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W Kruijer

Publications and source records attributed to W Kruijer.

At least 55 records · Page 3Linked to original sources
PubMed1993

Dominant negative retinoic acid receptor beta.

Induction of the retinoic acid receptor beta 2 (RAR beta 2) gene by retinoic acid (RA) is mediated by a RA response element (RARE), which represents a high affinity binding site for RAR/RXR heterodimers acting at this site as RA-inducible transcription activators. In RA resistant RAC65 cells, RAR beta 2 induction is blocked due to expression of a truncated RAR alpha acting as a dominant negative repressor. Here we show that exogenous expression of RAR but not RXR can restore RA-dependent RAR beta 2 promoter activation in RAC65 cells. Structure-function analysis of hRAR beta 2 mutants in RAC65 cells shows, that the transactivation function required to restore RAR beta 2 promoter activation is dependent on the DNA binding, dimerization and RA-dependent transactivation properties of hRAR beta 2, which are retained in a mutant (beta delta 409) lacking the F domain. By contrast, dominant repression of RA-dependent mRAR beta 2 promoter activation by hRAR beta 2 mutants is independent of the DNA binding or RA-dependent transactivation function but requires a region (residues 204-384) in hRAR beta 2 involved in heterodimerization with RXR. These data extend previous observations on structure-function of RARs and provides tools for studying the role of retinoids and RARs during vertebrate development.

Amino Acid Sequence↗
PubMed1992

Differential expression of a novel murine non-receptor protein tyrosine phosphatase during differentiation of P19 embryonal carcinoma cells.

Protein phosphorylation on tyrosine residues is one of the major mechanisms of cell signal transduction and is regulated by protein tyrosine kinases and protein tyrosine phosphatases. Here we report the molecular cloning of an additional member of the protein tyrosine phosphatase-family from differentiated murine P19 embryonal carcinoma cells. This non-receptor protein tyrosine phosphatase, P19-PTP, does not contain regulatory sequences, homologous to the ones found in other non-receptor PTPases. P19-PTP is differentially expressed during in vitro differentiation of P19 EC cells, in that P19-PTP mRNA could only be detected in embryoid bodies, derived from P19 cells.

Amino Acid Sequence↗
PubMed1992

EGF-induced jun B-expression in transfected P19 embryonal carcinoma cells expressing EGF-receptors is dependent on Jun D.

The TPA-inducible transcription factor AP-1, consisting of homo- or hetero-dimers of members of the Jun- and Fos-families, regulates transcription of a wide variety of genes containing the TPA response element (TRE). In P19 embryonal carcinoma (EC) cells, Jun D is the only component of AP-1 expressed, while in these cells until now none of the members of the jun- and fos-families have been found to be inducable by external stimuli. Here we demonstrate that Jun B is the only member of the Jun- and Fos-families that is induced by Epidermal Growth Factor (EGF) in transfected murine P19 EC cells, expressing functional human EGF receptors (hEGF-Rs). Induction of jun B can be mimicked in wild type P19 EC cells by the synergistic action of the phorbol ester TPA and the calcium ionophore A23187, through activation of signal transduction pathways, that are activated simultaneously by EGF. The EGF induced jun B expression in the hEGF-R expressing P19 EC cells is mediated by an inverted repeat (IR) sequence in the jun B promoter, previously shown to be responsive to both PKC and PKA signal transduction. Transactivation of the IR sequence by EGF can be blocked completely by prior expression of antisense Jun D, but not by antisense c-Jun. These studies therefore implicate Jun D in the regulation of immediate early gene expression by external stimuli.

Blotting, Northern↗
PubMed1992

Identification of specific gravity sensitive signal transduction pathways in human A431 carcinoma cells.

