Alendronate Induces Mineralization in Mouse Osteoblastic MC3T3-E1 Cells: Regulation of Mineralization-Related Genes

Abstract

Alendronate, an aminobisphosphonate, is an effective reagent to reduce fracture risk in osteoporotic patients. Although several studies suggest that bisphosphonates affect osteoblast differentiation, how they affect the genes relating to the mineralization step remains unknown. The present study was performed to clarify the effects of alendronate on mineralization and its related genes in mouse osteoblastic MC3T3-E1 cells. Alendronate at 10⁻⁸ and 10⁻⁷ M induced mineralization in MC3T3-E1 cells. As for the genes that suppress mineralization, alendronate enhanced the level of PC-1 mRNA in a dose-dependent manner in 7-day cultures in semiquantitative RT-PCR, although it reduced the levels of PC-1 mRNA in 21-day cultures. On the other hand, alendronate did not affect the levels of ANK, osteopontin and matrix Gla protein mRNA in both 7- and 21-day cultures. Moreover, alendronate reduced the level of osteocalcin mRNA at 10⁻⁷ and 10⁻⁶ M in 14-day cultures of these cells. As for the expression of alkaline phosphatase (ALP), an important positive regulator of mineralization in osteoblasts, alendronate enhanced the levels of ALP mRNA and protein at 10⁻⁷–10⁻⁵ M. In conclusion, low-dose alendronate induced mineralization in mouse osteoblastic cells. The regulation of PC-1, osteocalcin and ALP by alendronate might play some role in these effects.

