Fine-tuning nucleophosmin in macrophage differentiation and activation.

M-CSF-driven differentiation of peripheral blood monocytes is one of the sources of tissue macrophages. In humans and mice, the differentiation process involves the activation of caspases that cleave a limited number of proteins. One of these proteins is nucleophosmin (NPM1), a multifunctional and ubiquitous protein. Here, we show that caspases activated in monocytes exposed to M-CSF cleave NPM1 at D213 to generate a 30-kDa N-terminal fragment. The protein is further cleaved into a 20-kDa fragment, which involves cathepsin B. NPM1 fragments contribute to the limited motility, migration, and phagocytosis capabilities of resting macrophages. Their activation with lipopolysaccharides inhibits proteolytic processes and restores expression of the full-length protein that negatively regulates the transcription of genes encoding inflammatory cytokines (eg, NPM1 is recruited with NF-κB on the MCP1 gene promoter to decrease its transcription). In mice with heterozygous npm gene deletion, cytokine production in response to lipopolysaccharides, including CXCL1 (KC), MCP1, and MIP2, is dramatically enhanced. These results indicate a dual function of NPM1 in M-CSF-differentiated macrophages. Proteolysis of the protein participates in the establishment of a mature macrophage phenotype. In response to inflammatory stimuli, the full-length protein negatively regulates inflammatory cytokine production.

Transactivation of the epidermal growth factor receptor by heat shock protein 90 via Toll-like receptor 4 contributes to the migration of glioblastoma cells.

Extracellular heat shock protein HSP90α was reported to participate in tumor cell growth, invasion, and metastasis formation through poorly understood signaling pathways. Herein, we show that extracellular HSP90α favors cell migration of glioblastoma U87 cells. More specifically, externally applied HSP90α rapidly induced endocytosis of EGFR. This response was accompanied by a transient increase in cytosolic Ca(2+) appearing after 1-3 min of treatment. In the presence of EGF, U87 cells showed HSP90α-induced Ca(2+) oscillations, which were reduced by the ATP/ADPase, apyrase, and inhibited by the purinergic P(2) inhibitor, suramin, suggesting that ATP release is requested. Disruption of lipid rafts with methyl ß-cyclodextrin impaired the Ca(2+) rise induced by extracellular HSP90α combined with EGF. Specific inhibition of TLR4 expression by blocking antibodies suppressed extracellular HSP90α-induced Ca(2+) signaling and the associated cell migration. HSPs are known to bind lipopolysaccharides (LPSs). Preincubating cells with Polymyxin B, a potent LPS inhibitor, partially abrogated the effects of HSP90α without affecting Ca(2+) oscillations observed with EGF. Extracellular HSP90α induced EGFR phosphorylation at Tyr-1068, and this event was prevented by both the protein kinase Cδ inhibitor, rottlerin, and the c-Src inhibitor, PP2. Altogether, our results suggest that extracellular HSP90α transactivates EGFR/ErbB1 through TLR4 and a PKCδ/c-Src pathway, which induces ATP release and cytosolic Ca(2+) increase and finally favors cell migration. This mechanism could account for the deleterious effects of HSPs on high grade glioma when released into the tumor cell microenvironment.

Alpha-defensins secreted by dysplastic granulocytes inhibit the differentiation of monocytes in chronic myelomonocytic leukemia.

Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic disorder that occurs in elderly patients. One of the main diagnostic criteria is the accumulation of heterogeneous monocytes in the peripheral blood. We further explored this cellular heterogeneity and observed that part of the leukemic clone in the peripheral blood was made of immature dysplastic granulocytes with a CD14(-)/CD24(+) phenotype. The proteome profile of these cells is dramatically distinct from that of CD14(+)/CD24(-) monocytes from CMML patients or healthy donors. More specifically, CD14(-)/CD24(+) CMML cells synthesize and secrete large amounts of alpha-defensin 1-3 (HNP1-3). Recombinant HNPs inhibit macrophage colony-stimulating factor (M-CSF)-driven differentiation of human peripheral blood monocytes into macrophages. Using transwell, antibody-mediated depletion, suramin inhibition of purinergic receptors, and competitive experiments with uridine diphosphate (UDP)/uridine triphosphate (UTP), we demonstrate that HNP1-3 secreted by CD14(-)/CD24(+) cells inhibit M-CSF-induced differentiation of CD14(+)/CD24(-) cells at least in part through P2Y6, a receptor involved in macrophage differentiation. Altogether, these observations suggest that a population of immature dysplastic granulocytes contributes to the CMML phenotype through production of alpha-defensins HNP1-3 that suppress the differentiation capabilities of monocytes.

Colony-stimulating factor-1-induced oscillations in phosphatidylinositol-3 kinase/AKT are required for caspase activation in monocytes undergoing differentiation into macrophages.

The differentiation of human peripheral blood monocytes into resident macrophages is driven by colony-stimulating factor-1 (CSF-1), which upon interaction with CSF-1 receptor (CSF-1R) induces within minutes the phosphorylation of its cytoplasmic tyrosine residues and the activation of multiple signaling complexes. Caspase-8 and -3 are activated at day 2 to 3 and contribute to macrophage differentiation, for example, through cleavage of nucleophosmin. Here, we show that the phosphatidylinositol-3 kinase and the downstream serine/threonine kinase AKT connect CSF-1R activation to caspase-8 cleavage. Most importantly, we demonstrate that successive waves of AKT activation with increasing amplitude and duration are required to provoke the formation of the caspase-8-activating molecular platform. CSF-1 and its receptor are both required for oscillations in AKT activation to occur, and expression of a constitutively active AKT mutant prevents the macrophage differentiation process. The extracellular receptor kinase 1/2 pathway is activated with a coordinated oscillatory kinetics in a CSF-1R-dependent manner but plays an accessory role in caspase activation and nucleophosmin cleavage. Altogether, CSF-1 stimulation activates a molecular clock that involves phosphatidylinositol-3 kinase and AKT to promote caspase activation. This oscillatory signaling pathway, which is coordinated with extracellular receptor kinase 1/2 oscillatory activation, involves CSF-1 and CSF-1R and controls the terminal differentiation of macrophages.

Targeting apoptosis proteins in hematological malignancies.

The apoptotic machinery plays a key role in hematopoietic cell homeostasis. Terminally differentiated cells are eliminated, at least in part, by apoptosis, whereas part of the apoptotic machinery, including one or several caspases, is required to go through very specific steps of the differentiation pathways. A number of hematological diseases involve a deregulation of this machinery, which in most cases is a decrease in cell sensitivity to pro-apoptotic signals through over-expression of anti-apoptotic molecules. In some situations however, e.g. in the erythroid lineage of low grade myelodysplastic syndromes, cell sensitivity to apoptosis is increased in a death receptor-dependent manner and cell death pathways are inhibited only when these diseases progress into high grade and acute leukemia. Therapeutic strategies targeting the apoptotic machinery specifically block cell death inhibitors that are over-expressed in transformed cells, mainly Bcl-2-related proteins and Inhibitor of Apoptosis Proteins (IAPs). Another strategy is the activation of the extrinsic pathway to apoptosis, mainly through the death receptor agonist Tumor necrosis factor-Related Apoptosis Inducing Ligand (TRAIL) or agonistic antibodies targeting TRAIL receptors. The use of inhibitors of death receptors could make sense when these receptors are involved in excessive cell death or activation of survival pathways. Most of the drugs targeting apoptotic pathways introduced in clinics have demonstrated their tolerability. Their efficacy, either alone or in combination with other drugs such as demethylating agents and histone deacetylase inhibitors, is currently tested in both myeloid and lymphoid hematological diseases.