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N-Cadherin in Human Bone Marrow 1569 Antigens Were Washed Several Times and Dissolved by Boiling in SDS- Cadherin in Methylcellulose-Containing Cultures

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N-Cadherin in Human Bone Marrow 1569 Antigens Were Washed Several Times and Dissolved by Boiling in SDS- Cadherin in Methylcellulose-Containing Cultures RESEARCH ARTICLE 1567 N-cadherin is developmentally regulated and functionally involved in early hematopoietic cell differentiation Sabine Puch1, Sorin Armeanu1, Christine Kibler1, Keith R. Johnson2, Claudia A. Müller1, Margaret J. Wheelock2 and Gerd Klein1,* 1University Medical Clinic, Section for Transplantation Immunology and Immunohematology, 72072 Tübingen, Germany 2University of Toledo, Dept of Biology, Ohio 43606, USA *Author for correspondence (e-mail: [email protected]) Accepted 24 January 2001 Journal of Cell Science 114, 1567-1577 © The Company of Biologists Ltd SUMMARY The cadherins, an important family of cell adhesion Treatment of CD34+ progenitor cells with function- molecules, are known to play major roles during embryonic perturbing N-cadherin antibodies drastically diminished development and in the maintenance of solid tissue colony formation, indicating a direct involvement of architecture. In the hematopoietic system, however, little is N-cadherin in the differentiation program of early known of the role of this cell adhesion family. By RT-PCR, hematopoietic progenitors. N-cadherin can also mediate western blot analysis and immunofluorescence staining we adhesive interactions within the bone marrow as show that N-cadherin, a classical type I cadherin mainly demonstrated by inhibition of homotypic interactions of expressed on neuronal, endothelial and muscle cells, is bone-marrow-derived cells with N-cadherin antibodies. expressed on the cell surface of resident bone marrow Together, these data strongly suggest that N-cadherin is stromal cells. FACS analysis of bone marrow mononuclear involved in the development and retention of early cells revealed that N-cadherin is also expressed on a hematopoietic progenitors within the bone marrow subpopulation of early hematopoietic progenitor cells. microenvironment. Triple-color FACS analysis defined a new CD34+ CD19+ N- + cadherin progenitor cell population. During further Key words: Hematopoiesis, Cadherin superfamily, Catenin, Cell differentiation, however, N-cadherin expression is lost. differentiation, Cell adhesion INTRODUCTION of E- and P-cadherin. Switches from E- to N-cadherin expression can be observed during development, for example, Classical cadherins comprise a family of functionally and during neurulation (Hatta et al., 1986; Duband et al., 1988). structurally closely related molecules that mediate calcium- The importance of N-cadherin expression during early dependent cellular adhesive interactions (Kemler, 1992; Geiger development has been demonstrated in mice carrying a null and Ayalon, 1992). The classical cadherins including E-, P- mutation for N-cadherin. The mutant embryos die early during and N-cadherin possess similar extracellular domains for development around day 10 (Radice et al., 1997). An even homophilic cell binding and Ca2+-binding, as well as similar earlier embryonic lethality at the preimplantation stage has cytoplasmic domains for the association with the cytoskeleton been reported for E-cadherin-deficient mice (Larue et al., 1994; (Grunwald, 1993; Kemler, 1993). The connection to the Riethmacher et al., 1995). These two examples highlight the cytoskeleton is mediated via a group of molecules known as significance of the classical cadherins during embryonic the catenins, and for classical cadherin-mediated adhesive development. interactions, functional cadherin-catenin complexes are In the hematopoietic system, however, where common stem essential (Aberle et al., 1996). Cell adhesion mediated by cells constantly undergo self-renewal as well as differentiation cadherins occurs mainly in a homophilic homotypic manner, into the various myeloid, erythroid and lymphoid blood cells, but an involvement of cadherins in heterophilic and even there have been few reports on the expression and functions of heterotypic interactions has been demonstrated (Tang et al., cadherins on lymphocytes (Lee et al., 1994; Cepek et al., 1996; 1993; Cepek et al., 1994; Higgins et al., 1998). Cadherins Tsutsui et al., 1996; Muller et al., 1997; Kawamura-Kodama et have been shown to play important roles during embryonic al., 1999). Cell adhesion molecules of other families such as development, tumorogenesis and the maintenance of tissue integrins or members of the immunoglobulin superfamily have architecture (Takeichi, 1991; Takeichi, 1993; Ranscht, 1994). been more intensively analyzed (Springer, 1990). Recently we The members of the classical cadherin family show specific showed that E-cadherin can be detected on human bone spatiotemporal expression patterns during development