Vol. 186, No. 2, 1992
BIOCHEMICAL
July 31, 1992
THYROID
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 617-623
HORMONE MODULATES APOLIPOPROTEIN EXPRESSION IN HEPGZ CELLS
Andre Theriault,
B GENE
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 expressionin HepG2 cells by thyroid hormone. ApoB secretion rate in serum-free media was found to be significantly increasedin the presenceof 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 increasedapoB mRNA levels by about 25-36% as determinedby slot- and Northern-blot analysis of total RNA. ApoB synthesisrate was also found to be increasedboth in in vivo pulse-chaseexperiments(61%) and in in vitro translation studies (54.5%). Despite the 54.5-61% increasein apoB synthesiswith triiodothyronine, only a 30% increasein apoB secretion was noted suggestingthat 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 0 1992Academic Press.,Inc. posttranscriptionallevels.
Apolipoprotein Bloo (apoB) is a major componentof LDL and VLDL of humanplasma. 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 regulatedby diet (3,4) and drugs (5, 6). The mechanismsfor 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 suppressnet accumulationof apoB, appearsto act through a posttranscriptionalmechanismsincethe hormone doesnot 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 mechanismscontrolling the acute regulation of apoB production. The effect of thyroid hormoneon apoB geneexpressionand apoB production rate hasnot received much attention and little data is available on thyroid hormone regulation of apoB * To whom correspondenceshouldbe addressedat Departmentof Chemistry and Biochemistry, University of Windsor, 400 SunsetSt., 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
617
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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 tissuespecific 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 Sadenosylmethionine, 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. 618
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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 X100, 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). Immunoprecipitateswere analyzed by SDS-PAGE which was performed essentiallyas described(20). The gels were fixed and stained,and were preparedfor fluorography.
RESULTS HepG2 cells were previously shown to secretehigher levels of apoB in long-term serumfree cultures when treated with thyroid hormone (16). The stimulatory effect of thyroid hormone (T3) was found to be dose-dependentand was detectablein the first few days with high dosesof the hormone. The production rate of apoB in serum-freecultures of HepG2 cells in presenceof Tg was 0.12 pgimglh at 10 nM, 0.16 pg/mg/h at 20 nM, and 0.21 pgimgih at 50 nM T3. The mechanismsunderlying the stimulation of apoB production by T3 were investigated by measuringapoB mRNA levels in control and T3-treated HepG2 cells by slot-blot hybridization of total HepG2 RNA (Fig. 1A). The slot-blot assaywas standardizedby measuringactin mRNA and calculating the ratio of apoB mRNA signal to actin mRNA signal. Desitometric scanningof the apoB mRNA signals suggestedan increaseof 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 washybridized with apoB A - T3
m 8 -?
+ ‘l-3
.;
- T3
;
+ T3
0.5
1.0 Total
-
T.1
ApoB
2.0
4.0
HepG2
+ T.3 mRNA
RNA
-T3 Actin
8.0
(pg)
+ T.3 mRNA
Figure 1. Modulation of apoB mRNA levelsin HepG2 cellsby Thyroid Hormone. Total RNA wasextractedfrom HepG2cellsgrownin presence andabsenceof T3 for 48 h. (A) slotblot analysis;Total HepG2 RNA samples(1-8 pg) were blotted on nylon membranes and hybridized with a [32P] labeledapoB genomicclone (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 hormoneon in vitro translation of endogenousapoB 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 T3treated 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 somewhatlower level of stimulation of apoB mRNA levels (25 f 3.1%) wasdetected by the Northern-blot assay. The effect of T3 on apoB synthesiswas also investigated by both in vitro translation and
in vivu pulse-chase labeling studies. First, we used a HepG2 cell-free lysate system to demonstratein vitro synthesisof apoB and the effect of T3 on apoB mRNA translation. This cell-free lysate systemwas originally characterized in our laboratory and waspreviously usedto demonstratethe 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 samemedium containing 50 nM T3. The lysateswere translatedin
