- Email: [email protected]
Deletion of frizzled-related protein reduces voluntary running exercise performance in mice
Osteoarthritis and Cartilage (2009) 17, 390e396 ª 2008 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.joca.2008.07.018
International Cartilage Repair Society
Deletion of frizzled-related protein reduces voluntary running exercise performance in mice R. J. U. Lories M.D., Ph.D.y*, J. Peeters B.Sc.y, K. Szlufcik Ph.D.z, P. Hespel Ph.D.z and F. P. Luyten M.D., Ph.D.y y Laboratory for Skeletal Development and Joint Disorders, Division of Rheumatology, Department of Musculoskeletal Sciences, Katholieke Universiteit Leuven, Leuven, Belgium z Research Centre for Exercise and Health, Department of Biomedical Kinesiology, Katholieke Universiteit Leuven, Leuven, Belgium Summary Objective: To study the effect of frizzled-related protein (Frzb) deletion in mice on voluntary running wheel exercise performance and osteoarthritis. Methods: At the age of 7 weeks, Frzb/ and wild-type mice were grouped and a running wheel was introduced into the cage. At week 8, all mice were caged solitarily with a running wheel available. Mice were allowed free exercise for 6e12 months and distances run were recorded daily. Non-running mice were used as additional control group. X-rays of knees and hips were taken at different time points. At the end of the experiment, mice were sacrificed and joints were processed for histological evaluation. Cartilage damage, synovitis and osteophyte formation were scored. Muscle fiber composition of the soleus and extensor digitorum longus was studied by immunofluorescence. Results: At the age of 6 months, both female and male wild-type mice showed a significantly greater exercise performance than the Frzb/ mice (P < 0.05). At 1 year, the difference was still significant for male mice, but not for females. Running exercise did not significantly affect severity of osteoarthritis. No statistical differences in osteoarthritis severity were seen between Frzb/ mice and wild-type mice. No differences were seen in muscle composition between Frzb/ mice and wild-type mice. Conclusion: Absence of Frzb in mice reduced voluntary exercise performance in running wheels. These experiments demonstrate that the effects of genes in mice can also be evaluated using functional outcomes such as running wheel exercise performance, similar to evolving practice in human clinical trials. ª 2008 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved. Key words: Osteoarthritis, Mouse model, Frizzled-related protein, Exercise.
related protein 5 and 6 co-receptors can lead to accumulation and nuclear translocation of beta-catenin. WNT signaling can also take place through the phosphatidyl-inositol/ Ca2þ or protein kinase C pathways, or via c-jun-amino-terminal kinase activation12e14. Accumulating evidence suggests a role for WNT biology in bone and cartilage of patients with osteoarthritis15e18. Increased WNT signaling is associated with cartilage damage and increased bone density18,10. By acting as a decoy receptor, FRZB can inhibit WNT signaling. We have generated Frzb/ mice10. These mice do not show apparent developmental abnormalities in the skeleton, but are characterized by increased cartilage damage in injury, instability or inflammation-triggered osteoarthritis of the knee10. These observations are associated with increased activation of WNT signaling and enhanced expression of tissue-destructive enzymes such as matrix metalloproteinase-3 (MMP3). In addition, FRZB inhibits MMP3 activity. Frzb/ mice also show increased cortical thickness and enhanced periosteal bone formation after mechanical stimulation in vivo leading to stiffer long bones10. The altered biomechanical properties of the long bones may not only contribute to the development and progression of osteoarthritis by influencing the cartilageebone
Introduction The osteoarthritic diseases are complex disorders to which both genetic and environmental factors contribute. Non-synonymous polymorphisms in the frizzled-related protein (FRZB) gene have been associated with hip osteoarthritis in females1. This genetic association has subsequently been confirmed in different cohorts of patients with various forms of osteoarthritis, including a differential association between severe osteoarthritis and osteoporotic fractures of the hip2e6. FRZB, originally identified from a chondrogenic extract of bovine articular cartilage7, is a secreted antagonist of the wingless-type (WNT) signaling pathway8e10. The WNT family forms a group of at least 19 secreted glycoproteins that affect cell proliferation, differentiation and behavior during development, growth, homeostasis and disease (for review see Ref. 11). WNT ligands can signal through at least three different signaling cascades. Interaction between WNTs, frizzled receptors and lipoprotein *Address correspondence and reprint requests to: Dr Rik Lories, M.D., Ph.D., Division of Rheumatology, University Hospitals Leuven, Herestraat 49, B-3000 Leuven, Belgium. Tel: 32-16342542; Fax: 32-16-342543; E-mail: [email protected] Received 2 August 2007; revision accepted 24 July 2008.
390
391
Osteoarthritis and Cartilage Vol. 17, No. 3
Fig. 1. Running wheel exercise performance in Frzb/ and wild-type mice. (A) Median daily distance ran at the age of 6 months in male and female mice. (B) Median daily distance ran at the age of 1 year in male and female mice. (C) Mean monthly distance ran in male Frzb/ and wild-type mice. (D) Mean monthly distance ran in female Frzb/ and wild-type mice (*indicates P < 0.05 in Frzb/ vs wild-type mice; # indicates P < 0.05 in male vs female mice; n ¼ 10e13 mice at 6 months and n ¼ 5e7 at 1 year).
biomechanical unit, but may also provide protection against osteoporotic hip fracture indicating a role for FRZB as a molecular basis for the observed and hypothesized inverse relationship between osteoarthritis and osteoporosis19. Chemically or surgically induced models of osteoarthritis in rodents bear the important disadvantage that cartilage damage is caused by direct injury in a rapidly evolving process with dynamics that are very different from what is usually seen in human osteoarthritis. In contrast, spontaneous development of osteoarthritis upon aging in genetic mouse models is generally mild, in particular when genes are involved that do not code for structural components of the articular cartilage. The complex biology of the ‘‘joint organ’’ make it less likely that inactivation of a non-structural gene involved in osteoarthritis will lead to major phenotypes in mice upon aging. In addition, disease development and progression are known to be influenced by other factors such as weight, mobility and
exercise20. Therefore, functional outcome measurements such as walking distance are increasingly important in the assessment, study and understanding of human disease. With this in mind, we measured distances covered in a running wheel over a 6-month to 1-year period to evaluate voluntary walking performance and natural development of osteoarthritis in Frzb/ mice.
Materials and methods ANIMAL EXPERIMENTS Frzb/ mice were generated as previously described by targeted deletion of the first exon10. The mice were kept in a CD1/Swiss genetic background. Frzb/ and wild-type mice were initiated to running wheel exercise at the age of 7 weeks by group training. From the age of 8 weeks onwards, mice were caged solitarily in the presence of a custom-made running wheel and allowed to run freely. Distances ran by the mice were
Table I Weight of mice using the running wheel vs control mice (grams) 6-Month-old mice Wild-type mice
Males Females
1-Year old mice Frzb/ mice
Wild-type mice
Frzb/ mice
Runners
Controls
Runners
Controls
Runners
Controls
Runners
Controls
42.71 0.76 31.72 0.74
42.02 1.58 36.82 1.32*
42.12 1.12 33.89 1.09
43.00 0.98 35.73 1.98
40.53 1.21 39.28 1.47
46.42 2.28 42.57 2.01
44.24 2.18 39.23 1.32
49.59 1.93 44.96 3.02
*P < 0.001 runners vs controls; data are presented as mean standard error.
