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

Abstract

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 ␤-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.

CCAAT/Enhancer-binding protein homologous protein (CHOP) decreases bone formation and causes osteopenia

“…It is not unusual for intracellular and extracellular proteins to be necessary for a biological effect, but when expressed in excess to be inhibitory. For example, Hes-1 is necessary for adipogenesis, but its overexpression inhibits adipogenesis, twisted gastrulation and connective tissue growth factor are necessary for BMP effects on osteoblastogenesis, but when in excess they can be inhibitory [48][49][50][51]. Similarly, CHOP is necessary for normal osteoblastic function, but when in excess, and under specific conditions in vivo, it may interact with intracellular proteins necessary for osteoblastic function and become inhibitory.…”

Osteogenic BMPs promote tumor growth of human osteosarcomas that harbor differentiation defects

“…29 We previously demonstrated that CTGF is upregulated at the early stage of osteogenic differentiation. 29 We analyzed the basal CTGF expression in human OS cells.…”

“…29 We previously demonstrated that CTGF is upregulated at the early stage of osteogenic differentiation. 29 We analyzed the basal CTGF expression in human OS cells. As shown in Figure 2c, almost all of the tested primary OS lines exhibited higher expression levels of CTGF than that of hFOB1.19, whereas MG63 (and to a lesser extent, U2OS) also had high basal level of CTGF expression.…”

“…19,20,[24][25][26] We have further demonstrated that osteogenic BMPs regulate a distinct set of target genes, such as Id HLH factors and connective tissue growth factor (CTGF)/CCN2, during osteogenic differentiation. 19,20,[27][28][29] Blockade of stem cell differentiation may lead to tumorigenesis. 3,30 The stem cell features of tumor cells have been confirmed by a recent study, in which silencing of Ewing's sarcoma (EWS)-FLI-1 in Ewing's tumor cells leads to their recovery of MSC features, such as differentiating along adipogenic and osteogenic lineages, 31 whereas expression of the EWS/FLI-1 oncogene in murine MSCs results in EWSlike tumors.…”

A protocol for rapid generation of recombinant adenoviruses using the AdEasy system

“…The key step is the generation of recombinants in AdEasier cells. Once these recombinants are obtained, generation of adenoviruses in HEK-293 cells is virtually guaranteed [23][24][25][26][27] . Depending on the transfection efficiency of HEK-293 cells, one can expect titers in the initial lysate to be 10 8 -10 10 pfu ml À1 .…”