DUSP8 induces TGF-β–stimulated IL-9 transcription and Th9-mediated allergic inflammation by promoting nuclear export of Pur-α
Huai-Chia Chuang, … , Ming-Han Chen, Tse-Hua Tan
Huai-Chia Chuang, … , Ming-Han Chen, Tse-Hua Tan
Published November 1, 2023
Citation Information: J Clin Invest. 2023;133(21):e166269. https://doi.org/10.1172/JCI166269.
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Research Article Inflammation

DUSP8 induces TGF-β–stimulated IL-9 transcription and Th9-mediated allergic inflammation by promoting nuclear export of Pur-α

Abstract

Dual-specificity phosphatase 8 (DUSP8) is a MAPK phosphatase that dephosphorylates and inactivates the kinase JNK. DUSP8 is highly expressed in T cells; however, the in vivo role of DUSP8 in T cells remains unclear. Using T cell–specific Dusp8 conditional KO (T-Dusp8 cKO) mice, mass spectrometry analysis, ChIP-Seq, and immune analysis, we found that DUSP8 interacted with Pur-α, stimulated interleukin-9 (IL-9) gene expression, and promoted Th9 differentiation. Mechanistically, DUSP8 dephosphorylated the transcriptional repressor Pur-α upon TGF-β signaling, leading to the nuclear export of Pur-α and subsequent IL-9 transcriptional activation. Furthermore, Il-9 mRNA levels were induced in Pur-α–deficient T cells. In addition, T-Dusp8–cKO mice displayed reduction of IL-9 and Th9-mediated immune responses in the allergic asthma model. Reduction of Il-9 mRNA levels in T cells and allergic responses of T-Dusp8–cKO mice was reversed by Pur-α knockout. Remarkably, DUSP8 protein levels and the DUSP8–Pur-α interaction were indeed increased in the cytoplasm of T cells from people with asthma and patients with atopic dermatitis. Collectively, DUSP8 induces TGF-β–stimulated IL-9 transcription and Th9-induced allergic responses by inhibiting the nuclear translocation of the transcriptional repressor Pur-α. DUSP8 may be a T-cell biomarker and therapeutic target for asthma and atopic dermatitis.

Authors

Huai-Chia Chuang, Chia-Hsin Hsueh, Pu-Ming Hsu, Ching-Yi Tsai, Ying-Chun Shih, Hsien-Yi Chiu, Yi-Ming Chen, Wen-Kuang Yu, Ming-Han Chen, Tse-Hua Tan

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Figure 3

DUSP8 promotes IL-9 transcription in T cells by blocking Pur-α suppressor function.

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DUSP8 promotes IL-9 transcription in T cells by blocking Pur-α suppresso...
(A) Luciferase reporter activity of the Il-9 promoter. Jurkat T cells were cotransfected with the Il-9 promoter (–1,097 to +26)-luciferase construct plus the plasmid encoding either DUSP8 WT or phosphatase-dead (C246S) mutant. T cells were stimulated with TGF-β. n = 3. (B) Identification of Pur-α as a DUSP8-interacting protein by mass spectrometry-based proteomics. Matched Pur-α peptides were shown in red. (C) Luciferase reporter activity of the Il-9 promoter in TGF-β-stimulated Jurkat T cells cotransfected with Myc-Dusp8 and Flag-Pur-α plasmids. n = 3. (D) Ablation or reduction of Pur-α proteins in splenic T cells of Pur-α homozygous KO (Pur-α–/–) mice or heterozygous KO (Pur-α+/–) mice were verified by immunoblot analysis. (E) Real-time PCR of basal Il-9 mRNA levels in splenic T cells isolated from Pur-α heterozygous/homozygous KO and WT mice. Il-9 mRNA levels were normalized to Srp72 levels. n = 3. (F) The binding of Pur-α to the Il-9 promoter in TGF-β-stimulated murine T cells was analyzed by ChIP-Seq using anti-Pur-α antibody. (G) Luciferase reporter activity of the Il-9 promoter in TGF-β-stimulated Jurkat T cells cotransfected with Pur-α plasmid and the Il-9 promoter-driven luciferase reporter plasmid containing WT or individually mutated Pur-α-binding repeats. n = 3. WT and mutated sequences (Δ426, Δ1031, Δ1059) of the 3 putative Pur-α-binding elements (underlined) are shown at the bottom. (H) Real-time PCR of Il-9 mRNA levels in WT, T-Dusp8 cKO, or Dusp8fl/fl;Cd4-Cre;Pur-α+/– peripheral blood T cells stimulated with TGF-β (50 ng/mL) and IL-4 (20 ng/mL) for 8 hours. n = 3. Mean ± SEM are shown. *P < 0.05; **P < 0.01; ***P < 0.001 (1-way ANOVA and Tukey’s posthoc test). Data shown are representative of 3 independent experiments.