Autocrine activation of EGF receptor promotes oscillation of glutamate-induced calcium increase in astrocytes cultured in rat cerebral cortex

We previously reported that astrocytes cultured for more than 2 days in a defined medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) showed calcium oscillation in response to glutamate, whereas the response pattern was transient in the absence of the exogenous growth factors. In the present study, we found that astrocytes showed glutamate-induced calcium oscillation, even in growth factor-free medium, if the cells had been cultured for more than 5 days. The calcium oscillation promoted by the prolonged culture period was suppressed by an inhibitor of EGF receptor tyrosine kinase, but not by a neutralizing antibody to bFGF, indicating that the accumulation of an autocrine factor that activates the EGF receptor leads to calcium oscillation. Astrocytes in our culture system expressed EGF, transforming growth factor alpha (TGFalpha), bFGF and acidic fibroblast growth factor (aFGF). Exogenous aFGF, which induced astrocyte immediate early gene expression to the same extent as EGF or bFGF, did not affect calcium oscillation. Exogenous EGF and bFGF promoted astrocyte hypertrophic morphology and proliferation, as well as calcium oscillation. In contrast, these properties did not accompany calcium oscillation induced by the prolonged culture period. These results suggest that astrocytes possess the ability to promote their own calcium oscillation, which is independent of hypertrophic changes to reactive astrocytes.     

Epidermal Growth Factor and Basic Fibroblast Growth Factor Have Independent Actions on Mesencephalic Dopamine Neurons in Culture

Abstract: Epidermal growth factor (EGF) and basic fibro-blast growth factor (bFGF) are both trophic for dopamine neurons s in cultures of dissociated embryonic rat mesen-cephaion, but the significance of this apparent overlap in neurotrophic activity is not yet known. In this study, we investigated the mechanisms of action of these two growth factors and the potential relationship between them, Using a nuclease protection assay, we determined that bFGF mRNA was expressed in the cultures. Double-label immunocytochemistry revealed that bFGF immunore-active material could be detected in tyrosine hydroxylase immunoreactive neurons and glial fibrillary acidic protein immunoreactive astrocytes. EGF treatment increased bFGF mRNA content per culture dish. As we have previously demonstrated that EGF exerts its dopaminergic neurotrophic activity via an intermediate cell type, studies were designed to address whether the pathway by which EGF acts on dopaminergic neurons is mediated by the release of bFGF. However, the trophic action of EGF on dopamine neurons, represented by high-affinity neuronal dopamine uptake, could not be blocked by immunoneutralization of bFGF, suggesting that the actions of EGF were not mediated by bFGF release. The time course of the effects of EGF and bFGF on dopamine uptake were similar, with significant increases detectable only after 5 days in culture. Both growth factors were active in the picomolar-to-nannomolar range with maximal trophic activity between 0.4 and 2.5 n M. EGF, however, was the more potent mitogen under these conditions. When cultures were simultaneously incubated with maximal concentrations of EGF and bFGF, the effect on dopamine uptake was significantly greater than with either growth factor alone and, in fact, approximated the sum of the individual effects. On the basis of these results we conclude that these growth factors have independent effects on dopamine neurons of the mesencephalon.     

Epidermal growth factor modulates claudins and tight junctional functions in ovarian cancer cell lines

Ovarian adenocarcinomas, like human ovarian surface epithelial cells, form functional tight junctions. Tight junction molecules claudin-3 and claudin-4, which are the receptors of Clostridium perfringens enterotoxin (CPE), are abnormally upregulated in epithelial ovarian cancers of all subtypes including, mucinous cystadenocarcinoma and serous cystadenocarcinoma. Clostridium perfringens enterotoxin may be a novel tumor-targeted therapy for ovarian cancers. In epithelial ovarian cancers, overexpression of epidermal growth factor receptor has been observed and the exogenous ligand EGF induces epithelial-mesenchymal transition in ovarian surface epithelium. Epidermal growth factor (EGF) signaling modulates expression of claudins with changes of fence and barrier functions in various cell types. However, the regulation of tight junctions by EGF in ovarian cancers remains unclear. In the present study, to investigate the mechanisms of the regulation of tight junctions in ovarian cancers, ovarian cancer cell lines mucinous cystadenocarcinoma (MCAS) and serous cystadenocarcinoma (HUOA) were treated with EGF. Epidermal growth factor downregulated claudin-3 in MCAS and claudin-4 in HUOA by inducing degradation of the proteins with changes in structures and functions of tight junctions via the MEK/ERK or PI3K/Akt signaling pathway. In addition, in HUOA but not MCAS, EGF downregulated the cytotoxic effect of CPE via claudin-4. Thus, there were different mechanisms for regulation of claudins by EGF between subtypes of epithelial ovarian cancer cells in vitro. These results indicate that EGF may affect claudins and tight junctional functions in ovarian cancer cells during cancer progression.     

