High-Throughput Identification of MiR-145 Targets in Human Articular Chondrocytes.

MicroRNAs (miRNAs) play key roles in cartilage development and homeostasis and are dysregulated in osteoarthritis. MiR-145 modulation induces profound changes in the human articular chondrocyte (HAC) phenotype, partially through direct repression of SOX9. Since miRNAs can simultaneously silence multiple targets, we aimed to identify the whole targetome of miR-145 in HACs, critical if miR-145 is to be considered a target for cartilage repair. We performed RIP-seq (RNA-immunoprecipitation and high-throughput sequencing) of miRISC (miRNA-induced silencing complex) in HACs overexpressing miR-145 to identify miR-145 direct targets and used cWords to assess enrichment of miR-145 seed matches in the identified targets. Further validations were performed by RT-qPCR, Western immunoblot, and luciferase assays. MiR-145 affects the expression of over 350 genes and directly targets more than 50 mRNAs through the 3'UTR or, more commonly, the coding region. MiR-145 targets DUSP6, involved in cartilage organization and development, at the translational level. DUSP6 depletion leads to MMP13 upregulation, suggesting a contribution towards the effect of miR-145 on MMP13 expression. In conclusion, miR-145 directly targets several genes involved in the expression of the extracellular matrix and inflammation in primary chondrocytes. Thus, we propose miR-145 as an important regulator of chondrocyte function and a new target for cartilage repair.

Derepression of MicroRNA-138 Contributes to Loss of the Human Articular Chondrocyte Phenotype.

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.

Haem oxygenase-1 induction reverses the actions of interleukin-1ß on hypoxia-inducible transcription factors and human chondrocyte metabolism in hypoxia.

HO-1 (haem oxygenase-1) catalyses the degradation of haem and possesses anti-inflammatory and cytoprotective properties. The role of inflammatory mediators in the pathogenesis of OA (osteoarthritis) is becoming increasingly appreciated. In the present study, we investigated the effects of HO-1 induction in OA and healthy HACs (human articular chondrocytes) in response to inflammatory cytokine IL-1 ß (interleukin-1ß) under hypoxic conditions. Hypoxia was investigated as it is a more physiological condition of the avascular cartilage. Hypoxic signalling is mediated by HIFs (hypoxia-inducible factors), of which there are two main isoforms, HIF-1α and HIF-2α. Normal and OA chondrocytes were stimulated with IL-1ß. This cytokine suppresses HO-1 expression and exerts both catabolic and anti-anabolic effects, while increasing HIF-1α and suppressing HIF-2α protein levels in OA chondrocytes in hypoxia. Induction of HO-1 by CoPP (cobalt protoporphyrin IX) reversed these IL-1ß actions. The hypoxia-induced anabolic pathway involving HIF-2α, SOX9 [SRY (sex determining region Y)-box 9] and COL2A1 (collagen type II α1) was suppressed by IL-1ß, but importantly, levels were restored by HO-1 induction, which down-regulated TNFα (tumour necrosis factor α), MMP (matrix metalloproteinase) activity and MMP-13 protein levels. Depletion of HO-1 using siRNA (small interfering RNA) abolished the CoPP effects, further demonstrating that these were due to HO-1. The results of the present study reveal the different mechanisms by which HO-1 exerts protective effects on chondrocytes in physiological levels of hypoxia.

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.

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.

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.

Inhibition of hypoxia-inducible factor-targeting prolyl hydroxylase domain-containing protein 2 (PHD2) enhances matrix synthesis by human chondrocytes.

Human articular cartilage is an avascular tissue, and therefore it functions in a hypoxic environment. Cartilage cells, the chondrocytes, have adapted to this and actually use hypoxia to drive tissue-specific functions. We have previously shown that human chondrocytes enhance cartilage matrix synthesis in response to hypoxia specifically through hypoxia-inducible factor 2alpha (HIF-2alpha)-mediated up-regulation of master regulator transcription factor SOX9, which in turn drives expression of the main cartilage-specific extracellular matrix genes. HIF-alpha isoforms are themselves regulated by specific prolyl hydroxylase domain-containing proteins, which target them for proteosomal degradation. In fact, prolyl hydroxylase domains are the direct oxygen sensors because they require molecular oxygen as a co-substrate. Here, we have identified PHD2 as the dominant isoenzyme regulating HIF-2alpha stability in human chondrocytes. Moreover, specific inhibition of PHD2 using RNA interference-mediated depletion caused an up-regulation of SOX9 and enhanced extracellular matrix protein production. Depletion of PHD2 resulted in greater HIF-2alpha levels and therefore enhanced SOX9-induced cartilage matrix production compared with the levels normally found in hypoxia (1% oxygen) implying that PHD2 inhibition offers a novel means to enhance cartilage repair in vivo. The need for HIF-specific hydroxylase inhibition was highlighted because treatment with the 2-oxoglutarate analogue dimethyloxalylglycine (which also inhibits the collagen prolyl 4-hydroxylases) prevented secretion of type II collagen, a critical cartilage matrix component.

