Study the effect of Urokinase plasminogen activator on the osteogenic differentiation capacity of mesenchymal stem cell
Abstract
Background and Objective: Human mesenchymal stem cells (MSC) have great application in tissue engineering, specifically bone tissue engineering. There is an evidence that the receptor of urokinase plasminogen activator (uPA) involves in osteogenic differentiation of mesenchymal stem cells. The aim of this study was to identify the effect of uPA and its inhibitor on osteogenic differentiation of mesenchymal stem cells (MScs).
Methods: The human dental pulp stem cells were treated with different concentrations of uPA and its inhibitor (amiloride) in osteogenic medium. MTT assay was used to study the cell viability. The expression of osteogenic genes was assessed by quantitative real-time PCR. Alizarin red staining and alkaline phosphatase activity (Alp) were used to evaluate the osteogenic potential of MScs
Results: Amiloride at concentrations more than 0.125 mM has a toxic effect while uPA showed a significant increase in cell proliferation at 1, 2, 4, 8 nM. The alkaline phosphatase activity increased in the presence of uPA while decreased in the presence of amiloride. The data showed that cells treated with osteogenic medium in the presence of urokinase did not have a positive effect on osteogenic differentiation while amiloride decreased mineralization. Urokinase did not have a significant effect on osteopontin and osteocalcin gene expression while amiloride significantly reduced the expressions of osteogenic related genes.
Conclusion: urokinase is an essential factor in osteogenic differentiation of mesenchymal stem cells and its inhibition leads to disruption of this process, however treatment of cells with urokinase has no effect on osteogenic potential.
Keywords
urokinase plasminogen activator, Human mesenchymal stem cells, osteogenic differentiation
References
- 1. Franz‐Odendaal TA, Hall BK, Witten PE. Buried alive: how osteoblasts become osteocytes. Developmental dynamics: an official publication of the American Association of Anatomists. 2006;235(1):176-90.
- 2. Frost HM. Bone remodelling dynamics: Thomas; 1963.
- 3. Reuning U, Sperl S, Kopitz C, Kessler H, Kruger A, Schmitt M, et al. Urokinase-type plasminogen activator (uPA) and its receptor (uPAR): development of antagonists of uPA/uPAR interaction and their effects in vitro and in vivo. Current pharmaceutical design. 2003;9(19):1529-43.
- 4. Kalbasi Anaraki P, Patecki M, Larmann J, Tkachuk S, Jurk K, Haller H, et al. Urokinase receptor mediates osteogenic differentiation of mesenchymal stem cells and vascular calcification via the complement C5a receptor. Stem cells and development. 2014;23(4):352-62.
- 5. Crippa MP. Urokinase-type plasminogen activator. The international journal of biochemistry & cell biology. 2007;39(4):690-4.
- 6. Furlan F, Galbiati C, Jorgensen NR, Jensen JEB, Mrak E, Rubinacci A, et al. Urokinase plasminogen activator receptor affects bone homeostasis by regulating osteoblast and osteoclast function. Journal of Bone and Mineral Research. 2007;22(9):1387-96.
- 7. Huang J-m, Ren R-y, Bao Y, Guo J-c, Xiang W, Jing X-z, et al. Corrigendum: Ulinastatin Inhibits Osteoclastogenesis and Suppresses Ovariectomy-Induced Bone Loss by Downregulating uPAR. Frontiers in pharmacology. 2018;9:1128.
- 8. Goltzman D. Mechanisms of the development of osteoblastic metastases. Cancer: Interdisciplinary International Journal of the American Cancer Society. 1997;80(S8):1581-7.
- 9. Jankun J, Skrzypczak-Jankun E. Molecular basis of specific inhibition of urokinase plasminogen activator by amiloride. Cancer biochemistry biophysics. 1999;17(1-2):109-23.
- 10. Hinrichs GR, Mortensen LA, Jensen BL, Bistrup C. Amiloride resolves resistant edema and hypertension in a patient with nephrotic syndrome; a case report. Physiological reports. 2018;6(12):e13743.