Biomedical subjects

E Rassart

Publications and source records attributed to E Rassart.

At least 19 recordsLinked to original sources

PubMed2003

Differential expression of membrane and soluble adenylyl cyclase isoforms in cytotrophoblast cells and syncytiotrophoblasts of human placenta.

Adenylyl cyclase (AC) activity is ubiquitous in mammalian cells, and various forms of this enzyme exist that widely differ with regard to tissue distribution, abundance, and modes of regulation. Human placenta is made, among others, of cytotrophoblast cells and syncytiotrophoblasts. This latter is a polynucleate structure that originates from the differentiation of proliferative mononucleated cytotrophoblast cells, the placental stem cell, into syncytiotrophoblasts. In vitro, this differentiation process is associated with a concomitant increase in cellular levels of cAMP and enhanced expression of genes representative of syncytiotrophoblasts endocrine activity. Thus, in this study we evaluated the differential distribution of AC isoforms in cytotrophoblast cells and syncytiotrophoblasts by reverse transcription-polymerase chain reaction (RT-PCR) using total RNA or purified mRNA. Our results demonstrate that all membrane and soluble AC mRNA isoforms are present in both cell types. Interestingly in syncytiotrophoblasts, AC4 and AC8 mRNA are highly expressed, while AC1, AC2 mRNA are less abundant when compared to cytotrophoblast cells. Additionally, the soluble AC is expressed in both trophoblast cells, but its expression is greatly reduced in differentiated cells, syncytiotrophoblasts. The presence of these AC proteins in both cells was confirmed by Western blotting. Taken together, these data help us to characterize the different AC isoforms in human cytotrophoblast cells and syncytiotrophoblasts, and demonstrate that the AC isoforms expression seems to be mainly modulated in groups 1 and 2. Moreover, the important decrease of the soluble AC isoform in syncytiotrophoblasts as compared to cytotrophoblast cells could suggest an important role of this AC in the extravillous trophoblast formation.

Adenylyl Cyclases↗

PubMed2001

Structure-function relationships of human apolipoprotein D an immunochemical analysis.

Apolipoprotein D (apoD), a 169 amino acid member of the lipocalin family, is thought to be a transporter of small, hydrophobic ligands. A panel of 10 anti-apoD monoclonal antibodies (mAbs) was prepared and characterized in order to define apoD structure-function relationships. An apoD epitope map was constructed based on reactivity of the mAbs with apoD fragments. Three mAbs react with epitopes between apoD residues 7-78, seven mAbs with epitopes between residues 128-169, one mAb recognizes an epitope that straddles residues 99-102 and one mAb is specific for an epitope composed of non-contiguous apoD residues. Several pairs of mAbs whose respective epitopes are widely separated in apoD primary structure can compete for binding to immobilized apoD. This would be consistent with the compact beta-barrel tertiary structure that apoD is thought to adopt. None of the mAbs block the interaction of apoD with pregnenolone, a putative physiological ligand for apoD.

Antibodies, Blocking↗

PubMed2000

Low- and high-density lipoprotein metabolism in HepG2 cells expressing various levels of apolipoprotein E.

