Ribosomal RNA-based epitranscriptomic regulation of chondrocyte translation and proteome in osteoarthritis.

OBJECTIVE: Osteoarthritis-related cartilage extracellular matrix remodeling is dependent on changes in chondrocyte protein expression. Yet, the role of ribosomes in chondrocyte translation regulation is unknown. In this exploratory study, we investigated ribosomal RNA (rRNA) epitranscriptomic-based ribosome heterogeneity in human articular chondrocytes and its relevance for osteoarthritis. METHODS: Sequencing-based rRNA 2'-O-methylation profiling analysis (RiboMethSeq) was performed on non-OA primary human articular chondrocytes (n = 5) exposed for 14 days to osteoarthritic synovial fluid (14 donors, pooled, 20% v/v). The SW1353 SNORD71 KO cell pool was generated using LentiCRISPRv2/Cas9. The mode of translation initiation and fidelity were determined by dual-luciferase reporters. The cellular proteome was analyzed by LC-MS/MS and collagen type I protein expression was evaluated by immunoblotting. Loading of COL1A1 mRNA into polysomes was determined by sucrose gradient ultracentrifugation and fractionation. RESULTS: We discovered that osteoarthritic synovial fluid instigates site-specific changes in the rRNA 2'-O-me profile of primary human articular chondrocytes. We identified five sites with differential 2'-O-me levels. The 2'-O-me status of 5.8S-U14 (one of identified differential 2'-O-me sites; decreased by 7.7%, 95% CI [0.9-14.5%]) was targeted by depleting the level of its guide snoRNA SNORD71 (50% decrease, 95% CI [33-64%]). This resulted in an altered ribosome translation modus (e.g., CrPV IRES, FC 3, 95% CI [2.2-4.1]) and promoted translation of COL1A1 mRNA which led to increased levels of COL1A1 protein (FC 1.7, 95% CI [1.3-2.0]). CONCLUSIONS: Our data identify a novel concept suggesting that articular chondrocytes employ rRNA epitranscriptomic mechanisms in osteoarthritis development.

Unravelling the Basic Calcium Phosphate crystal-dependent chondrocyte protein secretome; a role for TGF-ß signaling.

OBJECTIVE: Basic Calcium Phosphate (BCP) crystals play an active role in the progression of osteoarthritis (OA). However, the cellular consequences remain largely unknown. Therefore, we characterized for the first time the changes in the protein secretome of human OA articular chondrocytes as a result of BCP stimulation using two unbiased proteomic analysis methods. METHOD: Isolated human OA articular chondrocytes were stimulated with BCP crystals and examined by Quantitative Reverse Transcription PCR (RT-qPCR) and enzyme-linked immune sorbent assay (ELISA) after twenty-four and forty-eight hours. Forty-eight hours conditioned media were analyzed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) and an antibody array. The activity of BCP dependent Transforming Growth Factor Beta (TGF-ß) signaling was analyzed by RT-qPCR and luciferase reporter assays. The molecular consequences regarding BCP-dependent TGF-ß signaling on BCP-dependent Interleukin 6 (IL-6) were investigated using specific pathway inhibitors. RESULTS: Synthesized BCP crystals induced IL-6 expression and secretion upon stimulation of human articular chondrocytes. Concomitant induction of catabolic gene expression was observed. Analysis of conditioned media revealed a complex and diverse response with a large number of proteins involved in TGF-ß signaling, both in activation of latent TGF-ß and TGF-ß superfamily members, which were increased compared to non-stimulated OA chondrocytes. Activity of this BCP driven TGF-ß signaling was confirmed by increased activity of expression of TGF-ß target genes and luciferase reporters. Inhibition of BCP driven TGF-ß signaling resulted in decreased IL-6 expression and secretion with a moderate effect on catabolic gene expression. CONCLUSION: BCP crystal stimulation resulted in a complex and diverse chondrocyte protein secretome response. An important role for BCP-dependent TGF-ß signaling was identified in development of a pro-inflammatory environment.

Synovial fluid from end-stage osteoarthritis induces proliferation and fibrosis of articular chondrocytes via MAPK and RhoGTPase signaling.

