Rapamycin activates TGF receptor independently of its ligand: implications for endothelial dysfunction.

Abstract

Rapamycin, the macrolide immunosuppressant and active pharmaceutic in drug-eluting stents (DES), has a well-recognized antiproliferative action that involves inhibition of the mTOR pathway after binding to the cytosolic protein FKBP12. TGF receptor-type I (TGFRI) spontaneous activation is inhibited by the association with FKBP12. We hypothesized that rapamycin, in addition to inhibition of mTOR signaling, activates TGFRI independent of TGFβ. Human umbilical vein endothelial cells (HUVECs) were treated with rapamycin (10 nmol/l) and/or TGFβ RI kinase inhibitor (TGFRIi, 100 nmol/l) for 24 h. Rapamycin induced SMAD phosphorylation (SMAD1, SMAD2, and SMAD5) and PAI-1 up-regulation, which was specifically abrogated by SMAD2 knockdown. TGFRIi efficiently blocked phosphorylation of SMAD2, but not SMAD1/5. Interestingly, the inhibitor did not alter cell proliferation arrest induced by rapamycin. Active TGFβsecretion was not affected by the treatment. Neutralizing TGFβ experiments did not influence SMAD2 phosphorylation or PAI-1 expression indicating that activation of this pathway is independent of the ligand.In addition, rapamycin induction of endothelial-to-mesenchymal transition (EndMT) was potentiated by IL-1β and efficiently blocked by TGFRIi. In vivo, the prothrombogenic effects of rapamycin and up-regulation of PAI-1 in murine carotid arteries were reduced by TGFRIi treatment. In conclusion, we provide evidence that rapamycin activates TGF receptor independent of its ligand TGFβ, in concert with promotion of PAI-1 expression and changes in endothelial phenotype. These undesirable effects, the prothrombogenic state, and activation of EndMT are SMAD2-dependent and independent of the therapeutic rapamycin-induced cell proliferation arrest.