(bone marrow stromal cell, BMSC) BMP9. (mouse embryonic fibroblasts MEFs) .BMP9. real time PCR BMP9. C3H10 MEFs BMP9 BMP9 BMP9

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1 Research Papers Progress in Biochemistry and Biophysics 29, 36(1): 1291~1298 wwwpibbaccn * 9 ** ( 416) 9(bone morphogenetic protein 9 BMP9) C3H1 (mouse embryonic fibroblasts MEFs) (bone marrow stromal cell, BMSC) BMP9 (alkaline phosphatase ALP) real time PCR BMP9 BMP9 C3H1 MEFs BMSC ALP BMP9 C3H1 MEFs BMP9 C3H1 ALP Runx2 (osteopontin, OPN) (osteocalcin, OC) mrna BMP9 Smad Runx2 BMP9 C3H1 BMP9 9 Q254 DOI: 13724/SPJ [11] (multipotent stem cells) BMP2 ( ) 77% [12] BMP7 4%~5% [11] [1,2] BMP9( GDF-2, growth differentiation factor 2) [13] BMPs [3~6] BMP9 [6, 7] [14, 15] BMPs BMP9 BMP9 BMP9 (bone morphogenetic proteins BMPs) β (transforming growth (mouse embryonic fibroblasts MEFs) factor beta TGFβ) 2 BMPs [8~1] BMP2 BMP7, * (38658) (29BB56) ** Tel: , luojinyong@sinacom BMP2 C3H1 BMP7 BMP2 BMP

2 1292 Prog Biochem Biophys 29; 36 (1) (bone marrow stromal cell BMSC) BMP9 BMP2 123 real time PCR C3H1 BMP9 T-25 3% BMP9 GFP RNA cdna 1 real time PCR ALP Runx2 11 OPN OC GAPDH p12sbe-luc p(6ose)-luc ALP Runx2 OPN OC BMP9 mrna GAPDH BMP2 GFP mrna = C3H1 /GAPDH (ATCC) MEFs BMSC 1 RNA real time PCR Table 1 The sequence of primers for real time PCR dntp RNA Gene Forward primer Reversed primer Takara M-MLV ALP CCCCATGTGATGGCGTAT CGGTAGGGAGAGCACAGC Promega (ALP) Runx2 CCGGTCTCCTTCCAGGAT GGGAACTGCTGTGGCTTC BD S OPN CCTCCCGGTGAAAGTGAC CTGTGGCGCAAGGAGATT (Alizerin Red S) C β- OC CCAAGCAGGAGGGCAATA TCGTCACAAGCAGGGTCA Sigma 2 Invitrogen GAPDH GGCTGCCCAGAACATCAT CGGACACATTGGGGGTAG DMEM (FBS) Hyclone real time PCR Takara 124 C3H1 6~8 T-25 3% BALB/c DMEM 2 p12sbe-luc p(6ose)-luc 25 6%~7% 1 μl 4 h DMEM 24 h 24 BMP9 BMP2( ALP C3H1 MEFS ) GFP ( ) 24 h BMSC 24 3% 48 h BMP9 BMP2 GFP 125 C3H1 5 7 ALP 1 mm 6% ( ) BMP9 GFP ALP 4% 8% 122 C3H1 MEFs 2 ml 25% 24 3% 2 min 8 ml 1% DMEM BMP9 BMP2 GFP 5 g 2 min C3H1 2 MEFs 5 μl 1 U/ml 1 mg/l 14 S PBS BALB/c PBS 3 1% 1 ml 5 μl 1 min 3 5 4% S 3 μl

3 29; 36 (1) ALP 3 BMP9 (H&E Trichrome Alcain blue ) BMP2 ( ) GFP( ) (x ± s) ALP BMP2 BMP9 q C3H1 MEFs BMSC ALP SAS82 ( 1) (3%) BMP9 2 ALP BMP2 ALP ( 1) BMP9 21 BMP9 ALP C3H1 MEFs BMSC ALP ALP BMP9 C3H1 MEFs BMSC 22 BMP9 C3H1 MEFs ALP ALP BMP9 C3H1 MFEs BMP9 BMP9 C3H1 MEFs C3H1 MEFs (C3H1 BMP9 ( ) 2 MEFs 14 ) S BMP9 C3H1 MEFs BMP9 C3H1 MEFs ALP ( 2) ( 3) (3%) BMP9 ALP BMP9 BMP2 BMP9 ( 3) BMP9 C3H1 C3H1 MEFs 23 BMP9 C3H1 MEFs MFEs ALP

4 1294 Prog Biochem Biophys 29; 36 (1) 24 BMP9 C3H1 ( OPN OC) ALP OPN OC BMP9 ALP 5 BMP9 C3H1 14 ( 4c 4d) ALP Runx2 Runx2 OPN OC mrna ( 4b) Runx2 (master GFP BMP9 ALP gene) BMP9 BMP9 7 ( 4a) Runx2 ALP 7 C3H1