OBJECTIVES: Alterations in the composition of synovial fluid have been associated with adverse effects on cartilage integrity and function. Here, we examined the phenotypic and proliferative behavior of human articular chondrocytes when cultured in vitro for 13 days with synovial fluid derived from end-stage osteoarthritis patients. MATERIALS AND METHODS: Chondrocyte proliferation and phenotypical changes induced by osteoarthritic synovial fluid were analyzed using DNA staining, RT-qPCR, immunostainings, and immunoblotting. The molecular mechanisms by which osteoarthritic synovial fluid induced fibrosis and proliferation were studied using a phospho-protein antibody array and luciferase-based transcription factor activity assays. Specific pathway inhibitors were used to probe the involvement of pathways in fibrosis and proliferation. RESULTS: Prolonged stimulation with osteoarthritic synovial fluid sustained chondrocyte proliferation and induced profound phenotypic changes, favoring a fibrotic over a chondrogenic or hypertrophic phenotype. A clear loss of chondrogenic markers at both the transcriptional and protein level was observed, while expression of several fibrosis-associated markers were upregulated over time. Phospho-kinase analysis revealed activation of MAPK and RhoGTPase signaling pathways by osteoarthritic synovial fluid, which was confirmed by elevated transcriptional activity of Elk-1 and SRF. Inhibitor studies revealed that ERK played a central role in the loss of chondrocyte phenotype, while EGFR and downstream mediators p38, JNK and Rac/Cdc42 were essential for fibrosis-associated collagen expression. Finally, we identified EGF signaling as a key activator of chondrocyte proliferation. CONCLUSIONS: Osteoarthritic synovial fluid promoted chondrocyte fibrosis and proliferation through EGF receptor activation and downstream MAPK and RhoGTPase signaling.

Impairment of the chondrogenic phase of endochondral ossification in vivo by inhibition of cyclooxygenase-2.

Many studies have reported on the effects of cyclooxygenase-2 (COX-2) inhibition on osteogenesis. However, far less is known about the effects of COX-2 inhibition on chondrogenic differentiation. Previous studies conducted by our group show that COX-2 inhibition influences in vitro chondrogenic differentiation. Importantly, this might have consequences on endochondral ossification processes occurring in vivo, such as bone fracture healing, growth plate development and ectopic generation of cartilage. The goal of our study was to investigate, in vivo, the effect of COX-2 inhibition by celecoxib on the cartilaginous phase of three different endochondral ossification scenarios. 10 mg/kg/day celecoxib or placebo were orally administered for 25 d to skeletally-immature New Zealand White rabbits (n = 6 per group). Endochondral ossification during fracture healing of a non-critical size defect in the ulna, femoral growth plate and ectopically-induced cartilaginous tissue were examined by radiography, micro-computed tomography (µ-CT), histology and gene expression analysis. Celecoxib treatment resulted in delayed bone fracture healing, alterations in growth plate development and progression of mineralisation. In addition, chondrogenic differentiation of ectopically-induced cartilaginous tissue was severely impaired by celecoxib. In conclusion, we found that celecoxib impaired the chondrogenic phase of endochondral ossification.

Hypertrophic differentiation during chondrogenic differentiation of progenitor cells is stimulated by BMP-2 but suppressed by BMP-7.

OBJECTIVE: Bone morphogenic protein (BMP)-2 and BMP-7 are clinically approved and their recombinant proteins are used for bone tissue regenerative purposes and widely evaluated for cartilage regeneration. Previous comparison of the in vitro chondrogenic characteristics of BMP-2 vs BMP-7 did not address hypertrophic differentiation and characterizing their chondrogenic properties with a focus in on chondrocyte hypertrophy was topic of investigation in this study. DESIGN: Equimolar concentrations of BMP-2 or BMP-7 were added to chondrogenic differentiating ATDC5, human bone marrow stem cells or rabbit periosteal explants. Expression of Col2a1, Sox9, Acan, Col10a1, Runx2, ALP, Mmp13, Mef2c and Bapx1/Nkx3.2 was determined by reverse transcription-quantitative PCR (RT-qPCR) and immunoblotting. Glycosaminoglycan content, cell proliferation capacity and ALP activity were analysed by colourimetric analyses. Expression of Bapx1/Nkx3.2 and Sox9 was targeted by transfection of target specific siRNA duplexes. RESULTS: BMP-2 dose-dependently increased chondrocyte hypertrophy during chondrogenic differentiation of progenitor cells, whereas BMP-7 acted hypertrophy-suppressive and chondro-promotive. Both BMPs did not influence cell proliferation, but they did increase total glycosaminoglycan content. In a candidate approach Bapx1/Nkx3.2 was found to be involved in the BMP-7 mediated suppression of chondrocyte hypertrophy in ATDC5 cells. CONCLUSIONS: BMP-2 and BMP-7 display opposing actions on the chondrogenic outcome of differentiating progenitor cells: BMP-2 acts a specific inducer of chondrocyte hypertrophy, while BMP-7 appears to increase or maintain chondrogenic potential and prevent chondrocyte hypertrophy. Our results pave the way for an application-dependent differential use of BMP-2 or BMP-7.

