Christopher L. Murphy

The identification of differentially expressed microRNA in osteoarthritic tissue that modulate the production of TNF-alpha and MMP13.

OBJECTIVE To identify differentially expressed microRNAs (miRNAs) in human osteoarthritic (OA) cartilage and bone tissue and to determine their relevance to chondrocyte function. METHODS Cartilage and bone was obtained from OA patients who underwent total knee joint replacement surgery or from post-mortem patients with no previous history of OA. MiRNA expression was quantified by real-time PCR (RT-PCR). Functional pathway analysis of miRNA was performed using Ingenuity Pathway Analysis. Primary chondrocytes were isolated by collagenase digestion and transfected with miRNA mimics and miRNA inhibitors using cationic lipid. Tumour Necrosis Factor-alpha (TNF-alpha) and Matrix metalloprotease 13 (MMP13) protein levels were measured by Enzyme-Linked ImmunoSorbent Assay (ELISA). RESULTS In total we identified 17 miRNA that showed greater than 4-fold differential expression between OA and normal cartilage, and 30 miRNA that showed greater than 4-fold differential expression in OA bone. Functional pathway analysis of the predicted gene targets for miR-9, miR-98, which were upregulated in both OA bone and cartilage tissue, and miR-146, which was downregulated in OA cartilage, suggested that these miRNA mediate inflammatory functions and pathways. Over-expression of miR-9, miR-98 or miR-146 in isolated human chondrocytes reduced interleukin-1 beta (IL-1 beta) induced TNF-alpha production. Furthermore, inhibition and over-expression of miR-9 modulated MMP13 secretion. CONCLUSIONS We have identified a number of differentially expressed miRNAs in late-stage human OA cartilage and bone. Functional analysis of miR-9, miR-98 and miR-146 in primary chondrocytes suggests a role in mediating the IL-1 beta induced production of TNF-alpha. MiR-9, upregulated in OA tissue, was found to inhibit secretion of the collagen type II-targeting metalloproteinase MMP13 in isolated human chondrocytes.

Control of human articular chondrocyte differentiation by reduced oxygen tension

Cell number is often a limiting factor in studies of chondrocyte physiology, particularly for human investigations. Chondrocytes can be readily proliferated in monolayer culture, however, differentiated phenotype is soon lost. We therefore endeavored to restore normal phenotype to human chondrocytes after serial passage in monolayer culture by manipulating cell morphology and oxygen tension towards the in vivo state. Third passage cells were encapsulated in alginate and exposed to either 20% or more physiologic 5% oxygen tensions. To assess cell phenotype, gene expression was measured using TaqMan real‐time PCR. Encapsulated, primary chondrocytes cultured in 20% oxygen were used as a positive reference. Passaged human chondrocytes were fibroblastic in appearance and had lost normal phenotype as evidenced by a decrease in expression of collagen II, aggrecan, and sox9 genes of 66, 6, and 14 fold, respectively; with concomitant high expression of type I collagen (22 fold increase). A partial regaining of the differentiated phenotype was observed by encapsulation in 20% oxygen; however, even after 4 weeks, collagen II gene expression was not fully restored. Collagen II and aggrecan expression were increased, on average, 3 fold, in 5% oxygen tension compared to 20% cultures. Furthermore, matrix glycosaminoglycan (GAG) levels were significantly increased in reduced oxygen. In fact, after 4 weeks in 5% oxygen, encapsulated third passage cells had collagen II expression fully regained and aggrecan and sox9 levels actually exceeding primary cell levels in 20% oxygen. Our results show that the phenotype of serially passaged human articular chondrocytes is more fully restored by combining encapsulation with culture in more physiological levels of oxygen. Sox9, an essential transcription factor for chondrocyte differentiation is strongly implicated in this process since its expression was upregulated almost 27 fold. These findings have implications for the optimal conditions for the in vitro culture of chondrocytes.

Chondrogenic differentiation of murine embryonic stem cells: effects of culture conditions and dexamethasone.

