Studies of Erg target genes in cartilage and identification of a novel gene

La famille ETS comprend une trentaine de gènes codant des facteurs de transcription impliqués dans de nombreux processus physiologiques et néoplasiques. Le gène Erg (ETS Related Gene) appartient à cette famille. Il est exprimé de manière précoce et transitoire au cours du développement embryonnaire dans la mise en place du cartilage. Afin d’étudier la fonction du gène Erg, des souris transgéniques ont été établies au laboratoire. Elles expriment une protéine Erg tronquée à effet trans-dominant négatif de manière spécifique dans les chondrocytes embryonnaires. Le phénotype des souris obtenues s’apparente à un vieillissement précoce du squelette. Dans ce contexte, nous nous sommes intéressés à l’identification de nouvelles cibles des protéines Erg par l’étude transcriptomique des chondrocytes sauvages et transgéniques. Certains gènes dont l’expression est modifiée chez les souris transgéniques codent des protéines de la matrice extracellulaire du cartilage suggérant une composition anormale de la matrice chez l’embryon. Par ailleurs, la recherche de gènes-cibles des protéines Erg a permis de mettre en évidence un nouveau gène exprimé dans les structures cartilagineuses chez l’embryon. La protéine correspondante possède de nombreux résidus Gla, la rattachant ainsi à la famille des protéines dépendantes de la vitamine K (VKD). Ce gène se nomme maintenant Ucma/GRP (Upper zone of Cartilage Matrix Associated/Gla Rich Protein). Nous avons mis en évidence l’existence de quatre types de transcrits suite à l’épissage alternatif des exons 2 et/ou 4. Enfin, nos premières expériences in vitro ont montré que les protéines Erg pourraient réprimer l’expression d’Ucma/GRP.The Ets family includes more than thirty nuclear transcription factors involved in many physiological and neoplasic processes. The Ets related Gene (Erg) belongs to this family. It is expressed during embryogenesis and involved in chondrogenesis. In order to study the Erg gene functions, a transgenic mouse model, which expressed in embryo chondrocytes a truncated Erg protein with transdominant effects, has been developed in our laboratory. These transgenic mice develop early-ageing associated phenotype.In this context, we focused on the identification of new targets of the Erg protein in embryo chondrocytes using the transcriptomic screening approach. We found out a set of modulated genes, most of which encode extracellular matrix proteins. This suggests a disturbed composition of the extracellular matrix during embryogenesis.Moreover, the research of the Erg target genes highlights a new gene expressed in mouse embryo cartilaginous structure. This gene named Ucma/GRP (Upper zone of cartilage matrix associated/Gla Rich Protein). The encoded proteins contain many Gla residues and therefore belong to the vitamin K-dependent (VKD) protein family. Further research works led us to identify four new alternatively spliced transcript variants of Ucma/GRP in mouse chondrocytes. Finally, our preliminary results of Ucma/GRP promoter regulation showed that Erg protein represses Ucma/GRP expression

Recherche des gènes régulés par les protéines Erg dans le cartilage et mise en évidence d'un nouveau gène

