Mechanosensitive phosphorylation of NFATC4 at S213/S217 drives fibroblast-to-myofibroblast transition and fibrosis
Safwen Kadri, Laura F. Mattner, Zhen Zeng, Sai Rama Sridatta Prakki, Arun Kumar Verma, Umut Cetin, Christoph H. Mayr, Meshal Ansari, Xin Wei, Sara Asgharpour, Anita A. Wasik, Nikolaus Kneidinger, Mircea-Gabriel Stoleriu, Jürgen Behr, Julien Polleux, Ali Önder Yildirim, Laurens J. De Sadeleer, Wim A. Wuyts, Gerald Burgstaller, Matthias Mann, Martin Mück-Häusl, Herbert B. Schiller
Safwen Kadri, Laura F. Mattner, Zhen Zeng, Sai Rama Sridatta Prakki, Arun Kumar Verma, Umut Cetin, Christoph H. Mayr, Meshal Ansari, Xin Wei, Sara Asgharpour, Anita A. Wasik, Nikolaus Kneidinger, Mircea-Gabriel Stoleriu, Jürgen Behr, Julien Polleux, Ali Önder Yildirim, Laurens J. De Sadeleer, Wim A. Wuyts, Gerald Burgstaller, Matthias Mann, Martin Mück-Häusl, Herbert B. Schiller
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Research Article Cell biology Pulmonology

Mechanosensitive phosphorylation of NFATC4 at S213/S217 drives fibroblast-to-myofibroblast transition and fibrosis

Abstract

Mechanosensitive feedback between tissue stiffness and cellular contractile forces instructs cell identity. To characterize phosphorylation-mediated mechanosensing, we charted the global phosphoproteome dynamics of primary human lung fibroblasts on fibronectin-coated polydimethylsiloxane substrates of defined stiffness. We identified a key signaling threshold at 2–8 kPa, above which cells activated cytoskeletal remodeling, ECM secretion, and transition to a CTHRC1+/ACTA2+ myofibroblast state, accompanied by stiffness-dependent phosphorylation of the transcription factor NFATC4 at S213/S217. In micro-CT staged pulmonary fibrosis tissues, NFATC4 expression increased progressively, colocalizing with CTHRC1 and ACTA2 in myofibroblasts. Transcription factor regulon inference from a multicohort pulmonary fibrosis atlas confirmed elevated NFATC4 activity in disease fibroblasts, revealing a core 119-gene NFATC4-dependent fibrotic program with CTHRC1 as a top target. Phosphomimetic S213D/S217D mutants drove myofibroblast differentiation on soft substrates independently of TGFB, while phospho-dead S213A/S217A mutants blocked differentiation even on stiff matrix with TGFB, establishing the phospho-switch as both necessary and sufficient. Stiff matrix and TGFB converged on this JNK- and calcineurin-dependent switch to amplify the fibrotic response. This positions NFATC4 S213/S217 as a mechanosensitive checkpoint for CTHRC1+ myofibroblast fate and a candidate therapeutic target in multiorgan fibrosis.

Authors

Safwen Kadri, Laura F. Mattner, Zhen Zeng, Sai Rama Sridatta Prakki, Arun Kumar Verma, Umut Cetin, Christoph H. Mayr, Meshal Ansari, Xin Wei, Sara Asgharpour, Anita A. Wasik, Nikolaus Kneidinger, Mircea-Gabriel Stoleriu, Jürgen Behr, Julien Polleux, Ali Önder Yildirim, Laurens J. De Sadeleer, Wim A. Wuyts, Gerald Burgstaller, Matthias Mann, Martin Mück-Häusl, Herbert B. Schiller

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Figure 1

Human lung fibroblasts activate mechanosensitive signaling pathways on stiff ECM substrates.

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Human lung fibroblasts activate mechanosensitive signaling pathways on s...
(A) Immunofluorescence of YAP1 (green) in CCL-151 cells seeded for 2 h on fibronectin-coated substrates of indicated stiffness. Original magnification, ×40. (B) Experimental workflow for phosphoproteomic analysis of CCL-151 cells on varying substrate stiffnesses. FN, fibronectin. (C) Bar graph showing the number of quantified phosphosites per condition. (D) PCA of the stiffness-regulated phosphoproteome. (E) Pie charts showing the proportion of singly, doubly, and triply phosphorylated peptides (top) and phosphorylated S, T, and Y residues (bottom). (F) Pie chart showing the proportion of known, previously unreported, and regulatory phosphosites in PhosphoSitePlus. (G) Box-and-whisker plots of MYL9 T18/S19 phosphopeptide intensities across stiffness conditions. Immunofluorescence with phosphospecific MYL9 T18/S19 antibody (green) confirms stiffness-dependent induction; phalloidin (magenta) marks actin; DAPI (blue) marks nuclei. Original magnification, ×40. (H) Box-and-whisker plots of PXN S126 phosphopeptide intensities across stiffness conditions. Immunoblot with phosphospecific PXN S126 and total PXN antibodies confirms stiffness-dependent induction; GAPDH loading control (n = 3). Band intensities were quantified by densitometry, normalized to GAPDH, and compared between conditions by unpaired 2-tailed Student’s t test. ***P = 0.0007. Box-and-whisker plots show median, IQR, and whiskers to 1.5× IQR.