Biomedical subjects

D Aberdam

Publications and source records attributed to D Aberdam.

At least 19 recordsLinked to original sources

PubMed2005

BMP-4 induces a Smad-dependent apoptotic cell death of mouse embryonic stem cell-derived neural precursors.

Embryonic ectoderm is fated to become either neural or epidermal, depending on patterning processes that occur before and during gastrulation. It has been stated that epidermal commitment proceeds from a bone morphogenetic protein-4 (BMP-4)-dependent inhibition of dorsal ectoderm neuralization. We recently demonstrated that murine embryonic stem (ES) cells treated with BMP-4 undergo effective keratinocyte commitment and epidermogenesis. Focusing on the precise role of BMP-4 in the early choice between neural and epidermal commitment, we show here that BMP-4 treatment of ES cells leads to a dramatic apoptotic death of Sox-1+ neural precursors with concomitant epidermal engagement. In addition, neutralization of the Smad pathway prevents both the BMP-4 apoptotic process and the inhibition of neural differentiation. Our results suggest that, in mammals, BMP-4, as an active inducer of epidermal commitment, interferes with the survival of neural precursors through induction of their apoptotic cell death.

3T3 Cells↗

PubMed2005

Epidermal stem cells.

The identification of adult epidermal stem cells that are capable of self-renewal and can reconstitute not only the epidermis but also the cutaneous appendages opens new perspectives for the treatment of a variety of human skin disorders including severe burns, cutaneous cancers, alopecia and acne. However, the implementation and improvement of these novel treatment strategies require a better understanding of the biology of stem cells, in particular regarding their isolation and the maintenance of their unique characteristics in culture. In this review, we summarize the main features of epidermal stem cells and we present the most recent advances in our understanding of the development and maintenance of these cells. In addition, we discuss some of the challenges and the potential clinical applications of epidermal stem cell technology.

Epidermal Cells↗

PubMed2001

Transposable B2 SINE elements can provide mobile RNA polymerase II promoters.

Short interspersed elements (SINEs) are highly abundant components of mammalian genomes that are propagated by retrotransposition. SINEs are recognized as a causal agent of human disease and must also have had a profound influence in shaping eukaryotic genomes. The B2 SINE family constitutes approximately 0.7% of total mouse genomic DNA (ref. 2) and is also found at low abundance in humans. It resembles the Alu family in several respects, such as its mechanism of propagation. B2 SINEs are derived from tRNA and are transcribed by RNA polymerase (pol) III to generate short transcripts that are not translated. We find here, however, that one B2 SINE also carries an active pol II promoter located outside the tRNA region. Indeed, a B2 element is responsible for the production of a mouse Lama3 transcript. The B2 pol II promoters can be bound and stimulated by the transcription factor USF (for upstream stimulatory factor), as shown by transient transfection experiments. Moreover, this pol II activity does not preclude the pol III transcription necessary for retrotransposition. Dispersal of B2 SINEs by retrotransposition may therefore have provided numerous opportunities for creating regulated pol II transcription at novel genomic sites. This mechanism may have allowed the evolution of new transcription units and new genes.

Animals↗

PubMed2000

Tumor cell budding and laminin-5 expression in colorectal carcinoma can be modulated by the tissue micro-environment.

Expression of laminin-5 alpha3, beta3 and gamma2 protein subunits was investigated in colorectal adenocarcinomas using immunostaining and confocal microscopy. The laminin-5 heterotrimer was found in basement membranes and as extracellular deposits in tumor stroma. In contrast to the alpha3 subunit, which was under-expressed, the gamma2 and beta3 subunits were detected in the cytoplasm of carcinoma cells dissociating (budding) from neoplastic tubules, suggestive of focal alterations in laminin-5 assembly and secretion. Laminin-5 gamma2 or beta3 subunit-reactive budding carcinoma cells expressed cytokeratins but not vimentin; they did not proliferate and were not apoptotic. Furthermore, expression of laminin-5 gamma2 and beta3 subunits in budding cells was associated with focal under-expression of the E-cadherin-beta-catenin complex. Results from xenograft experiments showed that budding activity in colorectal adenocarcinomas could be suppressed when these tumors grew at ectopic s.c. sites in nude mice. In vitro, cultured colon carcinoma cells, but not adenoma-derived tumor cells, shared the laminin-5 phenotype expressed by carcinoma cells in vivo. Using colon carcinoma cell lines implanted orthotopically and invading the cecum of nude mice, the laminin-5-associated budding was restored, indicating that this phenotype is not only determined by tumor cell properties but also dependent on the tissue micro-environment. Our results indicate that both laminin-5 alpha3 subunit expression and cell-cell cohesiveness are altered in budding carcinoma cells, which we consider to be actively invading. We propose that the local tissue micro-environment contributes to these events.

