Thyrotropin-Releasing Hormone (TRH) and Phorbol Myristate Acetate Decrease TRH Receptor Messenger RNA in Rat Pituitary GH3 Cells: Evidence That Protein Kinase-C Mediates the TRH Effect
Jiro Fujimoto, Richard E. Straub*, and Marvin C. Gershengorn Division of Endocrinology and Metabolism Departments of Medicine and Cell Biology and Anatomy New York Hospital Cornell University Medical College New York, New York 10021
In a previous report we showed that TRH-induced down-regulation of the density of its receptors (TRHRs) on rat pituitary tumor (GH3) cells was preceded by a decrease in the activity of the mRNA for the TRH-R, as assayed in Xenopus oocytes. Here we report the effects of TRH, elevation of cytoplasmic free Ca2+ concentration, phorbol myristate acetate (PMA), and H-7 [1-(5-isoquinolinesulfonyl)2-methylpiperazine dihydrochloride], an inhibitor of protein kinases, on the levels of TRH-R mRNA, which were measured by Northern analysis and in nuclease protection assays using probes made from mouse pituitary TRH-R cDNA, in GH3 cells. These agents were studied to gain insight into the mechanism of the TRH effect, because signal transduction by TRH involves generation of inositol 1,4,5-trisphosphate and elevation of cytoplasmic free Ca 2+ concentration, which leads to activation of Ca2+/calmodulindependent protein kinase, and of 1,2-diacylglycerol, which leads to activation of protein kinase-C. TRH (1 HM TRH, a maximally effective dose) caused a marked transient decrease in TRH-R mRNA that attained a nadir of 20-45% of control by 3-6 h, increased after 9 h, but was still below control levels after 24 h. Elevation of the cytoplasmic free Ca2+ concentration had no effect on TRH-R mRNA. A maximally effective dose of PMA (1 MM) caused decreases in TRH-R mRNA that were similar in magnitude and time course to those induced by 1 MM TRH. H-7 (20 MM) blocked the effects of TRH and PMA to lower TRH-R mRNA to similar extents. These data show that TRH and PMA decrease the levels of TRH-R mRNA in GH3 cells and are consistent with the idea that the effect of TRH is via a mechanism 0888-8809/91/1527-1532$03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society
that is mediated by protein kinase-C. (Molecular Endocrinology 5: 1527-1532, 1991)
INTRODUCTION Down-regulation of cell surface receptor density is an important mechanism for modulation of cell responsiveness. The number of receptors may be decreased by the action of its own ligand (homologous down-regulation) or by the action of other extracellular regulatory factors that interact with other cell receptors (heterologous down-regulation). It has been suggested that one mechanism of receptor down-regulation may involve internalization (1, 2) and perhaps subsequent intracellular degradation, another may involve receptor phosphorylation (3), and a third may be inhibition of the synthesis of receptors secondary to decreases in receptor mRNA levels (4-7). These mechanisms may be activated together to produce the final effect. TRH down-regulates the number of TRH receptors (TRHRs) on anterior pituitary cells (8, 9), and we have shown that this is preceded in rat pituitary tumor (GH3) cells by a decrease in the activity of the mRNA for the TRHR, as assayed in Xenopus laevis oocytes (10). The molecular mechanism(s) that mediates the decrease in TRH-R mRNA activity is unknown, but the cellular response to TRH has been shown to involve activation of at least two protein kinases, protein kinase-C (PKC) (11,12) and Ca2+/calmodulin-dependent protein kinase (13, 14). In the experiments reported here, we studied whether the decreases in TRH-R mRNA activity caused by TRH were secondary to decreases in mRNA levels, using probes developed using a mouse pituitary TRHR cDNA (15), and investigated a role for PKC and Ca 2+ / calmodulin-dependent protein kinase in mediating TRHinduced decreases in TRH-R mRNA in GH3 cells.
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MOL ENDO-1991 1528
Vol5No. 10
RESULTS AND DISCUSSION
TRH has been shown to down-regulate the number of TRH-Rs on GH3 cells in a time-dependent manner, with a half-maximal effect by 18 h and a maximal decrease by 30 h (8,10). We showed previously that TRH causes a transient decrease in TRH-R mRNA activity, as assayed in Xenopus oocytes, that attains a nadir between 3-6 h and increases toward the control value thereafter (10). Figure 1 {left panel) illustrates the effect of a maximally effective dose of TRH (1 ^M) on TRH-R mRNA levels assayed by Northern analysis in GH3 cells. A single dominant mRNA species of approximately 3.8 kilobases was observed. TRH caused a marked transient decrease in the TRH-R mRNA level, which attained a nadir by 3 h, remained low for an additional several hours, and then increased again after 6 h. To permit measurement of the changes in the levels of TRH-R mRNA more readily, we established a RNAase protection assay. Using this assay we confirmed that TRH caused decreases in TRH-R mRNA levels (Fig. 2). These data show that the previously reported decreases in endogenous TRH-R mRNA activity assayed in Xenopus oocytes (10) caused by TRH were due to decreases in the level of TRH-R mRNA. Signal transduction by TRH in GH3 cells uses the phosphatidylinositol 4,5-bisphosphate pathway (1618). Stimulation by TRH causes an inositol 1,4,5-trisphosphate-mediated elevation of the cytoplasmic free Ca2+ concentration, which leads to activation of Ca2+/ calmodulin-dependent protein kinase, and generation of 1,2-diacylglycerol leads to activation of PKC. Because signal transduction by TRH involves activation of PKC and activation of PKC by phorbol esters has been shown to affect the level of mRNAs for other receptors in other cell types (19), we studied the effects of phorbol myristate acetate (PMA) on TRH-R mRNA levels. Figures 1 and 2 illustrate the effect of 1 HM PMA on TRH-
TRH
PMA
a-actin C
1
3
5
16
C
3
6
16
Hours Fig. 1. Effects of TRH and PMA on TRH-R mRNA, as Analyzed by Northern Hybridization GH3 cells were incubated for the times indicated with 1 ^M TRH (left) or 1 HM PMA (right), poly(A)+-enriched RNA was isolated, and 70 or 33 ng, respectively, were analyzed. a-Actin mRNA was probed on the same filters. Data are from one of three similar experiments. C, Control.
