Yonago Acta medica 1997;40:133–136

Regulation of the Expression of Epidermal Growth Factor Receptor
mRNA with Thyroid Hormone L-3,5,3'-Triiodothyronine in Rat HepatomaCells
Yukio Satoh and Takeshi Sairenji
Department of Biosignaling, Faculty of Medicine, Tottori University, Yonago 683, JapanThe expression of epidermal growth factor (EGF) receptor mRNA was examined using
thyroid hormone L -3,5,3'-triiodothyronine (T 3) by Northern blot analysis in rat
hepatoma cells. The expression of full-length, but not truncated receptor mRNA wasenhanced and destabilized with T3. These results suggest that T3 induces not only theexpression of full-length EGF receptor mRNA but also accelerates their rate of turnover.
The 3' terminal region of full-length EGF receptor mRNA may have a role in thedestabilization of EGF receptor mRNA by T3, because truncated EGF receptor mRNAlacks only the 3' terminal region of full-length EGF receptor mRNA.
Key words: EGF receptor; EGF receptor mRNA; mRNA stability; regulation of gene expression;
thyroid hormone

The epidermal growth factor (EGF) receptor
mediates cell proliferation by EGF or transforming growth factor α (Carpenter and Cohen,1990). Normal rat liver cells express full-lengthand truncated EGF receptor messenger RNAs(mRNAs) (Petch et al., 1990; Satoh et al., 1996,1997). Full-length EGF receptor mRNA encodes a transmembrane EGF receptor tyrosine
kinase, c-ErbB1. Truncated EGF receptor
mRNA encodes a secreted EGF-binding proteinwith a yet unknown physiological function(Ullrich et al., 1984). Regulation of the gene
expression of EGF receptors has not been wellunderstood. Full-length and truncated EGF
receptor mRNAs initiate from the same 5' site(Ishii et al., 1985) and are subsequently
processed into different forms by alternative
splicing (Petch et al., 1990). Expression of
EGF receptors has been reported to be regulatedby thyroid hormone (Mukku, 1984; Vonderhaar
et al., 1986). Thyroid hormone is a ligand of cErbA, a member of the steroid/nuclear receptor
superfamily of ligand-regulated transcription
factors (Tsai and O’Malley, 1994). However,the EGF receptor promoter has not been reported to contain the thyroid hormone-responsive
element, while it was recently reported to have
putative estrogen-responsive elements (Yardenet al., 1996). Thyroid hormone was also
reported to decrease the length of the poly(A)tract of mRNA (Krane et al., 1991; Murphy etal., 1992).
Here we studied the effect of a thyroid
hormone, L-3,5,3'-triiodothyronine (T3) on theexpressions of full-length and truncated EGF
receptor mRNAs in rat hepatoma cells. We
found that full-length EGF receptor mRNA wasupregulated rapidly and subsequently
destabilized with T3.

Materials and Methods

Cell culture
AH66 cells provided by Dr. K. Sato (Department of Molecular Biology, Tottori University,Yonago, Japan) were cultured in Dulbecco’s

Abbreviations: DCC-NCS, dextran-charcoal-treated NCS; DRB, 5,6-dichlororibofuranosylbenzimidazole;
EGF, epidermal growth factor; kb, kilobase; mRNA, messenger RNA; NCS, newborn calf serum; T3, L-3,5,3'triiodothyronine

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Modified Eagle’s Medium (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) with 10% newborn calf serum (NCS; Filtron Ltd., Brooklyn,Australia) at 37˚C in a humidified atmospherecontaining 5% CO2.

medium containing 65 µM DRB (Harrold et al.,
1991; Sigma-Aldrich Japan K.K., Tokyo) and
3% DCC-NCS with or without 100 nM T3 andcells were cultured for an indicated time.

Northern blot analysis
Treatment of cells with thyroid hormone
and 5,6-dichlororibofuranosylbenzimidazole (DRB)
Cells were inoculated at 5 × 105 cells/dish in 9
cm plastic dishes and cultured for 48 h. The
cells were rinsed with serum-free medium andcultured in the medium containing 3% dextrancharcoal-treated NCS (DCC-NCS), which wasfree of thyroid hormone, with or without 100
nM T3 (Nacalai Tesque, Inc., Kyoto, Japan) for
3 h. For the measurement of stability of mRNA,the culture medium was changed to the fresh