Epidermal growth factor (EGF) activates a well characterized signal transduction cascade in human A431 epidermoid carcinoma cells. The influence of gravity on EGF-induced EGF-receptor clustering and early gene expression as well as on actin polymerization and actin organization have been investigated. Different signalling pathways induced by the agents TPA, forskolin and A23187 that activate gene expression were tested for sensitivity to gravity. EGF-induced c-fos and c-jun expression were decreased in microgravity. However, constitutive beta-2 microglobulin expression remained unaltered. Under simulated weightlessness conditions EGF- and TPA-induced c-fos expression was decreased, while forskolin- and A23187-induced c-fos expression was independent of the gravity conditions. These results suggest that gravity affects specific signalling pathways. Preliminary results indicate the EGF-induced EGF-receptor clustering remained unaltered irrespective of the gravity conditions. Furthermore, the relative filamentous actin content of steady state A431 cells was enhanced under microgravity conditions and actin filament organization was altered. Under simulated weightlessness actin filament organization in steady state cells as well as in EGF-treated cells was altered as compared to the 1 G reference experiment. Interestingly the microtubule and keratin organization in untreated cells showed no difference with the normal gravity samples. This indicates that gravity may affect specific components of the signal transduction circuitry.

Calcimycin↗
PubMed1992

Aggregation and cell cycle dependent retinoic acid receptor mRNA expression in P19 embryonal carcinoma cells.

Differentiation of P19 EC cells along different pathways into derivatives resembling cells of the three embryonic germ layers is accompanied by characteristic differences in modulation of expression of each of the three retinoic acid receptor genes, RAR alpha, -beta and -gamma. Differentiation induced by addition of RA to P19 EC cells cultured in monolayer is accompanied by a rapid increase in expression of both RAR alpha and -beta. Induction of RAR beta occurs in a characteristic biphasic manner, suggesting that multiple factors and/or different mechanisms are involved in controlling its expression. RAR beta mRNA is induced to a far higher level during early aggregation in the presence of RA than during early differentiation in monolayer, suggesting that the direction of differentiation depends on the number and/or ratio of alpha and beta type of RA receptors. Aggregation of P19 EC cells in the presence of RA, but not DMSO, is accompanied by repression of RAR gamma, suggesting that the expression of RAR beta and RAR gamma during neuroectodermal differentiation is mutually exclusive. The effects of RA on RAR expression are significantly greater in G1 than in S-phase of the cell cycle. These results extend previous observations that commitment to differentiation is cell cycle dependent and indicates that critical target gene regulation in response to RA has to take place in G1 for differentiation to occur.

Carrier Proteins↗
PubMed1992

Direct stimulation by estrogen of growth factor signal transduction pathways in human breast cancer cells.

Estrogen is thought to stimulate the proliferation of human breast tumors indirectly, through induced production of autocrine polypeptide growth factors. Constitutive production of such growth factors would lead to the loss of 17 beta-estradiol (E2)-dependence that is associated with progression of the disease. Our data, however, do not support this hypothesis and suggest that hormone-dependent breast tumor cell lines like MCF7 do not react to the growth factors which they produce. Moreover, we provide evidence that E2 directly stimulates proliferation by inducing, like many growth factors, the c-fos proto-oncogene. E2 by itself, however, is poorly mitogenic. This may be caused by the lack of induction of genes from the jun family, whose gene products are necessary for dimerization with the c-fos encoded protein, leading to an important step in growth factor signalling pathways; stimulation of TPA responsive element (TRE)-dependent transcriptional activity. In combination with insulin-like growth factors, efficient inducers of c-jun in these cells, E2 synergistically stimulates proliferation and TRE-activity. Constitutive TRE-activation may lead to loss of E2-dependence.

Animals↗
PubMed1992

Retinoic acid resistance of the variant embryonal carcinoma cell line RAC65 is caused by expression of a truncated RAR alpha.

