Connective Tissue Growth Factor (CTGF) Is Regulated by Wnt and Bone Morphogenetic Proteins Signaling in Osteoblast Differentiation of Mesenchymal Stem Cells (2004)

Osteoblast lineage-specific differentiation of mesenchymal stem cells is a well regulated but poorly understood process. Both bone morphogenetic proteins (BMPs) and Wnt signaling are implicated in regulating osteoblast differentiation and bone formation. Here we analyzed the expression profiles of mesenchymal stem cells stimulated with Wnt3A and osteogenic BMPs, and we identified connective tissue growth factor (CTGF) as a potential target of Wnt and BMP signaling. We confirmed the microarray results, and we demonstrated that CTGF was up-regulated at the early stage of BMP-9 and Wnt3A stimulations and that Wnt3A-regulated CTGF expression was β-catenin-dependent. RNA interference-mediated knockdown of CTGF expression significantly diminished BMP-9-induced, but not Wnt3A-induced, osteogenic differentiation, suggesting that Wnt3A may also regulate osteoblast differentiation in a CTGF-independent fashion. However, constitutive expression of CTGF was shown to inhibit both BMP-9- and Wnt3A-induced osteogenic differentiation. Exogenous expression of CTGF was shown to promote cell migration and recruitment of mesenchymal stem cells. Our findings demonstrate that CTGF is up-regulated by Wnt3A and BMP-9 at the early stage of osteogenic differentiation, which may regulate the proliferation and recruitment of osteoprogenitor cells; however, CTGF is down-regulated as the differentiation potential of committed pre-osteoblasts increases, strongly suggesting that tight regulation of CTGF expression may be essential for normal osteoblast differentiation of mesenchymal stem cells. Osteoblast lineage-specific differentiation of mesenchymal stem cells is a well regulated but poorly understood process. Both bone morphogenetic proteins (BMPs) and Wnt signaling are implicated in regulating osteoblast differentiation and bone formation. Here we analyzed the expression profiles of mesenchymal stem cells stimulated with Wnt3A and osteogenic BMPs, and we identified connective tissue growth factor (CTGF) as a potential target of Wnt and BMP signaling. We confirmed the microarray results, and we demonstrated that CTGF was up-regulated at the early stage of BMP-9 and Wnt3A stimulations and that Wnt3A-regulated CTGF expression was β-catenin-dependent. RNA interference-mediated knockdown of CTGF expression significantly diminished BMP-9-induced, but not Wnt3A-induced, osteogenic differentiation, suggesting that Wnt3A may also regulate osteoblast differentiation in a CTGF-independent fashion. However, constitutive expression of CTGF was shown to inhibit both BMP-9- and Wnt3A-induced osteogenic differentiation. Exogenous expression of CTGF was shown to promote cell migration and recruitment of mesenchymal stem cells. Our findings demonstrate that CTGF is up-regulated by Wnt3A and BMP-9 at the early stage of osteogenic differentiation, which may regulate the proliferation and recruitment of osteoprogenitor cells; however, CTGF is down-regulated as the differentiation potential of committed pre-osteoblasts increases, strongly suggesting that tight regulation of CTGF expression may be essential for normal osteoblast differentiation of mesenchymal stem cells. Osteoblast lineage-specific differentiation from the pluripotent mesenchymal stem cells is a well orchestrated process (1Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.D. Moorman M.A. Simonetti D.W. Craig S. Marshak D.R. Science. 