To determine the importance of hepatic apolipoprotein (apo) E in lipoprotein metabolism, HepG2 cells were transfected with a constitutive expression vector (pRc/CMV) containing either the complete or the first 474 base pairs of the human apoE cDNA inserted in an antisense orientation, for apoE gene inactivation, or the full-length human apoE cDNA inserted in a sense orientation for overexpression of apoE. Stable transformants were obtained that expressed 15, 24, 226, and 287% the apoE level of control HepG2 cells. The metabolism of low-density lipoprotein (LDL) and high-density lipoprotein-3 (HDL(3)), two lipoprotein classes following both holoparticle and cholesteryl esters (CE)-selective uptake pathways, was compared between all these cells. LDL-protein degradation, an indicator of the holoparticle uptake, was greater in low apoE expressing cells than in control or high expressing cells, while HDL(3)-protein degradation paralleled the apoE levels of the cells (r(2) = 0.989). LDL- and HDL(3)-protein association was higher in low apoE expressing cells compared to control cells. In opposition, LDL- and HDL(3)-CE association was not different from control cells in low apoE expressing cells but rose in high apoE expressing cells. In consequence, the CE-selective uptake (CE/protein association ratio) was positively correlated with the level of apoE expression in all cells for both LDL (r(2) = 0.977) and HDL(3) (r(2) = 0.998). We also show that, although in normal and low apoE expressor cells, 92% of LDL- and 80% HDL(3)-CE hydrolysis is sensitive to chloroquine suggesting a pathway linked to lysosomes for both lipoproteins, cells overexpressing apoE lost 60% of chloroquine-sensitive HDL(3)-CE hydrolysis without affecting that of LDL-CE. Thus, the level of apoE expression in HepG2 cells determines the fate of LDL and HDL(3).

Apolipoproteins E↗

PubMed2000

Presence of new alternative exons in human and mouse Fli-1 genes.

The mouse Fli-1 proto-oncogene is activated by proviral integration of four murine leukemia retroviruses and its human counterpart is translocated (11,22) in Ewing tumors. We have identified two alternative exons 1 by RACE analysis from a human neuroectodermal tumor. Exons 1a and 1b are located respectively 1.3 and 2.5 kb upstream from the published exon 1. Translation of these alternative messengers is predicted to generate very similar proteins. The sequence upstream from exon 1b showed functional promoter activity. Exon 1b was not conserved in the mouse but was detected in every analyzed human cell, whereas exon 1a was present only in a subset of them and also in various mouse cell lines. These results suggest that both mouse and human Fli-1 gene expression might be under the control of several independent promoter regions.

Alternative Splicing↗

PubMed2000

Apolipoprotein D.

Apolipoprotein D (apoD) is a 29-kDa glycoprotein that is primarily associated with high density lipoproteins in human plasma. It is an atypical apolipoprotein and, based on its primary structure, apoD is predicted to be a member of the lipocalin family. Lipocalins adopt a beta-barrel tertiary structure and transport small hydrophobic ligands. Although apoD can bind cholesterol, progesterone, pregnenolone, bilirubin and arachidonic acid, it is unclear if any, or all of these, represent its physiological ligands. The apoD gene is expressed in many tissues, with high levels of expression in spleen, testes and brain. ApoD is present at high concentrations in the cyst fluid of women with gross cystic disease of the breast, a condition associated with increased risk of breast cancer. It also accumulates at sites of regenerating peripheral nerves and in the cerebrospinal fluid of patients with neurodegenerative conditions, such as Alzheimer's disease. ApoD may, therefore, participate in maintenance and repair within the central and peripheral nervous systems. While its role in metabolism has yet to be defined, apoD is likely to be a multi-ligand, multi-functional transporter. It could transport a ligand from one cell to another within an organ, scavenge a ligand within an organ for transport to the blood or could transport a ligand from the circulation to specific cells within a tissue.

Animals↗

PubMed2000

Binding of an antibody mimetic of the human low density lipoprotein receptor to apolipoprotein E is governed through electrostatic forces. Studies using site-directed mutagenesis and molecular modeling.

Monoclonal antibody 2E8 is specific for an epitope that coincides with the binding site of the low density lipoprotein receptor (LDLR) on human apoE. Its reactivity with apoE variants resembles that of the LDLR: it binds well with apoE3 and poorly with apoE2. The heavy chain complementarity-determining region (CDRH) 2 of 2E8 shows homology to the ligand-binding domain of the LDLR. To define better the structural basis of the 2E8/apoE interaction and particularly the role of electrostatic interactions, we generated and characterized a panel of 2E8 variants. Replacement of acidic residues in the 2E8 CDRHs showed that Asp(52), Glu(53), and Asp(56) are essential for high-affinity binding. Although Asp(31) (CDRH1), Glu(58) (CDRH2), and Asp(97) (CDRH3) did not appear to be critical, the Asp(97) --> Ala variant acquired reactivity with apoE2. A Thr(57) --> Glu substitution increased affinity for both apoE3 and apoE2. The affinities of wild-type 2E8 and variants for apoE varied inversely with ionic strength, suggesting that electrostatic forces contribute to both antigen binding and isoform specificity. We propose a model of the 2E8.apoE immune complex that is based on the 2E8 and apoE crystal structures and that is consistent with the apoE-binding properties of wild-type 2E8 and its variants. Given the similarity between the LDLR and 2E8 in terms of specificity, the LDLR/ligand interaction may also have an important electrostatic component.

