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E J van Zoelen

Publications and source records attributed to E J van Zoelen.

At least 19 recordsLinked to original sources

Clubbed fingers: the claws we lost?

Clubbed digits resemble the human embryonic fingers and toes, which look like the digits of a claw. Clubbed digits, thus, may represent the return of the embryonic claw and may even represent the claws man has lost during evolution, if ontogenesis really recapitulates phylogenesis. We put forward the hypothesis that secondary clubbing, like gynecomastia, is caused by a pathologic condition, which alters hormone levels in the blood, leading to the activation of 'dormant' genes, resulting in the development of an organ. However, the nature of the diseases that cause clubbing suggests that these hormones may actually be cytokines, acting as hormones. The nature of these cytokines is not known. They may be identified by comparing their blood levels or the combination of their blood levels to the presence or absence of clubbing, but also to the degree of clubbing and its disappearance after treatment of the primary disease.

Animals↗

Fenamates: a novel class of reversible gap junction blockers.

The effect of fenamates on gap junctional intercellular communication was investigated in monolayers of normal rat kidney (NRK) fibroblasts and of SKHep1 cells overexpressing the gap junction protein connexin43 (Cx43). Using two different methods to study gap junctional intercellular communication, single electrode voltage-clamp step response measurements and dye microinjection, we show that fenamates are reversible blockers of Cx43-mediated intercellular communication. After adding fenamates to a confluent monolayer of electrically coupled NRK fibroblasts, the voltage step-induced capacitive current transient changed from a transient characteristic for charging multiple coupled cell capacitances to one characteristic for a single cell in isolation. The capacitance of completely uncoupled cells was 19.7 +/- 1.0 pF (mean +/- S.E.M.; n = 11). Junctional conductance between the patched cell and the surrounding cells in the monolayer changed from >140.7 +/- 9.6 nS (mean +/- S.E.M.; n = 14) to <1.4 +/- 0.4 nS (mean +/- S.E.M.; n = 11) after uncoupling. Electrical coupling could be restored to >51.8 +/- 4.2 nS (mean +/- S.E.M.; n = 11) by washout of the fenamates. Voltage-clamp step response measurements showed that the potency of fenamates in inhibiting electrical coupling decreases in the order meclofenamic acid > niflumic acid > flufenamic acid. The half-maximal concentration determined by dye-coupling experiments was 25 and 40 microM for meclofenamic acid and flufenamic acid, respectively. Inhibition of gap junctional communication by fenamates did not involve changes in intracellular calcium or pH, and was unrelated to protein kinase C activity or an inhibition of cyclooxygenase activity. Voltage-clamp step response measurements in confluent monolayers of SKHep1 cells that had been stably transfected with Cx43 revealed that fenamates are potent blockers of Cx43-mediated intercellular communication. In conclusion, fenamates represent a novel class of reversible gap junction blockers that can be used to study the role of Cx43-mediated gap junctional intercellular communication in biological processes.

Cell Communication↗

Superagonistic activation of ErbB-1 by EGF-related growth factors with enhanced association and dissociation rate constants.

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) are mitogenic hormones that exert their activity primarily by binding to the EGF receptor, also known as ErbB-1. We have recently characterized a set of EGF/TGFalpha chimeric molecules with similar high affinity for ErbB-1 as EGF and TGFalpha and shown that three of these chimeras induce mitogenic cell stimulation at already a 10-fold lower concentration than their wild-type counterparts (Lenferink, A. E., Kramer, R. H., van Vugt, M. J., Königswieser, M., DiFiore, P. P., van Zoelen, E. J., and van de Poll, M. L. (1997) Biochem. J. 327, 859-865). In the present study we show that these so-called superagonistic chimeras do not differ from EGF and TGFalpha in their ability to induce ErbB-1 tyrosine phosphorylation but are considerably more potent in activation of mitogen-activated protein kinase phosphorylation. Direct cell binding studies and analysis of ligand-receptor interaction by surface plasmon resonance measurements revealed that both the association rate constant (k(on)) and the dissociation rate constant (k(off)) of these superagonists is 3-5-fold higher in comparison with the wild-type ligands and nonsuperagonistic chimeras. These data indicate that the dynamic on and off rate constants for receptor binding may be more specific parameters for determining the mitogenic activity of peptide hormones than their constants for equilibrium receptor binding.

3T3 Cells↗

Non-linear antigenic regions in epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) studied by EGF-TGF alpha chimaeras.

