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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2707-2712.)
© 1997 American Heart Association, Inc.

Articles

Upregulation of Low Density Lipoprotein Receptor by Gemfibrozil, a Hypolipidemic Agent, in Human Hepatoma Cells Through Stabilization of mRNA Transcripts

Daisuke Goto; Tadayoshi Okimoto; Mayumi Ono; Hidenori Shimotsu; Kazuhiro Abe; Yoshio Tsujita; ; Michihiko Kuwano

From the Department of Biochemistry, Kyushu University School of Medicine, Fukuoka (D.G., T.O., M.O., M.K.) and Research Institute, Sankyo Company, Shinagawa, Tokyo (H.S., K.A., Y.T.), Japan.

Correspondence to D. Goto, Department of Biochemistry, Kyushu University School of Medicine, Fukuoka 812-82, Japan.

	Abstract

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Abstract Gemfibrozil reduces the plasmal levels of cholesterol and triglyceride in patients with hyperlipidemia by a mechanism that is not well understood. The present study evaluated the effect of gemfibrozil on the LDL receptor in human hepatoma cells compared with that of pravastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Exposure to gemfibrozil, 40 µmol/L, for 3 days increased the binding of ¹²⁵I-LDL to the surface of three lines of human hepatoma cell, HepG2, HuH7, and HLE by 1.5- to 2.0-fold. Similar findings were observed with pravastatin. Scatchard analysis with ¹²⁵I-LDL indicated an increased number of LDL receptors on the cell surface of HepG2 cells when treated with gemfibrozil and pravastatin. However, the gemfibrozil-treated cells exhibited no increase in the binding of ¹²⁵I-epidermal growth factor (EGF). Gemfibrozil increased the levels of LDL receptor mRNA and protein in HepG2 cells. The increase in LDL receptor activity induced by pravastatin was abolished by concomitant administration of mevalonic acid, 770 µmol/L. This effect was not seen with gemfibrozil, suggesting the mechanism differs for the two lipid-lowering drugs. To determine whether this increase in mRNA was due to transcriptional activation, we prepared HepG2 cells transfected with an LDL receptor promoter-reporter construct that contained a sterol regulatory element. The expression of LDL receptor regulated by the sterol regulatory element was increased by pravastatin, but not by gemfibrozil. We evaluated the stability of the mRNA in the presence of actinomycin D to explain the increase in the LDL receptor mRNA. Gemfibrozil prolonged the half-life of the mRNA for LDL receptor but not that for the EGF receptor. Stabilization of the LDL receptor mRNA is suggested to be the novel mode of action of gemfibrozil.

Key Words: gemfibrozil • pravastatin • low density lipoprotein receptor • mRNA stability

	Introduction

Top Abstract Introduction Methods Results and Discussion References

 
Derivatives of fibric acid such as bezafibrate, fenofibrate, and gemfibrozil effectively reduce total plasma levels of cholesterol and triglyceride and are clinically used as hypolipidemic agents.^{1 2 3} These agents appear to have four principal actions: production of a decline in plasma free fatty acid, activation of the lipolysis of such triglyceride-rich particles as VLDL via the activation of lipoprotein lipase, increase in the receptor-mediated metabolism of LDL in the liver, and acceleration of the centripetal transport of sterols.^{4 5 6 7}

A 34% reduction in the incidence of coronary heart disease was observed in dyslipidemic men who were treated with gemfibrozil in the Helsinki Heart Study, a randomized 5-year double-blind trail. Over the 5-year study, mean decreases in the serum total cholesterol level of 10% and in LDL cholesterol of 11% were produced compared with baseline levels.⁸ Agents that reduce the levels of HMG-CoA reductase such as pravastatin reduce the plasma levels of cholesterol.⁹ In one clinical trial, pravastatin significantly reduced the incidence of myocardial infarction and death from cardiovascular causes in men with moderate hypercholesterolemia who had no history of myocardial infarction.¹⁰ Goldstein et al¹¹ showed that the inhibition of cholesterol synthesis by HMG-CoA reductase inhibitors elicits an increase in the synthesis of LDL receptors, thus lowering the plasma levels of LDL cholesterol. After 4 weeks of treatment with gemfibrozil, mononuclear cells freshly isolated from patients with hyperlipidemia types IIa and IIb showed a 78% suppression of HMG-CoA reductase activity.¹² However, gemfibrozil failed to affect HMG-CoA reductase directly in homogenized or cultured mononuclear cells, which suggests that the observed inhibition of HMG-CoA reductase was not a direct effect of the drug on cellular metabolism of cholesterol.¹² We therefore conducted the present study to determine whether gemfibrozil would directly affect the metabolism of LDL receptors in cultured human hepatoma cells.

