Icariin Induces Osteoblast Differentiation and Mineralization without Dexamethasone in Vitro

Abstract

An effective method for preventing bone loss is by promoting osteoblast differentiation and bone formation. While dexamethasone has been routinely used as a classical inducer for osteoblast differentiation, limitations have been observed with its usage, including its varied effects on expression of osteoblast genes in different species and its potentials in suppressing osteoblastic differentiation and mineralization. In this study, we assessed the ability of flavonoid icariin in enhancing differentiation and mineralization of cultured rat primary osteoblasts in the absence of dexamethasone. It was found that, compared to the non-stimulated control, icariin at 10⁻⁵ M produced a higher alkaline phosphatase activity, more and larger areas of alkaline phosphatase-positive colonies (CFU-F_ALP) and mineralized nodules, more osteocalcin secretion and calcium deposition, higher levels of mRNA expression of alkaline phosphatase, osteoblastic transcription factors osterix and runt-related transcription factor 2, and collagen 1α, higher levels of protein expression of collagen 1α, alkaline phosphatese, osterix, and runt-related transcription factor 2. In addition, icariin at 10⁻⁵ M was always more potent than dexamethasone at its optimal concentration of 10⁻⁸ M on the above osteoblast differentiation and mineralization markers. Taken together, our studies demonstrated that icariin has a pronounced ability in promoting osteoblast differentiation in vitro in the absence of dexamethasone.

Key words

osteoblasts - differentiation - icariin - proliferation - dexamethasone

^* These two authors contributed equally to this work.

