title: A novel model system for characterization of phagosomal maturation, acidification, and intracellular collagen degradation in fibroblasts.
abstract: 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 alpha(2)beta(1) 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 alpha(2)beta(1) integrin after initial bead binding. beta-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.
authors.affiliation: Medical Research Council Group in Periodontal Physiology, the Faculty of Dentistry, and the Faculty of Medicine, Division of Respirology, University of Toronto, Toronto M5S 3E8, Ontario, Canada.
authors.forename: P D
authors.initials: PD
authors.lastname: Arora
authors.forename: M F
authors.initials: MF
authors.lastname: Manolson
authors.forename: G P
authors.initials: GP
authors.lastname: Downey
authors.forename: J
authors.initials: J
authors.lastname: Sodek
authors.forename: C A
authors.initials: CA
authors.lastname: McCulloch
mesh_terms: D000199:Actins / Q000378:metabolism; D000293:Adolescent; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015703:Antigens, CD / Q000378:metabolism; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002401:Cathepsin B / Q000378:metabolism; D002648:Child; D003094:Collagen / Q000378:metabolism*; D015638:Cytochalasin D / Q000494:pharmacology; D011992:Endosomes / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology; D018260:Gelsolin / Q000378:metabolism; D005881:Gingiva / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016023:Integrins / Q000378:metabolism; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism; D018942:Macrolides; D008264:Macrophages / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010588:Phagosomes / Q000502:physiology*; D038981:Receptors, Collagen; D013552:Swine; D013997:Time Factors
chemical_list: D000199:Actins; D000900:Anti-Bacterial Agents; D015703:Antigens, CD; D004791:Enzyme Inhibitors; D018260:Gelsolin; D016023:Integrins; D051907:Lysosomal Membrane Proteins; D018942:Macrolides; D008562:Membrane Glycoproteins; D019384:Nucleic Acid Synthesis Inhibitors; D038981:Receptors, Collagen; D015638:Cytochalasin D; C036978:concanamycin A; D003094:Collagen; D002401:Cathepsin B; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium