Biomedical subjects

J Boonstra

Publications and source records attributed to J Boonstra.

At least 37 records · Page 2Linked to original sources

PubMed1999

Growth factor-induced signal transduction in adherent mammalian cells is sensitive to gravity.

Epidermal growth factor (EGF) activates a well-characterized signal transduction cascade in a wide variety of cells. This activation leads to increased cell proliferation in most cell types. Among the early effects evoked by EGF are receptor clustering, cell rounding, and early gene expression. The influence of gravity on EGF-induced EGF receptor clustering and gene expression as well as on actin polymerization and cell rounding have been investigated in adherent A431 epithelial cells with the use of sounding rockets to create microgravity conditions. EGF-induced c-fos and c-jun expression decreased in microgravity. This was caused by alteration of the EGF receptor and protein kinase C-mediated signal transduction pathways. In contrast, neither the binding of EGF to the receptor nor the receptor clustering were changed under microgravity conditions. Because cell morphology was also modulated under microgravity conditions, and the growth factor-induced signal transduction cascades have been demonstrated to be linked to the actin microfilament system, it is tempting to suggest that the actin microfilament system constitutes the gravity-sensitive cell component.

Actins↗

PubMed1999

Function of trehalose and glycogen in cell cycle progression and cell viability in Saccharomyces cerevisiae.

Trehalose and glycogen accumulate in Saccharomyces cerevisiae when growth conditions deteriorate. It has been suggested that aside from functioning as storage factors and stress protectants, these carbohydrates may be required for cell cycle progression at low growth rates under carbon limitation. By using a mutant unable to synthesize trehalose and glycogen, we have investigated this requirement of trehalose and glycogen under carbon-limited conditions in continuous cultures. Trehalose and glycogen levels increased with decreasing growth rates in the wild-type strain, whereas no trehalose or glycogen was detected in the mutant. However, the mutant was still able to grow and divide at low growth rates with doubling times similar to those for the wild-type strain, indicating that trehalose and glycogen are not essential for cell cycle progression. Nevertheless, upon a slight increase of extracellular carbohydrates, the wild-type strain degraded its reserve carbohydrates and was able to enter a cell division cycle faster than the mutant. In addition, wild-type cells survived much longer than the mutant cells when extracellular carbon was exhausted. Thus, trehalose and glycogen have a dual role under these conditions, serving as storage factors during carbon starvation and providing quickly a higher carbon and ATP flux when conditions improve. Interestingly, the CO2 production rate and hence the ATP flux were higher in the mutant than in the wild-type strain at low growth rates. The possibility that the mutant strain requires this steady higher glycolytic flux at low growth rates for passage through Start is discussed.

Biomass↗

PubMed1999

Growth factor signalling.

Signalling between cells in the developing vertebrate embryo is essential for normal embryonic development. In the mid 1970's, signal transduction research started at the Hubrecht Laboratory with special emphasis on analysis of the signalling mechanisms that direct cell proliferation and differentiation. The introduction of in vitro model systems contributed tremendously to the success of the signal transduction research at the Hubrecht Laboratory. Initially neuroblastoma cell lines, and later embryonal carcinoma and embryonal stem cells played an important role in identification of the molecular key players in developmental signalling. For instance, embryonal carcinoma cells were used to identify and characterise polypeptide growth factors. Growth factor signalling research was extended to analysis of growth factor receptor activation. Moreover, the second messenger systems that are linked to growth factor receptors were studied, as well as the nuclear responses to growth factor receptor activation. Finally, the role of growth factor signalling in differentiation was established using embryonal carcinoma cells. Here, we will review work that was characteristic for the growth factor receptor signalling research that was done at the Hubrecht Laboratory between 1980 and the early 1990's.

Animals↗

PubMed1998

Actin polymerization is required for negative feedback regulation of epidermal growth factor-induced signal transduction.

