Biomedical subjects

Simon Saule

Publications and source records attributed to Simon Saule.

9 recordsLinked to original sources

PubMed2006

Nuclear addressing provides a clue for the transforming activity of amino-truncated CCN3 proteins.

CCN3 is a founding member of the CCN (Cyr61, Ctgf, Nov) family of cell growth and differentiation regulators. These secreted proteins are key regulators in embryonic development, and are associated with severe pathologies including fibrotic diseases and cancers. CCN3 was discovered as a MAV integration site in an avian nephroblastoma. Previous work established that the amino-truncated protein expressed in this tumor was inducing morphological transformation of chicken embryo fibroblasts, whereas the full-length secreted CCN3 protein was inhibiting cell growth. Amino-truncated variants were identified in cancer cell lines. Since the lack of signal peptide was expected to alter the fate of the truncated proteins, we hypothesized that modifications of CCN3 subcellular addressing could be responsible for the oncogenic activities of CCN3. The CCN proteins are composed of four structural modules (IGFBP, TSP1, VWC, and CT). We report that amino-truncated variants of CCN3 are addressed to the nucleus and that the carboxyterminal (CT) module of CCN3 is responsible for the nuclear addressing. Furthermore, our data identify nuclear CCN3 variants as potential transcriptional regulators. In this context, the CT module confers on nuclear CCN3 proteins a negative regulatory effect on transcription. We propose that the nuclear localization of amino-truncated CCN3 proteins be correlated to oncogenicity.

Animals↗

PubMed2005

Protein interaction mapping: a Drosophila case study.

The Drosophila (fruit fly) model system has been instrumental in our current understanding of human biology, development, and diseases. Here, we used a high-throughput yeast two-hybrid (Y2H)-based technology to screen 102 bait proteins from Drosophila melanogaster, most of them orthologous to human cancer-related and/or signaling proteins, against high-complexity fly cDNA libraries. More than 2300 protein-protein interactions (PPI) were identified, of which 710 are of high confidence. The computation of a reliability score for each protein-protein interaction and the systematic identification of the interacting domain combined with a prediction of structural/functional motifs allow the elaboration of known complexes and the identification of new ones. The full data set can be visualized using a graphical Web interface, the PIMRider (http://pim.hybrigenics.com), and is also accessible in the PSI standard Molecular Interaction data format. Our fly Protein Interaction Map (PIM) is surprisingly different from the one recently proposed by Giot et al. with little overlap between the two data sets. Analysis of the differences in data sets and methods suggests alternative strategies to enhance the accuracy and comprehensiveness of the post-genomic generation of broad-scale protein interaction maps.

Animals↗

PubMed2004

Pax6 interacts with cVax and Tbx5 to establish the dorsoventral boundary of the developing eye.

Dorsoventral pattern formation of the optic cup is essential for vertebrate eye morphogenesis and retinotectal topographic mapping. Dorsal and ventral aspects of the eye are distinct at early stages of development; cVax homeodomain protein expression is confined to the ventral optic cup, whereas Tbx5 (T-box transcription factor) expression domain becomes restricted to the dorsal region. Misexpression of cVax or Tbx5 induces profound defects in eye morphology and abnormal visual projections. In the Pax6-/- mutant Tbx5 fails to be expressed, and Vax1 and -2 are abnormally present in the entire optic vesicle. During eye development Pax6 becomes expressed in a gradient at the optic cup stage due to the specific activation of a highly conserved intronic alpha enhancer in the Pax6 locus. We observed that the highest level of Pax6 in the optic cup corresponds to the boundary between non-overlapping cVax and Tbx5 territories. To further investigate how these transcription factors control the patterning of the eye, we overexpressed Pax6 in the chick optic cup (E2) using in ovo electroporation. We observed that overexpression of Pax6 extends the Tbx5 and Bmp4 domains but reduces the cVax expression domains in the E3 chick eye. This results in an abnormal eye phenotype at E4. In addition, we showed that cVax and Tbx5 interact with Pax6 and modulate in an opposite manner the activity of the Pax6 alpha enhancer. Moreover, the Pax6/cVax interaction inhibits the transactivation properties of Pax6. These results demonstrate that Pax6 together with cVax and Tbx5 mediate dorsoventral patterning of the eye.