vitro in presenceof [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 comparedby quantitative densitometry. A significant stimulation of apoB mRNA translation was observedwith T3 (an average of 54.5 f 1.3%). The effect of T3 on apoB mRNA translation was apparently specific for apoB sinceno changesin mRNA activity for apoAI was observed. Fig. 2B showsthe in vitro synthesizedapoA1 immunoprecipitatedfrom T3-treated (lanes 1 and 2) and control untreatedlysates (lanes 3 and 4 ). No detectable increasein synthesisof apoA1 was found following T3 treatment asdeterminedby densitometricanalysisof the signals. Pulse-chaselabeling of HepG2 cells was also performed to investigate the effect of T3 on apoB synthesisin vivo and to study the rate of apoB secretionfrom the cells into the extracellular medium.Fig. 3 showsthe amount of apoB synthesizedby HepG2 cells in presenceand absenceT3 and the rate of depletion of intracellular apoB. An average of 61% increasein the incorporation of [35S]methionineinto immunoprecipitableapoB was apparentwith T3. When the radioactivity was chased,a gradual reduction in labeled apoB was noted. Table 1 showsthe amount of radioactive apoB depletedduring the chaseand the estimatedamounts of apoB lost or degraded.ApoB content was calculated by densitometric scanningof the bandsand expressedas number of scanningunits per mg of total protein. ApoB secretedas a percentageof the intracellular peak was 53.6% for 620
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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 wasintracellularly degraded.TX-treated cells secretedan average of 30% more labeledapoB than control untreated cells
DISCUSSION Taken together, our data suggestthat thyroid hormoneregulatesthe expressionof hepatic apoB at number of points. There appearsto be an increasein the level of apoB mFCNAindicating an effect on the rate of apoB gene transcription and/or increasedmRNA stability. Further to this mRNA effect, T3 stimulate the rate of apoB synthesisboth in vitro and in vivo. The increasein 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% enhancementin the rate of protein synthesis.This raisesthe possibility Table 1. Changes in intracellular
apoB during a 3 b chase
Peak to 40 min Intracellular Depleted Secreted Degraded
Peak
Peak to 180 min
-T3
+T3
-T3
fT3
3.89kO.73 2.14 0.15 1.99
6.3OrtO.35 2.74 0.37 2.37
3.89kO.73 3.61 1.94 1.61
6.3OkO.35 5.33 2.34 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-chaseexperiment, it is evident that the 61% increasein [?S]methionine incorporation into apoB in the presenceof T3 did not fully translate into a similar stimulation in apoB secretion. Only a 30% increase in apoB secretion wasnoted 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 correspondedto a 37% increase in apoB massas measuredby ELISA. Our resultson the effect of thyroid hormone on apoB production concur with a previous study investigating apoB secretionas a function of thyroid statusin rat liver. Davidson et al (15) alsoreported decreasedapoB100and apoB4gsecretionin hypothyroid rats. However, contrary to our results when a state of hyperthyroidism was induced, hepatic apoBluc synthesis virtually ceasedwhile apoB4s synthesiswas unchanged. This discrepancy between the responseof rat hepatocytesand HepG2 cells to excessthyroid hormonemay stem from the fact that T3 appears to have effects on apoB expressionthat is specific to the rat system. Modulation of gene expressionby thyroid hormone in other systemsinvolves several distinct mechanismsincluding increasedtranscription rate (21), increasedmRNA stability (22), and/or changesin the rate of protein degradation (23, 24). Similar mechanismsappear to be involved in the caseof apoB generegulation by thyroid hormone.
ACKNOWLEDGMENTS This work was supportedby a grant from the Heart and Stroke Foundation of Ontario (Grant No. AN1904). We would like to thank the excellent technical assistanceof Mrs Debbie Rudy.
REFERENCES 1. Janus,E.D., Nicholl, A.M., Turner, P.R., Magill, P., and Lewis, B. (1980) Eur. J. Clin. Invest. 10, 161-172. 2. Kissebah,A.H., Alfarsi, S., and Adams, P.W. (1981) Metabolism 30,856-868. 3. Ginsberg, H. N., Le, N.-A., and Gibson, J.C. (1985) J. Clin. Invest. 75,614-623. 4. Turner, J.D., Le, N.-A., and Brown, W.V. (1981) Am. J. Physiol. 241, E57-E63. 5. Grundy, S.M., and Vega, G.L. (1985) J. Lipid Res. 26, 1464-1475. 6. Arad, Y., Ramakrishnan,R., and Ginsberg, H.N. (1990) J. Lipid Res. 3 1,567-582. 7. Pullinger, C. R., North, J. D., Teng, B-B,, Rifici, V. A., de Brit, A. E. R., and Scott, J. (1989) J. Lipid Res.30, 1065-1077. 8. Dashti, N., Williams, D.L., and Alaupovic, P. (1989) J. Lipid Res. 30, 1365-1373. 9. Davis R.A., Thrift R.N., Wu C.C., and Howell K.E. (1990) J. Biol. Chem. 265, 10005-10011. 10. Sato, R., Imanaka, T., Takatsuki, A., Takano, T. (1990) J. Biol. Chem. 265, 11880-11884. 11. Sparks,J.D. and Sparks,C.E. (1990) J. Biol. Chem. 265, 8854-8862. 12. Theriault A., Cheung R.C., and Adeli K. (1991) Proceedings of the 9th International Symposiumon Atheroscleorosis.Chicago, Illinois, October 6-l 1. p. 27. 13. Barum, C. L. , Teng, B., Davidson, N.O. (1990) J. Biol. Chem. 265, 19263-19270 14. Davidson, N.O., Powell, L.M., Wallis, S.C., Scott, J. (1988) J. Biol. Chem. 263, 1348213485. 15. Davidson, N.O., Carlos, R.C., Drewek, M.J., and Parmer,T.G. (1988) J. Lipid Res.29, 15ll1522, 16. Adeli, K., and Sinkevitch, C. (1990) FEBS Letts. 263, 345-348. 622
