0013-7227/92/1304-1879$03.00/O Endocrinology Copyright 0 1992 by The Endocrine Society
Vol. 130, No. 4 Printed
in U.S.A.
Mechanism of Regulation of Thyrotropin-Releasing Hormone Receptor Messenger Ribonucleic Acid in Stably Transfected Rat Pituitary Cells* JIRO FUJIMOTO, C. S. NARAYANAN, MARCOS HEINFLINK, AND MARVIN
JONATHAN E. BENJAMIN, C. GERSHENGORN
Division of Endocrinology and Metabolism, Department New York Hospital, New York, New York 10021
of
Medicine,
Cornell
EGULATION of the level of receptor expression is a common mechanism for modulating cell responses to hormones, neurotransmitters, and growth factors. This may occur through several mechanisms (1, 2). One mechanism for regulation of receptor number is to modulate the rate of receptor synthesis secondary to changes in the level of receptor mRNA. Modulation of receptor mRNA can be caused by changes in the rate of transcription, mRNA degradation, or both. In rat pituitary GH3 cells and related cell lines, the number of TRH receptors (TRH-Rs) is down-regulated by TRH (3, 4). We found that TRH caused the level of TRH-R mRNA to be down-regulated also (5, 6). We were not able to determine whether the effect on TRH-R mRNA was caused by decreased transcription or increased degradation in GH3 cells, although our preliminary evidence was consistent with an effect on mRNA degradation (5). We Received October 14,199l. Address all correspondence and requests for reprints to: Dr. Marvin C. Gershengorn, Room A328, Department of Medicine, Cornell University Medical College, 1300 York Avenue, New York, New York 10021. * This work was supported by USPHS Grant DK-43036.
Medical
College and
enase (GAPDH) mRNA, an endogenous gene product. TRH stimulated the rate of transcription of mouse TRH-R DNA by approximately 2-fold, but did not affect total poly(A) RNA synthesis. Most importantly, TRH caused a a-fold increase in the rate of degradation of mouse TRH-R mRNA, but did not affect degradation of GAPDH mRNA. The half-lives of mouse TRH-R and GAPDH mRNAs were 3 and more than 20 h in control cells and 0.75 and more than 20 h in cells treated with 1 PM TRH for 1.5 h, respectively. These data show that the predominant effect of TRH on mouse TRH-R mRNA in GHmTRHR-1 cells is to enhance the rate of ita degradation. We suggest, therefore, that down-regulation of TRH-R mRNA caused by TRH in the parent GH3 cell line is secondary to increased TRH-R mRNA degradation. (Endocrinology 130: 1879-1&X34,1992)
ABSTRACT. We showed previously that the level of TRH receptor (TRH-R) mRNA in rat pituitary GH3 cells is downregulated by TRH. Here, we study the mechanism of regulation of TRH-R mRNA in a line of GHs cells that are stably transfected with mouse pituitary TRH-R cDNA (GH-mTRHR-1 cells). GH-mTRHR-1 cells were found to have 2.4 times the number of TRH-Ra and to stimulate a 2.5fold greater increase in inositol phosphates in response to TRH than the parent cell line and to show TRH-induced down-regulation of TRH-R number. GH-mTRHR-1 cells contained 26 f 1.6 molecules of mouse TRH-R mRNA/cell and 230 + 31 molecules of mRNA for the neomycin resistance gene (NEO) with which it was cotransfected. In GH-mTRHR-1 cells, TRH caused a dosedependent transient decrease in mouse TRH-R mRNA, with a nadir to 20% of control levels after 6 h. In contrast, TRH did not affect NE0 mRNA or glyceraldehyde phosphate dehydrog
R
University
have, therefore, developed a line of GH3 cells that are stably transfected with mouse pituitary TRH-R cDNA (7) (GH-mTRHR-1 cells) in which we can more readily study regulation of TRH-R mRNA. In this report we characterize these cells with regard to their responses to TRH and use them to begin to study the mechanism of regulation of TRH-R mRNA, in particular to determine whether mRNA regulation is caused by changes in mRNA synthesis or degradation. Materials
and Methods
GHa cells were stably transfected with the mouse pituitary TRH-R cDNA (7). The entire TRH-R cDNA contained in a NotI/EcoRV fragment was cloned directionally into the eukaryotic expression vector pcDNAINE0 (pcNEOmTRHR), in which transcription of the TRH-R DNA is driven by a cytomegalovirus promoter, and the neomycin resistance gene (NEO) is under control of a ROW sarcoma virus long terminal repeat promoter. GH, cells (3 x 10’) were seeded into loo-mm dishes in Ham’s F-10 medium supplemented with sodium bicarbonate (1.68 g/liter), HEPES (2.39 g/liter; pH 7.4), glucose 2.4 (g/liter), penicillin G (0.05 g/liter), 15% horse serum, and 2.5% fetal bovine serum (growth medium) and transfected with 1879
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pcNEOmTRHR (10 pg/ml) 24 h later, using the DEAE-dextran method (8). This subclone of GH, cells, which we have had growing in our laboratory for more than 1 yr, was chosen because it had a low number of endogenous TRH-Rs. After 48 h, Geneticin (G418; 400 pg/ml) was added, and the incubations were continued. Cultures were refed every 3 or 4 days with growth medium containing 400 rg G418/ml. After 5 weeks, several colonies had grown, and individual colonies were transferred to new dishes. The cell clone used in this report was named GH-mTRHR-1. TRH-R binding was measured under equilibrium conditions, using doses of [3H][Nj-Me-His]TRH ([3H]MeTRH), a high affinity agonist for the TRH-R, between 0.1-2.5 nM, as previously described (4). TRH stimulation of inositol phosphate formation was measured in cells after incubation in growth medium containing myo-[3H]inositol (1 &i/ml) for more than 24 h, as previously described (9). Cells were exposed to TRH (l-1000 nM) for 30 min in the presence of 10 mM LiCI, which inhibits dephosphorylation of inositol phosphates. Total cellular RNA was isolated using sodium dodecyl sulfate, potassium acetate, and acetic acid (10). The integrity of ribosomal RNA was assessedby gel electrophoresis. Nuclease protection assays (11) were used to measure TRH-R and NE0 mRNAs. The cRNA probe for TRH-R mRNA was transcribed from a plasmid containing a 430-nucleotide BglII-BgflI fragment of pBSmTRHR (7), as previously described (6). To measure NE0 mRNA, we cloned a 411-nucleotide EcoRI-BgZII fragment of the NE0 DNA from pcDNAINE0 into EcoRI- and BamHIdigested plasmid Bluescript (pBSNE0411). pBSNE0411 was digested with HindIII and transcribed using T3 polymerase with [32P]UTP to generate a [32P]cRNA, which was purified on a 6% urea polyacrylamide gel. This antisense probe contained 63 nucleotides complementary to vector DNA. Sample RNA (lo-30 pg) and [32P]cRNA (2.3 x lo5 cpm) were coprecipitated; redissolved in buffer containing 60% formamide, 900 mM NaCl, 6 mM EDTA, and 60 mM Tris-HCl, pH 7.4; heated at 85 C for 10 min; and incubated at 68 C overnight. The sample was diluted with buffer containing 200 mM NaCI, 10 mM MgC12, 10 mM Na acetate (pH 5.0), and 400 U RNAase-Tl and incubated at 37 C for 45 min. The sample was extracted with phenolchloroform, precipitated with ethanol, washed, redissolved in 90% formamide buffer, incubated at 85 C for 10 min, and electrophoresed on a 6% urea polyacrylamide gel. After drying the gel, autoradiograms were exposed for 16-96 h, and the bands were quantified by densitometry. Standard curves were generated with increasing amounts of mouse TRH-R RNA and NE0 RNA transcribed in uitro and were linear up to 70 and 30 pg RNA, respectively. Using 400 U RNAase-Tl for digestion in the assay appears to degrade endogenous rat TRH-R mRNA, and therefore, we cannot detect TRH-R mRNA in up to 70 c(g GH, cell RNA (data not shown). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (12) was analyzed by Northern gels and quantitated by autoradiography and densitometry. Transcription rates were measured using run-on assays (13, 14). In brief, approximately 8 x lo6 cells in a loo-mm dish were scraped into 5 ml 10 mM Tris-HCl (pH 8), 10 mM NaCI, 6 mM MgC12, 1 mM dithiothreitol, and 0.2% Triton X-100 and vortexed at low speed, and the nuclei were collected by centrifu-
