The immunomodulator FTY720 and its phosphorylated derivative activate the Smad signalling cascade and upregulate connective tissue growth factor and collagen type IV expression in renal mesangial cells.
1.--The immunomodulating agent FTY720 is a substrate for the sphingosine kinase and the phosphorylated form is able to bind to sphingosine 1-phosphate (S1P) receptors. In this study, we show that exposure of renal mesangial cells to phospho-FTY720 leads to a rapid and transient activation of several protein kinase cascades, including the mitogen- and stress-activated protein kinases. The nonphosphorylated FTY720 also increased MAPK phosphorylation, but with a reduced potency and a more delayed time course. In addition, phospho-FTY720 and FTY720 are able to increase phosphorylation of Smad proteins which are classical members of the transforming growth factor-beta (TGF-beta) signalling device, thus suggesting a crosstalk between FTY720 and TGF-beta signalling. 2.--Pretreatment with the S1P(3) receptor antagonist suramin inhibits FTY720 and phospho-FTY720-induced Smad phosphorylation, whereas pertussis toxin pretreatment, which blocks G(i/0) proteins, has no effect on Smad phosphorylation. 3.--Since TGF-beta is a potent profibrotic cytokine in mesangial cells and upregulates the connective tissue growth factor (CTGF) and collagen as important hallmarks in the fibrotic sequelae, we investigated whether FTY720 and phospho-FTY720 are able to mimic these effects of TGF-beta. Indeed, FTY720 and phospho-FTY720 markedly upregulate CTGF and collagen type IV protein expressions. In addition, the tissue inhibitor of metalloproteinase-1 is transcriptionally activated by FTY720, whereas cytokine-induced matrix metalloproteinase-9 is down-regulated by FTY720. 4.--Depletion of the TGF-beta receptor type II by the siRNA transfection technique blocks not only Smad phosphorylation but also CTGF upregulation. Similarly, Smad-4 depletion by siRNA transfection also abrogates CTGF upregulation induced by FTY720 and phospho-FTY720. 5.--In summary, our data show that FTY720 and phospho-FTY720 not only activate the Smad signalling cascade in mesangial cells, but also upregulate the expression of CTGF and collagen. These findings suggest that FTY720 may have additional effects besides the established immunomodulatory action and, importantly, a profibrotic activity has to be considered in future experimental approaches.
Xin
Cuiyan
C
Pharmazentrum frankfurt Izafes, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany.
Ren
Shuyu
S
Eberhardt
Wolfgang
W
Pfeilschifter
Josef
J
Huwiler
Andrea
A
D000818:Animals; D002478:Cells, Cultured; D024141:Collagen Type IV / Q000096:biosynthesis*; D055513:Connective Tissue Growth Factor; D005355:Fibrosis; D000068876:Fingolimod Hydrochloride; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D017874:Immediate-Early Proteins / Q000096:biosynthesis*; D007166:Immunosuppressive Agents / Q000378:metabolism / Q000494:pharmacology*; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis*; D020935:MAP Kinase Signaling System / Q000502:physiology; D008297:Male; D010766:Phosphorylation; D011409:Propylene Glycols / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D051785:Smad Proteins / Q000378:metabolism / Q000502:physiology*; D051898:Smad1 Protein / Q000378:metabolism; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000502:physiology; D015854:Up-Regulation
C526316:CCN2 protein, rat; D024141:Collagen Type IV; D017874:Immediate-Early Proteins; D007166:Immunosuppressive Agents; D036341:Intercellular Signaling Peptides and Proteins; D011409:Propylene Glycols; D051785:Smad Proteins; D051898:Smad1 Protein; C497400:Smad1 protein, rat; D051899:Smad2 Protein; C497784:Smad2 protein, rat; D051900:Smad3 Protein; C497787:Smad3 protein, rat; D016212:Transforming Growth Factor beta; D055513:Connective Tissue Growth Factor; D000068876:Fingolimod Hydrochloride; D013110:Sphingosine