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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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CD4-Positive T-Lymphocytes / cytology
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CD4-Positive T-Lymphocytes / metabolism
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Chromones / pharmacology
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Interleukin-17 / biosynthesis
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Interleukin-17 / genetics
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Lymphopoiesis / drug effects
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MAP Kinase Signaling System / drug effects
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Mice
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Mice, Inbred C57BL
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Morpholines / pharmacology
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Phosphatidylinositol 3-Kinases / physiology
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Phosphorylation / drug effects
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Protein Kinase Inhibitors / pharmacology
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Protein Processing, Post-Translational / drug effects
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Protein Serine-Threonine Kinases / antagonists & inhibitors
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Protein Serine-Threonine Kinases / deficiency
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Protein Serine-Threonine Kinases / physiology
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Proto-Oncogene Proteins c-akt / physiology
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Receptor, Transforming Growth Factor-beta Type I
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Receptor, Transforming Growth Factor-beta Type II
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Receptors, Transforming Growth Factor beta / antagonists & inhibitors
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Receptors, Transforming Growth Factor beta / deficiency
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Receptors, Transforming Growth Factor beta / physiology*
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Signal Transduction / drug effects
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Signal Transduction / physiology*
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Sirolimus / pharmacology
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Smad Proteins / physiology
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T-Lymphocytes, Regulatory / cytology
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T-Lymphocytes, Regulatory / drug effects
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TOR Serine-Threonine Kinases / physiology
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Th17 Cells / cytology
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Th17 Cells / metabolism*
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Transforming Growth Factor beta / physiology*
Substances
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Chromones
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Interleukin-17
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Morpholines
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Protein Kinase Inhibitors
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Receptors, Transforming Growth Factor beta
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Smad Proteins
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Transforming Growth Factor beta
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2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
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mTOR protein, mouse
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Akt1 protein, mouse
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-akt
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TOR Serine-Threonine Kinases
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Receptor, Transforming Growth Factor-beta Type I
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Receptor, Transforming Growth Factor-beta Type II
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Sirolimus