Linarin promotes osteogenic differentiation by activating the BMP-2/RUNX2 pathway via protein kinase A signaling

Li,Jia; Hao,Lingyu; Wu,Junhua; Zhang,Jiquan; Su,Jiansheng

doi:10.3892/ijmm.2016.2490

Linarin promotes osteogenic differentiation by activating the BMP-2/RUNX2 pathway via protein kinase A signaling

Authors:
- Jia Li
- Lingyu Hao
- Junhua Wu
- Jiquan Zhang
- Jiansheng Su
View Affiliations

Affiliations: Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Department of Prosthodontics, School of Stomatology, Tongji University, Shanghai 200072, P.R. China, Ministry of Education, Engineering Research Center of Modern Preparation Technology of TCM, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, P.R. China
Published online on: February 18, 2016 https://doi.org/10.3892/ijmm.2016.2490
Pages: 901-910
Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Linarin (LIN), a flavonoid which exerts both anti-inflammatory and antioxidative effects, has been found to promote osteogenic differentiation. However, the molecular mechanism of its effect on osteoblast differentiation was unclear. In the present study, LIN from Flos Chrysanthemi Indici (FCI) was isolated in order to investigate the underlying mechanisms of LIN on MC3T3-E1 cells (a mouse osteoblastic cell line) and the osteoprotective effect of LIN in mice which had undergone an ovariectomy (OVX). The results revealed that LIN enhanced osteoblast proliferation and differentiation in MC3T3-E1 cells dose‑dependently, with enhanced alkaline phosphatase (ALP) activity and mineralization of extracellular matrix. LIN upregulated osteogenesis-related gene expression, including that of ALP, runt‑related transcription factor 2 (RUNX2), osteocalcin (OCN), bone sialoprotein (BSP), and type I collagen (COL‑I). Pretreatment with noggin, a bone morphogenetic protein-2 (BMP-2) antagonist, meant that LIN-induced gene expression levels of COL-1, ALP, OCN, BSP and RUNX2 were significantly reduced, as shown by RT-qPCR. Western blot analysis showed that LIN dose‑dependently increased the protein levels of BMP-2 and RUNX2 and enhanced the phosphorylation of SMAD1/5. In addition, LIN dose‑dependently upregulated protein kinase A (PKA) expression. H-89 (a PKA inhibitor) partially blocked the LIN-induced protein increase in BMP-2, p-SMAD1/5 and RUNX2. We noted that LIN preserved the trabecular bone microarchitecture of ovariectomized mice in vivo. Moreover, pretreatment with LIN significantly lowered serum levels of ALP and OCN in ovariectomized mice. Our data indicated that LIN induced the osteogenic differentiation and mineralization of MC3T3-E1 osteoblastic cells by activating the BMP-2/RUNX2 pathway through PKA signaling in vitro and protected against OVX-induced bone loss in vivo. The results strongly suggest that LIN is a useful natural alternative for the management of postmenopausal osteoporosis.

