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

Abstract

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-b (TGF-b) signalling device, thus suggesting a crosstalk between FTY720 and TGF-b 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-b 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-b. 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-b 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. British Journal of Pharmacology (2006Pharmacology ( ) 147, 164-174. doi:10.1038; published online 21 November 2005 Keywords: FTY720; mesangial cell; Smad; TGF-b 2 ; CTGF; collagen Abbreviations: DMEM, Dulbecco's modified Eagle medium; ECL, enhanced chemiluminescence; FTY720, 2-amino-2-[2-(4-octyl-phenyl)ethyl]-1,3-propanediol hydrochloride; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL-1b, interleukin-1b; iNOS, inducible nitric oxide synthase; MAPK, mitogen-activated protein kinase; MMP-9, matrix metalloproteinase-9; NO, nitric oxide; PKB, protein kinase B; SAPK/JNK, stress-activated protein kinase/Nterminal c-Jun kinase; S1P, sphingosine 1-phosphate; TGF-b, transforming growth factor-b; TIMP-1, tissue inhibitor of metalloproteinase-1

FTY720 Ameliorates Th1-Mediated Colitis in Mice by Directly Affecting the Functional Activity of CD4+CD25+ Regulatory T Cells

“…Recently, such a direct interference with TGF␤ signaling was also proven for FTY720, since FTY720 led to a significant phosphorylation and thereby activation of Smad proteins. Thus, in different cell types these data underline the possibility of a cross-talk between FTY720 and TGF␤ signaling, which may also contribute to the observed induction of Treg capacities seen in our study (30). Further evidence to support possible overlapping signaling pathways of S1P and TGF␤ has recently been provided by our own study demonstrating a clear interaction between S1PRs and TGF␤ signaling on the level of Smad protein activation (28).…”

“…However, in the latter investigation it was also considered that modulation of migration might not be the end of the story regarding its impact on Treg. Indeed, FTY720 may additionally alter the functional activity of Treg directly or via "tolerogenic" DC (25)(26)(27)(28)(29)(30). Thus, a more complex mode of action may be supported by the fact that the affinity profile of FTY720-P on S1PRs is not identical to S1P.…”

Sphingosylphosphorylcholine acts in an anti-inflammatory manner in renal mesangial cells by reducing interleukin-1β-induced prostaglandin E2 formation

“…The finding that SPC stimulates Smad activation is in good agreement with our previous findings that S1P, and also the S1P mimetic FTY720 phosphate, are able to trigger the phosphorylation and activation of Smad proteins (33,34). It is well established that S1P and FTY720 phosphate bind to the same receptors (35, 36) (i.e., the S1P receptors), and it has been proposed that there is a physical interaction between one of the S1P receptors, most likely S1P 3 , and the TGFb receptor (33).…”

Thematic Review Series: Sphingolipids. Cross-talk at the crossroads of sphingosine-1-phosphate, growth factors, and cytokine signaling*

“…Cross-talk between S1P and TGFb signaling pathways has also been observed in renal mesangial cells (62,63). In these cells, S1P activates ERK1/2 and phosphorylates Smad1 and Smad2, albeit with different kinetics.…”

“…2). This model was also reinforced through studies with the immunomodulator FTY720 (63). Like S1P, both FTY720 and FTY720-P are able to activate MAPK and stress kinase pathways in renal mesangial cells, in addition to inducing phosphorylation of Smads 1, 2, and 3 (63).…”