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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

Binding of TTP to ARE located in the IL-22-3′-UTR as detected in vitro by RNA-EMSA.In vitro translated TTP was incubated together with a 32P-γ-ATP-labelled RNA oligonucleotide probe that includes the ARE5/6 region of the human IL-22-3′-UTR (see Fig. 1). In addition to this ‘wildtype’ (wt) oligonucleotide, a ‘mutated’ (mut) oligonucleotide was used lacking regular ARE sequences (see methods section). Reaction mixtures were subjected to native polyacrylamide gel electrophoresis. The brace indicates positions of retarded complexes present when the ‘wildtype’ but absent when the ‘mutated’ oligonucleotide was used. This retarded signal allegedly represents RNA/TTP complexes. Ctrl, denotes a control-in vitro translation setup with expression of an unrelated protein (firefly-luciferase) serving as control for unspecific protein/RNA interactions. Inset: Immunoblot analysis of in vitro translated TTP. One representative of three independently performed experiments is shown.
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f6: Binding of TTP to ARE located in the IL-22-3′-UTR as detected in vitro by RNA-EMSA.In vitro translated TTP was incubated together with a 32P-γ-ATP-labelled RNA oligonucleotide probe that includes the ARE5/6 region of the human IL-22-3′-UTR (see Fig. 1). In addition to this ‘wildtype’ (wt) oligonucleotide, a ‘mutated’ (mut) oligonucleotide was used lacking regular ARE sequences (see methods section). Reaction mixtures were subjected to native polyacrylamide gel electrophoresis. The brace indicates positions of retarded complexes present when the ‘wildtype’ but absent when the ‘mutated’ oligonucleotide was used. This retarded signal allegedly represents RNA/TTP complexes. Ctrl, denotes a control-in vitro translation setup with expression of an unrelated protein (firefly-luciferase) serving as control for unspecific protein/RNA interactions. Inset: Immunoblot analysis of in vitro translated TTP. One representative of three independently performed experiments is shown.

Mentions: Finally, in vitro binding assays were performed that demonstrate physical binding of TTP to an RNA sequence derived from the IL-22-3′-UTR but not to a mutated counterpart (Fig. 6). This RNA oligonucleotide was specifically selected and spans the region of human ARE5/6. Notably, the whole IL-22-3′-UTR sequence covered by this RNA oligonucleotide (45 nt) is conserved between mice and humans displaying 93.3% identity. Data altogether indicate that TTP is able to regulate reporter gene expression by interacting with adjacent IL-22-3′-UTR sequences and thus by destabilizing target mRNA.


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)

Binding of TTP to ARE located in the IL-22-3′-UTR as detected in vitro by RNA-EMSA.In vitro translated TTP was incubated together with a 32P-γ-ATP-labelled RNA oligonucleotide probe that includes the ARE5/6 region of the human IL-22-3′-UTR (see Fig. 1). In addition to this ‘wildtype’ (wt) oligonucleotide, a ‘mutated’ (mut) oligonucleotide was used lacking regular ARE sequences (see methods section). Reaction mixtures were subjected to native polyacrylamide gel electrophoresis. The brace indicates positions of retarded complexes present when the ‘wildtype’ but absent when the ‘mutated’ oligonucleotide was used. This retarded signal allegedly represents RNA/TTP complexes. Ctrl, denotes a control-in vitro translation setup with expression of an unrelated protein (firefly-luciferase) serving as control for unspecific protein/RNA interactions. Inset: Immunoblot analysis of in vitro translated TTP. One representative of three independently performed experiments is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4613560&req=5

f6: Binding of TTP to ARE located in the IL-22-3′-UTR as detected in vitro by RNA-EMSA.In vitro translated TTP was incubated together with a 32P-γ-ATP-labelled RNA oligonucleotide probe that includes the ARE5/6 region of the human IL-22-3′-UTR (see Fig. 1). In addition to this ‘wildtype’ (wt) oligonucleotide, a ‘mutated’ (mut) oligonucleotide was used lacking regular ARE sequences (see methods section). Reaction mixtures were subjected to native polyacrylamide gel electrophoresis. The brace indicates positions of retarded complexes present when the ‘wildtype’ but absent when the ‘mutated’ oligonucleotide was used. This retarded signal allegedly represents RNA/TTP complexes. Ctrl, denotes a control-in vitro translation setup with expression of an unrelated protein (firefly-luciferase) serving as control for unspecific protein/RNA interactions. Inset: Immunoblot analysis of in vitro translated TTP. One representative of three independently performed experiments is shown.
Mentions: Finally, in vitro binding assays were performed that demonstrate physical binding of TTP to an RNA sequence derived from the IL-22-3′-UTR but not to a mutated counterpart (Fig. 6). This RNA oligonucleotide was specifically selected and spans the region of human ARE5/6. Notably, the whole IL-22-3′-UTR sequence covered by this RNA oligonucleotide (45 nt) is conserved between mice and humans displaying 93.3% identity. Data altogether indicate that TTP is able to regulate reporter gene expression by interacting with adjacent IL-22-3′-UTR sequences and thus by destabilizing target mRNA.

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