TGF-ß2 Induces Ribosome Activity, Alters Ribosome Composition and Inhibits IRES-Mediated Translation in Chondrocytes.

Alterations in cell fate are often attributed to (epigenetic) regulation of gene expression. An emerging paradigm focuses on specialized ribosomes within a cell. However, little evidence exists for the dynamic regulation of ribosome composition and function. Here, we stimulated a chondrocytic cell line with transforming growth factor beta (TGF-ß2) and mapped changes in ribosome function, composition and ribosomal RNA (rRNA) epitranscriptomics. 35S Met/Cys incorporation was used to evaluate ribosome activity. Dual luciferase reporter assays were used to assess ribosomal modus. Ribosomal RNA expression and processing were determined by RT-qPCR, while RiboMethSeq and HydraPsiSeq were used to determine rRNA modification profiles. Label-free protein quantification of total cell lysates, isolated ribosomes and secreted proteins was done by LC-MS/MS. A three-day TGF-ß2 stimulation induced total protein synthesis in SW1353 chondrocytic cells and human articular chondrocytes. Specifically, TGF-ß2 induced cap-mediated protein synthesis, while IRES-mediated translation was not (P53 IRES) or little affected (CrPv IGR and HCV IRES). Three rRNA post-transcriptional modifications (PTMs) were affected by TGF-ß2 stimulation (18S-Gm1447 downregulated, 18S-ψ1177 and 28S-ψ4598 upregulated). Proteomic analysis of isolated ribosomes revealed increased interaction with eIF2 and tRNA ligases and decreased association of eIF4A3 and heterogeneous nuclear ribonucleoprotein (HNRNP)s. In addition, thirteen core ribosomal proteins were more present in ribosomes from TGF-ß2 stimulated cells, albeit with a modest fold change. A prolonged stimulation of chondrocytic cells with TGF-ß2 induced ribosome activity and changed the mode of translation. These functional changes could be coupled to alterations in accessory proteins in the ribosomal proteome.

Discovery of calcite as a new pro-inflammatory calcium-containing crystal in human osteoarthritic synovial fluid.

OBJECTIVE: We aimed to characterize calcium-containing crystals present in synovial fluid from patients with knee osteoarthritis (OA) using Raman spectroscopy, and specifically investigate the biological effects of calcite crystals. DESIGN: Thirty-two synovial fluid samples were collected pre-operatively from knee OA patients undergoing total joint arthroplasty. An integrated Raman polarized light microscope was used for identification of crystals in synovial fluid. Human peripheral blood mononuclear cells (PBMC's), human OA articular chondrocytes (HACs) and fibroblast-like synoviocytes (FLSs) were exposed to calcite crystals. Expression of relevant cytokines and inflammatory genes were measured using ELISA and real-time PCR. RESULTS: Various calcium-containing crystals were identified, including calcium pyrophosphate (37.5 %) and basic calcium phosphate (21.8 %), but they were never found simultaneously in the same OA synovial fluid sample. For the first time, we discovered the presence of calcite crystals in 93.8 % of the samples, while dolomite was detected in 25 % of the cases. Characterization of the cellular response to calcite crystal exposure revealed increased production of innate immune-derived cytokines by PBMC's, when co-stimulated with lipopolysaccharide (LPS). Additionally, calcite crystal stimulation of HACs and FLSs resulted in enhanced secretion of pro-inflammatory molecules and alterations in the expression of extracellular matrix remodeling enzymes. CONCLUSIONS: This study highlights the unique role of Raman spectroscopy in OA crystal research and identified calcite as a novel pro-inflammatory crystal type in OA synovial fluid. Understanding the role of specific crystal species in the OA joint may open new avenues for pharmacological interventions and personalized approaches to treating OA.