Adenocarcinoma↗

PubMed2000

Transcriptional regulation of laminin gene expression.

Laminins are the most abundant structural non-collagenous glycoproteins ubiquitously present in basement membranes. They are multidomain molecules consisting of of alpha, beta, and gamma chains. Although the precise functional differences between the laminin variants are not well understood, the diversity of laminin isoforms may reflect the formation of distinct basement membranes. The laminins display a remarkable restricted expression profile, suggesting a fine regulation of their genes. In this review, we focus on the most recent developments of laminin biology, centering on transcriptional and posttranscriptional controls. We discuss only those laminin chains whose gene organization and promoter elements have been characterized and proved to be functional. When possible, we correlate the effects of growth factors, cytokines, retinoids, and transcription factors on laminin gene expression with the identity of cis-acting elements in their genomic control regions.

Animals↗

PubMed2000

DNA conformation driven by AP-1 triggers cell-specific expression via a strong epithelial enhancer.

We report here the characterization of the regulatory region of the human LAMA3 gene, coding for the alpha3A chain of laminin-5. A 202 bp fragment is sufficient to confer epithelial-specific expression to a thymidine kinase promoter through the cooperative effect of three AP-1 binding sites. Remarkably, removal of the sequences located between the AP-1 sites does not modify the promoter activity in keratinocytes but allows strong expression in fibroblasts. Replacement of the deleted sequences by non-homologous ones fully restores the restricted enhancement in keratinocytes. Functional analysis and mutagenesis experiments demonstrate that a minimal distance between the AP-1 sites is required for the enhancer DNA fragment to adopt a particular conformation driven by the binding of Jun-Fos heterodimers. In non-permissive cells, this conformation leads to the anchorage of non-DNA-binding fibroblastic cofactors to form an inhibitory ternary complex. Therefore, our results describe for the first time an unusual conformation-dependent epithelial-specific enhancer.

3T3 Cells↗

PubMed2000

Differential expression of laminin-5 subunits during incisor and molar development in the mouse.

Rodent incisors are continuously growing teeth and enamel deposition is restricted to the labial side. In the present study, the expression of laminin-5 subunits (alpha3, beta3 and gamma2) has been analyzed by in situ hybridization in developing mouse lower incisors and compared to that reported in the molar. At the bud stage (E12), mRNAs for all subunits were detected in the whole epithelial thickening. At E14, when histogenesis had started, transcripts for alpha3 and gamma2 subunits were restricted to the outer dental epithelium (ODE), whereas the beta3 subunit was intensely expressed in the inner dental epithelium (IDE). A transient expression for alpha3 subunit was seen in the enamel knot area and disappeared at E15. Subsequently, all laminin-5 subunit genes were re-expressed in differentiating ameloblasts on the labial side. Similar patterns of transcription were observed in incisor and molar, suggesting that the differential expression of laminin-5 subunits in the IDE might be involved in the histogenesis of the IDE and ameloblast differentiation. At E16.5, cells of the IDE at the anterior extremity of the incisor and in the anterior part of the lingual IDE expressed transcripts for alpha3 and beta3 but not for gamma2 subunit. Similar expression patterns were observed in the enamel-free areas of the E18 molar. This specific expression might thus be related to cells that do not differentiate as functional ameloblasts. Throughout incisor development, intense expression for all laminin-5 subunits was restricted to the labial side of the cervical loop. The asymmetrical expression of laminin-5 might be related to incisor morphogenesis and to the differences in histogenesis and cytodifferentiation of the IDE that exist in the labial versus lingual aspect of the cervical loop.

