Regulation of apolipoprotein A-I gene expression in Hep G2 cells depleted of Cu by cupruretic tetramine

John Y. J. Wu, Jin J. Zhang, Yenshen Wang, Scott K. Reaves, Yi Ran Wang, Polin P. Lei, and Kai Y. Lei

Department of Nutritional Sciences, University of Arizona, Tucson, Arizona 85721

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Studies were designed to examine the regulation of apolipoprotein (apo) A-I gene expression in Cu-depleted Hep G2 cells. The cupruretic chelator N,N'-bis(2-aminoethyl)-1,3-propanediamine · 4 HCl (2,3,2-tetramine or TETA) was used to maintain a 77% reduction in cellular Cu in Hep G2 cells. After two passages of TETA treatment, the relative abundance of apoA-I mRNA was elevated 52%. In TETA-treated cells, the rate of apoA-I mRNA decay measured by an actinomycin D chase study was accelerated 108%, and the synthesis of apoA-I mRNA determined by a nuclear runoff assay was enhanced 2.5-fold in TETA-treated cells. All of those changes could be reverted toward the control values with Cu supplementation for only 2 days. In transient transfection assays, a 26.7% increase in chloramphenicol O-acetyltransferase (CAT) activity for the reporter construct -256AI-CAT was observed in the treated cells. However, the ability of apoA-I regulatory protein 1 (ARP-1) to repress the CAT activity was not affected by the depressed Cu status. In addition, gel retardation experiments demonstrated that Cu depletion enhanced the binding of hepatocyte nuclear factor 4 (HNF-4) and other undefined nuclear factors to oligonucleotides containing site A, one of three regulatory sites of the apoA-I gene promoter. Moreover, the relative abundance of HNF-4 mRNA was increased 58% in the Cu-depleted cells. Thus the observed increase in apoA-I gene transcription may be mediated mostly by an elevated level of the regulatory factor, HNF-4. In summary, the present findings established the mechanism by which a depressed cellular Cu status can enhance apoA-I mRNA production and subsequently increase apoA-I synthesis.

copper depletion; hepatocyte nuclear factor 4; N,N'-bis(2-aminoethyl)-1,3-propanediamine · 4 HCl

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

HIGH-DENSITY LIPOPROTEIN (HDL) is involved in the process of "reverse cholesterol transport" (16), which returns excess cholesterol from peripheral tissues back to the liver for excretion. Apolipoprotein (apo) A-I, the major protein component of HDL, is inversely correlated with the risk of cardiovascular disease (3, 20). Thus one possible approach to reduce the risk of cardiovascular disease is to increase plasma levels of apoA-I, especially in subjects with low apoA-I levels. To accomplish this goal, the molecular mechanism(s) that enhances apoA-I expression must be established.

The apoA-I gene is expressed mainly in the liver and small intestine (7, 30), and its promoter sequence is highly conserved in the region of -240 to -15 in both humans and rats (21). Three sites within this region, namely site A (-214 to -192), site B (-169 to -146), and site C (-136 to -119), have been shown to regulate the expression of human apoA-I gene (8, 25). Site A has been established to be a positive and negative response element for the transcription factors retinoid X receptor alpha  and apoA-I regulatory protein 1 (ARP-1), respectively (14, 19), which are members of the steroid-thyroid receptor superfamily. Another member of this family, hepatocyte nuclear factor 4 (HNF-4), has been found to interact with site A (25) and with site C (4) to enhance activity of the apoA-I gene promoter. A number of factors have been shown to bind to site B (18).

Hypercholesterolemia is a well-established symptom, during diet-induced Cu deficiency, in rats and other rodent species, as well as in nonhuman primates (15). In Cu-deficient rats (1), the plasma HDL protein level is markedly increased. This elevation in plasma HDL protein is associated with a more than twofold increase in plasma apoA-I pool size (10) and in vivo hepatic apoA-I synthesis in Cu-deficient rats (10). Similarly, an enhanced synthesis and secretion of apoA-I have been observed in freshly isolated hepatocytes derived from rats deficient in Cu (10). In addition, a twofold increase in apoA-I synthesis has been reported in Hep G2 cells depleted of Cu by a cupruretic chelator, N,N'-bis(2-aminoethyl)-1,3-propanediamine · 4 HCl (2,3,2-tetramine or TETA) (31). Thus a depressed Cu status, brought about by the use of either diet or chelator, appears to enhance hepatic apoA-I synthesis.

The present studies were designed to examine the regulation of apoA-I gene expression in Cu-depleted Hep G2 cells. Enhanced apoA-I mRNA abundance, synthesis, and degradation were observed in the Cu-depleted cells. The use of a reporter construct, -256AI-CAT, in a transient transfection study demonstrated that the apoA-I gene promoter activity was enhanced in these cells. Moreover, the increase in apoA-I gene expression may be mediated by the increased HNF-4 mRNA abundance and the enhanced binding of HNF-4 and other unknown factors to site A of the apoA-I gene promoter. Because most of the observed changes were rapidly normalized by Cu repletion, the increase in apoA-I gene expression appeared to have resulted specifically from Cu depletion.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Actinomycin D, yeast tRNA, proteinase K, and all reagents used for cell culture were purchased from Life Technologies (Grand Island, NY). Hep G2 cells were purchased from the American Type Culture Collection (Rockville, MD) at passage 75. Protease inhibitors were purchased from Boehringer Mannheim (Indianapolis, IN). All restriction enzymes, the pGEM-3Z vector, the pCAT-Enhancer vector, the pSV-beta -galactosidase control vector, the Prime-a-Gene and 5'-end-labeling systems, as well as the beta -galactosidase enzyme and the chloramphenicol O-acetyltransferase (CAT) enzyme assay systems, were purchased from Promega (Madison, WI). Oligonucleotides were synthesized by the University Biotech Center (University of Arizona).