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.

Six-transmembrane epithelial antigen of the prostate (STEAP1 and STEAP2)-differentially expressed by murine and human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have great potential for cell-based therapies. However, lack of cell-specific markers thwarts full realization of this as it prevents their identification in vivo, and subsequent purification. In the present study, to ensure cell purity multiple individual clones were derived from the bone marrow of BALB/b and BALB/c mice, and subsequently defined as MSCs by demonstrating their multipotentiality and self-renewal ability. In an effort to define the molecular signature of such MSCs and identify potentially cell-specific markers, an extensive genome-wide microarray analysis was performed comparing eight individual undifferentiated MSC clones to four different controls-corresponding differentiated MSC clones, bone marrow adherent cells, freshly isolated bone marrow cells, and embryonic fibroblasts. Strikingly, all MSC clones expressed differentially high levels of six-transmembrane epithelial antigen of the prostate (STEAP1 and STEAP2). Further, both STEAP members showed an extremely similar expression profile to stem cell antigen-1 (Sca-1) as demonstrated by two-dimensional hierarchical cluster analysis. Most importantly, differentially high levels of STEAP1 and STEAP2 proteins were also detected in human multipotent bone marrow adherent cultures. Thus, STEAPs may represent novel markers of MSCs in man as well as mice. Depletion of STEAP1 in human MSCs using RNAi resulted in decreased cell adhesion to tissue culture plastic. Further work is now needed to fully uncover its function in these cells, and to explore its potential as a marker of MSCs.

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 2alpha (HIF-2alpha)-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 special 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-2alpha, but were not regulated by SOX9. Therefore, hypoxia, and more specifically HIF-2alpha, promotes both SOX9-dependent and -independent factors important for cartilage homeostasis. HIF-2alpha may therefore represent a new and promising therapeutic target for cartilage repair.

Hypoxia-inducible factor 2alpha is essential for hypoxic induction of the human articular chondrocyte phenotype.

OBJECTIVE: To uncover the mechanism by which hypoxia enhances cartilage matrix synthesis by human articular chondrocytes. METHODS: The hypoxic response was investigated by exposing normal (nonarthritic) human articular chondrocyte cultures to 20% oxygen and 1% oxygen. Induction of the differentiated phenotype was confirmed at the gene and protein levels. In its first reported application in human articular chondrocytes, the RNA interference method was used to directly investigate the role of specific transcription factors in this process. Small interfering RNA directed against hypoxia-inducible factor 1alpha (HIF-1alpha), HIF-2alpha, and SOX9 were delivered by lipid-based transfection of primary and passaged human articular chondrocytes. The effect of each knockdown on hypoxic induction of the chondrocyte phenotype was assessed. RESULTS: Hypoxia enhanced matrix synthesis and SOX9 expression of human articular chondrocytes at both the gene and protein levels. Although HIF-1alpha knockdown had no effect, depletion of HIF-2alpha abolished this hypoxic induction. Thus, we provide the first evidence that HIF-2alpha, but not HIF-1alpha, is essential for hypoxic induction of the human articular chondrocyte phenotype. In addition, depletion of SOX9 prevented hypoxic induction of matrix genes, indicating that the latter are not direct HIF targets but are up-regulated by hypoxia via SOX9. CONCLUSION: Based on our data, we propose a novel mechanism whereby hypoxia promotes cartilage matrix synthesis specifically through HIF-2alpha-mediated SOX9 induction of key cartilage genes. These findings have potential application for the development of cartilage repair therapies.

Internal standards in differentiating embryonic stem cells in vitro.

Embryonic stem (ES) cell lines are important for use in developmental biology studies, and because these cells are totipotent, they may provide a much-needed source of differentiated cells for certain therapeutic applications. The phenotype of the ES cell in culture is often assessed by (semi)quantitative RNA analyses. In such cases, it is critical to use appropriate internal standards to correct for experimentally induced sources of error. This is particularly true for ES cell differentiation because it is heterogeneous in nature. We describe protocols for determining the suitability of housekeeping genes to act as internal controls in differentiating ES cell cultures. Such assessment is needed for every experimental condition under investigation. The protocol focuses on polymerase chain reaction; however, the principle and experimental design are applicable to any (semi)quantitative RNA assay.

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.

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.

Differentiating embryonic stem cells: GAPDH, but neither HPRT nor beta-tubulin is suitable as an internal standard for measuring RNA levels.