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gation at 180 X g for 5 min. RNA chains were labeled by incubation in 50 mM Tris-HCl (pH 8), 100 mM KCl, 12.5% glycerol, 6 mM MgC12, 1 mM MnC12, 0.1 mM EDTA, 2 mM dithiothreitol, 150 U/ml RNAsin, 2 mM ATP, 1 mM CTP, 1 mM GTP, and 1 PM [32P]UTP at room temperature for 30 min. The reaction mixture was treated with RNAase-free DNAase, extracted with phenol-chloroform-isoamyl alcohol, precipitated with ethanol, and then digested again with DNAase, reextracted, and reprecipitated. Labeled RNA was hybridized to 1 rg linearized plasmid DNA containing the mouse TRH-R cDNA immobilized on Nytran membranes. Hybridizations containing 3 x lo6 cpm 32P-labeled RNA were performed overnight at 42 C in buffer containing 750 mM NaCl, 50 mM Na phosphate (pH 7.0), 5 mM EDTA, 5 x Denhardt’s reagent, 50 Fg/ml salmon sperm DNA, 10% dextran sulfate, and 50% formamide. Final washes were performed with 30 mM NaCl, 2 mM Na phosphate (pH 7.0), and 0.2 mM EDTA at 65 C. Autoradiograms were exposed for 6 days. Poly(A) RNA was isolated on oligo(dT)-cellulose. Statistical analyses were performed by t test; differences were considered significant when P < 0.05. Materials
TRH was purchased from Beckman (Palo Alto, CA) or Sigma (St. Louis, MO). Plasmid Bluescript was obtained from Stratagene (La Jolla, CA), and plasmid pcDNAINE0 was from Invitrogen (San Diego, CA). [3H] [MeTRH was purchased from DuPont-New England Nuclear (Boston, MA), and my~-[~H] inositol from Amersham (Arlington Heights, IL). Restriction endonucleases were obtained from Boehringer Mannheim (Indianapolis, IN) and New England Biolabs (Beverly, MA). Oligo(dT)-cellulose was purchased from Collaborative Research (Bedford, MA). All chemicals were ultrapure grade from Sigma.
Results Figure 1 compares the specific binding of MeTRH and TRH stimulation of inositol phosphate accumulation, an index of TRH stimulation of second messenger formation (15, 16), in parental GH3 cells and GH-mTRHR-1 cells. There was no difference in the apparent equilibrium dissociation constants, which were 1.4 + 0.1 and 1.4 + 0.2 nM for the parental cells and GH-mTRHR-1 cells, respectively, but there was a 2.4-fold greater number of TRH-Rs in the GH-mTRHR-1 cells (340 + 50 fmol/mg protein) than in the parental cells (140 f 30 fmol/mg). Half-maximal stimulation of inositol phosphate accumulation occurred at the same concentration of TRH (-7 nM) in both cell lines, but the maximal amount of inositol phosphates accumulated was 2.5fold greater in GH-mTRHR-1 cells than in parental GH3 cells. A direct correlation between generation of second messenger molecules and the number of TRH-Rs has been made previously in GH cell clones (17, 18). Regulation by TRH of TRH-R number in GHmTRHR-1 cells is illustrated in Fig. 2. As in parental GH3 cells (data not shown) and other related GH cells
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MECHANISM
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0 ‘;;;
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1.0
1.5
MeTRH
2.0
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(nM)
10
100
1000
TRH (nM)
FIG. FIG. 1. Comparison of MeTRH binding and TRH stimulation of inositol phosphate accumulation in GH-mTRHR-1 and GHs cells. Left, Equilibrium binding of the indicated concentrations of [3H]MeTRH. Nonspecific binding was measured in the presence of 1 PM TRH and was subtracted from the total binding. Right, Cells prelabeled with myo-[SH]inositol were incubated in medium containing 10 mM mM LiCl and l-1000 nM TRH or without TRH for 30 min. The data are presented as disintegrations per min in inositol phosphates divided by lo6 dpm in inositol lipids so as to correct for differences in amounts or labeling of the lipid precursors. These data represent the mean f SE of duplicate or triplicate points in three to five experiments for GH-mTRHR-1 cells and GHI cells.
I 250
FIG. 2. Effect of TRH to down-regulate TRH-R number in GH-mTRHR-1 cells. Left, Cells were exposed for the indicated times to 100 nM TRH. Right, Cells were exposed to the indicated concentrations of TRH for 43 h. Equilibrium binding was performed with 1 nM [SH]MeTRH. These dam represent the mean k SE of duplicate points in two experiments.
E z m
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5 2 E s
0
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Time (3,5), TRH caused a time- and concentration-dependent down-regulation of the number of TRH-Rs in GHmTRHR-1 cells. There was no effect on the affinity of the receptor for TRH (data not shown). The decrease in TRH-R number in GH-mTRHR-1 cells of 30% was similar to that observed in the parent cell line (data not shown), although it has been reported that the decrease in TRH-R level in GH cells can be as much as 70% (3, 5). The effect of TRH on the levels of mouse TRH-R, NEO, and GAPDH mRNAs in GH-mTRHR-1 cells was determined. Unstimulated GH-mTRHR-1 cells contained 43 f 2.6 attomole (amol) mouse TRH-R mRNA/
24
(h)
46
TRH
(nM)
lo6 cells (or 26 + 1.6 molecules/cell) and 390 f 53 amol NE0 mRNA/lO’ cells (or 230 f 31 molecules/cell). Figure 3 illustrates the effects of stimulation of GHmTRHR-1 cells by TRH for 3 h on the levels of TRH-R and NE0 mRNAs. TRH caused the level of mouse TRHR mRNA to decrease in a concentration-dependent manner, with maximal down-regulation after 3 h to 35% of control levels, The concentration of TRH that elicited a half-maximal effect was approximately 1 nM, which is similar to that for other persistent effects of TRH in GH cells (15, 16). In contrast, TRH did not affect the level of NE0 mRNA consistently; in some experiments there was a small increase in NE0 mRNA. TRH did not affect
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MECHANISM
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0
3. Effects of TRH on mouse TRH-R and NE0 mRNAs in GHmTRHR-1 cells. Cells were incubated without TRH or with the indicated concentrations of TRH for 3 h. Total cell RNA was extracted and the levels of TRH-R and NE0 mRNAs were measured. Data represent the mean f SD of triplicate points from one of two similar experiments.
FIG.
the level of GAPDH mRNA (data not shown). Figure 4 illustrates the time course of down-regulation of TRHR mRNA caused by a maximally effective dose of TRH. As we found in the parent cell line (5, 6), the effect of TRH to decrease TRH-R mRNA was transient, with a nadir approximately 6 h after TRH addition and an increase toward control levels thereafter. The level of TRH-R mRNA, however, remained below the control value after 24 and 48 h. To determine whether the decrease in TRH-R mRNA
Endo. 1992 Voll30. No 4
was caused by a decrease in the rate of transcription, we determined the effect of TRH on transcription of mouse TRH-R DNA. We measured transcription rates after 1, 2 and 3 h of exposure to TRH. The effects of TRH were the same at all three time points, and we have combined these data. Figure 5 shows that TRH caused the transcription rate of mouse TRH-R DNA to increase by approximately 2-fold. This increase did not reflect a generalized enhancement of transcription, because the rate of synthesis of the total population of poly(A)enriched RNAs was not increased by TRH. Importantly, the effect of TRH on TRH-R DNA transcription could not account for its effect to decrease TRH-R mRNA. Based on the above data, it was likely that the effect of TRH was to increase the degradation of TRH-R mRNA. In these experiments the rate of mRNA degradation was assessed by inhibiting transcription with actinomycin-D. In preliminary experiments we showed that 5 pg actinomycin-D/ml inhibited the incorporation of [3H]uridine into RNA by more than 98%. Figure 6 illustrates the effect of TRH on the rates of degradation of mouse TRH-R mRNA and GAPDH mRNA. TRH caused an increase in the degradation rate of TRH-R mRNA, but had no effect on the rate of degradation of GAPDH mRNA. The half-lives of mouse TRH-R mRNA and GAPDH mRNA were 3 and more than 20 h in control cells and 0.75 and more than 20 h in cells treated with 1 pM TRH for 1.5 h, respectively. Discussion We developed a rat pituitary cell line, GH-mTRHR-1, that is stably transfected with a mouse pituitary TRHR cDNA. GH-mTRHR-1 cells contain approximately 40,000 TRH-Rs/cell and respond to TRH with the ex-
4. Time course of the effect of TRH on TRH-R mRNA. Cells were incubated without (Control) or with 1 pru TRH for the times indicated, RNA was extracted, and mouse TRH-R and NE0 mRNAs were assayed. Data are presented as a percentage of the control ratio of the amount of mouse TRH-R mRNA divided by NE0 mRNA in order to normalize the values for mouse TRH-R mRNA between experiments. Each bar represents the mean + SE of four or five replicates from two experiments. FIG.
Control
3 Time
6 after
TRH
24 40 addition (h)
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MECHANISM c
250
OF TRH RECEPTOR mRNA REGULATION
1
TRH
Control
FIG. 5. Effects of TRH on the rate of transcription of mouse TRH-R DNA and synthesis of total poly(A) RNA. Cells were incubated without or with 1 PM TRH for l-3 h, nuclei were isolated, and the rates of DNA transcription were measured by run-on assays, as described in Materials and Methods. Data represent the mean + SD of triplicate points from one of three experiments.
GAPDH
mRNA [yl
0
1
3
Tirnl
4
(h)
FIG. 6. Effects of TRH on the rates of degradation of mouse TRH-R and GAPDH mRNAs. Cells were incubated without or with 1 NM TRH for 1.5 h, and then actinomycin-D was added to a final concentration of 5 pg/ml. At the times indicated, RNA was extracted, and mouse TRH-R and GAPDH mRNAs were assayed. Data are presented as a percentage of the respective control value. The level of mouse TRH-R mRNA was 66 f 13% of the control level after exposure to TRH for 1.5 h.
petted generation of inositol lipid-derived second messengers. Down-regulation by TRH of the number of TRH-Rs and of TRH-R mRNA was similar to that observed in the parent GH3 cell line. Hence, GHmTRHR-1 cells appear to be a valid model in which to study regulation of TRH-R expression. It appears that the only TRH-R mRNA being measured by our modified nuclease protection assay in RNA extracted from GH-mTRHR-1 cells is mouse mRNA. This is so because under the stringent conditions of this modified assay rat TRH-R mRNA is not detected even in 3.5-7 times as much RNA isolated from parental GH3 cells. To measure endogenous rat TRH-R mRNA we used an assay method that has less stringent conditions
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for RNA digestion (6). It is likely, therefore, that there are differences in the nucleotide sequences of mouse and rat mRNAs in the region that is protected by the probe, and these base mismatches permit digestion by RNAase of mouse (probe)-rat (sample) hybrids under stringent conditions. In contrast, we are not certain whether we are measuring mouse or rat TRH-Rs, or both, on GHmTRHR-1 cells. The binding characteristics of the mouse and rat TRH-Rs are too similar to permit this differentiation (4,19), and no other means of distinguishing between them is available. We were able to measure the rate of transcription of the transfected mouse TRH-R DNA and the rate of degradation of mouse TRH-R mRNA in GH-mTRHR-1 cells. We could not measure these parameters for the endogenous rat TRH-R in parental GH3 cells. We are not certain why these measurements were possible in the transfected cells and not in untransfected cells. It is possible that GH-mTRHR-1 cells have higher levels of TRH-R mRNA than GH3 cells because transcription of mouse TRH-R DNA is being driven by the strong cytomegalovirus promoter, and the DNA may be present in a greater number of copies than the endogenous gene. We cannot compare the levels of TRH-R mRNA in GHmTRHR-1 and GH3 cells because we cannot measure the absolute level of endogenous rat TRH-R mRNA. Another possibility is that our probes, which were developed using mouse TRH-R cDNA (7), may have permitted greater sensitivity in measuring mouse TRH-R mRNA than rat mRNA. We chose to express the mouse TRH-R cDNA in GHB cells because we expected that posttranscriptional mechanisms that regulate the endogenous TRH-R mRNA would continue to be functional in transfected cells. Our previous experience with HeLa cells, monkey kidney (CV-1) cells, and rat glioma (C6) cells that were stably expressing the mouse TRH-R and with monkey kidney (COS-1) cells that were transiently transfected with TRH-R cDNA was that TRH-R mRNA regulation was not the same as that in pituitary cells (unpublished observations) (20). For example, in COS-1 cells, TRH caused an increase in TRH-R mRNA levels. We found that TRH regulation of mouse TRH-R mRNA is complex in GH-mTRHR-1 cells, involving effects on both synthesis and degradation. TRH increases the rate of transcription by 2-fold and increases the rate of degradation by 4-fold. Of note is that the &-adrenergic receptor mRNA is regulated in a biphasic fashion, also including stimulation of transcription and mRNA degradation (21, 22). The predominating effect of TRH for the first 6 h is that of enhanced degradation, as the level of TRH-R mRNA is decreased during this period. After 6 h, the mRNA level increases toward the control value. More detailed analyses at these later time points will be needed
