MAPK phosphatase 1 inhibition of p38α within lung myofibroblasts is essential for spontaneous fibrosis resolution
Sean M. Fortier, … , Anton M. Bennett, Marc Peters-Golden
Sean M. Fortier, … , Anton M. Bennett, Marc Peters-Golden
Published March 21, 2024
Citation Information: J Clin Invest. 2024;134(10):e172826. https://doi.org/10.1172/JCI172826.
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MAPK phosphatase 1 inhibition of p38α within lung myofibroblasts is essential for spontaneous fibrosis resolution

Abstract

Fibrosis following tissue injury is distinguished from normal repair by the accumulation of pathogenic and apoptosis-resistant myofibroblasts (MFs), which arise primarily by differentiation from resident fibroblasts. Endogenous molecular brakes that promote MF dedifferentiation and clearance during spontaneous resolution of experimental lung fibrosis may provide insights that could inform and improve the treatment of progressive pulmonary fibrosis in patients. MAPK phosphatase 1 (MKP1) influences the cellular phenotype and fate through precise and timely regulation of MAPK activity within various cell types and tissues, yet its role in lung fibroblasts and pulmonary fibrosis has not been explored. Using gain- and loss-of-function studies, we found that MKP1 promoted lung MF dedifferentiation and restored the sensitivity of these cells to apoptosis — effects determined to be mainly dependent on MKP1’s dephosphorylation of p38α MAPK (p38α). Fibroblast-specific deletion of MKP1 following peak bleomycin-induced lung fibrosis largely abrogated its subsequent spontaneous resolution. Such resolution was restored by treating these transgenic mice with the p38α inhibitor VX-702. We conclude that MKP1 is a critical antifibrotic brake whose inhibition of pathogenic p38α in lung fibroblasts is necessary for fibrosis resolution following lung injury.

Authors

Sean M. Fortier, Natalie M. Walker, Loka R. Penke, Jared D. Baas, Qinxue Shen, Jennifer M. Speth, Steven K. Huang, Rachel L. Zemans, Anton M. Bennett, Marc Peters-Golden

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Figure 2

Effect of gain- and loss-of-function of MKP1 on lung fibroblast phenotypic features.

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Effect of gain- and loss-of-function of MKP1 on lung fibroblast phenotyp...
(A) CRISPR/Cas9-mediated DUSP1 deletion using MKP1 or NT sgRNAs in HLF-derived MFs. Protein quantification by Western blotting of MKP1 and the fibrosis-associated genes αSMA, Col1A1, FN1, and CTHRC1 (top: representative blot; bottom: densitometric analysis). (B and C) Inducible MKP1 overexpression in human lung MFs. (B) Protein quantification by Western blotting of MKP1 and the fibrosis-associated genes αSMA, Col1A1, FN1, and CTHRC1 (top: representative blot; bottom: densitometric analysis). (C) αSMA stress fibers identified by immunofluorescence microscopy using an anti–αSMA-Cy3–conjugated antibody in MFs (Myofibs) overexpressing MKP1 or GFP and fibroblast (Fibs) controls (using the same protocol as in B). Nuclei were stained with DAPI. Scale bars: 20 μm (top row) and 10 μm (bottom row). (D) IPF fibroblasts harboring the same MKP1 overexpression construct as normal HLFs in B were treated with doxycycline for 48 hours to induce MKP1 or GFP overexpression. Protein quantification by Western blotting of MKP1 and the fibrosis-associated genes αSMA, Col1A1, FN1, and CTHRC1 (left: representative blots, right: densitometric analysis). (E) Apoptosis sensitivity in MKP1-overexpressing (or vehicle-treated) MFs further treated with an anti-Fas–activating antibody (100 ng/mL) for 24 hours. Apoptosis was determined by caspase-3/-7 activity assay (left) or annexin V expression (right). Dashed lines represent caspase-3/-7 activity or annexin V expression in untreated, undifferentiated fibroblasts stimulated with anti-Fas antibody. The sample number for each experiment (n) varied between 3 and 9 and is indicated by the number of data points in each histogram. Each blot grouping containing a protein (or proteins) of interested and its corresponding loading control were run on separate gels. Significance for densitometric analysis and apoptosis activity assays was determined by 2-tailed t test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Dox, doxycycline.