Abstract
Abstract: :
Purpose: Dermatan sulfate (DS) is the glycosaminoglycan that modifies decorin, a major proteoglycan component of the corneal stroma. Accumulation of unusually highly sulfated DS is a well-known feature of fibrotic tissue and may contribute to the inappropriate hydration of corneal scars. We have recently shown (J Biol Chem, 276, 44173) upregulation of highly sulfated DS proteoglycan during transforming growth factor-beta (TGF-ß)-induced transition of bovine keratocytes to myofibroblasts in vitro. The purpose of this study was to identify bovine genes involved in DS biosynthesis and to correlate their expression with changes in DS regulation in myofibroblasts. Methods:Keratocytes were isolated from fresh bovine eyes and transformed to myofibroblasts by culturing in the presence of TGF-ß. DSPG was isolated from these cultures, and the amount and distribution of sulfation was determined by fluorophore-assisted carbohydrate electrophoresis (FACE). Genes for DS biosynthesis were identified by degenerate random-primed reverse-transcriptase polymerase chain reaction (RT-PCR) or from bovine EST database using published human DNA sequences. Levels of messenger RNA for the synthetic enzymes, as compared to 18S RNA, were estimated by semi-quantitative multiplex RT-PCR. Results:Analysis by FACE indicated increased dermatan sulfate with increased 6-O-sulfation in cultures exposed to TGF-ß. Genes for three enzymes involved in DS biosynthesis were identified: chondroitin synthase (ChSy) (involved in chain elongation), chondroitin 4- sulfotransferase (C4ST), and chondroitin 6-sulfotransferase (C6ST). The mRNA pools for both ChSy and C6ST increased rapidly, i.e. in less than 6 hours, in response to TGF-ß. Conclusion:Our in vitro results suggest that changes in DS in corneal scar tissue could result from increases in mRNA pools for biosynthetic enzymes for sulfate chain elongation and sulfation. The rapidity of the increase in mRNA levels implies that the observed changes are at the transcriptional level.
Keywords: 374 cornea: stroma and keratocytes • 403 extracellular matrix • 423 growth factors/growth factor receptors