Redifferentiation of dedifferentiated human articular chondrocytes: comparison of 2D and 3D cultures.

OBJECTIVE: Three-dimensional (3D) cultures are widely used to redifferentiate chondrocytes. However, the rationale behind the choice for 3D above two-dimensional (2D) cultures is poorly systematically investigated and mainly based on mRNA expression and glycosaminoglycan (GAG) content. The objective was to determine the differential redifferentiation characteristics of human articular chondrocytes (HACs) in monolayer, alginate beads and pellet culture by investigating mRNA expression, protein expression, GAG content and cell proliferation. DESIGN: Dedifferentiated HACs from six individuals were redifferentiated in identical medium conditions for 7 days in monolayer, alginate beads or pellet culture. Read-out parameters were expression of chondrogenic and hypertrophic mRNAs and proteins, GAG content and cell proliferation. RESULTS: 3D cultures specifically expressed chondrogenic mRNAs [collagen type II (COL2A1), SRY (sex determining region Y)-box 9 (SOX9), aggrecan (ACAN)), whereas 2D cultures did not. Hypertrophic mRNAs (collagen type X (COL10A1), runt-related transcription factor 2 (RUNX2), matrix metalloproteinase 13 (MMP13), vascular endothelial growth factor A (VEGFA), osteopontin (OPN), alkaline phosphatase (ALP)) were highly increased in 2D cultures and lower in 3D cultures. Collagen type I (COL1A1) mRNA expression was highest in 3D cultures. Protein expression supports most of the mRNA data, although an important discrepancy was found between mRNA and protein expression of COL2A1 and SOX9 in monolayer culture, stressing on the importance of protein expression analysis. GAG content was highest in 3D cultures, whereas chondrocyte proliferation was almost specific for 2D cultures. CONCLUSIONS: For redifferentiation of dedifferentiated HACs, 3D cultures exhibit the most potent chondrogenic potential, whereas a hypertrophic phenotype is best achieved in 2D cultures. This is the first human study that systematically evaluates the differences between proliferation, GAG content, protein expression and mRNA expression of commonly used 2D and 3D chondrocyte culture techniques.

Inhibition of cyclooxygenase-2 impacts chondrocyte hypertrophic differentiation during endochondral ossification.

Skeletogenesis and bone fracture healing involve endochondral ossification, a process during which cartilaginous primordia are gradually replaced by bone tissue. In line with a role for cyclooxygenase-2 (COX-2) in the endochondral ossification process, non-steroidal anti-inflammatory drugs (NSAIDs) were reported to negatively affect bone fracture healing due to impaired osteogenesis. However, a role for COX-2 activity in the chondrogenic phase of endochondral ossification has not been addressed before. We show that COX-2 activity fulfils an important regulatory function in chondrocyte hypertrophic differentiation. Our data reveal essential cross-talk between COX-2 and bone morphogenic protein-2 (BMP-2) during chondrocyte hypertrophic differentiation. BMP-2 mediated chondrocyte hypertrophy is associated with increased COX-2 expression and pharmacological inhibition of COX-2 activity by NSAIDs (e.g., Celecoxib) decreases hypertrophic differentiation in various chondrogenic models in vitro and in vivo, while leaving early chondrogenic development unaltered. Our findings demonstrate that COX-2 activity is a novel factor partaking in chondrocyte hypertrophy in the context of endochondral ossification and these observations provide a novel etiological perspective on the adverse effects of NSAIDs on bone fracture healing and have important implications for the use of NSAIDs during endochondral skeletal development.

A sandwich enzyme linked immuno-sorbent assay for the determination of rat heart fatty acid-binding protein using the streptavidin-biotin system. Application to tissue and effluent samples from normoxic rat heart perfusion.

