Structure and expression of the mouse prealbumin gene

We cloned a genomic DNA fragment which covers the entire sequence of the mouse prealbumin gene and then studied the structure. The coding regions are separated into four exons by three introns, and these numbers, the sizes of the exons and the relative sites of the exon-intron junctions are all in complete agreement with those determined for the human gene. The sequences of four exons can be aligned perfectly with that of the previously determined mouse prealbumin cDNA. In addition to the exon regions, we found two highly conserved DNA regions between the mouse and human prealbumin genes, one in the 5'-flanking region of the gene and the other in the 3' end region of the first intron. These DNA regions contain several consensus glucocorticoid receptor-binding site sequences, and the latter also contains an enhancer sequence present in the immunoglobulin kappa-chain joining-constant kappa intron. RNA hybridizing to the mouse prealbumin cDNA was detected in the extracts from liver, brain, and kidney, but was not detected in testes, spleen, or heart. Little change was caused in the level of prealbumin mRNA in the liver by administration of dexamethasone to mice.     

Induction of apolipoprotein E gene expression in human and experimental atherosclerotic lesions

Hypercholesterolemia and atherosclerosis were induced in New Zealand White rabbits by cholesterol feeding. Apolipoprotein E mRNA levels in livers were found to be slightly increased, as determined by Northern blots. Apolipoprotein E gene expression was dramatically induced in rabbit atherosclerotic aortas with respect to healthy aortas. However, apolipoprotein E mRNA levels in atherosclerotic aortas were low as compared with the hepatic mRNA levels of the same animals. Interestingly, we also found a significant increase in apolipoprotein E expression in human atheromata with respect to healthy aorta from the same individual. This is the first report on apo E gene induction in human atherosclerotic lesions.     

Apolipoprotein E gene expression is reduced in apolipoprotein A-I transgenic mice

The levels of plasma apolipoprotein (apo) E, an anti-atherogenic protein involved in mammalian cholesterol transport, were found to be 2-3 fold lower in mice over-expressing human apoA-I gene. ApoE is mainly associated with VLDL and HDL-size particles, but in mice the majority of the apoE is associated with the HDL particles. Over-expression of the human apoA-I in mice increases the levels of human apoA-I-rich HDL particles by displacing mouse apoA-I from HDL. This results in lowering of plasma levels of mouse apoA-I. Since plasma levels of apoE also decreased in the apoA-I transgenic mice, the mechanism of apoE lowering was investigated. Although plasma levels of apoE decreased by 2-3 fold, apoB levels remained unchanged. As expected, the plasma levels of human apoA-I were almost 5-fold higher in the apoAI-Tg mice compared to mouse apoA-I in WT mice. If the over-expression of human apoA-I caused displacement of apoE from the HDL, the levels of hepatic apoE mRNA should remain the same in WT and the apoAI-Tg mice. However, the measurements of apoE mRNA in the liver showed 3-fold decreases of apoE mRNA in apoAI-Tg mice as compared to WT mice, suggesting that the decreased apoE mRNA expression, but not the displacement of the apoE from HDL, resulted in the lowering of plasma apoE in apoAI-Tg mice. As expected, the levels of hepatic apoA-I mRNA (transgene) were 5-fold higher in the apoAI-Tg mice. ApoE synthesis measured in hepatocytes also showed lower synthesis of apoE in the apoAI-Tg mice. These studies suggest that the integration of human apoA-I transgene in mouse genome occurred at a site that affected apoE gene expression. Identification of this locus may provide further understanding of the apoE gene expression.     

Recycling of apolipoprotein E in mouse liver

Following the internalization of low density lipoprotein (LDL) by the LDL receptor within cells, both the lipid and the protein components of LDL are completely degraded within the lysosomes. Remnant lipoproteins are also internalized by cells via the LDL receptor as well as other receptors, but the events following the internalization of these complexes, which use apolipoprotein E (apoE) as their ligand for receptor capture, have not been defined. There is evidence that apoE-containing beta-very low density lipoproteins follow differential intracellular routing depending on their size and apoE content and that apoE internalized with lipoproteins can be resecreted by cultured hepatocytes and fibroblasts. In the present studies, we addressed the question of apoE sparing or recycling as a physiologic phenomenon. Remnant lipoproteins (d < 1.019 g/ml) from normal mouse plasma were iodinated and injected into normal C57BL/6 mice. Livers were collected at 10, 30, 60, and 120 min after injection, and hepatic Golgi fractions were prepared for gel electrophoresis analysis. Golgi preparations were analyzed for galactosyltransferase enrichment (>40-fold above cell homogenate) and by appearance of the Golgi stacks and vesicles on electron microscopy. Iodinated apoE was consistently found in the Golgi fractions peaking at 10 min and disappearing by 2 h after injection. Although traces of apoB48 were present in the Golgi fractions, the apoE/apoB ratio in the Golgi was 50-fold higher compared with serum. Quantitatively similar results were obtained when the very low density lipoprotein remnants were injected into mice deficient in either apoE or the LDL receptor, indicating that the phenomenon of apoE recycling is not influenced by the production of endogenous apoE and is not dependent on the presence of LDL receptors. In addition, radioactive apoE in the Golgi fractions was part of d = 1.019-1.21 g/ml complexes, indicating an association of recycled apoE with either newly formed lipoproteins or the internalized complexes. These studies show that apoE recycling is a physiologic phenomenon in vivo and establish the presence of a unique pathway of intracellular processing of apoE-containing remnant lipoproteins.     