P19 embryonal carcinoma (EC) cells differentiate when treated with retinoic acid (RA). The P19 EC-derived mutant cell line RAC65 is resistant to the differentiation-inducing activity of RA. We show that these cells express a truncated retinoic acid receptor alpha(mRAR alpha-RAC65), probably due to the integration of a transposon-like element in the RAR alpha gene. This receptor lacks 71 C-terminal amino acids and terminates in the ligand-binding domain. In CAT assays in RAC65 cells, mRAR alpha-RAC65 fails to trans-activate the RAR beta promoter, which contains a RA-response element. In wild-type P19 EC cells mRAR alpha-RAC65 functions as a dominant-negative repressor of RA-induced RAR beta activation. Gel retardation assays demonstrate that mRAR alpha-RAC65 is still able to bind to the RA-response element of the RAR beta promoter, indicating that competition with functional RARs for the same binding site leads to the observed dominant-negative effect. In addition, in two RAC65 clones in which wild-type hRAR alpha was stably transfected RA-sensitivity was restored and in one RAR beta expression could be induced by RA. Taken together, these data show that the primary cause of RA-resistance of RAC65 cells is the expression of a defective RAR alpha, which prevents the trans-activation of RA-responsive genes and results in a loss of the ability to differentiate.

Animals↗
PubMed1992

Altered gravity conditions affect early EGF-induced signal transduction in human epidermal A431 cells.

Epidermal growth factor (EGF) activates a well-characterized signal transduction cascade in human A431 epidermal carcinoma cells. Among the early responses evoked by EGF are receptor clustering, cell rounding, and early gene expression. These processes have been studied under various gravity conditions. In addition, we have investigated signalling pathways as induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), forskolin, and A23187 that bypass the EGF receptor, but mimic the partial activation of signal transduction pathways. Hypergravity, simulated microgravity, and real microgravity have been obtained by means of centrifuge, fast-rotating clinostat, and sounding rocket, respectively. EGF-induced c-fos gene expression is suppressed in simulated microgravity (clinostatting) and even more so in real microgravity, while hypergravity increases early gene expression. This indicates that gravity inhibits early EGF-induced signal transduction. However, neither microgravity nor clinostatting affect EGF-induced EGF receptor clustering, suggesting that inhibition of EGF-induced signal transduction by microgravity and clinostatting is independent of EGF receptor clustering. EGF-induced cell rounding is enhanced under clinostatting, while hypergravity does not significantly influence this process. Furthermore, both under clinostatting and real microgravity, EGF- and TPA-induced c-fos expression is decreased, while forskolin and A23187-induced c-fos expression remains unaltered. These observations demonstrate that gravity affects specific components in the EGF-induced signal transduction circuitry, in particular the protein kinase C pathway which is common to EGF and TPA activated intracellular signalling.

Calcimycin↗
PubMed1992

Embryonic stem cells stably transfected with mRAR beta 2-lacZ exhibit specific expression in chimeric embryos.

Using the embryonic stem (ES) cell/chimera approach, we have studied the activity of the mouse retinoic acid receptor beta 2 (mRAR beta 2) promoter during ES cell differentiation and during embryonic development. Stable ES clones were isolated after introduction of a 1.8 kb mRAR beta 2-lacZ expression cassette. LacZ expression in these stable clones was specifically induced by retinoic acid (RA) in a similar fashion as the endogenous RAR beta 2 gene. Following introduction of three different ES clones into blastocysts, an integration-independent mRAR beta 2-lacZ expression pattern was obtained in chimeric embryos similar to that described by in situ hybridization and transgenic studies. Moreover, mRAR beta 2-lacZ expression was also detected at some additional sites not described before, e.g. body wall, ureter, mesonephric duct and optic stalk. Maternal RA administration at 8.5 days of pregnancy extended lacZ expression to more anterior and posterior regions. Transgenic mice were generated from germ-line transmission of the transfected ES cells; expression pattern and changes in expression upon RA induction in these transgenic embryos were identical to those in chimeric embryos. We conclude that by using the ES/chimera approach, the proximal 1.8 kb of the mRAR beta 2 promoter produces a reliable and reproducible expression pattern of the reporter gene, and that the ES cell/chimera approach is invaluable for the study of gene expression and regulation.

