Biomedical subjects

K Verschueren

Publications and source records attributed to K Verschueren.

At least 19 recordsLinked to original sources

PubMed2001

Smad-interacting protein 1 is a repressor of liver/bone/kidney alkaline phosphatase transcription in bone morphogenetic protein-induced osteogenic differentiation of C2C12 cells.

Up-regulation of liver/bone/kidney alkaline phosphatase (LBK-ALP) has been associated with the onset of osteogenesis in vitro. Its transcription can be up-regulated by bone morphogenetic proteins (BMPs), constitutively active forms of their cognate receptors, or appropriate Smads. The promoter of LBK-ALP has been characterized partially, but not much is known about its transcriptional modulation by BMPs. A few Smad-interacting transcriptional factors have been isolated to date. One of them, Smad-interacting protein 1 (SIP1), belongs to the family of two-handed zinc finger proteins binding to E2-box sequences present, among others, in the promoter of mouse LBK-ALP. In the present study we investigated whether SIP1 could be a candidate regulator of LBK-ALP transcription in C2C12 cells. We demonstrate that SIP1 can repress LBK-ALP promoter activity induced by constitutively active Alk2-Smad1/Smad5 and that this repression depends on the binding of SIP1 to the CACCT/CACCTG cluster present in this promoter. Interestingly, SIP1 and alkaline phosphatase expression domains in developing mouse limb are mutually exclusive, suggesting the possibility that SIP1 could also be involved in the transcriptional regulation of LBK-ALP in vivo. Taken together, these results offer an intriguing possibility that ALP up-regulation at the onset of BMP-induced osteogenesis could involve Smad/SIP1 interactions, resulting in the derepression of that gene.

Alkaline Phosphatase↗

PubMed2001

Transforming growth factor beta signalling in vitro and in vivo: activin ligand-receptor interaction, Smad5 in vasculogenesis, and repression of target genes by the deltaEF1/ZEB-related SIP1 in the vertebrate embryo.

The identification and characterization of components of the transforming growth factor beta (TGFbeta) signalling pathway are proceeding at a very fast pace. To illustrate a number of our activities in this field, we first summarize our work aiming at the selection from a large collection of single residue substitution mutants of two activin A polypeptides in which D27 and K102, respectively, have been modified. This work has highlighted the importance of K102 and its positive charge for binding to activin type II receptors. Activin K102E, which did not bind to high-affinity receptor complexes, may be a valuable beta chain, when incorporated in recombinant inhibin to unambiguously detect novel inhibin binding sites at the cell surface. We then illustrate how Smad5 knockout mice and an overexpression approach with a truncated TGFbeta type II receptor in the mouse embryo can contribute to the identification of a novel TGFbeta-->TbetaRII/ALK1-->Smad5 pathway in endothelial cells in the embryo proper and the yolk sac vasculature. We conclude with a summary of our results with a Smad-interacting transcriptional repressor but focus on its biological significance in the vertebrate embryo.

Activin Receptors↗

PubMed2001

The two-handed E box binding zinc finger protein SIP1 downregulates E-cadherin and induces invasion.

Transcriptional downregulation of E-cadherin appears to be an important event in the progression of various epithelial tumors. SIP1 (ZEB-2) is a Smad-interacting, multi-zinc finger protein that shows specific DNA binding activity. Here, we report that expression of wild-type but not of mutated SIP1 downregulates mammalian E-cadherin transcription via binding to both conserved E2 boxes of the minimal E-cadherin promoter. SIP1 and Snail bind to partly overlapping promoter sequences and showed similar silencing effects. SIP1 can be induced by TGF-beta treatment and shows high expression in several E-cadherin-negative human carcinoma cell lines. Conditional expression of SIP1 in E-cadherin-positive MDCK cells abrogates E-cadherin-mediated intercellular adhesion and simultaneously induces invasion. SIP1 therefore appears to be a promoter of invasion in malignant epithelial tumors.

Animals↗

PubMed2001

Self-representations and socioemotional competence in young children: a 3-year longitudinal study.

