Differences in Mammalian Target of Rapamycin Gene Expression in the Peripheral Blood and Articular Cartilages of Osteoarthritic Patients and Disease Activity
The gene expression of mTOR, autophagy-related ULK1, caspase 3, CDK-inhibitor p21, and TNFα was measured in the peripheral blood of osteoarthritic (OA) patients at different stages of the disease aiming to establish a gene expression profile that might indicate the activity of the disease and joint destruction. Whole blood of 65 OA outpatients, 27 end-stage OA patients, 27 healthy volunteers, and knee articular cartilages of 28 end-stage OA patients and 26 healthy subjects were examined. OA outpatients were subjected to clinical testing, ultrasonography, and radiographic and WOMAC scoring. Protein levels of p70-S6K, p21, and caspase 3 were quantified by ELISA. Gene expression was measured using real-time RT-PCR. Upregulation of mTOR gene expression was observed in PBMCs of 42 OA outpatients (“High mTOR expression subset”) and in PBMCs and articular cartilages of all end-stage OA patients. A positive correlation between mTOR gene expression in PBMCs and cartilage was observed in the end-stage OA patients. 23 OA outpatients in the “Low mTOR expression subset” exhibited significantly lower mTOR gene expression in PBMCs compared to healthy controls. These “Low mTOR” subset subjects experienced significantly more pain upon walking, and standing and increased total joint stiffness versus “High mTOR” subset, while the latter more often exhibited synovitis. The protein concentrations of p70-S6K, p21, and caspase 3 in PBMCs were significantly lower in the “Low” subset versus “High” subset and end-stage subjects. Increases in the expression of mTOR in PBMCs of OA patients are related to disease activity, being associated with synovitis more than with pain. 1. Introduction Osteoarthritis (OA) is a systemic condition that can affect single or multiple joints and involves degenerative changes in the articular cartilage, remodeling of the subchondral bone, and limited synovial inflammation [1]. At present, the disease course is generally monitored by clinical and radiographic changes, which show poor sensitivity. Therefore, there is a need to identify new approaches in indicating disease activity. Detection of gene expression changes measured in the whole blood is an emerging approach in OA research. Blood-based gene expression patterns recently obtained in transcriptome and microarray analyses appeared capable of distinguishing OA patients from control subjects [2, 3], already showing promising results. Moreover, the level of IL-1β gene expression in peripheral monocytes has been proposed for OA patient stratification, as upregulation of IL-1β was accompanied by
References
[1]
A. R. Poole, F. Guilak, and S. B. Abramson, “Etiopathogenesis of osteoarthritis,” in Osteoarthritis: Diagnosis and Medical/Surgical Management, R. W. Moskowitz, R. D. Altman, M. C. Hochberg, J. A. Buckwalter, and V. M. Goldberg, Eds., pp. 27–49, Williams & Wilkins, Lippincott, Pa, USA, 4th edition, 2007.
[2]
S. Mahr, G. R. Burmester, D. Hilke et al., “Cis- and trans-acting gene regulation is associated with osteoarthritis,” American Journal of Human Genetics, vol. 78, no. 5, pp. 793–803, 2006.
[3]
K. W. Marshall, H. Zhang, T. D. Yager et al., “Blood-based biomarkers for detecting mild osteoarthritis in the human knee,” Osteoarthritis and Cartilage, vol. 13, no. 10, pp. 861–871, 2005.
[4]
M. Attur, I. Belitskaya-Lévy, C. Oh et al., “Increased interleukin-1β gene expression in peripheral blood leukocytes is associated with increased pain and predicts risk for progression of symptomatic knee osteoarthritis,” Arthritis and Rheumatism, vol. 63, no. 7, pp. 1908–1917, 2011.
[5]
E. V. Tchetina, “Developmental mechanisms in articular cartilage degradation in osteoarthritis,” Arthritis, vol. 2011, Article ID 683970, 16 pages, 2011.
