Vol. 186, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

July 31, 1992 Pages 617-623

THYROID HORMONE MODULATES APOLIPOPROTEIN B GENE EXPRESSION IN HEPGZ CELLS

Andre Theriault, Godwin Ogbonna, and Khosrow Adeli*

Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada N9B 3P4

Received May 26, 1992

We have investigated the modulation of apolipoprotein B gene expression in HepG2 cells by thyroid hormone. ApoB secretion rate in serum-free media was found to be significantly increased in the presence of the hormone in long-term cultures (48 h, 37%). This stimulatory effect was dose-dependent. The mechanisms underlying the stimulatory effect of triiodothyronine on apoB production were investigated. Triiodothyronine increased apoB mRNA levels by about 25-36% as determined by slot- and Northern-blot analysis of total RNA. ApoB synthesis rate was also found to be increased both in in vivo pulse-chase experiments (61%) and in in vitro translation studies (54.5%). Despite the 54.5-61% increase in apoB synthesis with triiodothyronine, only a 30% increase in apoB secretion was noted suggesting that part of the increase in the intracellular apoB pool may be lost by degradation. Overall, apoB gene expression appears to be modulated by thyroid hormone at both transcriptional and posttranscriptional levels. 0 1992 Academic Press., Inc.

Apolipoprotein Bloo (apoB) is a major component of LDL and VLDL of human plasma.

Patients with increased plasma levels of apoB-containing lipoproteins may have higher

production rate of apoB (1, 2). Hepatic production rate of apoB-containing lipoproteins is known

to be regulated by diet (3,4) and drugs (5, 6). The mechanisms for the acute regulation of apoB

gene expression are now being unraveled. Free fatty acids such as oleate stimulate apoB

secretion without changing apoB mRNA levels (7, 8). Insulin, which is known to suppress net

accumulation of apoB, appears to act through a posttranscriptional mechanism since the hormone

does not alter apoB mRNA levels, despite a significant inhibition of the protein output (7, 8).

ApoB degradation (9, 10) and apoB mRNA translation (11,12) may be the key regulatory

mechanisms controlling the acute regulation of apoB production.

The effect of thyroid hormone on apoB gene expression and apoB production rate has not

received much attention and little data is available on thyroid hormone regulation of apoB

* To whom correspondence should be addressed at Department of Chemistry and Biochemistry, University of Windsor, 400 Sunset St., Windsor, Ontario, Canada N9B 3P4.

The abbreviations used are: apoB, apolipoprotein B100; PAGE, polyacrylamide gel electrophoresis; TCA, trichloroacetic acid; Hepes, 4-(2-hydroxyethyl)-1-piperazine-ethane sulfonic acid; SDS, sodium dodecyl sulfate; VLDL, very low density lipoprotein. T3, triiodothyronine.

0006-291X/92 $4.00

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617 All rights of reproduction in any fiwn reserved.

Vol. 186, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

synthesis and secretion in human liver. In the rat, thyroid hormone regulates the synthesis of

intestinal apoB4g by inducing posttranscriptional editing of apoB mRNA through creation of an

in-frame stop codon (13,14). This suggests a direct role for triiodothyronine (T3) in the tissue-

specific expression of apoB gene. In vivo studies in hypothyroid rats have shown reduced

hepatic synthesis of apoB4g in the hypothyroid state (15). Administration of T3 restored the

synthesis of apoB4g to control levels while abolishing the synthesis of apoBIo0 (15). These

responses to thyroid status were not accompanied by changes in apoB mRNA levels (15). No

further data is, however, available on the detailed effects of T3 on the transcriptional and

posttranscriptional regulation of apoB gene expression. We recently investigated thyroid

hormone (T3) modulation of apoB production by HepG2 cells and reported a positive regulatory

role for T3 (16). Cells grown in presence of T3 produced significantly higher levels of apoB than

control cultures (I 6). Here, we provide data on the effects of T3 on apoB mRNA levels, and

apoB synthesis and degradation rates.

