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

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

Morphological changes in wt and DN-Erg E18.5 chondrocytes in culture.

<p>Chondrocytes from freshly isolated from ribs of wt and DN-Erg transgenic mouse embryos (at E18.5) were cultured for up 20 days and stained with Oil red O. Phase-contrast images at days 0, 3, 9, 15 and 20 (with day 0, the day of plating) are shown (Scale bar, 50 µm).</p

Microarray analysis and gene ontology analysis of signalling pathways.

<p>A. Numbers of genes with differential expression between monolayer culture day 0 and day 20 in wt and DN-Erg embryo (E18.5) chondrocytes. Probe sets were filtered according to a 10-fold change cut-off. B. Hierarchical Clustering (HCl) diagram with clusters genes corresponding to the “extracellular matrix”, “metallopeptidase activity”, “Cartilage condensation and development”, “Ossification” annotations. C. Major signalling pathways predicted using Pathway-Express. Pathways listed are pathways with at least 5 or more genes which expression was modified during culture, as determined by Pathway Express. D. Hierarchical Clustering (HCl) tree with clusters of “Lipid metabolism process” and “Lipid transport” genes.</p

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

Expression of chondrogenic and adipogenic markers during monolayer culture of wt and DN-Erg chondrocytes.

<p>A. Expression of chondrogenic genes <i>Col2a1</i>, <i>Sox9</i>, <i>Col10</i>, and <i>Runx2</i>. Gene expression was evaluated by RT-qPCR. B. Expression of adipogenic genes <i>Adpn</i>, and <i>Pparγ</i>. Gene expression was evaluated by RT-qPCR. The reported target gene: <i>Hprt</i> transcript ratio in chondrocytes was normalised to the target gene:<i>Hprt</i> transcript ratio (set to 1) of freshly plated wt chondrocytes (day 0). Data represent the mean of at least 2 independent chondrocyte cultures from 2 distinct mice for each genotype.</p