Influence of PECAM-1 ligand interactions on PECAM-1-dependent cell motility and filopodia extension

Abstract

Platelet endothelial cell adhesion molecule (PECAM‐1) has been implicated in angiogenesis through processes that involve stimulation of endothelial cell motility. Previous studies suggest that PECAM‐1 tyrosine phosphorylation mediates the recruitment and then activation of the tyrosine phosphatase SHP‐2, which in turn promotes the turnover of focal adhesions and the extension of filopodia, processes critical to cell motility. While these studies have implicated PECAM‐1‐dependent signaling in PECAM‐1‐mediated cell motility, the involvement of PECAM‐1 ligand binding in cell migration is undefined. Therefore to investigate the role of PECAM‐1 binding interactions in cell motility, mutants of PECAM‐1 were generated in which either homophilic or heparin/glycosaminoglycan (GAG)‐mediated heterophilic binding had been disabled and then expressed in an endothelial cell surrogate. We found that the ability of PECAM‐1 to stimulate cell migration, promote filopodia formation and trigger Cdc42 activation were lost if PECAM‐1‐dependent homophilic or heparin/GAG‐dependent heterophilic ligand binding was disabled. We further observed that PECAM‐1 concentrated at the tips of extended filopodia, an activity that was diminished if homophilic, but not heparin/GAG‐mediated heterophilic binding had been disrupted. Similar patterns of activities were seen in mouse endothelial cells treated with antibodies that specifically block PECAM‐1‐dependent homophilic or heterophilic adhesion. Together these data provide evidence for the differential involvement of PECAM‐1‐ligand interactions in PECAM‐1‐dependent motility and the extension of filopodia.

Involvement of TIMP-1 in PECAM-1-mediated tumor dissemination

“…These pro-proliferative effects were mediated by soluble endothelial-derived factors that are dependent on PECAM-1 homophilic ligand interactions, but are independent of PECAM-1-dependent signaling. These results contrast with previous findings, which indicated that PECAM-1 promotes EC migration via both PECAM-1 homophilic and PECAM-1-dependent, GAG-mediated heterophilic ligand interactions, as well as tyrosine phosphorylation of the cytoplasmic domain and recruitment of the SHP-2 tyrosine phosphatase ( 35 , 52 , 53 ). Further analyses of the CM from tumor cell/EC co-cultures identified TIMP-1 as a PECAM-1-regulated endothelial factor, the targeting of which in the tumor cell/REN-MP system inhibited tumor cell proliferation.…”

“…The present study next aimed to confirm the co-culture findings obtained with primary MECs, and to determine the molecular basis of PECAM-1 activity in promoting tumor cell proliferation. Specifically, tumor co-culture studies were performed with REN cells, a non-PECAM-1 expressing human mesothelioma cell line, which has been used as an endothelial surrogate for studying the function of PECAM-1 ( 14 , 34 , 52 , 53 ). REN cells were transduced to express wild-type mouse PECAM-1 (REN-MP), or mouse PECAM-1 in which homophilic binding (REN-MP ΔHom ), GAG/heterophilic binding (REN-MP ΔHet ) or intracellular signaling (REN-MPY→F) had been disabled ( 53 ) ( Fig.…”

“…Cells were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 1.0 g/l glucose, 2 mM L-glutamine, 100 U/ml penicillin, 0.1 µ g/ml streptomycin and 10% fetal bovine serum (FBS; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). REN cells ( 51 ), a human mesothelioma cell line that has been used as a surrogate for studying ECs ( 14 , 34 , 52 , 53 ) were provided by Dr Steven Albelda (University of Pennsylvania), and were grown in RPMI-1640, (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 0.1 µ g/ml streptomycin and 10% FBS. Previously generated stably transduced REN cell lines expressing wild-type and mutant mouse PECAM-1 were employed ( 53 ) and were cultured in RPMI-1640 complete media containing 1 µ g/ml puromycin.…”

“…REN cells ( 51 ), a human mesothelioma cell line that has been used as a surrogate for studying ECs ( 14 , 34 , 52 , 53 ) were provided by Dr Steven Albelda (University of Pennsylvania), and were grown in RPMI-1640, (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 0.1 µ g/ml streptomycin and 10% FBS. Previously generated stably transduced REN cell lines expressing wild-type and mutant mouse PECAM-1 were employed ( 53 ) and were cultured in RPMI-1640 complete media containing 1 µ g/ml puromycin. Primary MECs were isolated from C57BL/6 mouse lungs using fluorescent-activated cell sorting (FACS) as previously described ( 54 ) and were cultured in M199 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 15% FBS, 50 µ g/ml endothelial growth factor (BD Biosciences, Franklin Lakes, NJ, USA), 100 µ g/ml heparin and 1 mM glutamine.…”

ITIM receptors: more than just inhibitors of platelet activation

“…33 Other factors, including receptor density, glycosylation and Ca 21 , can all influence PECAM-1ligand interactions. 34 This interaction is likely to be of low affinity under resting conditions, because platelets do not adhere to each other or to endothelial cells under healthy conditions, making platelet-platelet and platelet-endothelial cell interactions during thrombus formation the probable physiologically relevant context. Avidity and affinity are likely increased under these conditions.…”