We have previously shown that Syk-specific siRNA inhibits FcγRIIA mediated platelet phagocytosis in the human macrophage cell line, THP-1. Surprisingly, we observed that THP-1 cell-cell interactions were affected by Syk-specific siRNA suggesting that Syk may be important in the signaling pathway for physiological cell spreading. As reported by

Brown et al. (Nature 418:200, 2002
), homophilic ligation of PECAM-1 on macrophages triggers cell-cell detachment, prompting us to speculate that Syk may be involved in PECAM-1 mediated signaling. PECAM-1 is a member of the immunoglobulin superfamily that contains an immunoreceptor tyrosine inhibitory motif (ITIM) required for inhibitory signaling and cell spreading via binding protein tyrosine phosphatase. The structure of Syk reveals considerable flexibility in the relative orientation of its tandem SH2 domains providing a molecular basis for the potential involvement of Syk in a variety of signal transduction pathways. Thus, the five tyrosine residues present in the cytoplasmic domain of human PECAM-1 could serve as docking sites for recruitment of other intracellular signaling molecules such as the protein tyrosine kinase, Syk. The present studies test the hypothesis that Syk may mediate an activation influence on intercellular interaction upon PECAM-1 ligation.

METHODS: Association of Syk with PECAM-1 was studied in THP-1 cells by immunoprecipitation and immunoblotting with anti-PECAM-1 monoclonal antibody with or without Syk-specific siRNA. The effect of Syk-specific siRNA on THP-1 cells was evaluated by morphology, cell growth kinetics, Trypan Blue exclusion, annexin V staining, and DNA fragmentation analysis. A series of synthetic peptides including sequences within the ITIM region of PECAM-1 were evaluated for their ability to bind Syk directly in THP-1 lysates and to phosphorylate Syk intracellularly.

RESULTS: First, Syk protein was co-immunoprecipitated from THP-1 cell lysates by anti-PECAM-1 monoclonal antibody, and Syk-specific siRNA knocked down PECAM-1 associated Syk in THP-1 cells. Second, knock down of Syk by siRNA resulted in decreased cell spreading and cell proliferation. Trypan Blue exclusion, annexin V staining, and DNA fragmentation were identical in THP-1 cells transfected with control dsRNA and Syk-specific siRNA. Third, the synthetic peptide of residues 658-691 within the ITIM region of PECAM-1 that contains two phosphotyrosines (N-S-D-V-Q-pY-T-E-V-Q-V-S-S-A-E-S-H-K-D-L-G-K-K- D-T-E-T-V-pY-S-E-V-R-K) bound directly to Syk. Fourth, ligation of PECAM-1 on THP-1 cells by anti-PECAM-1 monoclonal antibody resulted in Syk phosphorylation. Fifth, the dual phosphotyrosine 658-691 PECAM-1 peptide triggered direct phosphorylation of Syk in permeabilized THP-1 cells.

CONCLUSIONS: Our novel observations suggest that signal transduction after PECAM-1 ligation may trigger an activation pathway through the tyrosine kinase, Syk, and facilitate intercellular adhesion. The decreased proliferation observed in THP-1 cells transfected with Syk-specific siRNA is not a result of cell death or apoptosis. Based on our findings, we propose that PECAM-1 may exhibit both ITIM (inhibitory) and ITAM (activation) properties and that the balance of phosphatase and Syk activity triggered by PECAM-1 homophilic binding may affect the fate of intercellular interaction. These processes are expected to influence the net degree of phagocytosis of opsonized platelets by the reticuloendothelial system in patients with immune thrombocytopenia.

Topics:
blood platelets, cd31 antigens, macrophages, phagocytosis, rna, small interfering, signal transduction, ligation, monoclonal antibodies, peptides, annexin a5

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2005, The American Society of Hematology
2004
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