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JunD/AP-1-mediated gene expression promotes lymphocyte growth dependent on interleukin-7 signal transduction.

Ruppert SM, Chehtane M, Zhang G, Hu H, Li X, Khaled AR - PLoS ONE (2012)

Bottom Line: 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.One of these growth promoters was the oncogene, Pim-1.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.

View Article: PubMed Central - PubMed

Affiliation: Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida, United States of America.

ABSTRACT
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.

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IL-7 signaling induces JNK activity and promotes JunD-containing AP-1 complexes.(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 Methods. (Right panel) In cells treated as described above, glucose uptake was assessed by measuring the accumulation of radiolabeled 2-DOG as stated in Methods. (*) 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. Results (A, B, and C) are representative of three or more independent experiments (values in graphs are mean ± SD).
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pone-0032262-g001: IL-7 signaling induces JNK activity and promotes JunD-containing AP-1 complexes.(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 Methods. (Right panel) In cells treated as described above, glucose uptake was assessed by measuring the accumulation of radiolabeled 2-DOG as stated in Methods. (*) 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. Results (A, B, and C) are representative of three or more independent experiments (values in graphs are mean ± SD).

Mentions: Previously, using an IL-7 dependent T-cell line, D1, and primary T-cells, we reported that HXKII gene expression was regulated by an IL-7 signal [39]. However, we found that inhibition of STAT5 did not prevent the expression of HXKII in response to IL-7 (Fig. S1). To demonstrate that a STAT5-independent, but still an IL-7 dependent signal, controlled HXKII gene expression, we nucleofected D1 cells with a chimeric IL-4/IL-7 wild-type receptor or with a chimeric IL-4/IL-7 receptor bearing a mutation in the STAT5 binding site, Y449. Normally, when Y449 is phosphorylated it binds STAT5; therefore mutation of this site would prevent STAT5 activation. D1 cells, nucleofected with the chimeric receptors, were cultured with human IL-4. Gene expression of HXKII was measured by quantitative PCR and glucose uptake was measured by 2-DOG uptake. We found that the IL-4-induced gene expression and glucose uptake in nucleofected cells was not affected by the Y449 mutation and that cells were comparable to those incubated with IL-7, suggesting that these events were STAT5-independent (Fig. 1A). Examination of the mouse HXKII promoter region showed that the proximal promoter region contained most of the transcription factors binding motifs that regulate HXKII expression [40]. Some of the recognized factors included SP-1, NF-Y, CREB, Glucose binding site, and the AP-1 complex. To study this, we examined the activity of the MAPK pathway and induction of AP-1 transcription factors in response to IL-7.


JunD/AP-1-mediated gene expression promotes lymphocyte growth dependent on interleukin-7 signal transduction.

Ruppert SM, Chehtane M, Zhang G, Hu H, Li X, Khaled AR - PLoS ONE (2012)

IL-7 signaling induces JNK activity and promotes JunD-containing AP-1 complexes.(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 Methods. (Right panel) In cells treated as described above, glucose uptake was assessed by measuring the accumulation of radiolabeled 2-DOG as stated in Methods. (*) 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. Results (A, B, and C) are representative of three or more independent experiments (values in graphs are mean ± SD).
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Related In: Results  -  Collection

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pone-0032262-g001: IL-7 signaling induces JNK activity and promotes JunD-containing AP-1 complexes.(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 Methods. (Right panel) In cells treated as described above, glucose uptake was assessed by measuring the accumulation of radiolabeled 2-DOG as stated in Methods. (*) 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. Results (A, B, and C) are representative of three or more independent experiments (values in graphs are mean ± SD).
Mentions: Previously, using an IL-7 dependent T-cell line, D1, and primary T-cells, we reported that HXKII gene expression was regulated by an IL-7 signal [39]. However, we found that inhibition of STAT5 did not prevent the expression of HXKII in response to IL-7 (Fig. S1). To demonstrate that a STAT5-independent, but still an IL-7 dependent signal, controlled HXKII gene expression, we nucleofected D1 cells with a chimeric IL-4/IL-7 wild-type receptor or with a chimeric IL-4/IL-7 receptor bearing a mutation in the STAT5 binding site, Y449. Normally, when Y449 is phosphorylated it binds STAT5; therefore mutation of this site would prevent STAT5 activation. D1 cells, nucleofected with the chimeric receptors, were cultured with human IL-4. Gene expression of HXKII was measured by quantitative PCR and glucose uptake was measured by 2-DOG uptake. We found that the IL-4-induced gene expression and glucose uptake in nucleofected cells was not affected by the Y449 mutation and that cells were comparable to those incubated with IL-7, suggesting that these events were STAT5-independent (Fig. 1A). Examination of the mouse HXKII promoter region showed that the proximal promoter region contained most of the transcription factors binding motifs that regulate HXKII expression [40]. Some of the recognized factors included SP-1, NF-Y, CREB, Glucose binding site, and the AP-1 complex. To study this, we examined the activity of the MAPK pathway and induction of AP-1 transcription factors in response to IL-7.

Bottom Line: 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.One of these growth promoters was the oncogene, Pim-1.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.

View Article: PubMed Central - PubMed

Affiliation: Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus