Characterization of Proteoglycans Synthesized by Cultured Corneal Fibroblasts in Response to Transforming Growth Factor β and Fetal Calf Serum*
- From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118
Abstract
A culture system was developed to analyze the relationship between proteoglycans and growth factors during corneal injury. Specifically, the effects of transforming growth factor β-1 (TGF-β1) and fetal calf serum on proteoglycan synthesis in corneal fibroblasts were examined. Glycosaminoglycan synthesis and sulfation were determined using selective polysaccharidases. Proteoglycan core proteins were analyzed using gel electrophoresis and Western blotting. Cells cultured in 10% dialyzed fetal calf serum exhibited decreased synthesis of more highly sulfated chondroitin sulfate and heparan sulfate compared with cells cultured in 1% dialyzed fetal calf serum. The amount and sulfation of the glycosaminoglycans was not significantly influenced by TGF-β1. The major proteoglycan species secreted into the media were decorin and perlecan. Decorin was glycanated with chondroitin sulfate. Perlecan was linked to either chondroitin sulfate, heparan sulfate, or both chondroitin sulfate and heparan sulfate. Decorin synthesis was reduced by either TGF-β1 or serum. At early time points, both TGF-β1 and serum induced substantial increases in perlecan bearing chondroitin sulfate and/or heparan sulfate chains. In contrast, after extended periods in culture, the amount of perlecan bearing heparan sulfate chains was unaffected by TGF-β1 and decreased by serum. The levels of perlecan bearing chondroitin sulfate chains were elevated with TGF-β1 treatment and were decreased with serum. Because both decorin and perlecan bind growth factors and are proposed to modulate their activity, changes in the expression of either of these proteoglycans could substantially affect the cellular response to injury.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grants EY110004, by departmental grants from the Research to Prevent Blindness, and by the Massachusetts Lions Eye Research Fund, Inc. A grant from the New England Corneal Transplant Fund assisted in supporting this research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Boston University School of Medicine, Rm. L903, 80 East Concord St., Boston, MA 02118. Tel.: 617-638-4536; Fax: 617-638-5337.
- Abbreviations:
- ECM
-
extracellular matrix
- CS
-
chondroitin sulfate
- CSPG
-
chondroitin sulfate proteoglycan
- FCS
-
fetal calf serum
- dFCS
-
dialyzed FCS
- DMEM
-
Dulbecco’s modified Eagle’s medium
- GAG
-
glycosaminoglycan
- HS
-
heparan sulfate
- HSPG
-
heparan sulfate proteoglycan
- KS
-
keratan sulfate
- mAb
-
monoclonal antibody
- PAGE
-
polyacrylamide gel electrophoresis
- TBS
-
Tris-buffered saline
- TGF-β
-
transforming growth factor β
-
- Received September 3, 1998.
- Revision received December 21, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