Embryonic stem (ES) cells are pluripotent cell lines that possess virtually unlimited self-renewal and differentiation capacity. Such characteristics make them potentially an invaluable cell source for diverse tissue-engineering applications. In vitro ES cell differentiation occurs spontaneously in three-dimensional structures termed "embryoid bodies" that mimic postimplantation embryonic tissue. HPRT, beta-tubulin, and GAPDH are commonly used as internal RNA standards in ES cell-derived gene transcription studies so that corrected sample mRNA levels can be obtained for (semi) quantitative gene expression data. However, if reliable data is to be obtained, it is essential that such housekeeping gene expression remains constant, and this has not been demonstrated for differentiating ES cell cultures, which represent a mixed and changing population of cells with time in culture. Therefore, in the present study, we tested the suitability of these housekeeping genes to act as true internal standards for differentiating murine ES cells cultured as embryoid bodies. PCR-amplified gene-specific products were quantified from digital images of ethidium bromide-stained gels using a computer software package. Both HPRT and beta-tubulin mRNA levels varied markedly in spontaneously differentiating and growth factor-supplemented (TGF-beta) ES cell cultures (p < 0.001, ANOVA), while GAPDH expression remained relatively constant (p > 0.2). Our results demonstrate the importance of fully validating housekeeping gene expression in in vitro ES cell gene transcription studies and suggest that GAPDH may be a suitable candidate to act as an internal RNA standard, while both HPRT and beta-tubulin appear to be inappropriate. Finally, we demonstrate enhanced mesodermal differentiation of ES cell-derived cultures by treatment with TGF-beta through significant upregulation of Brachyury T expression, with a concomitant decrease in expression of the undifferentiated ES cell marker Oct-4.

Sulphorhodamine-B-labelled albumin uptake around the ostium of the renal artery in rabbits: changes with age.

Because protein transport between blood and artery wall is important in atherogenesis, we have measured the uptake of fluorescently labelled albumin around the aorto-renal branch, an important site for lesions. Tracer concentrations in artery wall sections were quantified using digital fluorescence microscopy. Short-term experiments indicated endothelial permeability, while longer ones indicated the steady-state distribution of native proteins within the wall. In sexually immature rabbits (65-75 days), the permeability of the aorta was greater downstream of the renal ostium than upstream (p < 0.004). However, in mature rabbits (120-156 days), the permeability was greater, on average, at the upstream site compared to downstream. This change in pattern of uptake around the renal ostium with age appears to be due to increased uptake at the upstream site in mature animals, which was over fourfold greater than that measured at the equivalent site in immature animals (p < 0.01). In the renal artery itself, permeability was low and age independent. In mature animals, endothelial permeability did not correlate with steady-state wall concentrations, suggesting that the latter may be more dependent upon the rate of exit of protein and/or available space in the wall, rather than the rate of entry. The age-dependent changes in the spatial pattern of permeability correspond to the different distributions of spontaneous lipid accumulation in immature and mature vessels of both rabbits and humans, implying that permeability of the wall is important in atherogenesis.

A ratiometric method of autofluorescence correction used for the quantification of Evans blue dye fluorescence in rabbit arterial tissues.

Evans blue dye (EBD) conjugates with albumin in the circulation and is frequently used to measure vascular protein leakage. The fluorescence of the dye from tissue sections can be used to measure its uptake at very specific anatomical locations, but problems arise with dye quantification because tissue components also fluoresce; so-called autofluorescence. We have measured uptake of EBD by blood vessel walls at various points around the aorto-renal branch of rabbits. High resolution, digitised, fluorescence images of histological sections of artery wall allowed detailed microscopic analysis of EBD accumulation; and a ratiometric method was developed to enable autofluorescence to be separated from EBD fluorescence. When EBD-free tissue sections were illuminated with blue light, the ratio of red to green fluorescence was constant throughout the tissue (0.59 +/- 0.03, mean +/- S.D., n = 32). Therefore, at each individual pixel, the level of red autofluorescence could be determined by multiplying the green intensity at that pixel by the calculated red to green ratio. Since EBD fluorescence was detected only in the red region of the spectrum, intensity values of the dye alone were obtained from EBD-exposed tissue by subtracting the red autofluorescence estimated by this ratiometric method. In such cases the red to green fluorescence ratio was measured from adjacent sites known to be free of EDB (0.59 +/- 0.02, mean +/- S.D., n = 56). We were therefore able to increase the sensitivity of tracer quantification by complete elimination of background autofluorescence on a pixel-by-pixel basis. Use of EBD standards allowed calibration of corrected fluorescence intensities and calculation of mass transfer coefficients for albumin into the artery wall. Spatial variations in the permeability of the artery wall around the renal ostium were detected with the present high resolution technique, with an average mass transfer coefficient of (6.8 +/- 0.9) x 10(-8) cm s(-1) for all sites combined (n = 56). The present ratiometric method could potentially be applied to other quantitative fluorescence-based techniques.