The BMP7-Derived Peptide p[63-82] Reduces Cartilage Degeneration in the Rat ACLT-pMMx Model for Posttraumatic Osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is characterized by articular cartilage erosion, pathological subchondral bone changes, and signs of synovial inflammation and pain. We previously identified p[63-82], a bone morphogenetic protein 7 (BMP7)-derived bioactive peptide that attenuates structural cartilage degeneration in the rat medial meniscal tear-model for posttraumatic OA. This study aimed to evaluate the cartilage erosion-attenuating activity of p[63-82] in a different preclinical model for OA (anterior cruciate ligament transection-partial medial meniscectomy [anterior cruciate ligament transection (ACLT)-pMMx]). The disease-modifying action of the p[63-82] was followed-up in this model for 5 and 10 weeks. DESIGN: Skeletally mature male Lewis rats underwent ACLT-pMMx surgery. Rats received weekly intra-articular injections with either saline or 500 ng p[63-82]. Five and 10 weeks postsurgery, rats were sacrificed, and subchondral bone characteristics were determined using microcomputed tomography (µCT). Histopathological evaluation of cartilage degradation and Osteoarthritis Research Society International (OARSI)-scoring was performed following Safranin-O/Fast Green staining. Pain-related behavior was measured by incapacitance testing and footprint analysis. RESULTS: Histopathological evaluation at 5 and 10 weeks postsurgery showed reduced cartilage degeneration and a significantly reduced OARSI score, whereas no significant changes in subchondral bone characteristics were found in the p[63-82]-treated rats compared to the saline-treated rats. ACLT-pMMx-induced imbalance of static weightbearing capacity in the p[63-82] group was significantly improved compared to the saline-treated rats at weeks 5 postsurgery. Footprint analysis scores in the p[63-82]-treated rats demonstrated improvement at week 10 postsurgery. CONCLUSIONS: Weekly intra-articular injections of p[63-82] in the rat ACLT-pMMx posttraumatic OA model resulted in reduced degenerative cartilage changes and induced functional improvement in static weightbearing capacity during follow-up.

Multi-Omic Temporal Landscape of Plasma and Synovial Fluid-Derived Extracellular Vesicles Using an Experimental Model of Equine Osteoarthritis.

Extracellular vesicles (EVs) contribute to osteoarthritis pathogenesis through their release into joint tissues and synovial fluid. Synovial fluid-derived EVs have the potential to be direct biomarkers in the causal pathway of disease but also enable understanding of their role in disease progression. Utilizing a temporal model of osteoarthritis, we defined the changes in matched synovial fluid and plasma-derived EV small non-coding RNA and protein cargo using sequencing and mass spectrometry. Data exploration included time series clustering, factor analysis and gene enrichment interrogation. Chondrocyte signalling was analysed using luciferase-based transcription factor activity assays. EV protein cargo appears to be more important during osteoarthritis progression than small non-coding RNAs. Cluster analysis revealed plasma-EVs represented a time-dependent response to osteoarthritis induction associated with supramolecular complexes. Clusters for synovial fluid-derived EVs were associated with initial osteoarthritis response and represented immune/inflammatory pathways. Factor analysis for plasma-derived EVs correlated with day post-induction and were primarily composed of proteins modulating lipid metabolism. Synovial fluid-derived EVs factors represented intermediate filament and supramolecular complexes reflecting tissue repair. There was a significant interaction between time and osteoarthritis for CRE, NFkB, SRE, SRF with a trend for osteoarthritis synovial fluid-derived EVs at later time points to have a more pronounced effect.

Depletion of SNORA33 Abolishes ψ of 28S-U4966 and Affects the Ribosome Translational Apparatus.

Eukaryotic ribosomes are complex molecular nanomachines translating genetic information from mRNAs into proteins. There is natural heterogeneity in ribosome composition. The pseudouridylation (ψ) of ribosomal RNAs (rRNAs) is one of the key sources of ribosome heterogeneity. Nevertheless, the functional consequences of ψ-based ribosome heterogeneity and its relevance for human disease are yet to be understood. Using HydraPsiSeq and a chronic disease model of non-osteoarthritic primary human articular chondrocytes exposed to osteoarthritic synovial fluid, we demonstrated that the disease microenvironment is capable of instigating site-specific changes in rRNA ψ profiles. To investigate one of the identified differential rRNA ψ sites (28S-ψ4966), we generated SNORA22 and SNORA33 KO SW1353 cell pools using LentiCRISPRv2/Cas9 and evaluated the ribosome translational capacity by 35S-Met/Cys incorporation, assessed the mode of translation initiation and ribosomal fidelity using dual luciferase reporters, and assessed cellular and ribosomal proteomes by LC-MS/MS. We uncovered that the depletion of SNORA33, but not SNORA22, reduced 28S-ψ4966 levels. The resulting loss of 28S-ψ4966 affected ribosomal protein composition and function and led to specific changes in the cellular proteome. Overall, our pioneering findings demonstrate that cells dynamically respond to disease-relevant changes in their environment by altering their rRNA pseudouridylation profiles, with consequences for ribosome function and the cellular proteome relevant to human disease.