Epidermal growth factor (EGF) induces rapid actin filament assembly in the membrane skeleton of a variety of cells. To investigate the significance of this process for signal transduction, actin polymerization is inhibited by dihydrocytochalasin B (CB). CB almost completely abolishes EGF-induced actin polymerization, as assessed by quantitative confocal laser scanning microscopy. Under these conditions, EGF induces enhanced EGF receptor (EGFR) tyrosine kinase activity, as well as superinduction of the c-fos proto-oncogene. These data suggest that EGF-induced actin polymerization may be important for negative feedback regulation of signal transduction by the EGFR. The phosphorylation of Thr654 by protein kinase C (PKC) is a well-characterized negative feedback control mechanism for signal transduction by the EGFR tyrosine kinase. A synthetic peptide, corresponding to the regions flanking Thr654 of the EGFR, is used to analyze EGF stimulated PKC activity by incorporation of 32P into the peptide. Cotreatment of cells with CB and EGF results in a complete loss of EGF-induced phosphorylation of the peptide. These data suggest that actin polymerization is obligatory for negative feedback regulation of the EGFR tyrosine kinase through the C-kinase pathway.

Actins↗

PubMed1998

Glucose repression in Saccharomyces cerevisiae is related to the glucose concentration rather than the glucose flux.

Glucose plays an important regulatory role in the yeast Saccharomyces cerevisiae, which is mostly reflected at the transcriptional level by glucose repression. The signal that initiates glucose repression is unknown, but data indicate that it is located at or above the level of glucose 6-phosphate, suggesting the involvement of either the intracellular or extracellular glucose concentration or the glucose flux in triggering glucose repression. We have investigated the role of the glucose flux and the extracellular glucose concentration in glucose repression by growing the cells in continuous culture under nitrogen limitation. By a step-wise increase in the glucose feed concentration, the glucose flux and extracellular glucose concentrations were modulated in an accurate way. Furthermore, the glucose flux and glucose concentrations were modulated independently of each other by increasing the dilution rate or by the use of fructose as a substrate. Using these approaches we demonstrate that glucose repression is related to the extracellular (or intracellular) glucose concentration rather than the glucose flux. At external glucose concentrations lower than 14 mM, glucose repression of SUC2 gene transcription was not triggered, whereas glucose repression of this gene was activated when the glucose concentration exceeded 18 mM. A comparable effect was observed for the glucose-repressible carbon source fructose.

Dose-Response Relationship, Drug↗

PubMed1998

Impaired secretion of a hydrophobic cutinase by Saccharomyces cerevisiae correlates with an increased association with immunoglobulin heavy-chain binding protein (BiP).

This study focuses on the different efficiencies of secretion of two fungal cutinases by Saccharomyces cerevisiae, a wild-type cutinase (CY000) and a hydrophobic mutant cutinase (CY028). Both cutinases are placed under control of the GAL7 promoter, by which the expression levels can be regulated. Wild-type cutinase was secreted at up to 25 mg per g (dry weight), while CY028 was secreted at a level of 2 mg per g (dry weight); this difference is nearly independent of the expression level. Pulse-chase experiments revealed that whereas CY000 cutinase is secreted, CY028 is irreversibly retained in the cell. Immunogold labelling followed by electron microscopy revealed colocalization of CY028 with immunoglobulin heavy-chain binding protein (BiP) in the endoplasmic reticulum (ER). The increase of wild-type cutinase expression did not result in higher levels of the molecular chaperone BiP, but BiP levels are raised by increased induction of the hydrophobic mutant cutinase. Immunoprecipitation studies showed that in contrast to the wild-type cutinase, the hydrophobic mutant cutinase interacts with BiP. These results indicate that the introduction of two exposed hydrophobic patches in cutinase results in a higher affinity for BiP which might cause the retention of this mutant cutinase in the ER.

Blotting, Western↗

PubMed1998

Identification of connexin43 as a functional target for Wnt signalling.