With the help of 16 chimaeras between human epidermal growth factor (hEGF) and human transforming growth factor alpha (hTGF alpha), a detailed analysis was performed on the epitope recognized by two polyclonal antibodies raised against hEGF, and one polyclonal antibody raised against hTGF alpha. All three antibodies recognized essentially the same antigenic site, a non-linear and conformation-dependent sequence that is located near the second and fourth disulphide-bonded cysteines and that includes the start of the B-loop beta-sheet. The epitope recognized by the anti-hEGF antibodies was further characterized using 8 chimaeras between hEGF and an EGF-repeat from Drosophila Notch and was found to include Met(21), Ala(30) and Asn(32). All three polyclonal antibodies were able to neutralize the biological activity of the respective growth factor when tested on 32D murine haematopoietic progenitor cells transfected with ErbB-1, indicating that the receptor binding domain is shielded upon binding of the antibody.

Amino Acid Sequence↗

Antiestrogens specifically up-regulate bone morphogenetic protein-4 promoter activity in human osteoblastic cells.

Bone morphogenetic protein-4 (BMP-4) plays an important role in the onset of endochondral bone formation in humans, and a reduction in BMP-4 expression has been associated with a variety of bone diseases. Here we describe, by transient transfection assays in bone cells, that the human BMP-4 promoter recently characterized in our laboratory can be stimulated specifically by antiestrogens but not by estrogens or other steroid hormones. This activity is dependent on the presence of the estrogen receptor (ER)-alpha, although the promoter lacks a consensus estrogen-responsive element. No activity was observed in the presence of ERbeta, but synergy was observed when both ER subtypes were cotransfected. The observed stimulation of BMP-4 promoter activity by antiestrogens appeared bone cell specific and was reversed upon addition of estrogens. Since antiestrogens are known to be effective in hormone replacement therapies for postmenopausal women, this observation may help to develop new strategies for treatment and prevention of osteoporosis.

Adenocarcinoma↗

Expression of transforming-growth-factor (TGF)-beta receptors and Smad proteins in glioblastoma cell lines with distinct responses to TGF-beta1.

A panel of 6 human glioma cell lines was examined for TGF-beta1 responsiveness. U-178 MG and U-251 MG AgCl1 were significantly inhibited by TGF-beta1, while U-343 MGa 31L and U-343 MGa 35L were potently stimulated to proliferate. TGF-beta1 induced endogenous PAI-1 protein synthesis, Smad binding element/(CAGA)12-luciferase-reporter activity, as well as mRNA expression of Smad6 and Smad7 in all gliomas. Interestingly, TGF-beta1 differentially stimulated or inhibited the expression of TbetaR-I and TbetaR-II mRNA in the gliomas. Affinity cross-linking studies using 125I-TGF-beta1 revealed that the gliomas expressed TGF-beta-type-I(TbetaR-I) and -type-II(TbetaR-II) receptors, although binding to TbetaR-II in U-343 MGa 31L and U-251 MG AgCl1 was low to undetectable. Smad2 protein was abundantly present in U-178 MG, U-343 MG, and U-343 MGa 35L, while Smad3 was readily detectable in U-178 MG, U-343 MG, U-343 MGa 35L and U-251 MG AgCl1. In all gliomas, TGF-beta1 induced phosphorylation of Smad2. The level to which TGF-beta1 could activate the pathway leading to induction of the (CAGA)12-luciferase reporter seemed to correlate to the expression levels of TGF-beta receptors, Smad3 and Smad4 proteins. However, despite the plethora of data regarding TGF-beta1 signalling in the different glioma cell lines, the mechanism underlying the differential growth effects mediated by TGF-beta1 is still unclear. The results suggest that a complex balance between several components in the TGF-beta signalling pathway controls glioma responsiveness to TGF-beta1, and extend reports indicating that distinct signal transduction pathways are involved in growth inhibition and other cellular responses.

DNA, Neoplasm↗

Functional antagonism between activin and osteogenic protein-1 in human embryonal carcinoma cells.