5 29; 36 (1) (a) (c) ALP relative expression OPN relative expression d 3d 5d 7d 14d d 3d 5d 7d 14d (b) (d) Runx2 relative expression OC relative expression d 3d 5d 7d 14d d 3d 5d 7d 14d Fig 4 BMP9 induced expression of osteogenic gene in C3H1 cells C3H1 cells were infected with BMP9 or GFP (control) virus At d, 3 d, 5 d, 7 d, 14 d after infection, total RNA was collected and subjected to real time PCR analysis using primers corresponding to the target genes respectively; all samples were normalized for GAPDH (a) BMP9 was shown to induce ALP mrna expression in the early osteogenic differentiation stage of C3H1 (b) BMP9 promoted expression of Runx2 of C3H1 (c), (d) BMP9 induced expression of OPN and OC in the late osteogenic differentiation stage of C3H1 Data are the (x ± s) of triplicates experiments : GFP; : BMP9 25 BMP9 Smad Runx2 BMPs TGFβ-Smad [14] p12sbe-luc Smad 2 SBE(smad binding element) Smad [15] SBE BMP9 Smad Fig 5 BMP9 promoted luciferase activity controlled BMP9 Smad by BRE or OSE in C3H1 cells BMP9 TGFβ-Smad p(6ose)-luc Runx2 Runx2 [16] real time PCR BMP9 Runx2 p (6OSE)-Luc C3H1 BMP9 Runx2 26 BMP9 C3H1 ( 5) real time PCR BMP9 BMP9 Runx2 BMP9 GFP C3H1 Relative luciferase activity GFP BMP9 SBE reportor GFP BMP9 OSE reportor C3H1 cells were transfected with SBE or OSE reporter and then infected with BMP9 or GFP (control) virus At 24 h and 48 h after infection, cells were collected for luciferase assay BMP9 was shown to promote luciferase activity controlled by BRE or OSE element, these results demonstrated that BMP9 can activate canonic TGFβ-Smad pathway and promote osteogenic master gene Runx2 activity Data are the (x ± s) of triplicates experiments : 24 h; : 48 h BMP9

6 1296 Prog Biochem Biophys 29; 36 (1) GFP 5 Trichom Alcian blue H&E Alcian blue Trichome H&E Trichome BMP9 ( 6) ( bone matrix BM ) ( osteocyte OC ) (cartilage matrix CM) Fig 6 BMP9 induced entopic bone formation of C3H1 cells C3H1 cells were infected with BMP9 or GFP (control) virus The infected cells were collected at 8% confluence and subjected to subcutaneous injection into athymic mice At 5 weeks after implantation, animals were killed and bony masses were retrieved The retrieved samples were then decalcified and subjected to H&E staining, Trichrome staining, and Alcian Blue staining Representative images are shown For the Trichrome stain, decalcified bone matrix stained dark red, whereas cartilage matrix stained blue For the Alcian Blue stain, cartilage stained blue BM, bone matrix; OC, osteocyte; CM, cartilage matrix Magnification, 15 3 BMP9 ALP 7 OPN OC ( ) Runx2 [2~22] BMPs real time PCR Runx2 BMPs BMP9 BMP2 Runx2 [17~19] BMP7 BMP9 BMP9 BMP2 TGFβ-Smad [14] BMP9 BMP9 BMP2 TGFβ-Smad ALP BMP9 ALP Smad BMP9 (C3H1, MEFs, real time PCR BMP9 BMSC) ALP real Runx2 time PCR BMP9 C3H1 BMP9 ALP mrna BMP9 Runx2 BMP9 BMP9 ( C3H1 MEFs BMP2 ) TGFβ-Smad BMP9 Runx2 ALP BMP9 BMP9 BMP9