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to determine the chronic effects of TRH on TRH-R mRNA metabolism. The time course of the effects of TRH on mouse TRH-R mRNA levels in GH-mTRHR1 cells is similar to that on the endogenous mRNA in GH3 cells (5, 6). We think, therefore, that the observations made using the transfected gene accurately reflect the posttranscriptional mechanisms of regulation of the endogenous TRH-R mRNA. It is less likely that the effects observed on transcription of the transfected cDNA are simulating effects on the endogenous gene. The mouse TRH-R cDNA contains only 258 bases of 5’untranslated sequence (7) and, of course, does not contain the up-stream gene regulatory elements. It is very unlikely that the mouse cDNA recombined homologously with the endogenous gene and came under control of its normal regulatory elements. We think, therefore, that the increase in transcription of TRH-R DNA caused by TRH in GH-mTRHR-1 cells is probably a manifestation of a nonspecific increase in transcription driven by the cytomegalovirus promoter, as shown by others in GH3 cells (23). In conclusion, the predominant effect of TRH to lower mouse TRH-R mRNA levels in pituitary GH-mTRHR1 cells is caused by an increase in the rate of mRNA degradation. We suggest that a similar mechanism is involved in the decrease in endogenous TRH-R mRNA in parental GH3 cells. Acknowledgment
7. 8. 9. 10. 11.
12.
13. 14.
15. 16. 17.
18.
We thank Dr. Peter Guidon for his help with measurements of GAPDH mRNA. References 1. Collins S, Caron MG, Lefkowitz RI 1991 Regulation of adrenergic receptor responsiveness through modulation of receptor gene expression. Annu Rev Physiol53:497-508 2. Hadcock JR, Malbon CC 1991 Regulation of receptor expression by agonists: transcriptional and post-transcriptional controls. Trends Neurosci 14~242-247 3. Hinkle PM. Tashiian Jr AH 1975 Thvrotronin-releasing hormone regulates the number of its own receptors-in the GH; strain of pituitary cells in culture. Biochemistry 143845-3851 4. Gershengorn MC 1978 Bihormonal regulation of the thyrotropin releasing hormone receptor in mouse pituitary thyrotropic tumor cells in culture. J Clin Invest 623937-943 5. Oron Y, Straub RE, Traktman P, Gershengorn MC 1987 Decreased TRH receptor mRNA activity precedes homologous downregulation: assay in oocytes. Science 238:1406-1408 6. Fujimoto J, Straub RE, Gershengorn MC 1991 Thyrotropin-releas-
19.
20.
21. 22.
23.
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ing hormone (TRH) and phorbol myristate acetate decrease TRH receptor mRNA in rat pituitary GHs cells. Evidence that protein kinase C mediates the TRH effect. Mol Endocrinol5:1527-1532 Straub RE, Frech GC, Joho RH, Gershengom MC 1990 Expression clonine of a cDNA encodine the mouse nituitarv thvrotroninreleaskg hormone receptor. P&z Nat1 AcadSci USA 87~9514-9518 Cullen BR 1987 Use of eukaryotic expression technology in the functional analysis of cloned genes. Methods Enxymol 152:684704 Imai A, Gershengorn MC 1987 Measurement of lipid turnover in response to thyrotropin-releasing hormone. Methods Enzymol 141:100-101 Peppel K, Baglioni C 1990 A simple and fast method to extract RNA from tissue culture cells. Biotechniaues 9711-713 Williams DL, Newman TC, Shelness GS,-Gordon DA 1986 Measurement of apolipoprotein mRNA by DNA-excess solution hybridization with single-stranded probes. Methods Enzymol 128:671689 Tso JY, Sun X-H, Kao T-h, Reece KS, Wu R 1985 Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res 13:2485-2502 Ucker DS, Yamamoto KR 1984 Early events in the stimulation of mammary tumor virus RNA synthesis by ducocorticoids. Novel assays of-transcription rates. J Biol Chem 259~7416-7420 Yaffe BM. Samuels HH 1984 Hormonal reaulation of the srowth hormone gene. Relationship of the rate of tr&cription to the level of nuclear thyroid hormone-receptor complexes. J Biol Chem 2596284-6291 Gershengorn MC 1986 Mechanism of thyrotropin releasing hormone stimulation of pituitary hormone secretion. Annu Rev Physiol48:515-526 Drummond AH 1986 Inositol lipid metabolism and signal transduction in clonal pituitary cells. J Exp Biol 124~337-358 Imai A, Gershengorn MC 1985 Evidence for tight coupling of thyrotropin-releasing hormone receptors to stimulated inositol trisphosphate formation in rat pituitary cells. J Biol Chem 260:10536-10540 Ramsdell JS, Tashjian Jr AH 1986 Thyrotropin-releasing hormone (TRH) elevation of inositol trisphosphate and cytosolic free calcium is dependent on receptor number. Evidence for multiple rapid interactions between TRH and its receptor. J Biol Chem 261:53015306 Hinkle PM, Woroch EL, Tashjian Jr AH 1974 Receptor-binding affinities and biological activities of analogs of thvrotronin-releasing hormone in prolactin-producing pit&q cells in-culture. J Biol Chem 2493085-3090 Gershengorn MC, Thaw CN 1991 Regulation of thyrotropin-releasing hormone receptors is cell type specific: comparison of endogenous pituitary receptors and receptors transfected into nonpituitary cells. Endocrinology 128:1204-1206 Hadcock JR, Malbon CC 1988 Down-regulation of fl-adrenergic receptors: agonist-induced reduction in receptor mRNA levels. Proc Nat1 Acad Sci USA 85~5021-5025 Collins S, Bouvier M, Bolanowski MA, Caron MG, Lefkowitz RJ 1989 CAMP stimulates transcription of the &adrenergic receptor gene in response to short-term agonist exposure. Proc Nat1 Acad Sci USA 864853-4857 Yan G, Pan WT, Bancroft C 1991 Thyrotropin-releasing hormone action on the prolactin promoter is mediated by the POU protein Pit-l. Mol Endocrinol5:535-541
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Vol. 130, No. 4 Printed
in U.S.A.