Antibodies, Monoclonal↗

PubMed1999

Identification of a common site of provirus integration in radiation leukemia virus-induced T-cell lymphomas in mice.

The BL/VL(3) Kaplan radiation leukemia virus (RadLV-VL(3)) is a nondefective retrovirus that induces T cell lymphomas in several strains of mice. By using DNA probes derived from RadLV/VL(3) provirus-flanking sequences cloned from the BL/VL(3) cell line, we identified a DNA region rearranged in 5 of 19 tumors analysed (25%). All proviruses were integrated in the same 5'-to-3' orientation in a small DNA region called Kis1 (Kaplan integration site 1). This region was localized on distal mouse chromosome 2 in a region not previously identified as important to lymphomagenesis. The cells rearranged at the Kis1 locus represent a clonal subpopulation of the clonal tumor masses examined, indicating a probable role of Kis1 in tumor progression.

Animals↗

PubMed1999

Deletion of the C-terminal domain of apolipoprotein A-I impairs cell surface binding and lipid efflux in macrophage.

The contribution of the amphipathic alpha-helices of apoA-I toward lipid efflux from human skin fibroblasts and macrophage was examined. Four apoA-I mutants were designed, each by deletion of a pair of predicted adjacent helices. Three mutants lacked two consecutive central alpha-helices [Delta(100-143), Delta(122-165), and Delta(144-186)], whereas the final mutant lacked the C-terminal domain [Delta(187-243)]. When compared to recombinant wild-type apoA-I and mutants with central domain deletions, Delta(187-243) exhibited a marked reduction in its ability to promote either cholesterol or phospholipid efflux from THP-1 macrophages. This mutant also demonstrated a decreased ability to bind lipids and to form lipoprotein complexes. In contrast, the four mutants and apoA-I equally supported cholesterol efflux from fibroblasts, albeit with a reduced capacity when compared to macrophages. Delta(187-243) bound poorly to the macrophage cell surface when compared to apoA-I, and competitive binding studies with the central domain and C-terminal deletions mutants showed that only Delta(187-243) did not compete effectively with [(125)I]apoA-I. Omission of PMA during cholesterol loading enhanced cholesterol efflux to both apoA-I (1.5-fold) and the C-terminal deletion mutant (2.5-fold). Inclusion of the Sandoz ACAT inhibitor (58-035) during loading and, in the absence of PMA, increased and equalized cholesterol efflux to apoA-I and Delta(187-243). Surprisingly, omission of PMA during cholesterol loading had minimal effects on the binding of apoA-I or Delta(187-243) to the THP-1 cell surface. Overall, these results show that cholesterol efflux from cells such as fibroblasts does not require any specific sequence between residues 100 and 243 of apoA-I. In contrast, optimal cholesterol efflux in macrophages requires binding of the C-terminal domain of apoA-I to a cell surface-binding site and the subsequent translocation of intracellular cholesterol to an efflux-competent pool.

Amides↗

PubMed1999

The GATA-1 and Spi-1 transcriptional factors bind to a GATA/EBS dual element in the Fli-1 exon 1.

Fli-1 is a proto-oncogene which is rearranged in tumors induced by three different retroviruses, Cas-Br-E, F-MuLV, and 10A1. This gene is a member of the Ets gene family, a class of transcription factors that recognize and bind to a DNA motif known as the Ets binding site (EBS). Our laboratory has previously cloned and characterized the promoter region of both human and mouse Fli-1 genes. We had then identified several regulatory elements conserved between the two species. Two of them, an exon 1 GATA/EBS dual element and an EBS element located in the 5' end of intron 1, were analysed in the present study. EMSA analysis performed with nuclear extracts from different cell lines showed that the EBS element in intron 1 (EBSi) was bound by one potential Ets-related ubiquitous factor. The GATA/EBS element was bound by several factors that seemed Ets-related, one of which was found to be specifically expressed in hematopoietic cells. the GATA/EBS dual element was thus chosen for further analysis. A human Fli-1-derived genomic fragment containing the GATA/EBS led to enhanced transcription when positioned upstream of the SV40 promoter in the erythroleukemic HEL cell line. In addition, an increasing number of GATA/EBS oligonucleotides upstream of this same promoter resulted in a copy number-dependent increase in luciferase activity which was greatly reduced when the EBS consensus sequence was mutated. One of the factors binding to the GATA/EBS region was identified to be Spi-1 by supershift analysis and was also shown to bind to the EBS element of the human Ets-2 gene. Supershift analysis also demonstrated the binding of the GATA-1 factor to the GATA/EBS dual element. Our results suggest that Spi-1 and GATA-1 might play a key role in the regulation of Fli-1.

Animals↗

PubMed1999

Biomarkers of DNA damage in marine mammals.

Certain environmental contaminants found in marine mammals have been shown to cause DNA damage and cancer. The micronuclei (MN), sister chromatid exchange (SCE) and/or chromosome aberration (CA) assays were used to assess baseline (spontaneous) levels of DNA damage in blood lymphocytes of individuals of the relatively healthy and lightly contaminated Arctic beluga whale (Delphinapterus leucas), Sarasota Bay, FL, bottlenose dolphin (Tursiops truncatus) and Northwestern Atlantic grey (Halichoerus grypus) and harp (Phoca groenlandicus) seal populations. MN cell (MNC) frequencies ranged between 2 and 14/1000 binucleated (BN) cells and were statistically similar between species. In bottlenose dolphins, MNC frequency was correlated with age and was significantly higher in females than in males. No intraspecific variation in MNC frequency was found in beluga whales. Intraspecific variation was not tested in seals due to the small sample size. Frequencies of SCEs and total CAs, excluding gaps, ranged, respectively, between 1 and 15 SCE(s)/per cell and 4-6 CAs/100 cells in beluga whales. SCE and CA frequencies did not vary with age or sex in beluga whales. The MN, SCE and CA assays were found to be practical tools for the detection of DNA damage in marine mammals and could be used in the future to compare DNA damage between relatively lightly and highly contaminated populations.

Animals↗

PubMed1999

Modulation of apolipoprotein D and apolipoprotein E expression in rat hippocampus after entorhinal cortex lesion.

Apolipoprotein (apo) D is a member of the lipocalin family of proteins. Although its physiological function is unknown, apoD is thought to transport one or more small hydrophobic ligands. A second apolipoprotein, apoE is known to play an important role in lipid transport, and apoE genetic polymorphism has been shown to be associated with susceptibility to Alzheimer's disease. Both apoD and apoE are expressed in the central nervous system (CNS) and both proteins accumulate at sites of peripheral nerve injury due to increased local synthesis. The two proteins may have overlapping or complementary functions within nervous tissue. In order to define the role of apoD within the CNS, we have studied the regional distribution of apoD and apoE mRNA and protein within the normal rat brain and the changes in apoD and apoE expression in the hippocampus of rats after entorhinal cortex lesion (EC lesion). Within the brains of normal rats, apoD expression in the hippocampus was as high as 180-fold that of the liver. ApoD mRNA levels in other areas of the rat brain ranged from 40 to 120 times the hepatic levels. The distribution of apoE gene expression within the brain was similar to that of apoD, but was much lower than hepatic apoE expression. When rats were subjected to EC lesion, the apoD message increased by 54% at 4 days post lesion (DPL) in the ipsilateral region of hippocampus while apoE mRNA levels (ipsilateral and contralateral) decreased by 43%. At 6 to 8 DPL apoD mRNA in the ipsilateral hippocampus remained elevated (42% above controls) whereas the apoE mRNA levels increased to about 15% above those of controls. At 14 and 31 DPL, both apoD and apoE expression was similar to controls. The increase in immunoreactive apoD in hippocampal extracts was more dramatic. At 1 DPL, immunoreactive apoD levels were already 16-fold higher than those in extracts of non-lesioned animals and, at 31 DPL, levels were still 8-fold higher than those of control animals. Finally, we have demonstrated that the levels of apoD in the brains of apoE-deficient mice are 50-fold those of wildtype control mice. ApoD clearly has an important function within the CNS in both normal and pathological situations.

Animals↗

PubMed1999

Induction of micronuclei in vitro by organochlorine compounds in beluga whale skin fibroblasts.

Beluga whales (Delphinapterus leucas) inhabiting the St. Lawrence estuary are highly contaminated with environmental pollutants and have a high incidence of cancer. Environmental contaminants may be partly responsible for the high cancer incidence observed in this population. DNA damage plays an important role in the development of cancer. The micronuclei (MN) assay was used to test the genotoxic potential of organochlorine (OC) pesticides with and without external metabolic factor in skin fibroblasts of an Arctic beluga whale. Toxaphene, chlordane and p,p'-DDT induced significant (p<0. 05) concentration-response increases of micronucleated cells (MNCs). Statistically significant increases in MNCs, ranging from 1.7- to 5-folds when compared to control cultures, were observed for 0.05, 0. 5, 5 and 10 microg/ml toxaphene, 2, 5 and 10 microg/ml chlordane and 10 and 15 microg/ml p,p'-DDT. Presence of exogeneous metabolic factor (S9) completely abolished the MN induction potency of chlordane and p,p'-DDT, and toxaphene induced MN formation at higher concentrations (0.5 microg/ml) than without S9 mix. The ecotoxicological significance of MN induction by low concentrations of toxaphene is unknown and do not imply that toxaphene is involved in the etiology of cancer in St. Lawrence beluga whales. However, because of the known genotoxicity of toxaphene and the long lifespan of beluga whales, it cannot be excluded that toxaphene may pose a long-term genetic hazard to the more contaminated whales of this population.

Animals↗

PubMed1999

Bacterial expression and purification of the Fab fragment of a monoclonal antibody specific for the low-density lipoprotein receptor-binding site of human apolipoprotein E.

We report the bacterial expression and the purification of a monoclonal antibody (mAb) specific for an epitope that coincides with the LDL receptor (LDLr)-binding domain of human apolipoprotein E (apoE). This antibody resembles the LDLr in its primary structure and its specificity for apoE variants. The recombinant Fab (rFab) fragment of mAb 2E8, consisting of the entire light chain and the Fd portion of the heavy chain, was expressed in Escherichia coli and purified to homogeneity. Purification was facilitated by including a five-histidine carboxyl-terminal extension on the Fd chain. A 5- to 10-fold difference in yield of the antibody was observed when the plasmid was expressed in two different strains of E. coli. Typically 2-6 mg of rFab per liter of culture medium was recovered in the periplasm of the TG1 strain and less than 1 mg was recovered in the periplasm of the XL1-Blue strain. Culture temperatures above 35 degrees C or inclusion of sucrose in the medium reduced rFab yields. The 2E8 rFab was indistinguishable from Fab prepared from 2E8 hybridoma-generated IgG with respect to its affinity and fine specificity. We are using this system to express a panel of 2E8 variant Fabs that will be used as probes to establish the structural features responsible for the binding of apoE to the LDLr.

Animals↗

PubMed1999

Immunochemical evidence that cholesteryl ester transfer protein and bactericidal/permeability-increasing protein share a similar tertiary structure.

Cholesteryl ester transfer protein (CETP) plays an important role in plasma lipoprotein metabolism through its ability to transfer cholesteryl ester, triglyceride, and phospholipid between lipoproteins. CETP is a member of a gene family that also includes bactericidal/permeability-increasing protein (BPI). The crystal structure of BPI shows it to be composed of two domains that share a similar structural fold that includes an apolar ligand-binding pocket. As structurally important residues are conserved between BPI and CETP, it is thought that CETP and BPI may have a similar overall conformation. We have previously proposed a model of CETP structure based on the binding characteristics of anti-CETP monoclonal antibodies (mAbs). We now present a refined epitope map of CETP that has been adapted to a structural model of CETP that uses the atomic coordinates of BPI. Four epitopes composed of CETP residues 215-219, 219-223, 223-227, and 444-450, respectively, are predicted to be situated on the external surface of the central beta-sheet and a fifth epitope (residues 225-258) on an extended linker that connects the two domains of the molecule. Three other epitopes, residues 317-331, 360-366, and 393-410, would form part of the putative carboxy-terminal beta-barrel. The ability of the corresponding mAbs to compete for binding to CETP is consistent with the proximity of the respective epitopes in the model. These results thus provide experimental evidence that is consistent with CETP and BPI having similar surface topologies.

Antibodies, Monoclonal↗

PubMed1999

Oncogene activation in myeloid leukemias by Graffi murine leukemia virus proviral integration.

The Graffi murine leukemia virus (MuLV) is a nondefective retrovirus that induces granulocytic leukemia in BALB/c and NFS mice. To identify genes involved in Graffi MuLV-induced granulocytic leukemia, tumor cell DNAs were examined for genetic alterations at loci described as common proviral integration sites in MuLV-induced myeloid, lymphoid, and erythroid leukemias. Southern blot analysis revealed rearrangements in c-myc, Fli-1, Pim-1, and Spi-1/PU.1 genes in 20, 10, 3.3, and 3.3% of the tumors tested, respectively. These results demonstrate for the first time the involvement of those genes in granulocytic leukemia.

Animals↗

PubMed1998

Nuclear factors that bind to the U3 region of two murine myeloid leukemia-inducing retroviruses, Cas-Br-E and Graffi.

Cas-Br-E and Graffi are two myeloid leukemia-inducing murine viruses. Cas-Br-E induces, in NIH-Swiss mice, mostly non-T, non-B leukemia composed of very immature cells with no specific characteristics (Bergeron et al. (1993). Leukemia 7, 954-962). The Graffi murine leukemia virus causes exclusively myeloid leukemia, but the tumor cells are clearly of granulocytic nature (Ru et al. (1993). J. Virol. 67, 4722). We were interested to understand the role of the long terminal repeat (LTR) U3 region in the myeloid specificity of these two retroviruses. We used DNase I footprinting and gel mobility shift assays to identify a number of protein binding sites within Cas-Br-E and Graffi U3 regions. The pattern of protected regions is highly similar for the two viruses. Some factors identified in other murine leukemia viruses, like the core binding factor, also bind to Cas-Br-E and Graffi LTR; however, other binding sites seem specific for these two viruses. Only one difference between them was noted, at the 5' end of the U3 region. Transcriptional activity of both LTRs was also analyzed in various cell lines and compared with other murine leukemia viruses. The results show a slight myeloid specificity for the two LTRs, and indicate that the Graffi enhancer is quite strong in a broad range of cell types.

Animals↗

PubMed1998

Importance of central alpha-helices of human apolipoprotein A-I in the maturation of high-density lipoproteins.

