RasGAP is involved in signal transduction triggered by FGF1 in Xenopus oocytes expressing FGFR1.

The role of RasGAP was investigated in the model system of Xenopus oocytes expressing fibroblast growth factor receptor 1 (FGFR1) stimulated by fibroblast growth factor 1 (FGF1). The injection of the SH2-SH3-SH2 domains of RasGAP suppressed Ras activity, extracellular signal-regulated protein kinase 2 (ERK2) phosphorylation and Mos synthesis. The SH2 domain of Src, and PP2, an inhibitor of Src, also abolished Ras activity, ERK2 phosphorylation and Mos synthesis. In addition, Src activity was blocked by the SH2-SH3-SH2 domains of RasGAP. Immunoprecipitation of a chimera composed of the extracellular domain of the platelet-derived growth factor (PDGF) receptor and the intracellular domain of FGFR1 stimulated by PDGF-BB demonstrates the recruitment of phosphorylated RasGAP. This study shows that the transduction cascade induced by the FGFR1-FGF1 interaction in Xenopus oocytes involves RasGAP as a co-activator of Src to stimulate the Ras/mitogen-activated protein kinase cascade and Mos synthesis. It emphasises a new positive regulatory role for RasGAP in FGFR transduction.

Fibroblast growth factors 1 and 2 differently activate MAP kinase in Xenopus oocytes expressing fibroblast growth factor receptors 1 and 4.

The mitogen-activated protein kinase (MAP kinase) signalling cascade activated by fibroblast growth factors (FGF1 and FGF2) was analysed in a model system, Xenopus oocytes, expressing fibroblast growth factor receptors (FGFR1 and FGFR4). Stimulation of FGFR1 by FGF1 or FGF2 and FGFR4 by FGF1 induced a sustained phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2) and meiosis reinitiation. In contrast, FGFR4 stimulation by FGF2 induced an early transient activation of ERK2 and no meiosis reinitiation. FGFR4 transduction cascades were differently activated by FGF1 and FGF2. Early phosphorylation of ERK2 was blocked by the dominant negative form of growth factor-bound protein 2 (Grb2) and Ras, for FGF1-FGFR4 and FGF2-FGFR4. The phosphatidylinositol 3-kinase (PI3 kinase) inhibitors wortmannin and LY294002 only prevented the early ERK2 phosphorylation triggered by FGF2-FGFR4 but not by FGF1-FGFR4. ERK2 phosphorylation triggered by FGFR4 depended on the Grb2/Ras pathway and also involved PI3 kinase in a time-dependent manner.

Transduction cascades initiated by fibroblast growth factor 1 on Xenopus oocytes expressing MDA-MB-231 mRNAs. Role of Grb2, phosphatidylinositol 3-kinase, Src tyrosine kinase, and phospholipase Cgamma.

Xenopus oocytes expressing fibroblast growth factor receptors (FGFRs) from the hormone-independent breast cancer cells, MDA-MB-231, are used as a biological system to analyze the signalling cascades initiated by FGF1. FGF1 induces ERK2 phosphorylation and G2/M transition. These events are dependent on the Shc/Grb2/Ras pathway, on Src and PI3Kinase (PI3K), as shown by the use of SH2 domains or dominant negative proteins, and on PLC gamma and calcium as demonstrated by a PLC gamma inhibitory peptide and BAPTA-AM. FGF1 mobilizes Ins(1,4,5)P3-sensitive calcium stores, as recorded through the inhibition by caffeine of a chloride calcium-dependent current in expressing oocytes. This study shows that the transduction cascades induced by FGF1 on FGFRs from MDA-MB-231 cells represent the sum of Ras, Src, PI3K, and PLC gamma pathways. It emphasizes the mitogenic effect of the PLC gamma-calcium cascade.

Grb2 promotes reinitiation of meiosis in Xenopus oocytes.

The adaptor protein Grb2 plays a central role in cell proliferation and/or cell cycle progression. In this study, we investigate the role of Grb2 in signalling pathways involved in meiotic reinitiation. For that purpose, Xenopus Grb2 cRNA and its mutated forms or human Grb2 protein was microinjected into immature Xenopus oocytes. Reinitiation of meiosis was seen in unstimulated oocytes. Induction of the meiosis was time dependent and Ras dependent, and the presence in Grb2 of SH2 and SH3 domains was required. Several tyrosine phosphorylated proteins were solely detected in oocytes responsive to Grb2 injection. Our results are in favour of an unusual recruitment and initiation of the Grb2 transduction cascade independent of a receptor tyrosine kinase (RTK) stimulation.

Signal transduction pathways triggered by fibroblast growth factor receptor 1 expressed in Xenopus laevis oocytes after fibroblast growth factor 1 addition. Role of Grb2, phosphatidylinositol 3-kinase, Src tyrosine kinase, and phospholipase Cgamma.