Changes in Metabotropic Glutamate Receptor mRNA Levels Following Global Ischemia: Increase of a Putative Presynaptic Subtype (mGluR4) in Highly Vulnerable Rat Brain Areas

Abstract: Metabotropic glutamate receptors mediate their intracellular response by coupling to G proteins and may be divided into three subfamilies: mGluR1 and mGluR5, which stimulate phosphatidylinositol hydrolysis; mGluR2 and mGluR3, which are negatively coupled to cyclic AMP formation; and mGluR4 and mGluR6, which also inhibit forskolin-stimulated cyclic AMP formation. The mGluR4 subtypes may represent l -2-amino-4-phosphonobutyrate-sensitive presynaptic autoreceptors, and two alternatively spliced variants of the mGluR4 coding for two receptors with different C termini have been identified. Using in situ hybridization, we measured the levels of mGluR1–mGluR5 mRNA in regions of the rat brain 24 h after transient global ischemia, a time point when no neuronal damage can yet be observed morphologically. In the hippocampus, the mRNA levels for mGluR1, mGluR2, and mGluR5 were decreased, mGluR3 mRNA levels were unchanged, and the mGluR4 mRNA levels were strongly increased. The strongest increase appeared to be in the mRNA encoding mGluR4b. The mGluR4 mRNA was also increased in the parietal cortex, whereas the ventral posteromedial thalamic nucleus showed a small decrease in its mRNA content. These results suggest that vulnerable neurons react to an increased extracellular glutamate concentration by differential regulation of the mRNA for pre- and postsynaptically located metabotropic glutamate receptors.     

Cytokine-induced selective increase of high-molecular-weight bFGF isoforms and their subcellular kinetics in cultured rat hippocampal astrocytes

Cytokines such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and epidermal growth factor (EGF) are probable factors responsible for up-regulation of basic fibroblast growth factor (bFGF) expression in reactive astrocytes following brain damage, however the effect of these cytokines on the expression of each bFGF-isoform has not been elucidated. Western biot analysis revealed the expression of 18, 22 and 24-kD bFGF isoforms in cultured rat hippocampal astrocytes, and the expression of high molecular weight (HMW)-isoforms (22 and 24-kD isoforms) but not of 18-kD isoform was selectively increased by cytokines. Immunofluorescent analysis demonstrated that bFGF content in the cytoplasm of astrocytes is initially increased by cytokines followed by nuclear targeting and localization in agreement with the previous evidence that HMW-isoforms possess a nuclear targeting signal. The present results suggest the important role of HMW-bFGF isoforms in the response of nervous tissue to injury.     

Group I metabotropic glutamate receptors mediate phospholipase D stimulation in rat cultured astrocytes

We have studied the activation of phospholipase D (PLD) by glutamate in rat cultured astrocytes by measuring the PLD-catalyzed formation of [32P]phosphatidylbutanol in [32P]Pi-prelabeled cells, stimulated in the presence of butanol. Glutamate elicited the activation of PLD in cortical astrocytes but not in cortical neurons, whereas similar glutamate activation of phosphoinositide phospholipase C was found in both astrocytes and neurons. The extent of PLD stimulation by glutamate was similar in astrocytes from brain cortex and hippocampus, but no effect was found in cerebellar astrocytes. In cortical astrocytes, the glutamate response was insensitive to antagonists of ionotropic glutamate receptors and was reproduced by agonists of metabotropic glutamate receptors (mGluRs) with a rank order of agonist potency similar to that reported for group I mGluR-mediated phosphoinositide phospholipase activation [quisqualate > (S)-3,5-dihydroxyphenylglycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid]. The response to (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid was inhibited by the mGluR antagonist (S)-alpha-methyl-4-carboxyphenylglycine and, less potently, by 1-aminoindan-1,5-dicarboxylic acid and 4-carboxyphenylglycine, two antagonists of group I mGluRs that display higher potency on mGluR1 than on mGluR5. The mGluR5-selective agonist (RS)-2-chloro-5-hydroxyphenylglycine also activated PLD in astrocytes. These findings indicate the involvement of group I mGluRs, most likely mGluR5, in the glutamate activation of PLD in cultured rat cortical astrocytes.     

