Inhibition of Tyrosine Aminotransferase Gene Expression by Retinoic Acid

Chi-Jiunn Pan, Leslie Janice Yang Chou

L. Shelly,

Douglas

S. Rabin,

and

Human Genetics Branch National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20892

Regulation of tyrosine aminotransferase (TAT) gene expression was examined in RALA255-IOG, a simian virus-40 tsA mutant-immortalized adult rat hepatocyte line. At the nonpermissive temperature (40 C), these hepatocytes exhibited a differentiated phenotype and actively expressed the TAT gene, but only in the presence of dexamethasone (DEX). The glucocorticoid-mediated TAT expression was inhibited by cycloheximide, a protein synthesis inhibitor, and by RU488, a glucocorticoid antagonist, suggesting that glucocorticoid induction requires protein synthesis and may be mediated through hormone receptors. (Bu),cAMP (B&CAMP) or retinoic acid, individually or in combination, failed to increase TAT mRNA levels. However, BWAMP greatly potentiated the induction by DEX, whereas retinoic acid inhibited the induction by DEX or DEX/Bt,cAMP. Nuclear run-on assays demonstrated that the induction of TAT expression by DEX or DEX/Bt& in RALA255-IOG cells is regulated primarily at the transcriptional level. In contrast, retinoic acid antagonized the DEX- or DEX/Bt,cAMP-mediated induction without affecting the rate of TAT gene transcription. Instead, retinoic acid destabilized TAT mRNA. The half-life values of TAT mRNA in DEX/Bt$AMPand DEX/Bt,cAMP/retinoic acid-treated cells were approximately 235-270 min and 90-100 min, respectively. Our results indicate that inhibition of TAT expression by retinoic acid was regulated primarily at the posttranscriptional level. (Molecular Endocrinology 8: 572~58O,lgg2)

INTRODUCTION

Tyrosine aminotransferase (TAT) is the first and ratelimiting enzyme in the tyrosine degradative pathway (1). It is expressed almost exclusively in parenchymal cells of the liver; developmental onset of expression occurs 0888.8809/92/0572-0580503.00/O Molecular Endocrinology Copyright 0 1992 by The Endocrine

Society

shortly after birth (2). In addition to developmental and tissue-specific regulation, TAT expression is also under multiple hormonal control. Transcription of the TAT gene is stimulated by glucocorticoids, as well as the CAMP signalling pathway (3-5). Two glucocorticoid response elements, situated approximately 2.5 kilobases (kb) up-stream of the transcription initiation site of the TAT gene, that are essential for glucocorticoid induction have been functionally demonstrated for the gene (6). A CAMP response element that mediates CAMP induction is located 3.6 kb up-stream of the TAT transcription start site (7). TAT is also subject to negative control by the trans-dominant tissue-specific extinguisher locus Tse-1; the target for repression by Tse-1 is the CAMP response element (7, 8). Retinoic acid (RA) and its derivatives have been shown to be important in cell growth, differentiation, and the expression of a variety of mammalian genes (9, 10). In the presence of RA, F9 teratocarcinoma stem cells differentiate into parietal endoderm (1 I), promyelocytic leukemia cells differentiate into granulocytes (12), and developing chick limb buds establish an anterior-posterior axis (13). RA acts through specific receptors, and these receptor genes are members of the steroid/thyroid receptor supergene family (14-I 9). RAinduced expression has been demonstrated in the genes for GH (20) laminin Bl (21), RA P-receptor (22) and alcohol dehydrogenase (23). Recently, we showed that transcription of the hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene is stimulated by RA (24). Expression of PEPCK, like TAT, is regulated by glucocorticoids and CAMP. In this study we examined whether RA plays a role in the control of rat TAT gene expression. To investigate the effects of RA on TAT expression, an adult rat hepatocyte cell line, RALA255-IOG, immortalized by a simian virus-40 tsA255 mutant, was employed (25). Because the transforming large tumor antigen encoded by the A-gene is inactivated at the nonpermissive temperature of 40 C (26) RALA255IOG cells exhibit a phenotype comparable to that of fully differentiated liver cells. Previous studies have shown that glucocorticoid hormone is required for the

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optimal expression of many liver genes in these cells (24, 25, 27). Moreover, at the nonpermissive temperature, RALA255-1 OG ceils actively synthesize TAT and PEPCK in the presence of glucocorticoids and CAMP (24, 27). In the present study the effects of RA, alone or in combination with dexamethasone (DEX) and/or (Bu)& (Bt,cAMP), on TAT mRNA levels and rates of gene transcription were investigated. RA inhibited the glucocorticoid/cAMP-mediated induction of TAT mRNA, but did not significantly alter its rate of gene transcription. TAT mRNA half-life measurements indicated that RA appears to inhibit TAT expression by destabilizing the TAT mRNA.

