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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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HXKII gene expression is dependent upon JNK/JunD signaling.(A) HXKII gene expression in the IL-7 dependent T cell line, D1, was measured by quantitative PCR as described in Methods. 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 Methods, 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. Results 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. Results (A, B and C) are representative of three experiments performed in triplicate (values in graphs are mean ± SD).
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pone-0032262-g004: HXKII gene expression is dependent upon JNK/JunD signaling.(A) HXKII gene expression in the IL-7 dependent T cell line, D1, was measured by quantitative PCR as described in Methods. 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 Methods, 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. Results 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. Results (A, B and C) are representative of three experiments performed in triplicate (values in graphs are mean ± SD).

Mentions: In our previous study, we reported that HXKII gene expression increased after 2 hours of IL-7 re-addition to deprived cells [39]. We also found that a STAT5-independent mechanism with driving the synthesis of HXKII (Fig. S1 and Fig. 1A). Since the gene expression of JunD was dependent upon JNK and IL-7 (Fig. 2A), it was possible that the increase of HXKII gene expression that followed IL-7 re-addition was also associated with the activity of JNK. Therefore, the synthesis of HXKII was evaluated in D1 cells stimulated with IL-7 and treated with a JNK inhibitor. Quantitative PCR results demonstrated that HXKII synthesis was reduced by about 60% compared to vehicle control upon treatment with JNK inhibitor (Fig. 4A). D1 cells incubated with the p38 MAPK inhibitor showed little effect (Fig. 4A). This data confirmed that the IL-7-driven increase in HXKII gene expression is likely mediated by JNK. To establish a role for JunD-containing AP-1 complexes in HXKII gene expression, D1 cells were stimulated with IL-7 and treated with JunD siRNA. Shown in Figure 4B is a representative experiment in which loss of JunD reduced the IL-7-driven expression of HXKII. A graph alongside shows that treatment with JunD siRNA reduced JunD mRNA levels by approximately 60% as compared to the non-targeting control siRNA. Hence, JunD/AP-1 complexes were contributing to the synthesis of HXKII 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)

HXKII gene expression is dependent upon JNK/JunD signaling.(A) HXKII gene expression in the IL-7 dependent T cell line, D1, was measured by quantitative PCR as described in Methods. 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 Methods, 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. Results 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. Results (A, B and C) are representative of three experiments performed in triplicate (values in graphs are mean ± SD).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3285677&req=5

pone-0032262-g004: HXKII gene expression is dependent upon JNK/JunD signaling.(A) HXKII gene expression in the IL-7 dependent T cell line, D1, was measured by quantitative PCR as described in Methods. 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 Methods, 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. Results 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. Results (A, B and C) are representative of three experiments performed in triplicate (values in graphs are mean ± SD).
Mentions: In our previous study, we reported that HXKII gene expression increased after 2 hours of IL-7 re-addition to deprived cells [39]. We also found that a STAT5-independent mechanism with driving the synthesis of HXKII (Fig. S1 and Fig. 1A). Since the gene expression of JunD was dependent upon JNK and IL-7 (Fig. 2A), it was possible that the increase of HXKII gene expression that followed IL-7 re-addition was also associated with the activity of JNK. Therefore, the synthesis of HXKII was evaluated in D1 cells stimulated with IL-7 and treated with a JNK inhibitor. Quantitative PCR results demonstrated that HXKII synthesis was reduced by about 60% compared to vehicle control upon treatment with JNK inhibitor (Fig. 4A). D1 cells incubated with the p38 MAPK inhibitor showed little effect (Fig. 4A). This data confirmed that the IL-7-driven increase in HXKII gene expression is likely mediated by JNK. To establish a role for JunD-containing AP-1 complexes in HXKII gene expression, D1 cells were stimulated with IL-7 and treated with JunD siRNA. Shown in Figure 4B is a representative experiment in which loss of JunD reduced the IL-7-driven expression of HXKII. A graph alongside shows that treatment with JunD siRNA reduced JunD mRNA levels by approximately 60% as compared to the non-targeting control siRNA. Hence, JunD/AP-1 complexes were contributing to the synthesis of HXKII 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