Transcriptional and posttranscriptional regulation of rat hepatic 3 ...

THEJOURNALOF BIOLOGICAL CHEMISTRY Q 1988 by The American Soeiety for Biochemistry and Molecular Biology. Inc.

Vol. 263, No. 25, Issue of September 5, pp. 12448-12453,1988 Printed in U.S.A.

Transcriptional and Posttranscriptional Regulation of Rat Hepatic 3-Hydroxy-3-methylglutaryl-coenzymeA Reductase by Thyroid Hormones* (Received for publication, November 23, 1987)

W. Scott Simonet and Gene C. Ness$ From the DeDartment of Biochemistrv and Molecular Biology, College of Medicine, University of South Florida, Tampa, Florida 33612 ‘

The mechanisms by which thyroid hormones in- hepatic HMG-CoA reductase activity (3),mRNA, and protein crease hepatic 3-hydroxy-3-methylglutaryl-coenzyme (4).Administration of triiodothyronine (Ta)to these animals A (HMG-CoA) reductase mRNA levels were investiresultsin large increases in HMG-CoA reductase mRNA, gated in hypophysectomized rats. Feeding these rats a immunoreactive protein, and enzyme activity (3,4). diet supplemented with 0.6% desiccated porcine thyThyroid hormone-mediated increases in gene expression roid powder resulted in a &fold increasein the rate of are believed to involve interactions with a nuclear chromatintranscription of the HMG-CoA reductase gene as meas- associated non-histone receptor (5-8). These interactions reured by in vitro “run-on” transcription assays in iso- sult in increased levels of mRNA for several proteins. In lated rat liver nuclei. Time courses of change in reducgeneral, there is a variable lag period before accumulation of tase mRNA, showing the kinetics of approach to new mRNA occurs. The shortest lag time thus far reported is on steady-state levels, indicate that reductase mRNA is the order of 30 min for the mRNA encoded by the S-14gene also 4-6-fold more stable in thyroid hormone-treated (9). Other mRNAs require several hours or more for signifianimals thanin non-treated animals. Reductase mRNA decayed with a half-life of 2.5 h when mevinolin, a cant amounts to accumulate. For HMG-CoA reductase mRNA potent inhibitor of HMG-CoA reductase, andcolestipol, this lag period is approximately 36 h (4).There appear to a bile acid sequesterant, were removed from the diet exist two distinct motifs by which thyroid hormones promote increases in specific mRNAs. On the one hand, an increased of hypophysectomized rats. When these drugs were removed from the dietof thyroid hormone-treated hy- rate of transcription totally accounts for the observed inpophysectomized rats, reductase mRNA decayed with creases in mRNA (10-14).In these cases, the kinetics of a half-life of 15 h. Treating rats with only mevinolin mRNA accumulation are often hyperbolic. Onthe otherhand, and colestipol increased reductase mRNA levels with- certain thyroid-promoted mRNA accumulations involve inout stabilizing themRNA. creases in message stability in addition to an increase in Administration of cycloheximide to thyroidhormone transcription (15, 16). The kinetics of these responses are treated rats rapidly decreased HMG-CoA reductase often sigmoidal (15).We have recently demonstrated that the mRNA levels by destabilizing reductase mRNA and accumulation of HMG-CoA reductase mRNA in thyroid hordecreasing reductase gene transcription. Cyclohexi- mone-treated hypophysectomized rats is also sigmoidal (17). mide treatment had no effect on &actin gene transcripIn thispaper, we report that theprofound increase in HMGtion or steady state levels of &actin mRNA. These CoA reductase mRNA caused by thyroid hormone treatment results suggest that a short-lived protein(s) may me- of hypophysectomized rats is due to both an increase in diate the transcriptional and post-transcriptional ef- transcription and stabilization of the message. We also demfects of thyroid hormones on HMG-CoA reductase onstratethatthe thyroid hormone-promoted increases in mRNA levels. transcription and stabilization of HMG-CoA reductase mRNA are eliminated by cycloheximide treatment, suggesting that a short-lived protein(s) may mediate these effects of thyroid hormones. The regulation of cholesterol biosynthesis is under the control of the enzyme 3-hydroxy-3-methylglutarylcoenzyme EXPERIMENTALPROCEDURES A (HMG-CoA)’ reductase (1).The expression of this enzyme Animals-Normal and hypophysectomized male Sprague-Dawley is affected by a number of hormones (2), including triiodothyronine and glucocorticoids. Oneapproach to theinvestigation rats were obtained from Harlan Industries, Madison, WI. They were housed in a reverse cycle light controlled room with a 14-h light of hormonal regulation of gene expression by individual hor- period followed by a 10-h dark period. The animals were fed Purina mones is the use of hormone-deficient animals, particularly Rodent Laboratory Chow 5001 ad libitum. Where indicated, rats were hypophysectomized rats. These rats exhibitvery low levels of fed ground chow containing either2% colestipol and 0.04% mevinolin *This research was supported by United States Public Health Service Grant HL18094. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed. The abbreviations used are: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; T3, triiodothyronine; SDS, sodium dodecyl sulfate; acid; Pipes, Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic 1,4-piperazinediethanesulfonicacid.

