The expression of epidermal growth factor (EGF) receptor mRNA was examined using thyroid hormone L-3,5,3'-triiodothyronine (T3) by Northern blot analysis in rat hepatoma cells. The expression of full-length, but not truncated receptor mRNA was enhanced and destabilized with T3. These results suggest that T3 induces not only the expression 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 the destabilization of EGF receptor mRNA by T3, because truncated EGF receptor mRNA lacks only the 3' terminal region of full-length EGF receptor mRNA

Sairenji, Takeshi

Satoh, Yukio

Yonago Acta Medica

Tottori University Research Result Repository

133Yonago Acta medica 1997;40:133–136Regulation of the Expression of Epidermal Growth Factor ReceptormRNA with Thyroid Hormone L-3,5,3'-Triiodothyronine in Rat HepatomaCellsYukio Satoh and Takeshi SairenjiDepartment of Biosignaling, Faculty of Medicine, Tottori University, Yonago 683, JapanThe expression of epidermal growth factor (EGF) receptor mRNA was examined usingthyroid hormone L-3,5,3'-triiodothyronine (T3) by Northern blot analysis in rathepatoma 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 hormoneAbbreviations:  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'-triiodothyronineThe epidermal growth factor (EGF) receptormediates cell proliferation by EGF or trans-forming 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 en-codes a transmembrane EGF receptor tyrosinekinase, c-ErbB1.  Truncated EGF receptormRNA encodes a secreted EGF-binding proteinwith a yet unknown physiological function(Ullrich et al., 1984).  Regulation of the geneexpression of EGF receptors has not been wellunderstood.  Full-length and truncated EGFreceptor mRNAs initiate from the same 5' site(Ishii et al., 1985) and are subsequentlyprocessed into different forms by alternativesplicing (Petch et al., 1990).  Expression ofEGF receptors has been reported to be regulatedby thyroid hormone (Mukku, 1984; Vonderhaaret al., 1986).  Thyroid hormone is a ligand of c-ErbA, a member of the steroid/nuclear receptorsuperfamily of ligand-regulated transcriptionfactors (Tsai and O’Malley, 1994).  However,the EGF receptor promoter has not been report-ed to contain the thyroid hormone-responsiveelement, while it was recently reported to haveputative estrogen-responsive elements (Yardenet al., 1996).  Thyroid hormone was alsoreported 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 thyroidhormone, L-3,5,3'-triiodothyronine (T3) on theexpressions of full-length and truncated EGFreceptor mRNAs in rat hepatoma cells.  Wefound that full-length EGF receptor mRNA wasupregulated rapidly and subsequent lydestabilized with T3.Materials and MethodsCell cultureAH66 cells provided by Dr. K. Sato (Depart-ment of Molecular Biology, Tottori University,Yonago, Japan) were cultured in Dulbecco’sY. Satoh and T. Sairenji134Modified Eagle’s Medium (Nissui Pharma-ceutical Co. Ltd., Tokyo, Japan) with 10% new-born calf serum (NCS; Filtron Ltd., Brooklyn,Australia) at 37˚C in a humidified atmospherecontaining 5% CO2.Treatment of cells with thyroid hormoneand 5,6-dichlororibofuranosylbenzimida-zole (DRB)Cells were inoculated at 5 × 105 cells/dish in 9cm plastic dishes and cultured for 48 h.  Thecells were rinsed with serum-free medium andcultured in the medium containing 3% dextran-charcoal-treated NCS (DCC-NCS), which wasfree of thyroid hormone, with or without 100nM T3 (Nacalai Tesque, Inc., Kyoto, Japan) for3 h.  For the measurement of stability of mRNA,the culture medium was changed to the freshFig. 1.  Effect of T3 on the expression of EGF receptor mRNAs in AH66 cells.  Cells were culturedwith (+T3) or without (–T3) 100 nM T3 for 3 h at 37˚C and were then treated with DRB and culturedwith 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.medium containing 65 µM DRB (Harrold et al.,1991; Sigma-Aldrich Japan K.K., Tokyo) and3% DCC-NCS with or without 100 nM T3 andcells were cultured for an indicated time.Northern blot analysisRNA was extracted from the cells by the acidguanidinium thiocyanate-phenol-chloroformmethod (Chomozynski and Sacchi, 1987) andwas analyzed by Northern blot analysis asdescribed previously (Satoh et al., 1997).  TheRNA was denatured, electrophoresed and trans-ferred to a nylon membrane.  The membranewas hybridized with the probe, washed and ex-posed to an X-ray film.  The rat EGF receptorcDNA encoding extracellular domain (Petch etal., 1990) was provided by Dr. H. Shelton Earp(University of North Carolina at Chapel Hill,Regulation of EGF receptor with T3135North Carolina, USA) and used as the 32P-labeled probe for EGF receptor mRNA.  TheEGF receptor mRNA level was quantitated byan AMBIS 4000 image analyzer (JapanMedical Dynamic Marketing Inc., Tokyo,Japan).ResultsAH66 cells were cultured in the presence orabsence of T3 for 3 h, and then RNA synthesiswas 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 receptorcDNA probe (Fig. 1A).  Ten, 7 and 5 kb full-length and 2.7 kb truncated EGF receptormRNAs were detected in the cells as previouslydescribed (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 kbmRNA with T3 treatment was 10-fold higherthan T3-untreated cells at 0 h.  However, thelevel reduced rapidly to the level of T3-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 mRNAwas not affected by the T3 treatment as shown at0 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 T3induced the expression of the mRNA of the full-length EGF receptor and had accelerated turn-over.  However it did not have such an effect onthe truncated receptor mRNA.DiscussionIt has been reported that thyroid hormoneupregulates EGF receptor protein in cells of ratliver (Mukku, 1984) and mouse mammaryglands (Vonderhaar et al., 1986).  We foundthat the level of full-length EGF receptormRNA was upregulated with T3 in rat hepa-toma cell line, AH66 cells.  This is consistentwith the following reports: the enhancement ofEGF binding to the membranes of liver andmammary glands of euthyroid animals asopposed to hypothyroid controls (Mukku, 1984;Vonderhaar et al., 1986), the enhancement ofthe transcription of EGF receptor in T3-treatedhuman mammary cells (Fernandez-Pol et al.,1989) and the stimulation of T3 on the level of5.6 kb EGF receptor mRNA in human epi-dermoid cells (Kesavan et al., 1991).  Thehigher level of 10 kb mRNA in the T3-treatedcells may suggest that the expression of full-length EGF receptor mRNA is enhanced attranscriptional levels and/or alternative splicingsteps in the presence of T3.  We also observedthat the induced full-length EGF receptormRNA was unstable in the treatment of T3.  Ithas been reported that rat mRNAs wereunstable in the presence of a thyroid hormonethan in its absence (Krane et al., 1991; Murphyet al., 1992).  It has also been explained that theextension of polyadenylation in the cell nucleiis downregulated by thyroid hormone (Murphyet al., 1992; Sachs and Wahle, 1993).Full-length and truncated EGF receptormRNAs were produced by alternative splicing(Petch et al., 1990).  The expression of trun-cated EGF receptor mRNA was not affected bythe treatment of T3 (Fig. 1).  The mRNAs have acommon 5' terminal region but truncatedmRNA lacks a 3' terminal region of full-lengthform mRNA in rat hepatoma cells (Satoh et al.,1996).  We hypothesize that the 3' terminalregion of the full-length EGF receptor mRNAmay be involved in the turnover acceleration byT3 in the rat hepatoma cells.T3 exerts effects in hepatocellular regener-ations during cirrhosis and partial hepatectomy(Knopp et al., 1992; Sasaki et al., 1992; Huangand Liaw, 1995; Oren et al., 1996).  The experi-mental findings in the hepatocellular carcinomacell line, AH66 may have clinical implicationsparticularly relevant to liver diseases andhepatocellular carcinoma.Y. Satoh and T. Sairenji136Our results suggest that the mode of actionof thyroid hormone mediates the growth-promoting 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 Compre-hensive 10-Year Strategy for Cancer Control fromthe 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 ofRNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.  Anal Biochem1987;162:156–159. 3 Fernandez-Pol JA, Hamilton PD, Klos DJ.Transcriptional regulation of proto-oncogeneexpression by epidermal growth factor, trans-forming growth factor β1, and triiodothyronine inMDA-468 cells.  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Regulation of the Expression of Epidermal Growth Factor Receptor mRNA with Thyroid Hormone L-3,5,3'-Triiodothyronine in Rat Hepatoma Cells

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Regulation of the Expression of Epidermal Growth Factor Receptor mRNA with Thyroid Hormone L-3,5,3'-Triiodothyronine in Rat Hepatoma Cells

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