• References

• 1 Yamaguchi M. Isoflavone and bone metabolism: its cellular mechanism and preventive role in bone loss. J Health Sci 2002; 48: 209-222
• 2 Huang J, Yuan L, Wang X, Zhang TL, Wang K. Icaritin and its glycosides enhance osteoblastic, but suppress osteoclastic, differentiation and activity in vitro . Life Sci 2007; 81: 832-840
• 3 Ma HP, Ming LG, Ge BF, Zhai YK, Song P, Xian CJ, Chen KM. Icariin is more potent than genistein in promoting osteoblast differentiation and mineralization in vitro . J Cell Biochem 2011; 112: 916-923
• 4 Mok SK, Chen WF, Lai WP, Leung PC, Wang XL, Yao XS, Wong MS. Icariin protects against bone loss induced by oestrogen deficiency and activates oestrogen receptor-dependent osteoblastic functions in UMR 106 cells. Br J Pharmacol 2010; 159: 939-949
• 5 Bao JR, Yang JW, Li SF, Zhao W, Zhang Q, Yan Y. [Effects of icariin on ovariectomized osteoporotic rats]. Wei Sheng Yan Jiu 2005; 34: 191-193
• 6 Chen KM, Ge BF, Liu XY, Ma PH, Lu MB, Bai MH, Wang Y. Icariin inhibits the osteoclast formation induced by RANKL and macrophage-colony stimulating factor in mouse bone marrow culture. Pharmazie 2007; 62: 388-391
• 7 Ming LG, Chen KM, Xian CJ. Functions and action mechanisms of flavonoids genistein and icariin in regulating bone remodeling. J Cell Physiol 2013; 228: 513-521
• 8 Jorgensen NR, Henriksen Z, Sorensen OH, Civitelli R. Dexamethasone, BMP-2, and 1,25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype: validation of an in vitro model for human bone marrow-derived primary osteoblasts. Steroids 2004; 69: 219-226
• 9 Alm JJ, Heino TJ, Hentunen TA, Vaananen HK, Aro HT. Transient 100 nM dexamethasone treatment reduces inter- and intraindividual variations in osteoblastic differentiation of bone marrow-derived human mesenchymal stem cells. Tissue Eng Part C Methods 2012; 18: 658-666
• 10 Canalis E. Mechanisms of glucocorticoid action in bone. Curr Osteoporos Rep 2005; 3: 98-102
• 11 Cox RF, Jenkinson A, Pohl K, OʼBrien FJ, Morgan MP. Osteomimicry of mammary adenocarcinoma cells in vitro; increased expression of bone matrix proteins and proliferation within a 3D collagen environment. PloS One 2012; 7: e41679
• 12 Hintze V, Miron A, Moller S, Schnabelrauch M, Heinemann S, Worch H, Scharnweber D. Artificial extracellular matrices of collagen and sulphated hyaluronan enhance the differentiation of human mesenchymal stem cells in the presence of dexamethasone. J Tissue Eng Regen Med 2012;
• 13 Luppen CA, Smith E, Spevak L, Boskey AL, Frenkel B. Bone morphogenetic protein-2 restores mineralization in glucocorticoid-inhibited MC3 T3-E1 osteoblast cultures. J Bone Miner Res 2003; 18: 1186-1197
• 14 Ventura A, Brunetti G, Colucci S, Oranger A, Ladisa F, Cavallo L, Grano M, Faienza MF. Glucocorticoid-induced osteoporosis in children with 21-hydroxylase deficiency. Biomed Res Int 2013; 2013: 1-8
• 15 He Q, Huang HY, Zhang YY, Li X, Qian SW, Tang QQ. TAZ is downregulated by dexamethasone during the differentiation of 3 T3-L1 preadipocytes. Biochem Biophys Res Commun 2012; 419: 573-577
• 16 Jaiswal RK, Jaiswal N, Bruder SP, Mbalaviele G, Marshak DR, Pittenger MF. Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase. J Biol Chem 2000; 275: 9645-9652
• 17 Naito M, Omoteyama K, Mikami Y, Takahashi T, Takagi M. Inhibition of Wnt/beta-catenin signaling by dexamethasone promotes adipocyte differentiation in mesenchymal progenitor cells, ROB-C26. Histochem Cell Biol 2012; 138: 833-845
• 18 Hardy R, Cooper MS. Glucocorticoid-induced osteoporosis – a disorder of mesenchymal stromal cells. Front Endocrinol 2011; 2: 1-8
• 19 Lian JB, Shalhoub V, Aslam F, Frenkel B, Green J, Hamrah M, Stein GS, Stein JL. Species-specific glucocorticoid and 1,25-dihydroxyvitamin D responsiveness in mouse MC3 T3-E1 osteoblasts: dexamethasone inhibits osteoblast differentiation and vitamin D down-regulates osteocalcin gene expression. Endocrinology 1997; 138: 2117-2127
• 20 Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 1997; 89: 747-754
• 21 Lo KW, Kan HM, Ashe KM, Laurencin CT. The small molecule PKA-specific cyclic AMP analogue as an inducer of osteoblast-like cells differentiation and mineralization. J Tissue Eng Regen Med 2012; 6: 40-48
• 22 Fratzl-Zelman N, Horandner H, Luegmayr E, Varga F, Ellinger A, Erlee MP, Klaushofer K. Effects of triiodothyronine on the morphology of cells and matrix, the localization of alkaline phosphatase, and the frequency of apoptosis in long-term cultures of MC3 T3-E1 cells. Bone 1997; 20: 225-236
• 23 Barbara A, Delannoy P, Denis BG, Marie PJ. Normal matrix mineralization induced by strontium ranelate in MC3 T3-E1 osteogenic cells. Metabolism 2004; 53: 532-537
• 24 Chen KM, Ge BF, Ma HP, Liu XY, Bai MH, Wang Y. Icariin, a flavonoid from the herb Epimedium enhances the osteogenic differentiation of rat primary bone marrow stromal cells. Pharmazie 2005; 60: 939-942
