Cytokine regulation of proliferation and cell adhesion are correlated events in human CD34+ hemopoietic progenitors

Adhesive interactions with the extracellular matrix of the bone marrow (BM) stroma are of critical importance in the regulation of hematopoiesis. In part, these interactions are presumed to play an important role in retaining CD34+ hematopoietic progenitor cells (HPCs) within the BM environment, in close proximity with BM stromal cells and the cytokines they produce. Evidence of a more direct role for cell adhesion in the regulation of hematopoiesis is provided by recent data showing that adhesive interactions can also provide important costimulatory signals. We have previously shown that normal CD34+ HPCs express high levels of fibronectin (Fn) receptors very late antigen-4 (VLA-4) and VLA-5 in a low-affinity state, which do not allow HPCs to strongly adhere on immobilized Fn, and that cytokines such as interleukin-3, granulocyte-monocyte colony-stimulating factor, and stem cell factor transiently activate these receptors, providing HPCs with an adhesive phenotype on Fn. Thus, knowledge of the functional states of adhesion receptors is critical to our understanding of the physiological mechanisms responsible for the regulation of normal hematopoiesis. Herein, we show that combinations of cytokines that synergize to stimulate the proliferation of CD34+ HPCs result in additive stimulation of the adhesion of these cells to Fn. Thus, the activation level of Fn receptors expressed by normal CD34+ HPCs is highly correlated with their proliferative state, suggesting a functional link between these two events. Therefore, we propose a 2-step model with an initial activation of VLA-4 and VLA-5 generated by cytokine receptors that is followed by a secondary signal resulting from Fn binding to VLA-4 and VLA-5, which may cooperate with those generated by cytokine receptors.     

Paracrine regulation of germinal center B cell adhesion through the c-met-hepatocyte growth factor/scatter factor pathway

T cell-dependent humoral immune responses are initiated by the activation of naive B cells in the T cell areas of the secondary lymphoid tissues. This primary B cell activation leads to migration of germinal center (GC) cell precursors into B cell follicles where they engage follicular dendritic cells (FDC) and T cells, and differentiate into memory B cells or plasma cells. Both B cell migration and interaction with FDC critically depend on integrin-mediated adhesion. To date, the physiological regulators of this adhesion were unkown. In the present report, we have identified the c-met-encoded receptor tyrosine kinase and its ligand, the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF), as a novel paracrine signaling pathway regulating B cell adhesion. We observed that c-Met is predominantly expressed on CD38(+)CD77(+) tonsillar B cells localized in the dark zone of the GC (centroblasts). On tonsil B cells, ligation of CD40 by CD40-ligand, induces a transient strong upregulation of expression of the c-Met tyrosine kinase. Stimulation of c-Met with HGF/SF leads to receptor phosphorylation and, in addition, to enhanced integrin-mediated adhesion of B cells to both VCAM-1 and fibronectin. Importantly, the c-Met ligand HGF/SF is produced at high levels by tonsillar stromal cells thus providing signals for the regulation of adhesion and migration within the lymphoid microenvironment.     

Overexpression and activation of hepatocyte growth factor/scatter factor in human non-small-cell lung carcinomas

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the invasive growth of epithelial cells via the c-MET oncogene-encoded receptor. In normal lung, both the receptor and the ligand are detected, and the latter is known to be a mitogenic and a motogenic factor for both cultured bronchial epithelial cells and non-small-cell carcinoma lines. Here, ligand and receptor expression was examined in 42 samples of primary human non-small-cell lung carcinoma of different histotype. Each carcinoma sample was compared with adjacent normal lung tissue. The Met/HGF receptor was found to be 2 to 10-fold increased in 25% of carcinoma samples (P = 0.0113). The ligand, HGF/SF, was found to be 10 to 100-fold overexpressed in carcinoma samples (P < 0.0001). Notably, while HGF/SF was occasionally detectable and found exclusively as a single-chain inactive precursor in normal tissues, it was constantly in the biologically-active heterodimeric form in carcinomas. Immunohistochemical staining showed homogeneous expression of both the receptor and the ligand in carcinoma samples, whereas staining was barely detectable in their normal counterparts. These data show that HGF/SF is overexpressed and consistently activated in non-small-cell lung carcinomas and may contribute to the invasive growth of lung cancer.     

Basic fibroblast growth factor promotes the proliferation of human megakaryocyte progenitor cells

Basic fibroblast growth factor (bFGF), a multifunctional growth factor produced by bone marrow stromal cells, is known to be a potent modulator of hematopoiesis. Because bFGF is present in both human megakaryocytes (MKs) and platelets, we have hypothesized that this growth factor might affect human megakaryocytopoiesis. To test this hypothesis, either low density bone marrow (BM) cells (LDBM), a human BM subpopulation (CD34+ DR+) enriched for the colony-forming unit megakaryocyte (CFU-MK) or a BM subpopulation (CD34+ DR-) enriched for the more primitive burst-forming unit megakaryocyte (BFU-MK) were assayed in the presence of this growth factor. The effect of bFGF on MK colony formation differed according to the cell population assayed. bFGF alone had on MK colony-stimulating activity (MK-CSA) when either CD34+ DR+ or CD34+ DR- BM cells were cloned, but exhibited MK-CSA equivalent to that of interleukin-3 (IL-3) when LDBM cells were used as the target cell population. The MK-CSA of bFGF was inhibited by the addition of neutralizing antisera to either IL-3 and/or granulocyte-macrophage colony-stimulating factor (GM-CSF) but not IL-6. The addition of excess amounts of either IL-3 or GM-CSF to cultures containing bFGF plus anti-IL-3 or anti-GM-CSF reversed the inhibition by the corresponding antisera. The addition of bFGF and IL-3 to assays containing CD34+ DR+ or CD34+ DR- cells increased the size of both CFU-MK- and BFU-MK-derived colonies, respectively, when compared with assays containing IL-3 alone. This increase in MK colony size mediated by bFGF was not affected by addition of either an anti-GM-CSF or anti-IL-6 neutralizing antisera. When LDBM cells were assayed, bFGF alone increased CFU-MK-derived colony size when compared with control values. However, this potentiation of MK colony size by bFGF could be reversed by the addition of either anti-IL-3 or anti-GM-CSF but not anti-IL-6 antisera. In addition, the effects of bFGF and IL-3 on the size of MK colonies cloned from LDBM were not additive. These results suggest that bFGF affects human megakaryocytopoiesis by directly promoting MK progenitor cell proliferation and stimulating BM accessory cells to release growth factor(s) with MK-CSA, such as IL-3 and GM-CSF. We conclude that bFGF, likely produced by cellular components of the BM microenvironment, plays an important role in the control of human megakaryocytopoiesis.     

Regulation of adhesion and migration in the germinal center microenvironment

T cell dependent humoral immune responses are initiated by the activation of naive B cells in the T cell areas of the secondary lymphoid tissues. This primary B cell activation leads to migration of germinal center (GC) cell precursors into B cell follicles where they engage follicular dendritic cells (FDC) and T cells, and differentiate into memory B cells or plasma cells. Both B cell homing to the GC and interaction with FDC critically depend on integrin-mediated adhesion. We have recently indentified the c-met-encoded receptor tyrosine kinase and its ligand, the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF), as a novel paracrine signalling pathway regulating B cell adhesion (van der Voort et al., 1997, J. Exp. Med. 185, 2121-2131). The c-Met protein is expressed on B cells localized in the dark zone of the GC (centroblasts) and is induced by CD40 plus BCR ligation. Stimulation of c-Met with HGF/SF, which is produced at high levels by tonsillar stromal cells and FDC, leads to receptor phosphorylation and to enhanced integrin-mediated adhesion of B cells to both VCAM-1 and fibronectin. Interestingly, these responses to HGF/SF are promoted by heparan-sulfate proteoglycan forms of CD44 (CD44-HS). Like c-Met, CD44-HS is induced on B cells by CD40 ligation. It efficiently binds HGF/SF and strongly promotes signalling through c-Met. We conclude that integrin regulation during antigen specific B cell differentiation involves cross-talk between the HGF/SF-c-Met pathway and CD44-HS.     

Hepatocyte growth factor/scatter factor induces not only scattering but also cohort migration of human colorectal-adenocarcinoma cells

We presented earlier a 2-dimensional cell-motility assay using a highly metastatic variant (L-10) of human rectal-adenocarcinoma cell line RCM-1 as a motility model of tumor cells of epithelial origin. In this model, L-10 cells moved as coherent cell sheets when stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA), and we called this type of movement "cohort migration". Electron- and immunoelectron-microscope study of the migrating cell sheets demonstrated localized release from cell-cell adhesion only at the lower portion of the cells with loss of E-cadherin immunoreactivity, and this change was associated with increased tyrosine phosphorylation of the E-cadherin-catenin complex, including beta-catenin. In the present study, to obtain evidence to support the relevance of our model to carcinoma-cell movement in vivo, we sought a naturally occurring motogenic factor(s) able to induce this cohort migration. Among the factors examined, hepatocyte growth factor/scatter factor (HGF/SF) clearly induced cohort migration of L-10 cells. Additionally, not only L-10 but several other human colorectal-carcinoma cell lines showed this type of migration in response to HGF/SF, while yet others showed scattering-type motility. In this HGF/SF-induced migration, localized release from cell-cell adhesion was induced only at the lower portion of the cells, allowing them to extend leading lamellae, whereas close cell-cell contacts remained at the upper portion of the cells, as seen in TPA-induced cohort migration. Scattering-type cell lines tended to express more c-Met (receptor for HGF/SF) mRNA than the cell lines that showed cohort-type migration. LoVo, one of the scattering-type cell lines, expressed more c-Met protein and less E-cadherin than L-10, which showed cohort-type migration. HGF/SF treatment of LoVo reduced the amount of alpha-catenin complexed with E-cadherin more markedly than in L-10, but in both cell lines this reduction was not accompanied by increased tyrosine phosphorylation of beta-catenin, suggesting the presence of a mechanism other than phosphorylation for release from cell-cell adhesion during cell motility.     

Growth factor-dependent activation of alphavbeta3 integrin in normal epithelial cells: implications for tumor invasion

Integrin activation is a multifaceted phenomenon leading to increased affinity and avidity for matrix ligands. To investigate whether cytokines produced during stromal infiltration of carcinoma cells activate nonfunctional epithelial integrins, a cellular system of human thyroid clones derived from normal glands (HTU-5) and papillary carcinomas (HTU-34) was employed. In HTU-5 cells, alphavbeta3 integrin was diffused all over the membrane, disconnected from the cytoskeleton, and unable to mediate adhesion. Conversely, in HTU-34 cells, alphavbeta3 was clustered at focal contacts (FCs) and mediated firm attachment and spreading. alphavbeta3 recruitment at FCs and ligand-binding activity, essentially identical to those of HTU-34, occurred in HTU-5 cells upon treatment with hepatocyte growth factor/scatter factor (HGF/SF). The HTU-34 clone secreted HGF/SF and its receptor was constitutively tyrosine phosphorylated suggesting an autocrine loop responsible for alphavbeta3 activated state. Antibody-mediated inhibition of HGF/SF function in HTU-34 cells disrupted alphavbeta3 enrichment at FCs and impaired adhesion. Accordingly, activation of alphavbeta3 in normal cells was produced by HTU-34 conditioned medium on the basis of its content of HGF/SF. These results provide the first example of a growth factor-driven integrin activation mechanism in normal epithelial cells and uncover the importance of cytokine-based autocrine loops for the physiological control of integrin activation.     

Mobilization and homing of peripheral blood progenitors is related to reversible downregulation of alpha4 beta1 integrin expression and function

Despite the wide use of mobilized peripheral blood (PB) progenitor cells (PBPC) for clinical transplantation the mechanism(s) underlying their mobilization and subsequent engraftment are still unknown. We compared the adhesive phenotype of CD34(+) colony-forming cells (CFC) in bone marrow (BM) and PB of normal donors before and after administration of granulocyte colony-stimulating factor (G-CSF) for 5 d. G-CSF-mobilized PB CFC cells adhered significantly less to BM stroma, fibronectin, and to the alpha4 beta1 binding fibronectin peptide, CS1, because of decreased expression of the alpha4 integrin. Since incubation of BM CD34(+) cells for 4 d with G-CSF at concentrations found in serum of G-CSF- treated individuals did not affect alpha4-dependent adhesion, G-CSF may not be directly responsible for the decreased alpha4-mediated adhesion of PB CFC. Culture of G-CSF-mobilized PB CD34(+) cells with cytokines at concentrations found in BM stromal cultures upregulated alpha4 expression and restored adhesion of mobilized PB CFC to stroma, fibronectin, and CS1. Adhesion of cultured, mobilized PB CFC to stroma and CS1 could not be further upregulated by the beta1 activating antibody, 8A2. This indicates acquisition of a maximally activated alpha4 beta1 integrin once PB CFC have been removed from the in vivo mobilizing milieu. Thus, decreased alpha4 expression on CD34(+) CFC in PB may be responsible for the aberrant circulation of mobilized PB CD34(+) cells. Reexpression of a maximally activated alpha4 beta1 integrin on mobilized PB CFC removed from the mobilizing in vivo milieu may contribute to the early engraftment of mobilized PBPC.     

