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-β 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-β2 knockdown (RNAi). Expression of TGF-β isoforms, and collagen type II was evaluated by RT-qPCR and immunoblotting. TGF-β2 protein secretion was evaluated using ELISA and TGF-β 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-β2 mRNA expression and protein secretion as well as TGF-β bioactivity. Upon TGF-β2 isoform-specific knockdown, gene expression of chondrocyte marker COL2A1 was induced. TGF-β2 RNAi under physosmolarity enhanced TGF-β bioactivity. BMP bioactivity increased upon physosmotic treatment, but was not related to TGF-β2 RNAi. In contrast, dorsomorphin inhibited COL2A1 mRNA expression in human articular chondrocytes independent of the osmotic condition. Our data suggest a role for TGF-β 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.
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.
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.
tRNA splicing, T helper cell differentiation, and IL4 signaling, among others. Many CpG sites were selected by our machine learning algorithms, the majority of predictive capability being provided by 29 CpG sites selected in at least 5 iterations of the model. Over multiple slits of data into training and testing sets, the predictive modeling strategy achieved a mean error rate of 33.3% on previously unseen validation data. The average receiver operating characteristic (ROC) curve of the model demonstrated an AUC of 0.75 for prediction of rapid progression.
Osteoarthritis (OA) is a degenerative joint disease characterized by irreversible cartilage damage, inflammation and altered chondrocyte phenotype. Transforming growth factor-β (TGF-β) 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-β signaling via SMAD2/3 linker-modifications in chondrocytes. We found that both Interleukin (IL)-1β and OA-synovium conditioned medium negated SMAD2/3 transcriptional activity in chondrocytes. This inhibition of TGF-β signaling was enhanced if SMAD3 could not be phosphorylated on Ser213 in the linker region and the inhibition by IL-1β 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.
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