(Mevacor) and/or 0.5% desiccated porcine thyroid powder for up to 5 days. This approach allowed a steady-state level to be reached rather than a peak response whichfollows injections of thyroid hormones. Hypophysectomized rats were used in experiments 14-24 days after surgery and typically weighed125-150 g at this time. Normal rats weighed about 200 g a t the time of use. Determination of HMG-CoA Reductase Activity-Methods for the preparation of lysosome-free microsomes were those previously described (18). The protein concentrations of microsomal suspensions were determined by a biuret method (19). Microsomal protein (50100 rg) was preincubated for 10 min at 37 “C in 230 rl of a buffer

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Regulation of HMG-CoA Reductase mRNA by Thyroid Hormones containin 100 mM potassium phosphate (pH 7.4), 260 mM KCI, and 6.5 mM 6SH. Reactions were started by the addition of70 pl of a buffer containing 0.1 M potassium phosphate (pH7.4), 17 mM glucose 6-phosphate, 0.25 unit of lucose-6 hos hate dehydrogenase, 6.4 mM NADP, and 350 p M DL-f3-l4C]Hd)G-&A (specific activity = 3960 cpm/nmol). Incubation times ranged from 5 to 10 min. The reactions were terminated by the addition of 30 p1 of 2.4 N HCI, and further incubated for 10 min at 37 "C. The [14C]mevalonolactonewas separated from unreacted substrate on silica thin layer chromatography plates using a benzene/acetone (1:l)solvent. The labeled mevalonolactone was quantitated in a liquid scintillation spectrometer. Isolation of Rat Liver RNA and Qmntitation of HMG-CoA Reductase mRNA Levels by Dot Blot Hybridization Assays and Northern Analysis-RNA was prepared from 0.5-1.0 g of liver by a recently described, low temperature modification (20) of the guanidinium thiocyanate extraction procedure previously described (21). After four extractions with guanidine hydrochloride, followed by extraction of the RNA into sterile, diethyl pyrocarbonate treated water, the RNA was further fractionated by oligo(dT)-cellulose chromatography (22). Total poly(A') RNA was quantitated spectrophotometrically. Three volumes of6.15 M formaldehyde/lO X SSC (SSC = 0.15 M sodium chloride, 15 mM sodium citrate, pH 7.0) was added and the poly(A') RNA was denatured a t 65 "C for 15 min. Aliquots ranging from 0.1 to 2.0 pg of RNA were applied to nitrocellulose using a dot blot apparatus. Hybridizationswith 32P-labeled pRED227 and washes were performed as previously described (17). After washing, the filters were autoradiographed a t -70 "C for 3-24 h and the amount of HMGCoA reductase mRNA was determined by cutting out theindividual dots from the filter and countingin a liquid scintillation spectrometer. Before fractionation by oligo(dT) chromatography, aliquots of total rat liver RNA were taken for Northern blot analysis. Total tissue RNA (5-10 pg) was electrophoresed in 2.2 M formaldehyde-1.5% agarose denaturing gels. Lanes for staining were soaked in several changes of water for 2 hand stainedwith ethidium bromideto localize 28 S and 18 S rRNA species. RNA was transferred to nitrocellulose and probed as described above for dot blot filters. Isolntion of Rat Liver Nuclei-Nuclei were isolated from rat liver by a modification of a procedure previously described (23). Four to five grams of liver were homogenized in 25ml of ice-cold 0.32 M sucrose, 3 mM MgCI2, 1mM Hepes (pH 6.8), and thehomogenate was filtered through four layers of sterile cheese cloth. The nuclei were sedimented by centrifugation a t 750 X g for 10 