Thyroid hormone attenuates and augments hepatic gene expression

Transcription

1 Proc. Ntl Acd. Sci. USA Vol. 78, No. 8, pp , August 1981 Biochemistry Thyroid hormone ttenutes nd ugments heptic gene expression t pretrnsltionl level (triiodothyronine/heptic mrna/two-dimensionl gel electrophoresis) STEVEN SEELIG, CHEN LIuw, HOWARD C. TOWLE, AND JACK H. OPPENHEIMER Division of Endocrinology nd Metbolism, Deprtment of Medicine nd Deprtment of Biochemistry, University of Minnesot, Minnepolis, Minnesot Communicted byj. Edwrd Rll, April 13, 1981 ABSTRACT We hve ttempted to scertin the proportion of the rt heptic genome tht is under the selective influence of thyroid hormones nd to describe the response ptterns of individul mrna sequences in the trnsition between hypothyroidism nd euthyroidism nd between euthyroidism nd hyperthyroidism. Poly(A)+RNA ws extrcted from livers of thyroidectomized, intct, euthyroid rts nd of thyroidectomized rts rendered euthyroid nd hyperthyroid with dily doses oftriiodothyronine. The extrcted RNA ws trnslted in reticulocyte lyste system in the presence of [3S]methionine, nd the products were nlyzed by two-dimensionl gel electrophoresis. Triiodothyronine ttenutes s well s ugments the expression of certin genes t pretrnsltionl level. This could represent either direct or n indirect ction of the hormone. Triiodothyronine influences pproximtely 8% of the 231 mrna sequences visulized, stimulting ctivity in 11 nd inhibiting ctivity in 7 sequences. Trnsltionl ctivity of t lest one mrna sequence decresed in both thyroidectomized nd hyperthyroid nimls, compred to euthyroid levels. The reltionship of mrna response to receptor occupncy vried with exmples of liner nd mplified responses nd responses tht were mximl t less thn full nucler occupncy. Thyroid hormones re generlly believed to ct by selective stimultion of gene trnscription or pretrnsltionl events (1). Nevertheless, previous investigtors hve provided evidence tht severl enzymes decrese in response to thyroid hormone dministrtion (2). More recently, Ivrie et l. (3) demonstrted tht triiodothyronine (T3) inhibits the reltive rte of synthesis of certin proteins in GH3 cells, cell line derived from rt pituitry tumor nd mintined in tissue culture. Armstrong nd Feigelson (4) hve lso shown tht T3 inhibits the reltive rte of synthesis of histidinse in the rt liver. In none of these studies ws the mechnism of inhibition clrified. Prompted by these observtions, we undertook studies to determine whether such inhibition occurred t posttrnsltionl or t pretrnsltionl level. Both inhibitory nd stimultory effects of thyroid hormone on the genome were reflected in two-dimensionl gel electrophoresis ptterns of in vitro trnsltionl products of heptic poly(a)+rna obtined from rts ofdifferent thyroid sttus. These experiments llowed us to define 19 mrna sequences under the selective influence of T3 nd to document brod spectrum of chnges tht occur in the trnsitions between the hypothyroid nd euthyroid nd the euthyroid nd hyperthyroid sttes. METHODS Mle Sprgue-Dwley rts weighing 18 g, obtined from Tconic Frms (Germntown, NY), were surgiclly thyroidectomized by the supplier nd given 1,Ci (1 Ci = 3.7 X 11 The publiction costs ofthis rticle were defryed in prt by pge chrge pyment. This rticle must therefore be hereby mrked "dvertisement" in ccordnce with 18 U. S. C solely to indicte this fct. becquerels) of 131I fter 1 week of low-iodine intke. The nimls were considered to be dequtely hypothyroid only fter growth retrdtion ws demonstrted pproximtely 3-4 weeks fter 131I dministrtion. Thyroidectomized (Tx) nimls were given dily intrperitonel injection of 3 ng or 15,tg of L- T3 (Sigm) per 1 g of body weight for 12 dys, doses designed to chieve euthyroid or hyperthyroid sttus, respectively. Mlic enzyme (EC ) ctivity ws ssyed by the method of Hsu nd Lrdy (5). One unit of enzyme ctivity ws defined s the mount of enzyme required to reduce 1 nmol of NADP+ in 1 min. -Glycerophosphte dehydrogense (- GPD) (EC ) ws mesured by the method of Lee nd Lrdy (6). Protein ws mesured by the method of Lowry et l. (7). Totl cellulr RNA ws extrcted from frozen (-8C) liver with phenol/chloroform t ph 9 s described (8). Totl cellulr RNA smples were resuspended in wter, heted to 68C for 2 min, nd pplied to oligo(dt)-cellulose columns for isoltion of poly(a)+rna (9). Rbbit reticulocyte