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 9

Model of the NFATC4 S213/S217 phospho-switch controlling fibroblast-to-myofibroblast transition.

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Model of the NFATC4 S213/S217 phospho-switch controlling fibroblast-to-m...
On soft ECM (<2 kPa), JNK signaling and TGFB/SMAD2/3 inputs are absent, NFATC4 remains unphosphorylated and cytoplasmic, and myofibroblast genes such as ACTA2 and CTHRC1 are not expressed. On stiff ECM (>8 kPa) with TGFB, mechanosensitive JNK activation phosphorylates NFATC4 at S213/S217, driving nuclear translocation and transcriptional induction of the myofibroblast program, including ACTA2 and CTHRC1, while parallel SMAD2/3 activation enhances the fibrotic gene program to firmly establish the CTHRC1+/ACTA2+ myofibroblast state. Mechanistic evidence is shown in the bottom panel: the phospho-dead mutant (S213A/S217A, blocked posttranslational modification [PTM]) fails to induce ACTA2 or CTHRC1 even on stiff ECM with TGFB; conversely, the phosphomimetic mutant (S213D/S217D, mimicked PTM) drives robust myofibroblast differentiation on soft ECM in the complete absence of TGFB. P, phosphorylation.