Functional differences between two Tie2 ligands, angiopoietin-1 and -2, in regulation of adult bone marrow hematopoietic stem cells
Received 7 September 2009; received in revised form 6 November 2009; accepted 20 November 2009. published online 30 November 2009.
Objective
Angiopoietin-1 (Ang-1) plays a critical role in the maintenance of hematopoietic stem cells (HSCs) in the bone marrow (BM) through its binding to the Tie2 receptor. Ang-2, another Tie2 ligand, is known to be an antagonist of Tie2/Ang-1 signaling in angiogenesis; however, its function in regulation of HSCs remains unclear. Here, we investigated the functional differences between Ang-1 and Ang-2 in the maintenance of HSCs.
Materials and Methods
We treated mouse BM lineage−Sca-1+c-Kit+ side population+ cells with Ang-1 and/or Ang-2, and evaluated angiopoietin function by gene expression analysis, immunocytochemical staining of phosphorylated Akt, a colony-formation assay, and a long-term BM reconstitution assay.
Results
Gene expression analysis and BM transplantation assay revealed that Ang-1 upregulated expression of p57, p18, Itgb1, Alcam, Tie2, Hoxb4, and Bmi1 genes in HSCs, while Ang-2 antagonized the effects of Ang-1. Ang-1 enhanced the phosphorylation of Akt, while Ang-2 again reduced the effect of Ang-1. The colony assay demonstrated that neither Ang-1, nor Ang-2 influenced the colony formation of HSCs. BM transplantation assay, following in vitro cultivation of HSCs with angiopoietins, showed that Ang-1 maintained long-term repopulating activity of HSCs, while the addition of Ang-2 interfered drastically with the effects of Ang-1.
Conclusion
Gene expression analysis and BM transplantation assay demonstrated that Ang-1 maintained HSC activity in an in vitro culture. In contrast, Ang-2 reversed the effects of Ang-1/Tie2 signaling in the regulation of long-term HSCs. Our data suggest that Ang-1 is a dominant ligand for the Tie2 receptor in long HSCs in BM.
Department of Cell Differentiation, The Sakaguchi Laboratory of Developmental Biology, School of Medicine, Keio University, Tokyo, Japan
Offprint requests to: Fumio Arai, D.D.S., Ph.D., Department of Cell Differentiation, The Sakaguchi Laboratory of Developmental Biology, School of Medicine, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan
ABSTRACT