Activin A and osteogenic protein-1 (OP-1) exerted antagonistic effects on each other's responses on the human Tera-2 embryonal carcinoma cell line. OP-1 dose dependently inhibited activin A-induced activation of p3TP-Lux transcriptional reporter, containing part of the human plasminogen activator inhibitor-1 (PAI-1) promoter, while activin A inhibited OP-1-mediated alkaline phosphatase induction. Approximately equimolar concentrations of both growth factors resulted in 50% inhibition of the respective biological responses. Affinity cross-linking studies using 125I-activin A or 125I-OP-1 followed by receptor-immunoprecipitations revealed that both ligands bound to the activin type II receptor (ActR-II), but recruited different type I receptors. In addition, OP-1 competed with binding of 125I-activin A, and activin A competed with binding of 125I-OP-1 to ActR-II. Transient transfection studies showed that competition between activin A and OP-1 also occurred at the type I receptor (ActR-1) level; constitutively active (CA)-ActR-I inhibited CA-ActR-IB-mediated p3TP-Lux reporter induction. There was no competition between activin A and OP-1 for availability of Smad4, indicating that the concentration of this common signal transducer is not limiting for generating the observed biological responses. Overexpression of ActR-II abolished the inhibitory effect of OP-1 on activin A-induced p3TP-Lux activation and, surprisingly, led to OP-1-induced transcriptional reporter activity. Whereas the exact mechanism of competition is unclear, the role of ActR-II in the competition between activin A and OP-1 is discussed in light of the observed interference in downstream signaling by CA-ActR-I and CA-ActR-IB.

Activin Receptors, Type I↗

Homeobox proteins as signal transduction intermediates in regulation of NCAM expression by recombinant human bone morphogenetic protein-2 in osteoblast-like cells.

The role of homeobox genes in signaling of recombinant human bone morphogenetic protein-2 (rhBMP-2) was studied in osteoblast-like cells. Expression of several homeobox genes was decreased by rhBMP-2. The finding that this regulation of homeobox gene expression by rhBMP-2 was not dependent on protein synthesis suggests that homeobox proteins can act as direct intermediates in signal transduction of BMPs. Therefore, we studied the regulation of neural cell adhesion molecule (NCAM), which has previously been described as a target gene of both rhBMP-2 and homeobox proteins. We now show that in osteoblast-like cells, rhBMP-2 inhibits NCAM expression, while HOXC6 increases its expression, both acting via the same region of the promoter. As overexpression of HOXC6 could abolish effects of rhBMP-2 on NCAM promoter activity, these data show for the first time that members of the homeobox gene family may form direct functional intermediates in the signaling mechanism of the TGF-beta superfamily.

Animals↗

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↗

Altered regulation of platelet-derived growth factor receptor-alpha gene-transcription in vitro by spina bifida-associated mutant Pax1 proteins.

Mouse models show that congenital neural tube defects (NTDs) can occur as a result of mutations in the platelet-derived growth factor receptor-alpha gene (PDGFRalpha). Mice heterozygous for the PDGFRalpha-mutation Patch, and at the same time homozygous for the undulated mutation in the Pax1 gene, exhibit a high incidence of lumbar spina bifida occulta, suggesting a functional relation between PDGFRalpha and Pax1. Using the human PDGFRalpha promoter linked to a luciferase reporter, we show in the present paper that Pax1 acts as a transcriptional activator of the PDGFRalpha gene in differentiated Tera-2 human embryonal carcinoma cells. Two mutant Pax1 proteins carrying either the undulated-mutation or the Gln --> His mutation previously identified by us in the PAX1 gene of a patient with spina bifida, were not or less effective, respectively. Surprisingly, Pax1 mutant proteins appear to have opposing transcriptional activities in undifferentiated Tera-2 cells as well as in the U-2 OS osteosarcoma cell line. In these cells, the mutant Pax1 proteins enhance PDGFRalpha-promoter activity whereas the wild-type protein does not. The apparent up-regulation of PDGFRalpha expression in these cells clearly demonstrates a gain-of-function phenomenon associated with mutations in Pax genes. The altered transcriptional activation properties correlate with altered protein-DNA interaction in band-shift assays. Our data provide additional evidence that mutations in Pax1 can act as a risk factor for NTDs and suggest that the PDGFRalpha gene is a direct target of Pax1. In addition, the results support the hypothesis that deregulated PDGFRalpha expression may be causally related to NTDs.

Amino Acid Substitution↗

Identification of the minimal requirements for binding to the human epidermal growth factor (EGF) receptor using chimeras of human EGF and an EGF repeat of Drosophila Notch.