3
6
9
24
Time (hr) Fig. 2. Effects of TRH and PMA on TRH-R mRNA, as Measured in a RNAase Protection Assay GH3 cells were incubated for the times indicated with 1 HM TRH (•) or 1 HM PMA (•), total RNA was isolated, and 50 or 100 ^g were assayed. Data are the mean ± SE from five experiments.
R mRNA levels. PMA caused a decrease in TRH-R mRNA, with a time course similar to that of TRH. In these sets of experiments, TRH was more effective than PMA in decreasing TRH-R mRNA; TRH and PMA caused TRH-R mRNA to decrease to 20% and 45% of the control value, respectively. However, in other experiments, the effects of TRH and PMA were of similar magnitude (see below). These data demonstrate that TRH and PMA have similar effects on TRH-R mRNA and are consistent with the idea that the effect of TRH may be mediated by the lipid limb of the TRH signal transduction pathway via activation of PKC. To study more directly whether the effect of TRH to decrease TRH-R mRNA may be mediated by PKC, we used two approaches. We incubated GH3 cells with 1 fiM PMA for more than 16 h to deplete them of PKC (20), and we used H-7 ([1-(5-isoquinolinesulfonyl)2methylpiperazine dihydrochloride]), which is a protein kinase inhibitor (21). We have shown that GH3 cells pretreated with 1 HM PMA have reduced PKC activity, a reduced secretory response to phorbol esters, and show inhibition of the second phase of the secretory response to TRH, which is thought to be mediated by PKC (22). Nevertheless, it is possible that some actions of TRH will not be affected by PMA pretreatment even if they are mediated by PKC, because a major PKC isoenzyme in GH3 cells, PKCa, is not depleted by prolonged incubation with PMA (23, 24) (our unpublished data). Figure 3 illustrates the effects of TRH on TRH-R mRNA levels in cells preincubated with 1 HM PMA for 24 h, in which the acute effect of PMA was inhibited; TRH-R mRNA was 95.5 ± 1.5% (mean ± SE) of the control value in PMA-pretreated cells exposed to 1 IXM PMA for an additional 3 or 6 h. The effect of TRH to decrease TRH-R mRNA after 3 and 6 h was not inhibited. Hence, an attempt to deplete GH3 cells of PKC did not block the effect of TRH on TRH-R mRNA. In contrast, H-7, which by itself lowered TRH-R mRNA
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TRH Receptor mRNA
o -£ 100 o o
Control
"o
75
PMA —pretreated
(pg)
1
1000
•
Fig. 6. Nuclease Protection Assay for the Measurement of TRH-R mRNA The assay was performed, as described in Materials and Methods, using 200 U RNAase-T1/RNAase per tube. Mouse TRH-R RNA transcribed in vitro and GH3 cell RNA were assayed in parallel. Left panel, Autoradiograms of gel eletrophoresis. Right panel, Plots of densitometric analysis.