In a longitudinal study, the connections between children's self-representations at age 5 and their self-perceptions, socioemotional competence according to the teacher, and peer acceptance at age 8 were examined. The sample consisted of 60 children (33 boys, 27 girls). Self-representations at age 5 were assessed by the Puppet Interview (J. Cassidy, 1988). Results generally revealed the expected connections between the positiveness of self at age 5 and self-perceptions and socioemotional functioning 3 years later. These findings support the predictive validity of the Puppet Interview. Moreover, they suggest that young children do possess at least a rudimentary sense of being generally worthy and lovable, which can be assessed by using adequate, age-appropriate interviews.

Age Factors↗

PubMed2001

SIP1 (Smad interacting protein 1) and deltaEF1 (delta-crystallin enhancer binding factor) are structurally similar transcriptional repressors.

BACKGROUND: Smad proteins are intracellular mediators of transforming growth factor-beta (TGFbeta) signalling that regulate gene expression by interacting with different classes of transcription factors including DNA-binding multi-zinc finger proteins. One of these, Smad interacting protein 1 (SIP1), is a novel two-handed zinc-finger protein that displays strong similarity with the transcriptional repressor delta-crystallin enhancer binding factor (deltaEF1). Here, we summarize what is known about the mechanism of action of both proteins and their role in vertebrate embryogenesis. Our data are discussed together with the present knowledge on other zinc-finger containing Smad interacting proteins. METHODS: The activities and function of SIP1 have been analysed through documentation of expression patterns, the effect of over-expression of SIP1 on target-gene expression, and promoter studies using Xenopus embryos. Moreover, S1P1/Smad complexes and their association with target promoter DNA were analyzed in biochemical studies. RESULTS: SIP1 is a transcriptional repressor displaying overlapping DNA binding specificities with deltaEF1. An in vivo target of SIP1 in Xenopus is a gene required for the formation of mesoderm, Brachyury (XBra). Our data indicate that SIP1 is required to confine XBra gene expression to the mesoderm. Furthermore, the expression pattern in Xenopus invites us to speculate that SIP1 plays a role in specification/differentiation of neuroectoderm. Unlike deltaEF1, SIP1 can bind directly to activated receptor regulated Smads (R-Smads) and recruit them to the DNA. This indicates that Smads may modulate the activity of SIP1 as a transcriptional repressor. CONCLUSIONS: Our data point to a role of SIP1 in developmental processes regulated by members of the TGFbeta family such as induction of mesoderm (mediated through activin-like signalling) and inhibition of neuroectoderm formation (mediated by bone morphogenetic proteins [BMPs]). Whereas SIP1 could act in TGFbeta signal transduction by virtue of interaction with activated R-Smads, genetic studies in the mouse indicate that deltaEF1 may act in certain TGFbeta pathways-i.e., BMPs and growth and differentiation factors (GDFs)-as well. The molecular mechanisms by which these transcriptional repressors act, as well as the function of the SIP1/Smad interaction, remain to be elucidated. CLINICAL RELEVANCE: Mutations in components of the TGFbeta signalling pathways have been associated with disease and congenital malformations. We anticipate that identification of Smad interacting transcription factors including SIP1 and their targets will help us to understand the molecular basis of certain pathologies.

Animals↗

PubMed2000

The bone morphogenetic protein 2 signaling mediator Smad1 participates predominantly in osteogenic and not in chondrogenic differentiation in mesenchymal progenitors C3H10T1/2.

The role of the bone morphogenetic protein (BMP)-signaling mediator Smad1 in osteogenic or chondrogenic differentiation was investigated in murine parental mesenchymal progenitors C3H10T1/2 and its derivatives constitutively expressing BMP-2 (C3H10T1/2-BMP-2) and, therefore, undergo BMP-mediated osteogenic/ chondrogenic development. The functions of the three Smad1 domains, that is, the N-terminal (MH1) domain, the C-terminal (MH2) domain, and the midregional proline-rich linker domain, were documented and compared with full-length Smadl. We showed that expression of the MH2 domain in parental C3H10T1/2 cells was sufficient to initiate osteogenic differentiation. Interestingly, MH1 was sufficient to initiate transcription of osteogenic marker genes like the osteocalcin or parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) receptor. However, MH1 interfered with the histologically distinct formation of osteoblast-like cells. A dominant-negative effect on MH2-mediated osteogenic development in C3H10T1/2 cells was observed by the dose-dependent trans-expression of the midregional linker domain. Importantly, in contrast to osteogenic differentiation, Smad1 and its domains do not mimic or interfere with BMP-2-dependent chondrogenic development as monitored by the inability of MH2 to give rise to histologically distinct chondrocytes in parental C3H10T1/2 cells and by the inefficiency of the MH1 or linker domain to interfere with BMP-2-mediated chondrogenic differentiation.

