Regulation of glial cell cycle through neurotransmitter receptors and ion channelsExpression of functional neurotransmitter receptors and ionic channels in glia indicates that glial cells are also responsive elements of the mammalian central nervous system, and receptors and channels may have an important physiological role not only in neurons, but also in non-neuronal cells. Our objective is to understand the functional properties and the associated signal transduction pathways of glial neurotransmitter receptors, and their physiological role in the mature and developing brain. In previous work, we focused on glutamatergic and b-adrenergic receptors (GluRs and b-ARs, respectively) in oligodendrocyte progenitors (OPs), and demonstrated that activation of these receptors inhibits OP proliferation, but has opposite effects on cell differentiation. Glutamate inhibits, whereas norepinephrine stimulates OP differentiation. Inhibition of OP cell proliferation and differentiation is also observed with agents that block or reduce K+ channel activity (e.g. tetraethylammonium, TEA), indicating that also these ionic channels are involved in OP cell cycle progression.We demonstrated that the neurotransmitters glutamate and norepinephrine cause cell cycle arrest in G1 phase through an increase in the levels of the cyclin-dependent kinase inhibitors (cdkis) p27Kip1 and p21CIP1. In more recent studies, we analyzed the role of K+ channels in OP cell cycle progression. We found that the antiproliferative effects of the K+ channel blockers TEA, forskolin and dideoxyforskolin, and of the Na+ channel opener veratridine were due to OP cell cycle arrest in G1 phase. In fact: i) cyclin D accumulation in synchronized OP cells was not affected by K+ channel blockers or veratridine; ii) these agents prevented OP cell proliferation only if present during G1 phase, and iii) G1 blockers, such as rapamycin and deferoxamine, mimicked the anti-proliferative effects of K+ channel blockers. Blockage of K+ channels and membrane depolarization also caused accumulation of the cyclin dependent kinase inhibitors p27Kip1 and p21CIP1 in OP cells. The anti- proliferative effects of K+ channel blockers and veratridine were still present in OP cells isolated from INK4a-/- mice, lacking the cyclin dependent kinase inhibitors p16INK4a and p19ARF. Our results demonstrate that blockage of K+ channels and cell depolarization induce G1 arrest in OP cell cycle through a mechanism that may involve p27Kip1 and p21CIP1, and further support the conclusion that OP cell cycle arrest and differentiation are two uncoupled events. Current studies are aimed at identifying the intracellular pathways that link neurotransmitter receptor activation and G1 arrest in OP cells. Different antiproliferative agents can arrest the same cell type at distinct checkpoints in G1. Furthermore, the effects of antiproliferative agents on different molecular components of cell cycle machinery is linked to the position at which they arrest cells in G1. Therefore, we want to establish whether the intracellular signaling cascades triggered by GluR and b-AR activation and leading to G1 arrest are partially overlapping or completely independent. Preliminary results indicate that distinct neurotransmitter receptor systems and membrane channels differentially affect the activity of two cyclin-dependent kinases, cdk2 and cdk4. In particular, both GluR and b-AR agonists decreased cdk2 activity, whereas only b-AR agonists affected cdk4. These data indicate that the pathways of cell cycle regulation coupled to different neurotransmitter receptors and membrane channels diverge at the cdk level. Kainate receptor genes and their regulation in the brainKainate receptors represent a distinct molecular and pharmacological subtype of glutamate receptors. Two kainate receptor gene sub-families encode the subunits GluR5, 6 and 7, and KA1 and 2. These subunits bind the agonists kainate and domoate with high affinity. When expressed in Xenopus oocytes or heterologous mammalian cells, only GluR5-GluR7 form functional homomeric. KA1 and KA2 are inactive as homomeric ion channels, but associate with members of the GluR5-7 family to form functional heteroligomeric kainate-preferring receptors with biophysical properties and agonistselectivity distinct from homomeric GluR5-GluR7. In situ hybridization