cAMP response element-binding protein 1 controls porcine ovarian cell proliferation, apoptosis, and FSH and insulin-like growth factor 1 response.

The aim of the present study was to examine the role of cAMP response element-binding protein (CREB) and its phosphorylation in the regulation of ovarian cell proliferation and apoptosis, and of the response of proliferation and apoptosis to the upstream hormonal stimulators FSH and insulin-like growth factor (IGF) 1. In the first series of experiments, porcine ovarian granulosa cells, transfected or not with a gene construct encoding wild-type CREB1 (CREB1WT), were cultured with and without FSH (0, 1, 10 or 100ngmL-1). In the second series of experiments, these cells were transfected or not with CREB1WT or non-phosphorylatable mutant CREB1 (CREB1M1) and cultured with and without FSH (0, 1, 10 or 100ngmL-1) or IGF1 (0, 1, 10 and 100ngmL-1). Levels of total and phosphorylated (p-) CREB1, proliferating cell nuclear antigen (PCNA), a marker of proliferation, and BAX, a marker of apoptosis, were evaluated by western immunoblotting and immunocytochemical analysis. Transfection of cells with CREB1WT promoted accumulation of total CREB1 within cells, but p-CREB1 was not detected in any cell group. Both CREB1WT and CREB1M1 reduced cell proliferation and apoptosis. Addition of 10 and 100ngmL-1 FSH to non-transfected cells promoted CREB1 accumulation and apoptosis, whereas cell proliferation was promoted by all concentrations of FSH tested. FSH activity was not modified in cells transfected with either CREB1WT or CREB1M1. IGF1 at 100ngmL-1 promoted cell proliferation, whereas all concentrations of IGF1 tested reduced apoptosis. Transfection with either CREB1WT or CREB1M1 did not modify the effects of either FSH or IGF1, although CREB1M1 reversed the effect of IGF1 on apoptosis from inhibitory to stimulatory. These observations suggest that CREB1 is involved in the downregulation of porcine ovarian cell proliferation and apoptosis. The absence of visible CREB1 phosphorylation and the similarity between the effects of CREB1WT and CREB1M1 transfection indicate that phosphorylation is not necessary for CREB1 action on these processes. Furthermore, the observations suggest that FSH promotes both ovarian cell proliferation and apoptosis, whereas IGF1 has proliferation-promoting and antiapoptotic properties. The effect of FSH on CREB1 accumulation and the ability of CREB1M1 to reverse the effects of IGF1 on apoptosis indicate that CREB1 is a mediator of hormonal activity, but the inability of either CREB1WT or CREBM1transfection to modify the primary effects of FSH and IGF1 suggest that CREB1 and its phosphorylation do not mediate the action of these hormones on ovarian cell proliferation and apoptosis.

Calcium regulation of neuronal gene expression.

Plasticity is a remarkable feature of the brain, allowing neuronal structure and function to accommodate to patterns of electrical activity. One component of these long-term changes is the activity-driven induction of new gene expression, which is required for both the long-lasting long-term potentiation of synaptic transmission associated with learning and memory, and the activity dependent survival events that help to shape and wire the brain during development. We have characterized molecular mechanisms by which neuronal membrane depolarization and subsequent calcium influx into the cytoplasm lead to the induction of new gene transcription. We have identified three points within this cascade of events where the specificity of genes induced by different types of stimuli can be regulated. By using the induction of the gene that encodes brain-derived neurotrophic factor (BDNF) as a model, we have found that the ability of a calcium influx to induce transcription of this gene is regulated by the route of calcium entry into the cell, by the pattern of phosphorylation induced on the transcription factor cAMP-response element (CRE) binding protein (CREB), and by the complement of active transcription factors recruited to the BDNF promoter. These results refine and expand the working model of activity-induced gene induction in the brain, and help to explain how different types of neuronal stimuli can activate distinct transcriptional responses.

Magnitude of the CREB-dependent transcriptional response is determined by the strength of the interaction between the kinase-inducible domain of CREB and the KIX domain of CREB-binding protein.