392
R. J. U. Lories et al.: Frzb deletion affects running performance
recorded daily and average distances covered per day were calculated for the different time periods. All experiments were approved by the Ethics Committee for Animal Research (Katholieke Universiteit Leuven, Belgium).
HISTOMORPHOLOGICAL ANALYSIS OF THE JOINT At the indicated time points (26 and 52 weeks) mice were sacrificed. Knees and hips were dissected and fixed in 2% paraformaldehyde/phosphate buffered saline (PBS) overnight. Subsequently, they were decalcified in 0.5 M ethylenediaminetetraacetic acid (EDTA) in PBS, pH 7.5 for 10 days. Five paraffin embedded coronal sections (100 mm apart) of each knee and hip were stained with hematoxylin/eosin or safranin O/hematoxylin. Cartilage damage was scored using the Osteoarthritis Research Society International (OARSI) score system21. In addition, synovitis was scored taking into account different features: exsudate, hyperplasia of the synovial lining, sublining zone infiltration and villus formation. Each of the features was scored as 0 (absent), 1 (mild), 2 (clearly present), 3 (extensive). Presence or absence of osteophytes was also evaluated. Scores were attributed based on an evaluation of the whole joint.
myosin type IIa (1:20 dilution; N2.261 supernatant, Developmental Studies Hybridoma Bank, IA, USA) and slow myosin type I (1:20 dilution; A4.840 supernatant, Developmental Studies Hybridoma Bank) or isotype control antibodies (mouse IgG1 and IgM (Santa Cruz Biotechnologies, Santa Cruz, CA, USA). Thereafter, slides were washed three times and incubated for 60 min with wash buffer containing the secondary antibodies Alexa Fluor 350 anti-mouse IgG1 (Molecular Probes, Leiden, The Netherlands) and Fluorescein isothiocyanate (FITC) anti-mouse IgM (Southern Biotechnology Associates, Birmingham, AL, USA), each in a 1:500 dilution. Cover slips were mounted with Fluorescent Mounting Medium (DakoCytomation, Carpinteria, CA, USA). Two to three digital images were taken from each section with a Nikon Eclipse E1000 microscope (Nikon, Boerhavedorp, Germany) at a 20 magnification with appropriate band-pass filters for Alexa Fluor 350 and FITC. Morphometric analysis of double-labeled immunofluorescent images was performed with Lucia G Software (LIM, Prague, Czech Republic). Type I fibers were identified by green staining, type IIa fibers by intense blue staining and type IIb fibers by the absence of intense blue staining). At least 10 digital images were analyzed per muscle.
STATISTICS HISTOMORPHOLOGICAL ANALYSIS OF THE MUSCLE Muscle fiber typing was performed by means of immunofluorescent staining as previously described22 with specific anti-type-I and anti-type IIa myosin antibodies and digital morphometric analysis. Transverse sections (10 mm) were cut from the mid-belly area of soleus and Extensor digitorum longus (EDL) muscles at 20 C, brought to room temperature and fixed in PBS containing 4% paraformaldehyde and 0.1% Triton X-100 for 10 min and rinsed for 2 5 min in wash buffer (0.5% bovine serum albumin in PBS). Then, sections were incubated for 30 min in 10 mM NH4Cl, rinsed for 2 5 min in wash buffer and incubated in PBS containing 1% bovine serum albumin for 30 min. Sections were incubated overnight at 4 C in wash buffer containing two primary monoclonal antibodies directed against fast
Data were analyzed using either two-sided Student’s t test for unpaired samples or ManneWhitney U test when normal distribution of the data could not be assumed. For correlations, Spearman Rank non-parametric test was used.
Results REDUCED RUNNING PERFORMANCE IN FRZB/ MICE
In male mice, median daily distance ran was significantly lower in Frzb/ mice as compared to wild-type mice at the
Fig. 2. Histomorphology of the knee joint in a wild-type mouse after 1 year of free running. (A) Overview of the knee anatomy on the coronal section. Patella, femur and tibia are labeled. Arrows indicated zones of synovial hyperplasia. Two boxes highlight zones presented more in detail in (B) and (C) (magnification 25, safranin O/hematoxylin staining). In (B) damage to the superficial layer and loss of proteoglycan staining in the deeper layer of the articular cartilage is shown. Arrows indicate zones of proteoglycan loss, white arrowheads point toward synovitis and black arrowheads show synovial fibrosis. (C) Osteophyte formation indicated by arrowheads. Arrows indicate synovitis.
Osteoarthritis and Cartilage Vol. 17, No. 3 age of 6 months (5.1 km/day vs 7.3 km/day, ManneWhitney U test, P < 0.032, n ¼ 10 vs 12 mice) [Fig. 1(A)] and at the age of 1 year (3.3 km/day vs 7.7 km/day, ManneWhitney U test, P < 0.008, n ¼ 5 vs 6 mice) [Fig. 1(B)]. In female mice, median daily distance ran was significantly lower in Frzb/
393 mice as compared to wild-type mice at the age of 6 months (8.8 km/day vs 11.3 km/day, ManneWhitney U test, P < 0.042, n ¼ 11 vs 13 mice) [Fig. 1(A)]. At the age of 1 year the difference between female Frzb/ mice and wild-type mice was not significant (6.76 km/day vs
Fig. 3. X-ray analysis of osteoarthritis in running and control mice. (A) Representative images of the different scores used. All images are from wild-type mice. No specific features were found in Frzb/ mice. (B) X-ray scores in running and control male mice (n ¼ 10 control mice in each group; n ¼ 4 Frzb/ and 6 wild-type mice in the running group; both knees were scored for each mouse). (C) X-ray scores in running and control female mice (n ¼ 9 Frzb/ and 10 wild-type control mice; n ¼ 7 Frzb/ and 4 wild-type mice in the running group; both knees were scored for each mouse).
394
R. J. U. Lories et al.: Frzb deletion affects running performance
9.57 km/day, ManneWhitney U test, P > 0.05, n ¼ 7 vs 6 mice) [Fig. 1(B)]. Of note, running performance was significantly better in female mice than in male mice both in wildtype animals (ManneWhitney U test, P < 0.001 at 6 months, n ¼ 13 vs 12; P > 0.05 at 1 year, n ¼ 6 per group) and in Frzb/ mice (ManneWhitney U test, P < 0.024 at 6 months, n ¼ 11 vs 10; P < 0.03 at 1 year, n ¼ 7 vs 5). In male mice, differences between Frzb/ and wild-type mice were not apparent in the first month of running wheel exercise, but appeared quickly thereafter [Fig. 1(C)]. In contrast, in female mice, differences in running performance were already present in the first month [Fig. 1(D)]. Overall, running performance declined from the age of 8 to 9 months onwards. This effect appeared more outspoken in female than in male mice. The observed differences were not explained by differences in weight between Frzb/ and wild-type mice (Table I). However, treadmill activity did result in consistently lower mean weight of the mice as compared to non-running controls (Table I). This difference was only significant for female wild-type mice at the age of 6 months (Student’s t test P < 0.001). OSTEOARTHRITIS IN RUNNING MICE
We studied the presence of osteoarthritis-like changes in the knee and hip joints of running mice and agematched controls (Fig. 2). X-ray analysis at 12 months [Fig. 3(A)] did not show a significant increase or decrease in osteoarthritis severity scores between running mice and controls although scores tended to be higher in the exercise group [Fig. 3(B, C)] (median score 1 vs 1.5 in male wild-type, 1 vs 2 in female wild-type, 0.5 vs 1.5 in male Frzb/ mice, 0 vs 2 in female Frzb/ mice, ManneWhitney U test, P > 0.05, n ¼ 10 vs 6, 10 vs 4, 10 vs 4 and 9 vs 7 mice, respectively, with both knees evaluated for each mouse). Histomorphological scores for cartilage damage after voluntary exercise were not different between wildtype and Frzb/ mice in males or females (median 2.5 vs 2 and 2 vs 2.5, respectively, ManneWhitney U test, P > 0.05, n ¼ 12 vs 12 knees from 6 males per group and 8 vs 14 knees from 4 wild-type and 7 Frzb/ females) [Fig. 4(A)]. Although all cartilages of the knee joint could be affected, the medial tibial cartilage was most often and most severely involved. No significant differences were found between both groups in features of synovitis (ManneWhitney U test, P > 0.05) [Fig. 4(B, C)]. X-ray scores, cartilage damage and synovitis scores did not correlate with distances ran in the different groups (data not shown). Osteophyte formation was not different between both groups. No signs of osteoarthritis were seen in the hip joints (data not shown). MUSCLE COMPOSITION IN FRZB/ AND WILD-TYPE MICE
As differences in voluntary running performance were not explained by severity of osteoarthritis, we additionally checked whether the observed differences could be explained by the effect of Frzb deletion on muscle fiber composition in 8e12-week old mice [Fig. 5(A, B)]. As shown in Fig. 5(C, D), similar type I and type IIa muscle fiber surface and percentage were found in the soleus muscle of Frzb/ as compared to wild-type mice. Also type IIa and type IIb fiber surface and percentage were similar in the extensor digitorum longus muscle of Frzb/ and wild-type mice [Fig. 5(C, D)] (n ¼ 3 vs 3 and 3 vs 2 for male mice and 4 vs 3 for female mice).
Fig. 4. Histomorphological signs of osteoarthritis in Frzb/ and wild-type mice. (A) Cartilage damage scores and (B and C) synovitis scores in males (B) and females (C) after 1 year of running exercise. Data are shown as box-plots with medians, quartile and percentile 10e90 (n ¼ 12 vs 12 knees from 6 males per group and 8 vs 14 knees from 4 wild-type and 7 Frzb/ females).
Discussion FRZB was identified as a susceptibility gene for osteoarthritis1e6. We recently demonstrated that genetic deletion of Frzb in mice results in increased cartilage damage in
Osteoarthritis and Cartilage Vol. 17, No. 3 different induced models of osteoarthritis10. In addition, the knockout mice have a higher cortical thickness, bone density and mineral content as well as enhanced periosteal bone formation upon mechanical stimulation. This results in stiffer long bones. A triple mechanism for the role of FRZB in osteoarthritis susceptibility has been proposed: increased WNT activity with up-regulation of tissue-destructive enzymes, higher MMP activity due to defective antagonism, and enhanced strain in the boneecartilage biomechanical unit10. In this study, we evaluated the performance of Frzb/ and wild-type mice in running wheels over a 1-year period. Running exercise has previously been used to study osteoarthritis in rodents with conflicting results23e26. However, we find this experimental model of particular interest as its primary analysis mimics a clinical outcome that is also relevant in humans. Of interest, voluntary running exercise did not specifically protect against or increase osteoarthritis in this study. Genes identified to be of importance in osteoarthritis, can be divided into two categories: those that are predominantly contributing to the integrity of the articular cartilage structure and those that play a role in cartilage and bone biology. In genetic mouse models, the former group of genes such as the type II collagen gene27,28 will often show defects in skeletal development29. Recognizing the importance of other, potentially more clinically and therapeutically relevant genes, may be more difficult as the development and progression of osteoarthritis are clearly multifaceted. Therefore, the combination of induced models and the analysis of voluntary running performance as presented here as outcome parameter in long-term studies may prove very useful to highlight the role of specific genes for which genetic associations have been reported in this disease.
395 Despite X-ray and some histomorphological scores for features of osteoarthritis appearing somewhat higher in Frzb/ than in wild-type mice, these differences were not statistically significant. More interestingly, these scores did not appear to correlate with distances covered. Although the relatively small number of animals used in the study could account for these observations, it is clear that the model used also bears some potential drawbacks. It should be noted that the running mice did not develop apparent hip osteoarthritis. This is another limitation of the running wheel model as weight distribution and stress in the joint are likely to be different between animals that walk on four limbs and humans. However, the in vivo and long-term experimental study as presented here can be considered as an inclusive model to which different factors contribute. This bears the consequence that other factors not directly relevant to cartilage and bone biology may contribute to the observed phenotype. In particular, the neurological deficit reported in another strain of Frzb KO mice may be relevant here30. No differences in muscle composition were detected upon screening between the transgenic and wild-type strains. In view of the human disease, however, this apparent lack of change in the joint itself should not necessarily be considered a disadvantage as the development of osteoarthritis is probably also reaching beyond the borders of skeletal biology. Of interest, female mice showed a higher activity in the running wheels than their male counterparts. However, this was again not explained by differences in osteoarthritis development. The difference in weight may partially explain this observation. Of interest, the original association of polymorphisms in the FRZB gene with hip osteoarthritis was reported in females1. This gender specific association was
Fig. 5. Analysis of muscle fiber composition in Frzb/ and wild-type mice. (A) Immunofluorescent staining for type I (green) and type IIa (blue) fibers in the soleus muscle of wild-type mice and isotype control. (B) Immunofluorescent staining for type IIa (blue) and type IIb (background darkblue) in the extensor digitorum longus muscle of wild-type mice and isotype controls. (C) Muscle fiber surface in Frzb/ and wild-type male and female mice. (D) Number of muscle fibers in Frzb/ and wild-type male and female mice (individual data points are shown; dark squares represent Frzb/ and open circles represent wild-type mice; n ¼ 3 vs 3 and 3 vs 2 for male mice and 4 vs 3 for female mice; per mouse muscles from the right and left paw were analyzed).
396
R. J. U. Lories et al.: Frzb deletion affects running performance
confirmed as a risk factor for hip osteoarthritis in a subsequent study2. Further evidence suggests a female specific effect in knee osteoarthritis6. In contrast, other data suggest that the effect of FRZB may not be gender or hip specific3. In conclusion, this study supports the genetic association of FRZB with osteoarthritis and confirms our earlier observations using short-term induced models of the disease10. FRZB and WNT biology can therefore be considered as therapeutic targets for the treatment of osteoarthritis.
