JunD/AP-1-Mediated Gene Expression Promotes Lymphocyte Growth Dependent on Interleukin-7 Signal Transduction

Interleukin-7 (IL-7) is an essential cytokine for lymphocyte growth that has the potential for promoting immune reconstitution. This feature makes IL-7 an ideal candidate for therapeutic development. As with other cytokines, signaling through the IL-7 receptor induces the JAK/STAT pathway. However, the broad scope of IL-7 regulatory targets likely necessitates the use of other signaling components whose identities remain poorly defined. To this end, we used an IL-7 dependent T-cell line to examine how expression of the glycolytic enzyme, Hexokinase II (HXKII) was regulated by IL-7 in a STAT5-independent manner. Our studies revealed that IL-7 promoted the activity of JNK (Jun N-terminal Kinase), and that JNK, in turn, drove the expression of JunD, a component of the Activating Protein 1 (AP-1) transcription factors. Gel shifts showed that the AP-1 complex induced by IL-7 contained JunD but not c-Fos or c-Jun. Inhibition of JNK/JunD blocked glucose uptake and HXKII gene expression, indicating that this pathway was responsible for promoting HXKII expression. Because others had shown that JunD was a negative regulator of cell growth, we performed a bioinformatics analysis to uncover possible JunD-regulated gene targets. Our search revealed that JunD could control the expression of proteins involved in signal transduction, cell survival and metabolism. One of these growth promoters was the oncogene, Pim-1. Pim-1 is an IL-7-induced protein that was inhibited when the activities of JNK or JunD were blocked, showing that in IL-7 dependent T-cells JunD can promote positive signals transduced through Pim-1. This was confirmed when the IL-7-induced proliferation of CD8 T-cells was impaired upon JunD inhibition. These results show that engagement of the IL-7 receptor drives a signal that is more complex than the JAK/STAT pathway, activating JNK and JunD to induce rapid growth stimulation through the expression of metabolic and signaling factors like HXKII and Pim-1

HXKII gene expression is dependent upon JNK/JunD signaling.

<p>(A) HXKII gene expression in the IL-7 dependent T cell line, D1, was measured by quantitative PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032262#s4" target="_blank">Methods</a>. Cells were cultured with or without IL-7, or after an IL-7 pulse for 2 hours (IL-7 Re-Addition), in presence of a vehicle control, 20 µM JNK inhibitor, or 20 µM p38 inhibitor. RQ (Fold change) = 2^−ΔΔCt. (***) indicates P<0.001. (B) HXKII gene expression in D1 cells cultured with or without IL-7 and the non-targeting control (NT) or JunD siRNA, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032262#s4" target="_blank">Methods</a>, was measured as above. (*) indicates P = 0.0254. Efficacy of JunD siRNA upon JunD mRNA levels (right panel) was established through measuring total JunD gene expression by quantitative PCR. (*) indicates P = 0.0336. RQ (Fold change) = 2^−ΔΔCt. (C) Chromatin Immunoprecipitation (ChIP) was performed using nuclear lysates from D1 cells cultured with or without IL-7 for 18 hours. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032262#s2" target="_blank">Results</a> from the quantitative PCR, reported as cycle threshold (Ct) values, are shown in the table. The PCR-amplified 150 bp region of AP-1 promoter DNA from the HXKII gene was visualized by ethidium bromide staining in a non-denaturing agarose gel. Input DNA is shown as equivalent starting materials. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032262#s2" target="_blank">Results</a> (A, B and C) are representative of three experiments performed in triplicate (values in graphs are mean ± SD).</p

IL-7 inducible Pim-1 gene expression is dependent on JNK/JunD.

<p>(A) Pim-1 gene expression in the IL-7 dependent T cell line, D1, was measured by quantitative PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032262#s4" target="_blank">Methods</a>. D1 cells were cultured with or without IL-7 (+/− IL-7), in the presence of a vehicle control (Vehicle) or 20 µM JNK inhibitor (JNK Inh). In some samples, after an 18 hour deprivation, IL-7 was added back to the culture for 2 hours in the presence of a vehicle control (IL-7 Re-addition, Vehicle) or 20 µM JNK inhibitor (IL-7 Re-addition, JNK Inh). (*) indicates P = 0.0104. (B) Quantitative PCR of Pim-1 gene expression was performed using D1 cells cultured for 52 hours with IL-7 (+) and then 18 hours without IL-7 (−) in the presence of non-targeting control (NT) or JunD siRNA. A separate group was also deprived of IL-7, and then re-stimulated with IL-7 for two hours in the presence of siRNA (IL-7 Re-addition). RQ (Fold change in gene expression normalized to β-actin) = 2^−ΔΔCt. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032262#s2" target="_blank">Results</a> are representative of three experiments performed in triplicate (values in graphs are mean ± SD). (*) indicates P = 0.0140.</p

JNK activity promotes JunD expression.

<p>(A) Quantitative PCR evaluation of JunD expression in the IL-7 dependent T-cell line, D1. Cells were continuously cultured with IL-7 or without IL-7 for 18 hours, or withdrawn from IL-7 (18 hours) and then re-stimulated for two hours (IL-7 Re-addition). Re-stimulated cells were untreated, treated with DMSO (Vehicle Control), 20 µM JNK inhibitor (JNK inhibitor), or 20 µM p38 MAPK (p38) inhibitor. (***) indicates P<0.0001. (B) Western blot analysis of JunD protein and p38 MAPK protein content, as loading control, of whole cell lysates from D1 cells cultured as stated above. Relative JunD protein indicates JunD protein levels normalized to p38 MAPK (loading control) and compared to the untreated, IL-7 re-addition sample. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032262#s2" target="_blank">Results</a> (A, B) are representative of four independent experiments (values in graphs are mean ± SD).</p

Pim-1 protein is dependent on IL-7 and JNK/JunD.

<p>(A) Immunoblot analysis of Pim-1 was performed. Whole cell lysates were prepared from D1 cells cultured in the presence or absence of IL-7 for the indicated time points. p38 MAPK was measured as a loading control. (B) Immunoblot analysis of Pim-1 was performed. Whole cell lysates were prepared from D1 cells cultured in the presence (+) or absence (−) of IL-7 and/or with JNK inhibitor (20 µM), or 2–4 hour IL-7 stimulation after 18 hour deprivation, alone, or with 20 µM JNK inhibitor. p38 MAPK was used as a loading control. (C) Whole cell lysates of D1 cells were cultured for 52 hours with IL-7 in the presence of non-targeting control (NT) or JunD siRNA, then cells were withdrawn from IL-7 for 18 hours as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032262#s4" target="_blank">Methods</a>. A separate group was also deprived of IL-7, and then re-stimulated with IL-7 for two hours in the presence of siRNA (Re-addition). Whole cell lysates were subjected to SDS-PAGE and immunoblotted for Pim-1, JunD, and p38 as loading control. Tables indicate relative amounts of protein normalized to p38 content and shown relative to the IL-7 re-addition sample. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032262#s2" target="_blank">Results</a> are representative of three experiments performed.</p

A functional consequence of JNK/JunD signaling is the IL-7 dependent uptake of glucose.

<p>(A) D1 cells continuously cultured with or without IL-7 for 18 hours, or withdrawn (18 hours) and then stimulated with IL-7 for four hours (IL-7 Re-addition), were untreated or treated with DMSO (Vehicle Control), 20 µM MAPK inhibitor, 20 µM PD169316, 20 µM MEK1/2 inhibitor or 5 nM PI3K inhibitor, wortmannin. Glucose uptake was assessed by measuring the accumulation of radiolabeled 2-DOG as stated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032262#s4" target="_blank">Methods</a>. (*) indicates P = 0.0348. (B) D1 cells were treated similarly as those in (A) except that cells were pulsed for two hours and specific inhibitors for JNK (20 µM) or p38 MAPK (20 µM) were used. (*) indicates P = 0.0342. (C) D1 cells were cultured with or without IL-7 after introduction of non-specific control (NT) or JunD siRNA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032262#s4" target="_blank">Methods</a>. Glucose uptake was assessed as above by measuring the accumulation of radiolabeled 2-DOG. (*) indicates P = 0.0320. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032262#s2" target="_blank">Results</a> (A, B, and C) are representative of three or more experiments performed (values in graphs are mean ± SD).</p

Bioinformatics evaluation of IL-7 dependent gene expression transduced through JunD.

