Abstract
Thrombomodulin (TM) stimulates angiogenesis and protects vascular endothelial cells (ECs) via its fifth epidermal growth factor-like region (TME5); however, the cell surface receptor that mediates the pro-survival signaling activated by TM has remained unknown. We applied pull-down assay followed by MALDI-TOF MS and western blot analysis, and identified G-protein coupled receptor 15 (GPR15) as a binding partner of TME5. TME5 rescued growth inhibition and apoptosis caused by calcineurin inhibitor FK506 in vascular ECs isolated from wild type (WT) C57BL/6 mice. On the other hand, TME5 failed to protect ECs isolated from GPR15 knockout (GPR15 KO) mice from FK506-caused vascular injury. TME5 induced activation of extracellular signal-regulated kinase (ERK) and increased level of anti-apoptotic proteins in a GPR15 dependent manner. In addition, in vivo Matrigel plug angiogenesis assay found that TME5 stimulated angiogenesis in mice. TME5 promoted endothelial migration in vitro. Furthermore, TME5 increased production of NO in association with activated endothelial NO synthase (eNOS) in ECs. All these pro-angiogenesis functions of TME5 were abolished by knockout of GPR15. Our findings suggest that GPR15 plays an important role in mediating cytoprotective function as well as angiogenesis of TM.
Highlights
Thrombomodulin (TM), a membrane protein comprising of an N-terminal lectin-like domain (TMD1), a six-tandem epidermal growth factor (EGF)-like region, an O-glycosylation site-rich region, a transmembrane region, and a cytoplasmic tail, is constitutively expressed on vascular endothelial cells and acts as an anticoagulant by binding to thrombin via its fourth to sixth EGF-like regions (TME456)[1, 2]
These observations prompted us to investigate the function of Recombinant human soluble thrombomodulin (rTM) in vascular endothelial cells (ECs) and we found that rTM alleviated apoptosis of ECs caused by calcineurin inhibitors including tacrolimus (FK506) and cyclosporine A (CsA) and inflammatory cytokines
The immunoprecipitation of mixture of human umbilical vein endothelial cells (HUVECs) membrane proteins and V5-tagged TME5 by anti-V5 antibody followed by MALD-TOF MS analysis identified G-protein coupled receptor 15 (GPR15) as a candidate binding partner of TME5 (Table 1)
Summary
Thrombomodulin (TM), a membrane protein comprising of an N-terminal lectin-like domain (TMD1), a six-tandem epidermal growth factor (EGF)-like region, an O-glycosylation site-rich region, a transmembrane region, and a cytoplasmic tail, is constitutively expressed on vascular endothelial cells and acts as an anticoagulant by binding to thrombin via its fourth to sixth EGF-like regions (TME456)[1, 2]. Use of rTM counteracted with capillary leakage developed in association with SOS and ES These observations prompted us to investigate the function of rTM in vascular endothelial cells (ECs) and we found that rTM alleviated apoptosis of ECs caused by calcineurin inhibitors including tacrolimus (FK506) and cyclosporine A (CsA) and inflammatory cytokines. This anti-apoptotic effect was associated with up-regulation of myeloid cell leukemia sequence 1 (Mcl-1) proteins, www.nature.com/scientificreports/. The present study aimed to identify the cell surface expressed protein that interacts with TME5 and mediates endothelial cytoprotective as well as pro-angiogenic function of rTM
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