We have studied the role of amphipathic alpha-helices in the ability of apoA-I to promote cholesterol efflux from human skin fibroblasts and activate lecithin:cholesterol acyltransferase (LCAT). Three apoA-I mutants were designed, each by deletion of a pair of predicted adjacent central alpha-helices [Delta(100-143), Delta(122-165), Delta(144-186)], and expressed in Escherichia coli. This strategy was used to minimize disruption of the predicted secondary structure of the resulting protein. These three central deletion mutants have been previously shown to be expressed as stable folded proteins but to exhibit altered phospholipid-binding properties. When recombined with phospholipids to form homogeneous LpA-I containing equivalent amounts of POPC and tested for their ability to promote diffusional cholesterol efflux from normal [3H]cholesterol-labeled fibroblasts, each mutant and the wild-type recombinant protein (Rec.-apoA-I) promoted cholesterol efflux with very similar rates at all the concentrations tested. These experiments showed that all LpA-I could acquire cellular cholesterol with similar affinity and binding capacity. However, when the cell-incubated LpA-I were incubated with purified LCAT, two mutants, Delta(122-165) and Delta(144-186), appeared incapable of activating the enzyme. To directly determine their ability to activate LCAT, each mutant and the control were recombined with equivalent amounts of cholesterol and phospholipid and incubated with the purified enzyme. The results show that whereas deletion of residues 100-143 has little effect on LCAT activation, deletion of residues 122-165 or 144-186 results in an inability of the mutants to promote cholesterol esterification. In conclusion, our results show that no specific sequence in the central domain of apoA-I is required for efficient diffusional cholesterol efflux from normal fibroblasts; however, residues 144-186 appear critical for optimum LCAT activation and cholesteryl ester accumulation. Since deletion of residues 144-186 also perturbs phospholipid association and prevents the formation of large LpA-I particles [Frank, P. G., Bergeron, J., Emmanuel, F., Lavigne, J. P., Sparks, D. L., Denèfle, P., Rassart, E., and Marcel, Y. L. (1997) Biochemistry 36, 1798-1806], the data show that this pair of alpha-helices plays an important role in the maturation of HDL. Sequence analysis of these apoA-I helices further identifies specific residues that appear essential to this activity.

Amino Acid Sequence↗

PubMed1998

Effect of reduced low-density lipoprotein receptor level on HepG2 cell cholesterol metabolism.

Low-density lipoproteins (LDL) are taken up by both LDL receptor (LDLr)-dependent and -independent pathways. In order to determine the importance of these pathways in the activity of the various enzymes that are important in maintaining the cellular cholesterol level in hepatic cells, we created HepG2 cells expressing lower levels of LDLr. Thus HepG2 cells were transfected with a constitutive expression vector (pRc/CMV) containing a fragment of LDLr cDNA inserted in an antisense manner. Stable transformants were obtained that showed significant reductions of 42, 72 and 85% of LDLr protein levels compared with the control, as demonstrated by immunoblotting and confirmed by the LDL binding assay. The best inactivation was achieved with the construct containing the first 0.7 kb of LDLr cDNA. Incubating the different HepG2 cell subtypes with LDL showed similar association of apolipoprotein B (apo B) or cholesteryl esters from LDL with the cells, indicating that the LDLr deficiency did not significantly affect LDL uptake by the cell. However, apoB degradation was reduced significantly by 71-82% in the most LDLr-deficient HepG2 cells. We also found that 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoA red) activity is significantly increased by 32-35% in HepG2 cells expressing very low levels of LDLr that also demonstrate a significant decrease of 20% in acyl-CoA:cholesterol acyltransferase (ACAT) activity. However, these effects are moderate compared with those observed when cells were incubated in lipoprotein-depleted medium, where a >900% increase in HMGCoA red activity and a loss of 60% of ACAT activity was observed. Thus, in HepG2 cells, different levels of LDLr affect LDL-apoB degradation, but have very little effect on LDL association, HMGCoA red and ACAT activities, revealing that LDLr is more important in the clearance of LDL-apoB than in HepG2 cell cholesterol homoeostasis, a role that should be attributable to both LDLr-dependent and -independent pathways.

Apolipoproteins B↗