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgements The authors would like to thank Bea Weynants and Inge Derese for technical assistance with this study. Rik Lories is the recipient of a post-doctoral fellowship from the Flanders Research Foundation (FWO Vlaanderen). This work was supported by a GOA grant from KU Leuven and a FWO research grant G.0452.05. FWO Vlaanderen was not involved in data acquisition, manuscript writing or submission. References 1. Loughlin J, Dowling B, Chapman K, Marcelline L, Mustafa Z, Southam L, et al. Functional variants within the secreted frizzled-related protein 3 gene are associated with hip osteoarthritis in females. Proc Natl Acad Sci U S A 2004;101:9757e62. 2. Lane NE, Lian K, Nevitt MC, Zmuda JM, Lui L, Li J, et al. Frizzled-related protein variants are risk factors for hip osteoarthritis. Arthritis Rheum 2006;54:1246e54. 3. Min JL, Meulenbelt I, Riyazi N, Kloppenburg M, HouwingDuistermaat JJ, Seymour AB, et al. Association of the frizzled-related protein gene with symptomatic osteoarthritis at multiple sites. Arthritis Rheum 2005;52:1077e80. 4. Lories RJ, Boonen S, Peeters J, De Vlam K, Luyten FP. Evidence for a differential association of the Arg200Trp single-nucleotide polymorphism in FRZB with hip osteoarthritis and osteoporosis. Rheumatology (Oxford) 2006;45:113e4. 5. Rodriguez-Lopez J, Pombo-Suarez M, Liz M, Gomez-Reino JJ, Gonzalez A. Further evidence of the role of frizzled-related protein gene polymorphisms in osteoarthritis. Ann Rheum Dis 2007;66(8): 1052e5. 6. Valdes AM, Loughlin J, Oene MV, Chapman K, Surdulescu GL, Doherty M, et al. Sex and ethnic differences in the association of ASPN, CALM1, COL2A1, COMP, and FRZB with genetic susceptibility to osteoarthritis of the knee. Arthritis Rheum 2007;56:137e46. 7. Hoang B, Moos Jr M, Vukicevic S, Luyten FP. Primary structure and tissue distribution of FRZB, a novel protein related to Drosophila frizzled, suggest a role in skeletal morphogenesis. J Biol Chem 1996;271: 26131e7. 8. Lin K, Wang S, Julius MA, Kitajewski J, Moos Jr M, Luyten FP. The cysteine-rich frizzled domain of Frzb-1 is required and sufficient for modulation of Wnt signaling. Proc Natl Acad Sci U S A 1997;94: 11196e200.
9. Wang S, Krinks M, Lin K, Luyten FP, Moos Jr M. Frzb, a secreted protein expressed in the Spemann organizer, binds and inhibits Wnt-8. Cell 1997;88:757e66. 10. Lories RJ, Peeters J, Bakker A, Derese I, Tylzanowski P, Schrooten J, et al. Articular cartilage and biomechanical properties of the long bones in Frzb-knockout mice. Arthritis Rheum 2007;56:4095e103. 11. Clevers H. Wnt/beta-catenin signaling in development and disease. Cell 2006;127:469e80. 12. Slusarski DC, YangSnyder J, Busa WB, Moon RT. Modulation of embryonic intracellular Ca2þ signaling by Wnt-5A. Dev Biol 1997;182:114e20. 13. Kuhl M, Sheldahl LC, Park M, Miller JR, Moon RT. The Wnt/Ca2þ pathway: a new vertebrate Wnt signaling pathway takes shape. Trends Genet 2000;16:279e83. 14. Tada M, Concha ML, Heisenberg CP. Non-canonical Wnt signalling and regulation of gastrulation movements. Semin Cell Dev Biol 2002;13: 251e60. 15. Dell’Accio F, De Bari C, El Tawil NM, Barone F, Mitsiadis TA, O’dowd J, et al. Activation of WNT and BMP signaling in adult human articular cartilage following mechanical injury. Arthritis Res Ther 2006;8:R139. 16. Nakamura Y, Nawata M, Wakitani S. Expression profiles and functional analyses of Wnt-related genes in human joint disorders. Am J Pathol 2005;167:97e105. 17. Westendorf JJ, Kahler RA, Schroeder TM. Wnt signaling in osteoblasts and bone diseases. Gene 2004;341:19e39. 18. Krishnan V, Bryant HU, Macdougald OA. Regulation of bone mass by Wnt signaling. J Clin Invest 2006;116:1202e9. 19. Dequeker J, Aerssens J, Luyten FP. Osteoarthritis and osteoporosis: clinical and research evidence of inverse relationship. Aging Clin Exp Res 2003;15:426e39. 20. Bierma-Zeinstra SM, Koes BW. Risk factors and prognostic factors of hip and knee osteoarthritis. Nat Clin Pract Rheumatol 2007;3:78e85. 21. Pritzker KP, Gay S, Jimenez SA, Ostergaard K, Pelletier JP, Revell PA, et al. Osteoarthritis cartilage histopathology: grading and staging. Osteoarthritis Cartilage 2006;14:13e29. 22. Derave W, Eijnde BO, Ramaekers M, Hespel P. No effects of lifelong creatine supplementation on sarcopenia in senescence-accelerated mice (SAMP8). Am J Physiol Endocrinol Metab 2005;289:E272e7. 23. Otterness IG, Eskra JD, Bliven ML, Shay AK, Pelletier JP, Milici AJ. Exercise protects against articular cartilage degeneration in the hamster. Arthritis Rheum 1998;41:2068e76. 24. Lapvetelainen T, Nevalainen T, Parkkinen JJ, Arokoski J, Kiraly K, Hyttinen M, et al. Lifelong moderate running training increases the incidence and severity of osteoarthritis in the knee joint of C57BL mice. Anat Rec 1995;242:159e65. 25. Pap G, Eberhardt R, Sturmer I, Machner A, Schwarzberg H, Roessner A, et al. Development of osteoarthritis in the knee joints of Wistar rats after strenuous running exercise in a running wheel by intracranial self-stimulation. Pathol Res Pract 1998;194:41e7. 26. Lapvetelainen T, Hyttinen M, Lindblom J, Langsjo TK, Sironen R, Li SW, et al. More knee joint osteoarthritis (OA) in mice after inactivation of one allele of type II procollagen gene but less OA after lifelong voluntary wheel running exercise. Osteoarthritis Cartilage 2001;9:152e60. 27. Meulenbelt I, Bijkerk C, De Wildt SC, Miedema HS, Breedveld FC, Pols HA, et al. Haplotype analysis of three polymorphisms of the COL2A1 gene and associations with generalised radiological osteoarthritis. Ann Hum Genet 1999;63:393e400. 28. Ikeda T, Mabuchi A, Fukuda A, Kawakami A, Ryo Y, Yamamoto S, et al. Association analysis of single nucleotide polymorphisms in cartilagespecific collagen genes with knee and hip osteoarthritis in the Japanese population. J Bone Miner Res 2002;17:1290e6. 29. Li SW, Prockop DJ, Helminen H, Fassler R, Lapvetelainen T, Kiraly K, et al. Transgenic mice with targeted inactivation of the Col2 alpha 1 gene for collagen II develop a skeleton with membranous and periosteal bone but no endochondral bone. Genes Dev 1995;9:2821e30. 30. Mouse Genome Informatics. Frzbtm1Dgen Phenotypic Allele Detail. Mouse Genome Informatics; 2006.
