Bìol. Tvarin, 2017, Volume 19, Issue 2, pp. 70–78


Kh. V. Malysheva1,2,3, O. K. Pavlenko4, R. S. Stoika1,4, O. G. Korchynskyi1,3

This email address is being protected from spambots. You need JavaScript enabled to view it.

1Institute of Cell Biology NAS of Ukraine,
14/16 Drahomanov str., Lviv 79005, Ukraine

2Insitute of Animal Biology NAAS,
38 V. Stus str., Lviv 79034, Ukraine

3Centre for Innovative Research in Medical and Natural Sciences,
Rzeszow University and Medical Faculty,
1A Warshawna str., Rzeszow 35-310, Poland

4Ivan Franko National University of Lviv,
4 Grushevsky str., Lviv 79005, Ukraine

Rheumatoid arthritis (RA) is a chronic inflammatory disorder characterized by massive joint destruction. Bone erosion belongs to the most painful consequences of RA that are tightly associated with disease severity and poor functional outcome. It is well known that bone morphogenetic protein (BMP) and Wnt regulatory pathways are involved in cartilage and bone formation and maintenance. We hypothesize that pituitary tumor transforming gene 1 (PTTG1)/(PTTG)-binding factor 1 (PBF1) axis serves as a new negative regulator of bone homeostasis with involvement into RA progression and pathogenesis.

The aim of this study was to investigate the effect of small hairpin (sh) RNA-mediated knockdown of PTTG1 mRNA expression on the early stages of BMP-induced osteoblast differentiation. Analysis of the experiment results was performed by spectrophotometric measurement of alkaline phosphatase activity, which is widely used as a marker of early osteogenesis.

We have found that shRNA-mediated knockdown of PTTG1 mRNA expression potentiated early stages of osteoblast differentiaion in stable multi-clonal cultures of C2C12 and KS483 cell lines. The most pronounced effect was found at the action of anti-PTTG1_shRNA-1 whose stable expression stimulated osteoblast differentiation of C2C12 and KS483 cells (2.1 fold and 2.7 fold, respectively), whereas anti-PTTG1_shRNA-3 stable expression did not show any significant effect on the osteoblast differentiation of these cells. Thus, we demonstrated that PTTG1 is an important repressor of early stages of osteogenesis and can serve as an inhibitor of bone remodelling, in particular, during RA progression.