Alkaline Phosphatase↗

PubMed1999

New mode of DNA binding of multi-zinc finger transcription factors: deltaEF1 family members bind with two hands to two target sites.

SIP1, a Smad-interacting protein, and deltaEF1, a transcriptional repressor involved in skeletal and T-cell development, belong to the same family of DNA binding proteins. SIP1 and deltaEF1 contain two separated clusters of zinc fingers, one N-terminal and one C-terminal. These clusters show high sequence homology and are highly conserved between SIP1 and deltaEF1. Each zinc finger cluster binds independently to a 5'-CACCT sequence. However, high-affinity binding sites for full-length SIP1 and deltaEF1 in the promoter regions of candidate target genes like Xenopus Xbra2, and human alpha4-integrin and E-cadherin, are bipartite elements composed of one CACCT and one CACCTG sequence, the orientation and spacing of which can vary. Using transgenic Xenopus embryos, we demonstrate that the integrity of these two sequences is necessary for correct spatial expression of a Xbra2 promoter-driven reporter gene. Both zinc finger clusters must be intact for the high-affinity binding of SIP1 to DNA and for its optimal repressor activity. Our results show that SIP1 binds as monomer and contacts one target sequence with the first zinc finger cluster, and the other with the second cluster. Our work redefines the optimal binding site and, consequently, candidate target genes for vertebrate members of the deltaEF1 family.

Animals↗

PubMed1999

SIP1, a novel zinc finger/homeodomain repressor, interacts with Smad proteins and binds to 5'-CACCT sequences in candidate target genes.

Activation of transforming growth factor beta receptors causes the phosphorylation and nuclear translocation of Smad proteins, which then participate in the regulation of expression of target genes. We describe a novel Smad-interacting protein, SIP1, which was identified using the yeast two-hybrid system. Although SIP1 interacts with the MH2 domain of receptor-regulated Smads in yeast and in vitro, its interaction with full-length Smads in mammalian cells requires receptor-mediated Smad activation. SIP1 is a new member of the deltaEF1/Zfh-1 family of two-handed zinc finger/homeodomain proteins. Like deltaEF1, SIP1 binds to 5'-CACCT sequences in different promoters, including the Xenopus brachyury promoter. Overexpression of either full-length SIP1 or its C-terminal zinc finger cluster, which bind to the Xbra2 promoter in vitro, prevented expression of the endogenous Xbra gene in early Xenopus embryos. Therefore, SIP1, like deltaEF1, is likely to be a transcriptional repressor, which may be involved in the regulation of at least one immediate response gene for activin-dependent signal transduction pathways. The identification of this Smad-interacting protein opens new routes to investigate the mechanisms by which transforming growth factor beta members exert their effects on expression of target genes in responsive cells and in the vertebrate embryo.

Amino Acid Sequence↗

PubMed1999

Identification of two amino acids in activin A that are important for biological activity and binding to the activin type II receptors.

Activins are members of the transforming growth factor-beta family of growth and differentiation factors. In this paper, we report the results of a structure-function analysis of activin A. The primary targets for directed mutagenesis were charged, individual amino acids located in accessible domains of the protein, concentrating on those that differ from transforming growth factor-beta2, the x-ray crystal structure of which is known. Based on the activities of the recombinant activin mutants in two bioassays, 4 out of 39 mutant proteins (D27K, K102A, K102E, and K102R) produced in a vaccinia virus system were selected for further investigation. After production in insect cells and purification of these four mutants to homogeneity, they were studied in bioassays and in cross-linking experiments involving transfected receptor combinations. Mutant D27K has a 2-fold higher specific bio-activity and binding affinity to an ActRIIA/ALK-4 activin receptor complex than wild type activin, whereas mutant K102E had no detectable biological activity and did not bind to any of the activin receptors. Mutant K102R and wild type activin bound to all the activin receptor combinations tested and were equipotent in bioassays. Our results with the Lys-102 mutants indicate that the positive charge of amino acid 102 is important for biological activity and type II receptor binding of activins.