	Methods

Top Abstract Introduction Methods Results and Discussion References

 
Cells
Human hepatoblastoma HepG2 cells were purchased from Riken, Wako, Saitama, Japan. The human hepatic cancer cell lines, HuH7 and HLE, were obtained from American Type Culture Collection. All cells were propagated in DMEM supplemented with 10% fetal bovine serum.

Preparation of Lipoprotein-Deficient Serum (LPDS)
LPDS was prepared from fetal bovine serum by ultracentrifugation using the standard technique with the addition of potassium bromide to adjust density. Fetal bovine serum was fractionated by centrifugation at a density of 1.215 g/mL at 105 000g for 48 hours. The LPDS fraction was stored as described previously.¹³

Preparation of ¹²⁵I-LDL
LDL was isolated from plasma of healthy males (average age 29.8 years) by sequential ultracentrifugation as described.¹⁴ LDL was iodinated with Na¹²⁵I by McFarlane's iodine monochloride procedure as modified for lipoproteins. The activity of ¹²⁵I-LDL was 20 to 60 cpm/ng lipoprotein. The fraction soluble in trichloroacetic acid was below 0.5%.

Binding of ¹²⁵I-LDL to the Cell Surface
The hepatic cell lines were incubated in a medium containing 10% LPDS with or without various concentrations of gemfibrozil or pravastatin for 72 hours at 37°C. Cells were washed with ice-cold PBS and incubated in binding buffer (DMEM containing 20 mmol/L HEPES and 5 mg/mL bovine serum albumin) with 10⁵ cpm per well of ¹²⁵I-LDL for 2 hours at 4°C for binding assay and 37°C for internalization and degradation assay in the absence and presence of 100-fold excess amounts of unlabeled LDL. Specific binding activities for LDL binding were determined as described previously. Dextran sulfate–releasable radioactivities at 37°C were counted as internalization activity. Degradation of ¹²⁵I-LDL was measured as acid-soluble materials of culture medium. Triplicate determinations of binding, internalization, and degradation varied less than 10% of the mean value.^{14 15}

Western Blot Analysis
Antibody against human LDL receptor^{14 15} and antibody against human epidermal growth factor (EGF) receptor¹⁶ were used for Western blot analysis. Receptors were detected with enhanced chemoluminescence.^{15 16}

Northern Blot Analysis
Northern blot analysis was performed as described previously.¹⁷ Total RNA was isolated from the hepatoma cells and dissolved in sterile distilled water. The resulting RNA was fractionated on a 1% agarose gel containing 2.2 mol/L formaldehyde and transferred to a Nytran filter. The filter was hybridized to a ³²P-labeled cDNA probe in Hybrisol for 24 hours at 42°C. The filter was washed at 65°C in 2x SSC and 0.1% SDS and then washed in 0.2x SSC and 0.1% SDS at 65°C. Autoradiography was performed using FUJIX BAS2000.

Preparation of ¹²⁵I-EGF and Binding of ¹²⁵I-EGF to the Cell Surface
EGF (Toyobo Co) was iodinated by the chloramine T method as described previously.^{16 18} Hepatoma cells were incubated under the same conditions as for the binding of ¹²⁵I-LDL. After being washed with ice-cold PBS, the cells were incubated in binding buffer as ¹²⁵I-LDL, 10⁵ cpm per well at 4°C for 2 hours in the absence and presence of 100-fold excess amounts of unlabeled EGF. Specific binding activities for EGF binding were determined as described previously.¹⁸

Plasmid Construction
Plasmids were constructed by standard genetic engineering techniques.¹⁹ Four overlapping fragments of the LDL receptor regulatory sequence (-235 to -124) ²⁰ were synthesized on an Applied Biosystems model 380A DNA synthesizer and linked to the herpes simplex virus thymidine kinase (HSV TK) promoter extending from -32 to +20.²¹ The LDL receptor–HSV TK promoter fragment was inserted into the multiple restriction site region (HindIII/Sma I) of pUC0-CAT, a vector plasmid that contains a pUC vector backbone,²² the coding sequence of CAT and the SV40 polyadenylation sequences derived from pSV0CAT.²³ The structure of the resulting plasmid (pSAK-204) was verified by DNA sequence analysis and restriction mapping.

