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The interaction between physical activity and amount of baseline knee cartilage
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Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
611 THE INTERACTION BETWEEN PHYSICAL ACTIVITY AND AMOUNT OF BASELINE KNEE CARTILAGE A. Teichtahl y, Y. Wang z, S. Heritier z, A. Wluka z, B. Strauss y, J. Proietto x, J. Dixon k, G. Jones ¶, F. Cicuttini z. y Baker IDI/Monash Univ., Melbourne, Australia; z Monash Univ., Melbourne, Australia; x Univ. of Melbourne, Melbourne, Australia; k Baker IDI, Melbourne, Australia; ¶ Menzies Res. Inst., Tasmania, Australia Purpose: Conflicting reports of the effect of physical activity on knee cartilage may be due to the heterogeneity of populations examined, in particular, the underlying health of the knee joint. This study examined the influence of recreational and occupational physical activity on cartilage volume loss. Methods: 250 participants with no significant musculoskeletal disease were recruited. A gender-specific median cartilage volume split was used to define people in the lowest and highest 50% of baseline cartilage volume. Baseline recreational and occupational activity was examined by questionnaire, while cartilage volume was assessed by magnetic resonance imaging at baseline and 2.4 years later. Results: Significant interactions were demonstrable between physical activity and cartilage volume loss based on stratification of baseline cartilage volume (all p 0.03). There was a dose-response relationship between frequently performed baseline occupational activities and medial cartilage volume loss and in the low (B ¼ 0.2%/annum, 95% CI 0.0 to 0.04%/annum) and high (B ¼ 0.2%/annum, 95% CI 0.4 to 0.0%/ annum) baseline cartilage volume groups (p ¼ 0.001 for interaction). Individuals with low baseline cartilage volume who were active in their occupation and or recreational activity had greater medial cartilage volume loss than their more inactive counterparts (2.4%/annum vs 1.5%/ annum, p ¼ 0.02). Conclusions: Whereas people with less baseline cartilage volume are more at risk of structural knee damage with either or both heavy occupational and recreational workloads, individuals with high baseline cartilage volume may advantageously modify their risk for knee osteoarthritis by participating in more frequent occupational physical activities. 612 BIG TASKS FOR SMALL RNAS; A NEW CLASS OF RNAS IN THE PATHGENESIS OF OSTEARTHRITIS M.J. Peffers y, P.D. Clegg y, E. Boothroyd z, A. Cremer x, D.A. Durtel x, M.M. Caron x, T.J. Welting x. y Univ. of Liverpool, Neston, United Kingdom; z Queens Sch., Chester, United Kingdom; x Dept. of Orthopaedic Surgery, Maastricht Univ. Med. Ctr., Maastricht, Netherlands Purpose: Chondrocytes acquire a modified phenotype with ageing, resulting in increased risk of osteoarthritis (OA) due to alterations in the cartilage extracellular matrix (ECM). SnoRNAs direct chemical modification of RNA substrates and are involved in endoribonucleolytic prerRNA processing. The post-transcriptional 2’O-ribose methylation and pseudouridylation carried out by snoRNAs fine-tunes spliceosome and ribosome function, accommodating changing requirements for protein synthesis during health and disease. Control of snoRNA levels may be pivotal in regulating the transcriptional and translational capacity of high protein producing chondrocytes. This is interesting as in OA there is an imbalance between ECM protein anabolism and catabolism. To ensure continuous ECM deposition it is essential for a chondrocyte to control the number and quality of its ribosomes. We tested the hypothesis that the ribosome’s translational capacity alters with age and disease due to dysregulation of expression and function of specific snoRNAs; contributing to the development of the OA chondrocyte phenotype. Methods: Total RNA was extracted from human OA knee cartilage of young (n¼6; mean age ± SD 22.7 ± 4.1 years) normal and old n¼6; (66.4 ± 15.9 years) donors and hybridised onto Affymetrix miRNA 4.0 arrays. The probe set for Homo sapiens was used to determine differentially expressed snoRNAs. Relative ribosome number and chondrocyte marker gene expression was determined using qRT-PCR of 5.8,