Cell culture. Hep G2 cells were grown as monolayers under 5% CO2 in the normal medium (minimum essential medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin). A cupruretic chelator, TETA (Kodak, Rochester, NY), was used to deplete cellular Cu (31). Experimental cells were cultured in the normal medium (control) or the normal medium containing 20 µM TETA. These two media contained a basal Cu concentration of 0.63 µM (0.04 mg/l), as measured by atomic absorption spectrophotometry (9), which was comparable to that found in most commonly used media. The treatments lasted for two passages for most experiments. In some experiments, cells previously cultured in the TETA medium were treated with the Cu repletion medium (TETA plus Cu), which was the same as the TETA medium except that CuSO4 was added to bring the Cu concentration of the medium to 1.56 µM (0.1 mg/l) for the last 2 days of the treatment. All experiments were performed at the end of the second passage unless otherwise indicated.

Determination of total cellular content of Cu and DNA. Cells were washed twice with phosphate-buffered saline (PBS) and harvested. Three 100-mm plates of cells from the same treatment group were pooled to provide one measurement. Cells were washed twice with PBS and centrifuged at 500 g for 5 min. The cell pellet was resuspended in PBS and sonicated. An aliquot of the sonicant was used to measure the cellular Cu content by atomic absorption spectrophotometry (9). Another aliquot was used for the measurement of the cellular DNA content (27). Total cellular contents of Cu per microgram DNA are presented, since a linear relationship was established between the amount of cellular DNA and cell number, regardless of treatments (data not shown).

Construction of plasmids. The human apoA-I genomic clone was kindly provided by Dr. Lawrence Chan (Baylor College of Medicine, Houston, TX). A 3-kilobase Hind III-Hind III fragment (-2500 to +397) was isolated and subjected to Sma I and Pst I restriction enzyme digestion, respectively, to obtain Sma I-Hind III (-256 to +397) and Pst I-Hind III (-41 to +397) fragments. These two fragments were blunt-ended. pCAT-Enhancer vector was linearized by Hind III enzyme digestion and was also blunt-ended. The two apoA-I gene promoter fragments were then ligated to the blunt-ended pCAT-Enhancer vector. Constructs containing inserts in the correct orientation were selected by restriction enzyme digestion, verified by DNA sequencing, and designated as -256AI-CAT and -41AI-CAT, respectively.

Preparation of cytoplasmic extracts and nuclei. Cells were washed twice with PBS and harvested. Two 100-mm plates of cells from the same treatment group were pooled. Cell pellets were then gently vortexed with 2 ml of ice-cold Nonidet P-40 (NP-40) lysis buffer [10 mM tris(hydroxymethyl)aminomethane (Tris) · HCl, pH 7.4, 10 mM NaCl, 5 mM MgCl2, and 0.5% NP-40]. Mixtures were incubated for 5 min on ice and then centrifuged. The supernatant, which represented the cytoplasmic extract, was immediately used for the isolation of the cytoplasmic RNA. The nucleus pellet was washed once more with NP-40 lysis buffer, resuspended in 100 µl of nucleus storage buffer (50 mM Tris · HCl, pH 8.3, 40% glycerol, 5 mM MgCl2, and 0.1 mM EDTA), and stored in liquid nitrogen until it was used for the nuclear runoff experiment.

cDNA and oligonucleotide probes. The human apoA-I cDNA was kindly provided by Dr. Lawrence Chan (Baylor College of Medicine), and the rat HNF-4 cDNA was a gift from Dr. Frances Sladek (University of California, Riverside, CA). The human albumin cDNA was purchased from the American Type Culture Collection. All cDNA inserts were subcloned into the pGEM-3Z vector. Large scale preparations of plasmid DNA were performed with Qiagen-tip 500 column (Qiagen, Chatsworth, CA), according to the manufacturer's instructions.

For Northern blot analysis, a 900-base pair Pst I fragment was isolated from the human apoA-I cDNA, and a Pvu I-Cla I fragment (nucleotides 226-873) was purified from the rat HNF-4 cDNA. Both fragments were labeled with [alpha -32P]dCTP using the Prime-a-Gene labeling kit. The 18S oligonucleotide (5'-CAC CTC TAG CGG CGC AAT AC-3') (17) was 5'-end labeled with [gamma -32P]ATP and used to normalize the amount of RNA loaded.

For the nuclear runoff assay, the human apoA-I, albumin, and empty pGEM-3Z plasmid were linearized by EcoR I digestion. Ten micrograms of various DNA probes were denatured in NaOH (0.2 M final concentration) at room temperature for 10 min. The mixture was diluted in 10 vol of 6× saline sodium citrate buffer and blotted onto a nitrocellulose filter.

For the gel retardation assay, single-stranded site A oligonucleotides (the sense strand, 5'-ACT GAA CCC TTG ACC CCT GCC CT-3', and the antisense strand, 5'-AGG GCA GGG GTC AAG GGT TCA GT-3'), corresponding to the human apoA-I promoter sequence from -214 to -192 (25), were synthesized. Double-stranded oligonucleotides were formed by annealing the two single-stranded oligonucleotides in TES buffer (in mM: 10 Tris · HCl, pH 7.8, 1 EDTA, and 100 NaCl) and 5'-end labeled with [gamma -32P]ATP using T4 polynucleotide kinase. The labeled double-stranded site A oligonucleotides were adjusted with TES buffer to a final concentration of 0.1 ng/µl and used within 1 wk.

RNA isolation and Northern blot analysis. Total cellular RNA was isolated by the method of Chomczynski and Sacchi (5). This method was modified for the isolation of cytoplasmic RNA by mixing the freshly prepared cytoplasmic extract with an equal volume of prewarmed (65°C) 2× guanidinium thiocyanate denaturing solution (8 M guanidinium thiocyanate, 50 mM sodium citrate, pH 7.0, 1% sarkosyl, and 0.2 M 2-mercaptoethanol). The rest of the procedures were the same for the total or cytoplasmic RNA isolation. Northern blot analysis was carried out by using 20 µg of RNA/lane as previously described (10). Blots were first hybridized with either apoA-I or HNF-4 cDNA probes, autoradiographed, and then stripped. No carryover signal was observed even after 1 wk of exposure following stripping. The same blots were then hybridized with 18S probe. Band intensities of autoradiographs were quantified by a laser densitometer (Molecular Dynamics, Sunnyvale, CA). The relative mRNA abundance in the samples was expressed as arbitrary units of the mRNA (apoA-I or HNF-4) band per arbitrary unit of the 18S band of the same sample.

