IntroductionIn addition to the well-known cartilage extracellular matrix-related expression of Sox9, we demonstrated that chondrogenic differentiation of progenitor cells is driven by a sharply defined bi-phasic expression of Sox9: an immediate early and a late (extracellular matrix associated) phase expression. In this study, we aimed to determine what biological processes are driven by Sox9 during this early phase of chondrogenic differentiation.MaterialsSox9 expression in ATDC5 cells was knocked down by siRNA transfection at the day before chondrogenic differentiation or at day 6 of differentiation. Samples were harvested at 2 h and 7 days of differentiation. The transcriptomes (RNA-seq approach) and proteomes (Label-free proteomics approach) were compared using pathway and network analyses. Total protein translational capacity was evaluated with the SuNSET assay, active ribosomes were evaluated with polysome profiling, and ribosome modus was evaluated with bicistronic reporter assays.ResultsEarly Sox9 knockdown severely inhibited chondrogenic differentiation weeks later. Sox9 expression during the immediate early phase of ATDC5 chondrogenic differentiation regulated the expression of ribosome biogenesis factors and ribosomal protein subunits. This was accompanied by decreased translational capacity following Sox9 knockdown, and this correlated to lower amounts of active mono- and polysomes. Moreover, cap- versus IRES-mediated translation was altered by Sox9 knockdown. Sox9 overexpression was able to induce reciprocal effects to the Sox9 knockdown.ConclusionHere, we identified an essential new function for Sox9 during early chondrogenic differentiation. A role for Sox9 in regulation of ribosome amount, activity, and/or composition may be crucial in preparation for the demanding proliferative phase and subsequent cartilage extracellular matrix production of chondroprogenitors in the growth plate in vivo.

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Background:Osteoarthritis (OA) is mainly characterized by the progressive deterioration of articular cartilage. Recent studies support that type 2 diabetes (TD2) is a risk factor to develop OA [1, 2]. However, the molecular cartilage profile of patients combining these two diseases remains unclear, and a better understanding of the different OA phenotypes should be considered for the development of personalized medicine.Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is used to investigate the bimolecular distribution of proteins, lipids or metabolites through thein-situanalysis of tissue sections. Bottom-up proteomics focuses on the relative quantification of proteins. The combination of both technologies could be considered to reveal specific molecular profiles and help for patient classification.Objectives:The main goal of this study is to apply a multimodal mass spectrometry approach on cartilage to reveal specific lipidomic and proteomic profiles associated to TD2 patients.Methods:Human cartilages from OA (na=10) and OA/TD2 human patients (nb=10) were obtained from donors undergoing total knee joint replacement. Cartilage punches of 8*8mm were sectioned at 12 µm thickness for MALDI-MSI and bottom-up proteomics.For MALDI-MSI experiments (na=6; nb=6), norharmane matrix was sprayed over the samples for the detection of lipids. Experiments were then performed in positive ion polarity at 50 µm of lateral resolution using a RapifleX MALDI Tissue-typer instrument. LipostarMSI and in-house ChemomeTricks toolbox for MATLAB software were used for data processing and analysis.For bottom-up proteomics experiment (na=10; nb=10), proteins were extracted, separated using SDS-PAGE and digested prior to liquid chromatography separation coupled to an orbitrap MS Q-Exactive HF mass spectrometer. Proteome Discoverer, enrichR and Reactome software were used for data processing and analysis.Results:MALDI-MSI showed overall differences between OA and OA/TD2 patients based on their specific lipidomic profiles. In particular, sphingomyelin and phosphatidylcholine species were significantly more abundant in OA patients whereas lysolipids such as lysophosphatidylcholine species were mainly present in OA/TD2 patients, providing therefore phenotype-specific OA molecular panels. Additionally, we observed that phosphatidylcholine and sphingomyelin species were more present in the superficial layer of the cartilage whereas lysophosphatidylcholine species were more abundant in the deep layer (Fig. 1A, B).Proteomics experiments applied on cartilage enables the quantification of 114 proteins. Among those, 73 were overexpressed in OA samples whereas 41 were overexpressed in OA/TD2 patients. Among the differentially regulated proteins (Fig. 1C), phospholipase A2 was increased in the diabetic cohort, in line with the elevated level of lysolipids found in the imaging data. Our results also involved the fatty acid omega oxidation and the fatty acid biosynthesis pathways as relevant to explain this deregulation of the lipid metabolism.Conclusion:MALDI–MSI combined with proteomics experiments showed different profiles between OA and OA/TD2 patients and could be employed for patient classification.References:[1]Louati, K., et al., Association between diabetes mellitus and osteoarthritis: systematic literature review and meta-analysis. RMD Open, 2015.1(1): p. e000077.[2]Williams, M.F., et al.,Type 2 diabetes and osteoarthritis: a systematic review and meta-analysis.J Diabetes Complications, 2016.30(5): p. 944-50.Acknowledgments:The Dutch Province of Limburg and MUMC institutional grant.Disclosure of Interests:Maxime Eveque: None declared, Pieter Emans Shareholder of: Shareholder and cofounder start-up company Chondropeptix, Grant/research support from: Institution received grants from STW, ReumaNederland, InSciTE, Consultant of: Consultancy to Kiomed, Speakers bureau: Payment for lectures by Kiomed, Episurf, Britt Claes: None declared, Freek Bouwman: None declared, Ron M A Heeren: None declared, Berta Cillero-Pastor: None declared

injuries presented for medical (Littlewood et al 2013) or veterinary attention (Williams et al 2001). Tendinopathies are painful, mobility limiting, affect quality of life and carry a substantial financial cost associated with treatment, rehabilitation and lost productivity. A range of tendons are commonly affected in humans, such as Achilles, posterior tibial and rotator cuff tendons, whilst in horses, the superficial digital flexor tendon (SDFT) is over represented. It is increasingly recognised that tendon failure is the end result of a chronic, asymptomatic process of deterioration at the cellular and molecular level and once this occurs, mechanical integrity is never fully restored. MicroRNAs (miRs) are robust regulators of cellular pathways and physiological processes, modulating multiple gene expression. Altered miR expression has been demonstrated during ageing (Peffers et al 2015) and mechanical loading (Mendias et al 2012) of tendon. Identification of miRs dysregulated during ageing and disease offers the potential to more fully understand the disease process through identifying miR-regulated pathways which may be targeted to develop effective miR-based therapeutics. Equine SDFT is an ideal model to study human tendinopathy, being an elastic energy storing tendon sharing functional similarity with commonly injured human tendons. The longevity and use of horses makes them ideal for studying age and exercise related impacts on tendon integrity. This study aimed to identify miRs differentially expressed in equine SDFT during ageing and with naturally occurring tendinopathy. Methods: Total RNA was extracted from equine SDFT collected from a commercial abattoir. Thirteen horses aged 3-25 years were included, eight samples were classified as healthy with no gross evidence of tendinopathy and five as diseased. Samples of both healthy and diseased tendon tissue were collected from 2 horses. Following spectrophotometric determination of nucleic acid concentration and purity, 100ng RNA template was converted into cDNA. Expression of miR-: 29a, 34a, 34b, 34c, 181a, 181b, 181c, 181d, 199a, 199b, let-7b and let-7f , selected by us based on predicted and validated target genes known, or predicted, to be associated with tendinopathy, was measured by quantitative real time PCR, using 2.5ng cDNA template. Relative expression was calculated relative to Snord61. For analysis purposes, individuals were classed as young (3-9 years, n¼9) or old (19-25 years, n¼4). Statistical analysis was performed using GraphPad Prism 6. Results: Expression of miR-29a, previously shown to be associated with tendinopathy (Millar et al 2015), was significantly decreased in old versus young and diseased versus normal equine tendon. The expression of miR-34a was reduced in old horses, whilst 34b was upregulated. MiR-34a and 34b demonstrated higher expression in diseased than healthy tendons at the population level. The expression of miR-181b was significantly increased in older animals, and reduced in diseased tissue. The expression of miRs: mi...