The Wnt Signaling Inhibitor Dickkopf-1 Is Required for Reentry into the Cell Cycle of Human Adult Stem Cells from Bone Marrow

Abstract

Adult human mesenchymal stem cells from bone marrow stroma (hMSCs) differentiate into numerous mesenchymal tissue lineages and are attractive candidates for cell and gene therapy. When early passage hMSCs are plated or replated at low density, the cultures display a lag phase of 3-5 days, a phase of rapid exponential growth, and then enter a stationary phase without the cultures reaching confluence. We found that as the cultures leave the lag phase, they secrete high levels of dickkopf-1 (Dkk-1), an inhibitor of the canonical Wnt signaling pathway. The addition of recombinant Dkk-1 toward the end of the lag period increased proliferation and decreased the cellular concentration of ␤-catenin. The addition of antibodies to Dkk-1 in the early log phase decreased proliferation. Also, expression of Dkk-1 in hMSCs decreased during cell cycle arrest induced by serum starvation. The results indicated that high levels of Dkk-1 allow the cells to reenter the cell cycle by inhibiting the canonical Wnt/␤-catenin signaling pathway. Since antibodies to Dkk-1 also increased the lag phase of an osteosarcoma line that expressed the gene, Dkk-1 may have a similar role in some other cell systems.Human bone marrow contains two main populations of stem cells: hematopoietic stem cells usually identified by a CD 34 ϩ phenotype and a population of CD 34 Ϫ cells of mesenchymal origin. The population of human nonhematopoietic mesenchymal stem cells or marrow stromal cells (hMSCs) 1 can differentiate into numerous mesenchymal tissue lineages including osteoblasts, chondrocytes, adipocytes, and neural precursors (1-8). hMSCs are easily obtained from bone marrow aspirates and are readily separated from hematopoietic cells by virtue of their adherence to tissue culture plastic (1). Under the appropriate conditions, hMSCs can be propagated manifold in vitro while retaining their multipotentiality, a feature that makes them attractive candidates for stem cell and gene therapy (2, 5, 9 -12). Although some of the in vitro growth characteristics of hMSCs have been documented, the molecular mechanisms by which hMSCs regulate their own growth in culture are poorly understood. In particular, there is no apparent explanation for the observation that when early passage hMSCs are replated at low density, they display a lag period of 3-5 days, followed by a phase of rapid exponential growth, and then enter a stationary phase without reaching confluence (8,11,13).Preliminary observations (15) suggested to us that conditioned medium from cultures of hMSCs increased the rate of proliferation when added to freshly plated cultures of hMSCs.In the experiments described here, we demonstrate that hMSCs in the early log phase of growth synthesize and secrete dickkopf-1 (Dkk-1), an inhibitor of the canonical Wnt pathway (16 -18).The Wnt signaling pathway controls patterning and cell fate determination in the development of a wide range of organisms, from Drosophila to mammals (19). The signaling can occur by at least three different pathways (20). In the canonic...

A potential role for Dkk-1 in the pathogenesis of osteosarcoma predicts novel diagnostic and treatment strategies

“…Although the data presented here demonstrate that Dkk-1 may contribute to OS pathogenesis by preventing repair of the surrounding osteoid as the tumour expands, Dkk-1 may act in an autocrine manner on the tumour cells too. Noteworthy are the observations that exposure of MG63 and LS1 OS cells to high concentrations of BIO reduces proliferation ( Figure 2F), and immunosequestration of Dkk-1 transiently slowed the proliferation of MG63 cells in vitro (Gregory et al, 2003). It is unlikely that Dkk-1 can solely act as a mitogen, but its presence at a critical threshold may serve as a checkpoint that permits proliferation of OS cells.…”

“…The inhibitor Dkk-1 was depleted from the medium by antibody incubation and protein A/G-mediated depletion as previously described (Gregory et al, 2003). Immunodepletion was carried out using the rabbit anti-Dkk-1 polyclonal antiserum described in Gregory et al (2003) or the goat anti-Dkk-1 polyclonal acquired from R&D Systems.…”

“…Immunodepletion was carried out using the rabbit anti-Dkk-1 polyclonal antiserum described in Gregory et al (2003) or the goat anti-Dkk-1 polyclonal acquired from R&D Systems. Protein A (for rabbit) and protein G (for goat) were conjugated to sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ, USA).…”

“…Given that high levels of Dkk-1 are also expressed by rapidly dividing osteosarcoma (OS) cells in vitro (Gregory et al, 2003(Gregory et al, , 2005a, we measured the level of Dkk-1 in the serum of paediatric patients with OS (median age, 13.4 years) and found it to be significantly elevated when compared with healthy controls. The aim of this study therefore, was to investigate the significance of such a finding in OS.…”

Thiazolidinediones increase the wingless-type MMTV integration site family (WNT) inhibitor Dickkopf-1 in adipocytes: a link with osteogenesis

“…The major pathway for regulating cell fate and committing mesenchymal stem cells to osteoblasts is the canonical wingless-type MMTV integration site family (WNT) signalling pathway [7]. Activation of this pathway suppresses adipogenesis and promotes osteoblast formation.…”