COLLAGEN PHAGOCYTOSIS AND APOPTOSIS ARE INDUCED BY HIGH-LEVEL ALKALINE-PHOSPHATASE EXPRESSION IN RAT FIBROBLASTS

Study of fibroblast origins and lineages is complicated by the lack of unambiguous markers that could be used to identify discrete subpopula tions 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 T N-AP collagen-binding domain on cell function. Various levels of expre ssion were driven by early (strong) or late (weak) SV40 promoters in t he 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 o f membrane-anchored enzyme released by phosphatidyl inositol phospholi pase C. Low levels of TN-AP expression increased cell spreading slight ly, nearly doubled the percentage of collagen phagocytic cells (up to 80%), and increased the number of internalized collagen-coated fluores cence beads per cell. In cells transfected with PL-AP (i.e., no collag en-binding domain), collagen phagocytosis was not affected. internaliz ation of BSA beads was also not affected by either AP isozyme, indicat ing that AP was selective for integrin-mediated phagocytosis. In singl e cells, histochemically demonstrable TN-AP activity on cell membranes was colocalized with the binding of collagen beads, but this colocali zation 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-lev el TN-AP expression caused a fivefold reduction of cell proliferation and was associated with the development of cells with sub-G(1) DNA con tent, nuclear condensation, and nuclear budding. In AP-positive cultur es, there was a greatly increased number of floating cells; nick-label ing of DNA by terminal transferase and biotinylated dUTP showed a 15-f old increase of stained cells. These data indicate that low-level TN-A P expression enhances collagen phagocytosis, presumably through the TN -AP collagen-binding domain. High-level AP expression promotes cell de letion by apoptosis. We suggest that the expression of AP by fibroblas ts indicates a novel role for this enzyme in collagen degradation by p hagocytosis. (C) 1997 Wiley-Liss, Inc.