CCL18/PARC stimulates hematopoiesis in long-term bone marrow cultures indirectly through its effect on monocytes.

Chemokines play a role in regulating hematopoietic stem cell function, including migration, proliferation, and retention. We investigated the involvement of CCL18 in the regulation of bone marrow hematopoiesis. Treatment of human long-term bone marrow cultures (LTBMCs) with CCL18 resulted in significant stimulation of hematopoiesis, as measured by the total number of hematopoietic cells and their committed progenitors produced in culture. Monocytes/macrophages, whose survival was almost doubled in the presence of CCL18 compared with controls, were the primary cells mediating this effect. Conditioned media from CCL18-treated mature monocytes fostered colony-promoting activity that increased the number of colonies formed by hematopoietic progenitor cells. Gene expression profiling of CCL18-stimulated monocytes demonstrated more than 200 differentially expressed genes, including those regulating apoptosis (caspase-8) and proliferation (IL-6, IL-15, stem cell factor [SCF]). Up-regulation of these cytokines was confirmed on the protein expression level. The contribution of SCF and IL-6 in CCL18-mediated stimulatory activity for hematopoiesis was confirmed by SCF- and IL-6-blocking antibodies that significantly inhibited the colony-promoting activity of CCL18-stimulated conditioned medium. In addition to the effect on monocytes, CCL18 facilitated the formation of the adherent layer in LTBMCs and increased the proliferation of stromal fibroblast-like cells.

Regulation of CXCR4-mediated nuclear translocation of extracellular signal-related kinases 1 and 2.

Activation of the chemokine receptor CXCR4 by its agonist stromal cell-derived factor 1 (SDF-1) has been associated with cell migration and proliferation in many cell types, but the intracellular signaling cascades are incompletely defined. Here we show that CXCR4-dependent extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation was mediated through the Ras/Raf pathway, as demonstrated with a dominant-negative Ras mutant and pharmacological inhibitors. The Src inhibitor 4-amino-5-methylphenyl-7-(t-butyl)pyrazolo[3,4-d] pyrimidine (PP1) and the Rho-kinase (ROCK) inhibitor N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide dihydrochloride (Y27632) also attenuated SDF-1-induced ERK1/2 phosphorylation. Involvement of Src could furthermore be demonstrated by Src phosphorylation and by the shortened ERK1/2 phosphorylation in SYF cells, which are Src/Yes/Fyn-deficient compared with Src-reconstituted Src(++) cells. Membrane translocation of RhoA could be detected similarly. A large portion of the SDF-1-mediated ERK phosphorylation was detected in the nucleus, as shown by Western blotting and confocal microscopy, and resulted in the phosphorylation of the transcription factor Elk. It is interesting that the nuclear accumulation of ERK1/2 and Elk phosphorylation was completely blocked by dominant-negative Rho, Y27632, PP1, and latrunculin B, indicating that the Rho/ROCK pathway, Src kinase, and the actin cytoskeleton were required in this process. In accordance, neither nuclear ERK phosphorylation nor Elk phosphorylation were observed in SYF cells stimulated with SDF-1 but were reconstituted in Src(++) cells. In summary, these results demonstrate that Src, Rho/ROCK, and an intact cytoskeleton contribute to overall ERK1/2 activation in SDF-1-stimulated cells and are indispensable for nuclear translocation of ERK1/2 and activation of transcription factors.

Arrestin regulates MAPK activation and prevents NADPH oxidase-dependent death of cells expressing CXCR2.

Activation of CXCR2 IL-8 receptor leads to activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and rapid receptor endocytosis. Co-immunoprecipitation and co-localization experiments showed that arrestin and CXCR2 form complexes with components of the ERK1/2 cascade following ligand stimulation. However, in contrast to the activation of the beta2-adrenergic receptor, arrestin was not necessary for ERK1/2 phosphorylation or receptor endocytosis. In contrast, beta-arrestin 1/2 double knockout cells showed greatly enhanced phosphorylation of ERK1/2, as well as phosphorylation of the stress kinases p38 and c-Jun N-terminal protein kinase. The stimulation of stress kinases in arrestin double knockout cells could be attenuated in the presence of diphenylene iodonium (DPI), an inhibitor of the NADPH oxidase, suggesting that reactive oxidant species (ROS) participated in mitogen-activated protein kinase (MAPK) activation. ROS could indeed be detected in IL-8-stimulated beta-arrestin 1/2 knockout cells, and cytoplasmic Rac was translocated to the membrane fraction, which is a prerequisite for oxidant formation. The oxidative burst induced cell death within 6 h of IL-8 stimulation of these cells, which could be prevented in the presence of DPI. These results indicate a novel function for arrestin, which is protection from an excessive oxidative burst, resulting from the sustained stimulation of G-protein-coupled receptors that cause Rac translocation.

MK-383 (tirofiban) induces a GPIIb/IIIa receptor conformation which differs from the resting and activated receptor.

The platelet integrin alphaIIb beta3 (GPIIb/IIIa) acts as a receptor for fibrinogen, playing a critical role in platelet aggregation. GPIIb/IIIa antagonists, which block the receptor-ligand interaction, have been accused of causing occasional thrombocytopenia, probably via drug-induced platelet activation or immunogenic neoepitopes. We, therefore, analyzed the effects of the GPIIb/IIIa antagonist MK-383 (tirofiban) on platelet activation and GpIIb/IIIa conformation. At a concentration of 10(-7) mol/l, MK-383 completely inhibited fibrinogen binding to in vitro stimulated platelets. Simultaneously, the GPIIb/IIIa expression density increased, similar to that on activated platelets, but no effect on P-selectin expression or the formation of platelet-leukocyte aggregates could be observed, indicating that MK-383 binding did not induce general platelet activation. The GPIIb/IIIa receptor conformation was further analyzed by fluorescence resonance energy transfer analysis between fluorochrome-labeled antibodies against different GpIIb/IIIa epitopes. As a result, MK-383 induced a receptor conformation that differed from the resting as well as the activated receptor as induced by ADP or TRAP-6. This conformational modulation of GPIIb/IIIa presents an interesting mechanism which may be linked to receptor recruitment without inducing general platelet activation.