[6]
E. V. Tchetina, G. Squires, and A. R. Poole, “Increased type II collagen degradation and very early focal cartilage degeneration is associated with upregulation of chondrocyte differentiation related genes in early human articular cartilage lesions,” Journal of Rheumatology, vol. 32, no. 5, pp. 876–886, 2005.
[7]
E. V. Tchetina, M. Kobayashi, T. Yasuda, T. Meijers, I. Pidoux, and A. R. Poole, “Chondrocyte hypertrophy can be induced by a cryptic sequence of type II collagen and is accompanied by the induction of MMP-13 and collagenase activity: implications for development and arthritis,” Matrix Biology, vol. 26, no. 4, pp. 247–258, 2007.
[8]
E. V. Tchetina, J. Antoniou, M. Tanzer, D. J. Zukor, and A. R. Poole, “Transforming growth factor-β2 suppresses collagen cleavage in cultured human osteoarthritic cartilage, reduces expression of genes associated with chondrocyte hypertrophy and degradation, and increases prostaglandin E 2 production,” American Journal of Pathology, vol. 168, no. 1, pp. 131–140, 2006.
[9]
J. C. Rousseau and P. D. Delmas, “Biological markers in osteoarthritis,” Nature Clinical Practice Rheumatology, vol. 3, no. 6, pp. 346–356, 2007.
[10]
D. Grcevic, Z. Jajic, N. Kovacic et al., “Peripheral blood expression profiles of bone morphogenetic proteins, tumor necrosis factor-superfamily molecules, and transcription factor Runx2 could be used as markers of the form of arthritis, disease activity, and therapeutic responsiveness,” Journal of Rheumatology, vol. 37, no. 2, pp. 246–256, 2010.
[11]
B. D. Chang, K. Watanabe, E. V. Broude et al., “Effects of p21Waf1/Cip1/Sdi1 on cellular gene expression: implications for carcinogenesis, senescence, and age-related diseases,” Proceedings of the National Academy of Sciences of the United States of America, vol. 97, no. 8, pp. 4291–4296, 2000.
[12]
K. Fukuda, “Progress of research in osteoarthritis. Involvement of reactive oxygen species in the pathogenesis of osteoarthritis,” Clinical calcium, vol. 19, no. 11, pp. 1602–1606, 2009.
[13]
S. M. Dai, Z. Z. Shan, H. Nakamura et al., “Catabolic stress induces features of chondrocyte senescence through overexpression of caveolin 1: possible involvement of caveolin 1-induced down-regulation of articular chondrocytes in the pathogenesis of osteoarthritis,” Arthritis and Rheumatism, vol. 54, no. 3, pp. 818–831, 2006.
[14]
S. Sesselmann, S. S？der, R. Voigt, J. Haag, S. P. Grogan, and T. Aigner, “DNA methylation is not responsible for p21WAF1/CIP1 down-regulation in osteoarthritic chondrocytes,” Osteoarthritis and Cartilage, vol. 17, no. 4, pp. 507–512, 2009.
[15]
N. Hay and N. Sonnenberg, “Upstream and downstream of mTOR,” Genes & Development, vol. 18, no. 16, pp. 1926–1945, 2004.
[16]
J. Bohensky, S. Leshinsky, V. Srinivas, and I. M. Shapiro, “Chondrocyte autophagy is stimulated by HIF-1 dependent AMPK activation and mTOR suppression,” Pediatric Nephrology, vol. 25, no. 4, pp. 633–642, 2010.
[17]
M. S. Kim, Y. W. Ke, V. Auyeung, Q. Chen, P. A. Gruppuso, and C. Phornphutkul, “Leucine restriction inhibits chondrocyte proliferation and differentiation through mechanisms both dependent and independent of mTOR signaling,” American Journal of Physiology, vol. 296, no. 6, pp. E1374–E1382, 2009.
