Esther López-Bayghen

Zona Occludens-2 Inhibits Cyclin D1 Expression and Cell Proliferation and Exhibits Changes in Localization along the Cell Cycle

Here, we have studied the effect of the tight junction protein zona occludens (ZO)-2 on cyclin D1 (CD1) protein expression. CD1 is essential for cell progression through the G1 phase of the cell cycle. We have found that in cultures of synchronized Madin-Darby canine kidney cells, ZO-2 inhibits cell proliferation at G0/G1 and decreases CD1 protein level. These effects occur in response to a diminished CD1 translation and an augmented CD1 degradation at the proteosome triggered by ZO-2. ZO-2 overexpression decreases the amount of Glycogen synthase kinase-3beta phosphorylated at Ser9 and represses beta-catenin target gene expression. We have also explored the expression of ZO-2 through the cell cycle and demonstrate that ZO-2 enters the nucleus at the late G1 phase and leaves the nucleus when the cell is in mitosis. These results thus explain why in confluent quiescent epithelia ZO-2 is absent from the nucleus and localizes at the cellular borders, whereas in sparse proliferating cultures ZO-2 is conspicuously present at the nucleus.

Glutamate-dependent transcriptional regulation of GLAST/EAAT1 : a role for YY1

Glutamate is the major excitatory transmitter in the vertebrate brain and its extracellular levels are tightly regulated to prevent excitotoxic effects. The Na+‐dependent glutamate/aspartate transporter GLAST/EAAT1 is regulated in the short and in the long term by glutamate. A receptors‐independent change in its membrane translocation rate, accounts for an acute modulation in GLAST/EAAT1 transport. In contrast, activation of the α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate subtype of glutamate receptors represses the transcription of the chick glast gene. A glutamate responsive element has been mapped to the promoter region of this gene containing a bonafide binding site for the transcription factor Ying‐Yang 1. Using cultured chick cerebellar Bergmann glia cells, glutamate elicited a time and dose‐dependent increase in Ying‐Yang 1 DNA binding consistent with the negative response generated in a reporter gene construct controlled for Ying‐Yang 1. Over‐expression of this transcription factor leads to a substantial reduction in GLAST/EAAT1 transporter uptake and an important decrease in mRNA levels, all associated with the transcriptional repression of the chick glast promoter activity. These results provide evidence for an involvement of Ying‐Yang 1 in the transcriptional response to glutamate in glial cells and favor the notion of a relevant role of this factor in GLAST/EAAT1 transcriptional control.

Glutamate‐dependent transcriptional regulation of GLAST: role of PKC

The Na+‐dependent glutamate/aspartate transporter GLAST plays a major role in the removal of glutamate from the synaptic cleft. Short‐term, as well as long‐term changes in transporter activity are triggered by glutamate. An important locus of regulation is the density of transporter molecules present at the plasma membrane. A substrate‐dependent change in the translocation rate of the transporter molecules accounts for the short‐term effect, whereas the long‐term modulation apparently involves transcriptional regulation. Using cultured chick cerebellar Bergmann glial cells, we report here that glutamate receptors activation mediate a substantial reduction in the transcriptional activity of the chglast promoter through the Ca2+/diacylglicerol‐dependent protein kinase (PKC) signaling cascade. Overexpression of constitutive active PKC isoforms of mimic the glutamate effect. Accordingly, increased levels of c‐Jun or c‐Fos, but not Jun‐B, Jun‐D or Fos‐B, lower the chglast promoter activity. Serial deletions and electrophorectic mobility shift assays were used to define a specific region within the 5′ proximal region of the chglast promoter, associated with transcriptional repression. A putative glutamate response element could be defined in the proximal promoter stretch more likely between nts – 40 and − 78. These results demonstrate that GLAST is under glutamate‐dependent transcriptional control through PKC, and support the notion of a pivotal role of this neurotransmitter in the regulation of its own removal from the synaptic cleft, thereby modulating, mainly in the long term, glutamatergic transmission.

Glial glutamate transporters: New actors in brain signaling

Glutamate, the main excitatory amino acid in the vertebrate brain, is critically involved in most of the physiological functions of the central nervous system. It has traditionally been assumed that glutamate triggers a wide array of signaling cascades through the activation of specific membrane receptors. The extracellular levels are tightly regulated to prevent neurotoxic insults. Electrogenic Na+‐dependent glial glutamate transporters remove the bulk of the neurotransmitter from the synaptic cleft. An exquisitely ordered coupling between glutamatergic neurons and surrounding glia cells is fundamental for excitatory transmission. The glutamate/glutamine and astrocyte/neuron lactate shuttles provide the biochemical framework of this compulsory association. In this context, recent advances show that glial glutamate transporters act as signal transducers that regulate the expression of proteins involved in their compartmentalization with neurons in the so‐called tripartite synapse.

Cerebellar Bergmann glia: an important model to study neuron–glia interactions

The biochemical effects triggered by the action of glutamate, the main excitatory amino acid, on a specialized type of glia cells, Bergmann glial cells of the cerebellum, are a model system with which to study glia-neuronal interactions. Neuron to Bergmann glia signaling is involved in early stages of development, mainly in cell migration and synaptogenesis. Later, in adulthood, these cells have an important role in the maintenance and proper function of the synapses that they surround. Major molecular targets of this cellular interplay are glial glutamate receptors and transporters, both of which sense synaptic activity. Glutamate receptors trigger a complex network of signaling cascades that involve Ca(2+) influx and lead to a differential gene-expression pattern. In contrast, Bergmann glia glutamate transporters participate in the removal of the neurotransmitter from the synaptic cleft and act also as signal transducers that regulate, in the short term, their own activity. These exciting findings strengthen the concept of active participation of glial cells in synaptic transmission and the involvement of neuron-glia circuits in the processing of brain information.

