Synthesis of proteoglycans in rat mucosal keratinocytes

The synthesis of hyaluronic acid and proteoglycans by rat mucosal keratinocytes of an established cell line (CCL-10) has been investigated. Proliferating cultures at or near confluency were grown in the presence of [35S] sulfate or D-[1-3H] glucosamine for 24 h, and the glycosaminoglycan composition of cells and medium was determined. Characterization of the 35S-labelled glycosaminoglycans showed that heparan sulfate was the major component (approximately 90%) and that small amounts (approximately 10%) of galactosaminoglycans had also been synthesized. Analysis of cultures labelled with D-[1-3H] glucosamine demonstrated that hyaluronic acid was also present, most prominently in the medium where approximately one third of the radioactivity in the glycosaminoglycan pool was found in the hyaluronic acid fraction. [35S]-labelled proteoglycans extracted from the cell layer in the presence of protease inhibitors showed substantial heterogeneity upon chromatography on Sepharose CL-6B. In contrast, the proteoglycans in the medium gave a major peak which was eluted at a Kav of 0.28. Gel chromatography of the glycosaminoglycan chains in the latter, isolated after proteolytic digestion, indicated a molecular weight of 17,000.