title: 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.
authors.affiliation: Department of Neurology, University Hospital Regensburg, Regensburg, Germany.
authors.forename: Maruf
authors.initials: M
authors.lastname: Hasan
authors.affiliation: Department of Neurology, University Hospital Regensburg, Regensburg, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Neumann
authors.affiliation: Department of Neurology, St Josef-Hospital, Ruhr-University Bochum, Bochum, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Haupeltshofer
authors.affiliation: Department of Neurology, St Josef-Hospital, Ruhr-University Bochum, Bochum, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Stahlke
authors.affiliation: Department of Systems Medicine, University 'Tor Vergata', Rome, Italy.
authors.forename: Massimo Claudio
authors.initials: MC
authors.lastname: Fantini
authors.affiliation: Department of Neurology, University Hospital Regensburg, Regensburg, Germany.
authors.forename: Klemens
authors.initials: K
authors.lastname: Angstwurm
authors.affiliation: Department of Neurology, University Hospital Regensburg, Regensburg, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Bogdahn
authors.affiliation: Department of Neurology, St Josef-Hospital, Ruhr-University Bochum, Bochum, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Kleiter
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D020381:Interleukin-17 / Q000096:biosynthesis / Q000235:genetics; D038041:Lymphopoiesis / Q000187:drug effects; D020935:MAP Kinase Signaling System / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000502:physiology; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D020123:Sirolimus / Q000494:pharmacology; D051785:Smad Proteins / Q000502:physiology; D050378:T-Lymphocytes, Regulatory / Q000166:cytology / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases / Q000502:physiology; D058504:Th17 Cells / Q000166:cytology / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000502:physiology*
chemical_list: D002867:Chromones; D020381:Interleukin-17; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta; D020123:Sirolimus; D051785:Smad Proteins; D058570:TOR Serine-Threonine Kinases; D016212:Transforming Growth Factor beta; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C546843:mTOR protein, mouse; C514862:Akt1 protein, mouse
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