La famille ETS comprend une trentaine de gènes codant des facteurs de transcription impliqués dans de nombreux processus physiologiques et néoplasiques. Le gène Erg (ETS Related Gene) appartient à cette famille. Il est exprimé de manière précoce et transitoire au cours du développement embryonnaire dans la mise en place du cartilage. Afin d étudier la fonction du gène Erg, des souris transgéniques ont été établies au laboratoire. Elles expriment une protéine Erg tronquée à effet trans-dominant négatif de manière spécifique dans les chondrocytes embryonnaires. Le phénotype des souris obtenues s apparente à un vieillissement précoce du squelette. Dans ce contexte, nous nous sommes intéressés à l identification de nouvelles cibles des protéines Erg par l étude transcriptomique des chondrocytes sauvages et transgéniques. Certains gènes dont l expression est modifiée chez les souris transgéniques codent des protéines de la matrice extracellulaire du cartilage suggérant une composition anormale de la matrice chez l embryon. Par ailleurs, la recherche de gènes-cibles des protéines Erg a permis de mettre en évidence un nouveau gène exprimé dans les structures cartilagineuses chez l embryon. La protéine correspondante possède de nombreux résidus Gla, la rattachant ainsi à la famille des protéines dépendantes de la vitamine K (VKD). Ce gène se nomme maintenant Ucma/GRP (Upper zone of Cartilage Matrix Associated/Gla Rich Protein). Nous avons mis en évidence l existence de quatre types de transcrits suite à l épissage alternatif des exons 2 et/ou 4. Enfin, nos premières expériences in vitro ont montré que les protéines Erg pourraient réprimer l expression d Ucma/GRP.The Ets family includes more than thirty nuclear transcription factors involved in many physiological and neoplasic processes. The Ets related Gene (Erg) belongs to this family. It is expressed during embryogenesis and involved in chondrogenesis. In order to study the Erg gene functions, a transgenic mouse model, which expressed in embryo chondrocytes a truncated Erg protein with transdominant effects, has been developed in our laboratory. These transgenic mice develop early-ageing associated phenotype.In this context, we focused on the identification of new targets of the Erg protein in embryo chondrocytes using the transcriptomic screening approach. We found out a set of modulated genes, most of which encode extracellular matrix proteins. This suggests a disturbed composition of the extracellular matrix during embryogenesis.Moreover, the research of the Erg target genes highlights a new gene expressed in mouse embryo cartilaginous structure. This gene named Ucma/GRP (Upper zone of cartilage matrix associated/Gla Rich Protein). The encoded proteins contain many Gla residues and therefore belong to the vitamin K-dependent (VKD) protein family. Further research works led us to identify four new alternatively spliced transcript variants of Ucma/GRP in mouse chondrocytes. Finally, our preliminary results of Ucma/GRP promoter regulation showed that Erg protein represses Ucma/GRP expression.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Mémoire(s) : construction, interprétation, enjeux

Ce volume n°74 publie une série d’articles présentés au colloque de l’Association Française d’Etudes Canadiennes qui s’est tenu à l’université Rennes II en juin 2013 sur le thème « Mémoire(s) : construction, interprétation, enjeux ». Les organisateurs du colloque Marc Bergère, Hélène Harter, Catherine Hinault, Eric Pierre et Jean-François Tanguy rappellent en introduction la problématique qui avait réuni de nombreux chercheurs européens et canadiens. Pour ce volume, le comité scientifique a sélectionné sept articles apportant un éclairage récent sur le sujet de la mémoire, sous l’angle de la littérature anglophone et francophone du Canada

Delayed [18F]-FDG PET Imaging Increases Diagnostic Performance and Reproducibility to Differentiate Recurrence of Brain Metastases From Radionecrosis

International audiencePurpose Differentiating brain metastasis recurrence from radiation necrosis can be challenging during MRI follow-up after stereotactic radiotherapy. [F-18]-FDG is the most available PET tracer, but standard images performed 30 to 60 minutes postinjection provide insufficient accuracy. We compared the diagnostic performance and interobserver agreement of [F-18]-FDG PET with delayed images (4-5 hours postinjection) with the ones provided by standard and dual-time-point imaging. Methods Consecutive patients referred for brain [F-18]-FDG PET after inconclusive MRI were retrospectively included between 2015 and 2020 in 3 centers. Two independent nuclear medicine physicians interpreted standard (visually), delayed (visually), and dual-time-point (semiquantitatively) images, respectively. Adjudication was applied in case of discrepancy. The final diagnosis was confirmed histologically or after 6 months of MRI follow-up. Areas under the receiver operating characteristic curves were pairwise compared. Results Forty-eight lesions from 46 patients were analyzed. Primary tumors were mostly located in the lungs (57%) and breast (23%). The median delay between radiotherapy and PET was 15.7 months. The final diagnosis was tumor recurrence in 24 of 48 lesions (50%), with histological confirmation in 19 of 48 lesions (40%). Delayed images provided a larger area under the receiver operating characteristic curve (0.88; 95% confidence interval [CI], 0.75-0.95) than both standard (0.69; 95% CI, 0.54-0.81; P = 0.0014) and dual-time-point imaging (0.77; 95% CI, 0.63-0.88; P = 0.045), respectively. Interobserver agreement was almost perfect with delayed images (kappa = 0.83), whereas it was moderate with both standard (kappa = 0.48) and dual-time-point images (kappa = 0.61). Conclusions [F-18]-FDG PET with delayed images is an accurate and reliable alternative to differentiate metastasis recurrence from radiation necrosis in case of inconclusive MRI after brain stereotactic radiotherapy