Temporal and Depolarization-Induced Changes in the Absolute Amounts of mRNAs Encoding Metabotropic Glutamate Receptors in Cerebellar Granule Neurons In Vitro

Abstract: Cerebellar granule neurons in primary culture express metabotropic glutamate receptors (mGluRs) coupled to the stimulation of phosphoinositide hydrolysis and to the inhibition of cyclic AMP (cAMP) formation. To evaluate which mGluR mRNAs are expressed in granule neurons under different depolarizing conditions, we measured the absolute amounts of selected receptor mRNAs in neurons cultured for 3–13 days in the presence of either 10 or 25 m M KCl. mGluR-specific primer pairs and internal standards, corresponding to unique regions of mGluR1a, mGluR2, mGluR3, mGluR4, and mGluR5, were constructed and used in a competitive PCR-derived assay to quantify the corresponding mRNA levels. For phosphoinositide-coupled receptors, the absolute content of mGluR1a mRNA was three to 10 times higher than the content of mGluR5 mRNA. The expression of mGluR5 mRNA increased up to 9 days in vitro and was much higher in 10 m M than in 25 m M KCl. For the cAMP-coupled receptors, there was a large amount of mGluR4 mRNA and a much smaller content of the mGluR3 and mGluR2 mRNAs. Maintaining the granule neurons in vitro in 10 m M KCl increased the absolute amount of mRNAs encoding mGluR2 and mGluR4 at 9 and 13 days in vitro. In contrast, the content of the mGluR3 mRNA was consistently higher in neurons cultured in 25 m M KCl. These data are consistent with the possibility that in primary cultures of cerebellar neurons, phosphoinositide responses may be predominantly mediated by mGluR1a, rather than mGluR5, and that cAMP inhibition involves preferentially mGluR4 and mGluR3.     

Cultured Rat Astrocytes Give Rise to Neural Stem Cells

Previously, we reported the occurrence of neural stem cells (NSCs) around an area of damage after rat traumatic brain injury (TBI), but it was unclear if this was due to blastgenesis in astrocytes, or to NSCs migrating from the subventricular zone (SVZ). In this study, NSCs were isolated and cultured from cultured type 1 astrocytes taken from newborn rat cortex in which the subventricular zone and hippocampus had been discarded. All cultured type 1 astrocytes showed glial fibrillary acidic protein (GFAP) immunopositivity. Nestin immunopositive spheres were isolated from type 1 astrocytes and cultured in the presence of bFGF and EGF in the medium. Neurospheres differentiated into Tuj1-, GFAP- and A2B5-positive cells after 4 days of culture without bFGF and EGF. These results indicate that isolated neurospheres from brain cortex astrocytes can differentiate into neurons and glia and might contribute to neurogenesis and neuroplasticity.     

Basic fibroblast growth factor promotes epidermal growth factor responsiveness and survival of mesencephalic neural precursor cells.

Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) induce proliferation of neural precursor cells from several central nervous system regions in vitro. We have previously described two neural precursor cell populations from 13.5 days postcoitium (dpc) mesencephalon, one forming colonies in response to EGF, present in the ventral mesencephalon, and other forming colonies in response to EGF + bFGF, mainly present in the dorsal mesencephalon. In the present work, we show that 13.5 dpc dorsal mesencephalic cells required bFGF only for 1 h to form colonies in response to EGF alone, indicating that these two growth factors act in sequence rather than simultaneously. Absence of bFGF at the beginning of the culture gave rise to very few colonies, even after the addition of EGF + bFGF, suggesting that cells responsive to bFGF were very labile in the primary culture condition. This result is in contrast with cells pretreated with bFGF, which could survive for up to 5 days in the absence of bFGF or EGF, and then were capable of efficiently forming colonies in response to EGF. Basic FGF was also able to support survival of EGF-responsive neural precursors from both ventral and dorsal mesencephalon. The population requiring bFGF to form colonies in response to EGF was identified at different developmental stages (11.5-15.5 dpc), with higher contribution to the total number of neural precursors cells detected (EGF-responsive plus bFGF-responsive) at early stages and in the dorsal region. We show that the differentiation effect of bFGF resulted in the appearance of the mRNA coding for the EGF receptor. Our data suggest that bFGF-responsive neural precursors are the source of EGF-responsive neural precursors.     

