Influence of glucose on somatostatin synthesis and secretion in isolated cerebral cortical cells

Somatostatin-producing cerebral cortical cell cultures were grown in either high- (33 mM) or low-glucose (5 mM) medium and then exposed to short repetitive changes of high- or low-glucose Krebs-Ringer's bicarbonate buffer. Equivalent amounts of somatostatin were released in the high-to-high, the low-to-low, and the low-to-high paradigms. The high-to-low experiment produced a rapid rise in somatostatin release, followed by a decline. Cultures exposed to 2-deoxyglucose after high-glucose medium also released much greater amounts of immunoreactive somatostatin. Separate sets of cultures were grown in high- or low-glucose medium for up to 19 days. Cultures grown in high-glucose medium generally contained more somatostatin intracellularly than did those maintained in low glucose, although somatostatin in the medium was only different at day 19. These results identify extracellular glucose as an important determinant of cortical somatostatin production.     

The postnatal development of intrinsic properties and spike encoding at cortical GABAergic neurons

GABAergic neurons play a critical role in maintaining the homeostasis of brain functions for well-organized behaviors. It is not known about the dynamical change in signal encoding at these neurons during postnatal development. We investigated this issue at GFP-labeled GABAergic neurons by whole-cell recording in cortical slices of mice. Our results show that the ability of spike encoding at GABAergic neurons is improved during postnatal development. This change is associated with the reduction of refractory periods and threshold potentials of sequential spikes, as well as the improvement of linear correlations between intrinsic properties and spike capacity. Therefore, the postnatal maturation of the spike encoding capacity at GABAergic neurons will stabilize the excitatory state of cerebral cortex.     

The development and ultrastructure of previously dissociated foetal human cerebral cortical cells in vitro

Summary Cells from foetal human cerebral cortex were mechanically dissociated and subsequently maintained in vitro for periods ranging between three and twenty-eight days.The ultrastructure of these cells at different stages of their development in culture was extensively examined. Nuclear and cytoplasmic features were extremely variable and a wide range of cell types was evidently represented. Of the three principal cell types found i.e. neurons, neuroglia and mesenchymal cells, only a minority of cells was classified with confidence, particularly during the first two weeks in culture.Extensive intercellular junctions of the adhaerens variety, common after 14 days in vitro were present at an earlier stage of development than synaptic profiles. First indications of synapse formation were observed after 21 days in vitro and after 24 days presynaptic sites filled with synaptic vesicles and with well defined presynaptic and postsynaptic thickenings were found. The significance of some of the features observed are both considered and discussed.     

Ect2, an ortholog of Drosophila Pebble, regulates formation of growth cones in primary cortical neurons

In collaboration with Marshall Nirenberg, we performed in vivo RNA interference (RNAi) genome-wide screening in Drosophila embryos. Pebble has been shown to be involved in Drosophila neuronal development. We have also reported that depletion of Ect2, a mammalian ortholog of Pebble, induces differentiation in NG108-15 neuronal cells. However, the precise role of Ect2 in neuronal development has yet to be studied. Here, we confirmed in PC12 pheochromocytoma cells that inhibition of Ect2 expression by RNAi stimulated neurite outgrowth, and in the mouse embryonic cortex that Ect2 was accumulated throughout the ventricular and subventricular zones with neuronal progenitor cells. Next, the effects of Ect2 depletion were studied in primary cultures of mouse embryonic cortical neurons: Loss of Ect2 did not affect the differentiation stages of neuritogenesis, the number of neurites, or axon length, while the numbers of growth cones and growth cone-like structures were increased. Taken together, our results suggest that Ect2 contributes to neuronal morphological differentiation through regulation of growth cone dynamics.     

