There are various conceptually different strategies to improve bone regeneration and to treat osteoporosis, each with distinct inherent advantages and disadvantages. The use of RNA interference strategies to suppress the biological action of catabolic factors or antagonists of osteogenic proteins is promising, and such strategies can be applied locally. They are comparably inexpensive and do not suffer from stability problems as protein-based approaches. In this study, we focus on sclerostin, encoded by the SOST gene, a key regulator of bone formation and remodeling. Sclerostin is expressed by mature osteocytes but also by late osteogenically differentiated cells. Thus, it is difficult and requires long-term cultures to investigate the effects of SOST silencing on the expression of osteogenic markers using primary cells. We, therefore, selected a rat osteosarcoma cell line, UMR-106, that has been shown to express SOST and secrete sclerostin in a comparable fashion as late osteoblasts and osteocytes. We investigated the effects of differentiating supplements on SOST expression and sclerostin secretion in UMR-106 cells and found that addition of 100 ng/ml of bone morphogenetic protein (BMP)-2 strongly induced sclerostin secretion, whereas dexamethasone inhibited secretion. Effects of silencing SOST in UMR-106 cells cultured in various differentiation media including BMP-2 and/or dexamethasone were determined next with the aim to find promising test conditions for a readout system for the evaluation of future small interfering RNA release formulations for local induction of bone formation. We found a direct correlation between attenuated SOST expression and an increase in the osteogenic potential of UMR-106 cells. The combination of SOST silencing and BMP-2 could synergistically improve osteogenic factors. A lowered proliferation rate in silenced groups may indicate a faster switch to differentiation.

1.
Achiou, Z., H. Toumi, J. Touvier, A. Boudenot, R. Uzbekov, M.S. Ominsky, S. Pallu, E. Lespessailles (2015) Sclerostin antibody and interval treadmill training effects in a rodent model of glucocorticoid-induced osteopenia. Bone 81: 691-701.
2.
Atkins, G.J., P.S. Rowe, H.P. Lim, K.J. Welldon, R. Ormsby, A.R. Wijenayaka, L. Zelenchuk, A. Evdokiou, D.M. Findlay (2011) Sclerostin is a locally acting regulator of late-osteoblast/preosteocyte differentiation and regulates mineralization through a MEPE-ASARM-dependent mechanism. J Bone Miner Res 26: 1425-1436.
3.
Baron, R., M. Kneissel (2013) WNT signaling in bone homeostasis and disease: from human mutations to treatments. Nat Med 19: 179-192.
4.
Becker, C.B. (2014) Sclerostin inhibition for osteoporosis - a new approach. N Engl J Med 370: 476-477.
5.
Bonewald, L.F., S.E. Harris, J. Rosser, M.R. Dallas, S.L. Dallas, N.P. Camacho, B. Boyan, A. Boskey (2003) von Kossa staining alone is not sufficient to confirm that mineralization in vitro represents bone formation. Calcif Tissue Int 72: 537-547.
6.
Brabnikova Maresova, K., K. Pavelka, J.J. Stepan (2013) Acute effects of glucocorticoids on serum markers of osteoclasts, osteoblasts, and osteocytes. Calcif Tissue Int 92: 354-361.
7.
Büttner, M., S. Möller, M. Keller, D. Huster, J. Schiller, M. Schnabelrauch, P. Dieter, U. Hempel (2013) Over-sulfated chondroitin sulfate derivatives induce osteogenic differentiation of hMSC independent of BMP-2 and TGF-β1 signalling. J Cell Physiol 228: 330-340.
8.
Chan, B.Y., E.S. Fuller, A.K. Russell, S.M. Smith, M.M. Smith, M.T. Jackson, M.A. Cake, R.A. Read, J.F. Bateman, P.N. Sambrook, C.B. Little (2011) Increased chondrocyte sclerostin may protect against cartilage degradation in osteoarthritis. Osteoarthritis Cartilage 19: 874-885.
9.
Chen, C., H. Uludag, Z. Wang, H. Jiang (2012) Noggin suppression decreases BMP-2-induced osteogenesis of human bone marrow-derived mesenchymal stem cells in vitro. J Cell Biochem 113: 3672-3680.
