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 was performed the same conditions as the LTMC (Thalmeier et al., 1994). The by applying labeled cells on positive selection columns. Columns hematopoietic cell lines listed in Table 1 were purchased either were washed repeatedly with buffer and CD34+ cells could be eluted from the German Collection of Microorganisms and Cell Cultures by removing columns from the magnetic field. (DSMZ, Braunschweig, Germany) or from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured as Immunoblot/immunoprecipitation recommended by the suppliers. Protein extracts from CD34+ progenitor cells isolated by MiniMACS, from LTMC and the stromal cell line L88/5 and from different Reverse transcriptase and polymerase chain reaction hematopoietic cell lines were obtained by incubating the cells for 30 Total RNA was obtained from freshly isolated bone marrow minutes on ice in extraction buffer containing 1% Triton-X100, 1% mononuclear cells, from LTMC and from stromal or hematopoietic NP-40, 1 mM CaCl2, 1 mM MgCl2, 150 mM NaCl and 50 mM Tris- cell lines using the RNeasy total RNA kit (Qiagen, Hilden, Germany). HCl pH 7.6. Proteins were separated on 10% polyacrylamide gels and Potential DNA contaminations of the RNA preparations were transferred to nitrocellulose filters. Nonspecific protein binding sites removed by DNase digestion (Roche) followed by RNA precipitation were blocked with 2% blocking reagent (Roche). The filters were with ethanol. cDNA was synthesized from purified RNA by reverse probed for 1 hour with the primary antibodies against N-cadherin or transcription with MuMLV-reverse transcriptase (Roche) and the different catenins and subsequently washed for 15 minutes with oligo(dT)12-18 (Amersham-Pharmacia, Freiburg, Germany) for 1 hour PBS. Bound antibodies were detected by either peroxidase-conjugated at 37°C. PCR was performed with the cDNAs and a specific primer rabbit α-mouse or swine α-rabbit immunoglobulins (DAKO, pair which flank an extracellular domain of human N-cadherin Hamburg, Germany), followed by colorimetric reaction with the (base 942 to 1298 of the cDNA sequence EMBL M34064; Reid FastDAB system (Sigma). and Hemperly, 1990). Specificity of the selected primer pair was For nonradioactive immunoprecipitations, the cell lysates were first controlled with the HUSAR/GCG program (EMBL, Heidelberg, incubated with protein-G sepharose for 1 hour for preclearing the Germany). Amplification was carried out with 0.3 µM of each primer, lysates. After centrifugation, the immune complexes were formed by 0.4 mM of each dNTP and 2U AmpliTaq DNA polymerase (Perkin- addition of the antibodies to the supernatants, followed by addition of Elmer, Weiterstadt, Germany). The cycling program consisted of an protein G-sepharose. After rotation for 1 hour at 4°C, the precipitated N-cadherin in human bone marrow 1569 antigens were washed several times and dissolved by boiling in SDS- cadherin in methylcellulose-containing cultures. For these studies, the PAGE sample buffer. After separation in 10% polyacrylamide gels the monoclonal antibody GC-4, a murine IgG1, was used at different precipitated antigens were detected by immunoblotting using concentrations (stock solution 3.7 µg/µl). Isotype control IgG1 antibodies against α-catenin, β-catenin or N-cadherin. antibodies (stock solution 0.1 µg/µl) were from Caltag Laboratories (Burlingame, CA). The murine antibody W6/32.HL (stock solution Immunofluorescence staining 0.325 µg/µl) of the IgG2a subtype, which recognizes HLA-A, -B and 5 µm bone marrow cryostat sections, long term bone marrow cultures -C molecules, was used as another control antibody that binds to a and the stromal cell line L88/5 grown on glass cover slides, as well control antigen on the cell surface of progenitor cells. 2×103 isolated as cytospin preparations of different hematopoietic suspension cells CD34+ cells were preincubated with the respective antibodies at were fixed with an ice-cold acetone/methanol mixture (1:1) for 5 different concentrations in 100 µl RPMI 1640 for 30 minutes at 37°C + minutes, washed with PBS and blocked with 1% BSA in PBS and 5% CO2. As a positive control, CD34 cells were incubated in (blocking buffer) for 20 minutes. The samples were incubated for 45 RPMI 1640 medium without antibody treatment. Cell suspensions minutes with the primary antibodies diluted in blocking buffer. After were then mixed with 1.5 ml methylcellulose media containing washing three times with PBS, bound antibodies were detected