obases detectable in GH3 cells under all conditions examined. This does not exclude increased degradation as the mechanism for lowering TRH-R mRNA, however, as it is not unusual not to be able to detect mRNA degradation products in mammalian cells even under conditions of enhanced turnover (27,28). We attempted to determine the molecular pathway involved in this effect. We found no evidence that the Ca2+ limb of this pathway is involved in regulating the level of TRH-R mRNA. In contrast, we developed evidence that PKC appears to mediate this effect. Regulation of the mRNA levels of G-protein-coupled receptors appears to be a general phenomenon. For example, the mRNA for 02-adrenergic receptors, which couple to the adenylyl cyclase-cAMP pathway, is regulated in a biphasic manner by &>-adrenergic agonists in hamster DDTT MF-2 cells (29-31). There is a rapid (during the first 2 h) increase in mRNA, which appears to be secondary to increased transcription, followed by a decrease to below control levels after 4 h, which may be secondary to increased degradation. In DDTT MF-2 cells (4) and Chinese hamster fibroblasts transfected with the human j82-adrenergic receptor (3), the effect on /32-adrenergic receptor mRNA appears to be mediated by its second messenger, cAMP. The mRNAs for the receptors for TSH (6; but see Ref. 32) and LH/ CG (33), which also couple to adenylyl cyclase are down-regulated in a cAMP-mediated manner. Izzo, Jr., and colleagues (7) showed that an-adrenergic agonists, whose receptors, like those of TRH, are coupled by Gproteins to the phosphatidylinositol 4,5-bisphosphateCa2+-PKC signal transduction pathway, caused a transient decrease in a r adrenergic receptor mRNA in rabbit aortic smooth muscle cells. The time course and magnitude of this effect are similar to the decreases in TRH-
R mRNA caused by TRH and PMA in GH3 cells. Although Izzo and colleagues did not show that the effect of a r adrenergic agonists to decrease receptor mRNA was mediated by PKC (7), in other systems (34, 35) phorbol ester activation of PKC has been implicated in regulation of mRNAs. For example, phorbol esters have recently been shown to decrease the mRNA for the LH/CG receptor (36). Hence, regulation of the mRNAs for G-protein-coupled receptors appears to occur commonly and to involve both homologous and heterologous second messenger-mediated pathways. In conclusion, we showed that TRH decreases TRHR mRNA by a mechanism that appears to be mediated by PKC. Although these data are consistent with the idea that decreases in TRH-R density are secondary to a lowered rate of receptor synthesis, they do not show a cause and effect relationship. Moreover, other mechanisms, such as enhanced degradation of TRH-R protein, may also be causally important in the down-regulation process. Indeed, phosphorylation by PKC of the TRH-R and of a protein(s) that regulates TRH-R mRNA degradation may coordinately down-regulate TRH-R number.
MATERIALS AND METHODS GH3 cells were grown and harvested, as described previously (37), in 100-mm dishes. Cells were exposed to 1 MM TRH, 1 IIM PMA, 25 mM KCI, 25 rriM NaCI, 20 MM H-7, or a combination thereof in growth medium for the times indicated. Total cellular RNA was isolated using guanidine thiocyanate and centrifugation through CsCI (38) or using sodium dodecyl sulfate, potassium acetate, and acetic acid (39). The integrity of ribosomal RNA was assessed by gel electrophoresis. Poly(A)+enriched RNA was obtained by one round of chromatography
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TRH Receptor mRNA
on oligo(dT)-cellulose. For Northern hybridization analysis, 3070 fig poly(A)+-enriched RNA were electrophoresed on a 1.2% agarose gel containing 2 M formaldehyde. After capillary transfer, the Duralon-UV membrane (Stratagene, La Jolla, CA) was hybridized using 2 x 106 cpm/ml probe. The probe was the 3.8-kilobase mouse TRH-R cDNA insert that was excised from pBSmTRHR with restriction endonucleases Kpn\ and Sacll (or A/ofl) (15), gel purified, electroeluted, and labeled with [32P] CTP to a specific activity of 3.7 x 108 cpm/^tg by random priming (40). a-Actin cDNA, a gift from Dr. Peter Guidon of the Hospital for Special Surgery, was also labeled and used as a control in some blots. Hybridization with 32P-labeled cDNA probes was performed overnight at 42 C in buffer containing 750 ITIM NaCI, 50 mM Na phosphate (pH 7.0), 5 mM EDTA, 5 x Denhardfs reagent, 50 fig/rr\\ salmon sperm DNA, 10% dextran sulfate, and 50% formamide. Final washes were performed with 30 mM NaCI, 2 mM Na phosphate (pH 7.0), and 0.2 mM EDTA at 65 C. Autoradiograms were exposed for 4 96 h. A nuclease protection assay (41) was established. The cRNA probe was transcribed from a plasmid containing a 430base Sg/ll-8g/ll fragment of pBSmTRHR (15) cloned into the 8amHI site of plasmid Bluescript (Stratagene), digested with ftsal, transcribed using T3 polymerase with [32P]UTP, and purified on a 6% urea polyacrylamide gel. This antisense probe contained 170 bases complementary to the open reading frame of the TRH-R and 35 bases complementary to vector DNA. Sample RNA (25-100 Mg) and [32P]cRNA (2.3 x 105 cpm) were coprecipitated; redissolved in buffer containing 60% formamide, 900 mM NaCI, 6 mM EDTA, and 60 mM Tris-HCI (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 MgCI2,10 mM Na acetate (pH 5.0), and 200 U RNAase-T1 and incubated at 37 C for 45 min. The sample was extracted with phenol-chloroform, precipitated with ethanoi, 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. Figure 6 shows a standard assay in which increasing amounts of mouse TRH-R RNA transcribed in vitro and total GH3 cell RNA were measured. The assay was linear with mouse TRH-R RNA up to 1000 pg and with GH3 cell RNA up to 50 Mg (up to 200 ng in other assays). Materials TRH was purchased from Beckman (Palo Alto, CA). Guanidine thiocyanate was obtained from Fluka BioChemika (Ronkonkoma, NY). Restriction endonucleases were purchased from Boehringer Mannheim (Indianapolis, IN) and New England Biolabs (Beverley, MA). H-7 was obtained from Seikagaku America (Rockville, MD). PMA and other chemicals were ultrapure grade for molecular biology and were purchased from Sigma (St. Louis, MO).
