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We investigated the gene expression pattern of selected enzymes involved in DNA methylation and the effects of the DNA methylation inhibitor 5-azacytidine during in vitro and in vivo cartilage formation. Based on the data of a PCR array performed on chondrifying BMP2-overexpressing C3H10T1/2 cells, the relative expressions of Tet1 (tet methylcytosine dioxygenase 1), Dnmt3a (DNA methyltransferase 3), and Ogt (O-linked N-acetylglucosamine transferase) were further examined with RT-qPCR in murine cell line-based and primary chondrifying micromass cultures. We found very strong but gradually decreasing expression of Tet1 throughout the entire course of in vitro cartilage differentiation along with strong signals in the cartilaginous embryonic skeleton using specific RNA probes for in situ hybridization on frozen sections of 15-day-old mouse embryos. Dnmt3a and Ogt expressions did not show significant changes with RT-qPCR and gave weak in situ hybridization signals. The DNA methylation inhibitor 5-azacytidine reduced cartilage-specific gene expression and cartilage formation when applied during the early stages of chondrogenesis. In contrast, it had a stimulatory effect when added to differentiated chondrocytes, and quantitative methylation-specific PCR proved that the DNA methylation pattern of key chondrogenic marker genes was altered by the treatment. Our results indicate that the DNA demethylation inducing Tet1 plays a significant role during chondrogenesis, and inhibition of DNA methylation exerts distinct effects in different phases of in vitro cartilage formation.

The protein clusterin has been implicated in the molecular alterations that occur in articular cartilage during osteoarthritis (OA). Clusterin exists in two isoforms with opposing functions, and their roles in cartilage have not been explored. The secreted form of clusterin (sCLU) is a cytoprotective extracellular chaperone that prevents protein aggregation, enhances cell proliferation and promotes viability, whereas nuclear clusterin acts as a pro-death signal. Therefore, these two clusterin isoforms may be putative molecular markers of repair and catabolic responses in cartilage and the ratio between them may be important. In this study, we focused on sCLU and used established, pathophysiologically relevant, in vitro models to understand its role in cytokine-stimulated cartilage degradation. The secretome of equine cartilage explants, osteochondral biopsies and isolated unpassaged chondrocytes was analyzed by western blotting for released sCLU, cartilage oligomeric protein (COMP) and matrix metalloproteinases (MMP) 3 and 13, following treatment with the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α. Release of sulfated glycosaminoglycans (sGAG) was determined using the dimethylmethylene blue assay. Clusterin messenger RNA (mRNA) expression was quantified by quantitative real-time polymerase chain reaction. MMP-3, MMP-13, COMP, and sGAG release from explants and osteochondral biopsies was elevated with cytokine treatment, confirming cartilage degradation in these models. sCLU release was attenuated with cytokine treatment in all models, potentially limiting its cytoprotective function. Clusterin mRNA expression was down-regulated 7-days post cytokine stimulation. These observations implicate sCLU in catabolic responses of chondrocytes, but further studies are required to evaluate its role in OA and its potential as an investigative biomarker.

Supplemental material, revised-supplementary-material for A Synchronized Circadian Clock Enhances Early Chondrogenesis by M. Abdulhadi Alagha, Judit Vágó, Éva Katona, Roland Takács, Daan van der Veen, Róza Zákány and Csaba Matta in CARTILAGE

Additional file 7. This file contains the uncropped western blot membrane images presented in Figs. 2, 5 and 6.

Additional file 1: Figure S1. GAG release levels in the chondrocyte secretome upon cytokine exposure. Figure S2. Rate of apoptosis of chondrocytes under pro-inflammatory versus control conditions. Table S1. List of proteins classified as Enzymes based on GO annotations. Table S2. List of proteins classified as Receptors based on GO annotations. Table S3. List of proteins classified as Transporters based on GO annotations. Table S4. List of proteins that could not be classified into any of the previous categories based on GO annotations (listed as Unclassified). Table S5. List of proteins classified into the category of Structural/Adhesion/Junctional proteins based on GO annotations. Table S6. List of proteins classified into the category of Extracellular matrix proteins based on GO annotations.

Additional file 1: Table S1. Sequences of primer pairs and PCR condition. Table S2. List of transcript variants amplified by the GRIN primers. Figure S1. Specificity controls for anti-GluN antibodies employed in this study. Figure S2. Uncropped agarose gel images showing negative results for GRIN2C and GRIN2D (RT-PCR) and lack of GluN2A signals in the membrane fraction (western blot). Figure S3. Visualising GluN subunit expression using immunocytochemistry. Figure S4. AMPA, kainate, and metabotropic GluR, as well as GlyR subunit mRNA expression profiles in chondrogenic micromass cultures. Figure S5. Glutamate secretion into the medium during chondrogenesis. Figure S6. VGLUT1 and VGLUT2 expression profiles during chondrogenesis. Figure S7. Negative control experiments for Fura-2 based fluorescence calcium assays . Figure S8. Whole-cell patch-clamp measurements in chondrogenic cells. Figure S9. mRNA expression profiles of chondrogenic marker genes following gene silencing by GRIN1 siRNA.

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