Functional receptor for C3a anaphylatoxin is expressed by normal hematopoietic stem/progenitor cells, and C3a enhances their homing-related responses to SDF-1.

Complement has recently been implicated in developmental pathways and noninflammatory processes. The expression of various complement components and receptors has been shown in a wide range of circulating myeloid and lymphoid cells, but their role in normal hematopoiesis and stem cell homing has not yet been investigated. We report that normal human CD34(+) cells and lineage-differentiated hematopoietic progenitors express the complement anaphylatoxin C3a receptor (C3aR) and respond to C3a. Moreover, C3a, but not the biologically inactive desArg-C3a, induces calcium flux in these cells. Furthermore, we found that C3 is secreted by bone marrow stroma and that, although C3a does not influence directly the proliferation/survival of hematopoietic progenitors, it (1) potentiates the stromal cell-derived factor 1 (SDF-1)-dependent chemotaxis of human CD34(+) cells and lineage-committed myeloid, erythroid, and megakaryocytic progenitors; (2) primes SDF-1-dependent trans-Matrigel migration; and (3) stimulates matrix metalloproteinase-9 secretion and very late antigen 4 (VLA-4)-mediated adhesion to vascular cell adhesion molecule 1 (VCAM-1). Furthermore, we found that murine Sca-1(+) cells primed by C3a engrafted faster in lethally irradiated animals. These results indicate that normal human hematopoietic stem and progenitor cells express functional C3aR and that the C3aR-C3a axis sensitizes the responses of these cells to SDF-1 and thus may be involved in promoting their homing into the bone marrow via cross talk with the SDF-CXC chemokine receptor-4 (CXCR4) signaling axis. C3a is the first positive regulator of this axis to be identified.

CXCR4-SDF-1 signaling is active in rhabdomyosarcoma cells and regulates locomotion, chemotaxis, and adhesion.

We hypothesized that the CXC chemokine receptor-4 (CXCR4)-stromal-derived factor-1 (SDF-1) axis may be involved in metastasis of CXCR4(+) tumor cells into the bone marrow and lymph nodes, which secrete the alpha-chemokine SDF-1. To explore this hypothesis, we phenotyped by fluorescence-activated cell sorter analysis various human tumor cell lines for expression of CXCR4 and found that it was highly expressed on several rhabdomyosarcoma (RMS) cell lines. We also observed that cell lines derived from alveolar RMS, which is characterized by recurrent PAX3- and PAX7-FKHR gene fusions and is associated with a poor prognosis, expressed higher levels of CXCR4 than lines derived from embryonal RMS. Furthermore, transfer of a PAX3-FKHR gene into embryonal RMS cell activates CXCR4 expression. Because alveolar RMS frequently metastasizes to the bone marrow and lymph nodes, it seems that the CXCR4-SDF-1 axis could play an important role in this process. These findings prompted us to determine whether SDF-1 regulates the metastatic behavior of RMS cells. Accordingly, we found that, although SDF-1 did not affect proliferation or survival of these cell lines, it induced in several of them (1) phosphorylation of mitogen-activated protein kinase p42/44; (2) locomotion; (3) directional chemotaxis across membranes covered by laminin, fibronectin, or Matrigel; (4) adhesion to laminin, fibronectin, and endothelial cells; and (5) increased MMP-2 and diminished tissue inhibitors of metalloproteinases secretion. The small-molecule CXCR4-specific inhibitor, T140, effectively blocked the in vitro responses of RMS cells to SDF-1. On the basis of these observations we suggest that the CXCR4-SDF-1 axis may play an important role in tumor spread and metastasis of RMS cells to bone marrow and that molecular strategies aimed at inhibiting this axis could thus prove to be useful therapeutic measures.

Thrombopoietin, but not cytokines binding to gp130 protein-coupled receptors, activates MAPKp42/44, AKT, and STAT proteins in normal human CD34+ cells, megakaryocytes, and platelets.