Determination of apoA-I mRNA stability. Stability of apoA-I mRNA was estimated by actinomycin D chase, according to the method of Semenkovich et al. (22). Cells were treated for two passages as described above. For the Cu repletion treatment, Cu supplementation was started 3 days before confluency was expected in the second passage. One day before the expected confluency, the media were removed and the cells were rinsed twice with their respective treatment media without fetal bovine serum. Three plates from each treatment group were harvested at this time point to represent time zero of the actinomycin D chase. Then the remaining plates of cells were exposed to their respective treatment media, with 5 µg/ml actinomycin D and 3% bovine serum albumin and without fetal bovine serum. The concentration of actinomycin D used here was found to be effective in inhibiting the RNA synthesis in cultured Hep G2 cells (22) and exerted no adverse effect on cell viability (data not shown). At 8, 16, 24, and 48 h of the actinomycin D chase, three plates from each treatment group were harvested. The total cellular RNA was isolated and analyzed by Northern blot analysis. Relative levels of apoA-I mRNA were estimated as the ratio of apoA-I mRNA to 18S rRNA and plotted against the time of actinomycin D chase for each treatment. The apoA-I mRNA half-life was determined by the equation Half-life = 0.693 / [2.303 × slope of line (log10 mRNA vs. time)], as described by Semenkovich et al. (22).

Nuclear runoff transcription assays. Nucleus samples isolated as described above were thawed at room temperature; 100 µl of nuclei were mixed with 100 µl of runoff reaction buffer [in mM: 10 Tris · HCl, pH 8.0, 5 MgCl2, 300 KCl, 5 1,4-dithiothreitol (DTT), and 0.5 each of ATP, CTP, GTP, as well as 150 µCi of [alpha -32P]UTP (3,000 Ci/mmol, NEN) and 1 µl of RNasin] and incubated at 30°C for 30 min. The isolation of 32P-labeled RNA, hybridization, and washing were carried out as described by Distel et al. (6), except that 20 × 106 counts/min of radioactively labeled RNA were used in each assay. The filters were autoradiographed, and the band intensities were quantitated by a laser densitometer. The transcription rates for the apoA-I gene were expressed relative to those observed for the albumin gene.

Transfection and CAT assays. At the end of the second passage of TETA treatment, cells were split at 2 × 106 cells/100-mm plate for transfection. DNA transfection was performed according to the calcium-phosphate coprecipitation method, as described by Higuchi et al. (8). The various types of DNA used for transfection were 3 µg of pSV-beta -galactosidase control vector, 5 µg of reporter construct (-41AI-CAT or -256AI-CAT), 2 µg of pMT2-ARP-1 (14) where indicated, and pMT2 (13), which was used to make up a total of 15 µg DNA. Transfected cells were harvested 48 h after glycerol shock, resuspended in reaction buffer (250 mM Tris · HCl, pH 7.8, 5 mM EDTA), and frozen in a dry ice-ethanol bath. Cells were lysed by three quick freeze-thaw cycles and cell lysates were collected by centrifugation. Both beta -galactosidase and CAT activities were measured, by using the beta -galactosidase enzyme assay system and CAT enzyme assay system, respectively. The activity of individual apoA-I CAT construct was normalized to its corresponding beta -galactosidase activity and expressed as CAT activity/beta -galactosidase activity.

Gel retardation assays. At the end of the second passage of treatment, cells were harvested, washed twice with PBS, resuspended, and lysed by three cycles of quick freeze-thaw in lysis buffer [20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-KOH, pH 7.8, 100 mM KCl, 20% glycerol, 0.2 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml of aprotinin, leupeptin, and pepstatin]. Cellular debris were removed by centrifugation. Cell extracts were divided into aliquots and stored in liquid nitrogen. Various amounts of whole cell extracts were incubated with 10 mM HEPES-KOH, pH 7.9, 50 mM KCl, 1 mM EDTA, 7% glycerol, 1 mM DTT, 1 ng of unrelated single-stranded oligonucleotide, and 1.25 µg poly(dI-dC) at room temperature for 10 min. 32P-labeled site A probe (2 µl at 0.1 ng/µl) was then added, and the mixture was further incubated for 20 min. To establish the reaction specificity, a 100-fold excess of unlabeled site A oligonucleotides or unrelated transthyretin (TTR; -175 TCG ACC GAT ACT CTA ATC TCC CTA GGC) oligonucleotides (23) were added 10 min after the addition of 32P-labeled site A probe in some experiments. When antibodies were used, they were added 10 min after the addition of the probe [chicken ovalbumin upstream promoter-transcription factor (COUP-TF); this antiserum was a kind gift from Dr. M. J. Tsai, Baylor College of Medicine; HNF-4 antiserum was a kind gift from Dr. F. M. Sladek, University of California, Riverside]. Reaction mixtures were subjected to nondenaturing polyacrylamide gel electrophoresis as described by Widom et al. (25). Gels were dried and autoradiographed. The band intensity was quantitated by a laser densitometer.

Data analyses. One-way analysis of variance was used to analyze the following measurements: the cytoplasmic apoA-I mRNA, the promoter activities of apoA-I gene, the transcriptional rate of apoA-I gene, the cytoplasmic HNF-4 mRNA abundance, and the cellular Cu contents. Treatment means were further separated by Duncan's new multiple range test (28). The total cellular apoA-I mRNA decay curves were constructed by plotting the relative amount of apoA-I mRNA remaining within the cells at a specific time of the actinomycin D chase against the time of actinomycin D chase. These data were analyzed by two-way analysis of variance (28).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

TETA-mediated depletion of cellular Cu. TETA was found to be effective in the depletion of Cu from cultured Hep G2 cells in previous studies (31). In the present study, a 77% reduction in cellular Cu content was attained after a two-passage treatment with 20 µM TETA (TETA vs. control, 70.5 ± 11.3 vs. 306.0 ± 40.0 pg Cu/µg DNA; n = 4; P < 0.01). A similar magnitude of reduction was maintained for at least two more passages thereafter (data not shown). For comparison with previous observations, all experiments were performed at the end of the second passage, except for the transfection assays, which were initiated at the beginning of the third passage. A separated group of TETA-treated cells were cultured with the medium containing 1.56 µM Cu in the presence of same level of TETA (20 µM) for the last 2 days of the second passage. Such limited Cu repletion (TETA plus Cu) elevated the cellular Cu level to 176% (124.3 ± 8.8 pg Cu/µg DNA) of that of unrepleted TETA-treated cells. However, the cellular Cu content in the Cu-repleted cells was still significantly lower than that of the control cells. A prolonged period of Cu repletion (4 days) also failed to completely restore the Cu content to the normal level (data not shown), indicating that such limited Cu repletion could not fully overcome the Cu-depletion effect of TETA.

