Regulation of Basic Fibroblast Growth Factor Binding and Activity by Cell Density and Heparan Sulfate*
- From the Departments of Biochemistry and Ophthalmology, Boston University School of Medicine, Boston, Massachusetts 02118
Abstract
The role of cell density in modulating basic fibroblast growth factor binding and activity was investigated. A primary corneal stromal fibroblast cell culture system was used, since these cells do not constitutively express heparan sulfate proteoglycansin vivo except after injury. A 3–5-fold reduction in bFGF binding per cell was observed as cell density increased from 1000 to 35,000 cells/cm2. The cell density-dependent change in bFGF binding was not the result of altered FGFR expression as determined by equilibrium binding experiments and by immunoblot analysis. However, bFGF-cell surface receptor binding affinities were measured to be 10–20-fold higher at low cell densities than at intermediate and high cell density. bFGF-induced cell proliferation was also cell density-dependent, with maximal stimulation of proliferation 190–280% greater at intermediate densities (15,000 cells/cm2) than at other cell densities. This effect was specific to bFGF as serum, epidermal growth factor, and transforming growth factor-β did not exhibit the same density-dependent profile. Further, heparan sulfate proteoglycans and, specifically, syndecan-4 were implicated as the modulator of bFGF binding and activity. Pretreatment of cell cultures with heparinase resulted in reduced bFGF binding to the cells and abrogated bFGF induced proliferation. These data suggest a mechanism by which cell density regulates heparan sulfate proteoglycan expression and modulates the cellular response to bFGF. Modulation of heparan sulfate proteoglycan expression might be an important aspect of the regulation of stromal cell migration and proliferation during wound healing.
Footnotes
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↵* This work was supported by National Institutes of Health Grant EY110004, by departmental grants from Research to Prevent Blindness, and the Massachusetts Lions Eye Research Fund, Inc.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. Tel.: 617-638-4169; Fax: 617-638-5337; E-mail: nugent{at}med-biochem.bu.edu.
- Abbreviations:
- bFGF
-
basic fibroblast growth factor
- DMEM
-
Dulbecco’s modified Eagle’s medium
- FBS
-
fetal bovine serum
- FGFR
-
fibroblast growth factor receptor
- HSPG
-
heparan sulfate proteoglycan
- PBS
-
phosphate-buffered saline
- PAGE
-
polyacrylamide gel electrophoresis
- TGF-β
-
transforming growth factor-β
-
- Received December 3, 1998.
- Revision received February 8, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