RESULTS Characterization of Glucocotticoid Mediated induction

Hormone-

Earlier studies have shown that in the presence of a glucocorticoid hormone at 40 C, RALA255-10G adult rat hepatocytes synthesize large amounts of TAT and express high levels of TAT mRNA (27) (Fig. IA, D3). Removing DEX from the culture medium markedly decreased TAT mRNA levels (Fig. 1 A, C3 vs. D3). Switching these cells back to DEX-containing medium resulted in reexpression of the TAT gene (Fig. 1 A, C3D3). However, TAT mRNA levels in these cultures (C3D3) were lower than those in cultures maintained continuously in DEX-containing medium (D3), indicating that the contin-

ued presence of DEX was necessary for optimal expression of the rat TAT gene in hepatocytes in vitro. TAT mRNA induction was found to be extremely sensitive to cycloheximide. Cycloheximide at 0.1 pg/ml, which did not result in any visible morphological changes to the cells and inhibited total protein synthesis by only IO-20% after a 3-day incubation at 40 C, inhibited the DEX-induced expression of TAT (Fig. 1A, D3CX3 or C3D3CX3). This suggests that synthesis of a new protein(s) sensitive to cycloheximide inhibition is required for glucocorticoid-mediated activation of TAT expression in these cells. To demonstrate that the effects of glucocorticoid observed in RALA255-10G cells were mediated through glucocorticoid receptors, we examined the effects of RU486, a glucocorticoid antagonist, on DEXmediated gene expression. RLl486 blocks glucocorticoid action by binding to available receptors (28). In the presence of RU486, the DEX-mediated increase in TAT mRNA was prevented after 4 days (compare D4 to D4RU4, Fig. 1B) or 6 days (compare C2D4 to C2D4RU4, Fig. 1 B), indicating a receptor-mediated mechanism of action. RU486 had no effect on p-actin mRNA expression, which is apparently not regulated by glucocorticoids (Fig. 1 B). Effects of DEX, BbcAMP, Levels

and RA on TAT mRNA

TAT mRNA levels were analyzed by Northern blot analysis in cells maintained in the presence of various

-285

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/ L4

’ -D-Actin-

-1s

Fig. 1. Effects of DEX, Cycloheximide, and the Glucocorticoid Antagonist RU486 on TAT mRNA Expression RAlA255-10G cells were grown at 33 C in medium containing 0.1 PM DEX and shifted to a nonpermissive temperature of 40 C after 4-5 days of growth at 33 C (day 0). A, Effects of cycloheximide. On day 0, cultures were washed three times with PBS, treated with control medium (C), medium containing 0.1 PM DEX (D), or medium containing DEX plus 0.1 pg/ml cycloheximide (CX). RNA isolated from cultures before the temperature shift was the zero time control (CO). After an additional 3 days of growth at 40 C, some of the cultures (C3, D3, and D3CX3) were lysed for RNA isolation. To test the effects of DEX and cycloheximide on TAT mRNA expression in cells that had been grown in control medium for 3 days (C3), these cultures were switched to medium containing DEX (C3D3) or DEX plus CX (C3D3CX3) and incubated at 40 C for an additional 3 days before RNA isolation. B, Effect of RU486. On day 0, cultures were washed three times with PBS, treated with control medium (C) or medium containing 0.1 PM DEX (D), 1 PM RU486 (RU), or DEX and RU486. After an additional 4 days growth at 40 C, cells (C4, D4, RU4, and D4RU4) were lysed for RNA isolation (total of 4 days). Some of the cultures were grown at 40 C for 2 days in the control medium; then, they were either maintained continuously for 4 more days at 40 C in the control medium (C6) or switched to medium containing DEX (C2D4), RU486 (C2RU4), or DEX plus RU486 (C2D4RU4) before RNA isolation (total of 6 days). Poly(A)+ RNA (5 fig/lane) was separated by agarose gel electrophoresis, transferred to Nytran membrane, and hybridized to a uniformly labeled TAT or @actin probe, as described in Materials and Methods.

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Vol6 No. 4

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4%Actin PEPCK-

ISActinFig. 2. Effects of DEX, &CAMP, and RA on TAT mRNA Expression RALA255-1 OG cells were grown at 33 C in medium containing 0.1 PM DEX. After 4-5 days of growth at 33 C (day 0), cultures were washed three times with PBS, shifted to 40 C, and treated with control medium (C6) or medium containing 0.5 mM Bt*cAMP (A2), 0.1 PM DEX (D6), 10 PM RA (R6), DEX/BtscAMP (D6A2), 10 /.LM RA/BtacAfvlP (R6A2), DEX/lO /JM RA (D6R6), DEX/Bt&/ 10 PM RA (D6R6A2; A and B), or DEX/BtZcAMP/l PM RA (D6R6A2; B). RNA was isolated from cultures that had been grown at 40 C for an additional 6 days. DEX or RA was present from days O-6, and Bt2cAMP was present from days 4-6. Poly(A)+ RNA (4 rg/lane) was separated by agarose gel electrophoresis, transferred to Nytran membranes, and hybridized to an antisense TAT, PEPCK, or @-actin riboprobe, as described in Materials and Methods.