1. Carreira A. C., Alves G. G., Zambuzzi W. F., Sogayar M. C., Granjeiro J. M. Bone Morphogenetic Proteins: structure, biological function and therapeutic applications. Archives of Biochemistry and Biophysics, 2014, 561, pp. 64–73. https://doi.org/10.1016/j.abb.2014.07.011
2. Chen G., Deng C., Li Y. P. TGF-β and BMP signaling in osteoblast differentiation and bone formation. International Journal of Biological Sciences, 2012, 8 (2), pp. 272–288. https://doi.org/10.7150/ijbs.2929
3. Chen H., Senda T., Kubo K. Y. The osteocyte plays multiple roles in bone remodeling and mineral homeostasis. Medical Molecular Morphology, 2015, 48 (2), pp. 61–68. https://doi.org/10.1007/s00795-015-0099-y
4. Chien W., Pei L. A novel binding factor facilitates nuclear translocation and transcriptional activation function of the pituitary tumor-transforming gene product. Journal of Biological Chemistry, 2000, 275 (25), pp. 19422–19427. https://doi.org/10.1074/jbc.M910105199
5. Choy E. Understanding the dynamics: pathways involved in the pathogenesis of rheumatoid arthritis. Rheumatology, Oxford, 2012, 51, suppl. 5, pp. 3–11. https://doi.org/10.1093/rheumatology/kes113
6. Diarra D., Stolina M., Polzer K., Zwerina J., Ominsky M. S., Dwyer D., Korb A., Smolen J., Hoffmann M., Scheinecker C., van der Heide D., Landewe R., Lacey D., Richards W. G., Schett G. Dickkopf-1 is a master regulator of joint remodeling. Nature Medicine, 2007, 13 (2), pp. 56–63. https://doi.org/10.1038/nm1538
7. Fire A., Xu S., Montgomery M. K., Kostas S. A., Driver S. E., Mello C. C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998, 391, pp. 806–811. https://doi.org/10.1038/35888
8. Fujii M., Takeda K., Imamura T., Aoki H., Sampath T. K., Enomoto S., Kawabata M., Kato M., Ichijo H., Miyazono K. Roles of bone morphogenetic protein type I receptors and Smad proteins in osteoblast and chondroblast differentiation. Molecular Biology of the Cell, 1999, 10 (11), pp. 3801–3813. https://doi.org/10.1091/mbc.10.11.3801
9. Gabriel S. E. The epidemiology of rheumatoid arthritis. Rheumatic Disease Clinics of North America, 2001, 27 (2), pp. 269–281. https://doi.org/10.1016/S0889-857X(05)70201-5
10. Galli C., Piemontese M., Lumetti S., Manfredi E., Macaluso G. M., Passeri G. The importance of Wnt pathways for bone metabolism and their regulation by implant topography. European Cells & Materials, 2012, 24, pp. 46–59. https://doi.org/10.22203/eCM.v024a04
11. Golub E. E., Boesze-Batt K. The role of alkaline phosphatase in mineralization. Current Opinion in Orthopaedics, 2007, 8, pp. 444–448. https://doi.org/10.1097/BCO.0b013e3282630851
12. Hashizume M., Mihara M. The roles of interleukin-6 in the pathogenesis of rheumatoid arthritis. Arthritis, 2011, 2011, 765624.
13. Imai K., Chiba T., Maeda G., Morikawa M. The role of Wnt in rheumatoid arthritis and its therapeutic implication. Mini-Reviews in Medicinal Chemistry, 2009, 9 (3), pp. 318–323. https://doi.org/10.2174/1389557510909030318
14. Issack P. S., Heflet D. L., Lane J. M. Role of Wnt signaling in bone remodeling and repair. HSS Journal, 2008, 4 (1), pp. 66–70. https://doi.org/10.1007/s11420-007-9072-1
15. Johnson M. L., Kamel M. A. The Wnt signaling pathway and bone metabolism. Current Opinion in Rheumatology, 2007, 19 (4), pp. 376–382. https://doi.org/10.1097/BOR.0b013e32816e06f9
16. Jung S. M., Kim K. W., Yang C. W., Park S. H., Ju J. H. Cytokine-mediated bone destruction in rheumatoid arthritis. Journal of Immunology Research, 2014, 2014, pp. 263625.
17. Katagiri T., Yamaguchi A., Komaki M., Abe E., Takahashi N., Ikeda T., Rosen V., Wozney J. M., Fujisawa-Sehara A., Suda T. Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage. The Journal of Cell Biology, 1994, 127 (6 pt 1), pp. 1755–1766. https://doi.org/10.1083/jcb.127.6.1755