1999; 284: 143-147Crossref PubMed Scopus (18406) Google Scholar, 2Olsen B.R. Reginato A.M. Wang W. Annu. Rev. Cell Dev. Biol. 2000; 16: 191-220Crossref PubMed Scopus (794) Google Scholar, 3Aubin J.E. Rev. Endocr. Metab. Disord. 2001; 2: 81-94Crossref PubMed Scopus (411) Google Scholar). Although not well understood, bone morphogenetic proteins (BMPs) 1The abbreviations used are: BMP, bone morphogenetic proteins; CTGF, connective tissue growth factor; RNAi, RNA interference; TGFβ, transforming growth factor-β; FCS, fetal calf serum; GFP, green fluorescent protein; qPCR, quantitative real time PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; siRNA, small interfering RNA; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; ECM, extracellular matrix; ALP, alkaline phosphatase. play an important role in regulating osteoblast differentiation and subsequent bone formation (4Urist M.R. Nilsson O. Rasmussen J. Hirota W. Lovell T. Schmalzreid T. Finerman G.A. Clin. Orthop. 1987; 214: 295-304PubMed Google Scholar, 5Wozney J.M. Rosen V. Celeste A.J. Mitsock L.M. Whitters M.J. Kriz R.W. Hewick R.M. Wang E.A. Science. 1988; 242: 1528-1534Crossref PubMed Scopus (3438) Google Scholar). At least 15 types of BMPs have been identified in humans (6Hogan B.L. Genes Dev. 1996; 10: 1580-1594Crossref PubMed Scopus (1737) Google Scholar, 7Rosen V. Thies R.S. Trends Genet. 1992; 8: 97-102Abstract Full Text PDF PubMed Scopus (234) Google Scholar, 8Reddi A.H. Cytokine Growth Factor Rev. 1997; 8: 11-20Crossref PubMed Scopus (256) Google Scholar). BMPs belong to the TGF-β superfamily and play an important role in development (6Hogan B.L. Genes Dev. 1996; 10: 1580-1594Crossref PubMed Scopus (1737) Google Scholar, 9Kingsley D.M. Novartis Found. Symp. 2001; 232: 213-222PubMed Google Scholar). Genetic disruptions of BMPs have resulted in various skeletal and extraskeletal abnormalities during development (10Zhao G.Q. Genesis. 2003; 35: 43-56Crossref PubMed Scopus (308) Google Scholar). Through a comprehensive analysis of the osteogenic activity of the 14 types of human BMPs, we have recently demonstrated that BMP-2, BMP-6, and BMP-9 are the most potent inducers of osteogenic differentiation both in vitro and in vivo (11Cheng H. Jiang W. Phillips F.M. Haydon R.C. Peng Y. Zhou L. Luu H.H. An N. Breyer B. Vanichakarn P. Szatkowski J.P. Park J.Y. He T.C. J. Bone Jt. Surg. Am. 2003; 85: 1544-1552Crossref PubMed Scopus (837) Google Scholar, 12Kang Q. Sun M.H. Cheng H. Peng Y. Montag A.G. Deyrup A.T. Jiang W. Luu H.H. Szatkowski J.P. Vanichakarn P. Park J.Y. Luo J. Li Y. Haydon R.C. He T.-C. Gene Ther. 2004; 11: 1312-1320Crossref PubMed Scopus (521) Google Scholar). BMPs fulfill their signaling activity by interacting with the heterodimeric complex of two transmembrane serine/threonine kinase receptors, BMPR type I and BMPR type II (13Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (4040) Google Scholar, 14Wrana J.L. Cell. 2000; 100: 189-192Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). The activated receptor kinases phosphorylate the transcription factors Smads 1, 5, or 8, which in turn form a heterodimeric complex with Smad4 in the nucleus and activate the expression of target genes in concert with other co-activators (13Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (4040) Google Scholar, 14Wrana J.L. Cell. 2000; 100: 189-192Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). We have recently analyzed the gene expression profiles of the pre-osteoblast precursor cells that were stimulated with osteogenic BMPs versus non-osteogenic BMPs (15Peng Y. Kang Q. Cheng H. Li X. Sun M.H. Jiang W. Luu H.H. Park J.Y. Haydon R.C. He T.C. J. Cell. Biochem. 2003; 90: 1149-1165Crossref PubMed Scopus (169) Google Scholar). Our results demonstrated that osteogenic BMPs (e.g. BMP-2, BMP-6, and BMP-9) regulated a distinct set of downstream targets that may play a role in regulating BMP-induced osteoblast differentiation (15Peng Y. Kang Q. Cheng H. Li X. Sun M.H. Jiang W. Luu H.H. Park J.Y. Haydon R.C. He T.C. J. Cell. Biochem. 2003; 90: 1149-1165Crossref PubMed Scopus (169) Google Scholar, 16Peng Y. Kang Q. Luo Q. Jiang W. Si W. Liu B.A. Luu H.H. Park J.K. Li X. Luo J. Montag A.G. Haydon R.C. He T.C. J. Biol. Chem. 2004; 279: 32941-32949Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Cross-talk between BMP and Wnt signaling plays a critical role in development (17Capdevila J. Izpisua Belmonte J. Annu. Rev. Cell Dev. Biol. 2001; 17: 87-132Crossref PubMed Scopus (351) Google Scholar, 18Christian J.L. Curr. Opin. Cell Biol. 2000; 12: 244-249Crossref PubMed Scopus (69) Google Scholar). Both synergistic and antagonistic roles of BMPs and Wnt signaling have been observed during embryonic development (19Hartmann C. Tabin C.J. Development (Camb.). 2000; 127: 3141-3159Crossref PubMed Google Scholar, 20Hoppler S. Moon R.T. Mech. Dev. 1998; 71: 119-129Crossref PubMed Scopus (145) Google Scholar, 21Baker J.C. Beddington R.S. Harland R.M. Genes Dev. 1999; 13: 3149-3159Crossref PubMed Scopus (279) Google Scholar, 22Labbe E. Letamendia A. Attisano L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8358-8363Crossref PubMed Scopus (388) Google Scholar, 23Nishita M. Hashimoto M.K. Ogata S. Laurent M.N. Ueno N. Shibuya H. Cho K.W. Nature. 2000; 403: 781-785Crossref PubMed Scopus (405) Google Scholar). For example, Wnt3A and LEF1-deficient mice display similar phenotypes with the latter developing limb deformities (24Galceran J. Farinas I. Depew M.J. Clevers H. Grosschedl R. Genes Dev. 1999; 13: 709-717Crossref PubMed Scopus (405) Google Scholar). The Wnt family consists of a large number of secreted glycoproteins that are involved in embryonic development, tissue induction, and axis polarity (25Cadigan K.M. Nusse R. Genes Dev. 1997; 11: 3286-3305Crossref PubMed Scopus (2249) Google Scholar, 26Wodarz A. Nusse R. Annu. Rev. Cell Dev. Biol. 1998; 14: 59-88Crossref PubMed Scopus (1758) Google Scholar). The Wnt ligands initiate the signaling pathway by binding to the frizzled receptors and co-receptors LRP5/6, leading to phosphorylation of the disheveled protein, which, through its association with axin and the adenomatous polyposis coil tumor suppressor, prevents glycogen synthase kinase 3β from phosphorylating β-catenin. Antagonist Dkk1 competes with Wnt for binding to LRP5/6. Unphosphorylated β-catenin is stabilized by escaping recognition by β-TrCP, a component of a ubiquitin-protein isopeptide ligase. Free β-catenin translocates to the nucleus, where it engages transcription factors LEF/Tcf-4 to activate expression of downstream genes (27Nelson W.J. Nusse R. Science. 2004; 303: 1483-1487Crossref PubMed Scopus (2273) Google Scholar), such as c-Myc, peroxisome proliferator-activated receptor-δ, cyclin D1, and WISPs (28He T.C. Sparks A.B. Rago C. Hermeking H. Zawel L. da Costa L.T. Morin P.J. Vogelstein B. Kinzler K.W. Science. 