hormones, as indicated in Fig. 2. In the absence of added hormones(C6), RALA255-1 OGhepatocytes expressedlow or undetectablelevels of TAT mRNA (Fig. 2A). TAT mRNA levels were increasedat least 20-fold in the presenceof DEX (D6). Bt,cAMP alone (A2) did not affect TAT mRNA levels, but greatly potentiated the increasein TAT mRNA induced by DEX (D6A2), resultingin an additionallo- to 20-fold increasein TAT mRNA. In the presenceof RA alone (R6), TAT mRNA levels were not affected (Fig. 2A). However, the induction of TAT mRNA by DEX or DEX/Bt,cAMP was inhibitedby RA. TAT mRNA levels were approximately 3-fold lower in the presenceof DEX/RA (D6R6) or DEX/Bt*cAMP/ RA (D6R6A2)than in the presenceof DEX (D6) or DEX/ Bt$AMP (D6A2), respectively (Fig. 2A). The most effective concentration of RA to inhibit DEX/Bt2cAMPmediatedTAT inductionwas 10 PM(Fig. 2). RA at 1 PM gave little or no inhibition(Fig. 28); RA at 100 PM was toxic to RALA255-1 OGcells. We have previously demonstratedthat expressionof the hepatic enzyme PEPCK was induced by DEX and Bt$AMP and that RA greatly potentiated this induction (24). In this study membranes previously hybridized with the TAT probe were used to measure PEPCK mRNA levels. In contrast to TAT, a further increasein PEPCK mRNA levels was observed when RA was added to the cells maintainedin the presenceof DEX/

BtncAMP (compare PEPCK in D6A2 to D6R6A2, Fig. 2A). The expressionof the p-actin gene was not altered in the presenceof DEX, BtZcAMP, or RA (Fig. 2). Glucocorticoid and RA Receptor Levels in the Presence of DEX and RA In an earlier study we showed that expression of the RA P-receptor in RALA255-10G cells was increased by RA, although RA a-receptor mRNA levels remained the same in both the presenceand absenceof glucocorticoid (24). In the present study we show that glucocorticoid receptor mRNA levels were unchangedby RA (Fig. 3A). This was confirmed by analyzing glucocorticoid receptor levels and the hormone receptor dissociationconstant under the various experimental conditions.The results in Fig. 38 show that there was no significant difference in the number of receptors in cellsgrown in the absenceor presenceof RA or DEX. In addition, the receptor binding affinity (K,,) was not significantlyaltered by hormonetreatment (K,,,2-3 nM). Characterization of DEX-, Bt,cAMP-, and RARegulated TAT Expression To further characterize RA-mediated inhibitionof TAT expression, TAT mRNA levels were analyzed in RALA255-1 OGcells maintainedat 40 C for either 2 or

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RA Inhibits TAT Expression

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Transcription Rates of TAT in the Presence &CAMP, and RA

Receptor mRNA

(A)

C

D

R

DR

of DEX,

The inhibitionof DEX- or DEX/Bt&-mediated TAT induction by RA may be the consequence of a decreased rate of gene transcription, altered mRNA stability, or both. To distinguishamongthese alternatives, the transcription rates of the TAT gene were analyzed in isolated nuclei prepared from RALA255-10G cells grown under various experimental conditions. Transcription rates were analyzed in nuclei isolated from four separate preparations of cells (Fig. 5). TAT transcription was not detected in cells maintainedin the absence of hormones. In the presence of DEX, TAT transcription was markedly enhanced. Transcription rates of the gene were further increasedwhen cells were maintained in the presence of DEX/Bt2cAMP. However, the TAT gene transcription rate in cells exposedto DEX/Bt$AMP/RA was not significantlydifferent from that in cells exposed to DEX/&cAMP (Fig. 5). The transcriptional inhibitor cy-amanitinblocked the increasein TAT gene transcription. These results indicate that RA inhibited the DEX- or DEX/Bt,cAMPmediatedinduction of TAT without affecting its rate of gene transcription.

28S18S-

Receptor Concentration

Effects of RA on TAT mRNA Stability

C (4

D

R

DR

(4)

(2)

(4)

Fig. 3. Effects of DEX and RA on Glucocorticoid Receptor mRNA and Protein Levels RALA255-1OG cells were grown at 33 C in medium containing 0.1 PM DEX. After 4-5 days growth at 33 C, cultures were washed three times with PBS, treated with control medium (C) or medium containing 0.1 PM DEX (D), 10 PM RA (R), or DEX plus RA (DR), and shifted to 40 C (day 0). After an additional 6 days of growth at 40 C, cells were harvested for either Northern analysis (A) or receptor binding assay (B), as described in Materials and Methods. Numbers in parentheses are the numbers of cultures analyzed, and bars indicate the SEM.