min a t 4 "C. The nuclei were resuspended in 8-10 ml of ice-cold 2.1 M sucrose, 1 mM MgC12, 1 mM Hepes (pH 6.8), and centrifuged for 60 min a t 18,000 rpm in a Sorvall SS34 rotor a t 4 "C. The pelleted nuclei were resuspended in 0.32 M sucrose, 3 mM MgC12, 1 mM Hepes (pH 6.81, and centrifuged a t 750 X g for 10 min. These pelleted nuclei were resuspended in 1 ml of ice-cold buffer A (20 mM Tris-HCI (pH 7.9), 30% glycerol, 140 mM KCI, 5 mM MgC12, 1 mMMnC12, and 14 mM pmercaptoethanol) and were pelleted in a microcentrifuge tube. The volume of the pellet was estimated. Nuclei were stored a t -70 "C as a 50% suspension in buffer A. Labeling and Isolation of Nascent RNA Transcripts-Transcription reactions contained30% (v/v) packednuclei in buffer A supplemented with 1 mM each ATP, CTP, and GTP, 10 mM phosphocreatine, 100 pg/ml creatine phosphokinase, and 250 pCi of [c~-~'P]UTP (3000 Ci/ mmol) (24). Reaction volumes were 100 pl and were incubated at 26 "Cfor 45 min. The reaction was terminated on ice and RNase-free DNase was added to 100 pg/ml. An equal amount of high salt buffer (0.6 M NaCI, 50 mM Tris-HCI (pH 7.5), 20 mM MgCI2) was added and the reaction mixture was incubated at room temperature for 10 min. The solution was transfered to a tube containing2 ml ofprotease buffer (0.15 M NaC1, 10 mM Tris-HCI (pH 7.5), 1 mM EDTA, 1% SDS).Protease K was added to 200 pg/ml and incubation was continued at 37 "C for 30 min. Sodium acetate (pH 5.0) was added to 0.1 M along with 200 pg of yeast tRNA. The solution was extracted once with phenol a t 55 "C and once with 1:l phenol/chloroform at room temperature. The aqueous phase was precipitated with 2 volumes of ethanol. The pellet was redissolved in 400 pl of 0.1 M sodium acetate (pH 7.0) and reprecipitated three additional times to remove unincorporated nucleotides. The final pellet was rinsed with 70% ethanol, dried, and dissolved in 30 pl of 0.1 X SET (1 X SET = 1% SDS, 1 mM EDTA, 10 mM Tris-HCI, pH 7.5). The RNA solution was heated in a boiling water bath for 5 min and quick chilled on ice immediately before hybridization. Transcription Rate Analysis by Hybridization to Immobilized cDNA-Detection of HMG-CoA reductase and @-actin transcripts was carried out by hybridization to specific cDNA sequences. Filters

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containing 2.0 pg of either pBR322, pRED227, or &actin cDNA were prepared as described previously (25). Prior to hybridization with nuclear transcripts, the filters were treated for 16-24 h at 42 "C in 33% formamide 5 X Denhardt's solution (1X Denhardt's solution = 0.02% Ficoll, 0.62% polyvinylpyrrolidone, 0.02% bovine serum alburnin), 50 mM Pipes, (pH 7.0). 0.5 M NaCI, 0.4% SDS, and 200 pg/ml yeast tRNA. Hybridizations were performed for 4 days at 42 "C in 250 pl of 33% formamide, 3 X Denhardt's solution, 0.05 M Pipes, (pH 7.0). 0.5 M NaCI, and 0.4% SDS. Following hybridization, the filters were washed in five changes of 2 X SSC containing 0.1% SDS at room temperature. The filters were then washed for 2 h in buffer B (0.3 M NaCI, 40 mM EDTA, and 10 mM Tris-HCI, pH 7.5) containing 0.1% SDS a t 45 "C, followed by a second wash in the same buffer without SDS at 45 "C for 30 min. The filters were treated with buffer B containing 10 pg/ml RNase A and 1.0 pg/ml RNase T, at 37 "C for 30 min. Finally, the filters were washed for another hour in buffer B containing 0.1% SDS at45 "C. Radioactive RNA bound to thevarious filters was quantitated by liquid scintillation counting.