Elevation of cytoplasmic apoA-I mRNA abundance in Cu-depleted cells. TETA treatment resulted in a twofold increase in the synthesis of apoA-I protein, but not albumin or total protein, in Hep G2 cells (31). Such elevated apoA-I protein synthesis was possibly due to a specific increase in the cellular apoA-I mRNA level. To address such a possibility, the effect of TETA on cytoplasmic apoA-I mRNA abundance was examined by Northern blot analysis. Two passages of TETA treatment increased the apoA-I mRNA level (Fig. 1A) but exerted no effect on the cytoplasmic levels of 18S rRNA (Fig. 1A) or albumin mRNA (data not shown). The relative abundance of cytoplasmic apoA-I mRNA (normalized to 18S) in the TETA-treated cells was found to be 52% higher (P < 0.05) than the control cells (Fig. 1B). Thus TETA treatment resulted in the elevation of apoA-I mRNA level in Hep G2 cells.


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Fig. 1.   Effect of 20 µM N,N'-bis(2-aminoethyl)-1,3-propanediamine · 4 HCl (TETA) treatment for 2 passages on cytoplasmic apolipoprotein (apo) A-I mRNA abundance in Hep G2 cells. Cytoplasmic RNA was isolated at end of second passage. mRNA abundance was estimated by Northern blot analysis. A: representative lanes. B: autoradiographic signals from Northern blot analysis were quantitated by densitometric scanning. AU, arbitrary units. Values are means ± SE from 4 experiments. Data were analyzed by 1-way analysis of variance (ANOVA). * Significant treatment difference, P < 0.05.

Decay of apoA-I mRNA was enhanced by Cu depletion but normalized by Cu repletion. The increase in apoA-I mRNA steady-state level in Cu-depleted cells could be due to an enhanced mRNA stability, an increased transcription rate, or both. To determine whether the stability of apoA-I mRNA was altered by TETA treatment, a chase study was performed using actinomycin D, an inhibitor of new transcription. The mRNA decay curves were constructed by plotting the cellular apoA-I mRNA remaining at a given time during the actinomycin D chase against the time of chase (Fig. 2). Compared with control cells, TETA-treated cells possessed a steeper decay curve, indicating a faster mRNA decay. The slope of the decay curve for the TETA plus Cu cells was similar to that of the control cells, which demonstrated that the Cu repletion effectively normalized the apoA-I mRNA decay rate. The half-lives of apoA-I mRNA for the control, TETA, and TETA plus Cu treatments were found to be 44.5 ± 4.8, 21.4 ± 2.1, and 56.5 ± 9.0 h, respectively (means ± SE; n = 3; significant difference between TETA and other 2 values, P < 0.05). TETA treatment induced a 52% reduction in the half-life of apoA-I mRNA compared with control cells, whereas 2 days of Cu repletion completely normalized the apoA-I mRNA decay rate. In addition, at time zero of the actinomycin D chase, the abundance of apoA-I mRNA represented the steady-state level of total cellular apoA-I mRNA in the various treatments. The total cellular apoA-I mRNA level was found to be higher in TETA-treated cells than in control cells (Fig. 2), which was consistent with the cytoplasmic apoA-I mRNA abundance results depicted in Fig. 1. Furthermore, 2 days of Cu repletion reduced and restored the total cellular apoA-I mRNA abundance to that observed for the control cells (Fig. 2).


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Fig. 2.   Effect of 20 µM TETA treatment for 2 passages and subsequent Cu repletion on total cellular apoA-I mRNA decay in Hep G2 cells. A: control cells. B: cells treated with TETA. C: cells treated with TETA + Cu. Cells were cultured for 2 passages as described in MATERIALS AND METHODS. At various times of actinomycin D (AD) chase, total cellular RNA was isolated. Levels of apoA-I mRNA remaining in cells during AD chase were measured by Northern blot analysis. Relative abundance of apoA-I mRNA was plotted against time of AD chase. Values are means ± SE from 3 experiments. Data were analyzed by 2-way ANOVA; Cu effect (2 degrees of freedom), P < 0.0001; time effect (4 degrees of freedom), P < 0.0001; interaction (Cu × time), nonsignificant (P > 0.05).

Transcription of apoA-I gene was enhanced by Cu depletion but normalized by Cu repletion. The accelerated decay of apoA-I mRNA in TETA-treated cells contradicted our initial hypothesis that an enhanced apoA-I mRNA stability contributed to the increased apoA-I mRNA level in TETA-treated cells. Therefore, the elevated apoA-I mRNA level in TETA-treated cells was most likely due to an increase in mRNA synthesis. A nuclear runoff study was conducted to examine whether apoA-I gene transcription may be enhanced by Cu depletion. Because neither the synthesis of albumin protein (31) nor the albumin mRNA level (data not shown) was altered by TETA treatment in Hep G2 cells, the albumin was used as an internal reference. Both apoA-I and albumin cDNA were inserted into the pGEM-3Z vector; therefore, the empty pGEM-3Z vector was used as the negative control for the nonspecific hybridization. Figure 3A depicts the representative results of the nuclear runoff assay. In slots with the empty pGEM-3Z vector, there were no significant signals, indicating that the nonspecific background could be ignored and signals of apoA-I or albumin mRNA must be specific. The relative transcription rate for apoA-I gene (normalized to that of albumin) was elevated 151% in TETA-treated cells compared with control cells, whereas 2 days of Cu repletion almost normalized the transcription rate toward that of control cells (Fig. 3B). Indeed, transcription rates in the control cells and in the TETA plus Cu cells were not statistically different. Similar results were also observed in a separate study (data not shown; n = 3).


