HSC differentiation occurs in direct proximity to osteoblasts (OBs) with in the bone marrow cavity. Using a novel cell blotting system in which OB membranes were run on non-denaturing discontinuous gels and blotted with labeled HSC cell lines, two major adhesive bands were identified. The two proteins were identified as annexin II monomers and multimers.
Immunohistochemistry and laser capture and microarray studies demonstrated that annexin II is preferentially expressed at endosteal surfaces and endothelial cells. Antibody studies against annexin II blocked the adhesion of HSC to OB in vitro. siRNA knockdown and over-expression studies of annexin II resulted in either reduced or enhanced binding characteristics, respectively, as expected. These findings were verified using primary human CD34+ bone marrow cells and human OBs where the binding of the progenitor cells and CFU-activities were linked with annexin II. Peptide mapping demonstrated that the majority of the HSC binding activity resides in the N-terminal aspect of the peptide.
To assess the role of annexin II in hematopoietic reconstitution, lethally irradiated mice were transplanted with bone marrow cells in the presence or absence of antibody to annexin II, antibody to the p11 annexin II binding partner, or isotype matched controls. Additional groups included use of the N-terminal p1-12 annexin II synthetic peptide or control. Animals that did not receive any marrow cells died within 14 days. Of the mice transplanted with 2x10^5 bone marrow cells, 1/8 animals died at day 11, representing an 80% survival rate. In contrast, when mice were transfused with the same number of marrow cells together with antibody to annexin II, none of the mice survived; the control treated animals survived (n = 8). Equivalent antibody doses against the annexin II binding partner p11 decreased the survival rate of the treated animals compared to IgG controls. However this treatment was less effective in inhibiting survival compared to the antibody to annexin II. Like wise, the survival of mice treated with two different doses of the p1-12 peptides at two different cell concentrations was significantly relative to the groups treated with a scrambled peptide control. These data suggest that the N-terminal twelve amino acids of annexin II facilitate stem cell homing to the marrow. Together these in vitro and in vivo data suggest a novel role for annexin II in the marrow microenvironment.