Article

Biochemical characterization of Wnt-Frizzled interactions using a soluble, biologically active vertebrate Wnt protein

Johns Hopkins University, Baltimore, Maryland, United States
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 04/1999; 96(7):3546-51. DOI: 10.1073/pnas.96.7.3546
Source: PubMed

ABSTRACT Biochemical studies of Wnt signaling have been hampered by difficulties in obtaining large quantities of soluble, biologically active Wnt proteins. In this paper, we report the production in Drosophila S2 cells of biologically active Xenopus Wnt8 (XWnt8). Epitope- or alkaline phosphatase-tagged XWnt8 proteins are secreted by concentrated S2 cells in a form that is suitable for quantitative biochemical experiments with yields of 5 and 0.5 mg per liter, respectively. Conditions also are described for the production in 293 cells of an IgG fusion of the cysteine-rich domain (CRD) of mouse Frizzled 8 with a yield of 20 mg/liter. We demonstrate the use of these proteins for studying the interactions between soluble XWnt8 and various Frizzled proteins, membrane anchored or secreted CRDs, and a set of insertion mutants in the CRD of Drosophila Frizzled 2. In a solid phase binding assay, the affinity of the XWnt8-alkaline phosphatase fusion for the purified mouse Frizzled 8-CRD-IgG fusion is approximately 9 nM.

0 Bookmarks
 · 
61 Views
  • Source
  • [Show abstract] [Hide abstract]
    ABSTRACT: Lack of Sonic hedgehog (Shh) signaling, mediated by the Gli proteins, leads to severe pulmonary hypoplasia. However, the precise role of Gli genes in lung development is not well established. We show Shh signaling prevents Gli3 proteolysis to generate its repressor forms (Gli3R) in the developing murine lung. In Shh−/− or cyclopamine-treated wild-type (WT) lung, we found that Gli3R level is elevated, and this upregulation appears to contribute to defects in proliferation and differentiation observed in the Shh−/− mesenchyme, where Gli3 is normally expressed. In agreement, we found Shh−/−;Gli3−/− lungs exhibit enhanced growth potential. Vasculogenesis is also enhanced; in contrast, bronchial myogenesis remains absent in Shh−/−;Gli3−/− compared with Shh−/− lungs. Genes upregulated in Shh−/−;Gli3−/− relative to Shh−/− lung include Wnt2 and, surprisingly, Foxf1 whose expression has been reported to be Shh-dependent. Cyclins D1, D2, and D3 antibody labelings also reveal distinct expression patterns in the normal and mutant lungs. We found significant repression of Tbx2 and Tbx3, both linked to inhibition of cellular senescence, in Shh−/− and partial derepression in Shh−/−; Gli3−/− lungs, while Tbx4 and Tbx5 expressions are less affected in the mutants. Our findings shed light on the role of Shh signaling on Gli3 processing in lung growth and differentiation by regulating several critical genes.
    Developmental Biology 06/2004; 270(1):214-231. DOI:10.1016/S0012-1606(04)00169-1 · 3.64 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Mesenchymal stem cells (MSCs) are a potential source of chondrogenic cells for the treatment of cartilage disorders, but loss of chondrogenic potential during in vitro expansion and the propensity of cartilage to undergo hypertrophic maturation impede their therapeutic application. Here we report that the signaling protein WNT3A, in combination with FGF2, supports long-term expansion of human bone marrow-derived MSCs. The cells retained their chondrogenic potential and other phenotypic and functional properties of multipotent MSCs, which were gradually lost in the absence of WNT3A. Moreover, we discovered that endogenous WNT signals are the main drivers of the hypertrophic maturation that follows chondrogenic differentiation. Inhibition of WNT signals during differentiation prevented calcification and maintained cartilage properties following implantation in a mouse model. By maintaining potency during expansion and preventing hypertrophic maturation following differentiation, the modulation of WNT signaling removes two major obstacles that impede the clinical application of MSCs in cartilage repair.

Preview

Download
0 Downloads
Available from