Many proteins contain so-called epidermal growth factor (EGF)-like domains that share the characteristic spacing of cysteines and glycines with members of the EGF family. They are, however, functionally unrelated, despite the fact that the three-dimensional structure of these EGF-like domains, also, is often very similar to that of the EGF receptor agonists. In the present study, we linked an EGF-like repeat from the Drosophila Notch protein to the N- and C-terminal linear tail sequences of human EGF (hEGF), and we showed that this chimera (E1N6E) is unable to bind or activate the hEGF receptor. This recombinant protein was then used as a basic construct for identifying the minimal requirements for high affinity EGF receptor binding and activation. We selectively reintroduced a limited number of important hEGF-derived residues, and by using this unique approach, we were able to make hEGF/Notch chimeras that, compared with wild type hEGF, showed nearly 100% binding affinity and mitogenic activity on HER-14 cells expressing the hEGF receptor.

Animals↗

Differential endocytic routing of homo- and hetero-dimeric ErbB tyrosine kinases confers signaling superiority to receptor heterodimers.

Both homo- and hetero-dimers of ErbB receptor tyrosine kinases mediate signaling by a large group of epidermal growth factor (EGF)-like ligands. However, some ligands are more potent than others, although they bind to the same direct receptor. In addition, signaling by receptor heterodimers is superior to homodimers. We addressed the mechanism underlying these two features of signal tuning by using three ligands: EGF; transforming growth factor alpha (TGFalpha); and their chimera, denoted E4T, which act on cells singly expressing ErbB-1 as a weak, a strong, and a very strong agonist, respectively. Co-expression of ErbB-2, a developmentally important co-receptor whose expression is frequently elevated in human cancers, specifically potentiated EGF signaling to the level achieved by TGFalpha, an effect that was partially mimicked by ErbB-3. Analysis of the mechanism underlying this trans-potentiation implied that EGF-driven homodimers of ErbB-1 are destined for intracellular degradation, whereas the corresponding heterodimers with ErbB-2 or with ErbB-3, dissociate in the early endosome. As a consequence, in the presence of either co-receptor, ErbB-1 is recycled to the cell surface and its signaling is enhanced. This latter route is followed by TGFalpha-driven homodimers of ErbB-1, and also by E4T-bound receptors, whose signaling is further enhanced by repeated cycles of binding and dissociation from the receptors. We conclude that alternative endocytic routes of homo- and hetero-dimeric receptor complexes may contribute to tuning and diversification of signal transduction. In addition, the ability of ErbB-2 to shunt ligand-activated receptors to recycling may explain, in part, its oncogenic potential.

Animals↗

The oncogenic ErbB-2/ErbB-3 heterodimer is a surrogate receptor of the epidermal growth factor and betacellulin.

The ErbB-1 receptor tyrosine kinase binds to six different growth factors, whose prototype is the epidermal growth factor (EGF). Two homologous epithelial receptors, ErbB-3 and ErbB-4, bind all isoforms of another family of growth factors, the Neu differentiation factors (NDFs/neuregulins). The fourth member of the ErbB family, ErbB-2, acts as the preferred heterodimeric partner of ligand-occupied complexes of the three other ErbB proteins. Here we report that at high concentrations, EGF can induce cell growth and differentiation in the absence of ErbB-1. This function is shared by betacellulin, but not by three other ligands, including the transforming growth factor alpha (TGFalpha). The functional receptor was identified as a heterodimer between ErbB-3 and ErbB-2, a previously identified oncogenic complex. When singly expressed, neither ErbB-3 nor ErbB-2 can mediate signaling by EGF. In addition, when co-expressed, blocking either receptor by using site-specific antibodies inhibited EGF and betacellulin activities, indicating strict cooperativity between ErbB-3 and ErbB-2. Through analysis of chimeras between EGF and TGFalpha, we identified the middle portion of EGF (loop B) as the site that enables activation of ErbB-2/ErbB-3. In conclusion, cooperative and promiscuous binding of stroma-derived growth factors by the epithelium-expressed ErbB-2/ErbB-3 heterodimer may be significant to cancer development. The mechanistic implications of our results for a model that attributes receptor dimerization to ligand bivalency, as well as to a recently proposed mechanism of secondary dimerization, are discussed.

Animals↗

The use of nonhomologous scatchard analysis in the evaluation of ligand-protein interactions.

Scatchard plots are widely used for the graphical presentation of receptor-ligand binding data. When a combination of labelled and unlabelled ligand molecules is used in a binding assay, equations for Scatchard plots are readily available if the labelled and unlabelled ligands have similar binding affinities. In this article, Everardus van Zoelen, Roel Kramer, Herman van Moerkerk and Jacques Veerkamp present mathematical equations to obtain the binding characteristics of an unlabelled ligand in a Scatchard plot, which has a dissociation equilibrium constant different from that of the labelled ligand used.