Xenopus oocytes expressing fibroblast growth factor receptor 1 (FGFR1) were used as a biological model system to analyse the signal transduction pathways that are triggered by fibroblast growth factor 1 (FGF1). Germinal vesicle breakdown (GVBD) and phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2) occured 15 h after FGF1 addition. These events were Ras-dependent as they were blocked by a Ras dominant negative form. The Ras activity was promoted by three upstream effectors, growth factor-bound protein 2 (Grb2), phosphatidylinositol 3-kinase (PI3K) and Src cytoplasmic kinase. Ras activation was inhibited by a Grb2 dominant negative form (P49L), by PI3K inhibitors, including wortmannin, LY294002, the N-SH2 domain of p85alpha PI3K and by the SH2 domain of Src. Src activation induced by FGF1 was blocked by the SH2 domain of Src and PP2, a specific inhibitor of Src. The Grb2 adaptor was recruited by the upstream Src homology 2/alpha-collagen-related (Shc) effector, as the SH2-Shc domain prevented the GVBD and the ERK2 phosphorylation induced by FGF1. The importance of another signalling pathway involving phospholipase Cgamma (PLCgamma) was also investigated. The use of the PLCgamma inhibitory peptide, neomycin and the calcium chelator BAPTA-AM on oocytes expressing FGFR1 or the stimulation by PDGF-BB of oocytes expressing PDGFR-FGFR1 mutated on the PLCgamma binding site, prevented GVBD and ERK2 phosphorylation. This study shows that the transduction cascade induced by the FGFR1-FGF1 interaction in Xenopus oocytes represents the sum of Ras-dependent and PLCgamma-dependent pathways. It emphasizes the role played by PI3K and Src and their connections with the Ras cascade in the FGFR1 signal transduction.

Fibroblast and epidermal growth factor receptor expression in Xenopus oocytes displays distinct calcium oscillatory patterns.

Electrophysiological study performed with the voltage clamp technique was used to examine the intracellular calcium pathway activated by tyrosine kinase receptor members. Three FGF receptors from Pleurodeles PR1, PR3, PR4, homologs to human receptors, and the human EGF receptor were expressed in Xenopus oocytes. Under FGF1, FGF2 and FGF4 stimulation, PR1 and PR3 display a one phase inward chloride calcium dependent current superimposed by sustained oscillations, whereas PR4 did not show any oscillations. These currents were dependent on intracellular calcium mobilisation, as the responses were reduced by caffeine (10 mM). Solely PR4 responses were affected by an extracellular calcium depleted solution suggesting the involvement of concomitant extracellular and intracellular calcium intervention in the calcium chloride current, whereas PR1 and PR3 did not. Under EGF stimulation, the EGF receptor elicits a two component inward current composed of an undelayed rapid transient dependent on intracellular calcium store recruitment followed by a second slower current dependent on calcium influx. The specific pattern and amplitude of the calcium oscillations induced by the combinatorial action of growth factors on their receptors could be relevant in numerous calcium dependent cell functions.

Rat yolk sac explants as a system for studying the regulation of endodermal genes: down-regulation of the alpha-fetoprotein gene by dexamethasone and phorbol ester.

The visceral yolk sac is a fetal membrane with essential placental functions. It is the major site of synthesis of alpha-fetoprotein (AFP), the most abundant plasma protein in the fetus. We developed a system of rat yolk sac explants in serum-free culture medium to study the regulation of endodermal gene expression in yolk sac. The explanted yolk sac tissues retained their double-sided morphology for up to 48 hours. The epithelial cells of both layers remained tightly joined on a basement membrane as seen by light and electron microscopy. This probably accounts for the continued expression of several endodermal cell-specific markers. The levels of mRNA encoding AFP, vitamin D-binding protein (DBP), hepatocyte nuclear factor 1alpha and beta transcription factors did not change during the 48-hour culture period. This reflects the stability of the differentiation state of the yolk sac endodermal cells. Dexamethasone and phorbol ester (TPA) specifically reduced the AFP mRNA level without affecting that of DBP. This suggests that these transduction pathways are functional in the yolk sac during this period of gestation and could be involved in the physiological down-regulation of AFP gene expression before birth. All these results show that this serum-free culture of rat yolk sac explants is a valuable system for further investigating the action of natural compounds and pharmacological drugs on endodermal gene expression during the embryonic and fetal periods.

Ultrastructural localization of intracellular calcium stores in Xenopus ovarian follicles as revealed by cytochemistry and X-ray microanalysis.

The ultrastructural localization of calcium in full-grown ovarian follicles of Xenopus laevis was demonstrated after fixation in the presence of fluoride ions and by means of energy dispersive X-ray microanalysis. In hormonally untreated follicles (prophase I-arrested oocytes), two calcium sites were detected: follicle cells and oocyte pigment granules. In follicle cells, calcium containing deposits were preferentially associated with macrovilli, which ended by gap junctions. In human chorionic gonadotropin treated follicles (meiotically reinitiated oocytes), deposits were only seen in follicle cells. This is the first report of the cytochemical detection of intracellular Ca2+ in follicle cells of amphibians. The possible involvements of these Ca2+ stores in mediating the hormonal control of meiotic maturation are discussed.

Ca2+ oscillations induced by fibroblast growth factor 2 in Xenopus oocytes expressing fibroblast growth factor receptors.

Double electrode voltage clamp technique was used to follow precisely the calcium signalling pathway activated by FGF receptors from a normal and a carcinogenous cell environment. Functional FGF receptors were expressed in Xenopus oocytes following either the injection of PFR1 cRNA from Pleurodeles, an homologue of the human FGFR1 mRNA, or breast cancer MCF7 cells total mRNA. Cytosolic calcium oscillations were monitored through the endogenous Ca(2+)-dependent Cl- channel activity from both RNA injected systems, under FGF2 treatment. The Ca(2+)-dependent Cl- channel was demonstrated using the Cl- channel blocker SITS (250 microM) and by the determination of the reversal potential of the Cl- ions close to -20 mV. The FGF2-evoked Ca(2+)-dependent Cl- current was abolished by external application of genistein (10 microM, tyrosine kinase inhibitor), neomycin (10 mM, phosphatidylinositol turnover inhibitor), caffeine (10 mM, inhibitor of Ins(1,4,5)P3-mediated release of intracellular calcium), and injection of BAPTA (50 microM, calcium chelator) or heparin (2 micrograms/ml, inhibitor of the binding of Ins(1,4,5)P3). The recorded current was independent of extracellular Ca2+ but involved tyrosine kinase phosphorylation and intracellular Ins(1,4,5)P3 sensitive stores. External application of heparin enhanced the oscillatory Ca2+ rise, suggesting a role for the heparan sulfates in the regulatory mechanism of the FGF receptors. The similarities in the Ca(2+)-dependent Cl- current obtained in PFR1 and total MCF7 FGF receptors expressing oocytes are discussed.

Endogenous DHP-sensitive Ca(2+) channels in Pleurodeles oocytes.

The double electrode voltage-clamp technique was used to study voltage-dependent Ca(2+) channels in Pleurodeles oocytes. From a holding potential of -80 mV, Ba-current (IBa) (recorded in Cl-free solution, Ba(2+ = 40 mM) activated at -36.7 +/- 4 mV, peaked at -11.6 +/- 4 mV and reversed at 55 +/- 7 mV (n = 24). This current activated slowly (rise time was 0.98 +/- 0.2 s;n = 14 at -10 mV) and was not inactivated. Cadmium (Cd(2+), 500 microM) completely inhibited I(Ba). The effect of Cd(2+) was dose-dependent (EC(50) = 37 +/- 5 microM; n = 5). Moreover, IBa was insensitive to omega-conotoxin (10 microM) but interestingly this I(Ba) displayed dihydropyridine (DHP) sensitivity. Bay K 8644 (5 microM), a DHP activator, increased the peak current amplitude in a dose-dependent manner (EC(50) = 5.9 +/- 0.6 microM; n = 10) and shifted the threshold and the maximum of current/voltage relationship towards negative potentials by -10 mV. Nifedipine (5 microM), a DHP antagonist, decreased I(Ba) by 80% at HP of -80 mV (EC(50) = 1.2 +/- 0.2 microM; n = 6). We concluded that Pleurodeles oocytes possess High-Voltage Activated Ca(2+) channels with properties similar to L-type Ca(2+) channels.

Extracellular matrix of the regeneration chamber and plasma membranes of the epidermis during leg regeneration in an insect Carausius morosus.

An extracellular matrix (ECM) is found in the regeneration chamber during leg regeneration in the stick insect Carausius morosus. The material which surrounds the regenerate is organised into fibrils and it includes proteins distributed in a hydrated polysaccharide gel. The compounds which can be demonstrated are chitin unlinked to proteins, glycoproteins and unsulfated glycosaminoglycans such as hyaluronic acid and/or chondroitin. Molecules related to vertebrate fibronectin and collagen IV were observed on the apical surface of the epidermal cells of the regenerate. During leg regeneration, the basal lamina which normally secures the cells to each other is absent. However a condensation of material on the regenerate epidermal cells ensures their cohesion. The extracellular matrix in the regeneration chamber must be secreted by the cells of retracted epidermis and then by the epidermal cells of the regenerate, until these cells are able to secrete the cuticle for the next instar. The analysis of the epidermal cell surface does not seem to show any localization or any changes during the development of the regenerate.