View Figures

View References

1	Ducy P, Schinke T and Karsenty G: The osteoblast: a sophisticated fibroblast under central surveillance. Science. 289:1501–1504. 2000. View Article : Google Scholar : PubMed/NCBI
2	Teitelbaum SL: Bone resorption by osteoclasts. Science. 289:1504–1508. 2000. View Article : Google Scholar : PubMed/NCBI
3	Isenbarger DW and Chapin BL: Osteoporosis. Current pharmacologic options for prevention and treatment. Postgrad Med. 101:129–132. 136–137. 141–142. 1997. View Article : Google Scholar : PubMed/NCBI
4	Orija IB and Mehta A: Hormone replacement therapy: current controversies. Clin Endocrinol (Oxf). 59:6572003. View Article : Google Scholar
5	Canalis E, Economides AN and Gazzerro E: Bone morphogenetic proteins, their antagonists, and the skeleton. Endocr Rev. 24:218–235. 2003. View Article : Google Scholar : PubMed/NCBI
6	Sykaras N and Opperman LA: Bone morphogenetic proteins (BMPs): how do they function and what can they offer the clinician? J Oral Sci. 45:57–73. 2003. View Article : Google Scholar : PubMed/NCBI
7	Phimphilai M, Zhao Z, Boules H, Roca H and Franceschi RT: BMP signaling is required for RUNX2-dependent induction of the osteoblast phenotype. J Bone Miner Res. 21:637–646. 2006. View Article : Google Scholar : PubMed/NCBI
8	Lee MH, Kwon TG, Park HS, Wozney JM and Ryoo HM: BMP-2-induced Osterix expression is mediated by Dlx5 but is independent of Runx2. Biochem Biophys Res Commun. 309:689–694. 2003. View Article : Google Scholar : PubMed/NCBI
9	Pasqualucci L, Kitaura Y, Gu H and Dalla-Favera R: PKA-mediated phosphorylation regulates the function of activation-induced deaminase (AID) in B cells. Proc Natl Acad Sci USA. 103:395–400. 2006. View Article : Google Scholar : PubMed/NCBI
10	Skalhegg BS and Tasken K: Specificity in the cAMP/PKA signaling pathway. Differential expression, regulation, and subcellular localization of subunits of PKA. Front Biosci. 5:D678–D693. 2000. View Article : Google Scholar : PubMed/NCBI
11	Lo KW, Kan HM, Ashe KM and 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. 6:40–48. 2012. View Article : Google Scholar
12	Li H, Jeong HM, Choi YH, Kim JH, Choi JK, Yeo CY, Jeong HG, Jeong TC, Chun C and Lee KY: Protein kinase a phosphorylates Dlx3 and regulates the function of Dlx3 during osteoblast differentiation. J Cell Biochem. 115:2004–2011. 2014.PubMed/NCBI
13	He S, Choi YH, Choi JK, Yeo CY, Chun C and Lee KY: Protein kinase A regulates the osteogenic activity of Osterix. J Cell Biochem. 115:1808–1815. 2014. View Article : Google Scholar : PubMed/NCBI
14	Sassi AB, Harzallah-Skhiri F, Bourgougnon N and Aouni M: Antimicrobial activities of four Tunisian Chrysanthemum species. Indian J Med Res. 127:183–192. 2008.PubMed/NCBI
15	Liu Q, Liu H, Yuan Z, Wei D and Ye Y: Evaluation of antioxidant activity of chrysanthemum extracts and tea beverages by gold nanoparticles-based assay. Colloids Surf B Biointerfaces. 92:348–352. 2012. View Article : Google Scholar : PubMed/NCBI
16	He J, Chen F, Chen S, Lv G, Deng Y, Fang W, Liu Z, Guan Z and He C: Chrysanthemum leaf epidermal surface morphology and antioxidant and defense enzyme activity in response to aphid infestation. J Plant Physiol. 168:687–693. 2011. View Article : Google Scholar
17	Marongiu B, Piras A, Porcedda S, Tuveri E, Laconi S, Deidda D and Maxia A: Chemical and biological comparisons on super-critical extracts of Tanacetum cinerariifolium (Trevir) Sch Bip with three related species of chrysanthemums of Sardinia (Italy). Nat Prod Res. 23:190–199. 2009. View Article : Google Scholar
18	Kim YH, Lee YS and Choi EM: Linarin isolated from Buddleja officinalis prevents hydrogen peroxide-induced dysfunction in osteoblastic MC3T3-E1 cells. Cell Immunol. 268:112–116. 2011. View Article : Google Scholar : PubMed/NCBI
19	Suh KS, Rhee SY, Jung WW, Kim NJ, Jang YP, Kim HJ, Kim MK, Choi YK and Kim YS: Chrysanthemum zawadskii extract protects osteoblastic cells from highly reducing sugar-induced oxidative damage. Int J Mol Med. 32:241–250. 2013.PubMed/NCBI
20	Han S, Sung KH, Yim D, Lee S, Lee CK, Ha NJ and Kim K: The effect of linarin on LPS-induced cytokine production and nitric oxide inhibition in murine macrophages cell line RAW264.7. Arch Pharm Res. 25:170–177. 2002. View Article : Google Scholar : PubMed/NCBI
21	Lou H, Fan P, Perez RG and Lou H: Neuroprotective effects of linarin through activation of the PI3K/Akt pathway in amyloid-β-induced neuronal cell death. Bioorg Med Chem. 19:4021–4027. 2011. View Article : Google Scholar : PubMed/NCBI
22	Qiaoshan Y, Suhong C, Minxia S, Wenjia M, Bo L and Guiyuan L: preparative purification of linarin extracts from Dendranthema indicum flowers and evaluation of its antihypertensive effect. Evid Based Complement Alternat Med. 2014:3942762014. View Article : Google Scholar : PubMed/NCBI
23	Li J, Zhang X, Yu Q, Fu X and Wang W: One-step separation of four flavonoids from Herba Salviae Plbeiae by HSCCC. J Chromatogr Sci. 52:1288–1293. 2014. View Article : Google Scholar : PubMed/NCBI
