Wei Jiang

BACKGROUND: Bone morphogenic proteins (BMPs) are known to promote osteogenesis, and clinical trials are currently underway to evaluate the ability of certain BMPs to promote fracture-healing and spinal fusion. The optimal BMPs to be used in different clinical applications have not been elucidated, and a comprehensive evaluation of the relative osteogenic activity of different BMPs is lacking. METHODS: To identify the BMPs that may possess the most osteoinductive activity, we analyzed the osteogenic activity of BMPs in mesenchymal progenitor and osteoblastic cells. Recombinant adenoviruses expressing fourteen human BMPs (BMP-2 to BMP-15) were constructed to infect pluripotent mesenchymal progenitor C3H10T1/2 cells, preosteoblastic C2C12 cells, and osteoblastic TE-85 cells. Osteogenic activity was determined by measuring the induction of alkaline phosphatase, osteocalcin, and matrix mineralization upon BMP stimulation. RESULTS: BMP-2, 6, and 9 significantly induced alkaline phosphatase activity in pluripotential C3H10T1/2 cells, while BMP-2, 4, 6, 7, and 9 significantly induced alkaline phosphatase activity in preosteoblastic C2C12 cells. In TE-85 osteoblastic cells, most BMPs (except BMP-3 and 12) were able to induce alkaline phosphatase activity. The results of alkaline phosphatase histochemical staining assays were consistent with those of alkaline phosphatase colorimetric assays. Furthermore, BMP-2, 6, and 9 (as well as BMP-4 and, to a lesser extent, BMP-7) significantly induced osteocalcin expression in C3H10T1/2 cells. In C2C12 cells, osteocalcin expression was strongly induced by BMP-2, 4, 6, 7, and 9. Mineralized nodules were readily detected in C3H10T1/2 cells infected with BMP-2, 6, and 9 (and, to a lesser extent, those infected with BMP-4 and 7). CONCLUSIONS: A comprehensive analysis of the osteogenic activity of fourteen types of BMPs in osteoblastic progenitor cells was conducted. Our results suggest an osteogenic hierarchical model in which BMP-2, 6, and 9 may play an important role in inducing osteoblast differentiation of mesenchymal stem cells. In contrast, most BMPs are able to stimulate osteogenesis in mature osteoblasts.

Pluripotent mesenchymal stem cells (MSCs) are bone marrow stromal progenitor cells that can differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Several signaling pathways have been shown to regulate the lineage commitment and terminal differentiation of MSCs. Here, we conducted a comprehensive analysis of the 14 types of bone morphogenetic protein (BMPs) for their abilities to regulate multilineage specific differentiation of MSCs. We found that most BMPs exhibited distinct abilities to regulate the expression of Runx2, Sox9, MyoD, and PPARgamma2. Further analysis indicated that BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9 effectively induced both adipogenic and osteogenic differentiation in vitro and in vivo. BMP-induced commitment to osteogenic or adipogenic lineage was shown to be mutually exclusive. Overexpression of Runx2 enhanced BMP-induced osteogenic differentiation, whereas knockdown of Runx2 expression diminished BMP-induced bone formation with a decrease in adipocyte accumulation in vivo. Interestingly, overexpression of PPARgamma2 not only promoted adipogenic differentiation, but also enhanced osteogenic differentiation upon BMP-2, BMP-6, and BMP-9 stimulation. Conversely, MSCs with PPARgamma2 knockdown or mouse embryonic fibroblasts derived from PPARgamma2(-/-) mice exhibited a marked decrease in adipogenic differentiation, coupled with reduced osteogenic differentiation and diminished mineralization upon BMP-9 stimulation, suggesting that PPARgamma2 may play a role in BMP-induced osteogenic and adipogenic differentiation. Thus, it is important to understand the molecular mechanism behind BMP-regulated lineage divergence during MSC differentiation, as this knowledge could help us to understand the pathogenesis of skeletal diseases and may lead to the development of strategies for regenerative medicine.

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). 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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. 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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 (i.e. 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). 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

Marrow mesenchymal stem cells are pluripotent progenitors that can differentiate into bone, cartilage, muscle, and fat cells. Wnt signaling has been implicated in regulating osteogenic differentiation of mesenchymal stem cells. Here, we analyzed the gene expression profile of mesenchymal stem cells that were stimulated with Wnt3A. Among the 220 genes whose expression was significantly changed by 2.5-fold, we found that three members of the CCN family, CCN1/Cyr61, CCN2/connective tissue growth factor (CTGF), and CCN5/WISP2, were among the most significantly up-regulated genes. We further investigated the role of CCN1/Cyr61 in Wnt3A-regulated osteogenic differentiation. We confirmed that CCN1/Cyr61 was up-regulated at the early stage of Wnt3A stimulation. Chromatin immunoprecipitation analysis indicates that CCN1/Cyr61 is a direct target of canonical Wnt/beta-catenin signaling. RNA interference-mediated knockdown of CCN1/Cyr61 expression diminished Wnt3A-induced osteogenic differentiation. Furthermore, exogenously expressed CCN1/Cyr61 was shown to effectively promote mesenchymal stem cell migration. These findings suggest that tightly regulated CCN1/Cyr61 expression may play an important role in Wnt3A-induced osteoblast differentiation of mesenchymal stem cells.

Efficient osteogenic differentiation and bone formation from mesenchymal stem cells (MSCs) should have clinical applications in treating nonunion fracture healing. MSCs are adherent bone marrow stromal cells that can self-renew and differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. We have identified bone morphogenetic protein 9 (BMP-9) as one of the most osteogenic BMPs. Here we investigate the effect of insulin-like growth factor 2 (IGF-2) on BMP-9-induced bone formation. We have found that endogenous IGF-2 expression is low in MSCs. Expression of IGF-2 can potentiate BMP-9-induced early osteogenic marker alkaline phosphatase (ALP) activity and the expression of later markers. IGF-2 has been shown to augment BMP-9-induced ectopic bone formation in the stem cell implantation assay. In perinatal limb explant culture assay, IGF-2 enhances BMP-9-induced endochondral ossification, whereas IGF-2 itself can promote the expansion of the hypertropic chondrocyte zone of the cultured limb explants. Expression of the IGF antagonists IGFBP3 and IGFBP4 leads to inhibition of the IGF-2 effect on BMP-9-induced ALP activity and matrix mineralization. Mechanistically, IGF-2 is further shown to enhance the BMP-9-induced BMPR-Smad reporter activity and Smad1/5/8 nuclear translocation. PI3-kinase (PI3K) inhibitor LY294002 abolishes the IGF-2 potentiation effect on BMP-9-mediated osteogenic signaling and can directly inhibit BMP-9 activity. These results demonstrate that BMP-9 crosstalks with IGF-2 through PI3K/AKT signaling pathway during osteogenic differentiation of MSCs. Taken together, our findings suggest that a combination of BMP-9 and IGF-2 may be explored as an effective bone-regeneration agent to treat large segmental bony defects, nonunion fracture, and/or osteoporotic fracture.