10.
Delgado-Calle, J., J. Arozamena, J. Pérez-López, A. Bolado-Carrancio, C. Sañudo, G. Agudo, R. de la Vega, M.A. Alonso, J.C. Rodríguez-Rey, J.A. Riancho (2013) Role of BMPs in the regulation of sclerostin as revealed by an epigenetic modifier of human bone cells. Mol Cell Endocrinol 369: 27-34.
11.
Gamez, B., E. Rodriguez-Carballo, R. Bartrons, J.L. Rosa, F. Ventura (2013) MicroRNA-322 (miR-322) and its target protein Tob2 modulate Osterix (Osx) mRNA stability. J Biol Chem 288: 14264-14275.
12.
Hamann, C., M. Rauner, Y. Höhna, R. Bernhardt, J. Mettelsiefen, C. Goettsch, K.-P. Günther, M. Stolina, C.-Y. Han, F.J. Asuncion, M.S. Ominsky, L.C. Hofbauer (2013) Sclerostin antibody treatment improves bone mass, bone strength, and bone defect regeneration in rats with type 2 diabetes mellitus. J Bone Miner Res 28: 627-638.
13.
Jacobi, A., J. Rauh, P. Bernstein, C. Liebers, X. Zou, M. Stiehler (2013) Comparative analysis of reference gene stability in human mesenchymal stromal cells during osteogenic differentiation. Biotechnol Prog 29: 1034-1042.
14.
Kadar, K., M. Kiraly, B. Porcsalmy, B. Molnar, G.Z. Racz, J. Blazsek, K. Kallo, E.L. Szabo, I. Gera, G. Gerber, G. Varga (2009) Differentiation potential of stem cells from human dental origin - promise for tissue engineering. J Physiol Pharmacol 60(suppl 7): 167-175.
15.
Kato, Y., A. Boskey, L. Spevak, M. Dallas, M. Hori, L.F. Bonewald (2001) Establishment of an osteoid preosteocyte-like cell MLO-A5 that spontaneously mineralizes in culture. J Bone Miner Res 16: 1622-1633.
16.
Kato, Y., J.J. Windle, B.A. Koop, G.R. Mundy, L.F. Bonewald (1997) Establishment of an osteocyte-like cell line, MLO-Y4. J Bone Miner Res 12: 2014-2023.
17.
Keller, H., M. Kneissel (2005) SOST is a target gene for PTH in bone. Bone 37: 148-158.
18.
Kim, E.-K., S. Lim, J.-M. Park, J.K. Seo, J.H. Kim, K.T. Kim, S.H. Ryu, P.-G. Suh (2012) Human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by AMP-activated protein kinase. J Cell Physiol 227: 1680-1687.
19.
Klein-Nulend, J., A.D. Bakker, R.G. Bacabac, A. Vatsa, S. Weinbaum (2013) Mechanosensation and transduction in osteocytes. Bone 54: 182-190.
20.
Krishnan, V., H.U. Bryant, O.A. Macdougald (2006) Regulation of bone mass by Wnt signaling. J Clin Invest 116: 1202-1209.
21.
Kwong, F.N.K., S.M. Richardson, C.H. Evans (2008) Chordin knockdown enhances the osteogenic differentiation of human mesenchymal stem cells. Arthritis Res Ther 10: R65.
22.
Li, X., Y. Zhang, H. Kang, W. Liu, P. Liu, J. Zhang, S.E. Harris, D. Wu (2005) Sclerostin binds to LRP5/6 and antagonizes canonical Wnt signaling. J Biol Chem 280: 19883-19887.
23.
Martin, T.J., P.M. Ingleton, J.C.E. Underwood, V.P. Michelangeli, N.H. Hunt, R.A. Melick (1976) Parathyroid hormone-responsive adenylate cyclase in induced transplantable osteogenic rat sarcoma. Nature 260: 436-438.
24.
