Abstract
Key points microRNAs (miRs) are small non‐coding molecules that regulate post‐transcriptional target gene expression.miRs are involved in regulating cellular activities in response to mechanical loading in all physiological systems, although it is largely unknown whether this response differs with increasing magnitudes of load.miR‐221, miR‐222, miR‐21‐5p and miR‐27a‐5p were significantly increased in ex vivo cartilage explants subjected to increasing load magnitude and in in vivo joint cartilage exposed to abnormal loading. TIMP3 and CPEB3 are putative miR targets in chondrocytesIdentification of mechanically regulated miRs that have potential to impact on tissue homeostasis provides a mechanism by which load‐induced tissue behaviour is regulated, in both health and pathology, in all physiological systems. MicroRNAs (miRs) are small non‐coding molecules that regulate post‐transcriptional target gene expression and are involved in mechano‐regulation of cellular activities in all physiological systems. It is unknown whether such epigenetic mechanisms are regulated in response to increasing magnitudes of load. The present study investigated mechano‐regulation of miRs in articular cartilage subjected to ‘physiological’ and ‘non‐physiological’ compressive loads in vitro as a model system and validated findings in an in vivo model of abnormal joint loading. Bovine full‐depth articular cartilage explants were loaded to 2.5 MPa (physiological) or 7 MPa (non‐physiological) (1 Hz, 15 min) and mechanically‐regulated miRs identified using next generation sequencing and verified using a quantitative PCR. Downstream targets were verified using miR‐specific mimics or inhibitors in conjunction with 3′‐UTR luciferase activity assays. A subset of miRs were mechanically‐regulated in ex vivo cartilage explants and in vivo joint cartilage. miR‐221, miR‐222, miR‐21‐5p and miR‐27a‐5p were increased and miR‐483 levels decreased with increasing load magnitude. Tissue inhibitor of metalloproteinase 3 (TIMP3) and cytoplasmic polyadenylation element binding protein 3 (CPEB3) were identified as putative downstream targets. Our data confirm miR‐221 and ‐222 mechano‐regulation and demonstrates novel mechano‐regulation of miR‐21‐5p and miR‐27a‐5p in ex vivo and in vivo cartilage loading models. TIMP3 and CPEB3 are putative miR targets in chondrocytes. Identification of specific miRs that are regulated by increasing load magnitude, as well as their potential to impact on tissue homeostasis, has direct relevance to other mechano‐sensitive physiological systems and provides a mechanism by which load‐induced tissue behaviour is regulated, in both health and pathology.
Highlights
Mechanical loading is essential with respect to regulating the functional capabilities of physiological systems including the musculoskeletal, cardiovascular and nervous system; this is achieved, at the cell and tissue level, by adapting to changes in mechanical load and/or metabolic stress applied
The biomechanical integrity of articular cartilage is reliant on the biochemical composition of the extracellular matrix (ECM) (Gilbert & Blain, 2018), and maintenance of cartilage tissue homeostasis, effected by the chondrocytes, is dependent on mechanical load (Buckwalter et al 2005)
Application of moderate, physiological mechanical loads is essential for maintaining cartilage homeostasis by promoting anabolic activities such as increased production of ECM molecules, whereas abnormal, non-physiological joint loading, as characterized by either overload or insufficient load, disrupts the homeostatic balance, favouring catabolism and cartilage degeneration, comprising the hallmark of osteoarthritis (OA) (Felson, 2013)
Summary
Mechanical loading is essential with respect to regulating the functional capabilities of physiological systems including the musculoskeletal, cardiovascular and nervous system; this is achieved, at the cell and tissue level, by adapting to changes in mechanical load and/or metabolic stress applied. One of the major musculoskeletal tissues, articular cartilage, primarily functions to dissipate mechanical forces across the synovial joint surface and facilitates smooth, low-friction movement. A small number of miRs were identified as being mechanosensitive in chondrocytes (Dunn et al 2009; Guan et al 2011; Jin et al 2014; Yang et al 2016; Cheleschi et al 2017). These studies were performed on isolated cells devoid of a substantial ECM, a feature known to be critical for cell–matrix mechano-communications (Guilak et al 2006). Using articular cartilage as a model system, the present study aimed to identify miRs that respond to ‘physiological’ and ‘non-physiological’ mechanical loads and to investigate the regulation of their potential downstream target genes
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