RESULTS

HMG-CoA ReductasemRNALevelsAreIncreased to a Greater Extent Than Reductase Gene Transcription in Thyroid Hormone-treated Hypophysectomized Rats-In order to achieve the necessary sensitivity to accuratelydetermine rates of reductase gene transcription, we utilized the full-length hamster reductase cDNA, pRED227. On Northern blots (Fig. l),this probe recognized a single band migrating very close to the 28 S rRNA marker, suggesting that rat liver reductase mRNA has a size of about 5 kilobases. Under the conditions in which this gel was run, no detectable amount of precursor mRNA was seen. For purposes of quantitation, the dot blot format was routinely used. Typically, feeding hypophysectomized rats a diet containing 0.5%porcine thyroid powder for 5 days increased reductase mRNA levels 20-40-fold. Using pRED227 improved the signal-to-noise ratio of our "run-on" transcription assays so that the background (amount bound to a filter containing pBR322) was only 10-20% that of the counts bound to the pRED227 filter. To investigate the effects ofthyroid hormones on reductase A

B

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FIG. 1. Thyroid hormones increase hepatic HMG-CoA reductase mRNA. The autoradiograms of a dotblot of rat liver HMGCoA reductase poly(A') RNA ( A ) and a Northern blot of rat liver HMG-CoA reductase total RNA ( B )are shown. In the dot blot assay, increasing amounts of poly(A+) RNA (0.1-1.0 pg) were spotted from top to bottom. Column I shows mRNA from a hypophysectomized rat. Column 2 shows equal amounts of mRNA from a hypophysectomized rat fed 0.5% desiccated porcine thyroid powder for 5 days. The Northern blot shows the identical samples before selection for polyadenylated RNA. Lane I contained 10 pg of RNA from a hypophysectomized rat. Lune 2 contained 10 pg of RNA from a hypophysectomized rat fed 0.5% porcine thyroid powder for 5 days. The procedures for RNA isolation, gel electrophoresis, and hybridization to 32Plabeled pRED227 (full-length reductase cDNA) are described under "Experimental Procedures."

Regulation of HMG-CoA Reductase

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mRNA synthesis, the rate of transcription of the HMG-CoA reductase gene was measured using isolated rat liver nuclei. Fig. 2 shows the relationship between hepatic reductase mRNA levels and reductase gene transcription in hypophysectomized rats fed thyroid powder for 5 days. Reductase transcription rates were increased &fold in response to thyroid hormone treatment. Interestingly, cycloheximide treatment of thyroid hormone-treated rats reversed the increases in reductase mRNA and gene transcription. These findings are consistent with the effects of cycloheximide on other rat hepatic genes responsive to thyroid hormones (16). @-Actin mRNA and @-actin gene transcription were moderately increased by thyroid hormone treatment, but were unaffected by cycloheximide treatment. The timecourse of increase in reductase gene transcription, mRNA, and enzyme activity in hypophysectomized rats fed thyroid hormones is shown in Fig. 3. Reductase gene transcription reached a new steady-state level within 4 days after administration of the diet. This was followed by reductase mRNA which approached a new steady state with a half-life of 8-10 h. Reductase activity was still increasing after 7 days

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FIG. 3. Time course of increase in reductase gene transcription, mRNA, and activity in thyroid hormone-treated hypophysectomized rats. Hypophysectomized rats were placed on a rat chow diet supplemented with 0.5% desiccated porcine thyroid powder. The animals were killed at theindicated times and thelevels reductase mRNA of hepatic reductase gene transcription (W), (M), and reductase activity (A-A) were determined as described under “Experimental Procedures.” All values are the means f S.D. of at least two determinations and are expressed relative to the 168-h time point. The values at 168 hare 1330 & 270 cpm hybridized/lO’ cpm input of 32P-RNAfor reductase gene transcription, 5400 cpm/pg poly(A+) RNA for reductase mRNA, and 4.80 nmol/min/mg for reductase activity.

