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Effects of Phytoestrogen α-ZAL and Mechanical Stimulation on Proliferation, Osteoblastic Differentiation, and OPG/RANKL Expression in MC3T3-E1 Pre-Osteoblasts

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Abstract

Estrogen and mechanical strain are two important regulators of bone remodeling. α-zearalanol (α-ZAL) is a new oomycete phytoestrogen that is similar to 17-beta estradiol, but has less adverse effects. The aim of this study was to investigate cell proliferation, differentiation, osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL) expression in the pre-osteoblast MC3T3-E1 cell line in response to α- ZAL, mechanical strain, or a combination of both. Cell proliferation was assessed by flow cytometry, and alkaline phosphatase (ALP) activity was measured using a spectrophotometric detection kit. mRNA expression of ALP, runt-related transcriptional factor 2 (Runx2), OPG, and RANKL genes was determined by real-time Reverse Transcription-Polymerase Chain Reaction (real-time RT-PCR). Protein expression of Runx2, OPG, and RANKL was determined by Western blotting. α-ZAL alone (10−6–10−10 M) suppressed proliferation of MC3T3-E1 cells and promoted the activity and gene expression of ALP, an early marker of osteogenesis. It also enhanced Runx2 mRNA expression and the OPG/RANKL ratio. Mechanical strain alone promoted proliferation and differentiation of MC3T3-E1 cells. In addition, Runx2 protein, but not mRNA expression, was upregulated and the OPG/RANKL ratio increased. Although a combination of α-ZAL and mechanical strain inhibited cell proliferation, high concentrations of α-ZAL (10−6 M) combined with a high magnitude of strain (2500 με), significantly enhanced ALP activity. α-ZAL with a high magnitude of strain increased RUNX2 protein expression, but lowered the OPG/RANKL ratio. The OPG/RANKL ratio significantly increased in response to α-ZAL combined with a low magnitude of strain (1000 με). Thus, α-ZAL might be a promising candidate for the treatment of osteoporosis. Mechanical strain was effective in inducing osteogenic differentiation. Taken together, our data suggest that α-ZAL combined with a low, but not high magnitude of strain, may provide benefits for osteogenesis.

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References

  1. Astrid, L., W. Liane, S. Lothar, et al. Estrogen receptor and Wnt signaling interact to regulate early gene expression in response to mechanical strain in osteoblastic cells. Biochem. Biophys. Res. Commun. 394:755–759, 2010.

    Article  Google Scholar 

  2. Boyle, W. J., W. S. Simonet, and D. L. Lacey. Osteoclast differentiation and activation. Nature 423:337–342, 2003.

    Article  Google Scholar 

  3. Chi, H. K., Y. Lidan, and E. Clare. Oscillatory fluid flow-induced shear stress decreases osteoclastogenesis. Bone 39:1043–1047, 2006.

    Article  Google Scholar 

  4. Cotter, A., and K. D. Cashman. Genistein appears to prevent early postmenopausal bone loss as effectively as hormone replacement therapy. Nutr. Rev. 61(10):346–351, 2003.

    Article  Google Scholar 

  5. Dai, S. L., J. Duan, Y. Lu, et al. A new phytoestrogen α-Zearalanol markedly inhibits progression of atherogenesis in ovariectomized cholesterol-fed rabbits. Mol. Cell Cardiol. 34:24–27, 2002.

    Google Scholar 

  6. Donna, C. Prevention of osteoporosis: from infancy through older adulthood. Hong Kong Physiother. J. 30:6–12, 2012.

    Article  Google Scholar 

  7. Drissi, H., Q. Luc, I. R. Shakoor, et al. Transcriptional autoregulation of the bone related CBFα1/RUNX2 gene. Cell Physiol. 184:341–350, 2000.

    Article  Google Scholar 

  8. Ducy, P., R. Zhang, V. Geoffroy, et al. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89:747–754, 1997.

    Article  Google Scholar 

  9. Elisabetta, L., P. Letizia, T. Elisa, et al. Human estrogen receptor α gene is a target of Runx2 transcription factor in osteoblasts. Exp. Cell Res. 313:1548–1560, 2007.

    Article  Google Scholar 

  10. Fujita, M., T. Urano, K. Horie, et al. Estrogen activates cyclin-dependent kinases 4 and 6 through induction of cyclin D in rat primary osteoblasts. Biochem. Biophys. Res. Commun. 299:222–228, 2002.

    Article  Google Scholar 

  11. Gohel, A., M. B. McCarthy, and G. Gronowicz. Estrogen prevents gulcocorticoid-induced apoptosis in osteoblasts in vivo and in vitro. Endocrinology 140:5339–5347, 1999.

    Article  Google Scholar 

  12. Iacovino, J. R. Mortality outcomes after osteoporotic fractures in men and women. Insur. Med. 33:316–320, 2001.

    Google Scholar 

  13. Kaneuji, T., S. Nogami, W. Ariyoshi, et al. Regulatory effect on osteoclastogenesis of mechanical strain-loaded osteoblasts. Int. J. Oral Maxillofac. Surg. 40(10):1215, 2011.

