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Tristetraprolin regulation of interleukin-22 production.

Härdle L, Bachmann M, Bollmann F, Pautz A, Schmid T, Eberhardt W, Kleinert H, Pfeilschifter J, Mühl H - Sci Rep (2015)

Bottom Line: Furthermore, overexpression of TTP in HEK293 cells substantially decreased luciferase activity directed by the IL-22-3'-UTR.Transcript destabilization by TTP was ified upon cellular activation by TPA/A23187, an effect dependent on MEK1/2 activity.Accordingly, IL-22 mRNA half-life as determined in TPA/A23187-stimulated Jurkat T cells decreased under the influence of the MEK1/2 inhibitor U0126.

View Article: PubMed Central - PubMed

Affiliation: pharmazentrum frankfurt/ZAFES, University Hospital Goethe-University Frankfurt, Germany.

ABSTRACT
Interleukin (IL)-22 is a STAT3-activating cytokine displaying characteristic AU-rich elements (ARE) in the 3'-untranslated region (3'-UTR) of its mRNA. This architecture suggests gene regulation by modulation of mRNA stability. Since related cytokines undergo post-transcriptional regulation by ARE-binding tristetraprolin (TTP), the role of this destabilizing protein in IL-22 production was investigated. Herein, we demonstrate that TTP-deficient mice display augmented serum IL-22. Likewise, IL-22 mRNA was enhanced in TTP-deficient splenocytes and isolated primary T cells. A pivotal role for TTP is underscored by an extended IL-22 mRNA half-life detectable in TTP-deficient T cells. Luciferase-reporter assays performed in human Jurkat T cells proved the destabilizing potential of the human IL-22-3'-UTR. Furthermore, overexpression of TTP in HEK293 cells substantially decreased luciferase activity directed by the IL-22-3'-UTR. Transcript destabilization by TTP was ified upon cellular activation by TPA/A23187, an effect dependent on MEK1/2 activity. Accordingly, IL-22 mRNA half-life as determined in TPA/A23187-stimulated Jurkat T cells decreased under the influence of the MEK1/2 inhibitor U0126. Altogether, data indicate that TTP directly controls IL-22 production, a process counteracted by MEK1/2. The TTP-dependent regulatory pathway described herein likely contributes to the role of IL-22 in inflammation and cancer and may evolve as novel target for pharmacological IL-22 modulation.

No MeSH data available.


