Macropinocytosis inhibition attenuates profibrotic responses in lung fibroblasts and pulmonary fibrosis models
Ivan O. Rosas, Aaron K. McDowell-Sanchez, Santiago Sanchez, Juan D. Cala-Garcia, Alan R. Waich Cohen, Elisa Ruiz-Echartea, Scott A. Ochsner, Daniel C. Kraushaar, Lindsay J. Celada, Dandan Sun, Francesca Polverino, Cristian Coarfa, Neil J. McKenna, Konstantin Tsoyi
Ivan O. Rosas, Aaron K. McDowell-Sanchez, Santiago Sanchez, Juan D. Cala-Garcia, Alan R. Waich Cohen, Elisa Ruiz-Echartea, Scott A. Ochsner, Daniel C. Kraushaar, Lindsay J. Celada, Dandan Sun, Francesca Polverino, Cristian Coarfa, Neil J. McKenna, Konstantin Tsoyi
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Research Article Cell biology Pulmonology

Macropinocytosis inhibition attenuates profibrotic responses in lung fibroblasts and pulmonary fibrosis models

Abstract

Idiopathic pulmonary fibrosis (IPF) is a devastating chronic lung disorder with limited treatment options. Macropinocytosis is one of the key cellular processes involved in nutrient consumption from the extracellular environment under stress conditions. Here, we studied the role of macropinocytosis in experimental pulmonary fibrosis models. We found that macropinocytosis is increased in human lung fibroblasts (HLFs) derived from patients with IPF. The inhibition of macropinocytosis with 5-(n-ethyl-n-isopropyl)-amiloride (EIPA) inhibited profibrotic responses in IPF-derived and TGF-β1–stimulated HLFs and reduced pulmonary fibrosis in bleomycin-injured (Bleo-injured) mice. EIPA exerted its antifibrotic effects by regulating amino acid uptake, mammalian target of rapamycin complex 1 (mTORC1) activation and mesenchyme homeobox1 (MEOX1) expression in activated HLFs. Fittingly, genetic inhibition of macropinocytosis also ameliorated lung fibroblast activation and pulmonary fibrosis in mice. Using IPF-derived precision cut lung slices (PCLSs), we observed robust repression of profibrotic gene expression programs in EIPA-treated PCLSs across different fibroblast subpopulations. Finally, we found that imipramine (Imi), a tricyclic antidepressant approved by the FDA, effectively inhibited macropinocytosis and ameliorated profibrotic responses in lung fibroblasts, Bleo-injured mice, and IPF-derived PCLSs. Taken together, our results suggest that macropinocytosis inhibition can be considered as a potential therapeutic strategy to treat pulmonary fibrosis.

Authors

Ivan O. Rosas, Aaron K. McDowell-Sanchez, Santiago Sanchez, Juan D. Cala-Garcia, Alan R. Waich Cohen, Elisa Ruiz-Echartea, Scott A. Ochsner, Daniel C. Kraushaar, Lindsay J. Celada, Dandan Sun, Francesca Polverino, Cristian Coarfa, Neil J. McKenna, Konstantin Tsoyi

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Figure 3

Macropinocytosis inhibition regulates MEOX1 expression in activated lung fibroblasts.

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Macropinocytosis inhibition regulates MEOX1 expression in activated lung...
(A) Volcano plot representing treatment of MRC5 HLFs with and without EIPA in the presence of TGFB. Overlaid in yellow is a set of TGFβ1-induced genes from a separate RNA sequencing experiment in MRC5 HLFs treated with TGF-β1 (10 ng/mL) or vehicle. MRC-5 cells were treated with EIPA (12.5 μM) with or without TGF-β1 (10 ng/mL) for 24 hours. Then, RNA was isolated and subjected to bulk RNA sequencing, as described in Methods (n = 3). (B and C) Control HLFs were treated with EIPA (12.5 μM) and Rapalink-1 (10 nM) with or without TGF-β1 (10 ng/mL) for 24 hours. Then, MEOX1 was measured at mRNA and protein levels by qRT-PCR (B) and Western blot (C), respectively (n = 4 each condition). (D) Control HLFs were lentivirally transfected with Scr or shTSC1, as described in Methods. Then, cells were treated with vehicle (Veh) or EIPA (12.5 μM) for 24 hours. Then, cells were harvested and subjected to Western blot to measure MEOX1 expression. (E) Control HLFs were lentivirally transfected with empty vector (EV) or pLenti-MEOX1-DDK, as described in Methods. Then, cells were treated with TGF-β1 (10 ng/mL) for 24 hours or not. Protein levels of COL1 and α-SMA were measured by Western blot, as described in Methods (n = 4 each condition). Data are shown as the mean ± SEM. *P < 0.05, vs. unstimulated or Scr alone; †P < 0.05, vs. TGF-β1 alone; #P < 0.05, TGF-β1+EIPA; significant comparisons by hypergeometric test (A) or 1-way ANOVA (B–E).