Mechanism of Regulation of Thyrotropin-Releasing Hormone Receptor Messenger Ribonucleic Acid in Stably Transfected Rat Pituitary Cells* JIRO FUJIMOTO, C. S. NARAYANAN, MARCOS HEINFLINK, AND MARVIN
JONATHAN E. BENJAMIN, C. GERSHENGORN
Division of Endocrinology and Metabolism, Department New York Hospital, New York, New York 10021
of
Medicine,
Cornell
EGULATION of the level of receptor expression is a common mechanism for modulating cell responses to hormones, neurotransmitters, and growth factors. This may occur through several mechanisms (1, 2). One mechanism for regulation of receptor number is to modulate the rate of receptor synthesis secondary to changes in the level of receptor mRNA. Modulation of receptor mRNA can be caused by changes in the rate of transcription, mRNA degradation, or both. In rat pituitary GH3 cells and related cell lines, the number of TRH receptors (TRH-Rs) is down-regulated by TRH (3, 4). We found that TRH caused the level of TRH-R mRNA to be down-regulated also (5, 6). We were not able to determine whether the effect on TRH-R mRNA was caused by decreased transcription or increased degradation in GH3 cells, although our preliminary evidence was consistent with an effect on mRNA degradation (5). We Received October 14,199l. Address all correspondence and requests for reprints to: Dr. Marvin C. Gershengorn, Room A328, Department of Medicine, Cornell University Medical College, 1300 York Avenue, New York, New York 10021. * This work was supported by USPHS Grant DK-43036.
Medical
College and
enase (GAPDH) mRNA, an endogenous gene product. TRH stimulated the rate of transcription of mouse TRH-R DNA by approximately 2-fold, but did not affect total poly(A) RNA synthesis. Most importantly, TRH caused a a-fold increase in the rate of degradation of mouse TRH-R mRNA, but did not affect degradation of GAPDH mRNA. The half-lives of mouse TRH-R and GAPDH mRNAs were 3 and more than 20 h in control cells and 0.75 and more than 20 h in cells treated with 1 PM TRH for 1.5 h, respectively. These data show that the predominant effect of TRH on mouse TRH-R mRNA in GHmTRHR-1 cells is to enhance the rate of ita degradation. We suggest, therefore, that down-regulation of TRH-R mRNA caused by TRH in the parent GH3 cell line is secondary to increased TRH-R mRNA degradation. (Endocrinology 130: 1879-1&X34,1992)
ABSTRACT. We showed previously that the level of TRH receptor (TRH-R) mRNA in rat pituitary GH3 cells is downregulated by TRH. Here, we study the mechanism of regulation of TRH-R mRNA in a line of GHs cells that are stably transfected with mouse pituitary TRH-R cDNA (GH-mTRHR-1 cells). GH-mTRHR-1 cells were found to have 2.4 times the number of TRH-Ra and to stimulate a 2.5fold greater increase in inositol phosphates in response to TRH than the parent cell line and to show TRH-induced down-regulation of TRH-R number. GH-mTRHR-1 cells contained 26 f 1.6 molecules of mouse TRH-R mRNA/cell and 230 + 31 molecules of mRNA for the neomycin resistance gene (NEO) with which it was cotransfected. In GH-mTRHR-1 cells, TRH caused a dosedependent transient decrease in mouse TRH-R mRNA, with a nadir to 20% of control levels after 6 h. In contrast, TRH did not affect NE0 mRNA or glyceraldehyde phosphate dehydrog
R
University
have, therefore, developed a line of GH3 cells that are stably transfected with mouse pituitary TRH-R cDNA (7) (GH-mTRHR-1 cells) in which we can more readily study regulation of TRH-R mRNA. In this report we characterize these cells with regard to their responses to TRH and use them to begin to study the mechanism of regulation of TRH-R mRNA, in particular to determine whether mRNA regulation is caused by changes in mRNA synthesis or degradation. Materials
and Methods
GHa cells were stably transfected with the mouse pituitary TRH-R cDNA (7). The entire TRH-R cDNA contained in a NotI/EcoRV fragment was cloned directionally into the eukaryotic expression vector pcDNAINE0 (pcNEOmTRHR), in which transcription of the TRH-R DNA is driven by a cytomegalovirus promoter, and the neomycin resistance gene (NEO) is under control of a ROW sarcoma virus long terminal repeat promoter. GH, cells (3 x 10’) were seeded into loo-mm dishes in Ham’s F-10 medium supplemented with sodium bicarbonate (1.68 g/liter), HEPES (2.39 g/liter; pH 7.4), glucose 2.4 (g/liter), penicillin G (0.05 g/liter), 15% horse serum, and 2.5% fetal bovine serum (growth medium) and transfected with 1879
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pcNEOmTRHR (10 pg/ml) 24 h later, using the DEAE-dextran method (8). This subclone of GH, cells, which we have had growing in our laboratory for more than 1 yr, was chosen because it had a low number of endogenous TRH-Rs. After 48 h, Geneticin (G418; 400 pg/ml) was added, and the incubations were continued. Cultures were refed every 3 or 4 days with growth medium containing 400 rg G418/ml. After 5 weeks, several colonies had grown, and individual colonies were transferred to new dishes. The cell clone used in this report was named GH-mTRHR-1. TRH-R binding was measured under equilibrium conditions, using doses of [3H][Nj-Me-His]TRH ([3H]MeTRH), a high affinity agonist for the TRH-R, between 0.1-2.5 nM, as previously described (4). TRH stimulation of inositol phosphate formation was measured in cells after incubation in growth medium containing myo-[3H]inositol (1 &i/ml) for more than 24 h, as previously described (9). Cells were exposed to TRH (l-1000 nM) for 30 min in the presence of 10 mM LiCI, which inhibits dephosphorylation of inositol phosphates. Total cellular RNA was isolated using sodium dodecyl sulfate, potassium acetate, and acetic acid (10). The integrity of ribosomal RNA was assessedby gel electrophoresis. Nuclease protection assays (11) were used to measure TRH-R and NE0 mRNAs. The cRNA probe for TRH-R mRNA was transcribed from a plasmid containing a 430-nucleotide BglII-BgflI fragment of pBSmTRHR (7), as previously described (6). To measure NE0 mRNA, we cloned a 411-nucleotide EcoRI-BgZII fragment of the