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Differences in decorin expression by papillary and reticular fibroblasts in vivo and in vitro.

ABSTRACT: Immunostaining of adult human skin shows that the small dermatan sulphate proteoglycan decorin is abundant in the whole dermal layer but absent from the epidermis. In the papillary layer adjacent to the dermal-epidermal border, more decorin was detected than in the reticular layer of the dermis. Expression of decorin mRNA by cells in the papillary dermis could also be shown by in situ hybridization. In contrast, biglycan, another small chondroitin sulphate/dermatan sulphate proteoglycan, is found only at the dermal-epidermal border. Therefore the biosynthesis of these two proteoglycans by papillary and reticular fibroblasts from two different donors was compared in tissue culture. Papillary fibroblasts secrete up to 5.9 times more decorin than reticular fibroblasts, while the amounts of cell-associated decorin in both cell types are similar. By Northern blot analysis as well as by in situ hybridization it was shown that papillary fibroblasts contain more mRNA coding for decorin than do reticular cells. In addition, no mosaic pattern of decorin expression was found in the cultured cells. The expression and synthesis of biglycan compared with decorin was about 10 times lower and did not show any significant differences for the two cells types. The kinetics of secretion and the rate of endocytosis of decorin were similar for both types of fibroblasts. These results were found with fibroblasts between the 9th and 15th passage from a newborn subject as well as from a 78-year-old donor, indicating that the pattern of decorin synthesis is not age-dependent in the range investigated. These results further show that fibroblasts from different layers of the dermis have a specific pattern of synthesis of small chondroitin sulphate/dermatan sulphate proteoglycans, and they also maintain these patterns in cell culture.

SUBMITTER: Schonherr E

PROVIDER: S-EPMC1132364 | biostudies-other | 1993 Mar

REPOSITORIES: biostudies-other

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Project description:Decorin, a small leucine-rich proteoglycan, is a natural inhibitor of transforming growth factor beta (TGFbeta). Myofibroblast and haze formation in the cornea have been attributed to TGFbeta hyperactivity released from corneal epithelium following injury to eye. This study tested the hypothesis that decorin-gene transfer inhibits TGFbeta-driven myofibroblast and haze formation in the cornea. Human corneal fibroblast (HSF) cultures generated from donor human corneas were used. Decorin cDNA was cloned into mammalian expression vector. Restriction enzyme analysis and DNA sequencing confirmed the nucleotide sequence of generated vector construct. The decorin gene cloned into mammalian expression vector was introduced into HSF with lipofectamine transfection kit. Expression of decorin in selected clones was characterized with RT-PCR, immunocytochemistry and western blotting. Phage contrast microscopy and trypan blue exclusion assay evaluated the effects of decorin-gene transfer on HSF phenotype and viability, respectively. Real-time PCR, western blot and immunocytochemistry were used to analyze inhibitory effects of decorin-gene transfer on TGFbeta-induced myofibroblast formation by measuring differential expression of alpha smooth muscle actin (SMA), a myofibroblast marker, mRNA and protein expression. Analysis of variance (ANOVA) and the Bonferroni-Dunn adjustment for repeated measures were used for statistical analysis. Our data indicate that decorin-gene transfer into HSF do not alter cellular phenotype or viability. Decorin over-expressing HSF clones grown in the presence of TGFbeta1 under serum-free conditions showed a statistically significant 80-83% decrease in SMA expression (p value < 0.01) compared to naked-vector transfected clones or un-transfected HSF controls. Decorin-transfected, naked-vector transfected and un-transfected HSF grown in the absence of TGFbeta1 showed no or extremely low expression of SMA. Furthermore, decorin over-expression did not affect HSF phenotype and decreased TGFbeta-induced RNA levels of profibrogenic genes such as fibronectin, collagen type I, III, and IV that play important role in stromal matrix modulation and corneal wound healing. The results of study suggest that decorin-gene transfer effectively prevents TGFbeta-driven transformation of keratocyte and corneal fibroblast to myofibroblasts. We postulate that decorin-gene therapy can be used to treat corneal haze in vivo.

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Differences in decorin expression by papillary and reticular fibroblasts in vivo and in vitro.

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