Related in: MedlinePlus

MAP kinase signaling is essential to IL-22 mRNA induction.(a–c) Where indicated, Jurkat T cells were pretreated with MAP kinase inhibitors for 1 h prior to stimulation with TPA (100 ng/ml)/A23187 (10 μM) for 4 h. PD98059 (n = 7), 50 μM; U0126 (n = 3), 10 μM, FR180204 (n = 11), 10 μM; SP600125 (n = 3), 10 μM; SB203580 (n = 5), 10 μM. In addition, cell were kept as unstimulated control. All cultures were adjusted to a final concentration of 0.21% DMSO (vehicle for TPA/A23187 plus inhibitor). (a,c) IL-22 and IL-6 mRNA were determined by realtime PCR. Target mRNA was normalized to GAPDH. Data are depicted as % of TPA/A23187-stimulation (means ± SD; *p < 0.05, **p < 0.01). Fold-induction of mRNA by TPA/A23187 (compared to unstimulated control): 121.12 for IL-22 (n = 16; p < 0.001), 19.3 for IL-6 (n = 3; p < 0.001). Statistical analysis on raw data, one-way analysis of variance with post-hoc Bonferroni-correction. (b) Viability is shown as % of untreated control (means ± SD). (d) Jurkat T cells were kept as unstimulated control or stimulated with TPA (100 ng/ml)/A23187 (10 μM). All cultures were adjusted to a final concentration of 0.11% DMSO (vehicle for TPA/A23187). After indicated time points, ERK (p42/44) activation, as detected by p42/44 phosphorylation, was determined by immunoblot analysis. One representative of three independently performed experiments is shown. (e–g) Primary human T cells were kept as unstimulated control (Co) or stimulated with αCD3 (20 μg/ml) for 24 h. Where indicated, cells were pretreated with U0126 (10 μM) for 1 h. All cultures were adjusted to a final concentration of 0.1% DMSO (vehicle for U0126). (e,g) IL-22 (e, n = 6) or IL-10 (g, n = 5) mRNA, determined by realtime PCR, was normalized to that of GAPDH (means ± SEM versus unstimulated control; *p < 0.05, **p < 0.01). Statistical analysis on raw data, one-way analysis of variance with post-hoc Bonferroni-correction. (f) IL-22 secretion was determined by ELISA. Data are shown as means ± SEM (n = 4; *p < 0.05). Statistical analysis, one-way analysis of variance with post-hoc Bonferroni-correction.
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f7: MAP kinase signaling is essential to IL-22 mRNA induction.(a–c) Where indicated, Jurkat T cells were pretreated with MAP kinase inhibitors for 1 h prior to stimulation with TPA (100 ng/ml)/A23187 (10 μM) for 4 h. PD98059 (n = 7), 50 μM; U0126 (n = 3), 10 μM, FR180204 (n = 11), 10 μM; SP600125 (n = 3), 10 μM; SB203580 (n = 5), 10 μM. In addition, cell were kept as unstimulated control. All cultures were adjusted to a final concentration of 0.21% DMSO (vehicle for TPA/A23187 plus inhibitor). (a,c) IL-22 and IL-6 mRNA were determined by realtime PCR. Target mRNA was normalized to GAPDH. Data are depicted as % of TPA/A23187-stimulation (means ± SD; *p < 0.05, **p < 0.01). Fold-induction of mRNA by TPA/A23187 (compared to unstimulated control): 121.12 for IL-22 (n = 16; p < 0.001), 19.3 for IL-6 (n = 3; p < 0.001). Statistical analysis on raw data, one-way analysis of variance with post-hoc Bonferroni-correction. (b) Viability is shown as % of untreated control (means ± SD). (d) Jurkat T cells were kept as unstimulated control or stimulated with TPA (100 ng/ml)/A23187 (10 μM). All cultures were adjusted to a final concentration of 0.11% DMSO (vehicle for TPA/A23187). After indicated time points, ERK (p42/44) activation, as detected by p42/44 phosphorylation, was determined by immunoblot analysis. One representative of three independently performed experiments is shown. (e–g) Primary human T cells were kept as unstimulated control (Co) or stimulated with αCD3 (20 μg/ml) for 24 h. Where indicated, cells were pretreated with U0126 (10 μM) for 1 h. All cultures were adjusted to a final concentration of 0.1% DMSO (vehicle for U0126). (e,g) IL-22 (e, n = 6) or IL-10 (g, n = 5) mRNA, determined by realtime PCR, was normalized to that of GAPDH (means ± SEM versus unstimulated control; *p < 0.05, **p < 0.01). Statistical analysis on raw data, one-way analysis of variance with post-hoc Bonferroni-correction. (f) IL-22 secretion was determined by ELISA. Data are shown as means ± SEM (n = 4; *p < 0.05). Statistical analysis, one-way analysis of variance with post-hoc Bonferroni-correction.

Mentions: Recently, we reported on robust IL-22 mRNA and promoter induction detected in Jurkat T cells stimulated by TPA/A2318722 (see also Fig. 8a). Those studies22 likewise revealed suppression of TPA/A23187-induced IL-22 mRNA expression by the mitogen-activated protein kinase kinase (MEK)-1/2 inhibitor U012635. In order to extend those previous data, TPA/A23187-activated Jurkat T cells were coincubated with a panel of pharmacological inhibitors affecting the mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinases (ERK) signaling pathway. As shown in Fig. 7a, PD98059 (targeting MEK1/2 albeit with less potency compared to U0126) and FR180204 (targeting ERK1/2) as well as SB203580 (targeting p38 MAPK) but not SP600125 (targeting c-jun N-terminal kinases) significantly inhibited IL-22 mRNA expression. Those experiments were performed under conditions not affecting cell viability (Fig. 7b). Alike IL-22, also expression of related IL-6 was potently inhibited by U0126 in TPA/A23187-stimulated Jurkat T cells (Fig. 7c). Since U0126 was, by far, the most effective inhibitor of IL-22 expression (Fig. 7a and ref. 22) and TPA/A23187 potently activated the MEK/ERK pathway in Jurkat T cells (Fig. 7d), we chose to focus on this inhibitor in subsequent experiments. Notably, U0126 likewise suppressed IL-22 mRNA induction (Fig. 7e) and protein release (Fig. 7f) as well as IL-10 mRNA induction (Fig. 7g) by human αCD3-stimulated primary T cells.


Tristetraprolin regulation of interleukin-22 production.

Härdle L, Bachmann M, Bollmann F, Pautz A, Schmid T, Eberhardt W, Kleinert H, Pfeilschifter J, Mühl H - Sci Rep (2015)

MAP kinase signaling is essential to IL-22 mRNA induction.(a–c) Where indicated, Jurkat T cells were pretreated with MAP kinase inhibitors for 1 h prior to stimulation with TPA (100 ng/ml)/A23187 (10 μM) for 4 h. PD98059 (n = 7), 50 μM; U0126 (n = 3), 10 μM, FR180204 (n = 11), 10 μM; SP600125 (n = 3), 10 μM; SB203580 (n = 5), 10 μM. In addition, cell were kept as unstimulated control. All cultures were adjusted to a final concentration of 0.21% DMSO (vehicle for TPA/A23187 plus inhibitor). (a,c) IL-22 and IL-6 mRNA were determined by realtime PCR. Target mRNA was normalized to GAPDH. Data are depicted as % of TPA/A23187-stimulation (means ± SD; *p < 0.05, **p < 0.01). Fold-induction of mRNA by TPA/A23187 (compared to unstimulated control): 121.12 for IL-22 (n = 16; p < 0.001), 19.3 for IL-6 (n = 3; p < 0.001). Statistical analysis on raw data, one-way analysis of variance with post-hoc Bonferroni-correction. (b) Viability is shown as % of untreated control (means ± SD). (d) Jurkat T cells were kept as unstimulated control or stimulated with TPA (100 ng/ml)/A23187 (10 μM). All cultures were adjusted to a final concentration of 0.11% DMSO (vehicle for TPA/A23187). After indicated time points, ERK (p42/44) activation, as detected by p42/44 phosphorylation, was determined by immunoblot analysis. One representative of three independently performed experiments is shown. (e–g) Primary human T cells were kept as unstimulated control (Co) or stimulated with αCD3 (20 μg/ml) for 24 h. Where indicated, cells were pretreated with U0126 (10 μM) for 1 h. All cultures were adjusted to a final concentration of 0.1% DMSO (vehicle for U0126). (e,g) IL-22 (e, n = 6) or IL-10 (g, n = 5) mRNA, determined by realtime PCR, was normalized to that of GAPDH (means ± SEM versus unstimulated control; *p < 0.05, **p < 0.01). Statistical analysis on raw data, one-way analysis of variance with post-hoc Bonferroni-correction. (f) IL-22 secretion was determined by ELISA. Data are shown as means ± SEM (n = 4; *p < 0.05). Statistical analysis, one-way analysis of variance with post-hoc Bonferroni-correction.
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f7: MAP kinase signaling is essential to IL-22 mRNA induction.(a–c) Where indicated, Jurkat T cells were pretreated with MAP kinase inhibitors for 1 h prior to stimulation with TPA (100 ng/ml)/A23187 (10 μM) for 4 h. PD98059 (n = 7), 50 μM; U0126 (n = 3), 10 μM, FR180204 (n = 11), 10 μM; SP600125 (n = 3), 10 μM; SB203580 (n = 5), 10 μM. In addition, cell were kept as unstimulated control. All cultures were adjusted to a final concentration of 0.21% DMSO (vehicle for TPA/A23187 plus inhibitor). (a,c) IL-22 and IL-6 mRNA were determined by realtime PCR. Target mRNA was normalized to GAPDH. Data are depicted as % of TPA/A23187-stimulation (means ± SD; *p < 0.05, **p < 0.01). Fold-induction of mRNA by TPA/A23187 (compared to unstimulated control): 121.12 for IL-22 (n = 16; p < 0.001), 19.3 for IL-6 (n = 3; p < 0.001). Statistical analysis on raw data, one-way analysis of variance with post-hoc Bonferroni-correction. (b) Viability is shown as % of untreated control (means ± SD). (d) Jurkat T cells were kept as unstimulated control or stimulated with TPA (100 ng/ml)/A23187 (10 μM). All cultures were adjusted to a final concentration of 0.11% DMSO (vehicle for TPA/A23187). After indicated time points, ERK (p42/44) activation, as detected by p42/44 phosphorylation, was determined by immunoblot analysis. One representative of three independently performed experiments is shown. (e–g) Primary human T cells were kept as unstimulated control (Co) or stimulated with αCD3 (20 μg/ml) for 24 h. Where indicated, cells were pretreated with U0126 (10 μM) for 1 h. All cultures were adjusted to a final concentration of 0.1% DMSO (vehicle for U0126). (e,g) IL-22 (e, n = 6) or IL-10 (g, n = 5) mRNA, determined by realtime PCR, was normalized to that of GAPDH (means ± SEM versus unstimulated control; *p < 0.05, **p < 0.01). Statistical analysis on raw data, one-way analysis of variance with post-hoc Bonferroni-correction. (f) IL-22 secretion was determined by ELISA. Data are shown as means ± SEM (n = 4; *p < 0.05). Statistical analysis, one-way analysis of variance with post-hoc Bonferroni-correction.
Mentions: Recently, we reported on robust IL-22 mRNA and promoter induction detected in Jurkat T cells stimulated by TPA/A2318722 (see also Fig. 8a). Those studies22 likewise revealed suppression of TPA/A23187-induced IL-22 mRNA expression by the mitogen-activated protein kinase kinase (MEK)-1/2 inhibitor U012635. In order to extend those previous data, TPA/A23187-activated Jurkat T cells were coincubated with a panel of pharmacological inhibitors affecting the mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinases (ERK) signaling pathway. As shown in Fig. 7a, PD98059 (targeting MEK1/2 albeit with less potency compared to U0126) and FR180204 (targeting ERK1/2) as well as SB203580 (targeting p38 MAPK) but not SP600125 (targeting c-jun N-terminal kinases) significantly inhibited IL-22 mRNA expression. Those experiments were performed under conditions not affecting cell viability (Fig. 7b). Alike IL-22, also expression of related IL-6 was potently inhibited by U0126 in TPA/A23187-stimulated Jurkat T cells (Fig. 7c). Since U0126 was, by far, the most effective inhibitor of IL-22 expression (Fig. 7a and ref. 22) and TPA/A23187 potently activated the MEK/ERK pathway in Jurkat T cells (Fig. 7d), we chose to focus on this inhibitor in subsequent experiments. Notably, U0126 likewise suppressed IL-22 mRNA induction (Fig. 7e) and protein release (Fig. 7f) as well as IL-10 mRNA induction (Fig. 7g) by human αCD3-stimulated primary T cells.

