Key role for Rac in the early transcriptional response to ECM stiffness and the stiffness-dependent repression of ATF3

The Rho family GTPases, Rac and Rho, play critical roles in transmitting mechanical information contained within the extracellular matrix (ECM) to the cell. Rac and Rho have well described roles in regulating stiffness-dependent actin remodeling, proliferation and motility. However, much less is known about the relative roles of these GTPases in stiffness-dependent transcription, particularly at the genome-wide level. Here, we selectively inhibited Rac and Rho in mouse embryonic fibroblasts cultured on deformable substrata and used RNA sequencing to elucidate and compare the contribution of these GTPases to the early transcriptional response to ECM stiffness. Surprisingly, we found that the stiffness-dependent activation of Rac is dominant over Rho in the initial transcriptional response to ECM stiffness. We also identified Activating Transcription Factor 3 (ATF3) as a major target of stiffness/Rac-mediated signaling and show that ATF3 repression by ECM stiffness helps to explain how the stiffness-dependent activation of Rac results in the induction of cyclin D1. Overall design: Spontaneously immortalized MEFs pre-incubated with EHT1864 (Rac inhibitor), CT04 (Rho inhibitor), or vehicle (untreated control) were plated on soft (2-4 kPa) or stiff (20-25 kPa) fibroenctin-coated polyacrylamide hydrogels for 1 hr before collection, isolation of total RNA and analysis of the transcriptomes by RNASeq.