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Fig. 3.   Effect of 20 µM TETA treatment for 2 passages and subsequent Cu repletion on transcription rates of apoA-I gene in Hep G2 cells. Cells were cultured for 2 passages, and nuclei were isolated as described in MATERIALS AND METHODS. Transcription rates were estimated by a nuclear runoff assay. Human albumin cDNA and pGEM-3Z vector were used as normalization reference and nonspecific hybridization control, respectively. A: representative signals. B: autoradiographic signals were quantitated by densitometric scanning. Values are means ± SE from 3 experiments. Data were analyzed by 1-way ANOVA, and means with different letters, a or b, were significantly different (P < 0.05). Similar results were also observed in another study (n = 3 for each treatment).

Activity of apoA-I gene promoter was enhanced by Cu depletion. Transient transfection assays were performed to establish the influence of Cu status on apoA-I gene promoter activity. The promoter sequence for Hep G2-specific expression has been defined to be located between -256 and -41 upstream of the apoA-I gene transcription initiation site (21). Reporter constructs -256AI-CAT and -41AI-CAT were transfected into control and TETA-treated cells. A 27.4% increase in CAT activity (normalized to beta -galactosidase activity) for reporter construct -256AI-CAT was observed in TETA-treated cells compared with the untreated controls (Fig. 4). In contrast, the transfection of reporter construct -41AI-CAT produced essentially background levels of CAT activity in both control and TETA-treated cells (Fig. 4). The transcription factor ARP-1 has been reported to repress apoA-I gene expression in Hep G2 cells (14). To examine whether TETA treatment may exert a direct effect on the ability of ARP-1 to repress apoA-I expression, transfection assays were also performed in the presence of ARP-1 expression vector. Cotransfection with ARP-1 expression vector repressed the CAT activity for the reporter construct -256AI-CAT in both control and TETA-treated cells. However, ARP-1 cotransfection did not alter the induction of CAT activity caused by TETA treatment (Fig. 4). Thus TETA treatment did not exert a direct effect on the ability of ARP-1 to repress apoA-I gene expression.


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Fig. 4.   TETA treatment in Hep G2 cells enhances apoA-I gene promoter activity. Reporter construct -256AI-CAT (5 µg) was transfected into control or TETA-treated Hep G2 cells (2 passages) in presence (+) or absence (-) of 3 µg apoA-I regulatory protein 1 (ARP-1) expression vector. Reporter construct -41AI-CAT containing only basal promoter of apoA-I gene was used as a control. Normalized chloramphenicol O-acetyltransferase (CAT) activity was expressed as CAT activity/beta -galactosidase activity in extracts from these cells. Transfection study was performed in 3 experiments.

Cu depletion enhanced the binding of transcription factors to site A of the apoA-I promoter. Site A is a major regulatory element in the apoA-I gene promoter, and potential factors targeting site A of the apoA-I gene promoter have been identified (26). To establish that the enhanced apoA-I gene transcription and CAT activity of -256AI-CAT reporter construct associated with TETA treatment may have resulted from an altered ability of various transcription factors to bind to site A, gel retardation assays were conducted. Figure 5 depicts the autoradiograph of a typical gel retardation assay. When increasing amounts of cell extracts (3, 6, 12, 24, and 48 µg protein) were used, almost linear increases in the amount of site A probe being shifted were observed for both control and TETA treatment (Fig. 5, lanes 1-5 and 6-10, respectively). The binding of protein factors to site A was found to be specific, since the binding was abolished by the addition of 100-fold excess unlabeled site A oligonucleotides (Fig. 6, lanes 2 and 7) but not by the unrelated double-stranded oligonucleotides TTR (data not shown). Compared with those observed in controls, equal amounts of cell extracts derived from the TETA-treated cells exhibited binding activities at least twofold higher. Nearly identical results were obtained from two other independent experiments (data not shown). Thus TETA treatment increased the amount of site A factors, in Hep G2 cells, binding to site A. 


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Fig. 5.   Comparison of binding activity of whole cell extracts from control and TETA-treated Hep G2 cells to site A probe, using gel retardation assays. Preparation of whole cell extracts and gel retardation assays were described in MATERIALS AND METHODS. Lanes 1-5, increasing concentrations of control cell extracts (3, 6, 12, 24, and 48 µg proteins, respectively); lanes 6-10, increasing concentrations of extracts from TETA-treated cells (3, 6, 12, 24, and 48 µg protein, respectively). P, protein/site A probe complex; NS, nonspecific binding; FP, free site A probe. Similar results were obtained from 2 other independent studies.


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Fig. 6.   Comparison of hepatocyte nuclear factor 4 (HNF-4) and ARP-1/COUP-TF binding activities in control or TETA-treated Hep G2 cells. Whole cell extracts from control (lanes 1-5) and TETA-treated (for 2 passages; lanes 6-10) Hep G2 cells were used in gel retardation assays under various conditions. Lanes 1 and 6, cell extracts only; lanes 2 and 7, 100-fold excess of unlabeled site A oligonucleotides; lanes 3 and 8, presence of antiserum against HNF-4 (Ab-H); lanes 4 and 9, presence of antiserum against COUP-TF (Ab-C); lanes 5 and 10, presence of both antisera. Similar results were obtained from 2 other independent studies.

