When human HepG2 hepatoma cells were pulsed wtth 12S Nabeled high density lipoproteins (HDL) and chased In fresh medium, up to 65% of the radioactivity released was preclpltable with trlchloroacetlc acid. Cell-Internalized 12S I-HDL contributed to the release of acld-preclpitable material; when cells were treated with trypsln before the chase to remove 12S I-HDL bound to the outer cell membrane, 50% of the released material was still acld-preclpitable. Characterization of the radioactive material resecreted by trypsinlzed cells revealed the presence of particles that were similar In size and density to mature HDL and contained intact apollpoprotelns (apo) A-l and A-ll. The release of Internalized label occurred at 37°C but not at 4°C. Monensln, which Inhibits endosomal recycling of receptors, decreased the binding of 12S I-HDL to cells by 75%, Inhibited the release of Internalized radioactivity as acld-preclpitable material by 80%, and increased the release of acid-soluble material by 90%. In contrast, the lysosomal Inhibitor chloroqulne increased the association of 12 *I-HDL to cells by 25%, Inhibited the release of preclpltable material by 10%, and inhibited the release of acid-soluble radioactivity by 80%. Pre-lncubatlon with cholesterol caused a 50% Increase In the specific binding, Internallzatlon, and resecretion of HDL label. Cholesterol affected the release of acld-preclpitable label much more (+90%) than that of acid-soluble material (+20%). Taken together, these findings suggest that HepG2 cells can bind, Internalize, and resecrete HDL by a retroendocytotic process. Furthermore, the results wtth cholesterol and monensin Indicate that a regulated, recycling, receptor-like molecule Is Involved In the binding and Intracellular routing of HDL.
Arteriosclerosis – Wolters Kluwer Health
Published: Jul 1, 1990