Cotranslational inhibition of apoB-100 synthesis by cyclosporin A in the human hepatoma cell line HepG2.
Treatment of patients with cyclosporin A (CsA) increases low-density lipoprotein (LDL) cholesterol levels. We investigated whether an elevated hepatic secretion of apolipoprotein (apo) B-100-containing lipoproteins is responsible for the increase of LDL by using the human hepatoma cell line HepG2. Addition of CsA to the culture medium of HepG2 cells resulted in a dose- and time-dependent decrease in the secretion of apoB-100. Maximal inhibition (-50%), which was obtained at 5 mumol/L CsA, was achieved within 8 hours. The secretion of apoA-I, albumin, and [35S]methionine-labeled proteins was not affected by CsA. The reduced accumulation of apoB-100 in the culture medium could not be explained by changes in the uptake and degradation of LDL by HepG2 cells treated with CsA. In addition, [35S]methionine incorporation studies indicated that synthesis and/or secretion of newly synthesized apoB-100 decreased in the presence of CsA. CsA did not affect the apoB-100 mRNA level, indicating that CsA regulates the secretion of apoB-100 at the cotranslational or posttranslational level. The decreased secretion of apoB-100 was accompanied by a diminished secretion of triglycerides (-47%), cholesterol (-18%), and cholesteryl esters (-27%) in the presence of CsA. In contrast, the intracellular concentrations and the total amount of these lipids present in the culture medium and cells were not changed. This indicates that a possible limited availability of one of these lipids was not responsible for the decreased secretion of apoB-100 by CsA. Pulse-chase experiments showed that the amount of intracellular apoB-100 was already decreased by 50% after the 10-minute pulse period and that CsA did not affect the intracellular processing of apoB-100 once it was fully synthesized. Short pulse incubations in the presence of [35S]methionine showed a decrease in the intracellular amount of labeled apoB-100 after an incubation of only 2 through 4 minutes, indicating that the translation was not affected but that inhibition of the apoB-100 secretion by CsA occurred at the cotranslational level. Our results suggest that the elevated plasma LDL levels observed in patients treated with CsA are not caused by hepatic overproduction of apoB-100-containing lipoproteins.
Kaptein
A
A
Gaubius Laboratory IVVO-TNO, Institute of Ageing and Vascular Research, Leiden, the Netherlands.
de Wit
E C
EC
Princen
H M
HM
D016632:Apolipoprotein A-I / Q000096:biosynthesis / Q000235:genetics; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000096:biosynthesis* / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D016572:Cyclosporine / Q000494:pharmacology*; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000187:drug effects*; D012333:RNA, Messenger / Q000032:analysis; D012709:Serum Albumin / Q000235:genetics; D014407:Tumor Cells, Cultured
D016632:Apolipoprotein A-I; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D012333:RNA, Messenger; D012709:Serum Albumin; D016572:Cyclosporine