Acknowledgments We thank C. S. Narayanan for preparing the plasmid used to generate the cRNA for the nuclease protection assay.
Received June 6,1991. Rerevision received July 31,1991. Accepted July 31, 1991. Address requests for reprints to: Dr. Marvin C. Gershengorn, Room A328, 1300 York Avenue, New York, New York 10021. This work was supported by NIH Grant DK-43036. * Present address: Molecular Genetics Laboratory, Columbia University College of Physicians and Surgeons, Box 58, 722 West 168th Street, New York, New York 10032.
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REFERENCES 1. Chen W-J, Goldstein JL, Brown MS 1990 NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein receptor. J Biol Chem 265:3116-3123 2. Valiquette M, Bonin H, Hnatowich M, Caron MG, Lefkowitz RJ, Bouvier M 1990 Involvement of tyrosine residues located in the carboxyl tail of the human /32-adrenergic receptor in agonist-induced down-regulation of the receptor. Proc Natl Acad Sci USA 87:5089-5093 3. Bouvier M, Collins S, O'Dowd BF, Campbell PT, De Blasi A, Kobilka BK, MacGregor C, Irons GP, Caron MG, Lefkowitz RJ 1989 Two distinct pathways for cAMP-mediated down-regulation of the 0-adrenergic receptor. J Biol Chem 264:16786-16792 4. Hadcock JR, Malbon CC 1988 Down-regulation of /3adrenergic receptors: agonist-induced reduction in receptor mRNA levels. Proc Natl Acad Sci USA 85:5021-5025 5. Burnstein KL, Jewell CM, Cidlowski JA 1990 Human glucocorticoid receptor cDNA contains sequences sufficient for receptor down-regulation. J Biol Chem 265:7284-7291 6. Akamizu T, Ikuyama S, Saji M, Kosugi S, Kozak C, McBride OW, Kohn LD 1990 Cloning, chromosomal assignment, and regulation of the rat thyrotropin receptor: expression of the gene is regulated by thyrotropin, agents that increase cAMP levels, and thyroid autoantibodies. Proc Natl Acad Sci USA 87:5677-5681 7. Izzo Jr NJ, Seidman CE, Collins S, Colucci WS 1990 a r Adrenergic receptor mRNA level is regulated by norepinephrine in rabbit aortic smooth muscle cells. Proc Natl Acad Sci USA 87:6268-6271 8. Hinkle PM, Tashjian Jr AH 1975 Thyrotropin-releasing hormone regulates the number of its own receptors in the GH3 strain of pituitary cells in culture. Biochemistry 14:3845-3851 9. Gershengorn MC 1978 Bihormonal regulation of the thyrotropin releasing hormone receptor in mouse pituitary thyrotropic tumor cells in culture. J Clin Invest 62:937943 10. Oron Y, Straub RE, Traktman P, Gershengorn MC 1987 Decreased TRH receptor mRNA activity precedes homologous downregulation: assay in oocytes. Science 238:1406-1408 11. Sobel A, Tashjian Jr AH 1983 Distinct patterns of cytoplasmic protein phosphorylation related to regulation of synthesis and release of prolactin by GH cells. J Biol Chem 258:10312-10324 12. Drust DS, Martin TF 1984 Thyrotropin-releasing hormone rapidly activates protein phosphorylation in GH3 pituitary cells by a lipid-linked, protein kinase C-mediated pathway. J Biol Chem 259:14520-14530 13. Drust DS, Martin TFJ 1982 Thyrotropin-releasing hormone rapidly and transiently stimulates cytosolic calciumdependent protein phosphorylation in GH3 pituitary cells. J Biol Chem 257:7566-7573 14. Jefferson AB, Travis SM, Schulman H 1991 Activation of multifunctional Ca2+/calmodulin-dependent protein kinase in GH3 cells. J Biol Chem 266:1484-1490 15. Straub RE, Freeh GC, Joho RH, Gershengorn MC 1990 Expression cloning of a cDNA encoding the mouse pituitary thyrotropin-releasing hormone receptor. Proc Natl Acad Sci USA 87:9514-9518 16. Gershengorn MC 1986 Mechanism of thyrotropin releasing hormone stimulation of pituitary hormone secretion. Annu Rev Physiol 48:515-526 17. Gershengorn MC 1985 Thyrotropin-releasing hormone action: mechanism of calcium-mediated stimulation of prolactin secretion. Recent Prog Horm Res 41:607-653 18. Drummond AH 1986 Inositol lipid metabolism and signal transduction in clonal pituitary cells. J Exp Biol 124:337358
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MOL ENDO-1991 1532
19. Martinez-Valdez H, Thompson E, Cohen A 1988 Coordinate transcriptional regulation of a and chains of the Tcell antigen receptors by phorbol esters and cyclic adenosine 5'-monophosphate in human thymocytes. J Biol Chem 263:9561-9564 20. Ballester R, Rosen OM 1985 Fate of immunoprecipitable protein kinase c in GH3 cells treated with phorbol 12myristate 13-acetate. J Biol Chem 260:15194-15199 21. Hidaka H, Inagaki M, Kawamoto S, Sasaki Y 1984 Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 23:5036-5041 22. Rodriguez R, Imai A, Gershengorn MC 1987 Phosphatidylinositol depletion in GH3 rat pituitary cells inhibits sustained responses to thyrotropin-releasing hormone. Reversal with myo-inositol. Mol Endocrinol 1:802-807 23. Kiley S, Schaap