Bottom Line: Furthermore, overexpression of TTP in HEK293 cells substantially decreased luciferase activity directed by the IL-22-3'-UTR.Transcript destabilization by TTP was ified upon cellular activation by TPA/A23187, an effect dependent on MEK1/2 activity.Accordingly, IL-22 mRNA half-life as determined in TPA/A23187-stimulated Jurkat T cells decreased under the influence of the MEK1/2 inhibitor U0126.

View Article: PubMed Central - PubMed

Affiliation: pharmazentrum frankfurt/ZAFES, University Hospital Goethe-University Frankfurt, Germany.

ABSTRACT
Interleukin (IL)-22 is a STAT3-activating cytokine displaying characteristic AU-rich elements (ARE) in the 3'-untranslated region (3'-UTR) of its mRNA. This architecture suggests gene regulation by modulation of mRNA stability. Since related cytokines undergo post-transcriptional regulation by ARE-binding tristetraprolin (TTP), the role of this destabilizing protein in IL-22 production was investigated. Herein, we demonstrate that TTP-deficient mice display augmented serum IL-22. Likewise, IL-22 mRNA was enhanced in TTP-deficient splenocytes and isolated primary T cells. A pivotal role for TTP is underscored by an extended IL-22 mRNA half-life detectable in TTP-deficient T cells. Luciferase-reporter assays performed in human Jurkat T cells proved the destabilizing potential of the human IL-22-3'-UTR. Furthermore, overexpression of TTP in HEK293 cells substantially decreased luciferase activity directed by the IL-22-3'-UTR. Transcript destabilization by TTP was ified upon cellular activation by TPA/A23187, an effect dependent on MEK1/2 activity. Accordingly, IL-22 mRNA half-life as determined in TPA/A23187-stimulated Jurkat T cells decreased under the influence of the MEK1/2 inhibitor U0126. Altogether, data indicate that TTP directly controls IL-22 production, a process counteracted by MEK1/2. The TTP-dependent regulatory pathway described herein likely contributes to the role of IL-22 in inflammation and cancer and may evolve as novel target for pharmacological IL-22 modulation.

No MeSH data available.


Related in: MedlinePlus