EXPERIMENTAL PROCEDURES

Cell Culture. Cells (1 x 105 cells) were grown in 25 cm2 flasks at 37” C, 5% CO2 in complete medium (a-MEM [Eagle’s minimal essential medium], 10% fetal bovine serum) until about 75% confluency. Hormonal studies were performed using a serum-free medium developed in our laboratory and reported previously (16). Media apoB concentrations were determined in triplicate by an in-house avidin-biotin based enzyme-linked immunosorbant assay. Total cellular protein was determined by a Bio-Rad protein kit.

In Vitro Translation in HepGt Cell-Free Lysate. Near confluent HepG2 cultures grown in 80 cm2 flasks were depleted of methionine by incubation in methionine-free MEM for 60 min at 37 “C under 5% C02. A cell-free lysate was then prepared by a modification of the method of Brown et al. (17). Cells were washed twice with buffer A (150 mM RNase-free sucrose, 33 mM NI-I4Cl, 7 mM KCI, 1.5 mM Mg(OAc)2, and 30 mM Hepes, pH 7.4) and lysed in buffer A containing 150 mM lysolecithin. The lysed cells were suspended in translation buffer [lo0 mM Hepes, pH 7.4, 200 mM KCI, 7 mM NH&l, 0.5 mM Mg(OAc)2, 1 mM dithiotreitol, 1 mM ATP, 1 mM GTP, 40 uM of each of 19 amino acids minus methionine, 0.1 mM S- adenosylmethionine, 1 mM spermidine trihydrochloride, 10 mM creatine phosphate, 40 units/ml of creatine phosphokinase]. The extract was centrifuged at 4 “C, 12000 X g , for 1 min and the supematant was collected. Typically the lysate had a A280 absorbance of 15-20 units/ml. In vitro protein synthesis in HepG2 lysate was carried out in presence of 400 @i/ml of [35S]methionine, at 30 “C for 60 min. Radioactive incorporation was determined by TCA precipitation.

Slot- and Northern Blot Analysis. Total HepG2 RNA was extracted by the guanidinium thiocyanate as described by Chomczynski and Sacchi (IS). Total HepG2 RNA was blotted on Nytran membrane using a Bio-Rad slot-blot apparatus. The RNA filters were hybridized with an apoB DNA probe (pB27) or a full length human y-actin probe (pHFyA-1). The probes (500 ng) were labeled with 50 pCi of deoxycytidine -5’-triphosphate, [a-32P] by nick translation (according to the BRL protocol) to a specific activity of 1 x 108 cpm/pg DNA. Membranes were pre-hybridized for 5 h and hybridized for 30 h. The membranes were washed, air-dried and autoradiographed. For Northern blots, total RNA was electrophoresed in either 0.8% (for apoB) or 1.2% (for actin) agarose and blotted onto nylon membrane. The blots were then hybridized as above. The linearity of the slot-blot assay was established by using several RNA concentrations (0.5-8 pg). Over this range, the signals obtained were proportional to the amounts of RNA applied to the filter.

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In vivo pulse-chase labeling. HepG2 cells cultured in six-well plates (9 cm2/well) were incubated with MEM minus methionine for 60 min, pulsed in the same medium containing 34 pCi/ml of [35S]methionine & hormone, for 10 min, and then chased for 20, 40 and 180 min. At each point, the medium (extracellular fraction) was collected. The cells were washed and lysed in 50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 150 mM NaCI, 0.0625 M sucrose, 0.5% Triton X- 100, 0.5% deoxycholate, and protease inhibitor mixture.

Immunoprecipitations. In vitro translation products (100-200 ~1) and in viva labeled extracts (50-200 ~1) were immunoprecipitated essentially as described (19). Immunoprecipitates were analyzed by SDS-PAGE which was performed essentially as described (20). The gels were fixed and stained, and were prepared for fluorography.

RESULTS

HepG2 cells were previously shown to secrete higher levels of apoB in long-term serum-

free cultures when treated with thyroid hormone (16). The stimulatory effect of thyroid hormone

(T3) was found to be dose-dependent and was detectable in the first few days with high doses of

the hormone. The production rate of apoB in serum-free cultures of HepG2 cells in presence of Tg

was 0.12 pgimglh at 10 nM, 0.16 pg/mg/h at 20 nM, and 0.2 1 pgimgih at 50 nM T3.