D, Parker P, Hsieh L-L, Jaken S 1990 Protein kinase C heterogeneity in GHUCt rat pituitary cells. Characterization of a Ca2+-independent phorbol ester receptor. J Biol Chem 265:15704-15712 24. Akita Y, Ohno S, Yajima Y, Suzuki K 1990 Possible role of Ca2+-independent protein kinase C isozyme, nPKC, in thyrotropin-releasing hormone-stimulated signal transduction: differential down-regulation of nPKC in GH 4 d cells. Biochem Biophys Res Commun 172:184-189 25. Gershengorn MC, Thaw C 1983 Calcium influx is not required for TRH to elevate free cytoplasmic calcium in GH3 cells. Endocrinology 113:1522-1524 26. Tan KN, Tashjian Jr AH 1984 Voltage-dependent calcium channels in pituitary cells in culture. I. Characterization by 45 Ca2+ fluxes. J Biol Chem 259:418-426 27. Brawerman G1989 mRNA decay: finding the right targets. Cell 57:9-10 28. Nielsen DA, Shapiro DJ 1990 Insights into hormonal control of messenger RNA stability. Mol Endocrinol 4:953957 29. Collins S, Bouvier M, Bolanowski MA, Caron MG, Lefkowitz RJ 1989 cAMP stimulates transcription of the foadrenergic receptor gene in response to short-term agonist exposure. Proc Natl Acad Sci USA 86:4853-4857 30. Hadcock JR, Wang H, Malbon CC 1989 Agonist-induced destabilization of /8-adrenergic receptor mRNA. Attenua-
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tion of glucocorticoid-induced up-regulation of /?-adrenergic receptors. J Biol Chem 264:19928-19933 Collins S, Altschmied J, Herbsman O, Caron MG, Mellon PL, Lefkowitz RJ 1990 A cAMP response element in the /32-adrenergic receptor gene confers transcriptional autoregulation by cAMP. J Biol Chem 265:19330-19335 Huber GK, Concepcion ES, Graves PN, Davies TF 1991 Positive regulation of human thyrotropin mRNA by thyrotropin. J Clin Endocrinol Metab 72:1394-1396 Wang H, Segaloff DL, Ascoli M 1991 Lutropin/choriogonadotropin down-regulates its receptor by both receptormediated endocytosis and a cAMP-dependent reduction in receptor mRNA. J Biol Chem 266:780-785 Vyas S, Biguet NF, Mallet J 1990 Transcriptional and post-transcriptional regulation of tyrosine hydroxylase gene by protein kinase C. EMBO J 9:3707-3712 Scarpati EM, Sadler JE 1989 Regulation of endothelial cell coagulant properties. Modulation of tissue factor, plasminogen activator inhibitors, and thrombomodulin by phorbol 12-myristate 13-acetate and tumor necrosis factor. J Biol Chem 264:20705-20713 Wang H, Segaloff DL, Ascoli M 1991 Epidermal growth factor and phorbol esters reduce the levels of the cognate mRNA for the LH/CG receptor. Endocrinology 128:26512653 Rebecchi MJ, Gerry RH, Gershengorn MC 1982 Thyrotropin-releasing hormone causes loss of cellular calcium without calcium uptake by rat pituitary cells in culture. Studies using arsenazo III for direct measurement of calcium. J Biol Chem 257:2751-2753 MacDonald RJ, Swift GH, Przybyla AE, Chirgwin JM 1987 Isolation of RNA using guanidinium salts. Methods Enzymol 152:219-227 Peppel K, Baglioni C 1990 A simple and fast method to extract RNA from tissue culture cells. Biotechniques 9:711-713 Sambrook J, Fritsch EF, Maniatis T 1989 Molecular Cloning-A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor Williams DL, Newman TC, Shelness GS, Gordon DA 1986 Measurement of apolipoprotein mRNA by DNA-excess solution hybridization with single-stranded probes. Methods Enzymol 128:671-689
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Jiro Fujimoto, Richard E. Straub*, and Marvin C. Gershengorn Division of Endocrinology and Metabolism Departments of Medicine and Cell Biology and Anatomy New York Hospital Cornell University Medical College New York, New York 10021
In a previous report we showed that TRH-induced down-regulation of the density of its receptors (TRHRs) on rat pituitary tumor (GH3) cells was preceded by a decrease in the activity of the mRNA for the TRH-R, as assayed in Xenopus oocytes. Here we report the effects of TRH, elevation of cytoplasmic free Ca2+ concentration, phorbol myristate acetate (PMA), and H-7 [1-(5-isoquinolinesulfonyl)2-methylpiperazine dihydrochloride], an inhibitor of protein kinases, on the levels of TRH-R mRNA, which were measured by Northern analysis and in nuclease protection assays using probes made from mouse pituitary TRH-R cDNA, in GH3 cells. These agents were studied to gain insight into the mechanism of the TRH effect, because signal transduction by TRH involves generation of inositol 1,4,5-trisphosphate and elevation of cytoplasmic free Ca 2+ concentration, which leads to activation of Ca2+/calmodulindependent protein kinase, and of 1,2-diacylglycerol, which leads to activation of protein kinase-C. TRH (1 HM TRH, a maximally effective dose) caused a marked transient decrease in TRH-R mRNA that attained a nadir of 20-45% of control by 3-6 h, increased after 9 h, but was still below control levels after 24 h. Elevation of the cytoplasmic free Ca2+ concentration had no effect on TRH-R mRNA. A maximally effective dose of PMA (1 MM) caused decreases in TRH-R mRNA that were similar in magnitude and time course to those induced by 1 MM TRH. H-7 (20 MM) blocked the effects of TRH and PMA to lower TRH-R mRNA to similar extents. These data show that TRH and PMA decrease the levels of TRH-R mRNA in GH3 cells and are consistent with the idea that the effect of TRH is via a mechanism 0888-8809/91/1527-1532$03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society
