Thrombin induces endocytosis of endoglin and type-II TGF-β receptor and down-regulation of TGF-β signaling in endothelial cells

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Abstract
Thrombin activates protease-activated receptor 1 (PAR1) on endothelial cells (ECs) and is critical for angiogenesis and vascular development. However, the mechanism underlying the proangiogenic effect of thrombin has not been elucidated yet. Here, we report the discovery of a novel functional link between thrombin-PAR1 and transforming growth factor-β (TGF-β) signaling pathways. We showed that thrombin via PAR1 induced the internalization of endoglin and type-II TGF-β receptor (TβRII) but not type-I receptors in human ECs. This effect was mediated by protein kinase C-ζ (PKC-ζ) since specific inhibition of PKC-ζ caused an aggregation of endoglin or TβRII on cell surface and blocked their internalization by thrombin. Furthermore, acute and long-term pretreatment of ECs with thrombin or PAR1 peptide agonist suppressed the TGF-β–induced serine phosphorylation of Smad2, a critical mediator of TGF-β signaling. Moreover, activation of PAR1 led to a profound and spread cytosolic clustering formation of Smad2/3 and markedly prevented Smad2/3 nuclear translocation evoked by TGF-β1. Since TGF-β plays a crucial role in the resolution phase of angiogenesis, the down-regulation of TGF-β signaling by thrombin-PAR1 pathway may provide a new insight into the mechanism of the proangiogenic effect of thrombin.