Drug Interactions↗

Specific expression in mouse mesoderm- and neural crest-derived tissues of a human PDGFRA promoter/lacZ transgene.

The platelet-derived growth factor alpha-receptor (PDGFR-alpha) displays a lineage-specific expression pattern in the mouse embryo and is required for normal development of mesoderm and cephalic neural crest derivatives. The purpose of the present study was to demonstrate the in vivo promoter function of genomic DNA fragments representing the 5'-flanking part of the human PDGFRA gene. 2.2, 0.9 and 0.4 kb PDGFRA promoter fragments, ligated to a lacZ reporter gene, were microinjected into fertilized mouse eggs and transgenic mouse lines were established. The expression patterns were basically similar in the 2.2 and 0.9 kb lines and overlapped grossly the endogenous Pdgfra gene expression pattern. The transgenic line with the highest expression level was chosen for detailed analysis. Expression was, as expected, mainly confined to tissues of mesodermal and neural crest origin. No expression was found in epithelial tissues of endo- or ectodermal origin. The promoter fragments were also active in neuroepithelium and in certain neuronal cell types that did not faithfully express PDGFR-alpha mRNA, while they failed to specify reporter expression in PDGFR-alpha expressing O-2A progenitor cells and other glial elements of the central nervous system. Thus, the isolated human PDGFRA promoter contains most but not all of the regulatory elements that are necessary to establish tissue specific gene expression during development.

Animals↗

The platelet-derived growth factor alpha-receptor 1.5 kb transcript: target for molecular detection of testicular germ cell tumours of adolescents and adults.

An accurate and relatively simple method to detect testicular germ cell tumours of adolescents and adults (TGCTs) could be useful for early detection and may help to avoid unnecessary orchidectomies. We report a highly sensitive reverse-transcription polymerase chain reaction assay for the 1.5 kb transcript of the platelet-derived growth factor-alpha receptor for molecular diagnosis of TGCTs. As a simulation of the clinical application of the assay the approach was applied on through-cut-biopsies from orchidectomy specimens. In a series of 31 specimens, the 1.5 kb transcript was detected in all samples containing either carcinoma in situ, or an invasive TGCT, with mature teratoma as only exception. No expression was detected in normal parenchyma. On the basis of the through-cut-biopsies the assay shows a sensitivity of 0.87 and a specificity of 1.00. The positive and negative predictive values of the test are 1.00 and 1.00. For carcinoma in situ alone these values are 0.86, 1.00, 1.00, and 0.80, respectively. The figures at least equal the results obtained with the most sensitive morphological/enzyme-histochemical study of duplicate biopsies. We conclude that the 1.5 kb transcript of the platelet-derived growth factor-alpha receptor is a useful molecular marker for TGCTs, and therefore of interest in a clinical setting.

Adolescent↗

Superagonistic behaviour of epidermal growth factor/transforming growth factor-alpha chimaeras: correlation with receptor routing after ligand-induced internalization.

Human epidermal growth factor (EGF) and human transforming growth factor alpha (TGF-alpha) are structurally related polypeptide growth factors that exert their mitogenic activity through interaction with a common cell-surface receptor, the epidermal growth factor receptor (EGFR). The biological effect induced by these two ligands is quantitatively similar in most cases; in some test systems, however, TGF-alpha functions as a more potent form of EGF. In this study, we have compared EGF, TGF-alpha and ten previously described chimaeras of these two ligands in terms of their ability to generate a mitogenic response in cells carrying the human EGFR, and observed that three of the mutant growth factors (E3T, E4T and T3E4T) are mitogenic at concentrations 10-fold lower than that of either wild-type EGF or TGF-alpha. No difference in tyrosine kinase activity of the receptor towards an external substrate was observed after binding of the various mutants. It has been established before [Ebner and Derynck (1991) Cell Regulation 2, 599-612] that EGF and TGF-alpha differ in the processing of the receptor-ligand complex after internalization, as a result of their different pH sensitivities of receptor binding. Similar measurements on our chimaeric mutants revealed that the above superagonists show an enhanced pH dependence of binding in comparison with EGF. Furthermore, induction of receptor recycling by these superagonists is largely comparable with that induced by TGF-alpha. No superagonistic behaviour was observed on a cell-line containing an EGFR/erbB-2 chimaera which does not show ligand-induced internalization. These data show that EGF/TGFalpha chimaeras can be more active than the naturally occurring ligands, and that receptor recycling after ligand-induced internalization seems to be a prerequisite for this phenomenon.

Animals↗