that is mediated by protein kinase-C. (Molecular Endocrinology 5: 1527-1532, 1991)
INTRODUCTION Down-regulation of cell surface receptor density is an important mechanism for modulation of cell responsiveness. The number of receptors may be decreased by the action of its own ligand (homologous down-regulation) or by the action of other extracellular regulatory factors that interact with other cell receptors (heterologous down-regulation). It has been suggested that one mechanism of receptor down-regulation may involve internalization (1, 2) and perhaps subsequent intracellular degradation, another may involve receptor phosphorylation (3), and a third may be inhibition of the synthesis of receptors secondary to decreases in receptor mRNA levels (4-7). These mechanisms may be activated together to produce the final effect. TRH down-regulates the number of TRH receptors (TRHRs) on anterior pituitary cells (8, 9), and we have shown that this is preceded in rat pituitary tumor (GH3) cells by a decrease in the activity of the mRNA for the TRHR, as assayed in Xenopus laevis oocytes (10). The molecular mechanism(s) that mediates the decrease in TRH-R mRNA activity is unknown, but the cellular response to TRH has been shown to involve activation of at least two protein kinases, protein kinase-C (PKC) (11,12) and Ca2+/calmodulin-dependent protein kinase (13, 14). In the experiments reported here, we studied whether the decreases in TRH-R mRNA activity caused by TRH were secondary to decreases in mRNA levels, using probes developed using a mouse pituitary TRHR cDNA (15), and investigated a role for PKC and Ca 2+ / calmodulin-dependent protein kinase in mediating TRHinduced decreases in TRH-R mRNA in GH3 cells.
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MOL ENDO-1991 1528
Vol5No. 10
RESULTS AND DISCUSSION
TRH has been shown to down-regulate the number of TRH-Rs on GH3 cells in a time-dependent manner, with a half-maximal effect by 18 h and a maximal decrease by 30 h (8,10). We showed previously that TRH causes a transient decrease in TRH-R mRNA activity, as assayed in Xenopus oocytes, that attains a nadir between 3-6 h and increases toward the control value thereafter (10). Figure 1 {left panel) illustrates the effect of a maximally effective dose of TRH (1 ^M) on TRH-R mRNA levels assayed by Northern analysis in GH3 cells. A single dominant mRNA species of approximately 3.8 kilobases was observed. TRH caused a marked transient decrease in the TRH-R mRNA level, which attained a nadir by 3 h, remained low for an additional several hours, and then increased again after 6 h. To permit measurement of the changes in the levels of TRH-R mRNA more readily, we established a RNAase protection assay. Using this assay we confirmed that TRH caused decreases in TRH-R mRNA levels (Fig. 2). These data show that the previously reported decreases in endogenous TRH-R mRNA activity assayed in Xenopus oocytes (10) caused by TRH were due to decreases in the level of TRH-R mRNA. Signal transduction by TRH in GH3 cells uses the phosphatidylinositol 4,5-bisphosphate pathway (1618). Stimulation by TRH causes an inositol 1,4,5-trisphosphate-mediated elevation of the cytoplasmic free Ca2+ concentration, which leads to activation of Ca2+/ calmodulin-dependent protein kinase, and generation of 1,2-diacylglycerol leads to activation of PKC. Because signal transduction by TRH involves activation of PKC and activation of PKC by phorbol esters has been shown to affect the level of mRNAs for other receptors in other cell types (19), we studied the effects of phorbol myristate acetate (PMA) on TRH-R mRNA levels. Figures 1 and 2 illustrate the effect of 1 HM PMA on TRH-
TRH
PMA
a-actin C
1
3
5
16
C
3
6
16
Hours Fig. 1. Effects of TRH and PMA on TRH-R mRNA, as Analyzed by Northern Hybridization GH3 cells were incubated for the times indicated with 1 ^M TRH (left) or 1 HM PMA (right), poly(A)+-enriched RNA was isolated, and 70 or 33 ng, respectively, were analyzed. a-Actin mRNA was probed on the same filters. Data are from one of three similar experiments. C, Control.