McClung, M.R., A. Grauer, S. Boonen, M.A. Bolognese, J.P. Brown, A. Diez-Perez, B.L. Langdahl, J.-Y. Reginster, J.R. Zanchetta, S.M. Wasserman, L. Katz, J. Maddox, Y.-C. Yang, C. Libanati, H.G. Bone (2014) Romosozumab in postmenopausal women with low bone mineral density. N Engl J Med 370: 412-420.
25.
Moester, M.J.C., S.E. Papapoulos, C.W.G.M. Löwik, R.L. van Bezooijen (2010) Sclerostin: current knowledge and future perspectives. Calcif Tissue Int 87: 99-107.
26.
Padhi, D., M. Allison, A.J. Kivitz, M.J. Gutierrez, B. Stouch, C. Wang, G. Jang (2014) Multiple doses of sclerostin antibody romosozumab in healthy men and postmenopausal women with low bone mass: a randomized, double-blind, placebo-controlled study. J Clin Pharmacol 54: 168-178.
27.
Pierotti, S., L. Gandini, A. Lenzi, A.M. Isidori (2008) Pre-receptorial regulation of steroid hormones in bone cells: insights on glucocorticoid-induced osteoporosis. J Steroid Biochem Mol Biol 108: 292-299.
28.
Poole, K.E.S., R.L. van Bezooijen, N. Loveridge, H. Hamersma, S.E. Papapoulos, C.W. Lowik, J. Reeve (2005) Sclerostin is a delayed secreted product of osteocytes that inhibits bone formation. FASEB J 19: 1842-1844.
29.
Recker, R., C. Benson, T. Matsumoto, M. Bolognese, D. Robins, J. Alam, A.Y. Chiang, L. Hu, J.H. Krege, H. Sowa, B. Mitlak, S. Myers (2015) A randomized, double-blind phase 2 clinical trial of blosozumab, a sclerostin antibody, in postmenopausal women with low bone mineral density. J Bone Miner Res 30: 216-224.
30.
Ren, H., D. Liang, X. Jiang, J. Tang, J. Cui, Q. Wei, S. Zhang, Z. Yao, G. Shen, S. Lin (2015a) Variance of spinal osteoporosis induced by dexamethasone and methylprednisolone and its associated mechanism. Steroids 102: 65-75.
31.
Ren, Y., X. Han, S.P. Ho, S.E. Harris, Z. Cao, A.N. Economides, C. Qin, H. Ke, M. Liu, J.Q. Feng (2015b) Removal of SOST or blocking its product sclerostin rescues defects in the periodontitis mouse model. FASEB J 29: 2702-2711.
32.
Roudier, M., X. Li, Q.-T. Niu, E. Pacheco, J.K. Pretorius, K. Graham, B.-R.P. Yoon, J. Gong, K. Warmington, H.Z. Ke, R.A. Black, J. Hulme, P. Babij (2013) Sclerostin is expressed in articular cartilage but loss or inhibition does not affect cartilage remodeling during aging or following mechanical injury. Arthritis Rheum 65: 721-731.
33.
Schneider, H., B. Sedaghati, A. Naumann, M.C. Hacker, M. Schulz-Siegmund (2014) Gene silencing of chordin improves BMP-2 effects on osteogenic differentiation of human adipose tissue-derived stromal cells. Tissue Eng Part A 20: 335-345.
34.
Schoeman, M.A.E., M.J.C. Moester, A.E. Oostlander, E.L. Kaijzel, E.R. Valstar, R.G.H.H. Nelissen, C.W.G.M. Löwik, K.E. de Rooij (2015) Inhibition of GSK3β stimulates BMP signaling and decreases SOST expression which results in enhanced osteoblast differentiation. J Cell Biochem 116: 2938-2946.
35.
Semenov, M., K. Tamai, X. He (2005) SOST is a ligand for LRP5/LRP6 and a Wnt signaling inhibitor. J Biol Chem 280: 26770-26775.
36.
Stern, A.R., L.F. Bonewald (2015) Isolation of osteocytes from mature and aged murine bone. Methods Mol Biol 1226: 3-10.
37.
Stern, A.R., M.M. Stern, M.E. Van Dyke, K. Jähn, M. Prideaux, L.F. Bonewald (2012) Isolation and culture of primary osteocytes from the long bones of skeletally mature and aged mice. BioTechniques 52: 361-373.
