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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Osteoarthritis presents as a change in the chondrocyte phenotype and an imbalance between anabolic and catabolic processes. Age affects its onset and progression. Small nucleolar RNAs (SnoRNAs) ...direct chemical modification of RNA substrates to fine-tune spliceosomal and rRNA function, accommodating changing requirements for splicing and protein synthesis during health and disease. Articular cartilage from young, old and OA knees was used in a microarray study to identify alterations in snoRNA expression. Changes in snoRNAs in osteoarthritis-like conditions were studied in chondrocytes using interleukin-1 and osteoarthritic synovial fluid. SNORD26 and SNORD96A knockdown and overexpression were undertaken using antisense oligonucleotides and overexpression plasmids. We identified panels of snoRNAs differentially expressed due to ageing (including SNORD96A, SNORD44) and osteoarthritis (including SNORD26 and SNORD116). In vitro experiments using osteoarthritis-like conditions affected snoRNA expression. Knockdown or overexpression of SNORD26 or SNORD96A resulted in changes in chondrogenic, hypertrophic, rRNA and osteoarthritis related gene expression. We demonstrate that snoRNA expression changes in cartilage ageing, and osteoarthritis and in osteoarthritis-like conditions, and when the expression of these snoRNAs is altered this affects chondrogenic and hypertrophic gene expression. Thus, we propose an additional dimension in the molecular mechanisms underlying cartilage ageing and osteoarthritis through the dysregulation of snoRNAs.
Despite osteoarthritis (OA) and rheumatoid arthritis (RA) being typically age-related, their underlying etiologies are markedly different. We used 1H nuclear magnetic resonance (NMR) spectroscopy to ...identify differences in metabolite profiles in low volumes of OA and RA synovial fluid (SF). SF was aspirated from knee joints of 10 OA and 14 RA patients. 100 μL SF was analyzed using a 700 MHz Avance IIIHD Bruker NMR spectrometer with a TCI cryoprobe. Spectra were analyzed by Chenomx, Bruker TopSpin and AMIX software. Statistical analysis was undertaken using Metaboanalyst. 50 metabolites were annotated, including amino acids, saccharides, nucleotides and soluble lipids. Discriminant analysis identified group separation between OA and RA cohorts, with 32 metabolites significantly different between OA and RA SF (false discovery rate (FDR) < 0.05). Metabolites of glycolysis and the tricarboxylic acid cycle were lower in RA compared to OA; these results concur with higher levels of inflammation, synovial proliferation and hypoxia found in RA compared to OA. Elevated taurine in OA may indicate increased subchondral bone sclerosis. We demonstrate that quantifiable differences in metabolite abundance can be measured in low volumes of SF by 1H NMR spectroscopy, which may be clinically useful to aid diagnosis and improve understanding of disease pathogenesis.
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-β2) 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-β2 stimulation induced total protein synthesis in SW1353 chondrocytic cells and human articular chondrocytes. Specifically, TGF-β2 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-β2 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-β2 stimulated cells, albeit with a modest fold change. A prolonged stimulation of chondrocytic cells with TGF-β2 induced ribosome activity and changed the mode of translation. These functional changes could be coupled to alterations in accessory proteins in the ribosomal proteome.
Abstract Introduction Previous studies have shown that human articular chondrocytes in vitro are osmolarity-responsive and increase matrix synthesis under cartilage-specific physiological osmolarity. ...The effects of increased osmolarity on chondrogenesis of progenitor cells in vitro are largely unknown. We therefore aimed to elucidate whether hyperosmolarity facilitates their chondrogenic differentiation and whether Nfat5 is involved. Materials and methods ATDC5 cells and human bone marrow stem cells (hBMSCs) were differentiated in the chondrogenic lineage in control and increased osmolarity conditions. Chondrogenic outcome was measured by gene- and protein expression analysis. RNAi was used to determine the role of Nfat5 in chondrogenic differentiation under normal and increased osmolarity. Results Increasing the osmolarity of differentiation medium with 100 mOsm resulted in significantly increased chondrogenic marker expression (Col2a1, Col10a1, Acan, Sox9, Runx2 and GAGs) during chondrogenic differentiation of the two chondroprogenitors, ATDC5 and hBMSCs. Nfat5 knockdown under both control and increased osmolarity affected chondrogenic differentiation and suppressed the osmolarity-induced chondrogenic induction. Knockdown of Nfat5 in early differentiation significantly decreased early Sox9 expression, whereas knockdown of Sox9 in early differentiation did not affect early Nfat5 expression. Conclusions Increasing the osmolarity of chondrogenic culture media by 100 mOsm significantly increased chondrogenic gene expression during the course of chondrogenic differentiation of progenitor cells. Nfat5 may be involved in regulating chondrogenic differentiation of these cells under both normal and increased osmolarities and might regulate chondrogenic differentiation through influencing early Sox9 expression.
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 TGFβ-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)
-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)
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.
Although pathways controlling ribosome activity have been described to regulate chondrocyte homeostasis in osteoarthritis, ribosome biogenesis in osteoarthritis is unexplored. We hypothesized that U3 ...snoRNA, a non-coding RNA involved in ribosomal RNA maturation, is critical for chondrocyte protein translation capacity in osteoarthritis. U3 snoRNA was one of a number of snoRNAs with decreased expression in osteoarthritic cartilage and osteoarthritic chondrocytes. OA synovial fluid impacted U3 snoRNA expression by affecting U3 snoRNA gene promoter activity, while BMP7 was able to increase its expression. Altering U3 snoRNA expression resulted in changes in chondrocyte phenotype. Interference with U3 snoRNA expression led to reduction of rRNA levels and translational capacity, whilst induced expression of U3 snoRNA was accompanied by increased 18S and 28S rRNA levels and elevated protein translation. Whole proteome analysis revealed a global impact of reduced U3 snoRNA expression on protein translational processes and inflammatory pathways. For the first time we demonstrate implications of a snoRNA in osteoarthritis chondrocyte biology and investigated its role in the chondrocyte differentiation status, rRNA levels and protein translational capacity.
Initiation of and progression through chondrogenesis is driven by changes in the cellular microenvironment. At the onset of chondrogenesis, resting mesenchymal stem cells are mobilized in vivo and a ...complex, step-wise chondrogenic differentiation program is initiated. Differentiation requires coordinated transcriptomic reprogramming and increased progenitor proliferation; both processes require chromatin remodeling. The nature of early molecular responses that relay differentiation signals to chromatin is poorly understood. We here show that immediate early genes are rapidly and transiently induced in response to differentiation stimuli in vitro. Functional ablation of the immediate early factor EGR1 severely deregulates expression of key chondrogenic control genes at the onset of differentiation. In addition, differentiating cells accumulate DNA damage, activate a DNA damage response and undergo a cell cycle arrest and prevent differentiation associated hyper-proliferation. Failed differentiation in the absence of EGR1 affects global acetylation and terminates in overall histone hypermethylation. We report novel molecular connections between EGR1 and Polycomb Group function: Polycomb associated histone H3 lysine27 trimethylation (H3K27me3) blocks chromatin access of EGR1. In addition, EGR1 ablation results in abnormal Ezh2 and Bmi1 expression. Consistent with this functional interaction, we identify a number of co-regulated targets genes in a chondrogenic gene network. We here describe an important role for EGR1 in early chondrogenic epigenetic programming to accommodate early gene-environment interactions in chondrogenesis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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.
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).
Our data identify a novel concept suggesting that articular chondrocytes employ rRNA epitranscriptomic mechanisms in osteoarthritis development.
Osteoarthritis (OA) is a degenerative musculoskeletal disorder affecting the whole synovial joint and globally impacts more than one in five individuals aged 40 and over, representing a huge ...socioeconomic burden. Drug penetration into and retention within the joints are major challenges in the development of regenerative therapies for OA. During the recent years, polymeric nanoparticles (PNPs) have emerged as promising drug carrier candidates due to their biodegradable properties, nanoscale structure, functional versatility, and reproducible manufacturing, which makes them particularly attractive for cartilage penetration and joint retention. In this review, we discuss the current development state of natural and synthetic PNPs for drug delivery and OA treatment. Evidence from in vitro and pre-clinical in vivo studies is used to show how disease pathology and key cellular pathways of joint inflammation are modulated by these nanoparticle-based therapies. Furthermore, we compare the biodegradability and surface modification of these nanocarriers in relation to the drug release profile and tissue targeting. Finally, the main challenges for nanoparticle delivery to the cartilage are discussed, as a function of disease state and physicochemical properties of PNPs such as size and surface charge.
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