Molecular analysis of chondrocytes cultured in agarose in response to dynamic compression

<p>Abstract</p> <p>Background</p> <p>Articular cartilage is exposed to high mechanical loads under normal physiological conditions and articular chondrocytes regulate the composition of cartilaginous matrix, in response to mechanical signals. However, the intracellular pathways involved in mechanotransduction are still being defined. Using the well-characterized chondrocyte/agarose model system and dynamic compression, we report protocols for preparing and characterizing constructs of murine chondrocytes and agarose, and analyzing the effect of compression on steady-state level of mRNA by RT-PCR, gene transcription by gene reporter assay, and phosphorylation state of signalling molecules by Western-blotting. The mouse model is of particular interest because of the availability of a large choice of bio-molecular tools suitable to study it, as well as genetically modified mice.</p> <p>Results</p> <p>Chondrocytes cultured in agarose for one week were surrounded by a newly synthesized pericellular matrix, as revealed by immunohistochemistry prior to compression experiments. This observation indicates that this model system is suitable to study the role of matrix molecules and trans-membrane receptors in cellular responsiveness to mechanical stress. The chondrocyte/agarose constructs were then submitted to dynamic compression with FX-4000C™ Flexercell^®Compression Plus™ System (Flexcell). After clearing proteins off agarose, Western-blotting analysis showed transient activation of Mitogen-activated protein kinases (MAPK) in response to dynamic compression. After assessment by capillary electrophoresis of the quality of RNA extracted from agarose, steady-state levels of mRNA expression was measured by real time PCR. We observed an up-regulation of cFos and cJun mRNA levels as a response to compression, in accordance with the mechanosensitive character observed for these two genes in other studies using cartilage explants submitted to compression. To explore further the biological response of mouse chondrocytes to the dynamic compression at the transcriptional level, we also developed an approach for monitoring changes in gene transcription in agarose culture by using reporter promoter constructs. A decrease in promoter activity of the gene coding for type II procollagen, the most abundant protein in cartilage, was observed in response to dynamic loading.</p> <p>Conclusion</p> <p>The protocols developed here offer the possibility to perform an integrated analysis of the molecular mechanisms of mechanotransduction in chondrocytes, at the gene and protein level.</p

La synapsine I de cerveau de boeuf: une phosphoproteine du tissu nerveux. Structure et etude de son interaction avec les microtubules

SIGLEINIST T 75331 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

A unique tool to selectively detect the chondrogenic IIB form of human type II procollagen protein

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Human odontoblast-like cells produce nitric oxide with antibacterial activity upon TLR2 activation

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Dynamic Compression of Chondrocyte-Agarose Constructs Reveals New Candidate Mechanosensitive Genes

International audienceArticular cartilage is physiologically exposed to repeated loads. The mechanical properties of cartilage are due to its extracellular matrix, and homeostasis is maintained by the sole cell type found in cartilage, the chondrocyte. Although mechanical forces clearly control the functions of articular chondrocytes, the biochemical pathways that mediate cellular responses to mechanical stress have not been fully characterised. The aim of our study was to examine early molecular events triggered by dynamic compression in chondrocytes. We used an experimental system consisting of primary mouse chondrocytes embedded within an agarose hydrogel; embedded cells were pre-cultured for one week and subjected to short-term compression experiments. Using Western blots, we demonstrated that chondrocytes maintain a differentiated phenotype in this model system and reproduce typical chondrocyte-cartilage matrix interactions. We investigated the impact of dynamic compression on the phosphorylation state of signalling molecules and genome-wide gene expression. After 15 min of dynamic compression, we observed transient activation of ERK1/2 and p38 (members of the mitogen-activated protein kinase (MAPK) pathways) and Smad2/3 (members of the canonical transforming growth factor (TGF)-β pathways). A microarray analysis performed on chondrocytes compressed for 30 min revealed that only 20 transcripts were modulated more than 2-fold. A less conservative list of 325 modulated genes included genes related to the MAPK and TGF-β pathways and/or known to be mechanosensitive in other biological contexts. Of these candidate mechanosensitive genes, 85% were down-regulated. Down-regulation may therefore represent a general control mechanism for a rapid response to dynamic compression. Furthermore, modulation of transcripts corresponding to different aspects of cellular physiology was observed, such as non-coding RNAs or primary cilium. This study provides new insight into how chondrocytes respond to mechanical forces

Cartilage-Characteristic Matrix Reconstruction by Sequential Addition of Soluble Factors During Expansion of Human Articular Chondrocytes and Their Cultivation in Collagen Sponges

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Characterization of Different Sources of Human MSCs Expanded in Serum-Free Conditions with Quantification of Chondrogenic Induction in 3D

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The Lysine Specific Demethylase-1 Negatively Regulates the COL9A1 Gene in Human Articular Chondrocytes

International audienceOsteoarthritis (OA) is a degenerative disease of the joints which is associated with an impaired production of the cartilage matrix by the chondrocytes. Here, we investigated the role of Lysine-Specific Demethylase-1 (LSD1), a chromatin remodeling enzyme whose role in articular chondrocytes was previously associated with a catabolic activity and which is potentially involved during OA. Following a loss of function strategy and RNA sequencing analysis, we detail the genes which are targeted by LSD1 in human articular chondrocytes and identify COL9A1, a gene encoding the α1 chain of the cartilage-specific type IX collagen, as negatively regulated by LSD1. We show that LSD1 interacts with the transcription factor SOX9 and is recruited to the promoter of COL9A1. Interestingly, we observe that OA cartilage displays stronger LSD1 immunostaining compared with normal, and we demonstrate that the depletion of LSD1 in OA chondrocytes prevents the decrease in COL9A1 following Il-1β treatment. These results suggest LSD1 is a new regulator of the anabolic activity of articular chondrocytes potentially destabilizing the cartilage matrix, since it negatively regulates COL9A1, a gene encoding a crucial anchoring collagen molecule. This newly identified role played by LSD1 may thus participate in the alteration of the cartilage matrix during OA

Experimental design and dynamic compression profile.

<p>Chondrocytes cultured in agarose for 6 days underwent dynamic compression using the FX-4000C Flexercell Compression Plus System (Flexcell International). Chondrocyte-agarose constructs underwent cyclical compression ranging from 20 kPa to 40 kPa at a frequency of 0.5 Hz for 5, 15 or 30 min. Signalling proteins were analysed by Western blot at each of the three time points. DNA microarray analysis was performed to compare 30 min-compression constructs to uncompressed constructs.</p

Results from DNA microarray analysis: gene expression levels in compressed samples were compared to uncompressed control samples (fold change >2 and p-value >0.01).

<p>Results from DNA microarray analysis: gene expression levels in compressed samples were compared to uncompressed control samples (fold change >2 and p-value >0.01).</p