Identification of the pathogenic pathways in osteoarthritic hip cartilage: commonality and discord between hip and knee OA

Xu, Yaobo; Barter, M.J.; Swan, Daniel; Rankin, Kenneth; Rowan, Andrew D.; Santibanez‐Koref, Mauro; Loughlin, John; Young, David A.

doi:10.1016/j.joca.2012.05.006

Cited by 81 publications

(95 citation statements)

References 56 publications

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“…For both genes, the AEI observed in cartilage was small in magnitude but highly significant and was also observed in other joint tissues. As noted in the results, interrogation of our previously published microarray data of cartilage [15] revealed that GNL3 and SPCS1 were in the highest quartile of expressed genes. However, in this data set neither gene demonstrated a diffence in expression between OA cartilage and cartilage from non-OA controls.…”

Section: Discussionsupporting

confidence: 69%

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Allelic expression analysis of the osteoarthritis susceptibility locus that maps to chromosome 3p21 reveals cis-acting eQTLs at GNL3 and SPCS1

et al. 2014

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BackgroundAn osteoarthritis (OA) susceptibility locus has been mapped to chromosome 3p21, to a region of high linkage disequilibrium encompassing twelve genes. Six of these genes are expressed in joint tissues and we therefore assessed whether any of the six were subject to cis-acting regulatory polymorphisms active in these tissues and which could therefore account for the association signal.MethodsWe measured allelic expression using pyrosequencing assays that can distinguish mRNA output from each allele of a transcript single nucleotide polymorphism. We assessed RNA extracted from the cartilage and other joint tissues of OA patients who had undergone elective joint replacement surgery. A two-tailed Mann–Whitney exact test was used to test the significance of any allelic differences.ResultsGNL3 and SPCS1 demonstrated significant allelic expression imbalance (AEI) in OA cartilage (GNL3, mean AEI = 1.04, p = 0.0002; SPCS1, mean AEI = 1.07, p < 0.0001). Similar results were observed in other tissues. Expression of the OA-associated allele was lower than that of the non-associated allele for both genes.Conclusionscis-acting regulatory polymorphisms acting on GNL3 and SPCS1 contribute to the OA association signal at chromosome 3p21, and these genes therefore merit further investigation.

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Section: Discussionsupporting

confidence: 69%

“…Interrogation of our previously published microarray analysis of cartilage [15] revealed that GNL3 and SPCS1 were in the highest quartile of expressed genes, GLT8D1 , PBRM1 , NT5DC2 and POC1A fell between the upper and lower quartiles, and TMEM110 was within the lowest quartile.…”

Section: Resultsmentioning

confidence: 92%

Allelic expression analysis of the osteoarthritis susceptibility locus that maps to chromosome 3p21 reveals cis-acting eQTLs at GNL3 and SPCS1

et al. 2014

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“…Furthermore, the model can be helpful to further investigate the differences in the OA pathomechansim of chondrocytes from different joints (knee, hip, ankle) as shown by Xu et al in a study comparing hip and knee OA cartilage. 67 Concluding, the porcine chondrocyte micromass model displayed significant parallels to human cartilage and vice versa confirmed the applicability of known human cartilage markers and characteristics in the porcine micromass model. TNF-α treatment initiated a typical OA reaction pattern suggesting the porcine micromass model as suitable tissue platform for the evaluation of innovative substances and techniques for the treatment of OA.…”

Section: ■ Discussionmentioning

confidence: 52%

Suitability of Porcine Chondrocyte Micromass Culture To Model Osteoarthritis in Vitro

et al. 2014

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In vitro tissue models are useful tools for the development of novel therapy strategies in cartilage repair and care. The limited availability of human primary tissue and high costs of animal models hamper preclinical tests of innovative substances and techniques. In this study we tested the potential of porcine chondrocyte micromass cultures to mimic human articular cartilage and essential aspects of osteoarthritis (OA) in vitro. Primary chondrocytes were enzymatically isolated from porcine femoral condyles and were maintained in 96-multiwell format to establish micromass cultures in a high-throughput scale. Recombinant porcine tumor necrosis factor alpha (TNF-α) was used to induce OA-like changes documented on histological (Safranin O, collagen type II staining), biochemical (hydroxyproline assay, dimethylmethylene blue method), and gene expression level (Affymetrix porcine microarray, real time PCR) and were compared with published data from human articular cartilage and human micromass cultures. After 14 days in micromass culture, porcine primary chondrocytes produced ECM rich in proteoglycans and collagens. On gene expression level, significant correlations of detected genes with porcine cartilage (r = 0.90), human cartilage (r = 0.71), and human micromass culture (r = 0.75) were observed including 34 cartilage markers such as COL2A1, COMP, and aggrecan. TNF-α stimulation led to significant proteoglycan (-75%) and collagen depletion (-50%). Comparative expression pattern analysis revealed the involvement of catabolic enzymes (MMP1, -2, -13, ADAM10), chemokines (IL8, CCL2, CXCL2, CXCL12, CCXL14), and genes associated with cell death (TNFSF10, PMAIPI, AHR) and skeletal development (GPNMB, FRZB) including transcription factors (WIF1, DLX5, TWIST1) and growth factors (IGFBP1, -3, TGFB1) consistent with published data from human OA cartilage. Expression of genes related to cartilage ECM formation (COL2A1, COL9A1, COMP, aggrecan) as well as hypertrophic bone formation (COL1A1, COL10A1) was predominantly found decreased. These findings indicating significant parallels between human articular cartilage and the presented porcine micromass model and vice versa confirm the applicability of known cartilage marker and their characteristics in the porcine micromass model. TNF-α treatment enabled the initiation of typical OA reaction patterns in terms of extensive ECM loss, cell death, formation of an inflammatory environment through the induction of genes coding for chemokines and enzymes, and the modulation of genes involved in skeletal development such as growth factors, transcription factors, and cartilage ECM-forming genes. In conclusion, the porcine micromass model represents an alternative tissue platform for the evaluation of innovative substances and techniques for the treatment of OA.