OBJECTIVE: The development of megakaryocytes is regulated by thrombopoietin (TPO), which binds to the c-mpl receptor, and by several other cytokines such as interleukin (IL)-6, IL-11, leukemia inhibitory factor (LIF), cilliary neurotropic factor (CNTF), and oncostatin (OSM), which bind to gp130 protein-coupled receptors. We attempted to identify signal transduction pathways activated by these factors in normal human megakaryocytes. MATERIALS AND METHODS: To better understand the role of these factors in normal human megakaryopoiesis we studied their effect on 1) purified human bone marrow-derived CD34+ cells, 2) human alpha(IIb)beta3+ cells (shown by immunophenotypical and morphological criteria to be megakaryoblasts), which had been expanded ex vivo from CD34+ cells in chemically defined artificial serum, and 3) gel-filtered human peripheral blood platelets. Further, in an attempt to correlate the influence of these factors on cell proliferation and survival with activation of signal transduction pathways, we evaluated their effect on the phosphorylation of MAPK p42/44 and activation of PI-3K-AKT and JAK-STAT proteins in these various cell types. RESULTS: Using serum-free liquid cultures, we found that only TPO and IL-6 protected CD34+ cells and megakaryocytes from undergoing apoptosis (decrease in annexin-V binding, PARP cleavage, and activation of caspase-3). Moreover, only TPO when used alone and IL-6 only when used in combination with TPO, stimulated the growth of human colony-forming unit-megakaryocytes (CFU-Meg) in semisolid serum-free medium. We also observed that while TPO efficiently activated various signaling pathways in CD34+ cells, megakaryocytes, and platelets (MAPK p42/44, PI-3K-AKT, STAT proteins), IL-6 stimulated phosphorylation of STAT-1, -3, and -5 proteins only in CD34+ cells and megakaryoblasts. To our surprise, none of the other gp130 protein-related cytokines tested (IL-11, LIF, CNTF, and OSM) activated these signaling pathways in CD34+ cells, megakaryoblasts, or platelets. CONCLUSIONS: Our signal transduction studies explain why TPO, by simultaneously activating several signaling pathways, is the most potent megakaryopoietic regulator and why of all five gp130 protein-related cytokines tested, only IL-6, through activation of STAT proteins, plays a role in normal human megakaryopoiesis.

The Hematopoietic Microenvironment: Matrix Metalloproteinases in the Hematopoietic Microenvironment.

Matrix metalloproteinases (MMPs) are structurally and functionally related zinc-dependent endopeptidases capable of degrading the components of extracellular matrix (ECM) and basement membranes. MMPs participate in many physiological processes and have also been implicated in various pathological conditions including tumor invasion and metastasis. The functions of MMPs are known to be controlled by mechanisms leading to activation of their latent forms and through inhibition of both active and latent forms by natural tissue inhibitors of metalloproteinases (TIMPs). The complex relationships between MMPs and TIMPs within the bone marrow microenvironment during normal hematopoiesis as well as during leukemic growth and dissemination have not been extensively investigated. We reported that primary acute myelogenous leukemia (AML) blasts and leukemic KG-1 cells penetrate reconstituted basement membrane (Matrigel) in an in vitro invasion assay, secrete the gelatinases (MMP-2 and MMP-9) and express active MMP-2 on the cell surface. We also analyzed MMP/TIMP expression in normal bone marrow cells of the myeloid and stromal lineages and showed that MMP-2, MMP-9, TIMP-1 and TIMP-2 are produced in the bone marrow microenvironment. Furthermore, we examined the role of gelatinases in the transmigration of stem/progenitor cells from the bone marrow into peripheral blood. We found that steady-state bone marrow CD34(+) cells, unlike circulating peripheral blood CD34(+) cells, did not express MMP-2 and MMP-9 mRNA transcripts and proteins, and that various cytokines were able to upregulate expression of these MMPs in bone marrow CD34(+) cells and trans-Matrigel migration of these cells. Thus, we now have evidence that MMPs and TIMPs are constituents of the hematopoietic microenvironment although their roles in hematopoiesis have yet to be determined.