Wnt mediated signal transduction is considered to regulate activity of target genes. In Xenopus embryos, ectopic Wnt1 and Wnt8 expression induces gap-junctional communication. During murine brain formation, Wnt1 and the gap-junctional protein connexin43 (Cx43) are co-expressed at the mid/hindbrain border, while interference with Wnt1 or Cx43 expression during embryogenesis leads to severe brain defects in the mid/hindbrain region. In PC12 cells, Wnt1 expression leads to an apparent increase in cell-cell adhesion. We investigated the effects of Wnt1 overexpression on gap-junctional communication in PC12 cells. Wnt1 expressing clones displayed an increased electrical and chemical coupling. This coincides with an increased expression of Cx43 mRNA and protein, while other connexins, Cx26, Cx32, Cx37, Cx40 and Cx45, were not up-regulated. Also, induction of Wnt1 expression in a mammary epithelial cell line leads to an increase in gap-junctional communication and Cx43 protein expression. In transient transactivation assays in P19 EC cells we found that Wnt1 and Li+, an ion that mimics Wnt signalling, increased transcription from the rat Cx43 promoter, potentially via TCF/LEF binding elements, in a pathway separate from cAMP-induced Cx43 transactivation. The results demonstrate that Cx43 acts as a functional target of Wnt1 signalling, and Cx43 expression can be regulated by Wnt1 at the transcriptional level. Our data suggest that Wnt1-induced cell fate determination is likely to involve regulation of gap-junctional communication.

Animals↗

PubMed1997

Epidermal growth factor-induced activation and translocation of c-Src to the cytoskeleton depends on the actin binding domain of the EGF-receptor.

In the epidermal growth factor (EGF)-receptor signal transduction cascade, the non-receptor tyrosine kinase c-Src has been demonstrated to become activated upon EGF stimulation. In this paper we show that c-Src associates with the cytoskeleton and co-isolates with actin filaments upon EGF treatment of NIH-3T3 cells transfected with the EGF receptor. Immunofluorescence studies using CLSM show colocalization of F-actin and endogenous c-Src predominantly around endosomes and not on stress fibers and cell-cell contacts. Stimulation of EGF receptor-transfected NIH-3T3 cells with EGF induces an activation and translocation of c-Src to the cytoskeleton. These processes depend upon the presence of the actin binding domain of the EGF-receptor since in cells that express EGF-receptors lacking this domain, EGF fails to induce an activation and translocation to the cytoskeleton of c-Src. These data suggest a role for the actin binding domain of the EGF-receptor in the translocation of c-Src.

3T3 Cells↗

PubMed1997

The actin binding domain of the epidermal growth factor receptor is required for EGF-stimulated tissue invasion.

NIH-3T3 fibroblasts expressing epidermal growth factor receptors (EGFRs) lacking the actin binding domain (ABD) were analyzed for their EGF-induced capacity to invade a bone marrow stromal cell (BMSC) monolayer. The fibroblasts display a reduction in the percentage of cytoskeleton-associated EGFRs. Furthermore, EGF-induced tyrosine kinase activity is unaffected by the mutation. Cells expressing the mutant EGFRs hardly invade a BMSC monolayer upon EGF stimulation in contrast to cells expressing wild-type EGFRs. Using the same cells no difference was observed in PDGF-induced invasion, which ligand was as potent in both cell types as EGF was in wild-type cells. Inhibition of both the phosphatidyl inositol-3-kinase (PI-3-K) and lipoxygenase pathways in wild-type cells mimicked the effect of the ABD deletion. Our results point to an important role for the ABD of the EGFR in EGF-induced tissue invasion.

3T3 Cells↗

PubMed1997

Identification of an intracellular domain of the EGF receptor required for high-affinity binding of EGF.

Although all EGF receptors in EGF receptor-expressing cells are molecularly identical, they can be subdivided in two different classes that have either a high or a low affinity for EGF. Specifically the high-affinity class is associated with filamentous actin. To determine whether the interaction of the EGF receptor with actin induces its high-affinity state, we studied EGF-binding properties of an EGF receptor mutant that lacks the actin-binding site. Interestingly, we found that cells expressing this mutant receptor still display both high- and low-affinity classes of EGF receptors, indicating that the actin-binding domain does not determine the high-affinity binding state. By further mutational analysis we identified a receptor domain, within the tyrosine kinase domain, that regulates the affinity for EGF.