[18]
C. P. Sanchez and Y. Z. He, “Bone growth during rapamycin therapy in young rats,” BMC Pediatrics, vol. 9, article 3, 2009.
[19]
D. Cejka, S. Hayer, B. Niederreiter et al., “Mammalian target of rapamycin signaling is crucial for joint destruction in experimental arthritis and is activated in osteoclasts from patients with rheumatoid arthritis,” Arthritis and Rheumatism, vol. 62, no. 8, pp. 2294–2302, 2010.
[20]
B. Caramés, N. Taniguchi, S. Otsuki, F. J. Blanco, and M. Lotz, “Autophagy is a protective mechanism in normal cartilage, and its aging-related loss is linked with cell death and osteoarthritis,” Arthritis and Rheumatism, vol. 62, no. 3, pp. 791–801, 2010.
[21]
B. Carames, A. Hasegawa, N. Taniguchi, S. Miyaki, F. J. Blanco, and M. Lotz, “Autophagy activation by rapamycin reduces severity of experimental osteoarthritis,” Annals of Rheumatic Diseases, vol. 71, no. 4, pp. 575–581, 2012.
[22]
T. Laragione and P. S. Gulko, “mTOR regulates the invasive properties of synovial fibroblasts in rheumatoid arthritis,” Molecular Medicine, vol. 16, no. 9-10, pp. 352–358, 2010.
[23]
B. Raught, A. C. Gingras, and N. Sonenberg, “The target of rapamycin (TOR) proteins,” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 13, pp. 7037–7044, 2001.
[24]
B. Lu, E. Capan, and C. Li, “Autophagy induction and autophagic cell death in effector T cells,” Autophagy, vol. 3, no. 2, pp. 158–159, 2007.
[25]
Y. Wei, S. Sinha, and B. Levine, “Dual role of JNK1-mediated phosphorylation of Bcl-2 in autophagy and apoptosis regulation,” Autophagy, vol. 4, no. 7, pp. 949–951, 2008.
[26]
F. J. Blanco, R. Guitian, E. Vazquez-Martul, F. J. de Toro, and F. Galdo, “Osteoarthritis chondrocytes die by apoptosis. A possible pathway for osteoarthritis pathology,” Arthritis & Rheumatism, vol. 41, no. 2, pp. 284–289, 1998.
[27]
K. Kuhn, D. D. D’Lima, S. Hashimoto, and M. Lotz, “Cell death in cartilage,” Osteoarthritis & Cartilage, vol. 12, no. 1, pp. 1–16, 2004.
[28]
N. Yatsugi, T. Tsukazaki, M. Osaki, T. Koji, S. Yamashita, and H. Shindo, “Apoptosis of articular chondrocytes in rheumatoid arthritis and osteoarthritis: correlation of apoptosis with degree of cartilage destruction and expression of apoptosis-related proteins of p53 and c-myc,” Journal of Orthopaedic Science, vol. 5, no. 2, pp. 150–156, 2000.
[29]
M. Zhu, M. Chen, M. Zuscik et al., “Inhibition of β-catenin signaling in articular chondrocytes results in articular cartilage destruction,” Arthritis and Rheumatism, vol. 58, no. 7, pp. 2053–2064, 2008.
[30]
T. Aigner, M. Hemmel, D. Neureiter, et al., “Apoptotic cell death is not a widespread phenomenon in normal aging and osteoarthritis human articular knee cartilage: a study of proliferation, programmed cell death (apoptosis), and viability of chondrocytes in normal and osteoarthritic human knee cartilage,” Arthritis & Rheumatism, vol. 44, no. 6, pp. 1304–1312, 2001.
[31]
J. P. Pelletier, J. Martel-Pelletier, and S. B. Abramson, “Osteoarthritis, an inflammatory disease: potential implication for the selection of new therapeutic targets,” Arthritis & Rheumatism, vol. 39, no. 9, pp. 1535–1544, 1999.
