Hyaluronan Synthesis Mediates the Fibrotic Response of Keratocytes to Transforming Growth Factor β

TGF beta induces fibrosis in healing corneal wounds, and in vitro corneal keratocytes up-regulate expression of several fibrosis-related genes in response to TGF beta. Hyaluronan (HA) accumulates in healing corneas, and HA synthesis is induced by TGF beta by up-regulation of HA synthase 2. This study tested the hypothesis that HA acts as an extracellular messenger, enhancing specific fibrotic responses of keratocytes to TGF beta. HA synthesis inhibitor 4-methylumbelliferone (4MU) blocked TGF beta induction of HA synthesis in a concentration-dependent manner. 4MU also inhibited TGF beta-induced up-regulation of alpha-smooth muscle actin, collagen type III, and extra domain A-fibronectin. Chemical analogs of 4MU also inhibited fibrogenic responses in proportion to their inhibition of HA synthesis. 4MU, however, showed no effect on TGF beta induction of luciferase by the 3TP-Lux reporter plasmid. Inhibition of HA using siRNA to HA synthase 2 reduced TGF beta up-regulation of smooth muscle actin, fibronectin, and cell division. Similarly, brief treatment of keratocytes with hyaluronidase reduced TGF beta responses. These results suggest that newly synthesized cell-associated HA acts as an extracellular enhancer of wound healing and fibrosis in keratocytes by augmenting a limited subset of the cellular responses to TGF beta.