Adaptation of the protein translational apparatus during ATDC5 chondrogenic differentiation.

Introduction: Ribosome biogenesis is integrated with many cellular processes including proliferation, differentiation and oncogenic events. Chondrogenic proliferation and differentiation require a high cellular translational capacity to facilitate cartilaginous extracellular matrix production. We here investigated the expression dynamics of factors involved in ribosome biogenesis during in vitro chondrogenic differentiation and determined whether protein translation capacity adapts to different phases of chondrogenic differentiation. Materials: SnoRNA expression during ATDC5 differentiation was analyzed by RNA sequencing of samples acquired from day 0 (progenitor stage), 7 (chondrogenic stage) and day 14 (hypertrophic stage). RT-qPCR was used to determine expression of fibrillarin, dyskerin, UBF-1, Sox9, Col2a1, Runx2, Col10a1 mRNAs and 18S, 5.8S and 28S rRNAs. Protein expression of fibrillarin, dyskerin and UBF-1 was determined by immunoblotting. Ribosomal RNA content per cell was determined by calculating rRNA RT-qPCR signals relative to DNA content (SYBR Green assay). Total protein translational activity was evaluated with a puromycilation assay and polysome profiling. Results: As a result of initiation of chondrogenic differentiation (Δt0-t7), 21 snoRNAs were differentially expressed (DE). Hypertrophic differentiation caused DE of 23 snoRNAs (Δt7-t14) and 43 when t0 was compared to t14. DE snoRNAs, amongst others, target nucleotide modifications in the 28S rRNA peptidyl transferase center and the 18S rRNA decoding center. UBF-1, fibrillarin and dyskerin expression increased as function of differentiation and displayed highest fold induction at day 5-6 in differentiation. Ribosomal RNA content per cell was significantly increased at day 7, but not at day 14 in differentiation. Similar dynamics in translational capacity and monosomal ribosome fraction were observed during differentiation. Conclusion: The expression of a great number of ribosome biogenesis factors is altered during chondrogenic differentiation of ATDC5 cells, which is accompanied by significant changes in cellular translational activity. This elucidation of ribosome biogenesis dynamics in chondrogenic differentiation models enables the further understanding of the role of ribosome biogenesis and activity during chondrocyte cell commitment and their roles in human skeletal development diseases.

BMP7 increases protein synthesis in SW1353 cells and determines rRNA levels in a NKX3-2-dependent manner.

BMP7 is a morphogen capable of counteracting the OA chondrocyte hypertrophic phenotype via NKX3-2. NKX3-2 represses expression of RUNX2, an important transcription factor for chondrocyte hypertrophy. Since RUNX2 has previously been described as an inhibitor for 47S pre-rRNA transcription, we hypothesized that BMP7 positively influences 47S pre-rRNA transcription through NKX3-2, resulting in increased protein translational capacity. Therefor SW1353 cells and human primary chondrocytes were exposed to BMP7 and rRNA (18S, 5.8S, 28S) expression was determined by RT-qPCR. NKX3-2 knockdown was achieved via transfection of a NKX3-2-specific siRNA duplex. Translational capacity was assessed by the SUNsET assay, and 47S pre-rRNA transcription was determined by transfection of a 47S gene promoter-reporter plasmid. BMP7 treatment increased protein translational capacity. This was associated by increased 18S and 5.8S rRNA and NKX3-2 mRNA expression, as well as increased 47S gene promotor activity. Knockdown of NKX3-2 led to increased expression of RUNX2, accompanied by decreased 47S gene promotor activity and rRNA expression, an effect BMP7 was unable to restore. Our data demonstrate that BMP7 positively influences protein translation capacity of SW1353 cells and chondrocytes. This is likely caused by an NKX3-2-dependent activation of 47S gene promotor activity. This finding connects morphogen-mediated changes in cellular differentiation to an aspect of ribosome biogenesis via key transcription factors central to determining the chondrocyte phenotype.