18 and 28S rRNAs and of COL2A1, ACAN, SOX9, COL10A1, RUNX2, MMP13, ADAMTS5, COX-2, IL6, BAPX1 mRNAs. Total DNA content by SYBR Green detection and total protein was determined using BCA assay. Results: Normal samples correlated closely together, however OA samples clustered into three groups. When PCA was integrated with the Kellgren and Lawrence scores of OA donors the sub-populations were separated on OA severity. Analysis of the three subgroups identified 26 snoRNAs reduced in OA and 11 snoRNAs increased. These include 25 box C/D and 11 box H/ACA snoRNAs. To address the potential impact of aberrant snoRNAs expression on rRNA maturation, we determined the relative ribosome content in healthy and OA human articular chondorcytes (HAC). 18S and 5.8S rRNA (not 28S) levels were decreased in OA, together with a typical OA chondrocyte gene expression profile. Pre-rRNA levels were higher in OA, indicating aberrant pre-rRNA processing. In concert array results indicated that expression of U3 and U13 snoRNAs is deregulated in OA. In contrast to the majority of the snoRNAs these direct sitespecific endoribonucleolytic cleavage of pre-rRNA. To address a potential involvement of the inflammatory compound of OA, healthy HACs were exposed to IL1b. Similar to OA chondrocytes 18S and 5.8S rRNA decreased on exposure and pre-rRNA increased. To address the question whether the chondrocyte phenotype responds in an OAlike fashion as a result of alterations in the cell’s translation capacity we inhibited rRNA transcription using actinomycin D using normal HACs. 18S and 5.8S rRNA levels/cell were significantly downregulated whereas 28S rRNA levels remained unaffected. Due to ribosome depletion a reduction in total protein content/cell was observed, confirming functionally decreased translation capacity. The expression of RUNX2 and COL10A1 was upregulated, whereas COL2A1 expression was downregulated. Findings indicate that as a result of decreased chondrocyte ribosome content and translation capacity, chondrocytes phenotypically respond in an OA-like fashion. Conclusions:Since we found evidence for altered ribosome abundancy and auxiliary rRNA maturation machinery in ageing chondrocytes accompanied by differential OA cartilage-specific expression of snoRNAs, we believe that the translational capacity of the articular chondrocyte in OA is impaired, due to dysregulation of expression and function of specific snoRNAs, thereby contributing to the development of the OA chondrocyte phenotype. 613 IDENTIFICATION OF A NOVEL TARGET, FOXA2, IN THE ONSET AND DEVELOPMENT OF OSTEOARTHRITIS A.M. Ionescu y, L. Xu y, E. Kozhemyakina z, A. Lassar z, Y. Li y, K. Kaestner x, V. Rosen y. y HSDM, Boston, MA, USA; z HSM, Boston, MA, USA; x UPenn, Philadelphia, MA, USA Purpose: The proposed studies are anticipated to establish whether FoxA2 is a potential target for the treatment of OA. Methods: The following mouse lines were used: FoxA2flox/flox, FoxA3/, Prg4CreERt2-GFP and rtTA from Jax Labs. Results: In OA, irreversible degradation of the “permanent” articular cartilage by MMP13 is a key pathological event. Since I found that FoxA factors control MMP13 expression in skeletal development, it seemed possible they would to do the same in OA. Thus, we evaluated the expression of FoxA2 in both healthy and OA cartilage using FoxA2IRES-CreERT2/þ Tomato reporter mice. In healthy articular cartilage, the majority of cells positive for FoxA2 expression (red) are located on the tidemark (TM) or below, in the calcified zone of the articular cartilage (Fig.1). We also looked for co-localization of FoxA2 and MMP13 in articular cartilage by performing immunofluorescence for MMP13 on sections from FoxA2IRES-CreERT2/ þ;Tomato mice and found that FoxA2 positive cells (red) and MMP13 positive cells (green) overlap in the hypertrophic zone (overlay yellow) (Fig.1). Next, we looked at expression of FoxA2 in OA articular cartilage using the Destabilization of the Medial Meniscus (DMM) murine model of OA.