Activin Receptors, Type II↗

PubMed1999

Smad5 knockout mice die at mid-gestation due to multiple embryonic and extraembryonic defects.

Smad5 has been implicated as a downstream signal mediator for several bone morphogenetic proteins (BMPs). To understand the in vivo function of Smad5, we generated mice deficient in Smad5 using embryonic stem (ES) cell technology. Homozygous mutant embryos die between E9.5 and E11.5, and display variable phenotypes. Morphological defects are first detected at E8.0 in the developing amnion, gut and heart (the latter defect being similar to BMP-2 knockout mice). At later stages, mutant embryos fail to undergo proper turning, have craniofacial and neural tube abnormalities, and are edematous. In addition, several extraembryonic lesions are observed. After E9.0, the yolk sacs of the mutants contain red blood cells but lack a well-organized vasculature, which is reminiscent of BMP-4, TGF-beta1 and TGF-beta type II receptor knockout mice. In addition, the allantois of many Smad5 mutants is fused to the chorion, but is not well-elongated. A unique feature of the Smad5 mutant embryos is that ectopic vasculogenesis and hematopoiesis is observed in the amnion, likely due to mislocation of allantois tissue. Despite the expression of Smad5 from gastrulation onwards, and in contrast to knockouts of Smad2 and Smad4, Smad5 only becomes essential later in extraembryonic and embryonic development.

Animals↗

PubMed1999

[Grandparents: their experience of the relationship with the oldest grand-child and their psychological well-being].

The aim of the present study was to investigate the relationship between the satisfaction with, perceived responsibilities in, and the meaning of the relationship with the oldest grandchild, on the one hand, and the level of psychological well-being measured with the six-dimensional scale of Ryff, on the other hand. Questionnaires came from 255 Flemish retired grandparents with a mean age of 64.6 years. The relationship with the oldest grandchild was generally experienced as positive. Nearly all the grandparents agreed with items about satisfaction drawn from the relationship with the grandchild, about feeling responsible for helping him or her and about experiencing oneself as a valued elder. In general, grandparents reported well-being on all six dimensions. The experience of the relationship with the grandchild was found to be a unique additional predictor of well-being, even when background variables as grandparent's age, subjective health, financial situation and marital status were first brought into the step-wise regression-analyses. These variables explained 5 to 8% of the variance in the dimensions of well-being and 7% of the variance in the total well-being. The experience of the relationship with the oldest grandchild explained an additional 6 to 7% of the variance.

Aged↗

PubMed1998

Five-year-olds' behaviorally presented self-esteem: relations to self-perceptions and stability across a three-year period.

In this study (a) the connection between 5-year-olds' behaviorally presented self-esteem and their domain-specific self-perceptions and (b) the stability in behaviorally presented self-esteem across a 3-year period (between age 5 and age 8) were examined. The behavioral manifestations of self-esteem were rated by the children's teachers using the Behavioral Rating Scale of Presented Self-Esteem in Young Children (J. Haltiwanger & S. Harter, 1988). The children's domain-specific self-perceptions were assessed by the Pictorial Scale for Perceived Competence and Social Acceptance (S. Harter & R. Pike, 1984). Ninety-five 5-year-olds (50 boys and 45 girls) and their teachers participated. Follow-up data on behavioral self-esteem were available for 55 of the children (30 boys and 25 girls). The results showed, first, that 5-year-olds' presented self-esteem as rated by their teachers was positively and significantly related to the children's perceptions of competence, but not to their perceptions of social acceptance. Second, a high degree of stability in presented self-esteem was found between age 5 and age 8. Some implications of these results for educational practice are discussed.

Child↗

PubMed1997

Direct evidence for SIR2 modulation of chromatin structure in yeast rDNA.