6 days under various hormonal conditions (Fig. 4A). Maximal TAT expression in the presence of DEX/ B& was observed after a 6-day incubationat 40 C (D6A2). RA inhibited the DEX/Bt$AMP-mediated inductionafter both 2 days (compareD2A2 to D2R2A2) and 6 days (compare D6A2 to D6R6A2) of growth at 40 c.

A comparisonof the TAT mRNA levelsin RALA2551OGcells and adult rat liver by slot blot hybridization is shown in Fig. 48. The TAT mRNA level in these hepatocytes grown at 40 C for 6 days in the presenceof DEX/Bt$AMP was about 50% of the adult rat liver level.

To determine whether RA inhibits TAT expression by destablizing its mRNA, half-life values of TAT mRNA were analyzed in cells grown in medium containing DEX/Bt,cAMP or DEX/Bt,cAMP/RA. After a 6-day incubation at 40 C, cells were exposed to the transcriptional inhibitor actinomycin-D (data not shown) or 5,6dichlororibofuranosylbenzimidazole(DRB),which inhibits transcription by causingprematurechaintermination (29). RNA was extracted from the cellsat various times after the addition of the inhibitor. Northern blots were hybridized with the TAT probe, which was then removed, and filters were rehybridized with a p-actin probe. Autoradiograms of Northern blots were scanned,and the relative amountsof TAT mRNA were normalized with respect to @actin mRNA. Half-life measurementswere analyzed in three different cell preparations; the half-life of TAT mRNA in DEX/ BtZcAMP/RA-treated cultures was approximately 90100 min comparedto 235-270 min in cells exposed to only DEX/Bt&. Results from a representativeexperiment are shown in Fig. 6. These results indicate that RA has a destabilizingeffect on TAT mRNA under these conditions.

DISCUSSION

In the present study we demonstrate that TAT gene expression in RALA255-10G adult rat hepatocytes is regulated by RA in addition to its regulationby multiple other hormones. Although without effect on its own,

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Fig. 4. A, Expression of TAT mRNA at 40 C RALA255-10G cells were initially grown at 33 C in medium containing 0.1 PM DEX. After 4-5 days of growth at 33 C (day 0), cultures were treated with medium containing 0.1 PM DEX, DEX and 10 PM RA, DEX and 0.5 mM B@AMP, or DEX/BtacAMP/RA and incubated for an additional 2 (D2, D2R2, D2A2, or D2R2A2) or 6 (D6, D6R6, D6A2, or D6R6A2) days at 40 C. B& was present for 2 days (days O-2 or days 4-6). Total RNA (20 pg/lane) was separated by agarose gel electrophoresis, transferred to Nytran membranes, and hybridized to an antisense TAT or P-actin riboprobe, as described in Materials and Methods. B, Quantitation of TAT mRNA in RALA255-1OG cells grown at 40 C for 6 days in the presence of DEX/Bt&. DEX was added on day 0; BtsAMP was added on day 4. Poly(A)+ RNA from adult rat liver or RALA255-10G cells was applied to a Nytran membrane using a Slot-Blot (Schleicher and Schuell, Manifold II) and hybridized to an antisense TAT riboprobe, as described in Materials and Methods. The RNA was quantitated by scanning the autoradiograms of different exposures with the Bio-Rad Densitometer (BioRad).

8. -TAT

-put

Fig. 5. Comparison of Rates of Transcription of the TAT Gene in RALA255-1 OG Cells Grown in the Presence or Absence of DEX, Bt,cAMP, and RA RALA255-1OG cells were grown at 33 C in medium containing 0.1 FM DEX. After 4-5 days of growth at 33 C, cultures were washed three times with PBS, treated with control

medium (C) or medium containing 0.1 FM DEX (D), 10 PM RA (R), 0.5 mM B& (A), DEX/Bt$AMP (DA), RA/Bt,cAMP (RA), DEX/RA (DR), or DEX/Bt&/RA (DRA), and shifted to 40 C (day 0). After an additional 6 days of growth at 40 C, nuclei were isolated, and the rate of TAT gene transcription was determined, as described in Materials and Methods. DEX or RA was present from days O-6, and Bt2cAMP was present from days 4-6.

RA inhibits the DEX- or DEX/Bt,cAMP-mediated induction of TAT mRNA. These results are surprising in light of the opposing effect of RA on a related hepatic enzyme, PEPCK (24). RA potentiates the induction of PEPCK gene transcription by DEX and Bt& in RALA255-1 OG cells. The molecular mechanisms by which RA regulates gene expression are still poorly understood. RA acts through RA receptors, which show structural similarity to the steroid/thyroid hormone superfamily of receptors (14-19). The hormone receptors bind to short DNA sequences in the promoters of target genes, thereby activating or repressing transcription (30, 31). RA is known to exert its effects at the level of transcription, and RA receptor response elements have recently been identified in the V-flanking region of many genes. These include the GH gene (20) the laminin Bl gene (21), the RA p-receptor gene (22) the alcohol dehydrogenase gene (23), the PEPCK gene (32) and the cellular retinolbinding protein-l gene (33). There is also evidence that RA regulates gene expression at the posttranscriptional level. Suva et al. (34) demonstrated an increase in expression of the transcription factor zif268 mRNA by RA that appeared not to be fully accounted for by an increase in the rate of transcription of the gene. Similarly, posttranscriptional mechanisms appear to be involved in the increase in transforming growth factor-82 mRNA levels in cultured keratinocytes after the addition of RA (35). Results from this study suggest that both transcriptional and posttranscriptional components may be involved in the induction of TAT by DEX, Bt$AMP, and RA. TAT gene transcription was increased when cells were exposed to DEX and Bt,cAMP, indicating effects of these hormones at the level of transcription. How-