Elevations in the amount of HNF-4 binding to site A and cytoplasmic mRNA abundance of HNF-4 in Cu-depleted cells. Several members of the steroid-thyroid hormone receptor superfamily, which includes ARP-1 and HNF-4, have been implied to be involved in the regulation of apoA-I gene expression (12). To investigate whether they are influenced by TETA treatment in Hep G2 cells, gel retardation assays were conducted using antibodies against HNF-4 and COUP-TF. The anti-HNF-4 antibody interacted specifically with HNF-4, whereas the anti-COUP-TF antibody interacted with all of the members of the COUP-TF family, including ARP-1. As depicted in Fig. 6, the binding of cell extracts to site A was specific and could be competed against by 100-fold excess of unlabeled site A oligonucleotides (lane 1 vs. lane 2, lane 6 vs. lane 7) but not by unrelated oligonucleotides (data not shown). Both control and TETA-treated cells appeared to contain HNF-4 and members of COUP-TF-ARP-1 family, because, in the presence of their corresponding antibodies, supershifted complexes of antibody/specific nuclear protein/site A were detected. The double supershift assays (lanes 5 and 10), in which both antibodies were present, revealed that there were still significant amounts of unshifted protein/site A complex, which implied the presence of other factors capable of binding to site A. Although the protein/DNA complex shifted by COUP-TF antibody was only slightly higher in TETA-treated cells than in control cells, the amount of protein/DNA complex shifted by HNF-4 antibody was more than twofold higher (n = 3; P < 0.05) in TETA-treated cells (213 ± 17.9%) than in control cells (100 ± 13.1%), indicating that a higher level of HNF-4 protein was present in TETA-treated cells.

To confirm the contention that the level of HNF-4 protein was elevated by TETA treatment, which may in turn increase the apoA-I mRNA transcription, the steady-state level of HNF-4 mRNA was determined. Results from Northern blot analysis are depicted in Fig. 7A. The mRNA abundance of HNF-4 was clearly increased in the TETA cells, whereas the 18S reference was the same in both groups. A 58% increase in HNF-4 mRNA was observed in the TETA-treated cells compared with control cells (Fig. 7B).


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Fig. 7.   Effect of 20 µM TETA treatment for 2 passages on cytoplasmic HNF-4 mRNA abundance in Hep G2 cells. Cytoplasmic RNA was isolated at end of second passage. A: mRNA abundance was estimated by Northern blot analysis. B: values are means ± SE from 3 experiments. Data were analyzed by 1-way ANOVA. * Significant treatment difference, P < 0.05.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Increases in apoA-I production and secretion have been previously reported in Hep G2 cells depleted of Cu by TETA (31). The results of the present studies indicated that apoA-I mRNA abundance was elevated 52% in Hep G2 cells after a two-passage TETA treatment. We initially hypothesized that the increased apoA-I mRNA abundance may have resulted from either enhanced mRNA stability or elevated transcription. However, data presented in Fig. 2 suggest that TETA treatment reduced the stability of apoA-I mRNA. To induce the elevated apoA-I mRNA steady-state level, a higher magnitude of increase in transcription was expected to overwhelm the accelerated mRNA decay. Indeed, the transcription rate of apoA-I gene was found to be elevated 151% in Cu-depleted cells compared with control cells (Fig. 3). Thus the enhanced apoA-I gene transcription is the primary mechanism that led to the increase of steady-state apoA-I mRNA level in TETA-treated cells. In addition, both the elevated transcription rate and the accelerated mRNA decay found in the TETA cells reverted toward the control levels after 2 days of limited Cu repletion (Figs. 2 and 3). Because the repletion medium contained the same amount of TETA as the TETA medium, the alterations in transcription rate and mRNA decay rate appeared to be specifically related to the changes in medium Cu level and not to other adverse effects of TETA treatment. Although 2 days of Cu repletion caused only a small increase in cellular Cu level, which was still lower than the control value, the limited Cu repletion may have restored the Cu level in the cells or in a specific intracellular pool to above the critical level. Thus the depressed cellular Cu status associated with TETA treatment appeared to specifically enhance the turnover of apoA-I mRNA.

Simultaneous regulations of apoA-I mRNA synthesis and stability may not be unusual. Tam and Deeley (24) reported a fourfold increase in apoA-I mRNA level and an eightfold increase in apoA-I protein synthesis in Hep 3B cells treated with phenobarbital. Such a marked increase in apoA-I mRNA abundance was found to be mediated synergistically by the combination of a 2-fold increase in the transcription rate and a 1.6-fold increase in the mRNA half-life. However, in the present study, the changes in transcription and mRNA stability appeared not to contribute synergistically to the increase in apoA-I mRNA abundance. One possible explanation is that the decay of apoA-I mRNA in TETA-treated cells may be accelerated in response to the increase in apoA-I transcription as a normal compensatory mechanism. If this hypothesis is true, the degradation of apoA-I mRNA should be normalized whenever the steady-state abundance of apoA-I is returned to the tolerable range. In the present study, the apoA-I transcription rate of Cu-repleted cells was restored to that observed in the control cells (Fig. 3). Meanwhile, the mRNA half-life was normalized and even appeared to be slightly higher (not significant) in the Cu-repleted cells compared with the control cells (mRNA half-life, 56.5 ± 9.0 h for repleted vs. 44.5 ± 4.8 h for control). The alternative explanation is that enhancements of both apoA-I transcription and mRNA decay are relatively independent consequences of Cu depletion and result from an overall increase in cellular metabolism in response to the low-Cu status.

In TETA-treated cells, the synthesis of apoA-I protein was increased more than twofold in Cu-depleted Hep G2 cells (31). However, the apoA-I mRNA level was only increased ~1.5-fold (Fig. 1). This disparity suggested that a cotranslational control may be involved. Indeed, Cu deficiency appeared to enhance the hepatic apoA-I mRNA translational efficiency in rats (29). In the liver of Cu-deficient rats, higher percentages of cytoplasmic apoA-I mRNA were recovered from larger polysomal mRNA fractions that are considered to be translationally more active. This may have resulted from the observed increase in transcription of apoA-I mRNA, since freshly produced mRNA may have a higher affinity for ribosomes. In addition, higher percentages of hepatic cytoplasmic apoA-I mRNA were also recovered in the translationally less active ribosome-free fractions in Cu-deficient rats (29). This observation supports the contention that apoA-I mRNA degradation is accelerated by Cu deficiency, since the dissociation from ribosomes is a prerequisite step for mRNA to be degraded. Therefore, the accelerated apoA-I mRNA turnover observed in Cu-depleted Hep G2 cells provides a possible explanation for the observed enhancement of apoA-I gene expression in Cu-deficient rat liver.