Prediction of the Effect of the Osteoarthritic Joint Microenvironment on Cartilage Repair.

Osteoarthritis (OA) is characterized by progressive articular cartilage loss. Human mesenchymal stromal cells (MSCs) can be used for cartilage repair therapies based on their potential to differentiate into chondrocytes. However, the joint microenvironment is a major determinant of the success of MSC-based cartilage formation. Currently, there is no tool that is able to predict the effect of a patient's OA joint microenvironment on MSC-based cartilage formation. Our goal was to develop a molecular tool that can predict this effect before the start of cartilage repair therapies. Six different promoter reporters (hIL6, hIL8, hADAMTS5, hWISP1, hMMP13, and hADAM28) were generated and evaluated in an immortalized human articular chondrocyte for their responsiveness to an osteoarthritic microenvironment by stimulation with OA synovium-conditioned medium (OAs-cm) obtained from 32 different knee OA patients. To study the effect of this OA microenvironment on MSC-based cartilage formation, MSCs were cultured in a three-dimensional pellet culture model, while stimulated with OAs-cm. Cartilage formation was assessed histologically and by quantifying sulfated glycosaminoglycan (sGAG) production. We confirmed that OAs-cm of different patients had significantly different effects on sGAG production. In addition, significant correlations were obtained between the effect of the OAs-cm on cartilage formation and promoter reporter outcome. Furthermore, we validated the predictive value of measuring two promoter reporters with an independent cohort of OAs-cm and the effect of 87.5% of the OAs-cm on MSC-based cartilage formation could be predicted. Together, we developed a novel tool to predict the effect of the OA joint microenvironment on MSC-based cartilage formation. This is an important first step toward personalized cartilage repair strategies for OA patients. Impact statement We describe the development of a novel molecular tool to predict if an osteoarthritis joint microenvironment is permissive for cartilage repair or not. Such a tool is of great importance in determining the success of mesenchymal stromal cell-based cartilage repair strategies.

Ribosome dysfunction in osteoarthritis.

PURPOSE OF REVIEW: Translation of genetic information encoded within mRNA molecules by ribosomes into proteins is a key part of the central dogma of molecular biology. Despite the central position of the ribosome in the translation of proteins, and considering the major proteomic changes that occur in the joint during osteoarthritis development and progression, the ribosome has received very limited attention as driver of osteoarthritis pathogenesis. RECENT FINDINGS: We provide an overview of the limited literature regarding this developing topic for the osteoarthritis field. Recent key findings that connect ribosome biogenesis and activity with osteoarthritis include: ribosomal RNA transcription, processing and maturation, ribosomal protein expression, protein translation capacity and preferential translation. SUMMARY: The ribosome as the central cellular protein synthesis hub is largely neglected in osteoarthritis research. Findings included in this review reveal that in osteoarthritis, ribosome aberrations have been found from early-stage ribosome biogenesis, through ribosome build-up and maturation, up to preferential translation. Classically, osteoarthritis has been explained as an imbalance between joint tissue anabolism and catabolism. We postulate that osteoarthritis can be interpreted as an acquired ribosomopathy. This hypothesis fine-tunes the dogmatic anabolism/katabolism point-of-view, and may provide novel molecular opportunities for the development of osteoarthritis disease-modifying treatments.

BMP7 reduces the fibrocartilage chondrocyte phenotype.

