Dual and Opposing Regulation of MMP1 and MMP13 by Both Arms of miR-675 in Human Articular Chondrocytes.

BACKGROUND/AIMS: MicroRNAs (miRs) are transcribed as stem-loop precursors harboring two different miRs on either side of the structure. Both miRs can modulate levels of cellular transcripts based on sequence complementarity between the miR and the mRNA target. The miR of the current study, miR-675, is encoded in the H19 gene with high expression in fetal/placental tissues but low levels in most adult tissues except for skeletal muscle and articular cartilage. miR-675 has a supportive role in expression of the major collagen component of articular cartilage (COL2A1) but it is unknown which arm contributes to this effect. Objectives: To determine the active arm of miR-675 in human articular chondrocytes. To evaluate effects of overexpression of both arms of miR-675 on MMP1 and MMP13, two enzymes involved in breakdown of COL2A1. To investigate whether abundance of both arms of miR-675 is dynamic. METHODS: miR-arm activity was determined by association with the AGO2 complex using immunoprecipitation with an AGO2 specific antibody. miR overexpression and inhibition was used to identify indirect downstream effects on two targets of the Matrix-Metalloprotease family, MMP1 and MMP13. Data was evaluated by qPCR and enzymatic activity assays. Early passage human articular chondrocytes (up to passage 2) obtained from cartilage from both healthy and osteoarthritis affected tissue were used. To evaluate miR-675 levels in a different model, myotube differentiation was employed. RESULTS: We show that both arms of miR-675 have opposing effects on MMP1 and MMP13; however only one arm, miR-675-3' is active in human articular chondrocytes. We demonstrate that during myotube differentiation, high expression of both arms of miR-675 is observed as well as an increase in expression of MMP1. CONCLUSION: We show that both arms of miR-675 result in opposing effects on two downstream molecules MMP1 and MMP13. We propose that miR abundance may arise as response to direct target transcript levels and are thus dynamic to meet the requirements of the cellular environment.

miR-1247 functions by targeting cartilage transcription factor SOX9.

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.

Parathyroid hormone-related protein is induced by hypoxia and promotes expression of the differentiated phenotype of human articular chondrocytes.

PTHrP (parathyroid hormone-related protein) is crucial for normal cartilage development and long bone growth and acts to delay chondrocyte hypertrophy and terminal differentiation in the growth plate. After growth plate closure adult HACs (human articular chondrocytes) still produce PTHrP, suggesting a possible role for this factor in the permanent articular cartilage. However, the expression regulation and function of PTHrP in the permanent articular cartilage is unknown. Human articular cartilage is an avascular tissue and functions in a hypoxic environment. The resident chondrocytes have adapted to hypoxia and use it to drive their tissue-specific functions. In the present study, we explored directly in normal articular chondrocytes isolated from a range of human donors the effect of hypoxia on PTHrP expression and whether PTHrP can regulate the expression of the permanent articular chondrocyte phenotype. We show that in HACs PTHrP is up-regulated by hypoxia in a HIF (hypoxia-inducible factor)-1α and HIF-2α-dependent manner. Using recombinant PTHrP, siRNA-mediated depletion of endogenous PTHrP and by blocking signalling through its receptor [PTHR1 (PTHrP receptor 1)], we show that hypoxia-induced PTHrP is a positive regulator of the key cartilage transcription factor SOX9 [SRY (sex determining region on the Y chromosome)-box 9], leading to increased COL2A1 (collagen type II, α1) expression. Our findings thus identify PTHrP as a potential factor for cartilage repair therapies through its ability to promote the differentiated HAC phenotype.

MicroRNA Target Identification-Experimental Approaches.

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.

Regulation of human chondrocyte function through direct inhibition of cartilage master regulator SOX9 by microRNA-145 (miRNA-145).

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.