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

doi:10.1074/jbc.M003221200

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

From the 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 and the University Health Network Research Institute, Toronto M5G 2C4, Ontario, Canada

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 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.

Footnotes

↵* This work was supported by the Canadian Institutes of Health Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ To whom correspondence should be addressed: Rm. 244, Fitzgerald Bldg., University of Toronto, 150 College St., Toronto, Ontario M5S 3E8, Canada. Tel.: 416-978-6684; Fax: 416-978-5956; E-mail: christopher.mcculloch@utoronto.ca.
Published, JBC Papers in Press, August 16, 2000, DOI 10.1074/jbc.M003221200
Abbreviations:

FITC

fluorescein isothiocyanate

HRP

horseradish peroxidase

LDL

low density lipoprotein

DiI

1,1′-dioctadocyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate

BSA

bovine serum albumin

MEM

minimal essential medium

MES

4-morpholineethanesulfonic acid

Pipes

1,4-piperazinediethanesulfonic acid

BAPTA/AM

1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxymethyl ester
- Received April 14, 2000.
- Revision received July 12, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.