and marrow mononuclear cells, but the expression of E-cadherin in the adult tissues. Whereas E- and P-cadherin are often is restricted to distinct developmental stages of one single codistributed, N-cadherin is mostly found on cell types devoid cell lineage, the erythrocytes. Other bone-marrow-derived 1568 JOURNAL OF CELL SCIENCE 114 (8) cell types or stromal cells from the hematopoietic initial denaturation at 95°C for 1 minute, 33 cycles of denaturation microenvironment that support the maturation of the (94°C, 1 minute), annealing (60°C, 1 minute) and extension (72°C, 1 hematopoietic progenitor cells do not express E-cadherin minute) and a final polymerisation step at 72°C for 10 minutes. The (Armeanu et al., 1995; Bühring et al., 1996). PCR product of the expected size was analyzed by gel electrophoresis In this study we have analyzed the expression of N-cadherin, in a 2% agarose gel, purified using the PCR purification kit (Qiagen) another member of the classical cadherin family, on human and sequenced with the ABI PRISM DyeDeoxy terminator kit (Perkin-Elmer). bone-marrow-derived cells. N-cadherin is known to be expressed in neural tissues, but also in many nonneural cells, Antibodies such as muscle tissue, endothelial cells and fibroblasts Two different monoclonal antibodies against human N-cadherin were (Takeichi, 1988; Knudsen et al., 1990; Wheelock and Knudsen, used. The antibody 8C11 recognizes an extracellular domain, whereas 1991; Salomon et al., 1992; Soler et al., 1994; Knudsen et al., the antibody 13A9 is directed against a cytoplasmic domain of N- 1995). We show that nonhematopoietic bone marrow stromal cadherin (Knudsen et al., 1995; Sacco et al., 1995). For two-color cells and a subpopulation of hematopoietic progenitor cells, but FACS analysis the antibody 8C11 was biotinylated with a solution of not later developmental stages within the human bone marrow, 1 mg/ml N-thiosuccinimide biotin ester (Calbiochem, La Jolla, CA) µ µ express N-cadherin. The involvement of N-cadherin in the in dimethyl sulfoxide. 100 g/100 l of the antibody in sodium borate differentiation process of early progenitor cells was analyzed buffer, pH 9.3, were incubated for 2 hours at room temperature with 10 µl of the biotin ester. Biotinylation was stopped by addition of 20 by antibody treatment in a colony-forming assay. N-cadherin µl 1M glycine. The biotinylated antibody was purified by gel filtration is also functionally involved in adhesive homophilic- on a G-25 sephadex column (Amersham-Pharmacia). For cell sorting homotypic interactions of bone-marrow-derived cells. Since by FACS, for colony forming assays and for cell adhesion inhibition the expression of cytoplasmic catenins is a prerequisite for studies, the commercially available monoclonal antibody GC-4, cellular interactions mediated via classical cadherins, the which reacts with the N-terminal half of the extracellular domain of presence of α-, β- and γ-catenin was also analyzed in the N- both human and chicken N-cadherin, was used (Sigma, Deisenhofen, cadherin-positive cell types. Germany). The two rabbit antisera against human α- and β-catenin were also purchased from Sigma. The monoclonal antibody generated against mouse γ-catenin crossreacts with its human homolog and was available from Transduction Laboratories (Dianova, Hamburg, MATERIAL AND METHODS Germany). Cells and cell cultures Isolation of CD34+ progenitor cells by MiniMACS Bone marrow aspirates were obtained after informed consent from CD34+ progenitor cells were isolated from bone marrow mononuclear hematologically normal donors undergoing harvest for allogeneic cells using the MiniMACS cell isolation kit (Miltenyi Biotec, bone marrow transplantation. Bone marrow mononuclear cells were Bergisch Gladbach, Germany). Briefly, 5×107 mononuclear cells were isolated by collecting cells from the interface on a Percoll cushion resuspended in 300 µl buffer containing PBS with 0.5% BSA and 5 (1.077 g/ml) after density gradient centrifugation. Long term bone mM EDTA. 100 µl reagent A1 (human Ig) and 100 µl reagent A2 marrow cultures (LTMCs) were established in RPMI 1640 culture (monoclonal hapten-conjugated anti-CD34 antibody) were added and medium containing 12.5% FCS, 12.5% horse serum and 10−6 mol/l incubated for 15 minutes at 6°C. Labeled cells were washed carefully, hydrocortisone. The medium was changed every week. For centrifuged and resuspended in 400 µl buffer mentioned above. immunofluorescence staining, bone marrow mononuclear cells were 100 µl reagent B (colloidal superparamagnetic MACS microbeads grown in 24-well plates on glass cover slides. The human stromal cell conjugated to an anti-hapten antibody) were added and cells were line L88/5 (kindly provided by Prof. Dörmer, GSF, Institute for incubated again for 15 minutes at 6°C. Then the cells were washed Experimental Hematology, Munich, Germany), was cultured under and resuspended in 500 µl buffer. Magnetic separation
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