Collagen degradation in human lung fibroblasts: extent of degradation, role of lysosomal proteases, and evaluation of an alternate hypothesis.

Experiments were conducted to determine the extent and variability of collagen degradation in human fetal lung fibroblasts. Cells were incubated with [14C]proline, and degradation was measured by determining the hydroxy[14C]proline in a low molecular weight fraction relative to total hydroxy[14C]proline. Average (basal) degradation in stationary phase HFL-1 cells incubated for 8 h was 16 +/- 3%, and substantial alterations in the composition of the labeling medium, e.g., omitting serum and varying pH between 6.8 and 7.8, had no effect. Organic buffers slightly lowered degradation in a manner that was independent of pH. Collagen degradation in two other lung cell lines, Wl-38 and lMR-90, did not differ from the level in HFL-1. Degradation was significantly higher (23 +/- 5%) in HFL-1 cultures labeled for 24 h rather than 8 h, and pulse-washout experiments showed that the rate of degradation was not uniform: after an 8-h pulse, 11% of the hydroxy [14C]proline in the medium was in the low molecular weight fraction, but 31% was in this fraction after a 16-h washout. The lack of effect of either serum deprivation or elevated pH suggests that lysosomal proteases have no direct role in basal degradation; however, NH4Cl decreased the enhanced degradation observed in ascorbate deficiency to basal level, indicating that abnormal molecules synthesized under those conditions are degraded by lysosomal proteases. The appearance of small hydroxy[14C]proline-containing molecules was inhibited by alpha alpha'dipyridyl and cycloheximide in a dose-dependent and reversible manner, demonstrating that their production depends on enzymatic hydroxylation of proline and protein synthesis.