Rapamycin induces the TGFβ1/Smad signaling cascade in renal mesangial cells upstream of mTOR |
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Authors: | Bashier Osman Anke Doller El-Sayed Akool Martin Holdener Edith Hintermann Josef Pfeilschifter Wolfgang Eberhardt |
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Institution: | aPharmazentrum Frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany |
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Abstract: | The mTOR kinase inhibitor rapamycin (sirolimus) is a drug with potent immunosuppressive and antiproliferative properties. We found that rapamycin induces the TGFβ/Smad signaling cascade in rat mesangial cells (MC) as depicted by the nuclear translocation of phospho-Smads 2, -3 and Smad-4, respectively. Concomitantly, rapamycin increases the nuclear DNA binding of receptor (R)- and co-Smad proteins to a cognate Smad-binding element (SBE) which in turn causes an increase in profibrotic gene expression as exemplified by the connective tissue growth factor (CTGF) and plasminogen activator inhibitor 1 (PAI-1). Using small interfering (si)RNA we demonstrate that Smad 2/3 activation by rapamycin depends on its endogenous receptor FK binding protein 12 (FKBP12). Mechanistically, Smad induction by rapamycin is initiated by an increase in active TGFβ1 as shown by ELISA and by the inhibitory effects of a neutralizing TGFβ antibody. Using an activin receptor-like kinase (ALK)-5 inhibitor and by siRNA against the TGFβ type II receptor (TGFβ-RII) we furthermore demonstrate a functional involvement of both types of TGFβ receptors. However, rapamycin did not compete with TGFβ for TGFβ-receptor binding as found in radioligand-binding assay. Besides SB203580, a specific inhibitor of the p38 MAPK, the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC) and a cell-permeable superoxide dismutase (SOD) mimetic strongly abrogated the stimulatory effects of rapamycin on Smad 2 and 3 phosphorylation. Furthermore, the rapid increase in dichlorofluorescein (DCF) formation implies that rapamycin mainly acts through ROS. In conclusion, activation of the profibrotic TGFβ/Smad signaling cascade accompanies the immunosuppressive and antiproliferative actions of rapamycin. |
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Keywords: | FK506 binding protein p38 MAP kinase Rapamycin Renal fibrosis Smads TGFβ |
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