on the thyroid hormone-supplemented diet. The increase in reductase gene transcription clearly was not sufficient to account for the observed increase in reductase mRNA levels, suggesting that thyroid hormones might also act at a post-transcriptional step to cause reductase mRNA accumulation. Effects of Thyroid Hormones on Reductase mRNA DegraH H+T H+T+CHX dation-The transition from one steady-state level of an mRNA to anothersteady-state level is a function of both the rate of synthesis and the rate of degradation of the mRNA. Since the approach to thenew steady state is first order with 1.2 respect to therate of degradation and zero order with respect to the rateof synthesis (26, 27), it is possible to estimate the 1.0 half-life of the mRNA by observing its approach to the new 0.8 steady state. By observing time courses of change in reductase mRNA levels and approach to new steady states, we have 0.8 investigated the effects of thyroid hormones on the stability of reductase mRNA. 0.4 Because of the long half-life of thyroid hormones in the circulation, simply removing thyroid hormones from the diet 0.2 and observing the change in reductase mRNA levels would 0.0 provide an uninterpretable model. To circumvent this probB+T fI+T+CBX lem we chose to feed hypophysectomized rats and thyroid FIG. 2. Relative levels of hepatic HMG-CoA reductase ac- hormone-treated hypophysectomized ratsadiet suppletivity, mRNA, and rates of reductase gene transcription in mented with mevinolin and colestipol. Upon removal of these hypophysectomized rats treated with thyroid hormones. Hypophysectomized rats were placed on a normal rat chow diet ( H ) ,or rapidly metabolized hepatic cholesterol-lowering agents from a rat chow diet supplemented with 0.5% desiccated porcine thyroid the diet, the change in reductase mRNA and approach to new powder ( H + T)for 5 days. Where indicated (H + T + C H X ) ,thyroid steady-state levels was observed. Fig. 4A shows that normal hormone-induced animals were given a single injection of cyclohexi- rats accumulate reductase mRNA very rapidly upon adminmide (1.3 pg/g of body weight) 8 h before being killed. All rats were istration of a mevinolin- and colestipol-supplemented diet. killed at the 4th h of the dark cycle and the levels of ( A ) reductase Fifty percent of the maximum response is observed in about activity, 0; reductase mRNA, & and @-actin mRNA, and ( B ) 4 h. Because this time course was initiated at the beginning reductase gene transcription, & and @-actin gene transcription, were determined. All values are the means & S.D. of at least three of the dark cycle, the 4-h point and the new steady-state animals and are expressed relative to the thyroid hormone-treated points were at the diurnal high period of the feeding cycle. animals. The values for the thyroid hormone-treated animals ( H + Upon switching to a normal diet (Fig. 4B), reductase mRNA T) were 2.15 & 0.25 nmol/min/mg for reductase activity, 2200 f 300 levels decreased to 10% of control levels within 12 h. Within cpm/pg for reductase mRNA, 4770 f 535 cpm/pg for @-actinmRNA 3 h, reductase mRNA followed a normal exponential decay levels, 1320 f 160 cpm hybridized to reductase cDNA/1OS cpm input of 32P-RNAfor reductase transcription rate and 1580 f 360 cpm curve where the mRNA decreased with a half-life of 3 h. In hybridized to @-actincDNA/lOa cpm input of 32P-RNAfor @-actin both experiments, reductase activity closely paralleled the change in reductase mRNA levels. These results suggest that transcription rate.

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Regulation of HMG-CoA Reductase mRNA by Thyroid Hormones

mum FIG. 4. Time courses of change in HMG-CoA reductase mRNA and activity showing the approach to new steady-state levels. A , normal rats fed a diet supplemented with 0.04% mevinolin and 2% colestipol and B, normal rats fed the mevinolin and colestipol diet for 5 days and then switched to a normal diet. Rats were killed at the indicated times and thelevels of reductase mRNA ( 0 , O ) and activity (A,A) were determined as described under "Experimental Procedures." All values are the means & S.D.and are expressed relative to the 100-h time point in A , or the zero hour time point in B. The values for the 100-h time point in A and thezero hour time point in B were 15,300 k 1,200cpm/pg for reductase mRNA and 11.82 +- 0.2 nmol/min/mg for reductase activity.

