Control Of Apolipoprotein Research Articles

Control of apolipoprotein E secretion in the human hepatoma cell line KYN-2.

Even though it is known that apolipoprotein E (apoE) is deeply involved in major age-related disorders such as atherosclerosis or Alzheimer's disease (AD), the control of cell-specific apoE expression is still poorly understood. We compared the apoE secretion as previously described in astrocytic cell17 to hepatic cell apoE secretion. We used the human hepatoma cell line KYN-2 to better delineate the characteristics of apoE secretion and to validate it with respect to the classical human hepatoma cell line HepG2. Interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) significantly inhibited, while IL-2, IL-6 and tumour necrosis factor-alpha (TNF-alpha) were inactive on apoE secretion by KYN-2 as well as HepG2 cells. Cholesterol and 25-OH cholesterol had no effect, while forskolin exerted a significant inhibitory effect, on apoE secretion in KYN-2 cells. Our results suggest that the KYN-2 cell line represents an appropriate cell model, and in any case an alternative model to the HepG2 cell line, to study the control of apoE secretion. The response of KYN-2 cells to both cytokines and cholesterol differs from that found in astrocytoma cells, suggesting that blood variations of apoE concentrations in AD may not reflect the dysregulations taking place in the brain.

Control of apolipoprotein E secretion by 25-hydroxycholesterol and proinflammatory cytokines in the human astrocytoma cell line CCF-STTG1.

During the last few years the increased presence of proinflammatory cytokines and oxidation products in Alzheimer's disease (AD) has been largely described. Oxysterols, formed by hydroxylation of cholesterol, occur naturally in the brain and are increased in the serum of AD patients. Of these oxysterols, 25-hydroxycholesterol is the most potent regulator of gene transcription. It stimulates the synthesis of apolipoprotein E (apo E) in macrophages. Apo E plays a major role in the brain as a phospholipid and cholesterol carrier molecule in compensatory synaptogenesis. Cytokines might also be able to modulate apo E expression. Accordingly, this study examined the control of apo E secretion by several proinflammatory cytokines and oxysterols in the human astrocytoma cell line CCF-STTG1. A time-dependent stimulation of apo E secretion by 25-hydroxycholesterol was observed. Among several cytokines tested, only tumor necrosis factor (TNF)-alpha inhibited apo E secretion in basal conditions in CCF-STTG1 human astrocytoma cells. In the presence of 25-hydroxycholesterol, TNF-alpha reduced apo E secretion by 80%, while interleukins (IL) IL-1beta, IL-6, and IL-2 had no significant effects. In Alzheimer's disease, the increase in the concentrations of cytokines and the concomitant decrease of cholesterol concentration in the brain could contribute jointly to reduce apo E concentration, and in doing so accelerate neurodegeneration. Surprisingly, oxysterols would be able to limit this phenomenon.

Decreased intracellular degradation and increased secretion of apo B-100 in Hep G2 cells after inhibition of cholesteryl ester synthesis

Control of apolipoprotein B (apo B) secretion in hepatocytes occurs partly at the post-translational level. The key step in this process appears to be intracellular degradation of newly synthesized apo B. The aim of this paper was to investigate the mechanisms that regulate apo B secretion by Hep G2 cells, in response to the inhibition of Acyl-CoA Acyltransferase (ACAT) by the compound Sandoz 58035 (S-58035). S-58035 (20 μM) reduced cholesteryl ester synthesis from [ 14C]oleate by 95%, and increased significantly, in a dose-dependent manner, (2–100 μM) apo B secretion, either in control conditions (from 78±4.3 to 126±6.1 ng apo B-100/mg cell protein/4 h) or upon stimulation of apo B secretion by oleate (from 134±4.23 to 177±4.3 ng apo B/mg cell protein/4 h). This increased secretion of newly synthesized apo B-100 was confirmed by pulse experiments and by gradient ultracentrifugation of the media. Moreover pulse-chase experiments showed that the addition of S-58035 reduced intracellular degradation of apo B-100, both in control conditions and in the presence of oleate. S-58035 (20 μM) did not affect total cellular cholesterol content, but free cholesterol increased with a concomitant decrease of cholesteryl ester (−20%). S-58035 increased cellular triglyceride mass, which was observed in basal conditions (from 12.8±1.09 to 22.7±2.7 μg TG/mg cellular protein) and also in presence of oleate (from 48±0.53 to 59±6.3 μg TG/mg cellular protein). This effect is due to a stimulation of triglyceride synthesis, as determined by incorporation of [ 3H]glycerol into cellular triglycerides. From these data we conclude that, under our experimental conditions, triglyceride synthesis and/or availability is likely to control intracellular degradation of apo B.

Control of Apolipoprotein B mRNA Editing: Implications of mRNA Dynamics at Various Maturation Stages

Apolipoprotein (apo) B mRNA editing is a genetic regulatory mechanism whereby nucleotide 6666 in apoB-100 mRNA is converted from a C to a U. The end result of this change is the creation of a premature stop codon so that the translation product of the edited (apoB-48) mRNA contains 2152 amino acid residues instead of 4536 residues in the product (apoB-100) encoded by the unedited mRNA. ApoB mRNA editing is a post-transcriptional process that is expressed in a tissue-specific manner. Here we present a model for the control of apoB mRNA editing. The three variables in the model include editing activity, apoB mRNA transcription rate and mRNA degradation rate at various maturation stages for the mRNA, and a simple formula can be used to quantify the degree of apoB mRNA editing with respect to these variables at different apoB mRNA maturation stages. Time-dependent equations were solved numerically. Using this model it can be shown that, in addition to editing activity, the degradation kinetics of apoB mRNA can also serve as an efficient modulator of the degree of editing. The rate of apoB mRNA transcription has a transient effect on the degree of editing; varying the apoB mRNA degradation constant tends to change the degree of editing only at specific maturation stage. This model can render some previously proposed hypothesis (e.g. coupling of mRNA editing to splicing/polyadenylation) unnecessary and provides a rational basis for the design of experiments on specific aspects of apoB mRNA editing in the future; it may also be applicable to other types of RNA editing.

Identification and characterization of transcriptional regulatory regions associated with expression of the human apolipoprotein E gene.

Multiple cis-acting regulatory elements have been mapped within a 1-kilobase fragment spanning nucleotides -651 through +356 of the human apolipoprotein E gene using a transient expression system based on the chloramphenicol acetyltransferase gene as well as DNase I footprinting techniques. A 651-base pair 5'-flanking region of the human apolipoprotein E gene was capable of directing chloramphenicol acetyltransferase gene expression over a 48-fold range among the various cultured cell lines tested. Deletion analysis of this 651-base pair upstream region linked to either the chloramphenicol acetyltransferase gene or the intact apolipoprotein E structural sequences revealed at least three regulatory domains within the proximal 383 nucleotides. One of these domains contained a GC box transcriptional control element. Further analysis demonstrated that the other two domains contained enhancer-like activity. These enhancer-like elements were located within the nucleotides spanning -366 to -246 and -193 to -124. A third enhancer element was identified in the first intron, within nucleotides +44 to +262. Changing the distance of the three enhancer elements from the transcription start site and reversing their orientation did not significantly alter their effects on transcription rates. However, enhancer activity was influenced by the promoter and cell line that were used. DNase I footprinting assays showed that specific sequences within two of these elements (-193 to -124 and +44 to +262) bind proteins in nuclear extracts from HepG2 and Chinese hamster ovary cells. A protein footprint also was identified for a GC box element at nucleotides -59 to -45. Thus, control of apolipoprotein E gene expression is the result of a complex interaction of several different regulatory elements.