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RA Inhibits TAT Expression

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IO' 0

100

200

Y 300

TIME (MIN) Fig. 6. Half-Life of TAT mRNA in RALA255-1 OG Cells Grown in Medium Containing DEX/B@AMP or DEX/Bt2cAMP/RA Cells were grown at 33 C in medium containing 0.1 PM DEX. After 4-5 days of growth at 33 C (day 0), cultures were treated with medium containing 0.1 PM DEX and 0.5 mM BtZcAMP (0) or DEX, Bt*cAMP, and 10 PM RA (m) and shifted to 40 C for an additional 6 days. DEX or RA was present from days O-6, and Bt2cAMP was present from days 4-6. On day 6, both DEX/Bt2cAMP-treated and DEX/Bt2cAMP/RA-treated cultures were exposed to DRB (50 FM) for the times indicated and were then lysed for RNA isolation. Total RNA (20 wg/lane) was separated by agarose gel electrophoresis, transferred to Nytran membranes, and hybridized to an antisense TAT riboprobe. After removing bound TAT by washing the filters in 95% formamide (60 C for 30 min), the filters were rehybridized with a p-actin riboprobe. Autoradiograms of Northern bolts were quantitated by scanning the autoradiograms of different exposures with the Bio-Rad Densitometer. The relative amounts of TAT mRNA were normalized with respect to the P-a&in mRNA. Data points are derived from triplicate filters. Three independent experiments were performed. Data from a representative experiment are shown. Each point represents the mean + SEW

ever, the inhibition of DEX/Bt,cAMP-mediated induction of TAT mRNA by RA was not observed at the level of transcription, suggesting regulation at the posttranscriptional level. Measurements of TAT mRNA half-life using inhibitors of transcription indicated a half-life of 235-270 min in RALA255-1 OG cells exposed to DEX/ Bt$AMP compared to only 90-l 00 min in cells exposed to DEX/Bt$AMP/RA. These results suggest that RA has a destabilizing effect on TAT mRNA. Previous studies have determined the half-time of TAT mRNA degradation to be 2-3 h. This value was observed in rat liver (36), in hepatoma cells in the presence or absence of CAMP, and in DEX-treated cells (37). In this study TAT mRNA had a half-life of approximately 4 h in cells exposed to DEX and Bt,cAMP. We cannot exclude the possibility that in the presence of both DEX and Bt,cAMP, a small stabilization effect of these hormones on TAT mRNA was evident. However, determinations of TAT mRNA half-life values in this study were designed to analyze the effects of RA on

the apparent rate of TAT mRNA degradation in cells exposed to DEX/Bt&. It is clear from these studies that RA has a destabilizing effect on TAT mRNA in the presence of DEX and B&. Many examples of hormonal regulation through changes in mRNA stability have been documented. Estrogen greatly stabilizes vitellogenin mRNA in cultured Xenopus liver (38), and the combination of insulin, hydrocortisone, and PRL alters casein secretion in mammary cultures by affecting mRNA stability (39). Moreover, the stimulation of GH gene expression by glucocorticoids resulted from an increase in mRNA stability (40). Glucocorticoids stabilize PEPCK mRNA in addition to increasing the rate of transcription of the gene (41). Although the exact mechanisms responsible for the control of hormone stability are currently unknown, several hypotheses have been postulated (for a review, see Ref. 42). The rate of degradation of a target mRNA appears to be controlled through binding of factors to specific sequences in the mRNA. Estrogen is thought to induce a cytoplasmic estrogen-binding regulator that affects turnover of vitellogenin mRNA (43). The stability of some mRNA species appears to be related to the length of the 3’-poly(A) tract. For example, the increase in the half-life of GH mRNA by glucocorticoid is associated with an increase in the length of the 3’-poly(A) tail of its mRNA (44). Sequences in the 3’-region of the mRNA also appear to be involved in stabilization. A glucocorticoid-dependent stabilizing element has been identified in the 3’-noncoding sequence of the PEPCK mRNA (41). Fusion of this region to a heterologous mRNA confers glucocorticoid-dependent stabilization upon the chimeric mRNA. Stabilization of PEPCK mRNA by glucocorticoids is thought to occur by interaction of an induced factor with the element in the 3’-noncoding region of the mRNA. The mechanisms involved in RA destabilization of TAT mRNA in the presence of DEX/Bt,cAMP remain to be elucidated. We have shown that RA had an inhibitory effect on the induction of TAT mRNA by DEX and/or Bt,cAMP. Similarly, RA has recently been reported to have an inhibitory effect on the phorbol ester and calcium ionophore-induced expression of the T-cell growth factor interleukin-2 gene (45). The mechanisms by which RA appears to interfere with signal pathways of other hormones are currently unknown. These systems are complicated by the presence of multiple hormones, which appear to regulate gene expression at s.everal levels. Indeed, even the receptor type involved in RA-mediated inhibition in these hepatocytes remains unknown. It is possible that the subfamily of RA receptors, designated RXRs, which differ in ligand specificity and patterns of expression from the RARs (46), may be involved in transduction of the RA effect in these cells. Elucidation of the mechanisms involved in the regulation of TAT by RA should be possible using the temperature-sensitive hepatocyte model described in this paper.