DNA Transfection
HepG2 cells were grown in monolayers in medium A (Eagle's minimum essential medium containing 0.1 mmol/L nonessential amino acids [GIBCO], 2 mmol/L L-glutamine, 1 mmol/L pyruvic acid, 100 U/mL penicillin, and 100 mg/mL streptomycin sulfate) in 10% (vol/vol) fetal calf serum. To create stable transformants, HepG2 cells were transfected with plasmids and selected for G418 resistance. Cells were transfected by the calcium phosphate coprecipitation technique using 10 µg of pSAK204 and 2 µg of pRSV-neo.²⁴ After 3 to 4 weeks of selection, the resistant colonies were pooled and expanded in mass culture in the presence of 700 µg/mL G418.

CAT Assay
On day 0, the stable transfectants of the plasmid pSAK204 were plated at 7x10⁵ cells per well (six-well plate, Sumilon) and incubated for 72 hours. On day 3, each well received 1 mL of fresh medium A containing 5% calf LPDS. Therefore, on days 4 to 6, we added to each well 1 mL of medium supplemented with gemfibrozil or pravastatin dissolved in DMSO just before use. After incubation for 72 hours (day 7), cells were harvested for the measurement of CAT activity.²⁴ The protein concentration was then determined by the method of Bradford.²⁵

Stability of mRNA
HepG2 cells were treated for 12 hours with 10% LPDS and 22 µmol/L pravastatin. After 12 hours, actinomycin D (3.9 µmol/L) was added with or without 40 µmol/L gemfibrozil, followed by incubation for the indicated period of time. RNA was then harvested for the analysis of LDL receptor mRNA in the Northern blot method. Data are expressed as the percent fall in the level of LDL receptor mRNA over time when normalized by 28S rRNA.²⁶

	Results and Discussion

Top Abstract Introduction Methods Results and Discussion References

 
We first examined whether the activity of the LDL receptor in hepatoma cell lines was affected by gemfibrozil. Treatment with or without 40 µmol/L gemfibrozil for 3 days, which lacked cytotoxicity, led to a 1.6- to 2.2-fold increase in the binding of ¹²⁵I-LDL to the cell surface of the HepG2 cells (Table 1). We observed a similar increase in LDL binding in other hepatoma cell lines, HuH7 and HLE, in response to gemfibrozil, 4 or 40 µmol/L. Treatment with pravastatin, 2.2 or 22 µmol/L, also enhanced the binding of LDL to the hepatoma cells (Table 1). In contrast, the hepatoma cell lines showed no increase in EGF binding when treated with gemfibrozil, suggesting that an upregulation by this drug was specific for the LDL receptor. We examined whether increased LDL binding by gemfibrozil or pravastatin was due to either the receptor number or the receptor affinity, by Scatchard analysis. As seen in Fig 1, treatment with gemfibrozil, 40 µmol/L, and pravastatin, 22 µmol/L, increased the number of LDL receptors about 1.8-fold and 2.0-fold higher than the control. However, the affinities of LDL receptor were not changed after treatment with these drugs.

View this table: [in this window] [in a new window]   Table 1. Effect (Percent Binding) of Gemfibrozil or Pravastatin on Binding of 125I-LDL to the Cell Surface of Human Hepatic Cells

View larger version (20K): [in this window] [in a new window]   Figure 1. Saturation binding kinetics and Scatchard analysis for 125II-LDL binding to HepG2 cells incubated with or without gemfibrozil or pravastatin. HepG2 cells were incubated in medium containing 10% LPDS with no drug () or with 40 µmol/L gemfibrozil () or 22 µmol/L pravastatin () for 72 hours at 37°C. The cells were incubated with various doses of 125I-LDL in the absence or presence of an excess amount of unlabeled LDL for 2 hours in 4°C. Scatchard analysis was indicated from saturation curves as shown in the inset.

Stange et al¹² reported that gemfibrozil increased the binding of LDL to the cell surface of cultured mononuclear cells, but gemfibrozil was not able to affect LDL degradation. We thus tested internalization and degradation of LDL in the drug-treated hepatoma cell line HepG2. Both internalization and degradation activities of LDL were about 2.0-fold higher by treatment with gemfibrozil, 40 µmol/L, or pravastatin, 22 µmol/L, than those in untreated cells (Table 2). Degradation could be induced in the drug-treated hepatoma cells but not in the drug-treated mononuclear cells.

