Differential regulation of the IL-17 receptor by gammac cytokines: inhibitory signaling by the phosphatidylinositol 3-kinase pathway

Abstract

The gammac-family cytokine IL-2 activates signaling events that contribute to cell survival and proliferation, the best-studied of which are the STAT-5 and phosphatidylinositol 3-kinase (PI3K) pathways. The starting point of this study was to define genes regulated by the IL-2R-mediated PI3K pathway in T cells. Accordingly, we used an erythropoietin (EPO) receptor chimeric receptor system in which IL-2-dependent HT-2 T cells expressed a mutant EPO-IL-2Rbeta construct where Tyr-338 is mutated to Phe. Cells expressing this mutant IL-2Rbeta chain fail to induce phosphorylation of PI3K-p85alpha/beta or activate Akt, but mediate normal IL-2-dependent proliferation and activation of JAK1 and STAT-5A/B. Microarray analyses revealed differential regulation of numerous genes compared with cells expressing a wild-type IL-2Rbeta, including up-regulation of the IL-17 receptor subunit IL-17RA. Blockade of the PI3K pathway but not p70S6K led to up-regulation of IL-17RA, and constitutive Akt activation was associated with suppressed IL-17RA expression. Moreover, similar to the mutant EPO-IL-2Rbeta chimera, IL-15 and IL-21 induced IL-17RA preferentially compared with IL-2, and IL-2 but not IL-15 or IL-21 mediated prolonged activation of the PI3K p85 regulatory subunit. Thus, there are intrinsic signaling differences between IL-2 and IL-15 that can be attributed to differences in activation of the PI3K pathway.

FIGURE 1.

Differential gene expression mediated by the PI3K pathway through IL-2RβY338. A, schematic diagram of the EPOR-IL-2Rβ chimeric receptor construct. The location of JAK-binding site (Box1/Box2) and the pathways activated by individual tyrosine residues are indicated. B, p85 subunit of PI3K is activated through Tyr-338 (1Y). HT-2 cells expressing the indicated EPO-IL-2R chimeras were stimulated for 1 h with EPO or IL-2. Lysates were immunoprecipitated with α-p85α/β Abs, separated by SDS-PAGE, and immunoblotted with α-Ptyr (4G10) Abs (top) or p85α/β Abs (bottom). C, differentially expressed genes in HT-2.EPOβγ cells versus HT-2.EPOβ5Y/γ cells. Gene differences with a p value <0.01 are shown (222 total genes). D, selected genes enhanced in HT-2.EPOβ5Y/γ cells or HT-2.EPOβγ cells. E, IL-17RA surface expression is elevated in HT-2.EPOβ5Y/γ cells. The indicated cell lines were incubated in EPO for 24 h and IL-17RA expression was assessed by flow cytometry. Filled histograms, isotype control.

FIGURE 2.

IL-17RA is induced by IL-15 and IL-21 but not by IL-2. A, IL-2 dominantly suppresses IL-17RA surface expression mediated by IL-15 and IL-21. HT-2 cells were treated for 24 h with the indicated cytokines, and IL-17RA surface expression was assessed by flow cytometry. Filled histograms, isotype control. Average mean fluorescent intensity of IL-17RA expression relative to IL-2-treated cells was determined from five independent experiments (right panel). 2°, secondary Ab alone. B, IL-2 dominantly suppresses IL-17RA mRNA expression mediated by IL-15 and IL-21. HT-2 cells were treated for 24 h with the indicated cytokines, and IL-17RA mRNA levels were determined in triplicate by real-time RT-PCR. ^*, p < 0.05 compared with IL-2-treated control.

FIGURE 3.