Growth factors and cytokines modulate gene expression of cell-surface proteoglycans in human periodontal ligament cells

Cell-surface proteoglycans have been known to be involved in many functions including interactions with components of the extracellular microenvironment, and act as co-receptors which bind and modify the action of various growth factors and cytokines. The purpose of this study was to determine the regulation by growth factors and cytokines on cell-surface proteoglycan gene expression in cultured human periodontal ligament (PDL) cells. Subconfluent, quiescent PDL cells were treated with various concentrations of serum, bFGF, PDGF-BB, TGF-beta1, IL-beta1, and IFN-gamma. RT-PCR technique was used, complemented with Northern blot for syndecan-1, to examine the effects of these agents on the mRNA expression of five cell-surface proteoglycans (syndecan-1, syndecan-2, syndecan-4, glypican and betaglycan). Syndecan-1 mRNA levels increased in response to serum, bFGF and PDGF-BB, but decreased in response to TGF-beta1, IL-1beta and IFN-gamma. In contrast, syndecan-2 mRNA levels were upregulated by TGF-beta1 and IL-1beta stimulation, but remained unchanged with the other agents. Betaglycan gene expression decreased in response to serum, but was upregulated by TGF-beta1 and unchanged by the other stimulants. Additionally, syndecan-4 and glypican were not significantly altered in response to the regulator molecules studied, with the exception that glypican is decreased in response to IFN-gamma. These data demonstrate that the gene expression of the five cell-surface proteoglycans studied is differentially regulated in PDL cells lending support to the notion of distinct functions for these cell-surface proteoglycans.     

Lysophosphatidic acid stimulates astrocyte proliferation through LPA1

Lysophosphatidic acid (LPA) is an extracellular lipid mediator that regulates nervous system development and functions through multiple types of LPA receptors. Here we explore the role of LPA receptor subtypes in cortical astrocyte functions. Astrocytes cultured under serum-free conditions were found to express the genes of five LPA receptor subtypes, lpa1 to lpa5. When astrocytes were treated with dibutyryl cyclic adenosine monophosphate, a reagent inducing astrocyte differentiation or activation, lpa1 expression levels remained unchanged, but those of other LPA receptor subtypes were relatively reduced. LPA stimulated DNA synthesis in both undifferentiated and differentiated astrocytes, but failed to do so in astrocytes prepared from mice lacking lpa1 gene. LPA also inhibited [3H]-glutamate uptake in both undifferentiated and differentiated astrocytes; and LPA-induced inhibition of glutamate uptake was still observed in lpa1-deficient astrocytes. Taken together, these observations demonstrate that LPA1 mediates LPA-induced stimulation of cell proliferation but not inhibition of glutamate uptake in astrocytes.     

Astroglial-Conditioned Media and Growth Factors Modulate Proliferation and Differentiation of Astrocytes in Primary Culture

Astroglial conditioned media (ACM) influence the development and maturation of cultured nerve cells and modulate neuron-glia interaction. To clarify mechanisms of astroglial cell proliferation/differentiation in culture, incorporation of [methyl-³H]-thymidine or [5,6-³H]-uridine in cultured astrocytes was assessed. Cultures were pre-treated with epidermal growth factor (EGF), insulin (INS), insulin-like growth factor-I (IGF-I), and basic fibroblast growth factor (bFGF) and subsequently with ACM. DNA labeling revealed a marked stimulatory effect of ACM from 15 days in vitro (DIV) cultures in 30 DIV astrocytes after12 h pre-treatment with growth factors. The main effects were found after INS or EGF pre-treatment in 30 DIV cultures. ACM collected from 15 or 60 or 90 DIV increased RNA labeling of 15 and 30 DIV astrocyte cultures, being the highest value that of 30 DIV cultures added with ACM from 90 DIV. The findings of increased DNA labeling after EGF or INS pre-treatment in 30 DIV cultures, followed by addition of ACM from 15 DIV cultures, suggest that these phenomena may depend by extra cellular signal-regulated kinase 1 (ERK1) activation.     

Modulation of the epidermal growth factor receptor of mouse embryonic palatal mesenchyme cells in vitro by growth factors.

A single class of high-affinity receptors for EGF were detected on mouse embryonic palatal mesenchyme (MEPM) cells cultured in vitro. The degree of confluence of the cultured cells did not affect the number or affinity of the binding sites. Culture of MEPM cells in the presence of bFGF, IGF-II or TGF-beta 1 induced changes in 125I-EGF binding. TGF-beta 1 caused a marked reduction in binding to 40% of control levels. This reduction was achieved after 2 h and persisted for 24 h after addition of the growth factor. IGF-II induced a similar reduction but this effect was transitory; after a 12 h pretreatment with IGF-II, binding was restored to control levels. The effects of bFGF were biphasic. Initially, a short pre-treatment period (3-5 h) with bFGF caused a small reduction in 125I-EGF binding; longer periods of pre-incubation (24 h) resulted in a large increase in receptor number. Pre-incubation in medium containing both bFGF and TGF-beta 1 resulted in a decrease in EGF binding. Thus, TGF-beta 1 negated the large increase in receptor number induced by bFGF alone. Changes in receptor number were usually, but not always, directly related to changes in the biological activity of EGF, as assessed by a thymidine incorporation assay. This study highlights the possible interactive role of growth factors known to be present in the developing palate.