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MATERIALS

Vol6

AND METHODS

Cell Culture The RALA255-1 OG cell line (25) was grown and maintained in a-modified Minimal essential medium supplemented with DEX (0.1 FM), streptomycin (100 kg/ml), penicillin (100 U/ml), and 4% fetal bovine serum. Cells were grown initially at 33 C (permissive temperature) until reaching 90% confluency (3-5 days) and then shifted to 40 C (nonpermissive temperature) for further experimentation. Medium was changed every 2 days. PBS contains 150 mM NaCI, 1 .l mM KH2P0.,, and 4.3 rn& Na&iP04, pH 7.4. The antiglucocorticoid RU486 was kindly provided by Roussel-UCLAF (Romainville, France). TAT mRNA induction was found to be extremely sensitive to cycloheximide. For these reasons, the lowest level of cycloheximide (0.1 ualml) that inhibited induction of TAT. but did not result in any-iisible morphological changes to the cells, was used. After a 3-day incubation of cells in 0.1 Kg/ml cycloheximide, total protein synthesis, as measured by [35S]methionine (20 &i/ml; 40 C for 3 h) incorporation into total trichloroacetic acid-precipitable radioactivity, was inhibited by only 1 O-20%. To measure TAT mRNA stability, the transcription-blocking agents, actinomycin-D (1 pg/ml) and DRB (50 PM) were employed. At the concentrations used, both agents greatly inhibited the incorporation of [3H]uridine (20 &i/ml; 40 C for 1, 2, or 4 h) into total trichloroacetic acid-precipitable radioactivity (60-85%). Nucleic

Acid

Hybridization

Analysis

Livers were obtained from adult Sprague-Dawley rats (-200 g BW). Total RNA from adult rat liver or RALA255-1 OG cells was extracted by the guanidinium thiocyanate-CsCl method (47) and poly(A)+ RNA was purified by oligo(dT)-cellulose chromatography. RNA was separated by electrophoresis in 1.2% agarose gels containing 2.2 M formaldehyde (48), transferred to Nytran membranes (Schleicher and Schulell, Keene, NH), and hybridized to a uniformly labeled antisense riboprobe of rat TAT (49), rat glucocorticoid receptor (50), rat PEPCK (51), or rat p-actin (B. Patterson, NIH). Hybridization was performed at62Cin5XSSC(l xSSCis0.15~NaClandO.015~Na citrate), 50% formamide, 8 x Denhardt’s solution (1 x Denhardt is 0.02% each of Ficoll, polyvinylpyrrolidone, and BSA), 50 mM sodium phosphate (pH 6.5), 1% sodium dodecyl sulfate (SDS), and 200 pg/ml sonicated salmon sperm DNA. RNA blots were washed twice in 2 x SSC containing 0.5% SDS for 30 min at room temperature, and then four times in 0.1 x SSC containing 0.1% SDS for 60 min at 67 C. Relative levels of TAT mRNA were quantified by scanning autoradiograms of different exposures. Results were normalized to fl-actin mRNA levels. Densitometry was performed using a Bio-Rad densitometer (model 620, Bio-Rad Laboratory, Richmond, CA). Glucocorticoid

Receptor

Assay

The receptor assay was performed essentially as previously described (52). Briefly, approximately 10’ cells were washed and homogenized in- buwer containing 10 mM Tris-HCI (pH 7.4). 1.5 mM EDTA, 1 mM dithiothreitol. 3 mM sodium molvbdate, and 10% glycerol. The homogen&es were centrifuged at 100,000 x g for 1 h, and supernatant (cytosol) was stored at -70 C. [3H]DEX (l-20 nM; 49.2 Ci/mmol; New England Nuclear, Boston, MA) was used as the radioactive ligand for the receptor assay, and nonspecific binding was measured by the addition of 1 mM cold DEX to a reaction mixture containing 20 nM radioactive ligand. Binding of ligand to the receptors (cytosol) was carried out at 4 C for 18 h, and free ligand was removed with dextran-coated charcoal (0.1% dextran and 0.5% Norit SG charcoal). Binding data were analyzed using the computer program Ligand. Data were analyzed by Student’s t test.

