Expression of Complex Junction Proteins in Hematopoietic Progenitor Cells.

The interaction of hematopoietic stem cells with the microenvironment plays a pivotal role in regulating self-renewal and differentiation. N-cadherin-based adherens junctions have been reported to be involved in this process (Puch et al., 2001; Zhang et al., 2003). However, the molecular composition of such junctions in hematopoietic progenitor cells (HPC) remains to be defined as well as their function. CD34⁺ cells and CD34⁺/CD38⁻ cells were isolated from human umbilical chord blood (CB). In immunoblotting, CD34⁺ cells were positive for the transmembrane proteins N- and E-cadherin, cadherin 11 (low amounts) and the cytoplasmic plaque proteins α- and β-catenin, protein p120^ctn, vinculin, spectrin/fodrin and actin as markers for adherens junctions as well as for ezrin, moesin and drebrin, actin-binding proteins known to be enriched in cellular protrusions. Other classical cadherins such as VE-, P-, R-cadherin or cadherin-6 were negative. Moreover HPCs were positive for the intermediate filament cytoskeletal protein vimentin, the integral heparan sulphate proteoglycans syndecan-1 (CD138), syndecan-2 (fibroglycan), syndecan-3 (N-syndecan) and syndecan-4 (ryudocan, amphiglycan) and the gap junction protein connexin 43. CD34⁺/CD38⁻ cells showed positive reactions with antibodies against vimentin, ezrin, vinculin, α- and β-catenin and with a pan cadherin antibody, with no obvious differences to CD34⁺/CD38⁺ and Kg1a cells. In parallel, we established single- and double-immunohistochemistry with antigen-retrieval-treated, formalin-fixed, paraffin-embedded bone marrow using peroxidase, alkaline phosphatase and glucose oxidase coupled secondary antibodies. The architecture of human, rat and bovine bone marrow tissue was analyzed with a panel of antibodies against components of adhering, gap and tight junctions. Immunohistochemical sections of bone marrow incubated with the various antibodies remarkably showed the presence of all these antigens in their natural microenvironment. Intercellular junctions of rat and bovine bone marrow were also observed with transmission electron microscopy. Our results show that HPC are equipped with components of cadherin-catenin-based adherens junctions. Immunoprecipitation and immunolocalisation experiments are underway to further elucidate the homo- and heterotypic intercellular junctional complexes between HPC and stromal cells in vitro and in vivo.