Dissociated regulation of macrophage LDL receptor and apolipoprotein E gene expression by sterol

The control of apoE gene expression by sterols and the relationship between regulation of the apoE and low density lipoprotein (LDL) receptor genes were investigated in a human macrophage line. Incubation of THP1 cells in either LDL or acetylated LDL increased apoE mRNA levels 4- to 15-fold. In addition, the cellular abundance of these two mRNA species (apoE and LDL receptor) was inversely regulated by cellular cholesterol content over an identical dose-response relationship. Regulation of the LDL receptor and apoE genes could, however, be temporally dissociated in response to the accumulation or removal of lipoprotein-derived (exogenous) cholesterol and in response to perturbation of endogenous cellular cholesterol biosynthesis. In addition, we observed that the apoE gene responded more promptly to 25-hydroxycholesterol than to exogenous cholesterol. These data support the concept that the apoE gene be considered among the family of genes sensitively regulated by cellular sterol balance but suggest that the molecular mechanism accounting for the modulation of the LDL receptor and apoE genes are distinct, with the relationship between cell sterol balance and apoE gene regulation being more complex.     

Complex control of mouse apolipoprotein B gene expression revealed by targeted duplication

An elevated plasma level of apolipoprotein B-containing lipoproteins is a risk factor for atherosclerotic cardiovascular disease. Subtle genetic abnormalities in gene expression including an increased expression of the APOB gene may play an important role in determining overall risk. In an attempt to increase mouse Apob expression, we used gene targeting and duplicated approximately 65 kb of genomic DNA containing the Apob locus in its natural genomic position in mice. While we successfully generated mice carrying the Apob gene duplication, the amount of the total Apob mRNA was not increased in their liver. In the intestine, total Apob mRNA was reduced to half of the wild-type mice. Plasma lipids in the Apob duplication mice were not altered. Expression analyses showed that the proximal Apob gene in the duplicated locus was preferentially expressed in both tissues suggesting a limitation of tissue-specific enhancer function. The previously characterized distant intestinal control element was not duplicated, explaining the unequal ratio of intestinal Apob expression. While the existence of an additional liver-specific enhancer element is unknown, our findings suggest the presence of an additional enhancer outside the duplicated region, and that Apob gene expression is more complicated than previously thought.     

Hormonal regulation of human apolipoprotein E gene expression in HepG2 cells

• 1.1. Hormonal regulation of apolipoprotein E (apoE) gene expression by insulin and thyroid hormone was studied in a human hepatoma cell line, HepG2.
• 2.2. Changes at the mRNA level, mRNA translation, in vivo synthesis and secretion were monitored.
• 3.3. Both insulin and triiodothyronine were found to have no significant effect on apoE mRNA levels.
• 4.4. Insulin treatment caused an inhibition of: (a) the in vitro translation of endogenous apoE mRNA in a HepG2 cell-free system (25%), and (b) the incorporation of radioactivity into newly-synthesized apoE in an in vivo pulse-chase labeling experiment (32%).
• 5.5. Interestingly, apoE secretion rate was found to be significantly reduced with insulin (84%) suggesting that a major portion of newly-synthesized apoE may be shunted into a degradative pathway.
• 6.6. Using a similar experimental approach, triiodothyronine showed no significant effect on the rate of apoE synthesis or translation (6–15% decrease), however a slight reduction (20%) in secretion rate was shown.
• 7.7. Overall, apoE gene expression does not appear to be influenced by triiodothyronine significantly but is modulated by insulin at the translational and post-translational level.

