monensin and ethyl-protocatechuate

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.

Topics: 2,2'-Dipyridyl; Biological Transport; Brefeldin A; Canavanine; Cell Line; Cell Membrane Permeability; Chloroquine; Collagen; Cyclopentanes; Cysteine Proteinase Inhibitors; Endoplasmic Reticulum; Fibroblasts; Fibronectins; Golgi Apparatus; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Hydroxybenzoates; Lung; Monensin; Organelles; Protein Folding; Protein Synthesis Inhibitors

Human fetal skin fibroblasts (TIG-3S) were found to migrate into a denuded area in a cell monolayer when cultured in both serum-depleted and serum-supplemented media, unlike adult-donor skin fibroblasts which migrated well only when cultured in serum-supplemented medium. Therefore, a series of experiments was carried out to determine whether autocrine factors are involved in their migration. The migration of TIG-3S cells in serum-depleted medium was suppressed by the addition of suramin, a factor with growth factor antagonist properties, which suggests that growth factors are important for cell migration. The suramin-induced inhibition was reversed completely by adding excess basic fibroblast growth factor (bFGF) to the culture medium and partially by platelet-derived growth factor (PDGF). Treatment with neutralizing anti-PDGF antibody did not suppress TIG-3S cell migration, whereas neutralizing anti-bFGF antibody did, which indicates that bFGF is an autocrine and PDGF a paracrine factor involved in cell migration. Next, an experiment was performed to ascertain whether the extracellular matrix is involved in TIG-3S cell migration. Monensin, an inhibitor of extracellular matrix secretion, inhibited cell migration, which was reversed by adding excess type I collagen, but not excess plasma fibronectin. In addition, further evidence for the involvement of collagen was provided by the observation that ethyl-3,4-dihydroxybenzoate, a specific inhibitor of collagen synthesis, suppressed cell migration. These results suggest that the autonomous migration of TIG-3S human fetal skin fibroblasts is mediated by bFGF and type I collagen, which they produce and secrete.

Topics: Antioxidants; Cell Line; Cell Movement; Collagen; Culture Media; Extracellular Matrix; Fetus; Fibroblast Growth Factor 2; Fibroblasts; Fibronectins; Growth Substances; Humans; Hydroxybenzoates; Male; Monensin; Skin; Suramin