Thrombopoietin, steel factor and the ligand for flt3/flk2 interact to stimulate the proliferation of human hematopoietic progenitors in culture

We have tested the effects of steel factor (SF) the ligand for flt3/flk2 (FL) and thrombopoietin (TPO, Mpl ligand), on the proliferation of primitive human bone marrow progenitors in serum-deprived culture. Varying combinations of SF, FL and TPO supported formation of only few colonies from CD34+/c-Kit(low)/CD38neg/low cells. However, the addition of interleukin 3 (IL-3) to the three cytokines significantly increased the number of colonies. When this population of cells was tested in suspension culture for one week for production of colony-forming cells there was synergism among SF, FL and TPO. Addition of IL-3 to the three cytokines further increased the number of erythroid colony-forming cells. The effects of these four factors on CD34+/c-Kit(low)/CD38high cells were merely additive. Studies of individual CD34+/c-Kit(low)/CD38neg/low cells demonstrated the direct effects of SF, FL and TPO. In the presence of SF, FL and TPO, approximately half of the individual CD34+/c-Kit(low)/CD38neg/low cells proliferated in seven day suspension culture. Addition of IL-3 to the combination of SF, FL and TPO did not increase the frequencies of proliferating clones, but increased the size of individual clones. These observations suggest that SF, FL and TPO play important roles in survival and proliferation of primitive human hematopoietic progenitors.     

Importance of information following myocardial infarction: a study of the self-perceived information needs of patients and their spouse/partner compared with the perceptions of nursing staff

Time course studies revealed that the combination of Flt-3 ligand (FL), Steel factor (SF) and interleukin-3 (IL-3) did not elicit as large an amplification of the long-term culture-initiating cell (LTC-IC) population in serum-free cultures of CD34+ CD38- cord blood (CB) cells as was obtained in similar cultures of adult human CD34+ CD38- bone marrow (BM) cells (4- v 90-fold maximum increases), even though both total and colony-forming cell (CFC) numbers initially increased more rapidly in CB cultures. Multifactorial analysis of the short-term (10 d) effects of different cytokines identified FL and IL-6 in combination with the soluble IL-6 receptor (sIL-6R) as most important for expanding the CB LTC-IC population. In contrast, their counterparts in adult BM were most effectively stimulated by FL, SF and IL-3. For rapid generation of increased numbers of CFC, SF with either FL or IL-6/sIL-6R were found to be the most important contributors in cultures of CD34+ CD38- CB cells, whereas, in analogous BM cultures, IL-6/sIL-6R and TPO (in addition to FL, SF and IL-3) were required. These findings reinforce the principle of altered cytokine responsiveness as a hallmark of early haemopoietic cell differentiation and demonstrate how cytokine requirements may change during human ontogeny. Identification of conditions for optimizing the expansion of different subsets of primitive CB cells has additional important implications for clinical transplantation and gene transfer.     

Differential use of very late antigen-4 and -5 integrins by hematopoietic precursors and myeloma cells to adhere to transforming growth factor-beta1-treated bone marrow stroma