by recombinant cytokines. These media consisted of 0.9% incubating the samples with Cy3TM-conjugated rabbit α-mouse methylcellulose, 30% fetal bovine serum, 1% BSA, 10−4 M 2- antibodies (Dianova). Cell nuclei were identified by counterstaining mercaptoethanol, 2 mM L-glutamine, 50 ng/ml recombinant human with DAPI (1 µg/ml). Controls were performed by omitting the first stem cell factor, 10 ng/ml recombinant human GM-CSF, 10 ng/ml antibody. Photographs were taken on a Zeiss axiophot microscope. recombinant human interleukin 3 with or without 3 units/ml recombinant human erythropoietin (MethoCultTM H4434 with FACS analysis erythropoietin or MethoCultTM H4534 without erythropoietin; Cells were labeled with the individual antibodies as described recently StemCell Technologies Inc., Vancouver, Canada). Cultures were (Armeanu et al., 1995). For single-color immunofluorescence, the plated in duplicates in 30 mm Petri dishes and incubated in a fully antibody QBEND10 for CD34 (Immunotech, Hamburg, Germany) humidified atmosphere at 37°C and 5% CO2. Cell aggregates was used. The antibody W6/32. HL, which recognizes an epitope of containing more than 50 cells were scored as single colonies after 14 the heavy chain of MHC class I antigens, was used as a positive days of incubation under an Axiovert microscope (Zeiss, Oberkochen, control. As a negative control, the cells were labeled with the antibody Germany). Colonies were counted on the basis of morphological W6/32.HK, an inactive variant of W6/32.HL (Barnstable et al., 1978). criteria, and all colonies were pooled for reporting total CFU-C By excluding propidium iodide permeable cells only living cells were counts. analyzed. For two-color FACS analysis, the bone marrow cells were labeled Cell adhesion assays with the biotinylated 8C11 antibody and detected with streptavidin- The KG1a cell line, which has the strong tendency to form cell PE (SA-PE; Sigma) in combination with the FITC-conjugated aggregates in culture was used to analyze homotypic cell interactions. antibodies against CD3, CD19 and CD34 (Dianova). For three-color 5×105 KG1a cells were resuspended in 1ml of RPMI 1640 culture staining, Percoll-separated bone marrow cells were incubated in a first medium by repeated pipetting and placed in wells of a 24-well tissue step with the anti-N-cadherin antibody 8C11 (IgG1), the anti-CD34 culture plate. Inhibition studies were performed by incubating the antibody ICH3 (IgG2a; Caltag Laboratories, Burlingame, CA, USA) resuspended KG1a cells with the anti-N-cadherin antibody GC-4. The and the anti-CD19 antibody B4 (IgG2b; Caltag Laboratories). In a calcium-dependency of the cell aggregation process was analyzed by second step, cells were incubated with the isotype-specific second addition of 10 mM EDTA to the culture medium. During the following antibodies goat anti-mouse IgG1-Cy5, goat anti-mouse IgG2a-FITC hours, cell aggregation was observed under an Axiophot microscope and goat anti-mouse IgG2b-PE (Caltag Laboratories). Appropriate background stainings were performed with the corresponding control antibodies. For data acquisition a FACScan or a FACSCalibur flow cytometer (Becton Dickinson, Heidelberg, Germany) were used. Cell sorting For cell sorting, mobilized peripheral blood progenitor cell preparations obtained by leukapheresis were used (Brugger et al., 1996). These cell preparations were separated on a discontinuous Percoll gradient. The gradient consisted of 3 ml of each d1=1.054 g/ml, d2=1.066 g/ml and d3=1.077 g/ml Percoll solutions (Biochrom, Berlin, Germany). After centrifugation for 20 minutes at 1500 g, cells enriched at the interface between d1 and d2 were collected and washed twice with PBS. The isolated cells were labeled with the N- cadherin antibody GC-4 followed by incubation with FITC- Fig. 1. RT-PCR analysis of N-cadherin expression on human bone- conjugated sheep anti-mouse antiserum (Dianova). marrow-derived cells. mRNA coding for human N-cadherin was Cell sorting was performed on a FACSCalibur with the CellQuest detected by specific amplification of a 357 bp cDNA fragment software (Becton Dickinson). The sort window was set for the FITC- (arrowhead) in reverse-transcribed total RNA preparations derived labeled cells. Non-viable cells were excluded by using propidium from bone marrow mononuclear cells (lane 1), LTMC (lane 2), the iodide. In one run at least 104 cells were isolated in BSA-coated tubes stromal cell line L88/5 (lane 3) and from the myeloblastic cell line at a sort rate of 103cells/seconds. The