1998; 281: 1509-1512Crossref PubMed Scopus (4121) Google Scholar, 29He T.C. Chan T.A. Vogelstein B. Kinzler K.W. Cell. 1999; 99: 335-345Abstract Full Text Full Text PDF PubMed Scopus (1040) Google Scholar, 30Tetsu O. McCormick F. Nature. 1999; 398: 422-426Crossref PubMed Scopus (3291) Google Scholar, 31Shtutman M. Zhurinsky J. Simcha I. Albanese C. D'Amico M. Pestell R. Ben-Ze'ev A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5522-5527Crossref PubMed Scopus (1948) Google Scholar, 32Pennica D. Swanson T.A. Welsh J.W. Roy M.A. Lawrence D.A. Lee J. Brush J. Taneyhill L.A. Deuel B. Lew M. Watanabe C. Cohen R.L. Melhem M.F. Finley G.G. Quirke P. Goddard A.D. Hillan K.J. Gurney A.L. Botstein D. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14717-14722Crossref PubMed Scopus (469) Google Scholar, 33Xu L. Corcoran R.B. Welsh J.W. Pennica D. Levine A.J. Genes Dev. 2000; 14: 585-595Crossref PubMed Google Scholar). Increasing evidence suggests that Wnt signaling plays an important regulatory role in bone formation. Several Wnt genes (e.g. Wnt3A) are expressed in the developing limb and are implicated in mesenchymal chondrogenesis (34Gavin B.J. McMahon J.A. McMahon A.P. Genes Dev. 1990; 4: 2319-2332Crossref PubMed Scopus (433) Google Scholar, 35Kengaku M. Capdevila J. Rodriguez-Esteban C. De La Pena J. Johnson R.L. Belmonte J.C. Tabin C.J. Science. 1998; 280: 1274-1277Crossref PubMed Scopus (368) Google Scholar). Some Wnt proteins may play an important role in osteoblast differentiation and osteogenesis (36Bergwitz C. Wendlandt T. Kispert A. Brabant G. Biochim. Biophys. Acta. 2001; 1538: 129-140Crossref PubMed Scopus (47) Google Scholar, 37Fischer L. Boland G. Tuan R.S. J. Cell. Biochem. 2002; 84: 816-831Crossref PubMed Scopus (104) Google Scholar). Recently, Wnt co-receptor LRP5 has been shown to participate in bone mass accrual and that loss-of-function mutations in the LRP5 gene cause the low bone mass phenotype of the autosomal recessive disorder osteoporosis-pseudoglioma syndrome (38Gong Y. Slee R.B. Fukai N. Rawadi G. Roman-Roman S. Reginato A.M. Wang H. Cundy T. Glorieux F.H. Lev D. Zacharin M. Oexle K. Marcelino J. Suwairi W. Heeger S. Sabatakos G. Apte S. Adkins W.N. Allgrove J. Arslan-Kirchner M. Batch J.A. Beighton P. Black G.C. Boles R.G. Boon L.M. Borrone C. Brunner H.G. Carle G.F. Dallapiccola B. De Paepe A. Floege B. Halfhide M.L. Hall B. Hennekam R.C. Hirose T. Jans A. Juppner H. Kim C.A. Keppler-Noreuil K. Kohlschuetter A. LaCombe D. Lambert M. Lemyre E. Letteboer T. Peltonen L. Ramesar R.S. Romanengo M. Somer H. Steichen-Gersdorf E. Steinmann B. Sullivan B. Superti-Furga A. Swoboda W. van den Boogaard M.J. Van Hul W. Vikkula M. Votruba M. Zabel B. Garcia T. Baron R. Olsen B.R. Warman M.L. Cell. 2001; 107: 513-523Abstract Full Text Full Text PDF PubMed Scopus (1899) Google Scholar). Conversely, activating mutations of LRP5 result in the autosomal dominant high bone density trait (39Boyden L.M. Mao J. Belsky J. Mitzner L. Farhi A. Mitnick M.A. Wu D. Insogna K. Lifton R.P. N. Engl. J. Med. 2002; 346: 1513-1521Crossref PubMed Scopus (1366) Google Scholar, 40Little R.D. Carulli J.P. Del Mastro R.G. Dupuis J. Osborne M. Folz C. Manning S.P. Swain P.M. Zhao S.C. Eustace B. Lappe M.M. Spitzer L. Zweier S. Braunschweiger K. Benchekroun Y. Hu X. Adair R. Chee L. FitzGerald M.G. Tulig C. Caruso A. Tzellas N. Bawa A. Franklin B. McGuire S. Nogues X. Gong G. Allen K.M. Anisowicz A. Morales A.J. Lomedico P.T. Recker S.M. Van Eerdewegh P. Recker R.R. Johnson M.L. Am. J. Hum. Genet. 