Animals↗
PubMed1991

Transcriptional control of c-jun by retinoic acid.

The proto-oncogene c-jun, a major component of transcription factor AP-1, is expressed at very low levels in undifferentiated embryonal carcinoma (EC) end embryonic stem (ES) cells. Retinoic acid (RA) induced differentiation causes a strong increase in the levels of c-jun mRNA. In this paper we report the cloning and characterization of the mouse c-jun promoter. Our results show that RA treatment causes a strong enhancement in c-jun promoter activity, an effect probably mediated by the RA-receptor beta (RAR beta). Sequences located between -329 and -293 are responsible for the observed RA effect, and bind at least five different protein complexes, of which three are decreased upon RA treatment. These protein binding sites do not resemble RA-responsive elements (RARE's) found in the promoters of retinoic acid receptor beta (RAR beta) and laminin B1. Furthermore, we could not detect a direct interaction of RAR alpha and RAR beta to these sequences, indicating that RA-induced c-jun expression is an indirect effect of RAR action.

Animals↗
PubMed1991

Phosphorylation of nuclear protein is an early event in TGF beta 1 action.

Transforming growth factor beta (TGF beta) is a family of polypeptides that modulate growth and differentiation. TGF beta exerts its effects on target cells through interaction with specific cell surface receptors, but the signal transduction pathways are as yet largely unresolved. In this study we report that the growth inhibitory action of TGF beta on mink lung CCl 64 cells is associated with a rapid and transient phosphorylation of a number of nuclear proteins. In parallel, a transient expression of the immediate early gene jun B is observed. The expression of jun B can be inhibited by the protein kinase inhibitor H7 and can be augmented by the phosphatase inhibitor okadaic acid. Thus, protein phosphorylation can be a possible mechanism through which TGF beta 1 initiates early genomic responses.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Activation of junB by PKC and PKA signal transduction through a novel cis-acting element.

The product of the junB gene, a gene homologous to the proto-oncogene c-jun, is a component of transcription factor AP-1. JunB expression is modulated by a wide variety of extracellular stimuli, such as serum, growth factors, phorbol esters (TPA) and activators of protein kinase A (PKA). In order to study the molecular basis of this complex regulation, we have cloned the mouse junB gene from a genomic testis library, and characterized the junB promoter. Here we show that the junB promoter is activated by serum, TPA, and activated PKA. Sequences located between -91 and -44 are necessary for induction. These sequences contain a CAAT box, a G-C rich region and a previously undescribed inverted repeat (IR). The IR element can mediate induction by TPA and PKA when coupled to a heterologous promoter, and specifically binds a protein of 110 kD.

Animals↗
PubMed1991

Characterization of the mouse junD promoter--high basal level activity due to an octamer motif.

The product of the junD gene belongs to the Jun/Fos family of nuclear DNA binding transcription factors. This family regulates the expression of TPA responsive genes by binding to the TPA responsive element (TRE). Unlike its counterparts c-jun and junB, junD expression is hardly inducible by growth factors and phorbol esters. In fact, junD is constitutively expressed at high levels in a wide variety of cells. To unravel the molecular mechanisms underlying constitutive junD expression, we have cloned and characterized the mouse junD promoter. We show that the high constitutive expression is caused by multiple cis-acting elements in its promoter, including an SP1 binding site, an octamer motif, a CAAT box, a Zif268 binding site and a TRE-like sequence. The octamer motif is the major determinant of junD promoter activity, while somewhat smaller contributions are made by the TRE and Zif268 binding site. The SP1 and CAAT box are shown to be of minor importance. The junD TRE is in its behavior indistinguishable from previously identified TREs. However, the junD promoter is not TPA inducible due to the presence of the octamer motif.

Animals↗
PubMed1991

Characterization of a tyrosine kinase signaling pathway in undifferentiated P19 embryonal carcinoma cells.

