The synthesis and turnover of sulfate-labeled glycosaminoglycans(35S-GAGs) has been investigated in diploid human embryo fibroblasts during in vitro cellular aging. With progressive subcultivation, there was a decreased incorporation of Na2(35)SO4 into 35S-GAGs released to the medium, but not into those accumulated at the cell surface. The composition of 35S-GAGs found in extracellular medium, cell surface (removable by gentle proteolysis), and intracellular compartments of the culture after 48-hr labeling did not change significantly with progressive subcultivation. Pulse-labeled 35S-GAGs moved from intracellular to surface and extracellular compartments more slowly in late-passage cultures. Addition of 1 mM beta-xyloside to both early- and late-passage cultures produced a ten-fold enhancement of extracellular 35S-GAG production without a concomitant increase in surface-associated 35S-GAG. We interpret the data of this study to mean that secreted and cell-surface glycosaminoglycans represent different pools and that cellular aging has its effect primarily upon the secreted pool of glycosaminoglycans. Late-passage fibroblasts demonstrate marked decreases in proliferation, culture density, fibronectin matrix, and gap-junction formation. Our results suggest that glycosaminoglycan synthesis and composition are not intimately related to these parameters.

As constituents of both extracellular matrix and the cell surface, glycosaminoglycans are in a strategic position to influence several basic cell features. The localization and turnover of glycosaminoglycans was investigated in cultured normal human embryo fibroblasts of lung origin (IMR-90). Attention was directed particularly toward that compartment of the culture which could be released by gentle proteolysis (trypsin, 0.1 mg/ml, 15 min) and is considered to represent the cell surface. In the presence of NA2SO4, sulfated glycosaminoglycans (S-GAGs) of the cell surface were labeled rapidly, but within 30 min some 35S-GAG appeared in the extracellular medium. The intracellular pool of S-GAGs labeled during a 10-min period was lost during the first hr of chase with a half-life of 18 min, compared with 16 hr for S-GAGs labeled over a 48-hr period. Pulse-labeled S-GAGs of the surface turned over with an initial half-life of 60 min, compared with 7 hr for surface material labeled over a 48-hr period. These rapid movements of the early chase period were followed by similar movement at a much slower rate. The results are consistent with a model in which most of the S-GAGs synthesized in the cell move rapidly to the surface. The surface GAGs are then released immediately to the medium or accumulate at the membrane to be shed more slowly at a later time or to be degraded. The S-GAG which left the cell layer most rapidly during chase was dermatan sulfate, while heparan sulfate made up an increasing percentage of the cell layer as chase progressed. These cultures produce a fibrillar matrix of fibronectin, but the kinetics of this study suggest that the S-GAGs of the surface are membrane-bound, and an extracellular glycosaminoglycan matrix does not form.