Proteoglycan expression during transforming growth factor beta -induced keratocyte-myofibroblast transdifferentiation.

James L Funderburgh,Mary M Mann,Lolita Corpuz,Martha L Funderburgh,Mary R Roth

doi:10.1074/jbc.m107596200

Abstract

Keratocytes of the corneal stroma secrete a unique population of proteoglycan molecules considered essential for corneal transparency. In healing corneal wounds, keratocytes exhibit a myofibroblastic phenotype in response to transforming growth factor beta (TGF-beta), characterized by expression of alpha-smooth muscle actin. This study examined proteoglycan and collagen expression by keratocytes in vitro during the TGF-beta-induced keratocyte-myofibroblast transition. TGF-beta-treated primary bovine keratocytes developed myofibroblastic features, including actin stress fibers anchored to paxillin-containing focal adhesions, cell-associated fibronectin, alpha(5) integrin, and alpha-smooth muscle actin. Collagen I and III protein and mRNA increased in response to TGF-beta. Secretion of [(35)S]sulfate-labeled keratan sulfate proteoglycans decreased markedly in response to TGF-beta. Dermatan sulfate proteoglycans, however, increased in size and abundance. Protein and mRNA transcripts for normal stromal proteoglycans (lumican, keratocan, mimecan, and decorin) all decreased in response to TGF-beta, but protein expression and mRNA for biglycan, a proteoglycan present in fibrotic tissue, was markedly up-regulated. These results show that TGF-beta in vitro induces a proteoglycan expression pattern similar to that of corneal scars in vivo. This altered proteoglycan expression occurred coordinately with transdifferentiation of keratocytes to the myofibroblastic phenotype, implicating these cells as the source of fibrotic tissue in nontransparent corneal scars.

Highlights

Transparency of the cornea to light is a function of the ultrastructure of the corneal stroma, a tissue composed of multiple lamellae of parallel, highly regular collagen fibrils
Cytoskeleton-Matrix Interactions in Response to TGF-␤—We recently demonstrated that cultured primary bovine keratocytes maintained in low mitogen horse serum retain a morphology and proteoglycan expression characteristic of these cells in vivo [18, 19]
We found that cultures of primary bovine keratocytes exposed to TGF-␤ respond in a manner similar to that reported for rabbit keratocytes

Summary

Introduction

Transparency of the cornea to light is a function of the ultrastructure of the corneal stroma, a tissue composed of multiple lamellae of parallel, highly regular collagen fibrils. Inflammation, and several chronic pathological conditions lead to disruption of the stromal molecular architecture by the accumulation of a disorganized fibrotic extracellular matrix This scarring is often associated with loss of corneal transparency and can contribute to permanent loss of vision. In corneal scars resulting from trauma or from chronic pathologies, keratan sulfate is reduced and dermatan sulfate-containing proteoglycans are elevated [7,8,9,10,11] This characteristic alteration in stromal proteoglycans appears likely to be an important factor in the loss of corneal transparency. Growth of the cells in fetal bovine serum, rapidly reduced the KSPG synthesis by these cultures in a manner similar to that of healing wounds [19] This earlier study presented preliminary data suggesting that KSPGs were down-regulated by exposure of keratocytes to TGF-␤ [19]. The results suggest that secretion of proteoglycans characteristic of fibrotic scars is coordinated with the development of a myofibroblastic phenotype as a response to TGF-␤

Methods

Results

Conclusion