to express melatonin receptors. In oPT cultures, physiological concentrations of melatonin (10(-9) M) partially reversed (ca. 70%) the inductive effect of 0.1% serum on nuclear pCREB-ir. However, in contrast to studies applying forskolin, the induction of pCREB-ir by serum occurred in the absence of measurable changes in the concentration of cyclic AMP, indicating that components of serum are able to stimulate the phosphorylation of CREB in the oPT through mechanisms independent of cyclic AMP. Both adenosine and prostaglandin E2 (PGE2) also induced nuclear pCREB-ir in the absence of increased levels of cyclic AMP. These results demonstrate that transcriptional activities in the oPT which are under the control of CREB may be modulated by convergent cyclic AMP-dependent and cyclic AMP-independent pathways. Regulation of these pathways by melatonin and other factors present in serum may be an important control-point in the generation of seasonal neuroendocrine cycles.

Adenosine↗
PubMed1998

CREB in the mouse SCN: a molecular interface coding the phase-adjusting stimuli light, glutamate, PACAP, and melatonin for clockwork access.

The suprachiasmatic nucleus (SCN) is a central pacemaker in mammals, driving many endogenous circadian rhythms. An important pacemaker target is the regulation of a hormonal message for darkness, the circadian rhythm in melatonin synthesis. The endogenous clock within the SCN is synchronized to environmental light/dark cycles by photic information conveyed via the retinohypothalamic tract (RHT) and by the nocturnal melatonin signal that acts within a feedback loop. We investigated how melatonin intersects with the temporally gated resetting actions of two RHT transmitters, pituitary adenylate cyclase-activating polypeptide (PACAP) and glutamate. We analyzed immunocytochemically the inducible phosphorylation of the transcription factor Ca2+/cAMP response element-binding protein (CREB) in the SCN of a melatonin-proficient (C3H) and a melatonin-deficient (C57BL) mouse strain. In vivo, light-induced phase shifts in locomotor activity were consistently accompanied by CREB phosphorylation in the SCN of both strains. However, in the middle of subjective nighttime, light induced larger phase delays in C57BL than in C3H mice. In vitro, PACAP and glutamate induced CREB phosphorylation in the SCN of both mouse strains, with PACAP being more effective during late subjective daytime and glutamate being more effective during subjective nighttime. Melatonin suppressed PACAP- but not glutamate-induced phosphorylation of CREB. The distinct temporal domains during which glutamate and PACAP induce CREB phosphorylation imply that during the light/dark transition the SCN switches sensitivity between these two RHT transmitters. Because these temporal domains are not different between C3H and C57BL mice, the sensitivity windows are set independently of the rhythmic melatonin signal.

Animals↗
PubMed2004

Role of basic helix-loop-helix (bHLH) and CREB transcription factors in the regulation of Sertoli cell androgen-binding protein expression.

Differentiation of Sertoli cells is marked by the presence of novel gene products such as transferrin and androgen-binding protein (ABP). Transcriptional regulation of Sertoli cell differentiation is, in part, controlled through the binding of specific transcription factors to response elements within these genes promoters. Transferrin gene expression has been shown to be regulated by the binding and interactions of basic helix-loop-helix (bHLH) and cAMP response element binding protein (CREB) to an E-box and cyclic AMP response element (CRE), respectively. Interaction between the bHLH and CREB is facilitated through subsequent binding of CREB-binding protein (CBP)/p300. The hypothesis tested in the current study is that ABP expression is regulated by a similar mechanism. The ABP promoter activation was analyzed through the use of transfection assays, site-directed mutagenesis, and electromobility shift assays (EMSA). Transient transfections of rat Sertoli cells used a reporter construct containing the proximal 619 bp of the ABP promoter. Observations suggest that cAMP and follicle stimulating hormone (FSH) upregulate the expression of ABP. Mutational studies of the three E-boxes and the CRE of the 619-bp ABP promoter indicate that all of these elements are critical for stimulation of promoter activity. EMSA revealed a weak interaction between an E-box-2 and the CRE that are overlapping in the promoter. An artificial promoter that contains only an E-box and CRE was created to further test this hypothesis. The artificial promoter was stimulated by both FSH and cAMP. Experiments with mutants of the artificial promoter demonstrate that both response elements contribute to the optimal activation of the promoter construct. The overexpression of the bHLH inhibitor Id (i.e., inhibitor of differentiation) that binds bHLH proteins and eliminates DNA binding was found to suppress hormone activation of the ABP promoter. Combined observations of the ABP promoter and artificial promoter provide insight into a common mechanism for gene regulation in differentiated Sertoli cells involving a role for both the bHLH and CREB family of transcription factors.

Androgen-Binding Protein↗