Dual effects on HDL metabolism by cholesteryl ester transfer protein inhibition in HepG2 cells

Zhiping Huang, Akihiro Inazu, Masa-aki Kawashiri, Atsushi Nohara, Toshinori Higashikata, and Hiroshi Mabuchi

Molecular Genetics of Cardiovascular Disorders, Division of Cardiovascular Medicine, Graduate School of Medical Science, Kanazawa University, Kanazawa 920-8641, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cholesteryl ester transfer protein (CETP) promotes reverse cholesterol transport via exchange of cholesteryl ester and triglyceride among lipoproteins. Here, we focused on HDL metabolism during inhibition of CETP expression by using CETP antisense oligodeoxynucleotides (ODNs) in HepG2 cells. CETP secretion was decreased by 70% in mRNA levels and by 52% in mass 20 h after ODNs against CETP were delivered to HepG2 cells. Furthermore, as a consequence of the downregulation of CETP, the expression of scavenger receptor class B type I (SR-BI), an HDL receptor, was also reduced by ~50% in mRNA and protein levels, whereas the apolipoprotein A-I (apoA-I) expression and secretion were increased by 30 and 92%, respectively. In a functional study, the selective uptake of 125I-[14C]cholesteryl oleate-labeled HDL3 was decreased. Cholesterol efflux to apoA-I and HDL3 was significantly increased by 88 and 37%, respectively. Moreover, the CE levels in cells after antisense treatment were elevated by 20%, which was related to the about twofold increase of cholesterol esterification and increased acyl-CoA:cholesterol acyltransferase 1 mRNA levels. Taken together, these findings suggest that although acute suppression of CETP expression leads to an elevation in cellular cholesterol stores, apoA-I secretion, and cellular cholesterol efflux to apoA-I, the return of HDL-CE to hepatocytes via an SR-BI pathway was inhibited in vitro. Thus antisense inhibition of hepatic CETP expression manifests dual effects: namely, increased formation of HDL and suppression of catabolism of HDL-CE, probably via the SR-BI pathway.

scavenger receptor class B type I; reverse cholesterol transport; high-density lipoprotein


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MOST PROSPECTIVE EPIDEMIOLOGICAL STUDIES have found that high plasma HDL levels are a negative risk factor for atherosclerosis and that plasma LDL concentrations correlate with the risk of coronary heart disease. The ability of HDL to protect against atherosclerosis is widely attributed to its role in reverse cholesterol transport, whereby HDL promotes the flux of excess cholesterol from peripheral cells to the liver for excretion. Various receptor complexes, apolipoprotein, lipid transfer proteins, and lipoprotein-processing enzymes have been suggested to be involved in the process of reverse cholesterol transport.

Cholesteryl ester transfer protein (CETP) is a plasma glycoprotein that promotes reverse cholesterol transport via the exchange of cholesteryl ester (CE) and triglyceride (TG) among lipoproteins (46). Growing evidence indicates that CETP has a central role in lipoprotein metabolism. Subjects with genetic CETP deficiency have both elevated HDL cholesterol concentrations and decreased LDL cholesterol levels (21, 28). A decrease in the catabolic rate of HDL apolipoproteins has also been reported (20). Although some of these effects are mediated through the actions of circulating CETP, CETP may also have an indirect effect on HDL and LDL by changing other potential lipoprotein receptors or apolipoprotein expressions. This view was supported by some studies indicating that CETP regulates the expression and secretion of an LDL receptor (LDL-R) and apolipoprotein A-I (apoA-I) (41, 44).

Scavenger receptor class B type I (SR-BI), a cell surface glycoprotein, as an HDL receptor, mediates selective uptake of HDL-CE and is not associated with degradation of the protein component of HDL (1). Both CETP and SR-BI have been found to be associated with HDL metabolism (21, 29). To what extent SR-BI is capable of HDL-CE transport in the case of CETP deficiency is an interesting issue. Studies have demonstrated that CETP activity increases the selective uptake of HDL-CE via the SR-BI pathway (9). Additionally, CETP associated with cell plasma membranes appears to facilitate CE selective uptake (4). We therefore hypothesized that the effect of CETP on the uptake of HDL-CE may use the SR-BI pathway.

CETP mRNA is expressed in a number of human tissues, including liver, spleen, and adipose (10). All of these tissues express two forms of mRNA CETP: a full-length form that is normally secreted and a shortened form with an exon 9 sequence deletion (23). The short form of CETP is poorly secreted but retains the CE/TG binding sites and lipid-surface interaction sites required for lipid transfer activity (53). The widespread tissue distribution of CETP mRNA raises the possibility that CETP synthesized by various tissues may have local functions in lipid metabolism. In a recent report, Izem and Morton (24) showed a new function of CETP, whose biosynthesis mediates intracellular cholesterol homeostasis in adipose cells. These results provide a new incentive to explore the effects of CETP on HDL and LDL metabolism, because the expressions of many lipoprotein receptors, apolipoproteins, and enzymes such as SR-BI are also regulated by cellular cholesterol changes.