isolated cells were recovered KG1a (lane 4). No signal could be obtained from the more by centrifugation at 600 g for 20 minutes over a 10% BSA cushion. differentiated myeloblastic cell line KG1 (lane 5) or the epidermoid After cytospin preparation the cells were stained according to cell line A431 (lane 6), which was used as a negative control. No Pappenheim. specific amplifications were observed when the PCR reactions were run with RNA that had not been reverse-transcribed. The identity of Colony-forming unit-culture (CFU-C) assay the 357 bp cDNA fragment as an N-cadherin-specific sequence was The colony-forming capacity of CD34+ progenitor cells isolated by confirmed by sequencing the PCR products. Lane M represents a 100 MiniMACS was analyzed in the presence of antibodies against N- bp ladder. 1570 JOURNAL OF CELL SCIENCE 114 (8)

Fig. 2. Localization of N-cadherin expression on human bone-marrow- derived cells. The micrographs show indirect immunofluorescence stainings (a,c,d) and a phase contrast picture (b) of bone marrow stromal cells (a-c) and the cell line KG1a (d). The stromal cell line L88/5 (a) and the adherent stromal layer of a long term bone marrow culture (c) as well as a cytospin preparation of the KG1a cells (d) were labeled with an anti-N- cadherin antibody. On stromal cells, expression of N-cadherin was found to be enriched at sites of cell-cell contact (a,c), whereas on the myeloblastic KG1a cells N-cadherin expression was found distributed over the entire cell surface (d). Bar, 25 µm. equipped with time-lapse video recording. Single cells and cell times with pre-warmed RPMI-1640 culture medium. Bound aggregations were counted at time t=0 hours, t=2 hours and t=4 hours hematopoietic cells were fixed together with the stromal monolayer in defined fields of the wells. The degree of cell aggregation was in 4% formaldehyde for 10 minutes, stained with 1% crystal violet in determined according to the decrease in particle number: N0 – Nt/N0, PBS and analyzed on a Zeiss axiovert microscope. where Nt was the total particle number (aggregates plus single cells) at incubation time t, and N0 was the initial cell number in the cell suspension. At least five independent experiments were performed in RESULTS duplicates with the KG1a cells. For analysis of heterotypic cell interactions, the stromal cell line Restricted expression pattern of N-cadherin by L88/5 was cultured in 24-well plates at a concentration of 5×105 cells/ml. After formation of an almost confluent stromal cell layer, the bone-marrow-derived cells myeloblastic KG1a cell line was added at a cell density of 106 To form a first impression of N-cadherin expression by bone- suspension cells/ml, incubated for 1 hour at 37°C and washed three marrow-derived cells, polymerase chain reactions with a

Table 1. RT-PCR analysis of N-cadherin expression of hematopoietic cell lines Cell line N-cadherin expression Characteristics of the cell lines Myeloid cell lines KG1 −− Myeloblastic KG1a ++ Less differentiated subtype of KG1, CD34+ U937 −− Promyelocytic HL60 −− Myelomonocytic Erythroblastic/megakaryoblastic cell lines K562 −− Erythroblastic KU.812 −− Megakaryoblastic/basophilic HEL −− Erythroblastic/megakaryoblastic Myeloma cell lines U266 −− Multiple myeloma IM-9 −− Multiple myeloma Bone marrow stromal cell line L88/5 ++ SV40 transfected stromal cell Epidermoid cell line A431 −− Epithelial N-cadherin in human bone marrow 1571

Fig. 3. Western blot analysis of N-cadherin expression on bone- marrow-derived cells. Protein extracts from primary stromal cells of an LTMC (lane 1), the stromal cell line L88/5 (lane 2), the myeloblastic cell line KG1a (lane 3), the epithelial cell line A431 (lane 4), CD34+ progenitor cells isolated by MiniMACS (lane 5) and from unseparated bone marrow mononuclear cells (lane 6) were separated on a 10% polyacrylamide gel. After transfer to nitrocellulose, the filter was incubated with the anti-N-cadherin antibody. After colorimetric development, positive signals for N- cadherin (arrowhead) at 130 kDa were detected in the stromal, the myeloblastic and the CD34+ progenitor cell extracts. No signal was obtained in bone marrow mononuclear cell extracts. A431 as a negative control also showed no positive signal at 130 kDa. The molecular weight markers are indicated on the left side (kDa). specific primer pair for human N-cadherin were performed. The expected 357 bp cDNA fragment could be amplified after reverse transcription from RNA of freshly isolated bone marrow mononuclear cells, and of stromal cells from LTMC (Fig. 1, lanes 1,2). Both 357 bp products were sequenced and their identity with the human N-cadherin sequence was confirmed. A positive signal was also obtained with the human bone marrow stromal cell line L88/5 (Fig. 1, lane 3). We then analyzed a panel of human hematopoietic cell lines including myeloid (KG1a, KG1, U937, HL60), erythroid (K562, HEL), megakaryoblastic (KU.812) and myeloma (U266, IM-9) cell lines by RT-PCR (Table 1). Of these cell types, only the myeloblastic cell line KG1a gave a positive signal after RT-PCR with the N-cadherin-specific primers (Fig. 1, lane 4). However, the myeloblastic cell line KG1, which represents a more Fig. 4. (A) Immunoblot analysis of catenin expression of differentiated cell line compared with KG1a, did not express N- myeloblastic and stromal cell lines. Protein extracts of the KG1a cell cadherin (Fig. 1, lane 5). Thus it seemed likely that, in addition line (lanes 1-3) as well as of L88/5 stromal cells (lanes 4-6) were to bone marrow stromal cells, a subpopulation of developing separated on 10% polyacrylamide gels. After transfer to bone marrow mononuclear cells expresses N-cadherin on their nitrocellulose, the three different catenins were analyzed using cell surface. The epithelial cell line A431, which expressed E- antisera against α-catenin (lanes 1,4) and β-catenin (lanes 2,5) and a and P-cadherin, was used as a negative control for N-cadherin monoclonal antibody against γ-catenin (lanes 3,6). Specific bands at α β γ amplification by RT-PCR (Fig. 1, lane 6). 102 kDa ( -catenin), 92 kDa ( -catenin) and 84 kDa ( -catenin) Immunofluorescence staining of bone marrow stromal cells, could be detected in both protein extracts (arrowheads). The positions of four molecular weight standard molecules are indicated either of primary cultures or of the L88/5 stromal cell line, on the left. (B) Co-precipitation of N-cadherin and catenins reveals showed prominent staining signals with the anti-N-cadherin N-cadherin-catenin complexes. Cell lysates of L88/5 stromal cells antibody. The strong staining signals could be localized at sites were immunoprecipitated (IP) with antisera against α-catenin (lanes of cell-cell contacts of the stromal cells (Fig. 2a-c). Strong N- 1,4,6), β-catenin (lanes 2,5,7) or a preimmune serum (P; lane 3), and cadherin staining could also be observed on the cell surface of immunoblotted (WB) with either anti-α-catenin (lanes 1,2), anti-β- the myeloblastic KG1a cells after cytospin preparations. The catenin (lanes 3-5) or N-cadherin antibodies (lanes 6,7). 1572 JOURNAL OF CELL SCIENCE 114 (8) Immunoblotting of protein extracts from LTMC, the stromal cell line L88/5 and the myeloblastic KG1a cells revealed prominent bands of N-cadherin expression at 130 kDa (Fig. 3). As expected from the RT-PCR results, no signal was observed in the extract of the epithelial cell line A431. Although RNA preparations of bone marrow mononuclear cells showed a strong signal of N-cadherin expression by RT-PCR analysis, no positive signal for N-cadherin expression was detected in protein extracts of this heterogeneous cell population by immunoblotting. However, in cell extracts of CD34+ cells isolated by MiniMACS from bone marrow mononuclear cells, a prominent band of N-cadherin expression was observed (Fig. 3), suggesting that only a subpopulation of CD34+ hematopoietic progenitor cells expresses N-cadherin. Since cellular interactions mediated by classical cadherins are strongly dependent on close interactions with cytoplasmic catenins, the expression of α-, β- and γ-catenin in KG1a and L88/5 cells was analyzed by immunoblotting. Specific bands of 102 kDa for α-catenin, 92 kDa for β-catenin and 84 kDa for γ-catenin were observed in extracts of both cell lines (Fig. 4A). Immunoprecipitations of L88/5 stromal cell extracts with antibodies against α- and β-catenin revealed existing N-cadherin-catenin complexes. In these experiments, N-cadherin, as well as α- catenin and β-catenin, were co-precipitated with both antisera, suggesting that functional interactions of these three molecules exist in the stromal cells (Fig. 4B). Homophilic interactions of hematopoietic cells mediated by N-cadherin A homophilic homotypic interaction mediated by N-cadherin was observed with the myeloblastic CD34+ KG1a cells. This cell line has the strong tendency to form cell aggregates in culture. By pipetting these cells up and down, single cell Fig. 5. N-cadherin mediates KG1a cell aggregation. After pipetting, the myeloblastic