Ptdglc-Enriched Lipid Rafts Are a Novel Platform for Apoptosis Signaling in Human Neutrophilic Lineage Cells

Phosphatidylglucoside (PtdGlc) is a recently discovered type of cell surface glycophospholipid that was originally detected in human cord red cells. PtdGlc was also detected in early erythroblastic leukemia cells, astroglial cells from fetal rat brains and the HL-60 acute promyelocytic leukemia (APL) cell line, and thought to be associated with their differentiation (Biochim Biophys Acta. 1851: 90, 2015). Indeed, the recombinant anti-PtdGlc Fab antibody rGL-7 induced HL-60 cells to differentiate into neutrophilic cells along with inducing the phosphorylation of Lyn and Hck (PNAS 100: 7454, 2003). These activities were suppressed by methyl-β-cyclodextrin, which reduced endogenous cholesterol, suggesting that PtdGlc forms lipid raft-like domains on HL-60 cells. Another anti-PtdGlc monoclonal antibody DIM21 induced the formation of FAS-colocalized clusters of PtdGlc on plasma membranes and the apoptosis of human neutrophils[RJ1]. However, it is still unclear about the molecular connection of PtdGlc, inserted into the external leaflet of the membrane bilayer, with signal transduction molecules located at the cytoplasmic side. In this study, we investigated the differentiation effect of DIM21 by comparing the ones of dimethyl sulfoxide (DMSO) and all-trans retinoic acid (ATRA), and identified the extracellular proteins associate with PtdGlc-enriched domains on plasma membranes during the activation of apoptosis signaling in neutrophilic lineage cells.

HL60 cells or chronic neutrophilic leukemia (MOLM-20) cells were treated with DMSO or ATRA in the absence or presence of DIM21, and the expression of differentiation markers was determined. DIM21 induced the neutrophilic differentiation marker CD11b by itself, and synergistically enhanced DMSO-induced CD11b expression in HL-60 and MOLM-20 cells. DIM21 significantly enhanced annexin V positive DMSO-treated neutrophilic differentiated HL-60 (D-HL60) cells, but not MOLM-20 cells. FITC BrdU Flow analysis showed that the both ATRA and DIM21 induced the cell cycle accumulation in G0/G1 phase and reduced the percentage in S phase, suggesting that not only ATRA but also DIM21 is capable of reducing cell proliferation. Treatment with ATRA or DMSO increased the expression of PtdGlc and FAS in HL60, while DIM21 in itself is not capable of inducing the PtdGlc expression. To isolate the extracellular proteins associate with PtdGlc-enriched domains on plasma membranes, PtdGlc associated molecules on plasma membranes were labeled with FITC by the enzyme-mediated activation of radical sources (EMARS) method. Silver staining and western blotting analyses showed that the treatment with DIM21 enhanced the expression of several proteins in HL60, including FAS, integrin αM/β2 (CD11b/CD18). Furthermore, about five FITC-labeled proteins were immunoprecipitated with anti-FITC antibody and detected by sequenced SDS-PAGE. The shot-gun proteomics analysis detected an approximately equal number of FITC-labeled CD18 peptides in the resting and DIM21-stimulated HL60 cells. Other FITC-labeled proteins detected by proteomics analysis were included insulin receptor and Insulin like growth factor-1-receptor which is responsible in cell growth and survival control. These findings indicate that PtdGlc might have an intimate involvement in cell survival through the direct interaction with integrins and other transmembrane receptors. The novel strategies targeting PtdGlc by monoclonal antibody DIM21 warrant further exploration in patients with leukemia including APL.

[RJ1]