Key words

alendronate - osteoblast - mineralization

References

• 1 Anderson HC. Molecular biology of matrix vesicles.  Clin Orthop Res. 1995;  314 266-280
• 2 Balena R, Toolan BC, Shea M. et al . The effects of 2-year treatment with the aminobisphosphonate alendronate on bone metabolism, bone histomorphometry, and bone strength in ovariectomized nonhuman primates.  J Clin Invest. 1993;  92 2577-2586
• 3 Boivin GY, Chavassieux PM, Santora AC. et al . Alendronate increases bone strength by increasing the mean degree of mineralization of bone tissue in osteoporotic women.  Bone. 2000;  27 687-694
• 4 Boskey AL, Boyan BD, Schwartz Z. Matrix vesicles promote mineralization in a gelatin gel.  Calcif Tissue Int. 1997;  60 309-315
• 5 Breuil V, Cosman F, Stein L. et al . Human osteoclast formation and activity in vitro: effects of alendronate.  J Bone Miner Res. 1998;  13 1721-1729
• 6 Chavassieux PM, Arlot ME, Reda C. et al . Histomorphometric assessment of the long-term effects of alendronate on bone quality and remodeling in patients with osteoporosis.  J Clin Invest. 1997;  100 1475-1480
• 7 Cranney A, Wells G, Willan A. et al . Meta-analysis of alendronate for the treatment of postmenopausal women.  Endocr Rev. 2002a;  23 508-516
• 8 Cranney A, Wells G, Willan A. et al . Meta-analysis of risedronate for the treatment of postmenopausal women.  Endocr Rev. 2002b;  23 517-523
• 9 Ducy P, Desbois C, Boyce B. et al . Increased bone formation in OCN-deficient mice.  Nature. 1996;  382 448-452
• 10 Duque G, Rivas D. Alendronate has an anabolic effect on bone through the differentiation of mesenchymal stem cells.  J Bone Miner Res. 2007;  22 1603-1611
• 11 Fisher JE, Rogers MJ, Halasy JM. et al . Alendronate mechanism of action: geranylgeraniol, an intermediate in the mevalonate pathway, prevents inhibition of osteoclast formation, bone resorption, and kinase activation in vitro.  Proc Natl Acad Sci USA. 1999;  96 133-138
• 12 Fujita T, Izumo N, Fukuyama R. et al . Incadronate and etidronate accelerate phosphate-primed mineralization of MC4 cells via Erk1/2-Cbfa1 signaling pathway in a Ras-independent manner: further involvement of mevalonate-pathway blockade for incadronate.  Jpn J Pharmacol. 2001;  86 86-96
• 13 Garcia-Moreno C, Serrano S, Nacher M. et al . Effect of alendronate on cultured normal human osteoblasts.  Bone. 1998;  22 233-239
• 14 Giuliani N, Pedrazzoni M, Negri G. et al . Bisphosphonates stimulate formation of osteoblast precursors and mineralized nodules in murine and human bone marrow cultures in vitro and promote early osteoblastgenesis in young and aged mice in vivo.  Bone. 1998;  22 455-461
• 15 Im GI, Qureshi SA, Kenney J. et al . Osteoblast proliferation and maturation by bisphosphonates.  Biomaterials. 2004;  25 4105-4115
• 16 Harmey D, Hessle L, Narisawa S. et al . Concerted regulation of inorganic pyrophosphate and osteopontin by akp2, enpp1, and ank: an integrated model of the pathogenesis of mineralization disorders.  Am J Pathol. 2004;  164 1199-1209
• 17 Ho AM, Johnson MD, Kingsley DM. Role of mouse ank gene in control of tissue calcification and arthritis.  Science. 2000;  289 265-270
• 18 Idris AI, Rojas J, Greig IR. et al . Aminobisphosphonates cause osteoblast apoptosis and inhibit bone nodule formation in vitro.  2008;  82 191-201
• 19 Johnson K, Moffa A, Chen Y. et al . Matrix vesicle plasma cell membrane glycoprotein-1 regulates mineralization by murine osteoblastic MC3T3 cells.  J Bone Miner Res. 1999;  14 883-892
• 20 Kaji H, Naito J, Inoue Y. et al . Statin suppresses apoptosis in osteoblastic cells: role of transforming growth factor-β-Smad3 pathway.  Horm Metab Res. 2008;  40 746-751
• 21 Kaji H, Naito J, Sowa H. et al . Smad3 differently affects osteoblast differentiation depending upon its differentiation stage.  Horm Metab Res. 2006;  38 740-745
• 22 Liberman UA, Weiss SR, Broll J. et al . Effect of oral alendronate on bone mineral density and the incidence of fractures in postmenopausal osteoporosis.  N Engl J Med. 1995;  333 1437-1443
• 23 Luckmann SP, Hughes DE, Coxon FP. et al . Nitrogen-containing bisphosphonates inhibit the mevalonate pathway and prevent post-translational prenylation of GTP-binding proteins, including RAS.  J Bone Miner Res. 1998;  13 581-589
• 24 Luo G, Ducy P, McKee MD. et al . Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein.  Nature. 1997;  386 78-81
• 25 Mackie PS, Fisher JL, Zhou H. et al . Bisphosphonates regulate cell growth and gene expression in the UMR 106-01 clonal rat osteosarcoma cell line.  Brit J Cancer. 2001;  84 951-958
• 26 Mathov I, Plotkin LI, Sgarlata CL. et al . Extracellular signal-regulated kinases and calcium channels are involved in the proliferative effect of bisphosphonates on osteoblastic cells in vitro.  J Bone Miner Res. 2001;  16 2050-2056
• 27 Murshed M, Harmey D, Millan JL. et al . Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone.  Gene Dev. 2005;  19 1093-1104
• 28 Naito J, Kaji H, Sowa H. et al . Menin suppresses osteoblast differentiation by antagonizing the AP-1 factor, JunD.  J Biol Chem. 2005;  280 4785-4791
• 29 Nussbaum SR, Warrell Jr RP, Rude R. et al . Dose-response study of alendronate sodium for the treatment of cancer-associated hypercalcemia.  J Clin Oncol. 1993;  11 1618-1623
• 30 Plotkin LI, Weinstein RS, Parfitt AM. et al . Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and calcitonin.  J Clin Invest. 1999;  104 1363-1374
• 31 Reinholtz GG, Getz B, Pederson L. et al . Bisphosphonates directly regulate cell proliferation, differentiation, and gene expression in human osteoblasts.  Cancer Res. 2000;  60 6001-6007
• 32 Reszka AA, Halasy-Nagy JM, Masarachia PJ. et al . Bisphosphonates act directly on the osteoclast to induce caspase cleavage of Mst1 kinase during apoptosis. A link between inhibition of the mevalonate pathway and regulation of an apoptosis-promoting kinase.  J Biol Chem. 1999;  274 34967-34973
• 33 Rodan GA. Mechanism of action of bisphosphonates.  Annu Rev Pharmacol Toxicol. 1998;  38 375-388
• 34 Sowa H, Kaji H, Yamaguchi T. et al . Smad3 promotes alkaline phosphatase activity and mineralization of osteoblastic MC3T3-E1 cells.  J Bone Miner Res. 2002;  17 1190-1199
• 35 Stein GS, Lian JB. Molecular mechanisms mediating proliferation/differentiation interrelationships during progressive development of the osteoblast phenotype.  Endocr Rev. 1993;  14 424-442
• 36 Still K, Phipps RJ, Scutt A. Effects of risedronate, alendronate, and etidronate on the viability and activity of rat bone marrow stromal cells in vitro.  Calcif Tissue Int. 2003;  72 143-150
• 37 Terkeltaub R. Inorganic pyrophosphate generation and disposition in pathophysiology.  Am J Physiol. 2001;  281 C1-C11
• 38 Terkeltaub R, Rosenbach M, Fong F. et al . Causal link between nucleotide pyrophosphohydrolase overactivity and increased intracellular inorganic pyrophosphate generation demonstrated by transfection of cultured fibroblasts and osteoblasts with plasma cell membrane glycoprotein-1.  Arthritis Rheum. 1994;  37 934-941
• 39 Tsuchimoto M, Azuma Y, Higuchi O. et al . Alendronate modulates osteogenesis of human osteoblastic cells in vitro.  Jpn J Pharmacol. 1994;  66 25-33

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