Inhibiting a mRNA motif binding protein that mediates TGF-β1 upregulation of translation attenuates pulmonary fibrosis in mice

Abstract

In human lung cells, the profibrotic cytokine TGF-{beta}1 increases sialidase 3 (NEU3) protein by increasing NEU3 translation without increasing levels of NEU3 mRNA. To elucidate how TGF-{beta}1 regulates translation, we treated human lung fibroblasts (HLF) with TGF-{beta}1 and used proteomics and RNA-seq to determine the effect of TGF-{beta}1 on proteins, mRNAs, and mRNA polysome/monosome ratios. We identified 181 mRNAs where TGF-{beta}1 also increases translation to increase protein levels without significantly affecting mRNA levels. These mRNAs share a common 20 nucleotide motif. Deletion or insertion of this motif in mRNAs eliminates or induces the TGF-{beta}1 regulation of translation. At least 5 RNA-binding proteins including DDX3 bind the RNA motif, and TGF-{beta}1 regulates their protein levels and/or binding to the motif. Inhibiting DDX3, either by siRNA or small molecule inhibitors, reduced TGF-{beta}1 induced NEU3 levels. In the mouse bleomycin model of pulmonary fibrosis, injections of the DDX3 inhibitor RK-33 starting 10 days after bleomycin potentiated survival and reduced lung inflammation, fibrosis, and lung tissue levels of DDX3, TGF-{beta}1, and NEU3. Together, these results suggest that TGF-{beta}1 regulates RNA-binding proteins that interact with a mRNA motif that is necessary and sufficient for TGF-{beta}1 to regulate mRNA translation, and that blocking this effect can reduce fibrosis.