Regulation of 5-HT2A receptor mRNA in P11 cells

ELSEVIER

Behavioural Brain Research 73 (1996) 187-191

B[HAVIOURAL BRAIN RESEARCH

Regulation of 5 - H T 2 A receptor mRNA in P l l cells Robert C. Ferry, Perry B. Molinoff * Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6084, USA

Abstract

Pll cells were used as an in vitro model system to explore effects of exposure to 5-HT on levels of mRNA encoding 5-HT2A receptors. Exposure of cells to the agonist 5-HT resulted in a doubling of receptor mRNA levels, mRNA levels returned to control levels within 8-16 h. The stability of receptor mRNA transcripts was transiently increased, suggesting that a post-transcriptional process was responsible for the up-regulation of receptor mRNA. The 5-HT-induced increase in Levels on 5-HT/A receptor mRNA did not require de novo protein synthesis since the increase was not affected by prior treatment with the protein synthesis inhibitor cycloheximide. Results of studies in which the activity of protein kinase C was either increased with PMA or antagonized with bisindolylmaleimide indicated that protein phosphorylation was essential for increasing levels of 5-HT2A receptor mRNA. These findings suggest that PKC-dependent post-transcriptional and post-translational processes participate in regulating 5-HT2Areceptor mRNA expression.

Keywords: ActinomycinD; Cycloheximide;Phorbol ester; Post-transcriptional;Protein kinace C; Ribonucleaseprotection assay; Serotonin(5-HT); PMA, phorbol 12-myristate 13-acetate;PI, phosphoinositide;PKC, protein kinase C; 5-HT, 5-hydroxytryptamine(serotonin)

1. Introduction

Responses of a cell to external stimuli can be modulated by regulating the properties or density of receptors through which signals are transmitted. Characterization of regulatory phenomena that affects expression of 5-HT receptors has been complicated by identification of an increasing number of receptor subtypes that may activate the same or distinct second messenger systems, bind drugs and radioligands with overlapping specificity, and share similar patterns of distribution [ 13]. The 5-HT2A receptor is one of three subtypes which comprise the 5-HT 2 family of 5-HT receptors according to a nomenclature system that has recently been introduced [22]. 5-HT2A, 5-HT2B, and 5-HTzc receptors share greater than 60% amino acid sequence homology, have introns that interrupt their coding sequence, and couple preferentially to activation of phospholipase C. The members of this receptor family have similar affinities for a number of radioligands and this has complicated studies of the receptors carried out in vivo. * Corresponding author. Present address: CNS Drug Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway, Wallingford,CT 06492,USA. Fax: + 1 203 284-6127. E-mail:[email protected] 0166-4328/96/$09.50 © 1995 ElsevierScienceB.V. All rights reserved

SSDI 0166-4328 ( 96 ) 00094-1

Cells in culture expressing a single subtype of receptor represent simplified model systems with which to study regulation of receptor expression. P11 cells are a permanent cell line derived from the prolactin-secreting transplantable rat pituitary tumor 7315a [ 14]. Studies in our laboratory using P l l cells have demonstrated that the density of 5-HTzA receptors is decreased following exposure to agonists and partial agonists, as has been observed for 5-HTzA receptors in vivo [9]. To determine whether exposure to an agonist affects 5-HTaA receptor mRNA expression in P l l cells, we used a ribonuclease protection assay to measure levels of mRNA encoding the receptors following challenge with the endogenous neurotransmitter 5-HT.

2. Results and discussion

A recombinant vector containing cDNA encoding the i3 loop of the 5-HT2A receptor was generated using standard molecular biological techniques and used as a template to synthesize an antisense riboprobe specific to mRNA encoding the 5-HT2A receptor (Fig. 1A). Hybridization of this riboprobe to total RNA isolated from P l l cells and subsequent digestion with single-

