A novel model system for characterization of phagosomal maturation, acidification, and intracellular collagen degradation in fibroblasts

Intracellular collagen degradation by fibroblasts is an important but poorl y understood pathway for the physiological remodeling of mature connective tissues. The objective of this study was to determine whether gingival-fibr oblasts that express endogenous alpha (2)beta (1) integrin, the collagen:re ceptor, would exhibit the cellular machinery required for phagosomal matura tion and collagen degradation. There was a time-dependent increase of colla gen bead internalization and a time-dependent decrease of bead-associated a lpha (2)beta (1) integrin after initial bead; binding. beta -Actin and gels olin associated transiently with beads (0-30 min) followed by LAMP-1 (60-24 0 min) and cathepsin B (30-240 min). Cytochalasin D prevented phagosome for mation and also prevented the sequential fusion of early endosomes with lys osomes, Collagen bead-associated pH was progressively reduced from: 7.25 to 5.4, which was contemporaneous with progressive increases in degradation o f bead-associated collagen (30-120 min). Concanamycin blocked acidification of phagolysosomes and collagen degradation but not phagosome maturation, P hagosomal acidification was partly dependent on elevated intracellular calc ium. These studies demonstrate that the cellular machinery required for int racellular collagen degradation in fibroblasts closely resembles the vacuol ar system in macrophages.