International Cartilage Repair Society
Deletion of frizzled-related protein reduces voluntary running exercise performance in mice R. J. U. Lories M.D., Ph.D.y*, J. Peeters B.Sc.y, K. Szlufcik Ph.D.z, P. Hespel Ph.D.z and F. P. Luyten M.D., Ph.D.y y Laboratory for Skeletal Development and Joint Disorders, Division of Rheumatology, Department of Musculoskeletal Sciences, Katholieke Universiteit Leuven, Leuven, Belgium z Research Centre for Exercise and Health, Department of Biomedical Kinesiology, Katholieke Universiteit Leuven, Leuven, Belgium Summary Objective: To study the effect of frizzled-related protein (Frzb) deletion in mice on voluntary running wheel exercise performance and osteoarthritis. Methods: At the age of 7 weeks, Frzb/ and wild-type mice were grouped and a running wheel was introduced into the cage. At week 8, all mice were caged solitarily with a running wheel available. Mice were allowed free exercise for 6e12 months and distances run were recorded daily. Non-running mice were used as additional control group. X-rays of knees and hips were taken at different time points. At the end of the experiment, mice were sacrificed and joints were processed for histological evaluation. Cartilage damage, synovitis and osteophyte formation were scored. Muscle fiber composition of the soleus and extensor digitorum longus was studied by immunofluorescence. Results: At the age of 6 months, both female and male wild-type mice showed a significantly greater exercise performance than the Frzb/ mice (P < 0.05). At 1 year, the difference was still significant for male mice, but not for females. Running exercise did not significantly affect severity of osteoarthritis. No statistical differences in osteoarthritis severity were seen between Frzb/ mice and wild-type mice. No differences were seen in muscle composition between Frzb/ mice and wild-type mice. Conclusion: Absence of Frzb in mice reduced voluntary exercise performance in running wheels. These experiments demonstrate that the effects of genes in mice can also be evaluated using functional outcomes such as running wheel exercise performance, similar to evolving practice in human clinical trials. ª 2008 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved. Key words: Osteoarthritis, Mouse model, Frizzled-related protein, Exercise.
related protein 5 and 6 co-receptors can lead to accumulation and nuclear translocation of beta-catenin. WNT signaling can also take place through the phosphatidyl-inositol/ Ca2þ or protein kinase C pathways, or via c-jun-amino-terminal kinase activation12e14. Accumulating evidence suggests a role for WNT biology in bone and cartilage of patients with osteoarthritis15e18. Increased WNT signaling is associated with cartilage damage and increased bone density18,10. By acting as a decoy receptor, FRZB can inhibit WNT signaling. We have generated Frzb/ mice10. These mice do not show apparent developmental abnormalities in the skeleton, but are characterized by increased cartilage damage in injury, instability or inflammation-triggered osteoarthritis of the knee10. These observations are associated with increased activation of WNT signaling and enhanced expression of tissue-destructive enzymes such as matrix metalloproteinase-3 (MMP3). In addition, FRZB inhibits MMP3 activity. Frzb/ mice also show increased cortical thickness and enhanced periosteal bone formation after mechanical stimulation in vivo leading to stiffer long bones10. The altered biomechanical properties of the long bones may not only contribute to the development and progression of osteoarthritis by influencing the cartilageebone
Introduction The osteoarthritic diseases are complex disorders to which both genetic and environmental factors contribute. Non-synonymous polymorphisms in the frizzled-related protein (FRZB) gene have been associated with hip osteoarthritis in females1. This genetic association has subsequently been confirmed in different cohorts of patients with various forms of osteoarthritis, including a differential association between severe osteoarthritis and osteoporotic fractures of the hip2e6. FRZB, originally identified from a chondrogenic extract of bovine articular cartilage7, is a secreted antagonist of the wingless-type (WNT) signaling pathway8e10. The WNT family forms a group of at least 19 secreted glycoproteins that affect cell proliferation, differentiation and behavior during development, growth, homeostasis and disease (for review see Ref. 11). WNT ligands can signal through at least three different signaling cascades. Interaction between WNTs, frizzled receptors and lipoprotein *Address correspondence and reprint requests to: Dr Rik Lories, M.D., Ph.D., Division of Rheumatology, University Hospitals Leuven, Herestraat 49, B-3000 Leuven, Belgium. Tel: 32-16342542; Fax: 32-16-342543; E-mail: [email protected] Received 2 August 2007; revision accepted 24 July 2008.
390
391
Osteoarthritis and Cartilage Vol. 17, No. 3
Fig. 1. Running wheel exercise performance in Frzb/ and wild-type mice. (A) Median daily distance ran at the age of 6 months in male and female mice. (B) Median daily distance ran at the age of 1 year in male and female mice. (C) Mean monthly distance ran in male Frzb/ and wild-type mice. (D) Mean monthly distance ran in female Frzb/ and wild-type mice (*indicates P < 0.05 in Frzb/ vs wild-type mice; # indicates P < 0.05 in male vs female mice; n ¼ 10e13 mice at 6 months and n ¼ 5e7 at 1 year).
biomechanical unit, but may also provide protection against osteoporotic hip fracture indicating a role for FRZB as a molecular basis for the observed and hypothesized inverse relationship between osteoarthritis and osteoporosis19. Chemically or surgically induced models of osteoarthritis in rodents bear the important disadvantage that cartilage damage is caused by direct injury in a rapidly evolving process with dynamics that are very different from what is usually seen in human osteoarthritis. In contrast, spontaneous development of osteoarthritis upon aging in genetic mouse models is generally mild, in particular when genes are involved that do not code for structural components of the articular cartilage. The complex biology of the ‘‘joint organ’’ make it less likely that inactivation of a non-structural gene involved in osteoarthritis will lead to major phenotypes in mice upon aging. In addition, disease development and progression are known to be influenced by other factors such as weight, mobility and
exercise20. Therefore, functional outcome measurements such as walking distance are increasingly important in the assessment, study and understanding of human disease. With this in mind, we measured distances covered in a running wheel over a 6-month to 1-year period to evaluate voluntary walking performance and natural development of osteoarthritis in Frzb/ mice.
Materials and methods ANIMAL EXPERIMENTS Frzb/ mice were generated as previously described by targeted deletion of the first exon10. The mice were kept in a CD1/Swiss genetic background. Frzb/ and wild-type mice were initiated to running wheel exercise at the age of 7 weeks by group training. From the age of 8 weeks onwards, mice were caged solitarily in the presence of a custom-made running wheel and allowed to run freely. Distances ran by the mice were
Table I Weight of mice using the running wheel vs control mice (grams) 6-Month-old mice Wild-type mice
Males Females
1-Year old mice Frzb/ mice
Wild-type mice
Frzb/ mice
Runners
Controls
Runners
Controls
Runners
Controls
Runners
Controls
42.71 0.76 31.72 0.74
42.02 1.58 36.82 1.32*
42.12 1.12 33.89 1.09
43.00 0.98 35.73 1.98
40.53 1.21 39.28 1.47
46.42 2.28 42.57 2.01
44.24 2.18 39.23 1.32
49.59 1.93 44.96 3.02
*P < 0.001 runners vs controls; data are presented as mean standard error.
