Cell density-dependent changes of glycosphingolipid biosynthesis in cultured human skin fibroblasts

In this study, the glycosphingolipid biosynthesis was investigated in the sparse and the confluent cell populations of cultured human skin fibroblasts. The human skin fibroblast cell populations were metabolically pulse labeled with (14)C-galactose (48 h). The amounts of (14)C-radioactivity (cpm) incorporated into extracted and purified total cellular glycosphingolipid fractions were counted by beta-scintillation and the individual glycosphingolipid species were separated by high performance thin layer chromatography and visualized by autoradiography. The relative labeling (%) of individual newly synthesized glycosphingolipid species was detected by densitometric scanning of autoradiographic glycosphingolipid patterns. The incorporation of (14)C-label into total glycosphingolipids per cell increased significantly as the cell-density increased, referring to five fold higher rate of glycosphingolipid biosynthesis de novo in cells at confluency vs. sparse populations. The total newly synthesized glycosphingolipid pattern (100%) of sparse cell populations showed a significant predominance of the gangliosides (70%) over the neutral glycosphingolipids (30%), with ganglioside GM2 as the major species followed by monohexosyl-ceramide. Oppositely, the newly synthesized neutral glycosphingolipids (67%) predominated over the gangliosides (33%) in cells at confluency (contact inhibition). Cells reaching confluency were characterized by: (a) a dramatic increase of absolute amount of all newly synthesized neutral glycosphingolipid species, particularly the most abundant monohexosyl-ceramide and trihexosyl-ceramide, but also of the ganglioside GM3; (b) a drastic decrease of absolute amount of newly synthesized ganglioside GM2. The specific shift in newly synthesized glycosphingolipid pattern in cells reaching confluency suggests a down-regulation of biosynthetic pathway primarily at the level of N-acetylgalactosaminyl-transferase. A possible involvement of glycosphingolipids in cell density-dependent regulation of cell growth through establishment of the direct intermolecular intermembrane interactions is discussed.