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17. Brown, G. D., Peluso, R. W., Moyer, S. A., and Moyer, R. W. (1983) J. Biol. Chem. 258, 14309-14314. 18. Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156-159. 19. Firestone, G.L. and Winguth, SD. (1990) in Methods in Enzymology, Vol. 182, pp. 688. Academic Press, New York. 20. Laemmli, U. K. (1970) Nature (Lond.) 227,680-685. 21. Spindler S.R., Mellon, S.H., and Baxter, J.D. (1982) J. Biol. Chem. 257, 11627-11632. 22. Simonet, W.S. and Ness, G.C. (1988) J. Biol. Chem. 263, 12448-12453. 23. Shambaugh III, G.E., Balinsky, J.R., and Cohen, P.P. (1969) J. Biol. Chem. 244,5295-5308. 24. Chandbury, S., Chattetjee, D., and Sarkar, P.K. (1985) Brain Res. 339, 191-194.
623
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July 31, 1992
THYROID
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 617-623
HORMONE MODULATES APOLIPOPROTEIN EXPRESSION IN HEPGZ CELLS
Andre Theriault,
B GENE
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 expressionin HepG2 cells by thyroid hormone. ApoB secretion rate in serum-free media was found to be significantly increasedin the presenceof 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 increasedapoB mRNA levels by about 25-36% as determinedby slot- and Northern-blot analysis of total RNA. ApoB synthesisrate was also found to be increasedboth in in vivo pulse-chaseexperiments(61%) and in in vitro translation studies (54.5%). Despite the 54.5-61% increasein apoB synthesiswith triiodothyronine, only a 30% increasein apoB secretion was noted suggestingthat 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 0 1992Academic Press.,Inc. posttranscriptionallevels.
Apolipoprotein Bloo (apoB) is a major componentof LDL and VLDL of humanplasma. 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 regulatedby diet (3,4) and drugs (5, 6). The mechanismsfor 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 suppressnet accumulationof apoB, appearsto act through a posttranscriptionalmechanismsincethe hormone doesnot 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 mechanismscontrolling the acute regulation of apoB production. The effect of thyroid hormoneon apoB geneexpressionand apoB production rate hasnot received much attention and little data is available on thyroid hormone regulation of apoB * To whom correspondenceshouldbe addressedat Departmentof Chemistry and Biochemistry, University of Windsor, 400 SunsetSt., 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
617
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Copyright 0 1992 rights of reproduction
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by Acodernic Press, Inc. in any fiwn reserved.
Vol.
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No.
2, 1992
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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 tissuespecific 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 Sadenosylmethionine, 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. 618
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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 X100, 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). Immunoprecipitateswere analyzed by SDS-PAGE which was performed essentiallyas described(20). The gels were fixed and stained,and were preparedfor fluorography.
RESULTS HepG2 cells were previously shown to secretehigher levels of apoB in long-term serumfree cultures when treated with thyroid hormone (16). The stimulatory effect of thyroid hormone (T3) was found to be dose-dependentand was detectablein the first few days with high dosesof the hormone. The production rate of apoB in serum-freecultures of HepG2 cells in presenceof Tg was 0.12 pgimglh at 10 nM, 0.16 pg/mg/h at 20 nM, and 0.21 pgimgih at 50 nM T3. The mechanismsunderlying the stimulation of apoB production by T3 were investigated by measuringapoB mRNA levels in control and T3-treated HepG2 cells by slot-blot hybridization of total HepG2 RNA (Fig. 1A). The slot-blot assaywas standardizedby measuringactin mRNA and calculating the ratio of apoB mRNA signal to actin mRNA signal. Desitometric scanningof the apoB mRNA signals suggestedan increaseof 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 washybridized with apoB A - T3
m 8 -?