392
R. J. U. Lories et al.: Frzb deletion affects running performance
recorded daily and average distances covered per day were calculated for the different time periods. All experiments were approved by the Ethics Committee for Animal Research (Katholieke Universiteit Leuven, Belgium).
HISTOMORPHOLOGICAL ANALYSIS OF THE JOINT At the indicated time points (26 and 52 weeks) mice were sacrificed. Knees and hips were dissected and fixed in 2% paraformaldehyde/phosphate buffered saline (PBS) overnight. Subsequently, they were decalcified in 0.5 M ethylenediaminetetraacetic acid (EDTA) in PBS, pH 7.5 for 10 days. Five paraffin embedded coronal sections (100 mm apart) of each knee and hip were stained with hematoxylin/eosin or safranin O/hematoxylin. Cartilage damage was scored using the Osteoarthritis Research Society International (OARSI) score system21. In addition, synovitis was scored taking into account different features: exsudate, hyperplasia of the synovial lining, sublining zone infiltration and villus formation. Each of the features was scored as 0 (absent), 1 (mild), 2 (clearly present), 3 (extensive). Presence or absence of osteophytes was also evaluated. Scores were attributed based on an evaluation of the whole joint.
myosin type IIa (1:20 dilution; N2.261 supernatant, Developmental Studies Hybridoma Bank, IA, USA) and slow myosin type I (1:20 dilution; A4.840 supernatant, Developmental Studies Hybridoma Bank) or isotype control antibodies (mouse IgG1 and IgM (Santa Cruz Biotechnologies, Santa Cruz, CA, USA). Thereafter, slides were washed three times and incubated for 60 min with wash buffer containing the secondary antibodies Alexa Fluor 350 anti-mouse IgG1 (Molecular Probes, Leiden, The Netherlands) and Fluorescein isothiocyanate (FITC) anti-mouse IgM (Southern Biotechnology Associates, Birmingham, AL, USA), each in a 1:500 dilution. Cover slips were mounted with Fluorescent Mounting Medium (DakoCytomation, Carpinteria, CA, USA). Two to three digital images were taken from each section with a Nikon Eclipse E1000 microscope (Nikon, Boerhavedorp, Germany) at a 20 magnification with appropriate band-pass filters for Alexa Fluor 350 and FITC. Morphometric analysis of double-labeled immunofluorescent images was performed with Lucia G Software (LIM, Prague, Czech Republic). Type I fibers were identified by green staining, type IIa fibers by intense blue staining and type IIb fibers by the absence of intense blue staining). At least 10 digital images were analyzed per muscle.
STATISTICS HISTOMORPHOLOGICAL ANALYSIS OF THE MUSCLE Muscle fiber typing was performed by means of immunofluorescent staining as previously described22 with specific anti-type-I and anti-type IIa myosin antibodies and digital morphometric analysis. Transverse sections (10 mm) were cut from the mid-belly area of soleus and Extensor digitorum longus (EDL) muscles at 20 C, brought to room temperature and fixed in PBS containing 4% paraformaldehyde and 0.1% Triton X-100 for 10 min and rinsed for 2 5 min in wash buffer (0.5% bovine serum albumin in PBS). Then, sections were incubated for 30 min in 10 mM NH4Cl, rinsed for 2 5 min in wash buffer and incubated in PBS containing 1% bovine serum albumin for 30 min. Sections were incubated overnight at 4 C in wash buffer containing two primary monoclonal antibodies directed against fast
Data were analyzed using either two-sided Student’s t test for unpaired samples or ManneWhitney U test when normal distribution of the data could not be assumed. For correlations, Spearman Rank non-parametric test was used.
Results REDUCED RUNNING PERFORMANCE IN FRZB/ MICE
In male mice, median daily distance ran was significantly lower in Frzb/ mice as compared to wild-type mice at the
Fig. 2. Histomorphology of the knee joint in a wild-type mouse after 1 year of free running. (A) Overview of the knee anatomy on the coronal section. Patella, femur and tibia are labeled. Arrows indicated zones of synovial hyperplasia. Two boxes highlight zones presented more in detail in (B) and (C) (magnification 25, safranin O/hematoxylin staining). In (B) damage to the superficial layer and loss of proteoglycan staining in the deeper layer of the articular cartilage is shown. Arrows indicate zones of proteoglycan loss, white arrowheads point toward synovitis and black arrowheads show synovial fibrosis. (C) Osteophyte formation indicated by arrowheads. Arrows indicate synovitis.
Osteoarthritis and Cartilage Vol. 17, No. 3 age of 6 months (5.1 km/day vs 7.3 km/day, ManneWhitney U test, P < 0.032, n ¼ 10 vs 12 mice) [Fig. 1(A)] and at the age of 1 year (3.3 km/day vs 7.7 km/day, ManneWhitney U test, P < 0.008, n ¼ 5 vs 6 mice) [Fig. 1(B)]. In female mice, median daily distance ran was significantly lower in Frzb/
393 mice as compared to wild-type mice at the age of 6 months (8.8 km/day vs 11.3 km/day, ManneWhitney U test, P < 0.042, n ¼ 11 vs 13 mice) [Fig. 1(A)]. At the age of 1 year the difference between female Frzb/ mice and wild-type mice was not significant (6.76 km/day vs
Fig. 3. X-ray analysis of osteoarthritis in running and control mice. (A) Representative images of the different scores used. All images are from wild-type mice. No specific features were found in Frzb/ mice. (B) X-ray scores in running and control male mice (n ¼ 10 control mice in each group; n ¼ 4 Frzb/ and 6 wild-type mice in the running group; both knees were scored for each mouse). (C) X-ray scores in running and control female mice (n ¼ 9 Frzb/ and 10 wild-type control mice; n ¼ 7 Frzb/ and 4 wild-type mice in the running group; both knees were scored for each mouse).