KG1a cells are present as single suspension cells (a). However, suspensions were obtained (Fig. 5a). The KG1a after incubation in culture medium these cells start to form larger cell aggregates cells were incubated in fresh culture medium in that are clearly visible after 4 hours of incubation (b). The cell aggregation the presence or absence of the anti-N-cadherin process can be almost completely inhibited by treatment with 10 mM EDTA antibody GC-4 and recorded by time-lapse video. indicating the Ca2+-dependency of the process (c). Incubation of 5×105 KG1a After 4 hours of incubation without the antibody cells with 80µg/ml of the anti-N-cadherin antibody GC-4 for 4 hours also led to GC-4, large cell clusters could be observed (Fig. drastically diminished cell aggregation of KG 1a cells (d). The degree of cell 5b), whereas 4 hours of incubation in the presence aggregate formation or its inhibition by N-cadherin antibodies or EDTA was of 80 µg/ml GC-4 resulted in a drastic reduction determined by counting cell aggregates and single cells at t=2 hours and t=4 µ both in number and size of such aggregates (Fig. hours (e). Bar, 80 m. 5d), strongly suggesting that N-cadherin is involved in these interactions. Addition of 10 mM staining signals were more or less equally distributed over the EDTA to the culture medium almost completely blocked cell entire cell surface of these nonpolar suspension cells (Fig. 2d). aggregate formation, indicating a calcium-dependent Localization of N-cadherin expression in the native bone mechanism (Fig. 5c). By counting single cells and cell marrow was analyzed in cryostat sections of this tissue. Here, aggregates at t=2 hours and t=4 hours, the increase in cell individual cells or cell clusters were detected with the anti-N- aggregate formation and its inhibition by the N-cadherin cadherin antibody, but it was not possible to identify these N- antibody or EDTA was determined (Fig. 5e). cadherin+ cells in the marrow by morphological criteria (data Cellular adhesive interactions between hematopoietic not shown). progenitor and stromal cells were analyzed using the N- N-cadherin in human bone marrow 1573 10% of the CD19+ cells express N-cadherin (Fig. 6c). Co- expression with the mature T-lymphoid marker molecule CD3 was not observed (Fig. 6d). Other myeloid (CD13, CD14, CD15), erythroid (CD71, CD117, glycophorin A) or lymphoid (CD4, CD8) marker molecules were also not co-expressed with N-cadherin (data not shown). To determine whether the N-cadherin+ CD19+ cells overlap with CD34+ cells, triple-color FACS analyses of bone marrow mononuclear cells were performed. After gating on the N- cadherin+ cells, it could be shown that the majority of these cells express both CD34 and CD19 (Fig. 7a,b), defining a new CD34+CD19+N-cadherin+ cell population. For isolation of these N-cadherin-expressing cells, peripheral blood progenitor cell preparations were used. The amount of N-cadherin+ cells in these preparations is in the same range as in the bone marrow cell aspirates (data not shown). After labeling of the progenitor cell preparation with the antibody GC-4 and cell sorting on a FACSCalibur, a homogeneous cell population could be isolated that had the typical appearance of early hematopoietic progenitor cells (Fig. 7d). In the unsorted cell preparation very few cells with a similar morphology were detected (Fig. 7c). A potential role of N-cadherin in the differentiation pathways of early hematopoietic progenitor cells was analyzed by culturing isolated CD34+ cells in semi-solid medium containing recombinant SCF, GM-CSF, IL-3 and Epo in the Fig. 6. Determination of N-cadherin+ cells in the bone marrow mononuclear cell fraction.Two-color FACS analysis of isolated bone presence of anti-N-cadherin antibodies. After 14 days of culture, the mixed colonies formed were counted. Although the marrow mononuclear cells was performed with the biotinylated = antibody 8C11 against N-cadherin and the directly FITC-labeled number of total colonies in different experiments (n 10) varied antibodies against CD34 (b), CD19 (c) and CD3 (d) as well as a (depending on the donors), the outcome of the individual control antibody (a). The biotinylated anti-N-cadherin antibody was antibody inhibition experiments was comparable. In Fig. 8 detected with streptavidin-phycoerythrin (PE). Double labeling with the results of the different experiments are summarized: N-cadherin was found on 0.6% of the cells for CD34 and CD19, preincubation of the CD34+ cells with different concentrations