The yeast SIR2 gene maintains inactive chromatin domains required for transcriptional repression at the silent mating-type loci and telomeres. We previously demonstrated that SIR2 also acts to repress mitotic and meiotic recombination between the tandem ribosomal RNA gene array (rDNA). Here we address whether rDNA chromatin structure is altered by loss of SIR2 function by in vitro and in vivo assays of sensitivity to micrococcal nuclease and dam methyltransferase, respectively, and present the first chromatin study that maps sites of SIR2 action within the rDNA locus. Control studies at the MAT alpha locus also revealed a previously undetected MNase-sensitive site at the a1-alpha 2 divergent promoter which is protected in sir2 mutant cells by the derepressed a1-alpha 2 regulator. In rDNA, SIR2 is required for a more closed chromatin structure in two regions: SRR1, the major SIR-Responsive Region in the non-transcribed spacer, and SRR2, in the 18S rRNA coding region. None of the changes in rDNA detected in sir2 mutants are due to the presence of the a1-alpha 2 repressor. Reduced recombination in the rDNA correlates with a small, reproducible transcriptional silencing position effect. Deletion and overexpression studies demonstrate that SIR2, but not SIR1, SIR3 or SIR4, is required for this rDNA position effect. Significantly, rDNA transcriptional silencing and rDNA chromatin accessibility respond to SIR2 dosage, indicating that SIR2 is a limiting component required for chromatin modeling in rDNA.

Chromatin↗

PubMed1997

The C-terminal domain of Mad-like signal transducers is sufficient for biological activity in the Xenopus embryo and transcriptional activation.

We report the characterization of two vertebrate homologs of Drosophila mothers against dpp (Mad) isolated from the mouse and the Xenopus embryo, named MusMLP (mad-like protein) and XenMLP, respectively, together with a summary of their expression patterns in the embryo. Overexpression of XenMLP causes ventralization of Xenopus embryos and we demonstrate that the C-terminal domain is necessary and sufficient to confer this biological effect. This domain also has the potential for transcriptional activation, as shown in one-hybrid assays in mammalian cells. We further demonstrate that MLPs are multidomain proteins by showing a cis-negative effect of the N-terminal domain on the transactivation by the C-terminal domain and that the proline-rich, middle domain maximizes the activity of the C-terminal domain. We also mapped the MusMLP gene to a region on mouse chromosome 13 that corresponds to a region on human chromosome 5q that contains cancer-related genes.

Amino Acid Sequence↗

PubMed1996

Truncated activin type II receptors inhibit bioactivity by the formation of heteromeric complexes with activin type I. receptors.

Truncated activin type II receptors have been reported to inhibit activin receptor signaling in Xenopus embryos, although the mechanism of action for this effect has not been fully understood. In the present study we demonstrate that in P19 embryonal carcinoma cells both the induction of the activin responsive 3TP-lux reporter construct and the inhibition of retinoic acid-induced neuronal differentiation by activin are blocked by expression of a truncated activin receptor. To reveal the mechanism of action of truncated activin receptors, the interaction between different activin receptors has been investigated upon coexpression in COS cells followed by cross-linking of 125I-activin A and subsequent immunoprecipitation. Complexes between a truncated activin type IIA receptor and activin type IA and type IB receptors can be formed, as demonstrated by coimmunoprecipitation of these type I receptors with the truncated activin type IIA receptor. Other type I receptors known as ALK-1 and ALK-6 also coimmunoprecipitate with the truncated type IIA receptor, whereas ALK-3 and ALK-5 do not. Furthermore, the activin type IIB2 receptor does not coimmunoprecipitate with the truncated type IIA receptor, but decreases activin binding to the truncated type IIA receptor. In double immunoprecipitation experiments with cell lysates from COS cells, in which full-length activin type IIA and type IIB2 receptors were cotransfected, no interaction between these receptors was found. In contrast, homomeric complexes of full-length activin type IIA receptors were detected. These results implicate that truncated activin receptors can interfere with activin signaling by interacting with activin type I receptors. Additionally, truncated activin type IIB2 receptors might also interfere with type IIA receptor signaling by decreasing activin binding to the type IIA receptor and therefore might be more potent in inhibiting activin signal transduction. Furthermore, our data indicate that truncated type IIA receptors can interact with other type I receptors and as such might inhibit signal transduction by type I receptors other than activin type IA and type IB receptors.