Recently, the promoter region of apoA-I gene has been extensively examined by a number of research teams. Within the proximal (starting from -256) promoter, which was known to be sufficient to support the Hep G2-specific expression of apoA-I gene, several regulatory regions (sites A, B, and C) have been identified (12). A number of transcription factors were found to interact with these sites. HNF-4 was found to be an activator for apoA-I gene expression on its binding to the regulatory site A of the apoA-I gene promoter. ARP-1, a member of COUP-TF family, was established to be a transcription inhibitor. In the current study, Cu-depleted cells exhibited at least a twofold higher total binding activity of transcription factors to site A compared with the control cells (Fig. 5). The binding activity of HNF-4 to site A, as revealed in the supershifted band by using the HNF-4-specific antibody, was also increased twofold. In addition, the cytoplasmic mRNA abundance of HNF-4 was elevated 1.6-fold in Cu-depleted cells (Fig. 7). These observations strongly suggested that Cu depletion may induce a higher HNF-4 binding activity and promote apoA-I gene expression, although the mechanism responsible is yet to be established. In contrast, the binding activity of the COUP-TF family (including ARP-1) to site A was only slightly influenced by the Cu depletion (Fig. 6). In the transient transfection assays, the over-expression of ARP-1 depressed the -256AI-CAT promoter activity but failed to alter the stimulatory effect induced by Cu depletion (Fig. 4). These observations indicated that the regulation of apoA-I gene expression by Cu status was unlikely to be directly mediated by ARP-1. The binding activity of unidentified factors to site A may also be elevated in Cu-depleted cells, as revealed in Fig. 6. Thus the depressed cellular Cu status appeared to enhance the binding of HNF-4 and possibly certain other transcription factors, but not ARP-1, to site A of apoA-I gene promoter and in turn stimulated apoA-I gene expression.

The present data indicate that the depressed cellular Cu status stimulated apoA-I gene transcription as well as accelerated the decay rate and turnover of apoA-I mRNA that subsequently led to a new steady-state level. However, this mechanism may not fully apply to the regulation of hepatic apoA-I gene expression in the diet-induced Cu-deficient rat model. Nevertheless, marked increases in hepatic apoA-I synthesis were observed in Cu-depleted Hep G2 cells and Cu-deficient rats (10, 31). The increase in hepatic apoA-I synthesis was associated with hypercholesterolemia, elevated HDL, and increased use of fat as the major energy substrate in Cu-deficient rats (11). In view of the established functional diversity of HDL and the concept of HDL functional metabolic states (12), the observed increase in hepatic apoA-I synthesis and circulating HDL may have enhanced the transport of lipids to peripheral tissues and of cholesterol from peripheral tissues back to the liver (2). Subsequently, this accelerated flux of substrates from the liver to peripheral tissues may have contributed to the increased utilization of fat as the major energy substrate. Regardless of the biological consequences of the enhanced apoA-I synthesis, additional studies must be performed to establish whether apoA-I gene expression is influenced by cellular Cu status directly through responsive promoter sites or indirectly through changes in metabolites or hormones.

    ACKNOWLEDGEMENTS

We thank Dr. L. Chan (Baylor College of Medicine, Houston, TX) for providing the apoA-I cDNA and genomic DNA, Dr. F. M. Sladek (University of California, Riverside, CA) for providing the HNF-4 cDNA and antisera, and Dr. M.-J. Tsai (Baylor College of Medicine) for providing the COUP-TF antisera.

    FOOTNOTES

This work was partially supported by United States Department of Agriculture National Research Initiative Grant 92-01045 and funds from the University of Arizona Agricultural Experiment Station.

Current address of  Y. R. Wang: Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267.

Address for reprint requests: K. Y. Lei, Dept. of Nutritional Sciences, 309 Shantz Bldg., University of Arizona, Tucson, AZ 85721.

Received 18 February 1997; accepted in final form 24 June 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Al-Othman, A. A., F. Rosenstein, and K. Y. Lei. Copper deficiency alters plasma pool size, percent composition, and concentration of lipoprotein components in rats. J. Nutr. 122: 1199-1204, 1992[Medline].

2.   Carr, T. P., and K. Y. Lei. High density lipoprotein cholesteryl ester and protein catabolism in hypercholesterolemic rats induced by copper deficiency. Metabolism 39: 518-524, 1990[Medline].

3.   Castelli, W. P., R. J. Garrison, P. F. Wilson, R. D. Abbott, S. Kalousdian, and W. B. Kannel. Incidence of coronary heart-disease and lipoprotein cholesterol level: the Framingham study. J. Am. Med. Assoc. 256: 2835-2838, 1986[Abstract].

4.   Chan, J., H. Nakabayashi, and N. C. W. Wong. HNF-4 increases activity of the rat apo A-I gene. Nucleic Acids Res. 21: 1205-1211, 1993[Abstract].

5.   Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

6.   Distel, R. J., G. S. Robinson, and B. M. Spiegelman. Fatty acids regulation of gene expression. J. Biol. Chem. 269: 5937-5941, 1992[Abstract/Free Full Text].

7.   Elshourbagy, N. A., M. S. Boguski, W. S. L. Liao, J. S. Jefferson, J. I. Gordon, and J. M. Taylor. Expression of rat apolipoprotein A-IV and apolipoprotein A-I genes. Messenger-RNA induction during development and in response to glucocorticoids and insulin. Proc. Natl. Acad. Sci. USA 82: 8242-8246, 1985[Abstract].

8.   Higuchi, K., S. W. Law, J. M. Hoeg, U. K. Schumacher, N. Meglin, and H. B. Brewer, Jr. Tissue-specific expression of apolipoprotein A-I (apo A-I) is regulated by the 5'-flanking region of the human apo A-I gene. J. Biol. Chem. 263: 18530-18536, 1988[Abstract/Free Full Text].