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“…We had previously generated gene expression microarray data for hip cartilage from patients who had undergone joint replacement surgery due to either hip OA or a neck of femur (NOF) fracture (Xu et al 2012). This latter cartilage serves as a non-OA control.…”

Section: Resultsmentioning

confidence: 99%

“…To our knowledge, this is the first study to compare methylation patterns between cartilage from different joints and the differences we observed at the GDF5 locus may reflect global differences in DNA methylation patterns between cartilages from different joints. Such differential methylation by skeletal site could partly explain why there is a limited overlap between the genes aberrantly expressed in OA hip and OA knee cartilage (Xu et al 2012). The methylation differences between OA and control cartilage in the CpG island and between OA knee and hip cartilage in the 5ʹUTR are only small (3–12 %) but are similar to those observed at the MMP13 and SOX9 promoters in OA and control hip cartilage (Bui et al 2012; Kim et al 2013).…”

Section: Discussionmentioning

confidence: 99%

CpG methylation regulates allelic expression of GDF5 by modulating binding of SP1 and SP3 repressor proteins to the osteoarthritis susceptibility SNP rs143383

et al. 2014

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GDF5 encodes an extracellular signalling molecule that is essential for normal skeletal development. The rs144383 C to T SNP located in the 5ʹUTR of this gene is functional and has a pleiotropic effect on the musculoskeletal system, being a risk factor for knee-osteoarthritis (OA), congenital hip dysplasia, lumbar disc degeneration and Achilles tendon pathology. rs143383 exerts a joint-wide effect on GDF5 expression, with expression of the OA-associated T allele being significantly reduced relative to the C allele, termed allelic expression imbalance. We have previously reported that the GDF5 locus is subject to DNA methylation and that allelic imbalance of rs143383 is mediated by SP1, SP3 and DEAF1 transcriptional repressors. In this study, we have assayed GDF5 methylation in normal and osteoarthritic cartilage, and investigated the effect of methylation on the allelic imbalance of rs143383. We observed demethylation of the GDF5 5ʹUTR in OA knee cartilage relative to both OA (p = 0.009) and non-OA (p = 0.001) hip cartilage, with the most significant demethylation observed at the highly conserved +37 CpG site located 4 bp upstream of rs143383. Methylation modulates the level and direction of allelic imbalance of rs143383, with methylation of the +37 CpG dinucleotide within the SP1/SP3 binding site having an allele-specific effect on SP1 and SP3 binding. Furthermore, methylation attenuated the repressive effects of SP1, SP3 and DEAF1 on GDF5 promoter activity. This data suggest that the differential methylation of the +37 CpG site between osteoarthritic hip and knee cartilage may be responsible for the knee-specific effect of rs143383 on OA susceptibility.Electronic supplementary materialThe online version of this article (doi:10.1007/s00439-014-1447-z) contains supplementary material, which is available to authorized users.

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Identification of the pathogenic pathways in osteoarthritic hip cartilage: commonality and discord between hip and knee OA

Cited by 81 publications

References 56 publications

Allelic expression analysis of the osteoarthritis susceptibility locus that maps to chromosome 3p21 reveals cis-acting eQTLs at GNL3 and SPCS1

Allelic expression analysis of the osteoarthritis susceptibility locus that maps to chromosome 3p21 reveals cis-acting eQTLs at GNL3 and SPCS1

Suitability of Porcine Chondrocyte Micromass Culture To Model Osteoarthritis in Vitro

CpG methylation regulates allelic expression of GDF5 by modulating binding of SP1 and SP3 repressor proteins to the osteoarthritis susceptibility SNP rs143383

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