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

Naxin Guo,Xuan Li,Mary M Mann,Martha L Funderburgh,Yiqin Du,James L Funderburgh

doi:10.1074/jbc.m110.127183

Abstract

TGFβ induces fibrosis in healing corneal wounds, and in vitro corneal keratocytes up-regulate expression of several fibrosis-related genes in response to TGFβ. Hyaluronan (HA) accumulates in healing corneas, and HA synthesis is induced by TGFβ 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 TGFβ. HA synthesis inhibitor 4-methylumbelliferone (4MU) blocked TGFβ induction of HA synthesis in a concentration-dependent manner. 4MU also inhibited TGFβ-induced up-regulation of α-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 TGFβ induction of luciferase by the 3TP-Lux reporter plasmid. Inhibition of HA using siRNA to HA synthase 2 reduced TGFβ up-regulation of smooth muscle actin, fibronectin, and cell division. Similarly, brief treatment of keratocytes with hyaluronidase reduced TGFβ 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 TGFβ.

Highlights

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 [1,2,3,4,5]
Myofibroblasts exhibit reduced synthesis of keratan sulfate and abundant secretion of extracellular matrix-containing components identified in corneal scars
These results show that the potency of these compounds for blocking TGF␤-induced EDA-FN and ␣SMA accumulation correlates with their ability to block HA synthesis

Summary

Introduction

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) [1,2,3,4,5]. Primary bovine keratocytes were untreated (first four samples) or treated with 2 ng/ml TGF␤ in 1% platelet-poor horse serum for 4 days, and mRNA levels were determined by quantitative reverse transcriptase PCR as described under “Materials and Methods.” 4MU was present during treatment at the concentrations noted.

Results

Conclusion