The mRNA-binding protein DDX3 mediates TGF-β1 upregulation of translation and promotes pulmonary fibrosis
Wensheng Chen, Darrell Pilling, Richard H. Gomer
Wensheng Chen, Darrell Pilling, Richard H. Gomer
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Research Article Cell biology Immunology

The mRNA-binding protein DDX3 mediates TGF-β1 upregulation of translation and promotes pulmonary fibrosis

Abstract

Pulmonary fibrosis is potentiated by a positive feedback loop involving the extracellular sialidase enzyme neuraminidase 3 (NEU3) causing release of active TGF-β1 and TGF-β1 upregulating NEU3 by increasing translation without affecting mRNA levels. In this report, we elucidate the TGF-β1 upregulation of the translation mechanism. In human lung fibroblasts, TGF-β1 increased levels of proteins, including NEU3, by increasing translation of the encoding mRNAs without significantly affecting levels of these mRNAs. A total of 180 of these mRNAs shared a common 20-nucleotide motif. Deletion of this motif from NEU3 mRNA eliminated the TGF-β1 upregulation of NEU3 translation, while insertion of this motif in 2 mRNAs insensitive to TGF-β1 caused TGF-β1 to upregulate their translation. RNA-binding proteins including DEAD box helicase 3, X-linked (DDX3), bind the RNA motif, and TGF-β1 regulates their protein levels and/or binding to the motif. We found that DDX3 was upregulated in the fibrotic lesions in patients with pulmonary fibrosis, and inhibiting DDX3 in fibroblasts reduced TGF-β1 upregulation of NEU3 levels. In the mouse bleomycin model of pulmonary fibrosis, injections of the DDX3 inhibitor RK-33 potentiated survival and reduced lung inflammation, fibrosis, and tissue levels of DDX3, TGF-β1, and NEU3. These results suggest that inhibiting an mRNA-binding protein that mediates TGF-β1 upregulation of translation can reduce pulmonary fibrosis.

Authors

Wensheng Chen, Darrell Pilling, Richard H. Gomer

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Figure 2

The motifs in the NEU3 mRNA are necessary for TGF-β1–increased NEU3 translation, and the group 4 motif is sufficient for TGF-β1–induced translation of S-formylglutathione hydrolase and McKusick-Kaufman syndrome protein.

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The motifs in the NEU3 mRNA are necessary for TGF-β1–increased NEU3 tran...
(A) Human lung fibroblasts transfected with the indicated constructs were cultured without (control) or with TGF-β1 and then stained with anti-Myc-tag antibodies. Green indicates Myc-NEU3 staining and blue is DAPI counterstain. Bar is 100 μm. Images are representative of 3 independent experiments. (B and C) Human lung fibroblasts transfected with the indicated constructs were cultured without (control) or with TGF-β1 and then stained with anti–Myc-tag antibodies. Red indicates Myc-ESD or Myc-MKKS staining and blue is DAPI counterstain. Images are representative of 3 independent experiments. (D–F) Quantification of A–C. IF, immunofluorescence. (G) Myc-NEU3 in transfected and nontransfected human lung fibroblasts was confirmed by Western blotting with an anti–Myc-tag antibody. Top image is Coomassie blue–stained gel of cell lysates; bottom image is Western blot. (H and I) Myc-ESD and Myc-MKKS in transfected and nontransfected human lung fibroblasts were verified by Western blotting with an anti–Myc-tag antibody. Images in G–I are representative of 3 independent experiments. (J–L) Quantification of G–I. WB, Western blot. Values in D–F and J–L are mean ± SEM, n = 3. * P < 0.05, *** P < 0.001 (t tests). ESD, S-formylglutathione hydrolase; MKKS, McKusick-Kaufman syndrome protein.