The activity of the transcription factor CREB is regulated by extracellular stimuli that result in its phosphorylation at a critical serine residue, Ser133. Phosphorylation of Ser133 is believed to promote CREB-dependent transcription by allowing CREB to interact with the transcriptional coactivator CREB-binding protein (CBP). Previous studies have established that the domain encompassing Ser133 on CREB, known as the kinase-inducible domain (KID), interacts specifically with a short domain in CBP termed the KIX domain and that this interaction depends on the phosphorylation of Ser133. In this study, we adapted a recently described Escherichia coli-based two-hybrid system for the examination of phosphorylation-dependent protein-protein interactions, and we used this system to study the kinase-induced interaction between the KID and the KIX domain. We identified residues of the KID and the KIX domain that are critical for their interaction as well as two pairs of oppositely charged residues that apparently interact at the KID-KIX interface. We then isolated a mutant form of the KIX domain that interacts more tightly with wild-type and mutant forms of the KID than does the wild-type KIX domain. We show that in the context of full-length CBP, the corresponding amino acid substitution resulted in an enhanced ability of CBP to stimulate CREB-dependent transcription in mammalian cells. Conversely, an amino acid substitution in the KIX domain that weakens its interaction with the KID resulted in a decreased ability of full-length CBP to stimulate CREB-dependent transcription. These findings demonstrate that the magnitude of CREB-dependent transcription in mammalian cells depends on the strength of the KID-KIX interaction and suggest that the level of transcription induced by coactivator-dependent transcriptional activators can be specified by the strength of the activator-coactivator interaction.

Circadian behavior and plasticity of light-induced c-fos expression in SCN of tau mutant hamsters.

In hamsters homozygous for the circadian clock mutation tau, the photic history dramatically affects the magnitude of light-induced circadian phase shifts. The maximum amplitude of phase shifts produced by 1-h light pulses presented at CT 14 was less than 2 h in animals that had been in DD for 2 days, whereas animals that had been kept in DD for 49 days could be shifted by more than 8 h. In this study, the authors compared the effect of previous light history on the amplitude of circadian phase shifts and on c-fos expression in the SCN of tau mutant hamsters. Although the maximum amplitude of behavioral phase shifts was drastically different between animals that had been held for either 2 or 49 days in DD, maximal fos induction was not significantly different in these two groups. However, photic thresholds for light-induced behavioral phase shifts, c-fos mRNA, and Fos immunoreactivity were closely correlated within both groups, and these thresholds were lower (more sensitive to light) after 49 than after 2 days in DD. The correlation between phase shifting and Fos induction thresholds, under conditions where both responses are dramatically altered by the previous light history, demonstrates an association between changes in circadian behavioral phase-shifting responses of tau mutant hamsters and plasticity of light-induced c-fos expression in SCN. However, because the maximum amplitudes of Fos induction and phase shifting were not correlated in animals that had been in DD for 2 days, we speculate that the level of c-fos expression does not directly determine phase shift amplitude.

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

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

Visual sensitivities of nur77 (NGFI-B) and zif268 (NGFI-A) induction in the suprachiasmatic nucleus are dissociated from c-fos induction and behavioral phase-shifting responses.

Mammalian circadian rhythms are regulated by a pacemaker in the suprachiasmatic nucleus of the hypothalamus. Recent work from several laboratories has shown that light induces the IEGs, c-fos and jun-B, in the rodent suprachiasmatic nucleus. In hamsters, there is a strong correlation between circadian entrainment and the induction of c-fos and jun-B in the suprachiasmatic nucleus by light. Previous work has shown that the IEGs, nur77 and zif268, both of which encode transcription factors, are also light-inducible in the rat suprachiasmatic nucleus [Rusak, B., McNaughton, L., Robertson, H.A. and Hunt, S.P., Circadian variation in photic regulation of IEG mRNAs in rat suprachiasmatic nucleus cells, Mol. Brain Res., 14 (1992) 124-130.; Sutin, E.L. and Kilduff, T.S., Circadian and light-induced expression of IEG mRNAs in the rat suprachiasmatic nucleus, Mol. Brain Res., 15 (1992) 281-290.]. To characterize the photic-regulation of these genes in the suprachiasmatic nucleus of golden hamsters, we used in situ hybridization to measure nur77 and zif268 mRNA levels with 33P-labeled complementary RNA probes. 5-min monochromatic light pulses at CT19 induced a dramatic increase in both nur77 and zif268 mRNA levels. Peak mRNA levels occurred 45-60 min after light onset for both nur77 and zif268. In addition, the induction of both nur77 and zif268 mRNA levels was gated by the circadian pacemaker. Light pulses during subjective day (CT3 and CT9), which do not cause behavioral phase-shifts, did not significantly alter mRNA levels of either nur77 or zif268; whereas light pulses during the subjective night (CT14 and CT19), which induce phase-shifts, dramatically increased both nur77 and zif268 mRNA levels. In contrast to c-fos induction, which has a photic threshold indistinguishable from that of the behavioral phase-shifting response, nur77 and zif268 mRNA induction were found to have visual sensitivities greater than the phase-shifting response by 1-2 log units (10-100-fold). Although light and circadian phase regulate nur77 and zif268 expression in the SCN, these results demonstrate that their induction is not rate-limiting for photic entrainment of the hamster circadian system.

Positional cloning of the mouse circadian clock gene.