and immunohistochemical studies have demonstrated that, in different brain regions, specific combinations of members of the KA1-2:00 AMnd GluR 5-7 gene subfamilies are co-expressed, indicating that kainate receptor subunits assemble to form diverse subtypes of heteroligomeric channels. Functional homomeric and heteroligomeric kainate- preferring receptors are expressed in cultured neurons and glia, and kainate receptor synaptic currents have been found in various areas of the CNS. Presynaptic kainate receptors are involved in the modulation of neurotransmitter release. The GRIK5 gene encodes the kainate receptor subunit KA2. Since KA2 associates with other kainate receptor subunits, it follows that different functional receptor subtypes can be formed as a result of a stringent qualitative and quantitative control of GRIK5 expression. To gain an understanding of how kainate receptor subunit expression is regulated, we have previously cloned and characterized the structure of the GRIK5 gene, including its 5 untranslated region. We have also identified an intronic element of this gene, which displays functional features of a silencer. The GRIK5 promoter lacks consensus TATAA and CCAAT sequences, contains multiple transcription initiation sites and is GC-rich. Analysis in cultured cells (e.g. CG-4 oligodendrocytes) and in transgenic mice showed that 600bp of the GRIK5 proximal upstream region were sufficient to maintain tissue-specific expression of the reporter gene. Deletion of these 600bp abolished GRIK5 transcription in CG-4 cells, indicating that the gene transcriptional unit is comprised within this region. DNase I footprinting analysis of the 600bp region revealed a total of twelve potential transcription binding sites, including Inr, Sp1, Ap2 and MED1. Mutation of the sequences corresponding to the Sp1 sites did not modify the 3.4kb intron1 interrupts the GRIK5 5- untranslated region. We previously demonstrated that an intronic element displaying features of a silencer modulates GRIK5 transcription. Mutational analysis of the nuclear protein binding site within intron1 of GRIK5 defined an 11 nucleotide sequence (direct repeats composed of AGGTCA-like motifs) accounting for the negative activity. Mutation of the silencer element also abolished its inhibitory effect on GRIK5 transcription, as determined by reporter gene analysis. Further gel shift assays using nuclear extracts from both rat tissue and primary cells revealed that the DNA binding activity was: (i) much more abundant in neural than non- neural tissue, (ii) developmentally regulated, being highest in embryonic brain tissue and declining toward adulthood; and (iii) higher in non-differentiated than in differentiated neurons. In subsequent studies, we identified the family of proteins that repress GRIK5 transcription by binding to the intron1 silencer. We used the yeast one-hybrid system and cloned several cDNAs from a rat brain library. These cDNAs corresponded to orphan nuclear receptor proteins (ONRs), including COUP-TFI, Ear-2 and Nurr1. Gel shift studies with postnatal day 2 (P2) rat brain extract indicated the presence of these ONRs in the DNA-protein complex. Competition assays with GRIK5 binding site mutations showed that the recombinant clones exhibit differential binding characteristics and suggested that the DNA-protein complex from P2 rat brain may consist primarily of EAR2. Co-transfection assays showed that recombinant nuclear orphan receptors function as transcriptional repressors in both CV1 cells and rat CG4 oligodendrocyte cells. Direct interaction of the orphan receptors with, and relief of repression by TFIIB indicated likely role(s) in active and/or trans-repression. Our findings are thus consistent with the notion that multiple nuclear orphan receptors can regulate the transcription of a widely expressed neurotransmitter receptor gene by binding a common element in an intron and directly modulating the activity of the transcription machinery.
| Gallo, V; Ghiani, C A (2000) Glutamate receptors in glia: new cells, new inputs and new functions. Trends Pharmacol Sci 21:252-8 |
| Ghiani, C A; Yuan, X; Eisen, A M et al. (1999) Voltage-activated K+ channels and membrane depolarization regulate accumulation of the cyclin-dependent kinase inhibitors p27(Kip1) and p21(CIP1) in glial progenitor cells. J Neurosci 19:5380-92 |