NE0 DNA from pcDNAINE0 into EcoRI- and BamHIdigested plasmid Bluescript (pBSNE0411). pBSNE0411 was digested with HindIII and transcribed using T3 polymerase with [32P]UTP to generate a [32P]cRNA, which was purified on a 6% urea polyacrylamide gel. This antisense probe contained 63 nucleotides complementary to vector DNA. Sample RNA (lo-30 pg) and [32P]cRNA (2.3 x lo5 cpm) were coprecipitated; redissolved in buffer containing 60% formamide, 900 mM NaCl, 6 mM EDTA, and 60 mM Tris-HCl, pH 7.4; heated at 85 C for 10 min; and incubated at 68 C overnight. The sample was diluted with buffer containing 200 mM NaCI, 10 mM MgC12, 10 mM Na acetate (pH 5.0), and 400 U RNAase-Tl and incubated at 37 C for 45 min. The sample was extracted with phenolchloroform, precipitated with ethanol, washed, redissolved in 90% formamide buffer, incubated at 85 C for 10 min, and electrophoresed on a 6% urea polyacrylamide gel. After drying the gel, autoradiograms were exposed for 16-96 h, and the bands were quantified by densitometry. Standard curves were generated with increasing amounts of mouse TRH-R RNA and NE0 RNA transcribed in uitro and were linear up to 70 and 30 pg RNA, respectively. Using 400 U RNAase-Tl for digestion in the assay appears to degrade endogenous rat TRH-R mRNA, and therefore, we cannot detect TRH-R mRNA in up to 70 c(g GH, cell RNA (data not shown). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (12) was analyzed by Northern gels and quantitated by autoradiography and densitometry. Transcription rates were measured using run-on assays (13, 14). In brief, approximately 8 x lo6 cells in a loo-mm dish were scraped into 5 ml 10 mM Tris-HCl (pH 8), 10 mM NaCI, 6 mM MgC12, 1 mM dithiothreitol, and 0.2% Triton X-100 and vortexed at low speed, and the nuclei were collected by centrifu-
l l
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gation at 180 X g for 5 min. RNA chains were labeled by incubation in 50 mM Tris-HCl (pH 8), 100 mM KCl, 12.5% glycerol, 6 mM MgC12, 1 mM MnC12, 0.1 mM EDTA, 2 mM dithiothreitol, 150 U/ml RNAsin, 2 mM ATP, 1 mM CTP, 1 mM GTP, and 1 PM [32P]UTP at room temperature for 30 min. The reaction mixture was treated with RNAase-free DNAase, extracted with phenol-chloroform-isoamyl alcohol, precipitated with ethanol, and then digested again with DNAase, reextracted, and reprecipitated. Labeled RNA was hybridized to 1 rg linearized plasmid DNA containing the mouse TRH-R cDNA immobilized on Nytran membranes. Hybridizations containing 3 x lo6 cpm 32P-labeled RNA were performed overnight at 42 C in buffer containing 750 mM NaCl, 50 mM Na phosphate (pH 7.0), 5 mM EDTA, 5 x Denhardt’s reagent, 50 Fg/ml salmon sperm DNA, 10% dextran sulfate, and 50% formamide. Final washes were performed with 30 mM NaCl, 2 mM Na phosphate (pH 7.0), and 0.2 mM EDTA at 65 C. Autoradiograms were exposed for 6 days. Poly(A) RNA was isolated on oligo(dT)-cellulose. Statistical analyses were performed by t test; differences were considered significant when P < 0.05. Materials
TRH was purchased from Beckman (Palo Alto, CA) or Sigma (St. Louis, MO). Plasmid Bluescript was obtained from Stratagene (La Jolla, CA), and plasmid pcDNAINE0 was from Invitrogen (San Diego, CA). [3H] [MeTRH was purchased from DuPont-New England Nuclear (Boston, MA), and my~-[~H] inositol from Amersham (Arlington Heights, IL). Restriction endonucleases were obtained from Boehringer Mannheim (Indianapolis, IN) and New England Biolabs (Beverly, MA). Oligo(dT)-cellulose was purchased from Collaborative Research (Bedford, MA). All chemicals were ultrapure grade from Sigma.
Results Figure 1 compares the specific binding of MeTRH and TRH stimulation of inositol phosphate accumulation, an index of TRH stimulation of second messenger formation (15, 16), in parental GH3 cells and GH-mTRHR-1 cells. There was no difference in the apparent equilibrium dissociation constants, which were 1.4 + 0.1 and 1.4 + 0.2 nM for the parental cells and GH-mTRHR-1 cells, respectively, but there was a 2.4-fold greater number of TRH-Rs in the GH-mTRHR-1 cells (340 + 50 fmol/mg protein) than in the parental cells (140 f 30 fmol/mg). Half-maximal stimulation of inositol phosphate accumulation occurred at the same concentration of TRH (-7 nM) in both cell lines, but the maximal amount of inositol phosphates accumulated was 2.5fold greater in GH-mTRHR-1 cells than in parental GH3 cells. A direct correlation between generation of second messenger molecules and the number of TRH-Rs has been made previously in GH cell clones (17, 18). Regulation by TRH of TRH-R number in GHmTRHR-1 cells is illustrated in Fig. 2. As in parental GH3 cells (data not shown) and other related GH cells
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MECHANISM
OF TRH RECEPTOR mRNA REGULATION ,
0 ‘;;;
I
I
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I
GH-mTRHR-1
-.
250
0.0
0.5
1.0
1.5
MeTRH
2.0
2.5
3.0
0
1
(nM)
10
100
1000
TRH (nM)
FIG. FIG. 1. Comparison of MeTRH binding and TRH stimulation of inositol phosphate accumulation in GH-mTRHR-1 and GHs cells. Left, Equilibrium binding of the indicated concentrations of [3H]MeTRH. Nonspecific binding was measured in the presence of 1 PM TRH and was subtracted from the total binding. Right, Cells prelabeled with myo-[SH]inositol were incubated in medium containing 10 mM mM LiCl and l-1000 nM TRH or without TRH for 30 min. The data are presented as disintegrations per min in inositol phosphates divided by lo6 dpm in inositol lipids so as to correct for differences in amounts or labeling of the lipid precursors. These data represent the mean f SE of duplicate or triplicate points in three to five experiments for GH-mTRHR-1 cells and GHI cells.
I 250
FIG. 2. Effect of TRH to down-regulate TRH-R number in GH-mTRHR-1 cells. Left, Cells were exposed for the indicated times to 100 nM TRH. Right, Cells were exposed to the indicated concentrations of TRH for 43 h. Equilibrium binding was performed with 1 nM [SH]MeTRH. These dam represent the mean k SE of duplicate points in two experiments.
