The role of the Rho kinase isoforms in fibroblast contractility

Abstract

The Rho kinases, ROCK I and ROCK II, are homologous effectors of the small GTPase RhoA. Rho kinases are known to mediate contraction in non‐muscle cells such as fibroblasts. However, the specific contribution of each isoform has yet to be determined as current chemical inhibitors do not distinguish between the isoforms. To investigate whether ROCK I and ROCK II have distinct roles in fibroblast contraction, we transfected human foreskin fibroblasts (HFF) with lentiviral vectors containing shRNA targeting each isoform. We were able to reduce ROCK I protein expression in HFF cells by 65 percent, and ROCK II expression by 50 percent, as confirmed by Western blot. These transfected HFFs were then embedded in a type I collagen matrix to produce engineered tissues for quantifying cellular contractility. Tissues with reduced ROCK I expression showed a 75 percent decrease in contractile force compared to control tissues following stimulation with 10µM lysophosphatidic acid (LPA). Contractility of HFF tissues expressing decreased levels of ROCK II was comparable to control tissues. These results suggest that ROCK I may be preferentially activated by LPA in fibroblasts to cause contraction, while ROCK II may not be essential to fibroblast contractility.