2002; 70: 11-19Abstract Full Text Full Text PDF PubMed Scopus (1109) Google Scholar). These findings suggest that balanced function of LRP5 is critical to the normal oteoblastic proliferation and differentiation. Consistent with this notion is a recent report, in which overexpression of Wnt antagonist Dkk1 is associated with the presence of lytic bone lesions in patients with multiple myeloma (41Tian E. Zhan F. Walker R. Rasmussen E. Ma Y. Barlogie B. Shaughnessy Jr., J.D. N. Engl. J. Med. 2003; 349: 2483-2494Crossref PubMed Scopus (1306) Google Scholar). We have demonstrated that Wnt/β-catenin signaling is frequently activated in human osteosarcoma, the most common form of primary malignancy of bone (42Haydon R.C. Deyrup A. Ishikawa A. Heck R. Jiang W. Zhou L. Feng T. King D. Cheng H. Breyer B. Peabody T. Simon M.A. Montag A.G. He T.C. Int. J. Cancer. 2002; 102: 338-342Crossref PubMed Scopus (168) Google Scholar), suggesting that a tight regulation of Wnt/β-catenin activity is important for normal bone formation. The identification of early targets regulated by both BMP and Wnt signaling could lend insights into the molecular framework of early osteogenesis. In this report, we demonstrate that both BMP-9 and Wnt3A induce the activity of alkaline phosphatase (ALP), a well established early osteogenic marker, in mesenchymal stem cells. An expression profiling analysis of mesenchymal stem C3H10T1/2 cells stimulated by various osteogenic BMPs and Wnt3A revealed that under the most stringent analysis conditions CTGF is among the most significantly up-regulated genes by both BMP-9 and Wnt3A. CTGF (also known as Fisp12, Hcs24, ecogenin, βIG-M2, IGFBP8, and CCN2) is a member of the CCN (acronym for Cyr61, CTGF, and Nov) family that also includes Cyr61/CCN1, Nov/CCN3, WISP1/CCN4, WISP2/CCN5, and WISP3/CCN6 (43Lau L.F. Lam S.C. Exp. Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (584) Google Scholar, 44Moussad E.E. Brigstock D.R. Mol. Genet. Metab. 2000; 71: 276-292Crossref PubMed Scopus (447) Google Scholar, 45Blom I.E. Goldschmeding R. Leask A. Matrix Biol. 2002; 21: 473-482Crossref PubMed Scopus (332) Google Scholar, 46Brigstock D.R. Goldschmeding R. Katsube K.I. Lam S.C. Lau L.F. Lyons K. Naus C. Perbal B. Riser B. Takigawa M. Yeger H. Mol. Pathol. 2003; 56: 127-128Crossref PubMed Scopus (208) Google Scholar, 47Planque N. Perbal B. Cancer Cell Int. 2003; 3: 1-15Crossref PubMed Scopus (143) Google Scholar, 48Brigstock D.R. J. Endocrinol. 2003; 178: 169-175Crossref PubMed Scopus (452) Google Scholar). The CCN proteins are secreted cysteine-rich multimodular proteins (43Lau L.F. Lam S.C. Exp. Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (584) Google Scholar, 44Moussad E.E. Brigstock D.R. Mol. Genet. Metab. 2000; 71: 276-292Crossref PubMed Scopus (447) Google Scholar, 45Blom I.E. Goldschmeding R. Leask A. Matrix Biol. 2002; 21: 473-482Crossref PubMed Scopus (332) Google Scholar, 46Brigstock D.R. Goldschmeding R. Katsube K.I. Lam S.C. Lau L.F. Lyons K. Naus C. Perbal B. Riser B. Takigawa M. Yeger H. Mol. Pathol. 2003; 56: 127-128Crossref PubMed Scopus (208) Google Scholar, 47Planque N. Perbal B. Cancer Cell Int. 2003; 3: 1-15Crossref PubMed Scopus (143) Google Scholar, 48Brigstock D.R. J. Endocrinol. 