We used positional cloning to identify the circadian Clock gene in mice. Clock is a large transcription unit with 24 exons spanning approximately 100,000 bp of DNA from which transcript classes of 7.5 and approximately 10 kb arise. Clock encodes a novel member of the bHLH-PAS family of transcription factors. In the Clock mutant allele, an A-->T nucleotide transversion in a splice donor site causes exon skipping and deletion of 51 amino acids in the CLOCK protein. Clock is a unique gene with known circadian function and with features predicting DNA binding, protein dimerization, and activation domains. CLOCK represents the second example of a PAS domain-containing clock protein (besides Drosophila PERIOD), which suggests that this motif may define an evolutionarily conserved feature of the circadian clock mechanism.

Light, immediate-early genes, and circadian rhythms.

Many diverse behaviors exhibit clear circadian rhythms in their expression. In mammals, these rhythms originate from a neural circadian clock located in the suprachiasmatic nuclei (SCN). Recently, signaling pathways activated by light in the SCN have begun to be identified. A specific set of immediate-early genes is induced by light in the SCN, and their expression is correlated with the resetting of circadian behavioral rhythms. These light-regulated immediate-early genes offer multiple inroads into the biology of the SCN: first, they are functional markers for the activation of SCN neurons by light; second, they can direct us to the upstream light-activated (and clock-regulated) signal transduction pathways which mediate their induction; and finally, they encode transcription factor proteins which may play a role in the molecular mechanism of resetting the circadian clock.

Effects of aging on light-induced phase-shifting of circadian behavioral rhythms, fos expression and CREB phosphorylation in the hamster suprachiasmatic nucleus.

Aging is associated with a variety of alterations in circadian rhythms, including changes in the response to environmental stimuli. The underlying causes for these age-related changes in the circadian system remain unknown. Recent studies have demonstrated that light induces the expression of Fos and phosphorylation of the cyclic-AMP response element-binding protein in the rodent suprachiasmatic nuclei, the location of a master circadian pacemaker in mammals, suggesting that these transcription factors may mediate the effects of light on the circadian clock. The purpose of this study was to determine the effects of aging upon light-induced phase-shifting of circadian locomotor activity rhythms, Fos protein expression and cyclic-AMP response element-binding protein phosphorylation in the suprachiasmatic nuclei. Young (three to four months) and old (18-22 months) male golden hamsters free-running in constant darkness were exposed to 5-min monochromatic light pulses of different irradiance levels, at circadian time 19, after which either steady-state phase shifts of locomotor activity rhythms were measured, or else immunocytochemistry for Fos or for phospho-cyclic-AMP response element-binding protein was performed. Old hamsters were approximately 20 times less sensitive to the phase-shifting effects of light on the activity rhythm, and the photic irradiance threshold for Fos-like immunoreactivity induction in the suprachiasmatic nuclei was elevated when compared to young animals. Aging was also associated with a deficit in cyclic-AMP response element-binding protein phosphorylation by light. These data indicate that there are dramatic changes in light-activated molecular responses in the suprachiasmatic nuclei of old hamsters, and suggest that these molecular changes may underlie age-related changes in the effects of light on the circadian clock system.

Temporal and spatial changes in GATA transcription factor expression are coincident with development of the chicken optic tectum.

The molecular mechanisms specifying patterns of gene expression in the vertebrate brain, which in turn determine the developmental fates of specific neurons, are yet to be clearly defined. Individual members of a recently identified family of transcriptional regulatory proteins, the GATA factors, are required for the differentiation of certain hematopoietic cell lineages. We show here that two of the members of this gene family, GATA-2 and GATA-3, are expressed within discrete cell populations of the chicken optic tectum during embryogenesis, and that they have highly restricted patterns of expression in the developing chicken brain. Furthermore, the induction of GATA factor expression within specific cell layers parallels the well established spatial (rostral to caudal) and temporal pattern of optic tectum development. The observation that both the timing of appearance and the localization of expression of GATA-2 and GATA-3 are correlated with optic tectum development suggest that these transcription factors may be associated with the initiation of gene transcription required for the determination of specific neuronal fates within visual areas of the vertebrate brain.

Mutagenesis and mapping of a mouse gene, Clock, essential for circadian behavior.

In a search for genes that regulate circadian rhythms in mammals, the progeny of mice treated with N-ethyl-N-nitrosourea (ENU) were screened for circadian clock mutations. A semidominant mutation, Clock, that lengthens circadian period and abolishes persistence of rhythmicity was identified. Clock segregated as a single gene that mapped to the midportion of mouse chromosome 5, a region syntenic to human chromosome 4. The power of ENU mutagenesis combined with the ability to clone murine genes by map position provides a generally applicable approach to study complex behavior in mammals.

Regulation of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock.

