title: Mechanism of collagen phagocytosis by human gingival fibroblasts: importance of collagen structure in cell recognition and internalization.
abstract: Phagocytosis of extracellular collagen by fibroblasts appears to be the principal pathway of collagen degradation in the physiological turnover of connective tissues. To study the mechanism of collagen phagocytosis, subconfluent gingival fibroblasts were serum-starved and incubated for up to 16 h with collagen-coated fluorescent latex beads. Internalization of beads was measured either by flow cytometry or by image analysis. Phagocytosis was blocked by inactivation of protein kinase C with staurosporin, and was also decreased significantly (32%) when cells were pre-incubated for 6h with cycloheximide. Phagocytosis of collagen-coated beads appeared to be receptor-mediated, since internalization was inhibited threefold by the cell-attachment blocking peptide (GRGDSP). The process of internalization was influenced by the type of collagen and its molecular structure. Thus, internalization was decreased in the order: type I greater than V greater than III collagen, and internalization of type I collagen was reduced significantly by digestion with either bacterial (45%) or vertebrate (38%) collagenase. However, collagen denaturation, which facilitates binding to fibronectin, did not effect internalization. Although concanavalin A stimulated both phagocytosis (71%) and collagenase synthesis, PMA and IL-1, which also increase collagenase expression, did not affect phagocytosis, indicating that phagocytosis of collagen-coated beads does not require collagenase. Moreover, analysis of tissue inhibitor of metalloproteinase expression revealed no difference between phagocytic and non-phagocytic cells. Collectively, these results demonstrate that collagen phagocytosis is regulated through protein kinase C and is also dependent upon cellular recognition and collagen structure, but not on the expression of collagenase.
authors.affiliation: Faculty of Dentistry, University of Toronto, Ontario, Canada.
authors.forename: G C
authors.initials: GC
authors.lastname: Knowles
authors.forename: M
authors.initials: M
authors.lastname: McKeown
authors.forename: J
authors.initials: J
authors.lastname: Sodek
authors.forename: C A
authors.initials: CA
authors.lastname: McCulloch
mesh_terms: D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000502:physiology; D002448:Cell Adhesion / Q000502:physiology; D003094:Collagen / Q000737:chemistry / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D004790:Enzyme Induction; D005347:Fibroblasts / Q000187:drug effects* / Q000502:physiology; D005881:Gingiva / Q000187:drug effects / Q000502:physiology; D006023:Glycoproteins / Q000235:genetics*; D006801:Humans; D008666:Metalloendopeptidases; D003012:Microbial Collagenase / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D010587:Phagocytosis / Q000187:drug effects* / Q000502:physiology; D011487:Protein Conformation; D019311:Staurosporine; D019714:Tissue Inhibitor of Metalloproteinases
chemical_list: D000470:Alkaloids; D006023:Glycoproteins; D009842:Oligopeptides; D019714:Tissue Inhibitor of Metalloproteinases; C047981:arginyl-glycyl-aspartic acid; D003094:Collagen; D003513:Cycloheximide; D008666:Metalloendopeptidases; D003012:Microbial Collagenase; D019311:Staurosporine