38.
Suen, P.K., T.Y. Zhu, D.H.K. Chow, L. Le Huang, L.-Z. Zheng, L. Qin (2015) Sclerostin antibody treatment increases bone formation, bone mass, and bone strength of intact bones in adult male rats. Sci Rep 5: 15632.
39.
Sutherland, M.K., J.C. Geoghegan, C. Yu, D.G. Winkler, J.A. Latham (2004) Unique regulation of SOST, the sclerosteosis gene, by BMPs and steroid hormones in human osteoblasts. Bone 35: 448-454.
40.
Tian, X., W.S.S. Jee, X. Li, C. Paszty, H.Z. Ke (2011) Sclerostin antibody increases bone mass by stimulating bone formation and inhibiting bone resorption in a hindlimb-immobilization rat model. Bone 48: 197-201.
41.
Tracy, R.P., A. Andrianorivo, B.L. Riggs, K.G. Mann (1990) Comparison of monoclonal and polyclonal antibody-based immunoassays for osteocalcin: a study of sources of variation in assay results. J Bone Miner Res 5: 451-461.
42.
van Bezooijen, R.L., B.A.J. Roelen, A. Visser, L. van der Wee-Pals, E. de Wilt, M. Karperien, H. Hamersma, S.E. Papapoulos, P. ten Dijke, C.W.G.M. Löwik (2004) Sclerostin is an osteocyte-expressed negative regulator of bone formation, but not a classical BMP antagonist. J Exp Med 199: 805-814.
43.
Wang, Y.-H., Y. Liu, P. Maye, D.W. Rowe (2006) Examination of mineralized nodule formation in living osteoblastic cultures using fluorescent dyes. Biotechnol Prog 22: 1697-1701.
44.
Winkler, D.G., M.K. Sutherland, J.C. Geoghegan, C. Yu, T. Hayes, J.E. Skonier, D. Shpektor, M. Jonas, B.R. Kovacevich, K. Staehling-Hampton, M. Appleby, M.E. Brunkow, J.A. Latham (2003) Osteocyte control of bone formation via sclerostin, a novel BMP antagonist. EMBO J 22: 6267-6276.
45.
Woo, S.M., J. Rosser, V. Dusevich, I. Kalajzic, L.F. Bonewald (2011) Cell line IDG-SW3 replicates osteoblast-to-late-osteocyte differentiation in vitro and accelerates bone formation in vivo. J Bone Miner Res 26: 2634-2646.
46.
Wu, S.Y., G. Lopez-Berestein, G.A. Calin, A.K. Sood (2014) RNAi therapies: drugging the undruggable. Sci Transl Med 6: 240ps7.
47.
Yao, W., Z. Cheng, C. Busse, A. Pham, M.C. Nakamura, N.E. Lane (2008) Glucocorticoid excess in mice results in early activation of osteoclastogenesis and adipogenesis and prolonged suppression of osteogenesis: a longitudinal study of gene expression in bone tissue from glucocorticoid-treated mice. Arthritis Rheum 58: 1674-1686.
48.
Yu, E.W., R. Kumbhani, E. Siwila-Sackman, B.Z. Leder (2011a) Acute decline in serum sclerostin in response to PTH infusion in healthy men. J Clin Endocrinol Metab 96: E1848-E1851.
49.
Yu, L., M. van der Valk, J. Cao, C.-Y.E. Han, T. Juan, M.B. Bass, C. Deshpande, M.A. Damore, R. Stanton, P. Babij (2011b) Sclerostin expression is induced by BMPs in human Saos-2 osteosarcoma cells but not via direct effects on the sclerostin gene promoter or ECR5 element. Bone 49: 1131-1140.
50.
Yuasa, M., T. Yamada, T. Taniyama, T. Masaoka, W. Xuetao, T. Yoshii, M. Horie, H. Yasuda, T. Uemura, A. Okawa, S. Sotome (2015) Dexamethasone enhances osteogenic differentiation of bone marrow- and muscle-derived stromal cells and augments ectopic bone formation induced by bone morphogenetic protein-2. PLoS One 10: e0116462.
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