View this table: [in this window] [in a new window]   Table 2. Effect of Gemfibrozil or Pravastatin on Percent of Internalization and Degradation of 125I-LDL to the Cell Surface of HepG2 Cells

To examine whether LDL receptor mRNA levels were upregulated, cellular mRNA levels were examined by Northern blot analysis. Treatment with 4 µmol/L or 40 µmol/L gemfibrozil led to a 2.8- to 4.3-fold increase in LDL receptor mRNA levels in HepG2 cells when normalized by GAPDH mRNA levels. Pravastatin, 2.2 or 22 µmol/L, also induced an increase of 2.7- to 3.6-fold in mRNA levels (Fig 2A). Western blot analysis showed that 4 or 40 µmol/L gemfibrozil induced a 2.1- to 2.8-fold increase in the protein levels of LDL receptor and also that 2.2 or 22 µmol/L pravastatin induced a 2.3- to 3.1-fold increase (Fig 2B). By contrast, gemfibrozil and pravastatin could not increase the protein levels of EGF receptor. Pravastatin is a potent hypolipidemic drug that targets HMG-CoA reductase, a key enzyme in the synthesis of cholesterol.²⁷ Mevalonic acid virtually abolishes this inhibitory effect of pravastatin.^{9 27} To determine whether the upregulation by gemfibrozil is mediated via a mechanism similar to that of pravastatin, we measured LDL binding activity in HepG2 cells in the absence or presence of mevalonic acid. As shown in Table 3, the concomitant administration of mevalonic acid, 770 µmol/L, virtually abolished the upregulation of LDL receptor induced by pravastatin but not by gemfibrozil. Thus, the mechanisms of the upregulation of LDL receptors by the two drugs appeared to differ.

View larger version (63K): [in this window] [in a new window]   Figure 2. Effect of gemfibrozil and pravastatin on the expression of LDL receptor and EGF receptor. A, HepG2 cells were incubated in LPDS for 24 hours and evaluated for 72 hours after the exposure to gemfibrozil (4 or 40 µmol/L) or pravastatin (2.2 or 22 µmol/L). At the indicated times, cells were collected and Northern blot analysis was performed with 32P-labeled LDL receptor cDNA, EGF receptor cDNA, and GAPDH cDNA probes. The radioactivity of the corresponding area was measured with a BAS2000 Bioimage analyzer. The cellular mRNA levels of the LDL receptor and the EGF receptor were normalized by 28S rRNA level. B, HepG2 cells were incubated under the same conditions as in A. Samples (80 µg each) were subjected to 12% gel and electrophoretically transferred to nitrocellulose filters and then incubated with anti-LDL receptor antibody and anti-EGF receptor antibody.

View this table: [in this window] [in a new window]   Table 3. Effect (Percent Binding) of Mevalonic Acid on Gemfibrozil- or Pravastatin-Induced Upregulation of 125I-LDL Binding

The SRE from the human LDL receptor gene is capable of driving expression of the CAT gene in a sterol-responsive manner.²¹ We prepared a similar plasmid (pSAK204) that contained the SRE inserted upstream of a TATA box that was derived from the HSV TK gene. The reporter plasmid was introduced into HepG2 cells by transfection, together with a second plasmid that contained the gene for a resistance to neomycin (G418). The CAT activity of the G418-resistant cells was confirmed to be regulated by the sterol in the culture medium as previously reported.²¹ To examine whether the upregulation of the LDL receptor mRNA induced by gemfibrozil was due to an increase in the transcriptional activity, we measured CAT activity in the presence or absence of this drug. As shown in Fig 3, there appeared no increase in the transcriptional activity after incubation with 4 or 40 µmol/L gemfibrozil. In contrast, treatment with pravastatin apparently increased the CAT activity, probably reflecting a reduction in the cellular level of cholesterol by the HMG-CoA reductase inhibitor. Results suggest that gemfibrozil did not affect the transcriptional activity of the LDL receptor gene via the sterol regulatory sequence.

View larger version (49K): [in this window] [in a new window]   Figure 3. Effect of gemfibrozil and pravastatin on expression of CAT activity under control of synthetic LDL receptor promoter elements in transfected HepG2 cells. Transactivation assay was assessed by measuring CAT activity in extracts of HepG2 cells cotransfected with LDL receptor gene promoter-driven CAT constructs. CAT activity was determined after 72 hours' treatment with or without 4 or 40 µmol/L gemfibrozil or pravastatin. Activity is expressed as relative CAT activity when normalized by activity observed after 72 hours' incubation in LPDS alone. The value was the mean average of four independent assays, and bars represent ±SD. Relative CAT activity in the cells treated with 22 µmol/L pravastatin significantly (*P<.01) exceeded that in its absence.

