Activation of TGF-β-induced non-Smad signaling pathways during Th17 differentiation.

Abstract

Although transforming growth factor-β (TGF-β) has been shown to positively regulate the development of murine T helper type 17 (Th17) cells, which of the intracellular signaling pathways are involved is controversial. We examined Smad-dependent and -independent signaling molecules downstream of the TGF-β receptor (TGFβR) involved in Th17 differentiation of naive murine CD4(+)CD62L(+) T cells. During Th17 differentiation of wild-type T cells, Smad2/3 was phosphorylated, indicating activation of the canonical Smad pathway. T cells lacking TGFβRII did not differentiate into Th17, whereas T cells treated with a TGFβRI kinase inhibitor (SB-431542) or overexpression of inhibitory Smad7 retained a low amount of Th17 polarization despite absent Smad2/3 phosphorylation. Using protein antibody arrays we found an increase of expression and phosphorylation of the following Smad-independent signaling molecules in Th17-polarized wild-type T cells: AKT1(Tyr474), AKT2 (Ser474), ERK1-p44/42 MAPK(Tyr204), mTOR(Thr2446), p38 MAPK(Thr180), Rac1/cdc42(Ser71), SAPK/JNK(Tyr185) and SP1(Thr739). Pharmacological inhibition of AKT/mammalian target of rapamycin (mTOR) signaling with rapamycin or LY294002 decreased Th17 differentiation of wild-type T cells, and completely abolished interleukin-17 production in T cells with overexpression of Smad7. Rapamycin and LY294002 also decreased induced regulatory T cell differentiation, but only had minor additive effects to Smad7 overexpression. Finally, inhibitors of mitogen-activated protein kinase (MAPK) blocked in vitro polarization of Th17 cells. Our data show that Smad-dependent and -independent intracellular pathways contribute to murine Th17 differentiation.