The very late antigen (VLA)-4 and VLA-5 integrins mediate hematopoietic progenitor cell attachment to bone marrow (BM) stroma. Transforming growth factor-beta1 (TGF-beta1) is a cytokine present in the BM microenvironment that has been shown to regulate the synthesis of adhesion elements in several cell types. We have investigated whether TGF-beta1 action on human BM stromal cells affected the adhesion of progenitor cells involving integrins VLA-4 and VLA-5. Two precursor cell lines, pre-B Nalm-6 and the multipotential UT-7, attached to untreated primary stroma and to the human BM stromal cell line Str-5 preferentially using VLA-4. However, treatment of the stroma with TGF-beta1 resulted in a significant reduction in the participation of VLA-4 in mediating precursor cell adhesion to stroma and a concomitant increase in the utilization of VLA-5. This effect was not exclusive of normal BM stroma. Treatment with TGF-beta1 of stroma from multiple myeloma BM samples produced a substantial increase in VLA-5 use by the myeloma cell line NCI-H929 to adhere to this stroma. The differential use of VLA-4 and VLA-5 correlated with an increase in fibronectin surface expression by stromal cells in response to TGF-beta1. Adhesion assays to purified fibronectin using Nalm-6 cells showed a predominant utilization of VLA-4 at low concentrations of this ligand, whereas higher concentrations resulted in a preferential use of VLA-5. These results indicate that regulation of fibronectin expression on BM stromal cells by TGF-beta1 results in a modulation of the pattern of integrins used by the precursor and myeloma cells to adhere to BM stroma, which could have important consequences on the proliferation and differentiation of hematopoietic precursor cells as well as on the localization and growth of myeloma cells.     

O-glucuronidation, a newly identified metabolic pathway for topotecan and N-desmethyl topotecan

BACKGROUND AND OBJECTIVE: CD34+ hematopoietic progenitor cells (HPCs) constitute a heterogeneous population both in size and in immunological features. Lack of CD38, HLA-DR and lineage committed antigens as well as the co-expression of Thy-1 (CDw90) and c-kit receptor (CD117), are able to identify the so-called stem cells. A flow cytometric study was carried out to investigate the co-expression of Thy-1 and c-kit receptors, both members of Ig superfamily adhesion molecules, involved in cell to cell and cell to stroma interactions, on bone marrow (BM), mobilized peripheral blood (PB) and human umbilical cord blood (HUCB) CD34+ HPCs. DESIGN AND METHODS: Lysed whole blood from 15 BM, 25 mobilized PB and 25 HUCB samples were used to perform a five-dimensional flow cytometric evaluation of both CDw90 and CD117 on CD34+ cells. RESULTS: Few CD34+ cells co-expressed Thy-1 antigen in all three compartments (BM: 11.2 +/- 7.2%; PB: 6.2 +/- 3.6%; HUCB: 6 +/- 2.9%; BM vs PB < 0.04; BM vs HUCB < 0.008; PB vs HUCB ns). c-kit receptor was detected on the majority of CD34+ HPCs, particularly in HUCB (HUCB: 80.7 +/- 8.2%; BM: 72.3 +/- 13.1%; PB: 64.2 +/- 17%; HUCB vs BM < 0.03; HUCB vs PB < 0.0001; BM vs PB ns). CD34+Thy-1+ and CD34+c-kit+ HPCs generally displayed HLA-DR antigen, as expression of early cell commitment. However, the most immature CD34+Thy-1+HLA-DR- (HUCB: 1 +/- 0.6%; BM: 0.4 +/- 03%; PB: 0.7 +/- 0.5%; HUCB vs BM < 0.0001; BM vs PB < 0.04; HUCB vs PB ns) and CD34+c-kit+HLA-DR- HPCs (HUCB: 6.5 +/- 4.4%; BM: 6.3 +/- 4.8%; PB: 2.2 +/- 1.8%; HUCB vs BM ns; BM vs PB < 0.0001; HUCB vs PB < 0.0001) were mainly detected in HUCB. Finally, the greatest percentage of CD34+Thy-1+c-kit+ cells was found in BM (6.9 +/- 4.1%) followed by leukapheretic samples (4.4% +/- 2.7) and then by HUCB (3.7 +/- 1.2%; BM vs PB ns; BM vs HUCB < 0.001; HUCB vs PB ns). INTERPRETATION AND CONCLUSIONS: Since the blood release or HPCs is probably due to a perturbation of the adhesive interactions between these cells and the marrow stroma, the different pattern of Thy-1 and c-kit receptor expression on CD34+ HPCs found in the three hemopoietic compartments evaluated can lead to new knowledge about the mobilization kinetics in which the Ig superfamily adhesion molecules are involved.     