Pluripotent embryonic stem (ES) cells have the capability to differentiate to various cell types and may represent an alternative cell source for the treatment of cartilage defects. Here, we show that differentiation of ES cells toward the chondrogenic lineage can be enhanced by altering the culture conditions. Chondrogenesis was observed in intact embryoid body (EB) cultures, as detected by an increase in mRNA levels for aggrecan and Sox9 genes. Collagen IIB mRNA, the mature chondrocyte‐specific splice variant, was absent at day 5, but appeared at later time points. Dexamethasone treatment of alginate‐encapsulated EB cultures did not have a strong chondrogenic effect. Nor was chondrogenesis enhanced by alginate encapsulation compared to simple plating of EBs. However, disruption of day 5 EBs and culture as a micromass or pelleted mass, significantly enhanced the expression of the cartilage marker gene collagen type II and the transcription factor Sox9 compared to all other treatments. Histological and immunohistochemical analysis of pellet cultures revealed cartilage‐like tissue characterized by metachromatically stained extracellular matrix and type II collagen immunoreactivity, indicative of chondrogenesis. These findings have potentially important implications for cartilage tissue engineering, since they may enable the increase in differentiated cell numbers needed for the in vitro development of functional cartilaginous tissue suitable for implantation.

Regulation of Human Chondrocyte Function through Direct Inhibition of Cartilage Master Regulator SOX9 by MicroRNA-145 (miRNA-145)

Background: SOX9 is essential for cartilage. Results: miR-145 directly targets SOX9, and increased miR-145 levels reduce expression of SOX9 and the extracellular matrix genes critical to cartilage function. Conclusion: SOX9 is subject to significant post-transcriptional regulation by miR-145 in human chondrocytes. Significance: Our data give new insights into the mechanisms regulating SOX9 and identify miR-145 as a new target for cartilage repair. 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.

Hypoxia Promotes the Differentiated Human Articular Chondrocyte Phenotype through SOX9-dependent and -independent Pathways

The chondrocyte is solely responsible for synthesis and maintenance of the resilient articular cartilage matrix that gives this load-bearing tissue its mechanical integrity. When the differentiated cell phenotype is lost, the matrix becomes compromised and cartilage function begins to fail. We have recently shown that hypoxia promotes the differentiated phenotype through hypoxia-inducible factor 2α (HIF-2α)-mediated SOX9 induction of the main matrix genes. However, to date, only a few genes have been shown to be SOX9 targets, while little is known about SOX9-independent regulators. We therefore performed a detailed microarray study to address these issues. Analysis involved 35 arrays on chondrocytes obtained from seven healthy, non-elderly human cartilage samples. Genes were selected that were down-regulated with serial passage in culture (as this causes loss of the differentiated phenotype) and subsequently up-regulated in hypoxia. The importance of key findings was further probed using the technique of RNA interference on these human articular chondrocytes. Our results show that hypoxia has a broader beneficial effect on the chondrocyte phenotype than has been previously described. Of especial note, we report new hypoxia-inducible and SOX9-regulated genes, Gdf10 and Chm-I. In addition, Mig6 and InhbA were induced by hypoxia, predominantly via HIF-2α, but were not regulated by SOX9. Therefore, hypoxia, and more specifically HIF-2α, promotes both SOX9-dependent and -independent factors important for cartilage homeostasis. HIF-2α may therefore represent a new and promising therapeutic target for cartilage repair.

Type II Collagen Expression Is Regulated by Tissue-specific miR-675 in Human Articular Chondrocytes

miRNAs have been shown to be essential for normal cartilage development in the mouse. However, the role of specific miRNAs in cartilage function is unknown. Using rarely available healthy human chondrocytes (obtained from 8 to 50 year old patients), we detected a most highly abundant primary miRNA H19, whose expression was heavily dependent on cartilage master regulator SOX9. Across a range of murine tissues, expression of both H19- and H19-derived miR-675 mirrored that of cartilage-specific SOX9. miR-675 was shown to up-regulate the essential cartilage matrix component COL2A1, and overexpression of miR-675 rescued COL2A1 levels in H19- or SOX9-depleted cells. We thus provide evidence that SOX9 positively regulates COL2A1 in human articular chondrocytes via a previously unreported miR-675-dependent mechanism. This represents a novel pathway regulating cartilage matrix production and identifies miR-675 as a promising new target for cartilage repair.

