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 4

DUSP8 dephosphorylates Pur-α and induces its nuclear export.

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DUSP8 dephosphorylates Pur-α and induces its nuclear export. (A) Confoca...
(A) Confocal microscopy analysis of endogenous Pur-α and DUSP8 in T cells from T-Dusp8–cKO or WT mice. T cell nucleus was stained with DAPI. Scale bars: 25 μm. Mean ± SEM of Pur-α nuclear localization (green over blue, %) or DUSP8 nuclear localization (red over blue, %) from 3 images are shown at the bottom. (B) Coimmunoprecipitations of Myc-tagged DUSP8 with Flag-tagged Pur-α proteins in the lysates of indicated HEK293T transfectants. (C) FRET analysis of HEK293T cells transfected with indicated plasmids encoding CFP-fused Pur-α or YFP-fused DUSP8 proteins. n = 3. (D) In vitro phosphatase assays of purified DUSP8 WT or phosphatase-dead (C246S) mutant, using purified Flag-tagged Pur-α proteins as substrates. Phosphorylation of Pur-α was determined by immunoblot analyses using anti-pan-phospho-serine (p-Ser) and anti-pan-phospho-threonine (p-Thr) antibodies. (E) Mass spectrometry analysis of the tryptic peptides of Pur-α to identify the peptide containing phosphorylated Ser127 residue. (F) Luciferase reporter activity of the Il-9 promoter in TGF-β-stimulated Jurkat T cells cotransfected with Il-9-luciferase reporter plasmid and Pur-α WT or phospho-deficient mutant (S127A or T183A) plasmid. n = 4. (G) Confocal microscopy analysis of Pur-α WT or phospho-deficient mutant (S127A or T183A) in HEK293T cells. Cell nucleus was stained with DAPI. Scale bars: 25 μm. Mean ± SEM of Pur-α nuclear localization (green over blue, %) from 3 images are shown at the bottom. (H) Real-time PCR of IL-9 mRNA levels in human primary Th9 cells transfected with Pur-α WT or phospho-deficient mutant (S127A) plasmid. IL-9 mRNA levels were normalized to GAPDH levels. Means ± SEM are shown. P < 0.05; **P < 0.01; ***P < 0.001 (1-way ANOVA and Tukey’s posthoc test). Data shown (A–D and F–H) are representative of 3 independent experiments.