We next examined the stability of LDL receptor mRNA in the cells exposed to each drug. We first treated HepG2 cells with pravastatin to enhance the cellular levels of LDL receptor mRNA and evaluated the mRNA levels of the LDL and EGF receptors in the presence of actinomycin D with or without the addition of 40 µmol/L gemfibrozil. As shown in Fig 4, LDL receptor mRNAs were rapidly degraded, with a half-life of 1 to 1.5 hours in the absence of drugs, whereas gemfibrozil stabilized LDL mRNA, with a half-life exceeding 6 hours. In contrast, EGF receptor mRNAs were degraded at similar rates in the untreated as well as the gemfibrozil-treated cells, with a half-life of about 4 hours. Treatment with gemfibrozil, 200 µmol/L, also stabilized the LDL receptor mRNA at rates comparable to those seen with the dose of 40 µmol/L (data not shown). The same treatment did not affect the stability of EGF receptor mRNA. In contrast, pravastatin, 22 µmol/L, did not stabilize the LDL receptor mRNA (data not shown). Results suggest gemfibrozil upregulated the LDL receptor by stabilizing its mRNA. The mechanism of such upregulation appeared to differ from that of pravastatin. The latter agent enhances LDL receptor levels via the transcriptional activation of the LDL receptor gene.²⁸

View larger version (36K): [in this window] [in a new window]   Figure 4. Effect of gemfibrozil on stability of LDL receptor and EGF receptor mRNA. A, HepG2 cells were treated for 12 hours with pravastatin, 22 µmol/L, containing 10% LPDS. After 12 hours, actinomycin D (3.9 µmol/L) was added with or without 40 µmol/L gemfibrozil for the indicated periods of time, and RNA was harvested for Northern blot analysis of LDL receptor mRNA. Autoradiograms were scanned and normalized to 28S rRNA. B, Cellular mRNA levels of the LDL and EGF receptors were quantified when normalized by 28S rRNA and blotted over time after the addition of actinomycin D. LDL receptor mRNA in the absence () or presence () of gemfibrozil; EGF receptor mRNA in the absence () or presence () of gemfibrozil.

Stange et al¹² reported that gemfibrozil treatment of patients with primary hyperlipidemia reduced the level of total serum cholesterol and the activity of HMG-CoA reductase in mononuclear cells. However, gemfibrozil failed to affect the HMG-CoA reductase directly in mononuclear cell homogenate. Those authors suggested that the drug produced changes in LDL structure to affect LDL receptor binding rather than directly affecting the cellular metabolism of cholesterol.¹² In contrast, Franceschini et al²⁹ reported that neither the apolipoprotein B secondary structure nor the affinity of LDL for the LDL receptor of fibroblasts was affected by gemfibrozil treatment for 12 weeks of patients with type IIa hyperlipidemia, thus suggesting no apparent change in the LDL structure. Those authors showed that, while the LDL receptor activity in the mononuclear cells of patients increased threefold after gemfibrozil, the difference did not reach statistical significance (P<.05) because of the wide variation in the response observed among the patients.²⁹ We observed a significant increase in LDL binding to hepatoma cells after incubation with gemfibrozil for 72 hours. Whether an altered LDL receptor activity in cultured hepatoma cells after treatment with gemfibrozil would also be seen in the liver cells of such patients requires study. Although the LDL receptor gene was activated by pravastatin at the transcriptional level via SRE, gemfibrozil failed to induce an expression of the LDL receptor promoter-reporter system containing SRE. Our present results suggest that gemfibrozil increases the stability of the LDL receptor mRNA rather than the synthesis of mRNA, leading to upregulation of the receptor on the surface of hepatoma cells.

The stability of mammalian mRNA molecules is influenced by such stimuli as glucose, hormones, cytokines, growth factors, and physical damage.³⁰ Among the agents used in lowering lipid level, antiprogesterone drugs stabilize fatty acid synthetase mRNA induced by progesterone.³¹ Poly (A) and 3'-untranslated regions are often related to stabilization or destabilization of mRNAs, and the presence of the sequence AUUUA appears to be related to the stability of mammalian mRNAs.^{30 32} The human LDL receptor gene contains the AUUUA sequences in its 3'-untranslated region.³³ Further studies are required to determine whether the AUUUA sequences in the LDL receptor gene are related to the stability of the mRNA and to identify whether gemfibrozil may alter the stability of the mRNA via such sequence.

	Acknowledgments

 
This study supported in part by Kimura Memorial Cardiovascular Research Fund (M. Ono). We thank Masatoshi Fujishima and Mototaka Yoshinari in the Second Department of Internal Medicine, Kyushu University School of Medicine, for fruitful discussion. We also thank Kyoko Yamada, Tomoko Matsuguma, and Hiroko Maeda of our laboratory for their technical assistance and editorial advice.

Received December 4, 1996; accepted April 17, 1997.

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