Animals↗

PubMed1999

Spectroscopic and saturation magnetization properties of the manganese- and cobalt-substituted Fur (ferric uptake regulation) protein from Escherichia coli.

The Fur apoprotein has been purified and reconstituted with Co2+ and Mn2+ ions. These samples have been analyzed by UV-visible, EPR, and 1H NMR spectroscopies, by XAS, and by magnetization measurements. The apo-Fur protein is able to bind one metal dication (Co2+ or Mn2+) per monomer. A saturation magnetization study confirms the presence of a high-spin metal dication [Mn(II) S = 5/2 and Co(II) S = 3/2]. The two metal ions per Fur dimer are not in magnetic interaction (|J| < 0.1 cm-1 ). The UV-visible spectrum of the cobalt-substituted form (Co-Fur) presents two main bands at 660 nm and 540(br) nm with epsilon540 nm = 65 M-1 cm-1. The EPR spectrum gives the following g values: gx = 5.0(5), gy = 4.0(2), and gz = 2. 3(1), which are in accordance with a nearly axial (E/D < 0.11) site. The value of 55 cm-1 for the splitting (Delta) between the ground and the first excited state has been derived from an EPR saturation study and is in agreement with magnetization data. The EXAFS data of Co-Fur indicate a metal environment comprising five nitrogen/oxygen atoms at 2.11 A, the absence of sulfur, and the presence of histidines as ligands. 1H NMR of Co-Fur in H2O and D2O shows at least two exchangeable signals coming from histidine NH protons and shows the signature of carboxylate group(s). The combined spectroscopic data allow us to propose that the main metal site of Fur in Co-Fur contains at least two histidines, at least one aspartate or glutamate, and no cysteine as ligands and is in an axially distorted octahedral environment.

Apoproteins↗

PubMed1998

SEDEM, a software package for EXAFS data extraction and modelling.

A software package for extended X-ray absorption fine structure (EXAFS) data extraction and modelling, running on DOS- or Windows-operated PCs, is described. This package is written with the aim of giving the user a tool to undertake all steps of data processing and modelling, rather than making use of the most recent programming facilities. However, it remains easy to use, and self-explanatory to those who have already worked with EXAFS. It is divided into two main executable pieces of software. The first one is used to extract the EXAFS k(n)chi(k) function from the data and isolate the shell contributions by Fourier filtering. A tool to sum the spectra before EXAFS extraction is provided. The second one is designed to model the EXAFS spectra or the shell contributions, using amplitude and phase data either from McKale's tables, computed from the FEFF program or extracted from experimental reference spectra. This modelling program allows either an optimization of the simulation by a least-mean-square gradient algorithm, with a statistical evaluation of the result of optimization, or, in the case of a single shell, a direct determination of the four main parameters (neighbour distance and number, energy shift and Debye-Waller factor) by decorrelation of the phase and amplitude. In the presence of anharmonicity, the cumulant expansion of the radial distribution of distances is obtained from the phase and amplitude decorrelation. This package is in use at the Collaborative Research Group on Interfaces (CRG-IF) bending-magnet #32 X-ray line (BM-32) at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France.

Journal Article↗

PubMed1998

Human colonic cancer cells synthesize and adhere to laminin-5. Their adhesion to laminin-5 involves multiple receptors among which is integrin alpha2beta1.