lyste ws prepred by the method of Evns nd Lingrel (1). The lyste ws treted with micrococcl nuclese (P-L Biochemicls) t 3,ug/ml s described by Pelhm nd Jckson (11). In vitro trnsltionl ssys were performed s described (8). The rection mixtures were incubted for 9 min t 23C nd centrifuged t 1, x g for 1 hr to remove ribosomes. Incorportion of [3S]methionine into protein ws determined by 1% trichlorocetic cid precipittion of n liquot of the trnslted products (12). Smples of trnslted products contining n equl rdioctivity (25,-3, cpm) were then subjected to two-dimensionl gel electrophoresis. For two-dimensionl gel electrophoresis, the method of O'Frrell (13) ws used with minor modifictions. A smll liquot of trnslted products (9-25,ug of reticulocyte lyste protein) ws diluted in lysis buffer (9.5 M ure/5% mercptoethnol/2% Nonidet P-4/2% Ampholines). The rtio of Ampholines ws 1% ph nd 1% ph 5-7. This Ampholine rtio empiriclly yielded slightly broder rnge of proteins entering the isoelectric focusing gel with only modest loss in resolution. Isoelectric focusing ws performed for totl of56 V-hr, nd the focused gels were stored frozen t -8 C in equilibrtion buffer (.65 M -Tris, ph 6.8/2% NDodSOJ5% mercptoethnol/1% glycerol). Prior to use for the second dimension, isoelectric focused gels were thwed nd gently gitted for 1 hr. The first-dimension gels were fixed to the top of the second-dimension gels by using 1% grose in equilibrtion buffer. Second-dimension gels consisted of 4-em 4% crylmide (crylmide/biscrylmide, 37:1) stcking gel nd 22-cm liner 8-18% crylmide seprting gel. Stndrd mo Abbrevitions: Tx, thyroidectomized; T3, triiodothyronine; -GPD, - glycerophosphte dehydrogense.

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3 Biochemistry: Seelig et l. leculr weight mrkers were plced t ech end ofthe isoelectric focusing gel nd the smples were electrophoresed t 8 V for 1 hr nd 2 V for 2 hr. After electrophoresis, the proteins were fixed in 25% trichlorocetic cid for 3 min, stined with.2% Coomssie brillint blue R25 in 5% methnol/7% cetic cid for 6 hr, nd then destined in 2% methnol/7% cetic cid overnight. In some cses ['4C]methylted protein stndrds (Bethesd Reserch Lbortories, Rockville, MD) were used for moleculr weight mrkers in the second dimension. When lbeled proteins were used for moleculr weight mrkers, the gels were treted in the sme wy except tht Coomssie brillint blue R25 ws not used. No substntil difference between stined nd unstined gels were detected in the fluorogrms. The gels were prepred for fluorogrphy by using EN3HANCE (New Englnd Nucler) s described by the mnufcturers nd dried on Whtmn 3M pper with het under reduced pressure. The dried gels were subsequently exposed to Kodk SB5 noscreen medicl x-ry film for 2 hr. The exposed film ws developed s described by the mnufcturers. Eighteen nimls were nlyzed individully s described bove: seven Tx; three norml; four Tx injected with.3,ug of T3 per 1 g; nd four Tx injected with 15 ug of T3 per 1 g. Fluorogrms from ech niml were ssessed by visul inspection. The mjority of spot intensities were independent of thyroid sttus nd constnt; however, pproximtely four or five spots ppered to vry in rndom fshion with respect to thyroid sttus. The significnce of these vritions is not understood. Genetic, environmentl, or subtle technicl differences my ccount for these vritions. For identifiction of T3-dependent spots, only those spots tht were ltered in ech niml of the vrious thyroid sttes re noted. RESULTS Tble 1 summrizes the weight chnges nd enzymtic indices of thyroid function in the nimls studied. The effectiveness of the T3 regimen is demonstrted by restortion of the rte of weight gin nd by incresed levels of T3-responsive enzymes (cytosolic mlic enzyme nd mitochondril -GPD). Both enzyme ctivities pproximte previously reported vlues for euthyroid nd hyperthyroid sttes (14, 15); however, complete restortion of growth, mlic enzyme, nd -GPD ws not chieved with the lower T3 dose. The nimls injected with T3 t 15,ug/1 g filed to gin weight normlly, n observtion consistent with hypermetbolic stte. Representtive two-dimensionl gel electrophoresis ptterns of trnslted products from poly(a)+rna isolted from norml nimls nd Tx nimls re shown in Fig. 1. These two-dimensionl nlyses reveled dose-dependent nd exceedingly heterogeneous pttern of response to T3 