Animals↗

PubMed2004

Microphthalmia transcription factor induces both retinal pigmented epithelium and neural crest melanocytes from neuroretina cells.

Mitf encodes a basic helix-loop-helix transcription factor that plays an essential role in the differentiation of the retinal pigmented epithelium (RPE) and neural crest-derived melanocytes. As cells containing melanogenic enzymes (TRP2) are found in Mitf mouse mutants, it is not clear whether Mitf is a downstream factor or a master regulator of melanocyte differentiation. To further study the role of Mitf in committing cells to the melanocyte lineage, we express Mitf in the cultured quail neuroretina cells. This leads to the induction of two types of pigmented cells: neural crest-derived melanocytes, according to their dendritic morphology, physiology, and gene expression pattern are observed together with pigmented epithelial RPE-like cells. The expression of Mitf is lower in pigmented epithelial RPE-like cells than in neural crest-derived melanocytes. Accordingly, overexpression of Mitf in cultured quail RPE causes cells to develop into neural crest-like pigmented cells. Thus, Mitf is sufficient for the proper differentiation of crest-like pigmented cells from retinal cells and its expression level may determine the type of pigment cell induced.

Animals↗

PubMed2003

OTX2 activates the molecular network underlying retina pigment epithelium differentiation.

The retina pigment epithelium (RPE) is fundamental for the development and function of the vertebrate eye. Molecularly, the presumptive RPE can be identified by the early expression of two transcription factors, Mitf and Otx. In mice deficient for either gene, RPE development is impaired with loss of melanogenic gene expression, raising the possibility that in the eye OTX proteins operate either in a feedback loop or in cooperation with MITF for the control of RPE-specific gene expression. Here we show that Otx2 induces a pigmented phenotype when overexpressed in avian neural retina cells. In addition, OTX2 binds specifically to a bicoid motif present in the promoter regions of three Mitf target genes, QNR71, TRP-1, and tyrosinase, leading to their transactivation. OTX2 and MITF co-localize in the nuclei of RPE cells and physically interact, and their co-expression results in a cooperative activation of QNR71 and tyrosinase promoters. Collectively, these data suggest that both transcription factors operate at the same hierarchical level to establish the identity of the RPE.

Alternative Splicing↗

PubMed2003

Microphthalmia transcription factor analysis in posterior uveal melanomas.

PURPOSE: The protein encoded by the Microphthalmia gene (MITF) is a transcription factor essential for the development and survival of melanocytes. It serves as a master regulator in modulating extracellular signals. Because of its central role in melanocytes survival and to assess its potential use as a histopathological marker for melanoma, MITF expression was examined in human choroidal melanomas. METHODS: Fifty-seven paraffin-embedded sections of choroidal melanoma specimens and 1 choroidal melanoma cell line were analyzed using immunochemistry and RT-PCR. Normal choroids and normal choroidal melanocyte cells were used as control. RESULTS: Sixty-five percent of the tumoral specimens stained positively for MITF with a predominant nuclear pattern of reactivity. MITF-M and MITF-A isoforms were detected by RT-PCR in all specimens examined. Using a chimeric protein resulting from the fusion of each Mitf protein with the GFP, Mitf-M exhibited an exclusive nuclear staining whereas Mitf-A exhibited a mixed nuclear and cytoplasmic staining. No correlation between MITF-positivity and parameters such as cell type, largest tumor diameter, sclera invasion, mitotic figures was observed. In contrast, a significant negative association was found between MITF staining and the pigmentation (p=0.02) and a positive correlation between MITF staining and the proliferative marker Ki67 was found (p=0.02). CONCLUSION: MITF may be implicated in choroidal melanoma pigmentation and proliferation. Further analysis should provide new insights into the mechanisms underlying the molecular and cellular changes of choroidal melanomas.