[32]
S. R. Goldring and M. B. Goldring, “The role of cytokines in cartilage matrix degeneration in osteoarthritis,” Clinical Orthopaedics and Related Research, no. 427, pp. S27–S36, 2004.
[33]
L. Fraenkel, R. Roubenoff, M. LaValley et al., “The association of peripheral monocyte derived interleukin 1β (IL-1β), IL-1 receptor antagonist, and tumor necrosis factor-α with osteoarthritis in the elderly,” Journal of Rheumatology, vol. 25, no. 9, pp. 1820–1826, 1998.
[34]
I. R. Patel, M. G. Attur, R. N. Patel et al., “TNF-α convertase enzyme from human arthritis-affected cartilage: isolation of cDNA by differential display, expression of the active enzyme, and regulation of TNF-α,” Journal of Immunology, vol. 160, no. 9, pp. 4570–4579, 1998.
[35]
R. Altman, E. Asch, and D. Bloch, “Development of criteria for the classification and reporting of osteoarthritis. Classification of osteoarthritis of the knee,” Arthritis and Rheumatism, vol. 29, no. 8, pp. 1039–1052, 1986.
[36]
H. J. Mankin, H. Dorfman, L. Lippiello, and A. Zarins, “Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolic data,” Journal of Bone and Joint Surgery A, vol. 53, no. 3, pp. 523–537, 1971.
[37]
A. P. Hollander, I. Pidoux, A. Reiner, C. Rorabeck, R. Bourne, and A. R. Poole, “Damage to type II collagen in aging and osteoarthritis starts at the articular surface, originates around chondrocytes, and extends into the cartilage with progressive degeneration,” Journal of Clinical Investigation, vol. 96, no. 6, pp. 2859–2869, 1995.
[38]
J. H. Kellgren and J. S. Lawrence, “Radiological assessment of osteo-arthrosis,” Annals of the Rheumatic Diseases, vol. 16, no. 4, pp. 494–502, 1957.
[39]
N. Bellamy, WOMAC Osteoarthritis Index: A User’s Guide, University of Western Ontario, London, UK, 1995.
[40]
M. Backhaus, G. R. Burmester, T. Gerber et al., “Guidelines for musculoskeletal ultrasound in rheumatology,” Annals of the Rheumatic Diseases, vol. 60, no. 7, pp. 641–649, 2001.
[41]
World Health Organization Study Group: Assessment of Fracture Risk and is Application for Screening for Postmenopausal Osteoporosis, WHO, Geneva, Switzerland, 1994.
[42]
B. K. Son, R. L. Roberts, B. J. Ank, and E. R. Stiehm, “Effects of anticoagulant, serum, and temperature on the natural killer activity of human peripheral blood mononuclear cells stored overnight,” Clinical and Diagnostic Laboratory Immunology, vol. 3, no. 3, pp. 260–264, 1996.
[43]
S. Isotani, K. Hara, C. Tokunaga, H. Inoue, J. Avruch, and K. Yonezawa, “Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase a in vitro,” Journal of Biological Chemistry, vol. 274, no. 48, pp. 34493–34498, 1999.
[44]
C. G. Proud, “p70 S6 kinase: an enigma with variations,” Trends in Biochemical Sciences, vol. 21, no. 5, pp. 181–185, 1996.
[45]
D. C. Fingar, S. Salama, C. Tsou, E. Harlow, and J. Blenis, “Mammalian cell size is controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E,” Genes and Development, vol. 16, no. 12, pp. 1472–1487, 2002.
[46]
M. M. Bradford, “A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding,” Analytical Biochemistry, vol. 72, no. 1-2, pp. 248–254, 1976.
[47]
K. J. Livak, “Comparative Ct method. ABI Prism 7700 sequence detection system,” in User Bulletin No. 2. Foster City, PE Applied Biosystems, Foster City, Calif, USA, 1997.