The mechanisms underlying the stimulation of apoB production by T3 were investigated by

measuring apoB mRNA levels in control and T3-treated HepG2 cells by slot-blot hybridization of

total HepG2 RNA (Fig. 1A). The slot-blot assay was standardized by measuring actin mRNA and

calculating the ratio of apoB mRNA signal to actin mRNA signal. Desitometric scanning of the

apoB mRNA signals suggested an increase of 36.1 i 4.9% in apoB mRNA levels in T3-treated

cells. The stimulation of apoB mRNA levels by actin was further confirmed by Northern-blot

analysis (Fig. 1B). Total RNA from control and T3-treated HepG2 cells was hybridized with apoB

A

- T3 m 8 -? + ‘l-3

.; - T3

; + T3

0.5 1.0 2.0 4.0 8.0 Total HepG2 RNA (pg)

- T.1 + T.3 -T3 + T.3

ApoB mRNA Actin mRNA

Figure 1. Modulation of apoB mRNA levels in HepG2 cells by Thyroid Hormone. Total RNA was extracted from HepG2 cells grown in presence and absence of T3 for 48 h. (A) slot- blot analysis; Total HepG2 RNA samples (1-8 pg) were blotted on nylon membranes and hybridized with a [32P] labeled apoB genomic clone (pB27) insert or y-actin cDNA probe. Numbers denote the amount of RNA applied in Kg. (B)Northem-blot analysis was also performed by formaldehyde agarose gel electrophoresis of total HepG2 RNA, northern-blot transfer and hybridization with pB27 and y -actin probes.

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Ap0B-w

- T3 + T3

Figure 2. Effect of thyroid hormone on in vitro translation of endogenous apoB mRNA. Lysates prepared from T3-treated (50 nM, 48 h) HepG2 cells as well as untreated controls were assayed for the in vitro synthesis of apoB and apoA1 (as a control). The assay consisted of translation in the presence of [3%] methionine, immunoprecipitation, electrophoresis and fluorography. (A)shows immunoprecipitation with a monospecific apoB antibody (lanes 1 and 2, the immunoprecipitate of untreated control lysate; lanes 3 and 4. the immunoprecipitate of T3- treated lysate).(B) shows the immunoprecipitation with a monospecific apoA1 antibody (lanes 1 and 2, the immunoprecipitate of untreated control lysate; lanes 3 and 4, the immunoprecipitate of T3-treated lysate).

and actin cDNA probes. A similar but somewhat lower level of stimulation of apoB mRNA levels

(25 f 3.1%) was detected by the Northern-blot assay.

The effect of T3 on apoB synthesis was also investigated by both in vitro translation and

in vivu pulse-chase labeling studies. First, we used a HepG2 cell-free lysate system to

demonstrate in vitro synthesis of apoB and the effect of T3 on apoB mRNA translation. This

cell-free lysate system was originally characterized in our laboratory and was previously used to

demonstrate the effect of insulin on apoB mRNA translation in vitro (12). To study the effect of

T3 on apoB translation, lysates were prepared from HepG2 cells grown for 48 h in serum-free

media and cells grown in the same medium containing 50 nM T3. The lysates were translated in

vitro in presence of [35S] methionine, and the products were probed with a monospecific apoB

antibody. Fig. 2A shows the in vitro synthesized apoB immunoprecipitated from control

untreated lysates (lane 1 and 2) and T3-treated (lane 3 and 4). ApoB band intensities were

compared by quantitative densitometry. A significant stimulation of apoB mRNA translation was

observed with T3 (an average of 54.5 f 1.3%). The effect of T3 on apoB mRNA translation was

apparently specific for apoB since no changes in mRNA activity for apoAI was observed. Fig.

2B shows the in vitro synthesized apoA1 immunoprecipitated from T3-treated (lanes 1 and 2)

and control untreated lysates (lanes 3 and 4 ). No detectable increase in synthesis of apoA1 was

found following T3 treatment as determined by densitometric analysis of the signals.