394
R. J. U. Lories et al.: Frzb deletion affects running performance
9.57 km/day, ManneWhitney U test, P > 0.05, n ¼ 7 vs 6 mice) [Fig. 1(B)]. Of note, running performance was significantly better in female mice than in male mice both in wildtype animals (ManneWhitney U test, P < 0.001 at 6 months, n ¼ 13 vs 12; P > 0.05 at 1 year, n ¼ 6 per group) and in Frzb/ mice (ManneWhitney U test, P < 0.024 at 6 months, n ¼ 11 vs 10; P < 0.03 at 1 year, n ¼ 7 vs 5). In male mice, differences between Frzb/ and wild-type mice were not apparent in the first month of running wheel exercise, but appeared quickly thereafter [Fig. 1(C)]. In contrast, in female mice, differences in running performance were already present in the first month [Fig. 1(D)]. Overall, running performance declined from the age of 8 to 9 months onwards. This effect appeared more outspoken in female than in male mice. The observed differences were not explained by differences in weight between Frzb/ and wild-type mice (Table I). However, treadmill activity did result in consistently lower mean weight of the mice as compared to non-running controls (Table I). This difference was only significant for female wild-type mice at the age of 6 months (Student’s t test P < 0.001). OSTEOARTHRITIS IN RUNNING MICE
We studied the presence of osteoarthritis-like changes in the knee and hip joints of running mice and agematched controls (Fig. 2). X-ray analysis at 12 months [Fig. 3(A)] did not show a significant increase or decrease in osteoarthritis severity scores between running mice and controls although scores tended to be higher in the exercise group [Fig. 3(B, C)] (median score 1 vs 1.5 in male wild-type, 1 vs 2 in female wild-type, 0.5 vs 1.5 in male Frzb/ mice, 0 vs 2 in female Frzb/ mice, ManneWhitney U test, P > 0.05, n ¼ 10 vs 6, 10 vs 4, 10 vs 4 and 9 vs 7 mice, respectively, with both knees evaluated for each mouse). Histomorphological scores for cartilage damage after voluntary exercise were not different between wildtype and Frzb/ mice in males or females (median 2.5 vs 2 and 2 vs 2.5, respectively, ManneWhitney U test, P > 0.05, n ¼ 12 vs 12 knees from 6 males per group and 8 vs 14 knees from 4 wild-type and 7 Frzb/ females) [Fig. 4(A)]. Although all cartilages of the knee joint could be affected, the medial tibial cartilage was most often and most severely involved. No significant differences were found between both groups in features of synovitis (ManneWhitney U test, P > 0.05) [Fig. 4(B, C)]. X-ray scores, cartilage damage and synovitis scores did not correlate with distances ran in the different groups (data not shown). Osteophyte formation was not different between both groups. No signs of osteoarthritis were seen in the hip joints (data not shown). MUSCLE COMPOSITION IN FRZB/ AND WILD-TYPE MICE
As differences in voluntary running performance were not explained by severity of osteoarthritis, we additionally checked whether the observed differences could be explained by the effect of Frzb deletion on muscle fiber composition in 8e12-week old mice [Fig. 5(A, B)]. As shown in Fig. 5(C, D), similar type I and type IIa muscle fiber surface and percentage were found in the soleus muscle of Frzb/ as compared to wild-type mice. Also type IIa and type IIb fiber surface and percentage were similar in the extensor digitorum longus muscle of Frzb/ and wild-type mice [Fig. 5(C, D)] (n ¼ 3 vs 3 and 3 vs 2 for male mice and 4 vs 3 for female mice).
Fig. 4. Histomorphological signs of osteoarthritis in Frzb/ and wild-type mice. (A) Cartilage damage scores and (B and C) synovitis scores in males (B) and females (C) after 1 year of running exercise. Data are shown as box-plots with medians, quartile and percentile 10e90 (n ¼ 12 vs 12 knees from 6 males per group and 8 vs 14 knees from 4 wild-type and 7 Frzb/ females).
Discussion FRZB was identified as a susceptibility gene for osteoarthritis1e6. We recently demonstrated that genetic deletion of Frzb in mice results in increased cartilage damage in
Osteoarthritis and Cartilage Vol. 17, No. 3 different induced models of osteoarthritis10. In addition, the knockout mice have a higher cortical thickness, bone density and mineral content as well as enhanced periosteal bone formation upon mechanical stimulation. This results in stiffer long bones. A triple mechanism for the role of FRZB in osteoarthritis susceptibility has been proposed: increased WNT activity with up-regulation of tissue-destructive enzymes, higher MMP activity due to defective antagonism, and enhanced strain in the boneecartilage biomechanical unit10. In this study, we evaluated the performance of Frzb/ and wild-type mice in running wheels over a 1-year period. Running exercise has previously been used to study osteoarthritis in rodents with conflicting results23e26. However, we find this experimental model of particular interest as its primary analysis mimics a clinical outcome that is also relevant in humans. Of interest, voluntary running exercise did not specifically protect against or increase osteoarthritis in this study. Genes identified to be of importance in osteoarthritis, can be divided into two categories: those that are predominantly contributing to the integrity of the articular cartilage structure and those that play a role in cartilage and bone biology. In genetic mouse models, the former group of genes such as the type II collagen gene27,28 will often show defects in skeletal development29. Recognizing the importance of other, potentially more clinically and therapeutically relevant genes, may be more difficult as the development and progression of osteoarthritis are clearly multifaceted. Therefore, the combination of induced models and the analysis of voluntary running performance as presented here as outcome parameter in long-term studies may prove very useful to highlight the role of specific genes for which genetic associations have been reported in this disease.
395 Despite X-ray and some histomorphological scores for features of osteoarthritis appearing somewhat higher in Frzb/ than in wild-type mice, these differences were not statistically significant. More interestingly, these scores did not appear to correlate with distances covered. Although the relatively small number of animals used in the study could account for these observations, it is clear that the model used also bears some potential drawbacks. It should be noted that the running mice did not develop apparent hip osteoarthritis. This is another limitation of the running wheel model as weight distribution and stress in the joint are likely to be different between animals that walk on four limbs and humans. However, the in vivo and long-term experimental study as presented here can be considered as an inclusive model to which different factors contribute. This bears the consequence that other factors not directly relevant to cartilage and bone biology may contribute to the observed phenotype. In particular, the neurological deficit reported in another strain of Frzb KO mice may be relevant here30. No differences in muscle composition were detected upon screening between the transgenic and wild-type strains. In view of the human disease, however, this apparent lack of change in the joint itself should not necessarily be considered a disadvantage as the development of osteoarthritis is probably also reaching beyond the borders of skeletal biology. Of interest, female mice showed a higher activity in the running wheels than their male counterparts. However, this was again not explained by differences in osteoarthritis development. The difference in weight may partially explain this observation. Of interest, the original association of polymorphisms in the FRZB gene with hip osteoarthritis was reported in females1. This gender specific association was
Fig. 5. Analysis of muscle fiber composition in Frzb/ and wild-type mice. (A) Immunofluorescent staining for type I (green) and type IIa (blue) fibers in the soleus muscle of wild-type mice and isotype control. (B) Immunofluorescent staining for type IIa (blue) and type IIb (background darkblue) in the extensor digitorum longus muscle of wild-type mice and isotype controls. (C) Muscle fiber surface in Frzb/ and wild-type male and female mice. (D) Number of muscle fibers in Frzb/ and wild-type male and female mice (individual data points are shown; dark squares represent Frzb/ and open circles represent wild-type mice; n ¼ 3 vs 3 and 3 vs 2 for male mice and 4 vs 3 for female mice; per mouse muscles from the right and left paw were analyzed).
396
R. J. U. Lories et al.: Frzb deletion affects running performance
confirmed as a risk factor for hip osteoarthritis in a subsequent study2. Further evidence suggests a female specific effect in knee osteoarthritis6. In contrast, other data suggest that the effect of FRZB may not be gender or hip specific3. In conclusion, this study supports the genetic association of FRZB with osteoarthritis and confirms our earlier observations using short-term induced models of the disease10. FRZB and WNT biology can therefore be considered as therapeutic targets for the treatment of osteoarthritis.