Hypoxia Promotes the Production and Inhibits the Destruction of Human Articular Cartilage

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.

Hypoxia. HIF-mediated articular chondrocyte function: prospects for cartilage repair

In a chronically hypoxic tissue such as cartilage, adaptations to hypoxia do not merely include cell survival responses, but also promotion of its specific function. This review will focus on describing such hypoxia-mediated chondrocyte function, in particular in the permanent articular cartilage. The molecular details of how chondrocytes sense and respond to hypoxia and how this promotes matrix synthesis have recently been examined, and specific manipulation of hypoxia-induced pathways is now considered to have potential therapeutic application to maintenance and repair of articular cartilage.

In Vivo Analyses of Interactions between SecE and SecY, Core Components of the Escherichia coli Protein Translocation Machinery

We have carried out structure-function studies on the cytoplasmic membrane protein, SecE, a component of the Escherichia coli secretion machinery. SecE, along with SecY, form a complex in the cytoplasmic membrane essential for protein translocation. By directed mutagenesis, we altered highly conserved residues of the second cytoplasmic domain (CD2) and of the COOH-terminal periplasmic region (PD2) of SecE. These mutants, as well as previously constructed mutations in the third membrane-spanning segment of SecE (MSS3), were tested for their ability to complement a secE null mutation, for their effects on protein export in vivo, and for their ability to form a stable complex with SecY. Most single mutations at the conserved positions in CD2 caused secretion defects, but had little effect on growth at 37°C. Double mutations in CD2, or the introduction or removal of proline residues, affected growth and protein translocation more severely. Co-immunoprecipitations of SecE and SecY revealed that all mutant proteins, except those altered in PD2, destabilized the SecE-SecY complex. These results suggest that several regions contribute to the formation of a stable SecE-SecY complex but the elimination of a single contact point does not necessarily affect the functionality of the complex.

LRP-1-mediated endocytosis regulates extracellular activity of ADAMTS-5 in articular cartilage

Aggrecan is a major matrix component of articular cartilage, and its degradation is a crucial event in the development of osteoarthritis (OA). Adamalysin‐like metalloproteinase with thrombospondin motifs 5 (ADAMTS‐5) is a major aggrecan‐degrading enzyme in cartilage, but there is no clear correlation between ADAMTS‐5 mRNA levels and OA progression. Here, we report that post‐translational endocytosis of ADAMTS‐5 by chondrocytes regulates its extracellular activity. We found 2‐ to 3‐fold reduced aggrecanase activity when ADAMTS‐5 was incubated with live porcine cartilage, resulting from its rapid endocytic clearance. Studies using receptor‐associated protein (RAP), a ligand‐binding antagonist for the low‐density lipoprotein receptor‐related proteins (LRPs), and siRNA‐mediated gene silencing revealed that the receptor responsible for ADAMTS‐5 clearance is LRP‐1. Domain‐deletion mutagenesis of ADAMTS‐5 identified that the noncatalytic first thrombospondin and spacer domains mediate its endocytosis. The addition of RAP to porcine cartilage explants in culture increased the basal level of aggrecan degradation, as well as ADAMTS‐5‐induced aggrecan degradation. Notably, LRP‐1‐mediated endocytosis of ADAMTS‐5 is impaired in chondrocytes of OA cartilage, with ~90% reduction in protein levels of LRP‐1 without changes in its mRNA levels. Thus, LRP‐1 dictates physiological and pathological catabolism of aggrecan in cartilage as a key modulator of the extracellular activity of ADAMTS‐5.—Yamamoto, K., Troeberg, L., Scilabra, S. D., Pelosi, M., Murphy, C. L., Strickland, D. K., Nagase, H. LRP‐1‐mediated endocytosis regulates extracellular activity of ADAMTS‐5 in articular cartilage. FASEB J. 27, 511–521 (2013). www.fasebj.org