Nuclear

Run-On

Transcription

No. 4

Assay

Nuclei were isolated essentially as described by Clayton and Darnell, Jr. (53). Briefly, cells were homogenized at 4 C in 10 vol 10 mM Tris-HCI (pH 7.4), 10 mM NaCI, 3.5 mM MgCI,, 150 mM KCI, 14 mM P-mercaptoethanol, and 0.5% Nonidet P-40. Nuclei were pelleted by centrifugation at 1000 x g for 5 min, stored (2-4 x 10’ nuclei/ml) in 20 mM Tris-HCI (pH 7.4), 4 mM MnC12, 1 mM MgC12, 5 mM dithiothreitol, 0.5 mM spermidine, and 60% glycerol, and used within 1 week. Rates of RNA transcription were measured with 250 &i 132P1UTP (3000 Ci/ mmol; New England Nuclear Products, Boston: MA) in 40 rni Tris-HCI (pH 8.3). 150 mM NH&I. 7.5 mM MaCI?. and 1 mM each of ATP, CiP, and GTP containing 2-4 G lo’ nuclei in a total volume of 400 ~1 and incubated at 27 C for 35 min. NAmanitin, when present, was in a concentration of 1 pg/ml. RNA synthesis was terminated by incubation with RNase-free DNase (62.5 pg/ml) for 15 min at 37 C, followed by incubation with proteinase-K (1 mg/ml), heparin (3 mg/ml), 10 mM TrisHCI (pH 7.4), 15 mM EDTA, and 3% SDS for 3 h at 42 C. RNA was purified by phenol-chloroform extraction, trichloroacetic acid and alcohol precipitation, and DNase treatment, then solubilized in 10 mM Tris-HCI, pH 8, and 1 mM EDTA and used for hybridization to solid phase plasmid DNA. Plasmid pUC19 (10 pg/slot) or an equal amount of TAT genomic subclones RTATG-EH0.94 (contains exons B and C) and RTATG-EEl.05 (contains exons K and part of exon L) (54) were boiled in 0.2 N NaOH for 5 min, adjusted to 6 x SSC, and applied to a Nytran membrane using a slot blot (Schleicher and Schuell, Manifold II). Filters were UV crosslinked with a Stratagene cross-linker (Stratagene Cloning Systems, La Jolla, CA) and hybridized for 72 h at 42 C in 5 x SSC, 50% formamide, 10% dextran sulfate, 1% SDS, 5 x Denhardt’s solution, 200 pg/ml sonicated salmon sperm DNA, 100 pg/ml poly(A), and 5 x 1 O6 cpm/ml labeled nuclear RNA. After hybridization, filters were washed as described for Northern filters. Acknowledgments We thank G. Schutz for providing the TAT cDNA and genomic clones, Drs. R. Miesfeld and K. R. Yamamoto for the glucocorticoid receptor cDNA, Dr. R. W. Hanson for the PEPCK cDNA, and Dr. B. Patterson for the rat p-actin cDNA.

Received October 28, 1992. Accepted January Address requests for ing 10, Room 9S242, thesda, Maryland 20892.

