Extracellular matrix stiffness controls cardiac fibroblast proliferation via the nuclear factor-Y (NF-Y) transcription factor.

The proliferative expansion of cardiac fibroblasts (CF) contributes towards cardiac fibrosis, which results in myocardial stiffening, cardiac dysfunction, and heart failure. CF sense and respond to increased stiffness of their local extracellular matrix, modulating their phenotype towards increased collagen synthesis and higher proliferation, leading potentially to a vicious circle of positive feedback. Here we describe a novel mechanism that mediates increased CF proliferation in response to a pathologically stiff Exteracellular matrix (ECM). The mechanism we describe is independent of the well-characterised mechano-sensitive transcript factors, YAP-TEAD and MKL1-SRF, which our data indicate are only responsible for part of the genes induced by stiffened ECM. Instead, our data identify Nuclear Factor-Y (NF-Y) as a novel mechanosensitive transcription factor, which mediates enhanced CF proliferation in response to a stiff ECM. We show that levels of NF-YA protein, the major regulatory subunit of NF-Y, and NF-Y transcriptional activity, are increased by a stiff ECM. Indeed, NF-Y activity drives the expression of multiple cell-cycle genes. Furthermore, NF-YA protein levels are dependent on FAK signalling suggesting a mechanistic link to ECM composition. Consistent with its role as a mechano-sensor, inhibition of NF-Y using siRNA or dominant negative mutant blocks CF proliferation on plastic in vitro, which models a stiff ECM, whereas ectopic expression of NF-YA increases the proliferation of cells interacting under conditions that model a physiologically soft ECM. In summary, our data demonstrate that NF-Y is a biomechanically sensitive transcription factor that promotes CF proliferation in a model of pathologically stiffened ECM.

Combined role for YAP-TEAD and YAP-RUNX2 signalling in substrate-stiffness regulation of cardiac fibroblast proliferation.

Cardiac fibrosis is associated with increased stiffness of the myocardial extracellular matrix (ECM) in part mediated by increased cardiac fibroblast proliferation However, our understanding of the mechanisms regulating cardiac fibroblast proliferation are incomplete. Here we characterise a novel mechanism involving a combined activation of Yes-associated protein (YAP) targets RUNX Family Transcription Factor 2 (RUNX2) and TEA Domain Transcription Factor (TEAD). We demonstrate that cardiac fibroblast proliferation is enhanced by interaction with a stiff ECM compared to a soft ECM. This is associated with activation of the transcriptional co-factor, YAP. We demonstrate that this stiffness induced activation of YAP enhances the transcriptional activity of both TEAD and RUNX2 transcription factors. Inhibition of either TEAD or RUNX2, using gene silencing, expression of dominant-negative mutants or pharmacological inhibition, reduces cardiac fibroblast proliferation. Using mutants of YAP, defective in TEAD or RUNX2 activation ability, we demonstrate a dual role of YAP-mediated activation of TEAD and RUNX2 for substrate stiffness induced cardiac fibroblast proliferation. Our data highlights a previously unrecognised role of YAP mediated RUNX2 activation for cardiac fibroblast proliferation in response to increased ECM stiffness.