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

Abstract

TGF␤ induces fibrosis in healing corneal wounds, and in vitro corneal keratocytes up-regulate expression of several fibrosisrelated genes in response to TGF␤. Hyaluronan (HA) accumulates in healing corneas, and HA synthesis is induced by TGF␤ 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␤. HA synthesis inhibitor 4-methylumbelliferone (4MU) blocked TGF␤ induction of HA synthesis in a concentration-dependent manner. 4MU also inhibited TGF␤-induced up-regulation of ␣-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␤ induction of luciferase by the 3TP-Lux reporter plasmid. Inhibition of HA using siRNA to HA synthase 2 reduced TGF␤ up-regulation of smooth muscle actin, fibronectin, and cell division. Similarly, brief treatment of keratocytes with hyaluronidase reduced TGF␤ 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␤.Keratocytes of the corneal stroma are responsible for the synthesis of the stromal extracellular matrix and thus play an essential role in maintenance of corneal transparency. Under normal in vivo conditions, keratocytes are characteristically quiescent, exhibiting a dendritic morphology with extensive intercellular contacts. In response to stromal injury, such as trauma, keratectomy, or photoablation by excimer laser, keratocytes near the injured site become activated to fibroblasts, altering their morphology, becoming migratory and mitotic, reducing expression of the characteristic keratan sulfate-containing proteoglycans, and eventually transforming into ␣-smooth muscle actin (␣SMA) 4 -expressing myofibroblasts (1).Along with this transition, atypical components of the extracellular matrix are observed, including hyaluronan, biglycan, the extra domain A (EDA) splice form of fibronectin, collagen types I and III, and SPARC (secreted protein acidic and rich in cysteine) (1-5). These fibrotic matrix components contribute to the loss of transparency associated with corneal scars.TGF␤ has been identified as one of the most important factors involved in corneal fibrosis (6). A major function of TGF␤ is to regulate the expression of genes, the products of which contribute to the formation and degradation of extracellular matrix. In monolayer cultures, primary bovine and rabbit keratocytes maintain a dendritic morphology and express corneal keratan sulfate-containing proteoglycans when cultured in serum-free medium. In the presence of TGF␤, keratocytes differentiate into myofibroblasts (2, 3, 7). These cells become mitotic, exhibit a spread morphology, develop focal adhesions, express ␣5␤1 integrin, and develop prominent actin stress fibers conta...

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