The fibrocartilage chondrocyte phenotype has been recognized to attribute to osteoarthritis (OA) development. These chondrocytes express genes related to unfavorable OA outcomes, emphasizing its importance in OA pathology. BMP7 is being explored as a potential disease-modifying molecule and attenuates the chondrocyte hypertrophic phenotype. On the other hand, BMP7 has been demonstrated to relieve organ fibrosis by counteracting the pro-fibrotic TGFß-Smad3-PAI1 axis and increasing MMP2-mediated Collagen type I turnover. Whether BMP7 has anti-fibrotic properties in chondrocytes is unknown. Human OA articular chondrocytes (HACs) were isolated from end-stage OA femoral cartilage (total knee arthroplasty; n = 18 individual donors). SW1353 cells and OA HACs were exposed to 1 nM BMP7 for 24 h, after which gene expression of fibrosis-related genes and fibrosis-mediating factors was determined by RT-qPCR. In SW1353, Collagen type I protein levels were determined by immunocytochemistry and western blotting. PAI1 and MMP2 protein levels and activity were measured with an ELISA and activity assays, respectively. MMP2 activity was inhibited with the selective MMP-2 inhibitor OA-Hy. SMAD3 activity was determined by a (CAGA)12-reporter assay, and pSMAD2 levels by western blotting. Following BMP7 exposure, the expression of fibrosis-related genes was reduced in SW1353 cells and OA HACs. BMP7 reduced Collagen type I protein levels in SW1353 cells. Gene expression of MMP2 was increased in SW1353 cells following BMP7 treatment. BMP7 reduced PAI1 protein levels and -activity, while MMP2 protein levels and -activity were increased by BMP7. BMP7-dependent inhibition of Collagen type I protein levels in SW1353 cells was abrogated when MMP2 activity was inhibited. Finally, BMP7 reduced pSMAD2 levels determined by western blotting and reduced SMAD3 transcriptional activity as demonstrated by decreased (CAGA)12 luciferase reporter activity. Our data demonstrate that short-term exposure to BMP7 decreases the fibrocartilage chondrocyte phenotype. The BMP7-dependent reduction of Collagen type I protein expression seems MMP2-dependent and inhibition of Smad2/3-PAI1 activity was identified as a potential pathway via which BMP7 exerts its anti-fibrotic action. This indicates that in chondrocytes BMP7 may have a double mode-of-action by targeting both the hypertrophic as well as the fibrotic chondrocyte phenotype, potentially adding to the clinical relevance of using BMP7 as an OA disease-modifying molecule.

Osteoarthritis-Related Inflammation Blocks TGF-ß's Protective Effect on Chondrocyte Hypertrophy via (de)Phosphorylation of the SMAD2/3 Linker Region.

Osteoarthritis (OA) is a degenerative joint disease characterized by irreversible cartilage damage, inflammation and altered chondrocyte phenotype. Transforming growth factor-ß (TGF-ß) signaling via SMAD2/3 is crucial for blocking hypertrophy. The post-translational modifications of these SMAD proteins in the linker domain regulate their function and these can be triggered by inflammation through the activation of kinases or phosphatases. Therefore, we investigated if OA-related inflammation affects TGF-ß signaling via SMAD2/3 linker-modifications in chondrocytes. We found that both Interleukin (IL)-1ß and OA-synovium conditioned medium negated SMAD2/3 transcriptional activity in chondrocytes. This inhibition of TGF-ß signaling was enhanced if SMAD3 could not be phosphorylated on Ser213 in the linker region and the inhibition by IL-1ß was less if the SMAD3 linker could not be phosphorylated at Ser204. Our study shows evidence that inflammation inhibits SMAD2/3 signaling in chondrocytes via SMAD linker (de)-phosphorylation. The involvement of linker region modifications may represent a new therapeutic target for OA.

Sox9 Determines Translational Capacity During Early Chondrogenic Differentiation of ATDC5 Cells by Regulating Expression of Ribosome Biogenesis Factors and Ribosomal Proteins.

INTRODUCTION: In 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. MATERIALS: Sox9 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. RESULTS: Early 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. CONCLUSION: Here, 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.

Current Practice in Bicistronic IRES Reporter Use: A Systematic Review.