3
6
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24
Time (hr) Fig. 2. Effects of TRH and PMA on TRH-R mRNA, as Measured in a RNAase Protection Assay GH3 cells were incubated for the times indicated with 1 HM TRH (•) or 1 HM PMA (•), total RNA was isolated, and 50 or 100 ^g were assayed. Data are the mean ± SE from five experiments.
R mRNA levels. PMA caused a decrease in TRH-R mRNA, with a time course similar to that of TRH. In these sets of experiments, TRH was more effective than PMA in decreasing TRH-R mRNA; TRH and PMA caused TRH-R mRNA to decrease to 20% and 45% of the control value, respectively. However, in other experiments, the effects of TRH and PMA were of similar magnitude (see below). These data demonstrate that TRH and PMA have similar effects on TRH-R mRNA and are consistent with the idea that the effect of TRH may be mediated by the lipid limb of the TRH signal transduction pathway via activation of PKC. To study more directly whether the effect of TRH to decrease TRH-R mRNA may be mediated by PKC, we used two approaches. We incubated GH3 cells with 1 fiM PMA for more than 16 h to deplete them of PKC (20), and we used H-7 ([1-(5-isoquinolinesulfonyl)2methylpiperazine dihydrochloride]), which is a protein kinase inhibitor (21). We have shown that GH3 cells pretreated with 1 HM PMA have reduced PKC activity, a reduced secretory response to phorbol esters, and show inhibition of the second phase of the secretory response to TRH, which is thought to be mediated by PKC (22). Nevertheless, it is possible that some actions of TRH will not be affected by PMA pretreatment even if they are mediated by PKC, because a major PKC isoenzyme in GH3 cells, PKCa, is not depleted by prolonged incubation with PMA (23, 24) (our unpublished data). Figure 3 illustrates the effects of TRH on TRH-R mRNA levels in cells preincubated with 1 HM PMA for 24 h, in which the acute effect of PMA was inhibited; TRH-R mRNA was 95.5 ± 1.5% (mean ± SE) of the control value in PMA-pretreated cells exposed to 1 IXM PMA for an additional 3 or 6 h. The effect of TRH to decrease TRH-R mRNA after 3 and 6 h was not inhibited. Hence, an attempt to deplete GH3 cells of PKC did not block the effect of TRH on TRH-R mRNA. In contrast, H-7, which by itself lowered TRH-R mRNA
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1529
TRH Receptor mRNA
o -£ 100 o o
Control
"o
75
PMA —pretreated
(pg)
1
1000
•
Fig. 6. Nuclease Protection Assay for the Measurement of TRH-R mRNA The assay was performed, as described in Materials and Methods, using 200 U RNAase-T1/RNAase per tube. Mouse TRH-R RNA transcribed in vitro and GH3 cell RNA were assayed in parallel. Left panel, Autoradiograms of gel eletrophoresis. Right panel, Plots of densitometric analysis.
obases detectable in GH3 cells under all conditions examined. This does not exclude increased degradation as the mechanism for lowering TRH-R mRNA, however, as it is not unusual not to be able to detect mRNA degradation products in mammalian cells even under conditions of enhanced turnover (27,28). We attempted to determine the molecular pathway involved in this effect. We found no evidence that the Ca2+ limb of this pathway is involved in regulating the level of TRH-R mRNA. In contrast, we developed evidence that PKC appears to mediate this effect. Regulation of the mRNA levels of G-protein-coupled receptors appears to be a general phenomenon. For example, the mRNA for 02-adrenergic receptors, which couple to the adenylyl cyclase-cAMP pathway, is regulated in a biphasic manner by &>-adrenergic agonists in hamster DDTT MF-2 cells (29-31). There is a rapid (during the first 2 h) increase in mRNA, which appears to be secondary to increased transcription, followed by a decrease to below control levels after 4 h, which may be secondary to increased degradation. In DDTT MF-2 cells (4) and Chinese hamster fibroblasts transfected with the human j82-adrenergic receptor (3), the effect on /32-adrenergic receptor mRNA appears to be mediated by its second messenger, cAMP. The mRNAs for the receptors for TSH (6; but see Ref. 32) and LH/ CG (33), which also couple to adenylyl cyclase are down-regulated in a cAMP-mediated manner. Izzo, Jr., and colleagues (7) showed that an-adrenergic agonists, whose receptors, like those of TRH, are coupled by Gproteins to the phosphatidylinositol 4,5-bisphosphateCa2+-PKC signal transduction pathway, caused a transient decrease in a r adrenergic receptor mRNA in rabbit aortic smooth muscle cells. The time course and magnitude of this effect are similar to the decreases in TRH-
R mRNA caused by TRH and PMA in GH3 cells. Although Izzo and colleagues did not show that the effect of a r adrenergic agonists to decrease receptor mRNA was mediated by PKC (7), in other systems (34, 35) phorbol ester activation of PKC has been implicated in regulation of mRNAs. For example, phorbol esters have recently been shown to decrease the mRNA for the LH/CG receptor (36). Hence, regulation of the mRNAs for G-protein-coupled receptors appears to occur commonly and to involve both homologous and heterologous second messenger-mediated pathways. In conclusion, we showed that TRH decreases TRHR mRNA by a mechanism that appears to be mediated by PKC. Although these data are consistent with the idea that decreases in TRH-R density are secondary to a lowered rate of receptor synthesis, they do not show a cause and effect relationship. Moreover, other mechanisms, such as enhanced degradation of TRH-R protein, may also be causally important in the down-regulation process. Indeed, phosphorylation by PKC of the TRH-R and of a protein(s) that regulates TRH-R mRNA degradation may coordinately down-regulate TRH-R number.
MATERIALS AND METHODS GH3 cells were grown and harvested, as described previously (37), in 100-mm dishes. Cells were exposed to 1 MM TRH, 1 IIM PMA, 25 mM KCI, 25 rriM NaCI, 20 MM H-7, or a combination thereof in growth medium for the times indicated. Total cellular RNA was isolated using guanidine thiocyanate and centrifugation through CsCI (38) or using sodium dodecyl sulfate, potassium acetate, and acetic acid (39). The integrity of ribosomal RNA was assessed by gel electrophoresis. Poly(A)+enriched RNA was obtained by one round of chromatography
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TRH Receptor mRNA
on oligo(dT)-cellulose. For Northern hybridization analysis, 3070 fig poly(A)+-enriched RNA were electrophoresed on a 1.2% agarose gel containing 2 M formaldehyde. After capillary transfer, the Duralon-UV membrane (Stratagene, La Jolla, CA) was hybridized using 2 x 106 cpm/ml probe. The probe was the 3.8-kilobase mouse TRH-R cDNA insert that was excised from pBSmTRHR with restriction endonucleases Kpn\ and Sacll (or A/ofl) (15), gel purified, electroeluted, and labeled with [32P] CTP to a specific activity of 3.7 x 108 cpm/^tg by random priming (40). a-Actin cDNA, a gift from Dr. Peter Guidon of the Hospital for Special Surgery, was also labeled and used as a control in some blots. Hybridization with 32P-labeled cDNA probes was performed overnight at 42 C in buffer containing 750 ITIM NaCI, 50 mM Na phosphate (pH 7.0), 5 mM EDTA, 5 x Denhardfs reagent, 50 fig/rr\\ salmon sperm DNA, 10% dextran sulfate, and 50% formamide. Final washes were performed with 30 mM NaCI, 2 mM Na phosphate (pH 7.0), and 0.2 mM EDTA at 65 C. Autoradiograms were exposed for 4 96 h. A nuclease protection assay (41) was established. The cRNA probe was transcribed from a plasmid containing a 430base Sg/ll-8g/ll fragment of pBSmTRHR (15) cloned into the 8amHI site of plasmid Bluescript (Stratagene), digested with ftsal, transcribed using T3 polymerase with [32P]UTP, and purified on a 6% urea polyacrylamide gel. This antisense probe contained 170 bases complementary to the open reading frame of the TRH-R and 35 bases complementary to vector DNA. Sample RNA (25-100 Mg) and [32P]cRNA (2.3 x 105 cpm) were coprecipitated; redissolved in buffer containing 60% formamide, 900 mM NaCI, 6 mM EDTA, and 60 mM Tris-HCI (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 MgCI2,10 mM Na acetate (pH 5.0), and 200 U RNAase-T1 and incubated at 37 C for 45 min. The sample was extracted with phenol-chloroform, precipitated with ethanoi, 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. Figure 6 shows a standard assay in which increasing amounts of mouse TRH-R RNA transcribed in vitro and total GH3 cell RNA were measured. The assay was linear with mouse TRH-R RNA up to 1000 pg and with GH3 cell RNA up to 50 Mg (up to 200 ng in other assays). Materials TRH was purchased from Beckman (Palo Alto, CA). Guanidine thiocyanate was obtained from Fluka BioChemika (Ronkonkoma, NY). Restriction endonucleases were purchased from Boehringer Mannheim (Indianapolis, IN) and New England Biolabs (Beverley, MA). H-7 was obtained from Seikagaku America (Rockville, MD). PMA and other chemicals were ultrapure grade for molecular biology and were purchased from Sigma (St. Louis, MO).
Acknowledgments We thank C. S. Narayanan for preparing the plasmid used to generate the cRNA for the nuclease protection assay.
Received June 6,1991. Rerevision received July 31,1991. Accepted July 31, 1991. Address requests for reprints to: Dr. Marvin C. Gershengorn, Room A328, 1300 York Avenue, New York, New York 10021. This work was supported by NIH Grant DK-43036. * Present address: Molecular Genetics Laboratory, Columbia University College of Physicians and Surgeons, Box 58, 722 West 168th Street, New York, New York 10032.
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