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

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.

• Kadri, S.
• Mattner, L. F.
• Zeng, Z.
• Prakki, S. R. S.
• Verma, A. K.
• Cetin, U.
• Mayr, C. H.
• Ansari, M.
• Wei, X.
• Asgharpour, S.
• Wasik, A. A.
• Kneidinger, N.
• Stoleriu, M. G.
• Behr, J.
• Polleux, J.
• Yildirim, A. O.
• De Sadeleer, L. J.
• Wuyts, W. A.
• Burgstaller, G.
• Mann, M.
• Muck-Hausl, M.
• Schiller, H. B.