7 29; 36 (1) BMP9 C3H1 biomaterials J Cell Biochem, 22, 86(1): 9~98 BMP9 C3H1 23, 28(15 Suppl): 64~67 BMP9 BMP9 assessment Spine, 26, 31(1): 167~174 BMP2BMP9 TGF-β-Smad Smad Runx2 Maxillofac Implants, 1997, 12(6): 785~792 ( ALP OPN OC) BMP9 Gene, 25, 357(1): 1~8 1 Deng Z L, Sharff K A, Tang N, et al Regulation of osteogenic differentiation during skeletal development Front Biosci, 28, 13(1): 21~221 2 Caplan A I, Bruder S P Mesenchymal stem cells: building blocks for molecular medicine in the 21stcentury Trends Mol Med, 21, 7(6): 259~264 3 He T C Distinct osteogenic activity of BMPs and their orthopaedic applications J Musculoskeletal Neuronal Interactions, 25, 5 (4): 363~366 4 Luo J, Sun M H, Kang Q, et al Gene therapy for bone regeneration Curr Gene Ther, 25, 5(2): 167~179 5 Luu H H, Song W X, Luo X J, et al Distinct roles of bone morphogenetic proteins in osteogenic differentiation of mesenchymal stem cells J Orthop Res, 27, 25(5): 665~677 6 Peng H, Huard J Stem cells in the treatment of muscle and connective tissue diseases Curr Opin Pharmacol, 23, 3(3): 329~ Prockop D J, Gregory C A, Spees J L One strategy for cell and gene therapy: harnessing the power of adult stem cells to repair tissues Proc Natl Acad Sci USA, 23, 1(Suppl): 11917~ Govender S, Csimma C, Genant H K, et al Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures: a prospective, controlled, randomized study of four hundred and fifty patients J Bone Joint Surg Am, 22, 84(12): 2123~ A 觭 il Y, Springer I N, Broek V, et al Effects of bone morphogenetic protein-7 stimulation on osteoblasts cultured on different 1 Sandhu H S Bone morphogenetic proteins and spinal surgery Spine, 11 Kanayama M, Hashimoto T, Shigenobu K, et al A prospective randomized study of posterolateral lumbar fusion using osteogenic protein-1 (OP-1) versus local autograft wiith ceramic bone substitute: emphasis of surgical exploration and histologic 12 Hanisch O, Tatakis D N, Rohrer M D, et al Bone formation and osseointegration stimulated by rhbmp-2 following subantral augmentation procedures in nonhuman primates Int J Oral 13 Mu 觡 oz Sanjuán I, Brivanlou A H Neural induction, the default model and embryonic stem cells Nat Rev Neurosci, 22, 3 (4): 271~28 14 Cao X, Chen D The BMP signaling and in vivo bone formation 15 Laurent D, Christine M, Sabine M, et al Identification of BMP9 and BMP1 as functional activators of the orphan activin receptor-like kinase 1 (ALK1) in endothelial cells Blood, 27, 19(5): 1953~ Zhao M, Ko S Y, Liu J H, et al Inhibition of microtubule assembly in osteoblasts stimulates BMP-2 expression and bone formation through the transcription factor Gli2 Mol Cell Biol, 29, 29 (5): 1291~135 17,,, -2, 23, 83(15): 1345~1349 Dai K R, Xu X L, Tang T T, et al National Medical Journal of China, 23, 83(15): 1345~ Dai K R, Xu X L, Tang T T, et al Repairing of goat tibial bone defects with BMP-2 gene-modified tissue-engineered bone Calcif Tissue Int, 25, 77(1): 55~61 19 Koh J T, Zhao Z, Wang Z, et al Combinatorial gene therapy with BMP2/7 enhances cranial bone regeneration J Dent Res, 28, 87 (9): 845~849 2 Panos G, Ziros A, Efthimia K, et al Runx2: of bone and stretch Inter J Biochem Cell Biol, 28, 4(9): 1659~ Patricia D, Zhang R, Valerie G, et al Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation Cell, 1997, 89(5): 747~ Harvard T, Charles A Transcriptional regulation of the human Runx2/Cbfa1 gene promoter by bone morphogenetic protein-7 Mol Cell Endocrinol 23, 25(1-2): 121~129

8 1298 Prog Biochem Biophys 29; 36 (1) BMP9 Induces Osteogenic Differentiation of Multipotent Stem Cell * ZHANG Yan, WEN Wei, LUO Jin-Yong ** (Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education Chongqing Medical University, Chongqing 416, China) Abstract In order to validate and estimate the capability of BMP9 to induce osteogenic differentiation of multipotent stem cells, three multipotent stem cells (C3H1, MEFs and BMSC) were used as target cells, and BMP9 was introduced into these cells by using recombinant adenoviruses assay, the effect of BMP9 on osteogenic differentiation of multipotent stem cells was demonstrated by using luciferase reporter assay, alkaline phosphatase (ALP) quantitative assay, calcium deposition assay, real time PCR, animal experiment and histological staining assay The results demonstrated that BMP9 can induce ALP expression of C3H1, MEFs and BMSC by a dose dependent manner BMP9 can also stimulate calcium deposition of C3H1 and MEFs in vitro, the osteogenic markers (ALP, Runx2, osteopontin, osteocalcin) were increased after stimulated by BMP9 BMP9 can activate canonical TGFβ-Smad pathway, and promote the expression of osteogenic master gene Runx2 The animal experiment and histological staining assay show that BMP9 can induce ectopic bone formation in naked mice To sum up, BMP9 is a more powerful cell factor to induce osteogenic differentiation of multipotent stem cells Key words bone morphogenetic protein 9(BMP9), multipotent stem cells, signal transduction, differentiation, transforming growth factor DOI: 13724/SPJ *This work was supported by grants from The National Natural Science Foundation of China (38658), Natural Science Foundation Project of Chongqing Science and Technology Commission(29BB56) **Corresponding author Tel: , luojinyong@sinacom Received: March 31, 29 Accepted: July 14, 29