dministrtion. These ptterns represent reltive trnsltionl ctivity of vrious poly(a)+rna becuse equl mounts of rdioctivity were pplied to ech gel. A totl of 19 spots consistently chnged in intensity with the trnsition from the hypothyroid to the hyperthyroid stte. Eleven spots incresed in intensity, seven Proc. Ntl. Acd. Sci. USA 78 (1981) 4735 Tble 1. Indices of T3 ction -GPD, BW gin, Mlic enzyme, AA5/min/mg Tretment g/dy units/mg protein protein Tx (control).36 ±.8.25 ± ±.6 Tx +.3 ug T3 3.4 ± ± ±.6 Tx + 15 Ag T3 1.1 ± ± ±.93 Norml 5. ±.5 18 ± ±.4 T3 ws injected t dosges of.3 nd 15,ug/1 g dily for 12 dys. Vlues presented re the men ± SEM of four nimls. BW, body weight. spots decresed in intensity, nd one spot showed biphsic response. With respect to the mrna species tht incresed, three distinctive ptterns were observed. In euthyroid nimls, pproximtely 5% of the T3 receptors re occupied (16). Therefore, in liner response pttern, n equl increse is expected between the hypothyroid-euthyroid nd euthyroid-hyperthyroid trnsitions. Spots 4, 5, 11, nd 12 pper to disply this response pttern. In contrst, spots 3 nd 8 incresed drmticlly in the hypothyroid-euthyroid trnsition with only miniml chnge in the euthyroid-hyperthyroid trnsition. This cn be considered n exmple of euthyroid-sturble response pttern. Spots 2 nd 14 illustrte n pprent mplified response pttern comprble to wht hs been described for -GPD nd mlic enzyme (17) in tht there ws substntilly greter increse in the euthyroid-hyperthyroid trnsition stte thn in the hypothyroid-euthyroid trnsition. In ddition, severl mrna species showed substntil decline in ctivity. Spot 13 decresed in the hypothyroid-euthyroid trnsition. We cnnot be certin whether n dditionl decrese occurred in the euthyroid-hyperthyroid trnsition becuse the intensity of the spot in the euthyroid stte is fint. In contrst, no substntil chnge or slight increse occurred in spots 9 nd 1 in the hypothyroid-euthyroid trnsition; however, mrked decrese in these spots occurred in the euthyroid-hyperthyroid trnsition. Spot 15 ppers to represent unique pttern. In ll seven Tx nimls nd four hyperthyroid nimls, the spot ws essentilly bsent. In seven euthyroid nimls (three norml nd four Tx given T3 t.3 jig/1 g) it ws clerly present in incresed intensity. Thus, the response pttern of spot 15 is biphsic. The comprisons discussed re bsed on the chnges observed between Tx nimls (A) nd Tx nimls injected with T3 nd on the ssumption tht Tx niml injected with T3 t.3,g/1 g is in n pproximte euthyroid stte. Quntittive comprison of the norml niml illustrted (B) with the Tx-injected nimls is not possible becuse the smple ws subjected to isoelectric focusing in seprte experiment. However, the chnges observed between Tx (Tx control not shown) nd norml nimls nd the chnges observed between Tx (A) nd Tx injected with T3 t.3 jig/1 g (C) were qulittively identicl. This comprison lso suggests tht intensities of spots 4, 5, 6, FIG. 1 (on preceding pge). Representtive fluorogrms of two-dimensionl gel electrophoresis of [35S]methionine-lbeled trnslted products of heptic poly(a)+rna obtined from Tx nimls (A), euthyroid nimls (B), Tx nimls injected with T3 t.3,g/1 g (C), nd Tx nimls injected with T3 t 15,g/1 g (D). The trnslted products illustrted in A, C, nd D were subjected to isoelectric focusing t the sme time; the trnslted products in B were subjected to seprte isoelectric focusing in which three Tx nimls nd three norml nimls were compred. The direction of chnge in spot intensity shown by the comprison of A nd C ws identicl qulittively to tht observed between Tx control (not shown) nd B. Spots 1 nd 1 re doublets. Whether these doublets represent the sme mrna sequence or two seprte mrna sequences is not known. For both, the doublets pper to hve similr response pttern to T3. Two-dimensionl gel electrophoresis of immunoprecipitte of trnslted products from hyperthyroid niml obtined with mlic enzyme ntibody produced spot t the re of spot 2. Comprison of trnslted products before nd fter immunoprecipittion with mlic enzyme ntibody indictes tht the trnslted mlic enzyme migrtes in the second dimension with Mr slightly smller thn tht of the strek tht trnsverses the top portion of re 2. The isoelectric focusing rnge ws from ph 7.9 (left) to ph 4.5 (right); NDodSO4 migrtion ws from top to bottom, with the rnge 1,-12, dltons displyed.