The effect of alpha4 beta1-integrin binding sequences of fibronectin on growth of cells from human hematopoietic progenitors

Highly regulated interactions between adhesion receptors on progenitor cells and their extracellular matrix ligands are essential for the control of hematopoiesis in bone marrow stroma. We have examined the relationship between alpha4beta1-integrin-mediated adhesion and growth of CD34(+) cells by assessing their adhesive and migratory patterns of proliferation in a mixture of hematopoietic growth factors in the presence of different recombinant fragments of the HepII/IIICS region of fibronectin. CD34(+) cells were isolated from cord blood and placed in culture wells containing serum-free medium and growth factors. Wells were precoated with either the H120 fragment of fibronectin, which contains three alpha4beta1-integrin binding sites, or the H0 fragment, which lacks the two highest affinity alpha4beta1 binding sequences. Proliferation of single cells of CD34(+)38(+)DR+ and CD34(+)38(-)DR+ phenotypes occurred in contact with the H120 substrate and was associated with migration. Larger numbers of cells were used to quantitate proliferative responses. Cells growing in wells coated with H120 formed attachments to the base of the wells throughout the culture period. Higher total cell counts were consistently found in wells coated with H120 compared with H0 and bovine serum albumin controls. The difference was first apparent at day 8 of culture and reached a maximum at days 11 through 13, when expansion with H120 was a mean of 1.8-fold higher than that seen with H0 (P相似文献   

Fluorescein-labeled dextran concentration is increased in BAL fluid after ANTU-induced edema in rats

We identified the cell cycle status of CD34(+) cells of steady-state bone marrow (BM) and peripheral blood (PB) obtained from healthy volunteers, and those of apherasis PB samples collected from healthy donors who had been administered granulocyte colony-stimulating factor (G-CSF). More than 10% of CD34(+) cells in BM were in S+G2/M phase. In contrast, regardless of whether G-CSF treatment was performed, less than 2% of CD34(+) cells in PB were cycling. BM CD34(+) cells showed greater VLA-4 expression and adherence to stromal cells than PB CD34(+) cells. In addition, when cycling and dormant BM CD34(+) cells were analyzed separately, the cells in S+G2/M phase expressed more VLA-4 and adhered to the stromal cell monolayer more efficiently than the cells in G0/G1 phase. Furthermore, this adhesion of CD34(+) cells to the stromal cell layer was almost completely inhibited by anti-VLA-4 antibody. Taken together, these results suggest that CD34(+) progenitors in G0/G1 phase of the cell cycle differ from those in S+G2/M phase in adhesiveness mediated by VLA-4 in the hematopoietic microenvironment.     

Opposing actions of hepatocyte growth factor and basic fibroblast growth factor on cell contact, intracellular free calcium levels, and rat ovarian surface epithelial cell viability

Previous studies demonstrated that cell-to-cell contact stimulates a tyrosine phosphorylation signal transduction pathway that prevents rat ovarian surface epithelial (ROSE) cells from undergoing apoptosis. Hepatocyte growth factor (HGF), also know as scatter factor (SF), is expressed by ovarian stromal and thecal cells and has been shown to reduce cell contact in nonovarian tissues. The present studies were designed to determine whether HGF/SF promotes ROSE cells to dissociate and subsequently become apoptotic. Because an increase in intracellular free calcium ([Ca2+]i) is often an early event in the apoptotic cascade, the effects of HGF/SF on [Ca2+]i levels were also assessed. ROSE cells were cultured in serum-free medium with HGF/SF, basic fibroblast growth factor (bFGF), thapsigargin, Bay K, actinomycin D, cycloheximide, and/or BAPTA depending on the experimental design. Cell contact was assayed by time-lapse photography; [Ca2+]i levels were measured with Fluo-3, and apoptosis was assessed by in situ DNA staining. HGF/SF decreased cell contact within 1 h, increased [Ca2+]i levels by 3 h, and induced apoptosis by 6 h of culture. bFGF inhibited these HGF/SF-induced responses. The increase in [Ca2+]i appears to represent a point in the apoptotic cascade that commits ROSE cells to die. This concept is based on the observations that: 1) in the presence of the calcium chelator BAPTA, HGF/SF decreased cell contact but did not increase [Ca2+]i or apoptosis; 2) bFGF blocked HGF/SF-induced increase in [Ca2+]i; 3) bFGF did not attenuate HGF/SF's apoptotic action if exposed to cells after the increase in [Ca2+]i; and 4) RNA and protein synthesis were required for HGF/SF to increase [Ca2+]i, whereas the thapsigargin- and Bay K-induced increase in [Ca2+]i and apoptosis were independent of RNA/protein synthesis. These observations indicate that the components of the apoptotic cascade distal to the increase in [Ca2+]i are present within ROSE cells and are activated by a sustained elevation of [Ca2+]i. The present studies also show that when ROSE cells establish contact with 3T3 cells that express N-cadherin, [Ca2+]i levels are maintained at low basal levels. In contrast, cell contact with 3T3 cells that do not express N-cadherin results in elevated [Ca2+]i levels. Similarly, a synthetic N-cadherin peptide, which inhibits homophilic N-cadherin binding, increases [Ca2+]i levels. Taken together, these data indicate that homophilic N-cadherin binding between adhering cells plays an important role in maintaining calcium homeostasis. Further, these data support the concept that HGF/SF's ability to promote the dissociation of ROSE cells accounts in part for its ability to increase [Ca2+]i levels.     