Valproate-dependent transcriptional regulation of GLAST/EAAT1 expression: involvement of Ying-Yang 1.

Valproate, a widely used anti-epileptic drug also employed in the treatment of neurological diseases such as bipolar disorder and migraine, regulates the glutamatergic and GABAergic systems, although its effects in cell physiology have not been thoroughly characterized. High concentrations of glutamate reached during abnormal neurotransmission if not removed properly, become neurotoxic. Glutamate clearance is carried out by high affinity Na(+)-dependent glutamate transporter systems. The glutamate/aspartate transporter GLAST/EAAT1 plays the major role in glutamate removal and is regulated at different levels: transcription, post-translational modifications and cytoplasmic trafficking. The aim of this work was to gain insight into a plausible effect of valproate in GLAST function. Using cultured Bergmann glia cells from chick cerebellum we demonstrate here that valproate exposure elicits a dual regulatory effect on GLAST. In the short-term, valproate increases its Na(+)-dependent [(3)H]-d-aspartate uptake activity in a cytochalasin B-sensitive manner. Interestingly, a synergism between valproate and a histone deacetylase inhibitor was observed. Long-term valproate treatment up-regulates chglast promoter activity, GLAST mRNA levels, GLAST molecules at the plasma membrane and its uptake activity. Furthermore, valproate induces histone 3 lysine 14 acetylation and regulates Ying-Yang 1 (YY1) transcriptional repression on the chglast promoter. These results suggest that valproate elicits its effect through its histone deacetylase inhibitor properties.

GLAST: gene expression regulation by phorbol esters.

The gene expression regulation of the Na+-dependent high affinity glutamate/aspartate transporter GLAST expressed in cultured Bergmann glia cells from chick cerebellum was studied. A 679bp fragment of the chick GLAST cDNA was cloned and sequenced. Specific PCR primers were used to quantify chick GLAST mRNA levels. Treatment of the cells with the Ca2+/diacylglycerol dependent protein kinase C (PKC) activator, phorbol 12-tetradecanoyl-13-acetate (TPA) produced a decrease in transporter mRNA levels, without an effect in its mRNA half life, suggesting a transcriptional down regulation. Activation of the cAMP pathway results in a transient decrease in GLAST mRNA levels, in contrast with the TPA effect. These findings suggest that GLAST expression is under control of distinct signaling pathways.

The Tight Junction Protein ZO‐2 Blocks Cell Cycle Progression and Inhibits Cyclin D1 Expression

ZO‐2 is an adaptor protein of the tight junction that belongs to the MAGUK protein family. ZO‐2 is a dual localization protein that in sparse cultures is present at the cell borders and the nuclei, whereas in confluent cultures it is concentrated at the cell boundaries. Here we have studied whether ZO‐2 is able to regulate the expression of cyclin D1 (CD1) and cell proliferation. We have demonstrated that ZO‐2 negatively regulates CD1 transcription by interacting with c‐Myc at an E box present in CD1 promoter. We have further found that ZO‐2 transfection into epithelial MDCK cells triggers a diminished expression of CD1 protein and decreases the rate of cell proliferation in a wound‐healing assay.

Glutamate regulates kainate-binding protein expression in cultured chick Bergmann glia through an activator protein-1 binding site.

The expression of the chick kainate-binding protein, a member of the ionotropic glutamate receptor family, is restricted to the cerebellum, specifically to Bergmann glia. Glutamate induces a membrane to nuclei signaling involved in gene expression regulation. Exposure of cultured chick Bergmann glia cells to glutamate leads to an increase in kainate binding protein and mRNA levels, suggesting a transcriptional level of regulation. The 5′ proximal region of the chick kainate binding gene was cloned and transfected 4into Bergmann glia cells. Three main regulatory regions could be defined, a minimal promoter region, a negative regulatory region, and interestingly, a glutamate-responsive element. Deletion of this element abolishes the agonist effect. Moreover, electrophoretic mobility shift assays, cotransfection experiments, and site-directed mutagenesis clearly suggest that the glutamate effect is mediated through an AP-1 site by a Fos/Jun heterodimer. The present results favor the notion of a functional role of kainate-binding protein in glutamatergic cerebellar neurotransmission.

Glutamate regulates eEF1A phosphorylation and ribosomal transit time in Bergmann glial cells

Glutamate, the major excitatory transmitter in the vertebrate brain, is involved in neuronal development and synaptic plasticity. Glutamatergic stimulation leads to differential gene expression patterns in neuronal and glial cells. A glutamate-dependent transcriptional control has been established for several genes. However, much less is known about the molecular events that modify the translational machinery upon exposure to this neurotransmitter. In a glial model of cerebellar cultured Bergmann cells, glutamate induces a biphasic effect on [(35)S]-methionine incorporation into proteins that suggests that the elongation phase of protein biosynthesis is the target for regulation. Indeed, after a 15 min exposure to glutamate a transient increase in elongation factor 2 phosphorylation has been reported, an effect mediated through the activation of the elongation factor 2 kinase. In this contribution, we sought to characterize the phosphorylation status of the eukaryotic elongation factor 1A (eEF1A) and the ribosomal transit time under glutamate exposure. A dose-dependent increase in eEF1A phosphorylation was found after a 60 min glutamate treatment; this phenomenon is Ca(2+)/CaM dependent, blocked with Src and phosphatidyl-inositol 3-kinase inhibitors and with rapamicyn. Concomitantly, the ribosomal transit time was increased with a 15 min glutamate exposure. After 60 more minutes, the average time used by the ribosomes to complete a polypeptide chain had almost returned to its initial level. These results strongly suggest that glutamate exerts an exquisite time-dependent translational control in glial cells, a process that might be critical for glia-neuron interactions.