4 4736 Biochemistry: Seelig et l. Tble 2. T3-induced chnges in heptic poly(a)+ RNA Chnge Spot Tx-.3 ug.3-15 jig pi Mr X ? f f T ? ? ? ?4, l Tble shows visully detected increses ( I ), decreses (4,), nd bsence of obvious chnge (+.) in reltive spot intensity between Tx nimls nd Tx nimls injected with T3 t.3 pg/1 g nd between Tx nimls injected with T3 t.3 pg1 g nd Tx nimls injected with T3 t 15 1Ag/1 g.? indictes tht the direction of chnge suggested in the illustrtion cn be questioned on the bsis of the three other gels not presented. Approximte isoelectric focusing point (pi) nd Mr re identified for ech spot. nd 14 my not be fully restored in the nimls given T3 t.3 pag/1 g. The possibilities should be considered tht (i) T3 dose greter thn.3 iug/1 g is necessry to chieve full replcement; (ii) the niml is not fully equilibrted fter 12 dys of injection; nd (iii) pulstile T3 replcement does not result in equivlent restortion of ll response prmeters. Tble 2 summrizes the pttern of chnges observed in the Tx injected nimls nd gives the pproximte pi nd moleculr weight of the T3 responsive proteins. DISCUSSION T3 ttenutes s well s ugments the reltive ctivities of specific mrnas. Others hve shown tht T3 inhibits the synthesis or ctivity ofvrious proteins (2-4, 18). We hve demonstrted, by trnsltion of poly(a)+rna in reticulocyte lyste system, tht t lest some of the observed inhibition is consequence of diminished ctivity of specific mrna sequences. The heterogeneity in ptterns of response to T3 is remrkble. The observed effects on mrna ctivities could be consequence ofdirect hormone ction on specific gene expressed, of n indirect effect of T3 on nother heptic gene or nother orgn, or of combintion of these. Of prticulr interest in connection with these considertions is the well-recognized effect of T3 on pituitry growth hormone production. In hypothyroidism, the pituitry content nd plsm levels of growth hormone re diminished. Thus, it is conceivble tht subststil number of observed chnges in mrna sequences my represent the indirect effect of T3 stimultion on growth hormone production. Wheres this could ccount for some of the chnges in the hypothyroid-euthyroid trnsition, it ppers unlikely tht growth hormone-relted chnges re responsible for the ltertions tht chrcterize the euthyroid-hyperthyroid trnsition. Coulombe et l. (19) hve shown tht the pituitry Proc. Ntl. Acd. Sci. USA 78 (1981) content of growth hormone in hyperthyroidism is not incresed bove tht found in the euthyroid stte. To dte, two thyroid hormone-responsive mrna sequences hve been identified in the liver: 21 -globulin, n exported protein tht lso is under the control of cortisol, dihydrotestosterone, nd growth hormone (2, 21); nd mlic enzyme, which lso is under the control of dietry crbohydrte (8, 14). The chnges observed in the expression of the mrna sequence for both 21-globulin nd mlic enzyme illustrte the multihormonl nd multifctoril control ofgene expression nd provide bsis for understnding the complexity ofthe response pttern observed in our studies. The heterogeneity of poly(a)+rna response is reminiscent of the effects of T3 or dexmethsone on the reltive rtes of protein synthesis in GH3 or HTC cells, respectively (3, 22). Our studies estblish tht such heterogeneity is reflected t the pretrnsltionl level. This report revels selective response to T3 by number of specific mrna sequences in ddition to the