Intracellular degradation of type I collagen and fibronectin in human lung fibroblasts: evidence against degradation in pre-lysosomal compartments.

Fibroblasts degrade about 15% of newly synthesised collagen within the cell before it can be secreted. When the helical structure of collagen is disrupted, about 30% is degraded intracellularly. To determine if collagen degradation occurs in a pre-lysosomal compartment, the passage of type 1 collagen out of the endoplasmic reticulum or Golgi was inhibited by incubating human lung fibroblasts with brefeldin A or monensin. In both cases, the type I collagen retained within the cell was stable over a 20 h period. Disrupting the helical structure of collagen with cis-hydroxyproline, 2,2'-bipyridyl or ethyl 3,4-dihydroxybenzoate did not alter the stability of type I collagen in brefeldin or monensin-treated cells. Incubating permeabilised cells in the presence of GTP gamma S (guanosine 5'-(3-O-thio)triphosphate), which blocks transport out of the endoplasmic reticulum, also resulted in the stable retention of type I collagen. Addition of dithiothreitol to permeabilised cells failed to initiate intracellular degradation. Similar results were obtained with fibronectin. Both normal fibronectin and fibronectin in which canavanine replaced arginine were stable for 20 h in cells treated with brefeldin A or monensin. The degradation of native collagen is sensitive to inhibition by a cell-permeable cysteine proteinase inhibitor (ALLN) but is insensitive to chloroquine (which raises the pH of acidic intracellular compartments), whereas the degradation of abnormal collagen was sensitive to both ALLN and chloroquine. These results argue against the intracellular degradation of collagen or fibronectin in a pre-lysosomal compartment.