• 25 Zhu XF, Zhang RH, Sun SM, Wang YC, Wang PP, Yang L, Han L. Icariin inducing the differentiation of MC3 T3-E1 Subclone14 cells via estrogen receptor and p 38MAPK signaling pathway in vitro . Chin J Gerontol 2012; 5: 957-960
• 26 Chen TL, Hauschka PV, Cabrales S, Feldman D. The effects of 1,25-dihydroxyvitamin D3 and dexamethasone on rat osteoblast-like primary cell cultures: receptor occupancy and functional expression patterns for three different bioresponses. Endocrinology 1986; 118: 250-259
• 27 Hsieh TP, Sheu SY, Sun JS, Chen MH, Liu MH. Icariin isolated from Epimedium pubescens regulates osteoblasts anabolism through BMP-2, SMAD4, and Cbfa1 expression. Phytomedicine 2010; 17: 414-423
• 28 Zhao J, Ohba S, Shinkai M, Chung UI, Nagamune T. Icariin induces osteogenic differentiation in vitro in a BMP- and Runx2-dependent manner. Biochem Biophys Res Commun 2008; 369: 444-448
• 29 Rauch A, Seitz S, Baschant U, Schilling AF, Illing A, Stride B, Kirilov M, Mandic V, Takacz A, Schmidt-Ullrich R, Ostermay S, Schinke T, Spanbroek R, Zaiss MM, Angel PE, Lerner UH, David JP, Reichardt HM, Amling M, Schutz G, Tuckermann JP. Glucocorticoids suppress bone formation by attenuating osteoblast differentiation via the monomeric glucocorticoid receptor. Cell Metab 2010; 11: 517-531
• 30 Ming LG, Lv X, Ma XN, Ge BF, Zhen P, Song P, Zhou J, Ma HP, Xian CJ, Chen KM. The prenyl group contributes to activities of phytoestrogen 8-prenynaringenin in enhancing bone formation and inhibiting bone resorption in vitro . Endocrinology 2013; 154: 1202-1214
• 31 Ming LG, Chen KM, Xian CJ. Functions and action mechanisms of flavonoids genistein and icariin in regulating bone remodeling. J Cell Physiol 2013; 228: 513-521
• 32 Kagami H, Agata H, Sumita Y, Tojo A. Heterogeneous responses of human bone marrow stromal cells (Multipotent mesenchymal stromal cells) to osteogenic induction. Stem cells and cancer stem cells, Vol. 2. Amsterdam: Springer; 2012: 307-314
• 33 Chiang ZC, Yu SH, Chao AC, Dong GC. Preparation and characterization of dexamethasone-immobilized chitosan scaffold. J Biosci Bioeng 2012; 113: 654-660
• 34 Wang X, Schroder HC, Diehl-Seifert B, Kropf K, Schlossmacher U, Wiens M, Muller WE. Dual effect of inorganic polymeric phosphate/polyphosphate on osteoblasts and osteoclasts in vitro . J Tissue Eng Regen Med 2012;
• 35 Ma X, Zhang X, Jia Y, Zu S, Han S, Xiao D, Sun H, Wang Y. Dexamethasone induces osteogenesis via regulation of hedgehog signalling molecules in rat mesenchymal stem cells. Int Orthop 2013; 37: 1399-1404
• 36 Canalis E, Mazziotti G, Giustina A, Bilezikian JP. Glucocorticoid-induced osteoporosis: pathophysiology and therapy. Osteoporos Int 2007; 18: 1319-1328
• 37 Maniatopoulos C, Sodek J, Melcher AH. Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats. Cell Tissue Res 1988; 254: 317-330
• 38 Anderson RE, Kemp JW, Jee WS, Woodbury DM. Effects of cortisol and fluoride on ion-transporting ATPase activities in cultured osteoblastlike cells. In Vitro 1984; 20: 847-855
• 39 Chen TL, Cone CM, Feldman D. Glucocorticoid modulation of cell proliferation in cultured osteoblast-like bone cells: differences between rat and mouse. Endocrinology 1983; 112: 1739-1745
• 40 Chung CH, Golub EE, Forbes E, Tokuoka T, Shapiro IM. Mechanism of action of beta-glycerophosphate on bone cell mineralization. Calcif Tissue Int 1992; 51: 305-311
• 41 Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro . J Cell Biochem 1997; 64: 295-312
• 42 Schwarz RI. Procollagen secretion meets the minimum requirements for the rate-controlling step in the ascorbate induction of procollagen synthesis. J Biol Chem 1985; 260: 3045-3049
• 43 Graves S, Francis M, Gundle R, Beresford J. Primary culture of human trabecular bone: effects of L-ascorbate-2-phosphate. Bone 1994; 15: 132-133
• 44 Dai W, Dong J, Fang T, Uemura T. Stimulation of osteogenic activity in mesenchymal stem cells by FK506. J Biomed Mater Res A 2008; 86: 235-243
• 45 Abe Y, Aida Y, Abe T, Hirofuji T, Anan H, Maeda K. Development of mineralized nodules in fetal rat mandibular osteogenic precursor cells: requirement for dexamethasone but not for beta-glycerophosphate. Calcif Tissue Int 2000; 66: 66-69
• 46 Zhou J, Ming LG, Ge BF, Wang JQ, Zhu RQ, Wei Z, Ma HP, Xian CJ, Chen KM. Effects of 50 Hz sinusoidal electromagnetic fields of different intensities on proliferation, differentiation and mineralization potentials of rat osteoblasts. Bone 2011; 49: 753-761
• 47 Joseph EW, Pratilas CA, Poulikakos PI, Tadi M, Wang W, Taylor BS, Halilovic E, Persaud Y, Xing F, Viale A, Tsai J, Chapman PB, Bollag G, Solit DB, Rosen N. The RAF inhibitor PLX4032 inhibits ERK signaling and tumor cell proliferation in a V600E BRAF-selective manner. Proc Natl Acad Sci USA 2010; 107: 14903-14908
• 48 Ma XN, Liu XY, Yang YF, Xiao FJ, Li QF, Yan J, Zhang QW, Wang LS, Li XY, Wang H. Regulation of human hepatocellular carcinoma cells by Spred2 and correlative studies on its mechanism. Biochem Biophys Res Commun 2011; 410: 803-808