Bicistronic reporter assays have been instrumental for transgene expression, understanding of internal ribosomal entry site (IRES) translation, and identification of novel cap-independent translational elements (CITE). We observed a large methodological variability in the use of bicistronic reporter assays and data presentation or normalization procedures. Therefore, we systematically searched the literature for bicistronic IRES reporter studies and analyzed methodological details, data visualization, and normalization procedures. Two hundred fifty-seven publications were identified using our search strategy (published 1994-2020). Experimental studies on eukaryotic adherent cell systems and the cell-free translation assay were included for further analysis. We evaluated the following methodological details for 176 full text articles: the bicistronic reporter design, the cell line or type, transfection methods, and time point of analyses post-transfection. For the cell-free translation assay, we focused on methods of in vitro transcription, type of translation lysate, and incubation times and assay temperature. Data can be presented in multiple ways: raw data from individual cistrons, a ratio of the two, or fold changes thereof. In addition, many different control experiments have been suggested when studying IRES-mediated translation. In addition, many different normalization and control experiments have been suggested when studying IRES-mediated translation. Therefore, we also categorized and summarized their use. Our unbiased analyses provide a representative overview of bicistronic IRES reporter use. We identified parameters that were reported inconsistently or incompletely, which could hamper data reproduction and interpretation. On the basis of our analyses, we encourage adhering to a number of practices that should improve transparency of bicistronic reporter data presentation and improve methodological descriptions to facilitate data replication.

Discovery of bone morphogenetic protein 7-derived peptide sequences that attenuate the human osteoarthritic chondrocyte phenotype.

Treatment of osteoarthritis (OA) is mainly symptomatic by alleviating pain to postpone total joint replacement. Bone morphogenetic protein 7 (BMP7) is a candidate morphogen for experimental OA treatment that favorably alters the chondrocyte and cartilage phenotype. Intra-articular delivery and sustained release of a recombinant growth factor for treating OA are challenging, whereas the use of peptide technology potentially circumvents many of these challenges. In this study, we screened a high-resolution BMP7 peptide library and discovered several overlapping peptide sequences from two regions in BMP7 with nanomolar bioactivity that attenuated the pathological OA chondrocyte phenotype. A single exposure of OA chondrocytes to peptides p[63-82] and p[113-132] ameliorated the OA chondrocyte phenotype for up to 8 days, and peptides were bioactive on chondrocytes in OA synovial fluid. Peptides p[63-82] and p[113-132] required NKX3-2 for their bioactivity on chondrocytes and provoke changes in SMAD signaling activity. The bioactivity of p[63-82] depended on specific evolutionary conserved sequence elements common to BMP family members. Intra-articular injection of a rat medial meniscal tear (MMT) model with peptide p[63-82] attenuated cartilage degeneration. Together, this study identified two regions in BMP7 from which bioactive peptides are able to attenuate the OA chondrocyte phenotype. These BMP7-derived peptides provide potential novel disease-modifying treatment options for OA.

Uncovering pathways regulating chondrogenic differentiation of CHH fibroblasts.

Mutations in the non-coding snoRNA component of mitochondrial RNA processing endoribonuclease (RMRP) are the cause of cartilage-hair hypoplasia (CHH). CHH is a rare form of metaphyseal chondrodysplasia characterized by disproportionate short stature and abnormal growth plate development. The process of chondrogenic differentiation within growth plates of long bones is vital for longitudinal bone growth. However, molecular mechanisms behind impaired skeletal development in CHH patients remain unclear. We employed a transdifferentiation model (FDC) combined with whole transcriptome analysis to investigate the chondrogenic transdifferentiation capacity of CHH fibroblasts and to examine pathway regulation in CHH cells during chondrogenic differentiation. We established that the FDC transdifferentiation model is a relevant in vitro model of chondrogenic differentiation, with an emphasis on the terminal differentiation phase, which is crucial for longitudinal bone growth. We demonstrated that CHH fibroblasts are capable of transdifferentiating into chondrocyte-like cells, and show a reduced commitment to terminal differentiation. We also found a number of key factors of BMP, FGF, and IGF-1 signalling axes to be significantly upregulated in CHH cells during the chondrogenic transdifferentiation. Our results support postulated conclusions that RMRP has pleiotropic functions and profoundly affects multiple aspects of cell fate and signalling. Our findings shed light on the consequences of pathological CHH mutations in snoRNA RMRP during chondrogenic differentiation and the relevance and roles of non-coding RNAs in genetic diseases in general.

