Retroendocytosis of high density lipoproteins by the human hepatoma cell line, HepG2.
When human HepG2 hepatoma cells were pulsed with 125I-labeled high density lipoproteins (HDL) and chased in fresh medium, up to 65% of the radioactivity released was precipitable with trichloroacetic acid. Cell-internalized 125I-HDL contributed to the release of acid-precipitable material; when cells were treated with trypsin before the chase to remove 125I-HDL bound to the outer cell membrane, 50% of the released material was still acid-precipitable. Characterization of the radioactive material resecreted by trypsinized cells revealed the presence of particles that were similar in size and density to mature HDL and contained intact apolipoproteins (apo) A-I and A-II. The release of internalized label occurred at 37 degrees C but not at 4 degrees C. Monensin, which inhibits endosomal recycling of receptors, decreased the binding of 125I-HDL to cells by 75%, inhibited the release of internalized radioactivity as acid-precipitable material by 80%, and increased the release of acid-soluble material by 90%. In contrast, the lysosomal inhibitor chloroquine increased the association of 125I-HDL to cells by 25%, inhibited the release of precipitable material by 10%, and inhibited the release of acid-soluble radioactivity by 80%. Pre-incubation with cholesterol caused a 50% increase in the specific binding, internalization, and resecretion of HDL label. Cholesterol affected the release of acid-precipitable 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 with cholesterol and monensin indicate that a regulated, recycling, receptor-like molecule is involved in the binding and intracellular routing of HDL.
Kambouris
A M
AM
CSIRO, Division of Human Nutrition, Adelaide, SA, Australia.
Roach
P D
PD
Calvert
G D
GD
Nestel
P J
PJ
D000818:Animals; D002460:Cell Line; D011232:Chemical Precipitation; D006801:Humans; D007457:Iodine Radioisotopes; D008075:Lipoproteins, HDL / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D008985:Monensin / Q000494:pharmacology; D014238:Trichloroacetic Acid / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism*
D007457:Iodine Radioisotopes; D008075:Lipoproteins, HDL; D014238:Trichloroacetic Acid; D008985:Monensin; D014357:Trypsin