Pulse-chase labeling of HepG2 cells was also performed to investigate the effect of T3 on

apoB synthesis in vivo and to study the rate of apoB secretion from the cells into the extracellular

medium. Fig. 3 shows the amount of apoB synthesized by HepG2 cells in presence and absence T3

and the rate of depletion of intracellular apoB. An average of 61% increase in the incorporation of

[35S]methionine into immunoprecipitable apoB was apparent with T3. When the radioactivity was

chased, a gradual reduction in labeled apoB was noted. Table 1 shows the amount of radioactive

apoB depleted during the chase and the estimated amounts of apoB lost or degraded. ApoB content

was calculated by densitometric scanning of the bands and expressed as number of scanning units

per mg of total protein. ApoB secreted as a percentage of the intracellular peak was 53.6% for

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Chase Period (min)

Figure 3. Pulse-chase labeling of HepGZ cells. HepG2 cells were grown in serum-free media treated with and without T3 (50 nM) for 48 h. The cells were then pulsed for 10 min with [35S]methionine, washed, and chased with unlabeled methionine for 20, 40, and 180 min. The labeled media and cells were collected at the end of each chase period and used for immunoprecipitation of apoB. The immunoprecipitates were analyzed by electrophoresis and fluorography. (A) apoB immunoprecipitated from intracellular fractions. (B) apoB immunoprecipitated from extracellular fractions.

control and 43% for T3-treated cells over 3 h. ApoB recovery in cells plus media was 57% for

control cells and 52.5% for T3-treated cells at 180 min of chase. This indicated that in both cases

about half of the newly-synthesized apoB was intracellularly degraded. TX-treated cells secreted an

average of 30% more labeled apoB than control untreated cells

DISCUSSION

Taken together, our data suggest that thyroid hormone regulates the expression of hepatic

apoB at number of points. There appears to be an increase in the level of apoB mFCNA indicating

an effect on the rate of apoB gene transcription and/or increased mRNA stability. Further to this

mRNA effect, T3 stimulate the rate of apoB synthesis both in vitro and in vivo. The increase in

apoB synthesis rate could be partly explained by the increase in the concentration of apoB

mRNA levels with T3. However, the 2536% change in apoB mRNA levels does not totally

account for the 50-60% enhancement in the rate of protein synthesis. This raises the possibility

Table 1. Changes in intracellular apoB during a 3 b chase

Peak to 40 min Peak to 180 min -T3 +T3 -T3 fT3

Intracellular Peak 3.89kO.73 6.3OrtO.35 3.89kO.73 6.3OkO.35 Depleted 2.14 2.74 3.61 5.33 Secreted 0.15 0.37 1.94 2.34 Degraded 1.99 2.37 1.61 2.99

ApoB content is expressed as scanning unitsimg of protein. Intracellular peaks were calculated from apoB contents at the beginning of the chase period (20 min). Values represent the average of duplicate measurements of a typical experiment.

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that translational control may also be involved. From the pulse-chase experiment, it is evident

that the 61% increase in [?S]methionine incorporation into apoB in the presence of T3 did not

fully translate into a similar stimulation in apoB secretion. Only a 30% increase in apoB

secretion was noted in Tj-treated cells indicating that part of the newly-synthesized apoB chains

may have been channeled into a degradative pathway and may not have participated in the

secretion of apoB-containing lipoproteins. Interestingly, the increase in extracellular labeled

apoB (30%) with T3 closely corresponded to a 37% increase in apoB mass as measured by

ELISA. Our results on the effect of thyroid hormone on apoB production concur with a previous

study investigating apoB secretion as a function of thyroid status in rat liver. Davidson et al (15)

also reported decreased apoB100 and apoB4g secretion in hypothyroid rats. However, contrary to

our results when a state of hyperthyroidism was induced, hepatic apoBluc synthesis virtually

ceased while apoB4s synthesis was unchanged. This discrepancy between the response of rat

hepatocytes and HepG2 cells to excess thyroid hormone may stem from the fact that T3 appears

to have effects on apoB expression that is specific to the rat system.

Modulation of gene expression by thyroid hormone in other systems involves several

distinct mechanisms including increased transcription rate (21), increased mRNA stability (22),

and/or changes in the rate of protein degradation (23, 24). Similar mechanisms appear to be

involved in the case of apoB gene regulation by thyroid hormone.

ACKNOWLEDGMENTS

This work was supported by a grant from the Heart and Stroke Foundation of Ontario (Grant

No. AN1904). We would like to thank the excellent technical assistance of Mrs Debbie Rudy.

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