Embryonal carcinoma (EC) cells are the malignant stem cells of teratocarcinoma and have the capacity to proliferate in the absence of serum growth factors. As yet no receptor protein tyrosine kinases have been identified on undifferentiated EC cells and as a consequence tyrosine kinase signaling pathways could not be studied in these cells. We have used stably transfected P19 embryonal carcinoma cells expressing a well-characterized receptor protein tyrosine kinase, the human epidermal growth factor receptor (hEGF-R) to study protein tyrosine kinase signaling mechanisms in undifferentiated EC cells. Here we report that the ectopically expressed hEGF-R contains EGF-inducible autophosphorylation activity and is rapidly internalized and degraded upon ligand binding. In addition, the exogenous hEGF-R confers EGF-responsiveness to these cells in that inositol phosphate formation and cytoplasmic-free Ca2+ concentration are enhanced in response to EGF. Furthermore, the Na+/H+ exchanger is activated in response to EGF, leading to a sustained rise in intracellular pH. Our results show that undifferentiated P19 EC cells contain the necessary components of protein tyrosine kinase signal transduction machinery.

Adenosine Triphosphate↗
PubMed1991

Nuclear responses to protein kinase C signal transduction are sensitive to gravity changes.

A number of studies have suggested that gravity changes may influence mammalian cell growth and differentiation. To obtain insight in the molecular mechanisms underlying these effects, we have studied immediate early gene expression in response to activation of cytoplasmic signal transduction under microgravity conditions. In this paper we show that epidermal growth factor (EGF)- and 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced expression of the c-fos and c-jun protooncogenes is decreased in microgravity, while no effect of gravity changes was observed on A23187- and forskolin-induced expression of these genes. These decrease in c-fos expression was not due to delayed kinetics under microgravity. These results demonstrate that gravity differentially modulates distinctive signal transduction pathways.

Cell Nucleus↗
PubMed1991

Transcriptional regulation of retinoic acid receptor beta in retinoic acid-sensitive and -resistant P19 embryocarcinoma cells.

As in other embryocarcinoma (EC) cell lines retinoic acid (RA) rapidly induces expression of the nuclear retinoic acid receptor (RAR) beta in murine P19 EC cells, while RAR alpha is expressed constitutively. In the RA-resistant P19 EC-derived RAC65 cells, however, there is no such induction and an aberrant (smaller) RAR alpha transcript is expressed. RAR gamma 1 is expressed at low levels in both cell lines. To study the regulation of the RAR beta gene and the possible involvement of RAR alpha protein in transcriptional activation of the RAR beta gene we transfected these cells with a construct containing a 1.6 kb promoter fragment of the human RAR beta gene fused to the CAT gene. Upon transient assays in P19 EC cells CAT activity is enhanced rapidly by RA, to more than 100-fold in a concentration-dependent fashion. On the contrary no activity can be observed in the RA-resistant RAC65 cells; however, co-transfection of hRAR alpha, hRAR beta or hRAR gamma 1 restores the RA-dependent induction of CAT activity. These results clearly show that RAR alpha and RAR gamma 1 can transactivate the RAR beta gene; that RAR beta can stimulate its own expression and that resistance to RA in RAC65 cells is probably due to the altered RAR alpha transcript present in these cells.

Animals↗
PubMed1991

Octamer-dependent regulation of the kFGF gene in embryonal carcinoma and embryonic stem cells.

Expression of kFGF, which belongs to the family of fibroblast growth factor genes, is restricted to undifferentiated embryonal carcinoma and embryonic stem cells. Stem cell specific expression of kFGF is controlled by a distally localized enhancer, conferring both positive and negative regulation to the kFGF and tk promoters. This enhancer contains a consensus octamer binding sequence that controls positive regulation in EC and ES cells. The octamer sequence binds Oct1 and Oct4 in nuclear extracts from undifferentiated EC cells, while only Oct1 is bound in nuclear extracts from RA differentiated cells. These results suggest that the kFGF gene is a target for positive regulation by Oct4 and implicate Oct4 as target for regulation by the retinoic acid receptors.

Animals↗