188

Robert C Ferry, Perry B. Mol&off/BehaviouralBrain Research 73 (1996) 187-191

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Quantitate 5-HT2A protected probe with phosphorimage storage technology Fig. 1. Schematic diagram of the ribonuclease protection assay used to measure 5-HT2A receptor mRNA. A: cDNA encoding most of the i3 loop of the rat 5-HT:A receptor was amplified from a full-length receptor cDNA clone using the polymerase chain reaction. This fragment was ligated into the cloning vector pGEM7Z. Antisense radiolabeled transcripts generated from linearized plasmids using SP6 bacterial RNA polymerase were hybridized to total RNA isolated from Pll cells. Subsequent digestion of unhybridized riboprobes and size fractionation on polyacrylamide gels revealed a protected radiolabeled fragment whose intensity corresponded to the level of input 5-HTzA receptor mRNA. Quantification of the intensity of the radiolabeled fragment was accomplished using a BioRad phosphorimager. B: protected 5-HT2Aand cyclophilin riboprobe fragments following overnight hybridization with total RNA isolated from Pll cells and subsequent digestion with RNaseT1 and RNaseA. Lane 1, vehicletreated cells; lane 2, cells treated with 10 p,M 5-HT for 90 min.

strand nucleases resulted in a radiolabeled fragment of 183 bases (Fig. 1). A second riboprobe specific for the cytoskeletal gene cyclophilin was added to permit normalization of the 5-HT2A signal. Levels of cyclophilin were not altered by exposure to drugs used in our studies verifying that it was appropriate as an internal standard. Hybridization of riboprobes to full-length 5-HT2A receptor m R N A or total R N A from 5-HT2A receptor-rich tissue from rat forebrain served as positive controls,

while hybridization to yeast R N A or samples containing only buffer were negative controls [8]. Exposure to 5 - H T for 90 min resulted in a 2-fold increase in levels of 5-HT2A receptor m R N A (Fig. 1B). This effect could be prevented by co-incubation with the antagonist ketanserin (Table 1) confirming that the effect was receptor-mediated. Examination of the time course of effects of 5-HT on levels of receptor m R N A revealed that the effect of agonist was transient (Fig. 2). Similar

Robert C. Ferry, Perry B. Molinoff/Behavioural Brain Research 73 (1996) 187-191 Table 1 Regulation of levels of 5-HT2A receptor mRNA in P l l cells by agonists and modulators of protein kinase C

1 2 3 4 5 6

Treatment

5-HTzA receptor mRNA (% control)

Vehicle 5-HT (10 ~tM) 5-HT+Ketanserin (1 ktM) PMA (100 nM) 5-HT + bisindolylmaleimide (5 ~tM) 6-Fluoronorepinephrine (100 ~tM)

100 + 5 186+6 a 105+7 214+2 a 106+ 1 120_+ 1b

P l l cells were treated with drugs as indicated for 90 min. Ketanserin, a 5-HT2A receptor antagonist, or bisindolylmaleimide (Calbiochem, La Jolla, CA), a selective protein kinase C inhibitor, were added 15 min prior to the addition of 5-HT. Total RNA was isolated and 5-HTzA receptor mRNA measured using a ribonuclease protection assay. Data shown represent three or more determinations and represent means+_S.E. Ketanserin and bisindolylmaleimide had no effect on 5-HTzA receptor mRNA (data not shown) aP<0.001, vs. vehicle control.

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Fig. 2. Effect of agonist exposure on levels of 5-HTzA receptor mRNA. P l l cells were treated with 10 gM 5-HT for varying periods of time as indicated. Corresponding groups of cells were treated with vehicle to serve as controls. Total RNA was extracted, and 5-HT2A receptor mRNA levels were quantified using a ribonuclease protection assay. The data were normalized by comparison to the intensity of a cyclophilin signal in the same lane to control for variations in RNA loading. Data are means+_S.E, of three or more determinations. * P < 0.005 vs. matching vehicle control.

increases in levels of ill- [ 12] and fl2-adrenergic receptor mRNA [5] have been seen in rat C6 glioma and hamster DDT1 MF-2 cells, respectively, following exposure to the agonist isoproterenol. The increase in 5-HT2A receptor mRNA was greatest after 1.5 h and returned to basal levels within 8-16 h. ~r-Adrenergic receptors are present on P l l cells and are also coupled to PI hydrolysis [9,15]. Incubation of cells with the el-adrenergic receptor agonist 6-fluoronorepinephrine resulted in a heterologous increase in levels of 5-HTzA receptor mRNA (Table 1), potentially implicating metabolites of PI turnover in mediating the effect on 5-HTzA receptor mRNA. This observation suggests that PI-coupled receptor systems