Administration of anti-c-kit antibody into the cerebrospinal fluid leads to increased cell death in the developing cerebral cortex

It is generally believed that during development, neurons are usually produced in excess. Cell death occurs in the developing nervous system. The survival of the developing neurons depends on many factors derived from the target sites, of which the neuronal trophic factors are by far the best known. Stem cell factor (SCF) and its receptor, c-kit, is expressed in cells of nervous system during development and adulthood. Although the role of SCF/c-kit in the nervous system is so far not clear, in vitro studies indicate that SCF/c-kit is trophic to certain neurons derived from neural crest and cerebral cortex. In this study the effects of anti-c-kit antibody on cell death in the newborn chick cerebral cortex have been investigated. Injection of anti-c-kit antibody into the cisterna magnum increased the number of cell death and resulted in thinning of the cerebral cortex as compared to that from the control group. It is concluded that SCF/c-kit is essential for cortical progenitor cell survival in the cerebral cortex. Moreover, this method may be applied to the other factors and different CNS regions, allowing identification of factors involved in cell death. It additionally re-emphasizes the importance of further investigations into the potential roles of SCF/c-kit signaling in neurodegenerative diseases.     

Dual mode regulation of migration by lysophosphatidic acid in human gastric cancer cells

Lysophosphatidic acid (LPA), which interacts with at least three G protein-coupled receptors (GPCRs), LPA1/Edg-2, LPA2/Edg-4, and LPA3/Edg-7, is a lipid mediator with diverse effects on various cells. Here, we investigated the expression profiles of LPA receptors and patterns of LPA-induced migration in gastric cancer cells. Northern blot analysis revealed that various gastric cancer cells expressed variable levels of LPA1, LPA2, and LPA3 without a consistent pattern. Using a Boyden chamber assay, LPA markedly increased cell migration of LPA1-expressing cells, the effects of which were almost totally abrogated by Ki16425, an LPA antagonist against LPA1 and LPA3. In contrast, LPA by itself did not significantly induce migration in MKN28 and MKN74 cells, which exclusively expressed LPA2. However, when hepatocyte growth factor (HGF) was placed with LPA in the lower chamber, LPA induced migration of these cells in a dose-dependent manner. Immunoprecipitation analysis revealed that LPA induced transient tyrosine phosphorylation of c-Met in LPA2-expressing cells, which suggests that the transactivation of c-Met by LPA causes a cooperative migratory response with HGF to these cells. Our results indicate that LPA regulates the migration of gastric cancer cells in a receptor-specific manner and suggest that the expression pattern of LPA receptors may affect the metastatic behavior of gastric cancer.     

A novel signaling pathway of tissue kallikrein in promoting keratinocyte migration: Activation of proteinase-activated receptor 1 and epidermal growth factor receptor

Biological functions of tissue kallikrein (TK, KLK1) are mainly mediated by kinin generation and subsequent kinin B2 receptor activation. In this study, we investigated the potential role of TK and its signaling pathways in cultured human keratinocyte migration and in a rat skin wound healing model. Herein, we show that TK promoted cell migration and proliferation in a concentration- and time-dependent manner. Inactive TK or kinin had no significant effect on cell migration. Interestingly, cell migration induced by active TK was not blocked by icatibant or L-NAME, indicating an event independent of kinin B2 receptor and nitric oxide formation. TK's stimulatory effect on cell migration was inhibited by small interfering RNA for proteinase-activated receptor 1 (PAR₁), and by PAR₁ inhibitor. TK-induced migration was associated with increased phosphorylation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK), which was blocked by inhibition of protein kinase C (PKC), Src, EGFR and ERK. TK-induced cell migration and EGFR phosphorylation were blocked by metalloproteinase (MMP) inhibitor, heparin, and antibodies against EGFR external domain, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin (AR). Local application of TK promoted skin wound healing in rats, whereas icatibant and EGFR inhibitor blocked TK's effect. Skin wound healing was further delayed by aprotinin and neutralizing TK antibody. This study demonstrates a novel role of TK in skin wound healing and uncovers new signaling pathways mediated by TK in promoting keratinocyte migration through activation of the PAR₁-PKC-Src-MMP pathway and HB-EGF/AR shedding-dependent EGFR transactivation.     

