A Novel Model System for Characterization of Phagosomal Maturation, Acidification, and Intracellular Collagen Degradation in Fibroblasts

• Published in: The Journal of biological chemistry Vol. 275; no. 45; pp. 35432 - 35441
• Main Authors: Arora, Pamela D., Manolson, Morris F., Downey, Gregory P., Sodek, Jaro, McCulloch, Christopher A.G.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10-11-2000; American Society for Biochemistry and Molecular Biology
• Subjects: Actins - metabolism; Adolescent; Animals; Anti-Bacterial Agents - pharmacology; Antigens, CD - metabolism; Calcium - metabolism; Calcium - pharmacology; Cathepsin B - metabolism; Child; Collagen - metabolism; Cytochalasin D - pharmacology; Endosomes - metabolism; Enzyme Inhibitors - pharmacology; Fibroblasts - metabolism; Flow Cytometry; Fluorescein-5-isothiocyanate - pharmacology; Gelsolin - metabolism; Gingiva - metabolism; Humans; Hydrogen-Ion Concentration; Integrins - metabolism; Lysosomal Membrane Proteins; Lysosomes - metabolism; Macrolides; Macrophages - metabolism; Membrane Glycoproteins - metabolism; Microscopy, Electron; Microscopy, Fluorescence; Nucleic Acid Synthesis Inhibitors - pharmacology; Phagosomes - physiology; Receptors, Collagen; Swine; Time Factors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M003221200

Intracellular collagen degradation by fibroblasts is an important but poorly understood pathway for the physiological remodeling of mature connective tissues. The objective of this study was to determine whether gingival fibroblasts that express endogenous α2β1 integrin, the collagen receptor, would exhibit the cellular machinery required for phagosomal maturation and collagen degradation. There was a time-dependent increase of collagen bead internalization and a time-dependent decrease of bead-associated α2β1 integrin after initial bead binding. β-Actin and gelsolin associated transiently with beads (0–30 min) followed by LAMP-2 (60–240 min) and cathepsin B (30–240 min). Cytochalasin D prevented phagosome formation and also prevented the sequential fusion of early endosomes with lysosomes. Collagen bead-associated pH was progressively reduced from 7.25 to 5.4, which was contemporaneous with progressive increases in degradation of bead-associated collagen (30–120 min). Concanamycin blocked acidification of phagolysosomes and collagen degradation but not phagosome maturation. Phagosomal acidification was partly dependent on elevated intracellular calcium. These studies demonstrate that the cellular machinery required for intracellular collagen degradation in fibroblasts closely resembles the vacuolar system in macrophages.