24	Yang RS, Lin WL, Chen YZ, Tang CH, Huang TH, Lu BY and Fu WM: Regulation by ultrasound treatment on the integrin expression and differentiation of osteoblasts. Bone. 36:276–283. 2005. View Article : Google Scholar : PubMed/NCBI
25	Wedemeyer C, Xu J, Neuerburg C, Landgraeber S, Malyar NM, von Knoch F, Gosheger G, von Knoch M, Löer F and Saxler G: Particle-induced osteolysis in three-dimensional micro-computed tomography. Calcif Tissue Int. 81:394–402. 2007. View Article : Google Scholar : PubMed/NCBI
26	Bellows CG, Aubin JE and Heersche JN: Initiation and progression of mineralization of bone nodules formed in vitro: the role of alkaline phosphatase and organic phosphate. Bone Miner. 14:27–40. 1991. View Article : Google Scholar : PubMed/NCBI
27	Hallahan AR, Pritchard JI, Chandraratna RA, Ellenbogen RG, Geyer JR, Overland RP, Strand AD, Tapscott SJ and Olson JM: BMP-2 mediates retinoid-induced apoptosis in medulloblastoma cells through a paracrine effect. Nat Med. 9:1033–1038. 2003. View Article : Google Scholar : PubMed/NCBI
28	Franceschi RT and Xiao G: Regulation of the osteoblast-specific transcription factor, Runx2: responsiveness to multiple signal transduction pathways. J Cell Biochem. 88:446–454. 2003. View Article : Google Scholar : PubMed/NCBI
29	Martin TJ and Sims NA: Osteoclast-derived activity in the coupling of bone formation to resorption. Trends Mol Med. 11:76–81. 2005. View Article : Google Scholar : PubMed/NCBI
30	Schilling T, Ebert R, Raaijmakers N, Schütze N and Jakob F: Effects of phytoestrogens and other plant-derived compounds on mesenchymal stem cells, bone maintenance and regeneration. J Steroid Biochem Mol Biol. 139:252–261. 2014. View Article : Google Scholar
31	Lian JB and Stein GS: Concepts of osteoblast growth and differentiation: basis for modulation of bone cell development and tissue formation. Crit Rev Oral Biol Med. 3:269–305. 1992.PubMed/NCBI
32	Gordon JA, Tye CE, Sampaio AV, Underhill TM, Hunter GK and Goldberg HA: Bone sialoprotein expression enhances osteoblast differentiation and matrix mineralization in vitro. Bone. 41:462–473. 2007. View Article : Google Scholar : PubMed/NCBI
33	Jia TL, Wang HZ, Xie LP, Wang XY and Zhang RQ: Daidzein enhances osteoblast growth that may be mediated by increased bone morphogenetic protein (BMP) production. Biochem Pharmacol. 65:709–715. 2003. View Article : Google Scholar : PubMed/NCBI
34	Takuwa Y, Ohse C, Wang EA, Wozney JM and Yamashita K: Bone morphogenetic protein-2 stimulates alkaline phosphatase activity and collagen synthesis in cultured osteoblastic cells, MC3T3-E1. Biochem Biophys Res Commun. 174:96–101. 1991. View Article : Google Scholar : PubMed/NCBI
35	Miyazono K: Signal transduction by bone morphogenetic protein receptors: functional roles of Smad proteins. Bone. 25:91–93. 1999. View Article : Google Scholar : PubMed/NCBI
36	Lian JB, Javed A, Zaidi SK, Lengner C, Montecino M, van Wijnen AJ, Stein JL and Stein GS: Regulatory controls for osteoblast growth and differentiation: role of Runx/Cbfa/AMl factors. Crit Rev Eukaryot Gene Expr. 14:1–41. 2004. View Article : Google Scholar : PubMed/NCBI
37	Ducy P, Zhang R, Geoffroy V, Ridall AL and Karsenty G: Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell. 89:747–754. 1997. View Article : Google Scholar : PubMed/NCBI
38	Beavo JA and Brunton LL: Cyclic nucleotide research - still expanding after half a century. Nat Rev Mol Cell Biol. 3:710–718. 2002. View Article : Google Scholar : PubMed/NCBI
39	Wu X, Zeng LH, Taniguchi T and Xie QM: Activation of PKA and phosphorylation of sodium-dependent vitamin C transporter 2 by prostaglandin E2 promote osteoblast-like differentiation in MC3T3-E1 cells. Cell Death Differ. 14:1792–1801. 2007. View Article : Google Scholar : PubMed/NCBI
40	Siddappa R, Martens A, Doorn J, Leusink A, Olivo C, Licht R, van Rijn L, Gaspar C, Fodde R, Janssen F, et al: cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo. Proc Natl Acad Sci USA. 105:7281–7286. 2008. View Article : Google Scholar : PubMed/NCBI
41	Ohta Y, Nakagawa K, Imai Y, Katagiri T, Koike T and Takaoka K: Cyclic AMP enhances Smad-mediated BMP signaling through PKA-CREB pathway. J Bone Miner Metab. 26:478–484. 2008. View Article : Google Scholar : PubMed/NCBI
42	Xiao G, Jiang D, Thomas P, Benson MD, Guan K, Karsenty G and Franceschi RT: MAPK pathways activate and phosphorylate the osteoblast-specific transcription factor, Cbfa1. J Biol Chem. 275:4453–4459. 2000. View Article : Google Scholar : PubMed/NCBI
43	Kanazawa I, Yamaguchi T, Yano S, Yamauchi M, Yamamoto M and Sugimoto T: Adiponectin and AMP kinase activator stimulate proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells. BMC Cell Biol. 8:512007. View Article : Google Scholar : PubMed/NCBI
44	Turner RT, Vandersteenhoven JJ and Bell NH: The effects of ovariectomy and 17 beta-estradiol on cortical bone histomor-phometry in growing rats. J Bone Miner Res. 2:115–122. 1987. View Article : Google Scholar : PubMed/NCBI