Activin Receptors↗

PubMed1996

Follistatins neutralize activin bioactivity by inhibition of activin binding to its type II receptors.

Follistatin is an activin-binding protein, which inhibits activin bioactivity in several biological systems. In the present study it is demonstrated that preincubation of iodinated activin A with follistatin, purified from porcine follicular fluid, completely abolished the binding of activin to activin type IIA, IIB2 and IIB4 receptors, and consequently to activin type IB receptor, transiently transfected in COS cells. Binding of activin A to membrane proteins on the activin-responsive P19 embryonal carcinoma cells was also prevented by this follistatin preparation. The same results were obtained with a carboxy-terminally truncated form of follistatin (FS-288), which is only present in minor amounts in the purified follistatin preparation. Since FS-288 has a high affinity for heparan sulfate proteoglycans on the cell surface, we tested whether membrane-bound FS-288 presents activin A to the different activin receptors, thereby facilitating activin binding. FS-288 did bind to the cell surface of transfected COS cells, but inhibited the binding of activin A to its receptors IIA, IIB2 and IIB4. Furthermore, after addition of FS-288 to K562 erythroleukemia cells, the total binding of activin via cell surface-bound FS-288 was increased, whereas the binding of activin A to activin type II and type I receptors present on these cells was inhibited. These findings reveal that different forms of follistatin can neutralize activin bioactivity by interference with binding of activin to all known activin type II receptors, rather than that they inhibit the binding of the type I receptor to the activin/activin type II receptor complex. In addition, our studies indicate that cell surface-associated follistatin cannot present ligand to signalling receptors.

Activin Receptors↗

PubMed1996

Conformational restriction of Tyr and Phe side chains in opioid peptides: information about preferred and bioactive side-chain topology.

The side chain of Tyr and Phe was fixed into the gauche(-) or gauche(+) conformation by using the Tic Htc structures, and into the trans conformation by using an aminobenzazepine-type (Aba) structure. When incorporated into dermorphin or deltorphin II, the Tic and Htc analogues all showed a large decrease in both mu and delta affinities and activities. Fixation of Phe(3) in the trans rotamer resulted in a large increase in delta affinity in the dermorphin analogue, whereas in the [Aba(3)-Gly(4)] deltorphin II analogue, good delta affinity is maintained despite the removal of the Glu side chain. Whereas several authors propose a gauche(-) preferred conformation for the Phe(3) side chain, these results suggest a trans conformation at the delta receptor. The use of these conformationally constrained residues for evaluating the preferred solution conformation in the flexible N-terminal tripeptide Tyr-D-Ala-Phe is illustrated. The (1)H-nmr parameters--chemical shift, temperature dependence, and nuclear Overhauser effects to the D-Ala(2) methyl protons in the different analogues--provide direct evidence to confirm the proposed sandwich conformation in the native peptides.

Amino Acid Sequence↗

PubMed1996

Active complex formation of type I and type II activin and TGF beta receptors in vivo as studied by overexpression in zebrafish embryos.

We have investigated the involvement of activin receptors and TGF beta type I receptor in zebrafish development. Overexpression of either full-length or a truncated form of mouse ActR-IIA interferes with the development. Different splice variants of mouse ActR-IIB have distinct effects; ActR-IIB4 induces abnormal embryos, whereas ActR-IIB2 does not. Activin and TGF beta type I receptors can induce axis duplications. Co-expression of ActR-IA or ActR-IB with the type II activin receptors results in a synergistic increase of the frequency of axis duplication. Moreover, ActR-IIB2 is synergistic with ActR-IA and ActR-IB, demonstrating that ActR-IIB2 can interact with the zebrafish ligand. Overexpression of TGF beta R-I with ActR-IIA or ActR IIB4 results in a synergistic increase in frequency of abnormal embryos, whereas in combination with ActR-IIB2 no such increase occurs.

Activins↗