[48]
T. D. Spector, J. E. Dacre, P. A. Harris, and E. C. Huskisson, “Radiological progression of osteoarthritis: an 11 year follow up study of the knee,” Annals of the Rheumatic Diseases, vol. 51, no. 10, pp. 1107–1110, 1992.
[49]
P. A. Dieppe, J. Cushnaghan, and L. Shepstone, “The Bristol “OA500” Study: progression of osteoarthritis (OA) over 3 years and the relationship between clinical and radiographic changes at the knee joint,” Osteoarthritis and Cartilage, vol. 5, no. 2, pp. 87–97, 1997.
[50]
M. Dougados, A. Gueguen, M. Nguyen et al., “Longitudinal radiologic evaluation of osteoarthritis of the knee,” Journal of Rheumatology, vol. 19, no. 3, pp. 378–384, 1992.
[51]
M. Dougados, A. Gueguen, M. Nguyen et al., “Radiological progression of hip osteoarthritis: definition, risk factors and correlations with clinical status,” Annals of the Rheumatic Diseases, vol. 55, no. 6, pp. 356–362, 1996.
[52]
L. S. Lohmander, “What can we do about osteoarthritis?” Arthritis Research, vol. 2, no. 2, pp. 95–100, 2000.
[53]
S. O’Reily and M. Doherty, “Signs, symptoms, and laboratory tests,” in Osteoarthritis, K. D. Brandt, M. Doherty, and L. S. Lohmander, Eds., pp. 74–84, Oxford University Press, Oxford, UK, 1998.
[54]
X. Ayral, E. H. Pickering, T. G. Woodworth, N. Mackillop, and M. Dougados, “Synovitis: a potential predictive factor of structural progression of medial tibiofemoral knee osteoarthritis—results of a 1 year longitudinal arthroscopic study in 422 patients,” Osteoarthritis and Cartilage, vol. 13, no. 5, pp. 361–367, 2005.
[55]
M. A. D'Agostino, P. Conaghan, M. Le Bars et al., “EULAR report on the use of ultrasonography in painful knee osteoarthritis. Part 1: prevalence of inflammation in osteoarthritis,” Annals of the Rheumatic Diseases, vol. 64, no. 12, pp. 1703–1709, 2005.
[56]
P. G. Conaghan, M. A. D'Agostino, M. Le Bars et al., “Clinical and ultrasonographic predictors of joint replacement for knee osteoarthritis: results from a large, 3-year, prospective EULAR study,” Annals of the Rheumatic Diseases, vol. 69, no. 4, pp. 644–647, 2010.
[57]
P. P. Cheung, L. Gossec, and M. Dougados, “What are the best markers for disease progression in osteoarthritis (OA)?” Best Practice and Research, vol. 24, no. 1, pp. 81–92, 2010.
[58]
A. Shibakawa, H. Aoki, K. Masuko-Hongo et al., “Presence of pannus-like tissue on osteoarthritic cartilage and its histological character,” Osteoarthritis and Cartilage, vol. 11, no. 2, pp. 133–140, 2003.
[59]
H. Kristoffersen, S. Torp-Pedersen, L. Terslev et al., “Indications of inflammation visualized by ultrasound in osteoarthritis of the knee,” Acta Radiologica, vol. 47, no. 3, pp. 281–286, 2006.
[60]
X. Chevalier, T. Conrozier, and P. Richette, “Desperately looking for the right target in osteoarthritis: the anti-IL-1 strategy,” Arthritis Research & Therapy, vol. 13, no. 4, p. 124, 2011.
[61]
C. R. Scanzello, B. McKeon, B. H. Swaim et al., “Synovial inflammation in patients undergoing arthroscopic meniscectomy: molecular characterization and relationship to symptoms,” Arthritis and Rheumatism, vol. 63, no. 2, pp. 391–400, 2011.
[62]
E. V. Tchetina, K. Maslova, N. Demin, and V. A. Myakotkin, “Association of bone loss with upregulation of survival-related genes and concomitant downregulation of mammalian target of rapamycin (mTOR) and osteoblast differentiation-related genes in peripheral blood of osteoporotic postmenopausal women,” Annals of Rheumatic Diseases, vol. 68, supplement 3, p. 491, 2009.