    Article  Google Scholar 

  14. Lacey, D. L., E. Timms, H. L. Tan, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93:165–176, 1998.

    Article  Google Scholar 

  15. Lanyon, L., V. Armstrong, D. Ong, et al. Is estrogen receptor alpha key to controlling bones’ resistance to fracture? Endocrinology 182:183–191, 2004.

    Article  Google Scholar 

  16. Lee, K. C., H. Jessop, R. Suswillo, et al. The adaptive response of bone to mechanical loading in female transgenic mice is deficient in the absence of estrogen receptor-alpha and-beta. Endocrinology 182:193–201, 2004.

    Article  Google Scholar 

  17. Lee, M. H., Y. J. Kim, H. J. Kim, et al. BMP-2-induced Runx2 expression is mediated by Dlx5, and TGF-b1 opposes the BMP-2-induced osteoblast differentiation by suppression of Dlx5 expression. Biol. Chem. 278:34387–34394, 2003.

    Article  Google Scholar 

  18. Livak, K. J., and T. D. Schmittgen. Analysis of relative gene expression data using realtime quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408, 2001.

    Article  Google Scholar 

  19. Lorraine, A. F. Estrogen therapy for postmenopausal osteoporosis. Arq. Bras. Endocrinol. Metab. 50(4):705–719, 2006.

    Article  Google Scholar 

  20. Ludwika, K., L. Astrid, H. Sofia, et al. Mechanical regulation of osteoclastic genes in human osteoblasts. Biochem. Biophys. Res. Commun. 368:582–587, 2008.

    Article  Google Scholar 

  21. Marcus, R. Antiresorptive treatment of postmenopausal osteoporosis: comparison of study designs and outcomes in large clinical trials with fracture as an endpoint. Endocr. Rev. 23:16–37, 2002.

    Article  Google Scholar 

  22. Martin, R. B. Toward a unifying theory of bone remodeling. Bone 26:1–6, 2000.

    Article  Google Scholar 

  23. Meilin, W., H. Eric, M. Frederic, et al. Zfp521 antagonizes Runx2, delays osteoblast differentiation in vitro, and promotes bone formation in vivo. Bone 44:528–536, 2009.

    Article  Google Scholar 

  24. Okazaki, R., D. Inoue, M. Shibata, et al. Estrogen promotes early osteoblast differentiation and inhibits adipocyte differentiation in mouse bone marrow stromal cell lines the express estrogen receptor(ER) alpha or beta. Endocrinology 143:2349–2356, 2002.

    Article  Google Scholar 

  25. Papachroni, K. K., D. N. Karatzas, K. A. Papavassiliou, et al. Mechanotransduction in osteoblast regulation and bone disease. Trends Mol. Med. 15:208–216, 2009.

    Article  Google Scholar 

  26. Pérez-Casellas, L. A., X. Wang, et al. Nuclear Factor I transcription factors regulate IGF binding protein 5 gene transcription in human osteoblasts. Biochim. Biophys. Acta 1789(2):78–87, 2009.

    Article  Google Scholar 

  27. Reid, I. R. Anti-resorptive therapies for osteoporosis. Semin. Cell Dev. Biol. 19:473–478, 2008.

    Article  Google Scholar 

  28. Resca, E., G. Carnevale, A. Benelli, et al. Influence of ferutinin on bone metabolism in ovariectomized rats. II: role in recovering osteoporosis. J. Anat. 217(1):48–56, 2010.

    Article  Google Scholar 

  29. Ruimerman, R., P. Hilbers, B. van Rietbergen, and R. Huiskes. A theoretical framework for strain-related trabecular bone maintenance and adaptation. Biomechanics 38:931–941, 2005.

    Article  Google Scholar 

  30. Rumney, R. M. H., A. Sunters, G. C. Reilly, et al. Application of multiple forms of mechanical loading to human osteoblasts reveals increased ATP release in response to fluid flow in 3D cultures and differential regulation of immediate early genes. J. Biomech. 45(3):549–554, 2012.

    Article  Google Scholar 

  31. Sanaa, E., K. Gamal, A. M. Fayda, et al. Real-time PCR hTERT mRNA pattern in tumor core, edge, resection margin, and lymph nodes in laryngeal tumors: relation to proliferative index and impact on prognosis. Clin. Biochem. 38:873–878, 2005.

    Article  Google Scholar 

  32. Sanchez, C., O. Gabay, C. Salvat, et al. Mechanical loading highly increases IL-6 production and decreases OPG expression by osteoblasts. Osteoarthr. Cartil. 17:473–481, 2009.

    Article  Google Scholar 

  33. Schoppet, M., K. T. Preissner, L. C. Hofbauer, et al. RANK ligand and osteoprotegerin: paracrine regulators of bone metabolism and vascular function. Arterioscler. Thromb. Vasc. Biol. 22:549–553, 2002.

    Article  Google Scholar 

  34. Seelig, M. S., B. M. Altura, and B. T. Altura. Benefits and risks of sex hormone replacement in postmenopausal women. Am. Coll. Nutr. 23:482S–496S, 2004.

    Google Scholar 

  35. Shen, R., M. Chen, Y. J. Wang, et al. Smad6 interacts with Runx2 and mediates Smad ubiquitin regulatory factor 1-induced Runx2 degradation. Biol. Chem. 281:3569–3576, 2006.

    Google Scholar 

  36. Shireen, K., P. H. V. Calvin, G. Yuguang, et al. Collagen, type V, α1 (COL5A1) is regulated by TGF-β in osteoblasts. Matrix Biol. 23(7):445–455, 2004.

    Article  Google Scholar 

  37. Syed, F., and S. Khosla. Mechanisms of sex steroid effects on bone. Biochem. Biophys. Res. Commun. 328:688–696, 2005.

    Article  Google Scholar 

  38. Tang, D. Z., W. Hou, Q. Zhou, et al. Osthole stimulates osteoblast differentiation and bone formation by activation of beta-catenin-BMP signaling. J. Bone Miner. Res. 25:1234–1245, 2010.

    Article  Google Scholar 

  39. Tang, L. L., Y. L. Wang, J. Pan, and S. X. Cai. The effect of step-wise increased stretching on rat calvarial osteoblast collagen production. Biomechanics 37:157–161, 2004.

    Article  Google Scholar 

  40. Tang, L., H. Zhao, P. Shao, et al. Preliminary investigation of signal transduction pathway on expression levels of OPG/RANKL that mediated cellular responses to mechanical strain in MC3T3-E1 cells. Clin. Stomatol. 26:202–204, 2010.

    Google Scholar 

  41. Tatsuyama, K., Y. Maezawa, H. Baba, et al. Expression of various growth factors for cell proliferation and cytodifferentiation during fracture repair of bone. Eur. J. Histochem. 44(3):269–278, 2000.

    Google Scholar 

  42. Theoleyre, S., Y. Wittrant, S. K. Tat, et al. The molecular triad OPG/RANK/RANKL: involvement in the orchestration of pathophysiological bone remodeling. Cytokine Growth Factor Rev. 15:457–475, 2004.

    Article  Google Scholar 

  43. Wang, L., X. Zhang, Y. Guo, et al. Involvement of BMPs/Smad signaling pathway in mechanical response in osteoblasts. Cell. Physiol. Biochem. 26:1093–1102, 2010.

    Article  Google Scholar 

  44. Xiao, Z. S., S. G. Liu, T. K. Hinson, et al. Characterization of the upstream mouse Cbfa1/Runx2 Promoter. Cell Biochem. 82:647–659, 2001.

    Article  Google Scholar 

  45. Yan, Y., Y. Gong, Y. Guo, et al. Mechanical strain regulates osteoblast proliferation through integrin-mediated ERK activation. PLoS ONE 7(4):35709, 2012.

    Article  Google Scholar 

  46. Yan, Y., M. Song, C. Guo, et al. The effects of substrate-stretching strain on the BMP-2 mRNA expression in three kinds of mouse cell lines. Chin. J. Gerontol. 30:3092–3095, 2010.

    Google Scholar 

  47. Young, D. W., M. Q. Hassan, X. Q. Yang, et al. Mitotic retention of gene expression patterns by the cell fate-determining transcription factor Runx2. Proc. Nat. Acad. Sci. USA 104:3189–3194, 2007.

    Article  Google Scholar 

  48. Zaman, G., M. Z. Cheng, and H. L. Jessop. Mechanical strain activates estrogen response elements in bone cells. Bone 27:233–239, 2000.

    Article  Google Scholar 

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Acknowledgments

This work was supported by grant from National Nature Science Foundation of China (No. 10832012).

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Authors and Affiliations

  1. Institute of Medical Equipment of the Academy of Military Medical Sciences, Tianjin, 300161, China

    Lu Liu, Yong Guo, Zongming Wan, Caihong Shi, Jianyu Li, Ruixin Li, Qingxin Hao, Hao Li & Xizheng Zhang

  2. Logistics Institute of Chinese People’s Armed Police Forces, Tianjin, 300162, China

    Lu Liu, Zongming Wan & Jianyu Li

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  1. Lu Liu
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Correspondence to Xizheng Zhang.

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Associate Editor Mian Long oversaw the review of this article.

Lu Liu and Yong Guo contributed equally to this work.

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Liu, L., Guo, Y., Wan, Z. et al. Effects of Phytoestrogen α-ZAL and Mechanical Stimulation on Proliferation, Osteoblastic Differentiation, and OPG/RANKL Expression in MC3T3-E1 Pre-Osteoblasts. Cel. Mol. Bioeng. 5, 427–439 (2012). https://doi.org/10.1007/s12195-012-0244-9

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