TABLE I HMG-CoA reductase mRNA half-lives in various animals determined by observing the approach to new steady-state levels Animal condition"

HMG-CoA reductase mRNA half-life h

N (decay) 3 4 N MEV CTX (acc.) H (decay) 2.5 H + T (acc.) 9 H + T (decav) 15 N, normal rat; H, hypophysectomized rat; MEV+~CTX,fed 0.04% mevinolin and 2% colestipol for 5 days; T, fed 0.5% desiccated porcine thyroid powder for 5 days; acc., half-life determined from accumulation kinetics; decay, half-life determined from decay kinetics after removing MEV + CTX diet.

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hormones actpost-transcriptionally to stabilize reductase mRNA in the nucleus or cytoplasm. The half-lives of reducdecay in reductase mRNA levels are shown in hypophysectomized tase mRNA in animals under various hormonal conditions and thyroid hormone-treated hypophysectomized rats are summarized in Table I. rats (W) (U Hypophysectomized ). rats were fed a diet containing 2% Effects of Cycloheximide onthe Thyroid Hormone-promoted colestipol, 0.04% mevinolin, and either 0.5% thyroid powder (U or ) no thyroid powder (W) for 5 days. At time zero, Stimulation of HMG-CoA Reductase Gene Expression-Fig. 2 this diet was switched to a diet containing only 0.5% thyroid powder showed that administration of the protein synthesis inhibitor (U or) a normal rat chow diet (o"-o). Rats were killed cycloheximide to thyroid hormone-treated hypophysectomat the indicated times andthe levels of reductase mRNA were ized rats resulted in a 5-fold decrease in reductase mRNA determined as described under "Experimental Procedures." The inset shows the semi-log plot used to determine the half-life of HMG-CoA levels within 8 h. This decrease was at least partially attribreductase mRNA. The [mRNA], is the concentration at theindicated utable to a decrease in the transcription rate of the hepatic time, t and [mRNA]., is the concentration at 48 h (0)h or 72 h (0). reductase gene. Fig. 6 shows the effects of cycloheximide on the continued accumulation of reductase mRNA in thyroid mevinolin and colestipol have nosignificant stabilizing effects hormone-treated hypophysectomized rats. The period beon reductase mRNA, and thatwhen these agents areremoved tween 96 and 108 h was chosen to observe the effects of from the diet, their influence on reductase mRNA and activity cycloheximide because this was the period in which the maxlevels dissipate very rapidly. Bioassays indicate that hepatic imum rate of accumulation of reductase mRNA was observed mevinolin is reduced to barely detectable levels within 2 h (Fig. 3). Administration of cycloheximide not only inhibited the increase but also resulted in a rapid decrease in reductase after removal from a mevinolin-containing diet. When mevinolin and colestipol were removedfrom the diet mRNA levels, with a significant change noticed within 3 h. of hypophysectomized rats and thyroid hormone-treated hy- Reductase mRNA levels approached a new steady-state level pophysectomized rats,as shown in Fig.5, the kinetics of at 7 h, with a half-life of 1.5-2 h. Even if the new steady state reductase mRNA decay were noticeably different. The decay were zero, the half-life would not exceed 2 h. Cycloheximide of reductase mRNA in hypophysectomized rats resembled treatment did not alter @-actin mRNA levels. These results that observed in normal rats. Reductase mRNA approached suggest that cycloheximide treatment destabilizes reductase a new steady state by 12 h, with a half-life of 2.5 h. However, mRNA in addition to decreasing its rateof synthesis. in thyroid hormone-treated hypophysectomized rats, reducFor comparison to the previous decay curves depicted in tase mRNA approached a new steady state (20% of control) Fig. 5, we administered cycloheximide to thyroid hormonewith a half-life of 15 h. These results suggest that thyroid treated hypophysectomized rats which were switched from a FIG. 5 . Thyroid hormone treatment of hypophysectomized

rats stabilizes HMG-CoA reductase mRNA. The time courses of

Regulation of HMG-CoA Reductase mRNA by Thyroid Hormones

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mevinolin- and colestipol-containing diet to a normal diet at time zero. Fig. 7 shows that giving cycloheximide simultaneously with switching the diet resulted in a much faster decrease in reductase mRNAlevels than was previously observed without cycloheximide treatment. In this experiment reductase mRNA approached the value of the 9-h time point with a half-life of 2.5 h. Again, if the new steady state were zero, the half-life of reductase mRNA would be about3 h. No change in @-actinmRNA levels were observed over the duration of thistime course (datanot shown). Theseresults suggest that ongoing protein synthesis isrequired for thyroid hormones to exert their effects on reductase gene expression.