+ ‘l-3
.;
- T3
;
+ T3
0.5
1.0 Total
-
T.1
ApoB
2.0
4.0
HepG2
+ T.3 mRNA
RNA
-T3 Actin
8.0
(pg)
+ T.3 mRNA
Figure 1. Modulation of apoB mRNA levelsin HepG2 cellsby Thyroid Hormone. Total RNA wasextractedfrom HepG2cellsgrownin presence andabsenceof T3 for 48 h. (A) slotblot analysis;Total HepG2 RNA samples(1-8 pg) were blotted on nylon membranes and hybridized with a [32P] labeledapoB genomicclone (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 hormoneon in vitro translation of endogenousapoB 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 T3treated 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 somewhatlower level of stimulation of apoB mRNA levels (25 f 3.1%) wasdetected by the Northern-blot assay. The effect of T3 on apoB synthesiswas also investigated by both in vitro translation and
in vivu pulse-chase labeling studies. First, we used a HepG2 cell-free lysate system to demonstratein vitro synthesisof apoB and the effect of T3 on apoB mRNA translation. This cell-free lysate systemwas originally characterized in our laboratory and waspreviously usedto demonstratethe 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 samemedium containing 50 nM T3. The lysateswere translatedin
vitro in presenceof [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 comparedby quantitative densitometry. A significant stimulation of apoB mRNA translation was observedwith T3 (an average of 54.5 f 1.3%). The effect of T3 on apoB mRNA translation was apparently specific for apoB sinceno changesin mRNA activity for apoAI was observed. Fig. 2B showsthe in vitro synthesizedapoA1 immunoprecipitatedfrom T3-treated (lanes 1 and 2) and control untreatedlysates (lanes 3 and 4 ). No detectable increasein synthesisof apoA1 was found following T3 treatment asdeterminedby densitometricanalysisof the signals. Pulse-chaselabeling of HepG2 cells was also performed to investigate the effect of T3 on apoB synthesisin vivo and to study the rate of apoB secretionfrom the cells into the extracellular medium.Fig. 3 showsthe amount of apoB synthesizedby HepG2 cells in presenceand absenceT3 and the rate of depletion of intracellular apoB. An average of 61% increasein the incorporation of [35S]methionineinto immunoprecipitableapoB was apparentwith T3. When the radioactivity was chased,a gradual reduction in labeled apoB was noted. Table 1 showsthe amount of radioactive apoB depletedduring the chaseand the estimatedamounts of apoB lost or degraded.ApoB content was calculated by densitometric scanningof the bandsand expressedas number of scanningunits per mg of total protein. ApoB secretedas a percentageof the intracellular peak was 53.6% for 620
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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 wasintracellularly degraded.TX-treated cells secretedan average of 30% more labeledapoB than control untreated cells
DISCUSSION Taken together, our data suggestthat thyroid hormoneregulatesthe expressionof hepatic apoB at number of points. There appearsto be an increasein the level of apoB mFCNAindicating an effect on the rate of apoB gene transcription and/or increasedmRNA stability. Further to this mRNA effect, T3 stimulate the rate of apoB synthesisboth in vitro and in vivo. The increasein 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% enhancementin the rate of protein synthesis.This raisesthe possibility Table 1. Changes in intracellular
apoB during a 3 b chase
Peak to 40 min Intracellular Depleted Secreted Degraded
Peak
Peak to 180 min
-T3
+T3
-T3
fT3
3.89kO.73 2.14 0.15 1.99
6.3OrtO.35 2.74 0.37 2.37
3.89kO.73 3.61 1.94 1.61
6.3OkO.35 5.33 2.34 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-chaseexperiment, it is evident that the 61% increasein [?S]methionine incorporation into apoB in the presenceof T3 did not fully translate into a similar stimulation in apoB secretion. Only a 30% increase in apoB secretion wasnoted 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 correspondedto a 37% increase in apoB massas measuredby ELISA. Our resultson the effect of thyroid hormone on apoB production concur with a previous study investigating apoB secretionas a function of thyroid statusin rat liver. Davidson et al (15) alsoreported decreasedapoB100and apoB4gsecretionin hypothyroid rats. However, contrary to our results when a state of hyperthyroidism was induced, hepatic apoBluc synthesis virtually ceasedwhile apoB4s synthesiswas unchanged. This discrepancy between the responseof rat hepatocytesand HepG2 cells to excessthyroid hormonemay stem from the fact that T3 appears to have effects on apoB expressionthat is specific to the rat system. Modulation of gene expressionby thyroid hormone in other systemsinvolves several distinct mechanismsincluding increasedtranscription rate (21), increasedmRNA stability (22), and/or changesin the rate of protein degradation (23, 24). Similar mechanismsappear to be involved in the caseof apoB generegulation by thyroid hormone.
ACKNOWLEDGMENTS This work was supportedby a grant from the Heart and Stroke Foundation of Ontario (Grant No. AN1904). We would like to thank the excellent technical assistanceof Mrs Debbie Rudy.
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