189

can communicate with one another at the level of mRNA expression. Generation of cAMP following stimulation of flz-adrenergic receptors coupled to activation of adenylyl cyclase has been shown to be important in protein kinase A-dependent transcriptional regulation of 132-adrenergic receptor mRNA [4], demonstrating that intracellular mediators activated just down-stream of receptor activation can participate in homologous regulation of receptor mRNA. 5-HTzA receptors activate phospholipase C which metabolizes phosphatidylinositol bisphosphate to generate diacylglycerol and inositol trisphosphate, which directly and indirectly activate protein kinase C. In P11 cells, activation of protein kinase C with the phorbol ester PMA mimicked the effects of agonists on receptor mRNA levels, whereas bisindolylmaleimide, a potent and selective inhibitor of protein kinase C, blocked the 5-HT-induced increase in levels of receptor mRNA (Table 1). Thus, in a manner analogous to changes in flz-adrenergic receptor mRNA, second messengers produced as a result of 5-HT2A receptor activation participate in a protein kinase-dependent, feed-forward mechanism that positively regulates 5-HTzA receptor mRNA levels. Protein kinase C also appears to regulate 5-HT2A receptor-mediated responses in P l l cells through a negative feedback mechanism since exposure to PMA for 15 min completely desensitizes 5-HTzA receptor-mediated PI hydrolysis (data not shown). Protein kinase C has been implicated in desensitization of 5-HTzA receptor-mediated PI turnover in rat aortic smooth muscle cells [23]. The recent identification of consensus transcription factor binding sites for AP-1, AP-2, and SP-1 protein complexes in the promoter region of the 5-HTzA receptor gene [6], and the observed dependence on PKC for agonist-induced increases in levels of receptor mRNA, raised the possibility that the increase in mRNA was due to a PKC-mediated increase in the rate of transcription of the 5-HT2A receptor gene. Nuclear run-on assays revealed no change in the rate of transcription of the receptor gene [-8] from 5 min to 2.5 h after addition of 5-HT. In other experiments levels of receptor mRNA were measured at various times after the addition of the transcription inhibitor actinomycin D to determine the half-life of receptor mRNA. Exposure of P l l cells to 5-HT for 45 min resulted in a 2-fold increase in receptor mRNA stability; receptor mRNA half-life was 142 min in 5-HT-treated cells and 71 min in control cells [8]. This increase in mRNA stability is sufficient to account for the 2-fold increase in levels of receptor mRNA that was observed. Stability returned to basal levels after exposure to 5-HT for 2.5 h I-8] which accounts for the transient nature of the increase. The present observation of an increase in receptor mRNA levels accompanied by an increase in the stability of receptor mRNA is the first demonstration of a system in which the stability of mRNA for a neurotransmitter

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Robert C Ferry, Perry B. Molinoff/Behavioural Brain Research 73 (1996) 187-191

receptor is increased following exposure to agonists. Studies of cq-adrenergic [16], ml-muscarinic [20], and /~2-adrenergic receptors [11] have revealed agonistinduced decreases in receptor mRNA stability. Consensus AUUUA pentameric motifs for docking of mRNA-binding proteins which alter the stability of mRNA transcripts have been identified in the 3' untranslated region (UTR) of mRNAs encoding the GM-CSF, c-fos, and IL-3 proteins [10], as well as in mRNA encoding the /?2-adrenergic receptor [21]. A possibly related AU-rich region is present in the 3' UTR of the 5-HT/A receptor (Roland Ciaranello, personal communication), and several putative 5-HTzAreceptor mRNA binding proteins have been identified [18]. Cycloheximide did not prevent 5-HT from increasing receptor mRNA levels; exposure to 5-HT for 1 h caused a 60% increase in 5-HT2A receptor mRNA in both vehicle-treated cells and cells that had been pretreated with the protein synthesis inhibitor cycloheximide for 30 min (data not shown). Thus, 5-HT-induced increases in levels of receptor mRNA did not require synthesis of new proteins (a translational event) but rather was dependent on activation or recruitment of proteins present in the cell prior to agonist challenge (a posttranslational event). A post-translational process involving phosphorylation of preexisting protein(s) by protein kinase C is consistent with the present data which demonstrates that PKC is required for agonist-induced increases in receptor mRNA. Results of studies of the regulaton of 5-HT2A receptor mRNA in P l l cells differ in several instances from those observed in primary cell culture systems. In cerebellar granule cells and in myometrial smooth muscle cells, exposure to 5-HT results in a 1.5-fold [1] or 4-fold [24] increase in levels of receptor mRNA which persists for at least 24 h. Dependence of the increase in receptor mRNA on preexisting proteins also varies among cell lines because 5-HT- and DOI-stimulated increases in receptor mRNA in cerebellar granule [ 1] and myometrial cells [24] were reported to be sensitive to cycloheximide, whereas the 5-HT-induced increase in receptor mRNA in P l l cells is insensitive to cycloheximide. In myometrial smooth muscle cells agonist-induced increases in levels of receptor mRNA were accompanied by an increase in the rate of transcription as assessed by nuclear run-on assays rather than a change in receptor mRNA stability. Recent identification of a variety of consensus sites for transcription factor binding in the promoter of the 5-HT2A receptor gene, as well as 5-HTinduced promotor activation [6] will further our understanding of the regulation of 5-HTzA receptor mRNA expression. 3. Conclusions and future directions

Because varied responses to agonist challenge have been observed at the level of mRNA encoding 5-HTzA

receptors, no general rule regarding regulation of 5-HTzAreceptor mRNA has emerged. The data currently available suggest that multiple pathways regulate receptor mRNA expression, and that receptor mRNA may be regulated by cell-specific processes. Regulation of levels of receptor mRNA in P11 cells by hydrolysis of phosphoinositides and subsequent activation of a protein kinase C-dependent, post-transcriptional mechanism represents one of these pathways (Fig. 3). Arachidonic acid [7], Ca 2+ [17], and cAMP [2] should also be considered as candidate modulators of 5-HTEA receptor mRNA since 5-HTzA receptors also regulate these intracellular messengers. The observed ability of a heterologous receptor system (~l-adrenergic receptors) to regulate mRNA for 5-HTzA receptors and evidence of allele-specific expression of 5-HT2Areceptors [25] reveal additional levels of complexity in the control of 5-HTzA receptor expression. The importance of understanding processes which control 5-HTEA receptor

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(~~AAAAA 5-HT2A receptor mRNA Fig. 3. Transduction pathways involved in mediating agonist-induced increases in levels of 5-HTzA receptor mRNA in P l l cells. Exposure of P l l cells to 5-HT or the ~l-adrenergic receptor agonist 6-fluoronorepinephrine results in increases in levels of 5-HT2A receptor mRNA. Activation of PI hydrolysis via homologous (5-HT2A) or heterologous (%-adrenergic) receptor systems coupled to heterotrimeric G proteins leads to activation of phospholipase C (PLC) and subsequent cleavage of phosphatidylinositol bisphosphate (PIP2) to yield the second messengers diacylglycerol (DAG) and inositol trisphosphate (IP3). This ultimately results in increases in levels of 5-HT2A receptor mRNA. The ability of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) to mimic the effects of the agonists and of the PKC inhibitor bisindolylmaleimide to antagonize the effects of 5-HT indicates that PKC is required for the increase in receptor mRNA levels. PKC, either directly or through one or more intermediate steps, may regulate an mRNA binding protein since the effects of agonists on receptor mRNA were accompanied by changes in the stability of the receptor mRNA transcripts.

Robert C Ferry, Perry B. Molinoff/Behavioural Brain Research 73 (1996) 187 191

expression is suggested by the number of psychiatric illnesses in which 5-HT2A receptor density is altered [3,19,26] and the appearance of developmental abnormalities in 5-HT2A receptor-knockout mice [25].

Acknowledgment " The authors wish to thank Lihui Lu for expert technical assistance. This work was supported by USPHS grants MH 48125 and NS 18591.

