Effects of tunicamycin on the biosynthesis of procollagen by human fibroblasts.

The reported effects of tunicamycin upon collagen biosynthesis are conflicting, especially with regard to procollagen secretion. We have examined the possible causes for this paradox by employing human fibroblasts in cell culture. Confluent and exponentially dividing fibroblasts were slowly affected by tunicamycin; the confluent cells responded maximally about 15 h following exposure to the antibiotic. The slow response of confluent cells was not accelerated by using 0.1% Tween 80 or 0.5% dimethyl sulfoxide to potentially enhance entry of tunicamycin. When confluent cells were preincubated in tunicamycin for 16 to 18 h, 0.3 pg/ ml of the antibiotic was sufficient to impair ~-[6-%]glucosamine incorporation into the cell layer by about 85% compared to control cultures. Simultaneously, L[U-14C]proline incorporatioL .;as impaired by about 40% compared to controls. Gel filtration profiles of the media showed that tunicamycin suppressed the appearance of all radioactive components while precipitation studies and electrophoretic gels showed that the appearance of procollagen and fibronectin in the culture medium was about 20% of control values. Pulse-chase studies showed that the rate of procollagen and fibronectin secretion was about 20 to 30% of the control rate. Moreover, enhanced degradation of procollagen and fibronectin did not occur in the medium of tunicamycin-treated cells. Both fibronectin and procollagen were found to be underglycosylated; the underglycosylated procollagen was compared with control procollagen by immunoprecipitation, gel filtration, and gel electrophoresis. We conclude that tunicamycin impairs glycosylation of procollagen and that the decreased content of carbohydrate occurs concomitantly with severe retardation of the intracellular movement of the protein and marked retardation of its secretion from human fibroblasts.