Influences And Differential Signaling Of Three Chemotactic Molecules LTD4, S1P And SDF-1 On CD34+ Hematopoietic Progenitor Cells

Hematopoietic stem and progenitor cells have characters of migration, proliferation and self-renewal. According to previous investigations, there were three chemotactic ligands which can regulate the migration and homing of hematopoietic stem cells (HSC): SDF-1α, Leukotriene D4 (LTD4) and spingosine-1-phosphate (S1P). CXCR4, the receptor of SDF-1, were well known to be expressed on hematopoietic cells and regulate the trafficking and homing of hematopoietic stem cells. Although two main receptors of LTD4 were cloned and identified, many effects of LTD4 were mainly mediated through cysLT1, which is also expressed in CD34+ hematopoietic progenitor cells. We have shown that LTD4 induced chemotaxis and in vitro transendothelial migration of CD34+ cells. Up to date, five members of S1P receptors, S1P1-5, have been identified. We previously analyzed expression of S1P receptors in CD34+ hematopoietic progenitor cells and only S1P1 mRNA expression was found in human hematopoietic progenitor cells (HPCs). Furthermore, S1P can acts on hematopoietic progenitor cells as a chemotactic factor, attracting peripheral blood CD34+ cell in vitro. All the three molecular receptors belong to G protein coupled receptors (GPCRs).GPCRs are a large superfamily of receptors, being capable of transducing a wide range of extracellular signals across the plasma membrane of the cells into discrete intracellular messages. Hematopoietic stem cells are known to carry anarray of GRCRs, which regulate a variety of important functions. Although both alpha and beta gamma subunits can induce downstream signaling, the classification of G-protein has been defined by the amino acid sequence of the alpha subunits, resulting in a subsequent grouping of these proteins into four families: Gas, Gai, Gaq/11 and Ga12-13. The Gai familiy can be ADP-ribosylated by pertusis toxin (PTX), which uncoupled the G-protein from the activated receptors. Pertussis toxin has been instrumental in delineating signaling pathways dependent on G proteins.In the present study, we investigated the effect of LTD4 and S1P on proliferation and adhesion of human CD34+ progenitor cells, and compared the signal transduction pathways induced by the GRCRs of three chemotactic ligands.In an effort to investigate the possible effects of LTD4 on primitive Hematopoietic progenitors, we use a stroma-free liqid culture system. In synergy with SCF and IL-3, LTD4 increase the expansion of human CD34~+ cells. After liquid culture for 1 weeks, the total number of CD34~+ cells in the presence of LTD4 was higher than the untreated cells. We also assessed the effect of SDF-1 and S1P on the expansion of hematopoietic progenitor cells and there were no significant increase of the CD34~+ cell number in presence of SDF-1 or S1P.Proliferation experiments with varying cytokine combinations revealed an effect of LTD4 on cell expansion predominantly in samples containing IL-3. The presence of 100 nM LTD4 in the culture medium raise cell numbers in all conditions tested, particularly in samples with IL-3 a significant increase in absolute cell numbers was observed.Cysteinyl leukotrienes are produced by bone marrow stroma and endothelium. To further characterize a potential function of cysteinyl leukotrienes within the bone marrow microenvironment and in the process of transendothelial migration, we analyzed primary human BM stromal cells (isolated from donor aspirates) aswell as human BM endothelial cells (BMEC-1) for their ability to synthesize cysteinyl leukotrienes in vitro by a technique combining HPLC and a sensitive immunoassay. Biologically significant amounts of LTC4, LTD4, and LTE4 were found in the supematants of confluent layers of BM endothelium and also in primary, pure stroma, with LTD4 concentrations equalling 6.05 ± 1.95 nM (BMEC-1) and 5.11 ± 1.12 nM (stroma) per 10~6 cultured cells. Summarizing all 3 leukotrienes (LTC4 is very rapidly transformed to LTD4 and further on to LTE4), concentrations were as high as 21.35 ± 5.25 nM (endothelium) and 18.27 ± 4.13 nM (stroma) per 10~6 cells, respectively. Of note, significant effects on HPC proliferation in vitro were observed at a final LTD4 concentration of as low as 1 nM, demonstrating the physiological relevance of the observed data. Interestingly, in conditioned medium of stromal cells not deprived of hematopoietic cells by treatment with MPA almost no cysteinyl leukotrienes could be detected.On the other hand, when pure stromal layers were cocultured with BM CD34~+ cells (derived from the same donor source), leukotriene-levels were significantly reduced. This suggests leukotriene metabolism and/or modulation of leukotriene production by BM hematopoietic cells and supports previous findings that the quantity and profile of leukotrienes produced both in vitro and in vivo critically depend on cellular interactions and the composition of mixed cell populations.To investigate the influence of S1P on the adhesion of hematopoietic stem