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[10] Gillis, P. and Malter, J.S., The adenosine-uridine binding factor recognizes the AU-rich elements of cytokine, lymphokine, and oncogene mRNAs, J. Biol. Chem., 266 (t991) 3172-3177. [ 11] Hadcock, J.R., Wang, H.Y. and Malbon, C.C., Agonist-induced destabilization of fl-adrenergic receptor mRNA: attenuation of glucocorticoid-induced up-regulation of fl-adrenergic receptors, J. Biol. Chem., 264 (1989) 19928-19933. [12] Hosoda, K., Feussner, G.K., Rydelek-Fitzgerald, L., Fishman, P.H. and Duman, R.S., Agonist and cyclic AMP-mediated regulation of fll-adrenergic receptor mRNA and gene transcription in rat C6 glioma cells, J. Neurochem., 63 (1994). [13] Humphrey, P.P.A., Hartig, P. and Hoyer, D., A proposed new nomenclature for 5-HT receptors, Trends Pharmacol. Sci., 14 (1993) 233-236. [ 14] Ivins, K.J. and Molinoff, P.B., Serotonin-2 receptors coupled to phoshoinositide hydrolysis in a clonal cell line, Mol. Pharmacol., 37 (1990) 622 630. [15] Ivins, K.J. and Molinoff, P.B., Desensitization and down-regulation of 5-HT2 receptors in P l l cells, J. Pharmacol. Exp. Ther., 259 (1991) 423 429. [16] Izzo, N.J., Seidman, C.E., Collins, S. and Colucci, W.S., cq-Adrenergic receptor mRNA level is regulated by norepinephrine in rabbit aortic smooth muscle cells, Proc. Natl. Acad. Sei USA, 87 (1990) 6268-6271. [17] Kagaya, A., Mikuni, M., Kusumi, I., Yamamoto, H. and Takahashi, K., Serotonin-induced acute desensitization of serotonin2 receptors in human platelets via a mechanism involving protein kinase C, J. Pharmacol. Exp. Ther., 255 (1990) 305-311. [18] Kao, H.T. and Ciaranello, R.D., A brain-specific protein that binds to 5-HTlc and 5-HT2 mRNA, Soc. Neurosci. Abstr., 20:2 (1994) 1267. [19] Laruelle, M., Abi-Dargham, A., Casanova, M.F., Toti, R., Weinberger, D.R. and Klienman, J.E., Selective abnormalities of prefontal serotonergic receptors in schizophrenia, Arch. Gen. Psychiatry, 50 (1993) 810-818. [20] Lee, N.H., Earle-Hughes, J. and Fraser, C.M., Agonist-mediated destabilization of ml muscarinic acetylcholine receptor mRNA, J. Biol. Chem., 269 (1994) 4291-4298. [21] Ling-Yan, H., Tholanikunnel, B.G., Vakalopoulou, E. and Malbon, C.C., The Mr 35,000 fl-adrenergic receptor mRNA-binding protein induced by agonists requires both an AUUUA pentamer and U-rich domains for RNA recognition, J. Biol. Chem., 268 (1993) 25769-25775. [22] Martin, G.R. and Humphrey, P.P.A., Receptors for 5-hydroxytryptamine: current perspectives on classification and nomenclature, Neuropharmacology, 33 (1994) 261-273. [23] Roth, B.L., Nakaki, T., Chuang, D.-M. and Costa, E., 5-Hydroxytryptamine2 receptors coupled to phospholipase C in rat aorta: modulation of phosphoinositide turnover by phorbol ester, J. Pharmacol. Exp. Ther., 238 (1986) 480-485. [24] Rydelek-Fitzgerald, L., Wilcox, B.D., Teitler, M. and Jeffrey, J.J., Serotonin-mediated 5-HT2 receptor gene regulation in rat myometrial smooth muscle cells, Mol. Cell. Endoerinol., 92 (1993) 253-259. [25] Toth, M., Benjamin, D. and Shenk, T., Targeted disruption of the 5-HT2 receptor gene results in developmental abnormalities in mice, 5-HT Third IUPHAR Satellite Meeting on Serotonin, Abstract (1994) 37. [26] Yates, M., Leake, A., Candy, J.M., Fairbairn, A.F., McKeith, I.G. and Ferrier, I.N., 5-HT2 receptor changes in major depression, Biol. Psychiatry, 27 (1990) 489-496.