Similarities and differences in the function of regulatory elements at the 5' end of the human apolipoprotein B gene in cultured hepatoma (HepG2) and colon carcinoma (CaCo-2) cells.

The arrangement of regulatory elements along the apolipoprotein B promoter region (positions -898 to +1) has been examined in transient transfection experiments performed in HepG2 and Hep3B (hepatic) and CaCo-2 (intestinal) cell lines, all of which express the apoB gene, and also in Chinese hamster ovary cells, which do not express the gene. The overall distribution of positive and negative regulatory segments was very similar in the two hepatoma cell lines (HepG2 and Hep3B) but different from that observed in the colon carcinoma cells (CaCo-2). Thus, whereas 260 base pairs of 5'-flanking sequence were sufficient for maximal expression of the promoter in HepG2 cells, only 139 nucleotides were required for maximal expression in CaCo-2 cells. Promoter activity in Chinese hamster ovary cells was exhibited by short constructs, with maximal activity for the -85 construct. DNase I footprinting of the apolipoprotein B promoter region using hepatic and intestinal extracts revealed multiple sites of interaction between the DNA and nuclear proteins. Gel retention experiments using the region from -262 to -88 (the region of greatest contrast between HepG2 and CaCo-2 cells) revealed interesting variations in the relative abundance of various nuclear proteins between the two cell types. A major functional difference between HepG2 and CaCo-2 cells was localized to the region between -111 and -88, which harbors the sequence TGTTTGCT, a motif present in the promoter region of several liver-specific genes. The molecular basis for the functional differences between these two cell types may be attributable to a difference in the relative abundance of three proteins that bind to sequences between -111 and -88.     

The limits of the DNase I-sensitive domain of the human apolipoprotein B gene coincide with the locations of chromosomal anchorage loops and define the 5' and 3' boundaries of the gene

In eukaryotic cells, chromatin is organized as domains or loops that are generated by periodic attachment of the chromatin fiber to protein components of a nuclear matrix, or scaffold. These chromosomal loops may have a function in gene regulation. The length of the chromatin domain encompassing the human apolipoprotein B gene was studied by determining the locations of nuclear matrix attachment sites as well as the boundaries of the DNase I-sensitive domain in cells that express the gene (such as HepG2 and CaCo-2 cells) and in those that do not (HeLa cells). Three nuclear matrix attachment regions (MARs) of the human apolipoprotein B gene have been localized: a 3' -proximal MAR, between nucleotides +43,186 and +43,850; a 5' -proximal MAR, between nucleotides -2,765 and -1,801; and a 5' -distal MAR, between nucleotides -5,262 and -4,048. Both the 3' -proximal and the 5' -distal MARS were present in cells that express the gene (HepG2 and CaCo-2 cells) as well as in cells that do not (HeLa cells), whereas the 5' -proximal MAR was detected only in HepG2 cells. These MARs were located at the bases of chromosomal loops in histone-extracted nuclei in all three cell lines. Various classes of A/T-rich sequences resembling the recognition site for topoisomerase II were present within the MAR-containing fragments. The boundaries of the DNase I-sensitive domain coincide with the positions of the 3' -proximal and 5' -distal matrix attachment sites. These results suggest the existence of a 47.5-kilobase domain that represents a topologically sequestered functional unit containing the coding region and all known cis-acting regulatory elements of the human apolipoprotein B gene.     

Insulin decreases the secretion of apoB-100 from hepatic HepG2 cells but does not decrease the secretion of apoB-48 from intestinal CaCo-2 cells

We compared the acute effect of insulin on the human colonic intestinal epithelial cell line CaCo-2 and the transformed human hepatic cell line HepG2. Over 24 h, 100 nM and 10 µM insulin significantly inhibited the secretion of apolipoprotein (apo) B-100 from HepG2 cells to 63 and 49% of control, respectively. Insulin had no effect on the secretion of apoB-48 from CaCo-2 cells. There was no effect of insulin on the cholesterol ester or free cholesterol concentrations in HepG2 or CaCo-2 cells. HepG2 and CaCo-2 cells bound insulin with high affinity, leading to similar stimulation of insulin receptor protein tyrosine kinase activation. Protein kinase C or mitogen-activated protein kinase activity in the presence or absence of insulin was not correlated with apoB-48 production in CaCo-2 cells. Therefore, insulin acutely decreases the secretion of apoB-100 in hepatic HepG2 cells, but does not acutely modulate the production or secretion of apoB-48 from CaCo-2 intestinal cells.