1991. Revision received January 13, 13,1992. reprints to: Dr. Janice Y. Chou, BuildNational Institutes of Health, Be-

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RA Inhibits

579

TAT Expression

5. Schmid E, Schmid W, Jantzen M, Mayer D, Jastorff B, Schutz G 1987 Transcription activation of the tyrosine aminotransferase gene by glucocorticoids and CAMP in primary hepatocytes. Eur J Biochem 165:499-506 6. Jantzen H-M, Strahle U, Gloss B, Stewart F, Schmid W, Boshart M, Miksicek R, Schutz G 1987 Cooperativity of glucocorticoid response elements located far upstream of the tyrosine aminotransferase gene. Cell 49:29-38 7. Boshart M, Weih F, Schmidt A, Keith Fournier RE, Schutz G 1990 A cyclic AMP response element mediates repression of tyrosine aminotransferase gene transcription by the tissue-specific extinguisher locus Tse-1 Cell 61:905916 8. Boshart M, Weih F, Nichols M, Schutz G 1991 The tissuespecific extinguisher locus TSEl encodes a regulatory subunit of CAMP-dependent protein kinase. Cell 66:849859 9. Chytil F 1984 Retinoic acid: biochemistry, pharmacology, toxicology, and therapeutic use. Pharmacol Rev [Suppl2] 36:935-l 005 10. Soorn MB, Roberts AB 1984 Biological methods for analysis and assay of retinoids-relationships between structure and activitv. In: Soorn MB. Roberts AB, Goodman DS (eds) The ‘Retinoibs. Academic Press, New York, vol 1:235-279 11. Strickland S, Mahadavi V 1978 The induction of differentiation in teratocarcinoma stem cells by retinoic acid. Cell 151393-403 12. Breitman TR, Selonick SE, Collins SJ 1980 Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid. Proc Natl Acad Sci USA 77:2936-2940 13. Thaller C, Eichele G 1987 Identification and spatial distribution of retinoids in the developing chick limb bud. Nature 327:625-628 14. Giguere V, Ong ES, Segui P, Evans RM 1987 Identification of a receptor for the morphogen retinoic acid. Nature 330:624-629 M, Brand NJ, Krust A, Chambon P 1987 A 15. Petkovich human retinoic acid receptor which belongs to the family of nuclear receptors. Nature 330:444-450 16. Benbrook D, Lernhardt E, Pfahl M 1988 A new retinoic acid receptor identified from a hepatocellular carcinoma. Nature 333:669-672 17. Brand N, Petkovich M, Krust A, Chambon P, de The H, Marchio A, Tiollais P, Dejean A 1988 Identification of a second human retinoic acid receptor. Nature 332:850853 M, Zelent A, Chambon P 18. Krust A, Kastner P, Petkovich 1989 A third human retinoic acid receptor, hRAR-g. Proc Natl Acad Sci USA 86:5310-5314 M, Kastner P, Chambon P 19. Zelent A, Krust A, Petkovich 1989 Cloning of murine LY and p retinoic acid receptors and a novel receptor gamma predominantly expressed in skin. Nature 339:714-717 20 Bedo G, Santisteban P, Aranda A 1989 Retinoic acid regulates growth hormone gene expression. Nature 339:231-234 21. Vasios GW, Gold JD, Petkovich M, Chambon P, Gudas LJ 1989 A retinoic acid-responsive element is present in the 5’ flanking region of the laminin 81 gene. Proc Natl Acad Sci USA 86:9099-9103 22 de The H, de Mar Vivanco-Ruiz M, Tiollais P, Stunnenberg H, Dejean A 1990 Identification of a retinoic acid responsive element in the retinoic acid receptor p gene. Nature 343:177-l 80 G, Shean ML, McBride MS, Stewart MJ 1991 23. Duester Retinoic acid response element in the human alcohol dehydrogenase gene ADH3: implication for regulation of retinoic acid svnthesis. Mol Cell Biol 11 :1638-l 646 24. Pan C-J, Hoppner W, Chou JY 1990 Induction of phosphoenolpyruvate carboxykinase gene expression by retinoic acid in an adult rat hepatocyte line. Biochemistry 29:10883-l 0888

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Vol6 No. 4

MOL ENDO. 1992 580

46. 47.

48.

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2 promoter via cis-regularity sequences containing an octamer motif. Mol Cell Biol 9:4771-4778 Mangelsdorf DJ, Ong ES, Dyck JA, Evans RM 1990 Nuclear receptor that identifies a novel retinoic acid response pathwasy. Nature 345224-229 Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:52945299 Lehrach H, Diamond D, Wozney JM, Boedtker H 1977 RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16:4743-4751 Scherer G, Schmid W, Strange CM, Rowekamp A, Schutz G 1982 Isolation of cDNA clones coding for rat tyrosine aminotransferase. Proc Natl Acad Sci USA 79:7205-7208 Miesfled R, Rusconi S, Godowski PM, Maler BA, Okret S, Wikstrom A-C, Gustafsson J-A, Yamamoto KR 1986

The 49th Laurentian

51.

52. 53. 54.

Genetic complementation of a glucocorticoid receptor deficiency by expression of cloned receptor cDNA. Cell 46:389-399 Yoo-Warren H, Monahan JE, Short J, Short H, Bruzel A, Wynshaw-Boris A, Meisner HM, Samols D, Hanson RW 1983 Isolation and characterization of the gene coding for cytosolic phosphoenolpyruvate carboxykinase (GTP) from the rat. Proc Natl Acad Sci USA 80:3656-3660 Brandon DD, Markwick A, Chrousos GP, Loriaux DL 1988 Glucocorticoid resisitance in humans and nonhuman primates. Cancer Res [Suppl] 49:22038-22138 Clayton DF, Darnell Jr JE 1983 Changes in liver-specific compared to common gene transcription during primary culture of mouse hepatocytes. Mol Cell Biol 3:1552-l 561 Shinomiya T, Scherer G, Schmid W, Zentgraf H, Schutz G 1984 Isolation and characterization of the rat tyrosine aminotransferase gene. Proc Natl Acad Sci USA 81 :13461350

Hormone

Conference

The 49th Laurentian Hormone Conference will be held at Palmas de Mar, Puerto Rico, November 15-18, 1992. Topics will include prostate, neuroendocrine growth factors, intercellular signaling, and glucose regulation. For more information contact: Laurentian Avenue, New York, New York 10021.

Hormone

Conference,

Box 273-JES,

1230 York

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