title: Collagen degradation in human lung fibroblasts: extent of degradation, role of lysosomal proteases, and evaluation of an alternate hypothesis.
abstract: 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.
mesh_terms: D015082:2,2'-Dipyridyl / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D001206:Ascorbic Acid Deficiency / Q000378:metabolism; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008168:Lung / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism
chemical_list: D000643:Ammonium Chloride; D015082:2,2'-Dipyridyl; D003094:Collagen; D003513:Cycloheximide; D010447:Peptide Hydrolases