3T3 Cells↗

PubMed1997

Effects of different carbon fluxes on G1 phase duration, cyclin expression, and reserve carbohydrate metabolism in Saccharomyces cerevisiae.

By controlled addition of galactose to synchronized galactose-limited Saccharomyces cerevisiae cultures, the growth rate could be regulated while external conditions were kept constant. By using this method, the G1 phase duration was modulated and expression of cell cycle-regulated genes was investigated. The expression of the cyclin genes CLN1 and CLN2 was always induced just before bud emergence, indicating that this event marks the decision to pass Start. Thus, G1 phase elongation was not due to a slower accumulation of the CLN1 and CLN2 mRNA levels. Only small differences in CLN3 expression levels were observed. The maximal SWI4 expression preceded maximal CLN1 and CLN2 expression under all conditions, as expected for a transcriptional activator. But whereas SWI4 was expressed at about 10 to 20 min, before CLN1 and CLN2 expression at high growth rates, this time increased to about 300 min below a particular consumption rate at which the G1 phase strongly elongated. In the slower-growing cultures, also an increase in SWI6 expression was observed in the G1 phase. The increase in G1 phase duration below a particular consumption rate was accompanied by a strong increase in the reserve carbohydrate levels. These carbohydrates were metabolized again before bud emergence, indicating that below this consumption rate, a transient increase in ATP flux is required for progression through the cell cycle. Since Start occurred at different cell sizes under different growth conditions, it is not just a certain cell size that triggers passage through Start.

Carbohydrate Metabolism↗

PubMed1997

Expression of the parathyroid hormone-related peptide gene in retinoic acid-induced differentiation: involvement of ETS and Sp1.

Differentiation of P19 embryonal carcinoma (EC) and embryonal stem (ES)-5 cells with retinoic acid (RA) induces expression of PTH-related peptide (PTHrP) mRNA. In this study we have characterized a region between nucleotide (nt) -88 and -58 relative to the transcription start site in the murine PTHrP gene that was involved in this expression. Sequence analysis identified two partially overlapping binding sites for the Ets family of transcription factors and an inverted Sp1-binding site. Two major specific bands were detected in a bandshift assay using an oligonucleotide spanning nt -88 and -58 as a probe and nuclear extracts from both undifferentiated and RA-differentiated P19 EC cells. The lower complex consisted of Ets-binding proteins as demonstrated by competition with consensus Ets-binding sites, while the upper complex contained Sp1-binding activity as demonstrated by competition with consensus Sp1-binding sites. The observed bandshift patterns using nuclear extracts of undifferentiated or RA-differentiated P19 cells were indistinguishable, suggesting that the differentiation-mediated expression was not caused by the induction of expression of new transcription factors. Mutations in either of the Ets-binding sites or the Sp1-binding site completely abolished RA-induced expression of PTHrP promoter reporter constructs, indicating that the RA effect was dependent on the simultaneous action of both Ets- and Sp1-like activities. Furthermore, these mutations also abolished promoter activity in cells that constitutively expressed PTHrP mRNA, suggesting a central role for the Ets and Sp1 families of transcription factors in the expression regulation of the mouse PTHrP gene.

Animals↗

PubMed1997

Ultrastructural localization of cPLA2 in unstimulated and EGF/A23187-stimulated fibroblasts.