Increased Adipogenesis in Cultured Embryonic Chondrocytes and in Adult Bone Marrow of Dominant Negative Erg Transgenic Mice

<div><p>In monolayer culture, primary articular chondrocytes have an intrinsic tendency to lose their phenotype during expansion. The molecular events underlying this chondrocyte dedifferentiation are still largely unknown. Several transcription factors are important for chondrocyte differentiation. The Ets transcription factor family may be involved in skeletal development. One family member, the <em>Erg</em> gene, is mainly expressed during cartilage formation. To further investigate the potential role of Erg in the maintenance of the chondrocyte phenotype, we isolated and cultured chondrocytes from the rib cartilage of embryos of transgenic mice that express a dominant negative form of Erg (DN-Erg) during cartilage formation. DN-Erg expression in chondrocytes cultured for up to 20 days did not affect the early dedifferentiation usually observed in cultured chondrocytes. However, lipid droplets accumulated in DN-Erg chondrocytes, suggesting adipocyte emergence. Transcriptomic analysis using a DNA microarray, validated by quantitative RT-PCR, revealed strong differential gene expression, with a decrease in chondrogenesis-related markers and an increase in adipogenesis-related gene expression in cultured DN-Erg chondrocytes. These results indicate that Erg is involved in either maintaining the chondrogenic phenotype <em>in vitro</em> or in cell fate orientation. Along with the <em>in vitro</em> studies, we compared adipocyte presence in wild-type and transgenic mice skeletons. Histological investigations revealed an increase in the number of adipocytes in the bone marrow of adult DN-Erg mice even though no adipocytes were detected in embryonic cartilage or bone. These findings suggest that the Ets transcription factor family may contribute to the homeostatic balance in skeleton cell plasticity.</p> </div

Correlation of microarray data and RT-qPCR analysis.

<p>Comparison of fold change in the expression of 11 genes implicated in chondrogenesis or adipogenesis as determined by microarray analysis and RT-qPCR.</p><p>Fold change between day 0 and day 20 observed by microarray-analysis and RT-qPCR for wt and DN-Erg chondrocytes are shown. Expression levels of target genes obtained by RT-qPCR were normalised to <i>Hprt</i>. Upregulation is indicated by positive values and downregulation is indicated by negative values.</p

Cytological analysis and adipocyte quantification of femoral bone marrow of wt and DN-Erg mice.

<p>A. Sections of femorotibial joint of week 6- and week 40- wt or DN-Erg mouse. Bars = 1 mm. B. Quantification of adipocytes in bone marrow of DN-Erg and wt femoro-tibial section. The result was expressed as the mean of total adipocyte number per square millimeter of marrow tissue area in the analysed fields of at least three different mouse. C. Cytological examination of bone marrow in a femur section of 40 week-old wt and DN-Erg mice. Left, Magnification: ×2.5. Right: higher magnification view (×10).</p

Histological examination of 18.5 day-old embryos.

<p>A. Skeletal and cartilage preparations of wt (left) and DN-Erg (right) mice at E18.5. Cartilage stained with Alcian blue, bone with Alizarin red. DN-Erg E18.5 embryos did not show any overt abnormalities in cartilaginous or skeletal development. B. Distribution of chondrocytes on sections of rib. Paraffin-embedded sagittal sections of wt (left) and DN-Erg (right) newborn mice were stained with Alcian blue. Bars = 50 µm.</p