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Computational insights into the substrate recognition mechanism of cartilage extracellular matrix degradation

ABSTRACT: Graphical abstract Articular cartilage is connective tissue that forms a slippery load-bearing joint surface between bones. With outstanding mechanical properties, it plays an essential role in cushioning impact and protecting the ends of bones. Abnormal mechanical stimulation, such as repetitive overloading or chondral injury, induces excessive cartilage extracellular matrix (ECM) degradation, leading to osteoarthritis and other joint disorders. A disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5) is an aggrecanase that dominates the catalysis of aggrecan, the major proteoglycan in the cartilage ECM. Intriguingly, unlike its critical cleavage site Glu373–374Ala, another potential cleavage site, Glu419-420Ala, composed of the same amino acids in the aggrecan interglobular domain, is not a major cleavage site. It remains unclear how ADAMTS-5 distinguishes between them and hydrolyzes the correct scissile bonds. This research introduces a bottom-up in silico approach to reveal the molecular mechanism by which ADAMTS-5 recognizes the cleavage site on aggrecan. It is hypothesized that the sequence in the vicinity assists ADAMTS-5 in positioning the cleavage site. Specific residues were found to serve as binding sites, helping aggrecan bind more stably and fit into the enzyme better. The findings provide insight into the substrate binding and recognition mechanism for cartilage ECM degradation from a brand new atomic-scale perspective, laying the foundation for prophylaxis and treatment of related joint diseases.

SUBMITTER: Lai Y

PROVIDER: S-EPMC8526910 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The present study was developed to explore whether microRNA (miR)-760 targets heparin-binding EGF-like growth factor (HBEGF) to control cartilage extracellular matrix degradation in osteoarthritis. Both miR-760 and HBEGF expression levels were analysed in human degenerative cartilage tissues and in interleukin (IL)-1β/tumour necrosis factor (TNF)-α-treated chondrocytes in vitro. A series of knockdown and overexpression assays were then used to gauge the functional importance of miR-760 and HBEGF in OA, with qPCR and western immunoblotting analyses. Bioinformatics assays were used to identify putative miR-760 target genes, with these predictions then being validated through RNA pulldown and luciferase reporter assays. A murine anterior cruciate ligament transection model of OA was then established to prove the in vivo relevance of these findings. These experiments revealed that human degenerative cartilage tissues exhibited significant increases in miR-760 expression with a concomitant drop in HBEGF levels. IL-1β/TNF-α-treated chondrocytes also exhibited significant increases in miR-760 expression with a concomitant drop in HBEGF expression. When chondrocytes were transfected with either miR-760 inhibitor or HBEGF overexpression constructs, this was sufficient to interfere with degradation of the extracellular matrix (ECM). Moreover, miR-760 was confirmed to control chondrocyte matrix homeostasis by targeting HBEGF, and the overexpression of HBEGF partially reversed the effects of miR-760 mimic treatment on the degradation of the cartilage ECM. When OA model mice were administered an intra-articular knee injection of an adenoviral vector encoding a miR-760 mimic construct, cartilage ECM degradation was aggravated. Conversely, the overexpression of HBEGF in OA model mice partially reversed the effects of miR-760 overexpression, restoring appropriate ECM homeostasis. In summary, these data indicated that the miR-760/HBEGF axis plays a central role in orchestrating the pathogenesis of OA, making it a candidate target for therapeutic efforts in OA.

Project description:BackgroundMesenchymal stem cell therapy for osteoarthritis (OA) has been widely investigated, but the mechanisms are still unclear. Exosomes that serve as carriers of genetic information have been implicated in many diseases and are known to participate in many physiological processes. Here, we investigate the therapeutic potential of exosomes from human embryonic stem cell-induced mesenchymal stem cells (ESC-MSCs) in alleviating osteoarthritis (OA).MethodsExosomes were harvested from conditioned culture media of ESC-MSCs by a sequential centrifugation process. Primary mouse chondrocytes treated with interleukin 1 beta (IL-1?) were used as an in vitro model to evaluate the effects of the conditioned medium with or without exosomes and titrated doses of isolated exosomes for 48 hours, prior to immunocytochemistry or western blot analysis. Destabilization of the medial meniscus (DMM) surgery was performed on the knee joints of C57BL/6 J mice as an OA model. This was followed by intra-articular injection of either ESC-MSCs or their exosomes. Cartilage destruction and matrix degradation were evaluated with histological staining and OARSI scores at the post-surgery 8 weeks.ResultsWe found that intra-articular injection of ESC-MSCs alleviated cartilage destruction and matrix degradation in the DMM model. Further in vitro studies illustrated that this effect was exerted through ESC-MSC-derived exosomes. These exosomes maintained the chondrocyte phenotype by increasing collagen type II synthesis and decreasing ADAMTS5 expression in the presence of IL-1?. Immunocytochemistry revealed colocalization of the exosomes and collagen type II-positive chondrocytes. Subsequent intra-articular injection of exosomes derived from ESC-MSCs successfully impeded cartilage destruction in the DMM model.ConclusionsThe exosomes from ESC-MSCs exert a beneficial therapeutic effect on OA by balancing the synthesis and degradation of chondrocyte extracellular matrix (ECM), which in turn provides a new target for OA drug and drug-delivery system development.

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Computational insights into the substrate recognition mechanism of cartilage extracellular matrix degradation

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