The 85 kDa cytosolic phospholipase A2 is the key enzyme in the release of arachidonic acid. To gain insight into cytosolic phospholipase A2 action in mitogen-activated cells, the localization of the phospholipase was investigated in fibroblasts upon stimulation with epidermal growth factor and the calcium ionophore A23187. By the use of indirect immunofluorescence microscopy, staining of endogenous cytosolic phospholipase A2 resulted in a punctate labeling pattern randomly distributed throughout the cytoplasm of the cell. Immunogold electron microscopy revealed that this punctate labeling pattern exhibited the presence of the 85 kDa phospholipase A2 in small clusters. These clusters were found in the cytosol in the vicinity of all organellar membranes, except for the Golgi system. The enzyme showed no preference for the nuclear envelope, the endoplasmic reticulum or the plasma membrane. Stimulation of cells with epidermal growth factor or A23187 or both did not change the punctate immunofluorescence labeling pattern. Furthermore, a similar labeling pattern was observed by the artificial introduction of extremely low or high intracellular calcium concentrations. Even by electron microscopy, translocation of cytosolic phospholipase A2 to membranes was not observed after stimulation of cells with epidermal growth factor and A23187. From these results it is concluded that cytosolic phospholipase A2 is localized in clusters close to membranes in stimulated as well as unstimulated fibroblasts, without preference for a specific organellar membrane.

3T3 Cells↗

PubMed1996

Kinetic analysis of hexose uptake in Saccharomyces cerevisiae cultivated in continuous culture.

As the expression of the hexose transporters in Saccharomyces cerevisiae is regulated by the external fructose concentration, in vivo fructose transport was investigated at a number of different but constant fructose concentrations in a continuous culture. The in vitro uptake rate and kinetic parameters were studied by uptake of 14C-labelled fructose. From Eadie-Hofstee plots, based on the results of the in vitro uptake experiment, K(m) and Vmax for the different steady-state conditions were determined. These results demonstrate that as fructose concentrations increase, fructose consumption rates and K(m) increase and Vmax slightly decreases. However, remarkably a large discrepancy was observed between in vivo determined consumption rates in the continuous culture and the in vitro uptake rates from the 14C-labelled fructose uptake experiment on cells from an identical steady-state condition. In every condition the in vivo consumption rate was approx. 2-3 times higher than the in vitro uptake rate. A comparable discrepancy was observed for glucose uptake rates; however, in vivo and in vitro galactose uptake rates were identical. These observations imply that kinetic analyses of sugar transport mechanisms can only be performed correctly when accompanied by a comparison with in vivo determined consumption rates.

Carbon Radioisotopes↗

PubMed1996

Maximal epidermal growth-factor-induced cytosolic phospholipase A2 activation in vivo requires phosphorylation followed by an increased intracellular calcium concentration.

The 85 kDa cytosolic phospholipase A2 (cPLA2) preferentially catalyses the hydrolysis of arachidonic acid from the sn-2 position of phospholipids. cPLA2 can be activated by extracellular stimuli such as thrombin, platelet-derived growth factor and epidermal growth factor (EGF): A full activation of cPLA2 requires an increase of intracellular Ca2+ concentration and phosphorylation on Ser-505 by mitogen-activated protein (MAP) kinase. Because EGF can provoke an increase in intracellular [Ca2+] ([Ca2+]i) and activation of MAP kinase, we investigated the role of these pathways in EGF-induced activation of cPLA2. Characterization of two cell lines expressing different numbers of EGF receptors (HERc13 and HER14) revealed that both were activating MAP kinase in response to EGF, but only HER14 responded with an increase in [Ca2+]i. In this study we used both cell lines as a tool to clarify the role of each pathway in cPLA2 activation. We show that EGF stimulates cPLA2 activity in both cell lines in vitro as measured in cytosolic fractions, but only in HER14 in vivo as measured by 3H release from cells prelabelled with [3H]arachidonic acid. This latter activation can be restored in HERc13 cells by the addition of the ionophore A23187. Interestingly, this effect is only observed when EGF stimulation precedes A23187 addition. The phosphorylation of MAP kinase, however, was identical under identical conditions. We conclude that a maximal cPLA2 activation by EGF requires both, and in this order: MAP kinase activation followed by a rise in [Ca2+]i concentration.

3T3 Cells↗

PubMed1996

The Cdc25 protein of Saccharomyces cerevisiae is required for normal glucose transport.