E z m
225
5 2 E s
0
3
6
Time (3,5), TRH caused a time- and concentration-dependent down-regulation of the number of TRH-Rs in GHmTRHR-1 cells. There was no effect on the affinity of the receptor for TRH (data not shown). The decrease in TRH-R number in GH-mTRHR-1 cells of 30% was similar to that observed in the parent cell line (data not shown), although it has been reported that the decrease in TRH-R level in GH cells can be as much as 70% (3, 5). The effect of TRH on the levels of mouse TRH-R, NEO, and GAPDH mRNAs in GH-mTRHR-1 cells was determined. Unstimulated GH-mTRHR-1 cells contained 43 f 2.6 attomole (amol) mouse TRH-R mRNA/
24
(h)
46
TRH
(nM)
lo6 cells (or 26 + 1.6 molecules/cell) and 390 f 53 amol NE0 mRNA/lO’ cells (or 230 f 31 molecules/cell). Figure 3 illustrates the effects of stimulation of GHmTRHR-1 cells by TRH for 3 h on the levels of TRH-R and NE0 mRNAs. TRH caused the level of mouse TRHR mRNA to decrease in a concentration-dependent manner, with maximal down-regulation after 3 h to 35% of control levels, The concentration of TRH that elicited a half-maximal effect was approximately 1 nM, which is similar to that for other persistent effects of TRH in GH cells (15, 16). In contrast, TRH did not affect the level of NE0 mRNA consistently; in some experiments there was a small increase in NE0 mRNA. TRH did not affect
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MECHANISM
OF TRH RECEPTOR mRNA REGULATION
0
3. Effects of TRH on mouse TRH-R and NE0 mRNAs in GHmTRHR-1 cells. Cells were incubated without TRH or with the indicated concentrations of TRH for 3 h. Total cell RNA was extracted and the levels of TRH-R and NE0 mRNAs were measured. Data represent the mean f SD of triplicate points from one of two similar experiments.
FIG.
the level of GAPDH mRNA (data not shown). Figure 4 illustrates the time course of down-regulation of TRHR mRNA caused by a maximally effective dose of TRH. As we found in the parent cell line (5, 6), the effect of TRH to decrease TRH-R mRNA was transient, with a nadir approximately 6 h after TRH addition and an increase toward control levels thereafter. The level of TRH-R mRNA, however, remained below the control value after 24 and 48 h. To determine whether the decrease in TRH-R mRNA
Endo. 1992 Voll30. No 4
was caused by a decrease in the rate of transcription, we determined the effect of TRH on transcription of mouse TRH-R DNA. We measured transcription rates after 1, 2 and 3 h of exposure to TRH. The effects of TRH were the same at all three time points, and we have combined these data. Figure 5 shows that TRH caused the transcription rate of mouse TRH-R DNA to increase by approximately 2-fold. This increase did not reflect a generalized enhancement of transcription, because the rate of synthesis of the total population of poly(A)enriched RNAs was not increased by TRH. Importantly, the effect of TRH on TRH-R DNA transcription could not account for its effect to decrease TRH-R mRNA. Based on the above data, it was likely that the effect of TRH was to increase the degradation of TRH-R mRNA. In these experiments the rate of mRNA degradation was assessed by inhibiting transcription with actinomycin-D. In preliminary experiments we showed that 5 pg actinomycin-D/ml inhibited the incorporation of [3H]uridine into RNA by more than 98%. Figure 6 illustrates the effect of TRH on the rates of degradation of mouse TRH-R mRNA and GAPDH mRNA. TRH caused an increase in the degradation rate of TRH-R mRNA, but had no effect on the rate of degradation of GAPDH mRNA. The half-lives of mouse TRH-R mRNA and GAPDH mRNA were 3 and more than 20 h in control cells and 0.75 and more than 20 h in cells treated with 1 pM TRH for 1.5 h, respectively. Discussion We developed a rat pituitary cell line, GH-mTRHR-1, that is stably transfected with a mouse pituitary TRHR cDNA. GH-mTRHR-1 cells contain approximately 40,000 TRH-Rs/cell and respond to TRH with the ex-
4. Time course of the effect of TRH on TRH-R mRNA. Cells were incubated without (Control) or with 1 pru TRH for the times indicated, RNA was extracted, and mouse TRH-R and NE0 mRNAs were assayed. Data are presented as a percentage of the control ratio of the amount of mouse TRH-R mRNA divided by NE0 mRNA in order to normalize the values for mouse TRH-R mRNA between experiments. Each bar represents the mean + SE of four or five replicates from two experiments. FIG.
Control
3 Time
6 after
TRH
24 40 addition (h)
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MECHANISM c
250
OF TRH RECEPTOR mRNA REGULATION
1
TRH
Control
FIG. 5. Effects of TRH on the rate of transcription of mouse TRH-R DNA and synthesis of total poly(A) RNA. Cells were incubated without or with 1 PM TRH for l-3 h, nuclei were isolated, and the rates of DNA transcription were measured by run-on assays, as described in Materials and Methods. Data represent the mean + SD of triplicate points from one of three experiments.
GAPDH
mRNA [yl
0
1
3
Tirnl
4
(h)
FIG. 6. Effects of TRH on the rates of degradation of mouse TRH-R and GAPDH mRNAs. Cells were incubated without or with 1 NM TRH for 1.5 h, and then actinomycin-D was added to a final concentration of 5 pg/ml. At the times indicated, RNA was extracted, and mouse TRH-R and GAPDH mRNAs were assayed. Data are presented as a percentage of the respective control value. The level of mouse TRH-R mRNA was 66 f 13% of the control level after exposure to TRH for 1.5 h.
petted generation of inositol lipid-derived second messengers. Down-regulation by TRH of the number of TRH-Rs and of TRH-R mRNA was similar to that observed in the parent GH3 cell line. Hence, GHmTRHR-1 cells appear to be a valid model in which to study regulation of TRH-R expression. It appears that the only TRH-R mRNA being measured by our modified nuclease protection assay in RNA extracted from GH-mTRHR-1 cells is mouse mRNA. This is so because under the stringent conditions of this modified assay rat TRH-R mRNA is not detected even in 3.5-7 times as much RNA isolated from parental GH3 cells. To measure endogenous rat TRH-R mRNA we used an assay method that has less stringent conditions
1883
for RNA digestion (6). It is likely, therefore, that there are differences in the nucleotide sequences of mouse and rat mRNAs in the region that is protected by the probe, and these base mismatches permit digestion by RNAase of mouse (probe)-rat (sample) hybrids under stringent conditions. In contrast, we are not certain whether we are measuring mouse or rat TRH-Rs, or both, on GHmTRHR-1 cells. The binding characteristics of the mouse and rat TRH-Rs are too similar to permit this differentiation (4,19), and no other means of distinguishing between them is available. We were able to measure the rate of transcription of the transfected mouse TRH-R DNA and the rate of degradation of mouse TRH-R mRNA in GH-mTRHR-1 cells. We could not measure these parameters for the endogenous rat TRH-R in parental GH3 cells. We are not certain why these measurements were possible in the transfected cells and not in untransfected cells. It is possible that GH-mTRHR-1 cells have higher levels of TRH-R mRNA than GH3 cells because transcription of mouse TRH-R DNA is being driven by the strong cytomegalovirus promoter, and the DNA may be