18. Korchynskyi O. Adenoviral vectors: convenient tools for gene delivery to primary mammalian cells. Biotechnol Acta, 2012, 5 (5), pp. 16–26.
19. Korchynskyi O., Dechering K. J., Sijbers A. M., Olijve W., ten Dijke P. Gene array analysis of bone morphogenetic protein type I receptor-induced osteoblast differentiation. Journal of Bone and Mineral Research, 2003, 18 (7), pp. 1177–1185. https://doi.org/10.1359/jbmr.2003.18.7.1177
20. Korchynskyi O., Rutger L. van Bezooijen, Löwik C. W. G. M., ten Dijke P. Bone morphogenetic protein receptors and their nuclear effectors in bone formation. Bone Morphogenetic Proteins: From Discovery to Clinical Applications, Switzerland, Birkhauser Verlag, Basel, 2002, pp. 9–44.
21. Krishnan V., Bryant H. U., MacDougald O. A. Regulation of bone mass by Wnt signaling. Journal of Clinical Investigation, 2006, 116 (5), pp. 1202–1209. https://doi.org/10.1172/JCI28551
22. Krupak V. I., Malysheva Kh. V., Pavlenko O. K., Shafranska G. I., de Rooij K., Löwik C. W. G. M., Stoika R. S., Korchynskyi O. G. The PTTG1 is a novel inhibitor of osteogenic differentiation of mouse mesenchymal stem cells. The Animal Biology, 2016, vol. 18, no. 1, pp. 61–68. https://doi.org/10.15407/animbiol18.01.061
23. Kwan Tat S., Padrines M., Théoleyre S., Heymann D., Fortun Y. IL-6, RANKL, TNF-alpha/IL-1: interrelations in bone resorption pathophysiology, bone signaling pathways and treatment of osteoporosis. Cytokine & Growth Factor Reviews, 2004, 15 (1), pp. 49–60. https://doi.org/10.1016/j.cytogfr.2003.10.005
24. Lequerré T., Bansard C., Vittecoq O., Derambure C., Hiron M., Daveau M., Tron F., Ayral X., Biga N., Auquit-Auckbur I., Chiocchia G., Le Loët X., Salier J. P. Early and long-standing rheumatoid arthritis: distinct molecular signatures identified by gene-expression profiling in synovia. Arthritis Research & Therapy, 2009, 11 (3), pp. R99. https://doi.org/10.1186/ar2744
25. Logan C. Y., Nusse R. The Wnt signaling pathway in development and disease. Annual Review of Cell and Developmental Biology, 2004, 20, pp. 781–810. https://doi.org/10.1146/annurev.cellbio.20.010403.113126
26. MacDonald B. T., Tamai K., He X. Wnt/β-catenin signaling: components, mechanisms, and disease. Developmantal Cell, 2009, 17 (1), pp. 9–26. https://doi.org/10.1016/j.devcel.2009.06.016
27. McInnes I. B., Schett G. The pathogenesis of rheumatoid arthritis. The New England Journal of Medicine, 2011, 365 (23), pp. 2205–2219. https://doi.org/10.1056/NEJMra1004965
28. Molina-Jiménez F., Benedicto I., Murata M., Martín-Vílchez S., Seki T., Antonio Pintor-Toro J., Tortolero M., Moreno-Otero R., Okazaki K., Koike K., Barbero J. L., Matsuzaki K., Majano P. L., López-Cabrera M. Expression of pituitary tumor-transforming gene 1 (PTTG1)/securin in hepatitis B virus (HBV)-associated liver diseases: evidence for an HBV X protein-mediated inhibition of PTTG1 ubiquitination and degradation. Hepatology, 2010, 51 (3), pp. 777–787. https://doi.org/10.1002/hep.23468
29. Moore C. B., Guthrie E. H., Huang M. T., Taxman D. J. Short Hairpin RNA (shRNA): Design, Delivery, and Assessment of Gene Knockdown. Methods in Molecular Biology, 2010, 629, pp. 141–158.
30. Park J. Y., Pillinger M. H. Interleukin-6 in the pathogenesis of rheumatoid arthritis. Bulletin of the NYU Hospital for Joint Diseases, 2007, 65, suppl. 1, pp. 4–10.
31. Pederson L., Ruan M., Westendorf J. J., Khosla S., Oursler M. J. Regulation of bone formation by osteoclasts involves Wnt/BMP signaling and the chemokine sphingosine-1-phosphate. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105 (52), pp. 20764–20769. https://doi.org/10.1073/pnas.0805133106