Induction of c-kit molecules on human CD34+/c-kit < low cells: evidence for CD34+/c-kit < low cells as primitive hematopoietic stem cells

c-kit, a receptor for stem cell factor, has been widely accepted as a distinctive marker for hematopoietic stem cells. However, the level of c-kit expression on pluripotent hematopoietic stem cells is still controversial in mice and humans. We purified CD34+/c-kit < low cells (phenotypically c-kit-negative but only detectable at the message level) from human cord blood and examined their maturational steps in relation to the expression of c-kit molecules. When the CD34+/c-kit < low cells were cultured with cytokines (flt 3 ligand, interleukin 6 and interleukin 7) plus immobilized anti-CD34 monoclonal antibody (to crosslink CD34 molecules), c-kit molecules were clearly induced within 24 h. The c-kit expression gradually increased until day 8. When CD34+/c-kit(low) or CD34+/c-kit+ cells that had been induced from CD34+/c-kit < low cells were resorted and recultured using a methylcellulose culture system, they showed the same colony-forming ability as the freshly isolated CD34+/c-kit(low) or CD34+/c-kit+ cells, respectively. Furthermore, CD34+/c-kit < low cells have a similar hematopoietic potential to CD34+/c-kit(low) cells in assays for long-term culture initiating cell and colony-forming unit culture generated from long-term cultures. These findings suggest that CD34+/c-kit < low cells mature into CD34+/c-kit(low) and CD34+/c-kit+ cells, and acquire the reactivity to various humoral hematopoietic stimuli. Moreover, CD34+/c-kit < low cells showed a low level of rhodamine 123 retention, suggesting that CD34+/c-kit < low cells have multidrug resistance. Therefore, the CD34+/c-kit < low cells without colony-forming unit-granulocyte-erythroid-macrophage-megakaryocyte activity are also a pluripotent hematopoietic stem cell population, and the expression of c-kit on c-kit < low cells is the first maturational step of hematopoiesis.     

The sialomucin CD164 (MGC-24v) is an adhesive glycoprotein expressed by human hematopoietic progenitors and bone marrow stromal cells that serves as a potent negative regulator of hematopoiesis

Mucin-like molecules represent an emerging family of cell surface glycoproteins expressed by cells of the hematopoietic system. We report the isolation of a cDNA clone that encodes a novel transmembrane isoform of the mucin-like glycoprotein MGC-24, expressed by both hematopoietic progenitor cells and elements of the bone marrow (BM) stroma. This molecule was clustered as CD164 at the recent workshop on human leukocyte differentiation antigens. CD164 was identified using a retroviral expression cloning strategy and two novel monoclonal antibody (MoAb) reagents, 103B2/9E10 and 105.A5. Both antibodies detected CD164/MGC-24v protein expression by BM stroma and subpopulations of the CD34(+) cells, which include the majority of clonogenic myeloid (colony-forming unit-granulocyte-macrophage [CFU-GM]) and erythroid (blast-forming unit-erythroid [BFU-E]) progenitors and the hierarchically more primitive precursors (pre-CFU). Biochemical and functional characterization of CD164 showed that this protein represents a homodimeric molecule of approximately 160 kD. Functional studies demonstrate a role for CD164 in the adhesion of hematopoietic progenitor cells to BM stromal cells in vitro. Moreover, antibody ligation of CD164 on primitive hematopoietic progenitor cells characterized by the cell surface phenotype CD34(BRIGHT)CD38(-) results in the decreased recruitment of these cells into cell cycle, suggesting that CD164 represents a potent signaling molecule with the capacity to suppress hematopoietic cell proliferation.