Isolation of Nucleus Pulposus and Annulus Fibrosus Cells from the Intervertebral Disc.

Cells isolated from the intervertebral disc are often used for in vitro experimentation. Correctly separating the intervertebral disc tissue in annulus fibrosus and nucleus pulposus is particularly challenging when working with surplus material from surgery or specimens from donors with an advanced age. Moreover, lineage controls are only sparsely reported to verify tissue of origin. Here we describe an approach to intervertebral disc cell isolation from human and bovine origin.

A Membranome-Centered Approach Defines Novel Biomarkers for Cellular Subtypes in the Intervertebral Disc.

OBJECTIVE: Lack of specific marker-sets prohibits definition and functional distinction of cellular subtypes in the intervertebral disc (IVD), such as those from the annulus fibrosus (AF) and the nucleus pulposus (NP). DESIGN: We recently generated immortalized cell lines from human NP and AF tissues; these comprise a set of functionally distinct clonal subtypes. Whole transcriptome analyses were performed of 12 phenotypically distinct clonal cell lines (4× NP-Responder, 4× NP-nonResponder, 2× AF-Sheet forming, and 2× AF-nonSheet forming). Data sets were filtered for membrane-associated marker genes and compared to literature. RESULTS: Comparison of our immortal cell lines to published primary NP, AF, and articular chondrocytes (AC) transcriptome datasets revealed preservation of AF and NP phenotypes. NP-specific membrane-associated genes were defined by comparison to AF cells in both the primary dataset (46 genes) and immortal cell-lines (161 genes). Definition of AF-specific membrane-associated genes yielded 125 primary AF cell and 92 immortal cell-line markers. Overlap between primary and immortal NP cells yielded high-confidence NP-specific marker genes for NP-R (CLDN11, TMEFF2, CA12, ANXA2, CD44) and NP-nR (EFNA1, NETO2, SLC2A1). Overlap between AF and immortal AF subtypes yielded specific markers for AF-S (COLEC12, LPAR1) and AF-nS (CHIC1). CONCLUSIONS: The current study provides a reference platform for preclinical evaluation of novel membrane-associated cell type-specific markers in the IVD. Future research will focus on their biological relevance for IVD function in development, homeostasis, and degenerate conditions.

Increased TGF-ß and BMP Levels and Improved Chondrocyte-Specific Marker Expression In Vitro under Cartilage-Specific Physiological Osmolarity.

During standard expansion culture (i.e., plasma osmolarity, 280 mOsm) human articular chondrocytes dedifferentiate, making them inappropriate for autologous chondrocyte implantation to treat cartilage defects. Increasing the osmolarity of culture media to physiological osmolarity levels of cartilage (i.e., 380 mOsm), increases collagen type II (COL2A1) expression of human articular chondrocytes in vitro, but the underlying molecular mechanism is not fully understood. We hypothesized that TGF-ß superfamily signaling may drive expression of COL2A1 under physiological osmolarity culture conditions. Human articular chondrocytes were cultured in cytokine-free medium of 280 or 380 mOsm with or without siRNA mediated TGF-ß2 knockdown (RNAi). Expression of TGF-ß isoforms, and collagen type II was evaluated by RT-qPCR and immunoblotting. TGF-ß2 protein secretion was evaluated using ELISA and TGF-ß bioactivity was determined using an established reporter assay. Involvement of BMP signaling was investigated by culturing human articular chondrocytes in the presence or absence of BMP inhibitor dorsomorphin and BMP bioactivity was determined using an established reporter assay. Physiological cartilage osmolarity (i.e., physosmolarity) most prominently increased TGF-ß2 mRNA expression and protein secretion as well as TGF-ß bioactivity. Upon TGF-ß2 isoform-specific knockdown, gene expression of chondrocyte marker COL2A1 was induced. TGF-ß2 RNAi under physosmolarity enhanced TGF-ß bioactivity. BMP bioactivity increased upon physosmotic treatment, but was not related to TGF-ß2 RNAi. In contrast, dorsomorphin inhibited COL2A1 mRNA expression in human articular chondrocytes independent of the osmotic condition. Our data suggest a role for TGF-ß superfamily member signaling in physosmolarity-induced mRNA expression of collagen type II. As physosmotic conditions favor the expression of COL2A1 independent of our manipulations, contribution of other metabolic, post-transcriptional or epigenetic factors cannot be excluded in the underlying complex and interdependent regulation of marker gene expression. Dissecting these molecular mechanisms holds potential to further improve future cell-based chondral repair strategies.