An enzyme linked immuno-sorbent assay (ELISA) of the sandwich type for the determination of heart-type fatty acid-binding protein (H-FABPc) was developed, making use of the streptavidin-biotin system. The assay turned out to be virtually disturbance insensitive and showed a detection limit for H-FABPc of 0.2 micrograms/l with an intra- and inter-assay variation of 5% and 14%, respectively. The H-FABPc content of adult rat heart muscle was found to be 0.740 +/- 0.120 mg/g wet weight. The H-FABPc content of a number of skeletal muscles varied from 0.013 to 0.303 mg/g wet weight and was related to the content of type I muscle fibers of these tissues, suggesting a role for H-FABPc in intracellular fatty acid metabolism. The assay was further applied to study the release of H-FABPc from isolated rat heart during normoxic Langendorff perfusion, as compared to that of lactate dehydrogenase (LDH), into fluid derived from the right ventricular cavity (Qrv) and that from the interstitial space (Qi). Total release of H-FABPc per 15 min amounted to 0.015 +/- 0.010% but that of LDH to 0.080 +/- 0.040% of their total tissue content. Furthermore, for both H-FABPc and LDH 80% was released into Qi, which only accounted for 1-2% of total flow. These findings suggest that during normoxic perfusion of rat heart H-FABPc, and LDH are released from different cellular compartments and that the bulk amount of released intracellular proteins is transported via the lymph instead of being directly released into the bloodstream.

Assay of the binding of fatty acids by proteins: evaluation of the Lipidex 1000 procedure.

Fatty acid (FA) binding by fatty acid-binding protein (FABP) is frequently monitored with the so-called Lipidex 1000 assay, in which protein associated and non-protein bound FA are separated by selectively binding the latter to Lipidex 1000. Careful evaluation of this assay showed that the use of aqueous FA solutions resulted in a marked decrease (60 to 70%) of FA concentration due to their aspecific binding to the surface of the test-tube used. In addition, solutions of rat heart FABP in the micromolar range also showed a concentration decrease up to 80% due to protein binding to the surface of the test-tube. Introduction of detergents, Triton X-100 or Tween 20, limited the FA loss to less than 20% and totally eliminated FABP adsorption. Kinetic parameters for the binding of [1-14C]oleic acid by purified rat heart FABP, assayed in the presence of Triton X-100, were found to be similar to those assayed in the absence of detergent, when adequate corrections were made for losses of FA and FABP due to surface adsorption. Use of Tween 20 resulted in a substantial increase of the dissociation constant. The addition of 100 microM Triton X-100 to the assay medium considerably facilitates the determination of kinetic parameters of fatty acid-binding by proteins.

Release of fatty acid binding protein and lactate dehydrogenase from isolated rat heart during normoxia, low-flow ischemia, and reperfusion.

The present study was performed to monitor the effect of low-flow ischemia and reperfusion on changes in the protein permeability of the cardiomyocyte cell membrane and the endothelial cell layer for two cytoplasmic proteins, i.e., fatty acid binding protein (FABP) and lactate dehydrogenase (LDH), which differ appreciably with respect to physicochemical characteristics. To accomplish this, isolated rat hearts were Langendorff perfused with separate collection of vascular and interstitial effluents. Control hearts were perfused normoxically for 300 min, whereas experimental hearts were subjected to 60 min normoxia (N), 180 min low-flow ischemia (I), and finally 60 min normoxic reperfusion (R). Protein release was measured in 15 min interval fractions. During the first 240 min of perfusion 0.2% of total tissue FABP and 1.1% of total tissue LDH were detected in the effluents in both groups. Moreover, in each case 80% of released FABP and LDH was found in the interstitial effluent. During R, following I in the experimental group, appreciable amounts of both proteins were released (2.2 and 5.1% of total tissue contents for FABP and LDH, respectively). During this period the percentage of protein released in the vascular effluent increased significantly for both proteins. It is concluded that the combination of low-flow ischemia and reperfusion increases the protein permeability of both the cardiomyocyte cell membrane and the endothelial barrier. Since the release patterns of FABP and LDH with respect to time were similar during the entire perfusion protocol, it is tempting to state that protein release from tissue is a nonspecific effect of a noxious intervention.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel method to examine the phenotype of chondrocytes.