respectively. The double-positive cells are shown in the upper right of the anti-N-cadherin antibody GC-4 (6, 12, 24 and 48 µg/ml) quadrant (b,c). No double labeling could be observed for the T-cell and culturing these cells in the presence of the antibody marker CD3 (d). Here, as well as with the control IgG antibody resulted in a dose-dependent reduction of the number of (a), the single N-cadherin+ cells are found in the upper left quadrant. formed colonies compared with the control experiments. Three different controls were performed: (1) without any antibody cadherin+ KG1a and L88/5 cells. In 24-well plates, confluent treatment, the total number of colonies could be determined; monolayers of the L88/5 stromal cells were incubated with 106 (2) treatment with the IgG1-isotype control antibody; or (3) the KG1a cells/ml for 1 hour, and then the unbound cells were anti-MHC class I antibody that binds to the cell surface of the removed by washing. Strong adhesion of the KG1a cells to the CD34+ cells did not interfere with the colony formation stromal cell layer could be observed (data not shown). process, indicating that antibody treatment per se does not However, these adhesive interactions could not be inhibited, affect colony formation. By contrast, treatment of the CD34+ not even partially, by addition of the anti-N-cadherin antibody cells with the GC-4 anti-N-cadherin antibody in the range of GC-4, indicating that additional adhesion mechanisms must 6-48 µg/ml inhibited colony formation dose-dependently up to contribute to the strong adhesive interactions observed for 77%. CD34+ cells were also cultured in the presence of the KG1a and the stromal cell line L88/5. recombinant cytokines SCF, GM-CSF and IL-3, but without Epo. Here, only myeloid but no erythroid colonies were Involvement of N-cadherin in differentiation of early formed. However, addition of the anti-N-cadherin antibody hematopoietic cells resulted in a similar inhibition of colony formation as in the To determine the subpopulations of human bone marrow Epo-containing cultures (data not shown). Thus, the strong mononuclear cells that express N-cadherin, dual colour FACS inhibition of mixed colony formation by the anti-N-cadherin analyses were performed. Only 0.5-2% of these cells antibody clearly shows an involvement of N-cadherin during (depending on the individual donors) express N-cadherin early hematopoietic progenitor cell development. on their cell surface (Fig. 6a). As expected from our immunoblotting results (Fig. 3, lane 5), co-expression of N- cadherin was found with CD34, a marker molecule for early DISCUSSION hematopoietic progenitor cells. More than one third of the CD34+ cells showed expression of N-cadherin (Fig. 6b). A In the hematopoietic system, a finely tuned program of second co-expression pattern was found with CD19, which can adhesive interactions seems to be necessary to ensure the be used as a marker molecule for B-lymphoid cells. Around tightly controlled release of developing blood cells from the 1574 JOURNAL OF CELL SCIENCE 114 (8)

Fig. 7. Triple-color FACS analysis and isolation of N-cadherin+ progenitor cells. Bone marrow mononuclear cells were triple- stained with antibodies against N-cadherin, CD34 and CD19, and detected with Cy5-, FITC- and PE-conjugates. N-cadherin+ cells were gated (a), and the expression of CD34 and CD19 of these gated cells were determined (b). The majority of the N- cadherin expressing cells also express CD34 and CD19. N-cadherin+ cells were also isolated by cell sorting on a FACSCalibur flow cytometer. The micrographs show Pappenheim-stained cytocentrifuge preparations of N-cadherin+ cells isolated from periperal blood progenitor cells (d) as well as unseparated peripheral blood progenitor cell population (c). The N- cadherin+ cells possess a non-granular cytoplasm and the almost-round nucleus occupies most of the cell area typical for an early progenitor cell type (d). Around 104 N- cadherin+ cells could be isolated from 2.5×106 cells of the peripheral blood progenitor cell preparation. The detection of only few cells with a similar morphology (c, arrowhead) also showed that N-cadherin+ progenitor cells represent only a minority of the unseparated peripheral blood cells. Bar, 5 µm. bone marrow (Gordon, 1988; Petrides and Dittmann, 1990). progenitor cells and on non-hematopoietic stromal cells. The cadherin family has not received a great deal of attention Functionally, N-cadherin seems to be involved in the in this field despite the fact that these