9.   Hitachi, Ltd.. Instruction Manual for Model 180-70 Polarized Zeeman Atomic Absorption Spectrophotometer. Mountain View, CA: Nissei Sanyo America, Hitachi, 1980.

10.   Hoogeveen, C. A. J. M., S. K. Reaves, and K. Y. Lei. Copper deficiency increases hepatic apolipoprotein A-I synthesis and secretion but does not alter hepatic total cellular apolipoprotein A-I mRNA abundance in rats. J. Nutr. 125: 2935-2944, 1995[Medline].

11.   Hoogeveen, C. A. J. M., S. K. Reaves, P. M. Reid, B. L. Reid, and K. Y. Lei. Copper deficiency shifts energy substrate utilization from carbohydrate to fat and reduces percent body fat mass in rats. J. Nutr. 124: 1660-1666, 1994[Medline].

12.   Karathanasis, S. K. Apolipoprotein AI gene regulation by members of the steroid/thyroid hormone receptor superfamily of ligand dependent transcription factors. In: High Density Lipoprotein and Atherosclerosis III, edited by N. E. Miller, and A. R. Tall. Amsterdam: Elsevier, 1992, p. 21-31.

13.   Kaufman, R. J., M. V. Davies, V. Pathak, and J. W. B. Hershey. Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus. Mol. Cell. Biol. 9: 946-958, 1989[Medline].

14.   Ladias, J. A. A., and S. K. Karathanasis. Regulation of the apolipoprotein AI gene by ARP-1, a novel member of the steroid receptor superfamily. Science 251: 561-565, 1991[Medline].

15.   Lei, K. Y. Plasma cholesterol response in copper deficiency. In: Role of Copper in Lipid Metabolism, edited by K. Y. Lei, and T. P. Carr. Boca Raton, FL: CRC, 1990, p. 1-23.

16.   Miller, N. E., A. Laville, and D. Crook. Direct evidence that reverse cholesterol transport is mediated by high-density lipoprotein in rabbit. Nature 314: 109-111, 1985[Medline].

17.   Omiecinski, C. J., C. A. Redlich, and P. Costa. Induction and developmental expression of cytochrome P450IA1 messenger RNA in rat and human tissues: detection by the polymerase chain reaction. Cancer Res. 50: 4315-4321, 1990[Abstract].

18.   Papazafir, P., K. Ogami, D. P. Ramji, A. Nicosia, P. Monaci, C. Cladaras, and V. I. Zannis. Promoter elements and factors involved in hepatic transcription of the human apo A-I gene positive and negative regulations bind to overlapping sites. J. Biol. Chem. 266: 5790-5797, 1991[Abstract/Free Full Text].

19.   Rottman, J. N., R. L. Widom, B. Nadal-Ginard, V. Mahdavi, and S. K. Karathanasis. A retinoic acid response element from the apolipoprotein A-I gene distinguishes between two different retinoic acid response pathways. Mol. Cell. Biol. 11: 3814-3820, 1991[Medline].

20.   Rubin, E. M., R. M. Krauss, E. A. Spangler, J. G. Verstuyft, and S. M. Clift. Inhibition of early atherogenesis in transgenic mice by human apolipoprotein A-I. Nature 353: 265-267, 1991[Medline].

21.   Sastry, K. N., U. Seedorf, and S. K. Karathanasis. Different cis-acting DNA elements control expression of the human apolipoprotein AI gene in different cell types. Mol. Cell. Biol. 8: 605-614, 1988[Medline].

22.   Semenkovich, C. F., T. Coleman, and R. Goforth. Physiologic concentration of glucose regulate fatty acid synthase activity in Hep G2 cells by mediating fatty acid synthase mRNA stability. J. Biol. Chem. 268: 6961-6970, 1993[Abstract/Free Full Text].

23.   Sladek, F. M., W. Zhong, E. Lai, and J. E. Darnell, Jr. Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily. Genes Dev. 4: 2353-2365, 1990[Abstract].

24.   Tam, S. P., and R. G. Deeley. Regulation of apolipoprotein A-I gene expression by phenobarbital in the human hepatocarcinoma cell line, Hep3B. Atherosclerosis 105: 235-243, 1994[Medline].

25.   Widom, R. L., J. A. A. Ladias, S. Kouidou, and S. K. Karathanasis. Synergistic interactions between transcription factors control expression of the apolipoprotein AI gene in liver cells. Mol. Cell. Biol. 11: 677-687, 1991[Medline].

26.   Widom, R. L., M. Rhee, and S. K. Karathanasis. Repression by ARP-1 sensitizes apolipoprotein AI gene responsiveness to RXR alpha and retinoic acid. Mol. Cell. Biol. 12: 3380-3389, 1992[Abstract].

27.   Williams, D. L., T. C. Newman, G. S. Shelness, and D. A. Gordon. Measurement of apolipoprotein mRNA by DNA-excess solution hybridization with single-stranded probes. In: Methods in Enzymology, edited by J. P. Segrest, and J. J. Albers. Orlando, FL: Academic, 1986, vol. 128, p. 671-689.

28.   Winer, B. J. Statistical Principles in Experimental Design (2nd ed.). New York: McGraw Hill, 1971.

29.  Wu, J. Y. J. The Regulation of Apolipoprotein A-I Gene Expression: Dietary Copper and Zinc (PhD thesis). Tucson, AZ: Univ. of Arizona, 1996.

30.   Zannis, V. I., F. S. Cole, C. L. Jackson, D. M. Kurnit, and S. K. Karathanasis. Distribution of apolipoprotein A-I, apolipoprotein C-II, apolipoprotein C-III, and apolipoprotein E messenger RNA in fetal human-tissues time-dependent induction of apolipoprotein E messenger RNA by cultures of human monocyte-macrophages. Biochemistry 24: 4450-4455, 1985[Medline].

31.   Zhang, J. J., Y. Wang, and K. Y. Lei. Apolipoprotein A-I synthesis and secretion are increased in HepG2 cells depleted of copper by cupruretic tetramine. J. Nutr. 125: 172-182, 1995[Medline].


AJP Cell Physiol 273(4):C1362-C1370
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