Adult↗

PubMed2003

Expression of the c-kit receptor in choroidal melanomas.

The KIT gene encodes c-kit, a transmembrane receptor that has tyrosine kinase activity and plays a role in haematopoiesis, gametogenesis and melanogenesis. The c-kit protein is found in normal cutaneous and choroidal melanocytes, and there is evidence that expression is lost in melanoma. Expression of c-kit was analysed in 57 paraffin-embedded sections of choroidal melanoma specimens and three choroidal melanoma cell lines using immunochemistry and Western blotting. Of the tumour specimens, 75% stained positively for c-kit with a membrane pattern of reactivity. Of the six patients who underwent proton beam therapy before enucleation, five tumours exhibited no c-kit immunoreactivity and the other tumour demonstrated weak staining. Of the three melanoma cell lines used, c-kit expression was observed in only one. No correlations between c-kit positivity and parameters such as cell type, largest macroscopic tumour dimension, scleral invasion or pigmentation were observed. In contrast, a significant positive association was found between c-kit staining and mitotic activity (P = 0.02). However, c-kit expression did not significantly influence survival when evaluated by univariate analysis. In conclusion, c-kit is expressed in most choroidal melanoma tumours. Further analysis should provide new insights into the mechanisms underlying the molecular and cellular changes in choroidal melanomas.

Blotting, Western↗

PubMed2002

Activated MAPK/ERK kinase (MEK-1) induces transdifferentiation of pigmented epithelium into neural retina.

During vertebrate eye development, the optic vesicle originating from the neuroectoderm is partitioned into a domain that will give rise to the neural retina (NR) and another that will give rise to the retinal pigmented epithelium (RPE). Previous studies have shown that ectopic expression of FGFs in the RPE induces RPE-to-NR transdifferentiation. Similarly, a naturally occurring mutation of the transcription factor Mitf in mouse resulted in the formation of a second neural retina in place of the dorsal RPE, but the putative signaling pathway linking FGF to Mitf regulation is presently unknown. In cultures of neural crest-derived melanocytes, the MAPK pathway was recently shown to target the Mitf transcription factor for ubiquitin-dependent proteolysis, resulting in a rapid degradation and downregulation. In the present study, we show that ectopic expression of a constitutively activated allele of MEK-1, the immediate upstream activator of the MAPK ERK, in chicken embryonic retina in ovo, induces transdifferentiation of the RPE into a neural-like epithelium that is correlated with a downregulation of Mitf expression in the presumptive RPE.

Animals↗

PubMed2002

O-glycosylation of the nuclear forms of Pax-6 products in quail neuroretina cells.

Many transcription factors are demonstrated as being glycosylated with O-N-acetylglucosamine (GlcNAc) residue in transfected insect cell lines, but rarely in the original cells. For the first time, we demonstrate the O-GlcNAc modification of the p48/p46 Pax-6 gene (a developmental control gene involved in the eye morphogenesis) products in the quail neuroretina (QNR). In conjunction with a systematic PNGase F treatment, we used wheat germ agglutinin (WGA) binding, in vitro labeling with bovine galactosyltransferase, and labeling of cultured QNR with [14C]GlcNH2. Glycosylated forms of Pax-6 proteins were found in the nucleus of the neuroretina cells. WGA-selected Pax-6 proteins produced in the reticulocyte lysate were able to bind a DNA target, as well as to the unglycosylated form. The O-GlcNAc may, however, modulate protein interactions, mainly with other factors involved in the transcription process. Characterization of products released after reductive alkaline treatment of the proteins clearly demonstrates that N-acetylglucosamine is directly linked to serine or threonine residues. Examination of Pax-6 primary sequence allowed us to determine potential O-GlcNAc attachment sites. Most of these expected glycosylation sites appear to be located on the two DNA binding domains and on the carboxyterminal transactivation domain, while experimental evidence taken from WGA-selected proteins experiment points in favor of a main localization on the paired-box domain.

Acetylglucosamine↗