     

Tissue-specific expression of genes encoding apolipoprotein E and apolipoprotein A-I in rabbits

We determined the site of synthesis of apolipoprotein (apo) E and apo-A-I in rabbit by measuring in vitro translational activity of their mRNAs from the liver and from the intestine. Poly(A+) RNA isolated from liver and intestinal epithelium of rabbits fed either a chow diet or a cholesterol-rich diet was translated in vitro in the rabbit reticulocyte lysate system using [35S] methionine as the labeled precursor. Newly synthesized apolipoproteins were immunoprecipitated with specific antisera and quantitated after electrophoresed on 10% polyacrylamide slab gels in the presence of 0.2% sodium dodecyl sulfate. The levels of liver apo-E and apo-A-I mRNAs from chow-fed rabbits are 0.41 and 0.002% of total translatable mRNA, respectively. The level of liver apo-A-I mRNA in the rabbit is approximately 500-fold lower than the reported level of apo-A-I mRNA in rat and human livers. Rabbit intestinal apo-E and apo-A-I mRNAs levels are 0.0036 and 0.67%, respectively. Our results indicate that in rabbits apo-E is synthesized primarily in the liver and that apo-A-I is synthesized primarily in the intestine. When rabbits are fed a cholesterol-rich diet, liver and intestinal apo-E in mRNA levels and intestinal apo-A-I mRNA levels are not changed. In contrast, the liver apo-A-I mRNA level increases 5-fold in response to the cholesterol-rich diet. However, because the intestinal liver apo-A-I mRNA level is so low, the 5-fold induction only increases liver mRNA levels to 2.7% of the corresponding intestinal apo-A-I mRNA level.     

Bacterial expression and characterization of rat apolipoprotein E

Apolipoprotein (apo) E is a protein involved in both lipid metabolism and neuroprotection. Recently, it has been suggested that apoE may play a role in the regulation of food intake and body weight in rodents. However, rodent plasma apoE is difficult to purify in reasonable amounts due to numerous time-consuming steps. To circumvent this, we created a bacterial expression system for the efficient production of large amounts of rat apoE. We inserted rat apoE DNA into the pET30 expression vector and overexpressed the proteins in Escherichia coli strain BL21 (DE3). A histidine tag present at the N-terminus allowed for easy purification of the recombinant protein. The tag was removed with an IgA protease (Igase) from Neisseria gonorrhoeae leaving the mature form of the protein. The use of Igase was important as several more common proteases routinely cleave apolipoproteins at undesired sites. The recombinant protein was then compared both structurally and functionally to rat plasma apoE. This expression system will be highly useful for probing the ability of rat apoE to mediate food intake in rats.     

Cloning and regulation of messenger RNA for mouse apolipoprotein E

A cDNA clone for mouse apolipoprotein E has been identified from a mouse liver cDNA library by a combination of differential colony hybridization and hybrid selection-translation. The identity of the clone was unambiguously established by partial sequencing and comparison with human apolipoprotein E nucleotide and amino acid sequences. In conjunction with an in vitro translation assay for apolipoprotein E, the clone has been used to examine the relative levels of apolipoprotein E mRNA in various tissues of the mouse and the regulation of apolipoprotein E synthesis in response to a diet rich in saturated fat and cholesterol. In the tissues examined, the clone was found to hybridize to a polyadenylated RNA species of approximately 1400 nucleotides. Of the tissues involved in lipoprotein synthesis, liver is very rich (about 1% of total) in apolipoprotein E mRNA while intestine contains only trace amounts. Appreciable levels of active apolipoprotein E mRNA (up to 10% of that in liver) are also detected in peripheral tissues not associated with lipoprotein synthesis, including lung, kidney, spleen, and heart. Thus, extrahepatic apolipoprotein E synthesis may contribute significantly to the levels present in plasma, and a possible function in "reverse cholesterol transport" is considered. When mice were placed on a high lipid diet there was no discernible change in the level of apolipoprotein E mRNA in liver or intestine, although the level of the circulating protein increased about 3-fold. We conclude that in mice the effect of diet on apolipoprotein E levels in blood does not result from induction of mRNA in these tissues.     

Structure and expression of the mouse homologue of the XK gene

 The human Kx blood group antigen is carried by a 37 000 M _r apparent molecular mass membrane polypeptide which is deficient in rare individuals with the McLeod syndrome. The X-linked human XK gene is transcribed in many tissues including adult skeletal muscle and brain, sieges of disorders observed in McLeod syndrome. We report here the cloning of the orthologous mouse XK mRNA. Comparison of XK from human and mouse revealed 80% sequence similarity at the amino acid level. The mouse XK gene is organized in two exons and is expressed in many tissues, but its expression pattern is slightly different from that of the human gene. The presence in mouse erythrocyte membrane of a 43 000 M _r Kx-related protein was demonstrated by immunoblotting with a rabbit antiserum directed against the human protein. With non-reduced samples, a 140 000 M _r species was detected instead of the 43 000 M _r protein, suggesting that, as demonstrated in the Kx polypeptide might be complexed with another protein in mouse red cells, presumably the homologue of the human Kell protein of 93 000 M _r. Received: 22 February 1999 / Revised: 8 June 1999