<p>Three cell lines (GM12878, Helas3, and K562) were evaluated for genes containing putative JunD binding sites that are potentially IL-7-responsive, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032262#s4" target="_blank">Methods</a>. Gene functions were determined using gene ontology analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032262#s4" target="_blank">Methods</a>, and were separated into suppressive (red) or supportive actions (green).</p

IL-7 signaling induces JNK activity and promotes JunD-containing AP-1 complexes.

<p>(A) (Left panel) Quantitative PCR evaluation of HXKII gene expression in the IL-7 dependent T-cell line, D1, after culture with or without IL-7. D1 cells were also nucleofected with a chimeric IL-4/IL-7 receptor (IL-4/IL-7R WT), a chimeric IL-4/IL-7 receptor with a mutation in Y449 (IL4/IL7R Y449) or an empty vector (pcDNA), and stimulated with IL-4, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032262#s4" target="_blank">Methods</a>. (Right panel) In cells treated as described above, glucose uptake was assessed by measuring the accumulation of radiolabeled 2-DOG as stated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032262#s4" target="_blank">Methods</a>. (*) indicates a P value of <0.05. (B) To assess JNK kinase activity in response to IL-7, a kinase assay was performed. JNK was immunoprecipitated from whole cell lysates prepared from D1 cells cultured in the presence or absence of IL-7 for the times indicated and the capacity to phosphorylate the kit-supplied substrate, c-Jun, measured as indicator of kinase activity. As input control, pre-immunoprecipitation levels of JNK in lysates are shown. (C) Nuclear lysates prepared from D1 cells grown with or without IL-7 for 18 hours were assayed for AP-1 complex binding to DNA by EMSA, using a radiolabeled DNA probe containing the AP-1 consensus binding site. Supershifts were performed with antibodies specific for c-Fos, c-Jun or Jun-D. Competition was also performed using 10×, 100× or 1000× excess unlabeled AP-1 probe or AP-1 mutant probe. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032262#s2" target="_blank">Results</a> (A, B, and C) are representative of three or more independent experiments (values in graphs are mean ± SD).</p

Inhibition of JunD Prevents IL-7 Induced Proliferation of Primary Lymphocytes.

<p>(A) Lymph node T-cells were isolated from WT C57Bl/6 mice, cultured continuously with 150 ng/ml of IL-7 for 7 days (Continuous) or for 5 days, then deprived from IL-7 overnight, and IL-7 re-added for 24 hours (Stimulation) and treated with vehicle (DMSO) or 20 µM JNK inhibitor as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032262#s4" target="_blank">Methods</a>. Whole cell lysates were prepared from T-cells, subjected to SDS-PAGE and immunoblotted for Pim-1, JunD, and p38 MAPK as loading control. Tables indicate amounts of protein normalized to p38 MAPK content and shown relative to the IL-7 (Continuous) sample. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032262#s2" target="_blank">Results</a> are representative of two experiments performed. (B, C) Proliferation was measured by BrdU incorporation. Lymph node T cells were isolated from WT C57Bl/6 mice as described in (A) and treated with non-targeting control (NT) or JunD siRNA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032262#s4" target="_blank">Methods</a>. Cells were assessed for BrdU incorporation and surface expression of CD4 and CD8 as determined by flow cytometric analysis of BrdU-PE and CD4- or CD8-PerCp fluorescence. Dot blots show percentages representing the population of cells that are non-proliferating (BrdU negative), proliferating (BrdU positive), and CD4+ or CD8+ as indicated by the quadrants. Quadrants were established using controls. Gating was performed to remove autofluorescent cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032262#s2" target="_blank">Results</a> are representative of duplicate samples.</p