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
611 THE INTERACTION BETWEEN PHYSICAL ACTIVITY AND AMOUNT OF BASELINE KNEE CARTILAGE A. Teichtahl y, Y. Wang z, S. Heritier z, A. Wluka z, B. Strauss y, J. Proietto x, J. Dixon k, G. Jones ¶, F. Cicuttini z. y Baker IDI/Monash Univ., Melbourne, Australia; z Monash Univ., Melbourne, Australia; x Univ. of Melbourne, Melbourne, Australia; k Baker IDI, Melbourne, Australia; ¶ Menzies Res. Inst., Tasmania, Australia Purpose: Conflicting reports of the effect of physical activity on knee cartilage may be due to the heterogeneity of populations examined, in particular, the underlying health of the knee joint. This study examined the influence of recreational and occupational physical activity on cartilage volume loss. Methods: 250 participants with no significant musculoskeletal disease were recruited. A gender-specific median cartilage volume split was used to define people in the lowest and highest 50% of baseline cartilage volume. Baseline recreational and occupational activity was examined by questionnaire, while cartilage volume was assessed by magnetic resonance imaging at baseline and 2.4 years later. Results: Significant interactions were demonstrable between physical activity and cartilage volume loss based on stratification of baseline cartilage volume (all p 0.03). There was a dose-response relationship between frequently performed baseline occupational activities and medial cartilage volume loss and in the low (B ¼ 0.2%/annum, 95% CI 0.0 to 0.04%/annum) and high (B ¼ 0.2%/annum, 95% CI 0.4 to 0.0%/ annum) baseline cartilage volume groups (p ¼ 0.001 for interaction). Individuals with low baseline cartilage volume who were active in their occupation and or recreational activity had greater medial cartilage volume loss than their more inactive counterparts (2.4%/annum vs 1.5%/ annum, p ¼ 0.02). Conclusions: Whereas people with less baseline cartilage volume are more at risk of structural knee damage with either or both heavy occupational and recreational workloads, individuals with high baseline cartilage volume may advantageously modify their risk for knee osteoarthritis by participating in more frequent occupational physical activities. 612 BIG TASKS FOR SMALL RNAS; A NEW CLASS OF RNAS IN THE PATHGENESIS OF OSTEARTHRITIS M.J. Peffers y, P.D. Clegg y, E. Boothroyd z, A. Cremer x, D.A. Durtel x, M.M. Caron x, T.J. Welting x. y Univ. of Liverpool, Neston, United Kingdom; z Queens Sch., Chester, United Kingdom; x Dept. of Orthopaedic Surgery, Maastricht Univ. Med. Ctr., Maastricht, Netherlands Purpose: Chondrocytes acquire a modified phenotype with ageing, resulting in increased risk of osteoarthritis (OA) due to alterations in the cartilage extracellular matrix (ECM). SnoRNAs direct chemical modification of RNA substrates and are involved in endoribonucleolytic prerRNA processing. The post-transcriptional 2’O-ribose methylation and pseudouridylation carried out by snoRNAs fine-tunes spliceosome and ribosome function, accommodating changing requirements for protein synthesis during health and disease. Control of snoRNA levels may be pivotal in regulating the transcriptional and translational capacity of high protein producing chondrocytes. This is interesting as in OA there is an imbalance between ECM protein anabolism and catabolism. To ensure continuous ECM deposition it is essential for a chondrocyte to control the number and quality of its ribosomes. We tested the hypothesis that the ribosome’s translational capacity alters with age and disease due to dysregulation of expression and function of specific snoRNAs; contributing to the development of the OA chondrocyte phenotype. Methods: Total RNA was extracted from human OA knee cartilage of young (n¼6; mean age ± SD 22.7 ± 4.1 years) normal and old n¼6; (66.4 ± 15.9 years) donors and hybridised onto Affymetrix miRNA 4.0 arrays. The probe set for Homo sapiens was used to determine differentially expressed snoRNAs. Relative ribosome number and chondrocyte marker gene expression was determined using qRT-PCR of 5.8,