Novel interactions between NFATc1 (Nuclear Factor of Activated T cells c1) and STAT-3 (Signal Transducer and Activator of Transcription-3) mediate G protein-coupled receptor agonist, thrombin-induced biphasic expression of cyclin D1, with first phase influencing cell migration and second phase directing cell proliferation

Thrombin, a G protein-coupled receptor agonist, induced a biphasic expression of cyclin D1 in primary vascular smooth muscle cells. Although both phases of cyclin D1 expression require binding of the newly identified cooperative complex, NFATc1·STAT-3, to its promoter, the second phase, which is more robust, depends on NFATc1-mediated recruitment of p300 onto the complex and the subsequent acetylation of STAT-3. In addition, STAT-3 is tyrosine-phosphorylated in a biphasic manner, and the late phase requires NFATc1-mediated p300-dependent acetylation. Furthermore, interference with acetylation of STAT-3 by overexpression of acetylation null STAT-3 mutant led to the loss of the late phase of cyclin D1 expression. EMSA analysis and reporter gene assays revealed that NFATc1·STAT-3 complex binding to the cyclin D1 promoter led to an enhanceosome formation and facilitated cyclin D1 expression. In the early phase of its expression, cyclin D1 is localized mostly in the cytoplasm and influenced cell migration. However, during the late and robust phase of its expression, cyclin D1 is translocated to the nucleus and directed cell proliferation. Together, these results demonstrate for the first time that the dual function of cyclin D1 in cell migration and proliferation is temperospatially separated by its biphasic expression, which is mediated by cooperative interactions between NFATc1 and STAT-3.     

D2-class dopamine receptor inhibition of NMDA currents in prefrontal cortical neurons is platelet-derived growth factor receptor-dependent

NMDA receptor function is modulated by both G-protein-coupled receptors and receptor tyrosine kinases. In acutely isolated rat hippocampal neurons, direct activation of the platelet-derived growth factor (PDGF) receptor or transactivation of the PDGF receptor by D4 dopamine receptors inhibits NMDA-evoked currents in a phospholipase C (PLC)-dependent manner. We have investigated further the ability of D2-class dopamine receptors to modulate NMDA-evoked currents in isolated rat prefrontal cortex (PFC). We have demonstrated that, similar to isolated hippocampal neurons, the application of PDGF-BB or quinpirole to isolated PFC neurons induces a slow-onset and long-lasting inhibition of NMDA-evoked currents. However, in contrast to hippocampal neurons, the inhibition of NMDA-evoked currents by quinpirole in PFC neurons is dependent upon D2/3, rather than D4, dopamine receptors. In PFC slices, application of both PDGF-BB and quinpirole induced a phosphorylation of the PDGF receptor at the PLCgamma binding and activation site, Tyr1021. The PDGF receptor kinase inhibitor, tyrphostin A9, and the D2/3 dopamine receptor antagonist, raclopride, inhibited quinpirole-induced Tyr1021 phosphorylation. These finding suggest that quinpirole treatment inhibits NMDAR signaling via PDGF receptor transactivation in both the hippocampus and the PFC, and that the effects of quinpirole in these regions are mediated by D4 and D2/3 dopamine receptors, respectively.     

Phosphorylation of doublecortin by protein kinase A orchestrates microtubule and actin dynamics to promote neuronal progenitor cell migration

Doublecortin (DCX) is a microtubule-associated protein that is specifically expressed in neuronal cells. Genetic mutation of DCX causes lissencephaly disease. Although the abnormal cortical lamination in lissencephaly is thought to be attributable to neuronal cell migration defects, the regulatory mechanisms governing interactions between DCX and cytoskeleton in the migration of neuronal progenitor cells remain obscure. In this study we found that the G(s) and protein kinase A (PKA) signal elicited by pituitary adenylate cyclase-activating polypeptide promotes neuronal progenitor cells migration. Stimulation of G(s)-PKA signaling prevented microtubule bundling and induced the dissociation of DCX from microtubules in cells. PKA phosphorylated DCX at Ser-47, and the phospho-mimicking mutant DCX-S47E promoted cell migration. Activation of PKA and DCX-S47E induced lamellipodium formation. Pituitary adenylate cyclase-activating polypeptide and DCX-S47E stimulated the activation of Rac1, and DCX-S47E interacted with Asef2, a guanine nucleotide exchange factor for Rac1. Our data reveal a dual reciprocal role for DCX phosphorylation in the regulation of microtubule and actin dynamics that is indispensable for proper brain lamination.