Articular cartilage enables weight bearing and near friction-free movement in the joints. Critical to its function is the production of a specialized, mechanocompetent extracellular matrix controlled by master regulator transcription factor SOX9. Mutations in SOX9 cause campomelic dysplasia, a haploinsufficiency disorder resulting in severe skeletal defects and dwarfism. Although much is understood about how SOX9 regulates cartilage matrix synthesis and hence joint function, how this master regulator is itself regulated remains largely unknown. Here we identify a specific microRNA, miR-145, as a direct regulator of SOX9 in normal healthy human articular chondrocytes. We show that miR-145 directly represses SOX9 expression in human cells through a unique binding site in its 3'-UTR not conserved in mice. Modulation of miR-145 induced profound changes in the human chondrocyte phenotype. Specifically, increased miR-145 levels cause greatly reduced expression of critical cartilage extracellular matrix genes (COL2A1 and aggrecan) and tissue-specific microRNAs (miR-675 and miR-140) and increased levels of the hypertrophic markers RUNX2 and MMP13, characteristic of changes occurring in osteoarthritis. We propose miR-145 as an important regulator of human chondrocyte function and a new target for cartilage repair.

Objective: To determine the effects of hypoxia on both anabolic and catabolic pathways of metabolism in human articular cartilage and to elucidate the roles played by hypoxia‐inducible factors (HIFs) in these responses.Methods: Normal human articular cartilage from a range of donors was obtained at the time of above‐the‐knee amputations due to sarcomas not involving the joint space. Fresh cartilage tissue explants and isolated cells were subjected to hypoxia and treatment with interleukin‐1α. Cell transfections were performed on isolated human chondrocytes.Results: Using chromatin immunoprecipitation, we found that hypoxia induced cartilage production in human tissue explants through direct binding of HIF‐2α to a specific site in the master‐regulator gene SOX9. Importantly, hypoxia also suppressed spontaneous and induced destruction of human cartilage in explant culture. We found that anticatabolic responses were predominantly mediated by HIF‐1α. Manipulation of the hypoxia‐sensing pathway through depletion of HIF‐targeting prolyl hydroxylase–containing protein 2 (PHD‐2) further enhanced cartilage responses as compared to hypoxia alone. Hypoxic regulation of tissue‐specific metabolism similar to that in human cartilage was observed in pig, but not mouse, cartilage.Conclusion: We found that resident chondrocytes in human cartilage are exquisitely adapted to hypoxia and use it to regulate tissue‐specific metabolism. Our data revealed that while fundamental regulators, such as SOX9, are key molecules both in mice and humans, the way in which they are controlled can differ. This is all the more important since it is upstream regulators such as this that need to be directly targeted for therapeutic benefit. HIF‐specific hydroxylase PHD‐2 may represent a relevant target for cartilage repair.

MicroRNAs are a large and essential class of gene regulators that play key roles in development, homeostasis, and disease. They are necessary for normal skeletal development, and their expression is altered in arthritis. However, the specific role of individual microRNAs is only beginning to be unraveled. Using microRNA expression profiling in healthy human articular cartilage cells (chondrocytes), we identified miR-1247 expression as highly correlated with that of the differentiated cell phenotype. Transcribed from the DLK1-DIO3 locus, the function of miR-1247 is completely unknown. In mice its expression level was relatively high in cartilage tissue, and correlated with cartilage-associated microRNA miR-675 across a range of 15 different mouse tissues. To further probe miR-1247 function, overexpression and inhibition studies were performed in isolated human chondrocytes. Modulation of miR-1247 was found to exert profound phenotypic effects altering expression levels of cartilage master regulator transcription factor SOX9. SOX9 is essential for cartilage development and subsequent function throughout life, and mutations in this gene result in severe dwarfism. Putative miR-1247 binding sites were further investigated using luciferase reporter assays, which indicated binding of miR-1247 to a highly conserved region in the coding sequence of SOX9 but not in its 3′-UTR. Interestingly, depletion of SOX9 in human chondrocytes resulted in increased levels of the mature, processed microRNA, suggesting a negative feedback loop between miR-1247 and its target SOX9.

MicroRNAs (miRNAs) are small non-coding RNA molecules of 21–23 nucleotides that control gene expression at the post-transcriptional level. They have been shown to play a vital role in a wide variety of biological processes and dysregulated expression of miRNAs is observed in many pathologies. Understanding the mechanism of action and identifying functionally important mRNA targets of a specific miRNA are essential to unravelling its biological function and to assist miRNA-based drug development. This review summarizes the current understanding of the mechanistic aspects of miRNA-mediated gene repression and focuses on the different approaches for miRNA target identification that have been proposed in recent years.

Objective: To investigate the function of microRNA‐138 (miR‐138) in human articular chondrocytes (HACs).Methods: The expression of miR‐138 in intact cartilage and cultured chondrocytes and the effects of miR‐138 overexpression on chondrocyte marker genes were investigated. Targets of miR‐138 relevant to chondrocytes were identified and verified by overexpression of synthetic miRNA mimics and inhibitors, luciferase assays, chromatin immunoprecipitation, and RNA immunoprecipitation of native argonaute 2, using quantitative polymerase chain reaction, Western blotting, and luciferase assays.Results: Expression levels of miR‐138 were maintained at relatively low levels in intact human cartilage but were greatly increased upon loss of the differentiated phenotype in culture, with a concomitant decrease in the major cartilage extracellular matrix component COL2A1. We showed that miR‐138 is able to repress the expression of COL2A1 by directly targeting Sp‐1 and hypoxia‐inducible factor 2α (HIF‐2α), 2 transcription factors that are essential for COL2A1 transcription. We further demonstrated a direct association of these targets with miR‐138 in the RNA‐induced silencing complex and confirmed binding of Sp‐1 to the COL2A1 promoter region in HACs.Conclusion: We propose that an evolutionary pressure helps to suppress expression levels of miR‐138 in human cartilage, thus enabling expression of appropriate tissue‐specific matrix genes. Inhibition of miR‐138 may serve as a potential therapeutic strategy to maintain the chondrocyte phenotype or reduce the progression of dedifferentiation in cultured HACs.