Rapamycin induces the TGFbeta1/Smad signaling cascade in renal mesangial cells upstream of mTOR.
The mTOR kinase inhibitor rapamycin (sirolimus) is a drug with potent immunosuppressive and antiproliferative properties. We found that rapamycin induces the TGFbeta/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 TGFbeta(1) as shown by ELISA and by the inhibitory effects of a neutralizing TGFbeta antibody. Using an activin receptor-like kinase (ALK)-5 inhibitor and by siRNA against the TGFbeta type II receptor (TGFbeta-RII) we furthermore demonstrate a functional involvement of both types of TGFbeta receptors. However, rapamycin did not compete with TGFbeta for TGFbeta-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 TGFbeta/Smad signaling cascade accompanies the immunosuppressive and antiproliferative actions of rapamycin.
Osman
Bashier
B
Pharmazentrum Frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
Doller
Anke
A
Akool
El-Sayed
el-S
Holdener
Martin
M
Hintermann
Edith
E
Pfeilschifter
Josef
J
Eberhardt
Wolfgang
W
D000818:Animals; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D050527:Mesangial Cells / Q000187:drug effects* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000494:pharmacology*; D051785:Smad Proteins / Q000378:metabolism*; D051896:Smad Proteins, Receptor-Regulated / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases; D022061:Tacrolimus Binding Protein 1A / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D007166:Immunosuppressive Agents; D047908:Intracellular Signaling Peptides and Proteins; D017382:Reactive Oxygen Species; D018125:Receptors, Transforming Growth Factor beta; D051785:Smad Proteins; D051896:Smad Proteins, Receptor-Regulated; D016212:Transforming Growth Factor beta; C546842:MTOR protein, human; C546845:mTOR protein, rat; D017346:Protein Serine-Threonine Kinases; D058570:TOR Serine-Threonine Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II; D022061:Tacrolimus Binding Protein 1A; D020123:Sirolimus