molecules are known to developmental pathways of early CD34+ progenitor cells and be morphoregulatory proteins during development, and to have in cell adhesive interactions of these progenitors, because dynamic expression patterns (Takeichi, 1988; Takeichi, 1991). antibodies against N-cadherin interfered with these processes. The present study, together with a recent report (Armeanu et Blood cells develop in close interaction with the al., 1995), has demonstrated that cadherins are also involved in hematopoietic microenvironment, which consists of the cellular decisions of distinct progenitor cell types in the stromal cell compartment and a complex extracellular matrix hematopoietic system. Whereas E-cadherin is exclusively (Dexter et al., 1990; Dorshkind, 1990; Klein, 1995). expressed on CD34− committed progenitors of the human Components of the extracellular matrix with adhesive erythroid cell lineage in the bone marrow (Armeanu et al., functions for hematopoietic progenitors include different 1995), N-cadherin is found on a subpopulation of early CD34+ collagen types, thrombospondin, fibronectin and tenascin

Fig. 8. Inhibition of CFU-C formation by anti-N-cadherin antibodies. For each experiment, 2×103 CD34+ bone marrow cells were incubated for 14 days in a semi-solid culture medium supplemented with recombinant cytokines (rhSCF, rhGM-CSF, rhIL-3, rhEpo) in the presence of different concentrations of anti-N-cadherin antibodies (6, 12, 24 and 48 µg/ml), or anti-HL antibodies (10.8 µg/ml) that recognize an epitope of the heavy chain of MHC class I antigens as a control. Treatment of CD34+ cells with the HL- antibodies did not influence CFU-C formation as these cultures show CFU-C counts comparable to untreated control cultures, which were set to 100%. By contrast, anti-N-cadherin antibodies inhibited the formation of total CFU-C in a dose-dependent manner. The percentage of formed colonies in the presence of the respective antibody concentrations were determined. N-cadherin in human bone marrow 1575 (Verfaillie et al., 1994; Klein et al., 1993; Klein et al., 1995; Triple color FACS analysis revealed that the great majority Klein et al., 1997; Seiffert et al., 1998). On stromal cells, the of the N-cadherin+ cells are CD34+CD19+. Co-expression of vascular cell adhesion molecule 1 (VCAM-1) has been shown these two marker molecules have been reported to to mediate attachment of hematopoietic progenitor cells characterize an early B-lymphoid committed progenitor cell (Simmons et al., 1992). However, the VCAM-1-dependent type (LeBien, 2000). The fact that addition of antibodies mechanism is certainly not the only one responsible for against N-cadherin to isolated CD34+ progenitor cells adhesion of immature progenitors to the stroma, and other cell resulted in a dose-dependent inhibition of myeloid colony adhesion molecules on stromal cells might also have crucial formation was therefore interesting. In the cell cultures used, functions at distinct levels of maturation of the various blood the recombinant growth factors SCF, IL-3, GM-CSF and Epo cell lineages. N-cadherin might be such a candidate since it is responsible for the development of myeloid and erythroid expressed on the surface of the marrow stromal cells. An colonies were present. Our results show that under the enrichment of N-cadherin expression was observed at sites of appropriate conditions human CD34+CD19+N-cadherin+ stromal cell-cell contact, but N-cadherin expression was not progenitor cells still have the plasticity and potency to restricted to the sites of cell contact. A similar expression develop into the myeloid cell lineage. Two recent reports of pattern has been detected on endothelial cells, where N- murine hematopoietic progenitor cells have shown that: (1) cadherin is also not restricted to the basal-lateral site, but CD19+ pro-B-cells can also differentiate into dendritic cells is localized in a diffuse pattern on the cell membrane (Björck and Kincade, 1998); and (2) CD45R−CD19+ B-cell (Lampugnani and Dejana, 1997; Navarro et al., 1998). Such an progenitors show a limited myeloid potential (Montecino- expression pattern might enable the stromal cells to interact Rodriguez et al., 2001). In addition, CD34 and CD19 can be with hematopoietic progenitors in a heterotypic interaction. An detected on blast cells from myeloid leukemia patients N-cadherin-mediated adhesion mechanism between stroma (Ferrara et al., 1998; Puch, S., unpublished), suggesting that and invasive breast carcinoma cells has recently been reported expression of the CD19 molecule indicates only a spatio- (Hazan et al., 1997). However, we failed to inhibit bone temporally limited preference for development into the B-cell marrow stromal cell-hematopoietic progenitor cell interactions lineage. with the anti-N-cadherin antibody. Although this result does Colony formation can be obtained from single progenitors not rule out a potential involvement of N-cadherin in stromal without any obvious cell-cell adhesive interactions. Inhibition cell-hematopoietic progenitor cell binding, it suggests that of colony formation by addition of the anti-N-cadherin additional adhesive mechanisms are likely to be involved in the antibody therefore suggests that (1) N-cadherin can act as a strong cell adhesive interactions observed. signal-transducing molecule in addition to its adhesion- On developing blood cells, N-cadherin seems to be mediating function; and (2) that the antibody cannot serve as functionally involved in homotypic cell interactions. This was a substitute for the activating ligand. A similar phenomenon shown by inhibition of cell aggregation of KG1a cells with was observed for E-cadherin on erythroid progenitors. The anti-N-cadherin antibodies or, more indirectly, by Ca2+- development of these progenitors was also modulated by anti- depletion, proving calcium dependency. Thus, N-cadherin E-cadherin antibodies leading to the suggestion that E- could be responsible for a close interaction of early progenitor cadherin plays an important role as a signaling molecule cell types of the same differentiation state. The N-cadherin- during erythroid differentiation (Armeanu et al., 1995). Thus, expressing CD34+ KG1a cells and the bone marrow-derived different members of the cadherin family might serve as stromal cells express all three catenins, as shown by western differentiation-inducing signals at different stages of blotting. Classical cadherins require the cytoplasmic catenins individual hematopoietic lineages. On non-hematopoietic to be functionally active (Kemler, 1993). The co-precipitation cells, a number of studies have already indicated that of N-cadherin with α- and β-catenin from stromal cell lysates cadherins can play an active role in regulating cellular showed that intact N-cadherin-catenin complexes exist in these differentiation (Knudsen et al., 1998). cells. Two recent publications provided evidence that cadherins Of the developing blood cells, only a subpopulation of might also play a role for other bone-marrow-derived cell CD34+ hematopoietic progenitor cells express N-cadherin. types. Whereas one (Tsutsui et al., 1996) showed that N- This was shown by immunoblotting of purified CD34+ cadherin can be detected on T cell leukemia and lymphoma progenitor cells and by FACS analysis of the heterogeneous cell lines but not on normal blood leukocytes, the other (Cepek bone marrow mononuclear cell preparations. Only 0.5-2% of et al., 1996) reported that mature T cells express a cadherin these cells (depending on the donor) were shown to be N- molecule that they could detect by immunoprecipitation with cadherin+. Co-expression was found with CD34 and CD19 a pan-cadherin antiserum. However, the exact nature of this antigens, but not with other lymphoid, myeloid or erythroid member of the cadherin family was not determined and it will marker molecules, demonstrating that E- and N-cadherin are certainly be a major task for future studies to identify expressed on different progenitor cell populations. Cell sorting expression and functions of cadherins on circulating blood of N-cadherin+ cells on a FACSCalibur revealed a single cell cells. population that was morphologically identified as an early Taken together, the present study provides evidence that progenitor cell type. The restricted expression pattern of N- N-cadherin can contribute to the development of early cadherin on maturing blood cells is also reflected by the fact hematopoietic progenitor cells. Since N-cadherin is found with that screening several myeloid and erythroid cell lines revealed a different expression pattern compared with E-cadherin in that only the CD34+ cell line KG1a expressed N-cadherin the human bone marrow, it is suggested that the family of on the cell surface, whereas the other cell lines analyzed, cadherins play a more important role in the human including the KG1 cell line, do not. hematopoietic system than hitherto appreciated. 1576 JOURNAL OF CELL SCIENCE 114 (8)

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