Downregulation of TGF-β Signaling by Thrombin in Endothelial Cells.

Thrombin activates protease-activated receptor 1 (PAR1) on endothelial cells (ECs) and regulates many aspects of EC biology. A body of evidence has demonstrated that the thrombin-PAR1 pathway 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. Using mass spectrometry, confocal immunofluorescence microscopy, cell surface ELISA and a phospho-specific antibody against the serine-phosphorylated endogin, we demonstrated for the first time that thrombin via PAR1 induced the internalization of endoglin and type-II TGF-β receptor (TβRII) but not the type-I receptors in primary human ECs. This effect was mediated by protein kinase C-ζ (PKC-ζ) that was rapidly activated by thrombin in ECs since specific inhibition of PKC-ζ caused aggregation of endoglin and TβRII on cell surface and blocked their internalization in response to thrombin. Furthermore, acute (30 min) and long-term (24 h) pretreatment of ECs with thrombin or PAR1 peptide agonist suppressed the TGF-β-induced serine phosphorylation of Smad2/3, the critical mediators of TGF-β signaling. Moreover, activation of PAR1 led to a profound clustering formation of Smad2/3 and a more spread cytosolic localization, which sequestered Smad2/3 from nucleus and prevented their nuclear translocation evoked by TGF-β. Since TGF-β and its receptors play a crucial role in the resolution phase of angiogenesis, the downregulation of TGF-β signaling by thrombin-PAR1 pathway may provide a new insight into the mechanism of the proangiogenic effect of thrombin.