Brown C T, Nugent M A, Lau F W, Trinkaus-Randall V
Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
J Biol Chem. 1999 Mar 12;274(11):7111-9. doi: 10.1074/jbc.274.11.7111.
A culture system was developed to analyze the relationship between proteoglycans and growth factors during corneal injury. Specifically, the effects of transforming growth factor beta-1 (TGF-beta1) 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-beta1. 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-beta1 or serum. At early time points, both TGF-beta1 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-beta1 and decreased by serum. The levels of perlecan bearing chondroitin sulfate chains were elevated with TGF-beta1 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.
开发了一种培养系统，以分析角膜损伤期间蛋白聚糖与生长因子之间的关系。具体而言，研究了转化生长因子β-1（TGF-β1）和胎牛血清对角膜成纤维细胞中蛋白聚糖合成的影响。使用选择性多糖酶测定糖胺聚糖的合成和硫酸化。通过凝胶电泳和蛋白质印迹分析蛋白聚糖核心蛋白。与在1%透析胎牛血清中培养的细胞相比，在10%透析胎牛血清中培养的细胞合成的硫酸化程度更高的硫酸软骨素和硫酸乙酰肝素减少。TGF-β1对糖胺聚糖的量和硫酸化没有显著影响。分泌到培养基中的主要蛋白聚糖种类是核心蛋白聚糖和基底膜聚糖。核心蛋白聚糖被硫酸软骨素糖基化。基底膜聚糖与硫酸软骨素、硫酸乙酰肝素或硫酸软骨素和硫酸乙酰肝素两者相连。TGF-β1或血清均可降低核心蛋白聚糖的合成。在早期时间点，TGF-β1和血清均可诱导带有硫酸软骨素和/或硫酸乙酰肝素链的基底膜聚糖大量增加。相反，在培养较长时间后，带有硫酸乙酰肝素链的基底膜聚糖的量不受TGF-β1影响，但受血清影响而减少。TGF-β1处理可使带有硫酸软骨素链的基底膜聚糖水平升高，而血清则使其降低。由于核心蛋白聚糖和基底膜聚糖都能结合生长因子并被认为可调节其活性，因此这两种蛋白聚糖中任何一种表达的变化都可能显著影响细胞对损伤的反应。