RNA was extracted from the cells by the acid
guanidinium thiocyanate-phenol-chloroform
method (Chomozynski and Sacchi, 1987) and
was analyzed by Northern blot analysis asdescribed previously (Satoh et al., 1997). The
RNA was denatured, electrophoresed and transferred to a nylon membrane. The membrane
was hybridized with the probe, washed and exposed to an X-ray film. The rat EGF receptor
cDNA encoding extracellular domain (Petch etal., 1990) was provided by Dr. H. Shelton Earp
(University of North Carolina at Chapel Hill,Fig. 1. Effect of T3 on the expression of EGF receptor mRNAs in AH66 cells. Cells were cultured
with (+T3) or without (–T3) 100 nM T3 for 3 h at 37˚C and were then treated with DRB and cultured
with or without T3 for indicated times. RNA was extracted from the cells and was analyzed byNorthern blot. A: Upper panel; EGF receptor mRNA proved with the cDNA probe. Sizes of themRNA are shown on the left side. Lower panel; RNA (20 µg/lane) stained by ethidium bromide.
B: The levels of 10 and 2.7 kb EGF receptor mRNAs were quantitated and shown as a function oftime after the addition of DRB. The background level (13.9 cpm) of the same area was subtracted.
Data were reproduced three times by repeated experiments.
134Regulation of EGF receptor with T3

North Carolina, USA) and used as the 32Plabeled probe for EGF receptor mRNA. The
EGF receptor mRNA level was quantitated by
an AMBIS 4000 image analyzer (Japan
Medical Dynamic Marketing Inc., Tokyo,Japan).

Results
AH66 cells were cultured in the presence orabsence of T3 for 3 h, and then RNA synthesis
was stopped by the addition of DRB andcultured in the presence or absence of T3. Afterthe incubation, total cellular RNA was preparedat various time points and was subjected toNorthern blot analysis with the EGF receptor
cDNA probe (Fig. 1A). Ten, 7 and 5 kb fulllength and 2.7 kb truncated EGF receptor
mRNAs were detected in the cells as previously
described (Satoh et al., 1996, 1997). The bandsof the full-length EGF receptor mRNA weremuch darker in the presence of T3 (lane 1) thanin the absence of T3 (lane 6). The mRNA levelsof the 10 and 2.7 kb were quantitated by theimage analyzer (Fig. 1B). The level of 10 kb
mRNA with T3 treatment was 10-fold higher
than T 3-untreated cells at 0 h. However, thelevel reduced rapidly to the level of T 3 untreated cells by 2 h after the addition of DRB.
On the other hand, in the absence of T3, the lowlevel of 10 kb mRNA was detected and did notchange significantly up to 5 h after the additionof DRB. In contrast, the level of 2.7 kb mRNA
was not affected by the T3 treatment as shown at
0 h and did not change for 5 h in the presence ofDRB. The effect of T3 on the levels of 7 and 5kb mRNAs was similar to those of 10 kb mRNA(data not shown). These results indicate that T3
induced the expression of the mRNA of the fulllength EGF receptor and had accelerated turnover. However it did not have such an effect on
the truncated receptor mRNA.

Discussion
It has been reported that thyroid hormone
upregulates EGF receptor protein in cells of rat

liver (Mukku, 1984) and mouse mammaryglands (Vonderhaar et al., 1986). We foundthat the level of full-length EGF receptor
mRNA was upregulated with T3 in rat hepatoma cell line, AH66 cells. This is consistent
with the following reports: the enhancement of
EGF binding to the membranes of liver andmammary glands of euthyroid animals as
opposed to hypothyroid controls (Mukku, 1984;
Vonderhaar et al., 1986), the enhancement of
the transcription of EGF receptor in T3-treatedhuman mammary cells (Fernandez-Pol et al.,1989) and the stimulation of T3 on the level of
5.6 kb EGF receptor mRNA in human epidermoid cells (Kesavan et al., 1991). The
higher level of 10 kb mRNA in the T3-treatedcells may suggest that the expression of fulllength EGF receptor mRNA is enhanced attranscriptional levels and/or alternative splicing
steps in the presence of T3. We also observedthat the induced full-length EGF receptor
mRNA was unstable in the treatment of T3. It
has been reported that rat mRNAs wereunstable in the presence of a thyroid hormone
than in its absence (Krane et al., 1991; Murphy
et al., 1992). It has also been explained that theextension of polyadenylation in the cell nuclei
is downregulated by thyroid hormone (Murphyet al., 1992; Sachs and Wahle, 1993).
Full-length and truncated EGF receptor
mRNAs were produced by alternative splicing(Petch et al., 1990). The expression of truncated EGF receptor mRNA was not affected by
the treatment of T3 (Fig. 1). The mRNAs have acommon 5' terminal region but truncatedmRNA lacks a 3' terminal region of full-length
form mRNA in rat hepatoma cells (Satoh et al.,1996). We hypothesize that the 3' terminal
region of the full-length EGF receptor mRNA
may be involved in the turnover acceleration by
T3 in the rat hepatoma cells.
T3 exerts effects in hepatocellular regenerations during cirrhosis and partial hepatectomy(Knopp et al., 1992; Sasaki et al., 1992; Huangand Liaw, 1995; Oren et al., 1996). The experimental findings in the hepatocellular carcinoma
cell line, AH66 may have clinical implications
particularly relevant to liver diseases andhepatocellular carcinoma.