In the present study, we used antisense oligodeoxynucleotides (ODNs) to suppress hepatic CETP expression to explore the potential interaction between CETP and HDL metabolism in a human liver tumor cell line (HepG2).


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ODN design. The 21-nucleotide CETP antisense ODNs were designed to target the 3'-untranslated region of human CETP mRNA (GenBank accession no. M30185) (10, 48). The sense ODNs were designed as a negative control. The ODN sequences were as follows: antisense, 5'-aagccccatcccgacctcctt-3'; sense, 5'-aaggaggtcgggatggggctt-3'; positions 1621-1641 bp of the 3'-untranslated region of CETP cDNA. BLAST (Basic Local Alignment Search Tool) research has shown that these selected target sequences have a relatively low homology with all of the other known cDNA sequences. All of these ODNs were modified with phosphorothioate, synthesized on an automated solid-phase nucleotide synthesizer, and subsequently purified with HPLC (Greiner, Tokyo, Japan).

Cell culture and preparation. The hepatocarcinoma cell line HepG2 was purchased from American Type Culture Collection (Rockville, MD) and maintained in minimum essential medium (MEM) supplemented with 10% FBS (ICN Biomedicals, Irvine, CA) and 50 µg/ml penicillin-streptomycin at 37°C and in a 5% CO2 atmosphere to keep the pH at 7.4.

Fasting mononuclear blood cells were obtained by centrifugation using Ficoll-Paque (Pharmacia Biotech) from normal healthy volunteers and subjects with CETP deficiency for SR-BI and CD14 RT-PCR. The CETP and HDL cholesterol (HDL-C) levels were 2.4 ± 0.3 (µg/ml) and 65 ± 8 (mg/dl) in the normal group (means ± SE, n = 6), 1.4 ± 0.2 and 106 ± 17 in the heterozygous group (n = 3), and 0.2 ± 0.2 and 183 ± 7 in the homozygous group (n = 3), respectively.

CETP ODNs and CETP chemical inhibitor JTT-705 delivery. The HepG2 cells were plated in 6-well plates for transfection. ODNs were delivered into cultured HepG2 cells by means of cationic liposome Tfx-20 (Promega, Madison, WI). Sense or antisense ODNs and liposomes (12 µM) were mixed in Opti-MEM I (GIBCO-BRL, Gaithersburg, MD). The cultured cells at 70-80% confluence were rinsed twice with PBS before the addition of the ODNs-liposome mixture. The cells were exposed to the mixture for 12 h at 37°C, 5% CO2, and 100% humidity and were returned to the growth medium. After 8 h of incubation, the cells were rinsed twice with PBS before total RNA and cellular protein preparation. After 24 h of incubation, the growth medium and cells were collected for CETP mass determination and protein quantitation. Different concentrations of ODNs (0-8 µM) were determined.

The HepG2 cells were plated in 6-well plates and cultured to 70-80% confluence. After being washed with PBS, the cells were incubated with growth medium and a different concentration (0-30 µM) of chemical inhibitor JTT-705 obtained from Japan Tobacco (Tokyo, Japan) and dissolved in 2% DMSO for 24 h (35). Total RNA was used for RT-PCR.

Reverse transcription and amplification of cDNA. Total RNA was prepared from rinsed cells by using RNAzoI B reagent (TEL-TEST, Friendswood, TX). Two micrograms of total RNA were transcribed into cDNA by using an oligo(dT) primer and SuperScript reverse transcriptase II (GIBCO-BRL).

The concentrations of the various mRNAs were determined with a PCR assay consisting of coamplification of the specific primer sets and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Clontech, Palo Alto, CA) in the same PCR tube. PCR was performed in 50-µl reaction volumes using the Program Temp Control System PC-700 (Astec, Fukuoka, Japan) with primers as described in Table 1. Each reaction contained 2 µl of RT reaction product as a template DNA (corresponding to cDNA synthesized from 200 ng of total RNA), 20 pmol of each of the target oligonucleotides, 200 µM of dNTPs, 2.5 units of Taq DNA polymerase (Biotech International, Tokyo, Japan), and 1× reaction buffer (50 mM KCl, 10 mM Tris · HCl, pH 8.3, 1.5 mM MgCl2, and 0.01% gelatin). For each primer set, an increasing number of PCR cycles with otherwise fixed conditions was performed to determine the optimal number of cycles, which was chosen as the number halfway though the exponential phase. PCR products were separated in 2% agarose gel (Wako, Osaka, Japan) electrophoresed in 90 mM Tris-borate and 4 mM EDTA (pH 8.3) buffer and visualized by means of SYBR Green. Gels were transilluminated with UV light and analyzed by computerized densitometric scanning of the images with a Gel Scanner DIANA (Raytest, Straubenhardt, Germany) and ZERO-Dscan and ONE-Dscan system software (Scanalytics, Fairfax, VA). The target mRNA levels were adjusted for GAPDH mRNA and expressed as percentage changes from the sense treatment cells. In a human experiment, the SR-BI mRNA levels were adjusted for monocyte marker CD14 mRNA (42).