18 and 28S rRNAs and of COL2A1, ACAN, SOX9, COL10A1, RUNX2, MMP13, ADAMTS5, COX-2, IL6, BAPX1 mRNAs. Total DNA content by SYBR Green detection and total protein was determined using BCA assay. Results: Normal samples correlated closely together, however OA samples clustered into three groups. When PCA was integrated with the Kellgren and Lawrence scores of OA donors the sub-populations were separated on OA severity. Analysis of the three subgroups identified 26 snoRNAs reduced in OA and 11 snoRNAs increased. These include 25 box C/D and 11 box H/ACA snoRNAs. To address the potential impact of aberrant snoRNAs expression on rRNA maturation, we determined the relative ribosome content in healthy and OA human articular chondorcytes (HAC). 18S and 5.8S rRNA (not 28S) levels were decreased in OA, together with a typical OA chondrocyte gene expression profile. Pre-rRNA levels were higher in OA, indicating aberrant pre-rRNA processing. In concert array results indicated that expression of U3 and U13 snoRNAs is deregulated in OA. In contrast to the majority of the snoRNAs these direct sitespecific endoribonucleolytic cleavage of pre-rRNA. To address a potential involvement of the inflammatory compound of OA, healthy HACs were exposed to IL1b. Similar to OA chondrocytes 18S and 5.8S rRNA decreased on exposure and pre-rRNA increased. To address the question whether the chondrocyte phenotype responds in an OAlike fashion as a result of alterations in the cell’s translation capacity we inhibited rRNA transcription using actinomycin D using normal HACs. 18S and 5.8S rRNA levels/cell were significantly downregulated whereas 28S rRNA levels remained unaffected. Due to ribosome depletion a reduction in total protein content/cell was observed, confirming functionally decreased translation capacity. The expression of RUNX2 and COL10A1 was upregulated, whereas COL2A1 expression was downregulated. Findings indicate that as a result of decreased chondrocyte ribosome content and translation capacity, chondrocytes phenotypically respond in an OA-like fashion. Conclusions:Since we found evidence for altered ribosome abundancy and auxiliary rRNA maturation machinery in ageing chondrocytes accompanied by differential OA cartilage-specific expression of snoRNAs, we believe that the translational capacity of the articular chondrocyte in OA is impaired, due to dysregulation of expression and function of specific snoRNAs, thereby contributing to the development of the OA chondrocyte phenotype. 613 IDENTIFICATION OF A NOVEL TARGET, FOXA2, IN THE ONSET AND DEVELOPMENT OF OSTEOARTHRITIS A.M. Ionescu y, L. Xu y, E. Kozhemyakina z, A. Lassar z, Y. Li y, K. Kaestner x, V. Rosen y. y HSDM, Boston, MA, USA; z HSM, Boston, MA, USA; x UPenn, Philadelphia, MA, USA Purpose: The proposed studies are anticipated to establish whether FoxA2 is a potential target for the treatment of OA. Methods: The following mouse lines were used: FoxA2flox/flox, FoxA3/, Prg4CreERt2-GFP and rtTA from Jax Labs. Results: In OA, irreversible degradation of the “permanent” articular cartilage by MMP13 is a key pathological event. Since I found that FoxA factors control MMP13 expression in skeletal development, it seemed possible they would to do the same in OA. Thus, we evaluated the expression of FoxA2 in both healthy and OA cartilage using FoxA2IRES-CreERT2/þ Tomato reporter mice. In healthy articular cartilage, the majority of cells positive for FoxA2 expression (red) are located on the tidemark (TM) or below, in the calcified zone of the articular cartilage (Fig.1). We also looked for co-localization of FoxA2 and MMP13 in articular cartilage by performing immunofluorescence for MMP13 on sections from FoxA2IRES-CreERT2/ þ;Tomato mice and found that FoxA2 positive cells (red) and MMP13 positive cells (green) overlap in the hypertrophic zone (overlay yellow) (Fig.1). Next, we looked at expression of FoxA2 in OA articular cartilage using the Destabilization of the Medial Meniscus (DMM) murine model of OA.