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  • Title: Collagen phagocytosis and apoptosis are induced by high level alkaline phosphatase expression in rat fibroblasts.
    Author: Hui MZ, Tenenbaum HC, McCulloch CA.
    Journal: J Cell Physiol; 1997 Sep; 172(3):323-33. PubMed ID: 9284952.
    Abstract:
    Study of fibroblast origins and lineages is complicated by the lack of unambiguous markers that could be used to identify discrete subpopulations on the basis of functional attributes. We have studied the role of the membrane-anchored hydrolytic enzyme tissue-nonspecific alkaline phosphatase (TN-AP) and the placental alkaline phosphatase (PL-AP) in collagen phagocytosis and in the deletion of cells by apoptosis. Rat-2 cells, which do not constitutively express AP, were transfected with full-length rat TN-AP or PL-AP cDNAs to determine the impact of the TN-AP collagen-binding domain on cell function. Various levels of expression were driven by early (strong) or late (weak) SV40 promoters in the plasmid construct. Controls were transfected with plasmids that did not contain AP cDNA. AP expression in transfected cells was confirmed by Northern blotting, histochemical analysis, and SDS-PAGE analysis of membrane-anchored enzyme released by phosphatidyl inositol phospholipase C. Low levels of TN-AP expression increased cell spreading slightly, nearly doubled the percentage of collagen phagocytic cells (up to 80%), and increased the number of internalized collagen-coated fluorescence beads per cell. In cells transfected with PL-AP (i.e., no collagen-binding domain), collagen phagocytosis was not affected. Internalization of BSA beads was also not affected by either AP isozyme, indicating that AP was selective for integrin-mediated phagocytosis. In single cells, histochemically demonstrable TN-AP activity on cell membranes was colocalized with the binding of collagen beads, but this colocalization was not detected in cells transfected with PL-AP. Phagocytosis was inhibited by antibodies to the alpha 2 integrin and to AP but not by levamisole, an inhibitor of AP phosphohydrolytic activity. High-level TN-AP expression caused a fivefold reduction of cell proliferation and was associated with the development of cells with sub-G1 DNA content, nuclear condensation, and nuclear budding. In AP-positive cultures, there was a greatly increased number of floating cells; nick-labeling of DNA by terminal transferase and biotinylated dUTP showed a 15-fold increase of stained cells. These data indicate that low-level TN-AP expression enhances collagen phagocytosis, presumably through the TN-AP collagen-binding domain. High-level AP expression promotes cell deletion by apoptosis. We suggest that the expression of AP by fibroblasts indicates a novel role for this enzyme in collagen degradation by phagocytosis.
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