                              
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Table 1.   Primer sequences and conditions used for RT-PCR assays

CETP mass and apoA-I assay. The levels of CETP mass were determined with a sandwich ELISA kit (Chugai Pharmaceutical, Tokyo, Japan). This assay uses a monoclonal antibody (TP2) against CETP as the capturing antibody and a horseradish peroxidase-conjugated polyclonal rabbit anti-CETP antibody (26).

After sense and antisense treatments, cells were incubated for 30 min in methionine-free DMEM containing 16.5 mg/ml BSA. This was followed by an 8-h incubation in the same medium containing 50 µCi/ml [35S]methionine and 50 µM unlabeled L-methionine. The culture medium was harvested, and the cells were lysed. Immunoprecipitation with polyclonal antibodies against apoA-I [Biogenesis, Poole, UK] was performed with a protocol previously described (30). Immunoprecipitates were released by boiling for 5 min in SDS-PAGE buffer in the presence of 5% (vol/vol) 2-mercaptoethanol and were analyzed by SDS-PAGE 4-20% (wt/vol) gradient gel for apoA-I. Gel was dried, and an autoradiographic image was captured by a phosphor storage system BAS-2000 (Fuji Film, Tokyo, Japan) and analyzed with v1.0 Image Analysis Software (Fuji Film). Data are expressed as digital light units per milligram of cellular protein.

Western blot analysis. HepG2 cells were washed and scraped in PBS and lysed in 10 mM Tris · HCl (pH 7.3), 1 mM MgCl2, 0.5% Nonidet P-40, and 0.005% PMSF. Fifty-microgram pellets were suspended in a gel loading buffer plus 1.2% 2-mercaptoethanol and heated to 99°C for 5 min before loading (33). Proteins were separated by 8% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK) for immunoblotting. The membranes were blocked with a blocking buffer for 60 min at 22°C. The primary antibodies were diluted in a blocking buffer: SR-BI to 1:1,000 (Novus Biologicals, Littleton, CO), LDL-R to 1:200 (Progen Biotechnik, Heidelberg, Germany), and apoA-I to 1:1,000, and were incubated with blocked membranes at 4°C overnight. Membranes were washed four times for 10 min in a washing buffer. The secondary antibodies were diluted: horseradish peroxidase-conjugated anti-rabbit IgG to 1:1,000 (Amersham Pharmacia Biotech), and peroxidase-conjugated affinipure rabbit anti-chicken IgY to 1:2,000 (Jackson ImmunoResearch, West Grove, PA), and were incubated with membranes in the same manner as the primary antibodies. The membranes were washed four times for 30 min in a washing buffer and visualized using enhanced chemiluminescence (ECL) detection (Amersham Pharmacia Biotech); the filters were exposed to medical film (Konica, Tokyo, Japan) at room temperature for the indicated time. The relative intensities of the bands were measured using a Gel Scanner DIANA. Ponceau S staining of membranes was used to detect molecular mass standards (Amersham Pharmacia Biotech) and to verify that the specimens were loaded and transferred with comparable efficiency.

Cholesterol assay. Cellular cholesterol and cholesteryl ester contents were enzymatically measured after isopropyl alcohol extraction from CETP sense- and antisense-treated cells with a free cholesterol (FC) and total cholesterol (TC) kit (18). The cholesteryl ester (CE) levels were calculated by subtracting the FC from the TC measured in the cells.

Lipoprotein isolation and radiolabeling. HDL (1.063-1.210 g/ml), HDL2 (1.063-1.125 g/ml), and HDL3 (1.125-1.210 g/ml) were isolated from normal human plasma by sequential ultracentrifugation at 10°C and 40,000 rpm in a Beckman Ti 50.3 rotor (16, 17). The HDL3 labeled with [1-14C]cholesteryl oleate (Amersham Pharmacia Biotech) was prepared as described previously (28). Radioiodination of the protein moiety of [1-14C]cholesteryl oleate-HDL3 was performed by the iodine monochloride method as described by Goldstein et al. (13). Briefly, 3 mg of HDL3 protein were placed in a glass tube, the pH was adjusted with glycine buffer (pH 10), and then ~1 mCi of carrier-free Na125I and 10 µl of a 2.64 mM iodine monochloride solution were added to initiate the labeling process. After ~30 s, the mixture was passed through a 10-ml Sephadex G-25 column equilibrated with 150 mM NaCl-0.25 mM EDTA (pH 7.4) to separate radiolabeled material from free 125I. Thereafter, fractions corresponding to the radioiodinated HDL3 were pooled and exhaustively dialyzed overnight against saline buffer containing 97 mM potassium iodide, and then with saline buffer without potassium iodide. HDL, HDL2, HDL3, and [1-14C]cholesteryl oleate-HDL3 were dialyzed against 0.15 M NaCl and sterilized by filtration through 0.45-µM filter. Specific activities were 98,496 dpm/µg of protein for 125I-labeled HDL3 and 100,591 dpm/µg of CE for [1-14C]cholesteryl oleate-HDL3.

[3H]cholesterol efflux study. HepG2 cells were plated in 24-well plates. The cell monolayers at 60-70% confluence were labeled with 1 µCi/ml 1,2-3H(N) cholesterol (Amersham Pharmacia Biotech) in 0.4 ml of MEM supplemented with 10% FBS for 24 h. The cells were washed twice with PBS, and 0.2 ml of medium (Opti-MEM I containing 0.2% fatty acid-free BSA and the ODNs-liposome mixture) was added to each well. After 12 h of equilibration and antisense treatment, the cells were washed with PBS. MEM (0.2 ml) and 0.2% BSA, containing 10 µg/ml apoA-I, 50 µg/ml HDL2, or 50 µg/ml HDL3, were added. After an 8-h incubation at 37°C, the [3H]cholesterol content of the cells and the media was determined as follows. The efflux media were collected and precipitated at 6,000 g for 10 min to remove cell debits and cholesterol crystals, and the radioactivity in 0.2 ml of each supernatant was determined by liquid scintillation counting (LSC). The cells were finally lysed in 0.2 ml of 0.1 M sodium hydroxide and 0.1% SDS, and the radioactivity in an aliquot was determined. Cholesterol efflux was expressed as the percentage of the radioactivity released from cells into the medium relative to the total radioactivity in cells and medium.

Determination of influx of CE. HepG2 cells were cultured in 24-well plates until 70-80% confluence. After antisense treatment, cells were washed with PBS and then incubated with 0.2 ml of MEM containing 25, 50, and 150 µg/ml of 125I-[14C]cholesteryl oleate-labeled HDL3 for 8 h. At the indicated times, medium was removed, and cells were washed twice with PBS and then lysed by the addition of 0.2 ml of 0.1 M sodium hydroxide and 0.1% SDS.

To simplify the comparison of results obtained with 125I-[14C]cholesteryl oleate-labeled HDL3, the uptake of radiolabeled HDL3 is expressed as apparent HDL particle uptake (36).

Lipid analysis. Cells were treated as described in the cholesterol efflux study. At the end of the 8-h incubation, cells were washed twice with PBS and scraped. The cellular lipids were extracted with methanol-chloroform 2:1 (34). Lipid extracts were dried under an N2 stream and resuspended in 30 µl of isopropanol. An aliquot (10 µl) was separated by thin-layer chromatography (TLC) on silica gel plates (Merck, Darmstadt, Germany) run in hexane-diethyl ether-acetic acid-methanol (85:20:1:1). The plates were dried, and the lipid spots were visualized by exposure to iodine vapor. After complete disappearance of I2, spots corresponding to the position of FC and CE were scraped from plates and quantified by LSC.

Statistical analysis. All measurements were performed in triplicate for each experiment. All values are shown as means ± SE. Comparisons among three groups were made using one-way ANOVA followed by Dunnett's modified test. Differences were considered to be statistically significant at a value of P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effects on hepatocyte membrane receptor mRNA levels during CETP inhibition. After the treatment of HepG2 cells with CETP ODN compounds for 20 h, cellular RNA was extracted and mRNA levels were analyzed. The mRNA levels of CETP were reduced progressively in a dose-dependent manner (Fig. 1A). The maximum inhibition of CETP expression was observed at a 4 µM concentration. At this concentration, CETP mRNA levels were decreased by 70% (P < 0.0001, three different experiments performed in duplicate). Unless specifically noted, 4 µM ODNs were used in subsequent studies. As a consequence of the downregulation of CETP, the SR-BI mRNA levels were also reduced (Fig. 1B). In contrast, the LDL-R levels increased in a dose-dependent manner (Fig. 1C). To investigate whether inhibition of CETP activity in vitro could produce the same changes in SR-BI and LDL-R, CETP chemical inhibitor JTT-705 was used to inhibit the CETP activity of media in HepG2 in a dose-dependent manner. The results showed no change in CETP or SR-BI and LDL-R mRNA levels, but decreased secretion of apoB-containing lipoprotein was found (not shown), which suggests that inactivated CETP activity in cultured media is not the primary cause of changes in receptor expression.


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Fig. 1.   Dose-dependent effects of cholesteryl ester transfer protein (CETP) antisense treatment on CETP and scavenger receptor class B type I (SR-BI) and LDL receptor (LDL-R) mRNA expression. HepG2 cells were treated using sense or antisense compounds of CETP (0-8 µM) for 12 h and were lysed 8 h after transfection. mRNA expression levels were analyzed by means of densitometric scanning of target RT-PCR and GAPDH (G3PDH) RT-PCR products. S, sense oligonucleotides (ODNs); A, antisense ODNs.

Effects on hepatocyte membrane receptor protein levels during CETP inhibition. Antisense treatment altered not only the mRNA transcription but also protein expressions of CETP, SR-BI, and LDL-R. After antisense treatment for 12 h, the medium was replaced with fresh cultured media. After 8 h of incubation, the media and cells were collected for protein determination.

CETP mass was measured with an enzyme-linked immunosorbent assay. The CETP mass level was 0.250 ± 0.030 ng/mg cellular protein (means ± SE, P < 0.0001 vs. sense and control) in cells treated with antisense ODNs, 0.508 ± 0.025 in cells treated with sense ODNs, and 0.513 ± 0.047 in control cells (Fig. 2A). Immunoblot analysis was performed for SR-BI and LDL-R and apoA-I. After CETP antisense treatment, the levels of immunodetectable SR-BI decreased by 50% (P < 0.001, n = 5) in cells treated with antisense ODNs compared with cells treated with sense ODNs and control cells (Fig. 2B). In contrast, the amount of LDL-R increased by 70% (P < 0.001, n = 5) in cells treated with antisense ODNs. Thus changes in mRNA and protein levels were comparable. The immunoreactive band of apoA-I was increased by 30% (P < 0.01, n = 5) in cells treated with antisense ODNs. Moreover, the apoA-I levels in medium were increased by 92% (P < 0.001; Fig. 2C). These data indicated that CETP suppression stimulated apoA-I synthesis and secretion.


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Fig. 2.   Effect of CETP antisense treatment on CETP, SR-BI, LDL-R, and apolipoprotein (apo)A-I protein levels in HepG2 cells. A: HepG2 cells were transfected with Tfx-20 alone or Tfx-20 mixed with 4 µM sense or antisense ODNs for 12 h and were returned to growth medium. After 24 h of incubation, medium and cells were collected for CETP mass determination. Results are means ± SE of triplicate determinations. Similar data were obtained in a separate experiment. B: SR-BI, LDL-R, and apoA-I expressions were analyzed by Western blotting after CETP antisense treatment. Solubilized proteins (50 µg from each sample) were size-fractionated on an 8% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. Immunoblots were developed with an enhanced chemiluminescence system and exposed to Konica medical film for 20 s. Data represent a typical experiment. C: after sense and antisense treatment, cells were incubated for 30 min in methionine-free DMEM. This was followed by an 8-h incubation in the same medium containing [35S]methionine. Culture medium was collected and immumoprecipitated using anti-apoA-I antibodies. Proteins in immunoprecipitates were separated by SDS-PAGE and quantified as described in EXPERIMENTAL PROCEDURES. Results are means ± SE of triplicate determinations. Similar data were obtained in 2 separate experiments.

Effects of short-term CETP depletion by antisense treatment on the expression of lipid-related genes and transcription factors. We investigated the changes in several lipid-related genes and transcription factors. As shown in Fig. 3, the expression of the 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (-59%), lipid-related transcription factor sterol regulatory element-binding protein (SREBP)-1 (-40%) gene was downregulated like SR-BI (-52%), whereas upregulation was found in acyl-CoA:cholesterol acyltransferase (ACAT)1 (+41%) and retinoid X receptor (RXR)-alpha (+30%), like LDL-R (+70%). Furthermore, the gene expressions of ATP-binding cassette transporter (ABC)A1, lipoprotein lipase (LPL), LDL receptor-related protein (LRP), phospholipid transfer protein (PLTP), SREBP-2, and liver X receptor (LXR)-alpha were not affected (±10%) in CETP-depleted HepG2 cells (not shown). Partial changes of lipid-related genes and transcription factors indirectly suggested that CETP depletion modified cellular cholesterol metabolism and trafficking in HepG2 cells.


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Fig. 3.   Effects of CETP antisense treatment on lipid-related genes in mRNA levels. ACAT1, acyl-CoA:cholesterol acyltransferase 1; RXRalpha , retinoid X receptor-alpha ; SREBP-1, sterol regulatory element-binding protein-1; HMG, 3-hydroxy-3-methylglutaryl. HepG2 cells were transfected with Tfx-20 alone or Tfx-20 mixed with 4 µM sense or antisense ODNs for 12 h and returned to growth medium for 8 h. Total RNA was extracted from HepG2 cells. mRNA levels were assessed by RT-PCR as described. Expression of each gene was quantified and normalized using simultaneous amplification of a GAPDH (G3PDH) fragment. Results are means ± SE of triplicate determination. Similar data were obtained in 2 separate experiments. *P < 0.001.

Effects on cellular cholesterol flux during CETP inhibition. We evaluated the functional consequence of decreased SR-BI expression in response to downregulation of CETP expression. SR-BI has dual functions, which metabolize HDL via selective uptake and the efflux of FC. Thus both the efflux of FC and influx of CE were evaluated in HepG2 cells after CETP antisense treatment. As shown in Fig. 4, [14C]cholesteryl oleate-HDL3 uptake was significantly decreased in antisense-treated compared with sense-treated cells by -55, -45, and -34% at 25, 50, and 150 µg/ml HDL3, respectively (P < 0.0001), whereas uptake of 125I-HDL3 showed no difference between sense- and antisense-treated cells, suggesting that the selective uptake was decreased in antisense-treated cells compared with sense-treated cells. As shown in Fig. 5, efflux of [3H]cholesterol to apoA-I and HDL3 was increased by 88 and 37% in antisense treatment, respectively. Efflux to HDL2 did not differ between the two groups (not shown). The data of cholesterol efflux are not compatible with decreased SR-BI expression. The mechanism for this is not completely clear but may be related to increased expression of apoA-I, increased stability of ABCA1, or increased expression of an ABC transporter other than ABCA1.


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Fig. 4.   Uptake of 125I-[14C]cholesteryl oleate-labeled HDL3 in CETP antisense-treated cells. HepG2 cells were transfected with Tfx-20 mixed with 4 µM sense or antisense ODNs in Opti-MEM for 12 h. After a wash with PBS, cells were incubated with 25, 50, and 100 µg/ml 125I-[14C]cholesteryl oleate-labeled HDL3 in MEM for 8 h. Medium was removed. Cells were washed twice with PBS and then lysed by addition of 0.1 M NaOH and 0.1% SDS. Radioactivity was determined in the lysate. Results are means ± SE of triplicate determinations. Similar data were obtained in a separate experiment. *P < 0.0001 vs. CETP sense treatment.



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Fig. 5.   Effects of CETP antisense treatment on cholesterol efflux in HepG2 cells. HepG2 cells were labeled with [3H]cholesterol for 24 h in MEM and 10% FBS. After antisense treatment and equilibration for 12 h in 0.2% BSA and Opti-MEM, cells were incubated with 10 µg/ml apoA-I or 50 µg/ml HDL3 for 8 h in 0.2% BSA and MEM. Cholesterol efflux was expressed as the medium [3H]cholesterol radioactivity as a percentage of total [3H]cholesterol radioactivity (cells plus medium). Results are means ± SE of triplicate determinations. Similar data were obtained in a separate experiment.

Effects on cellular cholesterol during CETP inhibition. To determine the effect of CETP antisense treatment on the amount of cellular cholesterol, we measured the levels of cellular cholesterol in the HepG2 cells. As shown in Table 2, the levels of cellular TC and FC did not vary among cells with different treatments. In contrast, the levels of cellular CE were increased by 20% in antisense-treated cells compared with sense-treated cells. In addition, HMG-CoA reductase mRNA expression was decreased by 59%, and ACAT1 mRNA expression was increased by 41%, indicating that downregulated CETP secretion reduced cellular cholesterol biosynthesis and increased CE synthesis. In the next experiment, we assessed further the association of esterification of cellular FC and CETP. HepG2 cells were preincubated (24 h) with [3H]cholesterol to label the cellular pool of FC. After antisense treatment, cells were cultured with HDL or apoA-I for 8 h. In antisense-treated vs. sense-treated cells, the labeled cholesterol esterification was significantly increased about twofold irrespective of cholesterol efflux (Fig. 6). These results indicated that downregulated CETP synthesis increased esterification of cellular FC, which was not altered by efflux of the cellular cholesterol pool.

                              
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Table 2.   Cellular cholesterol changes after CETP antisense treatment



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Fig. 6.   Effect of CETP antisense treatment on cholesteryl ester synthesis in HepG2 cells. Cells were incubated with [1,2-3H]cholesterol for 24 h. After 12-h treatment with the indicated ODNs and after incubation (8 h) with apoA-I (10 µg/ml) or HDL (150 µg/ml) in 0.2% BSA and MEM, cells were harvested, lipids were extracted, and free cholesterol and cholesteryl ester were separated by TLC and quantified by liquid scintillation counting. Cholesteryl ester synthesis was expressed as cholesteryl esters as a percentage of free cholesterol. In antisense-treated vs. sense-treated cells, labeled cholesterol esterification was increased 1.8-fold at BSA phase, 2.1-fold at apoA-I phase, and 1.8-fold at HDL phase. Results are means ± SE of triplicate determinations. Similar data were obtained in a separate experiment.

SR-BI mRNA levels in subjects with CETP deficiency. Our studies suggested that suppressed CETP secretion is tightly connected with downregulated SR-BI in vitro. This prompted us to focus on SR-BI changes in subjects with CETP deficiency in ex vivo assay. Total RNA was extracted from mononuclear blood cells for SR-BI and CD14 RT-PCR. CD14 was selected as an internal control because of specific expression in monocytes (37). The SR-BI mRNA levels were 94.7 ± 3.9 in the normal group (means ± SE, n = 6), 61.7 ± 6.0 in the heterozygous group (n = 3), and 52.0 ± 7.6 in the homozygous group (n = 3), respectively. However, LDL-R mRNA levels did not differ among the three groups. When homozygotes, heterozygotes, and controls were included in the correlation analysis, the correlation coefficient (r) was -0.805 (P < 0.0001), with a strong inverse relation noted between SR-BI mRNA levels and HDL-C levels (Fig. 7).


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Fig. 7.   Relationship between SR-BI mRNA expression and HDL levels in subjects' mononuclear blood cells. Total RNA was extracted from human mononuclear blood cells, and RT-PCR was used to evaluate changes of SR-BI mRNA. mRNA expression levels were analyzed by means of the ratio of SR-BI to CD14 to Control. Median value in normal subjects was selected as 100%. Inset: A, a control subject; B, a CETP-deficient heterozygote; C, a CETP-deficient homozygote.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CETP is a plasma glycoprotein that mediates the transfer of neutral lipids among lipoproteins. New evidence indicates that CETP also mediates intracellular cholesterol homeostasis (24). We explored the effects of CETP on HDL and LDL metabolism via downregulated CETP expression using antisense ODNs in HepG2 cells. It was shown that CETP secretion is closely connected with cellular CE content and membrane receptor expressions of SR-BI and LDL-R, which influence HDL and LDL metabolism.

CETP mRNA is widely expressed in human tissues. CETP exists in two forms: a full-length form that is normally secreted and a truncated form derived from alternative splicing (23). Moreover, CETP has broad specificity for membrane surfaces and can transfer CE from biological membranes, including the endoplasmic reticulum (ER) (15). These findings are consistent with the idea that CETP might have a local function in lipid metabolism.

Differing from the circulating effect by which CETP transfers neutral lipids among lipoproteins, new evidence by Izem and Morton (24) indicates that CETP biosynthesis and cellular cholesterol homeostasis are tightly interconnected in a liposarcoma cell line. In that study, inhibition of CETP biosynthesis by antisense ODNs induced increased CE synthesis and decreased CE hydrolysis, indicating that a phenotype characterized by inefficient mobilization of CE stores led to CE accumulation. In the current study, in HepG2 cells, acute CETP depletion by antisense treatment leads to a mild increase in cellular CE mass. Increased CE could arise from increased esterification of FC and/or ineffective mobilization of CE for efflux. The latter possibility was tested by Izem and Morton. The results showed that CE accumulation is related to a defect in CE hydrolysis. We tested the primary possibility by prelabeling cells with cholesterol. The results indicated that the labeled cholesterol esterification was significantly increased about twofold irrespective of cholesterol efflux. Additionally, increased ACAT1 mRNA levels suggested that accumulation of cellular CE was partially related to increased CE synthesis via activated ACAT1-mediated esterification of FC. Moreover, FC mass was not changed in antisense-treated cells compared with sense-treated cells in our study. However, HMG-CoA reductase, an enzyme that is related to FC biosynthesis, was decreased at the transcriptional level. Thus CETP biosynthesis was tightly interconnected with cellular cholesterol metabolism in both hepatocytes and adipocytes, indicating that the relationship is irrespective of lipoprotein secretion. Furthermore, we show for the first time that CETP biosynthesis was also closely related to cholesterol flux, namely a decrease in selective uptake HDL3 cholesterol and an increase in cholesterol efflux to apoA-I and HDL3, as discussed below. CETP depletion changes in intracellular cholesterol homeostasis, the expressions of some lipid-related genes (Table 1), and cholesterol flux would explain the significant role of CETP in reverse cholesterol transport.

Plasma HDL provides protection against atherosclerosis by removing cholesterol from peripheral tissues and delivering cholesteryl esters to the liver for disposal as bile acids (reverse cholesterol transport). In the first step of this pathway, apoA-I accepts cholesterol and then converts it to HDL. We investigated the apoA-I secretion and efflux of cellular cholesterol in antisense-treated cells. It is thought that small, CE-poor pre-beta -HDL is the preferred acceptor of cellular cholesterol (25). In the current study, inhibition of CETP expression increased secretion of apoA-I and the apoA-I accumulation in the medium, which is compatible with the findings of Sawada et al. (41). Furthermore, mRNA levels of RXR-alpha , a factor of apoA-I transcriptional regulation (52), were found to have increased by 30% in our study. An increase in apoA-I facilitates the efflux of cellular cholesterol. On the other hand, cholesterol efflux to apoA-I and HDL3 was significantly increased in antisense-treated cells. Previous studies have demonstrated that ABCA1 expression increases apoA-I-mediated cholesterol efflux (49). However, the mRNA levels of ABCA1 were not changed by an increase in apoA-I secretion or cholesterol efflux to apoA-I in our study. Recently, ABCG1, like ABCA1, has been shown to regulate cellular cholesterol and phospholipid efflux (27). Thus we speculate that other ABC transporters, such as ABCG1, expressed in hepatocytes, may have a role in cholesterol efflux to apoA-I. Also, a possibility of increased stability of ABCA1 cannot be excluded. Taken together, the secretion of apoA-I and cellular cholesterol efflux to apoA-I were increased, suggesting that downregulated CETP expression facilitates HDL formation.

A reduction in SR-BI expression was observed as a consequence of the reduction in hepatic CETP expression, partly resulting in decreased selective uptake of [14C]cholesteryl oleate-HDL3. However, using a chemical inhibitor of CETP activity, we did not observe any changes in CETP or SR-BI expressions. Similarly, HDL-CE uptake by HepG2 cells was not affected by the presence of human plasma CETP alone in the medium (12). Thus, our findings strongly support our hypothesis, which is that the effect of blocking CETP biosynthesis on SR-BI is not via inhibition of plasma CETP activity. Previous studies have demonstrated that SR-BI expression was upregulated in response to depletion of cholesterol stores in apoA-I and hepatic lipase knockout mice (50) and in an adrenal cell line (Y1-BS1) (45). In contrast, rats given a 2-wk high-cholesterol diet had decreased SR-BI expression in hepatic parenchymal cells (11). These studies clearly indicated that SR-BI expression is modulated by feedback in response to changes in cellular cholesterol stores. SREBP-1 is a sterol transcription factor that induces SR-BI gene expression (32). In our present study, SREBP-1 was reduced at the transcriptional level after inhibition of CETP expression, indicating that it may be part of a negative feedback system related to an excessive cholesterol pool in the ER. It thus appears that HepG2 cells indeed reduce the expressions of SR-BI as a result of cholesterol loading via downregulated SREBP-1 during CETP inhibition.

Markedly elevated HDL concentrations and an increased diameter of HDL particles were seen in subjects with CETP deficiency (7). SR-BI has been clearly identified as an HDL receptor by a number of studies of cell and animal models. Overexpression of SR-BI was found to be associated with a marked decrease in plasma HDL cholesterol and accelerated catabolism of HDL and non-HDL cholesterol (29, 47). On the other hand, inactivated SR-BI expressions resulted in an increase in HDL particle size and an elevation in HDL-CE (38, 39). We examined the SR-BI expression of monocytes in subjects with CETP deficiency and found that SR-BI mRNA expression is significantly lower in subjects with CETP deficiency than in normal control subjects. The reduction in SR-BI expression may result in decreased HDL-CE catabolism, thereby partly accounting for increased levels of HDL-CE in subjects with CETP deficiency.

Another important receptor change was LDL-R, in which expressions were increased at mRNA and protein levels after CETP antisense treatment in HepG2 cells. However, in mononuclear blood cells of subjects with CETP deficiency, LDL-R mRNA expressions were not increased. The former results were compatible with those of an in vivo study by Sugano et al. (44). In their rabbit experiment, inhibition of CETP expression by means of antisense oligonucleotides resulted in elevated hepatic LDL-R mRNA levels. The relationship of increases in LDL-R and decreased levels of LDL was suggested by study of in vivo injection of isotope-labeled LDL in subjects with complete CETP deficiency who produced an increase in fractional catabolic rates of LDL (19). Upregulation of LDL-R may increase cholesterol uptake from medium, eventually inducing some indirect changes observed in gene expression (Fig. 3).

The mechanism of LDL-R regulation is complex. Not only cellular cholesterol levels but also an external signal, such as Ca2+ ionophores, would regulate LDL-R expression (3, 8). Like our results, in contrast to a decrease in the expression of the HMG-CoA reductase gene, Ca2+ channel blockers enhanced LDL-R expression via stimulated platelet-derived growth factor in human skin fibroblasts (6). These opposite expression patterns of LDL-R and HMG-CoA reductase were also found between LDL-R and SR-BI genes. Treatment with 17-ethinyl estradiol not only upregulated LDL-R but also downregulated SR-BI in liver parenchymal cells (11, 14). A more recent study showed that unsaturated fatty acids such as oleate increased cellular LDL-R activity by stimulation of cellular ACAT activity (40). There has been no report so far suggesting that CETP directly effects ACAT, although it is shown that ACAT-derived CE (cholesteryl oleate) was increased in VLDL-LDL of homozygous CETP deficiency (5). Our in vitro data are compatible with downregulated expression of hepatic LDL-R transcription in apoB/CETP double-transgenic mice (31). Thus we postulate that pathways other than SREBP-mediated sterol signaling can stimulate LDL-R expression after CETP antisense treatments. Downregulation of HMG-CoA reductase and SR-BI, possibly in response to an excess cholesterol pool in ER, can reduce cellular cholesterol via decreasing de novo cholesterol synthesis and cholesterol uptake, because upregulation of LDL-R increases cellular cholesterol by LDL-mediated cholesterol uptake. These opposite expression patterns by CETP may indicate that intracellular cholesterol traffic is altered and its balance is maintained by a tight feedback mechanism that prevents the overaccumulation of cholesterol to cytotoxic levels.

Although the role of CETP in the development of atherosclerosis is controversial, it is a major determinant of plasma HDL as well as LDL in humans (22). Genetic CETP deficiency results in elevated HDL levels and decreased LDL concentrations. Our studies may provide new insights into the molecular basis of the elevated HDL levels seen in CETP deficiency, via decreased HDL-CE catabolism and increased HDL formation in liver. However, CETP expression is relatively low in HepG2 cells, and further studies are needed to confirm the relevance of the current study in other hepatocyte models with higher CETP secretion or in CETP transgenic animal models (2).


    ACKNOWLEDGEMENTS

We thank Mihoko Mizuno and Yutaka Takino for their technical assistance.


    FOOTNOTES

This study was supported by Scientific Research Grants from the Ministry of Education, Science, and Culture in Japan (no. 09307010 to H. Mabuchi and no. 10770568 to A. Inazu).

Address for reprint requests and other correspondence: A. Inazu, Molecular Genetics of Cardiovascular Disorders, Division of Cardiovascular Medicine, Graduate School of Medical Science, Kanazawa Univ., Takara-machi 13-1, Kanazawa 920-8641, Japan (E-mail: inazua{at}mhs.mp.kanazawa-u.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published February 25, 2003;10.1152/ajpendo.00453.2002

Received 22 October 2002; accepted in final form 17 February 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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