and progenitor cells, we measure the adhesion of freshly isolated human CD34~+ cells. To prepare HUVEC monolayers, 1x10~4 cells were seeded on gelatine-coated (0.1 % in PBS) 96-well flat bottom tissue culture plates 48 hours before the assay and grown to confluency in endothelial cell growth medium + 10 % FCS. Before adding HPC, the monolayers were prestimulated with 10 ng/ml IL-1β and washed twice with 200 μl/well serum-free assay medium. Our results showed that after stimulated with S1P, the adhesion cell number of hematopoietic stem cell increase significantly. However, the number doesn't continue increase afterexposure to LTD4 for longer time.Since S1P exists ubiquitously in the plasma and bone marrow microenvironment, the effect of S1P on the proliferation of human CD34~+ cells were measured with liquid culture system and colony forming assay (CFA). Our results showed that S1P didn't increase the expansion and forming colonies number significantly. Furthermore, the survival rate of human hematopoietic stem and progenitor cells were measured with Annexin V staining by FACS. However, after exposure the human hematopoietic stem cells to S1P, the survival rate did not change significantly.For MAP kinase was a common signal molecule that involved in cell proliferation, we studied the effects of LTD4 on the phosphorylation status of MAP kinase. We found that LTD4 significantly increased the level of phosphorylation of MAP kinase in both human primitive CD34~+ cells and CD34+ cell lines. In CD34~+ KG1a cells, the phosphorylation was rapid and transient, reaching a maximum after 1 min approach the initial basal level after 5 min. To investigate whether PI3K-Akt signal was involve the effect induced by LTD4, phosphorylation of Akt was also measured with western blot. The results showed no significant phosphorylation in KG1a cells cultured with LTD4.To identify the signal transduction mechanism of the two chemotacitc molecules, we used Pertussis toxin (PTX), which modify certain heterotrimeric G proteins. Our results showed that PTX blocked phosphorylation of MAPK induced with SDF1 while failed to block the one induced with LTD4. These results show that LTD4 and SDF1 activated MAP kinase signal through different heterotrimeric G-proteins.Since both LTD4 and SDF-1 could induce intracellular free calcium in CD34~+ cells, we compare the effect activated by LTD4 and SDF-1 in presence of PTX or not. Our results showed LTD4 induced a stronger increase of intracellular free calcium. The cells showed weaker effect in response to SDF-1 given in an optimistically concentration. Calcium fluxes induced by LTD4 were completelyblocked by the specific cysLT1 receptors antagonists MK571 and only partially by PTX, whereas PTX can completely block the calcium fluxes induced by SDF-1.Pyk2 signal is known to involve the cell motility. For LTD4, SDF-1 and S1P involved migration of human progenitor cells, pyk2 signals were measure with western blotting. We showed that LTD4 and SDF-1 can induce rapid and transient phosphorylation of Pyk2 in human CD34~+ cells. The phosphorylation of Pyk2 activated by SDF-1 can be completely inhibited with PTX while the one by LTD4 was only partially blocked with PTX. Most recently, our results showed S1P could also increase migration of human stem cells and progenitor cells. Therefore, the pyk2 signal was also investigated. The results showed that pyk2 signal were phosphorylated after cultured with S1P and were fully blocked by preincubation with PTX.Actin polymerization in response to LTD4 and SDF-1 is an initial step in cell locomotion. To identify the PTX-insensitve G-protein responsible for the F-actin polymerization, the polymerized F-actin were measured in presence of PTX or not. Our results showed that In CD34~+ cells, the SDF-1 induced formation of stress fibres was significantly inhibited by PTX. The LTD4 induced formation of stress fibres was only partially blocked with PTX.Taken together, our findings suggest that: 1)there was high expression of cysLT1 (receptor of LTD4) in human hematopoietic stem and progenitor cells, and LTD4 could induce significant proliferations of human CD34~+ hematopoietic stem cells; 2) stromal cell and endothelial cells in bone marrow could produce LTD4 when hematopoiesis is absent (aplasia, "emergency reaction"), facilitating adhesion (homing) and supporting immediate human hematopoietic progenitor/stem cell growth; 3) S1P could increase the adhesion of hematopoietic stem cells, but didn't affect the proliferation and survival of HSC; 4) CysLT1 activated PTX-sensitive and PTX-insensitive G-proteins, which induced calcium influx and ERK1/2 phosphorylatin in human hematopoietic stem cells toregulate migration and proliferation of CD34~+ cells; 5) in HPC, proliferative effects were mediated by cysLT1, but not by CXCR4 and S1P1 which signals were mediated only by Gi-dependent GPCR; 6) Gq-mediated acti-vation of MAPK/Erk most likely involves the Ca++/Pyk2 path-way; 7)CXCR4 and S1P1 sig-naling only involves Gi proteins, which may not be sufficient to induce significant proliferation.