In the mature gut, laminin-5 is expressed at the basal aspect of the differentiating epithelial cells. In vitro, we show that three more or less differentiated human colonic cancer HT29 cell lines produce and deposit laminin-5; they predominantly synthesize and secrete the 440 kDa form of laminin-5 that comprises the unprocessed 155 kDa gamma2 chain, as determined by immunoprecipitation analysis. In contrast, the highly differentiated colon carcinoma Caco-2 cells produce almost no laminin-5. Using anti-integrin antibodies, we show that adhesion of the two colonic cancer cell lines to laminin-5 is mediated by multiple integrin receptors including those for alpha3beta1, alpha6beta1 and alpha6beta4 integrins like in other cell types. In addition, the implication of integrin alpha2beta1 in this adhesion process is demonstrated for the first time. This has been shown by cell adhesion inhibition experiments, solid phase assays and confocal analysis. Together with previous in situ observations, these data provide a baseline knowledge for the understanding of the regulation of laminin-5 in normal and pathological intestine.

Antibodies, Monoclonal↗

PubMed1998

Three activator protein-1-binding sites bound by the Fra-2.JunD complex cooperate for the regulation of murine laminin alpha3A (lama3A) promoter activity by transforming growth factor-beta.

Several lines of evidence suggest a role for laminin-5 in skin wound healing. We report here that transforming growth factor-beta (TGF-beta), which elicits various responses during cutaneous healing, stimulates transcription of the mouse laminin alpha3A (lama3A) gene. To identify the TGF-beta-responsive elements (TGFbeta-REs) on the lama3A promoter, we have generated a series of 5'-deletions of the promoter upstream of the beta-galactosidase reporter gene. Transient cell transfection assays using mouse PAM212 keratinocytes revealed that TGFbeta-REs lie between nucleotides -297 and -54 relative to the transcription start site. Insertion of the TGFbeta-RE in front of the unresponsive minimal SV40 promoter conferred TGF-beta inducibility. Computer analysis of the promoter sequence identified three canonical activator protein-1 (AP-1) sites located at nucleotides -277 (AP-1A), -125 (AP-1B), and -69 (AP-1C). Site-directed mutagenesis of either the AP-1A or AP-1C site did not drastically alter the basal activity of the lama3A promoter, but reduced TGF-beta responsiveness by 50%. Simultaneous mutation of these two AP-1 sites resulted in a 65% decline in the response to TGF-beta, suggesting a cooperative contribution of each site to the overall promoter activity. In contrast, mutation of the AP-1B site markedly reduced the basal activity of the lama3A promoter, indicating that this AP-1 site is essential for gene expression. Mobility shift assays demonstrated specific binding of Fra-2 and JunD to the AP-1 sites, suggesting for the first time a possible regulatory function for the Fra-2.JunD AP-1 complex in a basal keratinocyte-specific gene.

Animals↗

PubMed1998

X-ray absorption spectroscopy of a new zinc site in the fur protein from Escherichia coli.

The zinc K-edge X-ray absorption spectra of the Fur (ferric uptake regulation) protein isolated from Escherichia coli have been analyzed in frozen solution to determine details of the zinc coordination. The spectra of apoFur and of the cobalt-substituted protein have been analyzed and compared in order to see the influence of the cobalt incorporation on the geometry of the zinc site. EXAFS analysis gave for both samples (apoFur and CoFur) a tetrahedral environment for the zinc atom with two sulfur donor ligands at a distance of 2.3 A from the zinc and two N/O donor ligands at 2.0 A. The two sulfur donor ligands are probably two of the four cysteines present in each Fur monomer and could be Cys92 and Cys95, which are known from mutagenesis studies to be essential for Fur activity [Coy, M., Doyle, C., Besser, J., and Neilands, J. B. (1994) BioMetals 7, 292-298]. The distances obtained from our fits were always too short to be compatible with penta or hexa coordination. The typical pattern observed for the Fourier transform of the EXAFS oscillations suggests the presence of at least one imidazole ligand. The XANES of these two forms of the protein are similar but significantly different. This suggests a change of the conformation of the zinc site upon cobalt incorporation. The present study provides the first unambiguous evidence for the presence of a structural zinc site in the Fur protein from Escherichia coli.

Apoproteins↗

PubMed1998

Differential expression of laminin-5 subunits and integrin receptors in human colorectal neoplasia.

