Abstract
We have previously demonstrated consistent and high expression of the G protein-coupled receptors (GPCR) CXCR4, cysLT1 and S1P1 in human hematopoetic progenitor and stem cells (HPC). All of these receptors induced chemotactic responses in mobilized CD34+ HPC. As their ligands are present in the hematopoietic microenvironment, these GPCR may be involved in HPC homing and/or proliferation. Ligands for CXCR4 (SDF-1) and cysLT1 (cys-leukotrienes) are both produced by stromal cells, while the S1P1 ligand (sphingosine 1-phosphate, S1P) is released by megakaryocytes and platelets. As GPCR may activate multiple signaling pathways resulting in different functional effects, we compared the signaling capacities of CXCR4, cysLT1 and S1P1 in CD34+ HPC and CD34+ progenitor cell lines. At optimal ligand concentrations, cysLT1 mediated the strongest intracellular calcium fluxes, in contrast to CXCR4 (intermediate response) and S1P1 (almost absent calcium fluxes). Pertussis toxin (PTX) only partially inhibited cysLT1-mediated calcium signaling, indicating coupling of the receptor to both Gi and Gq proteins, while CXCR4-mediated responses were exclusively PTX-sensitive and therefore only Gi-dependent. Both cysLT1 and CXCR4 induced phosphorylation of the proliferation-related p44/42 (Erk) MAP kinase. Again, much stronger responses were mediated by cysLT1, which again were only partially PTX-sensitive. No Erk/MAP kinase activation was mediated by S1P1. We could demonstrate that pyk2, a kinase that links calcium signaling with Erk/MAP kinase activation, was involved in Gq-mediated signaling of cysLT1, as its phosphorylation was not inhibited by PTX. The fact that cysLT1 employs both Gi and Gq-mediated signaling pathways, while CXCR4 signaling was exclusively Gi-dependent, may explain why enhanced proliferation of CD34+ hematopoietic progenitor cells in cytokine-supplemented serum-free liquid cultures was only seen in response to ligands of cysLT1, but not in response to the CXCR4 ligand SDF-1. Activation of CXCR4 resulted in phosphorylation of Akt, which was not observed in response to ligands of cysLT1, while both GPCR induced translocation of NFkappaB to the nucleus. We conclude that in CD34+ HPC, Gi protein-mediated signaling of CXCR4 is mainly involved in cell migration, while Gi and Gq-mediated signaling of cysLT1 also affects cell proliferation predominantly by combined and profound activation of the Erk/MAP kinase pathway. The signaling capacity of S1P1 however was limited compared to the other GPCRs.
Regulation of MAP kinase activation induced by the stimulation of G protein-coupled receptors.
