Lipoprotein lipase-mediated uptake of lipoprotein in human fibroblasts: evidence for an LDL receptor-independent internalization pathway.
Lipoprotein lipase (LPL), a key enzyme in lipoprotein triglyceride metabolism, produces a marked increase in the retention and uptake of all classes of lipoproteins by cultured cells. It was previously shown that two different receptors are involved in mediating the LPL effects: heparan sulfate proteoglycans (HSPG) and the low density lipoprotein (LDL) receptor-related protein/alpha 2 macroglobulin receptor (LRP). By immunofluorescence we show here that cell surface-bound LPL displays a pattern that corresponds to the previously described distribution of cell surface HSPG. No evident relation to the distribution of bound activated alpha 2-macroglobulin (alpha 2M*) or to LRP was observed. By immunoelectron microscopy we found that after 30 min at 37 degrees C most of the detected alpha 2M* (70% of the total gold particles) was inside the cells and associated with endosomal vesicles. However, at the same time, 76% of the LPL remained at the cell surface, suggesting that, LPL is internalized by a slow endocytic process. Binding of triglyceride-rich lipoproteins (TRL) or LDL together with LPL led to a spectacular increase in bound lipoproteins, which completely colocalized with LPL. After incubation at 37 degrees C, LPL and 1,1'-dioctadecyl-3,3,3,'3'-tetramethylindocarbocyanine (DiI)-TRL formed large clusters on the cell surface. Immunofluorescene and quantitative immunoelectron microscopy provided evidence of co-internalization of LPL and apoE-containing TRL by a slow endocytic process. In the absence of LPL, the fibroblasts rapidly internalized DiI-LDL and showed fluorescence in central, lysosome-like vesicles. In contrast, when LPL was present, internalization of DiI-LDL involved small, widely distributed vesicles. This pattern slowly changed to one consisting of large perinuclear vesicles. LDL receptor-deficient fibroblasts internalized DiI-LDL, either with or without LPL, into small widely distributed vesicles and no central vesicles were seen. Chloroquine-treated normal fibroblasts internalized DiI-LDL in a pattern similar to that of receptor-deficient fibroblasts. Taken together our results suggest an alternative receptor-independent endocytosis pathway for LDL. This pathway is potentiated by LPL and is characterized by a slow uptake involving small vesicles that gradually reach lysosomes. We suggest that, through its interaction with HSPG, LPL provides high capacity binding sites for lipoproteins and a independent internalization pathway.
Fernández-Borja
M
M
Department of Biochemistry and Physiology, University of Barcelona, Spain.
Bellido
D
D
Vilella
E
E
Olivecrona
G
G
Vilaró
S
S
D001057:Apolipoproteins E / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D004705:Endocytosis / Q000502:physiology; D005347:Fibroblasts; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008071:Lipoprotein Lipase / Q000378:metabolism*; D008074:Lipoproteins / Q000378:metabolism*; D008077:Lipoproteins, LDL / Q000378:metabolism; D011485:Protein Binding; D011509:Proteoglycans / Q000378:metabolism; D011973:Receptors, LDL / Q000172:deficiency / Q000378:metabolism*; D014280:Triglycerides / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000378:metabolism
D001057:Apolipoproteins E; D002738:Chloroquine; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D008071:Lipoprotein Lipase; D008074:Lipoproteins; D008077:Lipoproteins, LDL; D011509:Proteoglycans; D011973:Receptors, LDL; D014280:Triglycerides; D000511:alpha-Macroglobulins