Cell-matrix interactions contribute to regulating the adhesion, growth, migration, and differentiation of epithelial intestinal cells. Alterations in matrix components and their cellular receptors have been found in tumours but their specific roles remain unclear. The tissue patterns of laminin-5 and alpha 3, beta 3 and gamma 2 subunits, as well as those of the alpha 3, alpha 6, beta 1, and beta 4 integrin chains, were determined by immunofluorescence on frozen sections of 12 colorectal mucosal samples from four patients, 15 adenomas, 29 adenocarcinomas, and eight metastases. Distinct patterns of laminin-5 and integrin expression were found along the mucosa-adenoma, and adenoma-carcinoma transitions. Expression of basement membrane laminin-5 and subunits was continuous and gradient-like in normal mucosa, enhanced at the periphery of adenomas, and discontinuous in places in carcinomas and metastases. Decrease of the alpha 3 integrin chain was found in adenomas, together with that of alpha 6 and beta 4 chains in carcinomas. A subpopulation of carcinoma cells dissociating (budding) from neoplastic tubules was found to accumulate the laminin-5 beta 3 gamma 2 heterodimer in the cytoplasm, with progressive loss of surface integrin expression. These results suggest that in colorectal cancer, an abnormal expression of laminin-5 subunits and integrin chains may identify a subset of carcinoma cells prone to invade focally and to contribute to disease aggressiveness.

Adenocarcinoma↗

PubMed1998

Expression and localization of laminin-5 subunits during mouse tooth development.

Tooth morphogenesis is regulated by epithelial-mesenchymal interactions mediated by the basement membrane (BM). Laminins are major glycoprotein components of the BMs, which are involved in several cellular activities. The expression and localization of the alpha3, beta3, and gamma2 laminin-5 subunits have been analyzed by in situ hybridization and immunohistochemistry during mouse molar development. Initially (E12), mRNAs of all subunits were detected in the entire dental epithelium and the corresponding proteins were located in the BM. During cap formation (E13-14), transcripts for the alpha3 and gamma2 subunits were localized in the outer dental epithelium (ODE), whereas the beta3 subunit mRNA was present in the inner dental epithelium (IDE). During the early bell stage (E16), immunoreactivity for all subunits disappeared from the BM along the IDE, although intense signals for beta3 mRNA were detectable in cells of the IDE. Subsequently, when the dentinal matrix was secreted by odontoblasts (E18-19.5), mRNAs of all three subunits were re-expressed by ameloblasts, and the corresponding proteins were detected in ameloblasts and in the enamel matrix. Tissue recombination experiments demonstrated that when E16 IDE or ODE was associated with E18 dental papilla mesenchyme, immunostaining for all laminin-5 subunits disappeared from the BM, whereas when cultured with non-dental limb bud mesenchyme, they remained positive after 48 hr of culture. These results suggest that the temporospatial expression of laminin-5 subunits in tooth development, which appears to be differentially controlled by the dental mesenchyme, might be related to the enamel organ histo-morphogenesis and the ameloblast differentiation.

Ameloblasts↗

PubMed1998

Expression and localization of laminin-5 subunits in the mouse incisor.

Laminin-5 is associated with several epithelial tissues and forms part of the anchoring filaments of hemidesmosomes. Recent data have shown that the expression of laminin-5 subunits is impaired in junctional epidermolysis bullosa (JEB), and, in these patients, enamel hypoplasia is commonly observed. Rodent incisors are continuously growing teeth with an asymmetry between their labial and lingual sides. Enamel matrix formation is restricted to the labial side. We have analyzed the changes in the expression and localization of laminin-5 subunits (alpha3, beta3, and gamma2) in lower incisors of the mouse. The apical loop located at the end of the labial side contained stem cells and showed expression for all laminin-5 subunits. In the anterior direction, the inner dental epithelial cells (IDE) transiently lost the immunoreactivity for all subunits, whereas the transcripts for the beta3 subunit remained in the IDE. All subunit mRNAs and proteins were expressed in ameloblasts facing predentine and also in secretory and maturation stage ameloblasts. Enamel matrix contained laminin-5. On the lingual side, the expression of laminin-5 subunits was continuous from the epithelial root sheath to the epithelial rests of Malassez in the periodontal ligament. These results suggest that spatial and temporal regulation of laminin-5 subunits correlates with the histogenesis of the dental organ, ameloblast differentiation, and enamel formation and also that laminin-5 plays a role in the adhesion between dental epithelial cells and the extracellular matrix (enamel or dentine) in areas where the dental basement membrane is absent.

Amelogenesis↗

PubMed1998

Acquired junctional epidermolysis bullosa associated with IgG autoantibodies to the beta subunit of laminin-5.

We report the case of a 72-year-old man with clinical features resembling those of non-lethal junctional epidermolysis bullosa associated with IgG autoantibodies to the beta chain of laminin-5. The patient presented with a sudden onset of blistering and severe fragility of the skin and mucous membranes resulting in atrophic scars. Electron microscopy showed that the blistering arose in the lamina lucida. Indirect immunofluorescence indicated that the autoantibodies bound to the dermal side of 1 mol/L NaCl-split skin, and both direct and indirect immunoelectron microscopy demonstrated antibody binding to the lamina densa. Postembedding immunogold electron microscopy also revealed labelling in the lamina lucida beneath the hemidesmosomes. On immunoblotting, we found the autoantibodies to comigrate with the beta chain of laminin-5. Following the nomenclature of inherited junctional epidermolysis bullosa with mutations of the laminin-5 gene, we propose the name acquired junctional epidermolysis bullosa for this newly recognized disease.

Aged↗

PubMed1998

Localization of antigens associated with adherens junctions, desmosomes, and hemidesmosomes during murine molar morphogenesis.

Epitheliomesenchymal interactions are known to play a crucial role during odontogenesis. Since epithelial cell-cell and cell-matrix interactions may also be involved in enamel organ histomorphogenesis, we investigated the localization of proteins associated with junctional complexes in mouse and rat first lower molars by indirect immunofluorescence. Adherens junctions were detected using antibodies directed against E-cadherin, beta-catenin, and plakoglobin (gamma-catenin). Desmosomes were localized with antibodies against desmoglein, and hemidesmosomes using antibodies against BP-230 and HD-1 proteins. When the inner dental epithelium differentiates, a decrease of E-cadherin, plakoglobin, and BP-230 is seen. An asymmetric distribution of plakoglobin, desmoglein, and BP-230 between the lateral and medial side of the tooth exists; desmoglein, which was first restricted to the gubernaculum dentis, progressively accumulated in the stellate reticulum, the stratum intermedium, and the basal pole of ameloblasts. The specific temporospatial distributions patterns of these antigens suggests a direct involvement of adherens junctions, desmosomes, and hemidesmosomes in the development of the murine first lower molar.

Animals↗

PubMed1997

Murine laminin alpha3A and alpha3B isoform chains are generated by usage of two promoters and alternative splicing.

We already identified two distinct laminin alpha3A and alpha3B chain isoforms which differ in their amino-terminal ends and display different tissue-specific expression patterns. In this study we have investigated whether these two different isoforms are products of the same laminin alpha3 (lama3) gene and transcribed from one or two separate promoters. Genomic clones were isolated that encompass the sequences upstream to the 5' ends of both the alpha3A and the alpha3B cDNAs. Sequence analysis of the region upstream to the alpha3A open reading frame revealed the presence of a TATA box and potential binding sites for responsive elements. By primer extension analysis, the transcription start site of the alpha3B mRNA isoform was defined. The sequences upstream to the alpha3B mRNA transcription start site do not contain a TATA box near the transcription initiation sites, but AP-1, AP-2, and Sp1 consensus binding site sequences were identified. The genomic regions located immediately upstream of the alpha3A and alpha3B transcription start sites were shown to possess promoter activities in transfection experiments. In the promoter regions, response elements for the acute phase reactant signal and NF-interleukin 6 were found, and their possible relevance in the context of inflammation and wound healing is discussed. Our results demonstrate that the lama3 gene produces the two polypeptides by alternative splicing and contains two promoters, which regulate the production of the two isoforms alpha3A and alpha3B.

3T3 Cells↗