The essential CDC25 gene product of Saccharomyces cerevisiae is the most upstream known component of the RAS/adenylate cyclase pathway. Cdc25 is a GTP-exchange protein involved in activating RAS in response to fermentable carbon sources. In this paper it is reported that the Cdc25 protein, in addition to its stimulatory role in the RAS/adenylate cyclase pathway, regulates glucose transport. Continuous culture studies and glucose uptake experiments showed that the cdc25-1 and the cdc25-5 temperature-sensitive mutants exhibit decreased glucose uptake activity at the restrictive temperature under both repressed and derepressed conditions as compared to the wild-type strain. Because the cdc25-1 mutant is not impaired in its cAMP metabolism, it is concluded that this effect on glucose transport is independent of cAMP levels. Furthermore, it is shown that the decrease in glucose uptake activity is not due to a decrease in protein synthesis or to an arrest in the G1 phase of the cell cycle. In addition to a defect in glucose uptake, the cdc25-5 mutant strain exhibited differences in glucose metabolism, probably due to the decreased cAMP level and hence decreased protein kinase A activity. Because the Cdc25 protein is localized at the membrane, these results indicate that Cdc25 is directly involved in glucose transport and may be in direct contact with the glucose transporters.

Adenosine Triphosphate↗

PubMed1996

Parathyroid hormone related peptide mRNA expression during murine postimplantation development: evidence for involvement in multiple differentiation processes.

In this study we describe the spatio-temporal expression of Parathyroid Hormone related Peptide (PTHrP) mRNA during murine postimplantation development from day 5.5 post coitum (pc) until day 12.5 pc. From day 5.5 pc and onwards PTHrP mRNA was detected in the trophoblast. In addition, at day 5.5 and 6.5 pc epithelial cells of the antimesometrial crypt and cells of the inner zone of the decidua directly adjacent to the implanted embryo expressed PTHrP mRNA. This supported a previous model in which parietal endoderm formation is regulated by a paracrine mechanism involving PTHrP expressing trophoblast cells and receptor expressing extra-embryonal endoderm cells. The first embryonal PTHrP mRNA expression was detected in the roof of the hindbrain at gestation day 10.5 pc. From day 11.5 pc and onwards PTHrP mRNA was detected in the otic vesicle, the semilateral channels, the roof of the hindbrain and later in the choroid plexus, in epithelial cells of the lung and heart ventricle, mesenchymal cells lining the nasal pit, the dermis of the snout and at all sites of endochondral bone formation. The widespread expression of PTHrP mRNA during embryogenesis in extra-embryonic and embryonic tissues suggests the involvement of the peptide in multiple growth and differentiation processes.

Animals↗

PubMed1996

EGF-receptor RNA metabolism in the nucleus of A431 cells.

Epidermal growth factor (EGF) receptor RNA has been shown to be localized around nucleoli in the nucleus of A431 cells (Sibon et al., Histochemistry 101, 223-232 (1994)). Here we have studied the functional implication of this localization. Inhibition of transcription by alpha-amanitin did not influence the localization and amount of EGF-receptor RNA around the nucleolus, indicating that these RNAs represent mainly completed transcripts. Localization of the EGF-receptor genes in A431 cells by in situ hybridization revealed that the majority of the receptor gene clusters are located at the periphery of the nucleus. Next to this virtually all cells studied contain at least one gene cluster in the vicinity of the nucleolus. From these data, it is tempting to suggest that EGF-receptor gene transcription occurs around the nucleolus. In order to obtain information on the site of EGF-receptor RNA splicing, the localization of exon and intron sequences of the EGF-receptor transcripts was studied using a new electron microscopical approach. These labeling studies revealed that both intron and exon sequences were present at the same site around the nucleolus. In addition, exon sequences were also located, around nucleolus separate from intron sequences. All together, these studies suggest that transcription and splicing of the EGF-receptor transcript occurs at the same defined site around the nucleolus in A431 cells.

3T3 Cells↗