Figure 1. Stiff matrix upregulates α₆-expression by ROCK-dependent activation of c-Fos/c-Jun transcription complex.

(a) IPF lung myofibroblasts were cultured on PA hydrogels with increasing stiffness (1, 5, 11 and 20 kPa). Levels of α₆-protein were determined by immunoblot and flow cytometry, respectively. In flow cytometry, non-immune rat IgG2a, κ was used as isotype IgG control. (b) Schematic shows the WT and mutated human α₆-promoters. Promoter activities were determined by luciferase assay. (c) Nuclear extracts from myofibroblasts cultured on soft and stiff matrix were incubated with immobilized oligonucleotides containing TREs. The TRE-binding activities of six AP-1 components as indicated were quantified by colorimetric enzyme-linked immunosorbant assay (ELISA). (d) The TRE-binding activity of Fra2 in nuclear extracts was quantified by colorimetric ELISA. Levels of Fra2 protein in cell lyates, cytoplasmic and nuclear fractions were determined by immunoblot. (e) Protein levels of phospho and total c-Fos and c-Jun under soft versus stiff matrix conditions were determined by immunoblot. (f) Effects of ROCK inhibitor Fasudil (Fasu) and ROCK-specific siRNAs on stiff matrix-induced phosphorylation of c-Fos and c-Jun. (g) The binding of c-Fos/c-Jun complex to the α₆-promoter under soft versus stiff matrix conditions was measured by quantitative chromatin immunoprecipitation. (h) Schematic shows sgRNA-mediated targeted expression of KRAB transcription repressor at the distal TRE1 and the proximal TRE2 regions in human α₆-promoter. Effects of CRISPRi-based disruption of c-Fos/c-Jun-dependent promoter activation on stiff matrix-induced α₆-expression were evaluated by immunoblot and flow cytometry analyses. Control (Ctrl) indicates cells transfected with empty vector. (i) Effects of c-Fos/c-Jun inhibitors (T-5224 and c-Jun peptides) on matrix stiffness-regulated α₆-expression were evaluated by immunoblot and flow cytometry. Results are the means ±s.d. of at least three separate experiments; *P<0.05; **P<0.01; one-way analysis of variance. a.u., arbitrary units.