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgements The authors would like to thank Bea Weynants and Inge Derese for technical assistance with this study. Rik Lories is the recipient of a post-doctoral fellowship from the Flanders Research Foundation (FWO Vlaanderen). This work was supported by a GOA grant from KU Leuven and a FWO research grant G.0452.05. FWO Vlaanderen was not involved in data acquisition, manuscript writing or submission. References 1. Loughlin J, Dowling B, Chapman K, Marcelline L, Mustafa Z, Southam L, et al. Functional variants within the secreted frizzled-related protein 3 gene are associated with hip osteoarthritis in females. Proc Natl Acad Sci U S A 2004;101:9757e62. 2. Lane NE, Lian K, Nevitt MC, Zmuda JM, Lui L, Li J, et al. Frizzled-related protein variants are risk factors for hip osteoarthritis. Arthritis Rheum 2006;54:1246e54. 3. Min JL, Meulenbelt I, Riyazi N, Kloppenburg M, HouwingDuistermaat JJ, Seymour AB, et al. Association of the frizzled-related protein gene with symptomatic osteoarthritis at multiple sites. Arthritis Rheum 2005;52:1077e80. 4. Lories RJ, Boonen S, Peeters J, De Vlam K, Luyten FP. Evidence for a differential association of the Arg200Trp single-nucleotide polymorphism in FRZB with hip osteoarthritis and osteoporosis. Rheumatology (Oxford) 2006;45:113e4. 5. Rodriguez-Lopez J, Pombo-Suarez M, Liz M, Gomez-Reino JJ, Gonzalez A. Further evidence of the role of frizzled-related protein gene polymorphisms in osteoarthritis. Ann Rheum Dis 2007;66(8): 1052e5. 6. Valdes AM, Loughlin J, Oene MV, Chapman K, Surdulescu GL, Doherty M, et al. Sex and ethnic differences in the association of ASPN, CALM1, COL2A1, COMP, and FRZB with genetic susceptibility to osteoarthritis of the knee. Arthritis Rheum 2007;56:137e46. 7. Hoang B, Moos Jr M, Vukicevic S, Luyten FP. Primary structure and tissue distribution of FRZB, a novel protein related to Drosophila frizzled, suggest a role in skeletal morphogenesis. J Biol Chem 1996;271: 26131e7. 8. Lin K, Wang S, Julius MA, Kitajewski J, Moos Jr M, Luyten FP. The cysteine-rich frizzled domain of Frzb-1 is required and sufficient for modulation of Wnt signaling. Proc Natl Acad Sci U S A 1997;94: 11196e200.
9. Wang S, Krinks M, Lin K, Luyten FP, Moos Jr M. Frzb, a secreted protein expressed in the Spemann organizer, binds and inhibits Wnt-8. Cell 1997;88:757e66. 10. Lories RJ, Peeters J, Bakker A, Derese I, Tylzanowski P, Schrooten J, et al. Articular cartilage and biomechanical properties of the long bones in Frzb-knockout mice. Arthritis Rheum 2007;56:4095e103. 11. Clevers H. Wnt/beta-catenin signaling in development and disease. Cell 2006;127:469e80. 12. Slusarski DC, YangSnyder J, Busa WB, Moon RT. Modulation of embryonic intracellular Ca2þ signaling by Wnt-5A. Dev Biol 1997;182:114e20. 13. Kuhl M, Sheldahl LC, Park M, Miller JR, Moon RT. The Wnt/Ca2þ pathway: a new vertebrate Wnt signaling pathway takes shape. Trends Genet 2000;16:279e83. 14. Tada M, Concha ML, Heisenberg CP. Non-canonical Wnt signalling and regulation of gastrulation movements. Semin Cell Dev Biol 2002;13: 251e60. 15. Dell’Accio F, De Bari C, El Tawil NM, Barone F, Mitsiadis TA, O’dowd J, et al. Activation of WNT and BMP signaling in adult human articular cartilage following mechanical injury. Arthritis Res Ther 2006;8:R139. 16. Nakamura Y, Nawata M, Wakitani S. Expression profiles and functional analyses of Wnt-related genes in human joint disorders. Am J Pathol 2005;167:97e105. 17. Westendorf JJ, Kahler RA, Schroeder TM. Wnt signaling in osteoblasts and bone diseases. Gene 2004;341:19e39. 18. Krishnan V, Bryant HU, Macdougald OA. Regulation of bone mass by Wnt signaling. J Clin Invest 2006;116:1202e9. 19. Dequeker J, Aerssens J, Luyten FP. Osteoarthritis and osteoporosis: clinical and research evidence of inverse relationship. Aging Clin Exp Res 2003;15:426e39. 20. Bierma-Zeinstra SM, Koes BW. Risk factors and prognostic factors of hip and knee osteoarthritis. Nat Clin Pract Rheumatol 2007;3:78e85. 21. Pritzker KP, Gay S, Jimenez SA, Ostergaard K, Pelletier JP, Revell PA, et al. Osteoarthritis cartilage histopathology: grading and staging. Osteoarthritis Cartilage 2006;14:13e29. 22. Derave W, Eijnde BO, Ramaekers M, Hespel P. No effects of lifelong creatine supplementation on sarcopenia in senescence-accelerated mice (SAMP8). Am J Physiol Endocrinol Metab 2005;289:E272e7. 23. Otterness IG, Eskra JD, Bliven ML, Shay AK, Pelletier JP, Milici AJ. Exercise protects against articular cartilage degeneration in the hamster. Arthritis Rheum 1998;41:2068e76. 24. Lapvetelainen T, Nevalainen T, Parkkinen JJ, Arokoski J, Kiraly K, Hyttinen M, et al. Lifelong moderate running training increases the incidence and severity of osteoarthritis in the knee joint of C57BL mice. Anat Rec 1995;242:159e65. 25. Pap G, Eberhardt R, Sturmer I, Machner A, Schwarzberg H, Roessner A, et al. Development of osteoarthritis in the knee joints of Wistar rats after strenuous running exercise in a running wheel by intracranial self-stimulation. Pathol Res Pract 1998;194:41e7. 26. Lapvetelainen T, Hyttinen M, Lindblom J, Langsjo TK, Sironen R, Li SW, et al. More knee joint osteoarthritis (OA) in mice after inactivation of one allele of type II procollagen gene but less OA after lifelong voluntary wheel running exercise. Osteoarthritis Cartilage 2001;9:152e60. 27. Meulenbelt I, Bijkerk C, De Wildt SC, Miedema HS, Breedveld FC, Pols HA, et al. Haplotype analysis of three polymorphisms of the COL2A1 gene and associations with generalised radiological osteoarthritis. Ann Hum Genet 1999;63:393e400. 28. Ikeda T, Mabuchi A, Fukuda A, Kawakami A, Ryo Y, Yamamoto S, et al. Association analysis of single nucleotide polymorphisms in cartilagespecific collagen genes with knee and hip osteoarthritis in the Japanese population. J Bone Miner Res 2002;17:1290e6. 29. Li SW, Prockop DJ, Helminen H, Fassler R, Lapvetelainen T, Kiraly K, et al. Transgenic mice with targeted inactivation of the Col2 alpha 1 gene for collagen II develop a skeleton with membranous and periosteal bone but no endochondral bone. Genes Dev 1995;9:2821e30. 30. Mouse Genome Informatics. Frzbtm1Dgen Phenotypic Allele Detail. Mouse Genome Informatics; 2006.