Chondrogenic differentiation and cartilage homeostasis requires a high cellular translational capacity to meet the demands for cartilaginous extracellular matrix production. Box C/D and H/ACA snoRNAs guide post-transcriptional 2′-O ribose methylation and pseudouridylation of specific ribosomal RNA (rRNA) nucleotides, respectively. How specific rRNA modifications influence rRNA function is poorly documented, but modifications are thought to tune rRNA folding and interaction with ribosomal proteins, which is critical for ribosome function. We hypothesise that chondrocyte translational capacity is supported by snoRNA-mediated post-transcriptional fine-tuning of rRNAs.

ATDC5 progenitor cells were differentiated into the chondrogenic lineage, resembling mature and mineralising chondrocytes after 7 or 14 days, respectively. UBF-1 (rRNA transcription factor), fibrillarin (box C/D methyltransferase) and dyskerin (box H/ACA pseudouridylase) expression displayed highest fold induction at day 5/6 in differentiation. Ribosomal RNA content per cell was increased at day 7, but not at day 14 in differentiation. These data suggest that ribosome biogenesis adapts to the chondrocyte's differentiation status. RNA-Seq of RNA species <200 nt revealed expression of at least 224 individual snoRNAs. Due to initiation of chondrogenic differentiation (Δt0-t7), 21 snoRNAs were differentially expressed (DE; FDRadj-p<0.05, logFC>1or<−1). Mineralization (Δt7-t14) induced DE of 23 snoRNAs. Comparing t0 with t14 resulted in DE of 43 snoRNAs. To anticipate on the biological relevance of DE snoRNAs, their rRNA target nucleotides were plotted in 18S, 5.8S and 28S rRNA secondary structures. This revealed that DE snoRNAs, amongst others, target nucleotide modifications in the 28S peptidyl transferase center and the 18S decoding center (DC). Snora40 was DE, targeting helix 27/18S rRNA. Helix 27 controls DC function. Helix 68 of 28S rRNA is part of the ribosome's E-site, therefore, DE snord36c and snora31 (targeting helix 68) could potentially fine-tune the translation mechanism. As a final example we found snord46 to be DE (target: helix 69/28S rRNA). Mutations in helix 69 have been shown to severely affect cell viability.

Our data show that increased demand for translational capacity during chondrogenic differentiation is associated with differential expression of snoRNAs, potentially controlling ribosome fidelity via site-specific rRNA-modifications. These data enable us to determine the role of individual snoRNAs in tuning the chondrocyte's translational properties and current efforts focus on confirming site-specific rRNA-modifications and determine their biological relevance.

Heterotopic ossi?cation is the abnormal formation of bone in soft tissues and is a frequent complication of hip replacement surgery. Heterotopic ossi?cations are described to develop via endochondral ossification and standard treatment is administration of indomethacin. It is currently unknown how indomethacin influences heterotopic ossi?cation on a molecular level, therefore we aimed to determine whether indomethacin might influence heterotopic ossi?cation via impairing the chondrogenic phase of endochondral ossification.

ATDC5, human bone marrow stem cells (hBMSCs) and rabbit periosteal agarose cultures were employed as progenitor cell models; SW1353, human articular chondrocytes and differentiated ATDC5 cells were used as matured chondrocyte cell models. All cells were cultured in the presence of (increasing) concentrations of indomethacin. The action of indomethacin was confirmed by decreased PGE2 levels in all experiments, and was determined by specific PGE2 ELISA. Gene- and protein expression analyses were employed to determine chondrogenic outcome.

Progenitor cell models differentiating in the chondrogenic lineage (ATDC5, primary human bone marrow stem cells and ex vivo periosteal agarose cultures) were treated with increasing concentrations of indomethacin and a dose-dependent decrease in gene- and protein expression of chondrogenic and hypertrophic markers as well as decreased glycosaminoglycan content was observed. Even when hypertrophic differentiation was provoked the addition of indomethacin resulted in decreased hypertrophic marker expression. Interestingly, when mature chondrocytes were treated with indomethacin, a clear increase in collagen type 2 expression was observed. Similarly, when ATDC5 cells and bone marrow stem cells were pre-differentiated to obtain a chondrocyte phenotype and indomethacin was added from this time point onwards, low concentrations of indomethacin also resulted in increased chondrogenic differentiation.

Indomethacin induces differential effects on in vitro endochondral ossification, depending on the chondrocyte's differentiation stage, with complete inhibition of chondrogenic differentiation as the most pronounced action. This observation may provide a rationale behind the elusive mode of action of indomethacin in the treatment of heterotopic ossifications.

Summary

Indomethacin has differential effects on chondrogencic outcome depending on differentiation stage