Abstract
Proteoglycans (PGs) comprise a group of extracellular matrix macromolecules which play an important role in matrix biology. In this study, normal human skin and gingival fibroblast cultures were incubated with transforming growth factor-beta 1 (TGF-beta 1), and the expression of three PGs, viz. biglycan (PGI), decorin (PGII), and versican (a large fibroblast proteoglycan) was examined. The results indicate that TGF-beta 1 (5 ng/ml) markedly increased the expression of biglycan (up to 24-fold) and versican (up to 6-fold) mRNAs and the enhancement of biglycan expression was coordinate with elevated type I procollagen gene expression in the same cultures. In contrast, the expression of decorin mRNA was markedly (up to approximately 70%) inhibited by TGF-beta 1. The response to TGF-beta 1 was similar in both skin and gingival fibroblasts, although the gingival cells were clearly more responsive to stimulation by TGF-beta 1 with respect to biglycan gene expression. Analysis of 35S-labeled proteoglycans in the culture media of skin and gingival fibroblasts also revealed stimulation of biglycan and versican production, and reduction in decorin production. Quantitation of both [35S]sulfate and [3H]leucine-labeled decorin in cell culture media by immunoprecipitation revealed a 50% reduction in decorin production in cell cultures treated with TGF-beta 1. This TGF-beta 1-elicited reduction was accompanied by an apparent increase in the size of the decorin molecules, although the size of the core protein was not altered, as judged by Western immunoblotting following chondroitinase ABC digestion. Analysis of the proteoglycans in the matrix and membrane fractions also revealed increased amounts of versican in cultures treated with TGF-beta 1. These results indicate differential regulation of PG gene expression in fibroblasts by TGF-beta 1, and these observations emphasize the role of PGs in the extracellular matrix biology and pathology.
Highlights
(PGII), and versican(a largefibroblast proteoglycan) was examined
Quantitation of both [36S]sulfate The entire core protein structure of the large chondroitin and [3H]leucine-labeleddecorin in cell culture media sulfate proteoglycan expressed by human fibroblasts, versiby immunoprecipitation revealed a 50%reduction in decorin production in cell cultures treated withTGF
In this study,we examined the effects of TGF-Pl on the abundance of mRNAs coding for three different PGcore proteins in human skin andgingival fibroblasts
Summary
TGF-PI Modulation of PG Core Protein mRNA Levels in Cultured Fibroblasts-Previous studieshavedemonstrated that TGF-Pl markedly increases type I procollagen mRNA steady-state levels in cultured human fibroblasts [10,11,12,13]. The levels of the two versican core protein mRNAs (-9 and 10kb, respectively), were relatively low, yet detectable, inbothskin fibroblast strains (Fig. 2). Hybridizations of 12 pg of total RNA isolated from untreated gingival cell cultures revealed a very low, yet detectable, signal with the biglycan cDNA, while characteristic mRNA transcripts were readily detected with decorin, versicanp, rocul[1]collagen, and GAPDH cDNAs (Fig. 3). Incubationof these cells for 24 h in the presence of TGF-Pl (5 ng/ml) dramatically increased the tlme(h) 1 3 6 12 4284 biglycan mRNA levels (-24-fold) (Fig. 3 and TableI).Similar, FIG. In gingival fibroblast cultures, immunoprecipitations revealed anincrease in the molecular weight of decorin as a result of TGF-61 treatment (Fig. 6)
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