[63]
F. Eckstein, S. Cotofana, W. Wirth et al., “Greater rates of cartilage loss in painful knees than in pain-free knees after adjustment for radiographic disease stage: data from the osteoarthritis initiative,” Arthritis and Rheumatism, vol. 63, no. 8, pp. 2257–2267, 2011.
[64]
M. Ishijima, T. Watari, K. Naito et al., “Relationships between biomarkers of cartilage, bone, synovial metabolism and knee pain provide insights into the origins of pain in early knee osteoarthritis,” Arthritis Research and Therapy, vol. 13, no. 1, article R22, 2011.
[65]
P. Levinger, M. K. Caldow, J. A. Feller, et al., “Association between skeletal muscle inflammatory markers and walking pattern in people with knee osteoarthritis,” Arthritis Care & Research, vol. 63, no. 12, pp. 1715–1721, 2011.
[66]
J. N. Belo, M. Y. Berger, M. Reijman, B. W. Koes, and S. M. A. Bierma-Zeinstra, “Prognostic factors of progression of osteoarthritis of the knee: a systematic review of observational studies,” Arthritis Care and Research, vol. 57, no. 1, pp. 13–26, 2007.
[67]
J. Bedson and P. R. Croft, “The discordance between clinical and radiographic knee osteoarthritis: a systematic search and summary of the literature,” BMC Musculoskeletal Disorders, vol. 9, article 116, 2008.
[68]
L. Gossec, S. Paternotte, J. F. Maillefert et al., “The role of pain and functional impairment in the decision to recommend total joint replacement in hip and knee osteoarthritis: an international cross-sectional study of 1909 patients. Report of the OARSI-OMERACT Task Force on total joint replacement,” Osteoarthritis and Cartilage, vol. 19, no. 2, pp. 147–154, 2011.
[69]
B. Moretti, A. Notarnicola, L. Moretti, et al., “I-ONE therapy in patients undergoing total knee arthroplasty: a prospective, randomized and controlled study,” BMC Musculoskeletal Disorders, vol. 13, article 88, 2012.
[70]
J. J. Lum, R. J. DeBerardinis, and C. B. Thompson, “Autophagy in metazoans: cell survival in the land of plenty,” Nature Reviews Molecular Cell Biology, vol. 6, no. 6, pp. 439–448, 2005.
[71]
K. von der Mark, T. Kirsch, A. Nerlich et al., “Type X collagen synthesis in human osteoarthritic cartilage: indication of chondrocyte hypertrophy,” Arthritis and Rheumatism, vol. 35, no. 7, pp. 806–811, 1992.
[72]
I. Girkontaite, S. Frischholz, P. Lammi et al., “Immunolocalization of type X collagen in normal fetal and adult osteoarthritic cartilage with monoclonal antibodies,” Matrix Biology, vol. 15, no. 4, pp. 231–238, 1996.
[73]
R. Dreier, “Hypertrophic differentiation of chondrocytes in osteoarthritis: the developmental aspect of degenerative joint disorders,” Arthritis Research and Therapy, vol. 12, no. 5, article 216, 2010.
[74]
D. Phander, B. Swoboda, and T. Kirsch, “Expression of early and late differentiation markers (proliferating cell nuclear antigen, syndecan-3, annexin VI, and alkaline phosphatase) by human osteoarthritic chondrocytes,” American Journal of Pathology, vol. 159, no. 5, pp. 1777–1783, 2001.
[75]
H. Drissi, M. Zuscik, R. Rosier, and R. O'Keefe, “Transcriptional regulation of chondrocyte maturation: potential involvement of transcription factors in OA pathogenesis,” Molecular Aspects of Medicine, vol. 26, no. 3, pp. 169–179, 2005.