IL-2 and IL-15 differentially activate the PI3K pathway but not the JAK-STAT-5 pathway or proliferation in HT-2 cells. A, IL-2, IL-15, and IL-21 activate JAK1 equivalently in HT-2 cells. HT-2 cells were stimulated for 15 min with the indicated cytokines. Lysates were immunoprecipitated with α-JAK1 Abs, separated by SDS-PAGE, and immunoblotted with α-PTyr (4G10) Abs (top) or α-JAK1 Abs (bottom). B, IL-2, IL-15 and IL-21 activate STAT-5 equivalently in HT-2 cells. HT-2 cells were stimulated for 15 min with the indicated cytokines. Nuclear extracts were subjected to EMSA with a ³²P-labeled STAT oligonucleotide (7) (which binds to STAT-1, 5, 6 and to a lesser extent STAT-3). FP, free probe. Supershifting with α-STAT-5 Abs was performed in lanes 4, 6, and 8. Arrows indicate shifted and supershifted complexes. C, IL-2, IL-15, but not IL-21 drive proliferation and inhibit apoptosis in HT-2 cells. HT-2 cells were cultured for 24, 48, or 72 h in the indicated cytokines and concentrations. Proliferation was assessed by [³H]thymidine incorporation in triplicate ± S.D., ^*, p < 0.05. Apoptosis was determined by FACS staining with propidium iodide and GFP-Annexin V, and apoptotic cells were defined as PI-negative/GFP-Annexin V-positive, as described in Ref. , . Data are representative of three experiments. D, signaling from PI3K via IL-2 and Y338 promotes increased cell size. HT-2 cells (top panel) or HT-2 cells expressing the indicated EPO-IL-2Rβ chimeras (bottom 3 panels) were cultured in the indicated cytokines for 24 h, and cell size was assessed by forward scatter analysis by flow cytometry.

FIGURE 4.

The PI3K pathway inhibits IL-17RA expression. A, sustained phosphorylation of p85 induced by IL-2 but not IL-15 or IL-21. HT-2 cells were cultured in IL-2, IL-15, or IL-21 for the indicated time points, and phosphorylation of the p85α/β was assessed as in Fig. 1. B, HT-2 cells express PTEN and SHIP. Lysates from HT-2, Jurkat T cells or the A20 B cell line were immunoblotted with Abs to PTEN or IKKγ (top panels) or immunoprecipitated with α-SHIP Abs and probed for SHIP (bottom panel). C, PI3K inhibitors up-regulate IL-17RA. HT-2 cells were incubated in IL-2 ± LY294002 or Wortmannin for 20 h. IL-17RA expression was assessed by flow cytometry. D, rapamycin inhibits p70^S6K but not IL-15-induced IL-17RA expression. HT-2 cells were incubated with IL-2 or IL-15 ± Rapamycin for 20 h. IL-17RA (top) or p70^S6K (bottom) were assessed by flow cytometry.

FIGURE 5.

A constitutively active form of Akt promotes IL-17RA expression. A, HT-2 cells stably express Myr-Akt. Lysates from 3 HT-2 subcloned stably transfected with Myr-Akt were immunoblotted with α-pAkt (Ser-473). B, Myr-Akt delays apoptosis induced by cytokine withdrawal. HT-2 cells expressing Myr-Akt (clones A26 and A39) were incubated with or without IL-2 for the indicated time points, and cell survival (PI-negative) or apoptosis (GFP-Annexin V-positive) cells were assessed by flow cytometry. C, expression of a constitutively active form of Akt prevents up-regulation of IL-17RA induced by cytokine withdrawal. HT-2 or HT-2.MyrAktA39 cells were deprived of IL-2 for the indicated times, and IL-17RA expression was assessed by flow cytometry.

FIGURE 6.

Regulation of IL-17RA in primary CD8+ T cells. Splenic CD8+ T cells were purified by magnetic beads and stimulated for 6 h with the indicated cytokines with DMSO (top) or LY294002 (bottom). IL-17RA was assessed by flow cytometry.

FIGURE 7.

IL-17 does not induce detectable signaling in HT-2 cells or primary T lymphocytes. A and B, IL-17 does not induce canonical target genes such as IL-6 or LIX/CXCL5. HT-2 cells, splenocytes, or purified CD8+ T cells were incubated with the indicated cytokines for 2-24 h, and conditioned supernatants were assayed in triplicate for IL-6 and LIX/CXCL5 by ELISA (36).