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Y. Satoh and T. Sairenji

Our results suggest that the mode of actionof thyroid hormone mediates the growthpromoting and developmental effects by thetransient induction of full-length EGF receptor.
Acknowledgments: This work was supported in partby a Grant-in-Aid for the Second-Term Comprehensive 10-Year Strategy for Cancer Control from
the Ministry of Health and Welfare of Japan.

References
1 Carpenter G, Cohen S. Epidermal growth factor.
J Biol Chem 1990;265:7709–7712.
2 Chomczynski P, Sacchi N. Single-step method of
RNA isolation by acid guanidinium thiocyanatephenol-chloroform extraction. Anal Biochem
1987;162:156–159.
3 Fernandez-Pol JA, Hamilton PD, Klos DJ.
Transcriptional regulation of proto-oncogene
expression by epidermal growth factor, transforming growth factor β1, and triiodothyronine inMDA-468 cells. J Biol Chem 1989;264:41514156.
4 Harrold S, Genovese C, Kobrin B, Morrison SL,Milcarek C. A comparison of apparent mRNAhalf-life using kinetic labeling techniques vsdecay following administration of transcriptional
inhibitors. Anal Biochem 1991;198:19–29.
5 Huang MJ, Liaw YF. Clinical associations between thyroid and liver diseases. J Gastroenterol
Hepatol 1995;10:344–350.
6 Ishii S, Xu YH, Stratton RH, Roe BA, Merlino
GT, Pastan I. Characterization and sequence ofpromoter region of the human epidermal growth
factor receptor gene. Proc Natl Acad Sci USA
1985;82:4920–4924.
7 Kesavan P, Mukhopadhayay S, Murphy S,Rengaraju M, Lazar MA, Das M. Thyroid hormone decreases the expression of epidermal
growth factor receptor. J Biol Chem 1991;266:10282–10286.
8 Knopp J, Brtko J, Filipcik P. Effect of triiodothyronine on rat liver polysome profiles andtranslational activity of mRNA after partial
hepatectomy. Endocr Regul 1992;26:67–72.
9 Krane IM, Spindel ER, Chin WW. Thyroidhormone decreases the stability and poly(A) tract
length of rat thyrotropin β-subunit messenger
RNA. Mol Endocrinol 1991;5:469–475.
10 Mukku VR. Regulation of epidermal growth

factor receptor levels by thyroid hormone. J Biol
Chem 1984;259:6543–6547.
11 Murphy D, Pardy K, Seah V, Carter D. Posttranscriptional regulation of rat growth hormone
gene expression: increased message stability andnuclear polyadenylation accompany thyroid hormone depletion. Mol Cell Biol 1992;12: 2624–2632.
12 Oren R, Dotan I, Papa M, Marravi Y, Aeed H,Barg J, et al. Inhibition of experimentallyinduced cirrhosis in rats by hypothyroidism.
Hepatology 1996;24:419–423.
13 Petch LA, Harris J, Raymond VW, Blasband A,Lee DC, Earp HS. A truncated, secreted form of
the epidermal growth factor receptor is encodedby an alternatively spliced transcript in normal rat
tissue. Mol Cell Biol 1990;10:2973–2982.
14 Sachs A, Wahle E. Poly(A) tail metabolism andfunction in eucaryotes. J Biol Chem 1993;268:22955–22958.
15 Sasaki H, Matsumoto S, Shijyo H, Shimizu M,Tohara K, Tsutsu N, et al. Gross ascites as a firstmanifestation of primary hypothyroidism due topost-treatment of radioiodine therapy for Graves’disease. Intern Med 1992;31:256–259.
16 Satoh Y, Oomae M, Ichii S. Studies on rat
epidermal growth factor (EGF) receptor: rapid
amplification and cloning of cDNA ends corresponding to 3' termini of full-length EGF
receptor mRNAs. Yonago Igaku Zasshi 1996;
47:218–227 (in Japanese with English abstract).
17 Satoh Y, Oomae M, Hoshikawa Y, Izawa M,Sairenji T, Ichii S. The stability of truncatedepidermal growth factor receptor mRNA is higher than that of the full-length receptor mRNA inrat hepatoma cells. Endocrine J 1997;44:403–408.
18 Tsai MJ, O’Malley BW. Molecular mechanismsof action of steroid/thyroid receptor superfamily
members. Annu Rev Biochem 1994;63:451–486.
19 Ullrich A, Coussens L, Hayflick JS, Dull TJ,
Gray A, Tam AW, et al. Human epidermal
growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431
epidermoid carcinoma cells. Nature 1984;309:418–425.
20 Vonderhaar BK, Tang E, Lyster RR, Nascimento
MCS. Thyroid hormone regulation of epidermal
growth factor receptor levels in mouse mammaryglands. Endocrinology 1986;119:580–585.
21 Yarden RI, Lauber AH, El-Ashry D, Chrysogelos
SA. Bimodal regulation of epidermal growth
factor receptor by estrogen in breast cancer cells.
Endocrinology 1996;137:2739–2747.

(Received July 25, Accepted August 25, 1997)

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