present in a greater number of copies than the endogenous gene. We cannot compare the levels of TRH-R mRNA in GHmTRHR-1 and GH3 cells because we cannot measure the absolute level of endogenous rat TRH-R mRNA. Another possibility is that our probes, which were developed using mouse TRH-R cDNA (7), may have permitted greater sensitivity in measuring mouse TRH-R mRNA than rat mRNA. We chose to express the mouse TRH-R cDNA in GHB cells because we expected that posttranscriptional mechanisms that regulate the endogenous TRH-R mRNA would continue to be functional in transfected cells. Our previous experience with HeLa cells, monkey kidney (CV-1) cells, and rat glioma (C6) cells that were stably expressing the mouse TRH-R and with monkey kidney (COS-1) cells that were transiently transfected with TRH-R cDNA was that TRH-R mRNA regulation was not the same as that in pituitary cells (unpublished observations) (20). For example, in COS-1 cells, TRH caused an increase in TRH-R mRNA levels. We found that TRH regulation of mouse TRH-R mRNA is complex in GH-mTRHR-1 cells, involving effects on both synthesis and degradation. TRH increases the rate of transcription by 2-fold and increases the rate of degradation by 4-fold. Of note is that the &-adrenergic receptor mRNA is regulated in a biphasic fashion, also including stimulation of transcription and mRNA degradation (21, 22). The predominating effect of TRH for the first 6 h is that of enhanced degradation, as the level of TRH-R mRNA is decreased during this period. After 6 h, the mRNA level increases toward the control value. More detailed analyses at these later time points will be needed
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to determine the chronic effects of TRH on TRH-R mRNA metabolism. The time course of the effects of TRH on mouse TRH-R mRNA levels in GH-mTRHR1 cells is similar to that on the endogenous mRNA in GH3 cells (5, 6). We think, therefore, that the observations made using the transfected gene accurately reflect the posttranscriptional mechanisms of regulation of the endogenous TRH-R mRNA. It is less likely that the effects observed on transcription of the transfected cDNA are simulating effects on the endogenous gene. The mouse TRH-R cDNA contains only 258 bases of 5’untranslated sequence (7) and, of course, does not contain the up-stream gene regulatory elements. It is very unlikely that the mouse cDNA recombined homologously with the endogenous gene and came under control of its normal regulatory elements. We think, therefore, that the increase in transcription of TRH-R DNA caused by TRH in GH-mTRHR-1 cells is probably a manifestation of a nonspecific increase in transcription driven by the cytomegalovirus promoter, as shown by others in GH3 cells (23). In conclusion, the predominant effect of TRH to lower mouse TRH-R mRNA levels in pituitary GH-mTRHR1 cells is caused by an increase in the rate of mRNA degradation. We suggest that a similar mechanism is involved in the decrease in endogenous TRH-R mRNA in parental GH3 cells. Acknowledgment
7. 8. 9. 10. 11.
12.
13. 14.
15. 16. 17.
18.
We thank Dr. Peter Guidon for his help with measurements of GAPDH mRNA. References 1. Collins S, Caron MG, Lefkowitz RI 1991 Regulation of adrenergic receptor responsiveness through modulation of receptor gene expression. Annu Rev Physiol53:497-508 2. Hadcock JR, Malbon CC 1991 Regulation of receptor expression by agonists: transcriptional and post-transcriptional controls. Trends Neurosci 14~242-247 3. Hinkle PM. Tashiian Jr AH 1975 Thvrotronin-releasing hormone regulates the number of its own receptors-in the GH; strain of pituitary cells in culture. Biochemistry 143845-3851 4. Gershengorn MC 1978 Bihormonal regulation of the thyrotropin releasing hormone receptor in mouse pituitary thyrotropic tumor cells in culture. J Clin Invest 623937-943 5. Oron Y, Straub RE, Traktman P, Gershengorn MC 1987 Decreased TRH receptor mRNA activity precedes homologous downregulation: assay in oocytes. Science 238:1406-1408 6. Fujimoto J, Straub RE, Gershengorn MC 1991 Thyrotropin-releas-
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ing hormone (TRH) and phorbol myristate acetate decrease TRH receptor mRNA in rat pituitary GHs cells. Evidence that protein kinase C mediates the TRH effect. Mol Endocrinol5:1527-1532 Straub RE, Frech GC, Joho RH, Gershengom MC 1990 Expression clonine of a cDNA encodine the mouse nituitarv thvrotroninreleaskg hormone receptor. P&z Nat1 AcadSci USA 87~9514-9518 Cullen BR 1987 Use of eukaryotic expression technology in the functional analysis of cloned genes. Methods Enxymol 152:684704 Imai A, Gershengorn MC 1987 Measurement of lipid turnover in response to thyrotropin-releasing hormone. Methods Enzymol 141:100-101 Peppel K, Baglioni C 1990 A simple and fast method to extract RNA from tissue culture cells. Biotechniaues 9711-713 Williams DL, Newman TC, Shelness GS,-Gordon DA 1986 Measurement of apolipoprotein mRNA by DNA-excess solution hybridization with single-stranded probes. Methods Enzymol 128:671689 Tso JY, Sun X-H, Kao T-h, Reece KS, Wu R 1985 Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res 13:2485-2502 Ucker DS, Yamamoto KR 1984 Early events in the stimulation of mammary tumor virus RNA synthesis by ducocorticoids. Novel assays of-transcription rates. J Biol Chem 259~7416-7420 Yaffe BM. Samuels HH 1984 Hormonal reaulation of the srowth hormone gene. Relationship of the rate of tr&cription to the level of nuclear thyroid hormone-receptor complexes. J Biol Chem 2596284-6291 Gershengorn MC 1986 Mechanism of thyrotropin releasing hormone stimulation of pituitary hormone secretion. Annu Rev Physiol48:515-526 Drummond AH 1986 Inositol lipid metabolism and signal transduction in clonal pituitary cells. J Exp Biol 124~337-358 Imai A, Gershengorn MC 1985 Evidence for tight coupling of thyrotropin-releasing hormone receptors to stimulated inositol trisphosphate formation in rat pituitary cells. J Biol Chem 260:10536-10540 Ramsdell JS, Tashjian Jr AH 1986 Thyrotropin-releasing hormone (TRH) elevation of inositol trisphosphate and cytosolic free calcium is dependent on receptor number. Evidence for multiple rapid interactions between TRH and its receptor. J Biol Chem 261:53015306 Hinkle PM, Woroch EL, Tashjian Jr AH 1974 Receptor-binding affinities and biological activities of analogs of thvrotronin-releasing hormone in prolactin-producing pit&q cells in-culture. J Biol Chem 2493085-3090 Gershengorn MC, Thaw CN 1991 Regulation of thyrotropin-releasing hormone receptors is cell type specific: comparison of endogenous pituitary receptors and receptors transfected into nonpituitary cells. Endocrinology 128:1204-1206 Hadcock JR, Malbon CC 1988 Down-regulation of fl-adrenergic receptors: agonist-induced reduction in receptor mRNA levels. Proc Nat1 Acad Sci USA 85~5021-5025 Collins S, Bouvier M, Bolanowski MA, Caron MG, Lefkowitz RJ 1989 CAMP stimulates transcription of the &adrenergic receptor gene in response to short-term agonist exposure. Proc Nat1 Acad Sci USA 864853-4857 Yan G, Pan WT, Bancroft C 1991 Thyrotropin-releasing hormone action on the prolactin promoter is mediated by the POU protein Pit-l. Mol Endocrinol5:535-541
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