well-recognized generlized increse in cellulr RNA content. Ifwe ssume tht the 231 sequences nlyzed re typicl of the popultion of the 11, different sequences estimted to exist in the heptic cell (23), minimum of 8.2% of totl poly(a)+rna is selectively modulted by T3 (19 of 231 spots visulized). In the hyperthyroid stte, pproximtely.4% ofthe totl [3S]methionine incorported is in mlic enzyme (8). We ssume tht spot 2 represents mlic enzymes becuse it is selectively precipitted by specific ntibody directed ginst mlic enzyme (unpublished dt). If we ssume tht the methionine content per protein is similr for the responsive poly(a)+rnas nd tht they re trnslted with equl efficiency in the reticulocyte lyste system, we cn infer tht severl T3-responsive poly(a)+rna sequences re considerbly more bundnt thn the sequence represented by spot 2. The low level ofmrna coding for mlic enzyme [undetectble in Tx nd.4% in the euthyroid niml (8)] s well s poor resolution of proteins in the region of mlic enzyme hindered our bility to identify chnge in mlic enzyme mrna in the Tx-euthyroid trnsition. The mechnism by which T3 induces the observed chnges is yet to be elucidted. Whtever model of T3 ction is postulted, it must ccount for three observtions: (i) generlized increse in totl cellulr RNA; (ii) selective increse in reltively smll popultion ofmrnas tht exceeds the generlized increse of mrna; nd (iii) selective decrese in the ctivity of severl mrna sequences. We thnk Ms. Nncy Rutledge nd Ms. Colleen Jhnel for their ssistnce in preprtion of this mnuscript nd Mr. Robert Gunville nd Ms. An Mrtinez for their technicl ssistnce. This work ws supported by Ntionl Institutes of Helth Grnts AM (J. HO.) nd AM26919 (H.C.T.) nd Ntionl Service Trining Grnt Awrd AM6478 (S.S.). 1. Oppenheimer, J. H. (1979) Science 23, Wolff, E. C. & Wolff, J. (1964) in The Thyroid Glnd, eds. Pitt- Rivers, R. & Trotter, W. R. (Butterworth, London), Vol. 1, pp Ivrie, R. D., Morris, J. A. & Eberhrdt, N. L. (198) Rec. Prog. Horm. Res. 36, Armstrong, E. G. & Feigelson, M. (198) J. Biol, Chem. 255, Hsu, R. Y. & Lrdy, H. A. (1969) Methods Enzymol. 13, Lee, Y.-P. & Lrdy, H. A. (1965)J. Biol Chem. 24, Lowry,. H., Rosebrough, N. J., Frr, A. L. & Rndll, R. J. (1951) J. Biol Chem. 193, Towle, H. C., Mrish, C. N. & Oppenheimer, J. H. (198) Biochemistry 19, Krystosek, A., Cwthon, M. L. & Kbt, D. (1975)J. BioL Chem. 25, Evns, M. J. & Lingrel, J. B. (1969) Biochemistry 8,

5 Biochemistry: Seelig et l. 11. Pelhm, H. R. B. & Jckson, R. J. (1976) Eur. J. Biochem. 67, Mns, R. J. & Novelli, G. D. (1961) Arch. Biochem. Biophys. 94, O'Frrell, P. H. (1975) J. Biod Chem. 25, Mrish, C. N., Kiser, F. E., Schwrtz, H. L., Towle, H. C. & Oppenheimer, J. H. (198)J. Clin. Invest 65, Oppenheimer, J. H., Shpiro, H. C., Schwrtz, H. L. & Surks, M. I. (1971) Endocrinology 88, Oppenheimer, J. H., Schwrtz, H. L. & Surks, M. I. (1974) Endocrinology 95, Oppenheimer, J. H., Coulombe, P., Schwrtz, H. L. & Guffeld, N. W. (1978) J. Clin. Invest 61, Proc. NtL Acd. Sci. USA 78 (1981) Tsi, J. S. & Smuels, H. H. (1974) Biochem. Biophys. Res. Commun. 59, Coulombe, P., Schwrtz, H. L. & Oppenheimer, J. H. (1978)J. Clin. Invest. 62, Kurtz, D. T. & Feigelson, P. (1977) Proc. NtL Acd. Sci. USA 74, Roy, A. K. & Dowbenko, D. J. (1977) Biochemistry 16, Ivrie, R. D. & O'Frrell, P. H. (1978) Cell 13, Towle, H. C., Dillmnn, W. H. & Oppenheimer, J. H. (1979)J. Biod Chem. 254,