32. Pei L. Activation of mitogen-activated protein kinase cascade regulates pituitary tumor-transforming gene transactivation function. Journal of Biological Chemistry, 2000, 275 (40), pp. 31191–31198. https://doi.org/10.1074/jbc.M002451200
33. Pei L., Melmed S. Isolation and characterization of a pituitary tumor-transforming gene (PTTG). Moelcular Endocrinology, 1997, 11 (4), pp. 433–441. https://doi.org/10.1210/mend.11.4.9911
34. Prezant T. R., Kadioglu P., Melmed S. An intronless homolog of human proto-oncogene hPTTG is expressed in pituitary tumors: evidence for hPTTG family. Journal of Clinical Endocrinology and Metabolism, 1999, 84 (3), pp. 1149–1152. https://doi.org/10.1210/jcem.84.3.5658
35. Rabelo Fde S., da Mota L. M., Lima R. A., Lima F. A., Barra G. B., de Carvalho J. F, Amato A. A. The Wnt signaling pathway and rheumatoid arthritis. Autoimmunity Reviews, 2010, 9 (4), pp. 207–210. https://doi.org/10.1016/j.autrev.2009.08.003
36. Rawadi G., Roman-Roman S. Wnt signalling pathway: a new target for the treatment of osteoporosis. Expert Opinion on Therapeutic Targets, 2005, 9 (5), pp. 1063–1077. https://doi.org/10.1517/14728222.9.5.1063
37. Salehi F., Kovacs K., Scheithauer B. W., Lloyd R. V., Cusimano M. Pituitary tumor-transforming gene in endocrine and other neoplasms: a review and update. Endocrine-Related Cancer, 2008, 15 (3), pp. 721–743. https://doi.org/10.1677/ERC-08-0012
38. Schett G., Gravallese E. Bone erosion in rheumatoid arthritis: mechanisms, diagnosis and treatment. Nature Reviews Rheumatology, 2012, 8 (11), pp. 656–664. https://doi.org/10.1038/nrrheum.2012.153
39. Smolen J. S., Aletaha D., Koeller M., Weisman M. H., Emery P. New therapies for treatment of rheumatoid arthritis. Lancet, 2007, 370 (9602), pp. 1861–1874. https://doi.org/10.1016/S0140-6736(07)60784-3
40. Stock M., Schäfer H., Fliegauf M., Otto F. Identifcation of Novel Target Genes of the Bone Specifc Transcription Factor Runx2. Journal of Bone and Mineral Research, 2004, 19 (6), pp. 959–972. https://doi.org/10.1359/jbmr.2004.19.6.959
41. Tfelt-Hansen J., Kanuparthi D., Chattopadhyay N. The emerging role of pituitary tumor transforminggene in tumorigenesis. Clinical Medicine & Research, 2006, 4 (2), pp. 130–137. https://doi.org/10.3121/cmr.4.2.130
42. Van der Horst G., van Bezooijen R. L., Deckers M. M., Hoogendam J.,Visser A., Löwik C. W., Karperien M. Differentiation of murine preosteoblastic KS483 cells depends on autocrine bone morphogenetic protein signalling during all phases of osteoblast formation. Bone, 2002, 31 (6), pp. 661–669. https://doi.org/10.1016/S8756-3282(02)00903-1
43. Wang Z., Yu R., Melmed S. Mice lacking pituitary tumor transforming gene show testicular and splenic hypoplasia, thymic hyperplasia, thrombocytopenia, aberrant cell cycle progression, and premature centromere division. Molecular Endocrinology, 2001, 15 (11), pp. 1870–1879. https://doi.org/10.1210/mend.15.11.0729
44. Yu R., Cruz-Soto M., Li C. S., Hui H., Melmed S. Murine pituitary tumor-transforming gene functions as a securin protein in insulin-secreting cells. Journal of Endocrinology, 2006, 191 (1), pp. 45–53. https://doi.org/10.1677/joe.1.06885
45. Zhang X., Horwitz G. A., Prezant T. R., Valentini A., Nakashima M., Bronstein M. D., Melmed S. Structure, expression, and function of human pituitary tumor-transforming gene (PTTG). Molecular Endocrinology, 1999, 13 (1), pp. 156–166. https://doi.org/10.1210/mend.13.1.0225
46. Zou H., McGarry T. J., Bernal T., Kirschner M. W. Identifcation of a vertebrate sister-chromatid separation inhibitor involved in transformation and tumorigenesis. Science, 1999, 285 (5426), pp. 418–422. https://doi.org/10.1126/science.285.5426.418

Download full text in PDF format

© 2016 Institute of Animal Biology