Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels

Abstract

A comparison was made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48 h. Radiolabeled glycosaminoglycans were then analyzed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulfate and dermatan sulfate. Hyaluronic acid was the main 3H-labeled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulfate was the major [35S]sulfated glycosaminoglycan in trypsin extracts of cells on plastic; dermatan sulfate was the minor component. Dermatan sulfate was the principal [35S]sulfated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum influenced the amounts of [35S]sulfated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulfate (75%) and dermatan sulfate (> 90%); whereas the collagenase extract was the main source of heparan sulfate (60%) and dermatan sulfate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulfated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulfated glycosaminoglycans to the network of native collagen fibers that formed the insoluble fraction of the collagen gel. Cells on collagen showed a 3X increase in dermatan sulfate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulfate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata, labeling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulfate from cells on collagen gels eluted at a lower salt concentration than did heparan sulfate from cells on plastic; with dermatan sulfate the opposite result was obtained, with dermatan sulfate from cells on collagen eluting at a higher salt concentration than dermatan sulfate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulfation.