CCN1/Cyr61 Is Regulated by the Canonical Wnt Signal and Plays an Important Role in Wnt3A-Induced Osteoblast Differentiation of Mesenchymal Stem Cells

Abstract

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/␤-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.

Implication of dysregulation of the canonical wingless-type MMTV integration site (WNT) pathway in diabetic nephropathy

“…It is emerging that anti-TGF-β antibody and a CTGF antisense oligonucleotide ameliorate renal fibrosis [43,44]. Our results suggest that an antagonist of WNT signalling may have advantages over those treatments targeting individual growth factors, because WNT signalling regulates the production of a spectrum of pathogenic factors of diabetic retinopathy, including VEGF, CTGF, ICAM-1, TNF-α, Twist homologue 1 (TWIST) and matrix metalloproteinase 7 [10,14,37,45]. This notion is substantiated by previous reports demonstrating that dickkopf homologue 1 (DKK1) and secreted frizzled-related protein 4 (SFRP4) were able to ameliorate renal fibrosis [14,46].…”

“…The canonical WNT signalling pathway is known to mediate inflammation, angiogenesis and fibrosis through upregulation of intercellular adhesion molecule 1 (ICAM-1), TNF-α, VEGF and CTGF, which are all products of WNT target genes [10,18]. It has been reported that WNT1 protein levels are increased in the podocytes of human kidney biopsies from patients with diabetic nephropathy [16].…”

“…Inactivation of the 'destructive complex' prevents the proteasomal degradation of the transcription factor β-catenin and promotes its accumulation and nuclear translocation. Once β-catenin translocates into the nucleus, it dimerises with T cell factor (TCF) and regulates transcription of WNT target genes, including those encoding cyclin D1, vascular endothelial growth factor (VEGF), c-Myc and connective tissue growth factor (CTGF) [8][9][10].…”

Wnt signaling and skeletal development

“…Wnt3a has been reported to induce ALP activity in C3H10T1/2 cells through transient transfection [53,87], conditional medium treatment [77,[95][96][97], purified protein treatment [96], adenoviral treatment [98], retroviral infection [99]; (in ST2 cells) transient transfection [53,87], conditional medium treatment [86,97]; (in C2C12 cells) transient transfection [87], or conditional medium treatment [97]. Wnt3a can also enhance ALP activity and BSP in conditional medium, enhance osteocalcin expression in ST2 cells in nondifferentiation medium, and increase mineralization in differentiation medium [86].…”

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 .…”