Tissue engineering of articular cartilage in order to restore the function of degenerated, diarthrodial joints is currently widely under investigation. The results obtained thus far indicate that proper control of the differentiation of the cells used for this purpose is essential to produce and maintain a hyaline-like matrix. In this study, a procedure is described by which differentiation of chondrocytes in vitro and ex vivo can be studied. The method involves quantitative assessment of mRNA for different collagens, which are markers for differentiation of chondrocytes, by competitive PCR. In a culture system employing human osteoarthritic chondrocytes, mRNAs for the alpha1-chains of collagen types I, II and X are quantified. The procedure is fast, specific and sensitive. However, several controls should be included to ascertain the reliability of the assessment.

Modulation of fatty-acid-binding protein content of rat heart and skeletal muscle by endurance training and testosterone treatment.

The effects of training and/or testosterone treatment and its aromatization to oestradiol on fatty-acid-binding protein (FABP) content and cytochrome c oxidase activity in heart, soleus and extensor digitorum longus (EDL) muscles were studied in intact adult female rats. One group of rats remained sedentary, whereas the others were trained for 7 weeks. Thereafter the trained rats were divided into control and testosterone-treated groups, with or without an aromatase inhibitor. Testosterone was administered by a silastic implant. Training was continued for 2 weeks. In untreated sedentary rats the immunochemically assayed FABP contents were 497 +/- 28, 255 +/- 49 and 58 +/- 17 micrograms/g wet weight for the heart, soleus, and EDL respectively. In the heart the FABP content was increased after training (29%), testosterone treatment (33%) or both manipulations (53%). In soleus muscle FABP increased only after testosterone treatment (16%), whereas in EDL no changes were found. Inhibiting the aromatase enzyme complex abolished the testosterone-induced effect on FABP content in soleus (suggesting an oestradiol effect) but not in heart muscle. Among the three muscles studied the FABP content was found to be related to the cytochrome c oxidase activity in a non-linear way. In conclusion, it is shown that the FABP contents and mitochondrial activities of heart and skeletal muscle are affected by training and sex hormones and that these effects are different for heart and skeletal muscles.

High-yield two-step chromatographic procedure for purification of fatty acid-binding protein from human heart.

A high-yield procedure for the purification of cytoplasmic fatty acid-binding protein from human heart (H-FABP) is described. H-FABP was purified by gel permeation chromatography on a Sephacryl S-200 column followed by anion-exchange chromatography on a Sepharose Q fast-flow column at pH 7.0. At this pH H-FABP binds strongly to the column and can be selectively eluted with a salt gradient. The two-step procedure showed a high degree of reproducibility. On average 50 mg of H-FABP was obtained from 150 g of human heart tissue, which corresponds to a recovery of about 50%. Purity was confirmed by gel electrophoresis and isoelectric focusing. Binding of oleic acid to purified H-FABP, using the Lipidex 1000 assay, revealed a maximal binding of 0.75 +/- 0.01 mol fatty acid/mol protein and a dissociation constant of 0.19 +/- 0.01 microM.

The polymer Polyactive as a bone-filling substance: an experimental study in rabbits.

The biocompatible, osteoconductive and resorbable polymer Polyactive (PA) was investigated for its performance as a bone-graft substitute. The model consisted of a 4 mm borehole, 1.5 cm distal of the major trochanter in both femurs of a rabbit, of which one was filled with a cylinder of porous PA. The other was left untreated. PA70/30 and PA60/40 were investigated, both before and after being incubated with allogenic bone marrow. Analyses were performed after 4, 8, 26 and 52 weeks and comprised dual energy X-ray absorptiometry (DXA) and image analysis of histological sections. DXA revealed an increased bone mineral density in the filled defects compared to the controls, both at the defect and immediately proximal and distal of the defect. Histology showed that gap-bridging had occurred within 8 weeks, with 80%-90% of the pores of PA70/30 and PA60/40 occupied by new bone, and an intimate bone-PA contact. PA70/30 seemed to be more suitable compared to PA60/40, in that the highest amount of bone was formed within the shortest period of time. Incubation of PA with allogenic bone marrow resulted in inflammatory reactions at the sites of implantation, which delayed bone growth, but did not prevent it. It was concluded that PA70/30 and PA60/40 are suitable bone-graft substitutes.