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The mechanism by which thyroid hormones promote accumulation of hepatic HMG-CoA reductase mRNA was investigated in hypophysectomized rats. Feeding these animals a diet supplemented with 0.5% desiccated porcine thyroid powder increased the rate of transcription of the rat liver HMG-CoA reductase gene &fold. Interestingly, the maximal degree of sterol-mediated suppression of reductase transcription is also &fold (28). The octanucleotide sequence which is 0.0 the site of this regulation has been identified as GTGCGGTG 0 3 6 Q 1 2 and begins at nucleotide -153 relative to themajor transcripHOURS tion initiation site inthe hamster gene. FIG.6. Cycloheximide causes a rapid decrease in the level In addition to their effect on transcription, thyroid horof hepatic HMG-CoA reductase mRNA. After 92 h on a diet mones were also demonstrated to stabilize reductase mRNA containing 0.5% porcine thyroid powder, hypophysectomized rats 4-6-fold. Similar findings have been reported for rat hepatic weregiven a single injection of cycloheximide (1.3 pg/g ofbody weight). Poly(A+) RNA was isolated at the indicated times after malic enzyme mRNA and also for rat growth hormone mRNA cycloheximide treatment,andthe levels of HMG-CoA reductase (15, 29). It is also believed that the increase in an mRNA mRNA ( A ) (M and )&actin mRNA ( B ) (U were ) deter- encoding for a protein (designated spot 14) in rats treated mined as described under “Experimental Procedures.” mRNA levels with T3is attributable to anincreased stability of the nuclear in nontreated control animals (o”--o) are also shown. The inset in precursor to the mature mRNA (30). The rapid degradation A shows the semi-log plot used to determine the half-life of HMG- of reductase mRNA observed in response to cycloheximide CoA reductase mRNA. The [mRNAIr is the concentration at the treatment suggests that thyroid hormones may be inducing indicated time, t and [mRNA], is the concentration at 7 h after cycloheximide treatment. All values are expressed relative to the 1-h the expression of a short-lived mRNA stabilizing protein. This protein may bind to specific sequences or secondary control animal. structures common to transcripts of certain thyroid hormone responsive genes. It is interesting that giving thyroid hormones to hypophysectomized rats did not mimic the half-life of reductase mRNA in thenormal rat, where reductase mRNA is almost as shortlived as in the hypophysectomized animal. Perhaps some other pituitarycontrolled hormone may influence the stability of reductase mRNA. Indeed, our recent studies suggest that glucocorticoids act post-transcriptionally to decrease reductase mRNA stability. The requirement for ongoing protein synthesis for mediating theeffects of thyroid hormones on HMG-CoA reductase gene transcription suggests the possible existence of a transcriptional regulatory protein which is inducbd by thyroid hormone treatment. Requirements for short-lived proteins murm 0.00 have also been recently reported for the T3-induced increases 0 2 4 8 0 1 0 in rat pituitary growth hormone gene transcription (31), as well as the transcription of several other rat hepatic thyroid Hours FIG. 7. Destabilization of hepatic HMG-CoA reductase hormone-sensitive genes (32). The similarities between HMG-CoA reductase and malic mRNA in thyroid hormone-treated rats by administration of cycloheximide. Hypophysectomized rats were fed a ground chow enzyme (15) in terms of regulation by thyroid hormones is diet containing 2% colestipol, 0.04% mevinolin, and 0.5% desiccated striking. In both cases, transcriptional and post-transcripthyroid powder for 5 days. At zero time, 4th h into the darkperiod, tional effects areobserved which are sensitive to inhibitors of the diet was replaced with a diet supplemented with 0.5% thyroid protein synthesis. Perhaps a similar type of regulation requirpowder and the rats were given a single injection of cycloheximide (1.3pg/g of body weight). The ratswere killed at theindicated times ing short-lived proteins as mediators may occur.

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after this switch and the levels of HMG-CoA reductase mRNA, U, and activity, A-A, were determined. The values for reductase mRNA (0)and activity (A) in control animals at 9 h are also given. The inset shows the semi-log plot used to determine the half-life of reductase mRNA as itapproaches its new steady state.

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Regulation of HMG-CoA Reductase mRNA by Thyroid Hormones 3. 4.

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