2003; 178: 169-175Crossref PubMed Scopus (452) Google Scholar). In this study, we sought to determine the functional role of CTGF in osteoblast differentiation induced by BMP-9 or Wnt3A. We demonstrated that the expression of CTGF was induced at the early stage of BMP-9 and Wnt3A stimulation and returned to basal levels 5 days after stimulation. Most interestingly, RNAi-mediated knockdown of CTGF expression diminished the BMP-9-induced, but not Wnt3A-induced, osteogenic differentiation of mesenchymal progenitor cells. However, the constitutive overexpression of CTGF inhibited the osteoblast differentiation process initiated by BMP-9 or Wnt3A. Our findings demonstrate that CTGF may play an important role in promoting the proliferation of the early osteoblast progenitor cells, and that its activity has to be down-regulated during the terminal differentiation of the committed osteoblasts. This indicates that a balanced regulation of the CTGF gene expression may be essential to osteogenic differentiation and normal bone formation. Cell Culture and Chemicals—HEK 293 and C3H10T1/2 cell lines were obtained from the ATCC (Manassas, VA). HCT116 parental line and its CTNNB1 knockout derivatives were provided by Ken Kinzler and Bert Vogelstein of The Johns Hopkins Medical Institutions, Baltimore, and were maintained as described previously (49Chan T.A. Wang Z. Dang L.H. Vogelstein B. Kinzler K.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8265-8270Crossref PubMed Scopus (116) Google Scholar). HEK 293 cells were maintained in complete DMEM supplemented with 10% fetal calf serum (FCS, Mediatech, Herndon, VA), 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5% CO2. C3H10T1/2 cells were maintained in basal medium Eagle in Earle's balanced salt solution, supplemented with 10% FCS, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5% CO2. Unless indicated otherwise, all chemicals were purchased from Sigma or Fisher. Recombinant Adenoviral Vectors Expressing BMPs, Wnt3A, Oncogenic β-Catenin, and CTGF—Recombinant adenoviruses (AdBMPs) expressing human BMP-2, -6, and -9 (also known as GDF-2) were generated as described previously (11Cheng H. Jiang W. Phillips F.M. Haydon R.C. Peng Y. Zhou L. Luu H.H. An N. Breyer B. Vanichakarn P. Szatkowski J.P. Park J.Y. He T.C. J. Bone Jt. Surg. Am. 2003; 85: 1544-1552Crossref PubMed Scopus (837) Google Scholar, 50He T.C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3276) Google Scholar). For construction of the adenoviral vectors expressing Wnt3A, the coding region of mouse Wnt3A (kindly provided by Roel Nusse of Stanford University) was PCR-amplified and subcloned into pAdTrack-CMV, resulting in pAdTrack-Wnt3A. An expression cassette containing an oncogenic S33Y mutation of human β-catenin was subcloned into pAdTrack, resulting in pAdTrack-β-Cat*. The coding region of mouse CTGF was PCR-amplified and subcloned into pAdTrack-CMV, resulting in pAdTrack-CTGF. These shuttle vectors were used to generate recombinant adenoviruses (i.e. AdWnt3A, Adβ-Cat*, and AdCTGF) as described previously (50He T.C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3276) Google Scholar). All PCR-amplified fragments and cloning junctions were verified by DNA sequencing. For a control, we used an analogous adenovirus expressing only GFP (i.e. AdGFP) as described previously (28He T.C. Sparks A.B. Rago C. Hermeking H. Zawel L. da Costa L.T. Morin P.J. Vogelstein B. Kinzler K.W. Science. 1998; 281: 1509-1512Crossref PubMed Scopus (4121) Google Scholar, 50He T.C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3276) Google Scholar). Details about vector construction are of C3H10T1/2 cells HCT116 lines were cell for in complete medium supplemented with FCS, and with a of Adβ-Cat*, AdWnt3A, or At the indicated time after RNA was RNA to the RNA were used for target and to to mouse gene known genes and The and of were the with the The microarray were and to by the genes with in all and by the genes that an for all The analysis was by the C. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). and of RNA were used to generate for The was a and II The were and used as of CTGF was by analysis to the of the mouse or human CTGF complete of the is at The analysis was by the DNA The was as °C for for at °C for °C for and °C for with a of and at °C for °C for and °C for by a at °C The of was verified by analysis and confirmed by the was used as a from to were for All were by the expression of RNAi-mediated of CTGF Gene generate we the in vitro of RNA from the target a of with a at the of was used to mouse CTGF or The used are as for mouse CTGF, and and for GFP, and The were to RNA in vitro transcription The were to recombinant the of the was to the cells were with at °C for 15 and with The cells were with and with fetal calf serum at for by cells with fetal calf serum containing a for the cells were with for by the cells with for at The presence of CTGF was under a the primary or with were used as of phosphatase activity was by the as described previously (11Cheng H. Jiang W. Phillips F.M. Haydon R.C. Peng Y. Zhou L. Luu H.H. An N. Breyer B. Vanichakarn P. Szatkowski J.P. Park J.Y. He T.C. J. Bone Jt. Surg. Am. 2003; 85: 1544-1552Crossref PubMed Scopus (837) Google Scholar, 12Kang Q. Sun M.H. Cheng H. Peng Y. Montag A.G. Deyrup A.T. Jiang W. Luu H.H. Szatkowski J.P. Vanichakarn P. Park J.Y. Luo J. Li Y. Haydon R.C. He T.-C. Gene Ther. 2004; 11: 1312-1320Crossref PubMed Scopus (521) Google Scholar, Y. Kang Q. Cheng H. Li X. Sun M.H. Jiang W. Luu H.H. Park J.Y. Haydon R.C. He T.C. J. Cell. Biochem. 2003; 90: 1149-1165Crossref PubMed Scopus (169) Google Scholar). Cell cells were in at and with or At 15 after the HCT116 cells were under a and all HCT116 cells were In to determine C3H10T1/2 cells were by or HCT116 cells, C3H10T1/2 cells were to the HCT116 cells at various At after C3H10T1/2 cells, cell migration was under both and results from are Cell cells were at in cell and were with a of or At 15 after the cells were with At 1, and after the at the was under both and results from are cells were into the of and were with or at a At after cells were with DMEM containing and in the DMEM, medium at 37 5% for In to set the migration C3H10T1/2 cells were and in DMEM, 5 cells in of DMEM, were into which of types I and The cells were to for in a 5% 37 °C The was and the were in to cells. The cells the were in 10% for and in for to the were by that with a The was a with and to The of cells were by the cells in high The for were in and results are of the of BMP and Wnt3A-induced Osteoblast of a comprehensive analysis of the osteogenic activity of the 14 types of human BMPs, we demonstrated previously (11Cheng H. Jiang W. Phillips F.M. Haydon R.C. Peng Y. Zhou L. Luu H.H. An N. Breyer B. Vanichakarn P. Szatkowski J.P. Park J.Y. He T.C. J. Bone Jt. Surg. Am. 2003; 85: 1544-1552Crossref PubMed Scopus (837) Google Scholar, 12Kang Q. Sun M.H. Cheng H. Peng Y. Montag A.G. Deyrup A.T. Jiang W. Luu H.H. Szatkowski J.P. Vanichakarn P. Park J.Y. Luo J. Li Y. Haydon R.C. He T.-C. Gene Ther. 2004; 11: 1312-1320Crossref PubMed Scopus (521) Google that BMP-2, BMP-6, and BMP-9 the to induce osteoblast differentiation of mesenchymal progenitor cells in vitro as well as in shown in BMP-9 and Wnt3A induced a in alkaline phosphatase