The antiviral protein viperin regulates chondrogenic differentiation via CXCL10 protein secretion.

Viperin (also known as radical SAM domain-containing 2 (RSAD2)) is an interferon-inducible and evolutionary conserved protein that participates in the cell's innate immune response against a number of viruses. Viperin mRNA is a substrate for endoribonucleolytic cleavage by RNase mitochondrial RNA processing (MRP) and mutations in the RNase MRP small nucleolar RNA (snoRNA) subunit of the RNase MRP complex cause cartilage-hair hypoplasia (CHH), a human developmental condition characterized by metaphyseal chondrodysplasia and severe dwarfism. It is unknown how CHH-pathogenic mutations in RNase MRP snoRNA interfere with skeletal development, and aberrant processing of RNase MRP substrate RNAs is thought to be involved. We hypothesized that viperin plays a role in chondrogenic differentiation. Using immunohistochemistry, real-time quantitative PCR, immunoblotting, ELISA, siRNA-mediated gene silencing, plasmid-mediated gene overexpression, label-free MS proteomics, and promoter reporter bioluminescence assays, we discovered here that viperin is expressed in differentiating chondrocytic cells and regulates their protein secretion and the outcome of chondrogenic differentiation by influencing transforming growth factor ß (TGF-ß)/SMAD family 2/3 (SMAD2/3) activity via C-X-C motif chemokine ligand 10 (CXCL10). Of note, we observed disturbances in this viperin-CXCL10-TGF-ß/SMAD2/3 axis in CHH chondrocytic cells. Our results indicate that the antiviral protein viperin controls chondrogenic differentiation by influencing secretion of soluble proteins and identify a molecular route that may explain impaired chondrogenic differentiation of cells from individuals with CHH.

Identification of TGFß-related genes regulated in murine osteoarthritis and chondrocyte hypertrophy by comparison of multiple microarray datasets.

OBJECTIVE: Osteoarthritis (OA) is a joint disease characterized by progressive degeneration of articular cartilage. Some features of OA, including chondrocyte hypertrophy and focal calcification of articular cartilage, resemble the endochondral ossification processes. Alterations in transforming growth factor ß (TGFß) signaling have been associated with OA as well as with chondrocyte hypertrophy. Our aim was to identify novel candidate genes implicated in chondrocyte hypertrophy during OA pathogenesis by determining which TGFß-related genes are regulated during murine OA and endochondral ossification. METHODS: A list of 580 TGFß-related genes, including TGFß signaling pathway components and TGFß-target genes, was generated. Regulation of these TGFß-related genes was assessed in a microarray of murine OA cartilage: 1, 2 and 6 weeks after destabilization of the medial meniscus (DMM). Subsequently, genes regulated in the DMM model were studied in two independent murine microarray datasets on endochondral ossification: the growth plate and transient embryonic cartilage (joint development). RESULTS: A total of 106 TGFß-related genes were differentially expressed in articular cartilage of DMM-operated mice compared to sham-control. From these genes, 43 were similarly regulated during chondrocyte hypertrophy in the growth plate or embryonic joint development. Among these 43 genes, 18 genes have already been associated with OA. The remaining 25 genes were considered as novel candidate genes involved in OA pathogenesis and endochondral ossification. In supplementary data of published human OA microarrays we found indications that 15 of the 25 novel genes are indeed regulated in articular cartilage of human OA patients. CONCLUSION: By focusing on TGFß-related genes during OA and chondrocyte hypertrophy in mice, we identified 18 known and 25 new candidate genes potentially implicated in phenotypical changes in chondrocytes leading to OA. We propose that 15 of these candidates warrant further investigation as therapeutic target for OA as they are also regulated in articular cartilage of OA patients.