Determinants of human apolipoprotein [a] secretion from mouse hepatocyte cultures

Abstract

Efforts to develop an in vitro model system to analyze apolipoprotein [a] (apo[a]) gene transcription, mRNA translation, and protein secretion have been complicated by the limited tissue and species distribution of apo[a] and the presence of regulatory DNA sequences remote from the apo[a] transcription start site. In the current study we examined primary hepatocytes cultured from apo[a] transgenic mice as a model system for analyzing apo[a] biogenesis. Hepatocytes from mice transgenic for a yeast artificial chromosome (YAC) encoding the entire apo[a] gene in its own genomic context (YAC-apo[a] hepatocytes) were unable to maintain apo[a] expression beyond 48 h of culture. This suggests that the apo[a] promoter was not active in cultured YAC-apo[a] hepatocytes. In contrast, apo[a] expression was maintained for at least 7 days in hepatocytes cultured from mice transgenic for an apo[a] cDNA under control of the mouse transferrin promoter (transferrin-apo[a] hepatocytes). Pulse-chase experiments established that more than 80% of apo[a] synthesized by both transferrin-apo[a] and YAC-apo[a] hepatocytes was degraded prior to secretion, independently of the coexpression of human apoB. Thus, low secretion efficiency appears to be a general characteristic of human apo[a] proteins in mouse liver. Apo[a] secretion was increased somewhat (from 18% to 32%) in the presence of lipoprotein-containing serum. Transformed cell lines derived from transferrin apo[a] hepatocytes retained characteristics of apo[a] secretion similar to those observed in primary cells. Primary and transformed apo[a] transgenic hepatocytes may provide valuable additional models with which to study posttranslational mechanisms regulating apo[a] secretion. -Wang, J., J. Boedeker, H. H. Hobbs, and A. L. White. Determinants of human apolipoprotein [a] secretion from mouse hepatocyte cultures.

Differential effect of combined lipase deficiency (cld/cld) on human hepatic lipase and lipoprotein lipase secretion

“…Immunoblotting of culture medium demonstrated the expression of both apoB-100 and apoB-48 for at least 4 days in culture (the longest time point analyzed; Fig. 1A ) (25). Immunoprecipitation of medium from cultures labeled for 16 h with [ 35 S]methionine confirmed the expression of apoB-100 and apoB-48, and also demonstrated secretion of the liver-specific proteins albumin and transferrin (Fig.…”

“…Hepatocytes from cld and het fetal mice were isolated and cultured as described in Materials and Methods. A: Aliquots of medium collected every 24 h between 1 and 4 days of culture were analyzed for the presence of mouse apoB by immunoblotting, as described (25). The positions of apoB-100, apoB-48, the 200-kDa marker, and a nonspecifically recognized protein (X) are indicated.…”