Mammalian circadian rhythms are regulated by a pacemaker within the suprachiasmatic nuclei (SCN) of the hypothalamus. The molecular mechanisms controlling the synchronization of the circadian pacemaker are unknown; however, immediate early gene (IEG) expression in the SCN is tightly correlated with entrainment of SCN-regulated rhythms. Antibodies were isolated that recognize the activated, phosphorylated form of the transcription factor cyclic adenosine monophosphate response element binding protein (CREB). Within minutes after exposure of hamsters to light, CREB in the SCN became phosphorylated on the transcriptional regulatory site, Ser133. CREB phosphorylation was dependent on circadian time: CREB became phosphorylated only at times during the circadian cycle when light induced IEG expression and caused phase shifts of circadian rhythms. These results implicate CREB in neuronal signaling in the hypothalamus and suggest that circadian clock gating of light-regulated molecular responses in the SCN occurs upstream of phosphorylation of CREB.

Regulation of jun-B messenger RNA and AP-1 activity by light and a circadian clock.

The suprachiasmatic nuclei (SCN) of the hypothalamus comprise the primary pacemaker responsible for generation of circadian rhythms in mammals. Light stimuli that synchronize this circadian clock induce expression of the c-fos gene in rodent SCN, which suggests a possible role for Fos in circadian entrainment. Appropriate light stimuli also induce the expression of jun-B messenger RNA in the SCN of golden hamsters but only slightly elevate c-jun messenger RNA levels. In addition, light increases the amount of a protein complex in the SCN that binds specifically to sites on DNA known to mediate regulation by the AP-1 transcription factor. The photic regulation of both jun-B messenger RNA expression and AP-1 binding activity is dependent on circadian phase: only light stimuli that shift behavioral rhythms induce jun-B and AP-1 expression. Thus, light and the circadian pacemaker interact to regulate a specific set of immediate-early genes in the SCN that may participate in entrainment of the circadian clock.

cis and trans regulation of tissue-specific transcription.

Analysis of both the cis-regulatory sequences which control globin gene switching as well as the trans-acting factors which bind to these sequences to elicit a differential, developmentally regulated response has lent insight into the general mechanisms responsible for tissue-specific gene regulation. We show here that the chicken adult beta-globin gene promoter sequences are intimately involved in competitive interaction with the beta/epsilon-globin enhancer to regulate differentially epsilon- versus beta-globin gene transcription. Secondly, we show that the family of GATA transcription factors directs gene regulation in a variety of discrete cell types, and describe potential cellular target genes for each member of the GATA factor family, as well as potential mechanisms whereby multiple GATA factors expressed in a single cell might be used to elicit differential transcriptional activities.

Glucose transporter expression in brain. cDNA sequence of mouse GLUT3, the brain facilitative glucose transporter isoform, and identification of sites of expression by in situ hybridization.

Complementary DNAs encoding the mouse GLUT3/brain facilitative glucose transporter have been isolated and sequenced. The predicted amino acid sequence indicates that mouse GLUT3 is composed of 493 amino acids and has 83 and 89% identity and similarity, respectively, to the sequence of human GLUT3. In contrast to human GLUT3 mRNA, which can be readily detected by RNA blotting in all human tissues that have been examined, mouse GLUT3 mRNA was only present at significant levels in brain. In situ hybridization showed differential expression of GLUT3 mRNA in several regions of adult mouse brain. Specific expression was observed in the hippocampus, with GLUT3 mRNA levels being higher in areas CA1 to CA3 than in the dentate gyrus. It was also detected in the Purkinje cell layer of the cerebellum and in the cerebral cortex, with higher expression in the piriform cortex than in other regions of the cortex. Antisera to mouse GLUT3 immunoblotted a series of proteins of 45-50 kDa in mouse brain plasma membranes. These results are consistent with GLUT3 being a neuronal glucose transporter.

Photic and circadian regulation of c-fos gene expression in the hamster suprachiasmatic nucleus.

Photic information entrains a circadian pacemaker located in the suprachiasmatic nucleus (SCN) of the mammalian hypothalamus to environmental light/dark cycles. To determine whether light regulates c-fos gene expression in the SCN, we have measured c-fos mRNA levels in the SCN of the golden hamster. We report that, during the subjective night, light causes a rapid increase in levels of c-fos mRNA in the SCN. Light pulses of 5 min duration are sufficient to induce c-fos mRNA, and the highest mRNA levels occur 30 min following the onset of light. The minimum level of illumination required to induce an increase in c-fos mRNA is indistinguishable from the minimum irradiance that produces a phase shift in the hamster's circadian rhythm of activity. In addition, the induction of c-fos mRNA in the SCN by light is itself under circadian regulation. Light induction of c-fos mRNA occurs only during the subjective night, at circadian times when photic phase shifting of activity occurs. Taken together, these data suggest that c-fos may be a molecular component of the photic pathway for entrainment of mammalian circadian rhythms.